增液润燥汤对干燥综合征小鼠颌下腺相关mRNA和蛋白及外周血Th17/Treg表达的影响

Effects of Zengye Runzao Decoction on Submandibular Gland Related mRNAs,Proteins and Th17/Treg in Peripheral Blood of Mice with Sjögren Syndrome

目的 观察增液润燥汤对干燥综合征(SS)模型小鼠颌下腺组织相关mRNA和蛋白及外周血辅助性T细胞17(Th17)/调节性T细胞(Treg)表达的影响。方法 将SPF级BALB/c小鼠随机分为对照组和造模组,造模组通过免疫诱导法建立SS小鼠模型,对照组不予处理,将成模小鼠随机分为模型组、白芍总苷组和增液润燥汤低、中、高剂量组,每组3只。白芍总苷组予白芍总苷溶液0.234 mg/g灌胃,增液润燥汤低、中、高剂量组予增液润燥汤4、8、12 mg/g灌胃,对照组和模型组予等体积蒸馏水灌胃,每日1次,连续60 d。计算小鼠日饮水量、唾液流率、颌下腺指数,HE染色观察小鼠颌下腺组织形态,qPCR检测颌下腺组织同源异形盒基因A1(HOXA1)、信号传导与转录激活因子3(STAT3)、白细胞介素-17(IL-17)、叉头框蛋白P3(FOXP3) mRNA和miR-181c-3p表达,Western blot检测颌下腺组织HOXA1、STAT3、p-STAT3、IL-17、FOXP3蛋白表达,流式细胞仪检测外周血Th17、Treg比例。结果 与对照组比较,模型组小鼠日饮水量增加,唾液流率、颌下腺指数降低,颌下腺组织淋巴细胞浸润明显,HOXA1、STAT3、IL-17 mRNA表达升高,FOXP3 mRNA和miR-181c-3p表达降低,HOXA1、STAT3、p-STAT3、IL-17蛋白表达升高,FOXP3蛋白表达降低,Th17比例、Th17/Treg升高,Treg比例降低,差异均有统计学意义(P<0.05);与模型组比较,增液润燥汤各剂量组小鼠日饮水量减少,唾液流率升高,颌下腺组织淋巴细胞浸润均有不同程度减轻,HOXA1、IL-17 mRNA表达降低,FOXP3 mRNA和miR-181c-3p表达升高,HOXA1蛋白表达降低,FOXP3蛋白表达升高,Th17比例、Th17/Treg降低,Treg比例升高,增液润燥汤高剂量组颌下腺指数升高,STAT3 mRNA、蛋白表达及p-STAT3、IL-17蛋白表达降低,差异均有统计学意义(P<0.05)。结论 增液润燥汤可能通过上调miR-181c-3p、FOXP3表达,下调HOXA1、p-STAT3、STAT3、IL-17表达,调节Th17/Treg细胞平衡,改善SS所致颌下腺功能和组织损伤。

Objective To explore the effects of Zengye Runzao Decoction on mRNAs and proteins related to submandibular gland tissue and the expression of Th17/Treg in peripheral blood of Sjögren syndrome(SS)model mice.Methods SPF Balb/c mice were randomly divided into control group and modeling group,the modeling group established an SS mouse model through immune induction,while the control group was not treated.The modeled mice were randomly divided into model group,total glucosides of Paeoniae Radix Alba group and Zengye Runzao Decoction low-,medium-and high-dosage groups,with 3 mice in each group.The total glucosides of Paeoniae Radix Alba group was given 0.234 mg/g total glucosides of Paeoniae Radix Alba solution by gavage,Zengye Runzao Decoction low-,medium-and high-dosage groups were given 4,8,12 mg/g Zengye Runzao Decoction by gavage,and the control group and model group were given the same volume of distilled water by gavage,once a day for 60 consecutive days.The daily water intake,salivary flow rate and submandibular index were calculated,the tissue morphology of submandibular gland was observed by HE staining,the expressions of HOXA1,STAT3,IL-17,FOXP3 mRNA and miR-181c-3p in submandibular gland tissue were detected by qPCR,Western blot was used to detect the expressions of HOXA1,STAT3,p-STAT3,IL-17 and FOXP3 protein in submandibular gland tissue,the proportion of Th17 and Treg cells in peripheral blood was detected by flow cytometer.Results Compared with the control group,the daily water intake of the model group mice increased,saliva flow rate and submandibular gland index decreased,lymphocyte infiltration in submandibular gland tissue was significant,the expressions of HOXA1,STAT3,IL-17 mRNA increased,the expressions of FOXP3 mRNA and miR-181c-3p decreased,the expressions of HOXA1,STAT3,p-STAT3,IL-17 protein increased,FOXP3 protein expression decreased,Th17 ratio and Th17/Treg increased,and Treg ratio decreased,with statistical significance(P<0.05).Compared with the model group,the daily water intake of Zengye Runzao Decoction groups decreased,the saliva flow rate increased,and the infiltration of lymphocytes in the submandibular gland tissue was reduced to varying degrees,the expressions of HOXA1 and IL-17 mRNA decreased,while the expressions of FOXP3 mRNA and miR-181c-3p increased,the expression of HOXA1 protein decreased,while the expression of FOXP3 protein increased,the ratio of Th17 and Th17/Treg decreased,and the Treg ratio increased.The submandibular gland index increased in Zengye Runzao Decoction high-dosage group,and the expressions of STAT3 mRNA and protein,as well as p-STAT3 and IL-17 protein decreased,with statistical significance(P<0.05).Conclusion Zengye Runzao Decoction may regulate Th17/Treg cell imbalance by up-regulating the expressions of miR-181c-3p,FOXP3 and down-regulating the expressions of HOXA1,p-STAT3,STAT3 and IL-17,and then improve the function and tissue injury of submandibular gland caused by SS.