Interleukin-22 Protects Mice From Acute Severe Pancreatitis-associated Lung Injury

ObjectiveTo investigate the potential protective effect of exogenous recombinant interleukin-22 (rIL-22) on L-arginine-induced acute severe pancreatitis (SAP)-associated lung injury and its possible signaling pathway.MethodsA total of 72 male Balb/c mice were deprived of food and received only water 12 h before the trial commenced. The mice were randomlyassigned tofour groups: normal control group (n=12), SAP group (n=36), treatment control group (phosphate-buffered saline (PBS) group, n=12) and treatment group (recombinant interleukin-22 (rIL-22) group, n=12). Mice in the SAP, PBS and rIL-22 groups were injected intraperitoneally (i.p.) twice with 20%L-arginine hydrochloride (Sigma-Aldrich; pH=7.0,4 g/kg bodyweight), at an interval of 1 h. The normal control group received physiological saline injections. PBS or rIL-22 (Miltenyi Biotech) (200 ng/per,5 times) was administered subcutaneouslyto mice at indicated times in the PBS and rIL-22 groups. Mice in the SAP group were killed at 24 h,48 h, and 72 h after the administration of L-arginine. The remaining mice were sacrificed at 72 h after the L-arginine injection.The survival rates in PBS and rIL-22 groups were calculated. Serum amylaselevels and myeloperoxidase (MPO) activity in the lung tissue, wet/dry proportions of the pancreatic and lungtissues in different groups were measuredafter the L-arginine administration. Histopathology of the pancreas and lung was evaluated by hematoxylin and eosin (H&E) staining. B cell lymphoma/leukemia-2 (Bcl-2), Bcl-xL and IL-22RA1 mRNA in the lung tissue were detected by real-time PCR. Expression and phosphorylation of STAT3 were analyzed by Western blotting.Results1. General situation in all groups and survival rates in PBS and rIL-22 groupsThe general situation of the normal control group was normal. Mice in SAP group and PBS group had reduced activity, arched back, piloerection, listlessness, polydipsia. The spirit of rIL-22 group was slightly worse and its survival rate 100.0% (12/12) was higher than that of PBS group 66.7%(12/12) (P< 0.05).2.Serum level of amylaseCompared with the normal control group, the level of serum amylase in the SAP group was increased markedly (P< 0.05). The highest level of serum amylase was detected at48 h after the injection of L-arginine. At 72 h after injection,the serum amylase level partially recovered. No significant decrease was found in the PBS group relative to the SAP group at 72 h after the injection (P> 0.05). However, the activity ofserum amylase in rIL-22 group was significantly lower than that of PBS group (P< 0.05).3.Myeloperoxidase activity analysisA gradual increase over time (24,48,72 h after the injection of L-arginine) was observed in the lung MPO activities relative to the normal control group (P< 0.05). The pre-treatment with PBS did not significantly affect the MPO activity compared to the SAP group at 72 h after the injection(P> 0.05). In contrast, rIL-22 significantly decreased the MPOactivities compared with the PBS group (P< 0.05).4. Wet/dry proportions of the pancreatic and lungtissuesCompared with the normal control group, the wet/dry proportions of the pancreatic and lungtissues in the SAP groupincreased progressively(all P<0.05). No significant decreases were found in PBS group compared with the SAP group at 72h after injection (P> 0.05). However, the wet/dry proportions of the pancreatic and lungtissues in rIL-22 group were both lower than those of PBS group (both P<0.05).5.Histologic characteristics of the pancreatic and lungtissuesMacroscopically, the pancreas was edematous 24 and 48 h after the L-arginine administration. By contrast, the pancreas was shrunken, with many saponification spots on theomentum majus and mesentery, some intestinal cavities expanding andbloody ascites,72 h after the injection of L-arginine and pre-treatment with PBS. Under the light microscope, mice in the SAP group 24 h after L-arginine injection exhibited interstitial edema and infiltration of a small number ofneutrophil and mononuclear cells. The acinar architecture and integrity were partially destroyed with focal parenchyma necrosis and hemorrhage. The vascular and pancreatic ductal structures appeared undamaged. At 48 h, interstitial edema, inflammatory cellular infiltration, parenchyma necrosis and hemorrhage were significantly aggravated. The severity of pancreatic destruction became maximal 72 h after L-arginine administration. About 70-80% of the pancreatic acinic cells had been destroyed and replaced by inflammatory and fibrotic cells. The pancreatic ducts were expanded and appeared more numerous because of a decrease in acini and shrinkage of the pancreatic tissue. Compared with the SAP group,PBS did not significantly reduce the pancreatic injury. However, the mice in the rIL-22 group showed slighter interstitial edema, acinar cell necrosis and cellular infiltration compared withthe PBS group.The macroscopic view of the lung showed significant edema and hemorrhage in the SAP and PBS groups. By contrast, pulmonary edema and hemorrhage in the rIL-22 group were not obvious.The H&E staining showed that no observable signs of lung damage in normal control group. The lungs in the mice 24 h and 48 h after the L-arginine injection showed no significant swelling, inflammation or necrosis. However, interstitial edema, patchy hemorrhage, thickened alveolar interstitium and infiltrations of inflammatory cells were markedly observed 72 h after the L-arginine injection. No significant difference in the degree oflung injury was found between the PBS and SAP groups at 72 h. In contrast,the pre-trcatment with rIL-22significantly reduced the degrees of edema, alveolar congestion and infiltration of inflammatory cells relative to the PBS group.6. Levels of Bcl-2, Bcl-xL and IL-22RA1 mRNA expression in the lung tissue in PBS group and rIL-22 groupCompared with the normal control group, the expression of Bcl-2 and Bcl-xL mRNAin the lung tissue in the SAP group showed a significant decrease at 48 h and 72 h after the L-arginine injection (P< 0.05). However, no significant decrease was found at 24 h after the injection relative to the normal control group (P> 0.05). IL-22RA1 mRNA expression significantly increased in the SAP group relative to that in the normal control group (P< 0.05). The lowest expression level was detected at48 h after the L-arginine injection. rIL-22 stimulated the expression of Bcl-2 and Bcl-xL mRNA, which could play a key role in preventing cells from apoptosis involved in tissue regeneration (P< 0.05). In addition, IL-22RA1 expression in the rIL-22 group was also significantly higher than in the PBS group(P< 0.05).7. Levels of STAT3 activation and total STAT3 expression in the lung tissue in PBS group and rIL-22 groupThe STAT3 activation in the lung tissue in rIL-22 group was higher than that in the PBS group. However, there was no obvious difference was found between the two groups as to the STAT3 expression.The ratio of p-STAT3 to STAT3 protein in rIL-22 group wassignificantly higher than that of the PBS group (P< 0.05).Conclusions1. High doses of L-arginine successfully induced SAP associated lung injury mice model.2. Exogenous recombinant IL-22 protected mice against L-arginine-induced SAP-associated lung injury by enhancing the expression of anti-apoptosis genes such as Bcl-2 and Bcl-xL through the STAT3 signaling pathway.