益气养血方对1L-1β诱导的骨关节炎软骨细胞模型β-catenin、WISP-1蛋白水平的影响

Effects of Yiqi Yangxue Decoction to protein level of β-catenin, WISP-1 in IL-1β induced Osteoarthritis chondrocyte model

目的探讨益气养血方对体外培养的1L-1β诱导的骨关节炎模型软骨细胞β-catenin、WISP-1蛋白及MMP-1、MMP-13蛋白表达水平的影响,揭示其治疗骨关节炎部分机制。方法将20只大鼠按照3d(day)-2t(time)-1h(hour)模式完成益气养血方含药血清组及生理盐水血清组的含药血清制备,另将40只SD大鼠膝关节软骨建立离体培养的软骨细胞体系,抽签法随机分为正常组和诱导组,正常组以10%FBS/DMEM培养,诱导组以含10 ng/mL IL-1β的10%FBS/DMEM进行干预,应用HE染色及免疫荧光检测进行形态学鉴定。将IL-1β诱导的软骨细胞分成8个组:益气养血方药物血清组(20%、10%、5%)、生理盐水血清组(20%、10%、5%)、阳性对照组(DKK-1 30ng/ml)和IL-1β(10ng/ml)诱导组,并设置凋亡阳性对照组(用DNA酶处理)及细胞培养基对照组,观察不同组间大鼠软骨细胞增殖情况;应用Western blot技术检测细胞β-catenin、WISP-1、MMP-1、MMP-13蛋白的表达。结果 1L-1β能诱导软骨细胞发生凋亡,益气养血方可抑制1L-1β诱导的骨关节炎模型软骨细胞凋亡,同时能下调β-catenin、WISP-1、MMP-1、MMP-13的蛋白表达水平。结论益气养血方可能通过下调β-catenin、WISP-1调控Wnt/β-catenin信号通路,抑制下游MMP-1、MMP-13水平,可能是益气养血方减轻关节软骨破坏的机制之一。

Objective To evaluate the effects of Yiqi Yangxue Decoction on protein expression levels of β-catenin、WISP-1 MMP-1 and MMP-13 in chondrocytes of osteoarthritis model induced by 1 l-1 β in vitro, to reveal part of its mechanism for treating osteoarthritis.Methods 20 SD rats were made according to 3 d(day)-2 t(time)-1 h(hour)and randomly divided into the serum containing medicine of Yiqi Yangxue Decoction and normal saline serum group. The articular chondrocytes of 40 SD rats were isolated and cultured.According to the drawing, they were randomly divided into normal group and induction group, normal group treated with 10% FBS/DMEM,induction group treated with IL-1β at concentration of 10 mg/L and 10% FBS/DMEM. Morphological identified by the HE staining and immunofluorescence detection. The chondrocytes induced by IL-1 β were divided into 8 groups: Yiqi Yangxue Decoction serum group(20%,10%,5%), saline serum group(20%,10%,5%), positive control group(DKK-1 30 ng/ml) and IL-1 β(10 ng/ml) group, and apoptotic positive control group(treated with DNA enzyme) and cell culture medium control group. Observation of chondrocyte proliferation in different groups;detected the the protein levels of β-catenin, WISP-1,MMP-1,MMP-13 in cartilage tissues by immunohistochemical Western Blot. Results Apoptosis of chondrocytes can be induced by 1 L-1β. Yiqi Yangxue Decoction can inhibit the apoptosis of chondrocytes, down-regulate the protein levels of β-catenin, WISP-1,MMP-1,MMP-13.Conclusion Yiqi Yangxue Decoction may regulate Wnt/β-catenin signaling pathway by down-regulating the protein levels of β-catenin、WISP-1, inhibition the MMP-1,MMP-13 of downstream factors’ levels, may be one of the mechanisms of Yiqi Yangxue Decoction to alleviate the destruction of articular cartilage.