摘要
目的:探讨清热解毒、凉血化瘀中药联合大鼠骨髓间充质干细胞(BMSCs)移植对急性肝衰竭大鼠(ALF)肝脏细胞凋亡的影响。方法:40只清洁级雄性SD大鼠,随机分为空白组5只,造模组35只,造模组采用D-氨基半乳糖(D-Gal N,1.2 g·kg^(-1))联合脂多糖(LPS,5μg·kg^(-1))建立急性肝衰竭大鼠模型,造模后12 h取存活大鼠随机分为模型组、复方甘草酸苷组、茵陈四苓颗粒组、干细胞移植组及茵陈四苓颗粒联合干细胞移植组(联合组),联合组给予干细胞门静脉移植及茵陈四苓颗粒灌胃(每次1.5 g·kg^(-1)),干细胞组给予单纯干细胞门静脉移植(1.0×106个/m L),余各组给予等体积生理盐水门静脉注射,茵陈四苓颗粒组给予茵陈四苓颗粒灌胃,复方甘草酸苷组给予复方甘草酸苷灌胃(每次7.5 mg·kg^(-1)),余各组给予等体积生理盐水灌胃,干预后120 h处死大鼠。检测各组大鼠血清丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST),总胆红素(TIBL)变化,苏木-伊红(HE)染色观察肝组织病理改变,计算肝脏炎症活动度,原位末端转移酶标记技术(TUNEL)检测肝脏细胞凋亡指数(AI),蛋白免疫印迹法(Western blot)检测大鼠肝组织半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达,实时荧光定量PCR(Real-time PCR)测定肝组织中B细胞淋巴瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax)mRNA的表达。结果:急性肝衰竭大鼠造模后肝功能异常,联合组与模型组、复方甘草酸苷组、茵陈四苓颗粒组及干细胞移植组比较,大鼠血清AST,ALT,TBIL降低,肝炎活动度及凋亡指数明显降低(P<0.05);联合组大鼠肝组织内Caspase-3蛋白的表达明显降低,Bax mRNA的表达降低,Bcl-2 mRNA表达明显升高,与模型组及各干预组比较差异有统计学意义(P<0.05)。结论:清热解毒凉血化瘀中药联合干细胞移植能够改善肝功能,通过下调Caspase-3蛋白表达,上调Bcl-2 mRNA,下调Bax mRNA的表达来抑制肝脏细胞凋亡,效果优于单纯干预,两者对肝细胞凋亡的抑制具有协同作用。
Objective: To study the effect of Chinese traditional medicine(TCM) of' cleaning heat, cooling blood, disintoxication and removing blood stasis' combined with stem cell transplantation in treating the rats of acute liver failure, in order to to explore the effect on apoptosis of hepatocytes. Method: A total of 40 clean SD rats were randomly divided into the blank group (5 rats) and the model group (35 rats). The acute liver failure was induced by D-GalN (1.2 g·kg^-1) and LPS (5 μg·kg^-1) through intraperitoneal injection. At 12 hours after modelling, the living rats were randomly divided into the model group, the Compound Glycyrrhizin Tablet group, the Yinchen Siling granule group, the stem cell transplantation group and the combined treatment group (stem cell transplantation combined with TCM). The rats in the combined treatment group and the stem cell group were injected with BMSCs through portal vein, and the rats in the other groups were injected with physiological saline. The combined treatment group and the Yinchen Siling granule group were given by gavage twice a day with tradition Chinese drugs named Yinchen Siling granule (1.5 g·kg^-1), the compound glycyrrhizin tablet group was given compound glycyrrhizin tablet (7.5 mg·kg^-1) twice a day, and the rats in the other groups were provided with the equal volume of physiological saline until the rats were killed. Rat blood samples and liver tissues were collected in 120 hours, and alanine aminotransferase (ALT), aspartic aminotransferase (AST), total bilirubin (TIBL) were determined to see whether liver function in each group was improved and the extent. The liver tissue pathological changes were observed by HE staining, the apoptosis index was detected by TUNEL staining, protein expressions of cysteine aspartate protease-3(Caspase-3) was determined by Western blot, and mRNA expressions of B-cell lymphoma-2(Bcl-2) and Bcl-2 associated X protein(Bax) were detected by Real-time PCR. Result: Liver function abnormality occurred in rats with acute hepatic failure. Compared with the model group, the positive control group, the Chinese medicine group and the stem cell transplantation group, the combined treatment group was statistically better, with decreases in AST, ALT and TBIL, hepatic inflammatory activity and apoptosis index (P〈0.05). Caspase-3 in each group, except for the blank group, was significantly increased, and the combined treatment group was statistically superior to all of the other groups (P〈0.05). mRNA expression of Bax in each group was gradually decreased, while Bcl-2 mRNA increased, whereas Bax mRNA expression of the combined group was statistically lower all the other groups, and Bcl-2 mRNA was higher than the other groups (P〈0.05). Conclusion: The combined treatment of TCM of' cleaning heat, cooling blood, disintoxication and removing blood stasis' and BMSCs transplantation alleviates liver dysfunction, protects liver tissues and inhibits the hepatic apoptosis, and the mechanism may be related to down-regulation of the expression of Caspase-3 protein, and up-regulation of the expression of Bcl-2 mRNA and down-regulation of the expression of Bax mRNA, with a better effect than using either of them alone; and both are synergistic to the suppression of hepatic apoptosis.
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2017年第19期160-166,共7页
Chinese Journal of Experimental Traditional Medical Formulae
基金
四川省科技厅2014年四川省应用基础研究计划项目(14JC0079)
泸州市科技局2013泸州市科技计划项目[2013-S-49(7/15)]
关键词
急性肝衰竭
清热解毒、凉血化瘀中药
干细胞
凋亡
acute liver failure (ALF)
Chinese traditional medicine of cleaning heat, disintoxication,cooling blood and removing blood stasis
bone marrow mesenchymal stem cells (BMSCs)
apoptosis