Preventive Effects Of MAGL On Glucocorticoids-Induced Osteonecrosis Of The Femoral Head And The Underlying Mechanisms

Background:Numerous studies have confirmed that the occurrence and development of osteonecrosis of the femoral head(ONFH)is closely related to bone mesenchymal stem cells(BMSCs).High doses of glucocorticoids(GCs)can induce BMSCs apoptosis and change their differentiation direction,resulting in ONFH.Monoacylglycerol lipase(MAGL),as one of the most important nodes in the cannabinoid system in vivo,can regulate oxidative stress injury,proliferation and differentiation of cells of various tissue types.Therefore,we proposed the hypothesis that MAGL can also regulate oxidative stress and apoptosis,change the differentiation direction of BMSCs,and thus preventing early steroid-induced femoral head necrosis.Based on this hypothesis,the following studies are made:Part one:Role of MAGL in regulation of glucocorticoids-induced oxidative stress and apoptosis in BMSCs and the underlying mechanismsObjective:To assess the impact of glucocorticoids on BMSCs and the changes in MAGL expression levels.To investigated whether there is any correlation between MAGL expression and glucocorticoids-induced osteonecrosis of the femoral head.To explore an association between glucocorticoids-induced oxidative stress and apoptosis and to test whether MAGL regulation can protect against glucocorticoids-induced oxidative stress and apoptosis in BMSCs and the underlying mechanisms.Methods:BMSCs were harvested from long bones of SD rats(at 4 weeks of age)and were used at passages 3-6.We then tested whether methylprednisolone sodium succinate(MP)-induced toxicity correlated with oxidative stress and apoptosis.BMSCs were incubated with α-MEM containing PBS or different concentrations of MP(10-6mol/L-104mol/L).The effect of MP on BMSCs proliferation was detected using CCK8 assay.The Western blotting was used to analyze the expression levels of oxidative stress and apoptosisrelated proteins.ROS assay and TUNEL assay was used to assess the levels of oxidative stress and apoptosis in BMSCs.To determine whether there was a direct association GCsinduced oxidative stress and apoptosis in BMSCs,NADPH oxidase(NOX)inhibitors DPI(10-4mol/L)and VAS2870(10-4mol/L)were added to medium containing MP.Thereafter,we compared the differences in the level of oxidative stress,the level of apoptosis,the expression levels of oxidative stress and apoptosis-related proteins between NOX inhibitors groups and MP group.In addition,Western blotting and immunofluorescence staining were used to verify the expression of MAGL in BMSCs under the intervention of MP with different concentration gradients.Next,we tested whether MAGL could regulate oxidative stress and apoptosis in BMSCs.MAGL inhibitor MJN 110(10-6mol/L),MAGL overexpression plasmid,and MAGL lentivirus were used to intervene cells,and then MP(10-4mol/L)was added to the medium.These groups were compared with the group that BMSCs only were treated with MP(104mol/L).Western blotting,ROS staining and TUNEL staining were used to compare the levels of oxidative stress and apoptosis,as well as the expressions of oxidative stress-related indicators and apoptosis related indicators.The Nrf2 pathway is a key pathway of oxidative stress in vivo.Western blotting and immunofluorescence analysis were used to compare the expression changes of the Nrf2 signaling pathway related proteins(Nrf2,Keap 1,HO 1 and NQO1)in BMSCs under different intervention conditions.Then the BMSCs which under MP treatment were transfect with Nrf2 agonist or Nrf2 plasmid,to compare the oxidative stress level,apoptosis level,oxidative stress-related indicators and the expression of apoptosis-related indicators in different groups of cells.Finally,the BMSCs incubated in the medium containing both MP and MJN110 were knocked out or inhibited the expression of Nrf2 to observe whether the Nrf2 pathway blockade would reverse the therapeutic effect of MJN110 on GCs-induced oxidative stress and apoptosis in BSMCs.LPS and MP were used to establish a GC-induced SD rat ONFH model.SD rats were randomized into four groups(n=8):(1)DMSO only(control group);(2)MP and LPS(model group);(3)model group rats treated with MJN110(10 mg/kgper day,i.p.injection)(pre-treatment group).Micro-CT analysis,HE and TUNEL staining were used to analyze the morphological changes of the femoral head and the apoptosis level of BMSCs in each group.Western blotting and immunohistochemical assay were used to analyze the effects of GCs on the expression of oxidative stress-related proteins,apoptosis-related proteins,Nrf2 pathway-related proteins and MAGL in bone tissues.Results 1:CCK8 experiment results showed that high dose of MP was cytotoxic to BMSCs and could significantly inhibit the proliferation of BMSCs.The results of ROS and TUNEL staining showed that as the concentration of MP increased,the levels of oxidative stress and apoptosis in BMSCs cells also increased significantly.Western blotting experiments also confirmed that the expression levels of oxidative stress-related proteins NOX1,NOX2,and NOX4 accompanied the rise of MP concentration or increasing time,and the apoptosis-related proteins expressions(Cleaved-Casp3,Cleaved-Casp3,BAX and et al.)showed the same trend as that of oxidative stress-related proteins.We also found that the expression level of MAGL increased with MP concentration.Compared with the control group,the bone trabecula in the femoral head of the model group was significantly reduced,and the bone parameters BV,BV/TV,and Tb.Th decreased significantly,while Tb.Sp increased in the model group.The bone tissue protein Western blotting test further confirmed that,the expression of oxidative stress-related indicators(NOX1,NOX2,and NOX4),apoptosis-related indicators(Cleaved-Casp3,Cleaved-Casp3,BAX and et al.)increased significantly in the model group samples.In addition,the tissue TUNEL staining showed that the apoptotic BMSCs in the femoral head of the model group were markblely more than that of the control group.Result 2:Western blotting experiment results confirmed that,compared with the MP group,the expression of oxidative stress related proteins NOX1,NOX2,NOX4 in BMSCs intervention the level was were significantly decreased in DPI group or VAS2870 group,and the expression of apoptosis-related proteins(Cleaved-Casp3,Cleaved-Casp9,BAX and et al.)showed the same trend.The results of ROS staining and TUNEL staining also confirmed that NOX inhibitors could significantly reduce the levels of oxidative stress and apoptosis in BMSCs.We also found that the expression of MAGL increased with MP intervention in BMSCs.Result 3:Western blotting results showed that MJN110 or shMAGL significantly inhibited the expression of MAGL.In the MAGL inhibitor treatment group,the high expression of NOX family proteins and apoptosis-related proteins were reduced.The intracellular ROS level also decreased after MJN110 treatment or shMAGL transfection.In addition,the results of the TUNEL experiment confirmed that the number of apoptotic BMSCs decreased after MAGL blockade.We further tested the modulation of MAGL overexpression on the level of oxidative stress and apoptosis.As expected,MAGL overexpression further increased the levels of oxidative stress and apoptosis in BMSCs upon MP stimulation.Result 4:Through Western blot experiments and cell immunofluorescence experiments,we confirmed that the expression of Nrf2,HO1 and NQO1 was significantly reduced in BMSCs under the intervention of MP,while the expression of Keap1 increased.In contrast,after the treatment of MJN110,the expression of Nrf2,HO1 and NQO1 increased significantly,while the expression of Keapl decreased.At the same time,MJN110 can reverse the effect of MP on the expression of the Nrf2 pathway related proteins.In further experiments,we found that,by adding Nrf2 agonists or overexpressing Nrf2,not only the expression levels of oxidative stress-related proteins can be reduced,but also the expression levels of apoptosis-related proteins decreased.The results of ROS staining and TUNEL staining confirmed that with the activation of Nrf2,the level of oxidative stress and apoptosis in BMSCs decreased significantly.Finally,the Nrf2 inhibitor ML385 and siNrf2 were used to suppress the expression of Nrf2 in the BMSCs.The results show that the inhibition or knockout of Nrf2 can reverse the beneficial effects of inhibiting MAGL on BMSCs.Under the intervention of ML385 or siNrf2,the level of oxidative stress and apoptosis in BMSCs cells rebounded significantly.Result 5:Through in vivo experiments,we observed that the preventive administration of MJN110 could significantly reduce pyknotic nuclei and empty bone lacunas in the femoral head.The results of CT analysis further confirmed that prophylactic administration of MJN110 not only increased BV,BV/TV and Tb.Th value also significantly reduces Tb.Sp value.The results of the TUNEL experiment showed that the pretreatment group had fewer apoptotic cells than the model group.In addition,through Western blotting and immunohistochemical staining,we found MAGL inhibition negatively regulates GCsinduced oxidative stress response and apoptosis through the Nrf2 pathway.Conclusion:High doses of GCs can induce oxidative stress and increase apoptosis level in BMSCs.Improving GCs-induced oxidative stress can reduce the level of apoptosis in BMSCs.MAGL inhibition could effectively improve GCs-induced apoptosis of BMSCs by reducing oxidative stress.The protective mechanism of MAGL inhibition on BMSCs was mediated through the Nrf2 signaling pathway.Part two:The impact of MAGL regulation on the differentiation direction of BMSCs in glucocorticoids interventionObjective:Through a series of in vitro experiments,to explore whether regulating MAGL can protect BMSCs from the impact of GCs that promote adipogenic differentiation and inhibit osteogenesis in BMSCs,and the potential molecular mechanisms.Through in vivo experiments,to observe whether the regulation of MAGL can attenuate femoral head necrosis by changing the differentiation direction of BMSCs.Methods:3-6 passages of BMSCs harvested from the long bones of SD rats were used for in vitro experiments.In order to verify the effect of dexamethasone(DEX)on BMSCs differentiation and MAGL expression.In vitro experiment,PBS or different concentration of DEX(5*10-8mol/L-10-6mol/L)were used for analyzing the effect of DEX on BMSCs osteogenic or adipogenic differentiation.Western blotting,RT-PCR,ALP staining,ALP activity detection,Alizarin Red S staining,Oil Red O staining and cellular immunofluorescence staining were used to detect the osteogenic differentiation level,adipogenic differentiation level,the expressions of osteogenic,adipogenic differentiation related indicators and MAGL in BMSCs.Next,Western blotting,RT-PCR,ALP staining,and ALP activity detection,Alizarin red S staining and oil red O staining were performed to analyze the regulatory effects of MAGL inhibitors(MJN110(10-6mol/L))and MAGL overexpression plasmid on BMSCs differentiation.After this step,cells were incubated with DEX alone or co-incubated with MJN110.To analyze whether the inhibitor of MAGL can reverse the effect of DEX on the differentiation of BMSCs.Then,2-arachidonoyl glycerol(2-AG),the main hydrolysate of MAGL in vivo,was used to repeat the above experiments,to verify once again the regulatory effect of MAGL on the differentiation direction of BMSCs.In order to determine the downstream receptor of MAGL regulating BMSCs differentiation.We blocked CB1 and CB2 expression by corresponding inhibitors or the siRNA and tested the osteogenic and adipogenic differentiation level of BMSCs by Western blotting,RT-PCR,ALP staining,ALP activity detection,Alizarin Red S staining,and Oil Red O staining.The PI3K signaling pathway plays an important role in the process of cell differentiation.Thus,Western blotting was used to compare the expression changes of related proteins(AKT,p-AKT,GSK3β,p-GSK3β)in the PI3K signaling pathway under different intervention conditions in BMSCs.We then tested whether CB2 inhibitors could reverse the regulation of MJN110 on PI3K signaling pathway related proteins.Finally,we added different concentrations of PI3K inhibitor LY294002 to the medium containing both DEX and MJN110.Through a series of vitro experiments,we observed whether LY294002 could reverse the therapeutic effect of MJN110 on BSMCs.LPS and MP were used to establish a GC-induced SD rat ONFH model.SD rats were randomized into five groups(n=8):(1)DMSO only(control group);(2)MP and LPS(model group);(3)model group rats pre-treated with MJN110(10 mg/kg per day,i.p.injection)(treatment group).Through Micro-CT analysis and H&E staining,the morphological changes of femoral heads in each group were analyzed.Immunofluorescence was used to analyze the effect of MAGL inhibition on the expressions of osteogenic and adipogenesisrelated proteins in bone tissue,as well as the level of bone formation and fat content in the tissue.TRAP staining was used to detect the number of osteoclasts in the femoral head of each group.In vitro cell TRAP test was used to detect the effect of MAGL inhibition on the formation of osteoclasts.Then through Western blotting,and immunohistochemical staining to verify whether MAGL could regulate the expressions of RANKL and OPG in BMSCs and promote GCs-induced ONFH by regulating bone resorption.Result 1:ALP staining showed that,there were fewer ALP-positive cells and the lower the activity of ALP with increasing concentrations of DEX.Alizarin red staining also showed that the number of calcium nodules was negatively correlated with the concentration of DEX intervention.On the contrary,the number of fat droplets in the cell would significantly increase.Western blotting and RT-PCR experiments further confirmed that the expression levels of osteogenic differentiation related indicators(ALP,RUNX2 and OCN)would decrease with increasing concentrations of DEX,while the expressions of adipogenic differentiation related indicators(PPARγ,CEBPβ and LPL)showed an opposite trend.We also found a significant positive correlation between the level of MAGL expression and the concentration of DEX intervention.Additionally,bone tissues immunofluorescence and cells immunofluorescence experiment results confirmed again the changes in the expression of the above osteogenic and adipogenic related indicators.Result 2:MAGL inhibitor(MJN110)was used to inhibit the expression of MAGL in BMSCs.Then BMSCs were induced to differentiate toward osteogenic lineage and adipogenic lineage.Western blotting and PCR analysis results showed that,compared with DMSO-treated BMSCs,the expression levels of osteogenic markers were significantly increased in MJN110 group,while the expression level of adipogenic markers were significantly reduced.In addition,the ALP staining positive cells in the MJN110 treatment group were more than control group,the ALP activity was also stronger.Alizarin Red S staining showed that,after MJN110 intervention,the more calcium nodules were observed,while Oil Red O staining revealed that the fat droplets significantly decreased.Next,BMSCs were treated with MAGL overexpression plasmid and we observed opposite results compared to MJN110 intervention.We next investigated whether MAGL blockade can reverse the effect of GCs on the differentiation of BMSCs.The results of Western blotting and PCR showed that compared with the DEX group,in the DEX+MJN110 treatment group,the expression of osteogenic related proteins significantly increased,at the same time,the number of ALP staining positive cells were more,the ALP activity was stronger,and the calcium nodules increased.Contrarily,the expression levels of adipogenic markers were significantly reduced,and the number of fat droplets was less then DEX group.Additionally,coadministration of DEX and 2 AG achieved similar effects as MJN110 intervention.MicroCT image results showed that after MJN110 treatment,the subchondral trabecular bone was effectively repaired and arranged in an orderly manner in the femoral head.Further bone parameter analysis confirmed the imaging findings.Compared with the model group,the BV,BV/TV,and Tb.Th values were significantly increased in the treatment group,while the Tb.Sp value was decreased.H&E staining showed that,after MJN110 treatment,the epiphyses became wider,bone formation was increased and the lipid droplets were reduced.In addition,immunofluorescence staining further confirmed that MAGL inhibition could reversed the effect of GCs on the differentiation of BMSCs,thereby partially promoted GCsinduced femoral head necrosis.Result 3:After the corresponding siRNA and inhibitors of CB1 and CB2 treatment,PCR analysis showed that under separate intervention,CB1 inhibitor treatment or siCB1 transfection could enhance osteogenic differentiation,but weaken adipogenic differentiation,while CB2 Blocking showed the opposite results.When both MAGL and CB1 were simultaneously blocked,the enhancement in osteogenic differentiation and the induction adipogenic differentiation were both greater than either intervention given alone.When both MAGL and CB2 were simultaneously blocked,the blockade of CB2 reversed the therapeutic effect of MJN110.The in vivo experiment results were consistent with the above in vitro results.Result 4:Through Western blot analysis,we suggested that the PI3K pathway-related proteins expression was inhibited by the treatment of DEX.The administration of MJN110 could activate the PI3K signaling pathway.However,the activation of PI3K pathway-related proteins with MJN110 was reversed by the CB2 inhibitor AM630.Next,LY294002 was used to inhibit the expression of PI3K in BMSCs.Western blotting results showed that LY294002 could partially up-regulate the decrease in the expression of adipogenic markers in a dosedependent manner.On the contrary,the expression level of osteogenic markers showed a downward trend with increasing LY294002 concentrations.In addition,we also observed that,after adding LY294002 to the medium containing MJN110 and DEX,the number of ALP positive cells and the activities of ALP were significantly reduced but fat droplets were significantly increased.These data confirm that MAGL inhibition regulates the differentiation of BMSCs and interferes with GCs by activating the PI3K/AKT/p-GSK3 βsignaling pathway.Result 5:Through tissue TRAP staining,we found that MJN110 treatment reduced the number of osteoclasts in the femoral head.The cells TRAP staining experiments also confirmed that,compared with the DEX group,the administration of MJN110 could indeed reduce the number of osteoclasts.We further tested the expression levels of RANKL and OPG in BMSCs under different culture conditions.The results showed that DEX significantly reduced the expression of OPG,but increased the expression of RANKL,which directly leaded to an increase in the ratio of RANKL/OPG.The results of immunohistochemistry also confirmed that the expression of RANKL was increased in the model group.The MJN110 treatment reversed this trend,which was consistent with the results of TRAP staining.These data indicate that,in the GCs-induced ONFH model,MAGL blockades can improve bone resorption caused by GCs by inhibiting osteoclastogenesis both directly and indirectly by acting on osteoclasts.Conclusion:MAGL inhibition can not only enhance BMSCs osteogenic differentiation and inhibit adipogenic differentiation,but also effectively reverse the effect of GCs on BMSCs differentiation by activating CB2.The activation of the PI3K/AKT/GSK3βsignaling pathway plays an important role in the biology progression.It is worth noting that,MAGL blockade not only promotes bone formation,but also weakens the bone resorption induced by GCs,and ultimately reduces GCs-induced ONFH.