Protective effect and mechanism of TLR4/NF-κB pathway regulated by miR-146a in intracerebral hemorrhage model rats / 中国免疫学杂志

Objective:To explore role of miR-146a in regulating TLR4/NF-κB pathway on inflammatory injury and neuropro-tection in intracerebral hemorrhage model rats and its possible mechanism.Methods:A total of 40 rats were selected and randomly divided into sham,model,over-expressing miR-146a adenovirus and negative virus injection groups,with 10 rats in each group.Garcia score was used for neurological function;HE staining was used to observe changes of brain tissues.ELISA was used to detect inflammatory factors levels.TLR4,NF-κB protein and gene expressions in brain tissues were detected by Western blot and RT-PCR.Results:Compared with model group,neural function score of overexpressed miR-146a adenovirus injection group was increased(P<0.05).Model group had abnormal cell morphology,edema and inflammation.Cell morphology,edema and inflammation were alleviated in overexpressed miR-146a adenovirus injection group.Inflammatory factors levels in model group were higher than sham group(P<0.05).Inflammatory factors levels in overexpressed miR-146a adenovirus injection group were lower than model group(P<0.05).TLR4,NF-κB protein and mRNA expressions in model group were increased than sham group(P<0.05).TLR4,NF-κB protein and mRNA expressions in overexpressed miR-146a adenovirus injection group were decreased than model group(P<0.05).Conclusion:miR-146a can improve neural function and reduce inflammatory injury in rats with intracerebral hemorrhage,possibly by inhibiting activation of TLR4/NF-κB signaling pathway and reducing inflammatory factors levels of brain tissues.

Study on the Effect and Mechanism of Hederagenin on Dextran Sodium Sulfate-Induced Ulcerative Colitis in mice / 中药新药与临床药理

Objective To study the effect and its mechanism of hederagenin(hed)on dextran sulfate sodium(DSS)-induced ulcerative colitis(UC)in mice.Methods(1)In vitro experiments:after treating RAW264.7 cells with different concentrations(0,2.5,5,10,20,40 μmol·L-1)of hed for 24 hours,the cell survival rate was detected by MTT assay.RAW264.7 cells were divided into:blank group,lipopolysaccharide(LPS)group(1 μg·L-1),LPS+2.5 μmol·L-1 hed group,LPS+5 μmol·L-1 hed group and LPS+10 μmol·L-1 hed group;an in vitro cellular inflammation model was established using LPS intervention for 24 hours and co-incubated with hed for 24 hours.The levels of interleukin 1β(IL-1β),IL-6 and tumor necrosis factor α(TNF-α)in the cell supernatant were determined by ELISA;the expression levels of TLR4/NF-κB pathway-related proteins in the cells were detected by Western Blot.(2)In vivo experiments:C57BL/6 mice were randomly divided into a blank group,a model group,a Salazosulfapyridine group(200 mg·kg-1),and an hed low-,medium-,and high-dosage groups(12.5,25,and 50 mg·kg-1),with 5 mice in each group.Mice were induced to establish UC model by drinking 3%DSS solution freely for 7 days.The UC model was then established by gavage once a day for 7 days.At the end of the administration,the Disease Activity Index(DAI)was evaluated;pathological changes in the colonic tissues of mice were observed by HE staining;the levels of IL-1β,IL-6,and TNF-α in the colonic tissue were measured by ELISA;and the expression levels of proteins related to the TLR4/NF-κB pathway in the colonic tissue were detected by Western Blot.Results(1)In vitro experiments:compared with the blank group(0 μ mol·L-1 group),there was no significant change in the cell survival rate in the 2.5-10 μmol·L-1 hed group(P＞0.05),and there was no significant toxicity effect on RAW264.7 cells.Compared with the blank group,the expression levels of IL-1β,IL-6,and TNF-α in RAW264.7 cells in the LPS group were significantly increased(P＜0.01);and the protein expression levels of TLR4 and p-NF-κ B/NF-κ B were significantly increased(P＜0.01).Compared with the LPS group,the expression levels of IL-1β and TNF-α in RAW264.7 cells in the hed 2.5,5,and 10 μmol·L-1 concentration groups were significantly decreased(P＜0.05,P＜0.01),and the protein expression levels of TLR4,p-NF-κB/NF-κB were significantly decreased(P＜0.05,P＜0.01);the IL-6 expression level of RAW264.7 cells in the hed 5 and 10 μmol·L-1 concentration groups was significantly reduced(P＜0.05,P＜0.01).(2)In vivo experiments:compared with the blank group,the body mass of mice in the model group was consistently reduced(P＜0.01),the DAI score was significantly elevated(P＜0.01),and the length of the colon was significantly shortened(P＜0.01);the colonic tissue showed obvious epithelial cell damage,and the histopathological scores were significantly elevated(P＜0.01);and the expression levels of the pro-inflammatory cytokines IL-1β,IL-6 and TNF-α were significantly increased(P＜0.01);protein expression levels of TLR4 and p-NF-κB/NF-κB were significantly increased(P＜0.01)in colon tissue.Compared with the model group,the body mass of mice in the low-,medium-and high-dose groups of hed were significantly increased(P＜0.05,P＜0.01),the DAI score was significantly decreased(P＜0.05,P＜0.01),the pathological damage of colon tissue improved to different degrees,and the protein expression levels of TLR4,p-NF-κB/NF-κB in the colonic tissue were significantly decreased(P＜0.05,P＜0.01);the colon length of mice in the medium-and high-dose groups of hed were significantly increased(P＜0.05,P＜0.01),and the expression levels and histopathological scores of IL-1β,IL-6,and TNF-α in colon tissue were significantly reduced(P＜0.05,P＜0.01).Conclusion Hed were able to effectively ameliorate colonic histopathological injury and reduce the levels of inflammatory factors in DSS-induced UC mice,and their mechanism of action may be related to the inhibition of the TLR4/NF-κB pathway.

Modulating effects of Astragalus polysaccharide on immune disorders via gut microbiota and the TLR4/NF-κB pathway in rats with syndrome of dampness stagnancy due to spleen deficiency / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The syndrome of dampness stagnancy due to spleen deficiency (DSSD) is relatively common globally. Although the pathogenesis of DSSD remains unclear, evidence has suggested that the gut microbiota might play a significant role. Radix Astragali, used as both medicine and food, exerts the effects of tonifying spleen and qi. Astragalus polysaccharide (APS) comprises a macromolecule substance extracted from the dried root of Radix Astragali, which has many pharmacological functions. However, whether APS mitigates the immune disorders underlying the DSSD syndrome via regulating gut microbiota and the relevant mechanism remains unknown. Here, we used DSSD rats induced by high-fat and low-protein (HFLP) diet plus exhaustive swimming, and found that APS of moderate molecular weight increased the body weight gain and immune organ indexes, decreased the levels of interleukin-1β (IL-1β), IL-6, and endotoxin, and suppressed the Toll-like receptor 4/nuclear factor-‍κB (TLR4/NF-‍κB) pathway. Moreover, a total of 27 critical genera were significantly enriched according to the linear discriminant analysis effect size (LEfSe). APS increased the diversity of the gut microbiota and changed its composition, such as reducing the relative abundance of Pseudoflavonifractor and Paraprevotella, and increasing that of Parasutterella, Parabacteroides, Clostridium XIVb, Oscillibacter, Butyricicoccus, and Dorea. APS also elevated the contents of short-chain fatty acids (SCFAs). Furthermore, the correlation analysis indicated that 12 critical bacteria were related to the body weight gain and immune organ indexes. In general, our study demonstrated that APS ameliorated the immune disorders in DSSD rats via modulating their gut microbiota, especially for some bacteria involving immune and inflammatory response and SCFA production, as well as the TLR4/NF-κB pathway. This study provides an insight into the function of APS as a unique potential prebiotic through exerting systemic activities in treating DSSD.

Hepatoprotective effects of Yinchenzhufu decoction on intrahepatic cholestasis liver injury in mice induced by ANIT via suppressing TLR4/NF-κB pathway / 中国临床药理学与治疗学

AIM: To investigate the hepatoprotective effects of Yinchenzhufu decoction (YCZFD) via TLR4/NF-κB pathway on the hepatic injury in alpha-naphthyisothiocyanate (ANIT)-induced intrahepatic cholestasis mice. METHODS: Male C57BL/6 mice were administered with YCZFD for consecutive 10 days, and intrahepatic cholestasis mice model was induced by orally given ANIT at dose of 75 g/kg on the seventh day. On the last day, the blood samples and liver samples were collected 2 hours after oral administration of YCZFD. The serum biochemical index, liver histopathology, the change of bile acid contents in mice liver, and the expression of relative proteins in TLR4/NF-κB pathway were determined. RESULTS: Compared with the model group, YCZFD treatment group significantly improved the hepatic necrosis and reduced the inflammatory cell infiltration. The plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bile acid (TBA), and total bilirubin (TBIL) levels were significantly decreased, and the free and conjugated bile acids were both reduced. The expression of TLR4, IL-1β, IL-6, TNF-α, and MCP-1 were significantly decreased. CONCLUSION: YCZFD presents hepatoprotective effects on the hepatic injury in ANIT-induced intrahepatic cholestasis mice, which may be related to inhibiting the TLR4/NF-κB pathway, reducing the intrahepatic bile acids, and suppressing the inflammatory reaction.

Effects of Jiedu-jiangzhuo Granules on Gut Microbiota and TLR4/NF-κB Pathway of Metabolic Syndrome Rats / 中国药学杂志

OBJECTIVE: To explore the effects of Jiedu-jiangzhuo granules on gut microbiota of metabolic syndrome rats and TLR4/NF-κB pathway. METHODS: SD rats were randomly divided into normal control group, model group, metformin group, experimental high-dose of Jiedu-jiangzhuo granules 2.4 g•kg-1•d-1 group and experimental low-dose of Jiedu-jiangzhuo granules 1.2 g•kg-1•d-1 group, with 12 rats in each group. Except for rats in the control group, the other rats were fed with high-fat/ sugar/salt diet for 12 weeks. When fed for 8 weeks, the rats in metformin group (0.103 g•kg-1•d-1), experimental high-dose group (2.4 g•kg-1•d-1) or experimental low-dose group (1.2 g•kg-1•d-1) were simultaneously gavaged with metformin or Jiedu-jiangzhuo granules for 4 weeks. Body weight, serum lipid indexes and lipopolysaccharide(LPS) were determined by biochemical method, liver coefficient and epididymal fat tissue coefficient of rats were detected. Total genomic DNA of gut microbiota was extracted from fecal samples, then DNA library was built. Community structures of fecal microbes and α-diversity were sequenced on the Illumina Miseq platform. RESULTS: At the end of experiment, the body weight, LPS, serum total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C) and fasting blood glucose(FBG) of rats in experimental high-dose group were lower than those in the model group and higher than those in the control group (P0.05). The liver coefficient and epididymal fat tissue coefficient of rats in experimental high-dose group were lower than those in the model group (P0.05). Besides, the NF-κB, IL-6, TNF-α levels of rats inexperimental high-dose group were lower than those in the model group, metformin group or low-dose group (P0.05). Chaol index of gut microbiota was higher than that in the other four groups (P0.05). The ratios of Firmicutes, Proteobacteria, Actinobacteria, Tenericutes and Prevotella, Phascolarctobacterium in experimental high-dose group were lower than those in model group (P<0.05), meanwhile the ratios of Bacteroidetes, Paraprevotella and Bacteroides were higher than those in model group (P<0.05). Finally, TLR4 and NF-κB proteins in colonic tissues of rats in experimental high-dose group were lower than those in model group(P<0.05). CONCLUSION:S There are significant evidences that Jiedu-jiangzhuo granules could play a role in lower of weight gain and blood lipids in metabolic syndrome rats by adjusting the gut microbiota and TLR4/NF-κB pathway.

MiR-590 Inhibits Endothelial Cell Apoptosis by Inactivating the TLR4/NF-κB Pathway in Atherosclerosis

PURPOSE: Previous study has well documented the anti-apoptotic effects of miR-590 on oxidized low-density lipoprotein (ox-LDL)-treated endothelial cells (ECs). However, the mechanism underlying the anti-apoptotic effects of miR-590 in ox-LDL-treated ECs remains to be further addressed. MATERIALS AND METHODS: ApoE(−/−) mice fed with a high-fat diet (HFD) and human aortic endothelial cells (HAECs) treated with ox-LDL were used as in vivo and in vitro models of atherosclerosis. The expressions of miR-590 and toll-like receptor 4 (TLR4) were detected by quantitative real-time PCR and Western blot, respectively. Atherosclerotic lesion analysis was performed using Evans blue and hematoxylin-eosin staining. Cell proliferation was assessed by MTT assay. Apoptosis was examined using flow cytometry analysis and Western blot analysis of Cleaved poly (ADP-ribose) polymerase (PARP) and Cleaved Caspase-3 levels. The effect of miR-590 on TLR4/nuclear factor kappa B (NF-κB) pathway was evaluated by Western blot. Binding between miR-590 and TLR4 was confirmed by luciferase reporter assay and Western blot. RESULTS: miR-590 was downregulated in the aorta tissues from HFD-fed apoE(−/−) mice and ox-LDL-treated HAECs. miR-590 overexpression inhibited atherosclerotic lesion in HFD-induced apoE(−/−) mice and promoted proliferation and inhibited apoptosis of ox-LDL-treated HAECs. Additionally, TLR4 was identified as a direct target of miR-590 in ox-LDL-treated HAECs. Moreover, anti-miR-590 reversed TLR4 knockdown-mediated promotion of cell proliferation and suppression of apoptosis in ox-LDL-treated HAECs. miR-590 overexpression suppressed the TLR4/NF-κB pathway, and inhibition of the TLR4/NF-κB pathway promoted cell proliferation and impeded apoptosis in ox-LDL-treated HAECs. CONCLUSION: miR-590 promoted proliferation and blocked ox-LDL-induced apoptosis in HAECs through inhibition of the TLR4/NF-κB pathway.

Effect of Sinomenine Hydrochloride on TLR4/NF-κB Pathway in Mice with Experimental Colitis / 中国药师

Objective:To study the effect of sinomenine hydrochloride on dextran sulfate sodium(DSS) induced experimental colitis in mice. Methods:The colitis model of BALB/c mice was established with DSS. The mice were randomly divided into six groups (n = 6):the normal control group and the model group(giving normal saline,0.2 ml),salicylazosulfapyridine treatment group (100 mg·kg-1) and sinomenine treatment groups (30,90,270 mg·kg-1). After continuous administration for 10 d,the disease activity index (DAI) and the tissue damage index in each group were evaluated. The mouse colons of TLR4 and Myd88 were detected using Western-blotting, and the expression of NF-κB in the colon tissues was detected using immunohistochemical method. Results:The DAI and the tissue damage index, and the expressions of TLR4, Myd88 and NF-κB in the colon tissues were significantly higher in the model group than those in the normal group (P < 0.01). Sulfasalazine treatment group and sinomenine at low,medium and high dose groups could reduce the DAI and the tissue damage score,and the expressions of TLR4, Myd88 and NF-κB in the colon tissues (P < 0.01) in a concentration-dependent manner. There was no significant difference between salicylazosulfapyridine treatment group and sinomenine at medium/high dose groups. Conclusion:Sinomenine reduces the expression of TLR4,Myd88 and NF-κB in mice with experimental colitis by regulating the TLR4/NF-k B signaling pathway to play a therapeutic role.

The optimal duration of ischemic preconditioning for renal ischemia-reperfusion injury in mice

PURPOSE: The aim of the present study was to investigate the protective effects of ischemic preconditioning for different periods of time and to elucidate the optimal safe ischemic preconditioning time for renal ischemia-reperfusion (I/R) injury in mice. METHODS: A total of 25 male C57BL/6 mice were randomly divided into 5 groups (sham, I/R, ischemic preconditioning [IP]-3, IP-5, and IP-7 groups), in which the kidney was preconditioned with IP of various durations and then subjected to I/R injury (the last 3 groups). To induce renal ischemia, the left renal pedicle was occluded with a nontraumatic microaneurysm clamp for 30 minutes followed by reperfusion for 24 hours. The effects of IP on renal I/R injury were evaluated in terms of renal function, tubular necrosis, apoptotic cell death and inflammatory cytokines. RESULTS: Results indicated that BUN and creatinine (Cr) levels increased significantly in the I/R group, but the elevations were significantly lower in IP groups, especially in the IP-5 group. Histological analysis revealed that kidney injury was markedly decreased in the IP-5 group compared with the I/R group, as evidenced by reduced renal necrosis/apoptosis. In addition, IP significantly inhibited gene expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and chemokines (monocyte chemoattractant protein-1). Western blot analysis indicated that the expression levels of Toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) were upregulated in the I/R group, while expression was inhibited in the IP groups. CONCLUSION: Five-minute IP had the greatest protective effect against I/R injury.

Protective effects of combining human umbilical cord mesenchymal stem cells and glucagon like peptide-1 receptor agonist on the islet inflammatory injury in rat model of type 2 diabetes mellitus / 中华内分泌代谢杂志

T2DM+LIRA group and T2DM+LIRA+UC-MSCs group (P<0. 05). The ratio of insulin positive area in pancreas tissue was obviously rised, while the ratio of glucagon positive area in pancreas tissue was clearly descended in T2DM+LIRA+UC-MSCs group. And the same difference in valuating islet cells apoptosis by TUNEL could be observed ( P<0. 05). The expression of NF-κB and TLR4 protein in pancreas tissue of T2DM+LIRA+UC-MSCs group were the least amongthefourgroups[(0.75±0.10)vs(0.60±0.08),(0.47±0.08),(0.31±0.04),P<0.05]and[(1.24± 0. 12) vs (0. 93 ± 0. 10), (0. 95 ± 0. 09), (0. 74 ± 0. 07), P<0. 05 ]. Conclusion The combined treatment of liraglutide with umbilical cord mesenchymal stem cells is superior over a single treatment of liraglutide or umbilical cord mesenchymal stem cells in improving the islet function in type 2 diabetes mellitus models, which may be related to the down modulating the TLR4/NF-κB inflammatory signaling pathway.