Structural Characterization and Anti-inflammatory Activities of Anticomplementary Polysaccharides from Rhododendron principis.

Rhododendron principis leaves have been used as "Dama", a Traditional Tibetan Medicine for treating inflammatory diseases. R. principis crude polysaccharides with anticomplementary activity demonstrated promising anti-inflammatory effects on acute lung injury induced by lipopolysaccharide. R. principis crude polysaccharides significantly decreased the levels of TNF-α and interleukin-6 in both serum and blood and bronchoalveolar lavage fluid in lipopolysaccharide-induced acute lung injury mice by intragastric administration (100 mg/kg). A heteropolysaccharide, ZNDHP, was obtained from R. principis crude polysaccharides with successive anticomplementary activity-guided separation. ZNDHP was characterized as a branched neutral polysaccharide with a backbone composed of → 2)-ß-Glcp-(1→, → 2,6)-α-Glcp-(1→, → 6,3)-ß-Galp-(1→, → 2,6)-α-Galp-(1→, → 6,2)-ß-Glcp-(1→, → 4)-α-Glcp-(1→, → 5)-ß-Araf-(1→, → 3,5)-α-Araf-(1→, and → 4,6)-ß-Manp-(1→, and the backbone structure was further confirmed by partial acid hydrolysis. In addition to anticomplementary and antioxidant activities, ZNDHP exhibited potent anti-inflammatory activity by significantly inhibiting the secretion of nitric oxide, TNF-α, interleukin-6, and interleukin-1ß of lipopolysaccharide-treated RAW 264.7 cells. However, all of these activities decreased greatly after partially hydrolyzing, indicating the importance of the multibranched structure for its bioactivity. Therefore, ZNDHP might be an important component of R. principis for treating inflammation.

Yixin Ningshen Tablet Alleviates Comorbidity of Myocardial Infarction and Depression by Enhancing Myocardial Energy Metabolism and Increasing Availability of Monoamine Neurotransmitter.

OBJECTIVE: To investigate the therapeutic effect of Yixin Ningshen Tablet (YXNS) on comorbidity of myocardial infarction (MI) and depression in rats and explore the underlying mechanism. METHODS: The Sprague-Dawley rats were randomly divided into 5 groups with 7 rats in each group according to their weights, including control, model, fluoxetine (FLXT, 10 mg/kg), low-dose YXNS (LYXNS, 100 mg/kg), and high-dose YXNS (HYXNS, 300 mg/kg) groups. All rats were pretreated with corresponding drugs for 12 weeks. The rat model of MI and depression was constructed by ligation of left anterior descending coronary artery and chronic mild stress stimulation. The echocardiography, sucrose preference test, open field test, and forced swim test were performed. Myocardial infarction (MI) area and myocardial apoptosis was also detected. Serum levels of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α (TNF-α), 5-hydroxytryptamine (5-HT), adrenocorticotrophic hormone (ACTH), corticosterone (CORT), and norepinephrine (NE) were determined by enzyme linked immunosorbent assay. The proteins of adenosine 5'-monophosphate -activated protein kinase (AMPK), p-AMPK, peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), and nuclear respiratory factor 1 (NRF1) in heart were detected by Western blot analysis. The expression levels of TNF-α, IL-6, indoleamine 2,3-dioxygenase (IDO1), kynurenine 3-monooxygenase (KMO), and kynureninase (KYNU) in hippocampus were detected by real-time quantitative polymerase chain reaction. RESULTS: Compared with the model group, the cardiac function of rats treated with YXNS improved significantly (P<0.01). Meanwhile, YXNS effectively reduced MI size and cardiomyocytes apoptosis of rats (P<0.01 or P<0.05), promoted AMPK phosphorylation, and increased PGC-1α protein expression (P<0.01 or P<0.05). HYXNS significantly increased locomotor activity of rats, decreased the levels of TNF-α, IL-6 and IL-1ß, and increased the serum levels of 5-HT, NE, ACTH, and CORT (all P<0.05). Moreover, HYXNS decreased the mRNA expressions of IDO1, KMO and KYNU (P<0.05). CONCLUSIONS: YXNS can relieve MI by enhancing myocardial energy metabolism. Meanwhile, YXNS can alleviate depression by resisting inflammation and increasing availability of monoamine neurotransmitters. It may be used as a potential drug to treat comorbidity of MI and depression.

Total glucosides of Paeony restores intestinal barrier function through inhibiting Lyn/Snail signaling pathway in colitis mice.

BACKGROUND: Inflammatory bowel disease (IBD) is an autoimmune disease. The pathogenesis of IBD is complicated and intestinal mucosal barrier damage is considered as the trigger factor for the initiation and recurrence of IBD. Total Glucosides of Paeony (TGP) has shown good inhibitory effects on immune-inflammation in clinic studies. However, its effect and mechanism on IBD are largely unknown. PURPOSE: The purpose of this study is to evaluate the effect and mechanism of TGP on IBD. STUDY DESIGN: DSS-induced colitis mouse model was used. TGP was given by gavage. Caco-2 cells were stimulated by outer membrane vesicles (OMV) to establish an in vitro model. METHODS: C57BL/6 mice were divided into normal control group, model group, mesalazine group, paeoniflorin (PA) group, high-dose group of TGP, and low-dose group of TGP. The model was induced with 2.5% DSS for 7 days, and TGP was intragastrically administered for 10 days. The therapeutic effect of TGP was evaluated by symptoms, histochemical analysis, RT-qPCR and ELISA. The mechanism was explored by intestinal permeability, Western blot and immunofluorescence in vivo and in vitro. RESULTS: Our results showed that TGP could significantly improve the symptoms and pathological changes, with reduced levels of TNF-α, IL-17A, IL-23 and IFN-γ in the colon tissues and serum under a dose-dependent manner. TGP also reduced the intestinal permeability and restored the protein expression of tight junction and adherens junction proteins of intestinal epithelial cells in vivo and in vitro. Furthermore, TGP could inhibit the expression of p-Lyn and Snail and prevent Snail nuclear localization, thereby maintaining tight and adherens junctions. CONCLUSION: TGP effectively improves the symptoms of DSS-induced colitis in mice, protects the intestinal epithelial barrier by inhibiting the Lyn/Snail signaling pathway, and maybe a promise therapeutic agent for IBD treatment.

Oral Nanoparticles of SNX10-shRNA Plasmids Ameliorate Mouse Colitis.

BACKGROUND: Our previous study found that deletion of Sorting nexin 10 (SNX10) can protect against colonic inflammation and pathological damage induced by dextran sulfate sodium (DSS). This inspired us that modulation of SNX10 expression in colonic epithelial cells might represent a promising therapeutic strategy for inflammatory bowel disease (IBD). METHODS: Effective delivery of siRNA/shRNA to silence genes is a highly sought-after means in the treatment of multiple diseases. Here, we encapsulated SNX10-shRNA plasmids (SRP) with polylactide-polyglycolide (PLGA) to make oral nanoparticles (NPs), and then applied them to acute and chronic IBD mice model, respectively. The characteristics of the nanoparticles were assayed and the effects of SRP-NPs on mouse IBD were evaluated. RESULTS: High-efficiency SNX10-shRNA plasmids were successfully constructed and coated with PLGA to obtain nanoparticles, with a particle size of 275.2 ± 11.4mm, uniform PDI distribution, entrapment efficiency of 87.6 ± 2.5%, and drug loading of 13.11 ± 1.38%, displayed dominant efficiency of SNX10 RNA interference in the colon. In both acute and chronic IBD models, SRP-NPs could effectively reduce the loss of mice body weight, relieve the intestinal mucosal damage and inflammatory infiltration, inhibit the expression of inflammatory cytokines IL-1ß, IL-23, TNF-α, and down-regulate the expression of toll-like receptors (TLRs) 2 and 4. CONCLUSION: Oral nanoparticles of SNX10-shRNA plasmid displayed dominant efficiency of SNX10 RNA interference in the colon and ameliorate mouse colitis via TLR signaling pathway. SNX10 is a new target for IBD treatment and nanoparticles of SNX10-shRNA plasmid might be a promising treatment option for IBD.

Sorting Nexin 10 Mediates Metabolic Reprogramming of Macrophages in Atherosclerosis Through the Lyn-Dependent TFEB Signaling Pathway.

RATIONALE: SNX10 (sorting nexin 10) has been reported to play a critical role in regulating macrophage function and lipid metabolism. OBJECTIVE: To investigate the precise role of SNX10 in atherosclerotic diseases and the underlying mechanisms. METHODS AND RESULTS: SNX10 expression was compared between human healthy vessels and carotid atherosclerotic plaques. Myeloid cell-specific SNX10 knockdown mice were crossed onto the APOE-/- (apolipoprotein E) background and atherogenesis (high-cholesterol diet-induced) was monitored for 16 weeks. We found that SNX10 expression was increased in atherosclerotic lesions of aortic specimens from humans and APOE-/- mice. Myeloid cell-specific SNX10 deficiency (Δ knockout [KO]) attenuated atherosclerosis progression in APOE-/- mice. The population of anti-inflammatory monocytes/macrophages was increased in the peripheral blood and atherosclerotic lesions of ΔKO mice. In vitro experiments showed that SNX10 deficiency-inhibited foam cell formation through interrupting the internalization of CD36, which requires the interaction of SNX10 and Lyn-AKT (protein kinase B). The reduced Lyn-AKT activation by SNX10 deficiency promoted the nuclear translocation of TFEB (transcription factor EB), thereby enhanced lysosomal biogenesis and LAL (lysosomal acid lipase) activity, resulting in an increase of free fatty acids to fuel mitochondrial fatty acid oxidation. This further promoted the reprogramming of macrophages and shifted toward the anti-inflammatory phenotype. CONCLUSIONS: Our data demonstrate for the first time that SNX10 plays a crucial role in diet-induced atherogenesis via the previously unknown link between the Lyn-Akt-TFEB signaling pathway and macrophage reprogramming, suggest that SNX10 may be a potentially promising therapeutic target for atherosclerosis treatment.

A network pharmacology approach to discover action mechanisms of Yangxinshi Tablet for improving energy metabolism in chronic ischemic heart failure.

ETHNOPHARMACOLOGICAL RELEVANCE: Most cardiovascular diseases ultimately result in heart failure, an intractable problem in modern medicine. Yangxinshi tablet (YXS) is a Chinese medicine formula that is used clinically to treat coronary heart disease. However, the active compounds, potential targets, and pharmacological and molecular mechanism of its anti-heart failure activity remain unclear. Therefore, further investigation is required. AIM OF STUDY: Active ingredients and potential targets of YXS for treating heart failure have been reported previously. However, the molecular functions or biological processes of YXS in energy metabolism have not been discovered. To date, no experimental study to validate the potential anti-heart failure mechanism of YXS. The aim of this study was to study the therapeutic effect of YXS on rats with chronic ischemic heart failure by evaluating rat cardiac function and exercise tolerance, and to explore its potential mechanism by network pharmacology, western blotting, quantitative RT-PCR and histological analysis. MATERIALS AND METHODS: In this investigation, chronic ischemic heart failure rats were randomly assigned to five groups: control group (sham operation), model group (0.5% CMC-Na), trimetazidine group (positive control) and two YXS groups (low- and high-dose groups). Experimental rats were treated by gavage with 10 mg/kg/d (clinical equivalent dose) trimetazidine (TMZ), 500 mg/kg/d (clinical equivalent dose) YXS and 1000 mg/kg/d YXS, respectively, for 5 weeks. The cardiac functions of rats were detected by High-Resolution In Vivo Imaging System. We elucidated novel understanding of the active compounds of YXS in rat plasma and predicted the energy metabolism related targets and processes for heart failure. Then, we validated experimentally the targets and mechanism of YXS on these pathological processes in vivo. RESULTS: It was found that YXS was able to effectively improve cardiac LVIDs, LVEDV, LVESV and EF, decrease myocardial oxygen consumption and reduce myocardial infarct size in rats with chronic ischemic heart failure was similar to that of TMZ. We identified 63 major candidate targets for YXS that are closely to heart failure progression. Enrichment analysis revealed key targets for YXS associated to oxygen delivery, glucose utilization, and mitochondrial biogenesis. Meanwhile, we validated that YXS could promote the expression of downstream HIF-1α, PGC1α and GLUT4 by increasing phosphorylation of PI3K, Akt, mTOR, rpS6 and AMPK. The results show that YXS could activate related PI3K/Akt/mTOR/rpS6/HIF-1α and AMPK/PGC1α/GLUT4 signaling pathways in chronic ischemic heart failure rats. Further experiments demonstrated that YXS increased mitochondrial biogenesis in chronic ischemic heart failure rats and improved exercise tolerance CONCLUSION: YXS treated chronic ischemic heart failure through activating its targets which play pivotal roles in oxygen delivery, glucose utilization and mitochondrial biogenesis to improve energy metabolism through a multi-component, multi-level, multi-target, multi-pathway and multi-mechanism approaches.

Protective effects of Wang-Bi tablet on bone destruction in collagen-induced arthritis by regulating osteoclast-osteoblast functions.

ETHNOPHARMACOLOGICAL RELEVANCE: Wang-bi tablet (WB) consists of 17 traditional Chinese medicines and has been used for treating rheumatoid arthritis (RA) in China for many years, however, its pharmacologic mechanism is not clear. AIM OF STUDY: The aim of this study was to investigate the therapeutic effect of WB on collagen-induced mouse arthritis and explored the underlying mechanism. MATERIALS AND METHODS: DBA/1 mice were used to establish a type II collagen-induced arthritis (CIA) model. From the day of arthritis onset, mice were treated daily by gavage with either total glucosides of paeony (TGP, 0.37  g/kg/d) or WB at a lower (1.11  g/kg/d, WBL) or higher dose of (3.33  g/kg/d, WBH) for 8 weeks. The severity of arthritis, levels of cytokines and the activation of signaling pathways were determined. RESULTS: Our results revealed that WB treatment effectively alleviated inflammatory symptoms and prevented bone erosions and joint destructions. It obviously decreased the serum concentration of pro-inflammatory cytokines TNF-α, IL-6 and IL-17α, while increased the concentration of anti-inflammatory cytokine IL-10. Interestingly, the proportion of splenic Treg cells were increased significantly. In vitro experiments showed that WB inhibited the differentiation of osteoclasts. Consistently, the mRNA levels of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CtsK), and the activation of NF-κB and JAK-STAT3 signaling pathways in the paws of CIA mice were inhibited by WB treatment. On the other hand, up-regulation of osteogenic genes Runx2, Osterix mRNA, and activation of Wnt/ß-catenin signaling pathway along with a decreased receptor activator of nuclear factor κB ligand (RANKL) expression were found in WB treated mice. CONCLUSION: Our results suggest that the therapeutic effect of Wang-bi tablet could be attributed to its inhibitory activity on NF-κB and STAT3 signaling pathway-mediated osteoclast differentiation, and its enhancement on Wnt/ß-catenin signaling pathway-mediated osteoblast functions.

Suppressive effects of Wang­Bi Tablet on adjuvant­induced arthritis in rats via NF­κB and STAT3 signaling pathways.

Rheumatoid arthritis (RA) severely affects the quality life of patients due to its high association with disability. Traditional Chinese medicines have been reported to exert notable therapeutic effects on RA. The Chinese medicinal prescription Wang­Bi Tablet (WB) has been successfully used to clinically treat RA for many years; however, its pharmacological mechanism of action is largely unclear. In the present study, adjuvant­induced arthritis (AIA) rats were used to evaluate the anti­inflammatory effects of WB and western blotting was used to explore the molecular mechanisms. The experimental results demonstrated that WB treatment significantly reduced arthritis score and hind­paw volume. Furthermore, synovial hyperplasia, inflammatory cell infiltration and joint destruction were ameliorated by WB. The expression levels of the proinflammatory cytokines interleukin (IL)­1ß, tumor necrosis factor­α and IL­6, were reduced in the joints of WB­treated rats. Western blotting revealed that WB could also inhibit excessive activation of nuclear factor (NF)­κB and Janus kinase (JAK)­signal transducer and activator of transcription 3 (STAT3) signaling pathways. These results indicated that the therapeutic effects of WB on AIA may be accomplished through inhibition of the NF­κB and JAK­STAT3 signaling pathways. These findings provide experimental evidence to support WB as a therapeutic agent for the treatment of patients with RA.

Protective effects of a traditional Chinese herbal formula Jiang-Xian HuGan on Concanavalin A-induced mouse hepatitis via NF-κB and Nrf2 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiang-Xian HuGan (JXHG) formulated by five natural products including Freshwater clam (Corbicula fluminea), Curcuma longa L., Ligustrum lucidum, Eclipta prostrata (L.) L. and Paeonia lactiflora Pall., has exhibited a great hepatoprotective effect. AIM OF THIS STUDY: We investigated the effect of JXHG on concanavalin A (ConA)-induced acute live injury in mice, and to elucidate its underlying molecular mechanisms. MATERIALS AND METHODS: Jiangkanling Capsule (900 mg/kg), low-dose JXHG (LJXHG, 700 mg/kg), high-dose JXHG (HJXHG, 1400 mg/kg) were administered to mice by oral gavage daily for 20 days prior to a single intravenous injection of ConA (20 mg/kg). Liver injury was evaluated by measuring the serum levels of enzymes and cytokines as well as liver histological analysis. We also measured the hepatic expression of cytokines at mRNA levels and the proteins related to NF-κB and Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathways. RESULT: Our results showed that JXHG pretreatment significantly alleviated ConA-induced live injury as evidenced by decreased serum levels of glutamic-pyruvic transaminase (ALT) and glutamic oxalacetic transaminase (AST), and reduced hepatocyte apoptosis and mortality. Furthermore, JXHG was able to significantly reduce the serum levels of proinflammatory cytokines, down-regulate the mRNA expression of interleukin-6 (IL-6) and interferon-γ (IFN-γ), and up-regulate IL-10 as well as superoxide-dimutase-1 (SOD1), glutathione reductase (GSR) and Glutathione peroxidase 2 (GPX2) mRNA in the liver tissues after Con A injection. In addition, JXHG pretreatment dramatically suppressed the phosphorylation of NF-κB p65 (p65), increased Nrf2 expression, and decreased the expression ratio of cleaved caspase-3/caspase-3 in liver tissues. CONCLUSION: These results suggest that JXHG protects against ConA-induced acute live injury through inhibiting NF-κB mediated inflammatory pathway and promoting Nrf2 mediated anti-oxidative stress signaling pathway.

SNX10 mediates alcohol-induced liver injury and steatosis by regulating the activation of chaperone-mediated autophagy.

BACKGROUND & AIMS: Alcoholic liver disease (ALD) is a major cause of morbidity and mortality worldwide. However, the cellular defense mechanisms underlying ALD are not well understood. Recent studies highlighted the involvement of chaperone-mediated autophagy (CMA) in regulating hepatic lipid metabolism. Sorting nexin (SNX)-10 has a regulatory function in endolysosomal trafficking and stabilisation. Here, we investigated the roles of SNX10 in CMA activation and in the pathogenesis of alcohol-induced liver injury and steatosis. METHODS: Snx10 knockout (Snx10 KO) mice and their wild-type (WT) littermates fed either the Lieber-DeCarli liquid alcohol diet or a control liquid diet, and primary cultured WT and Snx10 KO hepatocytes stimulated with ethanol, were used as in vivo and in vitro ALD models, respectively. Activation of CMA, liver injury parameters, inflammatory cytokines, oxidative stress and lipid metabolism were measured. RESULTS: Compared with WT littermates, Snx10 KO mice exhibited a significant amelioration in ethanol-induced liver injury and hepatic steatosis. Both in vivo and in vitro studies showed that SNX10 deficiency upregulated lysosome-associated membrane protein type 2A (LAMP-2A) expression and CMA activation, which could be reversed by SNX10 overexpression in vitro. LAMP-2A interference confirmed that the upregulation of Nrf2 and AMPK signalling pathways induced by SNX10 deficiency relied on CMA activation. Pull-down assays revealed an interaction between SNX10 and cathepsin A (CTSA), a key enzyme involved in LAMP-2A degradation. Deficiency in SNX10 inhibited CTSA maturation and increased the stability of LAMP-2A, resulting in an increase in CMA activity. CONCLUSIONS: SNX10 controls CMA activity by mediating CTSA maturation, and, thus, has an essential role in alcohol-induced liver injury and steatosis. Our results provide evidence for SNX10 as a potential promising therapeutic target for preventing or ameliorating liver injury in ALD. LAY SUMMARY: Alcoholic liver disease is a major cause of morbidity and mortality worldwide. Recent studies highlight the involvement of chaperone-mediated autophagy (CMA) in regulating hepatic lipid metabolism. Our study reveals that deficiency of sorting nexin (SNX) 10 increases the stability of LAMP-2A by inhibiting cathepsin A maturation, resulting in the increase of CMA activity and, thus, alleviates alcohol-induced liver injury and steatosis.