Study On The Mechanism Of Modified Duhuo Jisheng Mixture And Its Active Ingredients In Regulating Cartilage Degeneration And Synovial Inflammation In The Treatment Of Knee Osteoarthriti

Objective:To study the molecular mechanism of Jiawei Duhuo Jisheng Mixture and its effective components,Nodakenin and Baicalein,by regulating chondrocyte mitochondrial fission and cell apoptosis,to improve cartilage loss,synovial inflammation and osteophyte formation,thus delaying knee osteoarthritis(KOA).Methods:1 Therapeutic effect of Jiawei Duhuo Jisheng Mixture on KOA The KOA model of 8-week-old male C57BL/6 mice has been established after Destabilization medial meniscus(DMM)of the right knee joint after adaptive feeding for a week.The mice were divided into sham group,model group and model+Jiawei Duhuo Jisheng Mixture group with 6animals each.Mice were given 16.25 m L/kg intragastric administration,and the other two groups were given normal saline once a day.Eight weeks later,the articular cartilage was sampled,and the articular cartilage was stained with saffranine fast green and type II collagen immunohistochemical staining.OARSI score and statistical analysis of cartilage area were performed to evaluate the degeneration of articular cartilage.2 Study on quality control of Jiawei Duhuo Jisheng Mixture The chemical constituents of 5 bottles of different batches of Jiawei Duhuo Jisheng Mixture were identified by ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry,and the collected mass spectrometry data were analyzed with the database of traditional Chinese medicine components,chemical constituents and reference substances.3 Experimental study on the treatment of KOA with the effective component of Nodakenin in Jiawei Duhuo Jisheng Mixture Eight-week-old male C57BL/6 mice were divided into sham operation group,model group and model+Nodakenin group.Nodakenin was administered orally at 10 mg/kg,once a day.After 8weeks,the samples were taken,and the cartilage injury,synovitis and osteophyte formation were evaluated by small animal CT,saffranine fast green staining,immunofluorescence and other methods.In vitro study,the primary chondrocytes were stimulated by LPS,Nodakenin was used to intervene,and CCK8 was used to screen the concentration of Nodakenin.Mito SOX probe was used to detect mitochondrial ROS levels in chondrocytes.Serum inflammatory factors were detected by ELISA.The levels of COX 2,IL-1β,Collagen II,Drp1 and NLRP3 were detected by PCR,Western blot and immunofluorescence.4 Experimental study on the treatment of KOA with the effective component of Baicalein in Jiawei Duhuo Jisheng Mixture Firstly,the target of Baicalein and the target genes related to osteoarthritis(OA)were identified by network pharmacology.Then,the potential targets of Baicalein in the treatment of OA were predicted.GO enrichment analysis and KEGG signaling pathway analysis were performed on the target,and molecular docking was performed on the key target of baicalein treatment of OA.In addition,histopathology and molecular biology techniques were used to verify the molecular mechanism of Baicalein’s intervention in KOA by regulating apoptosis of chondrocytes in KOA model mice.Results:1 Therapeutic effect of Jiawei Duhuo Jisheng Mixture on KOA Articular cartilage staining showed that compared with model group,Jiawei Duhuo Jisheng Mixture could significantly reduce cartilage injury,increase articular cartilage area and reduce OARSI score.Immunohistochemical staining showed that the expression of collagen II increased in the articular cartilage of Jiawei Duhuo Jisheng Mixture group.2 Study on quality control of Jiawei Duhuo Jisheng Mixture The fingerprint of Jiawei Duhuo Jisheng Mixture has multiple peaks,and the positive and negative ion modes are stable.A total of 21 active components were identified;The content of paeoniflorin,nodakenin,kaempferol,baicalein,and glycyrrhizic acid in Jiawei Duhuo Jisheng Mixture was stable according to the identification of the control substance;The fingerprints of five batches are highly similar.3 Experimental study on the treatment of KOA with the effective component of Nodakenin in Jiawei Duhuo Jisheng Mixture In vivo experiments found that Nodakenin can inhibit osteophyte formation,improve bone volume,reduce bone trabecular separation,and increase the number of bone trabeculae;It can also reduce OARSI score and synovitis score,increase the area of tibial cartilage,and down-regulate the expression level of p-Drp1 and NLRP3 in cartilage.In vitro studies showed that Nodakenin can reduce the phosphorylation of Drp1 in chondrocytes,inhibit mitochondrial over-fission,ROS production and NLRP3 expression,thereby reducing inflammatory reaction and chondrocyte damage.4 Experimental study on the treatment of KOA with the effective component of Baicalein in Jiawei Duhuo Jisheng Mixture Network analysis showed that the 19 targets of Baicalein intervention in OA were mainly involved in oxidative stress response,cell apoptosis response and PI3K-Akt and p53 signal pathways;Molecular docking showed that BAX,BCL 2 and Caspase 3 in the apoptotic signal pathway had high binding affinity with Baicalein.Animal validation experiments show that Baicalein can significantly inhibit the expression of apoptosis-related proteins(BAX,BCL 2 and Caspase 3)in articular cartilage,thereby reducing articular cartilage injury,inhibiting synovial inflammation of joints,and improving subchondral bone microstructure.Conclusion:1 The preparation process of Jiawei Duhuo Jisheng Mixture is reliable,with rich and stable active ingredients.2 Jiawei Duhuo Jisheng Mixture and its effective components,Nodakenin and Baicalein,can reduce cartilage damage,inhibit synovial inflammatory reaction,improve subchondral bone microstructure and osteophyte formation,thus delaying the process of KOA disease.3 The therapeutic effect of Jiawei Duhuo Jisheng Mixture is closely related to the regulation of mitochondrial dynamic balance and apoptosis of chondrocytes.