诃子水提物通过上调PD-1/PD-L1表达调节胶原蛋白诱发关节炎(CIA)大鼠脾脏中细胞的细胞免疫减轻关节炎症

Terminalia chebula water extract reduces joint inflammation by regulating cellular immunity in spleen cells of collagen-induced arthritis(CIA)rats through up-regulation of PD-1/PD-L1

目的研究诃子水提取物(TCWE)对胶原蛋白诱发关节炎(CIA)大鼠细胞免疫及程序性死亡蛋白1/程序性死亡蛋白1配体1(PD-1/PD-L1)通路的影响。方法SD大鼠随机分为对照组、CIA组、TCWE处理组、甲氨蝶呤(MTX)处理组,每组15只。除对照组外,其余各组SD大鼠皮下注射Ⅱ型胶原蛋白制备胶原蛋白诱发关节炎(CIA)模型,TCWE组大鼠采用[20 mg/(kg·d)]TCWE处理,MTX组大鼠采用[1.67 mg/(kg·d)]MTX处理。处理14 d后,通过HE染色检查软骨形态,流式细胞术检测脾脏T淋巴细胞凋亡和调节性T细胞(Treg)/Th17细胞比率,反转录PCR检测脾脏中的维甲酸相关孤核受体γt(RORγt)和叉头盒转录因子P3(FOXP3)、PD-1和PD-L1的mRNA表达,免疫组织化学染色检测RORγt、FOXP3的表达和定位。Western blot法检测脾脏淋巴细胞的PD-1和PD-L1的蛋白表达,ELISA检测大鼠血清白细胞介素17(IL-17)和转化生长因子β(TGF-β)水平。结果与CIA组相比,TCWE组和MTX组的软骨和滑膜病变明显减轻,脾脏中的T淋巴细胞凋亡率增加,Treg/Th17细胞比率上调,RORγt的表达降低,FOXP3、PD-1和PD-L1的表达增加。血清IL-17水平降低,血清TGF-β水平升高。结论TCWE处理可能激活脾脏细胞的PD-1/PD-L1通路调节CIA大鼠脾脏中细胞的细胞免疫,减轻软骨损伤。

Objective To investigate the effect of Terminalia chebula water extract(TCWE)on the cellular immunity and PD-1/PD-L1 pathway in rats with collagen-induced arthritis(CIA).Methods SD rats were randomly divided into four groups:a control group,a CIA group,a TCWE group and a methotrexate(MTX)group,with 15 rats in each group.Except for the control group,SD rats in other groups were subcutaneously injected with typeⅡcollagen to establish the model of collagen-induced arthritis(CIA).The rats in the TCWE group were treated with 20 mg/(kg·d)TCWE and the rats in the MTX group were treated with 1.67 mg/(kg·d)MTX.After 14 days of treatment,the cartilage morphology was examined using hematoxylin-eosin(HE)staining,and splenic T lymphocyte apoptosis and Treg/Th17 cell ratio were detected by flow cytometry.The mRNA expressions of retinoid-related orphan nuclear receptorγt(RORγt),forkhead box P3(FOXP3),PD-1 and PD-L1 in spleen were detected by reverse transcription PCR.The expression and localization of RORγt and FOXP3 were detected by immunohistochemical staining.The protein expressions of PD-1 and PD-L1 in splenic lymphocytes were detected by Western blot,and the levels of serum interleukin 17(IL-17)and transforming growth factorβ(TGF-β)in rats were detected by ELISA.Results Compared with CIA group,the pathological changes of cartilage and synovium were significantly alleviated in the TCWE group and the MTX group.Both the apoptosis rate of T lymphocytes in spleen and the ratio of Treg/Th17 cells increased.The expression of RORγt decreased,while the expressions of FOXP3,PD-1 and PD-L1 increased in spleen lymphocytes.The level of serum IL-17 decreased,while the level of serum TGF-βincreased.Conclusion TCWE treatment may activate PD-1/PD-L1 pathway in spleen cells to regulate cellular immunity,thus reducing cartilage injury in CIA rats.