睡莲花总黄酮对四氯化碳诱导大鼠肝纤维化的防治作用

Effects of total flavonoids from Nymphaea candida on hepatic fibrosis induced by carbon tetrachloride in rats

目的研究睡莲花总黄酮(NCTF)对四氯化碳(CCl4)诱导大鼠肝纤维化的防治作用。方法 Wistar大鼠随机分为对照组、模型组、NCTF低、中、高剂量(50、100、200 mg/kg)组和秋水仙碱(0.2 mg/kg)组。除对照组大鼠sc花生油外,其余各组分别于大鼠背部sc 50%CCl4花生油溶液(0.1 mL/100 g),2次/周,连续12周。除对照组和模型组ig 0.5%羧甲基纤维素钠(CMC-Na)溶液外,其余各组于造模第2天按设定剂量ig药物(1.0 mL/100 g),1次/d,连续12周。实验结束后,收集大鼠血样、肝脏及脾脏组织,测定大鼠肝脾指数;通过HE染色和Masson染色观察肝组织病理变化;通过免疫组化法检测肝组织α-平滑肌肌动蛋白(α-SMA)的表达;通过全自动生化仪检测大鼠血清天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、碱性磷酸酶(ALP)、总蛋白(TP)、白蛋白(ALB)水平;通过生化法检测丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)和一氧化氮(NO)水平;通过放射免疫分析法检测血清透明质酸(HA)水平;采用酶联免疫吸附法(ELISA)测定血清层连蛋白(LN)、III型前胶原(PCIII)、IV型胶原(CIV)、肿瘤坏死因子(TNF-α)、白介素-1β(IL-1β)和白介素-6(IL-6)水平;采用碱水解法处理肝组织后测定其羟脯氨酸(Hyp)水平。结果与对照组相比,模型组大鼠精神萎靡、肝脾指数增大,肝组织炎性细胞浸润并呈现出明显的纤维化,血清ALT、AST、ALP、HA、LN、PCⅢ、CIV、MDA和NO水平显著升高(P<0.05、0.01),血清TP、ALB、GSH和SOD水平显著下降(P<0.01);肝组织α-SMA和Hyp表达明显上调(P<0.01)。与模型组比较,NCTF组能明显改善大鼠状态和肝脾指数(P<0.05、0.01),改善大鼠肝组织病理学损伤和纤维化;显著降低大鼠血清ALT、AST、ALP、MDA、NO水平及大鼠肝组织α-SMA、Hyp的表达(P<0.05、0.01),显著提高血清TP、ALB、GSH、SOD水平(P<0.05、0.01);并能显著抑制HA、LN、PCⅢ、CIV型蛋白表达及TNF-α、IL-1β、IL-6的水平(P<0.05、0.01)。结论 NCTF具有较好的抗肝纤维化作用,其作用机制与抗氧化、调节胶原合成、抑制促炎因子表达相关。

Objective To investigate the preventive effect of total flavonoids from Nymphaea candida(NCTF) on carbon tetrachloride(CCl4)-induced hepatic fibrosis in rats. Methods Wistar rats were randomly divided into control group, model group, NCTF groups(50, 100, 200 mg/kg) and colchicine group(0.2 mg/kg). Rats were subcutaneously injected with 50% CCl4 peanut oil solution(0.1 mL/100 g, twice a week for 12 weeks) to induce hepatic fibrosis. In addition to control and model group given with 0.5% CMC-Na, the other groups were intragastrically administered with drugs(1.0 mL/100 g, once a day for 12 weeks), all rats were put to death. Blood, hepatic and splenic tissue were collected to detect liver indexes. Pathological histology observation by Masson and HE staining were performed for other hepatic tissues. The expression of α-smooth muscle actin(α-SMA) in hepatic tissue was analyzed by immunohistochemical technique. The levels of serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), alkaline phosphatase(ALP), total protein(TP) and albumin(ALB), malondialdehyde(MDA), superoxide dismutase(SOD), glutathione(GSH) and nitric oxide(NO) were determined by automatic biochemical analyzer. The levels of serum laminin(LN), type III procollagen(PCIII), type IV collagen(CIV), tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6) were measured by enzyme-linked immunoassay assay(ELISA). Serum hyaluronic acid(HA) was detected by radioimmunoassay. Hepatic tissue hydroxyproline(Hyp) was measured by spectrophotometric method. Result Compared with the control group, the rats showed energielos, liver and spleen index of those were increased, as well as inflammatory cell infiltration and obvious fibrosis of hepatic tissue were appeared in the model group;The levels of ALT, AST, ALP, HA, LN, PCⅢ, CIV, MDA, NO, TNF-α, IL-1β and IL-6 in serum in model group were increased significantly(P < 0.05, 0.01);The levels of TP, ALB, GSH and SOD in serum were decreased significantly(P < 0.01);The expressions of α-SMA and Hyp in hepatic tissue were significantly improved(P < 0.01). Compared with the model group, the levels of ALT, AST, ALP, MDA, NO in serum in NCTF group were significantly reduced(P < 0.05, 0.01);The levels of HA, LN, PC III, CIV, TNF-α, IL-1β and IL-6 were significantly reduced(P < 0.05, 0.01);The levels of TP, ALB, GSH and SOD were significantly increased(P < 0.05, 0.01);The expressions of α-SMA and Hyp in hepatic tissue were significantly reduced(P < 0.05, 0.01). Compared with the model group, the pathological damage of hepatic tissue was significantly improved, the degeneration and necrosis of liver cells in rat were significantly reduced(P < 0.05). Conclusion NCTF has a better anti-hepatic fibrosis effect, its mechanism is related to antioxidant, regulation of collagen synthesis and inhibition of the proinflammatory factors expression.