| Acute lung injury (acute lung injury, ALI) is a pulmonary parenchymal disease. The main causes of ALI are sepsis, pneumonia, the inhalation of gastrointestinal contents, trauma and other clinical disease. ALI could lead to acute hypoxic respiratory insufficiency. Any age of the patients can suffer from ALI. Severe cases may progress to the acute respiratory distress syndrome (ARDS), and even become the multiple organ dysfunction syndromes (MODS). The mortality of ALI is as high as more than44%. Due to the immature lung tissue development of neonates, the resistance ability to the outside stimulation and damage is poor. During the neonatal period, some causes such as severe infection and intestinal obstruction can lead neonates to ALI, and even to the damage in multiple organs of the body. The clinical manifestations are progressive dyspnea, moist ralse and cough pink frothy sputum. The neonatal pulmonary vascular basement membrane structure is so thin, which easily leading to pulmonary hemorrhage and the consequence of ALI in newborn is more serious. So the prevention research for neonatal ALI has important clinical significance. At present, foreign scholars has found that hypoxia-inducible factor-1alpha (HIF-1α) has played a vital role in the pathogenesis of acute lung injury. In our study, the ALI models were induced by given LPS by intraperitoneal injection. And then we observed the expression of relative mediators, the outcome of the lung tissue pathology on each group penehyclindine hydrochloride given or not. The aim is to investigate the mechanisms of penehyclidine hydrochloride, which plays a protective role in ALI, and to find the relation between the protective effect of PHC and the expression of HIF-1α in LPS-induced acute lung injury in rats to provide a new theoretical basis for clinical prevention and treatment.ObjectiveThe aim is to investigate the protective mechanisms of penehyclidine hydrochloride in ALI, and to find the relation between the protective effect of PHC and the expression of HIF-la in LPS-induced acute lung injury in rats to provide a new theoretical basis for clinical prevention and treatment.Materials and Methods1. Study ObjectTwenty-four male and female7days old SD rats, weighing17-22g, obtained from Henan laboratory animal center.2.Methods2.1Animals modelsThe neonatal rat acute lung injury was induced by given LPS3mg/kg with1ml syringe by intraperitoneal injection. After injection the rats are put back to the cage, and6hour later the rats are given operation to get the simples. We can evaluate the successful neonate rat model of ALI by the optical microscope morphologic outcome in lung tissue and the changes of the inflammatory factors.2.2Animal groupTwenty-four7days old SD rats were divided into3groups at random:control group (group C) that was given saline0.3ml by intraperitoneal injection; ALI group, which was given LPS3mg/kg0.3ml by intraperitoneal injection; penehyclidine hydrochloride group (group P), which was received PHC2mg/kg half an hour before LPS3mg/kg0.3ml administration, with8animals each group. At6h post LPS treatment, the lung tissue were collected under anesthesia with ketamine hydrochloride100mg/kg, by intraperitoneal injection. 2.3Lung tissue sample collectionThe left lung lobe was taken for weighing and then placed into the70℃oven for baking72hours to calculate the lung wet/dry weight ratio. The right upper lobe was washed by DEPC water, then stored at the-80℃refrigerator to be used for RT-PCR analysis.The posterior lobe of the right lung was put out into the formaldehyde solution for24hours, paraffin embedding, cutting it into5um paraffin section. After disinfected in different concentrations of alcohol, we observed the tissue pathology on an optical microscope after the HE stain.The inferior lobe of right lung was collected and weighed. Then we added PBS (9times as the weight of lung lobe) with them, homogenizing by hand and centrifugated at the speed of1800r.p.m to get the supernatant. The supernatant was stored at-20℃refrigerator for the further measurement.2.4Detection Indexes2.4.1Measurement of IL-6, TNF-αand HIF-la by ElisaThe levels of IL-6, TNF-α and HIF-1α were measured by ELISA. The inferior lobe of right lung was collected and weighed. Then we added PBS as9times as the weight of lung lobe to them, homogenizing by hand and centrifugated at the speed of1800r.p.m to get the supernatant. To measure the protein concentration level of IL-6, TNF-αand HIF-1α in lung homogenate supernatant.2.4.2Analysis of HIF-1α mRNA-levels by RT-PCR.The right upper lobe was washed by DEPC water and cooled by liquid nitrogen as soon as they were dried with filter paper. After the design and synthesis of HIF-1α and GAPDH mRNA, the extraction of total RNA, reverse transcription of RNA, amplification HIF-1α and GAPDH and agarose gel electrophoresis, we can analyse the expression of the HIF-1α on the mRNA levels.2.4.3Measurement of lung wet/dry weight ratioThe left lung lobe was dried with filter paper and weighed, then put into the70℃oven for baking72hours to get its dry weight. The lung wet/dry weight ratio was then calculated.2.4.4Histological examination of lung tissueThe posterior lobe of the right lung was made into5um paraffin section. We can observe the tissue pathology on an optical microscope after the HE stain.3. Statistic analysisData processing was performed by SPSS17.0.All data were presented as mean±standard deviation (x±s). ANOVA and Student-Newman-Keuls q test were used for statistical analysis to compare values data among all groups. The inspection level is a=0.05.Results1. The general status observation:At6h post LPS treatment, the neonate rats of ALI group become less exercise and breathing faster compared with group C, and some of the ALI ones had a series of symptoms, they coughed up pink foamy sputum and had cyanosis. The neonate rats of PHC group also become less exercise and breathing faster, but they did not cough up pink foamy sputum and cyanosis. And the control group did not such symptoms.2. Lung histological morphological changes:With the observation by light microscope, the lung tissue structural of group C is integrity with normal alveolar structure, evenly alveolar space and smooth alveolar wall. In group ALI, the complete structure of the alveolar wall was destruct and the alveolar wall was ruptured. There were diffuse edema and a large number of white blood cells in the pulmonary interstitium. A mass of inflammatory cells and red blood cells exudation can be seen in the alveolar space. In group P, he alveolar structure is more complete and the pulmonary interstitial edema was significantly reduced compared with group ALI.3. The W/D ratio of group ALI and group was higher in group ALI and group P than that of group C, and there was a statistically significant difference (P<0.05).While the W/D ratio was lower in group P than that of group ALI, and there was a statistically significant difference (P <0.05).4. The concentration of TNF-α,IL-6and HIF-1α were higher in group ALI and group P than that of group P, and there was a statistically significant difference (P<0.05).Compared with group C, the expression of HIF-1α mRNA was higher in group ALI and group P, and there was a statistically significant difference (P<0.05). And the concentration of TNF-α, IL-6, HIF-la and the expression of HIF-1α mRNA were lower in group P than that of group ALI, and there was a statistically significant difference (P<0.05).ConclusionPenehychdine hydrochloride has a protective action on LPS-induced acute lung injury of neonatal rats. The mechanisms of the effect may be through down-regulating the expression of HIF-la in lung tissues and reduce the inflammatory response. |
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