Role And Mechanism Of MAPK Signaling Pathway In Inflammatory Response Of Diabetic Cardiomyopathy

Objective:In this study,we established a model of diabetic cardiomyopathy hypertrophy by high glucose and high lipid intervention in rat embryonic cardiomyocytes(H9c2).The MAPK signaling protein was identified to determine its effect on inflammatory responses in diabetic cardiomyopathy(DCM).Methods:The experiment was divided into three parts:(1)H9c2 cells were randomly divided into 6 parallel holes in each group by random number table method: The control group;high glucose and high fat A(glucose 33mmol/L+Sodium palmitate 250μmol/L);high glucose and high fat B(glucose 33mmol/L+Sodium palmitate 500μmol/L);high glucose and high fat C(glucose 33mmol/L+Sodium palmitate 1000μmol/L)at different time(12h,24 h,36h,48h)treatment group.The m RNA expression of Atrial natriuretic peptide(ANP)and α-Skeletal muscle actin(α-SKA)were measured by real-time PCR(RT-PCR);The cells were stained with hematoxylin-eosin staining(HE staining)to observe the effect of high glucose and high fat on hypertrophy of H9c2 cardiomyocytes;The activity of lactate dehydrogenase(LDH)in the cell culture medium was measured to observe the effect of high glucose and high fat on the injury of H9c2 myocardial cells;(2)Combined with the first part of the experiment,the H9c2 cells were randomly divided into 6 parallel holes in each group by random number table method: The control group;high glucose and high fat group(glucose 33mmol/L + sodium palmitate 500μmol/L)treatment for 24 hours.The activation of p-ERK/T-ERK,p-JNK/T-JNK and p-p38/T-p38 was detected by Western Blot,and the relationship between high glucose and high fat and MAPK family signal activation was observed.(3)Combined with the first two experiments,the H9c2 cells were randomly divided into 6 parallel holes in each group by random number table method:The control group;high glucose and high fat group;inhibitor group(SB203580+high glucose and high fat group).Flow cytometry was used to detect the content of Reactive oxygen species(ROS)in each group;The levels of Nitric Oxide(NO)in each group were detected by chemical detection;ELISA method was used to detect the secretion of 3-nitrotyrosine(3-NT),Interlenkin-6(IL-6)and Interlenkin-1β(IL-1β)in each group of cells,to observe the effect of high glucose and high fat on inflammatory pathway of H9c2 cells and the relationship between MAPK and inflammatory response.Results:1.High glucose and high fat treatment resulted in time and concentration-dependent hypertrophy and injury in H9c2 cells.Compared with the control group,after high glucose and high fat treatment,the expression of ANP increased gradually with the increase of concentration and time,it reached its peak in the high glucose and high fat B 36 h treatment group(P<0.01);Compared with the control group,high glucose and high fat promotes the expression of α-SKA,but the expression ofα-SKA in high glucose and high fat B 36 h treatment group reached the highest(P<0.05),which was consistent with the results of ANP;Compared with the control group,the surface area of H9c2 cells was in a time and concentration dependent manner,in the high glucose and high fat B 36 h treatment group,the surface area of the cells were the most significant(P<0.01),but the cell morphology of the high glucose and high fat C 36 h treatment group were abnormal;Meanwhile,the activity of LDH in supernatant of H9c2 cells was in a time and concentration dependent manner(P<0.05).According to this,the cells were treated with high glucose and high fat B group(glucose 33mmol/L + sodium palmitate 500μmol/L)for 36 h as the intervention concentration and time for subsequent experiments.2.The MAPK signaling pathway of H9c2 cells was markedly activated after high glucose and high fat treatment.Compared with the control group,the phosphorylation levels of ERK,JNK and p38 increased significantly in the high glucose and high fat group,and the levels of p-ERK and p-JNK increased by 1.39 times and 1.47 times(P<0.05)respectively,while the activation level of p-p38 increased by 3.34 times(P<0.01).3.High glucose and high lipid significantly induced changes in inflammatory responses and could be inhibited by p38 MAPK inhibitor.Compared with high glucose and high fat group,the inhibitor group(SB203580+high glucose and high fat group)could reduce the content of ROS and the secretion level of NO,3-NT,IL-6 and IL-1βcontent(P<0.05).Conclusion:1.High glucose and high fat can increase the expression of ANP and α-SKA,and increase the surface area of H9c2 cells,and increase the level of LDH in the supernatant of cells,which suggest that high glucose and high fat can induce the damage of H9c2 cells and promote the hypertrophy of cells.2.high glucose and high fat can increase the degree of activation of MAPK family,especially p38,suggesting that the activation of inflammatory signaling pathway in the process of diabetic cardiomyopathy.3.P38 inhibitors could inhibit the increase of ROS,NO,3-NT,IL-6 and IL-1 levels induced by high glucose and high fat,suggesting that p38 MAPK is an important factor that mediates the development of inflammatory response in the pathogenesis of high glucose and high fat-induced diabetic cardiomyopathy.