健脾补肾方对辐射损伤模型小鼠脾脏自噬小体及PI3K/Akt/mTOR通路的影响  

Effects of Jianpi Bushen Formula(健脾补肾方)on the Autophagosomes and PI3K/Akt/mTOR Pathway in Radiation Injured Model Mice

作　　者：肖静静 何东初 张梦云 张勇 丁晓娟 伍伟 杨文超 XIAO Jingjing;HE Dongchu;ZHANG Mengyun;ZHANG Yong;DING Xiaojuan;WU Wei;YANG Wenchao(General Hospital of Central Theatre Command,Wuhan,430070)

机构地区：[1]中部战区总医院,湖北省武汉市430070

出　　处：《中医杂志》2023年第24期2553-2560,共8页Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(81873153,81273905);湖北省中医药管理局2023-2024年度中医药重点项目(ZY2023Z010)。

摘　　要：目的探讨健脾补肾方对辐射所致免疫功能损伤的影响和可能作用机制。方法50只昆明种小鼠随机分为正常组、模型组、胸腺肽组和健脾补肾方大、小剂量组,每组10只。除正常组外其余各组小鼠采用6.0 Gy X线一次性全身照射建立辐射损伤小鼠模型。造模成功后健脾补肾方小、大剂量组小鼠分别给予13 g/(kg·d)、26 g/(kg·d)健脾补肾方灌胃,胸腺肽组小鼠给予胸腺肽肠溶片11.7 mg/(kg·d)灌胃,正常组、模型组小鼠用0.9%氯化钠溶液0.1 ml/(10 g·d)灌胃。各组均每日灌胃1次,连续7天。最后一次灌胃后,小鼠称重,处死后分离小鼠脾脏,称重并计算脾脏指数;ELISA法检测脾脏组织干扰素γ(IFN-γ)、白细胞介素2(IL-2)含量;透射电镜观察脾脏自噬小体;实时荧光定量PCR检测脾脏磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)、雷帕霉素靶蛋白(mTOR)mRNA表达水平;Western blot法检测并计算脾脏PI3K、Akt、mTOR蛋白表达及自噬相关蛋白微管相关轻链蛋白3(LC3)、Beclin1、p62蛋白表达。结果与正常组比较,模型组小鼠体质量、脾脏指数均显著下降(P<0.01),脾脏IFN-γ、IL-2含量显著降低(P<0.01),脾脏PI3K、Akt、mTOR mRNA及蛋白表达水平均显著下降(P<0.01),Beclin1蛋白表达、LC3-Ⅱ/LC3-Ⅰ值显著升高(P<0.01),p62蛋白表达水平显著下降(P<0.01),透射电镜显示脾脏自噬小体数量明显增多,细胞结构损伤程度重。与模型组比较,各药物干预组小鼠上述各指标均显著改善(P<0.05或P<0.01),健脾补肾方大剂量组小鼠脾脏线粒体可见部分完整脊,自噬体较多;健脾补肾方小剂量组、胸腺肽组小鼠脾脏线粒体结构较完整,自噬体较少。健脾补肾方小剂量组各指标改善效果优于健脾补肾方大剂量组(P<0.05或P<0.01),且与胸腺肽组各指标差异无统计学意义(P>0.05)。结论健脾补肾方可能通过增强脾脏PI3K/Akt/mTOR信号通路,抑制辐射诱导的自噬过度活化,从而减�Objective To explore the effects and possible mechanisms of Jianpi Bushen Formula(健脾补肾方)on radiation-induced immune function damage of mice.Methods Fifty mice were randomly divided into four groups:normal group,model group,thymosin group,high-and low-dose groups of Jianpi Bushen Formula,with 10 mice in each group.Except for the normal group,the mice in the other groups were irradiated with a single wholebody dose of 6.0 Gy X-rays to establish a radiation-injured mouse model.After the successful modeling,the low-and high-dose groups of the Jianpi Bushen Formula were given respectively 13 g/(kg·d)、26 g/(kg·d)of the formula by gavage,while the thymosin group was given 11.7 mg/(kg·d)of thymosin by gavage,and the normal group and model group were given 0.1 ml/(10g·d)of 0.9%sodium chloride solution by gavage.Each group was administered once a day for 7 consecutive days.After the last gavage,the mice were weighed,and their spleens were separated and weighed to calculate the spleen index.The levels of interferon gamma(IFN-γ)and interleukin 2(IL-2)in the spleen tissue were detected by enzyme linked immunosorbent assay(ELISA).The autophagosomes in the spleen were observed by transmission electron microscopy.The mRNA expression levels of phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt),and mammalian target of rapamycin(mTOR)in the spleen were detected by real-time fluores⁃cent quantitative PCR.The protein expression of PI3K,Akt,and mTOR in the spleen,as well as the expression of autophagy-related proteins microtubule-associated light chain protein 3(LC3),Beclin1,and p62 were detected by Western blot.Results Compared with the normal group,the model group showed significant decreases in body weight,spleen index,and levels of IFN-γand IL-2 in the spleen(P<0.01);the mRNA and protein expression levels of PI3K,Akt,and mTOR in the spleen were also significantly reduced(P<0.01);the expression of Beclin1 protein,the ratio of LC3-II/LC3-I significantly increased(P<0.01),while the level of p62 protein expr

关 键 词：辐射损伤 健脾补肾方 自噬 磷脂酰肌醇3-激酶 蛋白激酶B 雷帕霉素靶蛋白 

分 类 号：R285.5[医药卫生—中药学]