Effect Of Qigu Capsules Combined With Alendronate On The Proliferation And Differentiation Of Osteoclasts

OBJECTIVE: To investigate the effect of Astragalus capsule combined with alendronate on osteoclasts and thus the effect of this Chinese medicine on the treatment of osteoporosis through in vitro experiments.Methods:(1)RAW264.7 cells were cultured,and after the cells were plastered,osteoclasts were induced to differentiate using RANKL and M-CSF for 3 days,and the cells were divided into control group,astragalus group,alendron group,and astragalus-bone combined with alendron group,and then the cells were interfered with the corresponding preparations for 24h;(2)the semi-inhibitory concentration of each experimental group was calculated by CCK-8 cytotoxicity assay,and the optimal in vitro Experimental concentration;(ⅲ)by anti-tartrate acid phosphatase(TRAP)staining,three or more cells with positive nuclear staining were osteoclasts,and the effect of drugs on osteoclast proliferation and differentiation was investigated;(ⅳ)by osteoclast-specific gene Real-time pcr assay,the expression of osteoclast-related genes(NFATcl,c-Fos)was detected under different experimental groups;(v)The changes in the levels of osteoclast-related proteins(NFATc1,c-Fos,p-p65,p65,p-IκBα)were detected by protein immunoblot Western-Blot assay.Results:(1)When the concentration of Astragalus capsule increased from 5 μg/l to 50 μg/l,the cellular activity was gradually inhibited compared with the control group,and the strongest cellular activity was inhibited when the concentration of Astragalus capsule reached 100 μg/l(P<0.0001),while it had cytotoxic effect when the concentration of Astragalus capsule was >200 μg/l.Therefore,it was determined that the inhibitory effect on osteoclastogenesis was strongest at the concentration of Astragalus capsule at 100 μg/l and was not due to drug cytotoxicity;when the concentration of alendronate reached 10μmol/L,the cell activity was inhibited the strongest(P<0.0001)compared with the control group,and it had cytotoxic effect when it exceeded 100 μmol/L,therefore,it was determined that the concentration of alendronate at 10 μmol/L had the strongest inhibitory effect on osteoclastogenesis and was not due to drug cytotoxicity;therefore,based on the above results,the drug concentrations used in the combination group were,respectively,100 μg/l for astragalus capsules and 10 μmol/L for alendronate.(2)After co-culture of osteoclasts with grouping reagents,the number of TRAP-staining positive multinucleated cells were reduced and the cell size became smaller.Both the Astragalus group and Alendron group could inhibit the osteoclastic differentiation of RAW264.7 cells and affected the proliferation of osteoclasts;the rate of positive TRAP staining was significantly lower in the combination group compared with the Astragalus group.(3)real-time PCR assay of relevant expression revealed that the expression of NFATc1 was different between the astragalus bone group and the allan group,but the difference was not significant and not statistically significant(P > 0.05),and the relative mRNA expression of NFATc1 was different between the astragalus bone group and the control group compared to the control group(P < 0.05),and also different and statistically significant between the control group and the allan group(P < 0.05).c-Fos assay results showed that the Allan group and the Astragalus group,both had some inhibitory effect(P < 0.05),while the difference was more significant in the combination group compared with both the Astragalus group and the control group(P < 0.05).(4)As detected by Western-Blot assay,the expression level of osteoclast-related proteins was significantly reduced in the combination group compared with the control group.The expression of NFATc1,p-p65,p65 and p-IκBα was decreased in the astragalus group compared with the control group(P < 0.05),while there was no significant change in c-Fos(P > 0.05);the expression of NFATc1,p65 and p-IκBα was decreased in the allan group compared with the control group(P < 0.05),while there was no significant change in p-p65 and c-Fos(P > 0.05);the expression of NFATc I,c-Fos,p-p65,p65 and p-IκBα was decreased in the combination group compared with the control group(P < 0.05).CONCLUSION: The combination of Astragalus capsule with alendronate inhibits the proliferation and differentiation of osteoclasts by down-regulating the NF-κ B pathway,thus achieving the effect of preventing and treating osteoporosis.