Research On The Critical Role Of Mast Cells In STZ-induced Mice Diabetic Cardiomyopathy And Its Underlying Mechanisms

Objective:To investigate the protective effects of mast cell deficiency on STZ-induced diabetic cardiomyopathy and the possible mechanisms involved.Methods:1.In the present study,wide type(WT)mice(C57BL/6 mice)and mast cell-deficient mice(KitW-sh/W-sh mice)were used to build the STZ-induced diabetic cardiomyopathy mice model with or without the reconstitution of mast cells.At the same time,bone marrow-derived mast cells were isolated from wide type mice(C57BL/6 mice),TNF-?-/-mice and IL-6-/-mice,and prepared to apply for reconstitution.Thus,the in-vivo study was designed with 7 different groups of mice,including WT mice,WT + STZ mice,KitW-sh/W-sh mice,KitW-sh/W-sh + STZ mice,KitW-sh/W-sh + STZ + WT-BMMCs mice,KitW-sh/W-sh+ STZ + IL-6-/--BMMCs mice,KitW-sh/W-sh+ STZ + TNF-?-/--BMMCs mice.Then we did the following measurements:(1)The cardiac remodeling and cardiac function of the STZ-induced diabetic mice were detected by ultra-sound system,including interventricular septum(diastole),left ventricular internal diameter(diastole),left ventricular posterior wall(diastole)and fractional shortening.(2)The heart weight index(HWI)was used to assess the cardiac hypertrophy through the measurements of heart weights and body weights of mice.(3)Toluidine blue staining was used to stain mast cells in the mice heart tissues.(4)Picric sirius red staining was one of the most popular approaches for observing the expressions of collagen ? and collagen ? in the heart tissues.(5)RT-PCR were widely used to test the expressions of TNF-? and IL-6 in the heart tissues.(6)Meanwhile,we also detect the expressions of ICAM-1 and Lysyl oxidase,two markers related to fibrosis,by RT-PCR.(7)TUNEL staining was used to observe cell apoptosis,also in the heart tissues.2.Moreover,in the in-vitro studies,we preformed several experiments to further confirm the results in the KitW-sh/W-sh mice.Caruiomyocytes and myocardial fibroblasts isolated from wide type mice were treated with the BMMCs lysates extracted from WT-BMMCs,TNF-?-/--BMMCs and IL-6-/--BMMCs.Then we tested the expressions of smad 2,p-smad 2,smad 3,p-smad 3,?-SMA,collagen ? and collagen ? to figure out the impacts of mast cells on myocardial fibrosis by western blot.Also,we used the same way to detect the expressions of the markers of apoptosis,such as Bax,Bcl-2 and Cleaved caspase 3.Results:The blood glucose levels of mice with injection of STZ were all above 500 mg/dL for over 4 weeks.The weight loss was only shown in WT + STZ mice and KitW-sh/W-sh+ STZ + WT-BMMCs mice.The number of mast cells were highly raised in the heart tissues of STZ-induced WT mice,and after reconstitution of the BMMCs isolated from wide type mice,TNF-?-/-mice and IL-6-/-mice,there were also slight increased number of mast cells in the heart tissues.The analysis of ultrasonic cardiogram showed the increase of IVS,LVID,LVPW and the decrease of FS in WT +STZ mice,indicating the occurrence of cardiac remodeling and the impairment of cardiac function in these mice,but the injuries were ameliorated in mast cell-deficient mice.However,after reconstitution of WT-BMMCs,the protective and beneficial effects were hampered.In addition,based on the results of Picric sirius red staining,the myocardial fibrosis was attenuated in the heart tissues of KitW-sh/W-sh + STZ mice.RT-PCR analysis also presented that mast cells promoted the expressions of ICAM-1 and Lysyl oxidase in the heart tissues of diabetic mice through TNF-a.TUNEL staining showed the reduced number of apoptotic cardiomyocytes with the absence of mast cells in the heart tissues.Furthermore,the findings of the in-vitro studies demonstrated that mast cells could promote the expressions of p-smad 2,p-smad 3 to produce more collagen ? and collagen ? for myocardial fibrosis,and mast cells also induced the expressions of Bax and Cleaved caspase 3 through IL-6 for cardiomyocytes apoptosis.It is noteworthy that all the impacts of mast cells in the diabetic cardiomyopathy were associated with two cytokines released by mast cell,TNF-? and IL-6.Without the presence of TNF-? and IL-6,mast cells couldn't completely cause the unfavourable outcomes in diabetic cardiomyopathy.Conclusions:1.Mast cells played a significant role in the pathophysiologic process of diabetic cardiomyopathy,because the cardiac remodeling and the cardiac function did not deteriorate in mast cell-deficient mice even with the STZ injection.2.Mast cell deficiency,particularly without TNF-a and IL-6,was the key factors for the inhibition of myocardial fibrosis and the reduction of cardiomyocytes apoptosis.3.Based on the utility of KitW-sh/W-sh mice,TNF-?-/-mice and IL-6-/-mice in the present study,we got these results to provide new insights into our understanding of the relationships between mast cells and diabetic cardiomyopathy,and meanwhile established an important theoretical foundation for future clinical use of immune-related therapy(such as pharmacological stabilization of mast cells)in diabetic cardiomyopathy.