The Effects Of Microcystin-LR On Insulin Signaling Pathway Both In HL7702 Cell Line And Mouse Liver

Objective:Microcystins(MCs)are secondary metabolites produced by certain genera of cyanobacteria and are ubiquitous in the waterbody where bloom occurs.The increasingly serious eutrophication leads to frequent bloom,which further leads to the production and release of plenty of Microcystins into water.Microcystins are now big concerns to human health.Many studies have shown that microcystins are closely related to the occurrence of human primary liver cancer and colorectal cancer,and recent studies have suggested that MCLR may be one of the environmental pathogenic factors of diabetes.In our previous study,we found that exposure to different concentrations of MCLR for 24 hours can cause changes in phosphorylation levels of key proteins of insulin pathway in HL7702 and mouse liver tissues.Since the insulin signaling pathway responds quickly to insulin stimulation and has a very complex feedback mechanism,it is very important to explore the time-dependent effects of microcystins on the key proteins in insulin signaling pathway.Therefore,the purpose of this study was to explore the effects of different concentrations of MCLR,under short-term treatment,on insulin signaling related proteins and the characteristics of their action in liver,that would give us a more complete understanding of the toxic effects of microcystins on this field.Research contents:(1)in vitro experiment:HL7702 was used as material to investigate the effects of MCLR at different concentrations and time(30 min,1 h,6 h)on the activity of protein phosphatase 2A(PP2A)and the key signaling molecules of insulin pathway,including Akt,GSK3a,GSK3β and glycogen synthase(GS).(2)in vivo experiments:ICR male mice were used as materials to study the effects of different concentrations of MCLR on liver,pancreas,PP2A activity and key molecules(IRS1,Akt,GSK3a,GSK3β,and GS)of insulin pathway in liver.We used western blot and immunohistochemistry to verify these effects.Through comparative analysis of in vitro and in vivo experiment,we investigate the effects of short-term MCLR treatment on insulin signaling pathway,and further analyzed the possible mode of action in combination with the previous 24 h experimental results.Research methods:The HL7702 cells were exposed to different concentrations of MCLR(0,1,5,10 μM)and the effects of MCLR on the key molecules of insulin signaling pathway are examined by western blot and PP2A activity kit.The ICR male mice were injected(i.p)with different concentrations of MCLR(0,40,80,120 μg/kg)and the effects of MCLR on liver tissue were studied by using western blot,PP2A activity kit,immunohistochemical staining and HE staining.Research results:(1)MCLR treatment inhibited PP2A activity in HL7702 cells at different time points(30 min,1 h and 6 h)in a time and concentration dependent manner.(2)MCLR treatment induced phosphorylation of Akt in HL7702 cells in a concentration-dependent manner at 30 min,1 h,and 6 h exposure timepoint and affected the phosphorylation of GSK3 and GS.(3)ICR male mice were intraperitoneally injected with different concentrations of MCLR and we found that after 2 h exposure,mice in high-dose group(120 μg/kg)began to die while the group injected with 80 and 120 μg/kg MCLR all died after 5 h.(4)HE staining showed that the livers of mice in the high-dose group had obvious hepatocyte degeneration(turbidity and swelling)and liver hemorrhage compared with the control group.However,MCLRtreatment had no effect on pancreas.(5)The activity of PP2A in liver of ICR male mice treated with MCLR for 1 h decreased in a concentration dependent manner.Western blot results show that MCLR induce phosphorylation of Akt at Ser473 and Thr308,phosphorylation of GSK3α and GSK3β,and GS in a copcentration-dependent manner.Furthermore,high-dose group shows decline may be due to the result of cell necrosis and protein degradation itself,Results of phosphorylation of Akt(at Ser473),phosphorylation of GSK3β,and elevated GS protein levels were also verified by immunohistochemistry.(6)Protein extracted from the liver of ICR male mice treated with MCLR for 1 h was detected by immunoblotting.We found that Ser phosphorylation level of IRS 1 was significantly increased and total IRS1 protein level was decreased.Conclusions:(1)In HL7702 cells,short-term treatment of MCLR inhibits PP2A activity in a time-and concentration-deopendent manner.(2)Short-term treatment of MCLR is toxic to hepatocytes and mice liver,the phosphorylation level of Akt is significantly changed,indicating that Akt is affected by MCLR in a very short time,which suggest that it could be a sensitive indicator to detect the early effect of MCLR on insulin signaling pathway in liver.(3)After a short period of exposure,MCLR interferes with the insulin signaling pathway through disrupting the phosphorylation levels of IRS1,Akt,GSK3α,GSK3β and GS in liver.