| Gestational diabetes mellitus（GDM）can be defined as "diabetes diagnosed in the second or third trimester of pregnancy that was not clearly overt diabetes prior to gestation",which shows a rising prevalence worldwide.The list of maternal and fetal complications associated with GDM is lengthy,including macrosomia,stillbirth,fetal malformation,hypertension,polyhydramnios,neonatal respiratory syndrome,neonatal hypocalcemia and hypoglycemia,neonatal death,etc.Women who experience GDM during their pregnancies are at increased risk of developing type 2 diabetes mellitus（T2DM）in the years following delivery.Children of GDM-affected mothers are likewise at a higher risk of developing metabolic diseases and of being obese.The etiology of GDM has not been fully elucidated,but the pathogenesis of the disease is similar to diabetes which arises from insulin resistance.Oxidative stress and mitochondrial dysfunction are involved in the pathogenesis of diabetes and its complications.Oxidative stress refers to the imbalance between oxidation and anti-oxidation in vivo.Excessive reactive oxygen species and active nitrogen free radicals damage biological macromolecules such as proteins,lipids and DNA through oxidation,resulting in structural and functional damage of tissues and cells.In the condition of diabetes,hyperglycemia,hyperlipidemia and inflammation stimulate the production of a large number of ROS in different tissues.The long-term excessive accumulation of ROS exceeds the processing capacity of antioxidant enzymes in the body,resulting in chronic oxidative stress and mitochondrial dysfunction in cells of different tissues.The results showed that insulin resistance（IR）and related complications occurred in the islet.In addition,mitochondria are the main subcellular organs for the oxidative metabolism of lipids and glucose.The mitochondrial function of pancreatic in T2DM is reduced,leading to lipids accumulation and promoting glucose intolerance.Oxidative stress and mitochondrial dysfunction are the important factors of cell damage in diabetes mellitus.They interact with each other and participate in the occurrence of diabetes mellitus,resulting in cell dysfunction of various organs.The placenta is the basis of normal pregnancy,and poor placenta function can lead to perinatal complications.Some studies have found that placental oxidative stress and mitochondrial dysfunction are related to the occurrence and development of GDM.The placenta of GDM pregnant women had lipid toxicity,combined with metabolic histological changes,increased inflammatory factors and oxidative stress and decreased angiogenic regulatory factors.These changes may disrupt mitochondrial function,resulting in impaired placenta function and adverse maternal outcomes.Studying the changes of oxidative stress and mitochondria in placenta is conducive to exploring the occurrence,development and outcome of GDM,but the specific molecular mechanism of oxidative stress-mitochondrial damage in GDM placenta is still unclear.P66Shc is an oxidoreductase involved in intracellular oxidative stress and apoptosis.The increased expression of p66Shc has been reported in clinical trials and animal trials of diabetes mellitus and its complications.The expression of p66Shc mRNA in peripheral blood mononuclear cells（PBMCs）of diabetic patients increased.Oxidative stress induced mitochondrial fragmentation through p66Shc activation in diabetic nephropathy patients.Likewise,the overexpression of p66Shc is closely related to diabetic retinopathy,while inhibition of p66Shc expression and ROS production can alleviate diabetes-induced vascular dysfunction.In animal studies,mice with p66Shc knockout did not develop diabetes-related complications,such as kidney disease and neuropathy.As a sensor of oxidative stress,p66Shc protein can be directly translocated to mitochondria to promote the generation of ROS,while the excessive ROS and p66Shc cause mitochondrial swelling and further lead to mitochondrial dysfunction.Some studies have showed that p66Shc may also interact with mitochondrial dynamics.However,the mechanism of p66Shc mediated mitochondrial dynamics is still unclear.Mitochondrial dynamics refers to the fusion and fission of mitochondria.The morphology,size and distribution of mitochondria are finely regulated by pre-fusion proteins（Mfnl and OPA1）and pre-division proteins（Drpl and Fis1）.Drpl is a motor protein necessary for mitochondrial division.Overexpression of Drp1 can accelerate mitochondrial division,resulting in the generation of a large number of fragmented mitochondria,leading to mitochondrial dysfunction.It has been reported that p66Shc interferes with mitochondrial fragmentation and leads to oxidative damage and apoptosis of renal tubular epithelial cells by regulating Drpl in diabetic nephropathy.In summary,we speculate that oxidative stress and mitochondrial dysfunction may lead to placental dysfunction and dysplasia in GDM patients,resulting in perinatal complications and affecting fetal growth.P66Shc may be a key regulatory molecule in this mechanism.Currently,the expression and mechanism of p66Shc which is involved in oxidative stress-mitochondrial damage in GDM placenta have not been reported.We will discuss this research in three parts.Part 1.The expression of p66Shc in peripheral blood of gestational diabetes mellitus and its clinical significanceObject:To understand the difference of p66Shc expression in PBMCs between normal pregnant women and GDM pregnant women,and to explore the correlation between p66Shc expression and GDM.Methods:1.Fifty-one full-term pregnant women with GDM and 25 pregnant women with normal glucose tolerance（NGT）were enrolled.In the GDM pregnant women,26 pregnant women developed secondary perinatal complications of GDM in the third trimester（2-GDM）and 25 cases had no perinatal complications（1-GDM）.2.The weight and height of women in each group during early pregnancy were retrospectively collected,and their weight and height before delivery were also collected,and then body mass index（BMI）was calculated.Biochemical indexes in the second trimester of pregnancy were collected,including:OGTT value,fasting insulin（FINS）and HOMA-IR in the second trimester.Fasting blood glucose（FBG）,2-hour post-meal blood glucose（2hPBG）,hemoglobin Alc（HbA1c）,total cholesterol（TC）,triglyceride（TG）,high density lipoprotein（HDL）,low density lipoprotein（LDL）levels were detected before delivery.3.PBMCs were extracted before delivery.The mRNA expression of p66Shc in PBMCs of the three groups was detected by fluorescence real-time quantitative PCR（qRT-PCR）.4.The differences among the three groups were compared and the correlation between p66Shc mRNA in PBMCs and all clinical indicators was analyzed.Results:1.Comparison of clinical dataThere were no significant differences in age and gestational age among 2-GDM group,1-GDM group and NGT group.Pre-pregnancy BMI in 2-GDM group was significantly higher than that in 1-GDM group and NGT group（P=0.001,P<0.001）,and there was no significant different between 1-GDM group and NGT group.Late pregnant BMI of 2-GDM group was higher than that of 1-GDM group and NGT group（P<0.001,P<0.001）,but there was no significant difference between 1-GDM group and NGT group.The increase of BMI in 2-GDM group was higher than that in 1-GDM group and NGT group（P<0.05,P<0.05）.There was no significant different between 1-GDM and NGT group.2.Blood glucose levels and insulin sensitivity during middle pregnancy（24～2 8 weeks）Blood glucose level:OGTT-0h、OGTT-1h、OGTT-2h in 1-GDM group and 2-GDM group were higher than those in NGT group（P<0.001,P<0.001,P<0.001;P<0.001,P<0.001,P<0.001）.Insulin sensitivity:There was no significant difference in FINS levels among the three groups.HOMA-IR in 2-GDM group was higher than that in NGT group（P<0.01）.But there was no significant difference between 2-GDM group and 1-GDM group or between 1-GDM and NGT group.3.Blood glucose and lipid levels during late pregnancy and neonatal outcomes at deliveryBlood glucose levels:The level of FBG in 2-GDM group and 1-GDM group was significantly higher than that in NGT group,with statistical difference（P<0.0001,P<0.0001）.There was no difference between 2-GDM group and 1-GDM group（P=0.068）.The level of 2hPBG in 2-GDM group was higher than that in 1-GDM group and NGT group（P<0.0001,P<0.0001）,and there was no difference between 1-GDM group and NGT group（P=0.401）.HbAlc level in NGT group,1-GDM group and 2-GDM group showed a trend of gradual increase,and the difference between 2-GDM group and NGT group was statistically significant（P<0.01）.Serum lipid levels:TC,HDL levels among the three groups had no statistical significance.The level of TG in 2-GDM group was higher than that in NGT group（P<0.05）.The level of LDL in 2-GDM group was significantly higher than that in 1-GDM group and NGT group（P<0.01,P<0.001）.The level of LDL in 1-GDM group was higher than that in NGT group too（P<0.05）.Neonatal situation:The average weight of newborns in 2-GDM group and 1-GDM group was significantly higher than that in the NGT group（P<0.001,P<0.001）,and there was no significant difference between 1-GDM group and 2-GDM group.4.Expression of p66Shc mRNA in PBMCsThe mRNA level of p66Shc in 2-GDM group was significantly higher than that in 1-GDM group and NGT group（P<0.01,P<0.001）.The mRNA level of p66Shc in 1-GDM group was higher than that in NGT group,but the difference was not statistically significant.5.Correlation between p66Shc mRNA and the clinical indicatorsP66Shc mRNA was positively correlated with FBG,LDL and TG in 2-GDM group（r=0.412,P<0.05;r=0.58,P<0.01;r=0.412,P<0.05）.P66Shc mRNA was positively correlated with OGTT-0h、FBG、LDL in 1-GDM group（r=0.424,P<0.05;r=0.446,P<0.05;r=0.581,P<0.01）.There was no such correlation in the NGT group.Conclusion:The expression of p66Shc is up-regulated in peripheral blood of GDM pregnant women with complications.P66Shc may be involved in glucolipid metabolism,leading to the occurrence of GDM and its complications.Part 2.The relationship between p66Shc and oxidative stress in the placenta of gestational diabetes mellitusObject:1.To observe the pathological changes and ROS changes and investigate the oxidative stress in the placenta of GDM.2.To detect the expression of mitochondrial mitotic protein Drpl and investigate the changes of mitochondria in placenta of GDM.3.To clarify the expression of p66Shc in the placenta of GDM patients,and to analyze the correlation between p66Shc and Drp1.Methods:1.Placental tissues were collected from 15 GDM pregnant women with late-pregnancy complications of GDM（2-GDM group）,16 uncomplicated GDM pregnant women（1-GDM group）and 15 normal pregnant women during full term pregnancy.2.The pathological changes of placenta in 2-GDM group,1-GDM group and control group were observed by routine HE staining,and the level of ROS in placenta was detected by DHE staining.3.The mRNA expression of p66Shc in the placenta of the three groups was detected by qRT-PCR,and the protein expression of p66Shc in the placenta of the three groups was detected by immunohistochemistry.4.The mRNA expression of mitochondrial mitogenic protein Drp1 in the placenta of the three groups was detected by qRT-PCR,and the protein expression of Drp1 in the placenta of the three groups was detected by immunohistochemistry.5.The correlation between p66Shc mRNA and Drpl mRNA in placenta of 2-GDM was analyzed by Pearson correlation assay.Results:1.Pathological changes of placental tissue in pregnant women with GDMThe incidence of maternal and fetal vascular dysperfusion in placental pathological sections of 2-GDM group,1-GDM group and NGT group was 80%（12/15）,37.5%（6/16）and 20%（3/15）,respectively.There was significant difference between 2-GDM group and the other groups（P<0.05,P<0.01）.There was no statistical significance between 1-GDM and NGT group.2.Higher ROS level was showed in the placenta of 2-GDM groupROS level in placenta in 2-GDM group was significantly higher than that in 1-GDM group and NGT group（P<0.01,P<0.01）.There was no significant difference between 1-GDM group and NGT group.3.The mRNA and protein levels of p66Shc were highly expressed in the placenta of GDMThe expression of p66Shc mRNA in placenta of 2-GDM group was significantly higher than that of 1-GDM group and NGT group with statistical difference（P<0.05,P<0.05）.Immunohistochemical results showed that the protein expression of p66Shc in placenta of 2-GDM group was significantly higher than that of 1-GDM group and NGT group（P<0.05,P<0.05）,while there was no statistical difference between 1-GDM group and NGT group.4.Drpl mRNA and protein levels were highly expressed in the placenta of pregnant women with GDMThe expression of Drpl mRNA in placenta of 2-GDM group was significantly higher than that of 1-GDM group and NGT group with statistical significance（P<0.05,P<0.05）.Immunohistochemical results showed that the protein expression level of Drpl in placenta in 2-GDM group was significantly higher than that in 1-GDM and NGT groups（P<0.05,P<0.01）.The protein expression level of Drpl in 1-GDM group was higher than that in NGT group too（P<0.05）.5.The expression of p66Shc mRNA was positively correlated with Drpl mRNA in the placenta of GDM with complicationsPearson correlation test confirmed positive correlation between p66Shc mRNA and Drp1 mRNA（r=0.54,P<0.05）.Conclusion:1.There was significant oxidative stress in the placenta of GDM pregnant women with complications,which may be related to the up-regulation of p66Shc expression.2.The expression of Drpl in the placenta of GDM with complications was significantly higher than that of GDM without complications,indicating the presence of mitochondrial dysfunction in the placenta of GDM with complications3.The expression of p66Shc in the placenta of GDM pregnant women with complications was positively correlated with Drpl,suggesting that p66Shc may be involved in placental mitochondrial damage of GDM.Part 3.The role and mechanism of p66Shc in high glucose concentration-induced mitochondrial dynamics and oxidative damage in human chorionic trophoblast cellsObject:1.To detect the expression of p66Shc in JEG3 under high glucose stimulation.2.To detect the expression of Drpl and ROS in JEG3 under high glucose.3.To explore the relationship between the expression of p66Shc and oxidative stress-mitochondrial damagein JEG3,and to explore the role and mechanism of p66Shc in GDM.Methods:1.Human chorionic trophoblast cells JEG3 were cultured in vitro at normal glucose concentration（5.5mM）and high glucose concentration（15mM,30mM）for 24 h,respectively.The expression of Drpl in human chorionic trophoblast cells was determined by Western Blot.2.JEG3 was cultured in 5.5mM for 24 hours and in 30mM for 24 h,48 h and 72 h,respectively.The mRNA expressions of p66Shc and Drpl were determined by qRT-PCR,and the protein expressions of p66Shc and Drpl were determined by Western Blot.3.MitoTracker Red staining and DHE staining were used to detect mitochondrial status and ROS levels in JEG3 cells under different glucose concentrations.4.Untransfected JEG3 cells（normal blank group）and JEG3 cells transfected with plasmid p66Shc（overexpressed normal culture group）were cultured in 5.5mM glucose for 24 h.Untransfected JEG3 cells（high glucose blank group）and transfected si p66Shc cells（high glucose silencing group）were cultured in 30mM glucose for 24 h too.The expression of Drpl mRNA was detected by qRT-PCR,and the ROS expression in JEG3 cells was detected by DHE.MitoTracker Red staining were performed on the above-mentioned four groups to detect the level of mitochondrial membrane potential in JEG3 cells.Results:1.The effect of different glucose concentration on Drp1 expression in JEG3Compared with normal glucose（5.5mM）group,Drpl expression was significantly increased in 30mM glucose（P<0.01）,and there was no statistical difference between 5.5mM group and 15mM group.The high-glucose group with 30mM glucose concentration was selected as the follow-up experiment.2.The expression of p66Shc and Drpl in JEG3 was enhanced in high glucose environmentThe mRNA levels of p66Shc and Drpl in high glucose group were higher than those in normal culture group,the differences were statistically significant（P<0.05,P<0.05）.The protein levels of p66Shc and Drpl in high glucose group were also significantly higher than those in normal culture group（P<0.01,P<0.05）.3.The expression of p66Shc and Drp1 in JEG3 increased gradually with the increase of time in high glucose environmentWith the increase of time,the expression of p66Shc mRNA and Drpl mRNA gradually increased.There was a difference between normal culture group（0 h group）and the other groups.The longer it took,the more significant the difference performed（P<0.05,P<0.01,P<0.01;P<0.05,P<0.01,P<0.001）.Western Blot analysis also confirmed this trend.The subsequent experiments were cultured for 24 hours.4.ROS level of JEG3 is enhanced in high glucose environmentThe red fluorescence intensity of high glucose group was significantly higher than that of normal culture group,indicating that ROS expression was increased（P<0.05）.5.Mitochondrial dynamics in JEG3 under high glucose stimulationUnder normal glucose concentration（5.5mM）,the mitochondria of JEG3 were evenly distributed in the cytoplasm with long tubular or filamentous morphology.In the high concentration of glucose（30mM）,the mitochondria of JEG3 were dispersed in the form of dots or fragments（P<0.01）.6.The regulation of Drpl by p66Shc in JEG3Under normal culture conditions,the expression of Drpl mRNA in JEG3 cells after overexpressing p66Shc in normal culture group was increased compared with the blank group（P<0.01）.After the stimulation with high glucose,the expression of Drp1 mRNA in the high glucose blank group increased compared with the normal blank group,which was consistent with the previous experimental results.When p66Shc was silenced,the expression of Drpl in the high glucose silence group was decreased compared with the high glucose blank group under the same high glucose culture stimulation（P<0.05）.7.The regulation of ROS by p66Shc in JEG3Under the condition of normal sugar culture,ROS expression in JEG3 cells after overexpressing p66Shc in normal culture group was increased compared with the blank group（P<0.01）.After JEG3 was stimulated by high glucose,ROS expression in the high glucose blank group was enhanced compared with the normal blank group,which was consistent with the previous experimental results.After p66Shc was silenced,ROS expression in the high glucose silence group was decreased compared with the high glucose blank group under the same high glucose stimulation（P<0.01）.8.Effect of p66Shc on mitochondrial membrane potential in JEG3Under normal glucose concentration,the red fluorescence of JEG3 cells after overexpressing p66Shc in normal culture group was increased compared with the blank group（P<0.05）.Under high glucose concentration,the red fluorescence of JEG3 cells in the high glucose blank group was weaker than that in the normal blank group（P<0.01）.After silencing p66Shc,the red fluorescence of JEG3 cells in the high glucose blank group was higher than that in the high glucose blank group（P<0.05）.Conclusion:1.The oxidative stress and mitochondrial dysfunction in trophoblast cells were significantly increased under high glucose.2.High concentration of glucose stimulates the increased expression of p66Shc in trophoblast cells,which leads to mitochondrial damage by positively regulating the expression of Drpl and ROS.3.P66Shc may be an important regulatory molecule in the pathogenesis of GDM and its complications. |
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