| Objective:To elucidate the interaction between autophagy and apoptosis during steroid-induced femur head necrosis（SANFH）,and to explore the possible mechanism of regulating autophagy and apoptosis of osteoblasts in SANFH mediated by PI3K/AKT signaling pathway mediated by Gubi Tongxiao granules in vivo and in vitro.Methods:Part I:In vivo experimental study on the intervention of Gubi Tongxiao granules in rabbit model of SANFHLipopolysaccharide（LPS）combined with glucocorticoid（GCs）was used to establish a simulated SANFH animal model.The experimental group was given Gubi Tongxiao granules intragastric treatment,and the model and control group were given normal saline intragastric.Once a day,after 8 weeks of administration,the animals were killed and bilateral femoral heads were removed.HE staining was used to detect the pathological changes of rabbit bone tissue,and to evaluate the improvement effect of Gubi Tongxiao granules on hollow bone lacunae of rabbit bone tissue in model group.Immunohistochemical staining was used to observe the localization of Bcl-2 and Bax in bone tissue.The expressions of Beclin-1 and LC3-II in bone tissue were detected by Western blot.RT-PCR was used to detect mRNA levels of ALP,Runx2,Caspase-3 and Caspase-9 in rabbit bone tissue,and to evaluate the improvement effect of Gubi Tongxiao granules on autophagy and apoptosis of SANFH rabbit osteoblasts.Part II:In vitro study on the intervention of dexamethasone（Dex）induced osteoblasts by the medicated serum of Gubi Tongxiao granulesMouse embryonic osteoblasts（MC3T3-E1）were cultured in vitro and the SANFH cell model was constructed using Dex solution.The effect of Dex on MC3T3-E1 cell viability was detected by CCK-8 method,and autophagy and apoptosis of MC3T3-E1cells induced by Dex at different concentrations were detected by MDC/PI staining and Western blot,and to screen optimal time and concentration.CCK-8 to detect the effect of the drug-containing serum of Gubi Tongxiao granules on the vitality of MC3T3T-E1cells pretreated with or without Dex,and to screen the best intervention time and concentration of the drug-containing serum of Gubi Tongxiao granules.BCIP/NBT method and alidin red S method were used to detect the alkaline phosphatase and the mineralization of osteoblast,and to evaluate the effect of the medicated serum of Gubi Tongxiao granules on the osteogenic differentiation of MC3T3-E1 cells induced by Dex.MDC/PI staining was used to detect autophagy and apoptosis of MC3T3-E1 cells.mRNA expression levels of LC3-II,Beclin-1,Bax and Bcl-2 were detected by RT-PCR.The effects of the drug-containing serum of Gubi Tongxiao granules on autophagy and apoptosis of Mc3t3-e1 cells induced by Dex were evaluated.The protein expressions of PI3K,AKT,p-AKT,Bcl-2 and m TOR in cells with or without pathway inhibitors and the drug-containing serum of Gubi Tongxiao Granules were detected by Western blot,and the effect of the drug-containing serum of Gubi Tongxiao granules on the key proteins of PI3K/AKT signaling pathway was evaluated.Results:Part I:Gubi Tongxiao granules can reduce the rate of hollow bone lacunae in SANFH rabbit bone tissue,improve the autophagy and apoptosis of bone tissue,and effectively maintain bone mass（1）HE results showed that the tissue structure of femoral head in SANFH model was damaged,the area of bone trabeculae was significantly reduced and there were severe stenosis or fracture,and the number of empty bone lacunae was significantly increased（P<0.01）.Western blot results showed that expression levels of autophagy markers in bone tissue of SANFH rabbit model were significantly increased（P<0.01）.Immunohistochemical staining indicated that there were a large number of apoptotic cells in SANFH rabbit bone tissue.（2）Gubi Tongxiao granules could improve the damage of femoral head structure and reduce the number of hollow bone lacunae in SANFH model rabbits（P<0.01）.Beclin-1 and LC3-II expression of autophagy markers were decreased（P<0.01）,and autophagy level in bone tissue tended to be normal（P<0.05）.At the same time,it can regulate the expression of poptosis-related protein Bax and Bcl-2,improving the autophagy and apoptosis of cells.（3）mRNA expressions of Caspase-3 and Caspase-9 in SANFH rabbit bone tissue were significantly increased,while mRNA expressions of ALP and Runx2 were significantly decreased（P<0.01）.Gubi Tongxiao granules could decrease the mRNA expressions of Caspase-3 and Caspase-9,increase the mRNA expressions of ALP and Runx2（P<0.05）,inhibit cell apoptosis,promote the development of osteoblasts,and effectively maintain bone mass.Part II:The drug-containing serum of Gubi Tongxiao granules can affect the expression of pathway proteins,and reduce the autophagy and apoptosis.（1）The viability of MC3T3-E1 cells decreased in a time and dose-dependent manner in GCs environment.There was no significant difference in the viability of cells in different doses of Dex after 24h（P>0.05）,and the viability of cells in 10-5M group after 48h was decreased（P<0.01）.The cell viability of was significantly decreased after72h（P<0.01）.（2）MDC/PI staining and Western blot results showed that after 72h exposure to GCs,the autophagy level of MC3T3-E1 cells increased in the low-dose Dex(10-7M)group（P<0.01）,and the highest level appeared in the 10-6M group（P<0.01）.The autophagy level in the high-dose Dex(10-5M)group was decreased（P<0.05）.In addition,with the extension of time and the increase of Dex concentration,osteoblast proliferation and differentiation were abnormal,and apoptosis increased（P<0.05）.（3）Different volume fractions of Gubi Tongxiao granules medicated serum can promote the proliferation of MC3T3-E1 cells,and there is a certain dose-effect and aging relationship;The activity of MC3T3-E1 cells decreased significantly under the induction of 10-5M Dex（P<0.01）.The activity of osteoblasts was inhibited by Dex after the intervention with different volume fraction of Gubi Tongxiao granules medicated serum,especially the 5%of Gub Ttongxiao granules medicated serum（P<0.05）.（4）The results of BCIP/NBT and alizarin red S staining showed that MC3T3-E1cells under high Dex concentration were significantly less than those in the control group（P<0.01）,and the number and volume of orange red mineralized nodules in calcium salt deposits were smaller under microscope（P<0.01）.The osteoblast differentiation of MC3T3-E1 cells was improved after the intervention of 5%Gubi Tongxiao granules medicated serum culture,and the intracellular alkaline phosphatase and osteoblast mineralization level were significantly increased（P<0.05）.（5）After being exposed to 10-5M Dex for 72h,MC3T3-E1 cells showed large number of autophagy and late apoptotic cells（P<0.01）,and autophagy and apoptosis rates were significantly increased and apoptosis level was higher than autophagy level（P<0.05）.The serum containing Gubi Tongxiao granules could regulate the expression of Bax and Bcl-2 mRNA（P<0.01）,which competitively combined with autophagy markers Beclin-1 in target cells,apoptosis was effectively improved and autophagy tended to be normal（P>0.05）.（6）Western blot results showed that the drug-containing serum of Gubi Tongxiao granules could up-regulate the protein expressions of PI3K,AKT,P-Akt and Bcl-2 in MC3T3-E1 cells induced by Dex（P<0.05）.When the signaling pathway inhibitors were used,theprotein expressions was inhibited.（P<0.01）.However,when Gubi Tongxiao granules medicated serum and pathway inhibitor treated MC3T3-E1 cells,the expression level of P-Akt increased（P<0.05）,the expression of Bcl-2 increased,the m TOR stabilized within a certain range,and the autophagy and apoptosis were improved（P<0.05）.Conclusion:（1）In the pathological process of GCs-induced femoral head necrosis,there are local pathological abnormalities of the femoral head,excessive autophagy and apoptosis of bone cells,and the osteogenic effect is weakened.Gubi Tongxiao granules have obvious protective effect on autophagy and apoptosis of osteoblasts in GCs environment,promote proliferation and development of osteoblasts,and effectively maintain bone mass.（2）The activity of MC3T3-E1 cells decreased in a time-and dose-dependent manner in GCs environment.GCs can induce autophagy and apoptosis of osteoblasts in both directions.Low dose of GCs can activate autophagy,remove damaged organoids and maintain homeostasis of osteoblasts.However,under high dose of GCs,autophagy tends to be normal and apoptosis pathway is activated.（3）In the GCs environment,the osteogenic differentiation potential of mouse embryonic osteoblast MC3T3-E1 was impaired,the anti-apoptotic ability was decreased,autophagy and apoptosis levels were significantly increased;The development of osteoblasts tended to be normal,autophagy and apoptosis of osteoblasts were inhibited to varying degrees after the intervention of the medicated serum of Gubi Tongxiao granules.（4）After the PI3K/AKT signaling pathway was inactivated by inhibitors,the level of intracellular apoptosis was further increased.The medicated serum of Gubi Tongxiao granules could improve the phosphorylation level of pathway proteins,regulate the Bax/Bcl-2 complex,and stabilize the expression of autophagy proteins within a certain range.Down-regulating the high expression of Caspase mediated by GCs can reduce the apoptosis of osteoblasts,thus effectively delaying the process of femoral head necrosis. |
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