Ascitic fluid and serum from rats with acute pancreatitis injure rat pancreatic tissues and alter the expression of heat shock protein 60

Abstract

Acute pancreatitis (AP) is an inflammatory process in which cytokines and chemokines are involved. After onset, extrapancreatic stimuli can induce the expression of cytokines in pancreatic acinar cells, thereby amplifying this inflammatory loop. To further determine the role and mechanism of irritating agents in the pathogenesis of AP, rat pancreatic tissues were stimulated with ascitic fluid (APa) and serum (APs) from rats with AP or with lipopolysaccharide (LPS). In addition, the alteration of heat shock protein 60 (HSP60) expression was evaluated. Rat pancreas was removed and meticulously snipped to fragments. The snips were cultured for up to 48 h. During this period, the tissue viability as well as amylase and TNF-alpha levels in the supernatant and the HSP60 expression in the pancreatic tissue before and after stimulation by APa, APs, and LPS were assayed time-dependently. At different time-points during the culture, the viability and the amylase activity in the pancreatic tissue remained largely stable. After stimulation with APa, APs, or LPS for 1 h, the pancreatic tissues showed some damage, and this was followed by a sharp decrease in the viability accompanied by increased levels of amylase and TNF-alpha in the culture medium 2 or 4 h after stimulation (p < 0.05). In contrast, both the HSP60 mRNA and protein levels had a relatively high expression in the freshly prepared tissue fragments (0 h). As the culturing period was extended, the expression of HSP60 mRNA decreased only slightly; at the same time, the HSP60 protein levels decreased over a prolonged culture time, significantly so from 12 through 48 h (p < 0.05). After stimulation with APs, APa, or LPS, both the expression of HSP60 mRNA and protein in the tissue fragments increased slightly at 1 h and decreased significantly thereafter at 2 and 4 h (p < 0.05). APa, APs, or LPS induce injuries on isolated pancreatic tissues, accompanied by an altered HSP60 expression pattern in a time-dependent manner.

Down-regulation of HSP60 expression by RNAi increases lipopolysaccharide- and cerulein-induced damages on isolated rat pancreatic tissues

“…The secretory response of the freshly cultured pancreatic tissue to the stimulation of high-dose cerulein was compatible to that of the freshly cultured acinar cells and similar to the in vivo reactions of the pancreas after stimulation by superphysiologic doses of cerulein including the co-localization of lysosomal hydrolase, the intracellular activation of trypsinogen, and the subsequent acinar cell injury (Bhagat et al , 2008Jaworek et al 2008). We have recently found that throughout the 48 h of in vitro culturing, the fragments of isolated rat pancreas retain the viability and the function of secreting amylase (Li et al 2010), confirming the feasibility of using the pancreatic tissue snips in in vitro experiments.…”

“…Under aseptic conditions, the pancreas was removed from anesthetized SD rats and rinsed with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH7.4) saturated with oxygen by bubbling, as described previously (Li et al 2010). The pancreas was then carefully snipped into pieces of <0.5 mm in diameter and resuspended in 6 mL Dulbecco's modified Eagle medium (DMEM; Invitrogen, California, USA) supplemented with 20% fetal bovine serum and 1% of penicillin-streptomycin solution (Invitrogen-Gibco, California, USA).…”

“…Treating the normal pancreatic tissues with LPS or cerulein In a previous study, Li et al (2010) showed that the isolated pancreatic tissues were viable for up to 48 h of culture and that tissues at 4 h of culturing were optimal for the stimulating experiments. In this study, different concentrations of cerulein (with final concentrations of 10 −5 , 10 −7 , 10 −10 , and 10 −11 mol/L) or LPS (with final concentrations of 20 and 10 μg/mL) were administrated to the isolated tissue snips after 4 h culture, and the responses of the tissue snips to the toxicants were observed.…”

“…Our purpose is to determine if the HSP60 protective function is essential to pancreatic tissue in pathological conditions. Cerulein is a cholecystokinin (CCK) analogue and can evoke, when in overdose, pancreatitis-like changes in vivo (Sidhapuriwala et al 2009), whereas LPS is a major component of the Gram-negative bacterial cell wall and can trigger endotoxin-induced shock and multiple organ damage, including pancreatic injuries (Li et al 2010;Jaworek et al 2008;Vaccaro et al 2000). Hence, these are the appropriate agents to create the conditions for experimental AP in animals.…”

Cannabinoid HU210 Protects Isolated Rat Stomach against Impairment Caused by Serum of Rats with Experimental Acute Pancreatitis

“…no: ALX-210-314 for anti-CB1 and Cat. no: ALX-210-315 for anti-CB2, Enzo, Plymouth Meeting, PA, USA) as described previously [18]. The slides with sections of rat stomach and pancreas were incubated overnight at 4°C with anti-CB1 or anti-CB2 antibodies, and the biotin-labeled goat anti-rabbit IgG working fluid (Cat.…”