补肾益髓方含药骨髓间充质干细胞外泌体调控miR-223介导PTEN/PI3K/Akt通路抑制CPZ小鼠神经元凋亡的机制研究  

Study of the mechanism of exosomes derived from bone marrow mesenchymal stem cells treatment with Bu Shen Yi Sui Formula in inhibiting neuronal apoptosis by regulation of miR-223 mediated PTEN/PI3K/Akt pathway in cuprizone mice

作　　者：李春雨 刘思思[1] 李辰 李海馨 王蕾[1] LI Chunyu;LIU Sisi;LI Chen;LI Haixin;WANG Lei(School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China)

机构地区：[1]首都医科大学中医药学院,北京100069

出　　处：《环球中医药》2025年第3期457-467,共11页Global Traditional Chinese Medicine

基　　金：国家自然科学基金(82274450)。

摘　　要：目的探讨补肾益髓方(Bu Shen Yi Sui formula,BSYS)含药血清修饰的骨髓间充质干细胞来源外泌体(bone marrow mesenchymal stem cells derived exosomes,BMSCs-exos)通过miR-223介导磷酸酯酶与张力蛋白同源物(phosphatase and tensin homolog deleted on chromosome ten,PTEN)/磷脂酰肌醇-3-激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB/Akt)信号通路抑制双环己酮草酰二腙(cuprizone,CPZ)小鼠神经元凋亡的机制。方法72只C57BL/6小鼠随机分为6组:正常组、CPZ模型组、空白血清处理的BMSCs-exos(BMSCs-exos)低、高剂量组和BSYS含药血清处理的BMSCs-exos(BSYS-BMSCs-exos)低、高剂量组,每组12只。正常组小鼠给予正常饲料,造模组小鼠喂食含0.2%CPZ的饲料6周制备CPZ小鼠模型。第5周始,给药各组分别尾静脉注射低(0.25 mg/kg)、高剂量外泌体(0.5 mg/kg),一周2次,共干预2周。每周记录各组小鼠体质量的变化;转棒实验评价小鼠的运动功能;苏木精—伊红(hematoxylin-eosin,HE)染色和尼氏(Nissl)染色观察小鼠脑组织皮层神经元病理学变化;Fluoro-Jade B(FJB)染色观察神经元细胞凋亡情况;实时荧光定量聚合酶链反应(quantitative real time polymerase chain reaction,qRT-PCR)检测脑组织凋亡分子B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)mRNA、Bcl-2-关联X蛋白(Bcl-2-associated X,Bax)mRNA、miR-223-3p、PTEN mRNA表达;蛋白免疫印迹法(Western blotting,WB)检测Bax、Bcl-2、半胱氨酸天冬氨酸特异性蛋白酶3(Cysteine Aspartate Specific Protease-3,Caspase-3)及PTEN、PI3K、p-PI3K、Akt、p-Akt蛋白表达。结果与正常组比较,CPZ模型组小鼠体质量及运动协调能力显著降低(P<0.05);皮层神经元数量减少、形态结构损伤,皮层FJB阳性神经元数量显著减少(P<0.01);Bcl-2 mRNA、miR-223-3p降低(P<0.01),Bax、PTEN mRNA显著升高(P<0.01);Bax/Bcl-2、Caspase-3、PTEN蛋白表达显著升高(P<0.05),Bcl-2、p-PI3K/PI3K、p-Akt/Akt蛋白表达显著降低(P<0.01)。与CPZObjective This study investigates the mechanism on exosomes derived from bone marrow mesenchymal stem cells(BMSCs-exos)modified with BSYS-containing serum in inhibiting neuronal apoptosis through the miR-223 mediated phosphatase and tensin homolog deleted on chromosome ten(PTEN)/phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway in cuprizone(CPZ)mice.Methods A total of seventy-two C57BL/6 mice are randomly assigned to six groups:the normal group,the CPZ model group,the BMSCs-exos treated with blank serum(BMSCs-exos)low-dose group and the BMSCs-exos high-dose groups,the BMSCs-exos treated with BSYS-containing serum(BSYS-BMSCs-exos)low-dose and high-dose groups,with 12 mice in each group.Mice in the normal group are given a standard diet,while those in the model group are fed a diet containing 0.2%CPZ for 6 weeks to establish the CPZ mouse model.Starting from week 5,the mice in the treatment groups received intravenous injections of low-dose(0.25mg/kg)or high-dose(0.5mg/kg)exos twice weekly for 2 weeks.Body weight of the mice in every group was recorded weekly.The rotarod test was employed to assess motor function.Hematoxylin and eosin(HE)staining and Nissl staining were used to examine pathological changes in cortical neurons of the brain.Fluoro-Jade B staining is performed to assess neuronal apoptosis.Quantitative real-time PCR(qRT-PCR)measures the levels of apoptosis-related molecules,including Bcl2-associated X protein(Bax),B-cell lymphoma-2(Bcl-2),miR-223-3p,and PTEN mRNA in brain tissue.Western blotting(WB)detects protein expression of Bax,Bcl-2,Cysteine Aspartate Specific Protease-3(Caspase-3),PTEN,PI3K,p-PI3K,Akt,and p-Akt.Results Compared with the normal group,the CPZ model group mice exhibited significant reductions in body weight and motor coordination(P<0.05).Cortical neurons showed a decrease in number and structural damage.The number of FJB-positive neurons in the cortex was significantly reduced(P<0.01).Bcl-2 mRNA and miR-223-3p were significantly decreased(P<0.01),while Bax and P

关 键 词：多发性硬化 补肾益髓方 双环己酮草酰二腙小鼠 神经元凋亡 miR-223-3p 磷酸酯酶与张力蛋白同源物/磷脂酰肌醇-3-激酶/蛋白激酶B信号通路 

分 类 号：R285.5[医药卫生—中药学]