枳实含药血清对大鼠胃Cajal间质细胞内质网应激损伤的影响及机制

Effect and mechanism of drug-containing serum of Zhishi( immature bitter orange)on endoplasmic reticulum stress injury in rat gastric interstitial cells of cajal

目的探讨枳实含药血清对大鼠胃Cajal间质细胞(ICCs)内质网应激(ERS)损伤的影响及机制。方法采用酶解法分离培养ICCs并行c-kit免疫荧光染色法鉴定细胞。使用衣霉素(Tm)构建ICCs内质网应激模型,4苯基丁酸(4-PBA)抑制内质网应激。血清药理学方法制备枳实含药血清。取对数生长期的ICCs,随机分为四组,ICCs组正常培养,ERS模型组予1μg/m L Tm处理,抑制剂组予含1μg/m L Tm和10 mmol/L 4-PBA处理,枳实组予1μg/m L Tm和20%枳实含药血清,每组作用时间均为24 h。透射电子显微镜观察胃ICCs内质网的超微结构;实时荧光定量PCR法检测ERS相关因子葡萄糖调节蛋白78(GRP78)、激活转录因子6(ATF6) mRNA表达。结果成功分离、培养并鉴定ICCs。透射电子显微镜下正常ICCs的内质网结构正常,排列整齐;模型组ICCs细胞器减少,内质网肿胀,扩张、囊泡化改变、排列紊乱;枳实组ICCs内质网肿胀,呈局限性扩张。与ICCs组比较,ERS模型组GRP78、ATF6 mRNA表达升高(P均<0. 05);与ERS模型组比较,枳实组GRP78、ATF6 mRNA表达降低(P均<0. 05)。结论枳实含药血清可减轻ICCs细胞的ERS损伤,其机制可能与调控ERS ATF6通路有关。

Objective To explore the effect of drug-containing serum of Zhishi(immature bitter orange)on endoplasmic reticulum stress(ERS)injury in rat gastric interstitial cells of Cajal(ICCs).Methods ICCs were isolated by collagenase digestion and identified by immunofluorescence staining.Tunicamycin(Tm)was used to induce ICCs to establish ERS models,the 4-Phenylbutyric acid(4-PBA)was used to block autophagy.Rat drug-containing serum of Zhishi was prepared by using the serum pharmacology routine method.ICCs were randomly divided into four groups after subculture,including the blank group(normal culture),ERS group(the ICCs were stimulated with 1μg/mL Tm),4-PBA group(the ICCs were stimulated with 1μg/mL Tm and 10 mmol/L 4-PBA),and Zhsihi group(the ICCs were stimulated with 1μg/mL Tm and 20%drug-containing serum),and we continued to cultivate ICCs for 24 h.The ultrastructure of endoplasmic reticulum in ICCs was observed by transmission electron microscopy.The mRNA expression levels of GRP78 and ATF6 were detected by real-time fluorescent quantitative PCR.Results We isolated and identified ICCs successfully.Under transmission electron microscope,we observed that the endoplasmic reticulum of ICCs was normal and well-arranged in the blank group;the number of organelles reduced,and endoplasmic reticulum swelling,dilation,vesiculation changes and disordered arrangement were found in the ERS group;ICCs had endoplasmic reticulum swelling and limitation expansion in the Zhsihi group.Compared with the ICCs group,the mRNA expression of GRP78 and ATF6 increased in the ERS group(both P<0.05).Compared with the ERS group,the mRNA expression of GRP78 and ATF6 decreased in the Zhishi group(both P<0.05).Conclusion The drug-containing serum of Zhishi can reduce the ERS injury of ICCs possibly by regulating the ATF6 signaling pathway of ERS.