| Objective:To explor the effects of Bushen Huoxue Decoction on the proliferation of rabbit chondrocytes,expression of mRNA cytokines(IL-1,IL-6,IGF-1,TGF-β)and expression level of NF-kb p65 protein.Then discussing its effects on the comorbidities of Osteoporosis and Osteoarthritis and providing theory for Nourishing Kidney and Activating Blood to treat this comorbidities.Method:Thirty New Zealand female rabbits were randomly divided into three groups:A(OP model,n=10),B(OA model,n=10),group C(OP and OA model,n=10).The Group A was modeled according to Calvo method,Group B was modeled according Hulth method and Group C was modeled combined with the above two modeling methods for OP and OA modele.Each group was extracted from right knee articular cartilage tissue to separate cultured chondrocytes.MTT method was used to observe the effects of 7 groups serum which contain drugs on the proliferation of rabbit chondrocytes(Normal serum group,Low dose,Middle dose and High dose of Bushen Huoxue Decoction group,Glucosamine hydrochloride group,Alendronate sodium group and Glucosamine hydrochloride+Alendronate sodium group).The effect of the expression of IL-1、IL-6、IGF-1、TGF-βmRNA in rabbit cartilage cell were detected by PCR and the expression of NF-kb p65 protein were detected by Western blot.Results:1.The type II collagen immunohistochemical MOD showed a decreasing trend in cells of A、B.C groups with the incresase in the transfer of algebra(P1>P3>P5),and the difference between the P1,P3,P5 was significant(P<0.05).In the case of fixed cell algebra,there were significant differences in P1 and P3 between the three groups(P<0.05),but there was no difference in P5(F=2.180,P=0.148>0.05).There was significant difference between the two groups(P<0.05)in group A and group C compared with PI and P3.2.MTT detection shows:With the using of the drug to 7 days.(1)All dose of Bushen Huoxue Decotion compared with normal group the OD value was increased,there was significant difference(P<0.05)except from between the low dose group and the normal group(t=2.152,P=0.098>0.05).It can be seen that Bushen Huoxue Decotion has the effect of promoting chondrocyte proliferation.(2)High dose group compared with low dose group the OD value was increased,there was significant difference(P<0.05).There was difference between B and C groups(P<0.05)in the low dose group to the middle dose group and the middle dose group to the high dose group.Compared with the OD in the A,B,C groups,high dose group>middle dose group>low dose group,it can be seen that high dose group to promote the proliferation of chondrocytes the most obvious in three doses of Bushen Huoxue Decotion,and the effect of Bushen Huoxue Decoction on the proliferation of chondrocytes presents a dose-time-benefit relationship.(3)Compared with the alendronate sodium group and the glucosamine hydrochloride group,the effect of the high dose group was better than that of the alendronate sodium group and the glucosamine hydrochloride group(P<0.05)except from between the high dose group and the Glucosamine hydrochloride group in group A(t=1.566,p=0.192>0.05).There was no significant difference between the high dose group and the Glucosamine hydrochloride+Alendronate sodium group(P>0.05).It was found that the proliferation of chondrocytes in the high dose group was similar to the glucosamine hydrochloride+alendronate sodium group and were better than glucosamine hydrochloride and alendronate sodium group.PCR results shows:(1)Each dose of Bushen Huoxue Decoction group Compared with the normal group can decrease the IL-1mRNA expression in cartilage cells,the difference was significant(P<0.05)but there was no significant difference between the low dose group and the normal group in the OA group(t=2.866,P=0.058>0.05),of which the most obvious declines was in high dose group,compared with low,middle dose group the differences were significant(P<0.05);The expression of IL-mRNA in the high dose group was significantly lower than that in the Glucosamine hydrochloride group(P<0.05).High dose group compared with the Alendronate sodium group and the glucosamine hydrochloride+alendronate sodium group,there was no significant difference in OP and OA+OP group(P>0.05),but was significant difference in OA group(P<0.05);It can be seen that the effect of high dose group decrease the IL-1mRNA expression is equal to the glucosamine hydrochloride+alendronate sodium group and alendronate sodium group,better than glucosamine hydrochloride group.(2)A11 dose of Bushen Huoxue Decoction group compared with normal group had the effect of reducing the expression of IL-6mRNA in the cells,of which the most obvious effect was in high dose group,compared to the low,middle dose group,with significant difference(P<0.05);High dose group compared with alendronate sodium group reduced IL-6 mRNA effect more apparent,the differences were significant(P<0.05),high dose group compared with glucosamine hydrochloride+alendronate sodium group(P>0.05)there was no significant difference,high dose group compared with glucosamine hydrochloride group,in OA group there was no significant difference(t=1.399,P=0.211>0.05),but in OP and OA+OP group differences were significant(P<0.05).It can be seen that the high dose group effect of reducing IL-6 mRNA expression is equal to glucosamine hydrochloride+alendronate sodium group,better than alendronate sodium group and glucosamine hydrochloride group.(3)Compared with the normal group,the middle and high dose group could increase the expression of IGF-1mRNA,the difference was significant(P<0.05),and there was significant difference between the middle and high dose group(P>0.05).Compared with the alendronate sodium group,the high dose group had better effect of increase the expression of IGF-1 mRNA,and this difference was significant(P<0.05).The high dose group was compared with the Glucosamine hydrochloride group and the glucosamine hydrochloride+alendronate sodium group,there was no difference in A,B,C groups(P>0.05).(4)All dose of Bushen Huoxue Decoction group compared with normal group had the effect of increasing the expression of TGF-PmRNA in cells,the difference was significant(P<0.05).The effect of increasing TGF-βmRNA expression showed that high dose group>middle dose group>low dose group,and the comparison between each dose group,difference was significant(P<0.05);High dose group compared with alendronate sodium group,there was no significant difference(P>0.05),high dose group compared with glucosamine hydrochloride group in OA and OA+OP group there were no significant difference(P>0.05),there was significant difference in the OP group(t=2.455,P=0.049<0.05),high dose group compared with glucosamine hydrochloride+alendronate sodium group,in OA and OP group there were no significant difference(P>0.05),but was significant difference in OA+OP group(t=5.526,P=0.001<0.05).It was found that the high dose group raised TGF-PmRNA expression role in cells equal to the glucosamine hydrochloride group,alendronate sodium group,glucosamine hydrochloride+alendronate sodium group.4.Western-blot results show:Middle and high dose group compared with normal group have the role of decreasing NF-kb p65 protein in cells,has significant difference(P<0.05),which the role was most obvious in high dose group,compared with low,middle dose group have significant differ ence(P<0.05);High dose group decreaed the expression of NF-kb p65 protein was more obviously when compared with glucoseamine hydrochloride group and alendronate sodium group,there were significant difference(P<0.05);in OP group high dose group compared with glucosamine hydrochloride+alendronate sodium group differences were significant(t=7.597,p=0.002<0.05),but in OA and OA+OP group,there was no significant difference(p>0.05),so the high dose group decreased the expression of NF-kb p65 protein were better than glucosamine hydrochloride group and alendronate sodium group,equal to the glucosamine hydrochloride+alendronate sodium group.Conclusions:1.The animal models of A,B,C groups can extract chondrocytes,compared with group A,the ability of group C to secrete proteoglycan and type Ⅱcollagenase decreased significantly.2.Bushen Huoxue Decoction have the effect of promoting the proliferation of chondrocytes and presents a dose-time-benefit relationship,high dose group effect was most obvious.3.Bushen Huoxue Decoction,alendronate sodium,glucosamine hydrochloride,glucosamine hydrochloride+alendronate sodium can promote chondrocyte proliferation effect,its mechanism may be related to increasing the expression of IGF-1、TGF-β mRNA,decreasing IL-1、IL-6mRNA and NF-kb p65 protein expression in cartilage cells,to indirectly or directly to protect or repair of cartilage cell. |
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