CCR1 is a novel target in the treatment of NASH by regulating macrophage activation through mTORC1 signaling

The number of patients with non-alcoholic steatohepatitis (NASH) is dramatically increasing globally. Recently, specific chemokine receptors have garnered interest as therapeutic targets in NASH. In different murine models with NASH, the level of CCR1 in plasma and the expression of CCR1 in liver increased, suggesting that CCR1 may be involved in the progression of NASH. Recent studies have shown that a variety of chemokines and their receptors play a certain role in the development of NASH. However, the role of CCR1 in pathogenesis of NASH remains obscure. Therefore, this study aims to explore the effect of CCR1 on the development of NASH. The results suggest that the absence of CCR1 prevents hepatic steatosis and abnormal glucose metabolism in the progression of NASH, and these results emphasize the impacts of CCR1 on macrophage recruitment and fibrosis in mice with NASH. Thus, the findings of the current study provide a new strategy for the clinical treatment of NASH. Overall design: 1. Both 8-week-old male C57BL/6 (wild-type, WT) mice and CCR1 knockout (CCR1- KO) mice were fed with normal chow (NC) and high-fat, high cholesterol and cholate diet (CL) for 12 weeks, respectively. The weight of mice was measured every week. Glucose tolerance test (GTT) and pyruvate tolerance test (PTT) were performed after overnight fasting. After 12 weeks of feeding, the mice were sacrificed and the liver tissues were harvested. The hepatic macrophages were determined by flow cytometry. 2. Plasma triglyceride (TG), total cholesterol (TC), non-esterified free fatty acid (NEFA), insulin (INS), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hepatic TG, TC, NEFA and hydroxyproline (HYP) were examined using ELISA or spectrophotometer according to the instructions. 3. The mRNA levels of liver lipid metabolism, inflammatory response and fibrosis related genes were detected by real-time fluorescence quantitative PCR (RT-qPCR). 4. Western blotting (WB) was used to detect the expression of proteins related to lipid metabolism, inflammatory signaling pathway and fibrosis in liver. 5. Hematoxylin and eosin (H&E) staining, Sirius Red staining and immunohistochemical staining (IHC) were used to evaluate the histology of liver sections 6. Protein chip and bioinformatic analysis were used to analyze the cluster of inflammatory proteins.

非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）患者数量在全球范围内呈显著增长趋势。近年来，特定趋化因子受体作为NASH的治疗靶点受到广泛关注。在不同的NASH小鼠模型中，血浆中CC趋化因子受体1（CC chemokine receptor 1, CCR1）水平及肝脏中CCR1的表达均升高，提示CCR1可能参与NASH的疾病进展。现有研究表明，多种趋化因子及其受体在NASH的发生发展中发挥一定作用，但CCR1在NASH发病机制中的具体作用仍不明确。因此，本研究旨在探讨CCR1对NASH发生发展的影响。结果显示，敲除CCR1可抑制NASH进程中的肝脏脂肪变性与糖代谢异常，同时证实CCR1对NASH小鼠的巨噬细胞招募及纤维化进程具有调控作用。本研究结果为NASH的临床治疗提供了全新策略。

实验整体设计：
1. 将8周龄雄性C57BL/6野生型（wild-type, WT）小鼠与CCR1敲除（CCR1 knockout, CCR1⁻/⁻）小鼠分别饲喂正常饲料（normal chow, NC）及高脂高胆固醇胆酸盐饲料（high-fat, high cholesterol and cholate diet, CL），持续12周。每周称量小鼠体重。于隔夜禁食后进行葡萄糖耐量试验（glucose tolerance test, GTT）与丙酮酸耐量试验（pyruvate tolerance test, PTT）。饲喂12周后处死小鼠，采集肝脏组织。采用流式细胞术检测肝脏巨噬细胞水平。
2. 采用酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）或分光光度法，按照试剂盒说明书检测血浆甘油三酯（triglyceride, TG）、总胆固醇（total cholesterol, TC）、非酯化游离脂肪酸（non-esterified free fatty acid, NEFA）、胰岛素（insulin, INS）、丙氨酸氨基转移酶（alanine aminotransferase, ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase, AST），以及肝脏组织TG、TC、NEFA与羟脯氨酸（hydroxyproline, HYP）的含量。
3. 采用实时荧光定量聚合酶链式反应（real-time fluorescence quantitative PCR, RT-qPCR）检测肝脏脂质代谢、炎症反应及纤维化相关基因的mRNA表达水平。
4. 采用蛋白质印迹法（Western blotting, WB）检测肝脏组织中脂质代谢、炎症信号通路及纤维化相关蛋白的表达情况。
5. 采用苏木精-伊红（hematoxylin and eosin, H&E）染色、天狼星红染色及免疫组织化学染色（immunohistochemical staining, IHC）评估肝脏组织切片的组织学特征。
6. 采用蛋白质芯片技术与生物信息学分析，对炎症蛋白簇进行检测与分析。