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目的:探讨清痹片对KLF6-FRP调控体系的干预作用,为中医药治疗类风湿关节炎(RA)的机制研究和推广应用提供依据。方法:复制胶原诱导型关节炎(CIA)大鼠模型,随机分为空白组,模型组,清痹片低剂量组、中剂量组、高剂量组,并以甲氨蝶呤(MTX)治疗组作为阳性对照,致炎第21天起每天给药干预。分别于第35天和第49天各处死一半动物,取腕关节行免疫印迹法检测Kruppel样转录因子6(KLF6)、卵泡抑素相关蛋白(FRP),踝关节行免疫组化法实验检测转化生长因子β1(TGFβ1)、肿瘤坏死因子α(TNFα)、白细胞介素1β(IL-1β)、基质金属蛋白酶3(MMP-3),比较各组间各炎性蛋白表达的差异。结果:成功复制CIA模型,模型组KLF6、FRP、TGFβ1、TNFα、IL-1β、MMP-3蛋白表达水平较空白组升高(P<0.01);清痹片中、高剂量组KLF6蛋白表达水平与模型组相比较低而FRP较高(P<0.01),并显示出一定的量效依赖关系;清痹片中、高剂量组FRP蛋白表达水平与MTX组相比较高(P<0.01);清痹片各剂量组、MTX组TGFβ1、TNFα、IL-1β、MMP-3表达水平均低于模型组;清痹片高剂量组TNFα、IL-1β、MMP-3表达水平低于MTX组(P<0.01)。结论:清痹片可能通过提高TGFβ1的下游产物FRP表达水平以及直接或间接抑制KLF6及其下游靶基因TGFβ1、炎性细胞因子IL-1β、TNFα和MMP-3等炎症介质的基因表达,从而干预KLF6-FRP调控体系,控制相关因子对RA病变过程的影响。
Objective: To investigate the intervention effect of Qingbai Pian on KLF6-FRP regulatory system and provide the basis for the mechanism research and promotion and application of traditional Chinese medicine in the treatment of rheumatoid arthritis (RA). Methods: The rat model of collagen induced arthritis (CIA) was duplicated and randomly divided into blank group, model group, Qingbiao tablet low dose group, middle dose group and high dose group, and methotrexate (MTX) treatment group As a positive control, day 21 administration of inflammation daily dose of intervention. Half animals were sacrificed on day 35 and day 49 respectively. Kruppel-like transcription factor 6 (KLF6) and follistatin-related protein (FRP) were detected by Western blotting. The ankles were examined by immunohistochemistry (TGFβ1), tumor necrosis factor α (TNFα), interleukin 1β (IL-1β) and matrix metalloproteinase-3 (MMP-3) Results: The expression of KLF6, FRP, TGFβ1, TNFα, IL-1β and MMP-3 in model group were significantly higher than those in blank group (P <0.01) Compared with the model group, FRP was higher (P <0.01), and showed a dose-dependent relationship. The expression of FRP in Qingbai tablet medium and high dose groups was higher than that in MTX group (P <0.01). The expression levels of TGFβ1, TNFα, IL-1β and MMP-3 in Qingbian tablet group and MTX group were lower than those in model group. The expression levels of TNFα, IL-1β and MMP-3 in high dose group of Qingbai tablet group were lower than those in MTX group P <0.01). Conclusion: Qingbai tablet may interfere with the expression of FRP, a downstream product of TGFβ1, and directly or indirectly inhibit the gene expression of KLF6 and its downstream target genes such as TGFβ1, inflammatory cytokines IL-1β, TNFα and MMP-3 KLF6-FRP regulatory system, the impact of control factors on the pathological changes of RA.