The Therapeutic Effect And Mechanism Of Pentoxifylline In Acute Pancreatitic Rats

Acute pancreatitis (AP) is a complex disease associated with significant complications and a high rate of mortality. Although several mechanisms are put forward, experimental and clinical evidences have shown that pro-inflammatory cytokines and oxidative stress seem to be the most important early events in the pathophysiology of AP.Serum levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL), and intercellular adhesion moleculein-1 (ICAM-1), increase during the course of AP and they appear to be the driving force for the initiation of pancreatic tissue damage and impairment of the pancreatic microcirculation in AP. Blockade of cytokines production may ameliorate experimental AP. TNF-αcan be detected in plasma early in the course of AP. TNF-αmay be an initiator of the cytokine cascade, because it induces the synthesis and release of other cytokines, oxygen free radical and nitric oxide.Pentoxifylline (PTX) is a methylxanthine derivative with rheological properties that improve microvascular flow. It has been widely used in vascular perfusion disorders. It also exhibits selective and marked anti-inflammatory properties mediated by inhibition of TNF-αproduction. As an inhibitor of non-selected phosphodiesterase, PTX can block the transformation of cyclic adenosine monophosphate (cAMP) to AMP. Therefore, the intracellular TNF-αmRNA transcription is blocked. In the present study, PTX completely prevent the increase in serum TNF-αand attenuate inflammatory responses in taurocholate- induced pancreatitis.Mitogen-activated protein kinase (MAPK) plays an important role in the transduction of extracellular signals to the nucleus. Extracellular signal-regulated kinase 1/2 (ERK1/2), c-jun N-terminal kinase (JNK), p38 MAPK and big mitogen- activated protein kinase 1 (BMK1) are essential members of MAPK family. Many researchers indicated that JNK and p38 MAPK have a role in pancreas injury and inflammation. JNK and p38 MAPK are activated by lipopolysaccharide (LPS), tumor necrosis factor-α(TNF-α) and IL-1. Activated JNK and p38 MAPK transfer into the nucleus, activate transcription factors, and are involved in a wide range of cellular behaviors, including cell proliferation, differentiation, and apoptosis. Recent studies have demonstrated the role of p38 MAPK and JNK in the pathogenesis of AP.The apoptotic index of pancreatic acinar cell is negatively correlated with the severity of AP. The apoptosis of pancreatic acinar cell may prevent the worsening of AP due to diminishing leakage of pancreatic enzyme and extension of inflammation. Control of apoptosis could be a potent strategy for improvement of the outcome in AP. Some studies indicate that inhibition of MAPK activation reduces local and systemic inflammatory response in AP and induces the apoptosis of pancreatic acinar cell. The advance of AP is ameliorated because the systemic response is restrained.Objective:To investigate the expressions of phospho-p38 MAPK, phospho-JNK1, ICAM-1, TNF-α, and the rate of cell apoptosis in experimental acute pancreatitis. To study the protective effects and mechanism of PTX in AP and its potential therapeutic value in the future.Methods:1 AP model: The operation was performed in male Wistar rats, which were randomly divided into four groups. The rats were weighted, marked, watered freely and absolute diet for 24h. Anesthesia was induced with an intraperitoneal injection of 10% chloralhydrate (3ml/kg body weight). After midline laparotomy and transduodenal cannulation of the bili- pancreatic duct, the bili-pancreatic duct was closed by a small bulldog clamp, and then 3.5% sodium taurocholate in saline (0.1ml/100g) was infused at a rate of 0.20ml/min. Ten minutes later, the bulldog clamp was moved off. The model of AP was completed.2 Experimental groups: All Wistar rats were randomly divided into four groups: sham-operate (SO) group, AP group, AP-NS group, AP-PTX group, and every group was randomly divided into 5 units (1h, 3h, 6h, 9h, 12h). SO group received no sodium taurocholate during the laparotomy. AP-PTX group was given PTX (50mg/kg) by intraperitoneal injection. AP-NS group received equal quantity physiological saline in the same way. In addition, the normal pancreatic tissues came from six rats would be provided to detect the expressions of p-p38 MAPK and p-JNK1.3 The animals were killed at 1h, 3h, 6h, 9h and 12h respectively after the injection of PTX or physiological saline. Blood and pancreatic tissue were collected immediately.4 The level of serum amylase was detected by automatic biochemical analyzer. Paraffin sections were stained by HE to observe histological changes. The levels of serum TNF-αand ICAM-1 were detected respectively by enzyme-labeled immunosorbent assay (ELISA). The expressions of p-p38 MAPK and p-JNK1 were determined respectively by western blotting.5 The rate of pancreatic acinar cellular apoptosis in AP was detected by terminal deoynucleotidle transferase mediated d-UTP nick end labeling (TUNEL).Result:1 The level of serum amylaseThe level of serum amylase had no significant change at different time points in SO group. The levels of serum amylase in AP,AP-NS and AP-PTX were higher than that in SO group and increased gradually with time prolonged (P<0.01). Compared with AP group and AP-NS group respectively, the level of serum amylase was increased significantly in AP-PTX group (P<0.01).2 The consequence of histopathologyIn SO group, the pancreatic tissues swelled slightly and the structure of pancreas remained normal except local interstitial edema in light microscopy. Hemorrhagic ascites appeared in AP-NS group, and there were necrosis focus on the edematus pancreas. Histology studies of the pancreas using light microscopy showed swollen pancreatic acinar cell, interstitial edema, inflammatory infiltration of neutrophils and mononuclear cells into the pancreatic tissue, necrosis, diffuse hemorrhage and other pathological changes were found in pancreatic tissue of rats. The damage was aggravated gradually. In AP-PTX group, treatment with pentoxifylline markedly reduced these histological alterations in pancreas.3 The levels of serum TNF-αand ICAM-1The levels of serum TNF-αand ICAM-1 had no significant change at different time points in SO group. In AP, AP-NS and AP-PTX groups, the levels of serum TNF-αand ICAM-1 were higher than that in SO group and increased gradually with time prolonged respectively (P<0.01). The peak values of TNF-αwere (174.84±7.87) ng/ml, (179.56±6.79) ng/ml, and (163.46±7.51) ng/ml in AP, AP-NS and AP-PTX groups respectively at 6h. Compared with AP group and AP-NS group, the level of serum TNF-αwas decreased obviously at different time points in AP-PTX group (P<0.05), the level of serum ICAM-1 was decreased obviously at 3h, 6h, 9h and 12h in AP-PTX group (P<0.05).4 Expressions of phospho-p38 MAPK and phospho-JNK1The expression of phospho-p38 MAPK: The results showed that zone of phospho-p38 MAPK hybridization appeared at 38kD and zone ofβ-actin hybridization appeared at 43kD. The zone of phospho-p38 MAPK hybridization was indicated by Integral Optical Density (IOD). The IOD of phospho-p38 MAPK hybridization in AP group (0.9803±0.02001) was increased apparently compared with that in SO group (0.8933±0.03215, P<0.01), and reached the peak at 3h point (0.9975±0.005). Compared with AP-NS group (0.9953±0.0808), the IOD of phospho-p38 MAPK hybridization in AP-PTX group (0.8883±0.01767) was decreased significantly (P<0.01).The expression of phospho-JNK1: The results showed that zone of phospho-JNK1 hybridization appeared at 46 kD and zone ofβ-actin hybridization appeared at 43kD. The zone of phospho-JNK1 hybridization was indicated by IOD. The IOD of phospho-JNK1 hybridization in AP group (0.9100±0.06083) was increased apparently compared with that in SO group (0.6467±0.01528, P<0.01), and reached the peak at 1h point (0.9343±0.03151). Compared with AP-NS group (0.9367±0.02082), the IOD of phospho-JNK1 hybridization in AP-PTX group (0.6267±0.02309) was decreased significantly (P<0.01). 5 The apoptosis of pancreatic acinar cell Compared with SO group, there appeared numerous cell apoptosis in AP and AP-NS groups. But at 9h and 12h, cell necrosis appeared for major in AP and AP-NS groups. After pretreatment of PTX, the numbers of apoptotic cells increased obviously at different time points (P<0.05).Conclusion:1 The pancreatic function was damaged that was indicated by increasing the level of serum amylase in taurocholate-induced AP. Histological finding showed swollen pancreatic acinar cell, inflammatory infiltration, necrosis, diffuse hemorrhage and other pathological changes in pancreatic tissue of rats. Pretreatment of PTX, the level of serum amylase was descented and the appearance of histology was ameliorated obviously.2 Serum TNF-αand ICAM-1 levels were increased in AP, AP-NS and AP-PTX groups after the model of AP completed. In addition, Serum TNF-αlevel reached the peak value at 6h. Compared with AP group and AP-NS group, both cytokines decreased obviously in AP-PTX group. The outcome demonstrated that PTX could completely prevent the increasing of TNF-αand ICAM-1 in serum.3 The expressions of phosphor-p38 MAPK and phosphor- JNK1 increased significantly in AP. The outcome demonstrated that MAPK signal transduction pathway played a role in the course of AP. PTX could inhibit the expression of phosphor-p38 MAPK and phosphor-JNK1.4 There appeared numerous cell apoptosis at early stage of AP. PTX could induce the apoptosis of pancreatic acinar cell. Induction of cell apoptosis could attenuate inflammatory responses in AP.5 The results of the study showed that PTX might play a protective role in AP, and this effect might be related to PTX blockade of MAPK (p38 MAPK and JNK1) activation. Inhibition of MAPK activation could reduce the production of pro-inflammatory cytokines (TNF-αand ICAM-1) and then induce the apoptosis of pancreatic acinar cell.