Study On The Efficacy, Safety And Mechanism Of Total Flavonoids From Drynaria Fortunei In Treating Osteoarthritis Caused By Kidney Yang Deficienc

Research BackgroundOsteoarthritis(OA)is a chronic inflammatory joint disease characterized by degenerative lesions of articular cartilage.OA has a high incidence and seriously affects the quality of life of patients,thus causing a huge socioeconomic burden and becoming a critical clinical problem that needs to be addressed today.However,there is still a lack of safe and effective drugs for its treatment.Based on a review of the literature on Chinese medicine,it was found that the onset and development of OA is closely related to kidney-yang deficiency.Therefore,the Chinese medicinal materials of warmly invigorating kidney yang,like Rhizoma Drynariae,have good clinical therapeutic effect on it.Flavonoids possess anti-inflammatory and antioxidant effects,which offer special advantages and good application prospects for OA treatment.Previous clinical studies have found that Total Flavonoid of Rhizoma Drynariae(TFRD)can effectively reduce pain and improve joint function in patients with OA.However,the therapeutic effect and mechanism of TFRD against kidney-yang deficiency in OA need to be further investigated.Based on this objective,the current study was designed to investigate the beneficial effect of TFRD on patients with kidney-yang deficiency through a randomized controlled clinical trial.Whereafter,network pharmacology and metabolomics were used:to predict the mechanism of TFRD against OA,and validated by in vivo and in vitro experiments.Clinical Research StatusObjective The randomized controlled trial intends to investigate the effect of TFRD against KOA patients with kidney-yang deficiency.Methods Seventy-two KOA patients with kidney-yang deficiency were divided into treatment group and control group.In the control group,patients were prescribed oral glucosamine hydrochloride and subjective to patient education and functional exercise,while in addition to that,treatment group was meantime administrated oral TFRD(Chinese patent drug:Qianggu Capsule),with diclofenac sodium enteric-coated tablets(fotarine)against intolerable joint pain.The following outcome indicators were evaluated after 8-week intervention:the efficacy of TFRD against the kidney-yang deficiency syndrome(primary outcome indicator);quantitative scale for grading of OA,VAS score,WOMAC score,and NSAIDs use.Security indexes included incidences of abnormal liver and kidney function,and adverse reactions.Results Compared to the control group,the treatment group showed a remarkable reduce in kidney yang deficiency symptoms,quantitative scale for grading of OA,VAS score,WOMAC score and NSAIDs dose.Also,there were no significant differences in security indexes between two groups.Conclusion TFRD(Chinese patent drug:Qianggu Capsule)can effectively improve the TCM symptoms and joint function of KOA patients with kidney yang deficiency,with a good safety profile.Experimental Study StatusObjective Network pharmacology and metabolomics were used to predict the mechanism of TFRD against OA,and validated by in vivo and in vitro experiments.Methods The flavonoid components in Rhizoma Drynariae were determined by searching the TCMSP database,among which active components were screened by oral bioavailability,drug-like properties and previous literature.Liquid chromatography-mass spectrometry instrument(LC-MS)was utilized to detect whether these active ingredients were available in TFRD samples used for subsequent experiments.Further,the therapeutic targets of active ingredients against OA were obtained and key targets and pathways were identified.Meanwhile,the OA model rats were established by Hulth’s surgery and intervened with TFRD and glucosamine hydrochloride(positive control drug).After 28d intervention,these rats were humanely euthanized.Their knee joints were isolated and then fixed,decalcified and sections were made to assess drug efficacy by cartilage pathology,and serum and cartilage tissue were extracted for subsequent experiments.Rat sera were assayed by large-scale targeted metabolomics to find differential metabolites.Then,KEGG pathway enrichment was conducted on differential metabolites to predict the mechanism of TFRD against OA.Chondrocytes were isolated from suckling rats and identified by immunofluorescence stain for in vitro experiments.These cells were stimulated with inflammatory factor IL-1β to establish an in vitro model of OA,and then intervened with TFRD.RT-PCR,ELISA assay,Western Blot,immunofluorescence and other molecular biological experiments were used to verify the predicted mechanism.Results The network pharmacology suggested that COX-2,IL-6 and TNF-α might be the key targets of TFRD against OA,and their therapeutic mechanisms might be related to NFκB and AMPK signalling pathways and arachidonic acid metabolic pathways.Serum metabolomics showed that the inflammatory metabolites associated with arachidonic acid catabolism were elevated in OA model rats,but the content of arachidonic acid increased and the downstream inflammatory metabolites of arachidonic acid were decreased in TFRD intervention group,KEGG enrichment analysis revealed that TFRD intervention for OA was closely related to arachidonic acid pathway.Histopathology sections showed that TFRD were effective in improving the Mankin’s score of knee cartilage of OA model rats.RT-PCR and Western Blot found that TFRD could significantly inhibit the mRNA and protein expression of COX-2 in cartilage.After stimulation on chondrocytes with IL-1β,the expression of IL-6,TNF-α and COX-2 at the mRNA and protein levels was significantly increased,while TFRD could effectively inhibit their expression with a dose-dependent effect.At the same time,IL-1βstimulation of chondrocytes decreased AMPK phosphorylation and IκB-α expression and increased NFκB p65 nuclear translocation and phosphorylation,indicating that AMPK signalling pathway was inhibited and NFκB signalling pathway was activated;but TFRD could reverse these changes to some extent.Furthermore,A-769662 was used to activate the AMPK signalling pathway and JSH-23 to inhibit the NFκB signalling pathway in IL-1β-stimulated chondrocytes,which demonstrated that the mRNA expression of COX-2 was not markedly affected by the TFRD intervention.Conclusion The treatment of OA with TFRD was closely related to the inhibition of AMPK/NFκB signalling pathway-mediated inflammatory response in chondrocytes.