桔梗多糖对CCl4诱导的小鼠急性肝损伤的保护作用及机制研究

Protective effect of polysaccharides from Platycodon grandiflorum on CCl4-induced acute liver injury in mice and its regulatory mechanism

目的探讨桔梗多糖对CCl4诱导的小鼠急性肝损伤的保护作用及其调控机制。方法C57BL/6小鼠预先给予桔梗多糖(200、400 mg·kg^(-1))共21 d,腹腔注射0.5%CCl4(10 mg·kg^(-1))诱发急性肝损伤模型。生化试剂盒测定血清中谷草转氨酶(AST)和谷丙转氨酶(ALT)活力,肝组织中的丙二醛(MDA)和超氧化物歧化酶(SOD)水平;HE染色法评价肝脏病理组织学情况;免疫组化法(IHC)检测肝脏中内质网应激(ERS)标志性蛋白葡萄糖调节蛋白78(GRP78)的表达;酶联免疫吸附(ELISA)法测定各组小鼠肝组织中的肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素1β(IL-1β)的含量;Western blot法检测肝组织中蛋白激酶RNA样内质网激酶(PERK)、人转录激活因子5(ATF5)、硫氧还蛋白互作蛋白(TXNIP)和NOD样受体热蛋白结构域相关蛋白3(NLRP3)的表达情况。结果桔梗多糖可改善CCl4诱导的肝损伤小鼠的肝细胞多发坏死,大量炎性细胞浸润,细胞边界模糊不清现象;有效改善血清转氨酶的异常情况;显著提高SOD活力(P<0.01),显著降低MDA水平(P<0.01);桔梗多糖高剂量组(400 mg·kg^(-1))显著抑制肝组织中TNF-α、IL-6和IL-1β表达(P<0.05,P<0.01,P<0.001);桔梗多糖干预后小鼠肝组织中ERS相关蛋白GRP78表达水平显著降低(P<0.01),其高剂量组(400 mg·kg^(-1))相关PERK、ATF5、TXNIP和NLRP3的表达得到有效抑制(P<0.01)。结论桔梗多糖预处理可明显保护CCl_(4)诱导的小鼠急性肝损伤,改善炎性损伤和ERS,这种保护作用可能与调控PERK/TXNIP/NLRP3通路有关。

Objective To determine the protective effect of Platycodon grandiflorum polysaccharide on CCl4-induced acute liver injury in mice and its regulatory mechanism.Methods C57BL/6 mice were given Platycodon grandiflorum polysaccharides(200,400 mg·kg^(-1))for 21 days.The acute liver injury in mice was created by intraperitoneal injection of 0.5%CCl4(10 mg·kg^(-1)).The levels of AST,ALT in the serum,SOD and MDA in the liver tissue were measured via biochemical kits.The pathological condition of liver tissue was evaluated by HE staining.The expression of ERS marker protein GRP78 was detected by immunohistochemistry.The levels of TNF-α,IL-6 and IL-1βin the liver tissue was detected by ELISA.The expression levels of PERK,ATF5,TXNIP,and NLRP3were measured by Western blot.Results Compared with the model group,Platycodon grandiflorum polysaccharide ameliorated the multiple necrosis of hepatocytes in mice with CCl4-induced liver injury,infiltration of many inflammatory cells,and blurred cell boundaries.The abnormality of serum transaminase was effectively improved.The activity of SOD was obviously increased(P<0.01),and the level of MDA was significantly decreased(P<0.01).In the high-dose group of Platycodon grandiflorum polysaccharide(400 mg·kg^(-1)),the levels of TNF-α,IL-6 and IL-1βin the tissues of mice were effectively down-regulated(P<0.05,P<0.01,P<0.001).After Platycodon grandiflorum polysaccharide treatment,the expression level of ERS-related protein GRP78 was significantly decreased in the liver tissue(P<0.01).The protein expression of PERK and its downstream signaling pathway PERK,ATF5,TXNIP,and NLRP3 were effectively controlled in high dose group(400 mg·kg^(-1))(P<0.01).Conclusion Platycodon grandiflorum polysaccharide can protect against CCl_(4)-induced acute liver injury in mice and the regulation on inflammatory injury and ERS,which may be related to the regulation of PERK/TXNIP/NLRP3 pathway.