中药药理与临床

目的：观察银杏叶-赤芍提取物(GPE)防治非酒精性脂肪性肝病(NAFLD)大鼠血液流变学、炎症因子和内皮功能的影响，并采用主成分分析(PCA)和正交校正的偏最小二乘判别分析法(OPLS-DA)进一步筛选核心药效指标，探索其作用机制，为银杏叶-赤芍提取物防治NAFLD提供药理基础。方法：SD大鼠50只，按体质量随机分为正常对照组、模型对照组、易善复137 mg/kg组、银杏叶-赤芍提取物45、90mg/kg组，每组10只。采用高脂饲料喂养18周制备NAFLD模型。末次给药后，取下腔静脉血和肝脏。HE染色和油红O染色观察肝脏组织病理变化及脂肪变性；全血流变仪检测大鼠血液流变学指标；散斑血流实时成像系统测定大鼠尾部微循环血流量；酶联免疫法(ELISA)检测肝匀浆中转化生长因子β1(TGF-β1)、血清白细胞介素6(IL-6)、肿瘤坏死因子-α(TNF-α)等炎症细胞因子和一氧化氮(NO)、血管内皮素1(ET-1)、血栓素B2(TXB2)及6-酮-前列环素F1α(6-Keto-PGF1α)等血管内皮功能指标。PCA和OPLS-DA等多元统计方法用于进一步筛选核心药效指标。Western blot法检测大鼠肝脏中蛋白激酶B1(AKT1)、p38丝裂原活化蛋白激酶(p38 MAPK)磷酸化水平和细胞间黏附分子-1(ICAM-1)、血管内皮细胞黏附分子-1(VCAM-1)、血管内皮生长因子(VEGF)蛋白表达。结果：18周后，与正常对照组比较，模型对照组大鼠全血粘度、血浆粘度(PV)、血沉(ESR)、红细胞压积(HCT)、红细胞聚集指数(EAI)、血清TNF-α、IL-6、TGF-β1、ET-1、NO、TXB2含量、T/P比值明显升高(P<0.05或P<0.01)，红细胞变形指数(EDI)、大鼠尾部血流灌注分布、6keto-PGF1α水平显著降低(P<0.01)；与模型对照组比较，银杏叶-赤芍提取物能显著降低全血粘度、血浆粘度(PV)、血沉(ESR)、红细胞压积(HCT)红细胞聚集指数(EAI)、血清TNF-α、IL-6、TGF-β1、ET-1、NO、TXB₂及TXB₂/6-keto-PGF1α比值(P<0.05或P<0.01)，显著升高红细胞变形指数(EDI)、大鼠尾部血流灌注分布(P<0.05或P<0.01)。病理结果显示，银杏叶-赤芍提取物可减少肝细胞脂质沉积、改善肝脏组织脂肪样变。PCA和OPLS-DA分析结果提示，血沉(ESR)、低切变率全血粘度(LS-WBV)和TXB2/6-keto-PGF1α是银杏叶-赤芍提取物抗NAFLD的意义指标。Western blot检测表明银杏叶-赤芍提取物可显著下调AKT1、p38蛋白磷酸化的表达，下调VCAM-1、ICAM-1和VEGF蛋白表达。结论：银杏叶-赤芍提取物可通过缓解肝脏脂肪变性、改善血液流变学和微循环障碍，降低炎症反应，减轻血管内皮功能损伤防治NAFLD。其机制可能是通过抑制AKT1、p38蛋白磷酸化水平，降低VCAM-1、ICAM-1和VEGF蛋白表达，进而调节血液黏稠度，改善NAFLD“血瘀态”情况有关。

Objective: To observe the effects of Yinxingye (银杏叶)-Chishao (赤芍) extract on blood rheology， inflammatory factors， and endothelial function in the rat model of non-alcoholic fatty liver disease (NAFLD)， screen core pharmacodynamic indicators by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA)， and explore the therapeutic mechanisms， thus providing a pharmacological basis for Yinxingye-Chishao extract in the prevention and treatment of NAFLD. Methods: Fifty SD rats were randomized by body mass into a normal control group， a model control group， a YSF (phosphatidylcholine， 137 mg/kg) group， a Yinxingye-Chishao extract (45 mg/kg) group， and a Yinxingye-Chishao extract (90 mg/kg) group， with 10 rats in each group. An NAFLD model was established by feeding with a high-fat diet for 18 weeks. After the last administration， blood was collected from the inferior vena cava and the liver tissue was obtained. Hematoxylin-eosin (HE) staining and Oil Red O staining were used to observe hepatic pathological changes and steatosis. A blood rheometer was used to measure blood rheology in each group， and a laser Doppler flow imaging system was used to measure microcirculation blood flow in the tail of the rats. Enzyme-linked immunosorbent assay (ELISA) was adopted to measure the levels of inflammatory cytokines such as transforming growth factor β1 (TGF-β1) in the liver homogenate， interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in the serum， as well as endothelial function indicators including nitric oxide (NO)， endothelin-1 (ET-1)， thromboxane B2 (TXB2)， and 6-keto-prostaglandin F1α (6-keto-PGF1α). PCA and OPLS-DA were employed for further screening of core pharmacodynamic indicators. The phosphorylation levels of protein kinase B1 (AKT1) and p38 mitogen-activated protein kinase (p38 MAPK)， as well as the protein levels of intercellular adhesion molecule-1 (ICAM-1)， vascular cell adhesion molecule-1 (VCAM-1)， and vascular endothelial growth factor (VEGF) in rat livers， were determined by Western blot. Results: After 18 weeks， compared with the normal control group， the model control group showed increased whole blood viscosity， plasma viscosity (PV)， erythrocyte sedimentation rate (ESR)， hematocrit (HCT)， and erythrocyte aggregation index (EAI)， elevated serum levels of TNF-α， IL-6， TGF-β1， ET-1， NO， and TXB2， and increased TXB2/6-keto-PGF1α (T/P) ratio (P<0.01 or P<0.05)， but decreased erythrocyte deformability index (EDI)， tail blood perfusion distribution， and 6-keto-PGF1α level (P<0.01). Compared with the model control group， Yinxingye-Chishao extract reduced the whole blood viscosity， PV， ESR， HCT， and EAI， lowered the serum levels of TNF-α， IL-6， TGF-β1， ET-1， NO， and TXB2， and decreased the TXB2/6-keto-PGF1α ratio (P<0.01 or P<0.05)， while increasing the EDI and tail blood perfusion distribution (P<0.01 or P<0.05). Pathological results showed that Yinxingye-Chishao extract reduced hepatocellular lipid deposition and ameliorated hepatic steatosis. PCA and OPLS-DA indicated that ESR， low-shear whole blood viscosity (LS-WBV)， and the TXB2/6-keto-PGF1α ratio were significant indicators of the anti-NAFLD effect of Yinxingye-Chishao extract. Western blot analysis demonstrated that Yinxingye-Chishao extract significantly down-regulated the phosphorylation levels of AKT1 and p38 and the protein levels of VCAM-1， ICAM-1， and VEGF. Conclusion: Yinxingye-Chishao extract can alleviate hepatic steatosis， improve blood rheology and microcirculation disorders， reduce inflammatory responses， and mitigate endothelial dysfunction in the prevention and treatment of NAFLD. It may inhibit the phosphorylation levels of AKT1 and p38 and down-regulate the protein levels of VCAM-1， ICAM-1， and VEGF to modulate blood viscosity and alleviate Xueyu (血瘀) in NAFLD.