Protective Effect Of Ulinastatin On Autophagy-associated Proteins Expression In Acute Lung Injury Induced By Remote Limb Ischemia-reperfusion In Rats

Limb ischemia-reperfusion (LIR) was very common in clinical surgery, such as recanalization after artery injury or embolism, replantation of ampulated limbs, limb trauma and application of the tourniquet for a long time and so on. It’s worthy of attention that the disability rate and mortality rate caused by limb ischemia injury was very high in the earthquake. The key thing of saving ischemic limb was restoring circulation of blood as soon as possible, but a large number of studies had shown that LIR not only affected the survival and function of local ischemic tissue but also could result in systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). Lung was one of the most vulnerable organs, easy to cause acute lung injury (ALI).For LIR secondary remote organ, damage mechanism was unclear. In recent years, medical researchers focused on the aspect of injury and oxidative stress for the research of the mechanism upon LIR resulted in ALI and considered that the center of the link which caused the damage was not only lager production of reactive oxygen species (ROS), but also a series of chain reaction including activation of neutrophile granulocyte, generation of inflammatory mediators and increment of protein enzymes which all triggered by production of oxygen free radicals. Remote organ damage similar to LIR damage meant that the reperfusion period was serious than ischemia period and the degree of damage was associated with ischemia time. The research thought that under normal temperature skeletal muscle was the easiest ischemic tissue of body which could tolerate ischemia only 4 hrs.Medical experts considered apoptosis and necrosis as the pathological result of cells of ischemia-reperfusion (I/R) tissue. But as this topic was further researched, people considered that autophagy could be induced by a variety of damage and oxidative stress. In 1977, Hourdry and his colleagues found autophagy in dying cells for the first time, and put forward the hypothesis that autophagy may promote cell survival. The relationship between autophagy and apoptosis was a hot spot in the research of the life science, but the research concerning LIR-ALI and autophagy was little. In recent years, most of the research confirms that autophagy not only can protect cell of poor nutrition effectively, but also can promote cell survival. Also found that, under certain conditions, inhibiting autophagy can induce cell apoptosis, and raising autophagy can prevent apoptosis.Autophagy plays distinct roles in I/R process as a new method of programmed cell death (PCD), the formation of autophagy some is the key link of autophagy. The occurrence of autophagy needed the participation of many key autophagy related genes (ATG). And there are many measuring methods of expressing activity of autophagy at present; therein, the most detection method of autophagy is using western blot detect microtubule-associated protein light chain3 (LC3). LC3-I/II and Beclin-1 were two important molecules relating to autophagy and were widely used to study the occurrence and development of autophagy. LC3-II was considered to be specific molecular markers of autophagic cells death, especially the number of LC3-II levels was proportional to the number of autophagic vacuoles levels. Therefore, we could accord the level of LC3 to estimate the size of autophagy role. High expression of inflammatory markers induced autophagy in Pulmonary vascular cells, with conversion and aggregation of LC3-II increasing the amount of autophagy further increased thereby caused pulmonary bronchial epithelial cells death and tissue damage.Ulinastatin (UTI) is the extraction of glycoprotein from human urine, is a broad-spectrum enzyme inhibitor, low molecular UTI degradation products is still efficient inhibitory effects on the enzyme. UTI can inhibit a variety of hydrolytic enzyme activity and stabilize physiological function of cell membranes and lysosomal membrane; UTI can inhibit the excessive inflammatory reaction, improve circulation organ perfusion, protect tissues and organs function. Clinically, UTI was first used for the treatment of severe acute pancreatitis, now is widely used in the treatment of SIRS, MODS and ALI/ARDS, which can improve pulmonary gas exchange function, prevent lung function deterioration. Various kinds of injury and oxidative stress can be induced cell autophagy, and upregulating of autophagy may prevent apoptosis. Because Autophagy lysosome is acid hydrolase, we should speculate that UTI can inhibit the autophagy lysosome, regulate excessive autophagy and protect LIR-ALI effectively.The purpose of this study is to establish ALI-LIR model in rats, to observe the autophagic activation in different reperfusion time, to discuss the role of autophagy and to explore the proof of excessive autophagy inhibited by UTI in ALI-LIR. So we can provide effective therapeutic targets to reduce distant tissue and organ damage induced by LIR.Part 1:Study of Expression at Autophagy-associated Proteins Level in Rats with Acute Lung Injury Induced by Remote Limb Ischemia-reperfusionObjectiveTo explore the early expression of autophagy-associated proteins in lung tissues in acute lung injury (ALI) induced by remote limb ischemia-reperfusion (LIR) in rats.Method1.Twelve adult male Sprague-Dawly(SD) rats weighting 220～250 gram were made LIR models and divided randomly into two groups (6 in each group):Sham group and ischemia-reperfusion (I/R) group. After the rats were weighed and 3% sodium pentobarbital was injected into abdominal cavity of rats (the first dose of 40 mg/kg, maintenance dose of 20 mg/kg), we fixed anesthetized rats on the animal operating table. Sham group:bilateral femoral triangle skin was dissect, separated the femoral artery, used silk thread mark it and not ligate then sutured the wounds. I/R group:after separated the femoral artery, clipped femoral artery near inguinal ligament with non-invasive arteriole clip to make double lower limbs ischemia, sutured wounds then encircled and ligated the root of double lower limbs with moderately elastic rubber band to prevent collateral circulation, not only regarded could not touch palpable dorsals pedis pulse but also regarded feet cool and darken as a sign of ischemic successfully; opened the original incision after 3hrs, at this time removed arteriole clip to restore the blood of lower limbs to make it reperfusion for 4hrs, if pulses back and feet get red and warming occurred that meant that the LIR model was established successfully.2. We opened the chest at the end of 4hrs of reperfusion respectively, after that looking straight drew whole blood 3 to 4 ml by the puncture of apex afterwards putted them into centrifugal that’s spinning at 3000 rotations per minute at 4 degrees for 10 minute then taking the supernatant and cryopreserved lactate dehydrogenase (LDH) under test in 70 degrees below zero, bleeding all the time until the rats died.3. We putted double lung tissues in zero point nine percent sodium chloride solution under 4 degrees to soak, when lung tissues get white taked the left lung and placed in four percent paraformaldehyde more than 24 hours, then used paraffin to embed it and slice it after that specialist read the film which was treated by using immunofluorescence techniques adopting blind method under inverted fluorescence microscope to observe expression of immunofluorescence specific labeled antibody, Immunofluorescence intensity was Analyzed using Image J2x software.4. The expression of Beclinl protein and Atg5 mRNA in the lung tissues were detected by reverse transcription polymerase chain reaction (RT-PCR):Taked the right lung 100 milligrams and extracted total RNA by Trizol reagent kit, RNA synthesized cDNA using reverse transcription reaction, afterwards it was amplified in PCR.5. Expressions of LC3 protein were detected by Western blot method:We removed the remainder right lung tissue 100mg then saved it in the environment of 80 degrees below zero. The content of LC3 protein in the lung tissues were detected by immunoblotting (Western blot) and the ratio of protein content of LC3-II/GAPDH were detected too. After that the films were scanned or took photos.We analyzed stripe optical density values of the film by using gel image processing system.Result1. Measurement the serum lactate dehydrogenase (LDH) of rats:The level of serum LDH in I/R group (6.66±0.08)×103U/L were very higher than Sham group (2.35±0.03)×103U/L (P less than 0.01), at the same time showed that the rat’s models of LIR were established successfully.2. Immunofluorescence examination demonstrated disorder of lung tissue structure and the thickening of alveolar septum happened pulmonary consolidation in I/R group, but the above-mentioned damage was not observed significantly in Sham group. The level of immunefluorescence intensity in I/R group (9.58±0.50) is very higher than Sham group (5.54±0.23)(P less than 0.01), I/R group of lung tissue immune fluorescent labelled autophagy antibodies had specific expression.3. Measuring relative expression (CT value) of Beclinl and Atg5 mRNA regarding GAPDH genes as internal reference and 2-△△CT method was used to analyze the expression ratio of them:The ratio of Beclinl and Atg5 mRNA in I/R group and Sham group were 12.79 and 18.36 times respectively (P Less than 0.01).4. Detecting the contents of LC3 protein fluorescence banding figure by Western blot method and calculation the net optical density value and analysis the ratio of LC3-II and GAPDH:In Sham group, content of LC3-I, LC3-Ⅱ protein and the ratio of LC3-II and GAPDH in lung tissues of rats was 1699.65±50.85,5783.73±130.55 and 0.76±0.02 respectively (P less than 0.01); in I/R groups there were 6707.37±104.45,12961.46±500.96 and 1.72±0.07 and there were significantly higher compared with sham groups (P less than 0.01). But content of GAPDH protein in two groups were no significantly (P more than 0.05).Conclusion1. It was showed that the rat’s models of LIR were established successfully and ALI could be made effectively, so we could establish the rat’s models of LIR-ALI.2. Lung tissue immune fluorescent labeled autophagy antibodies had specific expression at the end of 4hrs of reperfusion after 3hrs of remote limb ischemia in rats.3. It was showed obviously that autophagy could be activated because the expression of Beclinl and Atg5 mRNA analyzed by 2-△△CT method were significantly higher; Also autophagy in lung tissue could be enhanced by LIR-ALI because of LC3-Ⅱ protein and LC3-II/GAPDH was obviously higher in I/R group.In a word, LIR lead to ALI could induce occurrence of autophagy, high expression of its relevant proteins and autophagy increase; continuous I/R easily caused autophagy inhibition.Part 2:Effect of Ulinastatin on Autophagy-associated Proteins Expression in Acute Lung Injury Induced by Remote LimbIschemia-reperfusion in RatsObjectiveTo investigate the protective effect of Ulinastatin (UTI) on expression of autophagy-associated proteins in lung tissues in LIR-ALI models in rats.Method1. Seventy-two adult male Sprague-Dawly (SD) rats weighting 220-250g were made LIR-ALI models and divided randomly into three groups (24 in each group): SO+NS group(sham operation and normal saline group), I/R+NS group(ischemia-reperfusion and normal saline group) and I/R+UTI group(ischemia-reperfusion and UTI group), each of the groups were divided 4 subgroups(0,2,4,8hr) respectively. As the methods of part 1, the rats were anesthetized and NS/UTI 1ml/5×104U/kg was injected through left internal jugular vein after 15 min at the end of 0,2,4,8hr of reperfusion after 3hrs of ischemia and the tissues of lung were removed.2. The serum LDH were detected with enzyme linked immunosorbent assay(ELISA) and the pathological changes of lung tissues were observed by using immunofluorescence techniques; The expression of Beclinl protein and Atg5 mRNA in the lung tissues were detected by reverse transcription polymerase chain reaction (RT-PCR) and analyzed by 2-△△CT method; Microtubules associated protein light chain 3(LC3) in the lung tissues were detected by Western blot test.Result1. The levels of serum LDH in I/R groups were very higher than Sham groups (P less than 0.01) at the same time showed that the rat’s models of LIR were also established successfully.2.. Immunofluorescence examination demonstrated disorder of lung tissue structure at time of 0,2,4 hours and the thickening of alveolar septum, point of 8 hours alveolar lumen narrowed considerably even happened pulmonary consolidation in I/R+NS groups, but the above-mentioned damage was not observed significantly in SO+NS and I/R+UTI groups.3. The expression of Beclinl protein and Atg5 mRNA in lung tissues were not significant in SO+NS groups (P more than 0.05), very higher at 4,8hrs in I/R+NS groups (P less than 0.01 or P less than 0.05) and obviously higher at 4hrs in I/R+UTI groups (P less than 0.01). Compared with SO+NS groups, There were significantly higher at the same time in I/R+NS and I/R+UTI groups respectively (P less than 0.01); and compared with I/R+NS groups, the expression at 4hr were significantly lower in I/R+UTI group (P less than 0.01).4. The expression of LC3-Ⅱ the ratio of LC3-Ⅱ and GAPDH in lung tissues were very higher at 2,4,8hr in SO+NS groups (P less than 0.01), very higher at 4,8hrs in I/R+NS groups (P less than 0.01) and obviously higher at 4,8hrs in I/R+UTI groups (P less than 0.01). Compared with SO+NS groups, There were significantly higher at the same time in I/R+NS groups respectively (P less than 0.01); and compared with I/R+NS groups, the expression at the same time were significantly lower in I/R+UTI groups (P less than 0.01).Conclusion1. The model of LIR-ALI can be duplicated effectively and repetitively.2. Lung tissue immune fluorescent labeled autophagy antibodies had specific expression at the end of 0,2,4,8hrs of reperfusion after 3hrs of remote limb ischemia in rats. The relative expression of Beclinl protein and Atg5 mRNA measured by semi-quantitative RT-PCR method increased after 4hrs of reperfusion, levels of LC3-II and LC3-Ⅱ/GAPDH detected by Western blot method were also increased. All of this illustrated that autophagy was further enhanced and increased rapidly at 4hrs; it was high at 8 hrs but decreased, this showed autophagy was lower.3. UTI could depress the expression of Beclinl protein and Atg5 mRNA and decrease the levels of LC3-Ⅱ and LC3-Ⅱ/GAPDH, it is illustrated that the occurrence and development of autophagy could be constrained by UTI. Furthermore, therapeutic interventions used by UTI should be made at the early period of autophagy, especially in 4 hours.In conclusion, UTI has notable protective effects on LIR-ALI by inhibiting the expression of autophagy-associated proteins in lung tissues in rats.