Establishment Of An Acute Necrotizing Pancreatitis-associated Liver Injury Model And Lung Injury Model Of Rats And The Study On Correlative Inflammatory Cytokines

Object：(1) To establish a rat model of severe acute pancreatitis-associated liver injury by occludingthe head of pancreas.(2) To establish a rat model of severe acute pancreatitis-associated lung injuryby occluding the head of pancreas.(3) To detected the levels of HIF-1α mRNA, iNOS RNA, NO,MDA and MPO in the lung of the rats with severe acute pancreatitis-associated lung injury. Andinvestigate the effect of HIF-1α, iNOS and NO in the SAP-LI, further provide the basis for treatmentof SAP-LI.Methods：(1)180rats were randomly divided into control group (n=60), sham-operated group (n=60),liver injury model group (n=60). Then divided each60rats into six groups randomly, include0h(n=10),6h (n=10),12h (n=10),18h (n=10),24h (n=10),36h (n=10). The head of pancreas wasoccluded by vessel forceps of16centimeters long for two hours in the liver injury model group. Thecontrol group and the sham-operated group were established at the same times. The blood samplets,liver tissue and pancreatic tissue were collected at each group. The levels of serum amylase (AMY),alanine aminotransferase (ALT), aspartate aminotransferase (AST) and the percentage of thepolymorph nuclear neutrophil were detected. Pathological changes of the liver and pancreatic wereobserved under light microscope.(2)210rats were randomly divided into control group (n=70),sham-operated group (n=70), SAP-LI model group (n=70). Then divided the70rats in every groupinto seven groups randomly. These Groups include0h (n=10),6h (n=10),12h(n=10),18h (n=10),24h (n=10),36h (n=10),48h (n=10). The head of pancreas was occluded by vessel forceps of16centimeters long for two hours in the SAP-LI model group. The rats in the sham-operated group weremade laparotomy at the relevant times and the pancreases were turned over slightly for10times andthen put the pancreas back. We did nothing to the control group but anesthetized for3hours. Theperipheral blood, pulmonary alveolar lavage fluid, lung tissue and pancreatic tissue were collected atthe relevant times. The level of AMY, the protein content of serum, the percentage of the polymorphnuclear neutrophil, lung tissue wet/dry weight ratio, the protein content in the pulmonary alveolarlavage fluid were determined. Pathological changes of the lung tissue and pancreatic tissue wereobserved under light microscope.(3)210rats were randomly divided into control group (n=70),sham-operated group (n=70), SAP-LI model group (n=70). Then divided the70rats in every groupinto seven groups randomly. These Groups include0h (n=10),6h (n=10),12h(n=10),18h (n=10),24h (n=10),36h (n=10),48h (n=10). The head of pancreas was occluded by vessel forceps of16centimeters long for two hours in the SAP-LI model group. The SAP-LI model was established byoccluding method. The blood of the rats, pulmonary alveolar lavage fluid, lung tissue were collected atthe relevant times. The protein content of serum, lung tissue wet/dry weight ratio, the protein contentin the pulmonary alveolar lavage fluid were determined. The content of HIF-1mRNA and iNOSmRNA in the lung tissue of the rats was determined by qPCR. And the content of NO, MDA and MPO was also determined. Pathological changes of the lung tissue and pancreatic tissue wereobserved under light microscope.Results:（1）The level of AMY [(5052.1±114.9)U/L], the percentage of the polymorph nuclearneutrophil[(75.2±5.8)%], ALT[(52.6±5.9)U/L] and AST [(629.5±57.4)] reached the peak value at12h. Compared with the other time groups, the liver injury group was significantly (P<0.05). Thetissue damage of pancreas became more and more serious from the beginning of the model. The injuryof the liver was the most serious at24h. Serum amylase showed a correlation with the percentage ofthe polymorph nuclear neutrophil，ALT and AST (r=0.796, P<0.01; r=0.710, P<0.01; r=0.875,P<0.01). The score of the pancreatic tissue pathology showed a correlation with the score of the livertissue pathology (rs=0.919, P<0.01).(2) The level of serum amylase (5052.1U/L±114.9U/L, P<0.01)and the percentage of the polymorph nuclear neutrophil (75.2%±5.8%, P<0.05) reached the peak valueat12h, and the protein content in the pulmonary alveolar lavage fluid/the protein content of serumratio reached the peak value at36h in the SAP-LI mold group (0.009021±0.000107, P<0.01). The lungtissue wet/dry weight ratio reached the minimum at36h in the SAP-LI mold group (1.2001±0.0443,P<0.01). Compared with the control group and the sham-operated group, the SAP-LI group wassignificantly (P<0.05). The tissue damage of pancreas became more and more serious from thebeginning of the model. The injury of the lung was the most serious at36h. Serum amylase showed acorrelation with the percentage of the polymorph nuclear neutrophil, lung tissue wet/dry weight ratio,the protein content in the pulmonary alveolar lavage fluid/the protein content of serum ratio (r=0.788,P<0.01; r=0.365, P=0.002). The percentage of the polymorph nuclear neutrophil showed a correlationwith the lung tissue wet/dry weight ratio (r=0.348, P=0.003). The lung tissue wet/dry weight ratioshowed a correlation with the pulmonary alveolar lavage fluid/the protein content of serum ratio(r=-0.717, P<0.01). The score of the pancreatic tissue pathology showed a correlation with the scoreof the lung tissue pathology (r=0.934, P<0.01).(3) The protein content in the pulmonary alveolarlavage fluid/the protein content of serum ratio reached the peak value at36h in the SAP-LI moldgroup (0.009021±0.000107, P<0.01). The lung tissue wet/dry weight ratio reached the minimum at36h in the SAP-LI mold group (1.2001±0.0443, P<0.01). The content of HIF-1α mRNA, iNOSmRNA and NO in the lung tissue of the rat reached the peak value at24h in the SAP-LI mold group(0.020279±0.000625, P<0.01;0.029780±0.000498, P<0.01;7.505±0.481, P<0.01). The content ofMDA and MPO in the lung tissue of the rat reached the maximum at36h in the SAP-LI mold group(6.819±0.810, P<0.01;5.089±0.523, P<0.05). Compared with the control group and thesham-operated group, the differences were considered statistically significant at p<0.05. The injury ofthe lung was the most serious at36h. The lung tissue wet/dry weight ratio showed a correlation withthe pulmonary alveolar lavage fluid/the protein content of serum ratio (r=-0.717, P<0.01). The lungtissue wet/dry weight ratio showed a correlation with the MPO and MDA (r=-0.636, P<0.01; r=-0.497, P<0.01). MDA showed a correlation with the protein content in the pulmonary alveolar lavage fluid/the protein content of serum ratio (r=0.661, P<0.01). NO showed a correlation with the MPO, MDA,iNOS, HIF-1α and the protein content in the pulmonary alveolar lavage fluid/the protein content ofserum ratio (r=0.721, P<0.01; r=0.529, P<0.01; r=0.695, P<0.01; r=0.642, P<0.01; r=0.781, P<0.01).HIF-1α showed a correlation with MPO, iNOS, MDA and the protein content in the pulmonaryalveolar lavage fluid/the protein content of serum ratio (r=0.387, P<0.01; r=0.99, P<0.01; r=0.269,P<0.05; r=0.413, P<0.01). iNOS showed a correlation with MPO, MDA and the pulmonary alveolarlavage fluid/the protein content of serum ratio (r=0.457, P<0.01; r=0.323, P<0.01; r=0.495, P<0.01).MPO showed a correlation with MDA and the protein content in the pulmonary alveolar lavage fluid/the protein content of serum ratio (r=0.595, P<0.01; r=0.884, P<0.01).Conclusion:（1）A new rats model of acute necrotizing pancreatitis-associated liver injury byoccluding the head of pancreas have been established. The injury of the liver was most serious at24h.The24h is the best time that we establish the model. It is the right model for the research of acutenecrotizing pancreatitis-associated liver injury at nosogenesis and therapy.(2) A new rats model ofsevere acute pancreatitis-associated lung injury by occluding the head of pancreas have beenestablished. The injury of the lung was the most serious at36h. The36h is the best time that weestablish the model. It is the right model for the research of the severe acute pancreatitis-associatedlung injury at nosogenesis and therapy（.3）The injury of the lung was most serious at36h. The contentof MDA and MPO in the lung reached the peak value at36h. The content of HIF-1α mRNA、iNOSmRNA and NO in the lung tissue of the rat reached the maximum at24h. So we conferred that HIF-1αplayed an important role in the initial process of lung injury. The indirect injury effect of iNOS andNO play an important role. HIF-1α, NO and iNOS are related with the activity of polymorph nuclearneutrophil, lipid peroxidation and the exudation of plasma protein in the lung tissue. The activity ofpolymorph nuclear neutrophil are related with lipid peroxidation. The activity of polymorph nuclearneutrophil and lipid peroxidation are related with the exudation of plasma protein in the lung tissue.