Notch1抑制干扰素刺激基因信号调控非酒精性脂肪性肝炎中肝细胞脂噬作用的机制

Notch1 inhibits the mechanistic role of STING signaling to regulate hepatocyte lipophagy in nonalcoholic steatohepatitis

目的:研究髓系Notch1特异性敲除抑制干扰素刺激基因(STING)信号调控肝细胞脂噬作用机制。方法:通过高脂饮食(HFD)构建小鼠非酒精性脂肪性肝炎(NASH)模型,分离小鼠骨髓来源巨噬细胞(BMMs)和原代肝细胞以构建共培养体系。12只Notch1^(FL/FL)小鼠随机分成2组:Notch1^(FL/FL)+正常饮食(NCD)组、Notch1^(FL/FL)+HFD组;12只Notch1^(M-KO)小鼠随机分成2组:Notch1^(M-KO)+NCD组、Notch1^(M-KO)+HFD组。分别留取小鼠血清标本进行血清丙氨酸转氨酶(ALT)、总胆固醇(TC)、甘油三酯(TG)检查,取肝组织样本进行HE染色、免疫荧光(IF)、免疫印迹、qRT-PCR检测,酶联免疫吸附法(ELISA)检测上清液肿瘤坏死因子(TNF)α水平。组间数据比较采用t检验。结果:成功构建小鼠NASH模型,及小鼠BMMs和原代肝细胞共培养体系。与Notch1^(FL/FL)+HFD组相比,Notch1^(M-KO)+HFD组血清ALT[(250.02±58.21)U/L与(370.70±54.57)U/L,t=3.705,P=0.004]、TG[(29.90±3.54)mg/g与(43.83±8.56)mg/g,t=3.685,P=0.004]和TC[(33.70±8.43)mg/g与(90.53±12.53)mg/g,t=9.917,P<0.001],明显升高;肝组织HE染色显示肝细胞气球样变明显,IF染色显示巨噬细胞浸润增加(t=7.346,P<0.001)。与Notch1^(FL/FL) BMMs共培养的肝细胞组相比,BODIPY探针显示,Notch1^(M-KO)组中肝细胞内脂滴(LDs)沉积明显增多(t=3.835,P<0.001);溶酶体相关膜蛋白1(LAMP1)和LDs共定位减少(t=7.103,P<0.001),微管相关蛋白轻链3(LC3)-II/LC3-I:(t=5.0,P=0.007)、自噬相关基因12(Atg12)(t=28.36,P<0.001)表达下降,p-62表达上升(t=3.253,P=0.03);且LC3和LDs共定位减少(t=5.24,P=0.0003)。与Notch1^(FL/FL)组相比,Notch1^(M-KO)组小鼠BMMs中p-STING(t=5.318,P=0.006)、p-TANK1结合激酶1(TKB1)(t=6.467,P=0.002)、p-干扰素调节因子3(IRF3)(t=14.61,P<0.001)、p-P65(t=12.7,P=0.002)蛋白表达上升,同时伴有干扰素(IFN)-β(t=7.978,P<0.001)、TNFα(t=8.496,P<0.001)、白细胞介素1β(IL-1β)(t=4.7,P<0.001)、趋化因子配体-10(t=4.428,P=0.001)炎性介质的mRNA表达明显上升。使用CRISPR/Cas9敲除Notch1^(M-KO)小鼠BMMs中STING基因,与CRISPR-Control组相比,STING-KO组BMMs中p-TKB1(t=2.909,P=0.044)、p-IRF3(t=10.96,P<0.001)、p-P65(t=7.091,P=0.002)蛋白表达下降,上清液中TNFα[(732.3±129.35)pg/ml与(398.17±47.15)pg/ml,t=4.204,P=0.014]释放减少。而与STING-KO BMMs共培养的肝细胞中LC3-II/LC3-I(t=7.546,P=0.001)上升,p-62(t=10.96,P<0.001)表达下降,且LC3和LDs共定位增多。结论:髓系Notch1特异性敲除通过激活巨噬细胞STING信号,增加炎性介质基因表达,抑制肝细胞自噬流及脂噬的发生,加重LDs沉积和促进NASH的进展。

Objective To study the mechanistic role of myeloid-specific Notch1 knockout inhibiting STING signaling to regulate hepatocyte lipophagy.Methods A mouse model of nonalcoholic steatohepatitis(NASH)was established using a high-fat diet(HFD)and mouse bone marrow-derived macrophages(BMMs).Primary hepatocytes were isolated to construct a co-culture system.Twelve Notch1^(FL/FL) mice were randomly divided into two groups:the Notch1^(FL/FL)+normal diet(NCD)and the Notch1^(FL/FL)+HFD group.Further,12 Notch1^(M-KO) mice were randomly divided into two groups:Notch1^(M-KO)+NCD,and Notch1^(M-KO)+HFD group.Serum alanine aminotransferase(sALT),total cholesterol(TC)and triglyceride(TG)were collected from mice serum samples.Liver tissue samples were collected for H&E staining,immunofluorescence(IF),Western blot and qRT-PCR.Tumor necrosis factor(TNF)-αwas detected in the supernatant by enzyme-linked immunosorbent assay(ELISA).The comparison of inter group data was conducted using a t-test.Results The mouse NASH model,mouse BMMs co-culture system,and primary hepatocytes were successfully constructed.Compared with the Notch1^(FL/FL)+HFD group,the Notch1^(M-KO)+HFD group showed a significant increase in serum ALT[(250.02±58.21)U/L vs(370.70±54.57)U/L,t=3.705,P=0.004],TG[(29.90±3.54)mg/g vs(43.83±8.56)mg/g,t=3.685,P=0.004],and TC[(33.70±8.43)mg/g vs(90.53±12.53)mg/g,t=9.917,P<0.001].HE staining of liver tissue showed remarkable balloon-like alterations in liver cells,while IF staining demonstrated increased macrophage infiltration(t=7.346,P<0.001).Compared with the hepatocyte group co-cultured with Notch1^(FL/FL) BMMs,the BODIPY probe showed a significant increase in lipid droplet(LDs)deposition in liver cells in the Notch1^(M-KO) group(t=3.835,P<0.001).The co-localization of lysosomal associated membrane protein 1(LAMP1),LDs(t=7.103,P<0.001),microtubule-associated protein light chain 3(LC3)-II/LC3-I(t=5.0,P=0.007),and autophagy associated gene 12(Atg12)(t=28.36,P<0.001)had decreased expression,while P-62 had increased expression(t=3.253,P=0.03),indicating a decrease in autophagic flow.Additionally,LC3 and LDs colocalization decreased(t=5.24,P=0.0003),indicating reduced lipophagy.Compared with the Notch1^(FL/FL) group,the Notch1^(M-KO) BMMS mouse group showed an increase in the expression of p-STING(t=5.318,P=0.006),p-TANK1 binding kinase 1(TKB1)(t=6.467,P=0.002),p-interferon regulatory factor 3(IRF3)(t=14.61,P<0.001),and p-P65(t=12.7,P=0.002)protein,accompanied by mRNA expression of the inflammatory mediators interferon(IFN)-β(t=7.978,P<0.001),TNFα(t=8.496,P=0.001),interleukin-1β(IL-1β)(t=4.7,P<0.001),and CXCL-10(t=4.428,P=0.001).The STING gene was knocked out in the BMMs Notch1^(M-KO) mice using CRISPR/Cas9.Compared with the CRISPR-Control group,the expression of P-TKB1(t=2.909,P=0.044),p-IRF3(t=10.96,P<0.001),p-IRF3(t=10.96,P<0.001),and p-P65(t=7.091,P=0.002)proteins was lower in the STING-KO BMMs group.The release of TNF-αin the supernatant was decreased(732.3±129.35 pg/ml vs.398.17±47.15 pg/ml,t=4.204,P=0.014).However,in hepatocytes co-cultured with STING-KO BMMs,LC3-II/LC3-I(t=7.546,P=0.001)increased,p-62(t=10.96,P<0.001)expression decreased,autophagic flow increased,and the colocalization of LC3 and LDs increased,lipophagy increased,and LDs deposition decreased.Conclusion Myeloid-specific Notch1 knockout can activate macrophages STING signaling,increase the expression of inflammatory mediator genes,inhibit the occurrence of autophagy flow and lipophagy in hepatocyte cells,and aggravate LDs deposition and NASH progression.