目的：研究髓系特异性Notch1基因敲除抑制STING信号调控肝细胞脂质自噬的机制作用。方法：采用高脂饮食（HFD）和小鼠骨髓源性巨噬细胞（BMMs）建立非酒精性脂肪肝（NASH）小鼠模型，并分离原代肝细胞构建共培养系统。将12只Notch1(FL/FL)小鼠随机分为两组：Notch1(FL/FL)+正常饮食（NCD）组和Notch1(FL/FL)+HFD组。此外，将12只Notch1(M-KO)小鼠随机分成两组：Notch1(M-KO)+NCD组和Notch1(M-KO)+HFD组。收集小鼠血清样品中的血清丙氨酸氨基转移酶（sALT）、总胆固醇（TC）和三酰甘油（TG）。收集肝组织样本进行H&E染色、免疫荧光（IF）、Western blot和qRT-PCR。通过酶联免疫吸附试验（ELISA）检测上清液中的肿瘤坏死因子（TNF）-α。使用t检验进行组间数据比较。结果：成功构建了小鼠NASH模型、小鼠BMMs共培养系统和原代肝细胞。与Notch1(FL/FL)+HFD组相比，Notch1(M-KO)+HFD组的血清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）和TC（（33.70 ± 8.43）mg/g vs （90.53 ± 12.53）mg/g，t = 9.917，P < 0.001）明显增加。肝组织的HE染色显示肝细胞中出现显著的球形变化，而IF染色显示巨噬细胞浸润增多（t = 7.346，P < 0.001）。与共培养Notch1(FL/FL)巨噬细胞的肝细胞组相比，Notch1(M-KO)组的BODIPY探针显示肝细胞内脂滴（LDs）沉积明显增加（t = 3.835，P < 0.001）。溶酶体相关膜蛋白1（LAMP1）、LDs（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）和自噬相关基因12（Atg12）（t = 28.36，P < 0.001）的共定位表达下降，而P-62的表达增加（t = 3.253，P = 0.03），表明自噬流下降。此外，LC3和LDs的共定位下降（t = 5.24，P = 0.0003），表示脂质自噬减少。与Notch1(FL/FL)组相比，Notch1(M-KO) BMMs小鼠组中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）和p-P65（t = 12.7，P = 0.002）蛋白的表达增加，伴随炎症介质干扰素（IFN）-β（t = 7.978，P < 0.001）、TNFα（t = 8.496，P = 0.001）、白介素-1β（IL-1β）（t = 4.7，P < 0.001）和CXCL-10（t = 4.428，P = 0.001）的mRNA表达增加。使用CRISPR/Cas9在BMMs Notch1(M-KO)小鼠中敲除STING基因。与CRISPR-Control组相比，STING-KO BMMs组中P-TKB1（t = 2.909，P = 0.044）、p-IRF3（t = 10.96，P < 0.001）、p-IRF3（t = 10.96，P < 0.001）和p-P65（t = 7.091，P = 0.002）蛋白的表达较低。上清液中TNF-α的释放减少（732.3 ± 129.35 pg/ml vs. 398.17 ± 47.15 pg/ml，t = 4.204，P = 0.014）。然而，在与STING-KO BMMs共培养的肝细胞中，LC3-II/LC3-I（t = 7.546，P = 0.001）增加，p-62（t = 10.96，P < 0.001）表达减少，自噬流增加，LC3和LDs的共定位增加，脂质自噬增加，LDs沉积减少。结论：髓系特异性Notch1基因敲除可激活巨噬细胞的STING信号通路，增加炎症介质基因的表达，抑制肝细胞的自噬流和脂质自噬，加重LDs沉积和NASH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)-α was 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-1 β (IL-1 β) (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-α in 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.