凉血化瘀解毒法对急性肝衰竭模型大鼠免疫功能、Toll样受体及VEGF表达的影响

Effect of Liangxue Huayu Jiedu Method on Immune Function,Toll-like Receptor and VEGF Expression in Rats with Acute Hepatic Failure

目的观察凉血化瘀解毒法对急性肝衰竭(ALF)模型大鼠免疫功能、Toll样受体及VEGF表达的影响。方法采用腹腔注射脂多糖(LPS)和D-氨基半乳糖(D-Gal)法诱导建立急性肝衰竭大鼠模型,按照数字表法随机分为模型组、中药组和阳性组,每组30只,另选择30只Wistar大鼠为空白组,空白组和模型组给予2 mL生理盐水灌胃,中药组给予2 mL的凉血化瘀解毒方灌胃,阳性组给予15 mg/kg复方甘草酸苷灌胃,分别于第2日和第5日处死15只大鼠,比较肝组织病理学、肝功能指标、炎症因子、Toll样受体及血管内皮生长因子(VEGF)表达变化。结果模型组第2日和第5日肝组织结构严重破坏,弥漫性片状坏死,炎性细胞浸润,可见较多红细胞填充;中药组和阳性组第2日肝损伤程度减轻,肝组织结构较清晰,细胞排列较整齐,红细胞和炎性细胞减少,第5日较模型组显著改善,肝小叶结构均有所恢复,可见增生肝细胞,炎性浸润明显减轻。模型组谷草转氨酶(GOT)、谷丙转氨酶(ALT)及总胆红素(TBIL)水平显著高于对照组,第2日和第5日中药组和阳性组GOT、ALT及TBIL水平显著降低,且第5日显著低于第2日(P<0.05)。模型组CD4+和NKT细胞含量显著低于对照组,CD8+含量高于对照组,第2日和第5日中药组和阳性组CD4+和NKT细胞含量显著升高,CD8+含量显著低于对照组,且第5日低于第2日(P<0.05)。模型组肿瘤坏死因子-α(TNF-α)、白细胞介素-2(IL-2)和白细胞介素-6(IL-6)水平显著高于对照组,第2日和第5日中药组和阳性组TNF-α、IL-2和IL-6水平显著降低,且第5日显著低于第2日(P<0.05)。模型组TLR2、TLR4和VEGF蛋白表达水平显著高于模型组,第2日和第5日中药组和阳性组TLR2、TLR4和VEGF蛋白表达水平显著降低,且第5日显著低于第2日(P<0.05)。模型组VEGF蛋白及mRNA表达水平显著高于模型组,第2日和第5日中药组和阳性组VEGF蛋白及mRNA表达水平显著降低,且第5日显著低于第2日(P<0.05)。结论凉血化瘀解毒方可显著改急性肝衰竭模型大鼠肝损伤程度,改善肝功能,其机制可能与提高免疫功能,减轻炎症反应,降低TLR2、TLR4和VEGF蛋白及mRNA表达水平有关。

Objective:To investigate the effects of Liangxue Huayu Jiedu method on immune function,Toll-like receptor and VEGF expression in rats with acute liver failure(ALF).Methods:90 rat models with ALF were in duced by intraperitoneal injection of LPS and D-Gal,and randomly divided into the model group,Chinese medicine group and positive group,30 cases in each.30 wistar rats were selected as the blank group.The blank group and the model group were given 2 mL of normal saline,Chinese medicine group 2 mL of Liangxue Huayu Jiedu formula,and the positive group 15 mg/kg compound licorice.15 rats were sacrificed on the 2 d and 5 d,respectively.The liver histopathology,liver function indexes,inflammatory factors,Toll-like receptors and VEGF expression were compared.Results:The hepatic tissue structure was severely damaged on the 2nd and 5th day in the model group,with diffuse patchy necrosis,inflammatory cell infiltration and polycyte infiltration.On the 2nd day,the degree of liver injury was reduced;the structure of hepatic tissue was clear;the arrangement of cells was neat;the number of red blood cells and inflammatory cells was reduced in Chinese medicine group and the positive group.On the 5th day,the structure of hepatic lobules was improved significantly compared with the model group,and the proliferative hepatocytes and inflammatory infiltration were obviously alleviated.The levels of GOT,ALT and TBIL in the model group were significantly higher than those in the control group.The levels of GOT,ALT and TBIL in the Chinese herbal medicine group and the positive group decreased significantly on the 2nd and 5th days,and were significantly lower on the 5th day than those on the 2nd day(P<0.05).The content of CD4+ and NKT cells in the model group was significantly lower than that in the control group,and the content of CD8+ was higher than that in the control group.The content of CD4+ and NKT cells in Chinese medicine group and the positive group increased significantly on the 2nd and 5th day,and the content of CD8+was significantly lower than that in the control group,and on the 5th day,the content of CD8+was lower than that in the control group(P<0.0).The levels of TNF-α,IL-2 and IL-6 in the model group were significantly higher than those in the control group.The levels of TNF-α,IL-2 and IL-6 in the Chinese herbal medicine group and the positive group decreased significantly on the 2nd and 5th days,and were significantly lower on the 5th day than those on the 2nd day(P<0.05).The expression levels of TLR2,TLR4 and VEGF in the model group were significantly higher than those in the model group.The expression levels of TLR2,TLR4 and VEGF inChinese medicine group and the positive group were significantly lower on the 2nd and 5th days,and were significantly lower on the 5th day than those on the 2nd day(P<0.05).The expression levels of VEGF protein and RNA in the model group were significantly higher than those in the model group.The expression levels of VEGF protein and RNA in the Chinese herbal medicine group and the positive group decreased significantly on the 2nd and 5th days,and were significantly lower on the 5th day than those on the 2nd day(P<0.05).Conclusion:Liangxue Huayu Jiedu Decoction can significantly improve the degree of liver injury and improve liver function in rats with AHF.The mechanism may be related to improving immune function,reducing inflammation,the expression of protein and mRNA in TLR2,TLR4 and VEGF.