MC-LR Regulates ERs And Autophagy In Mouse Ovarian Cells Via Oxidative Stress

ObjectiveMicrocystin-leucine arginine（MC-LR）is a cyclic heptapeptide intracellular toxin released by cyanobacteria that exhibits strong reproductive toxicity.However,little is known about its biotoxicity to the female reproductive system.Oxidative stress appears to play crucial roles in ERs and autophagy.However,no previous studies have combined ERs and autophagy by ROS mediated in MC-LR induced reproductive toxicity.Therefore,the aim of the present study is to investigate oxidative stress level and antioxidant ability in vitro and in vivo following treatment with MC-LR or N-acetyl-l-cysteine（NAC）.Furthermore,the study also analyzes unexplored molecular pathways by which oxidative stress acts on MC-LR-induced endoplasmic reticulum stress（ERs）and autophagy,and reveals the molecular mechanisms of the protective effects of NAC on MC-LR-induced reproductive toxicity.MethodsDetection of cell viability of murine ovarian granular cells（KK-1 cells）:KK-1cells were treated with MC-LR at final concentrations of 0,1,5,10,20,40 and 60μg/mL,or with NAC at final concentrations of 0,1,5,10,15,20 and 40 mM for 24hours.Then,the cell viability was calculated,and the half maximal inhibitory concentration IC₅₀ of MC-LR or the subsequent experimental concentration of NAC was determined via CCK-8 kit and microplate reader.KK-1 cells were exposed to various concentrations of MC-LR（0,8.5,17 and 34μg/mL）with or without NAC（10 mM）.After incubation for 24 hours,ROS content,SOD activity,cell apoptotic rate and GSH/GSSG were detected;Western Blotting was used to detect the accumulation of MC-LR and proteins expressions of endoplasmic reticulum stress and autophagy.C57BL/6 mice were exposed to various doses of MC-LR（0,12.5,25,40μg/kg·BW）with or without NAC（200 mg/kg·BW）.After exposed to 14 days,MDA content,ROS content,SOD activity and GSH/GSSG were detected;the morphological changes of mice ovaries were observed under microscope with hematoxylin and eosin staining;the ultrastructure of ovarian granulosa cells was observed using a transmission electron microscope;detection of apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labelling（TUNEL）method;Western Blotting was used to detect the accumulation of MC-LR and proteins expressions of endoplasmic reticulum stress and autophagy.ResultsThe IC₅₀ of MC-LR was calculated to be 34μg/mL after KK-1 cells exposed to MC-LR for 24 h.The cell viability after treatment with 10 mM of NAC was the highest,therefore,10 mM NAC was used for subsequent experiments.Results of Western Blotting showed that different concentrations of MC-LR entered KK-1 cells and C57BL/6 mice ovarian tissues.The bands of MC-LR gradually deepened with the increase of MC-LR concentrations.MC-LR induced the production of ROS in KK-1 cells and reduced the antioxidant capacity of KK-1 cells with the decrease of SOD activity and GSH/GSSG.However,the antioxidant NAC could alleviate the oxidative stress caused by MC-LR and enhance the antioxidant capacity;MC-LR could induce oxidative stress in C57BL/6 mice ovarian tissues via increasing levels of ROS and MDA and reducing levels of SOD and GSH/GSSG,while NAC pretreatment could protect against MC-LR-induced oxidative stress and enhance GSH/GSSG.However,a slight up-regulation in SOD activity was observed during the NAC pretreatment,but there was no significant difference when compared to the MC-LR（40μg/kg·BW）group.MC-LR induced C57BL/6 mice ovarian tissues pathological damage and ultrastructural damage.Pretreatment of NAC,an oxidative stress inhibitor,alleviated the pathological damage of mice ovarian tissues and ultrastructural damage of ovarian granulosa cells induced by MC-LR.MC-LR increased the relative expressions of endoplasmic reticulum stress-related proteins and autophagy-related proteins in KK-1 cells and C57BL/6mice ovarian tissues.In vitro,NAC pretreatment significantly decreased the expressions of endoplasmic reticulum stress-related proteins CHOP,XBP1,P-EIF2α,GRP78,P-PERK and autophagy-related proteins Beclin1,ATG12,ATG5-ATG12,ATG16 and LC3II/LC3I,when compared to 34μg/mL MC-LR group（P<0.05）;in vivo,the expressions of endoplasmic reticulum stress-related proteins and autophagy-related proteins Beclin1,ATG5-ATG12 and ATG16 were decreased significantly after pretreatment with NAC,when compared to 40μg/kg·BW MC-LR group（P<0.05）.NAC pretreatment significantly decreased the expressions of endoplasmic reticulum stress-related proteins and autophagy-related proteins.The expression of ovarian granulosa cells apoptotic（TUNEL-positive）was significantly increased in MC-LR group compared to control group.However,mice pretreated with NAC followed by MC-LR injection dramatically alleviated the cell apoptosis via inhibition of oxidative stress.Conclusion1.MC-LR induces oxidative stress in KK-1 cells and C57BL/6 mice ovaries.2.Oxidative stress mediates endoplasmic reticulum stress and autophagy induced by MC-LR in KK-1 cells and C57BL/6 mice ovaries.