B-cell lymphoma-2 phosphorylation at Ser70 site-related autophagy mediates puerarin-inhibited the apoptosis of MC3T3-E1 cells during osteoblastogenesis.

L I Xi, Lin Xiangquan, Chen Dongdong, Liu Hui
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Abstract

Objective: To explore the relationship between autophagy and apoptosis regulated by puerarin during osteoblastogenesis.

Methods: In this study, the effects of puerarin on the autophagic activity and apoptosis level of osteoblast precursors (MC3T3-E1 cells) was observed. Subsequently, the roles of puerarin on B-cell lymphoma-2 (Bcl-2) phosphorylation at different sites in osteoblast precursors were observed. The effect of puerarin on the interaction between Bcl-2 and autophagy regulatory molecule or pro-apoptotic molecule was also investigated using Co-immunoprecipitation assays. In addition, the effect of puerarin on mitochondrial membrane potential of osteoblast precursors was also identified by mitochondrial membrane potential fluorescence probe assays.

Results: Our results showed that puerarin can promote the autophagic activity and apoptosis level of MC3T3-E1 cells. In addition, puerarin promoted Bcl-2 phosphorylation at Ser70 site, and the dissociation of Bcl-2-Beclin1 complex. Moreover, puerarin could enhance the binding of Bcl-2-Bcl-2-Associated X (Bax) complex in MC3T3-E1 cells. Furthermore, puerarin increased the mitochondrial membrane potential of MC3T3-E1 cells.

Conclusions: Therefore, puerarin promotes Beclin1 into autophagy flux through Bcl-2 phosphorylation at Ser70, thereby enhancing autophagy of osteoblast precursors, which mediates its anti-apoptotic role during osteoblastogenesis. Furthermore, the dissociation of Bcl-2-Beclin1 complex is conducive to the binding of Bcl-2-Bax complex, which resists the apoptosis of osteoblast precursors viathe increased mitochondrial membrane potential.

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B细胞淋巴瘤-2在Ser70位点的磷酸化与自噬有关,在成骨过程中介导葛根素抑制MC3T3-E1细胞的凋亡。
目的:探讨葛根素在成骨细胞形成过程中调控自噬和细胞凋亡的关系:方法:观察葛根素对成骨细胞前体(MC3T3-E1细胞)自噬活性和细胞凋亡水平的影响:本研究观察了葛根素对成骨细胞前体(MC3T3-E1细胞)自噬活性和凋亡水平的影响。随后,观察了葛根素对成骨细胞前体不同位点 B 细胞淋巴瘤-2(Bcl-2)磷酸化的作用。还利用共免疫沉淀实验研究了葛根素对 Bcl-2 与自噬调控分子或促凋亡分子之间相互作用的影响。此外,葛根素对成骨细胞前体线粒体膜电位的影响也通过线粒体膜电位荧光探针测定得以确定:结果:葛根素能促进MC3T3-E1细胞的自噬活性和凋亡水平。此外,葛根素还能促进Bcl-2在Ser70位点的磷酸化和Bcl-2-Beclin1复合物的解离。此外,葛根素还能增强 MC3T3-E1 细胞中 Bcl-2-Bcl-2-Associated X(Bax)复合物的结合。此外,葛根素还能提高MC3T3-E1细胞的线粒体膜电位:因此,葛根素通过Bcl-2在Ser70处的磷酸化促进Beclin1进入自噬通路,从而增强成骨细胞前体的自噬,这是其在成骨过程中抗凋亡作用的介导因素。此外,Bcl-2-Beclin1 复合物的解离有利于 Bcl-2-Bax 复合物的结合,而 Bcl-2-Bax 复合物可通过线粒体膜电位的增加抵御成骨细胞前体的凋亡。
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