目的 基于线粒体动力学探讨白芍多糖对肝纤维化肝阴虚证大鼠的保护作用及机制。方法 雄性SD大鼠按照体质量随机分为对照组、模型组、水飞蓟宾（42 mg/kg）组、知柏地黄丸（408 mg/kg）组和白芍多糖低、高剂量（169、338 mg/kg）组，每组8只。连续给药干预6周，从给药第2周开始，除对照组外，其余大鼠采用ip 20%四氯化碳橄榄油溶液联合ig甲状腺片（30 mg/kg）建立肝纤维化肝阴虚证模型。检测大鼠体征评分、体质量、摄食量、摄水量、舌面干湿度和肛温；采用ELISA法检测大鼠血清环磷酸腺苷（cyclic adenosine monophosphate，cAMP）、环磷酸鸟苷（cyclic guanosine monophosphate，cGMP）水平并计算比值；采用比色法测定血清中丙氨酸氨基转移酶（alanine aminotransferase，ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase，AST）、γ-谷氨酰转肽酶（γ-glutamyl transpeptidase，γ-GT）、碱性磷酸酶（alkaline phosphatase，ALP）活性及总胆红素（total bilirubin，TBIL）、总胆汁酸（total bile acid，TBA）水平；采用苏木素-伊红（hematoxylin-eosin，HE）和Masson染色观察肝脏病理变化及肝纤维化程度；采用透射电镜观察肝脏线粒体超微结构；采用免疫荧光法检测肝脏活性氧（reactive oxygen species，ROS）水平；通过试剂盒测定肝脏三磷酸腺苷（adenosine triphosphate，ATP）水平；采用Western blotting检测大鼠肝脏纤连蛋白1（fibronectin 1，FN1）、I型胶原蛋白（collagen I，COL-I）、α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、细胞外信号调节激酶（extracellular regulated protein kinases，ERK）、p-ERK、p38、p-p38、线粒体裂变蛋白1（fission 1，Fis1）、线粒体动力相关蛋白1（dynamin-related protein 1，Drp1）、p-Drp1、视神经萎缩蛋白1（optic atrophy 1，Opa1）和线粒体融合蛋白2（mitofusin 2，Mfn2）表达水平。结果 与模型组比较，白芍多糖组大鼠体质量、大便评分、舌面干湿度显著升高（P＜0.05、0.01、0.001），精神状态评分、毛发评分、小便评分、总体体征评分、肛温、摄食和饮水量显著降低（P＜0.05、0.01、0.001），血清中cAMP水平降低且cGMP水平升高（P＜0.001），cAMP/cGMP值显著降低（P＜0.001），肝功能相关指标均显著降低（P＜0.05、0.01、0.001），肝组织病理损伤有所改善，胶原纤维面积显著降低（P＜0.001），肝纤维化蛋白α-SMA、COL-I和FN1表达水平均明显下降（P＜0.01、0.001），肝脏线粒体形态结构有所改善，肝脏ROS水平降低且ATP水平升高（P＜0.05、0.01、0.001），肝脏p-ERK/ERK、p-p38/p38、Fis1和p-Drp1/Drp1蛋白表达水平显著降低（P＜0.05、0.01、0.001），Opa1和Mfn2蛋白表达水平明显升高（P＜0.01、0.001）。结论 白芍多糖对肝纤维化肝阴虚证大鼠具有保护作用，其作用机制可能与抑制ERK/p38 MAPK通路调控线粒体动力学稳态有关。

Objective To study the protective effect and mechanism of Paeonia lactiflora polysaccharides on liver fibrosis with liver yin deficiency syndrome in rats based on mitochondrial dynamics. Methods Male SD rats were randomly divided into control group, model group, silybin (42 mg/kg) group, Zhibai Dihuang Wan (知柏地黄丸, 408 mg/kg) group and P. lactiflora polysaccharide low-, high-dose (169, 338 mg/kg) groups, with eight rats in each group. Drugs were continuously intervented for six weeks, starting from the second week of administration, except for the control group, all other rats were treated with an ip 20% carbon tetrachloride olive oil solution combined with ig thyroid tablets (30 mg/kg) to establish a liver fibrosis with liver yin deficiency syndrome model. Sign score, body weight, food intake, water intake, tongue surface moisture level and rectal temperature of rats were detected. Levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in serum were measured using ELISA, and ratio was calculated. Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (γ-GT), alkaline phosphatase (ALP) and levels of total bilirubin (TBIL), total bile acids (TBA) in serum were measured using colorimetric assays. Hematoxylin-eosin (HE) and Masson staining were used to observe the pathological changes and degree of liver fibrosis. Transmission electron microscopy was used to observe mitochondrial ultrastructure in liver. Immunofluorescence was used to detect reactive oxygen species (ROS) level in liver. Adenosine triphosphate (ATP) level was measured to using a kit. Western blotting was used to detect fibronectin 1 (FN1), collagen I (COL-I), α-smooth muscle actin (α-SMA), extracellular regulated protein kinases (ERK), p-ERK, p38, p-p38, fission 1 (Fis1), dynamin-related protein 1 (Drp1), p- Drp1, optic atrophy 1 (Opa1) and mitofusin 2 (Mfn2) protein expression levels in liver of rats. Results Compared with model group, the body weight, fecal consistency score and tongue surface moisture of rats in P. lactiflora polysaccharides group were significantly increased (P < 0.05, 0.01, 0.001), while mental state, coat luster, urine score, overall physical condition score, rectal temperature, food and water intake were significantly decreased (P < 0.05, 0.01, 0.001), cAMP level was decreased and cGMP level in serum was increased (P < 0.001), and cAMP/cGMP value was significantly reduced (P < 0.001), liver function related indicators were significantly reduced (P < 0.05, 0.01, 0.001), liver tissue pathological damage was improved, the collagen fiber area was significantly reduced (P < 0.001), and the expression levels of liver fibrosis proteins α-SMA, COL-I and FN1 were significantly decreased (P < 0.01, 0.001), the morphology and structure of mitochondria in liver were improved, ROS level in liver was decreased and ATP level was increased (P < 0.05, 0.01, 0.001), expression levels of p-ERK/ERK, p-p38/p38, Fis1 and p-Drp1/Drp1 proteins were significantly reduced (P < 0.05, 0.01, 0.001), while the expression levels of Opa1 and Mfn2 proteins were significantly increased (P < 0.01, 0.001). Conclusion P. lactiflora polysaccharides have a protective effect on liver fibrosis in rats with liver yin deficiency syndrome, and its mechanism may be related to the inhibition of ERK/p38 MAPK pathway to regulate mitochondrial dynamic homeostasis.

R285.5

国家自然科学基金资助项目（82074036）