摘要
目的:观察不同剂量解毒通络生津方对干燥综合征模型NOD/Ltj小鼠血清白细胞介素(IL)-6、肿瘤坏死因子-α(TNF-α)及颌下腺叉头框转录因子P3(FoxP3)表达的影响,探讨解毒通络生津方对NOD/Ltj小鼠免疫调节的作用机制。方法:将30只8周龄NOD/Ltj小鼠随机分为模型组,解毒通络生津方低、中、高剂量组和羟氯喹组共5组,12周龄开始每日分别灌服等量的生理盐水,含生药9、18、36 g·kg^(-1)的解毒通络生津方和60 mg·kg^(-1)的羟氯喹,同时选取6只ICR小鼠作为空白组,给予等量的生理盐水灌胃。实验期间记录各组小鼠第9、12、16周龄的日均饮水量、唾液流量,给药4周后摘取组织计算颌下腺指数、脾脏指数,苏木素-伊红(HE)染色观察颌下腺组织病理形态,Meso Scale Discovery(MSD)法检测小鼠血清IL-6、TNF-α、IL-10水平,免疫组化检测颌下腺FoxP3的表达和分布,蛋白免疫印迹法(Western blot)检测小鼠颌下腺FoxP3蛋白的表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测小鼠颌下腺FoxP3、TNF-α mRNA的表达。结果:与空白组ICR小鼠比较,模型组小鼠饮水量增多、唾液流量减少、颌下腺指数降低、颌下腺组织病理学评分升高,血清IL-6、TNF-α表达升高,IL-10表达降低,颌下腺FoxP3蛋白表达降低,颌下腺FoxP3 mRNA表达降低、TNF-α mRNA表达升高(P<0.05)。与模型组比较,解毒通络生津方低、中、高剂量组饮水量均明显减少,唾液流量均明显增加(P<0.05),且中、高剂量组疗效更显著;解毒通络生津方中剂量组颌下腺指数升高(P<0.05),解毒通络生津方高剂量组脾脏指数降低(P<0.05);解毒通络生津方低、中、高剂量组颌下腺组织病理评分均降低(P<0.05),高剂量组疗效更显著;解毒通络生津方低、中、高剂量组血清IL-6、TNF-α表达均降低,IL-10表达升高,颌下腺TNF-α mRNA表达降低(P<0.05);解毒通络生津方中、高剂量组颌下腺FoxP3蛋白表达升高,颌下腺FoxP3 mRNA表达升高(P<0.05)。结论:解毒通络生津方可能通过促进FoxP3+调节性T细胞(Treg)功能的稳定,抑制炎症细胞因子的分泌,从而缓解干燥综合征全身免疫炎症的进展。
Objective:To investigate the effect of different doses of Jiedu Tongluo Shengjin prescription(JTSP)on serum interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and forkhead box P3(FoxP3)in submandibular gland of NOD/Ltj mice with Sjögren's syndrome,and to explore the mechanism of JTSP on immune regulation in NOD/Ltj mice.Method:Thirty NOD/Ltj mice(eight weeks old)were randomly divided into model group,JTSP low-dose group,JTSP medium-dose group,JTSP high-dose group and hydroxychloroquine group,and were administrated with normal saline,JTSP 9,18,and 36 g·kg^(-1),and hydroxychloroquine 60 mg·kg^(-1) daily,respectively from the age of 12 weeks.Six ICR mice were given an equal amount of normal saline by gavage as the control group.During the experiment,daily water consumption and saliva secretion of mice at the age of 9,12,16 weeks were recorded.After 4 weeks of administration,submandibular gland and spleen tissues were dissected to calculate corresponding indexes.The pathological morphology of submandibular gland was observed by hematoxylin-eosin(HE)staining.Meso Scale Discovery(MSD)and immunohistochemistry were employed to detect the serum levels of IL-6,TNF-αand IL-10,and the expression and distribution of FoxP3 in submandibular gland,respectively.The protein expression of FoxP3 in mouse submandibular gland was determined by Western blot,and the mRNA expressions of FoxP3 and TNF-αwere determined by real-time polymerase chain reaction(Real-time PCR).Result:Compared with the control group,the model group presented increased daily water consumption,decreased saliva secretion,lowered submandibular gland index,elevated pathological score of submandibular gland,up-regulated serum IL-6 and TNF-αand mRNA expression of TNF-αwhile down-regulated serum IL-10 and protein and mRNA expressions of FoxP3 in submandibular gland(P<0.05).Compared with the conditions in model group,daily water consumption in JTSP groups was reduced while saliva secretion was increased,especially in medium-dose and high-dose groups(P<0.05),and there was an increase in the submandibular gland index of JTSP medium-dose group(P<0.05)while a decrease in the spleen index of JTSP high-dose group(P<0.05).Additionally,JTSP groups had lower pathological score of submandibular gland than the model group(P<0.05),especially high-dose group,as well as lower serum IL-6 and TNF-αand mRNA expression of TNF-αwhile higher serum IL-10(P<0.05).JTSP at medium and high doses up-regulated the protein and mRNA expressions of FoxP3 in submandibular gland(P<0.05).Conclusion:JTSP may inhibit the secretion of inflammatory cytokines by regulating the stability of FoxP3+regulatory T(Treg)cells,thus alleviating the systemic immune inflammation in Sjögren's syndrome.
作者
程丹丹
李永明
侯佳奇
薛鸾
CHENG Dandan;LI Yongming;HOU Jiaqi;XUE Luan(Yueyang Hospital of Integrated Traditional Chinese and Western Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 200437,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2023年第9期119-128,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81573925)。
