解毒通络生津方对干燥综合征NOD小鼠PD-1/PD-L1信号通路的影响

Effect of Jiedu Tongluo Shengjin Recipe on PD-1/PD-L1 Signal Pathway in NOD Mice with Sjogren’s Syndrome

目的观察解毒通络生津方对干燥综合征(SS)模型NOD小鼠PD-1/PD-L1通路的影响,探讨其作用机制。方法将24只8周龄雌性NOD小鼠随机分为3组,即模型组(Model)、解毒通络生津方组(TCM)、解毒通络生津方加抗PD-L1单抗阻断组(TCM+α-PD-L1),每组8只。另选8只8周龄雌性ICR小鼠作为正常对照组(Ctrl)。12周龄时开始中药干预,TCM组按照每天24.4 g·kg^(-1)解毒通络生津方灌胃,TCM+α-PD-L1组在每天给予24.4 g·kg^(-1)解毒通络生津方灌胃的基础上,腹腔注射抗PD-L1抗体,1周2次,每次100μg,Ctrl组和Model组给予同等剂量的生理盐水灌胃,实验期间记录各组小鼠第9、12、16、20周龄时的日均饮水量,干预8周后,小鼠统一麻醉下摘取颌下腺。HE染色观察各组小鼠颌下腺中炎症细胞浸润情况;流式细胞仪检测小鼠颌下腺CD4^(+)CD25^(+)Foxp3^(+)PD-1^(+)Treg细胞的表达;Western Blot检测小鼠颌下腺中PD-1、PD-L1和Foxp3蛋白的表达。结果随着小鼠周龄的增长,Ctrl组小鼠日均饮水量基本不变,Model组小鼠的日均饮水量有明显上升趋势,TCM组小鼠日均饮水量下降,TCM+α-PD-L1组小鼠日均饮水量上升;与Model组比较,TCM组炎症细胞浸润灶明显减少(P<0.001);与TCM组比较,TCM+α-PD-L1组炎症细胞浸润增多(P<0.001);与Ctrl组比较,Model组CD4^(+)CD25^(+)Foxp3^(+)PD-1^(+)Treg的表达降低(P<0.001),与Model组比较,TCM组CD4^(+)CD25^(+)Foxp3^(+)PD-1^(+)Treg表达升高(P<0.01);与TCM组比较,TCM+α-PD-L1组CD4^(+)CD25^(+)Foxp3^(+)PD-1^(+)Treg的表达降低(P<0.01);与Ctrl组比较,Model组小鼠颌下腺中PD-1、PD-L1和Foxp3蛋白的表达降低(P<0.01);与Model组比较,TCM组小鼠颌下腺中PD-1、PD-L1和Foxp3蛋白的表达升高(P<0.01);与TCM组比较,TCM+α-PD-L1组小鼠颌下腺中PD-1、PD-L1和Foxp3蛋白的表达降低(P<0.001)。结论解毒通络生津方可能通过PD-1/PD-L1信号通路促进SS模型小鼠Treg细胞活化,上调Foxp3的表达,增强Treg细胞抑制免疫炎症的作用,PD-1/PD-L1可能是解毒通络生津方治疗SS的一个作用靶点。

Objective To observe the effect of Jiedu Tongluo Shengjin recipe on PD-1/PD-L1 pathway in NOD mice with Sjogren’s syndrome(SS)and to explore its mechanism.Methods 24 female 8-week-old NOD mice were randomly divided into three groups:model group(Model),Jiedu Tongluo Shengjin recipe group(TCM),Jiedu Tongluo Shengjin recipe plus PD-L1 molecular blocking group(TCM+α-PD-L1),8 mice in each group.Eight 8-week-old female ICR mice were selected as normal control group(Ctrl).Began to intervene at 12 weeks of age.TCM group was solved according to 24.4 g·kg^(-1)per day.Jiedu Tongluo Shengjin recipe was given intragastrically to TCM+α-PD-L1 group on the basis of intragastric administration of 24.4 g·kg^(-1)Jiedu Tongluo Shengjin recipe every day,Anti-PD-L1 antibody was ip injected intraperitoneally twice a week,100μg at a time.Ctrl group and Model group were given the same dose of normal saline intragastrically.At 9,12,16,and 20 weeks of age,the daily water intake of all mice were recorded,After 8weeks of intervention,the submandibular gland was removed under unified anesthesia.The infiltration of inflammatory cells in submandibular gland of mice was observed by HE staining,and the expression of CD4^(+)CD25^(+)Foxp3^(+)PD-1^(+)Treg cells in submandibular gland of mice was detected by flow cytometry;The expression of PD-1、PD-L1 and Foxp3proteins by Western Blot.Results With the increase of the age of the mice,the daily drinking water of the Ctrl group remained unchanged,the daily drinking water of the Model group increased significantly,the daily drinking water of the TCM group decreased,and the daily drinking water of the TCM+α-PD-L1 group increased;The inflammatory cell infiltration was significantly decreased in TCM group compared with the Model group(P<0.001),and increased in TCM+α-PD-L1 group compared with the TCM group(P<0.001);the expression of CD4^(+)CD25^(+)Foxp3^(+)PD-1^(+)Treg was reduced in the Model group Compared with the Ctrl group(P<0.001),the expression of CD4^(+)CD25^(+)Foxp3^(+)PD-1^(+)Treg was increased in the TCM group Compared with the Model group(P<0.01),the expression of CD4^(+)CD25^(+)Foxp3^(+)PD-1^(+)Treg decreased in TCM+α-PD-L1 group compared with TCM group(P<0.01);the proteins expressions of PD-1,PD-L1,and Foxp3 in the submandibular glands were reduced in model group Compared with the Ctrl group(P<0.01),The protein expression of PD-1,PD-L1,and Foxp3 were expressed in the submandibular glands was increased in the TCM group Compared with the Model group(P<0.01),the proteins expressions of PD-1,PD-L1 and Foxp3 in the submandibular glands were decreased in the TCM+α-PD-L1 group Compared with the TCM group(P<0.001).Conclusion Jiedu Tongluo Shengjin Recipe may promote the activation of Treg cells,up-regulate the expression of Foxp3 and enhance the anti-inflammatory effect of Treg cells through PD-1/PD-L1 signaling pathway.PD-1/PD-L1 may be a target of Jiedu Tongluo Shengjin Recipe in the treatment of SS.