右归饮对糖皮质激素诱导的骨髓间充质干细胞凋亡的调节作用

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

目的:探讨右归饮对糖皮质激素(glucocorticoids,GCs)诱导的大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)凋亡的调控作用及机制。方法:将20只SD大鼠分为生理盐水组和右归饮组,每组10只,分别采用生理盐水及右归饮灌胃,每日一次。灌胃2周后,脱脊处死大鼠并取血,制备空白血清和含药血清,提取、培养大鼠BMSCs并进行分组给药:空白组(10%空白血清)、模型组(10%空白血清+地塞米松5×10^(−5)mol/mL)、中药组(10%含药血清+地塞米松5×10^(−5)mol/mL)、对照组(10%含药血清),分别用CCK-8法检测各组细胞增殖变化,ELISA法测定各组骨碱性磷酸酶(alkaline phosphatase,ALP)水平,茜素红染色比较各组红染钙矿化结节数量,Western blot检测BMSCs凋亡相关蛋白表达,并对量丰成分进行分子对接,根据结合能评价靶点与活性化合物的结合强度与活性。结果:对比各组不同时间点的吸光度(A_(450))比值,模型组较空白组均降低(P<0.05),而中药组、对照组较模型组均提高(P<0.05),对照组与空白组差异无统计学意义(P>0.05);ELISA结果表明模型组ALP水平显著低于空白组和对照组(P<0.05),中药组显著提高ALP水平(P<0.05),对照组与空白组差异无统计学意义(P>0.05);此外,模型组与各组相比钙矿化结节数目显著降低,对照组与空白组差异无统计学意义,两组均高于模型组与中药组;Western blot结果表明与空白组相比,模型组促凋亡蛋白Bax和cleaved caspase 3的表达水平均显著升高(P<0.01),抗凋亡蛋白Bcl-2的表达水平显著降低(P<0.01),中药组的结果则与之相反,对照组与空白组的表达无明显差异;分子对接结果表明右归饮主要通过epicatechin 5,7,3'-trimethyl ether、delphinin等调控BMSCs的凋亡,促进成骨分化。结论:右归饮含药血清可通过对Bax、cleaved caspase 3、Bcl-2的调控,保护GCs诱导的BMSCs凋亡,促进BMSCs增殖与成骨分化。

Objective:To investigate the effect of You-Gui Yin on glucocorticoid-induced apoptosis of rat bone marrow mesenchymal stem cells(BMSCs)and its possible mechanism.Methods:A total of 20 SD rats were divided into the normal saline group and the You-Gui Yin group,with 10 rats in each group.The rats were given normal saline and You-Gui Yin,respectively,by gavage for 2 weeks,once daily.After gavage,the rats were sacrificed by spinal removal,and blood was taken from the abdominal aorta and centrifuged to obtain blank serum and medicated serum.The rat BMSCs were extracted,cultured and then given different treatments:the blank group(10%blank serum),the model group(10%blank serum+dexamethasone 5×10^(−5) mol/mL),the traditional Chinese medicine group(10%medicated serum+dexamethasone 5×10^(−5)mol/mL),and the control group(10%medicated serum).CCK-8 method was used to detect cell proliferation in different groups.ELISA method was applied to detect bone alkaline phosphatase levels in each group.Alizarin red staining was used to compare red-stained calcium mineralized nodules in each group.The expression of apoptosis-related proteins in BMSCs was determined by Western blot,and molecular docking was performed to evaluate the binding strength and activity of the target and the active compounds.Results:The ratio of absorbance(A_(450))of the model group was significantly reduced in each time point(P<0.05),and the ratio of the traditional Chinese medicine group and the control group was significantly higher than that of the model group(P<0.05).There was no significant difference between the control group and the blank group(P>0.05);ELISA results 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),while 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(P>0.05).Moreover,the number of calcium mineralized nodules in the model group was decreased compared to other groups,and there was no significant difference between the control group and the blank group(P>0.05).The number of calcium mineralized nodules in these two groups was higher than that of the model group and the traditional Chinese medicine group.Western blot analysis indicated that compared with the blank group,the expressions of pro-apoptotic proteins Bax and cleaved caspase 3 in the model group were significantly increased(P<0.01),while the expression of anti-apoptotic protein Bcl-2 was decreased(P<0.01).The results of the traditional Chinese medicine group were opposite.The protein expression of the control group did not change significantly compared with the blank group.The molecular docking results showed that You-Gui Yin regulated the apoptosis of BMSCs mainly through epicatechin 5,7,3'-trimethyl ether,delphinin,etc.,thus promoting 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 caspase 3,and Bcl-2.