| Objective:To observe the cell morphology,detect the cell proliferation ability,detect the expression levels of ROS,SOD,MDANLRP3,Caspase-1,IL-18 and IL-1βm RNA,detect the expression level of GSDMD protein,explore the protective effect and molecular mechanism of Ghrelin on hepatocellular injury caused by high glucose,and expand the idea for the prevention and treatment of diabetic liver injury.Methods:AML-12 cells were used to establish a cell model of hepatocyte injury and administer Ghrelin(10-7mmol/L).The experiment was randomly divided into 4groups:blank control group（Con）,blank+Ghrelin group（G-Con）,high glucose group（HG）,and high glucose+Ghrelin group（G-HG）.Cell viability was measured by CCK8;morphological changes of cells in each group by inverted microscope;imagej software measuring cell diameter and area of each group;content of alanine aminotransferase（ALT）;content of malondialdehyde（MDA）and superoxide dismutase（SOD）;ROS measured by DCFH-DA for NLRP 3,Caspase-1,IL-18 and IL-1βm RNA,and GSDMD protein expression by immunofluorescence.Results:Compared with the control group,the cell viability of the HG group decreased with the increase of glucose concentration and the extension of culture time（P<0.05）.With the glucose concentration of 145 mmol/L,the decrease in the 48h group was the most significant.Further experiments were completed at this concentration.With the Ghrelin intervention,Compared with the control group,the cell viability of HG group decreased with time（P<0.05）,while the cell viability of G-HG group increased significantly（P<0.05）.Under optical microscope,Compared with the control group,HG group cells increased,blurred cell membrane edge,and decreased;after Ghrelin intervention,G-HG group cells showed regular polygons,and the number increased.Meanwhile,the changes of cell diameter and area of each group were measured by Imagej software,and the results showed that the average diameter and area of HG group were significantly higher than that of control group（P<0.01）,and the mean diameter and area of G-HG group were significantly lower compared with that of HG group（P<0.01）.Cell ALT results showed that ALT was significantly increased in the HG group（P<0.05）;ALT in the G-HG group（P<0.05）.The results of MDA and SOD showed that the MDA level decreased in the HG group（P<0.05）and in the HG group compared with the HG group（P<0.05）.The total ROS in cells was 3.42-fold in the HG group（P<0.01）and thirty-fold in the G-HG group with the HG group（P<0.01）.RT-q PCR results showed that the expression levels of NLRP 3,Caspase-1,IL-18 and IL-1βm RNA increased significantly in HG group（P<0.05）;after Ghrelin intervention,NLRP 3,Caspase-1,IL-18 and IL-1βm RNA were significantly decreased in G-HG group（P<0.05）.Immunofluorescence showed that the fluorescence intensity of GSDMD protein was higher in HG group than in control group,and the GSDMD protein was decreased after Ghrelin intervention.Conclusion:Ghrelin can reduce hepatocyte damage caused by high glucose,and the mechanism of Ghrelin can reduce hepatocellular damage is achieved by inhibiting oxidative stress,affecting the expression of NLRP3 inflammasome body,and inhibiting the classical pathway of NLRP3-Caspase-1-mediated pyroptosis. |
