The Role Of Oxidative Stress-induced JNK Signaling Pathway In Fetal Congenital Scoliosis Induced By Gestational Diabetes Mellitus

Gestational diabetes mellitus（GDM）is an endocrine and metabolic disease that occurs or is discovered for the first time due to abnormal maternal glucose metabolism after pregnancy-With the continuous improvement of people’s living standards in our country,the incidence of GDM has increased year by year in recent years,seriously endangering the health of pregnant women and fetuses,and even perinatal lives.Oxidative stress（OS）refers to a series of reactions caused by excessive production of reactive oxygen clusters（ROS）and reactive nitrogen free radicals（RNS）beyond the scavenging capacity of the body when the body is subjected to some harmful stimuli,which leads to a series of reactions caused by the imbalance between oxidation and antioxidation in the body,which is often accompanied by increased production of ROS and/or damage to the function of antioxidant defense system,which is the main cause of cell injury[1].Maternal hyperglycemia increases endogenous ROS production and impairs the antioxidant capacity of the intracellular antioxidant system,leading to oxidative stress.Since antioxidants can reduce cell apoptosis caused by maternal hyperglycemia,some researchers believe that excessive cell apoptosis caused by oxidative stress is the main mechanism of embryonic deformities caused by gestational diabetes mellitus[2,3].In gestational diabetes mellitus,activation of JNK1/2 in the embryo and yolk sac is associated with developmental abnormalities caused by apoptosis of embryonic cells.JNK activity in the embryos and yolk sacs of diabetic mice was inhibited by antioxidant administration,which prevented or remedied embryo malformation[2].This study with GDM pregnant rat model as the research object,detection of fetal rat spinal tissues oxidative stress indicators malondialdehyde（MDA）content,and determination of phosphorylation in fetal rat spinal tissue c-jun amino terminal kinase（p-JNK）and the expression of apoptosis inducing factor Bax protein,through specific inhibition of JNK activity,discuss JNK signaling pathway in gestational diabetes caused by the role of fetal congenital scoliosis,provide clinical prevention,diagnosis and condition monitoring of stronger theory basis and the research train of thought.Part 1 Construction of a rat model of gestational diabetes mellitusObjective:To explore the effect of different doses of streptozotocin（STZ）on the stability of GDM pregnant rat model,so as to provide a good experimental basis for the study of the mechanism of fetal congenital scoliosis caused by GDM.Methods:Forty-eight 8-week-old C57BL/6J pregnant rats（0.5 days of pregnancy）were randomly divided into 4 groups with 12 rats in each group.The body weight,random blood glucose and fasting blood glucose of each pregnant rat were recorded respectively.After 12 hours of fasting,STZ30mg/kg,40mg/kg,50mg/kg and the same amount of sodium citrate buffer were injected intraperitoneally for three consecutive days.Random blood glucose was measured on the 5.5,10.5,15.5 and 20.5 days of pregnancy,and the changes of body weight,food intake,water intake,coat color and mental state of pregnant rats were observed,and the modeling rate,negative conversion rate and mortality rate of gestational diabetes mellitus were compared.The pregnant rats were dissected on the 20₅ days of pregnancy,and the development of placenta,amniotic fluid and offspring were observed.Results:Female mice in STZ40mg/kg group showed obvious symptoms of "three more and one less" 5.5 days after the last administration,with the highest model formation rate（83.3%）and negative conversion rate（0%）.Compared with the control group on gestation day 20.5,STZ40mg/kg group showed statistically significant difference in the random blood glucose（18.2±3.0vs6.0±1.0）（P<0.05）,and the duration of hyperglycemia was long and stable.Conclusion:The mode of continuous intraperitoneal injection of STZ40mg/kg for three days is a relatively appropriate dose for the establishment of a pregnant rat model of GDM which has the advantages of high modeling rate,low mortality and sustained effect.Part 2 The Role of Oxidative Stress-induced JNK signaling pathway in Fetal Congenital Scoliosis Induced by Gestational Diabetes MellitusObjective:1.To observe the expression of oxidative stress markers and phosphorylated c-Jun amino-terminal kinase（p-JNK）protein in the spinal tissues of GDM pregnant rats,and to analyze the correlation between oxidative stress and JNK pathway.2.To observe the changes of apoptosis inducing factor BAX in the spinal tissue of pregnant rats with GDM.Methods:The levels of malondialdehyde（MDA）in the spinal homogenate of pregnant rats with gestational diabetes mellitus（GDM group）and normal pregnant rats（control group）were detected by colorimetry_The expression levels of p-JNK protein and Bax protein in the spinal tissues of pregnant rats in GDM group,SP group（JNK inhibitor SP600125）and control group were detected by Western blotting.Results:1.The MDA level in fetal spinal tissue of GDM group was significantly higher than that of control group,and the difference was statistically significant（P<0.05）.2.The expression level of p-JNK in fetal rat spinal tissue in GDM group was significantly higher than that in control group,with statistical significance（P<0.05）;The expression level of p-JNK in fetal spinal tissues in SP group was significantly lower than that in GDM group,with statistical significance（P<0.05）.3.There was a positive correlation between MDA level and p-JNK protein expression level in fetal spinal tissues of GDM group（r=0.773,P<0.05）.4.The expression level of Bax protein in fetal rat spinal tissue in GDM group was significantly higher than that in control group,with statistical significance（P<0.05）;The expression level of Bax in fetal spinal tissue of SP group was significantly lower than that of GDM group,with statistical significance（P<0.05）.Conclusion:1.Oxidative stress occurred in the fetal spinal tissues of pregnant rats with GDM,and the degree of oxidative stress was positively correlated with the activation of JNK protein,and the increased expression of p-JNK may be related to the spinal deformity of fetal rats caused by GDM.2.Activated JNK signaling pathway may lead to the high expression of the apoptotic inducing factor Bax in the spinal tissue of fetal spinal deformities of GDM pregnant rats,leading to the occurrence of fetal spinal deformities.