【摘要】 目的 从调控疤痕相关巨噬细胞（SAM）角度探讨扶正化瘀胶囊改善代谢相关脂肪性肝病肝纤维化的分子机制。方法 24只C57小鼠随机分为对照组（Con组）、模型组（Model组）和扶正化瘀胶囊组（FZHY组）。采用高脂饮食（HFD）联合CCl₄腹腔注射6周构建代谢相关脂肪性肝病肝纤维化小鼠模型。自造模第2周起，连续灌胃5周，FZHY组小鼠给予扶正化瘀胶囊灌胃，Con组和Model组以等量生理盐水灌胃。检测血清肝酶、肝脏甘油三酯（TG）及羟脯氨酸（Hyp）含量。观察肝组织HE及天狼星红染色情况。取Model组和FZHY组肝组织样本各3例，进行转录组测序探究扶正化瘀胶囊改善代谢相关脂肪性肝病肝纤维化的分子机制。Western Blot和RT-q PCR检测肝组织中SAM标志物白细胞分化抗原9（CD9）和髓系细胞触发受体2（TREM2）、SAM促纤维化功能基因包括转化生长因子-β1（TGF-β1）、血小板源性生长因子β（PDGFβ）、TNF超家族成员12（TNFSF12）及上游调控分子转录激活因子3（ATF3）的蛋白/基因表达。采用扶正化瘀胶囊含药血清干预脂多糖（LPS）联合TGF-β1诱导的促炎骨髓来源巨噬细胞（BMDM），免疫荧光、Western Blot和RT-q PCR检测TREM2和ATF3的表达。计量资料两组间比较采用成组t检验，多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。结果 FZHY组小鼠肝脏肝酶、肝组织TG和Hyp含量、NAS评分以及天狼星红染色阳性面积较Model组显著降低（P值均<0.01）。RNA-seq分析显示差异基因主要富集于趋化因子信号通路，扶正化瘀胶囊能够显著下调C-C基元趋化因子配体2、CX3C趋化因子配体1及其受体CX3C趋化因子受体1的表达（P值均<0.01）。扶正化瘀胶囊显著降低小鼠肝组织CD9和TREM2，以及ATF3的蛋白及基因表达水平（P值均<0.05）。扶正化瘀胶囊体外显著降低小鼠肝组织TGFβ1、PDFGB和TNFSF12 mRNA表达（P值均<0.01）。扶正化瘀胶囊减弱促炎BMDM细胞中TREM2荧光强度，并显著降低促炎BMDM中ATF3的m RNA和蛋白表达（P值均<0.05）。结论 扶正化瘀胶囊具有较好的改善小鼠代谢相关脂肪性肝病肝纤维化的作用，其作用机制可能与下调ATF3表达抑制SAM相关。更多还原

【Abstract】 Objective To investigate the molecular mechanism of Fuzheng Huayu Capsule in improving liver fibrosis in metabolic associated fatty liver disease by regulating scar-associated macrophages（SAMs）. Methods A total of 24 C57 mice were randomly divided into control group（Con group）, Model group, and Fuzheng Huayu Capsule group（FZHY group）. Mice were given a highfat diet and intraperitoneal injection of CCl₄ for six weeks to establish a model of liver fibrosis in metabolic associated fatty liver disease. The drug was given by gavage for 5 consecutive weeks since week 2 of modeling. FZHY group was administered Fuzheng Huayu Capsule via oral gavage, while the Con and Model groups received an equal volume of saline solution via oral gavage. The serum levels of liver enzymes were measured, as well as the levels of triglyceride（TG） and hydroxyproline（Hyp） in the liver. HE staining and picrosirius red staining were used to observe liver tissue. Three liver tissue samples were collected from the Model group and the FZHY group, and transcriptome sequencing was performed to investigate the molecular mechanism of Fuzheng Huayu Capsule in improving liver fibrosis in metabolic associated fatty liver disease. Western blot and RT-qPCR were used to measure the protein and/or mRNA expression levels of SAM markers（CD9 and triggering receptor expressed on myeloid cells 2 [TREM2]）, profibrogenic genes（transforming growth factor-β1 [TGFβ1], platelet-derived growth factor subunit beta [PDGFβ], and TNF superfamily member 12 [TNFSF12]）, and the upstream regulator activating transcription factor 3（ATF3） in liver tissue. The serum containing Fuzheng Huayu Capsule was used for the intervention of pro-inflammatory bone marrow-derived macrophages（BMDMs） induced by lipopolysaccharide and TGF-β1, and immunofluorescence assay, Western blot, and RT-qPCR were used to measure the expression levels of TREM2 and ATF3. The independent-samples t test was used for comparison of continuous data between two groups, and a one-way analysis of variance was used for comparison between multiple groups, while the least significant difference t-test was used for further comparison between two groups. Results Compared with the Model group, the FZHY group had significant reductions in the levels of liver enzymes, the levels of TG and Hyp in the liver, NAS score, and Sirius Red staining-positive area（all P<0.01）. The RNA-seq analysis showed that differentially expressed genes were mainly enriched in chemokine signaling pathways, and Fuzheng Huayu Capsule significantly downregulated the expression of CCL2, CX3 CL1, and CX3 CR1（all P<0.01）. Fuzheng Huayu Capsule significantly reduced the protein and mRNA expression levels of CD9, TREM2, and ATF3 in liver tissue（all P<0.05）. In vitro, Fuzheng Huayu Capsule significantly reduced the mRNA expression levels of TGFβ1, PDGFβ, and TNFSF12 in liver tissue（all P<0.01）. Fuzheng Huayu Capsule also attenuated TREM2 fluorescence intensity in proinflammatory BMDMs and significantly reduced the mRNA and protein expression levels of ATF3（all P<0.05）. Conclusion Fuzheng Huayu Capsule has a marked therapeutic effect on mice with liver fibrosis in metabolic associated fatty liver disease, possibly by downregulating the expression of ATF3 and inhibiting SAMs.更多还原