绿原酸调控内质网应激PERK-CHOP通路对安氟烷诱导大鼠肝毒性的保护作用

Protective effect of chlorogenic acid on enflurane-induced hepatotoxicity in rats by regulating the endoplasmic reticulum stress via the PERK-CHOP pathway

目的研究绿原酸(CGA)对安氟烷诱导大鼠肝毒性的保护作用,以及调控内质网应激蛋白激酶RNA样ER激酶(PERK)-C/EBP同源蛋白(CHOP)通路的机制。方法将60只SPF级SD雄性大鼠随机分为对照组、模型组、CGA低剂量组、CGA高剂量组、CGA高剂量+PERK激活剂组(CGA-H+CCT组),每组12只。检测大鼠血清中肝功能指标天门冬氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、碱性磷酸酶(ALP)的活性;检测血清中促炎因子肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、IL-6的水平;HE染色观察大鼠肝组织的病理变化;TUNEL染色检测大鼠肝细胞凋亡情况;Western blot检测大鼠肝组织中ER应激蛋白PERK、p-PERK、eIF2α、p-eIF2α、CHOP的表达情况;免疫组化法检测大鼠肝组织中BCL2、BAX蛋白水平。结果与对照组比较,模型组大鼠肝细胞数量减少,排列不规则,且出现肿胀和空泡,大鼠血清中肝功能指标AST、ALT、ALP、促炎因子TNF-α、IL-1β、IL-6水平、肝细胞凋亡率、PERK和eIF2α的磷酸化水平、CHOP和BAX蛋白表达均升高,BCL2蛋白表达降低(P<0.05);与模型组比较,CGA剂量组肝细胞数量增多且排列有序,有轻微肿胀,空泡减少,大鼠血清中肝功能指标AST、ALT、ALP、促炎因子TNF-α、IL-1β、IL-6水平、肝细胞凋亡率、PERK和eIF2α的磷酸化水平、CHOP和BAX蛋白表达均降低,BCL2蛋白表达升高(P<0.05),且CGA高剂量组与低剂量组比较,差异有统计学意义(P<0.05);与CGA高剂量组比较,CGA-H+CCT组中PERK的抑制剂CCT020312消除了CGA对上述各项指标的作用。结论CGA可以通过抑制内质网应激PERK-CHOP通路减轻安氟烷诱导的大鼠肝毒性。

Objective To investigate the protective effect of chlorogenic acid(CGA)on enflurane-induced hepatotoxicity in rats and the mechanism of regulating the endoplasmic reticulum stress protein kinase RNA like ER kinase(PERK)-C/EBP homologous protein(CHOP)pathway.Methods Sixty male SD rats at the SPF level were randomly divided into control group,model group,CGA low-dose group,CGA high-dose group,and CGA high-dose+PERK activator group(CGA-H+CCT group),with 12 rats in each group.The activities of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP)in rat serum were detected.Serum levels of proinflammatory factors,including tumor necrosis factor alpha(TNF-α),interleukin-1 beta(IL-1β)and IL-6 were detected.Hematoxylin and eosin(H&E)staining was performed to observe the pathological changes of rat liver.Terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)staining was performed to detect the apoptosis of rat hepatocytes.Western blot was performed to detect the expressions of ER stress proteins,including PERK,p-PERK,eIF2α,p-eIF2α,and CHOP in rat liver.Positive levels of BCL2 and BAX proteins in rat liver tissues were detected by immunohistochemistry.Results Compared with those of the control group,the number of hepatocytes in the model group decreased,which had irregular arrangement,swelling and vacuoles.Increased serum levels of AST,ALT,ALP,TNF-α,IL-1β,IL-6,apoptosis rate of hepatocytes,phosphorylated PERK and eIF2α,and protein expressions of CHOP and BAX were detected in the model group than those in control group,while the protein expression of BCL2 was downregulated(P<0.05).Compared with those of the model group,the number of hepatocytes in CGA groups increased,which were slightly swelling and arranged orderly,and the vacuoles were less observed.Decreased serum levels of AST,ALT,ALP,TNF-α,IL-1β,IL-6,apoptosis rate of hepatocytes,phosphorylated PERK and eIF2α,and protein expressions of CHOP and BAX were detected in the CGA groups than those in model group,while the protein expression of BCL2 was upregulated(P<0.05).Compared with CGA high dose group,CCT020312,the inhibitor of PERK in CGA-H+CCT group reversed the regulatory effect of CGA on the above indicators.Conclusion CGA can protect enflurane-induced hepatotoxicity by inhibiting the PERK-CHOP pathway via regulating ER stress in rats.