DADLE enhances viability and anti-inflammatory effect of human MSCs subjected to 'serum free' apoptotic condition in part via the DOR/PI3K/AKT pathway

Aim: Nutritional deprivation and inflammation-rich zones are the major causative reasons for poor survivability of transplanted mesenchymal stem cells (MSCs). Therefore in the present study, we demonstrated the cytoprotective and anti-inflammatory effects of activated delta (d)-opioid receptor (DOR) with synthetic peptide [D-Ala(2), D-Leu(5)]-enkephalin (DADLE) treatment on human MSCs cultured in serum-starved condition. Main methods: Cell viability was measured using MTT and Annexin V/PI assays. Expressions of pro-apoptotic (Bcl2) and anti-apoptotic genes (Bax/Bad), levels of activated p44/42 MAPK, Akt, PI3-kinase-p110 gamma and cleaved caspase-3 were determined by qPCR and western blot. Levels of secreted cytokines were measured by ELISA. Key findings: In comparison to the control, DADLE significantly increased cell survivability under serum deprived condition as confirmed by MTT (71% vs 45%) and Annexin V/PI assays (25.9% vs 3.7%). Significant upregulation of pro-apoptotic Bcl2 (similar to 2.1 folds), down-regulations of anti-apoptotic Bax/Bad (similar to 2.6/2.7 folds) as well as of cleaved caspase-3, increased expression of PI3kinase subunit p110 gamma and activation of Akt (Ser473) were observed following DADLE treatment in cells under 'serum deprivation' stress. In addition, DADLE treated hMSCs secreted increased levels of anti-inflammatory cytokines (IL10/IL4/TGF-beta) under serum deprived condition. LPS stimulated macrophages showed abated release of pro-inflammatory cytokines (IL1/TNF alpha/IL6) when grown in hMSC conditioned 'serum deprived' media treated with DADLE. Both the cytoprotective and antiinflammatory effects of DADLE were inhibited by the DOR specific antagonist naltrindole. Significance: The DOR signaling pathway improved cell viability and enhanced anti-inflammatory effect of hMSCs subjected to ` serum deprivation' stress that could have potential therapeutic benefits in reparative medicine.