Quercetin regulates depression-like behavior in CUMS rat models via TLR4/NF-κB signaling

Background:Depression is becoming increasingly prevalent around the world,imposing a substantial burden on individuals,families,as well as society.Quercetin is known to be highly effective in treating depression.However,additional research is needed to dissect the mechanisms of its anti-depressive effects.Methods:For this study,Sprague-Dawley(SD)rats were randomized into the control,model,quercetin,or fluoxetine group.The latter three groups were exposed to chronic unpredictable mild stress(CUMS)for 42 d.The first two groups received saline solution daily via oral gavage.Meanwhile,the quercetin group was orally administered a quercetin suspension(52.08 mg/kg)every day,while the fluoxetine group was orally administered a fluoxetine solution(2.08 mg/kg).Here,fluoxetine served as the positive control drug to compare the therapeutic effects of quercetin.The experimental period was 6 weeks.Depressive behaviors in rats were assessed through various physiological and behavioral measures.Additionally,pathological changes in hippocampal tissues were examined using Nissl staining.Serum cytokines were detected using an enzymelinked immunosorbent assay(ELISA),and immunohistochemistry was employed to quantify the levels and integral optical density(IOD)values of ionized calcium binding adaptor molecule-1(Iba-1)expression in the brain.Real-time fluorescence quantitative PCR(RT-qPCR)was utilized to evaluate the mRNA levels of inflammatory indicators as well as toll-like receptor 4(TLR4),and nuclear factor-κappa B P65(NF-κB P65)in hippocampus.Western blot(WB)technique was employed to observe the protein levels of TLR4,NF-κB P65,and phospho-NF-κB P65(p-NF-κB P65).Results:After 42 d of exposure to CUMS,rats exhibited a slow increase in body weight,a reduction in food intake,an abnormal preference for sugar water,and aberrant open-field behaviors.Pathological analysis revealed the disintegration,rupture,interruption,and disorganization of hippocampal neuronal cells after CUMS exposure,along with a decrease in Nissl bodies in the CA1 region.This was accompanied by the elevated expression of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and interleukin-6(IL-6)in the serum and the upregulation of IL-1β,IL-6,and TNF-αmRNA expression in the hippocampus.Increases in Iba-1-positive cells and the IOD values of Iba-1 were detected in hippocampal microglia.Furthermore,TLR4 and NF-κB P65 mRNA and protein levels were upregulated in hippocampal tissues.Quercetin,an antidepressant,could alleviate depression-like symptoms in rats and downregulate inflammatory factors associated with the TLR4/NF-κB signaling pathway in hippocampal microglia,and its therapeutic effect was comparable to fluoxetine.Conclusion:In rat models of CUMS,quercetin may act as an antidepressant by inhibiting inflammation in hippocampal microglia via TLR4/NF-κB signaling pathway.These results offer experimental and theoretical support for applying quercetin in the clinical management of depression.

Kuicolong-yu enema decoction retains traditional Chinese medicine enema attenuates inflammatory response ulcerative colitis through TLR4/NF-κB signaling pathway

BACKGROUND Ulcer colitis(UC)is a chronic,nonspecific,and noninfectious inflammatory bowel disease.Recently,Toll-like receptors(TLRs)have been found to be closely associated with clinical inflammatory diseases.Achieving complete remission in patients with intermittent periods of activity followed by dormancy is challenging.Moreover,no study has explored the mechanism by which Kuicolong-yu enema decoction retains traditional Chinese medicine enemas to attenuate the inflammatory response in UC.AIM To explore the mechanism by which Kuicolong-yu enema decoction retains traditional Chinese medicine enemas to attenuate the inflammatory response in UC.METHODS This prospective clinical study included patients who met the exclusion criteria in 2020 and 2021.The patients with UC were divided into two groups(control and experimental).The peripheral blood of the experimental and control groups were collected under aseptic conditions.The expression of TLR4 protein,NF-κB,IL-6,and IL-17 was detected in the peripheral blood of patients in the experimental group and control group before and 1 month after taking the drug.Linear co rrelation analysis was used to analyze the relationship between the expression level of TLR4 protein and the expression levels of downstream signal NF-κB and inflammatory factors IL-6 and IL-17,and P<0.05 was considered statistically significant.RESULTS There were no significant differences in the patient characteristics between the control and experimental groups.The results showed that the expression levels of TLR4 and NF-κB in the experimental group were significantly lower than those in the control group(P<0.05).The levels of IL-6 and IL-17 in the experimental group were significantly lower than those in the control group(P<0.05).The TLR4 protein expression in the experimental group was positively correlated with the expression level of downstream signal NF-κB and was positively correlated with the levels of downstream inflammatory cytokines IL-6 and IL-17(r=0.823,P<0.05).CONCLUSION Kuicolong-yu enema decoction retains traditional Chinese medicine enema attenuates the inflammatory response of UC through the TLR4/NF-κB signaling pathway.

β-arrestin 2 attenuates lipopolysaccharide-induced liver injury via inhibition of TLR4/NF-κB signaling pathwaymediated inflammation in mice

AIM To study the role and the possible mechanism of β-arrestin 2 in lipopolysaccharide(LPS)-induced liver injury in vivo and in vitro.METHODS Male β-arrestin 2^(+/+) and β-arrestin 2^(-/-)C57 BL/6 J mice were used for in vivo experiments, and the mouse macrophage cell line RAW264.7 was used for in vitro experiments. The animal model was established via intraperitoneal injection of LPS or physiological sodium chloride solution. Blood samples and liver tissues were collected to analyze liver injury and levels of pro-inflammatory cytokines. Cultured cell extracts were collected to analyze the production of pro-inflammatory cytokines and expression of key molecules involved in the TLR4/NF-κB signaling pathway.RESULTS Compared with wild-type mice, the β-arrestin 2 knockout mice displayed more severe LPS-induced liver injury and significantly higher levels of proinflammatory cytokines, including interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF)-α, and IL-10. Compared with the control group, pro-inflammatory cytokines(including IL-1β, IL-6, TNF-α, and IL-10) produced by RAW264.7 cells in the β-arrestin 2 si RNA group were significantly increased at 6 h after treatment with LPS. Further, key molecules involved in the TLR4/NF-κB signaling pathway, including phosphoIκBα and phosho-p65, were upregulated.CONCLUSION β-arrestin 2 can protect liver tissue from LPS-induced injury via inhibition of TLR4/NF-κB signaling pathwaymediated inflammation.

Study on inflammatory factors and curative effect of Rhizoma Imperata on community acquired pneumonia based on TLR4/NF-κB signaling pathway

Objective:To investigate the curative effect of Rhizoma Imperata in the treatment of patients with community-acquired pneumonia and its influence on inflammatory factors related to TLR4/NF-κB signaling pathway,so as to seek new drugs for the treatment of community-acquired pneumonia.Methods:120 patients with community-acquired pneumonia who were treated in Department of respiratory medicine,the First Affiliated Hospital of Hainan Medical College from November 2019 to September 2020 were collected and randomly divided into control group and experimental group,with 60 patients in each group.The control group was given cefuroxime sodium injection,and the experimental group was given cefuroxime sodium injection+Baimao root decoction.The levels of high-sensitivity C-reactive protein(CRP)and the level of serum CRP in the two groups before and after treatment were compared Objective To observe the expression of procalcitonin(PCT),IL-6,IL-8,TNF-αand TLR4/NF-κB mRNA levels,and to observe the effect of Radix Imperata Rubra on community-acquired pneumonia.Results:The fever,cough,expectoration subsided time and chest CT inflammation absorption time of the experimental group were shorter than those of the control group,the difference was statistically significant(P<0.05);the total effective rate of the experimental group(96.67%)was higher than that of the control group(85%),the difference was statistically significant(P<0.05);after treatment,the serum CRP,PCT,IL-6,IL-8,TNF-αin the two groups were lower than before,and the experimental group was significantly lower than the control group,the difference was statistically significant(P<0.05),There was statistical significance(P<0.05);after treatment,TLR4/NF-κB mRNA of the two groups were decreased,and the experimental group was significantly lower than the control group,the difference was statistically significant(P<0.05).Conclusion:In the treatment of cap,Rhizoma Imperata can reduce the levels of CRP,PCT,IL-6,IL-8 and TNF-α.The mechanism is related to inhibiting the activation of TLR4/NF-κB signaling pathway,so as to reduce the release of inflammatory factors and improve clinical symptoms.

Effects of Polysaccharides from Dicliptera chinensis(L.)Nees.on TLR4/NF-κB Signaling Pathway in Cell Model of Non-alcoholic Fatty Liver

[Objectives]To observe the effects of polysaccharides from Dicliptera chinensis(L.)Nees.on the expression of TLR/NF-κB pathway related proteins in HepG2 cells induced by oleic acid,and to explore the possible mechanism of polysaccharides from D.chinensis(L.)Nees.in the treatment of non-alcoholic fatty liver disease(NAFLD).[Methods]HepG2 cells were induced with oleic acid to establish a non-alcoholic fatty liver cell model.After intervention with 0.25 and 0.5 mg/mL of D.chinensis(L.)Nees.polysaccharides,the ALT and AST activity and TG and TC contents were detected with kits,and the changes in the expression of CDK5,TLR4,p-NF-κB and NF-κB were analyzed using Western-blotting.[Results]In the HepG2 cells induced with oleic acid,the ALT and AST activity increased significantly,the TG and TC contents increased significantly,and the expression levels of CDK5,TLR4 and p-NF-κB proteins up-regulated significantly.In the HepG2 cells intervened with D.chinensis(L.)Nees.polysaccharides,the activity of ALT and AST,the contents of TG and TC,and the expression levels of CDK5,TLR4 and p-NF-κB proteins all reduced significantly.[Conclusions]Polysaccharides from D.chinensis(L.)Nees.may interfere with NAFLD by inhibiting the TLR4/NF-κB pathway.

基于“微生物-肠-脑轴”探讨补肾通腑方对APP/PS1小鼠肠道菌群及LPS/TLR4/NF-κB通路的影响

目的探讨补肾通腑方调控肠道菌群,改善阿尔茨海默病(Alzheimer’s disease,AD)模型小鼠学习记忆能力的作用机制。方法以APP/PS1小鼠为研究对象,给予补肾通腑方治疗8周,采用Morris水迷宫法观察小鼠空间学习记忆能力变化;16S rDNA检测小鼠肠道菌群丰度、多样性变化;HE染色观察海马病理形态学变化;免疫荧光检测海马区小胶质细胞活化情况;Western blot检测TLR4、NF-κB、IL-6等炎症因子表达。结果与模型组相比,补肾通腑方可以缩短AD模型小鼠逃避潜伏期、游泳路径,增加跨越平台次数(P<0.05),提升肠道菌群多样性,调节肠道菌群丰度,促进海马神经元细胞损伤修复,降低iNOS/Iba1共表达,提高Arg1/Iba1共表达(P<0.01),促进小胶质细胞从M1型向M2型转化,下调TLR4、NF-κB、IL-6等促炎因子的表达。结论补肾通腑方改善AD模型小鼠学习记忆能力的作用机制可能与调节肠道菌群介导的LPS/TLR4/NF-κB通路,从而抑制促炎型小胶质细胞活化、减轻中枢神经系统炎症、改善海马区神经元细胞损伤有关。

基于TLR4 NF-κB通路-神经相关因子探究依达拉奉对急性脑梗死患者炎症反应与神经损伤的保护机制

目的基于Toll样受体4(TLR4)核因子-κB(NF-κB)通路-神经相关因子探究依达拉奉对急性脑梗死(ACI)患者炎症反应与神经损伤的保护机制。方法选取2020-07—2023-07保定市第一中心医院收治的110例ACI患者,以随机数字表法分为观察组、对照组,各55例,对照组给予阿替普酶溶栓,观察组给予阿替普酶溶栓联合依达拉奉治疗。比较2组疗效、神经功能[美国国立卫生研究院卒中量表(NIHSS)评分、改良Rankin评分]、TLR4 NF-κB通路指标(TLR4、NF-κB)、神经损伤相关因子[神经元特异性烯醇化酶(NSE)、中枢神经特异性蛋白(S-100β)、脑源性神经营养因子(BDNF)]、TLR4 NF-κB通路相关炎症因子[白介素-1β(IL-1β)、超敏C反应蛋白(hs-CRP)、肿瘤坏死因子(TNF-α)、五聚素3(PTX3)、脂蛋白相关磷脂酶A2(Lp-PLA2)]。结果观察组总有效率96.36%,高于对照组的83.64%(P<0.05)。治疗1、2周观察组NIHSS评分、改良Rankin评分均低于对照组(P<0.05),观察组TLR4、NF-κB均低于对照组(P<0.05)。相较于治疗前,2组治疗1、2周后S-100β、NSE水平明显下降,BDNF水平明显升高,观察组S-100β、NSE水平均低于对照组,BDNF水平高于对照组(P<0.05)。相较于治疗前,2组治疗1、2周后IL-1β、hs-CRP、TNF-α、PTX3、Lp-PLA2水平均明显下降,观察组IL-1β、hs-CRP、TNF-α、PTX3、Lp-PLA2水平均低于对照组(P<0.05)。结论依达拉奉对ACI患者的疗效显著,有利于缓解炎症反应,改善神经损伤,其保护机制可能与TLR4 NF-κB通路调控神经损伤、炎症反应相关因子有关。

Liu-Jun-Zi decoction alleviates chemotherapy-induced anorexia by regulating gut microbiota and TLR4/MyD88/NF-κB p65 signaling pathway

Background:Liu-Jun-Zi decoction(LJZD),a classical nourishing formula in China,has been proven to be effective in treating chemotherapy-induced anorexia.In this study,the mechanism of LJZD in alleviating chemotherapy-induced anorexia was discussed from the aspects of regulating gut microbiota,repairing intestinal barrier injury and inhibiting inflammatory pathways.Methods:A rat model of chemotherapy-induced anorexia was established using cisplatin.The study evaluated the therapeutic effects of LJZD by observing the weight,food intake,and intestinal pathology of rats.The impact of LJZD on gut microbiota and metabolites,specifically short-chain fatty acids,was investigated through gut microbiota analysis and targeted metabolomics.The anti-inflammatory and intestinal protective effects of LJZD were assessed by examining the expression of intestinal tight junction proteins associated with the inflammatory pathway.Results:LJZD alleviated cisplatin-induced inflammation and intestinal barrier disruption,as evidenced by upregulated expression of tight junction protein 1(TJ-1)and occludin,along with reduced serum levels of interleukin 6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and lipopolysaccharide.Additionally,LJZD alleviated microbiota imbalance and regulated the levels of short-chain fatty acids,especially increased the relative abundance of Coriobacteriales Incertae Sedis,Lactabacillus johnsonii F19785,Parasutterella,and reduced the Tyzzerella.In the hypothalamus,LJZD exerts suppressive effects on the toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor-κB(NF-κB)p65 signaling pathway,leading to a downregulation in the transcriptional activity of IL-6 and IL-1β,as well as Interleukin 6 receptors(IL-6R)and Interleukin-1βreceptors(IL-1R1)mRNA expression levels.Conclusion:In summary,LJZD alleviate chemotherapy-induced anorexia by modulating the gut microbiota,repairing the intestinal mechanical barriers,and suppressing the TLR4/MyD88/NF-κB p65 signaling pathway.

基于TLR4/MyD88/NF-κB通路探讨加味过敏煎对抗生素加重过敏性哮喘小鼠的抗炎机制

目的:探讨加味过敏煎对抗生素加重过敏性哮喘小鼠的干预作用及潜在作用机制。方法:将40只雄性BALB/c小鼠随机分为空白组、模型组(抗生素加重过敏性哮喘组)、中药组(加味过敏煎组,6.3 g·kg^(-1))、西药组(匹多莫徳联合氨茶碱组,给予匹多莫徳0.0378 g·kg^(-1)+氨茶碱0.2 g·kg^(-1)),每组10只。采用卵清蛋白(OVA)和氢氧化铝凝胶佐剂结合OVA雾化激发的方法建立过敏性哮喘小鼠模型,头孢哌酮舒巴坦钠滴鼻建立抗生素加重过敏性哮喘小鼠模型。雾化激发前30 min灌胃给药,连续给药7 d后检测小鼠肺组织病理变化;ELISA法检测小鼠血清中IgE、IL-4、IL-5、IL-13表达水平;Western blot法检测小鼠肺组织中TLR4、MyD88、p-NF-κB p65蛋白表达水平。结果:与空白组相比,抗生素加重过敏性哮喘组支气管结构重度异常,部分支气管上皮细胞脱落,其中还有大量蛋白黏液渗出,支气管周围见大量炎性细胞浸润,肺泡壁明显增厚,肺泡萎缩;血清中IgE、IL-4、IL-5、IL-13水平升高(P<0.01);TLR4、MyD88、p-NF-κB p65蛋白表达升高(P<0.01);与抗生素加重过敏性哮喘组相比,加味过敏煎组小鼠支气管结构正常,肺泡壁增厚情况显著改善,炎性细胞浸润减轻;血清中IgE、IL-4、IL-5、IL-13水平显著下降(P<0.01);TLR4、MyD88、p-NF-κB p65蛋白表达明显降低(P<0.01)。结论:加味过敏煎可改善抗生素加重过敏性哮喘气道炎症情况,其作用机制可能与调控TLR4/MyD88/NF-κB通路有关。

钩藤降压解郁方抑制TLR4/NF-кB信号通路对高血压并发抑郁症大鼠海马小胶质细胞极化的影响

目的探讨钩藤降压解郁方(钩藤、天麻、地龙、葛根等)调控TLR4/NF-кB信号通路对高血压并发抑郁症(HD)大鼠海马小胶质细胞极化的影响。方法将40只原发性高血压大鼠随机分为5组:模型组、阳性药组及中药(钩藤降压解郁方)高、中、低剂量组,每组8只;另取8只SD大鼠作为对照组。采用连续42 d慢性应激(CUMS)结合孤养的方式复制HD模型。造模同时给药干预,中药高、中、低剂量组分别给予钩藤降压解郁方29.61、14.81、7.40 g·kg^(-1)灌胃给药;阳性药组给予左旋氨氯地平0.45 mg·kg^(-1)+氟西汀1.8 mg·kg^(-1)灌胃给药;灌胃体积10 mL·kg^(-1),每天1次,连续42 d。采用无创血压计于给药前及每周最末日上午测量大鼠尾动脉收缩压;造模开始后的第2周和最后1周各进行1次糖水偏好行为学检测;造模结束后进行水迷宫实验;ELISA法测定血清炎性因子肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-10水平;HE染色法及尼氏染色法观察大鼠海马组织神经元病理变化;免疫荧光双染法检测海马区小胶质细胞M1(CD16)、M2(CD206)型表达情况;Western Blot法检测海马组织中TLR4、NF-κB p65蛋白的表达情况。结果与对照组比较,模型组大鼠第1~6周的尾动脉收缩压均显著升高(P<0.01);糖水偏好率显著下降(P<0.01);逃避潜伏期明显延长(P<0.05,P<0.01),穿越平台次数及目标象限停留时间占比显著降低(P<0.01);血清TNF-ɑ、IL-1β含量显著上升(P<0.01),IL-10含量显著下降(P<0.01);胞核深染,胞质固缩,细胞凋亡明显;海马小胶质细胞CD206/CD16荧光强度比明显降低(P<0.05);海马组织TLR4、NF-κB p65蛋白表达显著上调(P<0.01)。与模型组比较,给药组大鼠第1~6周的尾动脉收缩压均显著降低(P<0.01);糖水偏好率均明显上升(P<0.05);血清TNF-ɑ、IL-1β含量显著下降(P<0.01),IL-10含量显著上升(P<0.01);尼氏体丰富,细胞凋亡明显减少。阳性药组及钩藤降压解郁方高剂量组大鼠的逃避潜伏期明显缩短(P<0.05,P<0.01),穿越平台次数及目标象限停留时间占比明显增加(P<0.05);海马小胶质细胞CD206/CD16荧光强度比明显升高(P<0.05,P<0.01);海马组织TLR4、NF-κB p65蛋白表达明显下调(P<0.05,P<0.01)。结论钩藤降压解郁方可能通过抑制TLR4/NF-кB通路调节HD大鼠海马小胶质细胞极化状态,调控炎症因子分泌,减轻海马神经元损伤。

温针灸对兔膝骨性关节炎模型滑膜组织中TLR4/NF-κB信号通路诱导下滑膜修复作用机制的实验研究

目的通过观察温针灸对兔右膝骨性关节炎模型关节软骨滑膜组织中TLR4、NF-κB表达量的影响,探讨温针灸治疗膝骨性关节炎的机制。方法随机将40只兔分为空白组、模型组、双氯芬酸钠组、温针灸组,每组10只,采用右后肢石膏管型固定法制作膝骨性关节炎模型,于造模成功后第3 d开始进行干预治疗。空白组不做任何处理,常规饲养;模型组不治疗,每天以石膏固定1次,时间15 min。温针灸组给予患侧鹤顶、后三里、内外膝眼、阳陵泉5个穴位行温针灸治疗,15 min/1次,1次/d。双氯芬酸钠组将药品研末、溶解在纯净水中,并以15 mg/kg体重的浓度灌胃。6 d为1个疗程,观察2个疗程,治疗结束后取材。通过HE染色法观察各组膝关节软骨的病理变化,并进行Mankin′s评分;采用ELISA法检测各组兔膝关节滑膜组织中NF-κB、TLR4的表达水平。结果与空白组兔比较,各组兔膝关节Mankin′s评分、NF-κB、TLR4的含量均增高(P<0.05),与模型组兔相比,温针灸组和双氯芬酸钠组兔膝关节Mankin′s评分和NF-κB、TLR4的含量均有所下降(P<0.05)。结论温针灸可改善兔膝关节软骨退行性变,减轻炎症反应,其机制可能与改善兔膝关节软骨形态,降低滑膜组织中NF-κB、TLR4的表达量相关。

左归降糖舒心方调控TLR4/NF-κB通路抑制糖尿病心肌病小鼠心肌纤维化的机制研究

目的 基于Toll样受体4(Toll-like receptor 4,TLR4)/核因子κB(nuclear factor-κB,NF-κB)信号通路探讨左归降糖舒心方对糖尿病心肌病MKR小鼠心肌纤维化及炎症因子的影响。方法 以8周龄雄性MKR小鼠为实验对象,采用高脂饮食联合腹腔注射链脲佐菌素(streptozotocin,STZ)40 mg/kg构建糖尿病心肌病模型,随机分为中药高剂量组[33.67 g/(kg·d)左归降糖舒心方2 g/mL]、中药低剂量组[16.84 g/(kg·d)左归降糖舒心方2 g/mL]、西药联合组[0.23 g/(kg·d)二甲双胍联合1.5 mg/(kg·d)依那普利]和模型组(等容量蒸馏水);另设FVB小鼠为空白对照组(等容量蒸馏水)。每组10只,连续给药8周后取材。收集尾静脉血液检测空腹血糖水平;采用HE、Masson染色观察心肌病理改变,电镜观察心肌组织超微结构变化;采用ELISA检测小鼠血清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)及白细胞介素-1β(interleukin-1β,IL-1β)含量;采用Western blot法检测TLR4、NF-κB p56、p-NF-κB p56/NF-κB p56蛋白表达情况;RT-qPCR及Western blot检测小鼠心肌组织Ⅰ型胶原(collagen type I,CollagenⅠ)、Ⅲ型胶原(collagen typeⅢ,CollagenⅢ)及α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达水平。结果 与空白对照组比较,模型组小鼠空腹血糖及血清中TNF-α、IL-1β含量增高(P<0.01);心肌细胞结构紊乱,胶原含量增加,心肌细胞重度退行性变;心肌组织中TLR4、NF-κB p56、p-NF-κB p56/NF-κB p56蛋白表达上调(P<0.01),CollagenⅠ、CollagenⅢ、α-SMA mRNA及蛋白表达增加(P<0.05,P<0.01),中药高剂量组小鼠心肌组织CollagenⅠ、CollagenⅢ蛋白表达增加(P<0.01)。与模型组比较,中药高、低剂量组和西药联合组小鼠空腹血糖及血清中TNF-α、IL-1β含量均降低(P<0.01);心肌结构改善,胶原沉积减少;心肌组织中TLR4、NF-κB p56、p-NF-κB p56/NF-κB p56蛋白表达下调(P<0.01),CollagenⅠ、CollagenⅢ、α-SMA蛋白及mRNA表达下调(P<0.01)。与西药联合组比较,中药低剂量组小鼠空腹血糖及血清中TNF-α、IL-1β含量均升高(P<0.01),心肌结构无明显改善,胶原沉积无显著减少;心肌组织中TLR4、NF-κB p56、p-NF-κB p56/NF-κB p56蛋白表达上调(P<0.01),CollagenⅠ、CollagenⅢ、α-SMA蛋白及mRNA表达上调(P<0.01)。结论 左归降糖舒心方能抑制心肌炎性反应、改善心肌细胞结构,其作用机制可能与调控TLR4/NF-κB通路、下调细胞炎症因子表达、抑制心肌纤维化有关。

三黄糖肾康颗粒治疗糖尿病肾病患者的疗效观察及对TLR4、NF-κB、MCP-1的影响

目的:观察三黄糖肾康颗粒治疗糖尿病肾病的疗效及对血清Toll样受体4(TLR4)、核转录因子-kB(NF-κB)、人单核细胞趋化蛋白-1(MCP-1)的影响。方法:60例DN患者,随机分为观察组和对照组各30例。两组均予以西医基础治疗,观察组在对照组治疗基础上加用三黄糖肾康颗粒,两组疗程均为12周;评价疗效,ELISA检测血清TLR4、NF-κB、MCP-1含量。结果:观察组疗效优于对照组(P<0.05);两组治疗后血清TLR4、NF-κB、MCP-1均降低(P<0.05);与对照组比较,观察组TLR4、NF-κB、MCP-1降低更明显(P<0.05)。两组不良反应发生率差异无统计学意义(P>0.05)。结论:三黄糖肾康颗粒可提高西医常规治疗DN的疗效,调节TLR4、NF-κB、MCP-1水平是其部分作用机制。

基于TLR4/NF-κB信号通路探讨水蛭对大鼠肾纤维化的影响

目的 观察水蛭干预肾纤维化大鼠的疗效,并通过TLR4/NF-κB信号通路探讨其对炎症状态的干预机制。方法 将雄性SD大鼠50只,按随机数字表法分为五组:正常组、模型组、阳性对照组和水蛭低、高剂量组。以单侧输尿管梗阻建立肾纤维化模型。干预21天后,生化检测血肌酐、尿素氮,ELISA检测大鼠血清IL-1β、IL-6、TNF-α含量水平,HE、Masson染色光镜下观察大鼠肾脏病理改变,Western blot检测肾组织内TLR4、MyD88、NF-κB蛋白表达。结果 与正常组比较,模型组血Scr、BUN显著升高(P<0.01),血IL-1β、IL-6、TNF-α水平均显著升高(P<0.01),大鼠肾脏组织发生明显病理改变,大鼠肾脏组织中TLR4、MyD88、NF-κB蛋白表达均显著升高(P<0.01);与模型组比较,阳性对照组、水蛭低、高剂量组血清Scr、BUN、IL-1β、IL-6、TNF-α水平均显著降低(P<0.01),肾脏组织病理显著改善,肾脏组织中TLR4、MyD88、NF-κB蛋白水平均明显降低(P<0.05)。结论 水蛭能有效改善肾纤维化大鼠的肾功能,其机制可能是抑制TLR4/NF-κB信号通路从而抑制炎症反应发生,达到抗肾纤维化的目的。

基于TLR4/NF-κB/NLRP3信号通路探究白芍总苷对自身免疫性甲状腺炎大鼠炎症损伤的影响

目的探讨白芍总苷对自身免疫性甲状腺炎(AIT)大鼠炎症损伤及TLR4/NF-κB/NLRP3通路的影响。方法实验分为对照组(Control)、模型组(Model)、白芍总苷组(TGP)、TLR4抑制剂组(TLR4 inhibitor)和TGP+TLR4激动剂组(TGP+TLR4 agonist),每组各10只。除Control组外,其余各组大鼠采用皮下注射甲状腺球蛋白与弗氏佐剂诱导AIT大鼠模型。给药6周后,苏木精-伊红(HE)染色观察甲状腺组织病理学变化;酶联免疫吸附法(ELISA)测定血清TPOAb、TgAb、TSH、T3、T4、TNF-α、INF-γ、IL-1β、IL-18水平;RT-qPCR和Western blot检测甲状腺组织TLR4/NF-κB/NLRP3信号通路mRNA和蛋白表达。结果与Control组相比,Model组大鼠甲状腺滤泡上皮明显受损,TPOAb、TgAb、TSH、T3、T4、TNF-α、INF-γ、IL-1β、IL-18水平升高(P<0.01),TLR4/NF-κB/NLRP3信号通路mRNA和蛋白表达升高(P<0.01)。与Model组相比,TGP组、TLR4 inhibitor组大鼠甲状腺滤泡上皮损伤减轻,TPOAb、TgAb、TSH、T3、T4、TNF-α、INF-γ、IL-1β、IL-18水平降低(P<0.01),TLR4/NF-κB/NLRP3信号通路mRNA和蛋白表达降低(P<0.01)。与TGP组相比,TGP+TLR4 agonist组大鼠甲状腺滤泡上皮损伤加重,TPOAb、TgAb、TSH、T3、T4、TNF-α、INF-γ、IL-1β、IL-18水平升高(P<0.05或P<0.01),TLR4/NF-κB/NLRP3信号通路mRNA和蛋白表达增加(P<0.05或P<0.01)。结论TGP通过抑制TLR4/NF-κB/NLRP3信号通路,改善甲状腺组织炎症损伤发挥甲状腺保护作用。

miR-27a通过TLR4/NF-κB信号通路对类风湿关节炎滑膜细胞生物学行为的影响

目的:探讨miR-27a通过Toll样受体(TLR)4/NF-κB信号通路对类风湿关节炎(RA)滑膜细胞生物学行为的影响。方法:选择行膝关节置换术的30例RA患者(RA组)和同期因创伤急诊截肢的18例患者(对照组)的滑膜组织,采用qRT-PCR法检测miR-27a的表达。将RA成纤维样滑膜细胞MH7A分为4组:空白对照组,不进行任何处理;TNF-α组,加入终浓度为20μg/L的TNF-α处理24 h;TNF-α+miR-NC组,加入终浓度为20μg/L的TNF-α处理后转染miR-NC;TNF-α+miR-27a mimic组,加入终浓度为20μg/L的TNF-α处理后转染miR-27a mimic,采用qRT-PCR法检测组织或细胞中miR-27a的表达量,CCK-8法检测细胞增殖情况,克隆形成实验检测细胞克隆形成能力,Transwell法检测细胞侵袭和迁移能力,Annexin V/PI双染法检测细胞凋亡情况,双荧光素酶报告实验验证TLR4 mRNA与miR-27a的靶向关系,Western blot法检测细胞中TLR4、NF-κB、磷酸化TLR4(p-TLR4)和磷酸化NF-κB(p-NF-κB)蛋白的表达情况。结果:对照组和RA组滑膜组织中miR-27a的表达量分别为(1.00±0.08)和(0.36±0.05),RA组低于对照组(P<0.001)。与空白对照组比较,TNF-α组和TNF-α+miR-NC组细胞中miR-27a表达量下降,TNF-α+miR-27a mimic组miR-27a表达量升高;与TNF-α组和TNF-α+miR-NC组比较,TNF-α+miR-27a mimic组细胞中miR-27a表达量升高(P<0.05)。与空白对照组比较,TNF-α组和TNF-α+miR-NC组细胞增殖、克隆形成、侵袭和迁移能力增强,细胞凋亡率降低;与TNF-α组和TNF-α+miR-NC组比较,TNF-α+miR-27a mimic组细胞增殖、克隆形成、侵袭和迁移能力减弱,细胞凋亡率升高(P<0.05)。双荧光素酶报告实验证实TLR4是miR-27a的靶基因。与空白对照组比较,TNF-α组和TNF-α+miR-NC组p-TLR4/TLR4、p-NF-κB/NF-κB升高;与TNF-α组和TNF-α+miR-NC组比较,TNF-α+miR-27a mimics组p-TLR4/TLR4、p-NF-κB/NF-κB降低(P<0.05)。结论:miR-27a可能通过抑制TLR4/NF-κB信号通路,降低RA成纤维样滑膜细胞的增殖、侵袭和迁移能力,促进其凋亡。

五灵胶囊通过调控TLR4/NF-κB通路防治急性痛风性关节炎作用的研究

目的 探讨五灵胶囊对由单尿酸钠晶体(monosodium urate,MSU)诱导的急性痛风性关节炎(acute gouty arthritis,AGA)大鼠的治疗作用。方法 通过踝关节腔内注射MSU构建AGA大鼠模型。用缚线法、足趾容积测量仪、双足平衡测痛仪检测踝关节周径、足趾容积、双足痛觉;用HE染色观察大鼠右侧踝关节滑膜病理变化;用免疫吸附试验(enzyme-linked immunesorbent assay,ELISA)检测血清中炎症因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素1β(interleukin-1β,IL-1β)水平;用实时荧光PCR技术分别检测关节滑膜中的炎症因子及TLR4/NF-κB信号通路关键指标的表达情况。结果 与对照组比较,模型组的踝关节周径、足趾容积、对痛觉的敏感程度均显著升高(P<0.05),滑膜组织炎性浸润明显,血清中炎症因子TNF-α、IL-1β的水平均显著升高(P<0.05),滑膜组织中炎症因子及TLR4/NF-κB信号通路中关键指标的表达量均显著升高(P<0.05);与模型组比较,秋水仙碱和五灵胶囊各剂量组均可明显改善由MSU诱导的AGA大鼠踝关节周径、足趾容积及对痛觉的敏感程度(P<0.05),改善滑膜组织炎性浸润,抑制由MSU诱导的AGA大鼠血清中炎症因子的上调(P<0.05),抑制关节滑膜组织中IL-1β、TNF-α、IL-6及TLR4、MyD88、NF-κB、IκBα、NLRP3的表达(P<0.05)。结论 五灵胶囊可能通过减少炎症因子的释放,抑制TLR4/NF-κB信号通路,发挥防治AGA的作用。

电针对慢性前列腺炎大鼠TLR4/NF-κB/NLRP3通路蛋白表达的影响

目的观察电针对慢性前列腺炎大鼠前列腺Toll样受体4/核转录因子κB/NOD样受体蛋白3(TLR4/NF-κB/NLRP3)通路的影响,探讨电针治疗慢性前列腺炎的可能机制。方法将40只SPF级健康雄性SD大鼠随机分为空白组、模型组、电针组、抑制剂组、激动剂组,各8只。除空白组外,其余各组采用前列腺蛋白提取及免疫注射法制备大鼠慢性前列腺模型,于造模后1 d对电针组、抑制剂组及激动剂组大鼠行电针干预,穴取“白环俞”“会阳”,连续波,频率2 Hz,每日1次,每次20 min,7 d为1个疗程,每疗程后休息1 d,共治疗2个疗程。基于开野实验和糖水消耗实验开展行为学测试;取大鼠前列腺组织,计算前列腺湿重及前列腺指数;HE染色观察大鼠前列腺组织形态学变化;TUNEL法观察前列腺细胞凋亡;Western blot法检测前列腺组织中TLR4、NF-κB、NLRP3蛋白表达。结果与空白组比较,模型组大鼠水平运动评分、垂直运动评分和糖水消耗量均显著下降(P<0.05);与模型组比较,电针组大鼠水平运动评分、垂直运动评分和糖水消耗量显著增加(P<0.05);与电针组比较,抑制剂组大鼠水平运动评分、垂直运动评分和糖水消耗量显著增加,而激动剂组大鼠水平运动评分、垂直运动评分和糖水消耗量显著下降(P<0.05)。与空白组比较,模型组大鼠前列腺湿重和前列腺指数显著升高(P<0.05),前列腺细胞凋亡升高(P<0.05);与模型组比较,电针组大鼠前列腺湿重和前列腺指数显著下降(P<0.05),前列腺细胞凋亡显著下降(P<0.05);与电针组比较,抑制剂组大鼠前列腺湿重和前列腺指数显著下降(P<0.05),而激动剂组大鼠前列腺湿重和前列腺指数显著升高(P<0.05),且抑制剂组大鼠前列腺细胞凋亡进一步下降(P<0.05),而激动剂组大鼠前列腺细胞凋亡升高(P<0.05)。与空白组比较,模型组大鼠前列腺组织中TLR4、p-NF-κB/NF-κB、NLRP3蛋白表达显著增加(P<0.05),与模型组比较,电针组大鼠前列腺组织中TLR4、p-NF-κB/NF-κB、NLRP3蛋白表达显著减少(P<0.05);与电针组比较,抑制剂组大鼠前列腺组织中TLR4、p-NF-κB/NF-κB、NLRP3蛋白表达显著下降(P<0.05),而激动剂组大鼠前列腺组织中TLR4、p-NF-κB/NF-κB、NLRP3蛋白表达显著升高(P<0.05)。结论电针白环俞、会阳可明显改善慢性前列腺模型大鼠症状,降低细胞凋亡水平,其机制可能与下调TLR4/NF-κB/NLRP3通路蛋白表达,进而抑制前列腺细胞凋亡有关。

基于TLR4/NF-κB通路调补肺肾法干预细胞自噬对COPD肺血管重塑的影响和机制

目的基于Toll样受体4(TLR4)/核转录因子-κB(NF-κB)通路研究调补肺肾法干预细胞自噬对慢性阻塞性肺疾病(COPD)肺血管重塑的影响和机制。方法取SD大鼠采用香烟烟雾暴露结合反复细菌感染的方案建立COPD模型,随机分为3组,模型组、补肺益肾方(3.7 g/kg)组、补肺益肾方(3.7 g/kg)+脂多糖(LPS)(TLR4激活剂,15 mg/kg)组,每组12只,另取12只大鼠正常呼吸并气管滴注等剂量生理盐水作为对照组,经补肺益肾方、LPS对大鼠分组干预后,检测各组大鼠肺功能指标:潮气量(TV)、呼气峰流速(PEF)、第0.3秒用力呼气容积(FEV0.3)/用力肺活量(FVC)。以HE染色检测各组大鼠肺组织病理形态和肺血管重塑,比较其管壁厚度(WT)占血管直径(VD)百分比WT/VD(%)、管腔面积(LA)占血管总面积(TA)百分比LA/TA(%)。以免疫荧光染色检测各组大鼠肺组织内肺血管内皮标记物CD34表达。以酶标仪检测各组大鼠肺泡灌洗液(BALF)及血清促炎因子:肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-17水平。以免疫印迹法检测各组大鼠肺组织自噬及TLR4/NF-κB信号通路相关蛋白表达。结果与对照组相比,模型组大鼠肺组织呈现明显病理损伤及肺血管重塑症状,TV、PEF、FEV0.3/FVC、LA/TA、肺组织Beclin-1蛋白表达与LC3Ⅱ/LC3Ⅰ显著降低(P<0.05),WT/VD、CD34相对阳性表达、BALF及血清促炎因子TNF-α与IL-17水平、肺组织TLR4蛋白表达与p-NF-κB p65/NF-κB p65显著升高(P<0.05)。与模型组相比,补肺益肾方组大鼠肺组织损伤及肺血管重塑症状减轻,TV、PEF、FEV0.3/FVC、LA/TA、肺组织Beclin-1蛋白表达与LC3Ⅱ/LC3Ⅰ升高(P<0.05),WT/VD、CD34相对阳性表达、BALF及血清促炎因子TNF-α与IL-17水平、肺组织TLR4蛋白表达与p-NF-κB p65/NF-κB p65降低(P<0.05)。与补肺益肾方组相比,补肺益肾方+LPS组大鼠肺组织损伤及肺血管重塑症状加重,TV、PEF、FEV0.3/FVC、LA/TA、肺组织Beclin-1蛋白表达与LC3Ⅱ/LC3Ⅰ降低(P<0.05),WT/VD、CD34相对阳性表达、BALF及血清促炎因子TNF-α与IL-17水平、肺组织TLR4蛋白表达与p-NF-κB p65/NF-κB p65升高(P<0.05)。结论补肺益肾方可通过抑制TLR4/NF-κB信号而抑制COPD体内炎症,并增强自噬,进而减轻大鼠肺组织病理损伤和肺血管重塑,改善其肺功能。

基于TLR4/NF-κB通路研究白术-白芍药对对IBS-D模型大鼠的治疗作用

目的研究白术-白芍药对对腹泻型肠易激综合征(Diarrhea-predominant irritable bowel syndrome,IBS-D)模型大鼠TLR4/NF-κB信号通路相关靶标的影响,旨在阐述其治疗低度炎性反应的潜在机制。方法以番泻叶灌胃+慢性不可预知刺激建立IBS-D模型大鼠,并随机分为模型组、药对组、白芍组、白术组、西药组,每组6只。另取6只不造模大鼠,设为空白组。各药物组大鼠灌胃相应药液,连续治疗14 d。通过体重、大便含水量、AWR评分评价白术-白芍药对对模型大鼠症状的干预效果;各组大鼠腹主动脉取血,剖取回肠末端组织,通过qRT-PCR法、HE染色、Elisa、Westernblot法检测白术-白芍药对对IBS-D大鼠炎症以及TLR4/NF-κB信号通路相关靶标的调控。结果与模型组对比,药对能改善体重、降低大便含水量(P<0.01)、糖水偏好率(P<0.01),在40 mmHg、60 mmHg压力下降低AWR评分(P<0.01),改善回肠末端组织炎症病理状态,下调TLR4、NF-κB、IL-1βmRNA表达(P<0.01),下调血清TLR4、NF-κB、MyD88表达,上调血清IL-10表达,下调MyD88、p-NF-κB p65、IL-6蛋白表达(P<0.01)。结论白术-白芍药对改善IBS-D大鼠腹泻症状和内脏高敏感性,可能与抑制TLR4/NF-κB信号通路相关靶标,调控炎性反应细胞因子的表达水平有关。