枸杞多糖预处理对APAP诱导L02细胞损伤的保护作用

The Protective Effects of Lycium Barbarum Polysaccharides Pretreatment on the Acetaminophen-induced L02 Cell Injury

目的探讨枸杞多糖(lycium barbarum polysaccharides,LBP)预处理对对乙酰氨基酚(acetaminophen,APAP)诱导的正常人肝细胞(L02)损伤的保护作用及机制。方法将体外培养的L02细胞分为正常对照组、模型组(APAP组)、300、600μg·mL^(-1) LBP+APAP组、300、600μg·mL^(-1) LBP组,L02细胞经不同浓度的LBP预处理12 h后,300、600μg·mL^(-1) LBP+APAP组和模型组给予10 mmol·L^(-1) APAP诱导细胞24 h。CCK-8法检测细胞存活能力,光镜下观察各组细胞形态变化,qRT-PCR检测细胞色素P4502E1(cytochrome P4502E1,CYP2E1)和HNF4α的mRNA水平,Western blot检测IRE1α、eLF2α、Nrf2、Bax、Bcl-2、Caspase-12的蛋白表达。结果与正常对照组相比,模型组细胞经APAP诱导后,出现细胞损伤改变,CYP2E1 mRNA水平升高、HNF4αmRNA水平下降(P均<0.05),Nrf2蛋白水平下降(P<0.05),内质网应激相关蛋白(IRE1α、eLF2α)和凋亡相关蛋白(Bax、Bcl-2和Caspase-12)的表达均升高(P均<0.05)。LBP预处理可改善APAP诱导的L02细胞的损伤状态,降低CYP2E1 mRNA、升高HNF4αmRNA水平(P均<0.05),降低IRE1α、eLF2α、Bax、Bcl-2和Caspase-12的蛋白表达(P均<0.05)。结论LBP预处理对APAP诱导的L02细胞损伤具有一定的保护作用,其机制主要与减轻内质网应激和抑制细胞凋亡有关。

Objective To investigate the protective effects of lycium barbarum polysaccharides(LBP)pretreatment on the acetaminophen(APAP)-induced cell injury in human normal liver L02 cells and explore its mechanism.Methods L02 cells cultured in vitro were divided into normal control group,model group(APAP group),300,600μg·mL^(-1)LBP with APAP,300,600μg·mL^(-1)LBP group.L02 cells were pretreated with different concentrations of LBP for 12 h,the cells in the 300,600μg·mL^(-1) with APAP and the model group were treated with 10 mmol·L^(-1) APAP for 24 h.The viability of L02 cells induced by APAP was analyzed by cell counting kit-8(CCK-8)method.The morphological changes of cells in each group were observed under light microscope,qRT-PCR was used to detected the mRNA levels of CYP2E1 and HNF4α,Western blot was used to evaluate the expression levels of IRE1α,eLF2α,Nrf2,Bax,Bcl-2 and Caspase-12.Results Compared with the control group,APAP induced obvious cell injury in the model group,the levels of CYP2E1 mRNA was increased and HNF4αmRNA was decreased(P all<0.05).Moreover,Nrf2 protein level was decreased(P<0.05),the levels of endoplasmic reticulum stress related proteins(IRE1α,eLF2α)and apoptosis related proteins(Bax,Bcl-2,Caspase-12)were significantly increased(P all<0.05).However,LBP pretreatment could improve the cell injury in APAP-induced L02 cells,which reduced the mRNA level of CYP2E1 and increased the mRNA level of HNF4α(P all<0.05).Additionally,LBP pretreatment reduced protein levels of IRE1α,eLF2α,Bax,Bcl-2 and Caspase-12 in L02 cells(P all<0.05).Conclusion The results indicated that LBP pretreatment has protective effects on APAP induced L02 cell injury via reducing endoplasmic reticulum stress response and inhibiting apoptosis.