葛根素对自身免疫性心肌炎小鼠Th1细胞分化的影响及其机制  

Effect of puerarin on the differentiation of Th1 cells in a mouse model of experimental autoimmune myocarditis and its mechanism

作　　者：刘蒙 李玲[1] 王爽[1] LIU Meng;LI Ling;WANG Shuang(School of Basic Medicine,Qingdao University,Qingdao 266071,China)

机构地区：[1]青岛大学基础医学院,山东青岛266071

出　　处：《精准医学杂志》2025年第1期1-7,共7页Journal of Precision Medicine

基　　金：国家自然科学基金资助项目(82001754)。

摘　　要：目的探讨葛根素对于自身免疫性心肌炎(EAM)小鼠Th1细胞分化的影响及其机制。方法将BALB/c雄性小鼠随机分为A~D组,每组各5只。A组正常饲养20 d,B、C、D组在第0天和第7天时皮下注射心肌肌球蛋白肽(α-MyHC)和完全弗氏佐剂(CFA)构建EAM模型,第8天时,B组小鼠灌胃羧甲基纤维素钠,C组小鼠灌胃羧甲基纤维素钠+地塞米松,D组小鼠灌胃羧甲基纤维素钠+葛根素。第21天时,通过HE染色观察各组小鼠心脏组织的炎性细胞浸润情况,通过实时荧光定量逆转录聚合酶链式反应(RT-qPCR)检测各组小鼠心脏组织中IFN-γ、TNF-α和T-bet mRNA的相对表达量,通过Western blotting实验检测小鼠心脏组织中IFN-γ、TNF-α、T-bet蛋白相对表达量。从正常小鼠脾脏中分选出Naive CD4^(+)T细胞,分为a~d组,a组加入anti-CD28,b组加入anti-CD28、IL-4、IL-12和IL-2,c和d组在b组基础之上再分别加入12.5、25μmol/L葛根素,各组均处理72 h。通过流式细胞术检测Naive CD4^(+)T细胞中Th1/CD4^(+)T细胞比例,通过RT-qPCR技术检测Naive CD4^(+)T细胞中IFN-γ、TNF-α和T-bet mRNA的相对表达量,通过Western blotting实验检测Naive CD4^(+)T细胞中p-STAT4蛋白相对表达量,通过免疫荧光技术观察Naive CD4^(+)T细胞中p-STAT4的染色位置。结果HE染色结果显示,A、C、D组小鼠心脏组织炎性细胞浸润程度显著低于B组(F=228.50,q=20.98~36.72,P<0.05);RT-qPCR和Western blotting实验检测结果显示,A、C、D组小鼠心脏组织中T-bet、IFN-γ和TNF-αmRNA和蛋白相对表达量显著低于B组(F=6.10~1843.00,q=4.73~93.94,P<0.05)。流式细胞术结果显示,a、c、d组Naive CD4^(+)T细胞中Th1/CD4^(+)T细胞比例明显低于b组(F=116.40,q=9.49~26.08,P<0.05)。RT-qPCR结果显示,a和d组Naive CD4^(+)T细胞中T-bet、IFN-γ和TNF-αmRNA相对表达量明显低于b组(F=6.58~1072.00,q=4.21~23.24,P<0.05)。分子对接结果显示,葛根素与STAT4的结合能力为(-7.64±0.03)kcal/mol。Western blotting实验结�Objective To investigate the effect of puerarin(PUE)on the differentiation of Th1 cells in a mouse model of experimental autoimmune myocarditis(EAM)and its mechanism.Methods Male BALB/c mice were randomly divided into groups A,B,C,and D,with 5 mice in each group.The mice in group A were fed normally for 20 d,and those in groups B,C,and D were given subcutaneous injection of myocardial myosin peptide(α-MyHC)and complete Freund’s adjuvant(CFA)on days 0 and 7 to establish a model of EAM.On day 8,the mice in group B were given sodium carboxymethyl cellulose(CMC-Na)by gavage,those in group C were given CMC-Na+dexamethasone by gavage,and those in group D were given CMC-Na+PUE by gavage.On day 21,HE staining was used to observe inflammatory cell infiltration in cardiac tissue,RT-qPCR was used to measure the relative mRNA expression levels of IFN-γ,TNF-α,and T-bet in cardiac tissue,and Western blotting was used to measure the relative protein expression levels of IFN-γ,TNF-α,and T-bet in cardiac tissue.Na ve CD4^(+)T cells were isolated from the spleen of healthy mice and were divided into groups a,b,c,and d;the cells in group a were treated with anti-CD28,those in group b were treated with anti-CD28,IL-4,IL-12,and IL-2,and those in groups c and d were treated with 12.5 and 25μmol/L PUE in addition to the treatment in group b;the course of treatment was 72 h for all groups.Flow cytometry was used to measure the proportion of Th1/CD4^(+)T cells in na ve CD4^(+)T cells;RT-qPCR was used to measure the relative mRNA expression levels of IFN-γ,TNF-α,and T-bet in na ve CD4^(+)T cells;Western blotting was used to measure the relative protein expression level of p-STAT4 in na ve CD4^(+)T cells,and immunofluorescence assay was used to determine the position of p-STAT4 within na ve CD4^(+)T cells.Results HE staining showed that compared with group B,groups A,C,and D had a significantly lower degree of inflammatory cell infiltration in cardiac tissue(F=228.50,q=20.98-36.72,P<0.05);RT-qPCR and Western blotting showed that

关 键 词：葛根素 心肌炎 自身免疫疾病 TH1细胞 STAT4转录因子 信号传导 细胞分化 

分 类 号：R542.21[医药卫生—心血管疾病] R593.2[医药卫生—内科学]