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目的 基于Toll样受体4(TLR4)/核因子κB(NF-κB)信号通路探究补肾痹通方(BSBT)联合间充质干细胞(MSCs)对膝骨关节炎(KOA)关节软骨修复作用的影响。方法 选取2月龄SD大鼠50只，随机分为CON、M-KOA、M-MSC、M-F、M-MSC-F组，每组10只。除CON组外，其余4组采用改良Hulth法构建大鼠膝骨关节炎模型。造模成功后，M-KOA组(予以4 mL/100 g生理盐水灌胃)、M-MSC组(予以1×107/mL间充质干细胞0.1 mL关节腔内注射)、M-F组(予以BSBT32.68 g/kg灌胃)及M-MSC-F组(予以1×107/mL间充质干细胞0.1 mL关节腔注射，且给予BSBT 32.68 g/kg灌胃),除CON组外其余各组每天灌胃1次，持续3周。苏木精-伊红(HE)和甲苯胺蓝染色观察软骨组织病理变化；蛋白质免疫印迹(WB)法检测软骨组织β-连环蛋白(β-catenin)、SRY盒转录因子9(SOX9)、Runt相关转录因子2(RUNX2)、TLR4、NF-κB p65蛋白表达量情况；免疫荧光观察软骨组织Ⅱ型胶原蛋白(Collagen II)、骨形态发生蛋白7(BMP-7)荧光强度变化；酶联免疫吸附(ELISA)法检测血清肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)含量。结果 与CON组比较，M-KOA组大鼠软骨边缘损伤严重，细胞排列紊乱，出现大量炎性细胞浸润；软骨组织中Collagen II、BMP-7荧光表达下降(P<0.05),SOX9蛋白表达降低(P<0.05),β-catenin、RUNX2、TLR4和NF-κB p65蛋白表达升高(P<0.05);血清中TNF-α、IL-1β、IL-6水平升高(P<0.05)。与M-KOA组相比，M-MSC组、M-F组、M-MSC-F组大鼠软骨组织边缘损伤明显缓解，细胞排列较整齐，炎性细胞浸润情况明显减少；软骨组织中Collagen II、BMP-7荧光表达上升(P<0.05),SOX9蛋白表达增高(P<0.05),β-catenin、RUNX2、TLR4和NF-κB p65蛋白表达降低(P<0.05);血清中TNF-α、IL-1β、IL-6水平降低(P<0.05);且M-MSC-F组联合干预效果优于M-MSC组、 M-F组。结论 BSBT联合MSCs能有效改善KOA大鼠软骨的病理状况，抑制TLR4/NF-κB信号通路，缓解软骨的炎症损伤，达到治疗KOA的作用。
Abstract：Objective Based on the Toll-like receptor 4(TLR4)/nuclear factor κB(NF-κB) signaling pathway, to explore the effect of Bushen Bitong Formula(BSBT) combined with mesenchymal stem cells(MSCs) on articular cartilage repair in knee osteoarthritis(KOA). Methods 50 Sprague-Dawley rats, aged 2 months, were randomly allocated into 5 groups(n=10 per group): CON, M-KOA, M-MSC, M-F and M-MSC-F. All groups, except for the CON group, underwent the modified Hulth surgical procedure to induce a rat model of KOA. Following successful modeling, M-KOA(administered saline at a dosage of 4 mL/100 g via gavage), M-MSC(administered a single intra-articular injection of 0.1 mL MSCs, at a concentration of 1 × 107 cells/mL), M-F(administered BSBT at a dosage of 32.68 g/kg via gavage) and M-MSC-F(administered a single intra-articular injection of 0.1 mL MSCs, at a concentration of 1 × 107 cells/mL, in conjunction with BSBT at 32.68 g/kg via gavage), except for the CON group, all groups were gavaged once daily for 3 consecutive weeks. Cartilage pathology was evaluated using hematoxylin-eosin(HE) staining and toluidine blue staining. Protein expression of β-catenin, SRY-box transcription factor 9(SOX9), runt-related transcription factor 2(RUNX2), TLR4 and NF-κB p65 was quantified via western blotting. Immunofluorescence analysis was performed to assess Collagen II and Bone Morphogenetic Protein-7(BMP-7) expression. Serum concentrations of Tumor Necrosis Factor-alpha(TNF-α), Interleukin-1 beta(IL-1β) and Interleukin-6(IL-6) were measured using enzyme-linked immunosorbent assay(ELISA). Results In comparison to the CON group, the M-KOA group exhibited significant marginal cartilage damage, disorganized cellular arrangement and extensive infiltration of inflammatory cells. There was a notable reduction in the fluorescence expression of Collagen II and BMP-7 within the cartilage tissue(P<0.05), alongside a decrease in SOX9 protein expression(P<0.05). Conversely, there was an upregulation in the protein expression of β-catenin, RUNX2, TLR4 and NF-κB p65(P<0.05). Additionally, serum concentrations of TNF-α, IL-1β and IL-6 were elevated(P<0.05). Compared with M-KOA group, M-MSC, M-F and M-MSC-F groups demonstrated a significant reduction in marginal cartilage damage, a more orderly cellular arrangement, and a marked decrease in inflammatory cell infiltration compared to the M-KOA group. These groups also showed increased fluorescence expression of Collagen II and BMP-7 in cartilage tissue(P<0.05), enhanced SOX9 protein expression(P<0.05), and decreased protein expression of β-catenin, RUNX2, TLR4 and NF-κB p65(P<0.05). Furthermore, serum levels of TNF-α, IL-1β and IL-6 were reduced(P<0.05). Notably, the combined intervention in the M-MSC-F group proved to be more effective than the interventions in the M-MSC and M-F groups. Conclusion The combined application of BSBT and MSCs effectively ameliorates cartilage pathology in KOA rats by inhibiting the TLR4/NF-κB signaling pathway, attenuating inflammation-induced cartilage damage, and thereby exerting therapeutic efficacy against KOA.
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基本信息:
中图分类号:R285.5
引用信息:
[1]向文远,邓迎杰,方锐.基于TLR4/NF-κB信号通路探讨补肾痹通方联合间充质干细胞修复膝骨关节炎关节软骨的作用机制[J].新疆医科大学学报,2025,48(10):1448-1455.
基金信息:
新疆维吾尔自治区重点研发任务专项课题(2021B03006-2); 第二批“天山英才”-青年托举人才项目(2023TSYCQNTJ0050)
2025-10-15
2025-10-15