Changes and its significance of autophagy in rats with acute necrosis pancreatitis / 中华消化杂志

ABSTRACT

Objective To investigate the changes and significance of autophagy in rats with experimental acute necrosis pancreatitis (ANP).Methods According to method of random number,18 rats were randomly divided into control group,ANP group,ANP+rapamycin (RAP) group.The ANP rat model was established by intraperitoneal injection of 20％ L-arginine.The rats of ANP+RAP group were intraperitoneal injected with RAP 1.2 mg/kg at 30 minutes before modeling.The rats of control group were intraperitoneal injected with 0.9％ NaCl solution.The blood was drawed from the hearts nine hours after modeling for subsequent experiments.Serum levels of trypsinogen activation peptide (TAP),interleukin (IL-1),IL-6 and tumor necrosis factor (TNF) α were measured with enzyme-linked immunosorbent assay.The pancreatic tissues were pathologically scored.Autophagy-related structures in rat pancreatic acinar cells were observed by transmition electron microscopy.The expression of autophagy marker microtuble assciated protein 1 light chain 3 (LC3)-Ⅱ and Beclin-1 at mRNA and protein level were measured by quantitative real-time polymerase chain reaction (qRT-PCR),Western bloting and immunohistochemistry.The single factor analysis of variance was used for mean comparison among groups.Results A rat model of ANP was successfully established.Histopathological score of pancreas acinar cell necrosis of ANP+RAP group (2.19±1.38) was higher than that of ANP group (0.97±0.68),and the difference was statistically significant(F=33.75,P＜0.05).The results of Western blotting indicated that the protein expression of LC3-Ⅱ and Beclin-1 in ANP group (35.25±2.68 and 49.40±5.28)were higher than those in control group (1.54±0.16 and 0.78±0.06),furthermore the expressions in ANP+RAP group(123.53±3.21 and 76.41±3.80) were higher than those in ANP group,and the differences were statistically significant(F=2 045.54,326.87,both P＜0.01).Immunohistochemistry results also indicated that the LC3Ⅱ and Beclin-1 expression at protein level of ANP+RAP group (7 570.63±4 357.67 and 3 418.09±2 035.78) were higher than those of ANP group (1 926.53±1 414.44 and 536.11±403.10),and the differences were statistically significant (F=39.83,41.58,both P＜0.01).The expression of Beclin-1 at mRNA level of ANP group (107.12±29.10) was statistically higher than that of control group(7.01 ±3.39),and the difference was statistically significant (F=3.61,P＜0.05),but the expression of ANP+RAP group (97.63 ± 65.38)was no significant difference compared with ANP group.However,the expression of LC3-Ⅱ at mRNA level of ANP+ RAP group (4.37 ± 1.67) was statistically higher than that of ANP group (1.76 ± 1.59),and the difference was statistically significant(F=16.10,P＜0.05),but the expression of ANP group was no significant difference compared with control group (1.51 ±0.95).The result of electron microscopy showed that autophagy related structures increased in ANP group compared with that of control group,which of ANP+RAP group was more.The serum levels of TAP,IL-1 and IL-6 of ANP + RAP group were (36.47 ± 1.71) pmol/L,(122.88± 26.67) pg/mL and (107.39±13.95) pg/mL,which were all higher than those of ANP group ((25.63 ± 6.05) pmol/L,(98.06 ±9.29) pg/mL and (86.16± 7.20) pg/mL),and the differences were statistically significant (F=116.71,50.45,79.67; all P＜0.01).There was no significant difference in TNFα between ANP+ RAP group ((140.80±60.82) pg/mL) and ANP group ((105.23±6.95) pg/mL,F=14.76,P＞0.05).Conclusions Autophagy increased in rats with ANP.Promoting autophagy could significantly activate trypsinogen,aggravate pancreatic injury and increase inflammation reaction,which indicated that autophagy might involve in the pathogenesis of ANP through trypsinogen activation.