过氧化物酶体增殖物激活受体α在扩大肝切除后早期炎性反应中的保护作用

Protective effect of peroxisome proliferator-activated receptor-αin regulating the early inflammation response to extended hepatectomy

目的探讨大鼠扩大肝切除后早期炎性反应中的分子特征,筛选肝脏外科手术后肝衰竭的潜在治疗靶点。方法SD大鼠实施70%常规肝切除术(PH70)和80%扩大肝切除术(PH80),残存肝脏行转录组测序、筛选差异表达基因和进行加权基因共表达网络分析(WGCNA);实时定量聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)验证过氧化物酶体增殖物激活受体α(PPARα)的表达;继而在实施PH80大鼠中,使用PPARα激活剂WY14643(WY14643组)、PPARα拮抗剂GW6471(GW6471组)和二甲基亚砜组(DMSO组)预处理,苏木精-伊红(HE)染色和Suzuki标准评定肝脏病理损伤。自动生化分析仪检测血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST),酶联免疫吸附法检测肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)。组间差异比较采用t检验和单因素方差分析。结果转录组测序和WGCNA构建10个差异表达基因聚类模块,性状相关系数显示浅蓝色模块与PH80后1、12 h显著相关(皮尔森相关系数=0.36、0.36,P<0.05);选择浅蓝色模块中关键基因PPARα,显示PPARα在PH80后1 h表达显著降低(F=57.32,P<0.05),与炎性因子TNFα和IL-6峰值水平正相关(皮尔森相关系数=0.93、0.93,P<0.05);WY14643组较DMSO组,ALT[(543.1±201.9)U/L比(2514.7±158.2)U/L,t=8.90,P<0.05]、AST[(814.7±88.2)U/L比(1185.1±57.7)U/L,t=5.85,P<0.05]、TNFα[(107.3±2.0)U/L比(116.9±4.5)U/L,t=3.34,P<0.05]和IL-6水平[(2611.1±604.8)U/L比(3655.9±363.8)U/L,t=3.75,P<0.05]和Suzuki评分[(0.7±0.3)分比(1.6±0.2)分,t=3.67,P<0.05]均显著减低。结论转录组测序和WGCNA初步揭示了大鼠PH80后早期炎性反应中的分子特征,PPARα有望成为肝脏外科手术后肝衰竭的潜在治疗靶点。

Objective To analyze the molecular profiles of early inflammatory response following extended hepatectomy in rats and identify the therapeutic target of post-operative hepatic failure.Methods Major hepatectomy(PH70)and extended hepatectomy(PH80)were performed on the SD rat model,and the remnant livers were obtained dynamically for the high-throughput transcriptome analysis and weighted gene co-expression network analysis(WGCNA).The expression of peroxisome proliferator-activated receptor-α(PPARα)was detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)and Western blotting.Thereafter,the PH80 rats were administered PPARαagonist WY14643(WY14643 group),PPARαantagonist GW6471(GW6471 group)and dimethyl sulfoxide(DMSO group).Hepatic injury was assessed by hematoxylin and eosin(HE)staining and Suzuki criteria.Plasma samples were obtained to measure the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)by automatic chemistry analyzer,and tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)by enzyme linked immunosorbent assay.T-test and ANOVA were used for comparison between two groups or among more groups.Results Transcriptome sequencing and WGCNA identified 10 distinct gene co-expression modules.Interaction analysis identified that the lightcyan module was positively correlated with PH80 at 1 h and 12 h according to the correlation index.In the lightcyan module,PPARαwas selected,and down-regulated in the remnant liver at 1 h following PH80,and correlated with the levels of TNF-αand IL-6(Pearson correlation coefficient=0.93 and 0.93,P<0.05).Besides,administration of PPARαagonist attenuated hepatic inflammatory injury following PH80.ALT[(543.1±201.9)vs.(2514.7±158.2)U/L,t=8.90,P<0.05]and AST[(814.7±88.2)vs.(1185.1±57.7)U/L,t=5.85,P<0.05]levels,TNF-α[(107.3±2.0)vs.(116.9±4.5)U/L,t=3.34,P<0.05]IL-6[(2611.1±604.8)vs.(3655.9±363.8)U/L,t=3.75,P<0.05]levels,and histological Suzuki criteria(0.7±0.3 vs.1.6±0.2,t=3.67,P<0.05)in the WY14643 group were significantly reduced as compare with those in the DMSO group.Conclusion The molecular profiles after PH80 were identified by transcriptome sequencing and WGCNA,and PPARαcould be a potential therapy target for post-operative hepatic failure.