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Transcriptional factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, is generally regarded as a pro-survival factor. Here, we identify that besides its effect on autophagy induction, TFEB exerts a pro-apoptotic effect in response to the cyclopentenone prostaglandin 15-deoxy-∆-(12,14)-prostaglandin J2 (15d-PGJ2). Specifically, 15d-PGJ2 promotes TFEB translocation from the cytoplasm into the nucleus to induce autophagy and lysosome biogenesis via reactive oxygen species (ROS) production rather than mTORC1 inactivation. Surprisingly, TFEB promotes rather than inhibits apoptosis in response to 15d-PGJ2. Mechanistically, ROS-mediated TFEB translocation into the nucleus transcriptionally upregulates the expression of ATF4, which is required for apoptosis elicited by 15d-PGJ2. Additionally, inhibition of TFEB activation by ROS scavenger N-acetyl cysteine or inhibition of protein synthesis by cycloheximide effectively compromises ATF4 upregulation and apoptosis in response to 15d-PGJ2. Collectively, these results indicate that ROS-induced TFEB activation exerts a novel role in promoting apoptosis besides its role in regulating autophagy in response to 15d-PGJ2. This work not only evidences how TFEB is activated by 15d-PGJ2, but also unveils a previously unexplored role of ROS-dependent activation of TFEB in modulating cell apoptosis in response to 15d-PGJ2.

This work describes a base-mediated borylsilylation of benzylic ammonium salts to synthesize geminal silylboronates bearing benzylic proton under mild reaction conditions. Deaminative silylation of aryl ammonium salts was also achieved in the presence of LiOtBu. This strategy which is featured with high efficiency, mild reaction conditions, and good functional group tolerance provides efficient routes for late-stage functionalization of amines.

Lemairamin (also known as wgx-50), is isolated from the pericarps of the Zanthoxylum plants. As an agonist of α7 nicotinic acetylcholine receptors (α7nAChRs), it can reduce neuroinflammation in Alzheimer's disease. This study evaluated its antinociceptive effects in pain hypersensitivity and explored the underlying mechanisms. The data showed that subcutaneous lemairamin injection dose-dependently inhibited formalin-induced tonic pain but not acute nociception in mice and rats, while intrathecal lemairamin injection also dose-dependently produced mechanical antiallodynia in the ipsilateral hindpaws of neuropathic and bone cancer pain rats without affecting mechanical thresholds in the contralateral hindpaws. Multiple bi-daily lemairamin injections for 7 days did not induce mechanical antiallodynic tolerance in neuropathic rats. Moreover, the antinociceptive effects of lemairamin in formalin-induced tonic pain and mechanical antiallodynia in neuropathic pain were suppressed by the α7nAChR antagonist methyllycaconitine. In an α7nAChR antagonist-reversible manner, intrathecal lemairamin also stimulated spinal expression of IL-10 and β-endorphin, while lemairamin treatment induced IL-10 and β-endorphin expression in primary spinal microglial cells. In addition, intrathecal injection of a microglial activation inhibitor minocycline, anti-IL-10 antibody, anti-β-endorphin antiserum or μ-opioid receptor-preferred antagonist naloxone was all able to block lemairamin-induced mechanical antiallodynia in neuropathic pain. These data demonstrated that lemairamin could produce antinociception in pain hypersensitivity through the spinal IL-10/β-endorphin pathway following α7nAChR activation.

Graphical abstract, Highlights • Chitosan elicitation was conducted for flavonoid enhancement in hairy root cultures. • Chitosan elicitation gave a 7.08-fold increase in flavonoid yield over control. • Chalcone synthase and flavonoid 3′-hydroxylase might be two crucial genes. • Chitosan elicitation can facilitate the commercialization of plant in vitro cultures., Elicitation for phytochemical enhancement via cost-effective elicitors can overcome the limitation of commercial application faced by plant cell and organ culture technology. Chitosan is a natural, low-cost, and nontoxic elicitor that can trigger plant defense responses with the concomitant enhancement in phytochemical biosynthesis. In this work, the elicitation of Isatis tinctoria L. hairy root cultures by chitosan was conducted to enhance the production of pharmacologically active flavonoids. In comparison with control (2.31 ± 0.29 mg/g DW), a 7.08-fold enhancement of total flavonoids (16.35 ± 0.88 mg/g DW) was achieved in 24 day-old I. tinctoria hairy root cultures elicited by 150 mg/L chitosan for 36 h. Interestingly, the multiple hydroxyl-substituted flavonoids (rutin, quercetin, isorhamnetin, and isoliquiritigenin) were noticed to increase significantly in chitosan-elicited I. tinctoria hairy root cultures. Moreover, the transcription of associated genes involved in flavonoid biosynthesis pathway was significantly up-regulated underlying chitosan elicitation, among which chalcone synthase and flavonoid 3′-hydroxylase might play an important role in flavonoid enhancement. Additionally, extracts from chitosan-elicited I. tinctoria hairy root cultures exhibited higher antioxidant activities with lower IC50 values as compared with control. Overall, a cost-effective strategy via the simple chitosan elicitation is provided here to enhance the production of high-added value flavonoids in I. tinctoria hairy root cultures, which paves the way toward the successful commercialization of this in vitro culture system in the future.

Cynandione A, an acetophenone isolated from Cynanchum Wilfordii Radix, exhibits antineuropathic pain effect. This study further explored the target molecule and signaling mechanisms underlying cynandione-A-induced antineuropathic pain. Intrathecal injection of cynandione A significantly attenuated mechanical allodynia in neuropathic rats and substantially increased spinal expression of IL-10 and β-endorphin but not dynorphin A. Cynandione A treatment also enhanced expression of IL-10 and β-endorphin but not α7 nicotinic acetylcholine receptors (nAChRs) in cultured microglia. The IL-10 antibody attenuated cynandione-A-induced spinal or microglial gene expression of β-endorphin and mechanical allodynia, whereas the β-endorphin antiserum blocked cynandione-A-induced mechanical antiallodynia but not spinal or microglial IL-10 gene expression. The α7 nAChR antagonist methyllycaconitine significantly reduced cynandione-A-induced mechanical antiallodynia and spinal or microglial expression of IL-10 and β-endorphin. Furthermore, cynandione A stimulated microglial phosphorylation of PKA, p38, and CREB in an α7-nAChR-dependent manner, and treatment with their inhibitors attenuated cynandione-A-induced mechanical antiallodynia and spinal or microglial expression of IL-10 and β-endorphin. In addition, cynandione A stimulated spinal phosphorylation of the transcription factor STAT3, which was inhibited by methyllycaconitine, the PKA activation inhibitor or IL-10 antibody. The STAT3 inhibitor NSC74859 also abolished cynandione-A-induced mechanical antiallodynia and spinal expression of β-endorphin. These findings suggest that cynandione A suppresses neuropathic pain through α7-nAChR-dependent IL-10/β-endorphin signaling pathway in spinal microglia.

D‐Amino acid oxidase (DAAO) specifically catalyzes the oxidative deamination of neutral and polar D‐amino acids and finally yields byproducts of hydrogen peroxide. Our previous work demonstrated that the spinal astroglial DAAO/hydrogen peroxide (H2O2) pathway was involved in the process of pain and morphine antinociceptive tolerance. This study aimed to report mouse strain specificity of DAAO inhibitors on antinociception and explore its possible mechanism. DAAO inhibitors benzoic acid, CBIO, and SUN significantly inhibited formalin‐induced tonic pain in Balb/c and Swiss mice, but had no antinociceptive effect in C57 mice. In contrast, morphine and gabapentin inhibited formalin‐induced tonic pain by the same degrees among Swiss, Balb/c and C57 mice. Therefore, mouse strain difference in antinociceptive effects was DAAO inhibitors specific. In addition, intrathecal injection of D‐serine greatly increased spinal H2O2 levels by 80.0% and 56.9% in Swiss and Balb/c mice respectively, but reduced spinal H2O2 levels by 29.0% in C57 mice. However, there was no remarkable difference in spinal DAAO activities among Swiss, Balb/c and C57 mice. The spinal expression of glutathione (GSH) and glutathione peroxidase (GPx) activity in C57 mice were significantly higher than Swiss and Balb/c mice. Furthermore, the specific GPx inhibitor D‐penicillamine distinctly restored SUN antinociception in C57 mice. Our results reported that DAAO inhibitors produced antinociception in a strain‐dependent manner in mice and the strain specificity might be associated with the difference in spinal GSH and GPx activity., Effects of DAAO inhibitors (CBIO, benzoic acid and SUN) were compared between Swiss, Balb/c and C57 mice in formalin test. The results showed that the DAAO inhibitors significantly inhibited formalin‐induced tonic pain in Balb/c and Swiss mice, but had no effect in C57 mice.

An increasing number of studies suggest that angiotensin (Ang) Ⅱ contributes to the pathology of cerebrovascular disease possibly through injuring of the brain microvascular endothelial cells (BMEC), but the mechanisms involved are not well elucidated. Recent studies reveal the importance of Ang Ⅱ in reactive oxygen species induction and endothelial injury. Hence, the present study investigated the involvement of oxidative stress in Ang Ⅱ-induced BMEC injury. Our results showed that 10^(-6) mol/L Ang Ⅱ increased lactate dehydrogenase (LDH) leakage and inhibited the viability of primary cultured BMEC. Incubation with 10^(-6) mol/L of Ang Ⅱ for 48 hr increased the extracellular and intracellular malondialdehyde (IVIDA) content and decreased the activities of intracellular glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) significantly. These effects were abolished by pretreatment with the selective AT1 receptor antagonist losartan, but not with the AT2 selective antagonist PD 123319. Combining Losartan with PD 123319 neither diminished nor promoted the effect of losartan alone. These findings indicated that Ang Ⅱ induced injury of BMEC may result from oxidative stress by activating AT1 receptors. This injury may in turn lead to the development of cerebrovascular disease.

Bioactive phenolics primarily contribute to versatile health benefits of pigeon pea. For the first time, an UPLC-QqQ-MS/MS method was developed for the quantitative analysis of eleven bioactive phenolic compounds in pigeon pea natural resources (seeds, leaves, and roots) and in vitro cultures (calli and hairy roots). The proposed method could be achieved within 6 min of running time, and displayed the satisfactory linearity, sensitivity, precision, accuracy, and stability. According to analytical results, the distribution of eleven target compounds in different organs of pigeon pea was clarified. Also, it was surprisingly found that pigeon pea in vitro cultures exhibited superiority in contents of genistin and cajaninstilbene acid as compared with natural resources. Overall, the present work provided a rapid and sensitive analysis approach, which could be useful not only for quality control of pigeon pea natural resources, but also for applicability and safety evaluation of pigeon pea in vitro cultures.

Background Cinobufagin is the major bufadienolide of Bufonis venenum (Chansu), which has been traditionally used for the treatment of chronic pain especially cancer pain. The current study aimed to evaluate its antinociceptive effects in bone cancer pain and explore the underlying mechanisms. Methods Rat bone cancer model was used in this study. The withdrawal threshold evoked by stimulation of the hindpaw was determined using a 2290 CE electrical von Frey hair. The β-endorphin and IL-10 levels were measured in the spinal cord and cultured primary microglia, astrocytes, and neurons. Results Cinobufagin, given intrathecally, dose-dependently attenuated mechanical allodynia in bone cancer pain rats, with the projected Emax of 90% MPE and ED50 of 6.4 μg. Intrathecal cinobufagin also stimulated the gene and protein expression of IL-10 and β-endorphin (but not dynorphin A) in the spinal cords of bone cancer pain rats. In addition, treatment with cinobufagin in cultured primary spinal microglia but not astrocytes or neurons stimulated the mRNA and protein expression of IL-10 and β-endorphin, which was prevented by the pretreatment with the IL-10 antibody but not β-endorphin antiserum. Furthermore, spinal cinobufagin-induced mechanical antiallodynia was inhibited by the pretreatment with intrathecal injection of the microglial inhibitor minocycline, IL-10 antibody, β-endorphin antiserum and specific μ-opioid receptor antagonist CTAP. Lastly, cinobufagin- and the specific α-7 nicotinic acetylcholine receptor (α7-nAChR) agonist PHA-543613-induced microglial gene expression of IL-10/β-endorphin and mechanical antiallodynia in bone cancer pain were blocked by the pretreatment with the specific α7-nAChR antagonist methyllycaconitine. Conclusions Our results illustrate that cinobufagin produces mechanical antiallodynia in bone cancer pain through spinal microglial expression of IL-10 and subsequent β-endorphin following activation of α7-nAChRs. Our results also highlight the broad significance of the recently uncovered spinal microglial IL-10/β-endorphin pathway in antinociception.

TFEB (transcription factor EB), which is a master regulator of autophagy and lysosome biogenesis, is considered to be a new therapeutic target for Parkinson&rsquo, s disease (PD). However, only several small-molecule TFEB activators have been discovered and their neuroprotective effects in PD are unclear. In this study, a curcumin derivative, named E4, was identified as a potent TFEB activator. Compound E4 promoted the translocation of TFEB from cytoplasm into nucleus, accompanied by enhanced autophagy and lysosomal biogenesis. Moreover, TFEB knockdown effectively attenuated E4-induced autophagy and lysosomal biogenesis. Mechanistically, E4-induced TFEB activation is mainly through AKT-MTORC1 inhibition. In the PD cell models, E4 promoted the degradation of &alpha, synuclein and protected against the cytotoxicity of MPP+ (1-methyl-4-phenylpyridinium ion) in neuronal cells. Overall, the TFEB activator E4 deserves further study in animal models of neurodegenerative diseases, including PD.

Although emerging evidence suggests that the pathogenesis of Parkinson's disease (PD) is closely related to the aggregation of alpha-synuclein (α-syn) in the midbrain, the clearance of α-syn remains an unmet clinical need. Here, we develop a simple and efficient strategy for fabricating the α-syn nanoscavenger for PD via a reprecipitation self-assembly procedure. The curcumin analogue-based nanoscavenger (NanoCA) is engineered to be capable of a controlled-release property to stimulate nuclear translocation of the major autophagy regulator, transcription factor EB (TFEB), triggering both autophagy and calcium-dependent exosome secretion for the clearance of α-syn. Pretreatment of NanoCA protects cell lines and primary neurons from MPP+-induced neurotoxicity. More importantly, a rapid arousal intranasal delivery system (RA-IDDS) was designed and applied for the brain-targeted delivery of NanoCA, which affords robust neuroprotection against behavioral deficits and promotes clearance of monomer, oligomer, and aggregates of α-syn in the midbrain of an MPTP mouse model of PD. Our findings provide a clinically translatable therapeutic strategy aimed at neuroprotection and disease modification in PD.

The root of Chuanminshen violaceum is used as an important edible and medicinal plant in China. However, its leaves are generally considered byproducts, and therefore do not have a use. Thus, the phenolic compounds in the methanolic extracts (CVLMs) and the chemical characteristics of crude polysaccharides (CVLPs) from the leaves of C. violaceum and their in vitro antioxidant activities were explored. The results showed that chlorogenic acid and rutin were the major individual phenolic compounds in the leaves, which ranged from 1.22 &plusmn, 0.03 to 2.87 &plusmn, 0.04 mg/g DW, and from 2.25 &plusmn, 0.04 to 4.03 &plusmn, 0.05 mg/g DW, respectively. Meanwhile, the extraction yields of CVLPs from the leaves ranged from 4.73% to 5.41%. The CVLPs consisted of mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose, suggesting the existence of pectic polysaccharides. Furthermore, both CVLMs and CVLPs exhibited strong antioxidant activities. Chlorogenic acid and rutin were major contributors to the antioxidant activities of CVLMs, and the antioxidant activities of CVLPs were closely correlated to their &alpha, 1,4-D-galactosiduronic linkages. The results are beneficial for understanding the chemical properties and in vitro antioxidant activities of CVLMs and CVLPs. The leaves of C. violaceum have potential to be developed as natural antioxidants.

Chitosan, behaving as a potent biotic elicitor, can induce plant defense response with the consequent enhancement in phytoalexin accumulation. Accordingly, chitosan elicitation was conducted to promote the production of two phytoalexins, i.e. formononetin and calycosin (also known as health-promoting isoflavones), in Astragalus membranaceus hairy root cultures (AMHRCs). Compared with control, 12.45- and 6.17-fold increases in the yields of formononetin (764.19 ± 50.81 μg/g DW) and calycosin (611.53 ± 42.22 μg/g DW) were obtained in 34 day-old AMHRCs treated by 100 mg/L of chitosan for 24 h, respectively. Moreover, chitosan elicitation could cause oxidative burst that would induce the expression of genes (MPK3 and MPK6) related to mitogen-activated protein kinase signaling (MAPK) cascades, which contributed to the transcriptional activation of pathogenesis-related genes (β-1,3-glucanase, Chitinase, and PR-1) and eight biosynthesis genes involved in the calycosin and formononetin pathway. Overall, the findings in this work not only highlight a feasible chitosan elicitation practice to enhance the in vitro production of two bioactive isoflavones for nutraceutical and food applications, but also contribute to understanding the phytoalexin biosynthesis in response to chitosan elicitation.

Phenolic compounds are validated to be the pharmacologically active phytochemicals in Cajanus cajan (Linn.) Millsp. leaves (CCLs) and the medicinal product (Tongluo Shenggu capsules, TSCs) thereof. For the first time, C. cajan cell suspension cultures (CCSCs) were established as an effective in vitro platform for the production of phenolic compounds. Callus was induced from C. cajan leaves by 2.0 mg/L 2,4-D, 5.0 mg/L α-NAA, and 0.1 mg/L Kn, and then subcultured on MS solid medium supplemented with 1.5 mg/L 2,4-D, 3.0 mg/L α-NAA, and 0.25 mg/L Kn. Culture conditions of a selected high-productive callus line (CCCL VII) were systematically optimized in terms of biomass productivity and phenolic yield, and eleven target compounds were identified and quantified by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Under the optimal culture conditions (MS liquid medium, sucrose concentration 3.5 %, temperature 26 °C, inoculum size 3.0 %, and time 49 day), CCSCs produced the highest level of total phenolics (3462.99 μg/g DW) along with the maximum biomass productivity (10.03 g/L), which was higher than the phenolic yield in field-grown CCLs (3288.81 μg/g DW). Notably, CCSCs exhibited superiority in the content of the most valuable compound cajaninstilbene acid (2858.76 μg/g DW) as compared with CCLs (2114.60 μg/g DW). Also, CCSCs were discovered to be more abundant in genistin and genistein as against both CCLs and TSCs. This work concluded that CCSCs might be attractive alternative sources for the production of phenolic compounds with potential uses in pharmaceutical/nutraceutical industries.

Changes in temperature are known to cause a variety of physiological stress responses in insects and mites. Thermal stress responses are usually associated with the increased generation of reactive oxygen species (ROS), resulting in oxidative damage. In this study, we examined the time-related effect (durations for 1, 2, 3, and 5 h) of thermal stress conditions-i.e., relatively low (0, 5, 10, and 15 °C) or high (35, 38, 41, and 44 °C) temperatures-on the activities of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), glutathione S-transferases (GSTs), and total antioxidant capacity (T-AOC) of the predatory mite Neoseiulus cucumeris. Also the lipid peroxidation (LPO) levels of the predatory mite were measured under thermal stress conditions. The results confirmed that thermal stress results in a condition of so-called oxidative stress and the four antioxidant enzymes play an important role in combating the accumulation of ROS in N. cucumeris. CAT and POX activity changed significantly when the mites were exposed to cold and heat shock, respectively. The elevated levels of SOD and GSTs activity, expressed in a time-dependent manner, may have an important role in the process of antioxidant response to thermal stress. However, the levels of LPO in N. cucumeris were high, serving as an important signal that these antioxidant enzyme-based defense mechanisms were not always adequate to counteract the surplus ROS. Thus, we hypothesize that thermal stress, especially extreme temperatures, may contribute much to the generation of ROS in N. cucumeris, and eventually to its death.

The title Schiff base, C24H21N3O2, adopts an E configuration with respect to the central C=N bond. The pyrazole ring and the central benzene ring attached to the imino group are almost coplanar. The phenyl ring attached to the pyrazole unit is twisted by 39.3 (2)° with respect to the pyrazole ring plane. The phenoxy benzene ring makes a dihedral angle of 79.8 (2)° with the central benzene ring.

The complete molecule of the title compound, C16H14O3, is generated by crystallographic mirror symmetry, with two C atoms and one O atom lying on the mirror plane. The molecule adopts an E configuration about the C=C bond and the dihedral angle between the furan rings is 16.1 (2)°.

In the title compound, C24H18F3N3O, the mol­ecule exists in the enamine–keto tautomeric form. The pyrazolone ring makes dihedral angles of 31.8 (2), 45.4 (2) and 71.2 (2)° with the 1-phenyl, aniline and methylene-bound phenyl rings, respectively. An intra­molecular N—H⋯O hydrogen bond helps to stabilize the mol­ecular conformation.