养阴清热方通过下调JAK/STAT信号通路调节Treg/Th17平衡的实验研究  

Yangyin Qingre Formula Regulates Treg/Th17 Balance by Down-Regulating JAK/STAT Signaling Pathway

作　　者：掌琳惠 马元婧 缪蓉 王伟[2] 翁文馨 袁冬平[1] 龙军[1] 孙云霞[2] ZHANG Lin-hui;MA Yuan-jing;MIU Rong;WANG Wei;WENG Wen-xin;YUAN Dong-ping;LONG Jun;SUN Yun-xia(School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Department of Geriatrics,Jiangsu Province Hospital of Chinese Medicine,Nanjing 210004,China)

机构地区：[1]南京中医药大学药学院,江苏南京210023 [2]江苏省中医院老年科,江苏南京210004

出　　处：《南京中医药大学学报》2022年第9期810-818,共9页Journal of Nanjing University of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(81973823,81573929);国家自然科学基金青年科学基金项目(81904236);江苏省实验动物协会科研课题(DWXH201916)。

摘　　要：目的研究养阴清热方(YHF)对脂多糖(LPS)导致的小鼠Treg/Th17失衡的影响及机制。方法磁珠分选法提取小鼠脾脏CD4+T细胞,用LPS刺激CD4+T细胞,再以1、2、4 mg·mL^(-1)的YHF水提液分别进行干预。MTT法检测细胞增殖情况;流式细胞术检测CD4+T细胞亚群Treg/Th17比例;ELISA法检测细胞上清中细胞因子IL^(-1)0、TGF-β、IL-6、IL^(-1)2p70的含量;蛋白免疫印迹法(Western blot)检测细胞中Foxp3、RORγt及信号分子JAK/STAT的表达。结果HPLC检测鉴定出YHF水提液中8种有效成分,分别为羟基红花黄色素A、虎杖苷、芦丁、2,3,5,4'-四羟基二苯乙烯葡萄糖苷、白藜芦醇、槲皮素、大黄素和姜黄素。YHF水提液可以剂量依赖性地抑制LPS刺激的小鼠CD4+T细胞的增殖(P<0.05),增加Treg的比例(P<0.05),降低Th17的比例(P<0.01),促进培养上清中Treg相关的抗炎因子IL^(-1)0和TGF-β的分泌,抑制Th17相关的致炎因子IL-6和IL^(-1)2p70的分泌。1、2、4 mg·mL^(-1) YHF均能增加Foxp3蛋白的表达(P<0.05),减少RORγt蛋白的表达(P<0.01)。1、2、4 mg·mL^(-1) YHF均能降低JAK2、STAT3及其磷酸化蛋白的表达(P<0.05)。4 mg·mL^(-1) YHF与JAK2特异性抑制剂AG490联用可以增加培养上清IL^(-1)0和TGF-β含量(P<0.05),降低IL-6和IL^(-1)2p70含量(P<0.05),而且可以显著降低p-JAK2、p-STAT3和RORγt蛋白表达(P<0.01,P<0.001),增加Foxp3蛋白表达(P<0.01)。结论YHF可以有效抑制LPS诱导的小鼠CD4+T细胞增殖,通过抑制JAK2和STAT3信号分子表达,增加Foxp3的表达和Treg数量,减少RORγt的表达和Th17数量,调节Treg/Th17平衡及抗炎细胞因子和促炎细胞因子的分泌。OBJECTIVE To investigate the effect and mechanism of Yangyin Qingre formula(YHF)on Treg/Th17 imbalance induced by lipopolysaccharide(LPS)in mice.METHODS CD4^(+) T cells from mouse spleen were extracted by magnetic bead sorting method and CD4^(+) T cells differentiation was stimulated by LPS followed by intervention with 1,2,4 mg·mL^(-1) YHF.Cells proliferation was detected by MTT method.The ratio of Treg/Th17 of CD4^(+) T cells subsets was detected by flow cytometry.The expression of cytokines(IL^(-1)0,TGF-β,IL-6,IL^(-1)2p70)in cells supernatant was measured by ELISA.The expression of Foxp3,RORγt,and the signalling molecules JAK/STAT in the cells were detected by Western blot.RESULTS HPLC identified eight active ingredients of YHF,namely hydroxy saffron yellow pigment A,polydatin,rutin,2,3,5,4'-tetrahydroxystilbene glucoside,resveratrol,quercetin,emodin and curcumin.YHF dose-dependently inhibited the proliferation of LPS-stimulated mouse CD4^(+) T cells(P<0.05),increased the proportion of Treg(P<0.05)and decreased the proportion of Th17(P<0.01),promoted the secretion of Treg-related anti-inflammatory factors IL^(-1)0 and TGF-βin culture supernatant and inhibited the secretion of Th17-related pro-inflammatory factors IL-6 and IL^(-1)2p70 secretion.1,2,4 mg·mL^(-1) YHF increased the expression of Foxp3 protein(P<0.05)and decreased the expression of RORγt protein(P<0.01).1,2,4 mg·mL^(-1) YHF decreased the expression of JAK2,STAT3 and their phosphorylated proteins(P<0.05).The combination of 4 mg·mL^(-1) YHF with the JAK2-specific inhibitor AG490 increased culture supernatant IL^(-1)0 and TGF-βlevels(P<0.05),decreased IL-6 and IL^(-1)2p70 levels(P<0.05)and more significantly decreased p-JAK2,p-STAT3 and RORγt protein expression(P<0.01,P<0.001)and increased Foxp3 protein expression(P<0.01).CONCLUSION YHF can effectively inhibit the proliferation of inflammatory LPS-induced CD4^(+) T cells by suppressing the expression of JAK2 and STAT3 signaling molecules,increase the expression of Foxp3 and the number of Tre

关 键 词：动脉粥样硬化 养阴清热方 CD4+T细胞 调节性T细胞 辅助性T细胞17 JAK/STAT 

分 类 号：R285.5[医药卫生—中药学]