Crucial Role Of Group â…¡A Phospholipase A₂ In Pancreatitis-associated Adrenal Injury In Acute Necrotizing Pancreatitis

Crucial role of groupⅡA phospholipase A2 in pancreatitis-associated adrenal injury in acute necrotizing pancreatitis in ratsObjective:There is a clinical documented association between acute severe pancreatitis and adrenal insufficiency in patients. The underlying pathophysiology of adrenal insufficiency may be related to edema, ischemia, and necrosis of adrenal gland. We hypothesize this blunted adrenal response is due to hemorrhagic and necrosis of the adrenal parenchyma, and explore the mechanization of adrenal injury:(1) We establish the model of acute necrotizing pancreatitis in rats to observe the functional (serum corticosterone) as well as morphological and the adrenal fascicular zone ultrastructural changes; (2) sPLA2 is an inflammatory factor known to play a central role in the pathogenesis of acute pancreatitis. This study explored expression of sPLA2 in adrenal gland in acute necrotizing pancreatitis; (3) This study investigated the mechanistic role of sPLA2-ⅡA in the process of pancreatitis-associated adrenal injury in acute necrotizing pancreatitis.Methods:Pancreatitis model was induced by retrograde infusion of 5% sodium taurocholate into the bilio-pancreatic duct. In the controls, pancreas was reversed after a midline incision. Rats were killed at 0,3,6,12 and 24 hours time point. Adrenal weight, adrenal/body index, amylase were detected, and histology in pancreas and adrenal stained with H&E(hematoxylin and eosin stain) for light microscopy were analyzed. Serum corticosterone level was measured by an ELISA kit. Ultrastructural changes of adrenal cortical cells was obversed by TEM (transmission electron microscope). The sPLA2 activity of both serum and adrenal tissue was determined in full automatic biochemical analyzer. Immunohistochemical staining of groupⅡA secretory phospholipase A2 (sPLA2-ⅡA) was examined in section of adrenal tissue. The sPLA2-ⅡA protein analysis was quantitated by western blot. Pretreatment of sPLA2 inhibitor administered by intravenous injection 30 minutes before induction of pancreatitis. Effects of pretreatment of sPLA2 inhibitor (pku-mdl-101) in both pancreatitis and pancratitis-associated adrenal injury in rats were investigated by detecting those aspects. Results:1. The amylase, and pancreas pathologic score were increased significantly which indicate pancreatitis model was established successfully in pancreatitis rats. Adrenal/body index increased gradually in pancreatitis in rats. In pancreatitis rats, serum corticosterone levels was firstly stimulated from basic level to maximal level, declined subsequently, and reached to the bottom which was lower than the controls after 24 hours. Adrenal vessels and sinusoids opened obviously, and were hyperemia at 3 hours. Adrenal gland was damaged progressively, and microscopy showed hemorrhagic necrosis in pancreatitis animals after 24 hours. Under Electron microscopy, ultrastructure changes showed intercellular fissures, and swelling of mitochondria, endoplasmic reticulum bubble expansion and reduction of cytoplasmic lipid droplets in adrenal cortical cells. There were no significant changes in serum assay (amylase, corticosterone), pancreatic and adrenal pathology and ultrastructure in controls.2. After induction of acute necrotizing pancreatitis, sPLA2 activity in both serum and adrenal gland significantly increased firstly rapidly after 3h, to the peak after 6h,, the latter to decline from 6h to 24h. In Sham-operated rats, serum sPLA2 activity in both serum and adrenal gland maintained a low level, at each time point it was almost no significant changes. Compared with the corresponding sham-operated group at each time point, sPLA2 activity was significantly (P< 0.05). Immunohistochemistry of sPLA2-IIA showed that the adrenal cortex and medulla, the adrenal capsule, cytoplasm, and intercellular matrix was yellow, indicating that sPLA2-IIA is widely expressed in adrenal glands. sPLA2-IIA expression in adrenal cortical tissue during Pancreatitis group was significantly stronger than sham-operated group.3. Pretreatment of sPLA2 inhibitor significantly reduced the severity of pancreatitis (P＜0.05, vs ANP group) and adrenal histological injury (P<0.05, vs ANP group), improved the 24-hour serum corticosterone levels (P<0.05, vs ANP group), and effectively inhibited sPLA2 activity and sPLA2-IIA protein expression in adrenal glands (P＜0.05, vs ANP group)Conclusion:1. Pancreatitis-associated adrenal injury of function, morphology and ultrastructure was revealed in acute necrotizing pancreatitis model rats. Adrenal edema, hemorrhage, necrosis, and structure destruction, as well as the adrenal insufficiency took place with inflammation progress in acute necrotizing pancreatitis in rats. The adrenal injury may be responsible for the adrenal insufficiency in acute necrotizing pancreatitis.2. It showed weak expression of sPLA2-ⅡA in different adrenal zones in rats. The sPLA2 activity both in serum and adrenal tissue homogenate was significantly increased during acute necrotizing pancreatitis. Immunohistochemical expression of sPLA-ⅡA in adrenal cortex was significantly increased. It suggested that sPLA-IIA activation may be involved in pancreatitis-associated adrenal injury.3. The sPLA2 inhibitor attenuated the severity of pancreatitis and pancreatitis-associated adrenal injury. The observations indicate that sPLA2-ⅡA plays a crucial role in pancreatitis-associated adrenal injury in acute necrotizing pancreatitis.