Clinical Efficacy And Mechanism Of Wenyang Yisui Decoction Combined With Platelet-Rich Plasma In The Treatment Of Early Knee Osteoarthritis

BackgroundKnee osteoarthritis(KOA)is a chronic ailment resulting from the gradual deterioration and wear of the articular cartilage.Presently,both pharmacological and non-pharmacological therapies show promise in alleviating associated clinical symptoms and enhancing the overall quality of life.Nonetheless,these approaches fail to reverse the degenerative process of cartilage and may even introduce adverse effects in patients.Enter Wenyang Yisui Decoction—a meticulously formulated prescription borne out of the instructor’s extensive clinical experience—known to yield significant results,albeit lacking in specificity.Concurrently,the introduction of platelet-rich plasma(PRP)injections into the knee joint cavity has increasingly garnered recognition for its capacity to facilitate the repair of KOA cartilage tissue.However,its efficacy remains limited.Therefore,it becomes imperative to investigate the potential of synergistic effects and substantial therapeutic outcomes through an innovative combined treatment involving these two modalities.This clinical quandary holds promise as it aims to offer a fresh treatment paradigm for early-stage KOA while delving further into the underlying mechanisms of action.The first section of the study aims to conduct a clinical evaluation of the efficacy of Wenyang Yisui Decoction,the PRP treatment,and the combined administration of Wenyang Yisui Decoction and PRP in treating KOA.Additionally,the investigation seeks to determine whether there is a synergistic effect when these modalities are combined,thereby providing an initial exploration into their respective mechanisms of action.The second part of the research concentrates on the analysis of the active constituents present in Wenyang Yisui Decoction.It aims to identify the crucial targets for treating KOA and elucidate their biological functions and associated signaling pathways.This analysis is carried out through the utilization of network pharmacology,a cutting-edge approach that allows for a comprehensive understanding of the interplay between various compounds.By integrating the findings from the first and second parts,the third section of the study aims to validate the action pathways derived from the network pharmacology investigation.Specifically,it seeks to elucidate the mechanism of action underlying the combined administration of Wenyang Yisui Decoction and PRP in regulating autophagy,focusing on the PI3K/AKT/mTOR pathway.This verification process establishes a solid foundation for the clinical application and advancement of this treatment program for KOA.Together,these three sections form a cohesive research framework that combines clinical evaluation,network pharmacology analysis,and mechanistic investigations.This approach provides a comprehensive understanding of the therapeutic potential of Wenyang Yisui Decoction combined with PRP in the treatment of KOA and paves the way for further advancements in clinical practice.Part I Evaluation of the clinical curative effect of Wenyang Yisui Decocotion combined with platelet-rich plasmaObjectiveTo evaluate the clinical efficacy of Wenyang Yisui Decocotion combined with PRP in the treatment of early KOA and whether it has synergistic effect,and to explore its mechanism.MethodA total of 90 early-stage KOA patients(Kellgren Lawrance X≤Ⅱ)with liver and kidney deficiency and cold-dampness obstruction syndrome were collected and randomly divided into Chinese medicine group,PRP group and combination group.The traditional Chinese medicine group was given Wenyang Yisui Decocotion orally,twice a day,4 weeks a course of treatment;the PRP group was given knee joint cavity injection of PRP,2.5ml,once every two weeks(0,2,4 weeks),3 times a course of treatment;The combination group was given Wenyang Yisui Decocotion orally and PRP was injected into the knee joint.TCM clinic before and after treatment clinical symptom score,WOMAC score and Lequesne score were used to detect serum IL-1β,TGF-β,MMP-1 and MMP-3 levels before and after treatment.Results1.There was no significant difference in the general information of the patients before treatment,such as age,gender,BMI,and disease course(P>0.05).The patient’s routine laboratory tests and examinations(blood,urine,and stool routine,liver and kidney function,electrocardiogram,and chest X-ray)were normal.Before treatment,there was no significant difference in subjective indicators such as TCM clinical symptoms,WOMAC score,and Lequesne score among the three groups(P>0.05).Before treatment,there was no statistical difference in objective indicators such as serum IL-1β,TGF-β,MMP-1,MMP-3 among the three groups(P>0.05),and the three groups were comparable.2.Comparison of subjective indicators:After treatment,the three groups had statistical differences in subjective indicators(Chinese medicine clinical symptom score,WOMAC score,Lequesne index)compared with those before treatment(P<0.01).Comparison between groups after treatment:There was no statistical difference between the Chinese medicine group and the PRP group(P>0.05);the combination group had statistical differences compared with the Chinese medicine group or the PRP group(P<0.01).3.Comparison of objective indicators:After treatment,the objective indicators(IL-1β,TGF-β,MMP-3)of the three groups were significantly different from those before treatment(P<0.01).The three groups were compared before and after MMP-1 treatment There was no statistical difference(P>0.05).Comparison between groups after treatment:There was no statistical difference among the three groups after MMP-1 treatment(P>0.05);there was no statistical difference in objective indicators(IL-1β,TGF-β)between the Chinese medicine group and the PRP group(P>0.05),the combination group had a statistical difference compared with the Chinese medicine group or the PRP group(P<0.01);there was no statistical difference in the MMP-3 between the combination group and the Chinese medicine group,and the MMP-3 There was a statistical difference between them(P<0.05).Conclusion1.All three groups can effectively treat early KOA disease.However,Wenyang Yisui Decocotion combined with PRP is more effective than single use in relieving clinical symptoms,improving motor function,and relieving local discomfort,and has a synergistic effect.2.Wenyang Yisui Decocotion combined with PRP can reduce serum IL-1β and MMP-3 in patients with early KOA,and improve TGF-β,which is more effective than the two alone.Part 2 The key targets and pathways of Wenyang Yisui Decoction in the treatment of KOA on network pharmacologyObjectiveBased on the network pharmacology method,the possible mechanism and pathway of action of Wenyang Yisui Decocotion in the treatment of KOA were preliminarily explored,which provided a basis for the next step of animal experiments to verify its mechanism of action.Method1.Use TCMSP platform,PubChem,Swiss Target Prediction database and literature query to determine the active ingredients of Wenyang Yisui Decoction and related target screening;search the database Gene Cards,DRUGBANK,OMIM and TTD to collect KOA disease targets,and obtain the intersection of the two by Venn diagram Gene,String11.5 database to obtain target protein-protein interaction information.2.Use Cytoscape to draw ’Wenyang Yisui Decocotion-Active ComponentsTarget Network Diagram","Wenyang Yisui Decocotion-PPI Network Diagram of Common Targets of KOA Disease","Core Target Screening Diagram";AutoDock Vina Molecular docking simulation and visual mapping using Pymol software.3.Use the David database to perform GO analysis and KEGG analysis on the core targets to obtain the core cell components,molecular functions,biological processes and key signaling pathways,and screen out the main signaling pathways for the interaction between Wenyang Yisui Decocotion and KOA.Results1.The active ingredients of the Wenyang Yisui Decocotion mainly include efferentin,kaempferol,β-sitosterol,2-heptanone,peach aldehyde,coumaric acid methyl ether,cinnamyl acetate,genkwain,apigenin,etc.2.The key targets of Wenyang Yisui Decocotion in treating KOA are AKT1,ALB,IL6,TNF,IL1B,VEGFA,TP53,MAPK3,PTGS2,MMP9,JUN,CASP3,CXCL8,TLR4,etc.3.GO functional enrichment analysis Wenyang Yisui Decocotion mainly affects positive and negative regulation of RNA polymerase Ⅱ promoter transcription,inflammatory response,signal transduction,positive regulation of gene expression,positive regulation of cell proliferation,etc.;affects protein binding,Enzyme binding,ATP binding,DNA binding and other functions;affect cell membrane,cytosol,cytoplasm,nucleus and other components to interfere with KOA.4.KEGG pathway enrichment analysis showed that the main pathways of Wenyang Yisui Decocotion in treating KOA were Pathways in cancer,lipid and atherosclerosis,kaposi sarcoma-associated herpesvirus infection,PI3K-Akt signaling pathway,Human cytomegalovirus infection,Hepatitis B pathway,etc.ConclusionsThe effect of Wenyang Yisui Decocotion on treating KOA is multi-target and multi-pathway,and the PI3K-Akt signaling pathway is an important signal pathway.Part 3 Mechanism of Wenyang Yisui Decoction combined with platelet-rich plasma in the treatment of early knee osteoarthritis in rabbits based on PI3K/AKT/mTOR pathway regulation of autophagyObjectiveTo explore the regulation of autophagy by PI3K/AKT/mTOR,the mechanism of action of Wenyang Yisui Decoction combined with platelet-rich plasma in the treatment of early KOA by a rabbit model of KOA.Method36 female New Zealand white rabbits,aged 5 months,weighing 2.0±0.5kg,36 New Zealand white rabbits were randomly separated into 9 as the blank control group,and the remaining 27 were used for papain modeling.After 2 weeks of modeling,3 mice were randomly killed and 3 mice in the blank control group received Mankin’s score,micro-CT evaluated the success of modeling.The remaining 24 white rabbits with successful modeling were randomly divided into model group,traditional Chinese medicine group,PRP group and combination group,with 6 rabbits in each group.The traditional Chinese medicine group and the combined group were given intragastric administration of traditional Chinese medicine,the blank group,the model group,and the PRP group were given intragastric administration of equal volumes of normal saline,the PRP group and the combined group were given intra-articular injection of PRP at the same time,and the blank group,model group,and Chinese medicine group were given equal volumes of normal saline.Intra-articular injection of normal saline.Intragastric administration for 4 weeks is a course of treatment,and intra-articular injection is 3 times a course of treatment.Samples were collected 2 weeks after the intervention,behavioral evaluation by Lequese MG,HE staining,safranin-fast green staining,toluidine blue staining,masson staining,Mankin score,serum IL-1β and TGF-β detected by ELISA,Western-blot method Detect the expression of LC3B-Ⅰ/Ⅱ,PI3K,Akt,and mTOR protein in the cartilage tissue of the rabbit knee joint,detect the expression of PI3K,Akt,mTOR,and caspase-1 in the cartilage tissue of the rabbit by RT-PCR,and perform Micro-CT examination of the knee joint.Results1.Lequese MG behavioral evaluation of the rabbit knee joint:the Lequese MG score of the blank group was 0,and it was divided into 4 groups after modeling.The Lequesne MG score of the model group before intervention was significantly different from that of the blank group(P<0.01).There was no significant difference in Lequesne MG score between the model group and the traditional Chinese medicine group,PRP group and combination group before intervention(P>0.05).After the intervention,there was no change in the improvement of Lequese MG scores in the blank group(P>0.05);the model group,the traditional Chinese medicine group,the PRP group and the combination group all showed significant improvement(P<0.01).Compared with the model group,the scores of the traditional Chinese medicine group,the PRP group and the combination group improved significantly(P<0.05).Compared with the PRP group,the average improvement of Lequese MG scores in the traditional Chinese medicine group was better than that in the PRP group,but there was no significant difference in statistical analysis(P>0.05).Compared with the traditional Chinese medicine group and the PRP group,there was a significant difference between the combination group(P<0.01).2.Mankin score of cartilage tissue:Combined with histopathological analysis,Mankin score was performed on the cartilage tissue of rabbit knee joint.The results showed that there was a significant difference between the blank group and the model group,with statistical significance(P<0.05).There was a significant difference between the combined group and the Chinese medicine group and PRP group(P<0.01).There was no significant difference between the TCM group and PRP group(P>0.05).3.ELISA detection:Compared with the blank group,the IL-1β level of the model group was significantly higher than that of the blank group,and the difference was statistically significant(P<0.01).Two weeks after the drug intervention,the traditional Chinese medicine group,the PRP group and the combination group all showed different levels of reduction in serum IL-1β compared with the model group,and the combination group had the most significant decrease in IL-1β,which was statistically significant(P<0.01),followed by Chinese medicine group and PRP group,but there was no statistical difference between Chinese medicine group and PRP group(P>0.05).Compared with the blank group,the TGF-β level in the model group was significantly lower,and the difference was statistically significant(P<0.01).Two weeks after the drug intervention,serum TGF-β in the traditional Chinese medicine group,PRP group and combined group had different levels of increase compared with the model group,and the difference was statistically significant(P<0.05).The increase of TGF-β in the combination group was the most significant and had statistical significance(P<0.01),followed by the Chinese medicine group and the PRP group,but there was no statistical difference between the Chinese medicine group and the PRP group(P>0.05).4.Western-blot detection:Compared with blank group,LC3B-Ⅰ/Ⅱ protein expression was increased in model group after modeling,and the difference was statistically significant(P<0.01).Two weeks after the end of drug intervention,LC3B-Ⅰ/Ⅱ protein expression in TCM group,PRP group and combination group was significantly decreased compared with model group,with statistical significance(P<0.05).Compared with traditional Chinese medicine group or PRP group,the decrease in combination group was significant(P<0.05).There was no difference between TCM group and PRP group(P>0.05).Compared with the blank group,the expression of PI3K,Akt,and mTOR proteins in the model group decreased,and the difference was statistically significant(P<0.01).Two weeks after the drug intervention,the traditional Chinese medicine group,the PRP group and the combination group all showed different levels of reductions in PI3K,Akt,and mTOR compared with the model group,and the combination group had the most significant reduction in the protein expression of PI3K,Akt,and mTOR,which was statistically significant(P<0.05),followed by the Chinese medicine group and the PRP group,but there was no statistical difference between the Chinese medicine group and the PRP group(P>0.05).5.RT-PCR detection:Compared with the blank group,the expression of PI3K,AKT,mTOR and caspase-1 mRNA in the model group chondrocytes was significantly increased,and the difference was statistically significant(P<0.01).Compared with the traditional Chinese medicine group,PRP group and combination group,the expression of PI3K,AKT,mTOR and caspase-1 mRNA in the model group decreased,and the difference was statistically significant(P<0.01).Compared with the traditional Chinese medicine group,PRP group and combination group,the expression of PI3K,AKT,mTOR and caspase-1 mRNA in the combination group was significantly lower than that in the traditional Chinese medicine group and PRP group,and the difference was statistically significant(P<0.01).There was no significant difference in the expression of PI3K,AKT,mTOR and caspase-1 mRNA between the traditional Chinese medicine group and the PRP group(P>0.05).6.Micro-CT examination:anterior and lateral view showed that the femoral trochlea and tibial plateau of rabbit knee joint were smooth in blank group,rough in model group,and slightly rough in Chinese medicine group,PRP group and combined group.Taking the bottom of the growth plate on the side of the tibia as the baseline,the reconstructed cross-section with a thickness of 5mm below the baseline showed that the bone trabeculae in the model group were sparse and thinner.Compared with the blank group,some bone trabeculae in the Chinese medicine group,PRP group and combined group were sparse,but denser than the model group.The coronal plane showed that the number of subchondral bone trabeculae of the tibial bone in the model group was significantly reduced,the distribution was sparse,the arrangement was disordered,the fracture occurred,the trabecular space increased,the structural loss appeared in some areas,the bone lacunae appeared,and the cortical bone became thinner.Compared with the model group,the number of bone trabeculae in the traditional Chinese medicine group,PRP group and combination group was more,the distribution was denser,and the texture was more orderly.The combination group was slightly better than the Chinese medicine group or PRP group.The number of bone trabeculae in the combination group increased,and the bone trabeculae gradually became dense,and the subchondral bone structure was relatively complete.Conclusions(1)To verify that the pathogenesis of early KOA is related to the excessive activation of the PI3K/Akt/mTOR pathway to inhibit autophagy,by promoting IL-1βGeneration and reduction of TGF-β expression and promotion of caspase-1 transcription to promote cartilage degeneration.Wenyang Yisui Decoction,PRP,and their combination can treat KOA by inhibiting this pathway.(2)The onset of KOA and osteoporosis may have the potential to occur simultaneously.The two diseases can cause each other and aggravate each other.The Wenyang Yisui Decoction,PRP,and their combination can play an anti-osteoporosis role while treating KOA.(3)Wenyang Yisui Decoction combined with PRP can significantly inhibit the PI3K/Akt/mTOR signal pathway to promote autophagy expression,promote the conversion of LC3B-Ⅰ to LC3B-Ⅱ,inhibit the inflammatory factor IL-1β,with a rise of TGF-β expression,an inhibition of caspase-1 transcription,and a significant improvement of structural indicators related to osteoporosis.The combination of the two is more effective than single intervention,and the combination group is the preferred treatment for KOA.