| Part Ⅰ Assessment of Fetal Cardiac Function of Gestational Diabetes Mellitus using Dual-gate DopplerObjective:Gestational diabetes mellitus(GDM),as a common complication of pregnancy,has an increasing trend globally.GDM is associated with adverse fetal myocardial remodeling and fetal cardiac dysfunction.This study aims to comprehensively assess the effects of GDM on fetal cardiac systolic,diastolic,and global function by using multiple ultrasonic techniques.Both conventional method and Dual-gate Doppler method(DD)were conducted to evaluate fetal global and diastolic function,thereby to study the potentialities and the advantage of DD method in assessing fetal cardiac function.Method:This study selected 56 women diagnosed with gestational diabetes mellitus between 24 and 28 weeks of gestation(the GDM group),another 56 normal women matched to the gestational age of the GDM group were also enrolled(the control group).The ventricular transverse diameter and the interventricular septum thickness of each fetal were measured using M-mode echocardiography.The parameter in the evaluation of fetal cardiac systolic function were measured with M-mode echocardiography:including Left ventricular ejection fraction(LVEF),Left ventricular fractional shortening(LVFS),Mitral annular plane systolic excursion(MAPSE)and Tricuspid annular plane systolic excursion(TAPSE).Left ventricular global function parameters were measured using combined pulsed Doppler and Dual-gate Doppler method(DD)separately:including Isovolumetric contraction time(ICT),Isovolumetric relaxation time(IRT)and Ejection time(ET),and the Tei index was calculated.The parameters evaluating fetal cardiac diastolic function were measured using conventional method and DD method:mitral and tricuspid inflow velocities E-waves,A-waves on pulsed-wave Doppler imaging,and mitral and tricuspid annular velocities e’-waves,a’-waves on tissue Doppler imaging.The E/A,e’/a’ and E/e’ ratio were calculated afterwards.Results:1.Interventricular septum thickness were significant higher in the GDM group than that in the control group(P<0.0001),the ventricular transverse diameter were not differ significantly between groups(P>0.05);2.The results of fetal cardiac systolic function for both left and right ventricles:LVEF,LVFS,MAPSE and TAPSE showed no significantly difference between the two groups(P>0.05);3.The results of global function of left ventricular:ICT,IRT and Tei index were significant higher in the GDM group compared with control group(P<0.05).ET in the GDM group showed a trend of decreasing,but the difference was not statistically significant measured by combined pulsed Doppler(P>0.05),Notably,ET in the GDM group shower significant lower compared with control group measured by DD method(P<0.05);4.The results of fetal cardiac diastolic function for both left and right ventricles:The ratios of E/A and e’/a’ for both ventricles in GDM group were not significantly different from control group(P>0.05).There were no statistically significant differences in left E/e’ ventricles ratios between the two groups(P>0.05),while right ventricles E/e’ ratios were significantly lower in the GDM group compared with control group measured by conventional method(P<0.01).The E/e’ ratios of both left and right ventricular were significantly lower in the GDM group compared with control group measured by DD method(P<0.05).Conclusion:Maternal gestational diabetes might lead to the increase of fetal interventricular septal thickness and the LV-Tei index also the decrease of LV-E/e’ ratio and RV-E/e’ ratio.The results indicate that diabetic intrauterine environment may cause fetal cardiac hypertrophy and impaired global and diastolic function.The DD method is considered as a promising method,thus provide a reliable reference for early clinical detection of fetal cardiac dysfunction in GDM fetuses.Part Ⅱ Mechanism of myocardial injury in offspring of gestational diabetes ratsObjective:In the first part of the study,echocardiography examinations were performed on the fetuses of maternal with gestational diabetes.Maternal hyperglycemia induced fetal myocardial injury was observed,which was leading to fetal cardiac hypertrophy and fetal cardiac dysfunction.The mechanism of diabetic intrauterine environment on fetal myocardial injury remains unclear.In this part of the study,a rat model of gestational diabetes mellitus(GDM)was established,and RhoA/ROCK signaling pathway closely related to diabetic myocardial injury was selected for in-depth study in order to explore the possible mechanism of diabetic intrauterine environment on myocardial injury in offspring.Methods:1.In vivo experiments:Adult SD rats(1:1,male:female)were mated overnight.The pregnant rats were randomly divided into two groups,6 pregnant rats in each group.One group of pregnant rats were intraperitoneally injected with 30 mg/kg streptozotocin(STZ)solution on day 4 and day 5 of pregnancy,it was considered the GDM rat model was successful established when the random blood glucose was higher than 16 mmol/L(STZ group);The other group of pregnant rats were intraperitoneally injected with the same doses of sodium citrate buffer on day 4 and day 5 of pregnancy(Control group).Hearts from newborn rats were immediately collected on the day of birth,and the heart weight to body weight ratio were calculated.Then the myocardial tissues were taken for HE staining and Masson staining,and the myocardial collagen volume fraction(CVF)was calculated.The ultrastructure of the fetus’myocardial were observed by transmission electron microscope and the percentage of abnormal mitochondria was calculated.The mRNA expression levels of LDH,CK-MB and cTnI in myocardial tissues were detected using RT-qPCR.The protein expression levels of Fas,RhoA,ROCK1 and ROCK2 were detected by Western blot analysis;2.In vitro experiments:The H9c2 cardiomyocytes were set up into Control group and high glucose group(HG group).The cardiomyocytes of the Control group were cultured in DMEM medium(5.5 mmol/L glucose),and those of the HG group were cultured in high-glucose DMEM medium(33 mmol/L glucose).The cardiomyocytes were stained with phalloidin-rhodamine.The mRNA expression levels of LDH,CK-MB and cTnI in cardiomyocytes were detected using RT-qPCR,and the protein expression levels of Fas,RhoA,ROCK1 and ROCK2 were detected by Western blot analysis.Results:1.The high-glucose environment induces myocardial morphology changes:high-glucose induced part of cardiomyocyte hypertrophy in vitro,and the heart weight to body weight ratio were increased in offspring rats of maternal hyperglycemia(P<0.05);2.Maternal hyperglycemia causes myocardial histopathological changes in offspring rats:Compared with the control group,HE staining exhibited disordered arrangement of myocardial fibers and the myocardial tissues showed a minor level of necrosis and vacuolization occurring in STZ group.Masson staining showed the CVF of the myocardial interstitial was significantly increased in STZ group(P<0.01);3.Maternal hyperglycemia causes myocardial ultrastructural changes in offspring rats:Compared with the control group,part of myocardium mitochondrial swelling and the mitochondrial cristae partial fused in STZ group,the percentage of abnormal mitochondrial significantly increased(P<0.0001);4.The mRNA expression levels of LDH,CK-MB and cTnI,and the protein expression levels of Fas,RhoA,ROCK1 and ROCK2 in cardiomyocytes were significantly increased in high-glucose environment in vitro;Maternal hyperglycemia causes the mRNA expression levels of LDH,CK-MB and cTnI,and the protein expression levels of Fas,RhoA,ROCK1 and ROCK2 in myocardial tissues significantly increased in offspring of STZ group.Conclusion:1.The changes of myocardial histomorphology and molecular biology in offspring of gestational diabetes rats indicate that maternal hyperglycemia might leads to fetal myocardial injury;2.Fas pathway and RhoA/ROCK pathway are involved in myocardial injury in offspring of maternal hyperglycemia.Part Ⅲ The protective effect of irisin on myocardial injury in offspring of gestational diabetes ratsObjective:In the second part of the study,our experiment revealed that maternal hyperglycemia is related to fetal myocardial injury,Fas pathway and RhoA/ROCK pathway were involved in myocardial injury in offspring of gestational diabetes rats.Previous studies have shown that irisin improves glucose tolerance and insulin resistance,showing potential therapeutic value in studies on diabetes and obesity.In addition,irisin has played a protective role in myocardial injury in diverse cardiac disease models especially in diabetic cardiomyopathy.The purpose of this study is to explore the potential protective effect of irisin on myocardial injury in offspring of gestational diabetes rats,using both in vivo and in vitro approaches.So as to further explore the mechanism of the cardioprotective effects of irisin.Methods:1.In vivo experiments:Adult SD rats(1:1,male:female)were mated overnight.The pregnant rats were randomly divided into four groups,6 pregnant rats in each group.The first group of pregnant rats were intraperitoneally injected with 30 mg/kg streptozotocin solution on day 4 and day 5 of pregnancy(STZ group).The second group of pregnant rats were intraperitoneally injected with the same doses of sodium citrate buffer on day 4 and day 5 of pregnancy.After the successful establishment of GDM model,the third group of pregnant rats were intraperitoneally injected with 10μg/kg/day irisin since day 8 of pregnancy untill delivery day(STZ+irisin group),the fourth group of pregnant rats were intraperitoneally injected with 1 mg/kg/day RhoA inhibitor since day 8 of pregnancy untill delivery day(STZ+RhoA inhibitor group).Hearts from newborn rats were immediately collected on the day of birth,and the heart weight to body weight ratio were calculated.Then the myocardial tissues were taken for HE staining and Masson staining,and the myocardial collagen volume fraction(CVF)was calculated.The ultrastructure of the fetus’myocardial were observed by transmission electron microscope and the percentage of abnormal mitochondria was calculated.The mRNA expression levels of LDH,CK-MB and cTnI in myocardial tissues were detected using RT-qPCR.The protein expression levels of Fas,RhoA,ROCK1 and ROCK2 in myocardial tissues were detected by Western blot analysis;2.In vitro experiments:The H9c2 cardiomyocytes were set up into four groups.Control group,high glucose group(HG group),high glucose+irisin group(HG+irisin group)and High glucose+RhoA inhibitor group(HG+RhoA inhibitor group).The mRNA expression levels of LDH,CK-MB and cTnI in cardiomyocytes were detected using RT-qPCR.The protein expression levels of Fas,RhoA,ROCK1 and ROCK2 in cardiomyocytes were detected by Western blot analysis.Results:1.Contrast of heart mass index in offspring rats:①Compared with the control group,the heart weight to body weight ratio were increased in offspring rats of STZ group(P<0.05);②Compared with STZ group,the heart weight to body weight ratio in offspring rats of STZ+irisin group and STZ+RhoA inhibitor group showed a trend of decreasing,but the difference was not statistically significant(P>0.05);2.Contrast of myocardial histopathological changes in offspring rats:①Compared with the control group,histological analysis exhibited disordered arrangement of myocardial fibers in STZ group.The myocardial tissues showed a minor level of necrosis and vacuolization occurring,and the CVF of the myocardial interstitial was significantly increased(P<0.01);②Compared with STZ group,fetal cardiomyocyte injury was obviously alleviated in the STZ+irisin group and STZ+RhoA inhibitor group,which showed reduced myocardial tissue necrosis and vacuolization,and the CVF of the myocardial interstitial was significantly decreased(STZ vs STZ+irisin,P<0.05;STZ vs STZ+RhoA inhibitor,P<0.05);3.Contrast of myocardial ultrastructural changes in offspring rats:①Compared with the control group,part of myocardium mitochondria become swelling and vacuolization,the mitochondrial cristae partial fused in STZ group.The percentage of abnormal mitochondrial significantly increased(P<0.0001);②Compared with STZ group,the myocardium mitochondria swelling and mitochondrial cristae fused were alleviated in STZ+irisin group and STZ+RhoA inhibitor group,which showed the percentage of abnormal mitochondrial significantly decreased(STZ vs STZ+irisin,P<0.001;STZ vs STZ+RhoA inhibitor,P<0.0001);4.In vitro experiment:①Compared with the control group,the mRNA expression levels of LDH,CK-MB and cTnI,and the protein expression levels of Fas,RhoA,ROCK1 and ROCK2 in cardiomyocytes were significantly increased in HG group;②Compared with HG group,the mRNA expression levels of LDH,CK-MB and cTnI,and the protein expression levels of Fas,RhoA,ROCK1 and ROCK2 in cardiomyocytes were significantly decreased in HG+irisin group and HG+RhoA inhibitor group;5.In vivo experiments:①Compared with the control group,the mRNA expression levels of LDH,CK-MB and cTnI,and the protein expression levels of Fas,RhoA,ROCK1 and ROCK2 in myocardial tissues were significantly increased in STZ group;②Compared with STZ group,the mRNA expression levels of LDH,CK-MB and cTnI,and the protein expression levels of Fas,RhoA,ROCK1 and ROCK2 in myocardial tissues were significantly decreased in STZ+irisin group and STZ+RhoA inhibitor group.Conclusion:1.Irisin treatment during pregnancy could attenuate myocardial injure in offspring of gestational diabetes rats;2.Irisin might attenuates myocardial injury in offspring of maternal hyperglycemia via inhibition of RhoA/ROCK signaling pathway. |
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