The Study Of The C-jun N-terminal Kinase Pathway In Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM) is defined as an endocrine and metabolic disease resulting form glucose intolerance with glycometabolism disorder in varying degrees. It has first occur or detected during pregnancy. GDM is associated with short- and long-term morbidity in both the mother and the offspring.With the increasing prevalence of GDM year by year, the study on impact of GDM attracts extensive attention.Physiological levels of reactive oxygen species are important to maintain various cell functions, but over-loads of reactive oxygen species that exceed the capacity of the antioxidant system induce oxidative stress.Under destructive stimulus conditions, over-loads of reactive oxygen species and reactive nitrogen species can result in damage of the tissular or cellular structures and functions.In some researches, it has been confirmed that oxidative stress has participated in the development of diabetes mellitus and its complication by activating the c-Jun N-teminal Kinase (JNK) pathway. According to the domestic and abroad researches, JNK has play an important rola in insulin signaling pathway,βcell dysfunction, IR and apoptosis caused by various stress reaction. But the relationship between oxidative stress, activated JNK, apoptosis inducing factor Bax and GDM in pregnant rats had few report at the present. For this reason, it is necessary to investigate the effects of the JNK pathway on GDM rats, especially on p-JNK expression of uterine and placental tiusses. The study was designed to analyze the effects of the JNK pathway on GDM pregenant rats of uterine and placental tiusses through construction of GDM pregenant rats model, then try to expore the action of JNK in development and growth of GDM on uterus and placenta, try to provide some useful theories for clinic prevention, diagnosis and treatment.Part 1. Construction of streptozotocin-induced gestational diabetes mellitus on rats modelObjective:By injecting different dosage of streptozocin (STZ) explore the stability of model of gestational diabetes mellitus in rats. Methods:Forty-six Sprague-Dawley pregnant rats were selected and randomly assigned into four groups. The rats were injected respectively with STZ 35, 45, 60 mg/kg or equal dosage of citrate buffer solution intraperitoneally according to their assigned groups. Their fasting blood glucose was measured after STZ injecting 3, 9, 14 and 19 days later. The body weights, food consumption, amount of drinking water and urine volume of these rats were tested during the whole gestation period. Compared about the modeling rate,turning negative rate and mortality rate in the different groups. The rats in all groups underwent dissection to obtain the pancreatic tissue for pathological study by HE staining at their gestational ages of 19 days. Results: The pregnant rats of the 45 mg/kg group showed significant signs of polydipsia, polyuria, hyperphagia and weight loss after STZ injection. It has the highest making model success rate of 83.3% and the lowest turning negative rate. There was statistically significant difference between the STZ45mg/kg group and the control group in the level of the fasting blood glucose(mmol/L,21.8±3.0νs 5.9±1.2)(P<0.05). The state of hyperglycemia retained longer time and steadily. Conclusions: The optimum dose to construct ideal experimental animal model of streptozotocin-induced GDM is injected STZ 45 mg/kg intraperitoneally.Part 2. The study of the effect of oxidative stress and the expression of p-JNK in uterine and placental in GDM rats and the relationship with IRObjective:To investigate the oxidative stress and the expression of p-JNK in uterine and placental tissue in GDM rats induced by streptozotocin, in order to provide experimental basis in the research on maternal and fetal GDM. Methods:The level of malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant capacity(T-AOC) in uterine and placental tissue were detected by colorimetry in GDM rats (GDM group,n=10) and normal pregnant rats(normal pregnant group,n=10).The expression of p-JNK protein in uterine and placental tissue were detected by immunohistochemistry and western-blotting in GDM group,JNK inhibitor SP600125(SP group,n=10)and normal pregnant group. The fasting insulin (FINS), fasting plasma glucose(FPG) and insulin resistance index(HOMA-IR) were measured too. Results: (1)The level of FINS, FPG and HOMA-IR of GDM group was significantly higher than that in normal pregnancy group(P<0.05). The level of MDA of GDM group in uterine and placental tiusse was significantly higher than that in normal pregnancy group (P<0.05). The level of SOD and T-AOC of GDM group was significantly lower than that in normal pregnancy group (P<0.05). (2) The phosphorylated JNK (p-JNK) immunostaining was mainly localized to glandular epithelium and stromal cell in cell nuclei and endochylema.The p-JNK immunostaining was also mainly localized to chorion and materno-fetal interface decidual cell in cell nuclei and endochylema. The level of p-JNK protein were significantly increased in GDM group,compared with normal pregnancy group(P<0.05). Administration of SP600125 could significantly inhibit the expression of p-JNK protein. (3) The p-JNK protein was positively correlation to MDA in GDM group (P <0.05). P-JNK was negatively correlation to SOD and T-AOC in GDM group (P <0.05). There was no correlation in SP group and normal pregnancy group. (4)HOMA-IR was positively correlation to p-JNK protein in GDM group (P <0.05). There was no correlation in SP group and normal pregnancy group.Conclusions: Oxidative stress was closely related to the activation of JNK protein in GDM; the abnormal expression of p-JNK protein in uterine and placental tissue maybe one of the molecular mechanisms leading to gestational IR.Part 3. The study of the mechanism of apoptosis caused by p-JNK in uterine and placental in GDM ratsObjective:To investigate the changes of apoptosis inducing factor Bax in uterine and placental tissue in GDM rats induced by streptozotocin. Methods: The expression of Bax in uterine and placental tissue was detected by immune- histochemistry and western-blotting in GDM group, SP group and normal pregnant group. Results: The level of Bax was significantly increased in GDM group,compared with normal pregnancy group(P<0.05).Administration of SP600125 could significantly inhibit the expression of Bax. Conclusions: Oxidative stress was closely related to the activation of JNK protein in GDM; the abnormal expression of p-JNK protein in uterine and placental tissue may be lead to apoptosis and gestational IR and other complications.