| Objective: We sorted out the key formula of Professor Zhao Wenhai’s therapy of Knee Osteoarthritis(KOA)syndrome of kidney deficiency and blood stasis by analyzing the formula using data mining technologies.In vitro and in vivo research were done on the core formula’s impact on KOA as well as its molecular mechanism.Methods:1.Using the TCM inheritance assistance system(V2.5),compile the TCM prescriptions of patients diagnosed with KOA(kidney deficiency and blood stasis syndrome)from Professor Zhao Wenhai’s outpatient department at the Affiliated Hospital of Changchun University of Chinese Medicine from 2021 to 2023.Compute the drug frequency,four qi and five taste,channel conversion,and composition of the core prescription.Finally,combine this information with Professor Zhao Wenhai’s clinical medication habits to summarize the core formula.2.The TCMSP database was searched in order to identify the primary active components and possible targets of the core recipe.In order to determine the primary action targets of KOA,the data from five disease targets—OMIM,Drug Bank,Gene Cards,TTD,and Dis Ge NET—were analyzed concurrently.These targets were then intersected with the drug prediction targets.The STRING platform was used to build the PPI network,and Cytoscape3.9.0 was used to search for core targets.Using the Metascape platform,the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis and the Gene Ontology(GO)study were carried out for the primary targets.Lastly,Auto Dock and Py Mol were used to confirm the molecular docking of the primary active components and the key target.3.Destabilization of the medial meniscus(DMM)was used to create the rat model of KOA,which was then split into three groups: Sham,DMM,and core formula low-mediumhigh dosage.The medication was administered by gavage once day for four weeks after grouping.Utilizing histology,Micro-CT,and gait analysis,the anti-KOA action and associated signaling pathways of the core formula were observed.4.Primary chondrocytes were extracted from the rib cartilage of newborn mice using Alcian blue staining.number Real-time PCR(q RT-PCR)was used to investigate the effects of different core formulation dosages on the expression of genes associated with chondrocyte formation.In order to clarify the molecular mechanism of chondrocyte regulation,the antioxidant impact of the formula was further examined by measuring the levels of glutathione(GSH),superoxide dismutase(SOD),and reactive oxygen species(ROS).Results:1.137 TCM tastes were among the 382 prescriptions that were included after statistical analysis of the cases.36 TCM with frequency ≥50 were identified;the top 15 included cooked Radix Rehmanniae Praeparata,fevervine,Angelica Sinensis,Ligusticum wallichii,Red peony,Radix Achyranthis Bidentatae,safflower,peach kernels,Fructus aurantii,Radix bupleurum,Platycodon grandiflorum,Radix aconiti carmichaeli,cinnamon,cuttlebone,and zaocys dhumnade.medications that activate and remove blood stasis(1852 times,31.24%),vacuity tonifying(926 times,15.62%),wind-dampening(594 times,10.02%),warm-li(417 times,7.03%),and digestion(314 times,5.30%)were the most common among the 36 types of highfrequency medications.Warm,flat,cold,hot,and cool medication use frequencies among 137 TCM were 2997,1521,1307,221 and 110,respectively.Drugs that are tangy,bitter,sweet,sour,salty,and astringent were used 3464,3430,2626,434,372,and 227 times on average.Liver channel was the most popular medicine,followed by kidney,heart,and spleen channels.28 types of TCM prescription combinations,10 groups of core combinations,and 5 types of new prescriptions were obtained based on the rule of formula composition and drug cluster analysis.The core drugs included in these combinations were Radix Rehmanniae Praeparata,Red peony,Radix Achyranthis Bidentatae,and Angelica Sinensis.The fundamental method for treating kidney deficiency and blood stasis syndrome KOA developed by Professor Zhao Wenhai was distilled based on data mining regulations and the unique geographical features of Northeast China: Radix Achyranthis Bidentatae-Angelica Sinensis-Rehmanniae Praeparata(RAR).2.17 active components of RAR and 119 common target genes of RAR for the treatment of KOA were identified by network pharmacological analysis.By building a "drugcomposition-target-disease" network,the primary compounds of quercetin,kaempferol,baicalein,β-sitosterol,and stigasterol for the treatment of KOA were tested.Key gene targets implicated in the pathophysiology of KOA were identified as Jun,Rela,Tp53,Fos,Mapk1,Mapk14,Myc,Hif1 a,and Esr1.The anti-KOA impact of RAR was mostly linked to the cell’s response to oxidative stress and aging,according to GO analysis,and the MAPK signaling pathway was considerably enriched,according to KEGG enrichment.Using molecular docking,it was possible to combine Mapk1 and stigasterol because of their similar binding energies of-7.54 kcal/mol.3.In vivo,RAR may considerably lessen the disparity in rear foot stride length in DMM rats and successfully reduce the degree of joint swelling.The examination of the lower extremity load-bearing data revealed that the rats’ post-RAR treatment differences in hindpaw area,load-bearing paw percentage,and standing duration were all less than those of the DMM group.Following RAR therapy,the Micro-CT scan and safranin O-fast green staining of the knee joint demonstrated a reduction in osteophyte on the joint surface and a notable improvement in the subchondral bone’s trabecular architecture.The staining findings demonstrated that RAR might restore cartilage injury and decrease proteoglycan loss.Primary mouse rib chondrocytes were isolated in vitro,and the best RAR concentration gradients(0.1,0.2,and 0.4 mg/ml)on chondrocytes were evaluated.4.Studies investigating the mechanism of RAR against KOA discovered that RAR can considerably remove IL-1β the buildup of ROS and the rise of SOD and GSH content in the induced OA cell model,as well as stimulate the expression levels of Col2a1,Comp,and Acan in animal joint cartilage and chondrocytes.Furthermore,Mapk1’s m RNA and protein expression may be markedly suppressed by RAR.Conclusion:1.The anti-KOA core prescription RAR was compiled using data mining analysis of Professor Zhao Wenhai’s treatment of KOA(kidney deficiency and blood stasis syndrome)in order to give a theoretical foundation and medication reference for the TCM treatment of KOA.2.The primary target of the MAPK signaling pathway,Mapk1,was hypothesized to be the major target of RAR anti-KOA based on network pharmacology studies.Preliminary molecular docking validation was conducted,demonstrating the distinct therapeutic benefits of Chinese herbal compounds in the treatment of KOA with multi-target,multi-component,and multi-pathway approaches.3.Experiments conducted in vivo demonstrate that RAR may greatly reduce joint pain,enhance the gait of rats,improve the degree of joint swelling in DMM rats,and have a major therapeutic impact.Furthermore,in vitro tests shown that RAR significantly protects chondrocytes.4.RAR functions as a protective factor for cartilage and may stimulate cartilage anabolism both in vivo and in vitro.RAR simultaneously prevented ROS from building up in chondrocytes and performed an anti-oxidative stress function.Furthermore,the main objective of RAR against KOA is Mapk1. |
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