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Recently, a human mutation of OPG was identified to be associated with familial forms of osteoarthritis. This missense mutation (c.1205A = > T; p.Stop402Leu) occurs on the stop codon of OPG, which results in a 19-residue appendage to the C-terminus (OPG+19). The biochemical consequence of this unusual sequence alteration remains unknown. Here we expressed OPG+19 in 293 cells and the mutant OPG was purified to homogeneity by heparin affinity chromatography and size exclusion chromatography. We found that in sharp contrast to wildtype OPG, which mainly exists in dimeric form, OPG+19 had a strong tendency to form higher-order oligomers. To our surprise, the hyper-oligomerization of OPG+19 had no impact on how it binds cell surface heparan sulfate, how it inhibits RANKL-induced osteoclastogenesis and TRAIL-induced chondrocytes apoptosis. Our data suggest that in biological contexts where OPG is known to play a role, OPG+19 functions equivalently as wildtype OPG. The disease-causing mechanism of OPG+19 likely involves an unknown function of OPG in cartilage homeostasis and mineralization. By demonstrating the biochemical nature of this disease-causing OPG mutant, our study will likely help elucidating the biological roles of OPG in cartilage biology.

The economic burden of tumors is increasing, so there is an urgent need to develop new therapies for their treatment. Killing tumors by activating complement is an effective strategy for the treatment. We used the ABO blood group system and the corresponding antibodies to activate the killer cell capacity of the complement system. After the construction of a mouse model containing blood group A antibodies and inoculating colorectal cancer and breast cancer cells into the axillae of the mice, intratumoural injection using a lentivirus carrying a blood group antigen as a drug significantly reduced the tumor volume of the mice. Compared with the control group, the content of the C5b-9 complement membrane attack complex in the tumors of mice treated with the blood group A antigen was significantly increased, and the proportion of NK cells was also significantly increased. In vitro cell-based experiments proved that tumor cells expressing blood group A antigens showed significantly inhibited cell proliferation when added to serum containing blood group A antibodies. These results all prove that the ABO blood group antigen may become a powerful tool for the treatment of tumors in patients.

Shikonin induces glioma cell death via necroptosis, a caspase-independent programmed cell death pathway that is chiefly regulated by receptor-interacting serine/threonine protein kinase1 (RIP1) and 3 (RIP3). Chromatinolysis is considered as one of the key events leading to cell death during necroptosis. It is usually accompanied with nuclear translocation of AIF and formation of γ-H2AX. Cyclophilin A (CypA) is reported to participate in the nuclear translocation of AIF during apoptosis. However, it remains unclear whether CypA contributes to necroptosis and regulation of chromatinolysis. In this study, our results revealed for the first time that shikonin promoted time-dependent CypA activation, which contributed to nuclear translocation of AIF and γ-H2AX formation. In vitro studies showed that knockdown of CypA by siRNA or inhibition of CypA by its specific inhibitor, cyclosporine A (CsA), not only significantly mitigated shikonin-induced glioma cell death, but also prevented chromatinolysis. Mechanistically, activated CypA targeted mitochondria and triggered mitochondrial superoxide overproduction, which then promoted AIF translocation from mitochondria into the nucleus by depolarizing the mitochondria and intensified the formation of γ-H2AX by promoting intracellular accumulation of ROS. Additionally, the CypA in the nucleus can form DNA degradation complexes with AIF and γ-H2AX, which also promote the execution of chromatinolysis. Thus, we demonstrate that CypA contributes to shikonin-induced glioma cell necroptosis and promotion of chromatinolysis.

Studies have found that circRNA in exosomes is associated with oral squamous cell carcinoma (OSCC) progression. In this study, we examined the expression of circ_0000199 in circulating exosomes from patients with OSCC and its role in the evaluation of relapse and prognosis. Real‐time quantitative reverse transcription–polymerase chain reaction was performed to assess circ_0000199 expression in circulating exosomes from 108 patients with OSCC and 50 healthy people. Gain- and loss-functional experiments were performed to assess the role of circ_0000199 on cell proliferation and apoptosis in OSCC cells. Our results showed that the high level of circ_0000199 in circulating exosomes was significantly associated with betel quid chewing, tumor size, lymphatic metastasis, and TNM stage in patients with OSCC. In addition, the patients with high exosomal circ_0000199 had higher tumor recurrence rate and higher mortality rate than the patients with low exosomal circ_0000199. Overexpression of circ_0000199 promoted, while knockdown of circ_0000199 inhibited OSCC cell growth. Bioinformatics analysis predicted that circ_0000199 interacted with miR-145-5p and miR-29b-3p simultaneously, which were involved in multiple tumor‐related signaling pathways. In conclusion, upregulation of circ_0000199 in circulating exosomes from patients with OSCC is positively associated with poor survival outcome. Circulating exosomal circ_0000199 can be used as a biomarker and potential therapeutic target for OSCC.

The study’s objectives were to examine the effects of electrofusion on rabbit somatic cell nuclear transfer (SCNT) embryos, and to test melatonin as a protective agent against electrofusion damage to SCNT embryos. The levels of reactive oxygen species (ROS), the epigenetic state (H3K9me3), and the content of endoplasmic reticulum (ER) stress-associated transcripts (IRE-1 and CHOP) were measured. Melatonin was added during the preimplantation development period. The total blastocyst cell numbers were counted, and the fragmentation rate and apoptotic index were determined and used to assess embryonic development. Electrofusion increased (1) ROS levels at the 1-, 2-, 4-, and 8-cell stages; (2) H3K9me3 levels at the 2-, 4-, and 8-cell stage; and (3) the expression of IRE-1 and CHOP at the 8-cell, 16-cell, morula, and blastocyst stages. The treatment of SCNT embryos with melatonin significantly reduced the level of ROS and H3K9me3, and the expression levels of IRE-1 and CHOP. This treatment also significantly reduced the fragmentation rate and apoptotic index of blastocysts and increased their total cell number. In conclusion, the electrofusion of rabbit SCNT embryos induced oxidative stress, disturbed the epigenetic state, and caused ER stress, while melatonin reduced this damage. Our findings are of signal importance for improving the efficiency of SCNT and for optimizing the application of electrical stimulation in other biomedical areas.

Duck hepatitis A virus (DHAV) causes an infectious disease that mainly affects 1- to 4-week-old ducklings, resulting in considerable loss to the duck industry. Although there have been many studies on DHAV in recent years, the effects on host infection and pathogenesis of DHAV-1 remain largely unknown. This study investigated the effects of the DHAV-1 structural protein VP3 on DHAV-1 virus adsorption and apoptosis to explore the role of VP3 in the viral life cycle. The effects of DHAV-1 VP3 and an antibody against the protein on virion adsorption was analyzed by qRT-PCR. The results showed that the virus copy number for the rabbit anti-VP3 IgG-treated group was significantly lower than that for the negative control group but higher than that for the rabbit anti-DHAV-1 IgG-treated group. This result indicates that VP3 mediates DHAV-1 virus adsorption but that it is not the only protein that involved in this process. In addition, a eukaryotic recombinant plasmid, pCAGGS/VP3, was transfected into duck embryo fibroblasts (DEFs), and the apoptotic rate was determined by DAPI staining, the TUNEL assay and flow cytometry. DAPI staining showed nucleus fragmentation and nuclear edge shifting. TUNEL assay results revealed yellow nuclei, and flow cytometry indicated a significant increase in the apoptotic rate. In addition, qRT-PCR revealed increased in the transcriptional levels of the apoptotic caspase-3, −8 and −9, with the largest increase for caspase-3, followed by caspase-9 and caspase-8. Enzyme activity analysis confirmed these results. Furthermore, the VP3 protein decreased the mitochondrial membrane potential, and the transcriptional levels of the proapoptotic factors Bak, Cyt c and Apaf-1 in the mitochondrial apoptotic pathway were significantly upregulated. These data suggest that expression of VP3 in DEFs induces apoptosis and may primarily activate caspase-3-induced apoptosis through mitochondrion-mediated intrinsic pathways. The findings provide scientific data to clarify DHAV-1 infection and pathogenesis.

Pseudorabies virus (PRV) infection can elicit nervous system disorders. Curcumin has been reported to have neuroprotective effects. However, whether curcumin can protect neurons against PRV infection and the underlying mechanisms remain unclear. In the present study, for the first time, the protective effects of curcumin against PRV-induced oxidative stress, apoptosis, and mitochondrial dysfunction in rat hippocampal neurons and the brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) pathway were investigated. Results indicated that PRV with a titer of 3.06 × 106 TCID50 (50% tissue culture infective dose) induced oxidative damage of hippocampal neurons 2 h post-infection and that 10 μM curcumin improved the viability of PRV-infected hippocampal neurons. Blocking the BDNF/TrkB pathway reversed the neuroprotective effects of curcumin, which were imparted by decreasing the PRV-induced upregulation of nitric oxide synthase expression, repressing the PRV-activated mitochondrial apoptotic pathway, and mitochondrial dysfunction. To conclude, curcumin exhibited a neuroprotective role against PRV infection by upregulating the BDNF/TrkB pathway. This study provides insight into the anti-PRV neuroprotective application of curcumin and the underlying mechanism in the prophylaxis and treatment of neurological disorders caused by PRV infection.

Cryoablation has been used for the treatment of various sorts of solid visceral tumors, but few are reported on gastric tumor via endoscope, in terms of accurate control of ablation site, freezing depth and effective temperature. Thus, we developed a novel device, which could perform accurate cryoablation on the stomach via endoscope. This study aimed to evaluate the efficacy and safety of the device on porcine stomach. Results showed that the novel device could provide direct view of the operation space, allowing accurate and safe ablation of the stomach. Three minutes cryoablation caused a transmural, 1 cm radius gastric lesion. On serosal side, the temperature dropped to −64.2 °C, −34.1 °C, 26.1 °C at the center, 1 cm and 2 cm from center, respectively. Histopathology revealed acute ruptured cells with damaged glands in mucosa, partial disruption in muscularis propria and serosal slight exudation. Three months later, scar formed with complete recovery of gastric structure. No active bleeding or perforation of stomach, nor injury or adhesion of adjacent organs was observed. This endoscopic cryoablation device allowed safe, full-thickness cryoablation with effective temperature, which may provide an alternative treatment for gastric tumor.

We previously reported that oxidative stress induced by long-term tacrolimus treatment impairs mitochondrial function in pancreatic beta cells. In this study, we aimed to investigate the therapeutic potential of coenzyme Q10, which is known to be a powerful antioxidant, in mitochondrial dysfunction in tacrolimus-induced diabetic rats. In a rat model of tacrolimus-induced diabetes mellitus, coenzyme Q10 treatment improved pancreatic beta cell function. The administration of coenzyme Q10 improved insulin immunoreactivity within islets, which was accompanied by reductions in oxidative stress and apoptosis. Assessment of the mitochondrial ultrastructure by electron microscopy revealed that coenzyme Q10 treatment increased the size, number, and volume of mitochondria, as well as the number of insulin granules compared with that induced by tacrolimus treatment alone. An in vitro study using a pancreatic beta cell line showed that tacrolimus treatment increased apoptosis and the production of mitochondrial reactive oxygen species, while cotreatment with coenzyme Q10 effectively attenuated these alterations. At the subcellular level, tacrolimus-induced impairment of mitochondrial respiration was significantly improved by coenzyme Q10, as evidenced by the increased mitochondrial oxygen consumption and ATP production. Our data indicate that coenzyme Q10 plays an important role in reducing tacrolimus-induced oxidative stress and protects the mitochondria in pancreatic beta cells. These findings suggest that supplementation with coenzyme Q10 has beneficial effects in tacrolimus-induced diabetes mellitus.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly malignancies with in vivo study shows that monensin blunts PDAC xenograft tumor growth by suppressing cell proliferation via targeting EGFR pathway. Therefore, our findings demonstrate that monensin can be repurposed as an effective anti-pancreatic cancer drug even though more investigations are needed to validate its safety and anticancer efficacy in pre-clinical and clinical models.

Psoriasis is a chronic inflammatory skin disease with high morbidity, poor treatment methods and high rates of relapse. Keratinocyte hyperproliferation and shortened cell cycles are important pathophysiological features of psoriasis. As a known oncogene, Yes-associated protein (YAP) plays a role in promoting cell proliferation and inhibiting cell apoptosis; however, whether YAP is involved in the pathogenesis of psoriasis remains to be determined. Amphiregulin (AREG), a transcriptional target of YAP, was found to be upregulated in psoriasis, and overexpression of AREG promoted keratinocyte proliferation. In the present study, immunohistochemistry showed that YAP expression was elevated in the skin of psoriasis patients and in the Imiquimod (IMQ) mouse model of psoriasis. Knockdown of YAP in HaCaT cells inhibited cell proliferation, caused cell cycle arrest in G0/G1 phase and promoted apoptosis. These changes in YAP-knockdown HaCaT cells were related to changes in AREG expression. We concluded that YAP may play an important role in the regulation of abnormal keratinocyte proliferation via an AREG-dependent pathway and that YAP could be a new target in the treatment of psoriasis.

JS-K, a nitric oxide-releasing diazeniumdiolates, is effective against various tumors. We have discovered that JS-K was effective against Hepatitis B virus (HBV)-positive HepG2.2.15 cells. This study used iTRAQ to identify differentially expressed proteins following JS-K treatment of HepG2.2.15 cells. Silenced Transgelin (shTAGLN-2.15) cells were constructed, and the cell viability was analyzed by the CCK8 assay after treatment with JS-K. There were 182 differentially expressed proteins in JS-K treated-HepG2.2.15 cells; 73 proteins were up-regulated and 109 proteins were down-regulated. These proteins were categorized according to GO classification. KEGG enrichment analysis showed that Endocytosis, Phagosome and Proteoglycans were the most significant pathways. RT-PCR confirmed that the expression levels of TAGLN, IGFBP1, SMTN, SERPINE1, ANXA3, TMSB10, LGALS1 and KRT19 were significantly up-regulated, and the expression levels of C5, RBP4, CHKA, SIRT5 and TRIM14 were significantly down-regulated in JS-K treated-HepG2.2.15 cells. Western blotting confirmed the increased levels of USP13 and TAGLN proteins in JS-K treated-HepG2.2.15 cells. Molecular docking revealed the binding of JS-K to TAGLN and shTAGLN-2.15 cells were resistant to JS-K cytotoxicity, suggesting that TAGLN could be an important target in JS-K anti-HBV-positive liver cancer cells. These proteomic findings could shed new insights into mechanisms underlying the effect of JS-K against HBV-related HCC.

In view of the complicated pathophysiological process of vascular dementia (VD), drugs for the clinical treatment of VD mainly target related risk factors, while drugs with excellent efficacy in cognitive function are still relatively lacking. Imperatorin (IMP), an active constituent extracted from angelica dahuricae and notopterygium Notopterygii, which has anti-inflammatory, vasodilator, anticoagulant, block calcium channel, anticonvulsant, and anti oxygen free radical injury properties. Therefore,the present study examined its effects on VD rats and the underlying molecular mechanisms, in order to provide promising therapeutic methods. VD was established by modified ligation of perpetual two-vessel occlusion (2VO). After 2VO surgery, IMP (2.5, 5, and 10 mg/kg) was administered by intraperitoneal injection for 12 consecutive weeks to evaluate therapeutic effects. Cognitive function was verified by the Morris water maze. The neuronal morphological changes were examined via Hematoxylin–Eosin staining. Real-Time PCR and Western blot were used for detecting pro- and antiapoptotic biomarkers, and the hippocampus synaptic damage was examined by Transmission electron microscope. We revealed that 2VO-induced cognitive impairment, hippocampus CA1 neuron damage, apoptosis and synaptic damage. IMP-treatment significantly improved 2VO-induced cognitive deficits and hippocampus neuron damage. Molecular analysis revealed that IMP inhibited apoptosis through the down regulation of Bax, Caspase-3 and upregulation of Bcl-2. Meanwhile, IMP-treatment markedly improved synaptic ultrastructure morphology, increased the SAZ length, PSD thickness and up-regulated PSD-95 expression. Collectively, our findings demonstrated that IMP was effective in the treatment of 2VO-induced VD via inhibiting apoptosis of hippocampus neurons and reducing the synaptic plasticity destroy.

Although gentamicin is widely used as an antibiotic in clinical practice, it also has some side-effects, such as acute kidney injury, which is a common condition caused by the abuse of gentamicin. Sika deer antler protein (SDAPR) can antagonize drug-induced AKI. Since SDAPR is recognized as an effective part of velvet antler, its components were further separated. Two components named SDAP1 and SDAP2 were obtained. The protective effects of SDAPR, SDAP1 and SDAP2 on GM-induced cytotoxicity to HEK293 and its potential mechanisms were studied. MTT and xCELLigence Real-Time cell analysis showed that SDAPR, SDAP1 and SDAP2 could protect HEK293 cells from GM toxicity. Similarly, SDAPR, SDAP1 and SDAP2 can reduce ROS level, reduce oxidative stress and improve inflammation Further studies have shown that SDAPR, SDAP1 and SDAP2 upregulate the Nrf2/HO-1 pathway by increasing the expression of Nrf2 and HO-1, and down-regulate the NF-κB pathway by reducing the protein expression of NF-κB. Annexin V/PI flow cytometry and Hoechst 33258 staining showed that SDAPR, SDAP1 and SDAP2 inhibited GM-induced apoptosis in HEK293 cells. Western blot analysis showed SDAPR, SDAP1 and SDAP2 decreased expression level of Bax and Cleaved-caspase-3, and increased the expression level of Bcl-2. In addition, we examined the feasibility of SDAP1 and SDAP1 to avoid kidney injury in a GM mouse model. In conclusion, SDAPR, SDAP1 and SDAP2 can be used to prevent GM-induced HEK293 cytotoxicity, probably because they have strong anti-oxidative stress, anti-inflammatory and anti-apoptotic effects. And SDAP1 and SDAP2 can inhibit GM-induced acute kidney injury in mice.

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder and there is no effective cure for this devastating disease to date. Bushen Yizhi Formula (BSYZ-F), a Chinese herbal compound, has proved to be effective for AD. In this study, we further investigate the effective part of BSYZ-F, ethyl acetate extract components of BSYZ-F (BSYZ-E), protects scopolamine (SCOP)-induced cognitive impairment, which shows a similar effect to BSYZ-F. We also find that BSYZ-E could protect against SCOP-induced cholinergic system dysfunction. In neuron function level, BSYZ-E remarkably elevates protein levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). BSYZ-E also significantly mitigates SCOP-induced apoptosis, oxidative stress and nitrosative stress. Conclusively, BSYZ-E, the effective part of BSYZ-F, can provide neuroprotection against SCOP-induced cognitive impairment through a multifunctional strategy. These findings suggest that BSYZ-E might be developed as a therapeutic drug for AD by targeting multiple pathways of the pathogenesis.

High intensity focused ultrasound (HIFU) is a noninvasive thermal ablation technique for the treatment of benign and malignant solid masses. To improve the efficacy of HIFU ablation, we developed poly (lactide-co-glycolide) (PLGA) nanoparticles encapsulating perfluoropentane (PFP) and hematoporphyrin monomethyl ether (HMME) as synergistic agents (HMME+PFP/PLGA). Two-step biotin-avidin pre-targeting technique was applied for the HIFU ablation. We further modified the nanoparticles with streptavidin (HMME+PFP/PLGA-SA). HMME+PFP/PLGA-SA were highly dispersed with spherical morphology (477.8 ± 81.8 nm in diameter). The encapsulation efficiency of HMME and PFP were 46.6 ± 3.3% and 40.1 ± 2.6%, respectively. The binding efficiency of nanoparticles to streptavidin was 95.5 ± 2.5%. The targeting ability of the HMME+PFP/PLGA-SA nanoparticles was tested by parallel plate flow chamber in vitro. In the pre-targeting group (HMME+PFP/PLGA-SA), a large number of nanoparticles bound to the peripheral and surface of the cell. In the HIFU ablation experiment in vivo, compared with the other groups, the largest gray-scale changes and coagulation necrosis areas were observed in the pre-targeting (HMME+PFP/PLGA-SA) group, with the lowest energy efficiency factor value. Moreover, the microvessel density and proliferation index declined, while the apoptotic index increased, in the tumor tissue surrounding the coagulation necrosis area in the pre-targeting group. Meanwhile, the survival time of the tumor-bearing nude mice in the pre-targeting group was significantly longer than that in the HIFU treatment group. These results suggest that HMME+PFP/PLGA-SA have high potential to act as synergistic agents in HIFU ablation.

Epigallocatechin-3-gallate (EGCG), a main active catechin in green tea, was reported to attenuate renal injury and hypertension. However, its effects on salt-induced hypertension and renal injury remain unclear. In the present study, we explored its effects on hypertension and renal damage in Dahl rats with salt-sensitive hypertension. We found that EGCG could lower blood pressure after 6 weeks of oral administration, reduce 24 h urine protein levels and decrease creatinine clearance, and attenuate renal fibrosis, indicating that it could attenuate hypertension by protecting against renal damage. Furthermore, we studied the renal protective mechanisms of EGCG, revealing that it could lower malondialdehyde levels, reduce the numbers of infiltrated macrophages and T cells, and induce the apoptosis of NRK-49F cells. Considering that the 67 kD laminin receptor (67LR) binds to EGCG, its role in EGCG-induced fibroblast apoptosis was also investigated. The results showed that an anti-67LR antibody partially abrogated the apoptosis-inducing effects of EGCG on NRK-49F cells. In summary, EGCG may attenuate renal damage and salt-sensitive hypertension via exerting anti-oxidant, anti-inflammatory, and apoptosis-inducing effects on fibroblasts; the last effect is partially mediated by 67LR, suggesting that EGCG represents a potential strategy for treating salt-sensitive hypertension.

Ubiquinol-cytochrome c reductase hinge protein (UQCRH) is the hinge protein for the multi-subunit complex III of the mitochondrial electron transport chain and is involved in the electron transfer reaction between cytochrome c1 and c. Recent genome-wide transcriptomic and epigenomic profiling of clear cell renal cell carcinoma (ccRCC) by The Cancer Genome Atlas (TCGA) identified UQCRH as the top-ranked gene showing inverse correlation between DNA hypermethylation and mRNA downregulation. The function and underlying mechanism of UQCRH in the Warburg effect metabolism of ccRCC have not been characterized. Here, we verified the clinical association of low UQCRH expression and shorter survival of ccRCC patients through in silico analysis and identified KMRC2 as a highly relevant ccRCC cell line that displays hypermethylation-induced UQCRH extinction. Ectopic overexpression of UQCRH in KMRC2 restored mitochondrial membrane potential, increased oxygen consumption, and attenuated the Warburg effect at the cellular level. UQCRH overexpression in KMRC2 induced higher apoptosis and slowed down in vitro and in vivo tumor growth. UQCRH knockout by CRISPR/Cas9 had little impact on the metabolism and proliferation of 786O ccRCC cell line, suggesting the dispensable role of UQCRH in cells that have entered a Warburg-like state through other mechanisms. Together, our study suggests that loss of UQCRH expression by hypermethylation may promote kidney carcinogenesis through exacerbating the functional decline of mitochondria thus reinforcing the Warburg effect.

Epigallocatechin-3-gallate (EGCG), a main active catechin in green tea, was reported to attenuate renal injury and hypertension. However, its effects on salt-induced hypertension and renal injury remain unclear. In the present study, we explored its effects on hypertension and renal damage in Dahl rats with salt-sensitive hypertension. We found that EGCG could lower blood pressure after 6 weeks of oral administration, reduce 24 h urine protein levels and decrease creatinine clearance, and attenuate renal fibrosis, indicating that it could attenuate hypertension by protecting against renal damage. Furthermore, we studied the renal protective mechanisms of EGCG, revealing that it could lower malondialdehyde levels, reduce the numbers of infiltrated macrophages and T cells, and induce the apoptosis of NRK-49F cells. Considering that the 67 kD laminin receptor (67LR) binds to EGCG, its role in EGCG-induced fibroblast apoptosis was also investigated. The results showed that an anti-67LR antibody partially abrogated the apoptosis-inducing effects of EGCG on NRK-49F cells. In summary, EGCG may attenuate renal damage and salt-sensitive hypertension via exerting anti-oxidant, anti-inflammatory, and apoptosis-inducing effects on fibroblasts; the last effect is partially mediated by 67LR, suggesting that EGCG represents a potential strategy for treating salt-sensitive hypertension.

Non-alcoholic fatty liver disease (NAFLD) is characterized by increased uptake and accumulation of lipids in hepatocytes. Simple steatosis may progress to non-alcoholic steatohepatitis (NASH) with inflammation, hepatocellular injury and fibrosis. CCN1 is an important matrix protein that regulates cell death and promotes immune cell adhesion and may potentially control this process. The role of CCN1 in NASH remains unclear. We investigated the role of CCN1 in the pathogenesis of steatohepatitis. CCN1 upregulation was found to be closely related with steatosis in patients with NASH, obese mice and a FFA-treated hepatocyte model. Controlling the expression of CCN1 in murine NASH models demonstrated that CCN1 increased the severity of steatosis and inflammation. From the sequence results, we found that fatty acid metabolism genes were primarily involved in the MCD mice overexpressing CCN1 compared to the control. Then, the expression of fatty acid metabolism genes was determined using a custom-designed pathway-focused qPCR-based gene expression array. Expression analysis showed that CCN1 overexpression significantly upregulated the expression of fatty acid metabolism-associated genes. In vitro analysis revealed that CCN1 increased the intracellular TG content, the pro-inflammatory cytokines and the expression level of apoptosis-associated proteins in a steatosis model using murine primary hepatocytes. We identified CCN1 as an important positive regulator in NASH.

Mortality of glioblastoma multiforme (GBM) has not improved over the last two decades despite medical breakthroughs in the treatment of other types of cancers. Nanoparticles hold tremendous promise to overcome the pharmacokinetic challenges and off-target adverse effects. However, an inhibitory effect of nanoparticles by themselves on metastasis has not been explored. In this study, we developed transferrin-conjugated porous silicon nanoparticles (Tf@pSiNP) and studied their effect on inhibiting GBM migration by means of a microfluidic-based migration chip. This platform, designed to mimic the tight extracellular migration tracts in brain parenchyma, allowed high-content time-resolved imaging of cell migration. Tf@pSiNP were colloidally stable, biocompatible, and their uptake into GBM cells was enhanced by receptor-mediated internalisation. The migration of Tf@pSiNP-exposed cells across the confined microchannels was suppressed, but unconfined migration was unaffected. The pSiNP-induced destabilisation of focal adhesions at the leading front may partially explain the migration inhibition. More corroborating evidence suggests that pSiNP uptake reduced the plasticity of GBM cells in reducing cell volume, an effect that proved crucial in facilitating migration across the tight confined tracts. We believe that the inhibitory effect of Tf@pSiNP on cell migration, together with the drug-delivery capability of pSiNP, could potentially offer a disruptive strategy to treat GBM.

Hepatocellular carcinoma (HCC) is a highly fatal disease recognized as a growing global health crisis worldwide. Currently, no curative treatment is available for early-to-intermediate stage HCC, characterized by large and/or multifocal tumors. If left untreated, HCC rapidly progresses to a lethal stage due to favorable conditions for metastatic spread. Mechanochemical disruption of cellular structures can potentially induce phenotypic alterations in surviving tumor cells that prevent HCC progression. In this paper, HCC response to mechanical vibration via high-intensity focused ultrasound and a chemical disruptive agent (ethanol) was examined in vitro and in vivo. Our analysis revealed that mechanochemical disruption caused a significant overproduction of reactive oxygen species (ROS) in multiple HCC cell lines (HepG2, PLC/PRF/5, and Hep3B). This led to a decrease in cell viability and long-term proliferation due to increased expression and activity of death receptors TNFR1 and Fas. The cells that survived mechanochemical disruption had a reduced expression of cancer stem cell markers (CD133, CD90, CD49f) and a diminished colony-forming ability. Mechanochemical disruption also impeded HCC migration and their adhesion to vascular endothelium, two critical processes in hematogenous metastasis. The HCC transformation to a non-tumorigenic phenotype post mechanochemical disruption was confirmed by a lack of tumor spheroid formation in vitro and complete tumor regression in vivo. These results show that mechanochemical disruption inhibits uncontrolled proliferation and reduces tumorigenicity and aggressiveness of HCC cells through ROS overproduction and associated activation of TNF- and Fas-mediated cell death signaling. Our study identifies a novel curative therapeutic approach that can prevent the development of aggressive HCC phenotypes.

MicroRNAs (miRNAs) have been explored in many critical cellular processes, including proliferation and apoptosis. The purpose of this study was to detect the biological function and regulation of miR-99b-5p and miR-203a-3p in gastric cancer (GC). Here, we demonstrated that miR-99b-5p/203a-3p were downregulated in both GC tissues and cell lines. MiR-99b-5p/203a-3p overexpression reduced GC cell proliferation and cell cycle progression in vitro. Notably, we combined bioinformatics tools with biological validation assays to demonstrate that insulin-like growth factor 1 receptor (IGF-1R) is a direct co-target and functional mediator of miR-99b-5p/203a-3p in GC cells. Mechanistically, the AKT pathway, which is downstream of IGF-1R, is essential for the functional roles of miR-99b-5p/203a-3p in GC cells. Taken together, our data revealed that IGF-1R is a direct co-target of miR-99b-5p/203a-3p, and miR-99b-5p/203a-3p may function as tumor suppressive miRNAs by negatively regulating IGF-1R expression in GC cells.

Polymorphonuclear leukocytes (PMNs) are the most abundant cells of the innate immune system in humans, and spontaneous PMN apoptosis plays crucial roles in maintaining neutrophil homeostasis and resolving inflammation. However, the detailed mechanisms of spontaneous PMN apoptosis remain to be elucidated. By analysis of the public microarray dataset GSE37416, we identified a total of 3050 mRNAs and 220 long non-coding RNAs (lncRNAs) specifically expressed during PMN apoptosis in a time-dependent manner. By short time-series expression miner (STEM) analysis, Gene Ontology analysis, and lncRNA-mRNA co-expression network analyses, we identified some key molecules specifically related to PMN apoptosis. STEM analysis identified 12 gene profiles with statistically significance, including 2 associated with apoptosis. Protein-protein interaction (PPI) network analysis of the genes from 2 profiles and lncRNA-mRNA co-expression network analysis identified a 12-gene hub (including NFκB1 and BIRC3) associated with apoptosis, as well as 2 highly correlated lncRNAs (THAP9-AS1, and AL021707.6). We experimentally examined the expression profiles of two mRNA (NFκB1 and BIRC3) and two lncRNAs (THAP9-AS1 andAL021707.6) by quantitative real-time polymerase chain reaction to confirm their time-dependent expressions. These data altogether demonstrated that these genes are involved in the regulation of spontaneous neutrophil apoptosis and the corresponding gene products could also serve as potential key regulatory molecules for PMN apoptosis and/or therapeutic targets for over-reactive inflammatory response caused by the abnormality in PMN apoptosis.

This study aimed to investigate whether exosomes secreted by mouse GATA-4-expressing bone marrow mesenchymal stem cells (BMSCs) could induce BMSC differentiation into myocyte precursors, decrease cardiomyocyte apoptosis, and improve cardiac function following myocardial infarction (MI). BMSCs were transduced with a lentivirus carrying a doxycycline (DOX)-inducible GATA-4 or control lentivirus, and secreted exosomes from these BMSCs were collected and co-cultured with BMSCs or cardiomyocytes under hypoxic and serum free conditions. Furthermore, exosomes were injected into mice 48 h after MI. Cardiac function was evaluated by echocardiography at 48, 72, and 96 h after exosome treatment. Quantitative PCR showed that co-culture of BMSCs with GATA-4-BMSC exosomes increased cardiomyocyte-related marker expression. Co-culture of GATA-4-BMSC exosomes with cardiomyocytes in anoxic conditions decreased apoptosis as detected by flow cytometry. Injection of GATA-4-BMSC exosomes in mice 48 h after MI increased cardiac function over the next 96 h; increased cardiac blood vessel density and number of c-kit-positive cells and decreased apoptotic cardiomyocyte cells were also observed. Differential expression of candidate differentiation- and apoptosis-related miRNAs and proteins that may mediate these effects was also identified. Exosomes isolated from GATA-4-expressing BMSCs induce differentiation of BMSCs into cardiomyocyte-like cells, decrease anoxia-induced cardiomyocyte apoptosis, and improve myocardial function after infarction.

We investigated whether renal hedgehog interacting protein (Hhip) expression contributes to the progression of diabetic nephropathy (DN) and studied its related mechanism(s) in vivo and in vitro. Here, we show that Hhip expression is highly elevated in glomerular endothelial cells of adult type 1 diabetic (T1D) Akita and T2D db/db mouse kidneys as compared to non-diabetic control littermates. Hyperglycemia enhances reactive oxygen species (ROS) generation via NADPH oxidase 4 (Nox4) activation and stimulates renal Hhip gene expression, and that elevated renal Hhip gene expression subsequently activates the TGFβ1- Smad2/3 cascade and promotes endothelial to mesenchymal transition associated with endothelial cell fibrosis/apoptosis in vivo and in vitro. Furthermore, kidneys of low-dose streptozotocin-induced diabetic heterozygous Hhip deficient (Hhip+/−) mice displayed a normal albumin/creatinine ratio with fewer features of DN (glomerulosclerosis/fibrosis and podocyte apoptosis/loss) and less evidence of renal compensation (glomerular hypertrophy and hyperfiltration) as compared to diabetic wild type controls (Hhip+/+). Thus, our studies demonstrated that renal Hhip expression is associated with nephropathy development in diabetes and that hyperglycemia-induced renal Hhip expression may mediate glomerular endothelial fibrosis and apoptosis in diabetes, a novel finding.

The phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway plays a pivotal role in many cellular processes, including the proliferation, survival and differentiation of lung cancer cells. Thus, PI3K is a promising therapeutic target for lung cancer treatment. In this study, we applied free and open-source protein-ligand docking software, screened 3167 FDA-approved small molecules, and identified putative PI3Kα inhibitors. Among them, econazole nitrate, an antifungal agent, exhibited the highest activity in decreasing cell viability in pathological types of NSCLC cell lines, including H661 (large cell lung cancer) and A549 (adenocarcinoma). Econazole decreased the protein levels of p-AKT and Bcl-2, but had no effect on the phosphorylation level of ERK. It inhibited cell growth and promote apoptosis in a dose-dependent manner. Furthermore, the combination of econazole and cisplatin exhibited additive and synergistic effects in the H661 and A549 lung cancer cell lines, respectively. Finally, we demonstrated that econazole significantly suppressed A549 tumor growth in nude mice. Our findings suggest that econazole is a new PI3K inhibitor and a potential drug that can be used in lung cancer treatment alone or in combination with cisplatin.

Parthanatos is a newly discovered form of PARP-1-dependent programmed cell death. It has been reported to play an important role in several cancer or tumour cells; however, few studies have been performed in normal cells. Cadmium is a highly toxic pollutant and is reported to induce autophagy and apoptosis in multiple cell types. Although cadmium toxicity induces cell death, the underlying mechanism is not fully understood. Therefore, in this study we aimed to investigate the mechanism of Cadmium -induced cell damage using rat proximal tubular cell line NRK-52E and primary rat proximal tubular (rPT) cells. Our results indicated that parthanatos and the MAPK signalling pathway contribute to Cadmium-induced cell death, and that oxidative stress and mitochondrial damage play key roles in this process. In addition, parthanatos with oxidative stress has a synergistic effect on apoptosis, and JNK1/2 and p38 contribute to parthanatos.

Understanding the underlying molecular mechanisms of liver fibrosis is important to develop effective therapy. Herein, we show that focal-adhesion-kinse (FAK) plays a key role in promoting hepatic stellate cells (HSCs) activation in vitro and liver fibrosis progression in vivo. FAK activation is associated with increased expression of α-smooth muscle actin (α-SMA) and collagen in fibrotic live tissues. Transforming growth factor beta-1 (TGF-β1) induces FAK activation in a time and dose dependent manner. FAK activation precedes the α-SMA expression in HSCs. Inhibition of FAK activation blocks the α-SMA and collagen expression, and inhibits the formation of stress fibers in TGF-β1 treated HSCs. Furthermore, inhibition of FAK activation significantly reduces HSC migration and small GTPase activation, and induces apoptotic signaling in TGF-β1 treated HSCs. Importantly, FAK inhibitor attenuates liver fibrosis in vivo and significantly reduces collagen and α-SMA expression in an animal model of liver fibrosis. These data demonstrate that FAK plays an essential role in HSC activation and liver fibrosis progression, and FAK signaling pathway could be a potential target for liver fibrosis.

The Fas/FasL signaling pathway is one of the primary apoptosis pathways, but the involvement and regulatory mechanism of this pathway by autophagy remain unclear. Here we demonstrated that cadmium (Cd) activated the Fas/FasL apoptosis pathway in rat proximal tubular (rPT) cells; this was accompanied by simultaneous activation of autophagy resulted in reduced apoptosis. In this model, we induced autophagy through RAPA and further demonstrated that autophagy protects against activation of Fas/FasL signaling and apoptosis. The antiapoptotic effect of autophagy was blocked by 3-MA, an autophagy inhibitor. The interactions between Beclin-1 and Fas, FasL, FADD, caspase-8 and BID/tBID were relatively weak, with the exception of cleaved caspase-8, indicated that minimal interactions between these proteins and Beclin-1 are involved in maintaining the balance of autophagy and apoptosis. Beclin-1 precipitated with cleaved caspase-8 in a dose-dependent mannter, and the expression was increased by siRNA against Beclin-1. These data suggested that Beclin-1-mediated autophagy impairs the expression and function of cleaved caspase-8 to protect against Cd-induced activation of apopotosis through Fas/FasL signaling pathway.

Glioblastoma (GBM) is the most commonly diagnosed brain tumor that exhibit high mortality rate and chemotherapy resistance is a major clinical problem. Recent studies suggest that estrogen receptor beta (ERβ), may function as a tumor suppressor in GBM. However, the mechanism(s) by which ERβ contributes to GBM suppression and chemotherapy response remains unknown. We examined the role of ERβ in the DNA damage response of GBM cells, and tested whether ERβ sensitizes GBM cells to chemotherapy. Cell viability and survival assays using multiple epitope tagged ERβ expressing established and primary GBM cells demonstrated that ERβ sensitizes GBM cells to DNA damaging agents including temozolomide (TMZ). RNA-seq studies using ERβ overexpression models revealed downregulation of number of genes involved in DNA recombination and repair, ATM signaling and cell cycle check point control. Gene set enrichment analysis (GSEA) suggested that ERβ–modulated genes were correlated negatively with homologous recombination, mismatch repair and G2M checkpoint genes. Further, RT-qPCR analysis revealed that chemotherapy induced activation of cell cycle arrest and apoptosis genes were attenuated in ERβKO cells. Additionally, ERβ overexpressing cells had a higher number of γH2AX foci following TMZ treatment. Mechanistic studies showed that ERβ plays an important role in homologous recombination (HR) mediated repair and ERβ reduced expression and activation of ATM upon DNA damage. More importantly, GBM cells expressing ERβ had increased survival when compared to control GBM cells in orthotopic GBM models. ERβ overexpression further enhanced the survival of mice to TMZ therapy in both TMZ sensitive and TMZ resistant GBM models. Additionally, IHC analysis revealed that ERβ tumors had increased expression of γH2AX and cleaved caspase-3. Using ERβ-overexpression and ERβ-KO GBM model cells, we have provided the evidence that ERβ is required for optimal chemotherapy induced DNA damage response and apoptosis in GBM cells.

This study demonstrated that fenofibrate, a lipid-lowering drug, induced a significant time-dependent cytotoxicity of hepatoma Hep3B cells. Hep3B cells are significantly more sensitive to cell killing by fenofibrate than hepatoma HepG2, lung cancer CH27 and oral cancer HSC-3 cells. From the result of docking simulation, fenofibrate can bind excellently to the thioesterase domain of fatty acid synthase (FASN) binding site as orlistat, a FASN inhibitor, acts. The fenofibrate-induced cell cytotoxicity was protected by addition of palmitate, indicating that the cytotoxic effect of fenofibrate is due to starvation of Hep3B cells by inhibiting the formation of end product in the FASN reaction. Inhibition of lipid metabolism-related proteins expression, such as proteins containing thioesterase domain and fatty acid transport proteins, was involved in the fenofibrate-induced Hep3B cell death. Fenofibrate caused S and G2/M cell cycle arrest by inducing cyclin A/Cdk2 and reducing cyclin D1 and E protein levels in Hep3B cells. The anti-tumor roles of fenofibrate on Hep3B cells by inducing apoptosis and necroptosis were dependent on the expression of Bcl-2/caspase family members and RIP1/RIP3 proteins, respectively. These results suggest that fenofibrate has an anti-cancer effect in Hep3B cells and inhibition of lipid metabolism may be involved in fenofibrate-induced Hep3B cells apoptosis and necroptosis.

Retinal hypoperfusion injury is the pathophysiologic basis of ocular ischemic syndrome (OIS) which often leads to severe visual loss. In this study, we aimed to establish a rat model of retinal chronic hypoperfusion by bilateral common carotid artery occlusion (BCCAO) and observe changes in the retinal function and morphology. We found that model rats showed retinal arteriosclerosis, slight dilated retinal vein, small hemangiomas, hemorrhages, vascular segmental filling, and nonperfused areas after 2 weeks of BCCAO. In the model rats, the retinal circulation time was significantly prolonged by fluorescein fundus angiography (FFA), the latency of a and b waves was delayed and the amplitude was decreased significantly at each time point by electroretinogram (ERG), and the perfusion of the eyes continued to reduced. Morphologic and ultrastructural changes covered that the retinal ganglion cells (RGCs) presented obvious apoptosis and the thickness in the retinal layers were significantly thinner. Collectively, these findings suggested that BCCAO induced retinal hypoperfusion injury in the model rats, thus providing an ideal animal model for the study of OIS.

Anticancer activities of flavonoids derived from Tephroseris kirilowii (Turcz.) Holub. were evaluated in human cancer cells. We isolated and identified, for the first time, eight flavonoids from T. kirilowii and found that three of them (IH: isorhamnetin, GN: genkwanin, and Aca: acacetin) inhibited cell proliferation in a variety of human cancer cell lines. These active flavonoids caused cell cycle arrest at G2/M phase and induced apoptosis and autophagy in human breast cancer cells. Molecular docking revealed that these flavonoids dock in the ATP binding pocket of PI3Kγ. Importantly, treatment with these flavonoids decreased the levels of PI3Kγ-p110, phospho-PI3K, phospho-AKT, phospho-mTOR, phospho-p70S6K, and phospho-ULK. Pretreatment with PI3Kγ specific inhibitor AS605240 potentiated flavonoids-mediated inactivation of AKT, mTOR, p70S6K, ULK, and apoptosis. Taken together, these findings represent a novel mechanism by which downregulation of PI3Kγ-p110 and consequent interruption of PI3K/AKT/mTOR/p70S6K/ULK signaling pathway might play a critical functional role in these flavonoids-induced cell cycle arrest at G2/M phase, apoptosis, and autophagy. Our studies provide novel insights into the anticancer activities of selected flavonoids and their potential uses in anticancer therapy.

For a newly synthesized compound, identifying its target protein is a slow but pivotal step toward understand its pharmacologic mechanism. In this study, we systemically synthesized novel manzamine derivatives and chose 1-(9′-methyl-3′-carbazole)-3, 4-dihydro-β-carboline (MCDC) as an example to identify its target protein and function. MCDC had potent toxicity against several cancer cells. To identify its target protein, we first used a docking screen to predict macrophage migration inhibitory factor (MIF) as the potential target. Biochemical experiments, including mutation analysis and hydrogen-deuterium exchange assays, validated the binding of MCDC to MIF. Furthermore, MCDC was shown by microarrays to interfere with the cell cycle of breast cancer MCF7 cells. The activated signaling pathways included AKT phosphorylation and S phase-related proteins. Our results showed MIF as a potential direct target of a newly synthesized manzamine derivative, MCDC, and its pharmacologic mechanisms.

Arsenic resistance protein 2 (Ars2) is a component of the nuclear RNA cap-binding complex (CBC) that is important for some microRNA biogenesis and it is critical for cell proliferation and tumorigenicity. However, mechanism of Ars2-regulated cellular proliferation and tumorigenicity in glioblastoma has not been fully understood. Western blotting was used to detect the expressions of Ars2, p53, p21, and cleavage/activation of caspases-3 (C-Caspase 3). Microarray and Quantitative Real-time PCR (qRT-PCR) were performed to identify the Ars2-regulated microRNAs. Apoptosis assessed by flow cytometry analysis was used to evaluate the role of Ars2 in cells proliferation. The lentivirus-mediated gene knockdown approach was conducted to determine the function of Ars2. The orthotopic glioblastoma xenograft was used to demonstrate the role of Ars2 in glioblastoma growth in vivo. The high expression of Ars2 was observed in several glioblastoma cell lines and was significantly associated with poorer overall survival. Importantly, the overexpression of Ars2 promoted cell proliferation and colony formation in glioblastoma cells, whereas the depletion of Ars2 inhibited cell proliferation, colony formation, and tumor growth. Mechanistic study revealed that knockdown of Ars2 reduced the expression levels of miR-6798-3p, which was responsible for the up-regulation of p53 and p21, leading to apoptosis. Furthermore, the knockdown of Ars2 suppressed tumor growth in orthotopic glioblastoma xenograft model and significantly prolonged the survival time of the tumor-bearing mice. These findings identify a critical role for Ars2 in regulation of proliferation and tumorigenicity in glioblastoma and suggest that Ars2 could be a critical therapeutic target for glioblastoma intervention.

Aberrant expression of microRNAs hae been shown to be closely associated with glioblastoma cell proliferation, apoptosis and drug resistance. However, mechanisms underlying the role of mcroRNAs in glioblastoma cell growth and apoptosis are not fully understood. In this study, we report that miR-503 is overexpressed in glioblastoma tissue compared with normal human brain tissue. Mechanistically, miR-503 can be induced by TGF-β1 at the transcriptional level by binding the smad2/3 binding elements in the promoter. Ectopic overexpression of miR-503 promotes cell growth and inhibits apoptosis by targeting PDCD4. In contrast, inhibition of miR-503 reduces cell growth. Furthermore, miR-503 inhibitor augments the growth inhibitory effect of temozolomide in glioblastoma cells. These results establish miR-503 as a promising molecular target for glioblastoma therapy.

Combretastatin A-4 (CA4) is the lead compound of a relatively new class of vascular disrupting agents that target existing tumor blood vessels. Recent studies showed the CA4 might inhibit angiogenesis. However, the underlying molecular mechanisms by which CA4 exerts its anti-angiogenic effects are not fully understood. In this study, we revealed that CA4 inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and capillary-like tube formation of human umbilical vascular endothelial cells (HUVECs). In in vivo assay, CA4 suppressed neovascularization in chicken chorioallantoic membrane (CAM) model and decreased the microvessel density in tumor tissues of a breast cancer MCF-7 xenograft mouse model. In addition, CA4 decreased the expression level and secretion of VEGF both in MCF-7 cells and HUVECs under hypoxia, as well as the activation of VEGFR-2 and its downstream signaling mediators following VEGF stimulation in HUVECs. Moreover, VEGF and VEGFR-2 expression in tumor tissues of the mouse xenograft model were down-regulated following CA4 treatment. Taken together, results from the current work provide clear evidence that CA4 functions in endothelial cell system to inhibit angiogenesis, at least in part, by attenuating VEGF/VEGFR-2 signaling pathway.

The identification and characterization of long non-coding RNAs (lncRNAs) in diverse biological processes has recently developed rapidly. The large amounts of non-coding RNAs scale consistent with developmental complexity in eukaryotes, indicating that most of these transcripts may have functions in the regulation of biological processes and disorder in the organisms. In particular, Understanding of the overall biological significance of lncRNAs in cancers still remains limited. Here, we found a nuclear-retained lncRNA, termed Lnc_ASNR (apoptosis suppressing-noncoding RNA), which serves as a repressor of apoptosis. Lnc_ASNR was discovered in a set of microarray data derived from four kinds of tumor and adjacent normal tissue samples and displayed significant up-regulation in the tumor tissues. Using an RNA-pull down assay, we found that Lnc_ASNR interacted with the protein ARE/poly (U)-binding/degradation factor 1(AUF1), which is reported to promote rapid degradation of the Bcl-2 mRNA, an inhibitor of apoptosis. Lnc_ASNR binds to AUFI in nucleus, decreasing the cytoplasmic proportion of AUF1 which targets the B-cell lymphoma-2 (Bcl-2) mRNA. Taken together, the overall effect of Lnc_ASNR expression is thus a decrease in cell apoptosis indicating that Lnc_ASNR may play a vital role in tumorigenesis and carcinogenesis.

Heavy alcohol exposure causes profound damage to the adolescent brain, particularly the hippocampus, which underlie some behavioral deficits. However, the underlying molecular mechanisms remain inconclusive. The current study sought to determine whether binge alcohol exposure affects the hippocampus-related behaviors and key signaling proteins that may mediate alcohol neurotoxicity in adolescent rats. Alcohol exposure reduced the number of both NeuN-positive and doublecortin-positive cells in the hippocampus. Alcohol also induced neurodegeneration which was confirmed by ultrastructural analysis by electronic microscopy and was accompanied with the activation of microglia. Binge alcohol exposure impaired spatial learning and memory which was evaluated by the Morris water maze. However, alcohol did not alter the spontaneous locomotor activity which was determined by the open field test. GSK3β is a multi-function serine/threonine protein kinase regulating both neuronal survival and neurogenesis and plays an important role in various neurodegenerative disorders. We have previously shown that GSK3β is a key mediator of alcohol-induced neuron apoptosis in the developing brain. We showed here binge alcohol exposure caused GSK3β activation by inducing dephosphorylation at Ser9 without affecting the phosphorylation of Tyr216 in the hippocampus. Thus, GSK3β may be involved in binge alcohol exposure-induced neuronal damage to the adolescent hippocampus.

Methylglyoxal (MG) accumulation has been observed in human cerebrospinal fluid and body tissues under hyperglycaemic conditions. Recent research has demonstrated that MG-induces neuronal cell apoptosis, which promotes the development of diabetic encephalopathy. Our previous animal study has shown that luteolin, a natural flavonoid, attenuates diabetes-associated cognitive dysfunction. To further explore the neuroprotective properties of luteolin, we investigated the inhibitive effect of luteolin on MG-induced apoptosis in PC12 neuronal cells. We found that MG inhibited cell viability in a dose-dependent manner and induced apoptosis in PC12 cells. Pretreatment with Luteolin significantly elevated cell viability, reduced MG-induced apoptosis, inhibited the activation of the mTOR/4E-BP1 signaling pathway, and decreased pro-apoptotic proteins, Bax, Cytochrome C as well as caspase-3. Furthermore, we found that pretreatment with the mTOR inhibitor, rapamycin, significantly reduced the expression of the pro-apoptotic protein Bax. Therefore, these observations unambiguously suggest that the inhibitive effect of Luteolin against MG-induced apoptosis in PC12 cells is associated with inhibition of the mTOR/4E-BP1 signaling pathway.

HOXA11 antisense RNA (HOXA11-AS) has been shown to be involved in tumorigenesis and development of different cancers. However, the role of HOXA11-AS in non-small cell lung cancer (NSCLC) remains unclear. In this study, we firstly explored and confirmed the expression of HOXA11-AS in NSCLC tissues and cells. Cytometry, CCK-8, cell scratch, migration, Matrigel invasion and flow cytometry assays were performed to determine the biological impact of HOXA11-AS in vitro. Furthermore, a chick embryo chorioallantoic membrane (CAM) model of NSCLC was constructed to explore the effect of HOXA11-AS on tumorigenicity and angiogenesis in vivo. Additionally, bioinformatics analyses were performed to investigate the prospective pathways of HOXA11-AS co-expressed genes. As results, HOXA11-AS was markedly highly expressed in NSCLC tissues and cells. Furthermore, the proliferation, migration, invasion, tumorigenic and angiogenic ability of NSCLC cells were all inhibited and apoptosis was induced after HOXA11-AS knock-down. HOXA11-AS RNAi also led to cell cycle arrest on G0/G1 or G2/M phase. In addition, the non-small cell lung cancer pathway might be involved in regulating the co-expressed genes of HOXA11-AS in NSCLC. These results indicate that HOXA11-AS plays pivotal roles in NSCLC and it can become a novel therapeutic direction for treating NSCLC.