Study On The Mechanism Of Yougui Yin Inhibiting Apoptosis Of MLO-Y4 Osteocytes Based On Mitochondrial Autophagy Medi Ated By HIF-1? Receptor

Objective:By clarifying the interaction between mitochondrial autophagy and osteocyte ap optosis mediated by HIF-1? receptor,to explore whether the mechanism of Yougui Yin de coction inhibiting SANFH osteocyte apoptosis is related to mitochondrial autophagy media ted by HIF-1? receptor,so as to provide reliable theoretical and experimental basis for trad itional Chinese medicine in the prevention and treatment of SANFH.Methods:This study was divided into three parts,all of which were cultured with MLO-Y4 osteocytes in vitro.In experiment 1,Western blot was used to detect the effects of dexame thasone at different concentrations(1?M,10?M,100?M)on the expression of mitochondri al autophagy-related proteins BNIP3 and LC3 in MLO-Y4 osteocytes under normoxic and hypoxic conditions,and to determine the effect of hormones on HIF-1? mediated mitochon drial autophagy.Experiment 2 was divided into six groups: group A(blank control normox ic group),group B(blank control hypoxia group),group C(MLO-Y4 overexpression grou p transfected with HIF-1? under hypoxia),group D(HIF-1? infected siRNA group transfec ted with MLO-Y4 under hypoxia),group E(MLO-Y4 transfection HIF-1? gene overexpre ssion+10?M dexamethasone group under hypoxia),group F(MLO-Y4 transfection of HIF-1? cells si RNA+10?M dexamethasone group under hypoxia).The expression levels of B NIP3 and LC3 in HIF-1? cells and downstream mitochondrial autophagy proteins in each g roup were detected by Western blot,and the apoptosis of MLO-Y4 osteocytes in each grou p was detected by flow cytometry,so as to clarify the interaction between HIF-1? mediated mitochondrial autophagy and SANFH osteocyte apoptosis.In experiment 3,MLO-Y4 oste ocytes were divided into three groups: blank group,hormone group(10?M dexamethasone)and Yougui Yin decoction group(10?M dexamethasone+Yougui decoction).By using We stern blot to detect the expression levels of HIF-1?,BNIP3 and LC3,and flow cytometry t o detect the apoptosis of SANFH osteocytes,it is clear whether the mechanism of Yougui Yin inhibiting osteocyte apoptosisis related to mitochondrial autophagy mediated by HIF-1?.Results:(1)The expression levels of mitochondrial autophagy-related proteins HIF-1?,BNIP3 and LC3 in MLO-Y4 osteocytes were lower under normoxia.When 1?M dexameth asone was added,the expression level of HIF-1? increased significantly,and the expressio n trend of BNIP3 and LC3 protein was consistent with that of HIF-1? protein,and the diffe rence was statistically significant.When the concentration of local dexamethasone increase d to 10?M and 100?M,the protein expression levels of HIF-1?,BNIP3 and LC3 decreasedsignificantly compared with those at 1?M,and the difference was statistically significant(P<0.05).The expression levels of mitochondrial autophagy-related proteins HIF-1?,BNI P3 and LC3 in MLO-Y4 osteocytes were higher under hypoxia,but decreased with the incr ease of dexamethasone concentration.(2)The expression of HIF-1?,BNIP3 and LC3 pr otein in group B was significantly higher than that in group A,and the apoptosis rate of M LO-Y4 osteocytes in group B was significantly higher than that in group A(P<0.01).The expression of HIF-1?,BNIP3 and LC3 protein in group C was significantly higher than tha t in group B,and the apoptosis rate of osteocytes in group C was significantly lower than th at in group B(P<0.01).The expression levels of HIF-1? and BNIP3 in group D were signi ficantly lower than those in group B,and the apoptosis rate of osteocytes in group D was si gnificantly higher than that in group B(P<0.05).The protein expression level of LC3 in g roup D was slightly lower than that in group B,but there was no significant difference bet ween group D and group B(P>0.05).The protein expression levels of HIF-1?,BNIP3 and LC3 in group E were significantly lower than those in group C,and the apoptosis rate of os teocytes in group E was significantly higher than that in group C(P<0.05).The expression of BNIP3 protein in group F was significantly lower than that in group D(P<0.05).The pr otein expression levels of HIF-1? and LC3 in group D were lower than those in group D,b ut there was no significant difference(P>0.05).The apoptosis rate of osteocytes in group D was significantly higher than that in group D(P<0.05).The protein expression levels of H IF-1?,BNIP3 and LC3 in group E were significantly higher than those in group F,while th e rate of apoptosis in group E was significantly lower than that in group F(P<0.01).(3)The protein expression levels of HIF-1?,BNIP3 and LC3 in the hormone group were signi ficantly lower than those in the blank group,and the apoptosis rate of osteocytes in the hor mone group was significantly higher than that in the blank group(P<0.05).The protein ex pression levels of HIF-1?,BNIP3 and LC3 in Yougui Yin decoction group were significan tly higher than those in hormone group,and the apoptosis rate was significantly lower than that in hormone group(P<0.05).Conclusions:The increase of hormone concentration can significantly inhibit the expressio n of HIF-1? mediated osteocyte mitochondrial autophagy.Increasing the expression of HI F-1? can significantly increase the occurrence of osteocyte mitochondrial autophagy and in hibit hormone-induced osteocyte apoptosis.Yougui Yin decoction can significantly increas e the level of mitochondrial autophagy of osteocytes mediated by HIF-1?,thus protecting t he cells from apoptosis under exposure to hormone.This study clarified part of the mechan ism of YouguiYin decoction in the prevention and treatment of SANFH,and provided a rel iable experimental basis for clinical practice.However,the specific pathway still needs to be proved by further experiments.