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.

HSP110 aggravates ischemia-reperfusion injury after liver transplantation by promoting NF-κB pathway

Background:Ischemia-reperfusion injury(IRI)poses a significant challenge to liver transplantation(LT).The underlying mechanism primarily involves overactivation of the immune system.Heat shock protein 110(HSP110)functions as a molecular chaperone that helps stabilize protein structures.Methods:An IRI model was established by performing LT on Sprague-Dawley rats,and HSP110 was silenced using siRNA.Hematoxylin-eosin staining,TUNEL,immunohistochemistry,ELISA and liver enzyme analysis were performed to assess IRI following LT.Western blotting and quantitative reverse transcription-polymerase chain reaction were conducted to investigate the pertinent molecular changes.Results:Our findings revealed a significant increase in the expression of HSP110 at both the mRNA and protein levels in the rat liver following LT(P<0.05).However,when rats were injected with siRNAHSP110,IRI subsequent to LT was notably reduced(P<0.05).Additionally,the levels of liver enzymes and inflammatory chemokines in rat serum were significantly reduced(P<0.05).Silencing HSP110 with siRNA resulted in a marked decrease in M1-type polarization of Kupffer cells in the liver and downregulated the NF-κB pathway in the liver(P<0.05).Conclusions:HSP110 in the liver promotes IRI after LT in rats by activating the NF-κB pathway and inducing M1-type polarization of Kupffer cells.Targeting HSP110 to prevent IRI after LT may represent a promising new approach for the treatment of LT-associated IRI.

Pectolinarin inhibited LPS-stimulated inflammation in microglial BV_(2) cells via NF-κB signaling pathway

Background:Neuro-inflammation is regarded as one of the critical pathogenesis in neurodegenerative diseases,which is characterized by the activated microglial cells.Pectolinarin(Pec),a natural flavonoid that exists in many Chinese herbal medicines,has been reported to have various biological activities.However,the effects and mechanisms on neuro-inflammation are not clear.Methods:In this study,the inhibitory effects and mechanisms of Pec on neuro-inflammation were investigated in the LPS-stimulated microglial BV_(2) cells.BV_(2) microglial cells were treated with Pec or vehicle,followed by LPS.Enzyme-linked immunosorbent assay,real-time quantitative PCR,nitric oxide and reactive oxygen species assay,and western blot were performed to examine the effects of Pec on neuro-inflammatory responses.Results:We showed that Pec significantly inhibited the expression of tumor necrosis factorαand interleukin 6 in mRNA and protein levels induced by LPS.Moreover,the production of nitric oxide,iNOS,reactive oxygen species,and COX-2 were suppressed by Pec in LPS-stimulated microglial BV_(2) cells.In addition,Pec inhibited LPS-induced inflammation via nuclear factor kappa B signaling pathway,as evidenced by the reduction of the phosphorylation of inhibitor of nuclear factor kappa-B kinase,the degradation of IκBα,and the nuclear translocation of p65.Conclusion:Taken together,Pec exhibited anti-inflammatory effects in LPS-stimulated microglial BV_(2) cells via nuclear factor kappa B signaling pathway,which might provide therapeutic potential for neuro-inflammation and neurodegenerative diseases.

Ziwan-Taoren herb pair can exert an therapeutical effect in primary Sjogren’s syndrome through inhibiting the TLR/NF-κB pathway

Background:Ziwan and Taoren(ZT)is a classic medicine pair in the formula of Mai Dong Di Shao Decoction,has been used to treat primary Sjogren’s syndrome(pSS)for more than 20 years.But its action mechanism is still unknown.This study is aimed to reveal the potential mechanism of ZT treated pSS and discover its active compounds of ZT and therapeutic target for pSS.Methods:Firstly,the potential pathways of ZT for pSS treatment were predicted through network pharmacology and GO and KEGG enrichment analysis.Secondly,the inter-structural relationships between active compounds of ZT and target proteins were visualized using molecular docking techniques.Finally,efficacy and mechanism were conducted through in vivo experiments,such as water intake,spleen index,hematoxylin-eosin staining pathological changes,ELISA,Western Blot analysis,and immunofluorescence staining.Results:Nine active compounds were extracted from network pharmacology,including quercitrin,luteolin,kaempferol,β-sitosterol,isorhamnetin,galangin,hederagenin,diosmetin and gibberellin 7.Seven disease targets were identified:RELA,TP53,AKT1,interleukin(IL)6,MAPK1,ESR1,IL10;with RELA being the most core target.KEGG and GO enrichment analysis indicated that ZT may act through the TLR/NF-κB/RELA inflammatory mechanism process.preliminary results of molecular docking showed that ZT’s active compounds bind well to the RELA(p65)receptor.In vivo results demonstrated that a high dose of ZT significantly improved water intake and reduced lymphocytes infiltration in submandibular gland pathology in NOD mice.The expression content of AQP5 and vasoactive intestinal peptide in the submaxillary gland was significantly increased,while levels of inflammatory factors such as tumor necrosis factor-α,IL-6,and IL-1βalong with protein expressions including toll-like receptor4,p-p65 and p-IKKα/βin NF-κB pathway were reduced.Conclusions:The ZT treatment exhibits a promising efficacy in mitigating dryness symptoms of pSS,potentially attributed to its capacity for suppressing the TLR/NF-κB inflammatory signaling pathway.

Wedelolactone attenuates sepsis-associated acute liver injury by regulating the macrophage M1/M2 polarization balance through the PI3K/AKT/NF-κB signalling pathway

Background:Liver injury caused by sepsis seriously impairs the normal physiology of the liver.Wedelactone(WED)has an obvious anti-inflammatory effect against liver damage caused by various factors.Nevertheless,further research is needed to determine if WED might mitigate acute liver damage linked to sepsis by influencing macrophage polarization.Methods:We first assessed the effect of WED on lipopolysaccharides-triggered liver injury by biochemistry assay and tissue staining.Inflammatory factors were assessed using the ELISA kits.The expression of Cluster of Differentiation 86(CD86)and Cluster of Differentiation 206(CD206)was measured by immunofluorescence assay.The protein levels of inducible nitric oxide sythase(iNOS),Arginase 1(Arg-1),phosphatidylinositol 3-kinase(PI3K),protein kinase B(AKT),PI3K phosphorylation(p-PI3K),AKT phosphorylation(p-AKT),inhibitor of kappa B kinase(IKK),inhibitor of kappa B(IκB),and nuclear factor kappa-B(NF-κB)p65 were quantified by western blot analysis.Results:WED decreased the level of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP)and malondialdehyde,and increased the activity of superoxide dismutase(SOD)and glutathione peroxidase(GSH-PX).Moreover,WED exerted effective anti-inflammatory effects by decreasing the level of Tumor necrosis factor-α(TNF-α)and Interleukin 6(IL-6)and increasing the level of Interleukin 10(IL-10)in serum and cells.WED not only decreased CD86 and iNOS expression but also increased CD206 and Arg-1 expression.WED also downregulated the increased expression of PI3K,AKT,p-PI3K,p-AKT,IKK,and NF-κB p65 induced by lipopolysaccharides,while up-regulated the decreased expression of IκB.Besides,LY294002 with WED decreased the expression of protein PI3K,AKT,p-PI3K,p-AKT,IKK and NF-κB p65,and raised the expression of IκBα.Conclusion:Wedelolactone could attenuate sepsis-associated acute liver injury,and its mechanism may be associated with balancing pro-inflammatory and anti-inflammatory by the regulation of M1/M2 macrophage polarization via the PI3K/AKT/NF-κB signaling pathway.

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.

Jianpi Gushen Huayu decoction ameliorated diabetic nephropathy through modulating metabolites in kidney,and inhibiting TLR4/NF-κB/NLRP3 and JNK/P38 pathways

BACKGROUND Jianpi Gushen Huayu Decoction(JPGS)has been used to clinically treat diabetic nephropathy(DN)for many years.However,the protective mechanism of JPGS in treating DN remains unclear.AIM To evaluate the therapeutic effects and the possible mechanism of JPGS on DN.METHODS We first evaluated the therapeutic potential of JPGS on a DN mouse model.We then investigated the effect of JPGS on the renal metabolite levels of DN mice using non-targeted metabolomics.Furthermore,we examined the effects of JPGS on c-Jun N-terminal kinase(JNK)/P38-mediated apoptosis and the inflammatory responses mediated by toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)/NOD-like receptor family pyrin domain containing 3(NLRP3).RESULTS The ameliorative effects of JPGS on DN mice included the alleviation of renal injury and the control of inflammation and oxidative stress.Untargeted metabolomic analysis revealed that JPGS altered the metabolites of the kidneys in DN mice.A total of 51 differential metabolites were screened.Pathway analysis results indicated that nine pathways significantly changed between the control and model groups,while six pathways significantly altered between the model and JPGS groups.Pathways related to cysteine and methionine metabolism;alanine,tryptophan metabolism;aspartate and glutamate metabolism;and riboflavin metabolism were identified as the key pathways through which JPGS affects DN.Further experimental validation showed that JPGS treatment reduced the expression of TLR4/NF-κB/NLRP3 pathways and JNK/P38 pathway-mediated apoptosis related factors.CONCLUSION JPGS could markedly treat mice with streptozotocin(STZ)-induced DN,which is possibly related to the regulation of several metabolic pathways found in kidneys.Furthermore,JPGS could improve kidney inflammatory responses and ameliorate kidney injuries in DN mice via the TLR4/NF-κB/NLRP3 pathway and inhibit JNK/P38 pathwaymediated apoptosis in DN mice.

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。

萝卜硫素通过调节ALOX5/NF-κB信号通路调控巨噬细胞糖酵解抑制糖尿病肾病进展

目的探讨萝卜硫素(SFN)调节花生四烯酸5-脂氧合酶基因(arachidonic acid 5-lipoxygenase,ALOX5)/核因子kappa B(NF-κB)信号通路调节巨噬细胞糖酵解对糖尿病肾病(DN)进展的影响。方法生物信息学分析SFN治疗DN的靶基因。使用30 mmol/L高葡萄糖(HG)处理人近端肾小管上皮细胞系(HK-2细胞)诱导体外DN模型。将HK-2细胞分为如下组:正常糖(NG)组、HG组、HG+SFN(3 mmol/L)组、HG+ALOX5组、HG+SFN(3 mmol/L)+ALOX5组、HG处理的巨噬细胞+HK-2细胞组、HG+SFN(3 mmol/L)处理的巨噬细胞+HK-2细胞组、HG+ALOX5转染处理的巨噬细胞+HK-2细胞组、HG+SFN(3 mmol/L)+ALOX5转染处理的巨噬细胞+HK-2细胞组。CCK-8检测细胞活力,原位末端脱氧核苷酸转移酶标记(TUNEL)法检测细胞凋亡;葡萄糖和乳酸试剂盒检测各组细胞中葡萄糖和乳酸水平;Western blot检测各组细胞中ALOX5、NF-κB以及糖酵解相关蛋白己糖激酶-2(HK2)、丙酮酸激酶M2(PKM2)、葡萄糖转运蛋白1(GLUT1)的表达;使用链脲佐菌素(STZ)构建DN小鼠模型,DN小鼠给与SFN(0.5 mg/kg)治疗;检测小鼠各项生化指标,HE染色检测肾组织病理变化;Western blot检测小鼠肾脏巨噬细胞中糖酵解相关蛋白己糖激酶-2(HK2)、丙酮酸激酶M2(PKM2)、葡萄糖转运蛋白1(GLUT1)的表达。结果生物信息学分析结果显示ALOX5是SFN治疗DN的靶基因。与HG组相比,SFN处理增强HK-2细胞活力并抑制细胞凋亡(P<0.05);同时,SFN处理抑制HG诱导的巨噬细胞糖酵解相关蛋白的表达,减弱巨噬细胞介导的HK-2细胞损伤(P<0.05);Western blot结果表明SFN抑制ALOX5和NF-κB的表达(P<0.05);小鼠实验结果显示,SFN治疗改善DN小鼠肾功能和肾组织病理学改变,抑制肾组织中巨噬细胞糖酵解相关蛋白的表达(P<0.05)。结论SFN通过抑制ALOX5/NF-κB信号通路抑制巨噬细胞糖酵解从而改善DN进展。

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。

针刺对干眼兔角膜形态学及角膜组织NF-κB信号通路的影响

目的 探讨针刺对干眼兔角膜形态学及角膜组织NF-κB信号通路的影响,分析针刺对干眼的作用机制。方法 取雌雄不拘的健康新西兰兔24只,随机分为空白组、模型组、针刺组、假针刺组,每组6只。空白组为正常对照组,不加任何处理;其余3组动物每天800、 1100、1400和1800时于皮下注射氢溴酸东莨菪碱2.0 mg·kg-1,连续35 d直至实验结束。假针刺组:于造模后第22天给予假针刺治疗(睛明BL1、攒竹BL2、丝竹空SJ23、太阳穴EX-HN5、瞳子髎GB1),钝针头点刺穴位,不刺进穴位,每天1次,连续14 d。针刺组:于造模成功后第22天给予针刺治疗,穴位同假针刺组。造模后第0、21、28、35天分别进行角膜荧光素染色,第35天进行角膜共焦显微镜检查,之后处死动物,在光学显微镜、透射电子显微镜下观察角膜形态学变化,采用Western blot检测角膜组织NF-κB蛋白的表达。结果 与模型组相比,造模后第28、35天,针刺组、空白组兔角膜荧光素染色评分均减小,差异均有统计学意义(均为P<0.05)。造模后第35天共焦显微镜检查结果显示,模型组和假针刺组与其他组相比,兔角膜基质层出现大量球状免疫细胞和边界不清大小不规律的活化基质层细胞,可见具有不规则细胞间间隙的区域,存在炎症;针刺组基质层细胞形态得到改善,细胞呈轻微激活状态,角膜神经形态未见明显异常。造模后第35天光学显微镜检查结果显示,模型组和假针刺组兔角膜组织表面可见角化过度的扁平上皮细胞,淋巴细胞浸润,局灶上皮细胞层数增多,表面上皮细胞脱落;针刺组角膜上皮有接近4~6层上皮细胞,上皮脱落减少,与模型组相比,淋巴细胞浸润减少。造模后第35天透射电子显微镜检查结果显示,模型组和假针刺组兔角膜上皮细胞出现异常的微绒毛结构和上皮细胞缺失,细胞间隙增宽,粗面内质网严重扩张,桥粒解体伴随线粒体肿胀;针刺组上皮细胞微绒毛结构稀疏且短,仍见局部缺失,粗面内质网轻度扩张,线粒体未见明显肿胀。造模后第35天Western blot检测结果显示,与空白组相比,模型组、假针刺组p-NF-κB p65表达均上调(均为P<0.05);与模型组、假针刺组相比,针刺组p-NF-κB p65表达均下调(均为P<0.05)。结论 针刺可以抑制NF-κB信号通路从而发挥抗炎作用,改善兔干眼模型角膜炎症和损伤。

基于IKKβ/NF-κB通路探讨温胆汤对睡眠障碍小鼠的神经保护作用

目的 探讨温胆汤对睡眠障碍小鼠神经损伤的影响及其机制。方法 小鼠采用改良版水平转盘睡眠剥夺法构建失眠模型,造模成功后随机分为模型组、艾司唑仑片组(0.15 mg/kg)和温胆汤低、高剂量组(12.5、50 g/kg),每组6只,另取6只作为对照组。给药干预7 d后,HE染色观察大脑皮层组织、海马CA1区组织、下丘脑组织变化;尼氏染色观察神经元损伤情况;ELISA法检测脑组织和血清中神经丝轻链(NEFL)、神经特异性烯醇化酶(NSE)、S100钙结合蛋白B(S100B)、肿瘤坏死因子(TNF-α)、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)水平;免疫组化法检测脑组织中胶质纤维酸性蛋白(GFAP)表达;Western blot法检测脑组织中GFAP、磷酸化IκB激酶β(p-IKKβ)、磷酸化核转录因子-κB(p-NF-κB)蛋白表达。结果 与模型组比较,温胆汤高剂量组小鼠神经元细胞数量增加,结构完整,排列整齐,神经元细胞核皱缩变形数量减少,尼氏小体增多,血清和脑组织NEFL、NSE、S100B、TNF-α、IL-6、IL-1β水平降低(P<0.01),脑组织GFAP表达降低(P<0.01),脑组织p-IKKβ和p-NF-κB磷酸化水平均降低(P<0.01)。结论 温胆汤能够减少睡眠障碍小鼠的神经损伤,减少促炎介质释放,其机制可能与抑制IKKβ/NF-κB通路的激活有关。

脱氧胆酸通过ROS/NF-κB通路对Barrett食管细胞氧化应激的影响

目的 探讨脱氧胆酸(DCA)对人BAR-T细胞氧化应激的影响及机制。方法 体外培养人Barrett上皮细胞株BAR-T,采用不同浓度的DCA(100、200、300μmol/L)和不同作用时间(30 min、60 min、3 h、6 h)干预BAR-T细胞。采用Real-time PCR法和Western blotting法检测环氧酶-2(COX-2)的mRNA和蛋白表达;采用显微镜观察和流式细胞仪检测细胞内活性氧(reactive oxygen species, ROS)含量,并与200μmol/L DCA+5 mmol/L ROS清除剂N-乙酰半胱胺酸(NAC)组进行比较;采用细胞免疫荧光法检测细胞p65蛋白入核情况,并与200μmol/L DCA+100μmol/L NF-κB通路抑制剂吡咯烷二硫代甲酸铵(PDTC)组进行比较。结果 与对照组相比,DCA可以显著升高BAR-T细胞中ROS的含量,呈剂量依赖性,5 mmol/L NAC明显抑制DCA诱导的ROS释放。与对照组相比,相同干预时间下,DCA(200、300μmol/L组)均可以显著升高COX-2 mRNA表达。与1 h组相比,200、300μmol/L DCA 6 h组均可以显著升高COX-2 mRNA表达。与对照组比较,200μmol/L DCA可以显著升高COX-2蛋白表达。同时,200μmol/L DCA可以促进p65蛋白的入核,PDTC可以抑制DCA的作用。结论 DCA可能通过升高细胞内ROS水平,促进p65蛋白入核以激活NF-κB信号通路,进而上调COX-2的表达。

Nurr1通过抑制小胶质细胞NF-κB发挥抗炎作用

目的探索在细菌脂多糖(LPS)诱导的小鼠小胶质细胞(BV2细胞株)炎症反应中,核受体相关蛋白1(Nurr1)是否通过抑制NF-κB的表达及核转位发挥抗炎作用。方法培养小鼠BV2小胶质细胞株,将细胞分为Ctrl组、LPS组、C-DIM12(Nurrl特异性激动剂)组、siRNA干扰组、LPS+C-DIM12组。利用CCK-8检测LPS和C-DIM12的最适作用浓度,用Western blot观察NF-κB和Nurr1的表达变化,用酶联免疫吸附法(ELISA)检测培养液中炎症因子IL-6、IL-1β和TNF-α的水平,用免疫荧光观察siRNA干扰或C-DIM12激活Nurr1表达活性后,NF-κB及p-IκB在BV2细胞中的表达水平。结果LPS刺激BV2细胞后,NF-κB表达提高,炎症因子IL-1、IL-6、TNF-α释放量增加(P<0.05)。C-DIM12激活Nurr1后,免疫荧光染色技术和Western blot实验结果都显示NF-κB的表达显著下降,而小干扰RNA(siNurr1)沉默Nurr1表达后,NF-κB表达增加。在LPS激活的小胶质细胞中,NF-κB二聚体分子的结合分子p-IκB的磷酸化蛋白水平相对于对照组显著升高,而C-DIM12激活Nurr1后,p-IκB水平显著下降。结论Nurr1可以通过抑制小胶质细胞NF-κB表达水平并阻止NF-κB核位移发挥抗炎作用。Nurr1可能是一个新的潜在的治疗中枢神经系统炎症相关疾病的靶点。

清肠汤调节HGF/STAT3/NF-κB通路保护小鼠溃疡性结肠炎

目的研究清肠汤(Qingchang decoction,QCD)对溃疡性结肠炎(ulcerative colitis,UC)小鼠的保护作用以及对HGF/STAT3/NF-κB通路的影响。方法将40只C57BL/6小鼠分为正常组、葡聚糖硫酸钠(DSS)组、柳氮磺吡啶组(SASP)100 mg/kg、清肠汤组18 g/kg,除正常对照组以外的各组小鼠自由饮用2.5%的DSS溶液7 d,后替换为正常饮用水。正常对照组和模型组给予蒸馏水灌胃,其余各组灌胃相应药物。每天观察小鼠一般状况、体质量变化,10 d后处死小鼠,测量结肠长度,对结肠组织进行组织形态学分析,ELISA法测定小鼠结肠组织中的TNF-α、IL-1β和IL-6的浓度,Western blot、免疫荧光检测HGF、p-STAT3、p-NF-κB的表达水平。结果清肠汤可显著增加小鼠体质量和结肠长度,降低结肠病理评分,显著减少结肠TNF-α、IL-1β和IL-6的浓度,减低结肠组织HGF、p-STAT3、p-NF-κB的表达水平。结论清肠汤可能通过调节HGF/STAT3/NF-κB通路治疗DSS诱导的UC小鼠。

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。

清热祛浊胶囊对非酒精性脂肪肝炎小鼠NF-κB/NLRP3信号通路的调控机制研究

[目的]研究清热祛浊胶囊(QRQZ)对非酒精性脂肪肝炎(NASH)模型小鼠的治疗效果及对核转录因子-κB(NF-κB)/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)信号通路的影响。[方法]通过蛋氨酸和胆碱缺乏(MCD)饮食诱导建立NASH小鼠模型,并灌胃不同剂量的QRQZ。通过检测各组小鼠体质量、肝指数、丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、三酰甘油(TG)、总胆固醇(TC),肝苏木素-伊红(HE)染色及油红O染色评估QRQZ对NASH模型小鼠的治疗作用;通过检测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)水平评估QRQZ对NASH模型小鼠氧化应激水平;通过酶联免疫吸附测定(ELISA)检测白细胞介素-1β(IL-1β)、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平评估QRQZ对NASH模型小鼠炎症的影响;通过检测磷酸化核因子κB抑制蛋白(p-IκB)、磷酸化核转录因子κB P65(p-NF-κB P65)、NLRP3、凋亡相关斑点样蛋白(ASC)、活化的半胱氨酸天冬氨酸蛋白酶1(cleaved Caspase-1)、mature IL-1β的蛋白水平以及NLRP3、ASC、Caspase-1、IL-1β的mRNA表达来评估QRQZ对NASH小鼠NF-κB/NLRP3信号通路的影响。[结果] QRQZ可改善MCD引起的体质量减轻,降低肝指数,降低血清ALT、AST活性及TC、TG水平,同时改善肝组织病理学变化,提示QRQZ对NASH模型小鼠具有治疗作用;此外,QRQZ提升肝组织中SOD、GSH-Px活性,降低了MDA水平,降低了IL-6、IL-1β、iuTNF-α炎症因子水平,提示了QRQZ的抗氧化及抗炎作用;进一步研究发现QRQZ降低了IκB与P65的磷酸化水平,减少NLRP3、ASC、Caspase-1、IL-1β蛋白及基因表达。提示QRQZ干预抑制了NASH小鼠NF-κB/NLRP3信号通路活化。[结论] QRQZ对NASH模型小鼠具有治疗作用,并且可以提升抗氧化能力改善炎症,其作用机制可能与抑制NF-κB/NLRP3信号通路活化有关。

小檗碱通过NF-κB/iNOS-COX-2信号通路对睡眠剥夺大鼠认知功能的影响

目的探究小檗碱是否能够通过调控核因子(NF)-κB/诱导型一氧化氮合酶/环氧合酶(iNOS-COX)-2信号通路对睡眠剥夺(SD)大鼠认知功能产生影响。方法90只大鼠随机分为对照组、睡眠剥夺(SD)组、小檗碱低剂量组[小檗碱-L组,30 mg/(kg·d)]、小檗碱中剂量组[小檗碱-M组,60 mg/(kg·d)]、小檗碱高剂量组[小檗碱-H组,120 mg/(kg·d)]、艾司唑仑组[0.1 mg/(kg·d)],每组各15只,药物处理7 d后通过平台水环境法进行大鼠SD模型构建。通过Morris水迷宫实验和Y迷宫实验检测逃避潜伏期和行为正确率;酶联免疫吸附试验(ELISA)检测各组海马组织中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6、IL-17和血清中S100B、神经元特异性烯醇化酶(NSE)表达水平;试剂盒检测海马组织中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)水平;Western印迹检测海马组织中NF-κB p-p65、iNOS、COX-2蛋白表达水平。结果与对照组比较,SD组逃避潜伏期,海马组织中TNF-α、IL-6、IL-17、MDA水平,血清中S100B和NSE水平及海马组织中NF-κB p-p65、iNOS、COX-2蛋白水平明显升高,行为正确率和海马组织中SOD、GSH-Px水平明显降低(均P<0.05)。与SD组比较,小檗碱-L、M、H组和艾司唑仑组逃避潜伏期,海马组织中TNF-α、IL-6、IL-17、MDA水平,血清中S100B和NSE水平及海马组织中NF-κB p-p65、iNOS、COX-2蛋白水平均明显降低,行为正确率和海马组织中SOD、GSH-Px水平明显升高(均P<0.05)。小檗碱-H组与艾司唑仑组比较上述指标无明显差异(P>0.05)。结论小檗碱能够对SD大鼠认知功能产生保护作用,其机制可能与调控NF-κB/iNOS-COX-2信号通路,并减轻炎症和氧化应激损伤有关。

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。

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。