PCSK9介导NF-κB信号通路致小鼠肝脏脂肪变性的机制研究

Mechanism of Hepatic Steatosis Induced by NF-κB Signaling Pathway Mediated by PCSK9 in Mice

目的探讨PCSK9对小鼠肝脏脂肪变性的机制。方法以腺相关病毒8.2(adeno-associated virus 8.2,AAV8.2)为载体构建绿色荧光蛋白(green fluorescent protein,GFP)和人源PCSK9经尾静脉转染至小鼠肝脏表达。将36只C57BL/6J小鼠按照随机数字表法分为空白组、GFP组和PCSK9组,每组各12只,共饲养24周。通过苏木精-伊红(hematoxylin eosin,HE)染色观察肝脏组织形态,天狼星红染色(sirius red staining,SRS)观察肝脏纤维化程度,油红O(oil red O,ORO)染色观察肝脏脂质浸润程度。通过酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测血清PCSK9、白细胞介素(interleukin,IL)-1β,流式细胞术检测肝脏单核细胞胞内炎性细胞因子,包括IL-2、IL-4、γ-干扰素(interferon-γ,INF-γ)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、颗粒酶B(granulase B,GrB)。免疫组化检测肝组织巨噬细胞标志物CD68阳性细胞百分比。Western blot法检测PCSK9干预小鼠单核-吞噬细胞白血病细胞(RAW264.7)细胞胆固醇流出蛋白、清道夫受体相关蛋白、NF-κB信号通路相关蛋白表达。结果PCSK9组小鼠肝脏组织脂滴总量、脂肪变性、空泡化、纤维化程度明显增加或增强(P<0.001)。免疫组化检测结果显示,PCSK9组巨噬细胞标志物CD68阳性细胞百分比显著高于GFP组及空白组(P<0.05)。流式细胞术检测结果显示,PCSK9组IL-2、TNF-α在CD4+、CD8+细胞亚群百分比明显高于高脂GFP组(P<0.05),而IL-4、INF-γ、GrB在组间各细胞群中比较,差异无统计学意义(P>0.05)。Western blot法检测结果显示,与空白组比较,PCSK9显著降低LDLR水平,升高ATP结合盒转运体A1(ATP-binding cassette transporter A1,ABCA1)、清道夫受体B(scavenger receptor B,SRB或CD36)、Toll样受体4(Toll-like receptor 4,TLR4)、磷酸化IκBα激酶(phosphorylated inhibit kappa Bαkinase,p-IκBα)、磷酸化p65蛋白(phosphorylated p65,p-p65)蛋白水平(P<0.05)。结论PCSK9介导NF-κB信号通路致小鼠肝脏脂肪代谢失衡及炎性细胞因子浸润加速了肝脏脂肪变性。

Objective To investigate the mechanism of proprotein convertase subtilisin/kexin type 9|(PCSK9)on hepatic steatosis in mice.Methods Adeno-associated virus 8.2(AVV8.2)was used as veclor to constructed green fluorescent protein(GFP)and hu-man PCSK9 was transfected into mouse tail vein and express stably in liver.Thirty-six C57BL/6J mice were randomly divided into three groups:general diet blank group,high-fal GFP group and high-fat PCSK9 group,with 12mice in each group.After 24 weeks of feed-ing,the samples were taken to observe the morphology of liver tissue by hematoxylin eosin(HE)staining,the degree of liver fibrosis was observed by sirius red staining(SRS),and the degree of liver lipid infiltration was observed by oil red 0(ORO)staining.Serum PCSK9 and interleukin(IL)-1βwere detecled by enzyme linked immunosorbent assay(ELISA),and intraellular inflammatory cytokines in liver monocytes were detected by flow cytometry,including IL-2,IL-4,interferon-γ(INF-γ),tumor necrosis factor-α(TNF-α),granulase B(GrB).The percentage of macrophage CD68 positive cells in liver tissue was detected by immunohistochemistry.The expression of cholesterol eflux protein,scavenger receptor associated protein,and NF-KB signaling pathway associated protein in RAW264.7 cells treated with PCSK9 was detected by Wester blot.Results The total amount of lipid droplets,the degree of stealosis,blloon degeneration and the degree of fibrosis in liver tssue of mice in the high-fat PCSK9 group were signifeantly enhanced(P<0.001).Immunohistochemical results showed that the percentage of CD68 positive cells in the high-fat PCSK9 group was significantly higher than that in the high-fat GFP group and general diet blank group(P<0.05).The results of flow cytometry showed that the percentages of IL-2 and TNF-αin CD4^(+),CD8^(+)cell subsets was significantly higher in the high-fat PCSK9 than those in the high-fat GFP group(P<0.05),whereas the percentages of IL-4,INF-γand GrB in each cell subsets were not statistical difference between the groups(P>0.05).Compared with the general diet blank group,Western blot analysis showed that PCSK9 significantly reduced low density lipoprotein receptor(LDLR)protein levels and increased ATP-binding cassette transporter A1(ABCA1),scavenger receptor B(SRB)or CD36,Toll-like receptor 4(TLR4),phosphorylated inhibit kappa Bαkinase(p-IκBα),phosphorylated p65(p-p65)protein levels(P<0.05).Conclusion PCSK9mediates NF-κB signaling pathway induced hepatic lipo-metabolic imbalance and inflammation cytokines infiltrated accelerated hepatic steatosis in mice.