脐带间充质干细胞对内毒素血症诱发的大鼠急性肝功能损伤的影响

Effect of umbilical cord mesenchymal stem cells on acute liver injury induced by endotoxemia in rats

目的评价脐带间充质干细胞(hUC-MSCs)对内毒素血症诱发的大鼠急性肝功能损伤的影响及其与凋亡机制的关系。方法 6周龄雄性SD大鼠18只,随机分为3组,分别是对照组(C组)、内毒素血症组(M组)和内毒素血症+hUC-MSCs治疗组(M+cells组),每组6只。大鼠腹腔注射5 mg/kg脂多糖(LPS)诱导内毒素血症模型,并经尾静脉注射含20×106个hUC-MSCs。4 h时检测血清谷草转氨酶(AST)和谷丙转氨酶(ALT),ELISA方法检测肿瘤坏死因子(TNF-α)、白细胞介素6(IL-6),HE常规染色鉴定肝脏组织病理,Western Blot法检测肝脏组织抗凋亡蛋白Bcl-2、促凋亡蛋白Bax、凋亡信号调节激酶1(ASK1)、应激活化蛋白激酶即JUN氨基末端激酶(JNK)蛋白的表达。多组间比较采用单因素方差分析,相关分析选用pearson。结果(1)C组AST、ALT、TNF-α和IL-6浓度分别为(74.66±6.39)U/L、(40.07±6.07)U/L、(37.74±3.08)ng/L和(0.42±0.07)ng/L;与M组比较(310.75±9.13)U/L、(107.04±10.04) U/L、(160.32±4.88)ng/L和(0.90±0.09)ng/L,差异具有统计学意义(P?均?<0.05),M组AST、ALT、TNF-α、IL-6浓度分别为(310.75±9.13)U/L、(107.04±10.04) U/L、(160.32±4.88) ng/L和(0.90±0.09)ng/L,与M+cells组比较(204.49±15.36) U/L、(71.24±7.34) U/L、(117.61±9.37) ng/L和(0.60±0.10)ng/L,差异具有统计学意义(P?均?<0.05)。(2) C组大鼠肝细胞形态正常,可见肝小叶结构清晰,肝汇管区无炎性细胞浸润,M组大鼠肝小叶散在点状坏死肝细胞伴炎性浸润,肝细胞间隙散布增生的Kuffer细胞,M+cells组大鼠肝小叶炎性细胞浸润及肝细胞间隙Kuffer细胞浸润改善。(3)与C组比较,M组大鼠肝脏组织JUN、ASK1和Bax蛋白表达均增高(P?均?<0.05),Bcl-2蛋白表达降低(P <0.05);与M组比较,M+cells组大鼠肝脏组织JUN、ASK1和Bax蛋白表达降低(P?均?<0.05),Bcl-2蛋白增加(P <0.05)。(4)单因素相关分析显示大鼠血清ALT、AST与TNF-a指数呈正相关(r值分别为0.9580、0.9865,P均<0.05),大鼠血清ALT、AST与IL-6指数呈正相关(r值分别为0.9892、0.9630,P?均?<0.05),大鼠血清ALT、AST分别与BAX、ASK1、JNK指数均呈正相关(r值分别为0.9993、0.9851、0.7901、0.9864、0.9557、0.7128,P?均?<0.05),大鼠血清ALT、AST分别与BCL-2指数均呈负相关(r值分别为-0.8824、-0.9338,P?均?<0.05),大鼠血清TNF-α分别与BAX、ASK1、JNK指数均呈正相关(r值分别为0.9466、0.8958、0.6025,P均<0.05),大鼠血清TNF-α与BCL-2指数呈负相关(r=-0.6025,P?均?<0.05),大鼠血清IL-6分别与BAX、ASK1、JNK指数均呈正相关(r值分别为0.9941、0.9997、0.8679,P均<0.05),大鼠血清IL-6与BCL-2指数呈负相关(r=-0.8078,P?均?<0.05)。结论 hUC-MSCs具有减轻内毒素血症大鼠急性肝功能损伤的作用,其机制与抑制肝脏细胞凋亡相关。

Objective To evaluate the effect of umbilical cord mesenchymal stem cells(hUC-MSCs)on acute liver injury induced by endotoxemia in rats and its relationship with the mechanism of apoptosis.Methods 18 male SD rats were randomly divided into a control group(Group C),an endotoxemia group(Group M)and an endotoxemia with the treatment of hUC-MSCs group(Group M+cells).A rat model of endotoxemia was established by intraperitoneal injection of lipopolysaccharide(LPS,5mg/kg).20×10^6 hUC-MSCs was infused into the mice of Group M+cells through the tail vein.Four hours later,the serum AST,ALT,TNF-α and IL-6 were measured.The expression of JUN amino-terminal kinase(JNK),apoptosis signal regulated kinase 1(ASK1),Bcl-2 protein and Bax in the liver tissues was detected by Western Blot method.HE staining was used in histopathology,and then the liver histopathology was observed under a light microscope.Results(1)Compared with Group C,the concentration of AST,ALT,TNF-α and IL-6 in Group M and M+cells was higher than that in Group C(P<0 05).Compared with Group M,the concentration of AST,ALT,TNF-α and IL-6 in Group M+cells was lower than that in Group M(P<0.05).(2)The morphology of hepatocytes in Group C was normal and the structure of hepatic lobule was clear.There was no inflammatory cell infiltration in portal area of the liver.While,the scattered nodular necrosis of hepatocytes associated with inflammation could be seen in the hepatic lobules of rats in Group M.Less inflammatory cells infiltration in the hepatic lobules and infiltration of Kuffer cells were found in hepatocyte space in Group M+cells.(3)Compared with Group C,the expression of JUN,ASK1 and Bax protein in the liver tissues of Group M was significantly higher than that of Group C(P<0.05),while the expression of Bcl-2 protein was decreased(P<0.05).Compared with Group M,the expression of JUN,ASK1 and Bax protein in the liver tissues of Group M+cells was lower than that of Group M(P<0.05),while the expression of Bcl-2 protein was significantly increased(P<0.05).(4)Single factor analysis showed that serum ALT,AST were positively correlated with TNF-α index(r value were 0.9580,0.9865,P<0.05),and serum ALT,AST were positively correlated with IL-6 index(r value were 0.9892,0.9630,P<0.05).The serum ALT,AST were positively correlated with BAX,ASK1,JNK index(r value were 0.9993,0.9851,0.7901,0.9864,0.9557,0.7128,all P<0.05).Serum ALT and AST were negatively correlated with Bcl-2 index(r =-0.8824,-0.9338,all P<0.05),serum TNF-α was positively correlated with Bax,ASK1,JNK index(r = 0.9466,0.8958,0.6025,all P<0.05),and serum TNF-α was negatively correlated with Bcl-2 index(r =-0.6025,all P<0.05).Serum IL-6 was positively correlated with Bax,ASK1 and JNK indexes(r values were 0.9941,0.9997 and 0.8679,all P<0.05),while serum IL-6 was negatively correlated with Bcl-2 index(r =-0.8078,P<0.05).