补肾活血汤含药血清调控髓核细胞外泌体中miR-222-3p抑制髓核细胞凋亡的研究  

Effects of BuShen Huoxue Decoction-medicated serum on regulating miR-222-3p expression in exosomes and inhabiting nucleus pulposus cell apoptosis

作　　者：谭婉俊 吴官保[2] 彭兴宁 冯帅华[2] TAN Wanjun;WU Guanbao;PENG Xingning;FENG Shuaihua(Shaoyang Hospital of TCM,Shaoyang,Hunan 422001,China;The Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine,Changsha,Hunan 410006,China)

机构地区：[1]邵阳市中医医院,湖南邵阳422001 [2]湖南省中医药研究院附属医院,湖南长沙410006

出　　处：《湖南中医药大学学报》2025年第3期425-431,共7页Journal of Hunan University of Chinese Medicine

基　　金：湖南省自然科学基金面上项目(2022JJ30025);湖南省卫生健康委员会一般课题(W20243066)。

摘　　要：目的通过分析补肾活血汤含药血清调控髓核细胞外泌体中的miR-222-3p表达,探索其抑制髓核细胞凋亡的作用。方法(1)将髓核细胞随机分为PBS组、空白血清组和含药血清组。空白血清组培养液中加入10%普通血清,含药血清组培养液中加入10%补肾活血汤含药血清,PBS组加入等体积的PBS。3组分别培养48 h收集标本,提取外泌体;透射电镜鉴定外泌体形态结构,Western blot鉴定外泌体溶酶体相关膜蛋白3(CD63)、肿瘤易感基因101(TSG101)蛋白表达,Real-time PCR检测外泌体miR-222-3p的表达。(2)外泌体共培养肿瘤坏死因子α(TNF-α)诱导髓核细胞模型,将髓核细胞分为空白组、模型组、空白外泌体组、含药外泌体组、Anti-miR-222-3p外泌体组;共培养24 h后,用流式细胞术检测髓核细胞凋亡率,Real-time PCR检测各组髓核细胞miR-222-3p的表达,Western blot检测各组髓核细胞磷酸化蛋白53(p53)、细胞色素c(Cytc)、含半胱氨酸的天冬氨酸蛋白水解酶1(Caspase-1)蛋白的表达。结果(1)与空白血清组比较,含药血清组外泌体数量增多(P<0.05),含药血清组外泌体标志蛋白CD63、Tsg101相对表达量均增加(P<0.05),含药血清组髓核细胞外泌体中miR-222-3p表达降低(P<0.05);(2)与模型组及空白外泌体组比较,含药外泌体组及Anti-miR-222-3p外泌体组髓核细胞凋亡率进一步降低,髓核细胞miR-222-3p表达减少(P<0.05),p53、Cytc、Caspase-1蛋白相对表达量降低(P<0.05)。结论(1)补肾活血汤含药血清可有效促进大鼠髓核细胞分泌外泌体,并抑制髓核细胞外泌体中miR-222-3p的表达;(2)补肾活血汤含药外泌体可减轻TNF-α诱导的退行性髓核细胞的病理损伤,并减少髓核细胞凋亡,其机制可能与抑制miR-222-3p及凋亡相关蛋白p53、Cytc、Caspase-1表达密切相关。Objective To investigate the inhibitory effects of Bushen Huoxue Decoction-medicated serum on nucleus pulposus cell apoptosis by analyzing its regulation of miR-222-3p expression in exosomes derived from nucleus pulposus cell.Methods(1)Nucleus pulposus cells were randomly divided into PBS group,blank serum group,and medicated serum group.The blank serum group was supplemented with 10%normal serum,the medicated serum group with 10%Bushen Huoxue Decoction-medicated serum,and the PBS group with an equal volume of PBS.After 48 h,samples were collected,and exosomes were extracted.Transmission electron microscopy was used to identify the morphological structure of exosomes.Western blot was used to determine protein expressions of lysosome-associated membrane protein 3(CD63)and tumor susceptibility gene 101(TSG101)in exosomes.Real-time PCR was applied to examine the miR-222-3p expression in exosomes.(2)Exosomes were co-cultured with tumor necrosis factor-α(TNF-α)to induce nucleus pulposus cell models.The nucleus pulposus cells were divided into blank group,model group,blank exosome group,medicated exosome group,and Anti-miR-222-3p exosome group.After 24 h of co-culture,the apoptosis rate of nucleus pulposus cells was measured by flow cytometry,the expression of miR-222-3p in nucleus pulposus cells was determined by real-time PCR,and the protein expressions of p53,cytochrome C(Cytc),and cysteine-aspartic acid protegase 1(Caspase-1)were checked by Western blot among different groups.Results(1)Compared with the blank serum group,the medicated serum group showed increased number of exosomes(P<0.05),elevated relative protein expression levels of exosomal marker proteins CD63 and TSG101(P<0.05),and decreased miR-222-3p expression in exosomes derived from nucleus pulposus cells.(2)Compared with the model group and the blank exosome group,the apoptosis rate of nucleus pulposus cells further decreased,the miR-222-3p expression in nucleus pulposus cells decreased(P<0.05),and the relative protein expression levels of p53,Cytc,an

关 键 词：补肾活血汤 髓核细胞 外泌体 miR-222-3p 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]