补肾活血解毒方联合脐带间充质干细胞 外泌体移植对急性肝衰竭的影响

Effects of Bushen Huoxue Jiedu Recipe Combined with Umbilical Cord Mesenchymal Stem Cell Exosomes Transplantation on Acute Liver Failure

摘要:

〔摘 要〕 目的：探讨中药补肾活血解毒方联合大鼠脐带间充质干细胞外泌体（UCMSCs–EXO）移植调控转化生长因子 β1（TGF–β1）/Smad 通路对急性肝衰竭模型肝细胞凋亡和肝功能的影响。 方法：取健康成年 SD 大鼠，随机分组给药后制 备含药血清。取健康 SD 大鼠随机分为联合给药组、外泌体对照组、中药对照组、空白对照组和正常组，除正常组外均进 行造模成功后并给药处理，对大鼠肝功能指标进行测定。肝卵圆细胞和原代肝细胞培养后分别加入不同的含药血清，孵育 后诱导细胞凋亡。凋亡诱导结束后，细胞计数试剂盒 –8（CCK–8）法检测细胞活性，流式细胞仪检测细胞凋亡情况；免疫 荧光法检测凋亡肝细胞 TGF–β1 表达，Western blot 法对细胞中 Smad2/3 磷酸化（P–Smad2/3）、Smad7、TGF–β Ⅱ 型受体磷 酸化（P–TβRⅡ）和 TGF–β1 蛋白表达进行检测。 结果：联合给药组、外泌体对照组、中药对照组大鼠的谷丙转氨酶（ALT）、 谷草转氨酶（AST）水平均低于空白对照组大鼠（P ＜ 0.05），且联合给药组大鼠的 AST 和 ALT 水平低于外泌体对照组、 中药对照组大鼠（P ＜ 0.05）；联合给药组细胞活性高于外泌体对照组和中药对照组（P ＜ 0.05）；联合给药组凋亡细胞少 于外泌体对照组和中药对照组（P ＜ 0.05）；联合给药组的 TGF–β1 表达低于外泌体对照组、中药对照组细胞（P ＜ 0.05）； 联合给药组 P–TβRⅡ、P–Smad2/3、TGF–β1 蛋白表达低于外泌体对照组和中药对照组，Smad7 蛋白表达高于外泌体对照组 和中药对照组（P ＜ 0.05）。 结论：中药补肾活血解毒方结合 UCMSCs–EXO 能够改善急性肝衰竭大鼠肝功能并且能够通 过调控 TGF–β1/Smad 通路抑制急性肝衰竭模型肝细胞凋亡。

Abstract:

〔Abstract〕 Objective To investigate the effect of Bushen Huoxue Jiedu recipe combined with umbilical cord mesenchymal stem cell exosome (UCMSCs-EXO) transplantation on transforming growth factor β1 (TGF-β1) /Smad pathway on hepatocyte apoptosis and liver function in acute liver failure model. Methods Healthy adult SD rats were randomly assigned to prepare drugcontaining serum. Healthy SD rats were randomly divided into combined administration group, exosome control group, Chinese medicine control group, blank control group and normal group. Except for the normal group, all rats were successfully modeled and treated with medication, the liver function indexes of rats were measured. Hepatovale cells and primary hepatocytes were cultured with different drug-containing serum, and apoptosis was induced after incubation. After apoptosis induction, the cell activity was detected by cell counting kit-8 (CCK-8) method, and the cell apoptosis was detected by flow cytometry. The protein expression of TGF-β1 in apoptotic hepatocytes was detected by immunofluorescence, and the protein expression of phosphorylation of Smad2/3 (P-SMad2/3), Smad7, TGF-β Ⅱ receptor phosphorylation (P-TβRⅡ) and TGF-β1 were detected by Western blot. Results The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the combined administration group, exosome control group and Chinese medicine control group were lower than those in the blank control group (P < 0.05), and the levels of AST and ALT in combined administration group were lower than those in the exosome control group and Chinese medicine control group (P< 0.05). The cell activity of the combined administration group was higher than that of the exosome control group and Chinese medicine control group (P < 0.05). The number of apoptotic cells in the combined administration group was less than that in the exosome control group and Chinese medicine control group (P < 0.05). The expression of TGF-β1 in the combined administration group was lower than that in the exosome control group and Chinese medicine control group (P < 0.05). The protein expression levels of P-TβRⅡ, P-Smad2/3 and TGF-β1 in the combined administration group were lower than those in the exosome control group and Chinese medicine control group. The expression level of Smad7 in the combined administration group was higher than that in the exosome control group and Chinese medicine control group (P < 0.05). Conclusion Bushen Huoxue Jiedu recipe combined with UCMSCs-EXO can improve liver function in rats with acute liver failure and inhibit hepatocyte apoptosis in acute liver failure model by regulating TGF-β1 /Smad pathway.