3.3 益气养血方血清对体外培养的大鼠膝关节软骨细胞增殖情况的影响
在24h，48h两个时间点，空白对照组（培养基对照）与模型对照组（IL-1β诱导组）比较，空白对照组OD A450值明显高于模型对照组（P＜0.01），说明退行性软骨细胞诱导成功；各生理盐水血清组（5%、10%、20%）与模型对照组比较差异无统计学意义（P＞0.05）；与5%生理盐水血清组比较，10%、20%益气养血方血清组、阳性对照组（DKK-1组）高于5%生理盐水血清组（P﹤0.05，P﹤0.01）；与阳性对照组比较，10%、20%益气养血方血清组差异无统计学意义（P＞0.05）。实验结果显示，益气养血方血清对退变软骨细胞的增殖具有促进作用。见图13。
图13 益气养血方血清对体外培养的大鼠膝关节软骨细胞增殖情况的影响。用CCK8试剂盒测定24h和48h时各处理组细胞的A450，比较其生长状态。与模型对照组比较，**P<0.01；与5%生理盐水血清组比较，##P<0.01，#P<0.05。
3.4 Western blotting检测细胞β-catenin、WISP-1、MMP-1、MMP-13蛋白的表达
结果如图4所示，经过IL-1β诱导的大鼠软骨细胞中MMP-1、MMP-13、WISP-1和β-catenin蛋白表达量均上升，高于空白对照组，说明诱导大鼠退行性软骨细胞成功。检测发现，生理盐水血清各组中这四种蛋白表达量与诱导组一致，说明生理盐水血清对退行性软骨细胞没有治疗作用。药物血清组中，各蛋白表达量均有所下降。10%药物血清组，β-catenin、MMP-13、WISP-1和MMP-1的表达量与阳性药物组相似；20%药物血清组，四种蛋白的表达量均低于阳性药物组，说明20%的药物血清能明显抑制β-catenin、MMP-13、WISP-1和MMP-1的表达。
图4 益气养血方血清对大鼠软骨细胞β-catenin、WISP-1、MMP-1、MMP-13蛋白表达的影响。在药物干预后48h收集细胞，提取各组细胞总蛋白，Western blotting检测目的蛋白的表达情况。1、模型对照组（IL-1β诱导组）；2、5%生理盐水血清组；3、10%生理盐水血清组；4、20%生理盐水血清组；5、空白对照组；6、5%益气养血方血清组；7、10%益气养血方血清组；8、20%益气养血方血清组；9、阳性对照组（DKK-1组）。
4 讨论
益气养血方是云南省名老中医吴生元教授总结出的治疗骨关节炎的有效良方。该方具有补益气血、调补肝肾、活络止痛之功。益气养血方的临床研究显示：益气养血方能明显改善患者症状及关节功能，临床安全性高，依从性好 [[[]李兆福,彭江云,肖长虹,等.骨痹(骨关节炎)诊疗方临床验证总结[J].风湿病与关节炎.2012,1(1):16-21.]]。实验研究表明，益气养血方能上调人膝骨关节炎软骨聚集蛋白聚糖mRNA，下调COL-10 mRNA表达；对大鼠WIF1水平有上调作用，下调大鼠Wnt4水平，具有明显的抗炎作用，能促进软骨细胞的体外增殖，保护软骨基质，抑制关节软骨病变[[[]李兆福,刘维超,狄朋桃,等.益气养血方对膝骨关节炎退变软骨组细胞蛋白聚糖和X型胶原mRNA表达的影响.中华中医药学会第十六届全国风湿病学术大会论文集,2012:44-48.]-10]。为此，在前期研究基础上，进一步在体外实验模型—IL-1β诱导大鼠软骨细胞退变模拟骨关节炎模型上，研究益气养血方对白介素诱导性大鼠关节炎的作用及部分机制。IL-1β诱导的关节软骨退变模型是研究骨关节炎的理想模型，也是世界上公认的关节炎模型[[[]Ding QH, Zhong HM, Qi YY, et al. Anti-arthritic effects of crocin in interleukin-1β treated articular chondrocytes and cartilage in a rabbit osteoarthritic model[J].Inflamm Res, 2013, 62(1): 17-25.]-12]。
Wnt/β-catenin信号通路是一条广泛存在且高度保守的信号通路，主要分为经典Wnt信号通路（Wnt/β-catenin信号通路）、PCP通路及Wnt/Ca2 +通路，涉及肿瘤、骨病、肝病、血管疾病、皮肤科疾病以及多种免疫类疾病[[[] Kanehisa M，et al．KEGG: kyoto encyclopedia of genes and genomes [J].Nucleic Acids Res,2000,Jan 01: 27-30.]-14]。Wnt信号通路参与软骨发育、稳态平衡及软骨的退化，干扰Wnt通路可以促进软骨再生[[[] Maruotti N ,Corrado A,Neve A,et al.Systemic effects of Wnt signaling [J].Journal of cellular physiology, 2013, 228(7):1428-1432.]-17]。其中研究最多的为Wnt/β-catenin信号通路，当Wnt/β-catenin信号被激活，细胞质内的β-catenin向核内转移，与转录因子结合后激活下游靶基因表达，上调的Wnt/β-catenin可促进关节软骨细胞的过度成熟、分化及凋亡，从而加重细胞外基质的降解、关节软骨退变及骨赘生成，在骨关节炎的发生及发展中起重要作用[[[] Staal FJ, Clevers H. Tcf/Lef transcription factors during T-cell development:unique and overlapping functions[J]. Hematol J. 2000;1:3–6.]-20]。
Wnt/β-catenin信号通路的表达，受上游抑制因子的调节，如sFPR3、WISP-1、DKK1等[[[] Loughlin J, Dowling B, Chapman K, Marcelline L, et al. Functional variants within the secreted frizzledrelated protein 3 gene are associated with hip osteoarthritis in females. Proc Natl Acad Sci U S A. 2004;101:9757–62.]-23]，其中WISP-1在软骨发生退变时表达异常增高，促进下游的基质金属蛋白酶分泌使胶原蛋白与蛋白多糖的分解的表达导致关节软骨损伤，在滑膜内可使IL-6表达升高[[[] Blom AB, Brockbank SM, van Lent PL, et al. Involvement of the Wnt signaling pathway in experimental and human osteoarthritis: prominent role of Wnt-induced signaling protein 1. Arthritis Rheum. 2009;60(2):501–12.]]。在小鼠实验中，WISP-1在膝关节腺病毒中的高表达与关节软骨破坏程度成正相关[[[] VAN DEN BOSCH MH, BLOM AB, SLOETJES AW, et al. Induction of canonical Wnt signaling by synovial overexpression of selected Wnts leads to protease activity and early Osteoarthritis-Like cartilage damage[J]. Am J Pathol, 2015, 185(7):1970-1980.]]。当Wnt/β-catenin信号通路被激活后，下游靶基因如BMPs、MMPs、MYC被激活[[[] Liu F, Millar SE. Wnt/β-catenin signaling in oral tissue development and disease. [J]. J Dent Res, 2010, 89:381-330.]]。其中基质金属蛋白酶家族可与II 型胶原分子上的MMPs微电裂解位点结合使II 型胶原片段化，从而软骨细胞暴露于炎性环境中[[[] Hideto W. Aggrecan and its chondroitin sulfate in cartilage. Seikagaku. 2008;80(1):28-32]]。在关节软骨破坏严重的患者体内MMP-1、MMP-13呈高表达，可破坏关节软骨内稳态，加剧关节退变[[[] Wang M, Sampson ER, Jin HT, et al . MMP-13 is a critical target gene during the progression of osteoarthritis. Arthritis Res Ther, 2013, 15:R5.]-29]。
本研究结果表明，益气养血方对IL-1β诱导的关节软骨退变模型中软骨细胞凋亡有抑制作用，能下调β-catenin、WISP-1、MMP-1、MMP-13蛋白表达，减缓关节软骨退变。基于上述分析，推测益气养血方可能通过下调β-catenin、WISP-1，抑制相关Wnt/β-catenin信号通路激活，降低下游靶基因MMP-1、MMP-13的表达，可能是其保护关节软骨，减缓骨关节炎的病理发展的机制之一。
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