Effects And Underlying Mechanism Of Exogenous Hydrogen Sulfide On Glucocorticoid-induced Osteoporosis

Background:Glucocorticoids（GCs）lead to the increasing incidence of secondary osteoporosis.Previous studies found that free H₂S in patients of glucocorticoid-induced osteoporosis was lower than that of healthy people.Lack of endogenous H₂S could raise the risk of osteoporosis and bone fracture.Exogenous H₂S might positively affect osteoporosis.It is still unclear whether exogenous H₂S can reverse glucocorticoid-induced osteoporosis.This study mainly explored the protective effect and mechanism of exogenous H₂S on glucocorticoid-induced osteoporosis.Objective:1.To determine whether exogenous H₂S has a protective effect on dexamethasone-induced osteoporosis.2.To explore whether exogenous H₂S could reverse dexamethasone-induced injuries on osteoblasts.3.To investigate whether exogenous H₂S mediates the protection of osteoblasts by activating Wnt/β-catenin pathway.Methods:1.Dexamethasone-induced osteoporosis model was constructed by intraperitoneal injection of dexamethasone solution twice a week.Control group was injected with saline.Four weeks later,the femur was acquired and identified by HE staining.The tissue density of femur was detected by micro-CT.Three-dimensional reconstruction was performed to observe the trabecular thickness and degree of separation.Static parameters representing bone mass were analyzed,including the percentage of trabecular area（%Tb.A）,trabecular volume（%BV/TV）,trabecular number（Tb.N）,trabecular separation degree（Tb）.The levels of free H₂S in the blood of rats in each group were determined by ELISA.The expression of key enzymes of H₂S metabolism,CBS and CSE m RNA,were detected by q RT-PCR.2.When the GIOP was successfully constrcuted,rats were randomly divided into four groups:Control group,Dex group,Dex+GYY4137 group and GYY4137 group.After 12weeks,previous indicators as listed in Methods 1 were detected.3.Verify the effect of Dex on osteoblasts.Primary osteoblasts were extracted from SD rats and randomly divided into three groups at different concentration of Dex.Isolated osteoblasts were cultured for 48h,then cell proliferation was detected by CCK-8,MTT and Ed U.Hochest 33342 staining and Annexin-FITC/PI double staining were used to detect the apoptosis level of osteoblasts.The expression levels of BCL2 and Bax apoptotic proteins were detected by Western blot.4.Evaluate the effect of GYY4137 on osteoblasts.Primary osteoblasts were extracted from SD rats and randonmly divided into 4 groups（Control group,Dex group,Dex+GYY4137 group and GYY4137 groups）.After GYY4137 treatment,cell viability was detected by MTT and Ed U.Cell apoptosis of osteoblasts were detected by Hochest33342 staining and Annexin FITC/PI double staining.Apoptotic protein expression levels of BCL2 and Bax were detected by Western blot.Cell differentiation was detected by ALP staining.5.Explore the effect of GYY4137 on Wnt/β-catenin pathway.After extraction of primary osteoblasts,immunofluorescence was used to detect the expression levels of GSK-3βin osteoblasts.q RT-PCR was used to detect the m RNA levels of Wnt/β-catenin pathway activation markers（Ahr,Axin2,Nkd2,GFb3,and Wisp1）in osteoblasts.Results:1.After treatment with dexamethasone for 4 weeks,bone density of rats in the dexamethasone group decreased significantly compared with the control group（P<0.05）,and bone trabeculae in the dexamethasone group became thinner.Micro-CT showed that bone density of femur,tibia and part of vertebra decreased significantly in dexamethasone group（P<0.05）.CT three-dimensional reconstruction showed that the thickness of bone trabeculae was decreased and the degree of separation was increased in dexamethasone group.The bone trabecular volume（%BV/TV）and bone trabecular number（Tb.N）（P<0.01）of dexamethasone group decreased significantly,and the bone trabecular separation degree（Tb.Sp）of dexamethasone group increased significantly（P<0.01）.Bone trabecular thickness decreased significantly in dexamethasone group（Tb.Th）.In the dexamethasone group,H₂S in the serum of rats was significantly decreased compared with the control group（P<0.01）.2.After treatment with exogenous H₂S（GYY4137）in the dexamethason-induced rat osteoporosis model,there was no significant difference in bone mineral density between the single GYY4137 treatment group and the control group.Dex+GYY4137 group had significantly higher bone mineral density,higher bone trabecular thickness and lower bone trabecular separation degree than the Dex group（P<0.05）.Moreover,the number（Tb.N）,trabecular volume（%BV/TV）and free H₂S in serum of the Dex+GYY4137 group were significantly increased compared with the Dex group（P<0.05）.3.After cultured with dexamethasone for 24h,cell proliferation of primary osteoblasts was detected by CCK-8,MTT and Ed U method.The results showed that dexamethasone could inhibit the proliferation of osteoblasts in a dose-dependent manner,and further verified by Ed U experiment（P<0.01）.Alizarin red staining demonstrated that dexamethasone could reduce calcium salt deposition in bone.Expression of CBS and CSE protein in osteoblasts decreased at a dose-dependent manner of dexamethasone.4.After cultured with dexamethasone for 24h,Hochest 33342 staining and Annexin-FITC/PI double staining were used to further detect whether dexamethasone regulates the apoptosis of osteoblasts.Compared with control group,the results of Hochest33342 staining and Annexin-FITC/PI double staining showed that the apoptosis level of bone cells composed of dexamethasone increased.The apoptosis level of bone cells in Dex+GYY4137 group was significantly lower than Dex group.Since Caspase-3 plays an important role in cell apoptosis,we further tested the activity of Caspase-3 after combined treatment of Dex and GYY4137.We found that the Caspase-3 activity in the group treated with DEX was significantly increased compared with that in Control group.Dex-induced caspase-3 activity could be inhibited by GYY4137（P<0.05）.To further clarify the apoptosis level of osteoblasts treated with dexamethasone and GYY44137,we used Western blot to detect the expression levels of BCL2 and BAX.Consistent with the caspase-3 results,Dex could attenuate the expression of BCL2 and increase the expression of BAX.The BCL2/BAX ratio of osteoblasts was decreased in Dex group.Compared with Dex group,BCL2 was up-regulated while BAX was down-regulated in Dex+GYY4137group.BCL2/BAX ratio was increased in Dex+GYY4137.5.After the primary osteoblasts were cultured with dexamethasone,the m RNA levels of Wnt/β-catenin pathway activation markers Ahr,Axin2,Nkd2,GFb3 and Wisp1 in the Dex group were lower than those in the control group（P<0.05）.Compared with the Dex group,the m RNA expression levels of Ahr,Axin2,Nkd2,GFb3 and Wisp1 in the Dex+GYY4137 group increased（P<0.05）.The fluorescence intensity of GSK-3βin Dex group was significantly enhanced compared with that of the control group.Expression of GSK-3βin the Dex+GYY4137 group was decreased compared with that of the Dex group（P<0.05）.The expressions of Wnt3a,Wnt6 andβ-catenin in the Dex group were significantly down-regulated compared with the Control group（P<0.05）.The expressions of Wnt3a,Wnt6 andβ-catenin in the Dex+GYY4137 group were not significantly changed compared with the Control group,while significantly up-regulated compared with the Dex group（P<0.05）.Conclusion:Exogenous H₂S has a protective effect on the rat model of glucocorticoid-osteoporosis and osteoblasts.It promotes cell proliferation and differentiation of osteoblasts,suppresses cell apoptosis of osteoblasts by activateing Wnt/β-catenin pathway in.