基于BRD4/NF-κB/NLRP3通路介导的巨噬细胞焦亡探讨三石汤抑制痛风性关节炎的分子机制研究

目的:观察三石汤对BRD4/NF-κB/NLRP3通路介导的巨噬细胞焦亡的影响,从而阐明三石汤抑制痛风性关节炎的分子机制。方法:以佛波酯诱导THP-1人单核细胞株分化巨噬细胞,并分为空白组、模型组、三石汤低剂量、中剂量、高剂量和BRD4抑制剂组。除空白组外,余下各组以尿酸单钠结晶诱导构建痛风性关节炎细胞模型。分别采用CCK8检测巨噬细胞的活性,流式细胞术检测巨噬细胞焦亡水平,酶联免疫吸附法检测LDH的活性和IL-1β及IL-18的含量,Western blot检测BRD4/NF-κB/NLRP3通路中相关蛋白的表达。结果:与空白组比较,模型组巨噬细胞活性下降,焦亡水平、LDH活性、IL-1β与IL-18含量、BRD4、p-NF-κB p65、NLRP3、Caspase-1 p20和IL-1β蛋白的表达均显著的上调,差异均具有统计学意义(P<0.05)。与模型组相比,三石汤组巨噬细胞活性上调,焦亡水平、LDH活性、IL-1β与IL-18含量、BRD4、p-NF-κB p65、NLRP3、Caspase-1 p20和IL-1β蛋白表达均显著的下降,差异均具有统计学意义(P<0.05)。结论:三石汤通过抑制BRD4/NF-κB/NLRP3通路激活抑制巨噬细胞焦亡,从而改善痛风性关节炎的炎症水平。

基于NLRP3炎症小体探讨三石汤治疗痛风性关节炎的机制研究

目的:观察三石汤对P2X7R/PKR信号通路介导的巨噬细胞NLRP3炎症小体活化的影响,从而明确三石汤治疗痛风性关节炎的分子机制。方法:将THP-1巨噬细胞分为空白组、模型组、三石汤低剂量、中剂量、高剂量和抑制剂组。除空白组外,其余各组用尿酸单钠结晶诱导构建痛风性关节炎细胞模型。采用流式细胞术检测各组巨噬细胞ROS水平,以ELISA法检测各组MDA含量及SOD和GSH-PX的活性,以Western blot检测P2X7R/PKR信号通路和NLRP3炎症小体相关蛋白的表达。在此基础上,构建巨噬细胞与滑膜细胞共培养体系,用CCK8和流式细胞术分别检测滑膜细胞的活性及凋亡水平。结果:与空白组相比,模型组巨噬细胞ROS水平、MDA的含量、SOD和GSH-PX的活性显著增加,NLRP3、Mature IL-1β、Pro IL-1β、Mature IL-18、Pro IL-18、Mature Caspase-1、GSDMD-NT、P2X7R和p-PKR蛋白的表达水平显著上调,GSDMD-FL蛋白的表达显著下调,差异均具有统计学意义(P<0.05和P<0.01)。与模型组比较,三石汤能够降低巨噬细胞ROS水平、MDA的含量、SOD和GSH-PX的活性,下调巨噬细胞NLRP3、Mature IL-1β、Pro IL-1β、Mature IL-18、Pro IL-18、Mature Caspase-1、GSDMD-NT、P2X7R和p-PKR蛋白的表达,上调GSDMD-FL蛋白的表达,差异均具有统计学意义(P<0.05和P<0.01)。此外,与空白组相对比,模型组中滑膜细胞的活性降低,其凋亡的水平上升,组间比较差异均具有统计学意义(P<0.05)。而与模型组相对比,三石汤能够显著地增加滑膜细胞的活性,抑制细胞的凋亡水平,各组间比较差异均具有统计学意义(P<0.05和P<0.01)。结论:三石汤能够通过抑制P2X7R/PKR信号通路的激活从而降低NLRP3活化水平,达到治疗痛风性关节炎的作用。

Mechanism of Sanshi decoction inhibits macrophage pyroptosis by inhibiting BRD4/NF-κB/NLRP3 pathway in the treatment of gouty arthritis

Objective:To observe the effect of Sanshi decoction on BRD4/NF-κB/NLRP3 pathwaymediated macrophage pyroptosis,so as to elucidate the molecular mechanism of Sanshi decoction in the treatment of gouty arthritis.Methods:THP-1 was induced into macrophages with foboside and the divided into the control group,model group,low-dose,medium-dose,high-dose group of Sanshi decoction,and BRD4 inhibitor group.Except for the control group,the remaining groups were induced with monosodium urate crystals to construct a gouty arthritis cell model.The activity of macrophages was detected by CCK8,the level of macrophage pyroptosis was detected by flow cytometry,the activity of LDH,the content of IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay,and the expression of related proteins in the BRD4/NF-κB/NLRP3 pathway was detected by Western blot.Results:Compared with the control group,macrophage activity was decreased in the model group,and the level of pyroptosis,LDH activity,contents of IL-1β and IL-18,expression levels of BRD4,p-NF-kB p65,NLRP3,Caspase-1 p20,and IL-1β protein were significantly up-regulated,the differences were statistically significant(P<0.05 and P<0.01).Compared with the model group,macrophage activity was up-regulated in the Sanshi Decoction,and the level of pyroptosis,LDH activity,IL-1β and IL-18 contents,expression levels of BRD4,p-NF-kB p65,NLRP3,Caspase-1 p20,and IL-1β protein were significantly decreased with statistically significant differences(P<0.05 and P<0.01).Conclusion:Sanshi decoction inhibits macrophage pyroptosis by inhibiting BRD4/NF-κB/NLRP3 pathway activation,thus improving the inflammation level of gouty arthritis.

Mechanism of Sanshi decoction in the treatment of gouty arthritis by NLRP3 inflammasome

Objective:To observe the effect of Sanshi decoction on P2X7R/PKR pathway-mediated activation of macrophage NLRP3 inflammasome to elucidate the molecular mechanism of Sanshi decoction in the treatment of gouty arthritis.Methods:THP-1 macrophages were divided into control group,model group,low dose group,medium dose group,high dose group of Sanshi decoction and inhibitor group.The remaining groups were induced with monosodium urate crystals to establish a gouty arthritis cell model except the control group.Flow cytometry was used to detect macrophage ROS levels in each group,ELISA to detect MDA levels and SOD and GSH-PX activities in each group,and Western blot to detect P2X7R/PKR pathway and NLRP3 inflammasome-associated protein expression.We also used CCK-8 and flow cytometry to measure MH7A activity and apoptotic levels.Results:Compared with the control group,the ROS level,the content of MDA,the activities of SOD and GSH-PX were significantly increased,and the expression levels of NLRP3,full-length IL-1β,pro-IL-1β,full-length IL-18,pro-IL-18,full-length caspase-1,GSDMD-NT,P2X7R and p-PKR protein expression levels were significantly upregulated,and GSDMD-FL protein expression was significantly downregulated in the model group,and that the differences between them were statistically significant(P<0.05 and P<0.01).Compared with the model group,Sanshi decoction could reduce macrophage ROS levels,MDA content,SOD and GSHPX activities,and downregulate macrophage NLRP3,mature IL-1β,pro IL-1β,mature IL-18,pro IL-18,mature caspase-1,GSDMD-NT,P2X7R and p-PKR protein expression,and upregulate GSDMD-FL protein expression,with statistically significant differences(P<0.05 and P<0.01).In addition,MH7A activity was downregulated,and apoptosis level was upregulated in the model group in comparison with the control group,and differences were all significantly different(P<0.05).As compared to the model group,Sanshi decoction could significantly increase the activity of MH7A and inhibit the level of apoptosis,and that the differences between them were statistically significant(P<0.05 and P<0.01).Conclusion:Sanshi decoction can achieve the therapeutic effect of gouty arthritis by inhibiting P2X7R/PKR pathway activation,thus reducing the activation level of NLRP3.

三物黄芩汤对白塞病模型小鼠IFN-γ、IL-17A水平的影响

目的:探讨三物黄芩汤对白塞病(BD)模型小鼠IFN-γ、IL-17A水平的影响。方法:收集临床样本并分离单个核细胞,采用RNA-seq高通量测序并鉴别白塞病与健康受试者的差异基因。通过虾原肌球蛋白致敏构建BD小鼠模型并给予三物黄芩汤浓缩液灌胃治疗,ELISA和流式细胞术检测外周血和单个核细胞中IFN-γ、IL-17A的水平,RT-PCR法检测脾脏组织中TBX21基因的表达水平。结果:与健康受试者相比,BD患者共有31个差异基因,其中19个基因表达上调,12个基因表达下调。PPI分析结果显示差异基因中排名前5的基因分别为TBX21、STAT3、IL15、PRF1和IFNG。动物实验结果表明,与空白组相比,模型组外周血及PBMC中IFN-γ、IL-17A的水平明显增高(P<0.01),三物黄芩汤能明显降低小鼠外周血及PBMC中IFN-γ、IL-17A的水平(P<0.05)。RT-PCR结果显示,与空白组相比,模型组脾脏组织中TBX21 mRNA的表达水平明显增高,(P<0.01)。三物黄芩汤能降低小鼠脾脏组织中TBX21 mRNA的表达水平(P<0.05)。结论:三物黄芩汤通过靶向抑制TBX21 mRNA的表达水平,进而抑制INF-γ和IL-17的水平,达到治疗BD的作用。

基于中医证候研究白塞病患者肠道菌群的特征性变化及临床指标相关性的分析

目的:利用16S rRNA技术分析湿热证白塞病(BD)患者肠道菌群的特征及其与临床指标的相关性。方法:49例白塞病患者按照中医证型分为湿热组(n=19)和非湿热组(n=30),另选择43例健康受试者作为健康组。收集各组基本信息及实验室检查指标,并采集粪便样本通过16S rRNA检测各组肠道菌群的多样性和丰度。结果:湿热证BD与非湿热证相比,眼部受累的发生率更高(P<0.05)。湿热证BD患者WBC、CRP、ESR和IgG均明显高于非湿热证患者(P<0.05)。Alpha多样性结果显示与健康组和非湿热组比较,湿热组Ace、Chao1和Shannon指数明显降低,Simpson指数明显升高(P<0.05)。与非湿热组相比,湿热组Actinobacteria门丰度增加(P<0.05);Collinsella、Intestinimonas和Enterobacteriaceae_unclassified属水平丰度增加,Clostridiales_unclassified、Ruminococcaceae_UCG009、Ruminococcaceae_UCG007和Ruminococcus_gauvre auii_group属水平丰度降低(P<0.05)。Pearson相关性分析结果显示,Oscillospira丰度与WBC、CRP、ESR和IgG水平均呈现正相关,Eubacterium和Cetobacterium丰度与WBC、CRP、ESR和IgG水平均呈现负相关。结论:肠道菌群的改变参与了BD的发病,同时不同中医证型BD患者肠道中菌群存在差异,此外肠道中特定的菌属还与BD血清炎症、免疫指标密切相关。

基于网络药理学分析三物黄芩汤治疗白塞病的机制研究

目的:基于网络药理学方法分析三物黄芩汤的主要化合物并预测其治疗白塞病可能作用靶点和机制。方法:通过TCMSP、TCMID和文献检索三物黄芩汤的主要化合物及其相应的作用靶点;通过GeneCards数据库检索白塞病的疾病靶标;三物黄芩汤和白塞病的交集靶点通过String数据库和Cytoscape软件构建网络可视化;利用DAVID数据库对交集靶点进行GO和KEGG富集分析;通过构建BD小鼠模型,RT-PCR法验证三物黄芩汤对把核心靶基因的调控作用。结果:通过数据库共检索到三物黄芩汤主要活性化合物81个,药物-疾病-靶点50个。GO富集分析共得到531条生物功能信息,KEGG共富集到96条信号通路,其中富集程度排名前5的信号通路有Fluid shear stress and atherosclerosis、AGE-RAGE signaling pathway in diabetic complications、IL-17 signaling pathway、TNF signaling pathway、Malaria、Chagas disease(American trypanosomiasis)。RT-PCR结果显示,与空白组比较,BD小鼠脾脏组织中IL-4、IL-1β、IL-6、INF-γmRNA的表达明显上调,Caspase 3和Caspase 8 mRNA的表达显著下调,差异均具有统计学意义(P<0.01)。与模型组相比,三物黄芩汤组小鼠脾脏组织中IL-4、IL-1β、IL-6、INF-γmRNA的表达明显下调,Caspase 3和Caspase 8 mRNA的表达显著上调,差异均具有统计学意义(P<0.01)。结论:三物黄芩汤能够通过多靶点、多通路治疗BD,同时为深入揭示其作用机制和治疗提供了生物信息学基础。

加减金刚丸治疗强直性脊柱炎

强直性脊柱炎现已成为一种常见疾病,无论是病程前期的腰背痛还是病程后期的致残性都严重影响患者的生活质量,急需安全有效的治疗方法。本文介绍2例运用加减金刚丸治疗强直性脊柱炎,疗效显著。