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.

Fig. 1

The expression of HSP60 in pancreatic tissue in mouse with MAP and rats with SAP (paraffin sections were stained as described in the “Materials and methods” section; amplification ×400). In mice, MAP was induced by four hourly i.p. injections of cerulein, while SAP was induced in rats by retrograde injections of deoxycholate sodium into the biliopancreatic duct as described in the “Materials and methods” section. The pancreatic tissues from the animals with MAP or SAP showed varying degrees of edema, necrosis, and HSP60 staining (brown yellow). The experiments were performed in at least six animals

Fig. 2

Effects of LPS, APs, or APa on the viability of the pancreatic tissue (a) and on the amylase (b) and TNF-α levels (c) in the culture supernatant (mean ± SE, n = 6). Data are expressed as percentage of controls without stimulants. LPS was used in the final concentration of 10 µg/ml, while APs or APa were used as 100 µl per reaction. *p < 0.05 when compared to the control group

Fig. 3

Effects of APs, APa, or LPS on HSP60 mRNA expression of rat pancreas fragments (mean ± SE; n = 3). a Original recording for amplification curve of HSP60 mRNA expression in rat pancreatic fragments. b Relative expression of HSP60 mRNA was calculated as the individual ratio of the HSP60-amplified CT value against the respective GAPDH-amplified CT value. The data were expressed as percentage of control as described in the “Materials and methods” section. *p < 0.05 when compared to the controls

Fig. 4

Effects on HSP60 protein expression in rat pancreas fragments during culturing (mean ± SE, n = 3). a Western blot profiling of HSP60 protein expression in rat pancreatic fragments at different time-points of culture. b Quantitative analysis of the relative expression of HSP60 in rat pancreatic fragments, expressed as the gray scale of HSP60 band versus that of the corresponding β-actin band. The data are expressed as percentage of control (0 h). *p < 0.05, **p < 0.01 when compared to the control (0 h)

Fig. 5

Effect of LPS or APa on HSP60 protein expression in rat pancreas fragments (mean ± SE, n = 3). a Western blot profiling of HSP60 protein expression in rat pancreatic fragments after treatment with LPS or APa. b Relative expression value of HSP60 protein expression in the fragments, presented as the gray scale of the HSP60 band versus that of the corresponding β-actin band. Data were expressed as percentage of control with *p < 0.05 when compared to the controls