Morroniside ameliorates lipopolysaccharide-induced inflammatory damage in iris pigment epithelial cells through inhibition of TLR4/JAK2/STAT3 pathway

AIM:To investigate the effect of morroniside(Mor)on lipopolysaccharide(LPS)-treated iris pigment epithelial cells(IPE).METHODS:IPE cells were induced by LPS and treated with Mor.Cell proliferation was detected by cell counting kit(CCK)-8,apoptosis was detected by flow cytometry,the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,and IL-8 were measured by enzyme-linked immunosorbent assay(ELISA)kits,and the protein expression of TLR4,JAK2,p-JAK2,STAT3,and p-STAT3 was analyzed by Western blotting.In addition,overexpression of TLR4 and Mor treatment of LPS-stimulated IPE cells were also tested for the above indices.RESULTS:Mor effectively promoted the proliferation and inhibited the apoptosis of LPS-treated IPE cells.In addition,Mor significantly reduced the levels of TNF-α,IL-6,and IL-8 and significantly inhibited the expression of TLR4,p-JAK2,and p-STAT3 in LPS-treated IPE cells.The effect of Mor on LPS-treated IPE cells was markedly attenuated after overexpression of TLR4.CONCLUSION:These findings suggest that Mor may ameliorate LPS-induced inflammatory damage and apoptosis in IPE through inhibition of TLR4/JAK2/STAT3 pathway.

佛司可林通过TLR4/STAT3通路对新生大鼠缺血缺氧性脑损伤氧自由基水平和炎症反应的调节作用

目的研究佛司可林(Forskolin,FS)通过TLR4/STAT3通路对新生大鼠缺血缺氧性脑损伤氧(HIBD)自由基水平和炎症反应的调节作用。方法将75只7日龄SD大鼠15只/组随机分为假手术组、模型组、佛司可林低、中、高剂量治疗组,其中模型组和模型加药组采用Rice-Vannucci方法建立HIBD模型,假手术组只做创伤不作处理。术后第1天模型加药组大鼠分别接受不同剂量剂量的佛司可林腹腔注射;模型组与假手术组腹腔注射等体积量的生理盐水,1次/d注射7 d。ELISA检测各组大鼠血清中炎症因子水平;HE染色和凋亡TUNEL染色进行脑组织病理学分析;商业试剂盒检测丙二醛(MDA)、超氧化物歧化酶(SOD)和乳酸脱氢酶(LDH)水平;免疫组化分析细胞间黏附分子-1(ICAM-1)的表达;Western blot分析脑组织中STAT3、TLR4、NF-κB p65、TNF-α、cleaved-caspase-3/9的表达。通过尾静脉注射0.4 mg/kg脂多糖(LPS)激活TLR4通路后,再注射佛司可林分析对HIBD大鼠炎症因子、氧化应激指标及TLR4/STAT3通路蛋白的表达影响。结果与模型组相比,佛司可林治疗后,大鼠的脑组织损伤显著改善,凋亡细胞比例明显降低(P<0.05);同时脑组织液中MDA和LDH的水平明显降低,SOD含量显著上升,ICAM-1的表达和炎症因子IL-6、IL-8、IL-1β的释放明显受到抑制(P<0.05)。Western blot结果显示,随着佛司可林剂量增加,p-STAT3、TLR4、p-p65和TNF-α表达量显著降低(P<0.05)。此外佛司可林能够抑制LSP激活TLR4通路后p-STAT3、TLR4、pp65和TNF-α的表达,降低脑组织中IL-6的释放,提升SOD的水平含量(P<0.05)。结论佛司可林能够通过TLR4/STAT3通路改善新生大鼠缺血缺氧性脑损伤,降低氧自由基水平和抑制炎症反应。

沙眼衣原体持续感染对Hela细胞TLR4/IL-6/STAT3信号通路的影响

目的:分析沙眼衣原体(Chlamydia trachomatis,Ct)持续感染对靶细胞TLR4/IL-6/STAT3信号通路的影响。方法:利用Hela细胞分别建立Ct急性感染及持续性感染模型,通过qRT-PCR、ELISA等方法比较Ct感染过程中靶细胞TLR4、STAT3、IL-6转录水平及细胞因子IL-6分泌量的变化。结果:Ct感染后靶细胞TLR4、IL-6、STAT3转录水平及细胞因子IL-6分泌量均呈现时间相关性上调,且持续性感染状态下比急性感染状态下的上调更为显著;IL-6/STAT3的表达量与TLR4转录水平正相关。结论:Ct持续感染过程中TLR4的持续活化可大幅上调IL-6/STAT3信号通路表达,可能参与了Ct持续感染后慢性炎性损伤过程。

MyD88、TLR4和STAT3在肝细胞癌组织中的表达及意义

目的探讨TLR4、MyD88、STAT3在肝细胞癌组织中的表达及生物学意义。方法采用免疫组化方法检测TLR4、MyD88、STAT3在82例肝癌组织及其对应的癌旁肝组织中的表达水平。结合肝癌临床病理指标分析相关性。结果 TLR4、MyD88、STAT3蛋白在癌组织中的阳性表达率为76.8%(63/82)、67.1%(55/82)、69.5%(57/82),高于在癌旁肝组织中的阳性表达率25.9%(12/82)、12%(9/82)、8.5%(7/82),差异均具有统计学意义(P<0.05);在肝癌组织中TLR4、MyD88、STAT3的阳性表达呈正相关[TLR4和MyD88(r=0.612,P=0.011),MyD88和STAT3(r=0.578,P=0.002),TLR4和STAT3(r=0.542,P=0.016)]。MyD88和STAT3表达与性别、分化程度、有无HBV感染和肝硬化有关(P<0.05),而与肿瘤大小、有无静脉浸润无关(P>0.05)。结论 TLR4、MyD88、STAT3表达上调,导致肝癌细胞增殖和免疫逃逸是肝癌发生发展的重要分子机制。

Elevated retinol binding protein 4 levels are associated with atherosclerosis in diabetic rats via JAK2/STAT3 signaling pathway

BACKGROUND Atherosclerosis is a major cause of mortality worldwide and is driven by multiple risk factors,including diabetes,which results in an increased atherosclerotic burden,but the precise mechanisms for the occurrence and development of diabetic atheroscerosis have not been fully elucidated.AIM To summarize the potential role of retinol binding protein 4(RBP4) in the pathogenesis of diabetic atheroscerosis,particularly in relation to the RBP4-Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway.METHODS Male Wistar rats were randomly divided into three groups,including a control group(NC group),diabetic rat group(DM group),and diabetic atherosclerotic rat group(DA group).The contents of total cholesterol(TC), high-density lipoprotein cholesterol(HDL-c), triglycerides(TG), low-density lipoprotein cholesterol(LDLc), fasting insulin(FINS),fasting plasma glucose,and hemoglobin A1 c(HbA1 c)were measured.Moreover,the adipose and serum levels of RBP4,along with the expression levels of JAK2, phosphorylated JAK2(p-JAK2), STAT3,phosphorylated STAT3(p-STAT3), B-cell lymphoma-2(Bcl-2), and Cyclin D1 in aortic tissues were also measured.Besides,homeostasis model assessment of insulin resistance(HOMA-IR) and atherogenic indexes(AI) were calculated.RESULTS Compared with the NC and DM groups,the levels LDL-c,TG,TC,FINS,HOMAIR,RBP4,and AI were upregulated,whereas that of HDL-c was downregulated in the DA group(P <0.05);the mRNA levels of JAK2,STAT3,Cyclin D1,and Bcl-2 in the DA group were significantly increased compared with the NC group and the DM group;P-JAK2,p-JAK2/JAK2 ratio,p-STAT3,p-STAT3/STAT3 ratio,Cyclin D1,and Bcl-2 at protein levels were significantly upregulated in the DA group compared with the NC group and DM group.In addition,as shown by Pearson analysis,serum RBP4 had a positive correlation with TG,TC,LDL-c,FINS,HbA1 C,p-JAK2,p-STAT3,Bcl-2,Cyclin D1,AI,and HOMA-IR but a negative correlation with HDL-c.In addition,multivariable logistic regression analysis showed that serum RBP4,p-JAK2,p-STAT3,and LDL-c were predictors of the presence of diabetic atherosclerosis.CONCLUSION RBP4 could be involved in the initiation or progression of diabetic atherosclerosis by regulating the JAK2/STAT3 signaling pathway.

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.

Branched-chain fatty acids from goat milk alleviate ulcerative colitis via the TLR4/NF-κB/NLRP3 pathway

Branched-chain fatty acids(BCFAs)are new bioactive fatty acids with anti-inflammatory properties.However,the role of BCFAs in alleviating ulcerative colitis has not been clarified.Herein,we evaluated the protective effect of BCFAs from goat milk in mice with colitis induced using dextran sodium sulfate(DSS)and explored the corresponding mechanism.These results show that BCFAs extracted from goat milk can significantly alleviate weight loss in mice,and reduce the disease activity index and the activity of myeloperoxidase while increasing the content of antioxidant enzymes in colon tissue and reducing the oxidation stress response.These data also show that BCFAs can down-regulate the gene and protein expression of the toll-like receptor 4(TLR4)/nuclear factorκB p65(NF-κB p65)/NOD-like receptor thermal protein domain associated protein 3(NLRP3)signaling pathway,and at the same time significantly reduce the expression of pro-inflammatory factors tumor necrosis factorα(TNF-α),interleukin 1β(IL-1β),and IL-18 in colon tissue,and significantly increase the expression of the anti-inflammatory factor IL-10.In conclusion,these results demonstrated that BCFAs in goat milk exerted effects on colitis-related inflammatory cytokines and inhibited inflammation by inducing the TLR4/NF-κB/NLRP3 pathway to alleviate DSS-induced ulcerative colitis.This study provides evidence for the potential of BCFAs as bioactive fatty acids in food products and to ameliorate ulcerative colitis development in mice.

白桦酯酸介导STAT3信号通路调控肝纤维化进程

目的探讨白桦酯酸(betulinic acid,BA)介导STAT3信号通路改善肝纤维化的进程。方法Raw264.7细胞给予LPS(1 mg·L-1)孵育12 h,收集上清液。BA(25μmol·L-1)预处理HSCs 2 h后,给予上述上清液孵育0-6 h;不同浓度BA(0-25μmol·L-1)预处理HSCs 2 h后,给予上述上清液孵育1 h,分析BA对细胞外基质、炎性因子以及相关信号通路的影响。BA(50 mg·kg-1)预处理C57BL/6♂小鼠4 d后,腹腔注射10%CCl 4,24 h给予小鼠安乐死,收集小鼠血清和肝脏。结果Raw264.7细胞在LPS刺激后,测得细胞上清液IL-6含量因时间延长而增加,12 h后达最高值。取LPS刺激Raw264.7细胞12 h上清液活化HSCs,BA可明显降低活化HSCs中α-SMA、collagen-Ⅰ蛋白表达,降低TIMP-1及MMP-13比值,呈现时间和剂量依赖关系。BA可明显增加活化HSCs中STAT3磷酸化蛋白表达,明显降低TLR4和CD14蛋白表达,调控AMPK-LKB1磷酸化,呈现时间和剂量依赖性。结论BA可能介导炎性上清液活化HSCs中STAT3磷酸化蛋白表达,调控TLR4及AMPK/LKB1磷酸化,进而逆转肝纤维化。

TLR4促进苯肾上腺素诱导的心肌细胞肥大

目的:探究Toll样受体4(TLR4)基因对苯肾上腺素(PE)诱导的心肌细胞肥大的影响及其作用机制。方法:选择GEO数据库中人及小鼠心肌肥厚组织的测序数据,借助R软件进行分析,筛选出目的基因TLR4;使用PE处理原代心肌细胞,检测细胞中TLR4蛋白和mRNA水平;使用siRNA转染或者腺病毒感染的方式敲低或者过表达原代心肌细胞中TLR4蛋白和mRNA水平,检测其对PE诱导的心肌细胞肥大的影响;Western blot检测敲低或者过表达TLR4对IL-6/Stat3信号通路的影响。结果:共有32个基因在人和小鼠心肌肥厚组织中表达水平发生改变;PE处理原代心肌细胞后,TLR4蛋白和mRNA水平均显著升高;敲低TLR4能抑制PE诱导的心肌细胞肥大,而上调TLR4能促进PE诱导的心肌细胞肥大;Western blot结果表明,在PE处理下,敲低TLR4能引起心肌细胞中IL-6和p-Stat3表达水平降低,过表达TLR4能导致心肌细胞中IL-6和p-Stat3表达水平显著升高。结论:TLR4在心肌肥厚过程中高表达,敲低TLR4可能通过抑制IL-6/Stat3信号通路活性抑制PE诱导的心肌细胞肥大。

Elaidic acid-induced intestinal barrier damage led to gut-liver axis derangement and triggered NLRP3 inflammasome in the liver of SD rats

Previous studies have shown that trans fatty acids(TFA) are associated with several chronic diseases,the gut microbiota is directly influenced by dietary components and linked to chronic diseases.Our research investigated the effects of elaidic acid(EA),a typical TFA,on the gut microbiota to understand the underlying mechanisms of TFA-related chronic diseases.16S rDNA gene sequencing on faecal samples from Sprague-Dawley rats were performed to explore the composition change of the gut microbiota by EA gavage for 4 weeks.The results showed that the intake of EA increased the abundance of well-documented harmful bacteria,such as Proteobacteria,Anaerotruncus,Oscillibacter and Desulfovibrionaceae.Plus,EA induced translocation of lipopolysaccharides(LPS) and the above pathogenic bacteria,disrupted the intestinal barrier,led to gut-liver axis derangement and TLR4 pathway activation in the liver.Overall,EA induced intestinal barrier damage and regulated TLR4-MyD88-NF-κB/MAPK pathways in the liver of SD rats,leading to the activation of NLRP3 inflammasome and inflammatory liver damage.

The potential mechanism of Isodon suzhouensis against COVID-19 via EGFR/TLR4 pathways

Corona Virus Disease 2019(COVID-19)has brought the new challenges to scientific research.Isodon suzhouensis has good anti-inflammatory and antioxidant stress effects,which is considered as a potential treatment for COVID-19.The possibility for the treatment of COVID-19 with I.suzhouensis and its potential mechanism of action were explored by employing molecular docking and network pharmacology.Network pharmacology and molecular docking were used to screen drug targets,and lipopolysaccharide(LPS)induced RAW264.7 and NR8383 cells inflammation model was used for experimental verification.Collectively a total of 209 possible linkages against 18 chemical components from I.suzhouensis and 1194 COVID-19 related targets were selected.Among these,164 common targets were obtained from the intersection of I.suzhouensis and COVID-19.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enriched 582 function targets and 87 target proteins pathways,respectively.The results from molecular docking studies revealed that rutin,vitexin,isoquercitrin and quercetin had significant binding ability with 3 chymotrypsin like protease(3CLpro)and angiotensin converting enzyme 2(ACE2).In vitro studies showed that I.suzhouensis extract(ISE)may inhibit the activation of PI3K/Akt pathway and the expression level of downstream proinflammatory factors by inhibiting the activation of epidermal growth factor receptor(EGFR)in RAW264.7 cells induced by LPS.In addition,ISE was able to inhibit the activation of TLR4/NF-κB signaling pathway in NR8383 cells exposed to LPS.Overall,the network pharmacology and in vitro studies conclude that active components from I.suzhouensis have strong therapeutic potential against COVID-19 through multi-target,multi-pathway dimensions and can be a promising candidate against COVID-19.

丹参多酚酸盐对动脉粥样硬化大鼠TLR4/IL-6/STAT3通路的影响

目的:探讨丹参多酚酸盐对动脉粥样硬化(AS)大鼠Toll样受体4(TLR4)/白介素-6(IL-6)/信号传导及转录激活蛋白3(STAT3)通路的影响。方法:采用高脂饲料喂养结合腹腔注射小剂量脂多糖(LPS)的方法制备AS大鼠模型。取造模成功的大鼠随机分为模型组及丹参多酚酸盐低、中、高剂量组,每组10只;另设正常大鼠10只作为对照组。丹参多酚酸盐各剂量组大鼠分别腹腔注射10、20、40 mg/kg丹参多酚酸盐,连续8周;对照组和模型组大鼠给予等体积0.9%NaCl溶液。末次给药后,分离胸主动脉。HE染色后光镜下观察胸主动脉组织形态学改变;ELISA测定IL-6和肿瘤坏死因子-α(TNF-α)含量;PCR检测TLR4 mRNA表达;Western blot检测STAT3蛋白及其磷酸化表达水平。结果:与对照组相比,模型组大鼠胸主动脉内皮细胞肿胀,排列紊乱,IL-6和TNF-α含量显著升高(P<0.01),TLR4 mRNA表达和p-STAT3/STAT3蛋白表达比值均显著上调(P<0.01)。与模型组相比,丹参多酚酸盐各剂量组大鼠胸主动脉病变均明显减轻,以高剂量组的改善作用最为显著。与模型组相比,丹参多酚酸盐中、高剂量组大鼠胸主动脉组织IL-6和TNF-α含量显著降低(P<0.05,P<0.01),p-STAT3/STAT3蛋白表达比值明显下调(P<0.01);各剂量组TLR4 mRNA表达水平均显著下调(P<0.01)。丹参多酚酸盐高剂量组TNF-α含量、TLR4 mRNA表达水平及p-STAT3/STAT3蛋白表达比值较低、中剂量组明显降低(P<0.05,P<0.01),高剂量组IL-6含量较低剂量组亦明显降低(P<0.05)。结论:丹参多酚酸盐可明显减轻AS大鼠胸主动脉炎症反应,其作用机制可能与抑制TLR4/IL-6/STAT3通路有关。

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.

野蔷薇根总黄酮对类风湿关节炎模型大鼠血管损伤的改善作用及机制

目的研究野蔷薇根总黄酮对类风湿关节炎(RA)模型大鼠血管损伤的改善作用及其潜在机制。方法将雌性Wistar大鼠随机分为正常对照组、模型组、阿司匹林组(阳性对照,30mg/kg)和野蔷薇根总黄酮低、高剂量组(4.15、8.30g/kg,以生药量计),每组10只。除正常对照组外,其余各组大鼠采用胶原诱导+高脂饮食喂养建立RA模型。造模14 d后,各组大鼠灌胃相应药液/0.5%羧甲基纤维素钠溶液,每天1次,连续36d。定期评估大鼠的全身评分、关节炎指数和足爪肿胀数;末次给药后,检测大鼠血清中白细胞介素6(IL-6)、细胞间黏附分子1(ICAM-1)和血管细胞黏附分子1(VCAM-1)水平,观察大鼠胸主动脉血管组织病理学形态变化,检测胸主动脉血管组织中Toll样受体4(TLR4)蛋白表达水平和Janus激酶2(JAK2)、信号转导及转录激活因子3(STAT3)蛋白磷酸化水平。结果与正常对照组比较,模型组大鼠血清中IL-6、ICAM-1、VCAM-1水平和胸主动脉血管组织中TLR4蛋白表达水平及JAK2、STAT3蛋白磷酸化水平均显著升高(P<0.01),胸主动脉血管组织可见动脉粥样斑块(粥瘤)、胆固醇结晶、淋巴细胞浸润及少量未破裂的泡沫细胞聚集。与模型组比较,在第28天时,野蔷薇根总黄酮各剂量组大鼠的全身评分(低剂量组除外)、关节炎指数和足爪肿胀数均显著降低(P<0.05或P<0.01);在第49天时,野蔷薇根总黄酮低剂量组大鼠的全身评分、关节炎指数和足爪肿胀数均显著降低(P<0.05或P<0.01);野蔷薇根总黄酮各剂量组大鼠血清和血管组织中其余定量指标均显著逆转(P<0.05或P<0.01),血管组织病理损伤明显减轻。结论野蔷薇根总黄酮可改善RA模型大鼠的血管损伤,其作用可能与降低血管组织中TLR4蛋白表达,抑制IL-6/JAK2/STAT3信号通路活性有关。

肝细胞癌中MyD88和STAT3的表达及其意义

目的探讨髓样分化因子88(MyD88)和转录激活因子3(STAT3)在肝细胞癌(HCC)组织中的表达及其生物学意义。方法采用免疫组化方法检测MyD88和STAT3在82例肝癌组织及其对应的癌旁肝组织中的表达水平。结合肝癌临床病理指标分析它们的相关性。结果 MyD88和STAT3蛋白在癌组织中的阳性表达率分别为67.1%(55/82)和69.5%(57/82),高于癌旁肝组织中的11.0%(9/82)和8.5%(7/82),差异均具有统计学意义(P<0.05)。在肝癌组织中MyD88与STAT3的阳性表达呈正相关(r=0.578,P=0.002)。MyD88和STAT3表达与HCC患者的性别、癌的分化程度、有无HBV感染和肝硬化有关;上述4指标分组间差异具有显著性(P<0.05)。而与HCC肿瘤大小及有无静脉浸润无关;该2指标分组间差异无显著性(P>0.05)。结论 MyD88和STAT3表达上调,导致肝癌细胞增殖和免疫逃逸是肝癌发生发展的重要分子机制。

熊果苷调控TLR4的表达影响多发性骨髓瘤细胞RPMI-8226增殖、细胞周期和凋亡的实验研究

该次实验的目的是研究熊果苷是否通过调控Toll样受体4(TLR4)/JAK2/信号转导与转录因子3(STAT3)信号通路的激活从而影响多发性骨髓瘤细胞RPMI-8226增殖、细胞周期和凋亡。将RPMI-8226细胞分为对照NC组,低、中、高剂量组(50、100和200μg/mL熊果苷),si-NC组(转染si-NC),si-TLR4组(转染si-TLR4),pcDNA-NC+高剂量组(转染pc DNA-NC和200μg/mL熊果苷),pcDNA-TLR4+高剂量组(转染pc DNA-TLR4和200μg/mL熊果苷)。采用噻唑蓝溴化四唑(MTT)法检测细胞增殖。流式细胞仪检测细胞周期和细胞凋亡变化情况。Western blot检测细胞周期蛋白D1(Cyclin D1)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved-caspase-3)、TLR4、p-JAK2、p-STAT3蛋白的表达情况。q RT-PCR检测TLR4 mRNA表达水平。与NC组比较,50、100和200μg/mL熊果苷逐渐降低RPMI-8226细胞的Cyclin D1蛋白表达水平、增殖活性、S期细胞比例、TLR4 mRNA和TLR4蛋白表达水平,并逐渐提高G0/G1期细胞比例、Cleaved-caspase-3蛋白表达水平和凋亡率(均P<0.05),且熊果苷的作用效果随剂量的增加呈现增强趋势;并且200μg/mL熊果苷降低RPMI-8226细胞的p-JAK2、p-STAT3蛋白表达水平(P<0.05)。si-TLR4组RPMI-8226细胞的Cyclin D1蛋白表达水平、增殖活性、S期细胞比例比si-NC组低,G0/G1期细胞比例、Cleaved-caspase-3蛋白表达水平和凋亡率比si-NC组高(P<0.05)。pc DNATLR4可以逆转熊果苷对多发性骨髓瘤细胞RPMI-8226增殖、细胞周期、凋亡和p-JAK2、p-STAT3蛋白表达的影响。综上,熊果苷通过下调TLR4/JAK2/STAT3信号通路水平,抑制多发性骨髓瘤细胞RPMI-8226增殖、阻滞细胞周期和促进细胞凋亡。

基于反向对接法的黄连碱抗炎机制分子模拟研究

目的:基于分子反向对接技术,以黄连碱为研究对象,对TLR4/NF-κB、p38、JAK2/STAT3 3条通路上的蛋白进行反向筛选,以筛选出黄连碱的炎症靶标蛋白。方法:获取Toll样受体4/核因子(TLR4/NF-κB)、p38促分裂原活化蛋白激酶(P38 MAPK)和Janus激酶-信号转导转录激活因子(JAK2/STAT3)3条炎症通路上的30个蛋白的晶体学结构以及黄连碱的化学结构;利用AutoDockTools对所有蛋白的晶体学结构进行标准化处理;Auto Grid对蛋白的活性位点进行格子计算;利用AutoDock对黄连碱进行反向对接模拟实验;根据对接结果自由能高低进行排序,筛选出亲和力高的靶蛋白;对筛选得到的靶蛋白进行作用力分析并作图。结果:反向筛选得到4个与黄连碱具有高亲和性的靶蛋白,4个靶蛋白分别为PI3Kδ、PI3Kγ、IKKβ、PI3Kα。结论:黄连碱对PI3Kδ、PI3Kγ、IKKβ、PI3Kα靶蛋白具有竞争抑制的活性,提示黄连碱可以通过抑制该4个靶蛋白进而阻碍炎症信号传递,影响下游蛋白的表达,发挥抗炎作用。