中医正骨2025，Vol.37Issue(9)：16-25,10.
补肾痹通方联合骨髓间充质干细胞干预膝骨关节炎的效果及其作用机制研究
Efficacy and mechanism of Bushen Bitong Fang(补肾痹通方)combined with bone marrow mesenchymal stem cells against knee osteoarthritis
摘要
Abstract
Objective:To investigate the therapeutic efficacy of Bushen Bitong Fang(补肾痹通方,BSBTF)combined with bone marrow mesenchymal stem cells(BMMSCs)against knee osteoarthritis(KOA),and to explore its underlying mechanism.Methods:①Cell experi-ment.The chondrocytes were seeded into the lower chamber of 96-well Transwell chamber and allocated into blank control group,model group,BSBTF medicated serum group,BMMSC group,and combined intervention group.All chondrocytes but the ones in blank control group were intervened with interleukin-1β(IL-1β)at a final concentration of 10 ng/mL for 24 hours to induce in vitro KOA cell models.Following the model induction,in the upper chamber of the Transwell chamber,the chondrocytes in the BSBTF medicated serum group were further intervened with medium containing 15％BSBTF medicated serum,the ones in BMMSC group with an equal volume of BMMSC sus-pension,the ones in combined intervention group with both medium containing 15％BSBTF medicated serum and an equal volume of BMMSC suspension,while the ones in blank control group and model group with an equal volume of blank medium for another 24 hours.Af-ter the end of the intervention,the upper chamber of the Transwell chamber was removed for quantifying the chondrocytes and calculating the relative migration rate of chondrocytes in each group.In a parallel experiment,the chondrocytes were seeded into 96-well cell culture plates and subjected to the identical grouping,modeling,and intervention protocols.After 24-hour incubation,the apoptosis rate of chondro-cytes in each group was determined using flow cytometry,and the protein expression levels of key apoptosis-related genes,including B-cell lymphoma-2 associated X protein(BAX),B-cell lymphoma(Bel)-2,and cleaved-cysteine aspartic acid specific protease-3(cleaved-Caspase-3),were detected in each group by Western Blot.②Animal experiment.Forty rats were randomly assigned into sham-operated group,model group,BMMSC group,and combined intervention group,with 10 rats in each group.All rats but the ones in sham-operated group were subjected to surgeries on the right knee joints by using modified Hulth method to build KOA models,while the ones in sham-operated group were merely incised the skin and subcutaneous tissues at the corresponding site and then sutured after exposing the knee joint cavity.Four weeks after the modeling,the rats in BMMSC group and combined intervention group were intervened by right knee intra-articular injection of 100 μL BMMSC suspension(approximately 200 cells/pL),while the rest 2 groups by intra-articular injection of 100 μL sterile saline at the corresponding sites,once a week for consecutive 4 weeks.Additionally,the rats in combined intervention group were further intervened by intragastric administration with BSBTF decoction at a crude dose of 16.35 g/kg/day,once in the morning and evening,respectively,for consecutive 4 weeks.After the end of intervention,all rats were sacrificed.Four rats were randomly selected from each group,and their right knee joints were dissected for observing the histopathological changes of the knee articular cartilage.In addition,the right knee articular cartilage tissues were harvested from the remaining 6 rats in each group for detecting the mRNA expression levels of Toll-like receptor(TLR)4,myeloid differentiation primary response protein 88(MyD88),and nuclear factor-κB(NF-κB)p65,as well as the protein expression levels of TLR4,MyD88,NF-κB p65,and phosphorylated nuclear factor-κB(p-NF-κB)p65.Results:① Effects of BSBTF medicated serum combined with BMMSCs on the migration activity of chondrocytes.The relative migration rate of chondrocytes decreased in model group compared to blank control group(P=0.000),while it increased in BSBTF medicated serum group,BMMSC group,and com-bined intervention group compared to model group(P=0.000,P=0.010,P=0.016),with the greater value in combined intervention group compared to BSBTF medicated serum group and BMMSC group alone(P=0.000,P=0.012).(②Effects of BSBTF medicated serum combined with BMMSCs on the apoptosis of chondrocytes.The chondrocyte apoptosis rate was higher in model group compared to blank con-trol group(P=0.000),while,was lower in BSBTF medicated serum group,BMMSC group,and combined intervention group compared to model group(P=0.000,P=0.010,P=0.016),with the lower value in combined intervention group compared to BSBTF medicated serum group and BMMSC group alone(P=0.000,P=0.000).The relative protein expression levels of BAX and cleaved-Caspase-3,as well as the ratio of BAX to Bcl-2,were all higher,while that of Bcl-2 was lower in model group compared to blank control group(P=0.000,P=0.000,P=0.000,P=0.000);furthermore,the relative protein expression levels of BAX and cleaved-Caspase-3,as well as Bax/Bcl-2 ratio,were all lower,while that of Bcl-2 was higher in BSBTF medicated serum group,BMMSC group,and combined intervention group compared to model group(BAX:P=0.000,P=0.010,P=0.016;cleaved-Caspase-3:P=0.000,P=0.010,P=0.000;Bax/Bcl-2 ratio:P=0.000,P=0.010,P=0.000;Bcl-2:P=0.000,P=0.000,P=0.001),with the combined intervention group showing the lowest values in the rel-ative protein expression levels of BAX and cleaved-Caspase-3,as well as Bax/Bcl-2 ratio,and the highest value in that of Bcl-2 among the 3 treatment groups(P=0.000,P=0.020;P=0.002,P=0.022;P=0.000,P=0.000;P=0.010,P=0.049).③Histopathological find-ings in rat knee articular cartilage.The marked changes,manifesting as a rough articular cartilage surface with the presence of fissures and folds,an incomplete tide line,irregularly thickened subchondral bone,accompanied by unevenly distributed,and irregularly arranged chon-drocytes showing heterogeneous in size,were observed in the knee articular cartilage of rats in model group,while,compared with that of model group,the knee articular cartilage degeneration was attenuated in both BMMSC group and combined intervention group,with marked amelioration demonstrated in combined intervention group.(4)The mRNA and protein expression of TLR4-MyD88-NF-κB signaling pathway-related genes in rat knee articular cartilage tissues.The relative mRNA expression levels of TLR4,MyD88,and NF-κB p65 in rat knee artic-ular cartilage tissues were higher in model group compared to sham-operated group(P=0.000,P=0.000,P=0.000),while,were lower in both treatment groups(BMMSC group and combined intervention group)compared to model group(P=0.000,P=0.011,P=0.038;P=0.000,P=0.000,P=0.001),with the lower value observed in combined intervention group compared to BMMSC group(P=0.001,P=0.039,P=0.045).However,the relative protein expression level of NF-κB p65 in the knee articular cartilage tissues showed no significant difference among the 4 groups(F=1.797,P=0.226).In addition,the relative protein expression levels of TLR4,MyD88,and p-NF-κB p65 in the knee articular cartilage tissues were higher in model group compared to sham-operated group(P=0.000,P=0.000,P=0.000),while,were lower in BMMSC group and combined intervention group compared to model group(P=0.022,P=0.025,P=0.026;P=0.000,P=0.000,P=0.000),with the lower value observed in combined intervention group compared to BMMSC group(P=0.011,P=0.003,P=0.025).Conclusion:BSBTF combined with BMMSCs can promote the repair of cartilage tissues in KOA.It may work by in-hibiting the activation of TLR4-MyD88-NF-κB signaling pathway and suppressing the apoptosis of chondrocytes.关键词
骨关节炎,膝/补肾痹通方/间质干细胞/骨髓/软骨细胞/细胞凋亡/类Toll受体/髓系分化初级反应蛋白质88/NF-κB/动物实验Key words
osteoarthritis,knee/Bushen Bitong Fang/mesenchymal stem cells/bone marrow/chondrocytes/apoptosis/Toll-like receptors/myeloid differentiation primary response protein 88/NF-kappa B/animal experimentation引用本文复制引用
向文远,易林,张文豪,邓迎杰,方锐..补肾痹通方联合骨髓间充质干细胞干预膝骨关节炎的效果及其作用机制研究[J].中医正骨,2025,37(9):16-25,10.基金项目
"天山英才"培养计划科技创新领军人才项目(2022TSYCLJ0007) （2022TSYCLJ0007）
"天山英才"培养计划青年托举人才项目(2023TSYCQNTJ0050) （2023TSYCQNTJ0050）
