香砂六君子汤激活Wnt/β-catenin信号通路促进成骨分化防治PMOP的机制研究

The mechanism of Xiangsha Liujunzi Decoction activates Wnt/β-catenin signaling pathway promoting osteogenesis and preventing PMOP

目的 由Hedgehog信号通路探讨补肾方药左归丸对卵巢切除所致骨质疏松症(osteoporosis, OP)大鼠的作用机理。方法 选用雌性SD大鼠,随机分为空白对照组、假手术组、造模组。造模组切除双侧卵巢,假手术组不切除卵巢,仅切除卵巢周围少量脂肪组织。12周后,将造模组随机分为卵巢切除组(OVX组)、阳性对照组和左归丸组。阳性对照组灌服戊酸雌二醇混悬液,左归丸组灌服左归丸水煎液,OVX组灌服等体积纯水,给药13周后取材。通过Micro-CT检测各组骨密度(bone mineral density, BMD),免疫组化法(immunohistochemistry, IHC)检测各组胫骨骨髓基质细胞中Ihh、Ptch、Smo、Gli1、OPG、RANKL的蛋白表达。结果 假手术组骨密度(bone mineral density, BMD)高于OVX组、阳性对照组、左归丸组,且OVX组BMD较阳性对照组、左归丸组更低;OVX组Hedgehog信号通路关键因子Ihh、Ptch、Smo、GLi1蛋白表达水平以及RANKL/OPG比值较假手术组明显增高;阳性对照组和左归丸组Ihh、Ptch、Smo、GLi1蛋白表达水平及RANKL/OPG比值均低于OVX组,但仍高于假手术组。结论 左归丸可抑制卵巢切除所致的Hedgehog信号通路异常活跃,使其关键因子Ihh、Ptch、Smo、GLi1蛋白表达水平明显降低;进而降低RANKL/OPG比值,抑制破骨细胞(osteoclast, OC)活性,减少骨量流失。

Objective Research on the main mechanism of Xiangsha Liujunzi Decoction in preventing and treating postmenopausal osteoporosis.Methods SD female rats were randomly divided into 6 groups:blank group,model group,positive control group(Xian Ling Gu Bao capsule group),low,medium and high dose groups of Xiangsha Liujunji Decoction.Ovariectomy(OVX)method was used to make the model.After the model was successfully made,Chinese medicine was used to intervene.Materials were taken eight weeks later.MicroCT was used to detect bone tissue structure,biomechanical instrument was used to detect the maximum load of femur,ELISA was used to detect bone metabolism,and immunohistochemistry(IHC)was used to detect bone formation related proteins and pathway proteins.Results Compared with the model group,Xiangsha Liujunzi Decoction in each dose group could restore bone microstructure(P<0.05),especially in the high-dose group,so we used the high-dose group as the drug for this experiment;It also could significantly increase the maximum load on the femur of rats(P<0.05);Significantly increase and decrease the content of ALP and PINP in serumβ-CTX content level(P<0.01);Effectively increase the percentage of bone trabeculae(P<0.05);Promote the expression of Runx 2,the key protein regulating bone formation,and the osteogenic pathway related protein Wnt andβ-Catenin expression(P<0.05).Conclusion Xiangsha Liujunzi Decoction can restore the continuity and stability of the femoral trabecular bone of OVX rats,promote bone formation,improve bone density,and thus prevent and treat PMOP,and its mechanism may be related to the activation of Wnt/β-Catenin signaling pathway up-regulates the expression of Runx2,which contributes to bone differentiation.