Mechanisms Study Of Preventive And Therapeutic Effects Of L-arginine On IUGR Rats By Passive Smoking

Intrauterine growth retardation (IUGR) , also called fetal growth restriction (FGR) , is a significant complication of pregnancy. In addition to perinatal morbidity and mortality, epidemiological studies suggest that IUGR may contribute to adverse health effects in adulthood including cardiovascular disease, diabetes and other complications which may cause the development of intellect and physique of the infants. There are no generally accepted drugs to treat IUGR. The prevention and treatment of IUGR is one of the important topics in perinatal medicine. IUGR fetuses may exhibit symmetric or asymmetric patterns of growth. Asymmetric IUGR is mostly caused in the middle or late period of gestation by various harmful agents which lead to fetoplacental circulation disturbance and reduce fetal nutrient supply, then restrict the development of fetus. Asymmetric IUGR is the majority pattern of IUGR in clinical and often accompanies with other diseases such as preeclampsia. However, the aetiology of the majority of IUGR cases remains unexplained. The aims of the study are to find out the pathogenesis of IUGR, prevent the incidence of IUGR, and to find out the utility measure of IUGR treatment, eliminate the bad influence. The essential etiological factor of asymmetric IUGR is the deficiency of placental function. This study focused on factors concerned with placental circulation and their effects on each other. What effects perinatal IUGR have on the development of fetal brains? Can these be redressed by treatment in gestation such as the pathophysiology changes in placentas of IUGR, the damage of fetal brain and the incidence of IUGR? All of the above questions remain unanswered definitely, and wait for further study. Thus, circulating and locally released vasoactive molecules such as nitric oxide (NO) are therefore likely to be involved in the control of fetoplacentalhemody-namics. The reduced NO is concerned with the incidence of IUGR and it is confirmed that directly aspiration of NO gas cant decrease the damage of IUGR. L- arginine is the donor of NO and the relative and ( or) absolute deficiency of L- arginine probably is one of the causes of NO reduction. Can L - arginine increase the reduced NO level in IUGR? How about the relationship between the dose of L - arginine and the levels of NO and factors concerned with NO? Which pathway does L - arginine work? These questions should be answered before L -arginine used in clinical treatment. NO is an unstable polypeptide and would be harmful as free radical in some conditions. So different doses of L - arginine were used for IUGR rats and the mechanisms of L - arginine were studied.The endogenetic NO is synthesized from L - arginine through nitric oxide synthase (NOS) and NOS is the rate -limiting enzyme of this reaction. There are three kinds of NOS: neuronal NOS, inducible NOS and endothelial NOS. It is confirmed that the expression of iNOS and eNOS in placentas has some relationship with the incidence of IUGR. But the relationship between development of fetal brains and expression of nNOS and that between effects of L - arginine and expression of these NOS are still unknown. Growth factor is a species of endocrine factors that can adjust the growth and proliferation of cells. Vascular endothelial growth factor ( VEGF) and insulin like growth factor ( IGF) are concerned with NO and the function of development of vessels, so deeply concerned with placental circulation and fetal development. There are a species of insulin like growth factors including IGF - I , IGF - II , IGF receptors and IGF binding proteins. Study on expression of these factors are helpful to reveal the mechanisms of the effects of L - arginine on IUGR.The animal model by passive smoking rats has the similar pathophysiological changes with human IUGR, and can be used to appraise the curative effects of medicines. IUGR rat model was established by passive smoking from 8d to 20d of gestation. Different doses of L - arginine were injected to the enterocoelia of rats and the incidence of IUGR was observed. The placentas and fetal brains were observed by microscope. The levels of NO, MDA, SOD and the expression of nNOS, iNOS, eNOS, VEGF and IGFs in serum, placentas and fetal brains were measured to study the relationship between oxidate/antioxidae system,NO/NOS system, endocrine factors and the effects of L - arginine on IUGR. The effects and dosages of L - arginine were studied, too. Mechanisms of preventive and therapeutic effects of L - arginine were revealed to provide a theoretical basis for clinical treatment of L - arginine on IUGR.Methods1. Animal modelFemal Wistar rats ( weight: 200 - 260g) were randomly divided into 5 groups by pregnancy time; control group, model group, low dose ( LA) , middle dose ( MA) and high dose ( HA) of L - arginine treated groups( n=9). Model group and L - arginine treated groups were exposed to smoke for 2 h from 8d to 20d of gestation. Animals of control group were kept under same environmental conditions without smoke. Saline, L - arginine of 100mg/Kg, L - arginine of 200mg/Kg and L - arginine of 400mg/Kg were injected to enterocoelia of rats of control, LA, MA and HA groups respectively.2. Sample collection and treatmentOn 21d of gestation, 2ml blood samples were collected by heart puncturing from rats of each group. The placentas and fetal rats were taken out then. Tissues were collected and stored respectively according to the following methods;2.1 Blood samples were placed for 30 minutes and centrifuged at 3000 g/ min for 10 minutes. The serum was stored at - 70℃ for later detection.2.2 Placentas and fetal brains were put in RNase -free Eppendorf tubes, then put in liquid nitrogen immediately and stored at - 70℃.2. 3 Placentas and fetal brains were fixed in 4% formaldehydum polymeri-satum.3. Experiment Methods3.1 The numbers of alive, dead and resorption fetus were counted. The weight of body, brain and liver, the length of body and tail of fetal rats, the placentas weight were examined. The diagnostic standard of IUGR is generally assigned to the fetal rats measured with two standard deviations lower than the average fetal weight of control group.3. 2 Microscope exam of placentas and fatal brains pathology: The fixed tis-sues were dehydrated, embedded in paraffin, sectioned, stained with HE staining method.3. 3 The measurement of NO, MDA levels and SOD, NOS activities in serum, placentas and fetal brains; biochemical methods.3.4 The measurement of nNOS, iNOS, eNOS mRNA levels in placentas and fetal brains: RT - PCR.3.5 The measurement of nNOS, iNOS, eNOS protein levels in placentas and fetal brains: Western blot.3. 6 The measurement of nNOS, iNOS, eNOS, VEGF protein levels in placentas and fetal brains-.immonohistochemistry.3. 7 The measurement of VEGF,IGF - I ,IGF - Ⅱ,IGFBP3 levels in serum, placentas and fetal brains; ELISA.3. 8 The measurement of IGF - Ⅰ ,IGF - Ⅰ R mRNA levels in placentas and fetal brains; real time fluorescence quantitative RT - PCR.Results1. The effects of L - arginine on the growth and development of IUGR rats The number of dead and resorption fetus and the incidence of IUGR in model group were significantly higher than control group (P <0.01). The number of dead and resorption fetus and the incidence of IUGR in LA and MA groups were significantly lower than model group ( P <0.01), but there were no statistical difference between control group and treatment groups (P >0. 05). The number of dead and resorption fetus and the incidence of IUGR in HA group were significantly higher than control, LA and MA groups, and there were no statistical difference between HA and model group (P >0. 05).The average weight of body, brain and liver and length of body and tail of fetal rats in model groups were significantly lower than control group ( P < 0. 05 ). The average weight of body, brain and liver and length of body and tail of fetal rats in LA and MA groups were significantly higher than model group ( P < 0.05 ) but lower than control group ( P < 0. 01). The average weight of body, brain and liver and length of body and tail of fetal rats in HA group were signifi-cantly lower than control, model, LA and MA groups (P <0.01).2. L - arginine on pathological changes of placentas and fetal brains2.1 Gross examination;The placentas became hyacinthine and the edge of placentas became whiteness in model group instead of madder red color in control group. The ischemia and aging of placentas were improved in LA and MA groups. But the gross examination of placentas and fetal brains of HA group were similar to model group.2.2 Microscope examination:2.2. 1 Placenta; In control group, the formations of deciduas, villus and labyrinthus in placentas were clear. Vascular endothelial cells and decidual cells were integrated. The structure of villus and blood vessels were normal and there were plenty of sinusoids. In model group, calcify focus formed in labyrinthus and decidual cells degenerated. The intervillous space increased and there were fewer blood in the sinusoids. In LA and MA groups, the vascular endothelial cells and decidual cells were recovered. The structure of most of the intervillous space was normal and there were plenty of sinusoids. In HA group, decidual cells degenerated, intervillous space increased and there were fewer erythrocyte in the sinusoids.2.2. 2 Fetal brain; In control group, the structure of nerve cells were normal. The nuclei of glia cells were deep - stained and plasm of glia cells was poor. The vascular endothelial cells were integrated. In model group, the number of nerve cells decreased, part of nuclear membrane extincted, nuclei were not clear. Glial cells increased and some small vessels angiectasis. In LA and MA groups, Most of the nerve cells became normal, part of glial cells hyperplasia slightly and vascular endothelial cells were integrated. In HA group, nerve cells decreased, glial cells increased slightly and some small vessels angiectasis.3. The effects of L - arginine on NO, MDA levels and SOD activity in serum, placentas and fetal brainsCompared with model group, the NO levels of serum and placentas in LA and MA groups increased significantly and the NO levels of fetal brains decreased (P <0.01). The levels of MDA decreased and the activity of SOD in-creased in serum, placentas and fetal brains of LA and MA groups (P <0.01). But there were no statistical differences of levels of NO, MDA and SOD between HA and model group ( P > 0.05 ).4. The effects of L - arginine on the expression and activity of nNOS, iNOS and eNOS in serum, placentas and fetal brainsThe positive expression of nNOS in fetal brains was mainly in nerve cells by immunohistochemistry. The positive expression of iNOS was mainly in decidual cells, vascular endothelial cells and trophoblastic cells of placentas and nerve cells of fetal brains, but there were nearly no iNOS expression in fetal brains of control group. The positive expression of eNOS was mainly in vascular endothelial cells of placentas and fetal brains. Compared with model group, the protein and mRNA expression of nNOS and iNOS in LA and MA groups were decreased significantly by Western blot and RT-PCR (P<0.01). The protein and mRNA expression of eNOS in LA and MA groups were increased significantly ( P < 0.01). But there were no obvious differences of the expression and activity of nNOS, iNOS and eNOS between HA and model group (P>0.05).5. The effects of L - arginine on the expression of VEGF in serum, placentas and fetal brainsThe positive expression of VEGF was mainly in the decidual cells, vascular endothelial cells and trophoblastic cells of placentas and nerve cells and glial cells of fetal brains by immunohistochemistry. In serum and placentas, the expression of VEGF in LA and MA groups were increased significantly compared with model group (P <0. 01). But in fetal brains, the expression of VEGF in LA and MA groups were decreased significantly compared with model group ( P <0. 01). There were no obvious differences in the expression of VEGF between L - arginine treated groups and control group ( P >0.05).6. The effects of L - arginine on the expression of IGF - Ⅰ , IGF - Ⅱ , IGF - Ⅰ R and IGFBP3 in serum, placentas and fetal brainsCompared with model group, the expression of IGF - Ⅰ and IGF - Ⅱ in serum , placentas and fetal brains of L - arginine treated groups were increased significantly by ELISA, but the expression of IGFBP3 was decreased (P <0.01). The expression of IGF - Ⅰ and IGF -Ⅰ R mRNA in placentas and fetal brains of...