| Objective:To investigate the role of endogenous antioxidant signaling pathway Nrf2/ARE in acute pancreatitis associated lung injury (APALI) induced by cerulein with subsequent LPS administration in mice.Methods:Ninety-six Babl/c mice were randomly allocated to three experimental groups:the normal control group (CON group), the normal saline group (NS group) and the severe acute pancreatitis group (SAP group). Each group was randomly divided into4time units (3,6,12,24h) with8mice in each time unit. Twenty-four hours before the start of the experiment, the mice were deprived of food but allowed access to water. The CON group mice received no treatment. In the NS group, mice were injected intraperitoneally (i. p.)10mL/kg BW normal saline repeatedly every1h (7injections in total) and the injection dosage were doubled at the last time. In the SAP group, mice were injected intraperitoneally50μg/kg BW cerulein (10mL/kg of BW) repeatedly every1h (7injections in total) and10mg/kg of BW LPS (10mL/kg of BW, i. p.) after the last dose of cerulein immediately. Eight mice of each group were anesthetized with sodium pentobarbital (90mg/kg BW, i.m.), then were executed at3,6,12and24h after the last injection. Serum amylase was assayed by P-nitrophenyl malt seven glycoside method, C-reactive protein (CRP) were measured by enzyme linked immunosorbent assay (Elisa), histological score of the pancreas and lung and the wet-to-dry weight ratio of lung were evaluated. The severity of lesions was evaluated by pancreatic and lung histology. Expression of Nrf2, HO-1, and NQO1in lung tissue was evaluated by real-time PCR and western blot analysis, respectively.Results:Compared with CON and NS group, the serum amylase, CRP, histological score of the pancreas and lung and the wet-to-dry weight ratio of lung increased significantly in SAP group(P<0.05). In SAP group mice, the pathology of pancreas showed interstitial edema, minor inflammatory cells infiltration both around vessels and in the pancreatic mesenchyme at3h while lobules of it were still clear to observe. All the changes above were more obvious at6h. The organizational structure of the pancreas was more disturbed at12h and24h as there was no discernable normal lobular architecture, and massive acinar cell necrosis, hemorrhage, vascular necrosis, elastic fiber disintegration, thrombosis, together with microabscess could be seen in the necrotic pancreas; The pathology of lung showed edema, congestion, widened alveolar septa at3h and6h. Besides, RBC exudation, severe inflammatory cell infiltration, severe hemorrhage and alveolar collapse were observed at12h and24h. Ultrastructure of lung tissue showed complete mitochondrial outer membrane and distinct mitochondrial cristae in type II alveolar epithelial cells, and there was no evacuation phenomenon in laminated bodies in the control and NS group. In the SAP group, mitochondria in type II alveolar epithelial cells swelled with membranes indiscernible and most of mitochondrial cristae disappeared at3h and6h; At12h and24h, alveoli were clearly dilated and merged, a large number of red blood cells and active substance leaked into the alveolar space, vacuolar degeneration could be seen in lamellar bodies in type II alveolar epithelial cells. In SAP group mice, mRNA expression of Nrf2, HO-1and NQO1was slightly increased at3and6h compared with it of the CON group mice at the corresponding time point, however, it decreased somewhat at12and24h, but there were all no statistical significance. The expression of protein in the SAP group mice had a similar trend as its mRNA.Conclusion:The endogenous antioxidant signaling pathway Nrf2/ARE can be activated in SAP, but expression of Nrf2and its downstream antioxidant gene HO-1and NQO1were not increased significantly and cannot maintain adequate time. Objective:To investigate the effects of BML-111on acute pancreatitis associated lung injury (APALI) induced by cerulein with subsequent LPS administration in mice and its possible mechanisms.Methods:One hundred twenty-eight mice were randomly allocated to four experimental groups:the SAP group (SAP group), the BML-111pretreatment group (SAP+BML group), the BM-111control group (BML group) and the normal saline group (NS group). Each group was randomly divided into4time units (3,6,12,24h) with8mice in each time unit. Twenty-four hours before the start of the experiment, the mice were deprived of food but allowed access to water. In the NS and BML group, mice were injected intraperitoneally (i. p.)10mL/kg BW normal saline repeatedly every1h (7injections in total) and the injection dosage were doubled at the last time. In the SAP group, mice were injected intraperitoneally50μg/kg BW cerulein (10mL/kg of BW) repeatedly every1h (7injections in total) and10mg/kg of BW LPS (10mL/kg of BW, i. p.) after the last dose of cerulein immediately, and one hour before the first injection of cerulein, the mice received intraperitoneal injections of0.4%DMSO (the menstruum for BML-111,10mL/kg BW). In the BML-111pretreatment group, the mice were administered BML-111(dissolved in0.4%DMSO solution,1mg/kg,10mL/kg BW) one hour before the first cerulein administration instead of0.4%DMSO, and the other was same as the SAP group. In the BML-111control group, identical to the BML-111pretreatment group were performed, except that the normal saline was administered instead of cerulein and LPS. In the NS group, identical to the SAP group except that the normal saline was administered instead of cerulein and LPS. Eight mice of each group were anesthetized with sodium pentobarbital (90mg/kg BW, i.m.), then were executed at3,6,12and24h after the last injection. Serum levels of amylase was assayed by P-nitrophenyl malt seven glycoside method, and TNF-a, IL-1β and IL-10were measured by enzyme linked immunosorbent assay (Elisa). Histological score of the pancreas and lung and the wet-to-dry weight ratio of lung were evaluated. Myeloperoxidase (MPO), malondialdehyde (MDA) and superoxide dismutase (SOD) of lung tissue were assayed by colorimetric analysis. Expression of Nrf2, HO-1and NQOl in lung tissue was evaluated by real-time PCR and western blot analysis, respectively.Results:The findings revealed that injury of pancreas and lung was typically induced by cerulein. BML-11pretreatment significantly reduced the levels of serum amylase, TNF-a, IL-1β, lung MPO and MDA, the wet-to-dry weight ratio and the pathology injury scores of the lung, which increased in the SAP group. The expressions of Nrf2, HO-1, NQ01and activity of SOD in lung tissue increased in the SAP+BML group compared with those in the SAP group, and the serum levels of IL-10were markedly increased.Conclusion:BML-111may play a critical protective role on APALI induced by cerulein. The underlying mechanisms of protective role may be attributable to its antioxidant and anti-inflammatory effects through activation of Nrf2/ARE pathway. BML-111pretreatment protected APALI induced by cerulein and these effects may attribute to the antioxidant effect of the antioxidant enzymes that were induced by activation of Nrf2/ARE signaling pathways. In addition, anti-inflammatory effect of BML-111may also be one of the reasons for these protective effects on APALI. |
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