Mechanism Of FBXW7 Regulating The Development Of Diabetic Cardiomyopathy Through Rhoa/rho Kinase Signaling Pathway

Objective In recent years,the prevalence of diabetes mellitus(DM)has risen sharply in the world,and DM poses a serious threat to human life and health.One of the main causes of death in diabetic patients is cardiovascular complications.Diabetic cardiomyopathy(DCM)is an independent and specific cardiomyopathy recognized as a cardiovascular complication in diabetes,which is closely related to heart failure and increased mortality in diabetic patients.Myocardial fibrosis(MF)is one of the major pathological changes of DCM.The excessive proliferation of cardiac fibroblasts and the mass synthesis of matrix proteins and collagen fibers are the main causes of MF.This study examined the changes of F-box/WD repeat-containing protein7(FBXW7)and Rho A/Rho kinase signaling pathways in myocardial tissue of type 1 diabetes mellitus(T1DM)rats,and transfected human cardiac fibroblasts(HCFB)by lentivirus to further investigate the effect of FBXW7 expression on Rho A/Rho kinase signaling pathway in high glucose cultured HCFB cells.The influence of signaling pathways clarified the mechanism of FBXW7 regulating the development of MF through Rho A/Rho kinase signaling pathway,in order to provide a new theoretical basis for the treatment of DCM.Methods 1.Animal experiment 1.1 Establishment of the T1 DM rat model 113 SD male rats after 1 week adaptive feeding were randomly divided into non-diabetic group(ND group)40 and diabetic model group(DM group)73.Before the model was established,the rats were fasted for 16 h,DM group injected in STZ by intraperitonealinjection of 60 mg/kg the ice bath under the condition of the current allocation,while the ND group by intraperitoneal injection the same volume of buffer.After 72 h,all rats were fasted for 5~6 h,with a syringe needle punctured the tail vein blood glucose strips and connect to blood glucose,fasting blood glucose(FBG)detection values of all subjects.Successful animal model: compared with the control group,rats in DM groups FBG was more than 16.7 mmol/L,with polydipsia,polyphagia,polyuria,weight loss.In this experiment,56 models were successfully created.1.2 Relationship between FBXW7/Rho A/Rho kinase signaling pathways and myocardial fibrosis in T1 DM rats Rats in the ND group were randomly divided into 2,4,8,12,and 16 weeks of blank control group,8 rats in each group.The rats in the DM group were randomly divided into 2,4,8,12,and 16 weeks.Each group consisted of 8,9,9,13,and 17.Both the ND group and the DM group were kept in standard pellet feed and distilled water.After the specified period,rats anesthesia laparotomy,5~10 m L syringe aortic blood in a centrifuge tube,separation of serum.Remove the heart,separate the left heart,HE staining to observe the pathological changes of myocardial tissue.The volume of collagen was evaluated by Masson staining and hydroxyproline(HYP).Serum concentrations of FBXW7,transforming growth factor-?1(TGF-?1)and connective tissue growth factor(CTGF)were detected by ELISA.The activities of SOD and MDA in the serum were estimated by standard methods using commercially available kits.The expression level of FBXW7,Rho A and ROCK1 in the myocardium was determined by Western blotting and immunohistochemistry.2.Cell experiment 2.1 Cell transfection with lentivirus and grouping HCFB cells in the logarithmic growth phase and in good growth state were first divided into the low glucose group(5.5 mmol/L glucose concentration)and the high glucose group(25 mmol/L glucose concentration).The high glucose group was transfected with lentivirus and divided into FBXW7 overexpressing empty virus group,FBXW7 overexpression group,FBXW7 interfering empty virus group and FBXW7 interference group.2.2 The effect of interventing FBXW7 expression on Rho A/Rho kinase signaling pathway and related proteins The m RNA levels of FBXW7,Rho A and ROCK1 were detected by q RT-PCR.The expression of FBXW7,Rho A and ROCK1 protein was detected by Western blotting.The content of HYP in cells was detected by alkaline hydrolysis method.The content of TGF-?1 and CTGF was detected by Elisa method.The content of SOD and MDA in the cells was measured.Results 1.Animal experiment 1.1 The general characteristics of DM rats During the experiment,the rats in the normal control group were generally in good condition,strong,shiny,with autonomous activities,sensitive to the outside world,and no death.Rats in the model group gradually showed polydipsia,polyphagia,polyuria and weight loss,wilting,dull and dull fur,aggravation of sputum odor.And with the prolongation of time,polydipsia,polyphagia,polyuria and weight loss were aggravated,18 rats showed crystal whitening,the general condition is getting worse and worse.There were 56 successful models and 5 deaths.With the prolongation of diabetes,the body weight of ND rats increased significantly and FBG was normal.Compared with the ND group,the weight,heart and left ventricle weight of the DM group were significantly lower than those of the ND group(P<0.01),and the FBG was significantly increased(P<0.01).In addition,the cardiac index and left ventricular index of the DM group were not significantly changed at 2 and 4 weeks(P>0.05),but the rats at 8,12 and 16 weeks were significantly higher than the ND group(P<0.01).1.2 Relationship between FBXW7/Rho A/Rho kinase signaling pathways and myocardial fibrosis in T1 DM rats HE staining revealed that the myocardial texture of the ND group was clear,the myocardial cells were arranged neatly,the nucleus was evenly distributed,no degeneration and necrosis,and the myocardial cells in each DM group showed different degrees of focalcardiomyocyte degeneration,necrosis,fibrous tissue proliferation,etc..Compared with ND group,collagen deposition area and collagen content in myocardial tissue of DM group were significantly increased at 4~16 weeks(P<0.01,P<0.05),showing a continuous increasing trend,both of which have a significant positive correlation with the extension of time.(r=0.997,P<0.01;r=0.997,P<0.01).The results of Elisa showed that the serum FBXW7 level in the DM group increased significantly(P<0.01)at 2~4 weeks,and decreased in the 8th week DM group,but it was still significantly higher than the normal control group(P<0.01).The levels of TGF-?1 and CTGF in the serum of DM group increased gradually from 2 to 16 weeks,and increased significantly at 8~16 weeks(P<0.01),which was significantly positively correlated with the prolongation of time(r=0.994,P<0.01;r=0.990,P<0.01).Compared with the ND group,the SOD activity of the DM group decreased significantly(P<0.01),and there was a significant negative correlation with the time(r=-0.965,P<0.01);the MDA level was significantly increased(P<0.01),and time was significantly positively correlated(r=0.992,P<0.01).Western blotting and immunohistochemistry showed that the expression of Rho A and ROCK 1 protein in the DM group was significantly higher than that in the ND group(P<0.01),and the expression of Rho A and ROCK 1 Significantly increased with the prolongation of the cycle(P<0.05),there was a significant positive correlation with time prolongation(r=0.967,P<0.01;r=0.990,P<0.01).The expression of FBXW7 in the myocardium of the DM group was significantly increased compared with the ND group at 2~12 weeks(P<0.01),but the expression of FBXW7 was significantly decreased in the DM group at 12 and 16 weeks compared with the 8 week DM group.(P<0.01).2.Cell experiment 2.1 Cell transfection with lentivirus The cells in each group grew well and showed no obvious death.A large amount of green fluorescence was observed under the fluorescence microscope,and the lentivirus infection efficiency was about 80%.The lentivirus transfection was successful.2.2 Intervention of FBXW7 expression on Rho A/Rho kinase signaling pathway and relatedproteins The results of q RT-PCR showed that the expression of FBXW7 in the high glucose group was significantly decreased compared with the low glucose group(P<0.05),while the expression of Rho A and ROCK1 was significantly increased(P<0.01).With the overexpression of FBXW7,the expression of Rho A and ROCK1 was significantly decreased(P<0.01);while the down-regulation of FBXW7 was accompanied by a significant increase in Rho A and ROCK1(P<0.01).The results of Western blotting were consistent with the results of q RT-PCR.Compared with the low glucose group,the expression of FBXW7 in the high glucose group was significantly decreased(P<0.05),while the Rho A and ROCK1 were significantly increased(P<0.01,P<0.05).The expression of Rho A and ROCK1 was significantly decreased in FBXW7 overexpression(P<0.05).The down-regulation of FBXW7 was accompanied by a significant increase in Rho A and ROCK1(P<0.05).Compared with the low glucose group,the intracellular collagen content of the high glucose group was significantly increased(P<0.01).As FBXW7 was overexpressed,intracellular collagen content decreased significantly(P<0.01).The down-regulation of FBXW7 expression was accompanied by a significant increase in intracellular collagen content(P<0.01).The results of Elisa showed that the levels of TGF-?1 and CTGF in the high glucose group were significantly higher than those in the low glucose group(P<0.01).The content of TGF-?1 and CTGF in the cells decreased significantly with the overexpression of FBXW7(P<0.01);while the down-regulation of FBXW7 expression was accompanied by a significant increase in intracellular TGF-?1 and CTGF levels(P<0.01).Compared with the low glucose group,the SOD activity in the high glucose group was significantly decreased(P<0.01),while the MDA content was significantly increased(P<0.05).With the overexpression of FBXW7,the intracellular SOD activity was significantly increased(P<0.05).MDA content was significantly decreased(P<0.01);while FBXW7 expression was down-regulated with a significant decrease in SOD activity(P<0.01),and MDA content was significantly increased(P<0.01).Conclusion1.FBXW7 and Rho A/Rho kinase signaling pathway are involved in the development of myocardial fibrosis in diabetic rats.2.FBXW7 has a negative correlation with the expression levels of Rho A and ROCK1,and overexpression of FBXW7 can inhibit the activation of Rho A/Rho kinase signaling pathway.3.FBXW7 can alleviate oxidative stress by inhibiting the activation of Rho A/Rho kinase signaling pathway,thereby improving diabetic myocardial fibrosis.4.Activation of FBXW7 high expression may become a new idea for the treatment of diabetic myocardial fibrosis.