Mechanisms of Shugan Lipi Decoction (疏肝理脾汤) in Treating Rats with Metabolic Cssociated Steatohepatitis Based on TIM-3-Regulated Macrophage Polarization

Author：WANGYa1, SHI Yuanyuan2, ZHONG Hangyu3, ZHANG Tao1

Unit：1.The First Hospital of Hunan University of Chinese Medicine, Changsha Hunan 410007, China; 2.Yulin Chinese Medicine Hospital, Yulin Shaanxi 719000, China; 3.Changsha Ninth Hospital, Changsha Hunan 410007, China

Quote：引用：王雅，石媛媛，钟航宇，张涛.基于TIM-3调控巨噬细胞极化探讨疏肝理脾汤干预代谢相关脂肪性肝炎大鼠作用机制[J].中医药导报,2026,32(4):23-29,66.

DOI：10.13862/j.cn43-1446/r.2026.04.005

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Abstract：

Objective: Based on the mechanism of immune regulatory protein Tim-3 regulating macrophage polarization, this study aims to explore the targets and immunological mechanisms of Shugan Lipi decoction intervention in metabolic associated steatohepatitis (MASH) rats through animal and cell experiments. Methods: Totally 30 male SD rats were randomly divided into Shugan Lipi decoction group, model group, and blank group, with 10 rats in each group. The blank group was given conventional feed, while the Shugan Lipi decoction group and model group were given methionine choline deficient feed for 4 weeks to construct a MASH animal model. After successful modeling, all three groups were given a normal diet. The Shugan Lipi decoction group was intervened with Shugan Lipi decoction [6.18 g/kg (calculated based on crude drug)], while the other two groups were intervened with an equal amount of physiological saline. After 4 weeks of intervention， Masson staining was used to evaluate liver tissue pathology. Western blotting and immunohistochemical were used to evaluated TIM-3 expression. LPS was used to induce polarization of RAW264.7 macrophages, and the RAW264.7 macrophages were divided into a control group (20% blank plasma+1 μg/mL LPS co culture) and a traditional Chinese medicine group (20% Shugan Lipi decoction drug containing plasma+1μg/mL LPS co culture). Additionally, a blank group containing only CCK-8 in culture medium without plasma was established. After intervention for 12, 24, and 48 h, cell viability was detected by CCK-8 assay. Inflammatory factors were detected by enzyme linked immunosorbent assay (ELISA), and M1/M2 phenotype markers were detected by flow cytometry. Results: The blank group showed normal hepatocyte structure in rat liver tissue, while the model group and the Shugan Lipi decoction group exhibited hepatocyte steatosis, ballooning degeneration, and inflammatory cell infiltration. Compared with the blank group, the expression of TIM-3 in liver tissue of the model group and the Shugan Lipi decoction group was significantly reduced (P<0.01). Compared with the model group, the expression of TIM-3 in liver tissue was significantly enhanced in the Shugan Lipi decoction group (P<0.01). Compared with the blank group, the viability of RAW264.7 cells in the control group and the traditional Chinese medicine group was significantly enhanced at 12, 24, and 48 h of intervention (P<0.01). Compared with the control group, the viability of RAW264.7 cells in the traditional Chinese medicine group was significantly enhanced at 12, 24, and 48 h of intervention (P<0.01). In the control group and the traditional Chinese medicine group, pairwise intra-group comparisons were conducted at 12, 24, and 48 h, with significant enhancement in activity observed at all time points (P<0.01). The supernatant of RAW264.7 cells in each group was detected, and the expression levels of TNF-α, IL-6, as well as the positive rates of CD80 and CD86 in the traditional Chinese medicine group were significantly decreased compared with the control group after intervention for 12 h and 48 h (P<0.01). The expression levels of IL-10, TGF-β, as well as the positive rates of CD206 and Arg-1 in the traditional Chinese medicine group were significantly increased compared with the control group (P<0.01). At 12, 24, and 48 h of intervention, pairwise comparisons within the control group and the traditional Chinese medicine group showed that over time, the levels of IL-6 and TNF-α continuously increased (P<0.01), while the levels of IL-10 and TGF-β continuously decreased (P<0.01). At 48 h of intervention, the positive rates of CD80 and CD86 in both the control group and the traditional Chinese medicine group were higher than those in the same group at 12 h (P<0.01), whereas the positive rates of Arg-1 and CD206 were lower than those in the same group at 12 h (P<0.01). Conclusion: Shugan Lipi decoction can improve the pathological process of MASH by up-regulating TIM-3 expression, regulating macrophage M1/M2 polarization balance, inhibiting pro-inflammatory cascade reactions, and enhancing anti-inflammatory repair function.

Key words：metabolic associated steatohepatitis; Shugan Lipi decoction; T-cell immunoglobulin mucin-3; macrophage polarization; inflammation; rat

摘要：目的：基于T细胞免疫球蛋白黏蛋白-3（TIM-3）调控巨噬细胞极化的机制，从动物实验、细胞实验探讨疏肝理脾汤干预代谢相关脂肪性肝炎（MASH）大鼠的作用靶点及免疫学机制。方法：将30只雄性SD大鼠随机分为疏肝理脾汤组、模型组、空白组，每组10只，空白组予常规饲料，疏肝理脾汤组、模型组予蛋氨酸-胆碱缺乏饲料4周，构建MASH动物模型。造模成功后，3组均给予正常饮食，疏肝理脾汤组给予疏肝理脾汤[6.18 g/kg（按生药计）]，其余2组给予等量生理盐水。干预4周后，Masson染色评估肝组织病理，蛋白质印迹（Western blotting）法、免疫组化法评估TIM-3表达。脂多糖（LPS）诱导RAW264.7巨噬细胞极化，分为对照组（20%空白血浆+1 μg/mL LPS共培养）、中药组（20%疏肝理脾汤含药血浆+1 μg/mL LPS共培养），另设一组不加血浆，只含CCK-8的培养基空白组。分别在干预12、24、48 h后，细胞计数试剂盒-8（CCK-8）检测法检测细胞活性，酶联免疫吸附试验（ELISA）检测炎症因子，流式细胞术检测M1/M2表型标志物。结果：空白组大鼠肝组织的肝细胞结构正常，模型组、疏肝理脾汤组大鼠肝组织可见肝细胞脂肪变和气球样变，炎症细胞浸润。与空白组比较，模型组及疏肝理脾汤组大鼠肝组织TIM-3表达显著降低（P<0.01）；与模型组比较，疏肝理脾汤组肝组织TIM-3表达显著增强（P<0.01）。与空白组比较，干预12、24、48 h对照组、中药组RAW264.7细胞活性显著增强（P<0.01）；与对照组比较，干预12、24、48 h中药组RAW264.7细胞活性显著增强（P<0.01）；干预12、24、48 h对照组、中药组进行组内两两比较，差异均有统计学意义（P<0.01）。各组RAW264.7细胞上清液检测，干预12、48 h中药组TNF-α、IL-6表达水平，CD80、CD86阳性比例均低于对照组（P<0.01），中药组IL-10、TGF-β表达水平，CD206、Arg-1阳性比例均高于对照组（P<0.01）；干预12、24、48 h对照组、中药组进行组内两两比较，随时间延长，IL-6及TNF-α水平均持续升高（P<0.01），IL-10及TGF-β水平均持续降低（P<0.01）；干预48 h对照组、中药组CD80、CD86阳性比例均高于同组干预12 h（P<0.01），Arg-1，CD206阳性比例均低于同组干预12 h（P<0.01）。结论：疏肝理脾汤能通过上调TIM-3蛋白表达，调控巨噬细胞M1/M2极化平衡，抑制促炎级联反应并增强抗炎修复功能，从而改善MASH病理进程。

关键词：代谢相关脂肪性肝炎；疏肝理脾汤；Ｔ细胞免疫球蛋白黏蛋白-3；巨噬细胞极化；炎症；大鼠

Release time：2026-04-26

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