The Effect Of Ecdysterone On The Expression Of TNF-αmRNA And SP-A In Lung Tissue Of Acute Lung Injury Rats

BackgroundAcute lung injury (ALI) is a runaway inflammation response with pathological characteristics of the injury of pulmonary vascular endothelial cells and alveolar epithelial cells when after the body suffered from severe trauma, shock, severe infections, which is an early stage of acute respiratory distress syndrome (ARDS), and the clinical manifestations of progressive dyspnea and hypoxemia. ALI/ARDS pathogenesis is complex and not yet fully elucidated in current, but after a lot of basic research shows that changes in oxidative stress, inflammatory response, and endothelial cell dysfunction are involved in the pathogenesis, especially, the Balance and imbalance between inflammatory and anti-inflammatory response play an important role in the development of the disease. It is generally believed that the pathogenesis of ALI is the activation of inflammatory cells and the release of a series of inflammatory mediators in the effect of pathogenic factor, resulting in damage to the lung tissue. Then the inflammatory mediators activate more inflammatory cells to release more cytokines that further enlarge and strengthen the body damage signal, which form the inflammatory cascade effect. Finally, it caused excessive or out of control of the systemic inflammatory response syndrome (SIRS). In clinically, the main reason for ALI is a bacterial infection caused by the release of endotoxin, and the pathogenic main component is the lipopolysaccharide of the bacterial cell wall adventitia. Starting the intracellular signal transduction system when the LPS and receptor combined with each other and it will promote the release of various inflammatory cytokines such as TNF-a, etc. Interaction of these inflammatory cells and inflammatory mediators can damage the alveolar cells and endothelial cells, and increased vascular permeability. And it will reduce the secretion of pulmonary surfactant and breakdown the barrier of alveolar epithelial. Therefore, the experimental observe the dynamic changes of inflammatory mediators and the level of anti-inflammatory substances by rats with intraperitoneally injection of LPS to copy the model of ALITumor necrosis factor-α (TNF-α) is a pro-inflammatory immune molecules in the initiation and the key role of inflammation. And it is the polypeptide having a variety of functions, which mainly release by activated neutrophils, macrophages, endothelial cells and other cells. In currently known, when the TNF-a combined with the corresponding receptor, it can Play a toxic effect by stimulate inflammatory cell adhesion and release a large number of inflammatory mediator, such as oxygen free radicals, and stimulate monocytes macrophages to increase the production of IL-1, IL-2, IL-6, IL-8and other inflammatory cytokines. Thus, TNF-a is a good indicator to observed the development of ALI.Surfactant protein (SP) is a specific class of lipoprotein complexes which mainly secreted by alveolar type II epithelial cells and non-ciliated bronchiolar epithelial cells. Surfactant protein A (SP-A) is the richest content and most biologically active protein in SP. It has a lot of function, for example it can maintain the normal structure and function of the alveoli regulate the intrapulmonary immune and inflammation reaction, take park in regulating the synthesis, metabolism of PS, and enhance the bactericidal capacity of phagocytic cell by combined with microbiological specificity. Also it can inhibit the synthesis and release of various cytokines and inflammatory mediators. Now many people think that the SP-A content decreased in patients with ALI for endogenous metabolic abnormalities that can aggravate ALI.The clinical research in treatment of ALI/ARDS with drug has become a hot topic, but it has not yet to find an effective and reliable therapeutic drugs. thus, it is necessary to find the protective drugs, to treatment of ALI/ARDS.Ecdysterone (EDS) is a kind of steroid compound extensively existing in the nature. EDS is a hormone regulating the growing and metabolism of insects and was first studied by entomologists. Afterwards, a lot of similar substances were separated from many kinds of plants and also showed strong physiological activity in higher animals. For example, it can increase the synthesis of nucleic acid and protein, promote the angiogenesis of ischemia district, establish the collateral circulation and promote wound healing, etc.In recent years, studies have shown that EDS can improve lung tissue edema, decreased pulmonary vascular permeability, etc. This effect may have some therapeutic in patient with ALI. In order to further investigate the EDS treatment mechanism of ALI, we make the rat model of ALI by using of lipopolysaccharide to observe the level of TNF-a and SP-A in the lung tissue, illustrating the effect and possible mechanism of EDS in treatment of ALI.ObjectiveObserving the effects of diverse dosages of EDS on the expressions of TNF-a and SP-A in rats with ALI meanwhile comparing the correlation between EDS and the expressions of them. Discussing the mechanisms of EDS.in protecting lungs against ALI induced by the LPS in rats.Methods 1. The models and groupings of experimental animals:Establishing the ALI models by injecting the LPS into the abdomens of Wister rats. A total of120adult male Wister rats in SPF level, weighing170-220gram, were randomly divided into model group(24rats, group L),control group(24rats, group N), and treatment groups(EDS with different dosages, group T) which included LPS+EDS20mg/kg (group T1,24rats),LPS+EDS30mg/kg (group T2,24rats),and LPS+EDS40mg/kg (group T3,24rats).According to the different observation times(2h,4h,24h) after being injected the LPS,3subgroups were established for each group possessed8rats. LPS of8mg/kg was injected into each rat in model and treatment groups to duplicate the models of ALI while the rats in control group were injected with physiological saline. Rats in treatment groups were injected with the corresponding dosages of EDS through caudal veins at1h after the establishment of models. The rats in model and control groups were given the physiological saline.2. Collection and detection for specimens:Anaesthetize all rats with3%napental (30mg/kg) through abdomen anesthesia, then executed them by bleeding through abdominal aorta. Opening thoracic cavity, and extracting the lungs. Dying the Inferior lobe of right lungs with HE as the pathological specimens, pathology abnormalities were scored. Figuring out the W/D of the superior lobe of right lungs, TNF-a mRNA expression in homogenate of left lungs were detected through RT-PCR. The concentrations of TNF-a in homogenate of left lungs were detected through ELISA. SP-A expressions were tested through Western Blot.Resultsl.The general conditions observation:The rats of the NS group breathed a little more quickly and recovered soon..No symptoms like cyanosis were observed. The rats acted agilely to avoid catching. The rats of LPS group had series of symptoms such as accelerated breath frequency, less activity, incapability of escaping while being caught which displayed the most seriously at the time point of4h after the injection of LPS.. While in the T group, the symptoms of polypnea was slighter than that of the rats of LPS group. Some rats in L group come up with cyanosis. And the rats were more active that could escape rapidly while being catched in the T group. There were similar state in each T group at the time point of2h, but the T3group situation were better than the T1,T2group at the time of4h and24h.2. Pathological change:The pathology of the right lung in rats was observed under HE staining200times microscope. The structure of lung tissue in rats was integrated within the N group, while no edema in the alveolar septum and he infiltration of inflammatory cell was observed. There were a large number of neutrophil infiltrations and destruction of alveolar wall and hemorrhage in the lung tissue of L group. It means that the ALI model of rats was succeeded. The change of pathology of the lung tissue was significantly reduced in each T groups, comparing the L group. Five-category classification based on lung injury, interstitial inflammation, alveolar hemorrhage, pulmonary edema, atelectasis and hyaline membrane formation, the severity of Pathological changes between the groups were L>TI>T2>T3>N(P<0.05). The severity of Pathological changes between the time point within each group were4h>24>2h (P<0.05).3. The change of Wet/Dry weight ratio of lung tissue:Compared with N group,the change of W/D weight ratio of lung tissue was Significantly increased in the L and T groups at different time point, while the ratio of W/D between the time point within each group were4h>24>2h (P<0.05).Compared with L group,the change of W/D weight ratio of lung tissue was Significantly declined in the each T groups at time point of4h and24h.There was no statistical significance between L group and T1/T2groups at2hour, while T3lower than L group.(P<0.05)。The trend of W/D in the same group within different time were4h>24h>2h. 4. The content of SP-A in lung tissue:The content of SP-A in lung tissue of rat were not significant difference in N group at each time point, while were higher than T group and L group. Compared with L group, The content of SP-A in lung tissue was significantly increased in each T groups at different time point(P<0.05).The content of SP-A in lung tissue in each T group were T3>T2>T1(P<0.05) at the time point of4h and24h. There was no statistical significance between T2group and Tlgroups at2hour, while T3higher than T1/T2group.(P<0.05).The trend of SP-A in the same group within different time were2h>24h>4h.(P<0.05)5. The content of TNF-α in lung tissue:The expression of TNF-α in lung tissue of rat were very weak and no significant difference in N group at each time point, while there were lower in N group than other groups (P<0.05). Compared with L group, The content of TNF-α in lung tissue was significantly decreased in each T groups at different time point(P<0.05).Compared with each of T groups, there were significantly lower in T3group than T2/T1group at the time point of4h and24h (P<0.05), while there were no significant difference with T2group at2h.. The expression of TNF-α in T2group were significantly lower than T1group at2h and4h (P<0.05),but there were no significant difference at24h.The content of SP-A in lung tissue in each T group were T3>T2>T1(P<0.05) at the time point of4h and24h. There was no statistical significance between T2group and T1groups at2hour, while T3higher than T1/T2group (P<0.05).The trend of TNF-α in the same group within different time were2h>24h>4h (P<0.05). The trend of expression of TNF-αin lung tissue in the same group within different time were4h>2h>24h (P<0.05)6. The expression of TNF-α mRNA in lung tissue:The result of TNF-α mRNA expression in lungs tissue by RT-PCR showed that the expressions of TNF-αmRNA were very weak and no significant difference in N group at different time point. The expression of TNF-αmRNA in lung tissue in T and L groups were significantly more strengthen in compared with N group. Compared with L group, he expression of TNF-amRNA in lung tissue was significantly decreased in each T groups at different time point(P<0.05). The trend of expression of TNF-amRNA in lung tissue in the each other of T group within different time were T3<T2<T1(P<0.05),while in the same group within different time were4h>2h>24h.(P<0.05)Conclusions1. Models of ALI could be duplicated by injecting LPS into abdomens of rats.2. LPS injection could give rise to pulmonary edema in rats. Microscope displayed that the injuries of lung tissues were severe. TNF-a mRNA expression was enhanced while the concentration of SP-A declined.。And after injection of LPS4hours, the degree of lung injury was heaviest than2hours and24hours. The lung injury time trend was4h>2h>24h.3. EDS could play a role in pulmonary protection which was dependent on concentrations. The mechanisms of EDS in pulmonary protection may lie in depressing the expression of TNF-α mRNA as well as promoting the synthesis and release of SP-A.