Effects Of Aspirin/Glucocorticoids On Mesenchymal Stem Cells Apotosis And Related Pathway

Mesenchymal stem cells(MSCs) are multiopotential progenitor cells and have been used for regenerative medicine.However,their therapeutic potential remains limited by the marked loss through cell death of transplanted MSCs.Thus identifying factors that cause MSC death and understanding the mechanisms through which such actions may be mediated would have a significant therapeutic impact in promoting the use of MSC based therapy for regenerative medicine.Our previous studies demonstrated hypoxia/serum deprivation,both component of the ischemic microenvironment within the myocardium,induced MSCs apoptosis.In further research,we demonstrated that LPA and lovastatin protect MSCs from hypoxia/ischemia-induced apoptosis,but aspirin,a widely used anti-inflammatory drug,inhibits MSCs proliferation.Objective:Here we have aimed to elucidate the effects of aspirin and hydrocortisone, two anti-inflammation drugs,on the apoptosis of bone marrow-derived mesenchymal stem cells and the role of correlated signal-pathways in the above actions.Original Points:The effect of aspirin on the apoptosis of MSCs and role of Wnt/β-catenin pathway in aspirin's action1.Whether aspirin induces apoptosis in MSCs.2.Whether mitochrondrial/caspase-3 pathway and Wnt/β-catenin pathway change during aspirin-induced MSC apoptosis.3.Whether Wnt/β-catenin pathway mediates aspirin-induced MSC apoptosis. The effect of hydrocortisone on the hypoxia and serum deprivation(hypoxia/SD) induced MSC apoptosis and the involvement of ERK1/2 and Akt signal-pathways.1.Whether hydrocortisone reverses hypoxia/SD induced apoptosis in MSCs.2.Whether ERK1/2 and Akt involve in anti-apoptosis effect of hydrocortisone in MSCs under hypoxia/SD condition.3.Whether hydrocortisone inhibites the inflammatory factors in MSC under hypoxia/SD condition and to investigate the relationship of its anti-inflammation and anti-apoptosis effects.Material and Methods:The apoptosis of MSCs was assessed by Hoechst 33342 and Annexin V-FITC/PI Apoptosis Kit.Expression of protein and protein phosphorylation were assessed by Western blot analysis.Expression of mRNA was detected by Real-time Quantitative PCR.Caspase-3 activity was detected by using the Caspase-3/CPP32 Colorimetric Assay Kit.Results:Aspirin induced apoptotic morphological changes,cytochrome c release from the mitochondria,and caspase-3 activation in MSCs.Aspirin increased GSK-3βactivity by decreasing its phosphorylation on ser⁹ residue,accompanied by an increase in phosphorylatedβ-catenin and down-regulation of the expression of cyclin D1, which suggested aspirin inhibited Wnt/β-catenin pathway in MSCs.Stimulating Wnt/β-catenin pathway by both Wnt 3a and GSK-3βinhibitors(LiCl and SB 216763), blocked aspirin-induced apoptosis and protected mitochondrial function,as demonstrated by decreased cytochrome c release and caspase-3 activity.Aspirin initially caused a time dependent decrease in COX-2 expression but subsequently,and unexpectedly,elevated the latter.The stimulation of COX-2 expression by aspirin was further enhanced following stimulation of the Wnt/β-catenin pathway.Administration of the COX-2 inhibitor NS-398,suppressed elevated COX-2 expression and promoted aspirin-induced apoptosis.These results demonstrated that aspirin induced MSCs apoptosis occurs through the activation of the mitochondria/caspase-3 proapoptotic pathway,and this in turn appears to be dependent on Wnt/β-catenin inhibition by aspirin.In addition,the parallel enhancement of cyclin D1 and COX-2 expression by agonists of the Wnt pathway,that also protect against aspirin-induced apoptosis of MSCs.In the condition of hypoxia and serum deprivation,a significant apoptosis occurred in MSCs.Hydrocortisone,a natural glucocorticoids,significantly decreased the apoptosis and caspase-3 activation induced by hypoxia/SD.The effect of hydrocortisone on the intracellular signal pathways was also investigated. Hydrocortisone increased ERK1/2 and Akt activity by increasing these phosphorylation,while administration of the ERK1/2 inhibitor U0126 and PI3K inhibitor Wortmanin reversed the inhibiting action of hydrocortisone on caspase-3 activation under hypoxia/SD condition.In addition,hydrocortisone dose-dependently inhibited the mRNA expression of inflammatory factor IL-1βand TNF-α,but 10^-10mol/L(weak inhibition effect on inflammatory factors expression) and 10^-6 mol/L(strong inhibition effect on inflammatory factors expression) have similar anti-apoptotic effect on MSCs.These results demonstrated that hydrocortisone protects MSCs from hypoxia/SD-induced apoptosis which was involved in caspase-3 activation.Both ERK1/2 and Akt are involved in the anti-apoptosis effect of hydrocortisone under hypoxia/SD conditions.Moreover,its anti-inflammation effect seems not to be related to its anti-apoptotic effect in our experiment.Conclusion:These results demonstrate that Wnt/β-catenin pathway is a key modulator of aspirin-induced apoptosis in MSCs via regulation of mitochrondrial/caspase-3 function,while ERK1/2 and Akt are involved in the protection of hydrocortisone in hypoxia/SD-induced apoptosis.More importantly,our findings suggest that aspirin may have the influence on MSC survival under certain conditions and should therefore be used with caution when considering MSCs transplantation for regenerative medicine in patients with concomitant chronic inflammatory diseases such as arthritis.Glucocorticoids could be a potential drug for replacement therapy.