| Part I: Established acute necrotizing pancreatitis induced lung injury rat model and studied on the expression and role of NADPH oxidase in lungObjective: To understand the situation of lung injury in ANP rats model,we established the acute necrotizing pancreatitis rats model,and measured the expression level of NADPH oxidase(NOX)in lungMethods: Adult male SPF SD rats(n=16,7–8 weeks,200-250 g),and maintained at room temperature under a 12 h light-dark cycle,with free access to standard laboratory rodent chow and sterile.All of the rats were randomly assigned into 2 groups,including Sham operation(SO)group,and acute necrotizing pancreatitis(ANP)group.Freshly prepared 5% STC solution(50 mg/kg)was retrogradely infused into biliarypancreatic duct through the duodenum,and then isotonic saline solution(20 m L/kg)was injected into the back subcutaneously to compensate for fluid loss.The rats were sacrificed after 12 h at the ANP modeling.All blood samples were collected through the posterior vena cava.The levels of serum LIPA and AMY were detected by automatic biochemical analyzer.A small amount of pancreatic tissue and right middle lung tissue were taken to measure the dry-wet ratio of pancreas and lung respectively.The tissues from the lung and pancreas were fixed with paraformaldehyde,embedded in paraffin and sectioned(5 cm)for hematoxylin-eosin(HE)staining.Pathological changes of lung and pancreas tissues were observed and scored in light mic ROScope.DNA fragmentation during lung tissue apoptosis was detected by TUNEL staining.Meanwhile,the expression of reactive oxygen species(ROS)in lung tissues was detected by immunofluorescence,and the expression levels of NOX2 and NOX4 in lung tissues were detected by Western Blot.Results: Compared with the SO group,the levels of serum AMY and LIPA were significantly increased in the ANP group(P<0.05).Pathological injury of lung and pancreas tissues was significantly aggravated,and pathological score was significantly increased in the ANP group(P<0.05).The ratio of dry to wet of lung tissue and pancreas tissue increased significantly in the ANP group(P<0.05).The apoptosis of lung tissue was also increased in the ANP group(P<0.05).The expression levels of ROS and NOX2 and NOX4 in lung tissues were significantly increased in the ANP group(P<0.05).Conclusion:1.After 12 h of retrograde injection of STC,the rats were showed pancreatic necrosis and lung tissue damage.2.After 12 h of retrograde injection of STC,the levels of NADPH oxidase and ROS in the lung tissues increased.Part Ⅱ: Study on the effect and mechanism of NOX in acute necrotizing pancreatitis in rats with lung injuryObjective: Use NADPH oxidase inhibitors Apocynin to intervene in ANP rats,in order to explore Apocynin protective effect of ANP rats with lung injury and its mechanism.Methods: Adult male SPF SD rats(n=16,7–8 weeks,200-250 g),and maintained at room temperature under a 12 h light-dark cycle,with free access to standard laboratory rodent chow and sterile.All of the rats were randomly assigned into 3 groups(n=8)including Sham operation group(SO),acute necrotizing pancreatitis(ANP)and Apocynin intervention group(APO).In ANP group,freshly prepared 5% STC solution(50 mg/kg)was retrogradely infused into biliary-pancreatic duct through the duodenum,and then isotonic saline solution(20 m L/kg)was injected into the back subcutaneously to compensate for fluid loss.In APO group,30 min before the model of ANP,Apocynin was injected(50 mg/ kg,i.v.)and the optimal dose was selected based on previous studies.In SO and ANP group,rats received a volume-matched 10% DMSO substituted for Apocynin.All blood samples were collected through the posterior vena cava.After centrifuging at 1500 g × 10 min,the supernatants were collected and stored at-80 °C.Serum amylase(AMY)and lipase(LIPA)levels were measured using an automatic biochemistry analyzer with standard techniques.Serum levels of IL-1β,TNF-α and IL-6 were determined by ELISA kits.Paraffin-embedded samples from the pancreas and lungs were sectioned and stained with HE.Pathological changes of the pancreas and lung tissues were observed and scored in light mic ROScope.The expression levels of MPO,NLRP3,Caspase-1,IL-1β and p-NF-κB p65 in lung tissues were also detected by immunofluorescence.Lung tissue protein was extracted and the expression levels of NLRP3,ASC,pro-Caspase-1,cleaved-Caspase-1,p-IκBα and p-NF-κB p65 were detected by western-blot.TUNEL assay was used to detect the level of apoptosis in lung tissue.Results:1.Compared with the SO group,the levels of serum AMY and LIPA in the ANP group were significantly increased(P<0.05).the expression levels of IL-1β、TNF-αand IL-6 in the serum of the rats were significantly increased in the ANP group by compared with the SO group(P<0.05).The scores of pancreatic and pulmonary histopathology in ANP group were significantly increased by compared with the SO group(P<0.05).Compared with the SO group,the expression levels of NLRP3,Caspase-1,IL-1β and p-NF-κB p65 in lung tissues were increased in ANP group(P<0.05).The level of MPO in lung in ANP group was significantly increased by compared with the SO group(P<0.05).The apoptosis index of lung cells in ANP group was significantly higher than that in SO group(P<0.05).Compared with the SO group,the expression levels of NLRP3,ASC,cleaved-Caspase-1,p-IκBα and p-NF-κB p65 in lung tissues in the ANP group were significantly increased(p <0.05).2.Compared with ANP group,the levels of serum AMY and LIPA in APO group were decreased(P<0.05).The expression levels of IL-1β、TNF-α and IL-6 in serum of APO group decreased significantly(P<0.05).Compared with ANP group,pancreatic and pulmonary histopathology scores were significantly lower than that of ANP group,and the degree of injury was significantly reduced(P<0.05).Compared with the ANP group,the expression levels of NLRP3,Caspase-1,IL-1β and p-NF-κB p65 in lung tissues of the APO group were significantly reduced(P<0.05).The expression levels of NLRP3,ASC,cleaved-Caspase-1,p-IκBα and p-NF-κB p65 in the lung tissue in APO group were significantly decreased by compared with ANP group(p <0.05).Compared with ANP group,the level of apoptosis in APO group was significant decreased(P<0.05).Conclusion:The possible mechanism of Apocynin alleviated ANP rats with lung injury may relate to the effect of the inhibition the NADPH oxidase activation and by inhibiting of the NF-κB signaling pathway activation,and then reduce the production of ROS,reduce the activation of NLRP3 and the apoptosis. |
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