| Objective:This study validated the clinical efficacy and safety of artificial tiger bone powder in treating knee osteoarthritis(KOA)through meta-analysis.Based on this,a rat KOA model and an osteoarthritis(OA)cell model were constructed to observe the effect of artificial tiger bone powder on cartilage degeneration in the OA chondrocyte model and rat KOA model.The molecular mechanism of AMPK on PINK 1/Parkin signaling mediated mitophagy was explored from both animal and cell experiments.Method:1.Evidence based research:The search period is from the establishment of the database to January 2024.Foreign language databases such as Embase,Web of Science,Cochrane Library,Pubmed,as well as Chinese databases such as CNKI,China Biomedical Literature Database,Chongqing VIP Database,and Wanfang Database have published relevant literature.The literature has been strictly screened according to the inclusion and exclusion criteria,and the quality has been evaluated using the Cochrane system evaluation method.Data analysis has been completed using Revman 5.4 software.2.Cell experiments:(1)Identification of primary rat chondrocytes;(2)Cell proliferation and toxicity technology(CCK-8)for detecting IL-1 β The effect of serum containing artificial tiger bone powder on chondrocyte viability was investigated,and Western blot technology was used to detect the expression of MMP-13,a metabolic marker for cartilage degradation,to validate the rat chondrocyte OA model Observation of the effects of different drug containing serum concentrations and intervention time on cell viability in rat chondrocyte OA model using CCK-8 technology,Western blot detection of MMP-13 expression,and screening of the optimal drug containing serum concentration to inhibit MMP-13 expression.(4)Using siRNA technology to knock out the expression of AMPK in primary rat chondrocytes,flow cytometry,Western blot,PCR,fluorescence staining and other techniques were used to observe the expression of cartilage degradation metabolic markers,mitochondrial fluorescence staining,mitochondrial membrane potential,ATP,ROS,and mitophagy related markers in the blank group,model group,transfection group,transfection+model group,tiger bone powder drug containing serum group,and transfection+model+tiger bone powder drug containing serum group.3.Animal experiments:A rat KOA model was constructed by meniscus tear surgery,and artificial tiger bone powder was administered orally to the model rats.The rats were divided into high,medium,and low dose groups,and metformin was used as the positive group.The model group and blank group were given physiological saline by gavage.After gastric lavage,HE staining and safranin green staining were used to observe the degree of cartilage damage in the knee joint of rats.Transmission electron microscopy was used to observe the ultrastructural changes of mitochondria in each group.Immunohistochemistry,Western blot,and PCR techniques were used to observe the expression of MMP-13,a metabolic marker for cartilage degradation.Western blot and PCR techniques were used to observe the expression of mitophagy related proteins.Result:1.This study included a total of 13 clinical literature and 1269 patients.Meta analysis showed that:(1)the total effective rate was better in the experimental group than in the control group;(2)The efficacy of reducing VAS scores in KOA patients was similar between the experimental group and the control group;(3)The WOMAC score of the experimental group was better than that of the control group;(4)The Lequesne score showed similar therapeutic effects in reducing the Lequesne score of KOA patients between the experimental group and the control group;(5)The incidence of adverse reactions was significantly lower in the experimental group than in the control group.2.The primary rat cells adhere to the wall and grow in a spindle or polygonal shape.The cell nucleus and cytoplasm are normal.The cytoplasm of rat chondrocytes is light blue,while the nucleus is dark blue.Type II collagen fluorescence staining shows green fluorescence in the cells,which is consistent with the phenotype of rat chondrocytes.3.Different concentrations of IL-1 β After pretreatment of rat chondrocytes for 24 hours,there was no significant effect on the viability of rat chondrocytes.Western blot analysis revealed that compared with the blank group,MMP-13,a biomarker of cartilage breakdown metabolism,was expressed in IL-1 β The highest concentration is at 20ng/m L.4.The serum concentrations of 15% and 20% artificial tiger bone powder containing medicine can promote the proliferation of rat chondrocytes,but have no significant effect on the cell viability of the rat OA chondrocyte model;The optimal inhibitory effect of 15%concentration of artificial tiger bone powder containing serum on MMP-13,a biomarker of cartilage degradation metabolism,was observed in a rat OA chondrocyte model after 24 hours of intervention.5.After knocking out AMPK using siRNA technology,the expression of AMPK decreased in the transfection group and the transfection plus model group,while the model group showed a decrease in IL-1 expression β Under induction,the expression of AMPK decreased,and in the transfection group,transfection plus model group,and model group,the intensity of mitochondrial fluorescence staining,mitochondrial membrane potential decreased,ATP generation decreased,ROS increased.The expression of mitophagy pathway proteins PINK 1and Parkin decreased,while the expression of autophagy protein LC 3B decreased.The expression of mitophagy markers TOMM 20 and VDAC 1 increased,and the expression of cartilage breakdown metabolism marker MMP-13 increased.6.After intervention with serum containing artificial tiger bone powder,the tiger bone powder group and mitochondrial membrane potential slightly recovered,ATP generation increased,ROS decreased,MMP-13,TOMM20,VDAC1 expression decreased,AMPK,PINK 1,Parkin,and LC 3B expression increased;The transfection with model and tiger bone meal group showed a slight recovery in mitochondrial fluorescence staining intensity,mitochondrial membrane potential,increased ATP generation,decreased ROS,decreased expression of MMP-13,TOMM 20,and VDAC 1,and increased expression of AMPK,PINK 1,Parkin,and LC 3B,but no significant increase in AMPK expression was observed.7.After intervention with artificial tiger bone powder in the rat KOA model,the medium dose group showed the best therapeutic effect,with a decrease in the expression of MMP-13,a metabolic marker for cartilage matrix degradation,a slight recovery in the number and morphology of mitochondria in chondrocytes,a decrease in the expression of AMPK,PINK 1,and Parkin proteins in the mitophagy pathway,a decrease in the expression of autophagy protein LC3 B,and an increase in the expression of mitophagy markers TOMM 20 and VDAC1.Conclusion:1.Artificial tiger bone powder has good clinical efficacy and safety in the treatment of KOA.2.After AMPK knockout in rat chondrocytes,PINK 1/Parkin mediated mitophagy levels were reduced to varying degrees in rat chondrocytes and rat chondrocyte OA models.3.Artificial tiger bone powder containing serum can inhibit the expression of MMP-13,a metabolic marker for cartilage breakdown,through mitophagy mediated by the AMPK/PINK1/Parkin pathway.After knocking out AMPK,the drug containing serum of artificial tiger bone powder can reverse the activity of downstream pathway PINK 1/Parkin and its mediated decrease in mitophagy levels,achieving the goal of inhibiting the expression of MMP-13,a biomarker of cartilage catabolism,and reducing chondrocyte catabolism.This suggests that artificial tiger bone powder may have multiple targets in regulating mitophagy.4.In the rat KOA model,the model group showed upregulation of MMP-13 expression,increased degradation of cartilage matrix,and activation of AMPK/PINK 1/Parkin mediated mitophagy.Artificial tiger bone powder can inhibit cartilage matrix degradation and reduce AMPK/PINK 1/Parkin mediated mitophagy levels.Artificial tiger bone powder inhibits cartilage matrix degradation by regulating AMPK/PINK 1/Parkin mediated mitophagy. |
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