基于转录组学与加权基因共表达网络分析探讨降脂轻身胶囊介导NF-κB/NLRP3/Caspase-1信号轴改善高脂血症小鼠的作用及机制

Exploring the Mechanism of Jiangzhi Qingshen Capsules Mediating NF-κB/NLRP3/Caspase-1 Signaling Axis to Improve Hyperlipidemia in Mice Based on Transcriptomics and Weighted Gene Co-expression Network Analysis

目的基于转录组学与加权基因共表达网络分析(WGCNA)探讨降脂轻身胶囊介导NF-κB/NLRP3/Caspase-1信号轴改善高脂血症的作用机制。方法将40只C57BL/6小鼠随机分为对照组、模型组、瑞舒伐他汀组(1.3 mg·kg^(-1))及降脂轻身胶囊低、高剂量组(0.6、2.4 g·kg^(-1)),每组8只。除对照组给予普通饲料外,其余各组给予高脂饲料维喂养,以复制高脂血症模型。各给药组小鼠每日按上述剂量灌胃给药,每日1次,连续干预10周。称取小鼠肝脏质量并计算肝脏指数、Lee’s指数、肝脏胫骨比值;采用HE染色、油红O染色法观察肝脏组织病理学变化;ELISA法检测血清生化指标:总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、白细胞介素(IL)-18、IL-1β;采用博奥晶典小鼠真核转录组芯片V4对肝脏组织进行转录组测序,并进行芯片表达信息与血脂水平性状信息的加权基因共表达网络分析;分析得到降脂轻身胶囊干预高脂血症的核心基因群(潜在作用靶点),并进行蛋白质互作(PPI)网络分析及GO功能、KEGG通路富集分析;采用RT-qPCR法检测肝脏组织NF-κB、NLRP3、Caspase-1、IL-18、IL-1βmRNA表达水平;免疫荧光法检测肝组织中NF-κB、NLRP3、Caspase-1蛋白表达。结果与模型组比较,降脂轻身胶囊能明显降低高脂血症小鼠的体质量、肝湿质量、肝指数、Lee’s指数、肝脏胫骨比值(P<0.05);有效减轻高脂血症小鼠肝组织炎性浸润及脂肪变性,明显减少脂质沉积面积(P<0.05);明显降低血清TG、TC、LDL-C、ALT、AST、IL-18、IL-1β水平(P<0.05);明显下调肝组织中NF-κB、NLRP3、Caspase-1蛋白及mRNA表达(P<0.05),降低肝组织中IL-18、IL-1βmRNA表达水平(P<0.05)。转录组学与加权基因共表达网络分析显示,降脂轻身胶囊对血脂的调控作用与炎症通路密切相关。结论降脂轻身胶囊能够调节NF-κB/NLRP3/Caspase-1信号轴抑制高脂血症小鼠肝组织炎症反应,进而改善高脂血症小鼠的血脂水平。

Objective Based on transcriptomics and weighted gene co-expression network analysis(WGCNA),this study aims to explore the mechanism of Jiangzhi Qingshen Capsules mediating the nuclear transcription factor(NF)-κB/NOD like receptor heat protein domain related protein 3(NLRP3)/cysteine protease-1(Caspase-1)signaling axis to improve hyperlipidemia.Method Forty C57BL/6 mice were randomly divided into control group,model group,rosuvastatin group(1.3 mg·kg^(-1)),Jiangzhi Qingshen Capsules low-and high-dose groups(0.6 and 2.4 g·kg^(-1)),with eight mice in each group.Except the control group was given regular feed,other groups were given high-fat feed to replicate the hyperlipidemia model.The treatment groups were orally administered the corresponding drug once a day for 10 consecutive weeks.The mass of rat liver was taken to calculate the liver index,Lee’s index and ratio of liver weight to tibia length.Hematoxylin eosin(HE)staining and oil red O staining were used to observe pathological changes in liver tissue.Serum biochemical indicators including total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),alanine aminotransferase(ALT),aspartate aminotransferase(AST),interleukin(IL)-18 and IL-1βwere detected by ELISA.The transcriptome sequencing of liver tissue was performed using Bo’ao Jingdian mouse eukaryotic transcriptome chip V4,then WGCNA of chip expression information and the trait data of blood lipid levels was carried out.The core gene groups(potential targets)of Jiangzhi Qingshen Capsule intervention in hyperlipidemia were obtained,and they were further used to perform protein-protein interaction(PPI)network analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)enrichment analysis.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the mRNA levels of NF-κB,NLRP3,Caspase-1,IL-18,and IL-1βin liver,and immunofluorescence was used to detect the protein expression of NF-κB,NLRP3 and Caspase-1 in liver.Results Compared with model group,Jiangzhi Qingshen Capsules effectively reduced the body weight,liver wet weight,liver index,Lee's index and ratio of liver weight to tibia length(P<0.05),effectively alleviate inflammatory infiltration and steatosis in liver tissue of hyperlipidemic mice,and significantly reduce lipid deposition area(P<0.05).Moreover,Jiangzhi Qingshen Capsules obviously improved serum TC,LDL-C,ALT,AST,IL-18,and IL-1βlevels(P<0.05),effectively down-regulated the protein and mRNA expression of NF-κB,NLRP3 and Caspase-1 in liver tissue(P<0.05),and decreased the mRNA levels of IL-18 and IL-1β(P<0.05).Transcriptomics and WGCNA showed that the regulatory effect of Jiangzhi Qingshen Capsules on blood lipids may be closely related to the inflammatory pathway.Conclusion Jiangzhi Qingshen Capsules can inhibit inflammatory reaction in liver tissue of hyperlipidemic mice by mediating the NF-κB/NLRP3/Caspase-1 signaling axis,thereby improving blood lipid levels.