Melatonin Reduces Oxidative Damage In Mouse Granulosa Cells Via Restraining JNK-dependent Autophagy

Oxidative stress-induced granulosa cell(GCs)injury is believed to be a common trigger for mammalian follicular atresia.Emerging evidence indicates that excessive autophagy occurs in mammalian cells with oxidative damage.Recent studies have shown that activation of c-Jun N-terminal protein kinase(JNK)can promote the expression of BECN1,a key protein of autophagy,and inhibit the binding of BCL-2 with BECN1,which then leading to autophagy.N-acetyl-5-methoxytrypamine(melatonin)is a kind of antioxidant which have a large number in follicles.Studies have shown that MT can effectively inhibit the level of GCs death caused by oxidative stress.However,its mechanism still remains unknown.It has been confirmed that MT can inhibit the activity both of JNK and autophagy.Therefore,MT-JNK pathway might be an important way to control the viability of granulosa cells under oxidative damage.To verify this hypothesis,we explored the specific mechanism of how melatonin regulated GC in oxidative stress,focusing on JNK and autophagy.The research mainly include the following aspects:(1)To determined the relationship between the inhibition of MT on cell viability and JNK activity by detecting the cell viability assay and JNK activity;(2)Autophagy detection by Western Blotting and the accumulation of GFP-MAP1LC3B spots to make clear the regulation of MT on GC antophagy;(3)To elucidated the relationship between cell autophagy and the resistance of MT on oxidative damage by blocking cell autophagy after MT pretreatment during oxidative stress;(4)By determining the expression and combination of BCL-2 and BECN1,the downstream factors of JNK,to further clarify the molecular mechanism of how MT-JNK pathway regulating autophagic cell death on granulosa cells.The results are as follows:1.MT counteracted oxidative injury in mouse GCs via inhibiting the JNK signaling.Using an established invitro model for the induction of oxidative damage in GCs,we found that we found that H2O2 significantly decreased cell survival in a dose-dependent manner,10 μM MT for 24 h,however,exhibited an apparent resistance to H2O2-induced cell death.Moreover,GCs pretreated with MT showed an abolishment on the activation of JNK.However,MT could not further restore the viability of cells pretreated with the JNK inhibitor,suggesting that melatonin may inhibit oxidative damage through the JNK pathway.2.The suppression of JNK by MT protects GCs from oxidative stress-induced autophagic deathImmunoblotting results showed that treatment of MT markedly reduced the accumulation of MAP1LC3B-II,Accordingly,if was accompanied by compromised degradation of SQSTM1.By monitoring GCs transfected with GFP-MAP1LC3B expressing vector,we found that both MT and SP600125 blocked the formation of autophagic punta upon oxidative stress.Moreover,no additional decline in autophagosome formation was observed in MT-pretreated cells despite JNK inhibitor administration.Therefore,our data suggested that MT provides a preventive effect on oxidative stress-induced autophagic flux by antagonizing the JNK signaling.To investigate whether the suppression of JNK-dependent autophagy is responsible for MT-mediated GC protection upon oxidative stress,cells were treated with MT,SP600125 or the autophagy inhibitor 3-methyladenine(3-MA)prior to H2O2 exposure.Results showed that the cell viability loss during oxidative stress was reversed by the inhibition of autophagy and/or JNK.In accordance with this,MT displayed similar level of suppression in H2O2-induced cell death.Moreover,SP600125 and/or 3-MA did not significanly change cell viability in GCs treated with MT.In particular,the inhibition of autophagy failed to further improve GC survival following the co-treatment of MT and SP600125.Therefore,our data suggested that MT provides a preventive effect on oxidative stress-induced autophagic GC death by antagonizing the JNK signaling.3.MT inhibits JNK-dependent upregulation of BECN1 expression during autophagy(1)MT inhibits JNK-dependent upregulation of BECN1 expression during autophagyAs a key protein of autophagy,the expression of BECN1 reflect the formation of autophagosomes.Then we examined the expression of BCL-2 and BECN1,both of which belong to the downstream effectors of the JNK signaling.As a result,both qRT-PCR analysis and Western blotting results showed that BECN1 were significantly increased upon oxidative stimulation,whereas both MT and SP600125(JNK inhibitor)counteracted H2O2-induced transcriptional activation of BECN1.Notably,SP60025 provided no additional inhibitory effects on BECN1 expression in GCs pretreated with MT,indicating a potential downregulation of BECN1 by MT through the JNK pathway.However,no evident induction of BCL-2 was observed under the same conditions.Nevertheless,these data suggested the possibility that the MT-JNK signaling might regulate autophagy with a BECN1-dependent manner.(2)MT-mediated JNK inhibition facilitates the formation of BCL-2/BECN1 compleximmunoprecipitation experiments showed that both MT and JNK inhibitors significantly inhibited the dissociation of BCL-2 from BECN1 induced by H2O2 exposure.Importantly,the JNK inhibitor failed to further improve BECN1/BCL-2 interaction in cells pretreated with MT.Therefore,the formation of BCL-2/BECN1 complex might be an essential step in MT-mediated suppression of autophagy through the JNK signaling pathway.4.The protective effect of MT on GC survival by inhibiting JNK-induced autophagy is independent of ROS scavengingGCs were treated with specific antagonists targeted to the downstream antioxidants(hereafter referred as antioxidant inhibitors,or AOI)of MT.The results showed that AOI abolished MT-mediated inhibition of oxidative stress despite SP600125 administration.Moreover,AOI exerts no evident influence on MT-induced suppression of autophagosome formation and autophagic GC death in cells with H2O2 exposure.Collectively,these data further confirmed that the MT-JNK signaling directly inhibits autophagic death in GCs suffering oxidative damage.Taken together,our findings uncover a novel function of melatonin in preventing GCs from oxidative damage by targeting JNK-mediated autophagy,which might contribute to develop therapeutic strategies for patients with ovulation failure-related disorders.