Research Of Prevention And Treatment Mechanisms Of Fuyuan Capsule On The Cartilage Matrix Degradation Of Degenerative Osteoarthritis

ObjectiveTo study the protective effects of FuYuan capsule and its two main active ingredients, which introduced the therapy of nourishing liver and kidney also resolving stasis and dredging collaterals, on the extracellular matrix of osteoarthritis. To illuMinate its molecular mechanism through the urokinase-type plasminogen activator (uPA)systerm and IκBα-NF-κB signaling pathway.Methods1 Forty-eight New Zealand rabbits were divided into 6 groups randomly, 8 rabbits in each group : normol group and model group(saline10ml/d),Glucosamine hydrochloride (90mg/kg/d), Fyc low dose group(1350 mg/kg/d),middle dose group (2700 mg/kg/d) and high dose group (5400 mg/kg/d).Rabbit osteoarthritis model was induced by combined treatment of D-galactose injection in dose of 30mg/kg/d and plaster immobilization,for 6周eeks . Rabbits were treated according to grouping at the same day of modeling, gavage , daily , for 10周eeks. Morphology of cartilage were detected by light microscope and HE staining.Then cartilage sections were analyzed by immunohistochemistry to test uPA,uPAR and PAI. The expresss of uPA,NF-κB were analyzed by Western blotting .2 Rabbit cartilage tissues were cultured in 24 well comboplate , divided into blank group, 20% blank seruM group, TNF-αmodel group(10 ug / L,1h), uPA inhibitor amiloride group 0.5mmol / L, 5%, 10%, 20% Fyc containing seruM groups. Except the blank group, each group was stimulated by TNF-α(10 ug / L,1h) to induce the cartilage matrix degradation. Then disposed according to grouping. glycosaminoglycan(GAG) that had been released into the mediuM were measured by 1,9-dimethylmethylene blue method to evaluate the extent of proteoglycan degradation. Hydroxyproline that had been released into the mediuM were measured by alkali hydrolyzation method to evaluate the extent of collagen degradation.3 Rabbit cartilage cells were cultured for experimentation and its activity were detected by MTT. According to the result of MTT, 7 groups were divided: blank group, TNF-αmodel group, Glucosamine hydrochloride (25 mg/m1) group,20% FuYuan capsule seruM containing group,Icariin group (12.5μg/ml),arasaponin R1 group (125μg/ml),Icariin (12.5μg/ml) combined arasaponin R1 (125μg/ml) group. The content of uPA, NF-κB(p65) mRNA were determined by reverse transcription polymerase chain reaction (RT-PCR). The activities of NF-κB(P65) combined DNA were determined by electrophoretic mobility shift assays (EMSA). IκBαwere detected by Western blotting.Results1 After treatment of OA model 10 weeks, analyse Mankin's score of each group. Model group was 8.73±0. 94, Glucosamine hydrochloride group was 3.46±0.78, Fyc high administration was 3.28±0.46. Fyc high administration could markedly decrease the pathological damage. There were significant differences in Mankin's score among high dose FuYuan capsule and model group (P<0.01), not worse than the Glucosamine hydrochloride group(P>0. 05).Immunohistochemical staining indicated that the expression intensities and positive cell ratio of uPA, uPAR and PAI increased in OA group 79.61±2.78 , 60.55±1.35 , 52.33±2.87 compared with the model group(P<0.01), while FuYuan capsule treatment could reduce uPA 36.45±1.35 and uPAR 31.35±1.18 expression with the best effect in high dose group(P<0.01). PAI 66.57±1.25 decreased after FuYuan capsule treatment( P<0.01 ) . Not worse than the Glucosamine hydrochloride group 35.38±2.31,30.4±1.34,72.35±1.52(P>0.05).Western blot test results show that the uPA/GAPDH, NF-κB/GAPDH in model group were 1.13692±0.2451,0.89828±0.1650,which in glucosamine hydrochloride group were 0.25147±0.0537,0.36386±0.0458, in Fuyuan capsule group of high-dose were 0.26003±0.0427,0.25315±0.0336. These results indicate that the expresssion of uPA,NF-κB were reduce significantly after high dose FuYuan capsule treatment(P<0.01). The inhibiting effect to NF-κB in middle and high dose FuYuan capsule is better than the Glucosamine hydrochloride group(P<0. 05)。2 The study of rabbit cartilage tissue matrix degradation metabolites GAG, Hyp shows that blank group were 1.32±0.18,0.05±0.01,TNF-αmodel group were 4.76±0.39, 0.89±0.07, increased significantly than blank group (P <0.01). The contents of GAG and Hyp in uPA inhibitor amiloride group were 4.69±0.30,0.41±0.06, in glucosamine hydrochloride group were 3.15±0.59,0.72±0.13, which in 20% FuYuan capsule containing seruM group were 2.92±0.34, 0.64±0.07, all significantly decrease compared with the model group (P <0.01). The results suggest that FuYuan capsule could inhibite the cartilage matrix proteoglycan and collagen degradati in osteoarthritis chondrocytes.3 MTT showed that FuYuan capsule, icariin ,arasaponin R1 ,icariin and arasaponin R1 could significantly promote proliferation of cartilage cells in 48h compared with blank control group (P<0.01). FuYuan capsule group were better than the glucosamine hydrochloride group(P<0.05).RT-PCR showed that uPA, NF-κB(p65)mRNA expression in blank group were 0.225±0.016,0.202±0.014,while in TNF-αgroup were 0.429±0.018,0.463±0.021 which increased significantly compared with blank group(P<0.01)The uPA, mRNA expression of above remedy groups were 0242±0.019,0.254±0.012,0.273±0.013,0.247±0.015,0.236±0.014, NF-κB(p65)mRNA were 0.243±0.017,0.229±0.012,0.281±0.011,0.262±0.015,0.232±0.014 which indicate that the expresssion of uPA, NF-κB(p65)mRNA were reduced significantly than the TNF-αgroup(P<0.01). There were no significant difference among each remedy group.(P>0. 05).EMSA to evaluate the binding activity between NF-κB(p65)and DNA showed that absorbance value in blank group were 47.72±8.19, while in TNF-αgroup were 141.25±31.83,which increased significantly when compared with blank group(P<0.01).The absorbance value in remedy groups were 84.28±19.04,52.85±11.42,86.66±35.23,91.15±28.67,71.47±10.57 respectively,which indicate that FuYuan capsule containing seruM group has the best effect, compared with the TNF-αgroup and Icariin combined arasaponin R1 group(P<0.01). Icariin combined arasaponin R1 group show synergistic effect, compared with the TNF-αgroup(P<0.01).Western Blotting was performed to evaluate the extent of IκBα. There was no significant difference between TNF-αgroup and blank group(P>0.05). While glucosamine hydrochloride, Fyc, icariin, arasaponin R1, and icariin combined with arasaponin R1 group could significantly increase the extent of IκBα, which respectively reached 0.739±0.028,0.763±0.021,0.824±0.035,0.793±0.024,0.832±0.023, than TNF-αgroup 0.374±0.032(P<0. 05 or P<0.01),but no significant difference among all treatment groups(P>0. 05). Conclusions1 Fuyuan capsule can significantly improve pathological changes in OA rabbit model and lower Mankin's ' s score. FuYuan capsule could reduce the expression of uPA and uPAR while increase PAI in cartilage cells of experimental osteoarthritis of rabbits, which maybe potential mechanisms underlying the effect of FuYuan capsule for OA.2 FuYuan capsule containing seruM can significantly decrease the expression of GAG and Hyp in rabbit cartilage tissue. FuYuan capsule could inhibite the degradation of collagen and proteoglycan in the cartilage matrix.3 FuYuan capsule and its two main active ingredients, icariin and arasaponin R1 could protect chondrocytes from damage through increasing the express of IκBα, inhibiting the NF-κB(P65) activity,and then reducing the expresssion of uPA.