PCSK9在萝卜硫素改善棕榈酸诱导的肝细胞自噬流中的作用研究

Role of PCSK9 in sulforaphane attenuating palmitic acid-induced autophagic flux in hepatic cells

目的探究萝卜硫素(sulforaphane,SFN)对肝细胞损伤及自噬的影响及机制。方法用200μmol/L棕榈酸(palmitic acid,PA)处理人正常肝细胞株(HHL5)24 h建立肝细胞损伤模型组(PA组),用5μmol/L的SFN与200μmol/L PA共同处理细胞24 h为实验组(PA+SFN组),溶剂处理为对照组。CCK-8检测细胞增殖活力;CELLROXTM DEEP RED探针装载细胞后,流式细胞仪检测活性氧簇(reactive oxygen species,ROS)水平;试剂盒检测细胞内丙二醛(Malondialdehyde,MDA)水平;qPCR检测炎症标志物IL-1β、TNF-α转录水平;蛋白免疫印迹检测自噬相关蛋白SQSTM1及LC3Ⅱ蛋白表达水平;提取细胞总RNA进行全基因转录组测序,分析数据筛选差异基因,并对差异基因进行KEGG通路富集分析;用PCSK9 siRNA转染HHL5细胞,qPCR检测PCSK9转录水平,流式细胞仪检测ROS水平,蛋白免疫印迹检测SQSTM1及LC3Ⅱ蛋白表达水平。结果相对于PA组,SFN干预后HHL5细胞增殖活力升高,MDA含量以及ROS水平降低;PA组SQSTM1、LC3Ⅱ蛋白表达升高,SFN干预后SQSTM1表达降低,LC3Ⅱ表达进一步升高;转录组数据分析发现差异基因主要富集于自噬通路,并筛选出目标基因PCSK9;相对于对照组,PA组的PCSK9转录水平升高,SFN干预后PCSK9转录水平降低;PA组在PCSK9敲降后,与未敲降的PA组比,细胞内ROS水平有所降低,和SFN降低ROS趋势一致,PCSK9敲降后自噬蛋白SQSTM1表达降低,LC3Ⅱ表达水平升高,和SFN恢复自噬流的趋势一致。结论SFN可以改善PA引起的肝细胞损伤,其机制可能与其抑制PCSK9转录从而调控自噬流有关。

Objective To determine the effect and underlying mechanism of sulforaphane(SFN)on hepatocyte injury and autophagy.Methods HHL5 cells were treated with 200μmol/L palmitic acid(PA)for 24 h to establish a hepatocyte injury model.Then the model was treated with 5μmol/L SFN and co-cultured with 200μmol/L PA for 24 h.So there were 3 groups of cells,that is,control group,PA group,and PA+SFN group.Cell viability,malonaldehyde(MDA)content,and production of reactive oxygen species(ROS)were measured with CCk-8 assay,MDA reagent kit,and CellROXTM Deep Red Reagent,respectively.qPCR was used to detect the transcription levels of IL-1βand TNF-α,and Western blotting was employed to measure the protein expression of SQSTM1 and LC3II.The total RNA of the cell was extracted by TRIzol reagent to sequence the whole gene transcriptome,and the RNA sequence was analyzed to figure out differentially expressed genes.And the results were subjected to Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.Furthermore,HHL5 cells were pretreated with PCSK9 siRNA for 24 h,then the mRNA level of PCSK9 was measured by qPCR,and the expression of SQSTM1 and LC3 II were measured by Western blotting.Results Compared with the PA group,the cell survival rate was increased,and the levels of MDA and ROS were decreased significantly in the PA+SFN group(P<0.05).The transcriptional levels of IL-1βand TNF-αwere reduced obviously in the PA+SFN group(P<0.05).The expression of SQSTM1 was decreased and that of LC3II was increased in the PA+SFN group.KEGG enrichment analysis suggested that differential expressed genes were enriched on the autophagy pathway.PCSK9 was selected as our candidate gene.Compared with the control group,the transcriptional level of PCSK9 was increased in the PA group,while was decreased significantly in the PA+SFN group(P<0.05).The level was elevated in the PA group but decreased after SFN intervention.After PCSK9 knockdown,the ROS level in the PA group was decreased compared with that in the PA group without siPCSK9 treatment,which was consistent with the trend of SFN-induced ROS reduction.After PCSK9 knockdown,the expression level of LC3II was increased,which was consistent with the trend of SFN-induced autophagy flux recovery.Conclusion SFN can attenuate hepatocyte injury induced by PA,which may be related to the inhibition of PCSK9 at transcriptional level,thus regulating autophagic flux.