肝宁方含药血清对毒胡萝卜素诱导大鼠肝细胞内质网应激的影响  

Effect of medicated serum of Ganning Decoction on hepatocyte endoplasmic reticulum stress of rats innduced by thapsigargin

作　　者：朱朝阳[1] 王建超 银艳桃 文彬[3] 邓鑫[2] ZHU Chaoyang;WANG Jianchao;YIN Yantao;WEN Bin;DENG Xin(Nancheng Branch of Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine, Nanning 530011, Guangxi, China;Guangxi University of Chinese Medicine, Nanning 530011, Guangxi, China;Ruikang Hospital Affiliated to Guangxi University of Chinese medicine, Nanning 530000, Guangxi, China)

机构地区：[1]广西中医药大学附属瑞康医院南城分院,广西南宁530011 [2]广西中医药大学,广西南宁530000 [3]广西中医药大学附属瑞康医院,广西南宁530011

出　　处：《现代中西医结合杂志》2020年第36期3991-3995,4014,共6页Modern Journal of Integrated Traditional Chinese and Western Medicine

基　　金：广西医疗卫生适宜技术开发与推广应用课题(s201503);国家自然科学基金资助项目(81360532)。

摘　　要：目的研究肝宁方含药血清对毒胡萝卜素诱导的大鼠肝细胞BRL-3A内质网应激的影响及其相关机制。方法将30只SD大鼠平分为3组,其中10只予肝宁方25 g/(kg·d)灌胃,另2组大鼠以等量生理盐水灌胃,均2次/d,连续灌胃10 d后取血离心,获得含肝宁方大鼠血清及正常大鼠血清。取大鼠肝细胞BRL-3A细胞株,分为正常组、模型组、肝宁方组。正常组加入正常大鼠血清培养,模型组加入1μmol/L毒胡萝卜素及正常大鼠血清培养,肝宁方组加入1μmol/L毒胡萝卜素及含肝宁方大鼠血清培养。CCK8法检测BRL-3A细胞存活率,Fura-3/AM钙离子荧光探针测定BRL-3A细胞胞浆内Ca2+水平,谷草转氨酶(ALT)、谷丙转氨酶(AST)试剂盒检测BRL-3A细胞中ALT、AST水平;RT-PCR法检测BRL-3A细胞内质网应激相关基因GRP78、PERK、CHOP、Bcl-2 mRNA表达水平。结果与正常组比较,模型组、肝宁方组BRL-3A细胞存活率及Bcl-2 mRNA表达水平均明显降低(P均<0.05),细胞胞浆内Ca2+水平、细胞中ALT和AST水平及GRP78、PERK、CHOP mRNA表达水平均显著升高(P均<0.05);肝宁方组RL-3A细胞存活率及Bcl-2 mRNA表达水平均显著高于模型组(P均<0.05),细胞胞浆内Ca2+水平、细胞中ALT和AST水平及GRP78 mRNA、PERK mRNA、CHOP mRNA表达水平均明显低于模型组(P均<0.05)。结论肝宁方可减弱内质网的Ca2+流失,抑制内质网应激相关的GRP78、PERK、CHOP mRNA表达,上调Bcl-2mRNA表达,保护BRL-3A细胞并提高其存活率。Objective It is to study the effect of medicated serum of Ganning Decoction on thapsigargin-induced endoplasmic reticulum stress of rat liver cells BRL-3A and its related mechanisms.Methods 30 SD rats were divided into 3 groups,10 of which were given Ganning Decoction 25 g/(kg·d)by gavage,and the other two groups were given the same amount of normal saline by gavage,both times/d.After continuous intragastric administration for 10 days,blood was collected and centrifuged to obtain rat serum containing Ganning Decoction and normal rat serum.The BRL-3A cell line of rat liver cells were taken and divide into normal group,model group,and Ganning Decoction group.The normal group was cultured with normal rat serum,the model group was cultured with 1μmol/L thapsigargin and normal rat serum,and the Ganning Decoction group was cultured with 1μmol/L thapsigargin and the rat medicated serum containing Ganning Decoction.The survival rate of BRL-3A cells was detected by CCK8 method,the level of Ca2+in the cytoplasm of BRL-3A cells was detected by Fura-3/AM calcium ion fluorescent probe,and the levels of aspartate aminotransferase(ALT)and alanine aminotransferase(AST)in BRL-3A cells were detected by ALT and AST kits,the expression levels of related genes of endoplasmic reticulum stress GRP78,PERK,CHOP,Bcl-2 mRNAs in BRL-3A cells were detected by RT-PCR method.Results Compared with the normal group,the BRL-3A cell survival rate and Bcl-2 mRNA expression level in the model group and the Ganning Decoction group were significantly reduced(all P<0.05),the levels of intracytoplasmic Ca2+,ALT and AST,and GRP78,PERK,CHOP mRNA expression were significantly increased(all P<0.05);the survival rate of RL-3A cells and the expression of Bcl-2 mRNA in the Ganning Decoction group were significantly higher than those in the model group(all P<0.05),the level of Ca2+in the cytoplasm,the levels of ALT and AST in cells,and the expression levels of GRP78 mRNA,PERK mRNA,and CHOP mRNA were significantly lower than those of the model group(all P<0.0

关 键 词：肝宁方 大鼠肝细胞 内质网应激 

分 类 号：R-332[医药卫生]