Study On The Material Basis And Effect Of Eucommia Ulmoides-Achyranthes Bidentata Against Osteoarthritis

Objective:To clarify the synergistic effect of the crude aqueous extracts of Eucommia ulmoides-Achyranthes bidentata pair against osteoarthritis,explore the material basis of its effect,and screen the key monomers/monomer groups that may play a role in the pair.Method:1.The rat OA model was constructed using MIA,and the blank group,model group and drug administration group were set up.The behavioral condition of rats was evaluated.The diameter and mechanical pain threshold of the affected limb was determined.The levels of IL-1β,IL-6,MMP-3,MMP-13,NO,and TNF-α in the serum of rats were measured.The joint sections were stained with HE and safranin O-fast green,and the expression of MMP-3 and MMP-13 in articular cartilage were determined by immunohistochemistry,and the synergistic effect of drugs on water extracts in the treatment of OA was evaluated overall.2.The compound database of crude aqueous extracts was established,HPLCTOF/MS technique was used to qualitatively analyze the crude aqueous extracts and screen the higher content or the indicative components of Eucommia ulmoidesAchyranthes bidentata.UPLC-MS/MS technique was used to determine the in vitro content and in vivo blood incorporation.The ATDC5 cell membrane chromatographic column was prepared,and the incorporated monomers were injected sequentially into the HPLC-TOF/MS system to investigate the binding of monomers to cell membranes.3.Primary chondrocytes were isolated from C57 neonatal mouse joints,and stimulated by LPS to establish an inflammation model.and the monomers were matched and combined to verify the synergistic effect on the two inflammatory models of primary chondrocytes and ATDC5 cells.RNA-seq technology was used to compare the differential genes between the primary chondrocytes model group and the monomeric matching drug delivery group and predict the mechanism of action against OA.Results:1.In the MIA rat model,the results of each evaluation index were as follows: In the evaluation of the behavioral index,the behavioral scores of the P-Middle group and P-High group decreased significantly(P<0.01);In the determination of mechanical pain threshold,the threshold of P-Low group,P-Middle group,and P-High group increased significantly,which had a pain relief effect(P<0.01);In the measurement of joint diameter,the joint swelling in P-middle group and P-high group was significantly relieved(P<0.01);In the expression of cytokines,the P-High group significantly inhibited the secretion of IL-1β,MMP-3,MMP-13 and NO(P<0.05,P<0.01);The levels of TNF-α and IL-6 did not differ between the blank and the model;The pathological treatment was evaluated.The severity of symptoms was reduced in the PMiddle group and P-high group according to OASRI and Modified mankin’s score criteria(P<0.05,P<0.01).2.A total of 35 compounds were identified in the crude water extracts,and 10 components were selected to compare the content changes before and after matching.and The 10 components were eucommiol,genipin,geniposidic acid,geniposide,pinoresinol diglucoside,β-ecdysterone,ginsenoside Ro,achyranthoside C,and achyranthoside D.The contents of ginsenoside Ro,chikusetsusaponin Ⅳa,and achyranthoside C were increased;the contents of geniposidic acid and pinoresinol diglucoside were unchanged;and the levels of eucommiol,geniposide,β-ecdysterone,genipin,and achyranthoside D were decreased in vitro.In vivo,the contents of geniposidic acid,geniposide,pinoresinol diglucoside,and β-ecdysterone were reduced;the contents of eucommiol and ginsenoside Ro were unchanged;and those of achyranthoside D,chikusetsusaponin Ⅳa,and achyranthoside C increased compared to the corresponding levels in the internal control.The CMC screening of 9 monomers into the blood showed that chikusetsusaponin Ⅳa had an affinity effect on the ATDC5 cell membrane.3.RNA-seq technology was used to compare the differences between the inflammation model group and the drug administration group.Compared with the model group,284 differential genes were found in monomer compatibility,including60 down-regulated genes and 224 up-regulated genes.According to the KEGG pathway analysis,the differential genes were concentrated in 69 signaling pathways.Among them,PI3K-Akt and JAK-STAT pathways The combination of chikusetsusaponin Ⅳa and pinoresinol diglucoside significantly inhibited the secretion of i NOS,MMP-3,and MMP-13(P<0.05,P<0.01)in the ATDC5 cell models,and significantly inhibited the secretion of MMP-13,i NOS,IL-1β and MMP-3(P<0.05,P<0.01)in the primary chondrocytes model,and had a synergistic effect in inhibiting i NOS,IL-1β,and MMP-3.Using RNA-seq technology was used to compare the differences between the inflammatory model group and the administration group.Compared with the model group,a total of 284 differential genes were found in monomeric matching,including 60 down-regulated genes and 224 up-regulated genes.According to the KEGG’s pathway analysis,the differential genes were concentrated in 69 signaling pathways,among which PI3K-Akt and JAK-STAT signaling pathways were associated with OA.Conclusion:Eucommia ulmoides-Achyranthes bidentata pair has a synergistic effect on crude water extract in the treatment of OA,and the main synergistic effect may be due to the interaction between pinoresinol diglucoside in Eucommia ulmoides and chikusetsusaponin Ⅳa in Achyranthes bidentata.