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| 骨疏方调节FoxO3a/Wnt+O4+D4:R4+D4:Q4+O4+D4:R4+O+D4:P4 |
| Impact of Gushu formula on H2O2 induced osteoblast injury by regulating the FoxO3a/Wnt signaling pathway |
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| DOI：10.3969/j.issn.1006-7108.2024.10.004 |
| 中文关键词: 骨桂枝 软骨细胞 坏死性凋亡 膝骨关节炎 信号通路疏方 过氧化氢 成骨细胞 FoxO3a/Wnt信号通路 骨质疏松症 |
| 英文关键词:Gushu formulacassia twig chondrocyte necroptosis knee osteoarthritis signaling pathway hydrogen peroxide osteoblasts FoxO3a/Wnt signaling pathway osteoporosis |
| 基金项目:武汉市医学科国家自然科学基金面上项目（82274545）；江苏省医学重点学科/实验室建设单位项目（JSDW202252）；江苏省中医院科主任学术提升专项（Y2022ZR26）研项目（WZ19C17） |
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| 中文摘要: |
| 目的 探讨骨疏方调节FoxO3a/Wnt信号通路对过氧化氢（H2O2）诱导成骨细胞损伤的影响。方法 CCK-8法检测不同浓度（0.01、0.1、1、10、100 μmol/L）的骨疏方对H2O2诱导的MC3T3-E1细胞活力。随后设置对照组、H2O2（150 μmol/L H2O2）组、骨疏方组、骨疏方+空载体组、骨疏方+FoxO3a过表达组。CCK-8法、ELISA分别检测细胞活力、超氧化物歧化酶（SOD）、丙二醛（MDA）；成骨诱导分化后，碱性磷酸酶（ALP）试剂盒及茜素红分析ALP活性、细胞骨矿化结节；qRT-PCR检测成骨相关基因OPN mRNA、Runx2 mRNA以及FoxO3a mRNA表达水平；Western blot检测通路相关蛋白FoxO3a、Wnt2、β-连环蛋白（β-catenin）表达水平。结果 与对照组比较，H2O2组MDA、FoxO3a表达显著增加，细胞活力、SOD、ALP活性、骨矿化结节数量、OPN mRNA、Runx2 mRNA、Wnt2、β-catenin表达显著降低（P＜0.05）；与H2O2组比较，骨疏方组MDA、FoxO3a表达显著降低，细胞活力、SOD、ALP活性、骨矿化结节数量、OPN mRNA、Runx2 mRNA、Wnt2、β-catenin表达显著增加（P＜0.05）；与骨疏方+空载体组比较，骨疏方+FoxO3a过表达组MDA、FoxO3a表达显著增加，细胞活力、SOD、ALP活性、骨矿化结节数量、OPN mRNA、Runx2 mRNA、Wnt2、β-catenin表达显著降低（P＜0.05）。结论 骨疏方可改善H2O2诱导成骨细胞损伤，可能与抑制FoxO3a/Wnt信号通路有关。 |
| 英文摘要: |
| Objective To investigate the impact of Gushu formula on hydrogen peroxide (H2O2) induced osteoblast injury by regulating the FoxO3a/Wnt signaling pathway. Methods CCK-8 method was applied to detect the effects of different concentrations (0.01，0.1，1，10，100 μmol/L) of Gushu formula on H2O2 induced MC3T3-E1 cell viability. Subsequently，a control group，H2O2 (150 μmol/L H2O2) group，Gushu formula group，Gushuformula+empty body group，and Gushu formula+FoxO3a overexpression group were set up. CCK-8 method and ELISA were applied to detect cell viability，superoxide dismutase (SOD)，and malondialdehyde (MDA)，respectively； after osteogenic induction and differentiation，alkaline phosphatase (ALP) assay kit and alizarin red were applied to analyze ALP activity and cellular bone mineralization nodules；qRT-PCR was applied to detect the expression levels of osteogenic related genes OPN mRNA；Runx2 mRNA，and FoxO3a mRNA；Western blot was applied to detect the expression levels of pathway related proteins FoxO3a；Wnt2；and β-catenin. Results Compared with the control group，the expression of MDA and FoxO3a in the H2O2 group was greatly increased，the cell viability，SOD and ALP activities，number of bone mineralization nodules，the expression of OPN mRNA，Runx2 mRNA，Wnt2，β-catenin were greatly reduced (P<0.05)；compared with the H2O2 group，the expression of MDA and FoxO3a in the Gushu formula group was obviously reduced，the cell viability，SOD and ALP activities，number of bone mineralization nodules，the expression of OPN mRNA，Runx2 mRNA，Wnt2，β-catenin were obviously increased (P<0.05)；compared with the Gushuformula+empty-load group，the expression of MDA and FoxO3a in the Gushu formula + FoxO3a overexpression group was obviously increased，the cell viability，SOD and ALP activities， number of bone mineralization nodules，the expression of OPN mRNA，Runx2 mRNA，Wnt2， β-catenin were greatly reduced (P<0.05). Conclusion Gushu formObjective To investigate the mechanism of cassia twig in treating the necroptosis of KOA mouse chondrocytes based on RIPK1/RIPK3 signaling pathway. Methods Forty-eight male C57 mice were randomly divided into normal group，model group，Nec-1 group (5mg/(kg·d))，cassia twig low-dose group (0.43 g/(kg·d))，cassia twig medium-dose group (0.65 g/(kg·d)) and cassia twig high-dose group (2.6 g/(kg·d)). The mouse model of KOA was established by medial meniscus instability method，and the corresponding intragastric administration was performed after the modeling. After 28 days of drug intervention，the serum and cartilage tissues of mice were taken，and the histopathologic morphology of the knee joint of mice was observed by HE staining and saffranine O-solid green staining. The expression of RIPK3 in mouse cartilage was analyzed by immunofluorescence. The expression of RIPK1，RIPK3，p-RIPK1，p-RIPK3，TNF-α，IL-1β，IL-6 and other relatedmRNA and proteins were detected by RT-qPCR，ELISA and Western Blot. Results Compared with the normal group, the articular cartilage edge of the model group mice was severely damaged，and the mRNA and protein expressions of RIPK1，RIPK3，p-RIPK1，and p-RIPK3 were significantly increased (P<0.05). The content of TNF-α，IL-1β，and IL-6 significantly increased (P<0.05). The cartilage structure of the low-dose，medium dose，high-dose， and Nec-1 groups of cassia twig tended to be normal，and the mRNA and protein expressions of RIPK1，RIPK3，p-RIPK1，and p-RIPK3 were significantly reduced (P<0.05). The content of IL-1β， IL-6 and TNF-αsignificantly decreased (P<0.05). Conclusion By regulating RIPK1/RIPK3 signaling pathway，cassia twig can inhibit the necroptosis of knee cartilage cells，thereby improving the inflammatory environment of the knee joint in mice and delaying the pathological process of KOA.ula can improve H2O2 induced osteoblast damage，which may be related to the inhibition of FoxO3a/Wnt signaling pathway. |
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