Gastrodin Activates GATA-1 To Regulate Endoplasmic Reticulum Stress And Ameliorates Dexamethasone-induced Osteoblasts Apoptosis

Objective(s):In clinical practice,glucocorticoids are frequently used as immunosuppression and anti-inflammatory drugs,and small doses of glucocorticoids can increase bone production,but large doses of glucocorticoids used over an extended period cause osteoporosis and osteonecrosis because they reduce osteoblast viability and function.An essential physiological process for cells to maintain normal is endoplasmic reticulum stress.When cells experience prolonged and excessive endoplasmic reticulum stress,the endoplasmic reticulum is unable to maintain cellular normal,which will cause cells to undergo apoptosis and raise the risk of osteoporosis.However,the mechanism of osteoblast apoptosis induced by glucocorticoid-induced endoplasmic reticulum stress is still poorly understood.Therefore,how to alleviate endoplasmic reticulum stress,reduce apoptosis of osteoblasts,and reduce bone loss is particularly important.GATA-1 is a member of zinc-finger transcription factors,and research has demonstrated that transcription factors connected to endoplasmic reticulum stress can bind to GATA-1,suggesting that GATA-1 may be involved in endoplasmic reticulum stress regulation.Currently,gastrodin has been proven to reduce oxidative stress and endoplasmic reticulum stress.Thus,this study will explore the relationship between transcription factor GATA-1 and endoplasmic reticulum stress-related proteins was explored and whether gastrodin may activate GATA-1,control endoplasmic reticulum stress levels to prevent dexamethasone-induced osteoblast apoptosis and bone loss,it provides a preliminary basis for new targets for clinical osteoporosis prevention and treatment and gene care.Methods:1.To observe whether endoplasmic reticulum stress and GATA-1 are altered by dexamethasone-induced apoptosis in MC3T3-E1 cells: A dexamethasone-induced bone damage model was created using MC3T3-E1 cells,CCK8 was used to evaluate the drug’s effects on cell viability,western blot detected the apoptosis-related protein Caspase-3,endoplasmic reticulum stress-related protein Bip,Chop expression and the transcription factor GATA-1,and further determined the concentration of dexamethasone-induced apoptosis.The glucocorticoids osteoporosis model was constructed by intraperitoneal injection of glucocorticoids in C57BL/6J mice and the effect of dexamethasone on bone was detected by Masson staining.2.To observe whether suppression of endoplasmic reticulum stress improves dexamethasone-induced apoptosis: The effect of 4-phenylbutyric acid on cell viability was detected by CCK8,the expression of apoptosis-related proteins Caspase-3、Bax 、Bcl-2,and endoplasmic reticulum stress-related proteins Bip and Chop protein were detected by Western blot,Hoechst further detected the effect and effective concentration of 4-phenylbutyric acid on improving the apoptosis induced by MC3T3-E1 cells by dexamethasone.3.To observe the changes of endoplasmic reticulum stress level and apoptosis when overexpressing GATA-1 and knocking down GATA-1:Construct overexpression GATA-1 and knockdown GATA-1 was constructed,transfected into MC3T3-E1 cells,the expression position change of GATA-1 was detected by immunofluorescence,the expression of apoptosis-associated protein and endoplasmic reticulum stress-related protein was detected by Western blot,and whether the addition of dexamethasone could affect its expression.4.Exploration of the relationship between GATA-1 and endoplasmic reticulum stress-related protein Bip:Chromatin immunoprecipitation and mass spectrometry analysis were used to detect the interaction between GATA-1 and Bip.5.To observe whether gastrodin affects osteoprotective and GATA-1 expression:MC3T3-E1 cells were pretreated with gastrodin to detect GATA-1 expression position changes by immunofluorescence,Western blot detected apoptosis-associated protein and endoplasmic reticulum stress-related protein expression changes.A model of dexamethasone-induced osteoporosis was constructed with C57BL/6J male mice,and the expression changes of GATA-1 and Bip in each group were detected by immunohistochemistry and immunofluorescence,paraffin sections were stained by HE staining and Masson staining to detect changes in the structure of femur tissue in mice.Results:1.With the increase of dexamethasone concentration,the viability of MC3T3-E1 cells gradually decreased(P<0.05),apoptosis-related protein Caspase-3 protein expression was gradually increased(P<0.05),and endoplasmic reticulum stress-related proteins Bip and Chop protein expression levels were gradually increased with increasing drug concentration(P<0.05),the expression of the transcription factor GATA-1 was also upregulated,and Hoechst 、 Annexin V-FITC apoptosis assay observed 5μM dexamethasone can indeed lead to apoptosis,suggesting 5μM dexamethasone can induce apoptosis and endoplasmic reticulum stress in MC3T3-E1 cells.2.The endoplasmic reticulum stress inhibitor 4-phenylbutyric acid either inhibited nor increased at concentration of 0.1μM,cell viability begins to gradually decreased at 0.5–100μM(P<0.05),after pretreatment with 5μM dexamethasone and 0.1μM 4-phenylbutyric acid,Caspase-3 and Bax protein expression were downregulated(P<0.05),and Bcl-2 expression was upregulated(P<0.05),the results of the Hoechst test also demonstrated that dexamethasone-induced apoptosis can be decreased by pretreatment with 0.1μM 4-phenylbutyric acid.This shows that endoplasmic reticulum stress inhibitors can lessen osteoblast apoptosis and enhance dexamethasone-induced endoplasmic reticulum stress.3.After overexpression of GATA-1,the expression of endoplasmic reticulum stress-related proteins Bip,Chop,apoptosis related proteins Caspase-3,Bax and other proteins were up-regulated(P<0.05),while the expression of apoptosis related protein Bcl-2 was down regulated(P<0.05),but the addition of 5μM dexamethasone after GATA-1 overexpression could reduce the protein expression of endoplasmic reticulum stress and apoptosis(P<0.05).After knockdown of GATA-1,Bip and Chop were down regulated(P<0.05),while caspase-3 was up-regulated(P<0.05),and the level of ER stress was significantly inhibited,but after knockdown of GATA-1,5μM dexamethasone treatment for 24 h could increase the level of ER stress(P<0.05)and reduce the expression of apoptotic proteins(P<0.05).4.The results of the CHIP test demonstrated that the transcription factors GATA-1 could successfully bind to GRP78/Bip promoter,and transcription factor GATA-1could regulate Bip transcription.5.Dexamethasone plus 5μM gastrodin pretreatment in MC3T3-E1 cells can decrease GATA-1 protein expression(P<0.05),endoplasmic reticulum stress-related protein(P<0.05),and apoptosis-related protein(P<0.05),and reduce the nuclear entry of GATA-1.Immunofluorescence and immunohistochemistry results together in vivo experiments also show that GATA-1 and Bip expression results are consistent,and gastrodin could reduce the trabecular bone loss caused by dexamethasone.Conclusion(s):1.Dexamethasone causes endoplasmic reticulum stress,induces osteoblast apoptosis,and increases bone loss.2.Dexamethasone increases the expression of transcription factor GATA-1 and promotes its nuclear entry.3.Transcription factor GATA-1 binds to Bip promoter sequence.4.Gastrodin regulates GATA-1,reduces endoplasmic reticulum stress levels,reduces osteoblast apoptosis,and reduces bone loss.