Effect of You-Gui Yin on glucocorticoid-induced apoptosis of bone marrow mesenchymal stem cells

摘要

Objective:To investigate the effect of You-Gui Yin on glucocorticoid-induced apoptosis of rat bone marrow mesenchymal stem cells and its possible mechanism.Methods:20 SD rats were divided into normal saline group and You-Gui Yin group.Ten rats in each group were given normal saline and You-Gui Yin by gavage for 2 weeks,once daily.After the gavage,the rats were sacrificed by spinal removal,blood was taken from the abdominal aorta and centrifuged to obtain blank serum and medicated serum.The rat bone marrow mesenchymal stem cells were extracted and cultured and administered in groups:blank group(10%blank serum),Model group(10%blank serum+dexamethasone 5×10-5mol/ml),traditional Chinese medicine group(10%medicated serum+dexamethasone 5×10-5mol/ml),control group(10%medicated serum),CCK-8 method was used to detect cell proliferation changes in different groups,ELISA method was used to detect bone alkaline phosphatase levels in each group,Alizarin red staining was used to compare red-stained calcium mineralized nodules in each group,Western Blot Detect the expression of apoptosis-related proteins in BMSCs,and perform molecular docking of the quantitative components to evaluate the binding strength and activity of the target and the active compound.Results:The ratio of absorbance(A450)in each time period of the model group was significantly reduced(P0.05);ELISA method showed that the ALP level of the model group was significantly lower than that of the blank group and the control group(P<0.05),the traditional Chinese medicine group significantly increased the ALP level(P<0.05),there was no significant difference between the control group and the blank group;Observation of the number of calcium mineralized nodules under alizarin red staining microscope suggests that the number of calcium mineralized nodules in the model group is significantly lower than that of each group,and there is no significant difference between the control group and the blank group.Both groups are higher than the model group and traditional Chinese medicine Group;Western Blot indicated that compared with the blank group,the expression levels of proapoptotic proteins Bax and Cleaved-casepase-3 in the model group were significantly increased(P<0.01),and the expression levels of anti-apoptotic protein Bcl-2 were decreased(P<0.01),on the contrary in the traditional Chinese medicine group,the expression of the control group did not change significantly compared with the blank group;the molecular docking results showed that You-Gui Yin mainly regulates the apoptosis of BMSCs through Epicatechin 5,7,3'-trimethyl ether,Delphinin,etc.promote bone formation.Conclusion:You-Gui Yin medicated serum can protect BMSCs from apoptosis induced by GCs and promote BMSCs proliferation and osteogenic differentiation by regulating Bax,Cleaved-caspase3,and Bcl-2.