Effect Of Hydrogen Sulfide On NO/iNOS In Neonatal Rat With Hyperoxia Lung Injury

15 million premature infants would be born in the whole world each year, including more than 1 million infants dying of complications related to premature delivery. Premature delivery has become the leading cause of neonatal death causes. In recent years, advances in perinatal medicine and neonatal intensive care technology significantly improve the survival rate of premature infants especially the low birth weight infants. At the same time, long-term complications associated with premature delivery vary and increase significantly. In the long-term complications associated with premature delivery, the incidence of bronchopulmonary dysplasia(BPD) showed a increasing trend. BPD seriously affects the quality of life and the prognosis of the premature. Current treatments for BPD are still lack of effective measures. Therefore, looking for early interventions to mitigate hyperoxia-induced lung injury are particularly important. In pathology, BPD was showed alveolar and pulmonary vascular developmental disorders. The pathogenesis was related to hyperoxia-induced lung injury with chronic inflammation and antioxidant defense damage. Present studies suggest that hyperoxic lung injury plays an important role in the pathogenesis of BPD.The discovery of endogenous signaling molecule gas opened up a new field for the study of the pathogenesis of lung injury. In 1990 s, hydrogen sulfide(H2S), the third signal of endogenous biologically active gas molecular, has physical properties and biological activity similar with nitric oxide(NO) and carbon monoxide(CO). Previous studies of H2 S were concentrated on its toxicity. In recent years, physiological effects and pharmacological effects of H2 S had been extensively studied. A large number of animal studies had confirmed that endogenous H2 S participates and plays a protective effect role in regulating pulmonary hypertension and pulmonary vascular remodeling, fibrosis, the occurrence and development of acute lung injury caused by oleic acid, cigarette smoke, etc. In clinical studies, there was a certain relevance between the concentration of H2 S and the severity of the asthma and chronic obstructive pulmonary disease. Our previous experiments found that exogenous H2 S has a protective effect on hyperoxia-induced lung injury of neonatal rats.Inducible nitric oxide synthase(i NOS) is not expressed under physiological state, while i NOS could be highly expressed in vascular smooth muscle cells, pulmonary macrophages, in neutrophils and other inflammatory cells under pathological state by infection, high concentrations of oxygen, inflammation. The i NOS can catalytic synthesis a mass of NO. When NO exists in a high concentration, NO damages cells by three ways as the following:(1)NO activates the multiple signaling cascade, promotes the accumulation of inflammatory cells and releases inflammatory cytokines causing explosive inflammation.(2) NO inhibits the activity and function of various enzymes in the respiratory chain affecting cellular energy metabolism.(3) The main thing is to form a strong and oxygen free radical peroxynitrite anion oxide, which can oxidative, attack biofilm and interfere with cellular energy metabolism.Most studies suggested that NO was involved in the pathogenesis of hyperoxia- induced lung injury. This present study aimed to observe the effect of exogenous H2 S on the i NOS expression and the content of NO on hyperoxia-induced lung injury in neonatal rat lung tissue.In further, we explore the protection mechanism of H2 S on hyperoxia-induced lung injury, and provide a theoretical basis for the clinical remedy of H2 S on treating BPD in preterm infants.ObjectiveBy inhalating high concentration of oxygen, we establish neonatal rats of hyperoxia-induced lung injury model. Observing the changes of the i NOS m RNA expression and the content of NO by increasing or decreasing the concentration of exogenous H2 S, we explore the protection mechanism of H2 S on hyperoxia-induced lung injury.Materials and Methods 1 Experimental mice groups and hyperoxia-induced lung injury model making80 neonatal and Full-term natural birth Sprague-Dawley(SD) rats were divided into 4 groups using random number table:(1) the control group;(2) hyperoxia group;(3) Na HS +hyperoxia group,(4) PPG + hyperoxia group, 20 rats were in each group. Na HS + hyperoxia group and PPG + hyperoxia group were intraperitoneal injected Na HS(28 umol/kg) and PPG(50 mg/kg) respectively. While the control group and hyperoxia group were given equal normal saline intraperitoneal injection(the injection was started from the first day of the experiment, and each group was given different drug injection in the next 7 days). Neonatal SD rats and breastfeeding rats are placed in the same cage.The control group was feeding in the air condition, the rest three groups were placed in oxygen tank(60cm × 50 cm × 30cm), inputing the constant oxygen, regulating the rate of oxygen flow rate(5l/min), using digital CYS-1 oxygen meter dynamic detecting oxygen concentration to make oxygen concentration above 90%, using sodium lime to absorb carbon dioxide, keeping the temperature(22℃~25℃) and humidity around 65%. Open the tank for 1 hour daily, add water and word, replace the sodium lime, and exchange the breastfeeding rats to avoiding mothers’ feeding and nursing ability going down. In the whole experiment, all experimental animals were raised in the same room, and full grain fodder and water were supplied by the laboratory animal center. 2 The collection of rats lung samplesAfter 7 days, at the prescribed time, neonatal rats were fixed in the supine position on operation stage using medical adhesive tape. Rats were given intraperitoneal injection of sodium pentobarbital 90 mg/kg. Open the abdomen and cut off the abdominal aorta until bleeding to death, open chest quickly and expose the heart and lung thoroughly, separate and clear the lung carefully, ligate right bronchial, remove the right lung measuring lung wet weight ratio(W/D). Fixe left lower lobe more than 48 hours in 4% formaldehyde for pathological HE staining. The left upper lobe was used to making lung tissue homogenate to detect the content of MDA, NO, i NOS. The rest of the lung was frozen at-80℃ for detectiving the expression of i NOS m RNA.The expression of i NOS was detective by Real time-PCR.Result 1 Protective effects of hydrogen sulfide on hyperoxia-induced lung injury 1.1 Pathology changes of neonatal rats’ lung in each groupPathological changes of lung were observed at high magnification(10×40) field by optical microscopy. The control group:neonatal rat alveolar structure was regular and normal, alveolar septa was infiltrated without inflammatory cell. Hyperoxia group: a lot of inflammation cell such as leukocytes(mainly neutrophils) were infiltrated in alveolar septa, and some red blood cells were in the alveolar exudation in partial field. Hyperoxia+Na HS group: Compared with hyperoxia group, the infiltration of inflammatory was decreased significantly, the alveolar interval was narrow. Hyperoxia+PPG group: the infiltration of leukocyte became more obviously, alveolar interval was broaden severely, in some sections of the pathological image, the lung tissue structure was disordered. 1.2 The ratio of lung wet and dry(W/D) in each groupCompared with control group, the W/D of hyperoxia group was increased obviously, with statistical significance(P<0.01). Compared with hyperoxia group, hyperoxia+Na HS group the W/D was decreased obviously(P<0.01), but still higher than the control group(P<0.01);Hyperoxia+PPG group the W/D was significantly higher than any group(P<0.01). 1.3 The content of MDA in each groupCompared with control group, the content of MDA in hyperoxia group was increased obviously, with statistical significance(P < 0.01). Compared with hyperoxia group, hyperoxia+Na HS group the content of MDA was decreased obviously(P < 0.01), but still higher than the control group(P < 0.01); Hyperoxia+PPG group the content of MDA was significantly higher than any group(P<0.01). 2 The impact of hydrogen sulfide on i NOS/NO in hyperoxia-induced lung injury 2.1 The activity of i NOS in each groupCompared with control group, the activity of i NOS in hyperoxia group was increased obviously, with statistical significance(P < 0.01). Compared with hyperoxia group, hyperoxia+Na HS group the activity of i NOS was decreased obviously(P < 0.01), but still higher than the control group(P < 0.01); Hyperoxia+PPG group the activity of i NOS was significantly higher than any group(P<0.01). 2.2 The content of NO in each group.Compared with control group, the content of NO in hyperoxia group was increased obviously, with statistical significance(P < 0.01). Compared with hyperoxia group, hyperoxia+Na HS group the content of NO was decreased obviously(P<0.01), but still higher than the control group(P<0.01); Hyperoxia+PPG group the content of NO was significantly higher than any group(P<0.01). 2.3 The expression of i NOS m RNA in each groupCompared with control group, the expression of i NOS m RNA in hyperoxia group was increased obviously, with statistical significance(P<0.01). Compared with hyperoxia group, hyperoxia+Na HS group the expression of i NOS m RNA was decreased obviously(P<0.01), but still higher than the control group(P<0.01); Hyperoxia+PPG group the content of NO was significantly higher than any group(P<0.01).Conclusion1.i NOS / NO is involved in the pathogenesis of neonatal rats with hyperoxiainduced lung injury2. Exogenous hydrogen sulfide exerts its protective effect against hyperoxia-induced lung injury through downregulating the expression of i NOS m RNA and reducing the production of NO in rats lung tissue.