The Mechanism Of The Anti-senescence Effect Of Melatonin Through Endoplasmic Reticulum Stress On Canine Adipose-derived Mesenchymal Stem Cells

Adipose-derived Mesenchymal Stem Cells?ADSCs?is a kind of adult stem cell with multiple differentiation potentials.ADSCs transplantation showed good therapeutic effect in severe traumatic diseases,autoimmune diseases,metabolic diseases and degenerative diseases.Furthermore,because of its easy access,no tumorigenicity,no ethical dispute,and less harmful for donor animals,which makes it a hotspot and an ideal seed cell of stem cell therapy research.In veterinary clinical,canine ADSCs transplantation also has a wide application prospect.However,the inevitable senescence of ADSCs caused by the abundant cultivation in vitro will restrict their survival rate and the curative effect.Melatonin is reported to exert biorhythm regulation,anti-oxidation,and anti-senescence effects in various animal and cell models.Therefore,in this study,by using senescent cADSCs after multiple generations in vitro as the cell model,we investigated the effect and mechanism of melatonin on ADSCs senescence.The obtained results were as follows:1.Serial subcultivation in vitro could cause cADSCs senescence.And,melatonin could slow the senescence by reducing ERS in cADSCs.Senescence phenotype and ERS level of c ADSCs were tested by senescence-associated?-galactosidase staining,in vitro inducing differentiation tests,telomerase activity analyse,relative telomerase length analyse,real-time PCR and western blot and other detection methods.The results showed that cADSCs had notable increased senescence phenotype,such as increased positive rate of senescence-associated?-galactosidase staining,decreased telomerase activity,decreased differentiation potential,increased P16 protein expression level and increased ERS at around P11 passage.The senescence phenotype and ERS of cADSCs was alleviated by adding melatonin in vitro culture system.Moreover,ERS inhibitor 4-PBA could inhibit c ADSCs senescence phenotype,while ERS activator TM promoted cADSCs senescence phenotype.These results showed that melatonin was able to alleviate cADSCs senescence by inhibiting ERS.2.Melatonin could activate the rhythmic expression of Nrf2 gene in c ADSCs.With qRT-PCR detection,expression of circadian clock genes Per2 and Bmal1,anti-senescence related gene Nrf2 and ERS related gene Grp78 were found losing of rhythmicity in senescent cADSCs in vitro.By adding melatonin to the culture system,rhythm expression of Bmal1,Nrf2 and Grp78 genes could be activated.However,by adding MT1/MT2 melatonin receptor inhibitor luzindole to the culture system,the mRNA expression levels and rhythm expression amplitude of Bmal1 and Nrf2 genes were decreased.It is suggested that melatonin may regulate the rhythmical expression of the circadian clock genes and Nrf2 via the MT1/MT2 receptor pathway.3.Melatonin could activate the Nrf2 and ER-associated degradation?ERAD?of cADSCs.With qRT-PCR,biluciferase activity detection,immunohistochemistry and western blot detection,we found that melatonin could promote Nrf2 gene expression,nuclear translocation and activate the ERAD pathway.Moreover,by treating cADSCs with MT1/MT2 inhibitor Luzindole and activator Ramelteon,we found Luzindole inhibited,while Ramelteon activated the activation of Nrf2.Nrf2 knock down blocked the activation of ERAD related genes expression in melatonin treated cADSCs.Treatment of ERAD inhibitors MG132 and NMS-873 blocked the inhibitory effects on ERS in melatonin-treated cADSCs.These results proved that melatonin can activate Nrf2 through the MT1/MT2 receptor pathway,activate the ERAD pathway,and inhibit ERS.4.Melatonin could activate the Nrf2,inhibit the NF-?B pathway and senescence-associated secretory phenotype?SASP?in cADSCs.With qRT-PCR,biluciferase activity detection and western blot detection,we found that melatonin and Nrf2 activator Oltipraz could inhibit the the NF-?B pathway and SASP,while Luzindole treatment could block the inhibitory effect of melatonin treatment.Furthermore,Nrf2 knock down blocked the inhibitory effect of melatonin on the NF-?B signaling pathway and SASP.These results proved that melatonin can inhibit the NF-?B signaling pathway and SASP by activating Nrf2 through MT1/MT2receptor pathway.5.Melatonin pretreatment could improve the curative effect of cADSCs transplantation for canine acute liver injury.The canine acute liver injury model was constructed by intraperitoneal injection of CCl₄,and melatonin pretreated or untreated cADSCs were transplanted respectively.It was found that the survival rate of cADSCs and curative effect was better in melatonin pretreated transplantation group than untreated cADSCs transplantation group,by food and water intake,serum biochemistry,tissue frozen section and paraffin section HE staining detection and other detection methods.In conclusion,in this study,the senescence model of c ADSCs in vitro was established,and the anti-senescence mechanism of melatonin for cADSCs was explored.We proved that melatonin could activate Nrf2 through MT1/MT2 receptor pathway,activate ERAD,inhibit NF-?B,inhibit ERS,and alleviate cADSCs senescence.Furthermore,the anti-senescence effects of melatonin were verified by cADSCs transplantation in canine acute liver injury model.This study provided theoretical foundation and technical reference for the veterinary clinical application of cADSCs.