The combination of Schisandrin C and Luteolin synergistically attenuates hepatitis B virus infection via repressing HBV replication and promoting cGAS-STING pathway activation in macrophages.

BACKGROUND: HBV infection can result in severe liver diseases and is one of the primary causes of liver cell carcinoma-related mortality. Liuwei Wuling tablet (LWWL) is a traditional Chinese medicine formula, with a protecting liver and decreasing enzyme activity, usually used to treat chronic hepatitis B with NAs in clinic. However, its main active ingredients and mechanism of action have not been fully investigated. Hence, we aimed to screen the active ingredient and effective ingredient combinations from Liuwei Wuling tablet to explore the anti-herpatitis B virus activity and mechanism. METHODS: Analysis and screening of effective antiviral components in LWWL by network pharmacology, luteolin (Lut) may be a compound with significant antiviral activity. The mechanism of antiviral action of Lut was also found by real-time PCR detection and western blotting. Meanwhile, we established a co-culture model to investigate the antiviral mechanism of Schisandrin C (SC), one of the main active components of Schisandra chinensis fructus (the sovereign drug of LWWL). Next, HBV-infected mice were established by tail vein injection of pAAV-HBV1.2 plasmid and administered continuously for 20 days. And their antiviral capacity was evaluated by checking serum levels of HBsAg, HBeAg, levels of HBV DNA, and liver levels of HBcAg. RESULTS: In this study, we conducted network pharmacology analysis on LWWL, and through in vitro experimental validation and data analysis, we found that luteolin (Lut) possessed obviously anti-HBV activity, inhibiting HBV replication by downregulating hepatocyte nuclear factor 4α (HNF4α) via the ERK pathway. Additionally, we established a co-culture system and proved that SC promoted activation of cGAS-STINIG pathway and IFN-ß production in THP-1 cells to inhibit HBV replication in HepG2.2.15 cells. Moreover, we found the combination of SC and Lut shows a greater effect in inhibiting HBV compared to SC or Lut alone in HBV-infected mice. CONCLUSION: Taken together, our study suggests that combination of SC and Lut may be potential candidate drug for the prevention and treatment of chronic hepatitis B.

Dachaihu decoction ameliorates septic intestinal injury via modulating the gut microbiota and glutathione metabolism as revealed by multi-omics.

ETHNOPHARMACOLOGICAL RELEVANCE: Dachaihu decoction (DCH), a classic formula for Yangming and Shaoyang Syndrome Complex recorded in "Treatise on Cold Damage", has been widely used in treating intestinal disorders and inflammatory diseases with few side effects in China. However, the mechanism of DCH on septic intestinal injury (SII) remains to be explored. AIM OF THE STUDY: This study aimed to clarify the mechanism of DCH on SII. MATERIALS AND METHODS: SII model of rat, established by cecal ligation and puncture (CLP), was used to study the effect of DCH on SII. 24 h mortality was recorded. Histological changes were observed by H&E staining. The expression of tight junction protein ZO-1 (ZO-1) and mucin2 (MUC2) was determined by immunohistochemical analysis. Secretory IgA (sIgA), diamine oxidase (DAO) and intestinal fatty acid binding protein (iFABP) were determined by enzyme-linked immunosorbent assay (ELISA). IL-1ß, IL-6 and TNF-α were measured by ELISA and quantitative Real-time PCR (RT-qPCR). The gut microbiota was analyzed by 16S rRNA sequencing. The potential targets and pathways of DCH in treating SII were analyzed by integrative analysis of transcriptomic and metabolomic methods. Total glutathione (T-GSH), GSH, GSSG (reduced form of GSH), GSH peroxidase (GPX), superoxide dismutase (SOD), malonaldehyde (MDA) and indicators of hepatic and renal function were measured by biochemical kits. RESULTS: Medium dose of DCH improved 24 h mortality of SII rats, reduced the pathological changes of ileum, and increased the expression levels of ZO-1, MUC2 and sIgA. DCH decreased DAO, iFABP of serum and IL-1ß, IL-6, TNF-α of ileum. DCH improved α- and ß-diversity and modulated the structure of gut microbiota, with Escherichia_Shigella decreased and Bacteroides and Ruminococcus increased. GSH metabolism was identified as the key pathway of DCH on SII by integrative analysis of transcriptome and metabolome. GSH/GSSG and the most common indicators of oxidative stress, were validated. Antioxidative T-GSH, GSH, GPX and SOD were increased, while MDA, the mark of lipid peroxidation was downregulated by DCH. Eventually, DCH was proved to be safe and hepato- and nephro-protective. CONCLUSION: DCH ameliorated septic intestinal injury possibly by modulating the gut microbiota and enhancing glutathione metabolism of SII rats, without hepatotoxicity and nephrotoxicity.

[Effect of Erjing Pills on alleviating neuroinflammation of AD rats based on TLR4/NF-κB/NLRP3 pathway and its mechanism].

This paper aimed to study the effect of Erjing Pills on the improvement of neuroinflammation of rats with Alzheimer's di-sease(AD) induced by the combination of D-galactose and Aß_(25-35) and its mechanism. SD rats were randomly divided into a sham group, a model control group, a positive drug group(donepezil, 1 mg·kg~(-1)), an Erjing Pills high-dose group(9.0 g·kg~(-1)), and an Erjing Pills low-dose group(4.5 g·kg~(-1)), with 14 rats each group. To establish the rat model of AD, Erjing Pills were intragastrically administrated to rats for 5 weeks after 2 weeks of D-galactose injection. D-galactose was intraperitoneally injected into rats for 3 weeks, and then Aß_(25-35) was injected into the bilateral hippocampus. The new object recognition test was used to evaluate the learning and memory ability of rats after 4 weeks of intragastric administration. Tissues were acquired 24 h after the last administration. The immunofluorescence method was used to detect the activation of microglia in the brain tissue of rats. The positive expressions of Aß_(1-42) and phosphory protein Tau~(404)(p-Tau~(404)) in the CA1 area of the hippocampus were detected by immunohistochemistry. The levels of inflammatory factors interleukin-1ß(IL-1ß), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6) in the brain tissue were determined by enzyme-linked immunosorbent assay(ELISA). Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB)/nucleotide-binding oligomerization domain-like receptors 3(NLRP3) pathway-associated proteins in the brain tissue were determined by Western blot. The results showed that as compared with the sham group, the new object recognition index of rats in the model control group decreased significantly, the deposition of Aß_(1-42) and p-Tau~(404) positive protein in the hippocampus increased significantly, and the levels of microglia activation increased significantly in the dentate gyrus. The levels of IL-1ß, TNF-α, and IL-6 in the hippocampus of the model control group increased significantly, and the expression levels of TLR4, p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus increased significantly. Compared with the model control group, the Erjing Pill groups enhanced the new object recognition index of rats, decreased the deposition of Aß_(1-42) and the expression of p-Tau~(404) positive protein in the hippocampus, inhibited the activation of microglia in the dentate gyrus, reduced the levels of inflammatory factors IL-1ß, TNF-α, and IL-6 in the hippocampus, and down-regulated the expression levels of TLR4, p-NF-κB P65/NF-κB P65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus. In conclusion, Erjing Pills can improve the learning and memory ability of the rat model of AD presumably by improving the activation of microglia, reducing the expression levels of neuroinflammatory factors IL-1ß, TNF-α, and IL-6, inhibiting the TLR4/NF-κB/NLRP3 neuroinflammation pathway, and decreasing hippocampal deposition of Aß and expression of p-Tau, thereby restoring the hippocampal morphological structure.

Schisandrin C enhances cGAS-STING pathway activation and inhibits HBV replication.

ETHNOPHARMACOLOGICAL RELEVANCE: Schisandra Chinensis (Turcz.) Baill. is a long-term used traditional Chinese medicine with the functions of tonifying the kidney and calming the heart, tonifying qi and engendering fluid. It can be used to treat insomnia and dreaminess, spermatorrhea, coughs, as well as liver and kidney deficiency of Yin or Yang Syndrome. Modern pharmacological studies have shown that Schisandra Chinensis regulates host immunity and exhibits anti-cancer, antiviral and liver-protecting effects. However, the specific mechanism by which Schisandra Chinensis modulates antiviral immunity is unknown. AIM OF THE STUDY: We sought to explore the therapeutic effect of the active components of Schisandra Chinensis on anti-viral immunity and further investigate the underlying mechanism. MATERIALS AND METHODS: Immunoblotting, quantitative real-time PCR, enzyme-linked immunosorbent assay, immunofluorescence, and immunoprecipitation were used to investigate the effect of schisandrin C (SC), one of the most abundant and biologically active components of Schisandra Chinensis, on the activation of cGAS-STING signaling pathway and the underlying mechanism. In addition, CMA-mediated STING activation and hydrodynamic injection-mediated HBV-replicating mouse model were used to investigate the effect of SC on the activation of STING signaling pathway and its antiviral effect in vivo. RESULTS: SC promoted cGAS-STING pathway activation, accompanied by increased production of interferon ß (IFN ß) and downstream gene expression. Moreover, SC also exerted anti-HBV effects, reducing HBeAg, HBcAg, HBsAg, and HBV DNA levels in hydrodynamic injection-mediated HBV-replicating mouse model and elevating the production of IFN ß and expression of interferon-stimulated genes (IFIT1, ISG15, and CXCL10). Mechanistically, SC could facilitate the interaction between TANK-binding kinase 1 (TBK1) and STING, which is important for IRF3 phosphorylation and production of IFN ß. CONCLUSIONS: Our study confirmed that SC enhances cGAS-STING pathway activation and inhibits HBV replication, as well as provides clues for chronic hepatitis B and other infectious diseases treated by SC.

Combined-Acupoint Electroacupuncture Induces Better Analgesia via Activating the Endocannabinoid System in the Spinal Cord.

Electroacupuncture (EA) therapy has been widely reported to alleviate neuropathic pain with few side effects in both clinical practice and animal studies worldwide. However, little is known about the comparison of the therapeutic efficacy among the diverse EA schemes used for neuropathic pain. The present study is aimed at investigating the therapeutic efficacy discrepancy between the single and combined-acupoint EA and to reveal the difference of mechanisms behind them. Electroacupuncture was given at both Zusanli (ST36) and Huantiao (GB30) in the combined group or ST36 alone in the single group. Paw withdrawal mechanical threshold (PWMT) was measured to determine the pain level. Electrophysiology was performed to detect the effects of EA on synaptic transmission in the spinal dorsal horn of the vGlut2-tdTomato mice. Spinal contents of endogenous opioids, endocannabinoids, and their receptors were examined. Inhibitors of CBR (cannabinoid receptor) and opioid receptors were used to study the roles of opioid and endocannabinoid system (ECS) in EA analgesia. We found that combined-acupoint acupuncture provide stronger analgesia than the single group did, and the former inhibited the synaptic transmission at the spinal level to a greater extent than later. Besides, the high-intensity stimulation at ST36 or normal stimulation at two sham acupoints did not mimic the similar efficacy of analgesia in the combined group. Acupuncture stimulation in single and combined groups both activated the endogenous opioid system. The ECS was only activated in the combined group. Naloxone totally blocked the analgesic effect of single-acupoint EA; however, it did not attenuate that of combined-acupoint EA unless coadministered with CBR antagonists. Hence, in the CCI-induced neuropathic pain model, combined-acupoint EA at ST36 and GB30 is more effective in analgesia than the single-acupoint EA at ST36. EA stimulation at GB30 alone neither provided a superior analgesic effect to EA treatment at ST36 nor altered the content of AEA, 2-AG, CB1 receptor, or CB2 receptor compared with the CCI group. Activation of the ECS is the main contributor of the better analgesia by the combined acupoint stimulation than that induced by single acupoint stimulation.

Dietary Supplementation of Probiotic Bacillus subtilis Affects Antioxidant Defenses and Immune Response in Grass Carp Under Aeromonas hydrophila Challenge.

This study investigated whether Bacillus subtilis can provide protection for grass carp against oxidative stress damage induced by Aeromonas hydrophila. A total of 240 healthy grass carp (Ctenopharyngodon idellus) (average weight of 71.42 ± 4.36g) were randomly divided into four groups with three replicates: control group, A. hydrophila group, B. subtilis + A. hydrophila group, and A. hydrophila + B. subtilis group. After challenge with A. hydrophila, the lipid oxidative damage, antioxidant defenses, and the gene expression of inflammatory cytokines of the grass carp were investigated. Our results showed that A. hydrophila caused lipid oxidative damage, led to significant decreases in antioxidant defenses, and induced inflammatory responses of grass carp. However, the grass carp group fed the probiotic B. subtilis diet for 42 days before the challenge and the group fed the probiotic B. subtilis diet immediately after the challenge both showed (i) a reduced level of oxidative stress with a decrease in the level of MDA; (ii) an increase in antioxidant defenses, including an increase in total antioxidant capacity (T-AOC), increased activities of SOD and CAT, increased levels of GSH, and upregulated gene expression of antioxidant enzymes (SOD, CAT, and Gpx); and (iii) an improved immune response with the level of antiinflammatory cytokines IL-10 messenger RNA (mRNA) upregulated and the levels of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-8 mRNA downregulated. Based on this study, B. subtilis can provide effective protection of fish against oxidative stress damage induced by A. hydrophila infection.

Effects of coordination and manipulation therapy for patients with Parkinson disease.

PURPOSE: To determine the effects of a new exercise training regimen, i.e. coordination and manipulation therapy (CMT), on motor, balance, and cardiac functions in patients with Parkinson disease (PD). MATERIALS AND METHODS: We divided 36 PD patients into the CMT (n = 22) and control (n = 14) groups. The patients in the CMT group performed dry-land swimming (imitation of the breaststroke) and paraspinal muscle stretching for 30 min/workday for 1 year. The control subjects did not exercise regularly. The same medication regimen was maintained in both groups during the study. Clinical characteristics, Unified Parkinson's Disease Rating Scale (UPDRS) scores, Berg balance scale (BBS) scores, mechanical balance measurements, timed up and go (TUG) test, and left ventricular ejection fraction (LVEF) were compared at 0 (baseline), 6, and 12 months. Biochemical test results were compared at 0 and 12 months. The primary outcome was motor ability. The secondary outcome was cardiac function. RESULTS: In the CMT group, UPDRS scores significantly improved, TUG test time and step number significantly decreased, BBS scores significantly increased, and most mechanical balance measurements significantly improved after 1 year of regular exercise therapy (all p < 0.05). In the control group, UPDRS scores significantly deteriorated, TUG test time and step number significantly increased, BBS scores significantly decreased, and most mechanical balance measurements significantly worsened after 1 year (all P < 0.05). LVEF improved in the CMT group only (P = 0.01). CONCLUSIONS: This preliminary study suggests that CMT effectively improved mobility disorder, balance, and cardiac function in PD patients over a 1-year period.

Effect of Coriolus versicolor polysaccharides on the hematological and biochemical parameters and protection against Aeromonas hydrophila in allogynogenetic crucian carp (Carassius auratus gibelio).

The effect of dietary intake of Coriolus versicolor Polysaccharides (CVP) on the hematological and biochemical indices of Allogynogenetic crucian carp (Carassius auratus gibelio) was investigated. Fish were fed CVP supplemented diets (0, 0.25, 0.5, 1.0, 2.0 or 4.0 g CVP kg(-1)) for 56 days. The RBC, WBC counts, hemoglobin content, ESR in blood and TP, ALT, AST, ALP, GLU, CHO, TG, and BUN in serum were measured on day 0, 14, 28, 42, and 56. After feeding of 56 days, fish were infected with Aeromonas hydrophila and mortalities were recorded. The results indicated that feeding crucian carp with suitable dose of CVP enhanced the RBC, WBC counts, hemoglobin and TP content, ALP activity, and decreased the ESR, ALT, AST, GLU, CHO, TG and BUN. There was no effect in fish at low dose (0.25 g kg(-1)). Unexpectedly, the higher CVP dose used here (2.0 and 4.0 g kg(-1)) has a negative effect in fish. The results of challenge experiment indicated that a moderate level of CVP in the diet (1.0 g kg(-1)) was the most effective to enhance the survival of fish after infected with A. hydrophila. In summary, the use of CVP, as dietary supplements, can improve the innate defense of crucian carp providing resistance to pathogens.

Ginkgo biloba extract EGb 761 and Wisconsin Ginseng delay sarcopenia in Caenorhabditis elegans.

Previously we reported that the standardized Ginkgo biloba extract EGb 761 extended life span and increased stress resistance in Caenorhabditis elegans. In this study, pharmacological modulation of age-dependent muscle degeneration, or sarcopenia, was determined. Transgenic C. elegans strain (PD4251) expressing green fluorescent protein (GFP)-MYO-3, localized in body wall muscles and vulval muscle nuclei, were fed with EGb 761 or Wisconsin Ginseng, and muscle integrity was analyzed by quantification of GFP fluorescence. Both EGb 761 and Wisconsin Ginseng significantly delayed sarcopenia. Ginseng was more effective in worms of more advanced age, which is consistent with the ultrastructural changes observed by transmission electron microscopy. Furthermore, both agents ameliorated age-associated decline of locomotive behaviors including locomotion, body bend, and pharyngeal pumping. These results suggest that pharmacological extension of life span is a consequence of maintaining functional capacity of the tissue, and that C. elegans is a valid model system for testing therapeutic intervention for delaying the progress of sarcopenia.

Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans.

Amyloid-beta (Abeta) toxicity has been postulated to initiate synaptic loss and subsequent neuronal degeneration seen in Alzheimer's disease (AD). We previously demonstrated that the standardized Ginkgo biloba extract EGb 761, commonly used to enhance memory and by AD patients for dementia, inhibits Abeta-induced apoptosis in neuroblastoma cells. In this study, we use EGb 761 and its single constituents to associate Abeta species with Abeta-induced pathological behaviors in a model organism, Caenorhabditis elegans. We report that EGb 761 and one of its components, ginkgolide A, alleviates Abeta-induced pathological behaviors, including paralysis, and reduces chemotaxis behavior and 5-HT hypersensitivity in a transgenic C. elegans. We also show that EGb 761 inhibits Abeta oligomerization and Abeta deposits in the worms. Moreover, reducing oxidative stress is not the mechanism by which EGb 761 and ginkgolide A suppress Abeta-induced paralysis because the antioxidant L-ascorbic acid reduced intracellular levels of hydrogen peroxide to the same extent as EGb 761, but was not nearly as effective in suppressing paralysis in the transgenic C. elegans. These findings suggest that (1) EGb 761 suppresses Abeta-related pathological behaviors, (2) the protection against Abeta toxicity by EGb 761 is mediated primarily by modulating Abeta oligomeric species, and (3) ginkgolide A has therapeutic potential for prevention and treatment of AD.

Soy isoflavone glycitein protects against beta amyloid-induced toxicity and oxidative stress in transgenic Caenorhabditis elegans.

BACKGROUND: Epidemiological studies have associated estrogen replacement therapy with a lower risk of developing Alzheimer's disease, but a higher risk of developing breast cancer and certain cardiovascular disorders. The neuroprotective effect of estrogen prompted us to determine potential therapeutic impact of soy-derived estrogenic compounds. Transgenic C. elegans, that express human beta amyloid (Abeta), were fed with soy derived isoflavones genistein, daidzein and glycitein (100 microg/ml) and then examined for Abeta-induced paralysis and the levels of reactive oxygen species. RESULTS: Among the three compounds tested, only glycitein alleviated Abeta expression-induced paralysis in the transgenic C. elegans. This activity of glycitein correlated with a reduced level of hydrogen peroxide in the transgenic C. elegans. In vitro scavenging effects of glycitein on three types of reactive oxygen species confirmed its antioxidant properties. Furthermore, the transgenic C. elegans fed with glycitein exhibited reduced formation of beta amyloid. CONCLUSION: These findings suggest that a specific soy isoflavone glycitein may suppress Abeta toxicity through combined antioxidative activity and inhibition of Abeta deposition, thus may have therapeutic potential for prevention of Abeta associated neurodegenerative disorders.

Expression of the small heat-shock protein Hsp16-2 in Caenorhabditis elegans is suppressed by Ginkgo biloba extract EGb 761.

EGb 761, a standardized extract of Ginkgo biloba leaves, has been shown to have antioxidative properties. We have previously demonstrated that EGb 761 increases stress resistance and mean life span in the model organism Caenorhabditis elegans. In this study, the molecular mechanism of EGb 761 on alleviating effects of oxidative stress is further investigated using transgenic C. elegans expressing a jellyfish green fluorescent protein (GFP)-tagged inducible small heat-shock protein gene (hsp-16-2). The expression of hsp-16-2 induced by the pro-oxidant juglone and by heat shock was significantly suppressed by 86% and 33%, respectively, in the transgenic nematode fed with EGb 761. These effects of EGb 761 correlate with its ability to increase mean survival rate of the nematode in response to acute oxidative and thermal stresses, as well as to attenuate the basal levels of hydrogen peroxide in the organism. Thus, we interpret the suppression of hsp-16-2/GFP expression as an indication that EGb 761 decreases cellular stress resulting from exogenous treatments, therefore leading to a decreased transcriptional induction of the reporter transgene. These results support the hypothesis that EGb 761 augments the natural antistress system of C. elegans, thus increasing stress resistance and life span.

Ginkgo biloba extract EGb 761 increases stress resistance and extends life span of Caenorhabditis elegans.

EGb 761, a standardized extract of Ginkgo biloba leaves, has been used in clinical trials for its beneficial effects on brain functions. In mammals, EGb 761 has been shown to enhance cognition, stress resistance, and longevity, but its molecular and cellular mechanisms are not known. In the present investigation, we used the model organism Caenorhabditis elegans to evaluate pharmacological effects of EGb 761 on aging. We tested the theory that EGb 761 augments the natural antioxidant system of C elegans, and thus increases stress resistance and longevity. We found that treatment of the wild-type worms with EGb 761 extended their median life span by 8%. Amongst several purified components of EGb 761, the flavonoid tamarixetin showed the most dramatic effect: it extended the median life span by 25%. Furthermore, EGb 761 increased the wild type's resistance to acute oxidative and thermal stress by 33% and 25%, respectively. Treatment of the prematurely aging mutant worms mev-1 with EGb 761 increased their resistance to acute oxidative and thermal stress by 33% and 11%, respectively. It appears that oxidative stress, a major determinant of life span, as well as other types of stress, can be successfully counteracted by the Ginlkgo biloba extract EGb 761.