二甲双胍通过调控TLR4/NF-κB信号通路对大鼠创伤后骨关节炎软骨退变的作用机制

doi:10.3969/j.issn.1006-6233.2025.12.013

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摘要 目的: 探讨二甲双胍(MET)通过调控Toll样受体4(TLR4)/核因子κB(NF-κB)信号通路对大鼠创伤后骨关节炎(PTOA)软骨退变的作用机制。方法: 将60只雄性SD大鼠随机分为对照组(Control组)、模型组(PTOA组)、MET组、TLR4激活剂组(LPS组)、MET+LPS组,每组12只。Control组仅切开髌骨外侧皮肤后缝合,其余各组采用切断交叉韧带并部分切除半月板的方法建立PTOA大鼠模型。造模成功后,MET组和MET+LPS组大鼠灌胃200mg/kg的MET, LPS组和MET+LPS组灌胃50μg/kg的LPS,Control组和PTOA组灌胃等体积的生理盐水,每日1次,连续给药4周。末次给药后,采用HE染色、番红O-固绿染色观察大鼠关节软骨形态,并进行国际骨关节炎研究学会(OARSI)评分;ELISA检测大鼠软骨组织促炎症因子(TNF-α、IL-6、IL-1β)水平;RT-qPCR检测大鼠软骨组织凋亡相关基因(C-caspase-3、Bax、Bcl-2)及SOX9、MMP-13、ADAMTS5 mRNA表达水平;Western blot检测大鼠软骨组织TLR4、MyD88、NF-κB p65、p-NF-κB p65蛋白表达水平。结果: 与Control组相比,PTOA组的软骨损伤及退变严重,OARSI评分及软骨组织促炎症因子水平、C-caspase-3、Bax、 MMP-13、ADAMTS5 mRNA及TLR4、MyD88、p-NF-κB p65/NF-κB p65表达显著升高(P<0.05),Bcl-2、SOX9 mRNA表达显著降低(P<0.05)。与PTOA组相比,MET组的软骨损伤及退变明显减轻,OARSI评分及软骨组织促炎症因子水平、C-caspase-3、Bax、MMP-13、ADAMTS5 mRNA及TLR4、MyD88、p-NF-κB p65/NF-κB p65表达显著降低(P<0.05),Bcl-2、SOX9 mRNA表达显著升高(P<0.05);LPS组的上述指标结果则相反(P<0.05)。MET逆转了LPS对PTOA大鼠软骨退变、炎症及软骨细胞凋亡的促进作用。结论: MET可通过抑制TLR4/NF-κB信号通路,减轻关节炎症,抑制软骨细胞凋亡,减少软骨细胞外基质降解,从而改善PTOA大鼠软骨退变。

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关键词 ：创伤后骨关节炎, 二甲双胍, TLR4/NF-κB信号通路, 软骨退变

Abstract：Objective: To explore the mechanism of action of metformin (MET) in regulating Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway and its effect on cartilage degeneration in post-traumatic osteoarthritis (PTOA) in rats. Methods: Totally 60 male Sprague-Dawley (SD) rats were randomly divided into five groups: control group, post-traumatic osteoarthritis (PTOA) group, MET group, TLR4 activator group (LPS group), and MET+LPS group, with 12 rats in each group. In the control group, only the skin lateral to the patella was incised and sutured. For the other groups, the PTOA rat model was established by transecting the cruciate ligaments and partially resecting the meniscus. After successful modeling, rats in the MET group and MET+LPS group were gavaged with 200mg/kg of metformin, while rats in the LPS group and MET+LPS group were gavaged with 50μg/kg of lipopolysaccharide (LPS). Rats in the control group and PTOA group were gavaged with an equal volume of saline, once daily for 4 consecutive weeks. After the last gavage, the morphology of articular cartilage in rats was observed using hematoxylin and eosin (HE) staining and safranin O-fast green staining, and the Osteoarthritis Research Society International (OARSI) score was assessed. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) in rat cartilage tissue. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to detect the expression levels of apoptosis-related genes (C-caspase-3, Bax, Bcl-2) and mRNA expression levels of SOX9, MMP-13, and ADAMTS5 in rat cartilage tissue. Western blot analysis was conducted to detect the protein expression levels of TLR4, MyD88, NF-κB p65, and p-NF-κB p65 in rat cartilage tissue. Results: Compared with the control group, the PTOA group exhibited severe cartilage damage and degeneration, with significantly increased OARSI scores, levels of pro-inflammatory cytokines in cartilage tissue, and expression of C-caspase-3, Bax, MMP-13, ADAMTS5 mRNA, as well as TLR4, MyD88, and p-NF-κB p65/NF-κB p65 (P<0.05). Additionally, the expression of Bcl-2 and SOX9 mRNA was significantly decreased in the PTOA group (P<0.05). In comparison to the PTOA group, the MET group showed significantly reduced cartilage damage and degeneration, with decreased OARSI scores, levels of pro-inflammatory cytokines in cartilage tissue, and expression of C-caspase-3, Bax, MMP-13, ADAMTS5 mRNA, as well as TLR4, MyD88, and p-NF-κB p65/NF-κB p65 (P<0.05). Furthermore, the expression of Bcl-2 and SOX9 mRNA was significantly increased in the MET group (P<0.05). Conversely, the LPS group exhibited opposite results for these indicators (P<0.05). MET reversed the promoting effects of LPS on cartilage degeneration, inflammation, and chondrocyte apoptosis in PTOA rats. Conclusion: MET can ameliorate cartilage degeneration in PTOA rats by inhibiting the TLR4/NF-κB signaling pathway, reducing joint inflammation, inhibiting chondrocyte apoptosis, and decreasing the degradation of chondrocyte extracellular matrix.

Key words： Post-traumatic osteoarthritis (PTOA) Metformin TLR4/NF-κB signaling pathway Cartilage degeneration

基金资助:2021年四川省医学(青年创新)科研项目,(编号:S21327)

通讯作者: 郭明刚

引用本文:

袁志发, 刘蕈萁, 赵雪松, 向守刚, 郭明刚. 二甲双胍通过调控TLR4/NF-κB信号通路对大鼠创伤后骨关节炎软骨退变的作用机制[J]. 河北医学, 2025, 31(12): 2020-2026.
YUAN Zhifa, LIU Xunqi, ZHAO Xuesong, et al. Mechanism of Metformin in Regulating the TLR4/NF-κB Signaling Pathway and Its Effect on Cartilage Degeneration in Post-Traumatic Osteoarthritis in Rats. HeBei Med, 2025, 31(12): 2020-2026.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2025.12.013 或 http://www.hbyxzzs.cn/CN/Y2025/V31/I12/2020

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