| Part 1Objective:To study the effects of sodium palmitate（PA）,high glucose（HG）and sodium palmitate combined with high glucose on fibrosis of human glomerullar mesangial cells（HMCs）and its mechanism.The effects of oxidative stress and phosphorylation of MAPK pathway on HMCs cell fibrosis were investigated.Methods:HMCs cells were grouped as follows :（1）cells were treated with different concentrations of PA（40,80,160,320 μmol/L）for 24 h,and the control group was set up;Lipid deposition in cells was observed by oil red staining.H2 DCFDA was used to detect intracellular ROS expression.（2）Divided into control group,PA,HG and PA+HG groups.In addition to oil red staining and ROS,living cell imaging was used to detect changes in cell morphology.The expression of Col4 protein was observed by immunofluorescence assay.The expression of Col4,TGF-β and Fn was detected by PCR.Western blot was used to determine the protein expressions of p22 phox,p47phox,NOX4,ERK,p-ERK,JNK,p-JNK,P38,p-P38,TGF-β and FN in HMCs cells.Results:Oil red staining showed that the lipid deposition in the cells increased with the increase of PA concentration（40,80,160,320μmol/L）compared with the control group.Reactive oxygen species（ROS）results showed that compared with the control group,increased PA concentration（40,80,160,320 μmol/L）significantly increased ROS levels in cells.The living cell imaging results showed that compared with the control group,the morphology of HMCs cells could be changed in PA,HG and PA+HG groups.Compared with the control group,no obvious lipid deposition was found in HG group,but increased in PA and PA+HG groups.Compared with PA group,lipid deposition was increased in PA+HG group,but the difference was not statistically significant.ROS results showed that compared with the control group,ROS levels in PA,HG and PA+HG groups were significantly increased.Compared with PA group,ROS level in PA+HG group increased,but the difference was not statistically significant.Immunofluorescence showed that compared with the control group,the expression of Col4 protein increased in PA,HG and PA+HG groups.Compared with PA group,Col4 protein expression was increased in PA+HG group.PCR results showed that compared with the control group,the m RNA expressions of TGF-β and FN in PA,HG and PA+HG groups were significantly increased.Compared with PA group,m RNA expression of TGF-β and FN increased in PA+HG group.WB results showed that compared with the control group,the protein expressions of ERK,JNK and P38 were not significantly changed,while the protein expressions of p22 phox,p47phox,NOX4,ERK,p-ERK,JNK,p-JNK,P38,p-P38,TGF-β and FN were significantly increased in PA,HG and PA+HG groups.Compared with PA group,the protein expressions of p22 phox,p-JNK and FN were increased in PA+HG group.Conclusion:Sodium palmitate alone or in combination with high glucose can induce HMCs cell fibrosis,with obvious lipid deposition,increased intracellular ROS production,and increased phosphorylation level of MAPK pathway.However,the synergistic effect of sodium palmitate combined with high glucose is not obvious,indicating the main role of sodium palmitate in HMCs cell fibrosis.Part 2Objective:To investigate the role of oxidative stress and phosphorylation of MAPK pathway in PA-induced HMCs cell fibrosis,and to explore the effect and mechanism of Ginsenoside Rg1（Rg1）in improving HMCs cell fibrosis.Methods:HMCs cells were cultured in vitro and divided into control group,PA group,Rg1（5,10 μmol/L）group and Apocynin（50 μmol/L）group.The changes of cell morphology were detected by living cell imaging.Lipid deposition in cells was observed by oil red staining.Reactive oxygen species（ROS）detection kit was used to detect intracellular ROS expression.Col4 protein expression was detected by immunofluorescence assay.The expression of TGF-β and Fn was detected by PCR.Western blot was used to determine the protein expressions of p22 phox,p47phox,NOX4,ERK,p-ERK,JNK,pJNK,P38,p-P38,TGF-β and FN in HMCs cells.Results:Compared with the control group,Apocynin and Rg1（5 and 10μmol/L）reversed the morphological changes of HMCs cells in the PA group.Apocynin and Rg1（5 and10μmol/L）significantly reduced lipid deposition in HMCs compared with the model group.ROS results showed that Apocynin and Rg1（5 and 10μmol/L）significantly decreased ROS production in HMCs cells compared with the model group.Immunofluorescence results showed that Apocynin and Rg1（5 and 10μmol/L）significantly decreased Col4 expression in HMCs cells compared with the model group.PCR results showed that Apocynin and Rg1（5,10μmol/L）significantly reduced the expression of TGF-β and Fn in HMCs cells compared with the model group.WB results showed that compared with the control group,PA group increased the expression of p22 phox,p47phox,NOX4,ERK,p-ERK,JNK,p-JNK,P38,p-P38,TGF-β and FN proteins,while the expression of ERK,JNK and P38 proteins did not change significantly.Compared with the model group,Apocynin and Rg1（5 and 10μmol/L）significantly reduced the expression of p22 phox,p47phox,NOX4,ERK,p-ERK,JNK,p-JNK,P38,p-P38,TGF-β and FN.Conclusion:Ginsenoside Rg1 may reduce the fibrosis of HMCs cells by reducing the oxidative stress response and the phosphorylation of MAPK pathway related proteins. |
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