Study On The Mechanism Of Autophagy And Gap Junctional Intercellular Communication In Cadmium Induced Rat Hepatocyte Injury

Cadmium(Cd),is one of the common pollutants in the environment and causes damage to multiple organs of human and animal.The liver is an important detoxifying organ and is one of the major target organs for cadmium toxicity damage in the body.Autophagy is one of the major pathways for cells to remove damaged organelles and proteins,which is essential for the cell homeostasis.Gap junction intercellular communication(GJIC)is one of the direct communication ways between cells in multicellular organisms.Studies have shown that the autophagy level was increased and the GJIC function was decreased by cadmium,and its internal mechanism has not yet been clarified.In the present study,the rat liver cell line BRL 3 A cells as a research cells.We established the time gradient injury induced by cadmium model.We aim to explore the role of the autophagy and the GJIC in cadmium induced BRL 3A cells injury at different time and the mechanisms of GJIC regulating Cd-induced autophagy.To further clarify the mechanism provide a theoretical basis for the Cd-induced hepatotoxicity.1.Time-effects of cadmium induced BRL 3 A cells injuryTo investigate the toxic damage of cadmium on BRL 3A cells,and according to the result of the effect of 5 ?mol/L cadmium on the BRL 3A cell index obtained by RTCA,the time gradient model of Cd-injured cells was established at different time(0 h,1.5 h,3 h,6 h,12 h and 24 h).The change of cell nucleus morphology and cell ultrastructure were observed using hoechst 33258 staining and the transmission electron microscope respectively.The results showed that the mitochondria was damaged at 1,5 h and the nucleus morphology was changed at 3 h by cadmium compared with the control group.With the extension of time,the injury gradually increased and showed a significant time-effect relationship within 24 hours.2.Cadmium-induced injury and autophagy in BRL 3A cellsTo explore the changes of autophagy level in cadmium induced BRL 3A cells injury,the number of autophagosomes was observed using the transmission electron microscopy,the phenomenon of LC3 puncta were observed using immumofluorescence and the expression of autophagy-related proteins were detected using Western blot.The results showed that the autophagic level of cells increased and then decreased when cells were treated with 5 ?mol/L cadmium.The peak expression of autophagy marker protein LC3 II was seeen at 6 h and the difference was extremely significant(P<0.01)compared with control group.Co-treated with Cd and chloroquine(CQ)autophagy inhibitor,the expression level of P62 and LC3 II were significantly increased compare with CQ-only or Cd-only treatments respectively(P<0.05 or P<0.01).This results showed that cadmium promoted autophagy and meanwhile blocked the autophagy flux.3.Effect of GJIC on cadmium-induced BRL 3 A Cells InjuryTo investigate the changes of GJIC during Cd-induced injury on BRL 3A cells,the GJIC function was detected by scratch tracing method,the distribution and the distribution of connexin Cx43 was observed by immunofluorescence and the expression of GJIC-related protein was detected by Western blot.The results showed that 5 ?mol/L cadmium inhibited the function of GJIC during cadmium exposure,the inhibition was weakened while Cd-exposed for 3 h,the expression of Cx43 protein was increased relatively and the fluorescence spots were enhanced linearly and arranged on the cell membrane.The inhibitory effect on GJIC function was enhanced after Cd-exposed for 6 h,with the decressing of protein Cx43 and p-Cx43 were expression(P<0.01)and the Cx43 fluorescence spots transferred from the cell membrane to the cytoplasm.To further investigate the role of GJIC on Cd-induced hepatotoxicity injury,BRL 3A cells were treated with 5 ?mol/L Cd for 6 h with GJIC inhibitor GA,or GJIC accelerators ATRA,Cx43 inhibitors Dynasore and p-Cx43 inhibitor Ro318220.The results showed that compared with the cadmium only group,co-treatment of Cd and ATRA could significantly increase the diffusion distance of LY(P<0.01)and promote the linear distribution and expression of Cx43 on the cell membrane,but the expression level of Cx43 protein was decreased(P<0.01)and the protein expression level of p-Cx43 was increased(P<0.01).Combined with GA significantly inhibited the diffusion distance of LY(P<0.01)and reduced the distribution and expression of Cx43 on the membrane,the expression levels of Cx43 and p-Cx43 were decreased significantly or extremely significantly(P<0.05 or P<0.01).10 ?mol/L Dynasore could significantly inhibited the diffusion distance of LY(P<0.01),200 nmol/L Ro318220 could significantly inhibited the diffusion distance of LY(P<0.01),the combined treatment with cadmium significantly inhibited the diffusion distance of LY.Western blot results showed that the combination of Dynasore and Cd significantly reduced the expression of Cx43 and p-Cx43(P<0.01)compared with cadmium treatments group,but the combination of Ro318220 and cadmium significantly increased the expression of Cx43(P<0.01)and the level of p-Cx43 protein significantly decreased(P<0.01).In order to clarify the effect of GJIC on cadmium cell damage,we extend the cadmium exposure time to 12 and 24 h.Compared with cadmium treatment group,ATRA could aggravated Cd-induced cell injury,and the GA or Dynasore,Ro318220 could exacerbaied Cd-induced the cell damage,respectively.In summary,GJIC function could inhibited and would be get compensatory enhanced at 3 h by cadmium treatment.Cd-induced inhibition of GJIC is closely related to the distribution and expression of Cx43 on the plasma membrane,but not directly related to the overall expression level of Cx43 and positively correlated with the expression level of p-Cx43.Cell damaged was reduced by prolonged inhibition of GJIC by 5 ?mol/L cadmium.On the contray,it is promoted.4.GJIC Regulated cadmium-induced BRL 3 A Cells AutophagyAfter Cd-induced injury for 6 h,The accelerant and inhibitor of GJIC and Cx were added,the number of autophagosomes was observed using the transmission electron microscopy,the phenomenon of LC3 puncta were observed using immumofluorescence and the expression of autophagy-related proteins were detected using Western blot.The results showed that compared with the cadmium treatment group,the number of autophagosomes and the expression and distribution of LC3 were significantly reduc,ed in ATRA and Cd co-treatment group.The number of autophagosomes and the expression and distribution of LC3 were significantly enhanced in GA and Cd co-treatment group.GJIC accelerant ATRA could significantly inhibited(P<0.01)the expression of Atg7,Beclin-1,P62 and LC3 ? on Cd-induced BRL 3 A cells,inhibitor GA did the opposite.The expression of Beclin-1,P62,and LC3II were significantly increased(P<0.05 or P<0.01)by Cx protein-related inhibitors.These results indicated that the inhibition of GJIC could promoted the expression level of autophagy,and the promotion of GJIC could inhibited the expression level of autophagy.