Effects Of MTOR-dependent Or Independent Autophagy On Apoptosis In Rat Cerebral Cortex Induced By Methylmercury

Objective: To study the regulation of mTOR and Vps34-Beclin1 complex pathway on autophagy in cerebral cortex neurotoxicity induced by MeHg,explore the effects of autophagy on apoptosis,and provide experimental basis for further studies of the mechanism in MeHg neurotoxicity.Methods: The whole animal experiment was used in this study.Firstly,32 Wistar rats were divided into 4 groups: control group,4,8,or 12 μmol/kg MeHg group.The rats in control group were intragastrically administrated with saline,and rats in MeHg group were intragastrically administrated with 4,8,or 12 μmol/kg MeHg,respectively.Once a day for 4 weeks.After 24 hours of the last exposure,4 rats in each group were anesthetized and fixed by left ventricular perfusion to observe the pathological damage of rat cerebral cortex by HE staining.The other 4 rats in each group were used to undergo the following experiments: the ultra-structure changes were observed by transmission electron microscope,the autophagy fluorescence intensity was detected by MDC staining,the apoptosis rate was detected by flow cytometry,or the proteins expression level of Beclin1,p62,LC3,Bax,Bcl-2 and cleaved Caspase-3 were detected by Western blotting.On this basis,mTOR inhibitor Rapa or Vps34 inhibitor 3-MA were used to explore the effects of autophagy on apoptosis in MeHg-induced neurotoxicity.Twenty-four Wistar rats were divided into 6 groups.The first group was control group: saline was given intragastrically after subcutaneous injection of saline for 2 hours,the second group was Rapa group: saline was given intragastrically after subcutaneous injection of 1 mg/kg Rapa for 2 hours,the third group was 3-MA group: saline was given intragastrically after subcutaneous injection of 10 mg/kg 3-MA for 2 hours,the fourth group was MeHg group:12 μmol/kg MeHg was given intragastrically after subcutaneous injection of saline for 2hours,the fifth group was Rapa pretreatment group: 12 μmol/kg MeHg was given intragastrically after subcutaneous injection of 1 mg/kg 3-MA for 2 hours,or the sixth group was 3-MA pretreatment group,12 μmol/kg MeHg was given intragastrically after subcutaneous injection of 10 mg/kg 3-MA for 2 hours.The rats were exposed once a day and pretreated every other day for 4 weeks.After 24 hours of the last exposure,the autophagy fluorescence intensity was detected by MDC staining,and the autophagy and apoptosis-related indicators were detected by flow cytometry,RT-q PCR,or Western blotting method.Results: 1.MeHg induces autophagy and apoptosis in rat cerebral cortex,and with the increase of MeHg concentration,the levels of autophagy and apoptosis gradually increase.HE staining found that as the concentration of MeHg increased,pathological damage of cerebral cortex gradually increased.Under transmission electron microscope,it was observed that MeHg group tended to blur the boundary of cell membrane,incomplete nuclear membrane,severe damage of mitochondria and more autophagosomes.The fluorescence intensity of autophagy increased significantly after exposure to MeHg,which was consistent with the change of Beclin1 and LC3,while p62 decreased gradually.In addition,the apoptotic rate gradually increased after MeHg exposure.MeHg increased Bax and cleaved Caspase-3,or inhibited Bcl-2.2.The m RNA and protein of mTOR or Vps34-Beclin1 complex pathway related factors are involved in the regulation of MeHg neurotoxicity.Promoting autophagy can reduce the level of apoptosis in rat cerebral cortex.On the contrary,inhibition of autophagy can increase the level of apoptosis in rat cerebral cortex.The results of MDC fluorescence intensity and flow cytometry showed that MeHg enhanced the autophagy fluorescence intensity and apoptosis rate.Compared with 12 μmol/kg MeHg group,3-MA pretreatment reduced the fluorescence intensity of autophagy and increased the level of apoptosis.Rapa pretreatment enhanced autophagy fluorescence intensity and inhibited apoptosis.In addition,the m RNA and protein expressions of mTOR and Bcl-2 were inhibited by MeHg,while Vps34 and Beclin1 were enhanced by MeHg.In addition,MeHg increased the protein expressions of autophagy and apoptosis-related factors such as LC3,cleaved Caspase-3 and MAPKs(Erk,p38,or JNK).Compared with the 12 μmol/kg MeHg group,Rapa pretreatment increased autophagy and reduced apoptosis,as shown in the following:compared with the 12 μmol/kg MeHg group,the Rapa pretreatment decreased the m RNA and protein of mTOR and Bcl-2,increased the protein expressions level of autophagy-related factors such as LC3,and decreased the proteins of apoptosis-related factors such as S6K1,4EBP1 and MAPKs(Erk,p38,or JNK).The 3-MA pretreatment group inhibited autophagy and increased the level of apoptosis at the same time.Compared with the 12 μmol/kg MeHg group,the 3-MA pretreatment inhibited m RNA and protein of autophagy and apoptosis-related factors such as Vps34 and Bcl-2,and increased the proteins expression of Bax,cleaved Caspase-3 and JNK.Conclusions: 1.MeHg can induce autophagy and apoptosis in rat cerebral cortex.2.MeHg can induce autophagy through inhibiting mTOR pathway,and Rapa may alleviate apoptosis by further inhibiting mTOR.3.MeHg can induce autophagy through activating the Vps34-Beclin1 complex pathway,and 3-MA may inhibit autophagy and aggravate apoptosis by Vps34 inhibition.