茵陈四苓颗粒联合骨髓间充质干细胞移植对急性肝衰竭大鼠NF-κB、iNOS及TNF-α的影响研究

Effect of Yinchen Siling Granule combined with Bone Mesenchymal Stem Cells Transplantation on NF-κB,iNOS and TNF-α in Rats with Acute Liver Failure

目的探讨茵陈四苓颗粒联合骨髓间充质干细胞(BMSCs)移植对急性肝衰竭大鼠炎症介质谷丙转氨酶(ALT)、谷草转氨酶(AST)、内毒素(ET)及相关通路核因子-κB(NF-κB)、i NOS及肿瘤坏死因子-α(TNF-α)的影响。方法 1)全骨髓贴壁法制备大鼠BMSCs并体外培养;2)取清洁级雄性大鼠60只,随机分为正常组、模型组、阳性对照组、中药组、干细胞组、中药及干细胞联合组。除正常组外其余各组予以硫代乙酰胺灌胃造模;3)造模后中药组及联合组每日予以茵陈四苓颗粒灌胃;干细胞组及联合组予以尾静脉注射干细胞悬液。72 h后处死大鼠,收集血标本,检测肝脏ALT、AST及血浆ET水平,制作病理切片,免疫组化检测NF-κB表达,RT-PCR检测TNF-α、i NOS的基因转录。结果造模后各治疗组肝脏生化、TNF-α、i NOS的基因转录水平、NF-κB的细胞数目较模型组明显降低(P <0.05),肝脏组织坏死程度减轻,可见部分增生肝细胞,炎性浸润减轻。其中以联合组效果最佳(P <0.05)。结论茵陈四苓颗粒联合BMSCs治疗,可减轻急性肝衰竭大鼠肝损伤,有效抑制肝脏炎症反应,对急性肝衰竭有一定治疗作用。

Objective: To investigate the effects of Yinchen Siling Granule combined with bone mesenchymal stem cells(BMSCs) transplantation on inflammatory mediators and related pathways NF-κB,iNOS and TNF-α in rats with acute liver failure(ALF). Methods: 1)BMSCs was prepared with whole bone marrow adherent method and cultured in vitro;2)60 clean grade male rats were randomly divided into the normal group,the model group,positive control group,Chinese medicine group,stem cells group,the combined group. Except the normal group,the other groups were given thioacetamide intragastric injection;3)after the establishment of the model,the Chinese medicine group and the combined group were intragastrically perfused with Yinchen Siling Granule every day;at the same time,stem cells group and the combined group were injected with stem cells suspension by caudal vein. After 72 hours,the rats were killed and the blood samples were collected to detect the levels of ALT,AST and plasma endotoxin. The pathological sections of liver were made;the expression of NF-κB was detected by immunohistochemistry,and the gene transcription of TNF-α and iNOS was detected by RT-PCR. Results: The levels of liver biochemistry,TNF-α,iNOS gene transcription and the number of NF-κB cells in each treatment group were significantly lower than those in the model group(P < 0.05),among which the combined group had the best effect(P < 0.05). Conclusion: The transplantation of Yinchen Siling Granule combined with BMSCs can alleviate liver injury and inhibit hepatic inflammatory reaction,with a certain therapeutic effect on acute liver failure.