Zuogui Pill Regulates NPY Affecting Cartilage-Subchondral Bone Signal Interaction To Prevent Postmenopausal Osteoarthritis

ObjectiveThe aim of this study was to explore the theory of Zuogui Pill in preventing postmenopausal osteoarthritis(OA),by establishing postmenopausal osteoporosis(PMOP)and OA comorbidity model by ovariectomy,thus revealing the mechanism of postmenopausal OA,and providing new approaches for preventing postmenopausal OA in Traditional Chinese Medicine(TCM)Method1.Theoretical study:by summarizing the TCM theory of "nephridosis,main bone and bone marrow" and subchondral bone/cartilage research systematically,the reconstruction of subchondral bone with its crosstalk on cartilage was comprehensively analyzed,and the mechanism of Zuogui Pill to prevent postmenopausal OA was analyzed from the perspective of bone remodeling and cartilage-subchondral bone crosstalk.2.Experimental study:(1)The study established PMOP and postmenopausal OA comorbidity model by ovariectomy,and divided into Zuogui Pill low dose group(OVX+ZL)and Zuogui Pill high dose group(OVX+ZH),model group(OVX),alendronate group(OVX+AS),and sham group.After 3 months drug's intervention,left femurs were obtained for HE and safranin orange/fast green(SO/FG)pathological staining,and micro-CT was used to observe cartilage-subchondral bone microstructure.(2)Gene dataset GSE82107 was downloaded from the GEO database.Corrplot package and principal component analysis(PCA)were used for matrix quality assessment.GE02R was used to analyze and screen for differentially expressed genes(DEGs).ClueGO,DAVID v6.8 and Gene Enrichment Analysis Database(GSEA)were used to perform enrichment analysis to identify neuropeptide receptors related to OA.The gene co-expression network was calculated by WGCNA,and protein-protein interaction network was established with neuropeptide receptors to confirm the core clusters.Toxicological genome database(CTD)and receiver operating characteristic curve(ROC)were used to analyze the relationship between neuropeptide receptors and musculoskeletal diseases and evaluate their diagnostic efficacy.(3)Western blot was used to detect the expression of NPY,NPY1R,NPY2R and RUNX2,OPG,RANKL in subchondral bone tissue.(4)Western blot was used to detect the expression of NPY,NPY1R and NPY2R in cartilage tissue.Result1.Theoretical study:the basic pathogenesis of postmenopausal OA is deficiency of genuine yin,consumption of essence and marrow.Nourishing yin and tonifying the kidney,supplementing essence to replenish marrow is the basic method for preventing postmenopausal OA,and Zuogui pill has the effect to prevent postmenopausal OA by intervening cartilage-subchondral bone interaction.2.Experimental study:(1)HE staining showed that the structure of trabecular bones in the OVX group were sparse,disordered,discontinuous,and absent;the cartilage layer was disordered,and thin.The tidal line was blurred or even disappeared.OVX+AS,OVX+ZL and OVX+ZH group have improved pathology.The number of trabecular bones is increased,with reduced space,and the structure is more complete.Chondrocytes are neatly distributed,with little cell hyperplasia and tide line is relatively complete.SO/FG staining showed that the surface of the articular cartilage was rough and exfoliated in OVX group,and the cartilage matrix staining was reduced.While the articular cartilage structure is relatively complete,with neatly distributed cells,and the cartilage matrix is well stained in OVX+AS,OVX+ZL and OVX+ZH group.Micro-CT images showed that the number of trabeculae in the OXV group was significantly reduced,the distribution was sparse,the spacing was increased,the structure was missing,with a large blank area appeared,and the thickness of the cartilage was thin.Compared with the OVX group,the trabecular bone density of the OVX+AS group,OVX+ZL group and OVX+ZH group was increased,the spacing was reduced;the fracture was reduced,and the cartilage thickness was increased.(2)The gene expression matrix has the same clustering distribution of samples with the same phenotype.At the same time,the results of PCA analysis showed that the samples treated differently had obvious differences.The consistency of biological duplication was high and the differences between groups were obvious.A total of 1676 DEGs were identified in GSE82107,including 218 up-regulated DEGs and 1458 down-regulated DEGs.ClueGO,DAVID,and GSEA analysis found that OA-related gene enrichment functions include "positive regulation of hormone secretion","bone remodeling","bone growth",and "cartilage development" were statistically significant.This study found that the MEgreen module has a high correlation with OA,and NPY1R,NPY2R and MEgreen gene modules have a closer interaction.The study also found that NPY1R and NPY2R are involved in musculoskeletal and other related physiological and pathological processes.The CTD results show that NPY1R is closely related to OP or developmental bone disease,metabolic bone disease,PMOP,chondropathy,and bone resorption,or other diseases;NPY2R is related to developmental bone disease,musculoskeletal abnormalities,OP,and bone disease.ROC analysis showed that both NPY1R and NPY2R have value as biomarkers to identify OA lesions.(3)The results in western blot showed that the expression of RUNX2 and OPG in subchondral bone tissue of the OVX group were decreased(P<0.05),while the expression of RANKL was increased(P<0.05).The OVX+AS and OVX+ZH group had higher expressions of RUNX2 and OPG(P<0.05),and RANKL was decreased(P<0.05);but the difference expressions of the above-mentioned protein in OVX+ZL group were not statistically significant.In addition,the expression of NPY,NPYI R,and NPY2R in the subchondral bone tissue of the OVX group were decreased(P<0.05).The expressions of NPY,NPY1R,and NPY2R in the OVX+AS and OVX+ZH group increased(P<0.05);there was no statistical difference in the expressions of the above in OVX+ZL group.(4)The expression of NPY,NPY1R and NPY2R in cartilage tissue of OVX group were decreased(P<0.05).The expression of NPY,NPY1R,and NPY2R were increased in OVX+AS and OVX+ZH group(P<0.05);The expression of NPY,NPY2R protein expression were increased in OVX+ZL group(P>0.05),and NPY1R was increased(P<0.05).Conclusion1.Zuogui pill improves the structure and thickness of subchondral trabecular bone in ovariectomy SD rats.2.NPY1R and NPY2R are closely related to OA pathology,and NPY1R and NPY2R have a strong effect on the evaluation and identification of its pathology.3.Zuogui Pill increases the expression of NPY and its receptors NPY1R and NPY2R protein in subchondral bone,and promotes the bone formation in subchondral bone(increasing the expression of RUNX2),regulating the RANKL/OPG signaling pathway,and inhibits bone resorption and helps the balance of bone remodeling and prevent subchondral OP in postmenopausal OA.4.Zuogui Pill promotes the expression of NPY and its receptors NPY1R and NPY2R in cartilage tissue,thus preventing cartilage degradation and promoting cartilage repair in postmenopausal OA.