Role Of Macrophages In Acute Lung Injury In Obese Rats With Acute Pancreatitis

Part?:Establishment of acute lung injury model rats with acute pancreatitisObjective: To establish acute lung injury(ALI)model in obese rats with acute pancreatitis(ANP)by feeding high-fat diet and retrograde injection of 5% sodium taurocholate into biliopancreatic duct.Methods: Thirty SPF SD rats,weighing 200-220 g,were randomly divided into five groups: normal group(N group,n=6),standard diet normal group(S-N group,n=6),standard diet ANP6 h group(S-ANP6 h group,n=6),high fat diet normal group(H-N group,n=6),high fat diet ANP6 h group(H-ANP6 h group,n=6).The rats of N group were adapted for two days before killed for materials.The rats of S-N and S-ANP6 h groups were fed with common diet for eight weeks,the rats of H-N and H-ANP6 h groups were fed with high fat for eight weeks.The rats of S-N and H-N groups were fed without any treatment,S-ANP6 h and H-ANP6 h groups were retrograde injection of 5% sodium taurocholate solution(STC)to establish the ANP model after eight weeks.The rats were injected of saline(20ml/kg)for subcutaneous after the ANP model establishment.The rats were killed and blood was taken from inferior vena cava six hours later after the establishment of ANP model.The serum levels of AMY,LIP,TG and TC were detected by automatic biochemical analyzer.The pancreas and lung tissue of rats were paraffin-embedded and stained with HE method,pathological changes of pancreas and lung tissues were observed and scored under light microscope.Results: After 8 weeks of feeding,the body weight of rats in H group was significantly higher than that in S group,and the difference was statistically significant(P<0.05).The adipose tissue in abdominal cavity of H group was more than that in S group,and the saponification spot in abdominal cavity of H-ANP6 h group was more than that of S-ANP6 h group.The serum levels of TG and TC in H-N group were higher than those in S-N group,and the difference were statistically significant(P<0.05).The pathological of pancreas and lung tissue in H-ANP6 h group were more serious than that in H-N groups,and pathological score was significantly higher than that in H-N group(P<0.05).Conclusion: Acute lung injury model of obesity with acute pancreatitis can be established by feeding high-fat diet combined with retrograde injection of sodium taurocholate into biliopancreatic duct.Part?:Comparison of lung injury between obese rats with acute pancreatitis and non-obese rats with acute pancreatitisObjective: After feeding with high-fat diet or standard diet for 8 weeks,the acute pancreatitis(ANP)model was established by retrograde injection of 5% sodium taurocholate solution into biliopancreatic duct.The acute lung injury(ALI)pathological changes of rats fed with high-fat and standard diet were compared.Methods: Forty-eight SPF SD rats,weighing 200-220 g,were randomly divided into eight groups: standard diet normal group(S-N group,n=6),standard diet ANP3 h group(S-ANP3 h group,n=6),standard diet ANP6 h group(S-ANP6 h group,n=6),standard diet ANP12 h group(S-ANP12 h group,n=6),high fat diet normal group(H-N group,n=6),high fat diet ANP3 h group(H-ANP3 h group,n=6),high fat diet ANP6 h group(H-ANP6 h group,n=6),high fat diet ANP12 h group(H-ANP12 h group,n=6).The rats in S-N and H-N groups were fed with standard diet or high-fat diet for 8 weeks respectively,and then were killed directly.The S-ANP and H-ANP rat model were retrograde injection with sodium taurocholate solution into the biliopancreatic duct after 8 weeks of feeding standard diet or high-fat diet at corresponding time points(3h,6h,12h)respectively.The blood were taken from inferior vena cava and serum levels of AMY,LIP,TG,TC and FFA were detected by automatic biochemical.The right middle lobe of lung was taken to calculate the wet-dry ratio of lung tissue.The rat pancreas and lung tissues were paraffin-embedded and sectioned,staining with HE methods.The pathological changes of pancreas and lung tissues were observed and scored under light microscopy.The serum contents of MDA and SOD were measured by kits.The levels of CD68~+CD11c~+,MPO,IL-1?,IL-6 and IL-10 in lung tissues were detected by immunofluorescence method.The levels of TNF-? in lung tissues were detected by immunohistochemistry method.Results: Compared with rats fed with the same diet,the pathological damage of pancreas and lung tissue was the most serious,the lung wet-dry ration,the serum MDA levels,lung tissue CD68~+CD11c~+ and MPO levels were highest,while the serum SOD levels was the lowest in S-ANP12 h and H-ANP12 h groups,the difference had statistical significance(all P < 0.05).Compared with S-ANP group,the pathological change of pancreas and lung tissue were more serious,the levels of lung tissue CD68~+CD11c~+,MPO and serum MDA were higher,the level of serum SOD were lower in the H-ANP group,the difference had statistical significance(all P<0.05).Conclusion: Obesity can aggravate the degree of acute lung injury(ALI)during the acute pancreatitis(ANP),which may be related to the more severely inflammatory response in lung tissue during the acute pancreatitis with obesity.Part ?: The role of macrophages in acute lung injury in obese rats with acute pancreatitisObjective: To establish acute pancreatitis(ANP)model in obese rats and explore the role and possible mechanism of macrophages in acute lung injury(ALI)in obese rats with acute pancreatitis.Methods: Twenty-four SPF SD rats,weighing 200-220 g,were randomly divided into four groups: high fat diet normal group(H-N group,n=6),high fat diet sham operation 12 h group(H-SO12 h group,n=6),high fat diet ANP 12 h group(H-ANP12 h group,n=6),high fat diet pretreatment ANP 12 h group(H-ANPT12 h group,n=6).The rats of H-N group were fed with high-fat diet for 8 weeks and then killed directly.The rats of H-SO12 h group were fed with high-fat diet for 8 weeks and sacrificed 12 h later after laparotomy.The ANP model was prepared by retrograde injection of 5% sodium taurocholate solution into biliopancreatic duct after feeding with high-fat diet for 8 weeks.The rats of H-ANP12 T group,TLR4 inhibitor(TAK-242)was intraperitoneally injected 30 minutes before the establishment of ANP model.The blood were taken from inferior vena cava and serum AMY,LIP were detected by automatic biochemical instrument.The right middle lobe of lung were taken to detected lung wet-dry ratio.The pancreas and lung tissues were taken for paraffin embedding and stained with HE methods.Pathological changes and scores of pancreas and lung tissues were observed under light microscope.The levels of SP-A,SP-D,MPO,IL-1?,IL-6,IL-10,CD68~+CD11C~+,CD68~+CD206~+,CD68~+TLR4~+,CD68~+NLRP3~+ and TLR4~+NLRP3~+ in lung tissue were detected by immunofluorescence method.The levels of TNF-?,NF-?B in lung tissue were detected by immunohistochemical method.The levels of SP-A,SP-D in lung tissue were detected by Western bloot method.Results: Compared with H-N group,lung pathological damage were aggravated,lung wet-dry ration increased,levels of SP-A and SP-D decreased in H-ANP12 h group,and the difference was statistically significant(all P<0.05).Also,the levels of MPO,IL-1?,IL-6,CD68~+CD11C~+,CD68~+TLR4~+,CD68~+NLRP3~+ and TLR4~+NLRP3~+ in lung tissue of H-ANP12 h group were increased,and the difference were statistically significant(all P<0.05).Compared with H-ANP12 h group,lung pathological damage were alleviated,lung wet-dry ration decreased,the levels of SP-A and SP-D increased in H-ANPT12 h group,and the difference were statistically significant(all P<0.05).Also,the levels of MPO,IL-1?,IL-6,CD68~+CD11C~+,CD68~+TLR4~+,CD68~+NLRP3~+ and TLR4~+NLRP3~+ in lung tissue of H-ANP12 h group were decreased,and the difference were statistically significant(all P<0.05).Conclusion: Release of a large number of inflammatory factors in lung tissue can lead to acute lung injury in obesity with acute pancreatitis,which may be related to the activation of TLR4/NLRP3 signaling pathway in macrophages of lung tissue.