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We previously reported that pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, significantly ameliorated Alzheimer’s disease (AD)-associated cognitive defects in APP/PS1 and SAMP8 mice by inhibiting Aβ aggregation and tau hyperphosphorylation, suggesting a potential therapeutic effect of PF11 in the treatment of AD. In the present study we further evaluated the therapeutic effects of PF11 on relieving cognitive impairment in a rat model of sporadic AD (SAD). SAD was induced in rats by bilateral icv infusion of streptozotocin (STZ, 3 mg/kg). The rats were treated with PF11 (2, 4, 8 mg·kg(−1)·d(−1), ig) or a positive control drug donepezil (5 mg·kg(−1)·d(−1), ig) for 4 weeks. Their cognitive function was assessed in the nest building, Y-maze, and Morris water maze tests. We showed that STZ icv infusion significantly affected the cognitive function, tau phosphorylation, and insulin signaling pathway in the hippocampus. Furthermore, STZ icv infusion resulted in significant upregulation of the calpain I/cyclin-dependent protein kinase 5 (CDK5) signaling pathway in the hippocampus. Oral administration of PF11 dose-dependently ameliorated STZ-induced learning and memory defects. In addition, PF11 treatment markedly reduced the neuronal loss, protected the synapse structure, and modulated STZ-induced expression of tau phosphorylation by regulating the insulin signaling pathway and calpain I/CDK5 signaling pathway in the hippocampus. Donepezil treatment exerted similar beneficial effects in STZ-infused rats as the high dose of PF11 did. This study highlights the excellent therapeutic potential of PF11 in managing AD.

Background Microglial activation is a prominent feature of neuroinflammation, which is present in almost all neurodegenerative diseases. While an initial inflammatory response mediated by microglia is considered to be protective, excessive pro-inflammatory response of microglia contributes to the pathogenesis of neurodegeneration. Although autophagy is involved in the suppression of inflammation, its role and mechanism in microglia are unclear. Methods In the present study, we studied the mechanism by which lipopolysaccharide (LPS) affects microglial autophagy and the effects of autophagy on the production of pro-inflammatory factors in microglial cells by western blotting, immunocytochemistry, transfection, transmission electron microscopy (TEM), and real-time PCR. In a mouse model of neuroinflammation, generated by intraventricular injection of LPS (5 μg/animal), we induced autophagy by rapamycin injection and investigated the effects of enhanced autophagy on microglial activation by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Results We found that autophagic flux was suppressed in LPS-stimulated N9 microglial cells, as evidenced by decreased expression of the autophagy marker LC3-II (lipidated form of MAP1LC3), as well as increased levels of the autophagy adaptor protein SQSTM1. LPS significantly decreased Vps34 expression in N9 microglial cells by activating the PI3KI/AKT/MTOR pathway without affecting the levels of lysosome-associated proteins and enzymes. More importantly, overexpression of Vps34 significantly enhanced the autophagic flux and decreased the accumulation of SQSTM1 in LPS-stimulated N9 microglial cells. Moreover, our results revealed that an LPS-induced reduction in the level of Vps34 prevented the maturation of omegasomes to phagophores. Furthermore, LPS-induced neuroinflammation was significantly ameliorated by treatment with the autophagy inducer rapamycin both in vitro and in vivo. Conclusions These data reveal that LPS-induced neuroinflammation in N9 microglial cells is associated with the inhibition of autophagic flux through the activation of the PI3KI/AKT/MTOR pathway, while enhanced microglial autophagy downregulates LPS-induced neuroinflammation. Thus, this study suggests that promoting the early stages of autophagy might be a potential therapeutic approach for neuroinflammation-associated diseases.

Cyclosporine (CsA) is characterized by a narrow therapeutic window and high interindividual pharmacokinetic variability, particularly in juvenile patients. The aims of this study were to build a population pharmacokinetic model of CsA in Chinese children with hematopathy who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) and to identify covariates affecting CsA pharmacokinetics. A total of 86 Chinese children aged 8.4 ± 3.8 years (range 1.1–16.8 years) who received allo-HSCT were enrolled. Whole blood samples were collected before allo-HSCT. Genotyping was performed using an Agena MassARRAY system. A total of 1010 trough plasma concentration values of CsA and clinical data were collected. The population pharmacokinetic model of CsA was constructed using nonlinear mixed-effects modeling (NONMEM) software. The stability and performance of the final model were validated using bootstrapping and normalized prediction distribution errors. We showed that a one-compartment model with first-order elimination adequately described the pharmacokinetics of CsA. The typical values for clearance (CL) and volume of distribution (V) were 42.3 L/h and 3100 L, respectively. Body weight, postoperative days, CYP3A4*1 G genotype, estimated glomerular filtration rate and coadministration of triazole antifungal drugs were identified as significant covariates for CL. Weight and postoperative days were significant covariates for the V of CsA. Our model can be adopted to optimize the CsA dosing regimen for Chinese children with hematopathy receiving allo-HSCT.

Ethnopharmacological relevance Xiaochaihutang (XCHT), one of famous Chinese herbal prescription for treating Shaoyang symptom, has been used successfully in depressive disorders for many years. Our laboratory has demonstrated that XCHT remarkably alleviated various depressive behaviors induced by several depressive animal models, but previous studies only focused on one or several protein targets, lacked dynamic change and interrelation of proteins. Therefore, potential protein targets and mechanisms are required further systematic investigation. Aim of the study To discover and assess the differentially expressed proteins (DEPs) of hippocampus after oral administration of XCHT in corticosterone (CORT) induced model of depression by using isobaric tags for relative and absolute quantification (iTRAQ) analysis. Materials and methods The antidepressant effects of XCHT were assessed by two behavioral despair models (forced swimming test and tail suspension test) in CORT induced model of depression. The DEPs of hippocampus after XCHT treatment were investigated by iTRAQ analysis. Potential protein targets and mechanisms were assessed by gene ontology (GO), Kyoto encyclopedia of gene and genomes (KEGG) and protein-protein interaction (PPI) network. Results Our data demonstrated XCHT could significantly improve depressive behaviors. A total of 241 DEPs were identified after XCHT treatment, including 68 up regulation and 173 down regulation proteins. GO enrichment results indicated that XCHT mainly regulated intracellular structural proteins involved in cellular response to stress, transferase activity and steroid hormone. KEGG enrichment analysis showed that endocytosis might be the principal pathway of XCHT on depression. PPI analysis predicted cell division cycle and apoptosis regulator protein 1 (Ccar1) and Calretinin (Calb2) might play the central roles in XCHT's antidepressant network. Conclusion Our results indicate that XCHT plays the important roles in antidepressant action by restoring DEPs, which results in the dysregulation of hippocampal neurogenesis, neurotransmitter and steroid hormone. The current results wish to provide novel perspectives for revealing the potential protein targets of XCHT on depression.

The polarization of microglia/macrophages after cerebral ischemia is critical for post-stroke damage/recovery. Previously, we found that pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, has neuroprotective effects on permanent and transient cerebral ischemia in rats. This study aimed to investigate the effects and potential mechanisms of PF11 on microglia/macrophage polarization following transient cerebral ischemia in rats. In vivo data showed that oral administration of PF11 (12 mg/kg) significantly attenuated cognitive deficits and sensorimotor dysfunction, infarct volume and brain edema in transient middle cerebral artery occlusion (tMCAO)-treated rats, as well as reduced the loss of neurons and the over-activation of microglia in penumbra of ipsilateral striatum and cortex. Notably, the proportion of M2 microglia/macrophages in the total activated microglia/macrophages peaked on day 14 after tMCAO in rats, while PF11 promoted its peak advancing to day 3 post-tMCAO, which allowing the damaged brain to enter the repair period more quickly. Furthermore, PF11 increased the expression of anti-inflammatory markers and decreased the expression of pro-inflammatory markers in ipsilateral striatum and cortex. In addition, in vitro data showed that PF11 inhibited the induction of M1 microglia by oxygen glucose deprivation/re-oxygenation (OGD/R)-induced neurons, and promoted the polarization of microglia to M2 phenotype in a Jumonji domain-containing protein 3 (Jmjd3)-dependent manner. Moreover, PF11 promoted the protection of M2 microglia and attenuated the exacerbation of M1 microglia on OGD/R-induced neuronal damage. Taken together, these results indicate that PF11 protects ischemic neurons by promoting M2 microglia/macrophage polarization in a Jmjd3-dependent manner, ultimately facilitating the functional recovery following transient cerebral ischemia.

Ethnopharmacological relevance Aconiti Lateralis Radix Praeparata (Chinese name: Fuzi), the root of Aconitum carmichaelii Debx., is a representative medicine for restoring yang and rescuing patient from collapse. However, less studies had been reported on the reproductive toxicity and genotoxicity of Fuzi. According to the principle of reducing toxicity and preserving efficiency, only processed products of Fuzi are commonly applied in clinic, including Baifupian, Heishunpian and Danfupian. However, whether processing could alleviate the reproductive toxicity and genotoxicity of Fuzi had not been revealed. Aim of the study To assess the effect and possible mechanism of Fuzi and its processed products on reproductive toxicity and genotoxicity in male mice. Materials and methods Aqueous extracts of Fuzi and its processed products (Baifupian, Heishunpian and Danfupian, 5.85 g/kg) were administrated by gavage once daily for fourteen consecutive days. The reproductive toxicity was evaluated by testis weight, testis ratio, testis histopathology, sperm count, sperm viability rate and sperm deformity rate. The genotoxicity was evaluated by comet assay and micronucleus test in sperm, peripheral blood cell and bone marrow cell. Possible mechanisms of attenuating toxicity by processing were analyzed by detecting the level of testosterone, superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and catalase (CAT). Results Fuzi significantly caused different degrees of reproductive toxicity and genotoxicity, specifically reducing the weight and testicular coefficient of testis, causing obvious pathological changes in testicular tissue, reducing sperm count and sperm viability rate, increasing sperm deformity rate and DNA damage in sperm/peripheral blood cells/bone marrow cells. Moreover, Fuzi decreased the level of testosterone, SOD, GSH and CAT, while increased the level of MDA in serum. Notably, the reproductive toxicity and genotoxicity induced by the processed products, especially Heishunpian and Danfupian, were significantly lowered compared to Fuzi. Processing could increase the level of testosterone, SOD, GSH, CAT and decrease the level of MDA compared to Fuzi. Conclusion Fuzi and its processed products had reproductive toxicity and genotoxicity, but the toxicity of processed products was significantly weakened compared to Fuzi. The protective mechanism of processing to reduce the toxicity of Fuzi might be related to increasing the level of testosterone and decreasing oxidative stress.

We have reported that pseudoginsenoside-F11 (PF11) can significantly improve the cognitive impairments in several Alzheimer's disease (AD) models, but the mechanism has not been fully elucidated. In the present study, the effects of PF11 on AD, in particular the underlying mechanisms related with protein phosphatase 2A (PP2A), were investigated in a rat model induced by okadaic acid (OA), a selective inhibitor of PP2A. The results showed that PF11 treatment dose-dependently improved the learning and memory impairments in OA-induced AD rats. PF11 could significantly inhibit OA-induced tau hyperphosphorylation, suppress the activation of glial cells, alleviate neuroinflammation, thus rescue the neuronal and synaptic damage. Further investigation revealed that PF11 could regulate the protein expression of methyl modifying enzymes (leucine carboxyl methyltransferase-1 and protein phosphatase methylesterase-1) in the brain, thus increase methyl-PP2A protein expression and indirectly increase the activity of PP2A. Molecular docking analysis, structural alignment and in vitro results showed that PF11 was similar in the shape and electrostatic field feature to a known activator of PP2A, and could directly bind and activate PP2A. In conclusion, the present data indicate that PF11 can ameliorate OA-induced learning and memory impairment in rats via modulating PP2A.

We have previously found that transcription factor EB (TFEB), as a master regulator of autophagy and lysosome biogenesis, provides neuroprotective effects on cerebral ischemia-induced neuronal damage by activation of autophagy-lysosomal pathway (ALP). We have also reported that Pseudoginsenoside F11 (PF11), an ocotillol-type saponin isolated from Panax quinquefolium L., significantly attenuates the ischemic injury of rats subjected to permanent middle cerebral artery occlusion (pMCAO), possibly by alleviating the autophagic/lysosomal defects. The present study aims to investigate whether the beneficial effect of PF11 on ALP dysfunction induced by permanent ischemic stroke is based on its regulation of TFEB nuclear translocation in pMCAO rats and the oxygen-glucose-deprived (OGD) primary neurons. Meanwhile, the role of calcineurin, a serine/threonine protein phosphatase, during this process in which PF11 regulated TFEB transcriptional activity was also explored. The data showed that PF11 exerted significant protective effects on pMCAO-induced injury and decreased OGD-induced neuronal death. The nuclear localization of TFEB was decreased at 24 h after pMCAO. Notably, PF11 (6, 12 mg/kg, i.v.) significantly increased TFEB nuclear expression and Tfeb mRNA level at 24 h following pMCAO. OGD treatment promoted TFEB aggregation and nuclear translocation until 6 h, and the nuclear localization of TFEB was decreased at 12 h. Similarly, PF11 (30, 100 μM) could also promote the translocation of TFEB into nuclear in primary neurons at 12 h after OGD treatment. Moreover, PF11 attenuated OGD-induced lysosomal dysfunction and abnormal accumulation of autophagosomes and substrates. These in vitro effects could be abolished by neuronal-specific knocking down of TFEB via transfecting primary neurons with lentivirus encoding shTfeb. Further studies indicated that cyclosporine (10 μM), an inhibitor of calcineurin, could significantly diminish the effects of PF11 on TFEB nuclear translocation and ALP dysfunction in OGD-treated neurons. In summary, these results demonstrate that PF11 attenuates the dysfunction of ALP in permanent cerebral ischemia by promoting the calcineurin-mediated nuclear translocation of TFEB and further identifies an autophagic mechanism of PF11 against cerebral ischemia.

Ethnopharmacological relevance Cisplatin (CP), one of the most commonly used antitumor drugs in clinic, could induce reproductive and genetic toxicity. Traditional Chinese medicine believed that this side effect might be caused by the deficiency of both qi and blood. Panax notoginseng (Burk.) F. H. Chen (PN) is a traditional precious Chinese medicine for nourishing blood and hemostasis, which had the synergistic antitumor and reducing toxicity effects. However, the protective effect and mechanism of PN on CP-induced reproductive and genetic toxicity were still unknown. Aim of the study: This study was designed to illuminate the possible protective effect and mechanism of PN on CP-induced reproductive and genetic toxicity. Materials and methods Network pharmacology was first applied to analyze the potential components and targets of PN against CP-induced reproductive and genetic toxicity. Then, the results of network pharmacology were validated in a mouse model of reproductive and genotoxicity induced by CP. Body weight, testis weight, epididymis weight, sperm count, sperm viability and sperm morphology were used to assess protective effects of PN on CP-induced reproductive toxicity. Tail moment in peripheral blood cells and micronucleus in bone marrow cells were used to assess protective effects of PN on CP-induced genetic toxicity. Finally, possible protective targets obtained from network pharmacology, including 8-hydroxy-2-deoxyguanosine (8-OHdG), malondialdehyde (MDA), total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px), were experimentally validated by ELISA. Results One hundred and nineteen components of PN and sixty-eight targets of reproductive/genetic toxicity were acquired and constituted as the component-target network. Network pharmacology analysis showed alleviating oxidative stress might play important role in therapeutic mechanism of PN. In verified experiments, PN significantly improved the decline of body weight, testis weight and epididymis weight, increased sperm count and viability, decreased abnormal sperm morphology rate induced by CP in mice. Moreover, PN also significantly decreased the tail moment in peripheral blood cells and micronucleus formation rate in bone marrow cells in CP-induced mice. Finally, not only the decrease of T-SOD and GSH-Px level but also the increase of 8-OHdG and MDA level in serum were restored under PN treatment. Conclusion Current study found that PN could improve CP-induced reproductive and genetic toxicity, which were probably attributed to alleviating oxidative stress. This finding provided the new perspective for understanding the therapeutic effect of PN on CP-induced reproductive and genetic toxicity and facilitating the clinical use of PN.

To investigate the fecal microbiota changes in patients with spleen (Pi)-deficiency (SPD), a common Chinese medicine syndrome with digestive and absorptive disturbances and to provide insight into the relationship between Chinese medicine syndrome and gut dysbiosis.Fecal microbiotas from the stool samples of 53 SPD patients and 35 healthy subjects were analyzed via 16S rRNA gene polymerase chain reaction (PCR)-denaturation gradient gel electrophoresis (DGGE). SPD-related marker genes from 20 SPD patients and 20 healthy subjects were identified through gene sequencing, while some genes were quantified using quantitative PCR (qPCR). Discriminant analysis was conducted using SPSS software, and the canonical discriminant function formula for Pi-deficiency was established.Alterations in microbiota diversity and composition between the SPD and healthy groups were demonstrated via 16S rRNA gene PCR-DGGE combined with multivariate statistical analysis. Fecal microbiota changes were also observed among different SPD-subtype patients. Eight SPD-related markers were found, and putative species corresponding to these markers were identified through gene sequencing, which may have potential associations with the common digestive dysfunctions in SPD patients. qPCR methods were established for two of these markers, which were significantly altered in the SPD patients. The canonical discriminant function formula was calculated for SPD, and the validity rates of these markers were over 85%.Fecal microbiotas are altered in patients with SPD, which may provide insight for further studies on clinically diagnosing and treating SPD. The results may also provide data to gain a better understanding of Traditional Chinese Medicine syndrome and gut dysbiosis.

Propensity to relapse, even after long-term abstinence, is a crucial feature of methamphetamine (METH) abuse. We and other laboratories have reported that acute treatment of oxytocin (OXT), a hormone and neuropeptide, could inhibit reinstatement of METH seeking in animal studies. However, the effects of repeated OXT treatment on METH reinstatement as well as underlying mechanisms are still unclear. In the present study, the effects of repeated OXT treatment during abstinence on context- or restraint stress-induced reinstatement were investigated using the mice conditioned place preference (CPP) paradigm. After three intermittent injections of METH (2 mg/kg, i.p.) to induce CPP, mice received a daily bilateral intra-hippocampus injection of OXT (0.625, 1.25 or 2.5 μg) for 8 consecutive days before the context- or restraint stress-induced reinstatement test. Meanwhile, adult hippocampal neurogenesis (AHN) level was detected using immunostaining. To further clarify the role of AHN underlying OXT's effects on METH-CPP reinstatement, temozolomide (TMZ, 25 mg/kg, i.p.) was employed to deplete AHN prior to OXT treatment. The data showed that repeated OXT treatment (1.25 and 2.5 μg, intra-hippocampus) significantly inhibited both context- and restraint stress-induced METH-CPP reinstatement and concomitantly promoted AHN in a dose-dependent manner. Notably, TMZ pre-treatment markedly abolished all the above-mentioned effects of OXT, suggesting that AHN was closely involved in OXT's inhibition on reinstatement induced by both triggers. Taken together, the present study indicated that repeated OXT treatment during abstinence could inhibit both context- and restraint stress-induced METH-CPP reinstatement possibly by promoting AHN in mice, which provided a better understanding for OXT's beneficial effects on METH addiction.

Mounting attention has been focused on defects in macroautophagy/autophagy and the autophagy-lysosomal pathway (ALP) in cerebral ischemia. TFEB (transcription factor EB)-mediated induction of ALP has been recently considered as the common mechanism in ameliorating the pathological lesion of myocardial ischemia and neurodegenerative diseases. Here we explored the vital role of TFEB in permanent middle cerebral artery occlusion (pMCAO)-mediated dysfunction of ALP and ischemic insult in rats. The results showed that ALP function was first enhanced in the early stage of the ischemic process, especially in neurons of the cortex, and this was accompanied by increased TFEB expression and translocation to the nucleus, which was mediated at least in part through activation by PPP3/calcineurin. At the later stages of ischemia, a gradual decrease in the level of nuclear TFEB was coupled with a progressive decline in lysosomal activity, accumulation of autophagosomes and autophagy substrates, and exacerbation of the ischemic injury. Notably, neuron-specific overexpression of TFEB significantly enhanced ALP function and rescued the ischemic damage, starting as early as 6 h and even lasting to 48 h after ischemia. Furthermore, neuron-specific knockdown of TFEB markedly reversed the activation of ALP and further aggravated the neurological deficits and ischemic outcome at the early stage of pMCAO. These results highlight neuronal-targeted TFEB as one of the key players in the pMCAO-mediated dysfunction of ALP and ischemic injury, and identify TFEB as a promising target for therapies aimed at neuroprotection in cerebral ischemia. Abbreviations: AAV, adeno-associated virus; AIF1/IBA1, allograft inflammatory factor 1; ALP, autophagy-lysosomal pathway; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; CsA, cyclosporin A; GFAP, glial fibrillary acidic protein; LAMP, lysosomal-associated membrane protein; LC3, microtubule-associated protein 1 light chain 3; MAP2, microtubule-associated protein 2; mNSS, modified Neurological Severity Score; MTOR, mechanistic target of rapamycin kinase; OGD, oxygen and glucose deprivation; pMCAO, permanent middle cerebral artery occlusion; RBFOX3/NeuN, RNA binding fox-1 homolog 3; SQSTM1, sequestosome1; TFEB, transcription factor EB; TTC, 2,3,5-triphenyltetrazolium chloride.

There is growing evidence that uridine may act as an endogenous neuromodulator with a potential signaling role in the central nervous system in addition to its function in pyrimidine metabolism. We previously found that acute morphine treatment significantly increased uridine release in the dorsal striatum of mice, indicating that uridine may contribute to morphine-induced neurobehavioral changes. In the present study, we analyzed the mechanism involved in morphine-induced uridine release and the role of uridine in morphine-induced neurobehavioral changes. Uridine release in the dorsal striatum of mice was assessed by in vivo microdialysis coupled with high performance liquid chromatography (HPLC) after morphine treatment. Western blotting and immunofluorescence were used to evaluate the expression of uridine-related proteins. Morphine-induced neurobehavioral changes were assessed by locomotor activity, behavioral sensitization and conditioned place preference (CPP) test. The expression of NT5E, an extracellular enzyme involved in formation of nucleosides, including uridine, was specifically knocked down in the dorsal striatum of mice using adeno-associated virus (AAV)-mediated short hairpin RNA (shRNA). The results indicated that both acute and chronic morphine administration significantly increased uridine release in the dorsal striatum, and this was associated with upregulation of NT5E but not other uridine-related proteins. Inhibition of NT5E with APCP or shRNA markedly inhibited morphine-induced uridine release in the dorsal striatum and related neurobehavioral changes, including hyperlocomotor activity, behavioral sensitization and CPP. Our data give a better understanding of the contribution of NT5E to morphine-induced uridine release and neurobehavioral changes, and identify NT5E as a potential target for treating morphine abuse.

Uridine is a potential endogenous neuromodulator studied for several decades for its antiepileptic effect, but the results were controversial. One remarkable feature of uridine is its regulatory action on the dopaminergic pathways. In this study, the changes in uridine and dopamine (DA) release were examined in the mouse corpus striatum after pilocarpine (PC) intraperitoneal injection. Then, the effect of uridine pre-treatment on DA release and expression of dopamine receptor (DR) was determined. The results revealed an increased uridine release initially, followed by a downward trend after an injection of 400-mg/kg PC. However, the DA release continuous increased significantly. The expression of dopamine receptor-1 (D1R) increased in a dose-dependent manner while that of dopamine receptor-2 (D2R) decreased significantly. Prophylactic administration of uridine significantly relieved the high-frequency and high-amplitude expression induced by PC as well as dose-dependently reversed the PC-induced changes in DA and DRs levels. These findings suggested that uridine produced an antiepileptic effect, which might have been mediated in part by interfering with the dopaminergic system.

Procaspase Activating Compound-1 (PAC-1) and its derivative WF-210 induce apoptosis in cancer cells by activating procaspase-3 to caspase-3. The aim of this study was to extend current knowledge about the mechanisms of PAC-1 and WF-210, particularly about their effects on tumor angiogenesis. PAC-1 and WF-210 restrained VEGF-induced human umbilical vascular endothelial cells (HUVECs) proliferation, invasion, and tube formation. PAC-1 and WF-210 abrogated VEGF-induced vessel sprouting from rat aortic rings and inhibited vascular formation in the Matrigel plug assay. PAC-1 and WF-210 suppressed phosphorylation of VEGFR2 and its downstream protein kinases c-Src, FAK, and AKT in both HUVECs and U-87 cells. When given to mice bearing subcutaneous or orthotopic xenograft, PAC-1 and WF-210 inhibited the tumor growth and tumor angiogenesis. Further tests showed that PAC-1 and WF-210 inhibited stemness and induced autophagy flux of U-87 cells. This study revealed mechanisms of PAC-1 and WF-210 other than inducing apoptosis, which provides additional support for their using in the clinic.

Pseudoginsenoside-F11 (PF11), an ocotillol-type ginsenoside, has been reported to exert neuroprotective effects on ischemic stroke induced by permanent and transient middle cerebral artery occlusion in experimental animals. The aim of the present study was to investigate the effect of PF11 on thromboembolic stroke in rats and its possible mechanisms on thromboinflammation. PF11 (4, 12, 36 mg/kg) was injected intravenously (i.v.) once a day for 3 consecutive days to male Wistar rats followed by embolic middle cerebral artery occlusion (eMCAO). The results showed that PF11 significantly reduced the cerebral infarction volume, brain edema and neurological deficits induced by eMCAO. Meanwhile, the thromboinflammation in the ischemic hemisphere was observed at 24 h after eMCAO, as indicated by the increased number of microvascular thrombus and inflammatory response. Moreover, eMCAO resulted in the up-regulation of platelet glycoprotein Ibα (GPIbα) and VI (GPVI), as well as the activation of contact-kinin pathway. Notably, PF11 significantly reversed all these changes. Furthermore, PF11 prevented the eMCAO-induced loss of tight junction proteins and up-regulation of matrix metalloproteinase-9 (MMP-9), thus leading to the alleviation of blood-brain barrier (BBB) damage. In conclusion, the present study revealed that thromboinflammation was induced in the ischemic hemisphere of rats after eMCAO and PF11 exerted marked protective effects against thromboembolic stroke by attenuating thromboinflammation and preventing BBB damage. This research further identifies the potential therapeutic role of PF11 for ischemic stroke.

The high affinity of positively charged nanoparticles to biological interfaces makes them easily taken up by tumor cells but limits their tumor permeation due to non-specific electrostatic interactions. In this study, polyion complex coated nanoparticles with different charge reversal profiles were developed to study the influence of charge reversal profile on tumor penetration. The system was constructed by polyion complex coating using micelles composed of poly (lysine)- b -polycaprolactone (PLys- b -PCL) as the cationic core and poly (glutamic acid)- g - methoxyl poly (ethylene glycol) (PGlu- g -mPEG) as the anionic coating material. Manipulation of charge reversal profile was achieved by controlling the polymer chain entanglement and electrostatic interaction in the polyion complex layer through glutaraldehyde-induced shell-crosslinking. The delayed charge reversal nanoparticles (CTCL30) could maintain negatively charged in pH 6.5 PBS for at least 2 h and exhibit pH-responsive cytotoxicity and cellular uptake in an extended time scale. Compared with a faster charge reversal counterpart (CTCL70) with similar pharmacokinetic profile, CTCL30 showed deeper penetration, higher in vivo tumor cell uptake and stronger antitumor activity in vivo (tumor inhibition rate: 72.3% vs 60.2%, compared with CTCL70). These results indicate that the delayed charge reversal strategy could improve therapeutic effect via facilitating tumor penetration. Statement of Significance Here, the high tumor penetration capability of PEG-coated nanoparticles and the high cellular uptake of cationic nanoparticles were combined by a delayed charge reversal drug delivery system. This drug delivery system was composed of a drug-loading cationic inner core and a polyion complex coating. Manipulation of charge reversal profile was realized by varying the crosslinking degree of the shell of the cationic inner core, through which changed the strength of the polyion complex layer. Nanoparticles with delayed charge reversal profile exhibited improved tumor penetration, in vivo tumor cell uptake and in vivo tumor growth inhibition effect although they have similar pharmacokinetic and biodistribution behaviors with their instant charge reversal counterpart.

Tyrosine kinase inhibitors (TKIs) have demonstrated clinical benefits in the treatment of several tumour types. However, the emergence of TKI resistance restricts the therapeutic effect. This study uses non-small cell lung cancer (NSCLC) to explore the mechanisms contributing to TKI resistance in tumours. Biological phenotypes and RNA microarray expression data were analysed in NSCLC cells with and without TKI pretreatment. Specific inhibitors and siRNAs were used to validate the direct involvement of an AKT/FOXM1/STMN1 pathway in TKI resistance. Patients’ tissues were analysed to explore the clinical importance of FOXM1 and STMN1. In vitro and in vivo studies showed that TKIs induced the enrichment of cancer stem cells (CSC), promoted epithelial to mesenchymal transition (EMT), and conferred multidrug resistance on NSCLC cells in a cell type- and TKI class-dependent manner. Mechanistically, TKIs activated an AKT/FOXM1/STMN1 pathway. The crucial role of this pathway in TKI-induced enrichment of CSC and drug resistance was verified by silencing FOXM1 and STMN1 or blocking the AKT pathway. Additionally, overexpression of STMN1 was associated with upregulation of FOXM1 in advanced NSCLC patients, and STMN1/FOXM1 upregulation predicted a poor outcome. Our findings elucidate an additional common mechanism for TKI resistance and provide a promising therapeutic target for reversing TKI resistance in NSCLC.

Ethnopharmacological relevance Hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis is often observed in the pathophysiology of depression. Antidepressant therapy can restore hippocampal neurogenesis to rescue the HPA axis regulation defects. Xiaochaihutang (XCHT), a famous Chinese herbal formula, has been used clinically in depressive disorders in China. Our previous studies have demonstrated XCHT improved depressive-like behaviors in chronic unpredictable mild stress rat, but the underlying mechanisms of XCHT on hippocampal neurogenesis and the HPA axis were still unclear. Materials and methods We used chronic corticosterone (CORT)-induced mouse model of anxiety/depression to investigate antidepressant-like effects of XCHT by several physical and behavioral testing, including body weight, coat state, open field test, elevated plus maze, tail suspension test and forced swimming test. The integrity of negative feedback function on HPA axis was assessed by the dexamethasone (DEX) suppression test. In addition, Ki-67 and doublecortin (DCX) were performed to assess hippocampal cell proliferation and neurogenesis by immunohistochemistry. Chemical profile of active constituents in brain after oral administration of XCHT was revealed by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Results Our results showed that oral administration of XCHT (2.3, 7 and 21 g/kg) for 30 days remarkably normalized chronic CORT-induced the slowness in weight gain, the deterioration in coat state, the escape behavior in open field test and elevated plus maze, and the increase of immobility time in tail suspension test and forced swimming test. Moreover, XCHT significantly reversed chronic CORT-induced the reduction of DEX-induced plasma corticosterone/c-Fos suppression and Ki-67/DCX positive cells. Finally, a total 13 potential active constituents in brain were identified by UPLC-MS/MS after oral administration of XCHT, including 10 prototype components and 3 metabolites. Conclusions Our findings showed that XCHT could remarkably alleviate chronic CORT-induced anxiety/depression-like behaviors, which were probably attribute to promoting hippocampal neurogenesis and remodeling the integrity of the negative feedback loop on HPA axis. The constituents identified in brain might contribute to understanding the therapeutic basis of XCHT on depression.

Ischemic stroke is a cerebrovascular disease with high morbidity, high mortality, and high disability, representing a serious threat to human life and health. Clinically, the extensive injury caused by ischemic stroke results from ischemia-reperfusion (I/R) injury thrombolytic treatment. However, there are few reports on the use of medications in the subacute stage of cerebral I/R. Baicalein (5,6,7-trihydroxyflavone) is a biologically active ingredient extracted from the root of Scutellaria baicalensis Georgi. In the present study, we investigated the therapeutic effect of baicalein administered in the subacute phase of cerebral I/R injury in a rat model of ischemia induced by occlusion of the middle cerebral artery (MCA). Rats were treated daily with baicalein (200 mg/kg, i.g.) in the subacute phase (24 h after reperfusion) for 7 days. The results showed that baicalein significantly reduced neurobehavioral deficits and decreased brain infarct volume from 18.99% to 7.41%. Immunofluorescence analysis of the ischemic penumbra showed that baicalein significantly reduced expression of the M1 marker, cluster of differentiation (CD) 16 and CD86, and increased expression of the M2 marker, CD 163 and CD206, indicating that baicalein inhibited M1 transformation and promoted M2 transformation of microglia/macrophage to inhibit neuroinflammation. Moreover, baicalein suppressed NF-κB signaling by reducing IκBα phosphorylation and nuclear translocation of NF-κB/p65, which decreased the release of the pro-inflammatory factors IL-6, IL-18, and TNF-α. In addition, baicalein reduced phosphorylation of JNK, ERK and p38, which are involved modulation of microglia/macrophage M1/M2 polarization. Western blot analysis of apoptosis- and autophagy-related proteins showed that baicalein increased the Bcl-2/Bax ratio and reduced caspase-3 expression to decrease neuronal apoptosis and ameliorate neuronal loss. Baicalein also decreased the LC3-II/LC3-I ratio and promoted phosphorylation of the PI3K/Akt/mTOR signaling pathway which implied inhibition of autophagy. These observations suggest that baicalein exerts neuroprotective effects by reducing neuroinflammation, apoptosis and autophagy, and protects against cerebral I/R injury in the subacute phase in vivo.

Scope The anti-neuroinflammatory effect of a novel quinolyl-substituted analogue of resveratrol (RV01) on lipopolysaccharide (LPS)-induced microglial activation is investigated, as well as the possible underlying mechanisms. Methods and results Cell viability is measured using an MTT assay. Nitric oxide (NO) release is determined by nitrite assay. The interaction between RV01 and inducible nitric oxide synthase (iNOS) is studied using molecular docking. Free radical scavenging activity and reactive oxygen species (ROS) production are determined by DPPH reduction assay and DCFH-DA assay. Pretreatment with RV01 (1-30 µm) prior to LPS (1 µg mL-1 ) stimulation decreased NO release and iNOS expression without observable cytotoxicity. RV01 reduced the mRNA levels and secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). RV01 also inhibited LPS-induced ROS production and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. Furthermore, RV01 decreases the protein expression of toll-like receptor 4 (TLR4) and inhibits the LPS-induced activation of the mitogen-activated protein kinase (MAPK) and nuclear transcription factor-κB (NF-κB) signaling pathways. Additionally, conditioned medium from microglia co-treated with LPS and RV01 alleviates the death of SH-SY5Y cells induced by conditioned medium from activated N9 microglial cells. Lastly, a mouse neuroinflammation model is further used to confirm the effect of RV01 in vivo. Conclusion These results show that RV01 suppresses microglia-mediated neuroinflammation and protects neurons from inflammatory damage, which indicates that RV01 has great potential as a nutritional preventive strategy for neuroinflammation-related diseases.

// Xinwei Liu 1, 2 , Lihui Wang 1, 2 , Wei Cui 1 , Xiangzhong Yuan 1 , Lulu Lin 1 , Qi Cao 1 , Nannan Wang 1, 2 , Yi Li 1, 2 , Wei Guo 1 , Xun Zhang 1 , Chunfu Wu 1, 2 , Jingyu Yang 1, 2 1 Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, P.R. China 2 Benxi Institute of Pharmaceutical Research, Shenyang Pharmaceutical University, Benxi, P.R. China Correspondence to: Chunfu Wu, email: wucf@syphu.edu.cn Jingyu Yang, email: yangjingyu2006@gmail.com Keywords: disulfiram/copper, ALDH1A1, cancer stem cell, NSCLC, recurrence Received: February 03, 2016 Accepted: July 26, 2016 Published: August 16, 2016 ABSTRACT The existence of cancer stem cells (CSCs) in non-small cell lung cancer (NSCLC) has profound implications for cancer therapy. In this study, a disulfiram/copper (DSF/Cu) complex was evaluated in vitro and in vivo for its efficacy to inhibit CSCs, which drive recurrence of NSCLC. First, we investigated whether DSF/Cu could inhibit ALDH-positive NSCLC stem cells in vitro and tumors derived from sorted ALDH-positive CSCs in vivo . DSF/Cu (0.5/1 μmol/l) significantly inhibited the expression of stem cell transcription factors (Sox2, Oct-4 and Nanog) and reduced the capacities of NSCLC stem cells for self-renewal, proliferation and invasion in vitro . Regular injections with DSF/Cu (60/2.4 mg/kg) reduced the size of tumors derived from sorted ALDH-positive stem cells. Two other NOD/SCID xenograft models were used to determine whether DSF/Cu could target NSCLC stem cells and inhibit tumor recurrence in vivo . DSF/Cu treatment eliminated ALDH-positive cells and inhibited tumor recurrence, which was reflected by reduced tumor growth in recipient mice that were inoculated with tumor cells derived from DSF/Cu-treated cells or primary xenografts. RNA interference and overexpression of ALDH isozymes suggested that ALDH1A1, which plays a key role in ALDH-positive NSCLC stem cells, might be the target of the DSF/Cu complex. Collectively, our data demonstrate that DSF/Cu targets ALDH1A1 to inhibit NSCLC recurrence driven by ALDH-positive CSCs. Thus, the DSF/Cu complex may represent a potential therapeutic strategy for NSCLC patients.

Apple polyphenols (AP) have attracted much attention due to their various bioactivities. In this study, the protective effect of AP against chronic ethanol exposure-induced neural injury as well as the possible mechanisms were investigated. Body weight, daily average food intake and daily average fluid intake were measured and daily average ethanol consumption was calculated. The influences of AP on motor behavior and memory were detected by locomotor activity test, rotarod test, beam walking test, and Y maze test and novel object recognition test, respectively. The changes of blood ethanol concentration and the oxidative stress were also measured. AP improved chronic ethanol exposure-induced the inhibition of body weight and the decrease of daily average food intake, but did not influence the daily average fluid intake and the daily average ethanol intake, indicating that the improve effect of AP did not result from the decrease of ethanol intake. Motor activity and motor coordination were not influenced after chronic ethanol exposure though the blood ethanol concentration was higher than that in control group. AP improved significantly chronic ethanol-induced the memory impairment and the hippocampal CA1 neurons damage. Further studies found that AP decreased the contents of NO and MDA and increased the levels of T-AOC and GSH in the hippocampus of rats. These results suggest that AP exerts a protective effect against chronic ethanol-induced memory impairment through improving the oxidative stress in the hippocampus.

Stroke is the leading cause of long-term motor disability and cognitive impairment beside the acute brain injury. Recently, neurogenesis has become an attractive strategy for the chronic recovery of stroke. Our previous study showed that pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, isolated from leaves of Panax pseudoginseng subsp., exerted neuroprotective effects on stroke by alleviating autophagy/lysosomal defects and repressing calcium overload. The present study investigated whether PF11 improved long-term functional recovery and promoted neurogenesis after ischemic stroke induced by transient middle cerebral artery occlusion (tMCAO) in mice. The data showed that PF11 (16, 32 mg/kg, p.o.) administrated once daily one week before tMCAO significantly reduced brain infarction and brain edema on day 3 after tMCAO. Also, PF11 attenuated the mortality, sensorimotor dysfunction, cognitive impairment and hippocampal atrophy of stroke mice. Moreover, the migration of neuroblasts and the generation of newborn neurons in ipsilateral striatum and dentate gyrus (DG) were significantly enhanced by PF11. In line with this, PF11 prevented the decreased survival rate of newborn neurons on day 42 after tMCAO. In addition, PF11 promoted proliferation and differentiation of neural stem cells in vitro. Furthermore, PF11's pro-neurogenic effect was attributed to its activation of the BDNF/TrkB, which was evidenced by that the pharmacological effects of PF11 was abolished by ANA-12, a specific inhibitor of BDNF receptor. Thus, the present study showed that PF11 could improve long-term neurological impairment and promote neurogenesis after stroke possibly through activating BDNF/TrkB pathway, indicating its potential role on treating ischemic stroke, especially chronic recovery.

Ethnopharmacological relevance Cisplatin (CP) is the classical chemotherapeutic drug for various cancer, but it also accompanies reproductive toxicity and genotoxicity. Liuwei Dihuang Pill (LW) is the traditional Chinese medicine prescription for treating Kidney-Yin deficiency syndrome, which has been reported to prevent and treat various diseases. However, the protective effect of LW on CP-induced reproductive toxicity and genotoxicity has not been reported. Aim of the study To investigate the potential protective effect and mechanism of LW on CP-induced reproductive toxicity and genotoxicity in male mice. Materials and methods Mice were given LW (0.4, 1.2 and 3.6 g/kg) or Vitamin C (0.1 g/kg) once daily by oral gavage for thirteen consecutive days. Then, CP (3.00 mg/kg) was given intraperitoneal injection once daily for five consecutive days starting on the ninth day. The protective effects of LW against CP-induced reproductive toxicity and genotoxicity were evaluated by body weight, testis ratio, sperm count, sperm viability, sperm abnormal morphology type, micronuclei test, testicular histopathology, serum malondialdehyde (MDA), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) level. Results The results demonstrated that LW could significantly increase CP-induced the reduction of sperm count and sperm viability, then decrease abnormal sperm type rate and micronucleus rate. Moreover, LW also could improve testicular abnormal histopathologic morphology induced by CP exposure. Meanwhile, LW decreased serum MDA level and increased T-SOD, GSH-Px and CAT level compared to CP group. Conclusion our findings show that LW has protective effects on CP-induced reproductive toxicity and genotoxicity. LW decreases serum MDA level and increases T-SOD, GSH-Px and CAT level, which indicates that antioxidant activity may be the potential mechanism of LW to resist reproductive toxicity and genotoxicity.

Oxidative stress and neuroinflammation are thought to be the two key early events during the process of mild cognitive impairment (MCI). Therefore, effective regulation of oxidative stress and neuroinflammation is an important aspect of preventing and improving MCI. We previously found that pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, markedly reduced cognitive impairment in APP/PS1 mice and oAβ1–42-injected mice. In the present study, we further evaluate the effect of PF11 on learning and memory dysfunction in d ‑galactose ( d ‑gal)-treated mice model of MCI. C57BL/6 mice received daily subcutaneous injections of d ‑gal (100 mg/kg) and oral administration of PF11 (2, 4, 8, 16 mg/kg) for 9 weeks. We observed that PF11 significantly alleviated d ‑gal-induced cognitive impairment, attenuated the loss of neuron and the over-activation of microglia in hippocampus of d ‑gal-treated mice. The elevated levels of nod-like receptor protein 3 (NLRP3) inflammasome in hippocampus of d ‑gal-treated mice were reduced by PF11 through reducing the accumulation of advanced glycation endproducts (AGEs) and the expression of the receptor of advanced glycation endproducts (RAGE). Moreover, PF11 significantly decreased H2O2 and malondialdehyde (MDA) levels, improved superoxide dismutase (SOD) activity and increased glutathione (GSH) level in d ‑gal-treated mice. Finally, d ‑gal treatment reduced the level of nuclear factor erythroid-related factor 2 (Nrf2) and glutathione S-transferase (GST) in hippocampus, which could reverse by PF11. Together, our findings indicated that PF11 exerts a protective effect against MCI-like pathological changes.

Neutrophils have been traditionally considered as the major mediators of harmful inflammatory responses in ischemic stroke, whereas accumulating evidence indicates that neutrophils can be polarized into an N2 phenotype. Similar to M2 microglia, N2 neutrophils contribute to resolution of inflammation and may participate in neuroprotection. However, it remains unclear whether N2 neutrophils protect ischemic neurons and whether they are associated with long-term outcomes after transient cerebral ischemia in rats. The present study proved that N2 neutrophils protected against oxygen glucosedeprivation/re-oxygenation (OGD/R)-induced primary cortical neuron injury via brain-derived neurotrophic factor/tropomyosin-related kinase B (BDNF/TrkB) signaling. In addition, in vivo studies revealed that transient middle cerebral artery occlusion (tMCAO)-induced injury exhibited spontaneous recovery over time in rats. Moreover, neutrophils could infiltrate the ipsilateral brain parenchyma from the periphery after transient cerebral ischemia. Pearson's correlation analysis indicated that the proportion of N2 neutrophils in ipsilateral brain parenchyma was negatively correlated with the number of degenerating neurons, modified Neurological Severity Score (mNSS), brain water content and infarct volume, and positively correlated with the number of surviving neurons and grip strength. In summary, the present study shows that N2 neutrophils likely participate in spontaneous recovery after transient cerebral ischemia by inhibiting ischemic neuron damage in rats, which indicates that N2 neutrophils may represent promising therapeutic target for promoting recovery after ischemic stroke.

Methamphetamine (METH) causes memory changes, but the underlying mechanisms are poorly understood. Epigenetic mechanisms, including DNA methylation, can potentially cause synaptic changes in the brain. Oxytocin (OT) plays a central role in learning and memory, but little is known of the impact of OT on METH-associated memory changes. Here, we explored the role of OT in METH-induced epigenetic alterations that underlie spatial and cognitive memory changes. METH (2.0 mg/kg, i.p.) was administered to male C57BL/6 mice once every other day for 8 days. OT (2.5 μg, i.c.v.) or aCSF was given prior to METH. Spatial and cognitive memory were assessed. In Hip and PFC, synaptic structures and proteins were examined, levels of DNA methyltransferases (DNMTs) and methyl CpG binding protein 2 (MECP2) were determined, and the DNA methylation status at the Synaptophysin (Syn) promoter was assessed. METH enhanced spatial memory, decreased synapse length, downregulated DNMT1, DNMT3A, DNMT3B, and MECP2, and induced DNA hypomethylation at the Syn promoter in Hip. In contrast, METH reduced cognitive memory, increased synapse thickness, upregulated DNMT1, DNMT3A, and MECP2, and induced DNA hypermethylation at the Syn promoter in PFC. OT pretreatment specifically ameliorated METH-induced learning and memory alterations, normalized synapse structures, and regulated DNMTs and MECP2 to reverse the DNA methylation status changes at the Syn promoter in Hip and PFC. DNA methylation is an important gene regulatory mechanism underlying METH-induced learning and memory alterations. OT can potentially be used to specifically manipulate METH-related memory changes.

Alzheimer's disease (AD) is a common neurodegenerative disease which is characterized by aggregation of amyloid beta (Aβ) and hyperphosphorylated tau. We previously reported that pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, improved cognitive function and reduced Aβ aggregation in APP/PS1 mice, a familial AD model. Here, we chose senescence-accelerated mouse prone 8 (SAMP8) mice, a widely used model of aging, to investigate the effect of PF11 on sporadic AD. PF11 was orally administered to male 6-month-old SAMP8 mice for 3 months. Consistent with previous studies, SAMP8 mice showed several AD-type pathologies including cognitive impairment, Aβ deposition and tau hyperphosphorylation. We found increased protein levels of cytoplasmic amyloid precursor protein (APP) and β-site APP cleavage enzyme 1 (BACE1) in the hippocampus and cortex of SAMP8 mice. The protein level of demethylated protein phosphatase 2A (PP2A) was elevated in SAMP8 animals and the protein level of leucine carboxyl methyltransferase 1 (LCMT-1) was reduced. PF11 attenuated learning and memory impairments in the novel object recognition test and Morris water maze. PF11 promoted the transport of APP from cytoplasm to plasma membrane and decreased the abnormally high expression of BACE1 in hippocampus and cortex of SAMP8 mice. The elevated protein level of demethylated PP2A and the reduced expression of LCMT-1 in hippocampus and cortex of SAMP8 were also attenuated by PF11. Together, our findings indicate that PF11 has beneficial effects on AD-like pathological changes in SAMP8 mice and may act by inhibiting amyloidogenic processing of APP and attenuating tau hyperphosphorylation.

Neuropathic pain is a global medical concern, characterized by spontaneous pain, heat hyperalgesia and mechanical allodynia. The condition has been associated with alterations in the voltage‑gated sodium channels, Nav1.8 and Nav1.9, in nociceptive neurons termed nociceptors. However, an explanation for the contribution of these channels to the phenotype observed in neuropathic pain remains to be elucidated. The changes induced by chronic constriction injury (CCI) to Nav1.8 and Nav1.9 mRNA and protein levels, as well as electrical currents in injured and contralateral non‑injured dorsal root ganglion (DRG) neurons are described in the present study. A marked downregulation was observed for each Nav isoform transcript and protein expressed in injured neurons with the exception of the Nav1.9 protein, which exhibited no change, while in contralateral non‑injured neurons, the levels of protein and mRNA remained unchanged. Nav isoform functional analysis was then performed in L(4‑6) DRG neurons 14 days after CCI. The Nav1.8 current density was markedly decreased in injured DRG neurons following CCI. The voltage‑dependent activation of the Nav1.8 channel in these neurons was shifted to depolarized potentials by 5.3 mV, while it was shifted to hyperpolarized potentials by 10 mV for inactivation. The electrophysiological function of Nav1.9 was not affected by CCI. The present study demonstrated that ectopic discharge following CCI, which was likely induced by a positive shift in the Nav1.8 current inactivation curve in injured neurons, enhanced the excitability of the neurons by facilitating tetrodotoxin‑resistant sodium channels into the fast inactivation state and did not occur as a result of a compensatory redistribution in the contralateral uninjured neurons.

A sensitive and versatile, ultra-high performance, liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method coupled to pre-column derivatization for the simultaneous determination of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine (DA), norepinephrine (NE), homovanillic acid (HVA), γ-aminobutyric acid (GABA) and glutamic acid (Glu) was developed and validated in rat plasma. The analytes were dansylated under strong alkaline conditions after protein precipitation extraction, which were analyzed on a BEH C18 column using a gradient elution. The lower limit of quantification (LLOQ) values for 5-HT, 5-HIAA, DA, NE, HVA, GABA and Glu were 1.00, 1.00, 0.991, 0.992, 1.02, 1000, and 5030 pmol/mL, respectively. Good linearity was obtained (r > 0.99) and the intra- and inter-day precisions of the method (relative standard deviation, RSD%) were lower than 12%. The method was novel, sensitive and specific which can provide an alternative method for the quantification of neurotransmitters and their metabolites in plasma samples.

Retinoid X receptor (RXR) and Histone deacetylase (HDAC) are considered important targets for cancer therapy due to their crucial roles in genetic or epigenetic regulations of cancer development and progression. Here, we evaluated the potential of dual targeting of RXR and HDAC using DW22 as a novel therapeutic approach to cancer treatment. We found that the co-expression of RXR-α and HDAC1 was frequently appeared in lung cancer and breast cancer tissues and cell lines. RXR was activated by DW22 in RXRα and HDAC1 overexpressed A549 and MDA-MB-435 cell lines. Meanwhile, DW22 inhibited the activity of HDAC by decreasing its expression in A549 and MDA-MB-435 cell lines, but not in RXRα and HDAC1 deficient cell lines. Moreover, DW22 suppressed cell growth, induced cell differentiation, prompted cell apoptosis and arrested cell cycle in A549, MDA-MB-435 or HL60 cell lines. Treatment human umbilical vascular endothelial cells (HUVECs) with DW22 suppressed migration, invasion and tube formation through decreasing VEGF expression. The up-regulation of Ac-H3 and p21, and down-regulation of VEGF caused by DW22 was markedly attenuated by silencing of HDAC1. Furthermore, knockdown of RXRα by siRNA completely blocked DW22-induced cell differentiation, but partially attenuated DW22-caused inhibition of cell proliferation, induction of cell apoptosis, and suppression of cell migration, invasion and tube formation. Moreover, intravenous administration of DW22 significantly retarded tumor growth of A549 and MDA-MB-435 xenograft mice models, and induced no substantial weight loss and gross toxicity. In addition, DW22 also reduced cell proliferation, angiogenesis, and induced cell apoptosis in vivo. Collectively, our data demonstrates that dual targeting of RXR and HDAC using DW22 possesses pleiotropic antitumor activities both in vitro and in vivo, providing a novel therapeutic approach for cancer treatment.

A novel series of chalcone derivatives were synthesized and their biological activities against HIF-1 were evaluated. Among these compounds, 5d exhibited clearly inhibitory effects on HIF-1 by downregulating the expression of HIF-1α under hypoxic conditions. Meanwhile, it also significantly suppressed VEGF-induced migration and invasion of Hep3B and HUVEC cells in nontoxic concentrations. Additionally, tube formation assay demonstrated its anti-angiogenesis activity. Moreover, the in vivo study indicated that compound 5d could retard tumor growth of Hep3B xenograft models and reduced CD31 and MMP-2 expression in tumor tissues. Finally, in acute intravenous toxicity, 5d was well tolerated and was found to be non-toxic up to 200 mg/kg in Swiss mice. These findings support the further investigation on the anti-invasive and anti-angiogenic potential of this class of compounds as HIF-1 inhibitor.

Autophagy, a cellular survival mechanism, is thought to allow the recycling of cellular breakdown products when cancer cells are subjected to chemotherapy, thus decreasing drug-induced apoptosis. Disulfiram (DSF), a drug widely used to control alcoholism, possesses anticancer activity by inducing apoptosis in vitro and in vivo in a copper (Cu)-dependent manner. Our previous studies proved that DSF/Cu exerts increased anti-tumor effects on non-small cell lung cancer (NSCLC) xenograft models, and inhibits NSCLC recurrence driven by ALDH-positive cancer stem cells. The present study is designed to investigate whether DSF/Cu can induce autophagy in NSCLC cells and to determine the relationship between autophagy and apoptosis. First, we observed that DSF/Cu induced significant cytotoxicity and caspase-dependent apoptosis in NSCLC cells, accompanied by the formation of vacuoles in the cytoplasm. Next, we investigated the levels of autophagic markers, including LC3II (microtubule-associated protein 1 light chain 3), ATG5 and p62 with or without chloroquine by Western blot. In addition, we observed co-localization of LC3 and the lysosomal protein LAMP2 (lysosome-associated membrane protein 2) after treatment with DSF/Cu. The results showed that DSF/Cu induced autophagy. Finally, we demonstrated that DSF/Cu-induced apoptosis was greatly enhanced when autophagy was suppressed with Atg5 siRNA or 3-MA in NSCLC cells. This synergistic effect of DSF/Cu and 3-MA was further confirmed in the NSCLC xenograft model. Taken together, our results show that DSF/Cu stimulates autophagy in NSCLC cells, which is an impediment to DSF/Cu-induced apoptosis.

Disulfiram (DSF) in combination with copper (Cu) has been reported to override drug resistance in cancer cells, and DSF combined with chemotherapy based on the microtubule inhibitor vinorelbine appears to prolong survival in non-small cell lung cancer patients. Here, we investigated the mechanisms underlying these findings. DSF/Cu reversed the microtubule inhibitor resistance in A549/Taxol and KB/VCR cells in vitro, and had anti-tumor effects in A549/Taxol and KB/VCR xenograft mice. DSF/Cu and DSF reduced the cancer stem cell (CSC) characteristics of drug-resistant A549/Taxol and KB/VCR cells, including sphere formation, colony generation and migration, and DSF/Cu was more effective than DSF alone. DSF/Cu also decreased the aldehyde dehydrogenase (ALDH) activity and the expression of P-gp and stem cell transcription factors in A549/Taxol and KB/VCR cells. Knockdown of ALDH2 attenuated the CSC characteristics of resistant cancer cells and enhanced their sensitivity to Taxol or VCR. Importantly, DSF/Cu treatment inhibited the expression of ALDH2 in vitro and in vivo. Our findings suggest that DSF/Cu reverses microtubule inhibitor resistance in cancer cells by suppressing ALDH2 expression, and Cu improves the activity of DSF.

Uridine, a potential endogenous neuromodulator, has been demonstrated to interact with the dopaminergic system and to regulate dopamine-related behaviors. The present study investigated the effects of uridine on morphine-induced hyperactivity and behavioral sensitization and on modulating dopaminergic neurotransmission in mice, which may help to understand how uridine and its metabolites act as modulators of the GABAA receptors. The results showed that either systemic (30 or 100mg/kg) or central (30, 100 or 300nM) uridine administration significantly attenuated the hyperactivity induced by acute morphine treatment in mice. Intracerebroventricular administration of uracil and β-alanine also inhibited morphine-induced hyperactivity. Uridine, a known modulator of the GABA receptors, increased the extracellular levels of GABA in the brain. In addition, the GABAA receptors antagonist bicuculline significantly attenuated the effects of uridine on morphine-induced hyperactivity, suggesting that the GABAA receptors potentially mediate the effects of uridine and its metabolites on morphine-related activity. It was also observed that morphine-induced locomotor sensitization was abolished after chronic uridine treatment. In vivo microdialysis demonstrated that uridine reversed morphine-induced dopamine release in the dorsal striatum of morphine-sensitized mice. In conclusion, these data suggest that the therapeutic effects of uridine and its metabolites on morphine-induced hyperactivity and established behavioral sensitization may be mediated in part by interfering with the dopaminergic system possibly via agonistic effects at GABAA receptors.

Our previous study revealed that intracerebroventricular oxytocin (OT) markedly inhibited the restraint stress-priming conditioned place preference (CPP) reinstatement induced by methamphetamine (MAP) via the glutamatergic system. In this study, the effect of microinjection with OT into mesocorticolimbic regions, the medial prefrontal cortex (mPFC) and the dorsal hippocampus (DHC), on the restraint stress-priming CPP reinstatement were further studied. The results showed that a 15-min restraint stress significantly reinstated MAP-induced CPP, which was inhibited by the microinjection of OT (0.5 and 2.5μg/μl/mouse) into the mPFC. Atosiban (Ato), a selective inhibitor of OT receptor, could absolutely block the effect of OT (2.5μg/μl/mouse). The reinstatement was inhibited by microinjecting with OT (2.5 but not 0.5μg/μl/mouse) into the DHC, which could not be reversed by Ato. Western blotting results showed that the levels of GLT1, VGLUT2, NR2B, p-ERK1/2 and p-CREB expressions in the mPFC were increased and CaMKII was decreased markedly after the stress-priming MAP-induced CPP reinstatement test. OT blocked the changing levels of GLT1, VGLUT2, NR2B, p-CREB and CaMK II, which were reversed by Ato, but failed to affect the elevated expression of p-ERK1/2. In DHC, the levels of VGLUT2, p-ERK1/2 and CREB expressions were reduced during the stress-induced reinstatement, which could be reversed by OT and further abolished by Ato. The present results suggest that mPFC and DHC play differential roles in restraint stress-priming CPP reinstatement induced by MAP and OT via OT receptor affects the reinstatement in which the glutamatergic system is involved.

Pseudoginsenoside-F11 (PF11), an ocotillol-type ginsenoside, has been shown to possess significant neuroprotective activity. Since microglia-mediated inflammation is critical for induction of neurodegeneration, this study was designed to investigate the effect of PF11 on activated microglia. PF11 significantly suppressed the release of ROS and proinflammatory mediators induced by LPS in a microglial cell line N9 including NO, PGE2, IL-1β, IL-6 and TNF-α. Moreover, PF11 inhibited interaction and expression of TLR4 and MyD88 in LPS-activated N9 cells, resulting in an inhibition of the TAK1/IKK/NF-κB signaling pathway. PF11 also inhibited the phosphorylation of Akt and MAPKs induced by LPS in N9 cells. Importantly, PF11 significantly alleviated the death of SH-SY5Y neuroblastoma cells and primary cortical neurons induced by the conditioned-medium from activated microglia. At last, the effect of PF11 on neuroinflammation was confirmed in vivo: PF11 mitigated the microglial activation and proinflammatory factors expression obviously in both cortex and hippocampus in mice injected intrahippocampally with LPS. These findings indicate that PF11 exerts anti-neuroinflammatory effects on LPS-activated microglial cells by inhibiting TLR4-mediated TAK1/IKK/NF-κB, MAPKs and Akt signaling pathways, suggesting its therapeutic implication for neurodegenerative disease associated with neuroinflammation.

Scorpion venoms are complex mixtures of dozens or even hundreds of distinct proteins, many of which have diverse bioactivities. In this study, after bioassay-driven chromatographic purification, a new dual-function peptide with analgesic and antitumor activities was isolated and designated BmK AGAP-SYPU2. The first 12 amino acid residues were sequenced with Edman degradation. The cDNA was cloned by using rapid amplification of cDNA ends from the cDNA pool from scorpion glands. The amino acid sequence of BmK AGAP-SYPU2 was then deduced, and is consistent with the molecular mass measured with MALDI-TOF-MS. A preliminary pharmacological analysis revealed the following: in the dose-effect curve plotted with the mouse-twisting test, BmK AGAP-SYPU2 showed analgesic activity with an ED50 value of 1.42 mg/kg; in the time-effect curves plotted with a hot-plate procedure, BmK AGAP-SYPU2 had similar effects to those of the painkiller morphine, except for its longer duration. BmK AGAP-SYPU2 also showed antitumor activity against Ehrlich ascites tumor and S-180 fibrosarcoma models in vivo. Sequence alignment and homology modeling showed that BmK AGAP-SYPU2 is highly conserved relative to other scorpion α-toxins. However, a few different amino acids endow it with unique molecular properties, which may be responsible for its specific bioactivities. BmK AGAP-SYPU2, a new scorpion neurotoxin with dual functions, is a potential candidate drug amenable to exploitation and modification.

Intracerebroventricular (icv) streptozotocin (STZ) injection decreases cerebral insulin signal pathway function and produces multiple effects that resemble the molecular, pathological, and behavioural features of Sporadic Alzheimer’s disease (SAD). We previously reported that yonkenafil (yonk), the analogue of sildenafil and a novel PDE5 inhibitor exerts an anti-amyloidogenesis effect by regulating the Aβ level and inhibiting the expression of β-amyloid precursor protein in the APP/PS1 transgenic mice model. In this study, the effects of yonk on cognitive behaviors as well as the pathological features in streptozotocin-induced SAD rat model were investigated. The results demonstrated that administration of yonk at doses of 3 and 10 mg/kg for three weeks significantly improved cognitive deficits, attenuated STZ-induced neuronal death, inhibited the over-activation of microglia and astrocytes and the levels of pro-inflammatory markers, as well as decreased PDE5 protein expression in the hippocampus. Furthermore, yonk (3 mg/kg) notably prevented changes in tau hyperphosphorylation, decreased IRS-1and JNK phosphorylation and increased the GSK3β (ser9) phosphorylation induced by STZ. In summary, these data suggested that yonk significantly reversed STZ-induced memory deficits by inhibiting the over-activation of microglia and astrocytes, as well as ameliorated the levels of pro-inflammatory makers and tau hyperphosphorylation through regulating GSK3β signalling pathway.

Ethnopharmacological relevance Xiaochaihutang (XCHT), as a classical herbal formula for the treatment of “Shaoyang syndrome” has been demonstrated to exert an antidepressant effect in multiple animal models of depression as shown in our previous studies. However, the effects of XCHT on social isolation (SI)-reared mice have not been investigated. This study aims to explore the effects of XCHT on depressive/anxiety-like behaviors of SI-reared mice, and its implicated mechanisms, including alterations in the monoaminergic system, neurogenesis and neurotrophin expression. Materials and methods Male C57 BL/6 J mice (aged 4 weeks after weaning) were reared isolatedly for 8 weeks and XCHT (0.8, 2.3, 7.0 g/kg) were given by gavage once a day. Forced swimming test (FST), tail suspension test (TST), open field test (OFT), elevated-plus maze test (EPM) and intruder-induced aggression test were used to explore the effects of XCHT on depressive/anxiety-like behaviors of SI-reared mice after administration of XCHT for 6 weeks. HPLC-MS/MS was performed to quantify the levels of neurotransmitters in the hippocampus by in vivo microdialysis, while western immunoblotting was used to evaluate the action of XCHT on the synthesis, transport and degradation of monoamine neurotransmitters. Immunofluorescence was used to study the effects of XCHT on neurogenesis and neurotrophin expression, including Ki-67, DCX, BrdU and BDNF. Results Our results showed that administration of XCHT (0.8, 2.3 and 7.0 g/kg) for 6 weeks significantly attenuated the increase in immobility time in TST and FST, improved the anxiety-like behaviors in OFT and EPM, and improved the aggressive behaviors of SI-reared mice. XCHT significantly elevated monoamine neurotransmitters levels and inhibited 5-HT turnover (5-HIAA/5-HT) in hippocampal microdialysates of SI-reared mice. In addition, we found XCHT enhanced monoamine neurotransmitter synthesis enzymes (TPH2 and TH) expressions, inhibited serotonin transporter (SERT) expression and decreased monoamine neurotransmitter degradation enzyme (MAO A ) expression in the hippocampus of SI-reared mice for the first time. Moreover, XCHT significantly augmented hippocampal neurogenesis and BDNF expression in hippocampus of SI-reared mice. Conclusions Our results showed for the first time that XCHT improved depressive/anxiety-like behaviors of SI-reared mice by regulating the monoaminergic system, neurogenesis and neurotrophin expression. The findings indicate that XCHT may have a therapeutic application for early-life stress model of depression and in turn provide further evidence supporting XCHT a novel potential antidepressant from a distinct perspective.

Summary Aims Pseudoginsenoside-F11 (PF11), an ocotillol-type ginsenoside, has been reported to exert wide-ranging neuroprotective properties. The aim of this study was to investigate the effect and potential mechanisms of PF11 on the autophagic/lysosomal pathway following ischemic stroke. Methods Male Sprague-Dawley rats underwent permanent middle cerebral artery occlusion (pMCAO). Cerebral ischemia outcome, TUNEL staining, Fluoro-Jade B staining were carried out 24 hours poststroke. The autophagic/lysosomal-related proteins were measured. Results A single administration of PF11 significantly decreased the infarct area, reduced the brain water content, and improved neurological functions, even 4 hours after the onset of pMCAO. Meanwhile, PF11 lessened the ischemic insult-mediated loss of neurons and activation of astrocytes and microglia. Furthermore, PF11 attenuated pMCAO-induced accumulations of autophagosomes and apoptosis. We further observed a remarkable effect of PF11 in reversing the ischemic insult-induced accumulation of autophagosomes (LC3-II) and abnormal aggregation of autophagic proteins (SQSTM1 and ubiquitin). Furthermore, PF11 was capable of improving lysosomal function and lysosome/autophagosome fusion following pMCAO, and this change was reversed by the lysosomal inhibitor chloroquine. Also, the improvement of ischemic outcome and the antiapoptotic effect induced by PF11 was reversed by CQ. Conclusion These findings indicate that the autophagic flux is impaired in a rat model of pMCAO, and that PF11 exerts an excellent protective effect against ischemic stroke by alleviating autophagic/lysosomal defects.

Antidepressants increase hippocampal neurogenesis by activating the glucocorticoid receptor (GR), but excessive GR activation impairs hippocampal neurogenesis, suggesting that normal GR function is crucial for hippocampal neurogenesis. Baicalin was reported to regulate the expression of GR and facilitate hippocampal neurogenesis, but the underlying molecular mechanisms are still unknown. In this study, we used the chronic corticosterone (CORT)-induced mouse model of anxiety/depression to assess antidepressant-like effects of baicalin and illuminate possible molecular mechanisms by which baicalin affects GR-mediated hippocampal neurogenesis. We found that oral administration of baicalin (40, 80 or 160 mg/kg) for 4 weeks alleviated several chronic CORT-induced anxiety/depression-like behaviors. Baicalin also increased Ki-67- and DCX-positive cells to restore chronic CORT-induced suppression of hippocampal neurogenesis. Moreover, baicalin normalized the chronic CORT-induced decrease in GR protein levels, the increase in GR nuclear translocation and the increase in GR phosphorylation at Ser203 and Ser211. Finally, chronic CORT exposure increased the level of FK506-binding protein 51 (FKBP5) and of phosphorylated serum- and glucocorticoid-inducible kinase 1 (SGK1) at Ser422 and Thr256, whereas baicalin normalized these changes. Together, our findings suggest that baicalin improves anxiety/depression-like behaviors and promotes hippocampal neurogenesis. We propose that baicalin may normalize GR function through SGK1- and FKBP5-mediated GR phosphorylation.

Xiao-Chai-Hu-Tang (XCHT) has been proven to be effective for the clinical treatment of depression. However, the mechanisms of definite antidepressant-like effects and detailed metabolic biomarkers were still unclear in this prior study. Here, we have investigated the metabolic profiles and potential biomarkers in a chronic unpredictable mild stress model after treatment with XCHT. Metabonomics based on ultra-high performance liquid chromatography coupled with mass spectrometry was used to profile the metabolic fingerprints of serum obtained from a rat model with chronic unpredictable mild stress with and without XCHT treatment. The model rats showed a significant decrease in sucrose preference and food consumption, and these depression-like symptoms were significantly improved by XCHT. Through principal component analysis (PCA), nine potential biomarkers of tryptophan, uric acid, phenylalanine, cholic acid and lysophosphatidylcholine (C18:0 LPC, C16:0 LPC, C16:1 LPC, C18:1 LPC, C20:4 LPC) were characterized as potential biomarkers involved the pathogenesis of depression. The therapeutic effect of XCHT on depression may involve in amino acid metabolism, lipid metabolism, oxidative stress and inflammation response. The present investigation highlights that metabonomics is a valuable tool for studying the essence of depression as well as evaluating the efficacy of the corresponding drug treatment.

The medial prefrontal cortex (mPFC) and the dorsal hippocampus (DHC) play significant roles in stimulant-induced neurobehavioral effects. Methamphetamine (MAP)-induced hyperactivity has been reported to be involved in the regulation of the glutamatergic system. The present study examined whether the glutamatergic and GABAergic systems in the mPFC and DHC were involved in MAP-induced hyperactivity in mice. A combined kainic acid (KA) or N-methyl-d-aspartate (NMDA) lesion and microdialysis technique targeting both the mPFC and DHC were used. The results showed that both KA- and NMDA-induced lesions of the mPFC facilitated MAP-induced hyperactivity, while neither KA- nor NMDA-induced lesions of the DHC had a similar effect. MAP increased the extracellular glutamate (Glu) levels in the mPFC and reduced Glu levels in the DHC. GABA levels in both of these regions were reduced. A KA or NMDA lesion of the mPFC inhibited the Glu reduction in the DHC, and the same lesion of the DHC inhibited the Glu increase in the mPFC induced by MAP. A NMDA lesion of the mPFC blocked GABA reduction in the DHC, but a lesion of DHC enhanced the GABA decrease in the mPFC induced by MAP. Furthermore, a NMDA lesion of DHC increased the vesicular glutamate transporter-2 (VGLUT2) expression in the mPFC following MAP-administration. These findings indicate that glutamatergic as well as GABAergic systems in these two regions are involved in MAP-induced hyperactivity. Moreover, there may be an inhibitory role in these two regions, especially mediated by NMDA receptors, in MAP-induced abnormal behavior and neurotransmission responses.

Blood-brain barrier (BBB) disruption is a major consequence of cerebral ischemia/reperfusion. Several studies have reported the neuroprotection of pinocembrin on cerebral ischemia in vivo and in vitro, but the effects of pinocembrin on BBB and its underlying mechanisms are not clear. In this study, we investigated the effects of pinocembrin on BBB functions in the global cerebral ischemia/reperfusion (GCI/R) model in rats. Neurological scores and brain edema were evaluated. BBB permeability was assessed by detecting the concentrations of Evan's blue (EB) and fluorescein sodium (NaF) in brain tissue. The pathological changes of BBB ultrastructure were observed by transmission electron microscopy. Cerebral blood flow (CBF) was measured by laser Doppler flowmetry. The effects of pinocembrin on primary cultured rat cerebral microvascular endothelial cells (RCMECs) against oxygen-glucose deprivation/reoxygenation (OGD/R) were also investigated. The results showed pinocembrin decreased neurological score and lessened brain edema induced by GCI/R. Pinocembrin also reduced the concentrations of EB and NaF in brain tissue of the GCI/R rats. And pinocembrin alleviated the ultrastructural changes of cerebral microvessels, astrocyte end-feet and neurons, and improved CBF in the GCI/R rats. In addition, pinocembrin increased the viability and mitochondrial membrane potential of cultured RCMECs induced by OGD/R. In conclusion, these data demonstrate that pinocembrin alleviates blood-brain barrier injury induced by GCI/R in rats.

Diseases related to ethanol abuse, especially binge drinking, are becoming one of the most costly health problems in the world. Ethanol-induced DNA damage plays a key role in the etiology of these diseases. New compounds are expected to offer new options against ethanol-induced genotoxicity. It was found, for the first time, that resveratrol and three analogues with 3,5-dimethoxyl groups in the A-ring, such as (E)-4-(3,5-dimethoxystyryl)phenol (RV32), or with a quinolyl in the B-ring, such as (E)-5-[2-(quinolin-4-yl)vinyl]benzene-1,3-diol (RV01) and (E)-4-(3,5-dimethoxystyryl)quinoline (RV02), strongly inhibited ethanol-induced oxidative DNA damage in human peripheral lymphocytes in vitro. Resveratrol and RV32 with more hydroxyl groups in structures showed stronger direct scavenging activity of hydroxyl radicals than RV01 and RV02. Moreover, all compounds reduced hydroxyl radical generation by regulating the mRNA expression of alcohol dehydrogenase 1B and acetaldehyde dehydrogenase 2. Further studies proved resveratrol and three analogues activated the base excision repair system in transcriptional and protein levels in DNA repair process. Both 3,5-dimethoxyl groups and quinolyl modification may enhance such activity. In summary, resveratrol and its three analogues revealed significant protective activity against ethanol-induced oxidative DNA damage in human peripheral lymphocytes, which demonstrates their potential for use in prevention and treatment of the diseases related to ethanol abuse.

Local perfusion of morphine produces increased levels of extracellular ascorbic acid (AA) in the nucleus accumbens (NAc) of freely moving rats. However, the pathways that regulate morphine-induced AA release in the NAc are unclear. In the present study, we used high performance liquid chromatography with electrochemical detection (HPLC-ECD) to examine the effects of intra-ventral tegmental area (VTA) administration of a GABA(A) agonist and antagonist on morphine-induced increases in AA of the NAc. Also, using high performance liquid chromatography with fluorescent detection (HPLC-FD) and HPLC-ECD, the releases of γ-aminobutyric acid (GABA) and dopamine (DA) in the NAc induced by intra-VTA administration of a GABA(A) agonist and antagonist were also investigated. The results obtained showed that morphine (1 mM), locally perfused into the NAc, significantly increased AA release in the NAc and also GABA release. Intra-VTA infusion of bicuculline (150 ng/rat), a GABA receptor antagonist, not only abolished the enhanced extracellular AA and GABA levels produced by local perfusion of morphine but also decreased the basal release of extracellular GABA and increased the basal release of extracellular DA in the NAc. Muscimol (100 ng/rat), a GABA receptor agonist, affected the basal release of GABA and DA, but not the basal AA levels, or the morphine-induced changes in AA and GABA levels. These findings suggest that the GABA(A) receptors in the VTA play an important role in the modulation of morphine-induced AA release in the NAc, and the effect of morphine on AA release in the NAc is partially regulated by the GABA(A) receptor-mediated action of DA afferents from the VTA.

In this study, the hepatoprotective effects of apple polyphenols (AP, Appjfnol) against CCl(4)-induced acute liver damage in Kunming mice as well as the possible mechanisms were investigated. Mice were treated with AP (200, 400, and 800 mg/kg, ig) for seven consecutive days prior to the administration of CCl(4) (0.1%, intraperitoneally). The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) concentrations in the hepatic homogenate, and histopathological changes in mouse liver sections were determined. Levels of ferrous sulfate-L-cysteine (FeSO(4)-L-Cys)-induced lipid peroxidation and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were also determined in vitro. AP significantly prevented the increase in serum ALT and AST levels in acute liver injury induced by CCl(4) and produced a marked amelioration in the histopathological hepatic lesions coupled to weight loss. The extent of MDA formation was reduced; the SOD activity was enhanced, and the GSH concentration was increased in the hepatic homogenate in AP-treated groups compared with the CCl(4)-intoxicated group. AP also exhibited antioxidant effects on FeSO(4)-L-Cys-induced lipid peroxidation in rat liver homogenate and DPPH free radical scavenging activity in vitro. These results indicate that AP has a significant protective effect against acute hepatotoxicity induced by CCl(4) in mice, which may be due to its free radical scavenging effect, inhibition of lipid peroxidation, and its ability to increase antioxidant activity.

Accumulating evidence has shown the neuroactive properties of oxytocin (OT), a neurohypophyseal neuropeptide, and its ability to reduce the abuse potential of drugs. The present study investigated the effects of OT on the conditioned place preference (CPP) induced by methamphetamine (MAP, 2.0 mg/kg, i.p.) in mice and the possible role of glutamatergic neurotransmission in the reinstatement of CPP. The results showed that OT (0.1, 0.5, 2.5 microg, i.c.v.) significantly inhibited the acquisition and facilitated the extinction of MAP-induced CPP and abolished the reinstatement of CPP induced by restraint stress. This effect of OT could be attenuated by atosiban (Ato, 2.0 microg, i.c.v.), a selective OT-receptor antagonist. OT failed to block the expression and the reinstatement of CPP induced by MAP challenge. Extracellular glutamate (Glu) levels in the medial prefrontal cortex (mPFC) were determined using microdialysis coupled to a high-performance liquid chromatography (HPLC) with a fluorescence detection system. The results indicated that OT markedly inhibited extracellular Glu levels induced by restraint stress in CPP mice, but not those induced by MAP priming. Ato also attenuated the effects of OT on the changes in Glu levels. Therefore, these findings suggest that OT inhibits drug reward-related behaviors induced by MAP via the OT receptor, and OT blocks the reinstatement of CPP, at least partially, by interfering with the glutamatergic system in the mPFC.