Study On The Mechanism Of Tongbi Jiangu Recipe Inhibiting Inflammation Mediated Cartilage Degen-eration In Knee Osteoarthritis

Objective To explore the mechanism of Tongbi Jiangu prescription based on NF-κB signaling pathway to inhibit inflammation and delay cartilage degeneratio n in knee osteoarthritis,and provide experimental support and theoretical basis for further clinical application and basic research of Tongbi Jiangu Recipe.Methods 1.Summarize and sort out the academic thought of Professor Li Shenghua,a famous traditional Chinese Medicine professor in Gansu Province,on syndrome differentiation and treatment of knee osteoarthritis 2.Using network pharmacology and molecular docking technology,multiple databases and literature research were used to explore the core active components,key targets and signal pathways of Tongbi Jiangu Recipe in the treatment of knee osteoarthritis.3.10 SD rats were randomly selected from 60 SD rats as the spare group,10 as the blank group,and the remaining 40 rats were used to establish th e KOA model by the modified Hulth method.After successful modeling,they were randomly divided into model group,low-dose,medium-dose and high-dose groups of Tongbi Jiangu Recipe.After corresponding interventions,the general conditions of the rats were observed;the general observation of knee joint anatomy and Pelletier score;HE staining of articular cartilage and Mankin score;ELISA was used to detect the expressions of TNF-α,IL-1β,IL-10,COX-2,MDA and SOD in serum of rats in each group;Western Blot detection of p-NF-κBp65,IKK-β,p-IκB-α,NLRP3,Caspase-1,i NOS,MMP-3,ADAMTS-4 proteins in cartilage tissue of rats in each group Expression;RT-PCR was used to determine the gene expression of IL-1β,TNF-α,IL-10,NF-κBp65,IKK-β,IκB-α,NLRP3 and Caspase-1 in cartilage tissue of rats in each group.4.The chondrocytes were cultured in vitro,and the cell morphology was observed under microscope;the chondrocytes were identified by toluidine blue and type Ⅱ collagen staining;KOA chondrocyte model was established by lipopolysaccharide;effect of activity.The modeled chondrocytes were divided into model group,low-dose,medium-dose and high-dose groups of Tongbi Jiangu Recipe,and the unmodeled chondrocytes were set as blank group;ELISA,Western Blot,immunofluorescence and RT-PCR were used to detect inflammation,protein The related indexes of each group were detected at the gene level,and the mechanism of Tongbi Jiangu Recipe in inhibiting inflammation-mediated chondrocyte degeneration was explored.5.The chondrocyte culture system was established,and lipopolysaccharide was used to make KOA chondrocyte model;the effect of quercetin monomer of Tongbi Jiangu Recipe on the activity of KOA chondrocytes was detected by CCK-8 method.The modeled chondrocytes were divided into model group,quercetin low-dose,medium-dose and high-dose groups,and the unmodeled chondrocytes were set as blank group;the changes of cell morphology and ultrastructure in each group were observed by transmission electron microscope;ELISA,Western Blot,RT-PCR detection of protein and gene expression of each group of indicators.Results 1.According to the etiology of knee osteoarthritis,Professor Li Shenghua,a famous traditional Chinese medicine in Gansu Province,put forward the idea of "kidney deficiency and evil invasion" to treat KOA.According to the regional characteristics of northwest Gansu and partial wind cold,he pointed out that kidney deficiency is the root cause,wind cold invasion is the main external factor,and the pathogenesis is the syndrome of "deficiency and excess,deficiency and arthralgia,and deficiency and excess mixed".Joint dystrophy,inability to move,no honor,pain;Wind and cold attack,muscle and pulse block,obstruction leads to pain.It is further proposed that tonifying the liver and kidney,dispelling wind and dispersing cold,dredging arthralgia and relieving pain are the methods to treat the disease,based on which Tongbi Jiangu Recipe is developed by the research group under the guidance of the theory of "kidney deficiency and evil invasion".2.The results of network pharmacology data mining showed that there are 111 active ingredients in Tongbi Jiangu Recipe for KOA,the core active ingredients are quercetin,kaempferol,β-sitosterol,and the key targets are IL-1β,MMP-3,TNF-α.In the GO enrichment analysis,the results of the molecular function and biological process enrichment analysis were consistent with the rules of the PPI network analysis,and also with the pathogenesis of KOA inflammation.KOA-related genes are enriched in this process,causing the occurrence of knee osteoarthritis,and the genes involved in the enrichment are mostly genes involved in Tongbi Jiangu Recipe.KEGG enrichment analysis showed that the relevant signaling pathways were NF-κB,JAk-STAT and Wnt signaling pathways;molecular docking showed that the core active components had good binding activity to key targets.According to the KEGG enrichment anal ysis bubble chart,the NF-κB signaling pathway is at the top,and many studies have also confirmed that the NF-κB signaling pathway plays an important role in the inflammatory response of KOA.Therefore,based on the NF-κB signaling pathway and key targets,we used in vitro cell and in vivo animal experiments to explore and elucidate the mechanism of Tongbi Jiangu Recipe in the treatment of KOA by regulating inflammatory factors and inhibiting cartilage degeneration.3.In the animal experiment,the general observation of knee joint of KOA rats was compared from the degree of cartilage surface smoothness,the presence or absence of defects and vegetation,etc,and the Pelletier score was used to quantify.The results showed that the model group was worse than the blank group(P<0.05);Compared with the other groups,Tongbi Jiangu Recipe in each dose group was better than the model group(P<0.05);among the low,middle and high dose groups,the middle dose group was the most obvious.The cartilage tissue conditions of each group were compared from the tangent layer,transition layer,and radiation layer by HE staining,and Mankin score was performed on them.The results were consistent with the gross anatomy observation and Pelletier score.The general conditions of the rats were compared among the groups in terms of fur brightness and softness,the sensitivity of tapping the cage,activities,and diet.The results were consistent with the gross anatomy observation and HE staining.ELISA results showed that,compared with the blank group,the serum levels of IL-1β,TNF-α,COX-2 and MDA in the model group were significantly increased,while the levels of IL-10 and SOD were significantly decreased(P<0.05),the contents of IL-1β,TNF-α,COX-2 and MDA in serum of each dose group of Tongbi Jiangu Recipe were lower than those of the model group,and the contents of IL-10 and SOD were higher than those of the model group(P<0.05);in the high-dose group,the expression in the middle-dose group was the most significant.Western Blot results showed that compared with the blank group,the protein contents of p-NF-κBp65,p-IκB-α,IKK-β,i NOS,NLRP3,Caspase-1,MMP-3 and ADAMTS-4 in the model group were all increased(P <0.05);compared with the model group,Tongbi Jiangu Recipe could reduce the protein expressions of p-NF-κBp65,p-IκB-α and IKK-β in the NF-κB signaling pathway in each dose group(P<0.05);down-regulated The downstream factors of this pathway were NLRP3 inflammasome and Caspase-1 expression(P<0.05);meanwhile,the protein expressions of i NOS,MMP-3 and ADAMTS-4,which were mediated by inflammation and closely related to cartilage degeneration,were decreased(P<0.05).RT-PCR results showed that,compared with the blank group,except for the decreased expression of IκB-α and IL-10 in the model group,all other indexes were increased(P<0.05).All can improve the gene expression of IL-1β,TNF-α,IL-10,NF-κBp65,IKK-β,IκB-α,NLRP3,Caspase-1 in KOA cartilage tissue(P<0.05),and the middle dose group is the best.4.In the cell experiment,the third-generation chondrocytes had the same shape,clearly visible nuclei,and grew well;the cells were identified as chondroc ytes by toluidine blue and typeⅡcollagen staining;CCK-8 results showed that Tongbi Jiangu Recipe freeze-dried powder The optimal intervention time and concentration for KOA chondrocytes were 48 h and 150 μg/m L,respectively;ELISA,Western Blot,immunofluorescence,RT-PCR were used to detect the NF-κB signaling pathway and its downstream inflammatory factors from the inflammation,protein and gene levels,and The results of the indicators closely related to chondrocyte degeneration mediated by inflammatory factors are confirmed by animal experiments,that is,Tongbi Jiangu Recipe can inhibit the inflammatory response through the NF-κB signaling pathway,thereby delaying the degeneration of chondrocytes.5.The optimal intervention time and concentration of quercetin in Tongbi Jiangu Recipe on lipopolysaccharide-induced KOA chondrocytes were 24 h and 100μmol/L,respectively;ELISA,Western Blot,RT-PCR detection results were consistent with the above animal and cell experiments,which is consistent with the results observed by transmission electron microscopy.Conclusions 1.Tongbi Jiangu Recipe is the clinical experience of the research group under the guidance of the theory of "kidney deficiency and evil invasion".It is the essence of using the theory to treat KOA,and it is also the concrete presentation of the treatment of KOA by the theory,method,prescription and medicine.2.Using network pharmacology and molecular docking technology,applying multiple databases and combining with literature research,it shows that Tongbi Jiangu Recipe may play a role in inhibiting inflammation and delaying cartilage degeneration based on the NF-κB signaling pathway and key targets.3.Animal experiments show that Tongbi Jiangu Recipe can inhibit the degeneration of articular cartilage in KOA rats from a macroscopic perspective and reduce the Pelletier score;from a microscopic perspective,it can improve the HE staining and Mankin score of cartilage tissue.From the molecular level,it was found that Tongbi Jiangu Recipe can regulate the expression of downstream inflammatory factors through the NF-κB signaling pathway,reduce the inflammatory response,and reduce the degradation of type Ⅱ collagen and proteoglycan in the cartilage matrix mediated by inflammation,t hereby inhibiting inflammation,delaying cartilage degeneration.4.Cell experiments further confirmed from the three aspects of NF-κB signaling pathway,inflammation and chondrocyte degeneration,Tongbi Jiangu Recipe can inhibit inflammation through NF-κB signaling pathway,delay chondrocyte degeneration,and finally achieve the purpose of treating KOA.5.The conclusion of Tongbi Jiangu Recipe monomer quercetin experiment is consistent with the aforementioned animal and cell experiments.From the dimension of the single quercetin in this recipe,the mechanism of Tongbi Jiangu Recipe in inhibiting inflammation-mediated cartilage degeneration in KOA was expounded,which provided reference and ideas for exploring the most essential mechanism of traditional Chinese medicine compound recipes.