外源性TSLP蛋白对脓毒症肝损伤小鼠的作用及肝细胞自噬的影响

Effect of exogenous TSLP on sepsis-induced liver injury and hepatocyte autophagy

目的探究外源性胸腺基质淋巴细胞生成素(thymic stromal lymphopietin,TSLP)蛋白对脓毒症肝损伤(sepsis-induced liver injury,SILI)小鼠的作用及肝细胞自噬的影响。方法采用盲肠结扎穿孔术(CLP)制作SILI模型,将30只C57BL/6J小鼠随机分为假手术组(Sham组)、模型组[CLP+磷酸缓冲盐溶液(PBS)组]及外源性TSLP蛋白(重组小鼠TSLP蛋白,rTSLP)干预组(CLP+rTSLP组)。造模10 h处死小鼠,采集成功造模标本(每组n=6),检测三组小鼠血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)及乳酸脱氢酶(LDH)水平;苏木精-伊红染色法(HE)观察肝组织损伤情况并评分;应用实时荧光定量PCR技术(qRT-PCR)及蛋白免疫印迹(Western blot)检测小鼠肝脏白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)炎症因子表达。Western blot检测小鼠苄氯素1(Beclin1)、微管相关蛋白(LC3)Ⅱ自噬相关标记物及PI3K/Akt信号通路表达情况。结果rTSLP蛋白能降低血清ALT、AST和LDH水平(P<0.05),改善肝脏组织损伤(P<0.05),同时也能降低IL-1β、IL-6和TNF-α炎症因子的mRNA和蛋白表达水平(P<0.05),增强肝细胞自噬与PI3K/Akt信号通路水平的表达(P<0.05)。结论外源性TSLP蛋白通过上调肝细胞自噬,改善小鼠SILI,其机制可能是通过激活PI3K/Akt信号通路发挥作用。

Objective To explore the effect of exogenous thymic stromal lymphopoietin(TSLP)protein on sepsis-induced liver injury(SILI)and hepatocyte autophagy.Methods Thirty C57BL/6J mice were randomly divided into sham operation group(Sham),control group[cecal ligation and puncture(CLP)+phosphate buffer solution(PBS)]and exogenous TSLP protein intervention group[CLP+recombinant mouse TSLP protein(rTSLP)].CLP was used to perform SILI models.After 10 h of modeling,the mice were killed for the samples collection.The serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and lactate dehydrogenase(LDH)of the three groups were detected.Tissue damage were examined by HE staining and were scored.IL-6,IL-1βand TNF-αin liver tissues were detected by qRT-PCR and Western blot.The expressions of autophagy-related markers Beclin1 and LC3II,and PI3K/Akt signaling pathway were detected by Western blot.Results Exogenous TSLP reduced the levels of serum ALT,AST and LDH(P<0.05),attenuated liver histopathological injury(P<0.05),also decreased the mRNA and protein expression levels of IL-6,IL-1βand TNF-α(P<0.05),and increased the expressions of autophagy and PI3K/Akt signaling pathway in hepatocytes(P<0.05).Conclusions Exogenous TSLP protein can improve SILI by up-regulating hepatocyte autophagy,which may play a role by activating PI3K/Akt signaling pathway.