摘要
目的:基于蛋白多糖降解探讨加味四妙方对单钠尿酸盐(MSU)诱导的大鼠痛风性关节炎(GA)的作用及其作用机制。方法:将6只SD大鼠随机分为空白组和模型组,每组3只,模型组向关节腔内注射0.1 mL MSU溶液(100 mg/mL)建立GA模型,对照组注射等体积生理盐水。24 h后对两组大鼠的滑膜组织进行转录组测序,筛选出差异表达基因。体外培养家兔膝关节软骨细胞,甲苯胺蓝染色、RT-PCR法检测不同代数软骨细胞中蛋白多糖(PGs)的表达。采用Western blot与qRT-PCR法检测不同浓度MSU(200、500、1000µmol/L)处理后MMP-3的蛋白及mRNA表达。将33只SD大鼠随机分为空白组、模型组、给药组,每组11只。膝关节注射0.1 mL MSU溶液(100 mg/mL)制备大鼠痛风性关节炎模型,免疫组化检测滑膜组织中MMP-3的表达,番红固绿染色法检测大鼠关节软骨细胞蛋白多糖的表达。结果:MMP-3是显著上调的差异表达基因,与Jak/STAT信号通路、TGF-β信号通路、趋化因子信号通路、IL-17信号通路等密切相关。体外验证发现,与空白组比较,经250、500、1000µmol/L MSU刺激24 h后,MMP-3蛋白表达水平明显升高(P<0.05);与空白组比较,MMP-3 mRNA表达水平经250µmol/L MSU刺激后明显升高(P<0.01),经500、1000µmol/L MSU刺激后其表达显著升高(P<0.001)。与空白组比较,经250、500µmol/L MSU刺激后,各组软骨细胞蛋白多糖表达均显著降低(P<0.05);与250µmol/L MSU组比较,10%和20%加味四妙方含药血清(MSM)组均能有效逆转上述结果(P<0.001)。与500µmol/L MSU组比较,20%MSM能够有效上调蛋白多糖的表达(P<0.05)。与空白组比较,模型组软骨细胞蛋白多糖mRNA表达显著降低(P<0.001);与模型组相比,10%MSM与20%MSM均能有效增加其mRNA表达(P<0.001,P<0.05)。与空白组比较,模型组大鼠膝关节肿胀明显(P<0.05,P<0.01);与模型组比较,各给药组能明显抑制其关节肿胀(P<0.05,P<0.01)。与空白组比较,模型组MMP-3表达增加且关节软骨表层蛋白多糖丢失;与模型组相比,各给药组对上述现象均有明显改善作用。结论:加味四妙方通过抑制MMP-3生成调控蛋白多糖降解从而治疗痛风性关节炎。
Objective:To investigate the effects and mechanisms of modified Simiao Formula(MSM)on monosodium urate(MSU)-induced gouty arthritis(GA)based on proteoglycan degradation.Methods:6 SD rats were randomly divided into blank group and model group,with three rats in each group.0.1 mL MSU solution(100 mg/mL)was injected into the joint cavity in the model group to establish the GA model,and the same volume of saline was injected into the joint cavity in the control group.Transcriptome sequencing was performed on synovial tissues of rats in both groups after 24 h to screen out differentially expressed genes.Knee joint chondrocytes of rabbit were cultured in vitro.The expression of proteoglycans(PGs)in different generation of chondrocytes was detected by toluidine blue staining and RT-PCR.Western blot and qRT-PCR were used to detect the protein and mRNA expression of MMP-3 after it was treated with different concentrations of MSU solution(200,500 and 1000μmol/L).33 SD rats were randomly divided into blank group,model group and drug administration group,with 11 rats in each group.Gouty arthritis models were prepared by injecting 0.1 mL MSU solution(100 mg/mL)into each knee joint.MMP-3 expression in the synovial tissue was detected by immunohistochemistry,and the expression of proteoglycans in rat articular chondrocytes was detected by Safranin O-Fast Green staining.Results:MMP-3 is a significantly up-regulated differentially expressed gene,which is closely related to Jak/STAT signaling pathway,TGF-βsignaling pathway,chemokine signaling pathway,and IL-17 signaling pathway.In vitro experiments revealed that,compared with the blank group,after 24 h of stimulation with 250,500 and 1000μmol/L MSU,MMP-3 protein expression level was significantly increased(P<0.05).Compared with the blank group,the MMP-3 mRNA expression level was significantly increased after stimulation with 250μmol/L MSU(P<0.01),and even more significantly increased after stimulation with 500 and 1,000μmol/L MSU(P<0.001).Compared with the blank group,chondrocyte proteoglycan expression was significantly reduced in all other groups after stimulation with 250 and 500μmol/L MSU(P<0.05).Both 10%and 20%modified Simiao Formula medicated serum groups could effectively reverse these results compared with the 250μmol/L MSU group(P<0.01).Compared with the 500μmol/L MSU group,20%MSM group could effectively upregulate proteoglycan expression(P<0.05).Chondrocyte proteoglycan mRNA expression was significantly decreased in the model group compared with the blank group(P<0.001),and both 10%MSM and 20%MSM groups could effectively increase their mRNA expression compared with the model group(P<0.001,P<0.05).Compared with the blank group,the knee joint swelling of rats in the model group was obvious(P<0.05,P<0.01),and compared with the model group,each administration group could significantly inhibit the joint swelling(P<0.05,P<0.01).Compared with the blank group,the model group showed increased expression of MMP-3 and loss of proteoglycans in the surface layer of articular cartilage,and compared with the model group,every administration group significantly improved the above phenomenon.Conclusion:modified Simiao Formula can regulate proteoglycan degradation by inhibiting MMP-3 production,and therefore,it can treat gouty arthritis.
作者
秦冬儿
颜星宇
梁涛
唐璠
牛玉珍
朱方方
杨凡
时乐
QIN Donger;YAN Xingyu;LIANG Tao;TANG Fan;NIU Yuzhen;ZHU Fangfang;YANG Fan;SHI Le(Nanjing University of Chinese Medicine,Nanjing 210000,China)
出处
《中医药信息》
2023年第6期1-7,22,共8页
Information on Traditional Chinese Medicine
基金
国家自然科学基金项目(81703835)。
关键词
蛋白多糖
痛风性关节炎
基质金属蛋白酶
软骨细胞
加味四妙方
单钠尿酸盐
Proteoglycan
Gouty arthritis(GA)
Matrix metalloproteinase(MMP)
Chondrocyte
Modified Simiao Formula(MSM)
Monosodium urate(MSU)