The Expression Of Heat Shock Protein In Acute Lung Injury Induced By Lipopolysaccharide And The Mechanism Of Rhubard's Protective Effect

The Expression of Heat Shock Protein in Acute Lung Injury Induced by Lipopolysaccharide and The Mechanism of Rhubard's Protective EffectIntroductionIt is well established that acute respiratory distress syndrome(ARDS) is a commom severe case in clinics which is specialized by aggregation of neutrophil in lung, intravenous blood cogulation and pulmonary edema induced by enhanced permeability of pulmonary capillary, thus lead to death due to hypoxemia.Heat shock protein(HSP) is a large family of stress protein with endogenous protective effect, in which HSP70 is the most essential one. It is widely existed in most common animals which is remarkably increased after cellular stress. Besides, studies has indicated that rhubarb has protective effect against ALI induced by lipopolysaccharide (LPS), in that rhubarb can not only inhibit the production of NO and the activity of iNOS, but also decrease the activity of PLA2 and PAF, thus can protect lung and decrease the damage of ALI induced by LPS.In the present study, whether HSP70 has the relation to the protection of rhubarb to lung is not clear. also the time regularity of the expression of HSP70 induced by ALI in animal model is still not illustrated. In order to confirm that HSP70 has the relation to the protective effect of lung by rhubarb after ALI and provide the theoretic base of treating treat acute lung injury, this experimental studied the expression of HSP70 induced by LPS in lung injury in rhubarb precaution group and rhubarb therapy group through the technique of immunohistochemistry and Western blotting. Animals and Group:a total of 60 healthy wistar rats, were divide into 4 groups randomly, including control group, ALI group, rhubarb preventive medication group and rhubarb therapeutic group. After narcotism,0.9% normal sodium (NS) or LPS was injected through internal jugular vein, the blood gas was analyzed with the blood taken from the abdominal aorta, then the rats were sacrificed by cervical dislocation, then the specimens of lung were taken out to determine the pulmonary coefficient and HE-staining and immunohistochemistry (SP method) as well as Western-Blotting were made for the following procedures.Results analysis:positive materials was detected in cytoplasm or nucleus by immunohistochemistry, positive cells were identified and counted in 10 randomly selected fields in the section of each groups under microscope at a 400-fold magnification then the ratio of positive cells was calculated. The stripe of western-blotting was analyzed by gray assessment. The ratio of positive cell, western-blotting gray values, blood gas analytic results and pulmonary coefficients are all expressed by mean±standard deviation(χ±SD),the data were analyzed using SPSS for windows 11.0 with a significant level of P＜0.01 or P＜0.05.Results1,Blood gas analysisThe difference in every time interval in the control group is not clear. Compared with the control group, he results in LPS group decrease, especially in the 1,2 hours. PO2 of the rats injuried by LPS was ameliorated significantly in preventive medicationgroup.2,Pulmonary coefficients:In the LPS group, the pulmonary coefficients increases and are different from control group, those in preventive medicationl group and treatment medical group are lower than the injury group with significant differences. 3,Macroscopic findings and HE stainingIn LPS group, patching-like necrosis, congestion of the whole lungs, the edema of pulmonary septums, hemorrhage in alveolar space and bronchi can be observed. In the preventive medication group and treatment group, those findings are disappeared or amelinated, especially in the preventive medication group. In LPS group, infiltration of a large amount of red blood cells and neutrophils, edema around the blood vessels and the bronchi, the formation of hyaline membrane can be observed at later intervals. In the preventive medication group and treatment medical group, histopathology changes can be improved significantly except infiltration of inflammatory cells.4,immunohistochemical stainingIn the normal group, HSP70 antigen mainly distributes at the epithelial cells of the bronchi and bronchiole, positive staining can be detected in the alveolar cell and macrophages. In the LPS group, enhanced positive dyeing can be observed in macrophages and infiltrative neutrophils, the positive result of the alveolar epithelial cells increases slightly. Compared with the control group, the differences beween the ratio of positive cells are significant. In the preventive medication group and treatment medical group, the amount of positive cells increase.5,western-blottingHSP70 strap locates at about 70KD, The amount of HSP70 in the preventive medical group and treatment medical group are deeper and wider than the control group and LPS group. In addition, the differences between the preventive medical group and treatment medical group are significant.(P<0.01).DiscussionAcute pulmonary injury specialized by the accumulation of neutrophil in the lung, intravascular coagulation, edema of lung caused by increasing permeability of pulmonary capillary which induce hypoxemia. LPS can also increase the biological effect of various active factors mediated by itself and aggravate the injury of lung and clinical symptom. Heat shock protein (HSP) can improve the ability for stress, protect the cells by preventing the influence of harmful factors, in addition, decrease the fatality of animal. In our experiment, increasing expressions with HSP70 can be detected in preventive medication group, the ratio of positive cells by immunohistochemistry and the result of Western-blotting are both higher than the LPS group. Moreover, the results of blood gas analysis, pulmonary coefficients and the pulmonary pathologic impairment are all improved, although the degree of pathological amelioration in therapeutic medication group is less than preventive medication group, which shows that the expression of HSP70 can be induced by rhubarb and the preventive effect of lung by rhubarb is through enhancing the expression of HSP70. in addition, our experimental result also suggests a significant application of rhubarb for the treatment of ALI in clinics.ConclusionOur studying suggests that pulmonary injury is more obvious in LPS group and the injury was having an outstanding amelioration in pulmonary function in preventive medication group and a higher expression of HSP70 in therapeutic medication group than in LPS group, furthermore HSP70 has a strong positive expression in tunica mucosa bronchiorum, mucous membrane of bronchiole and blood vessel endothelium, which suggest that HSP70 probably play a pivotal role in the decreasing of permeability of blood vessels and keeping the normal ventilation. Our study also showed that injection of rhubarb before LPS can increase a higher expression of HSP70 in lung which cause an obvious pulmonary protection. While the expression of HSP70 has a slight increase in the therapeutic medication group and has an outstanding differences with preventive medication group, which demonstrate that the injection of rhubarb at the same time of ALI can not obviously improve the function of lung. The experimental results explains a significant application of rhubarb for the treatment of ALI in clinics and also suggests that the protective function of rhubarb to ALI is probably through inducing the high expressions of HSP70.