Reddy L Vinod Kumar, Sen Dwaipayan
Cellular and Molecular Therapeutics Laboratory, Centre for Biomaterials, Cellular and Molecular Theranostics, Vellore Institute of Technology (VIT) University, Vellore 632014, Tamil Nadu, India,.
Life Sci. 2017 Dec 15;191:195-204. doi: 10.1016/j.lfs.2017.10.024. Epub 2017 Oct 19.
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 (δ)-opioid receptor (DOR) with synthetic peptide [D-Ala, D-Leu]-enkephalin (DADLE) treatment on human MSCs cultured in serum-starved condition.
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-p110γ and cleaved caspase-3 were determined by qPCR and western blot. Levels of secreted cytokines were measured by ELISA.
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 up-regulation of pro-apoptotic Bcl2 (2.1 folds), down-regulations of anti-apoptotic Bax/Bad (2.6/2.7 folds) as well as of cleaved caspase-3, increased expression of PI3kinase subunit p110γ 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-β) under serum deprived condition. LPS stimulated macrophages showed abated release of pro-inflammatory cytokines (IL1/TNFα/IL6) when grown in hMSC conditioned 'serum deprived' media treated with DADLE. Both the cytoprotective and anti-inflammatory effects of DADLE were inhibited by the DOR specific antagonist naltrindole.
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.
营养缺乏和炎症丰富区域是移植间充质干细胞（MSCs）存活率低的主要原因。因此，在本研究中，我们证明了用合成肽[D-丙氨酸，D-亮氨酸] - 脑啡肽（DADLE）激活δ-阿片受体（DOR）对血清饥饿条件下培养的人MSCs的细胞保护和抗炎作用。
使用MTT和Annexin V/PI测定法测量细胞活力。通过qPCR和蛋白质印迹法测定促凋亡基因（Bcl2）和抗凋亡基因（Bax/Bad）的表达、活化的p44/42 MAPK、Akt、PI3-激酶-p110γ和裂解的caspase-3的水平。通过ELISA测量分泌细胞因子的水平。
与对照相比，MTT（71％对45％）和Annexin V/PI测定法（25.9％对3.7％）证实，DADLE在血清剥夺条件下显著提高了细胞存活率。在“血清剥夺”应激下的细胞中，DADLE处理后观察到促凋亡Bcl2显著上调（约2.1倍）、抗凋亡Bax/Bad下调（约2.6/2.7倍）以及裂解的caspase-3，PI3激酶亚基p110γ表达增加和Akt（Ser473）活化。此外，DADLE处理的hMSCs在血清剥夺条件下分泌的抗炎细胞因子（IL10/IL4/TGF-β）水平增加。当在经DADLE处理的hMSC条件“血清剥夺”培养基中生长时，LPS刺激的巨噬细胞显示促炎细胞因子（IL1/TNFα/IL6）的释放减少。DADLE的细胞保护和抗炎作用均被DOR特异性拮抗剂纳曲吲哚抑制。
DOR信号通路改善了“血清剥夺”应激下hMSCs的细胞活力并增强了抗炎作用，这可能在再生医学中具有潜在的治疗益处。
