Chaihu Longgu Muli Decoction relieving temporal lobe epilepsy in rats by inhibiting TLR4 signaling pathway through miR-146a-3p and miR-146a-5p

Objective To explore the effect and mechanism of Chaihu Longgu Muli Decoction(柴胡龙骨牡蛎汤,CHLGMLD)in rats with temporal lobe epilepsy(TLE).Methods A total of 80 Sprague-Dawley(SD)male rats were randomized into control(CON),model(MOD),carbamazepine(CBZ,0.1 g/kg),CHLGMLD low dose(CHLGMLD-L,12.5 g/kg),and high dose(CHLGMLD-H,25 g/kg)groups,with 16 rats in each group.TLE rat models were established in the four groups with the use of lithium-pilocarpine except for the CON group.After the successful establishment of TLE models,all drugs were administered through gavage,and distilled water was given to rats in the CON and MOD groups for four weeks.The frequency and duration of seizures before and after treatment were recorded for the evaluation of the alleviation degree.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miR-146a-3p and miR-146a-5p.The expression levels of toll-like receptor 4(TLR4),interleukin-1 receptor-associated kinase 1(IRAK1),tumor necrosis factor(TNF)receptor-associated factor 6(TRAF6),TAK1-binding protein(TAB),nuclear factor-kappa B(NF-κB),and interleukin-1 beta(IL-1β)in hippocampus were tested by immunofluorescence assay.Correlation analysis between the above factors and expressions of miR-146a-3p and miR-146a-5p were performed separately.Results CHLGMLD decreased the frequency(P<0.05)and duration(P<0.01)of seizures in rats.CHLGMLD down-regulated the expression levels of miR-146a-5p and miR-146a-3p(P<0.05),and inhibited the expression levels of TLR4,IRAK1,TRAF6,TAB,NF-κB,and IL-1β(P<0.01).The correlation analysis revealed that the expression levels of TLR4,IRAK1,TRAF6,TAB,NF-κB,and IL-1β were positively correlated with the expression levels of miR-146a-3p and miR-146a-5p detected by qRT-PCR,respectively(P<0.01).Conclusion CHLGMLD can inhibite the TLR4 signaling pathway by lowering the expression levels of miR-146a-3p and miR-146a-5p to alleviate hippocampal dentate gyrus inflammation in TLE rats,thus relieving seizures.

Effects of Liancao-Xieli capsule on intestinal mucosal inflammatory factors and TLR4/PI3K/Akt/mTOR signaling pathway in mice with ulcerative colitis

Objective:To observe the effect of Liancao-Xieli capsule on intestinal mucosal inflammatory factors and TLR4/PI3K/Akt/mTOR signaling pathway in mice with ulcerative colitis(UC);Methods:40 male C57BL/6 mice were randomly divided into the control group,model group,Liancao-Xieli group and mesalazine group,with 10 mice in each group.In addition to the control group,the remaining three groups of mice were induced by 3%dextran sulfate sodium(DSS)to induce acute UC model.During the modeling period,mice in each group were given corresponding drugs and normal saline by gavage.At the end of the experiment,HE staining was used to observe the pathological changes of colonic tissue in each group,and ELISA was used to detect the inflammatory factors(TNF-α,IL-6,IL-1β,IL-8,IL-17,and INF-γ)in serum and colonic tissue.The expression levels of TLR4/PI3K/Akt/mTOR signaling pathway related proteins were also detected by Western blot;Results:Compared with the model group,Liancao-Xieli capsule could significantly increase the colon length and decrease the score of colon histopathology in UC mice(P<0.01).In addition,the levels of TNF-α,IL-6,IL1β,IL-8,IL-17,and INF-γwere significantly reduced in serum and colon tissue,and the expressions of TLR4,PI3K,p-Akt and p-mTOR were significantly down-regulated in LiancaoXieyi group when compared with the model group(P<0.01).While the expressions of Akt and mTOR were not significantly affected in Liancao-Xieyi group(P>0.05);Conclusion:LiancaoXieli capsule can reduce the secretion of inflammatory factors,improve the intestinal mucosal damage and inflammatory response in UC by inhibiting the activation of TLR4/PI3K/Akt/mTOR signaling pathway。

To investigate the effect of Shenqi Tiaoshen Formula on CSE induced inflammatory response of MH-S cells based on TLR4/NF-kB/NLRP3 pathway

Objective:To study the effects of Shenqi Tiaoshen Formula(SQTS)on the inflammatory response of MH-S cells induced by cigarette smoking extract(CSE)and its mechanism based on TLR4/NF-kB/NLRP3 pathway.Methods:MH-S cells were used as subjects to evaluate cell viability by CCK-8 method.The levels of TNF-α,IL-1βand IL-6 in the supernatant were detected by ELISA.ROS were detected by DCFH-DA fluorescence probe.Western blotting was used to detect the expression of TLR4/NF-kB/NLRP3 pathway protein,and TAK-242,a TLR4 inhibitor,was used to verify the role of SQTS in the TLR4/NF-kB/NLRP3 pathway.Results:Compared with blank group,the cell survival rate of CSE group was decreased,and the contents of inflammatory cytokines TNF-α,IL-1βand IL-6 were increased(P<0.05),ROS fluorescence expression level was significantly increased(P<0.01),TLR4/NF-kB/NLRP3 pathway protein expression was significantly increased(P<0.05);Compared with CSE group,the survival rate of cells in SQTS groups was increased,and the expression levels of the above indexes were decreased(P<0.05),and TLR4/NF-kB/NLRP3 pathway protein decreased in TAK-242 groups(P<0.05).Conclusion:SQTS can reduce the inflammatory response of MH-S cells induced by CSE by inhibiting TLR4/NF-kB/NLRP3 pathway.

Huoxin Pill Reduces Myocardial Ischemia Reperfusion Injury in Rats via TLR4/NFκB/NLRP3 Signaling Pathway

Objective:To explore the protective effect of Huoxin Pill(HXP)on acute myocardial ischemia-reperfusion(MIRI)injury in rats.Methods:Seventy-five adult SD rats were divided into the sham-operated group,model group,positive drug group(diltiazem hydrochloride,DH),high dose group(24 mg/kg,HXP-H)and low dose group(12 mg/kg,HXP-L)of Huoxin Pill(n=15 for every group)according to the complete randomization method.After 1 week of intragastric administration,the left anterior descending coronary artery of the rat's heart was ligated for 45 min and reperfused for 3 h.Serum was separated and the levels of creatine kinase(CK),creatine kinase isoenzyme(CK-MB)and lactate dehydrogenase(LDH),superoxide dismutase(SOD),and malondialdehyde(MDA),hypersensitive C-reactive protein(hs-CRP)and interleukin-1β(IL-1β)were measured.Myocardial ischemia rate,myocardial infarction rate and myocardial no-reflow rate were determined by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride(TTC).Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine(BATMAN)databases were used to screen for possible active compounds of HXP and their potential therapeutic targets;the results of anti-inflammatory genes associated with MIRI were obtained from GeneC ards,Drugbank,Online Mendelian Inheritance in Man(OMIM),and Therapeutic Target Datebase(TTD)databases was performed;Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment were used to analyze the intersected targets;molecular docking was performed using AutoD ock Tools.Western blot was used to detect the protein expression of Toll-like receptor 4(TLR4)/nuclear factor kappa-B(NFκB)/NOD-like receptor protein 3(NLRP3).Results:Compared with the model group,all doses of HXP significantly reduced the levels of LDH,CK and CK-MB(P<0.05,P<0.01);HXP significantly increased serum activity of SOD(P<0.05,P<0.01);all doses of HXP significantly reduced the levels of hs-CRP and IL-1β(P<0.05,P<0.01)and the myocardial infarction rate and myocardial no-reflow rate(P<0.01).GO enrichment analysis mainly involved positive regulation of gene expression,extracellular space and identical protein binding,KEGG pathway enrichment mainly involved PI3K-Akt signaling pathway and lipid and atherosclerosis.Molecular docking results showed that kaempferol and luteolin had a better affinity with TLR4,NFκB and NLRP3 molecules.The protein expressions of TLR4,NFκB and NLRP3 were reduced in the HXP group(P<0.01).Conclusions:HXP has a significant protective effect on myocardial ischemia-reperfusion injury in rats,and its effect may be related to the inhibition of redox response and reduction of the inflammatory response by inhibiting the TLR4/NFκB/NLRP3 signaling pathway.

Hepatic protective effects of Shenling Baizhu powder, a herbal compound, against inflammatory damage via TLR4/NLRP3 signalling pathway in rats with nonalcoholic fatty liver disease

Objective: High-fat diet(HFD) and inflammation are two key contributors to nonalcoholic fatty liver disease(NAFLD). Shenling Baizhu powder(SLBZP), a classical herbal compound, has been successfully used to alleviate NAFLD. However, its specific mechanisms are not fully understood. In this study, we assessed the anti-NAFLD effect of SLBZP in vivo.Methods: Rats were fed an HFD with or without SLBZP or with probiotics. At the end of week 16, an echo magnetic resonance imaging(EchoMRI) body composition analyser was used to quantitatively analyse body composition;a micro-computed tomography(micro-CT) imaging system was used to evaluate whole body and liver fat;and the Moor full-field laser perfusion imager 2 was used to assess liver microcirculation, after which, all rats were sacrificed. Then, biochemical indicators in the blood and the ultrastructure of rat livers were evaluated. Protein expression related to the liver Toll-like receptor 4(TLR4)/Nod-like receptor family pyrin domain-containing 3(NLRP3) signalling pathway was assessed using Western blot analysis. Further, high-throughput screening of 29 related inflammatory factors in liver tissue was performed using a cytokine array.Results: SLBZP supplementation reduced body weight, serum free fatty acid, and insulin resistance index(P<0.05). It also ameliorated liver microcirculation and ultrastructural abnormalities. EchoMRI and micro-CT quantitative analyses showed that treatment with SLBZP reduced fat mass and visceral fat(P<0.05 and P<0.01, respectively). In addition, SLBZP decreased the expression of lipopolysaccharide(LPS)-activated TLR4/NLRP3 signalling pathway-related proteins and altered the expression levels of some inflammatory cytokines in liver tissues.Conclusion: SLBZP can inhibit NLRP3 inflammasome activation and interleukin-1 b release by suppressing LPS-induced TLR4 expression in rats with HFD-induced NAFLD. Thus, SLBZP may be beneficial for the prevention and treatment of inflammatory damage and associated diseases.

加味少腹逐瘀汤抑制TLR4/TRIF/IRF3信号通路改善子宫内膜异位症大鼠炎性微环境

目的探讨加味少腹逐瘀汤对子宫内膜异位症大鼠炎性微环境的影响与作用机制。方法随机选取成年雌性SD大鼠,采用自体移植法制备子宫内膜异位症大鼠模型,并分为模型组、加味少腹逐瘀汤低剂量、中剂量及高剂量组,另取SD大鼠作为对照组,每组10只。比较各组大鼠体质量及热痛潜伏期;HE染色观察大鼠异位内膜组织病理形态学变化;TUNEL染色检测大鼠异位内膜细胞凋亡;ELISA检测大鼠异位内膜组织中白介素-1β(IL-1β)、IL-6和肿瘤坏死因子α(TNF-α)水平;Western blot检测异位内膜组织中TLR4、TRIF、IRF3及p-IRF3蛋白表达。结果与对照组相比,模型组热痛潜伏期明显缩短,异位内膜组织病理损伤与纤维化明显,IL-1β、IL-6、TNF-α水平显著升高,TLR4、TRIF及p-IRF3蛋白表达水平显著升高。加味少腹逐瘀汤治疗后,大鼠热痛潜伏期显著延长,异位内膜组织病理损伤不同程度减轻,细胞凋亡明显减少,IL-1β、IL-6、TNF-α水平显著降低,TLR4、TRIF及p-IRF3蛋白表达水平显著降低。结论加味少腹逐瘀汤能够改善子宫内膜异位症大鼠的炎性微环境,其作用机制可能与抑制TLR4/TRIF/IRF3信号通路有关。

ATF4 is directly recruited by TLR4 signaling and positively regulates TLR4-trigged cytokine production in human monocytes

Toll-like receptors （TLRs） are sentinels of the host defense system, which recognize a large number of microbial pathogens. The host defense system may be inefficient or inflammatory diseases may develop if microbial recognition by TLRs and subsequent TLR-triggered cytokine production are deregulated. Activating transcription factor 4 （ATF4）, a member of the ATF/CREB transcription factor family, is an important factor that participates in several pathophysiological processes. In this report, we found that ATF4 is also involved in the TLR-mediated innate immune response, which participates in TLR4 signal transduction and mediates the secretion of a variety of cytokines. We observed that ATF4 is activated and translocates to the nucleus following l ipopolysaccharide （LPS） stimulation via the TLR4-MyD88-dependent pathway. Additionally, a cytokine array assay showed that some key inflammatory cytokines, such as I L-6, I L-8 and RANTES, are positively regulated by ATF4. We also demonstrate that c-Jun directly binds to ATF4, thereby promoting the secretion of inflammatory cytokines. Taken together, these results indicate that ATF4 acts as a positive regulator in TLR4-triggered cytokine production.

解毒活血方灌肠对溃疡性结肠炎小鼠TLR4相关通路蛋白及炎症因子表达的影响

目的探讨解毒活血方灌肠治疗对溃疡性结肠炎(UC)小鼠Toll样受体4(TLR4)、β干扰素TIR结构域衔接蛋白(TRIF)、干扰素调节因子3(IRF3)通路蛋白及相关炎症因子表达的影响。方法雄性C57BL/6小鼠80只,根据随机数字表法分为中药组、西药组、模型组和空白组,每组20只。中药组、西药组、模型组小鼠饮用3%葡聚糖硫酸钠(DSS)溶液7天,空白组小鼠正常饮水。造模成功后中药组以12.24 g/kg中药蒸馏水混悬液灌肠,西药组给予1.33 g/kg美沙拉秦灌肠剂灌肠,模型组、空白组给予等量生理盐水灌肠,每天1次,连续给药7天。记录小鼠体重、粪便性状及便血情况,计算疾病活动指数(DAI),检测血清白细胞介素18(IL-18)、白细胞介素10(IL-10)含量,采用Western Blot检测结肠组织TLR4、TRIF、IRF3通路蛋白表达及实时荧光定量PCR法检测mRNA水平。结果与空白组比较,模型组DAI升高(P<0.01),血清IL-10降低(P<0.01),IL-18升高(P<0.01),结肠组织TLR4、TRIF、IRF3蛋白及mRNA表达升高(P<0.01);与模型组比较,西药组、中药组结肠病理改善,DAI降低(P<0.01),血清IL-10升高(P<0.01),中药组IL-18降低(P<0.01),西药组TLR4蛋白、中药组TLR4、TRIF、IRF3蛋白表达降低(P<0.01),西药组、中药组TLR4、TRIF、IRF3mRNA表达降低(P<0.01);与西药组比较,中药组结肠病理改善,DAI降低(P<0.01),血清IL-10升高(P<0.01),IL-18降低(P<0.05),中药组TLR4、IRF3蛋白表达降低(P<0.05),TLR4、IRF3mRNA表达降低(P<0.05)。结论解毒活血灌肠方可下调TLR4、TRIF、IRF3信号通路,抑制促炎因子释放,增加抗炎因子水平,从而减轻肠道炎症反应,促进结肠黏膜修复。

Whey protein peptide PEW attenuates hyperuricemia and associated renal inflammation in potassium oxonate and hypoxanthine-induced rat

Pro-Glu-Trp(PEW),a whey protein-derived peptide,has been previously shown in vitro to have antihyperuricemic potential.The current study further evaluated the roles and the underlying mechanism of PEW in the management of hyperuricemia(HUA)in rat induced by potassium oxonate(PO)and hypoxanthine.Results revealed that PEW significantly reduced the levels of uric acid(UA),creatinine(Cr),and blood urea nitrogen(BUN)in serum,and effectively suppressed the activities of xanthine oxidase(XOD)associated with UA synthesis and modulated the expression of organic ion transporters related to UA excretion.Moreover,PEW alleviated UAinduced renal inflammation by regulating oxidative stress,suppressing the level of pro-inflammatory cytokines,and inhibiting the activation of NOD-like receptor family pyrin domain-containing 3(NLRP3)inflammasome and Toll-like receptor 4/myeloid differentiation factor 88/NF-kappaB(TLR4/MyD88/NF-κB)signaling pathway.Taken together,these relults indicated that PEW improved HUA and renal inflammation by inhibiting UA synthesis,promoting renal UA excretion and suppressing NLRP3 inflammasome and TLR4/MyD88/NF-κB signaling pathways.