Severe Acute Pancreatitis Model Group Research Articles

Electroacupuncture at Acupoint ST36 (Zusanli) Improves Intestinal Motility Dysfunction Via Increasing the Proportion of Cholinergic Neurons in Rat Ileal Myenteric Ganglia after Severe Acute Pancreatitis.

Using a severe acute pancreatitis (SAP) rat model, the mechanism of electroacupuncture (EA) were studied on the intestinal function of pancreatitis. The SAP models were established by injecting 30% L-ornithine at hourly intervals, and were divided into two groups (14 in each): SAP model group, which was not treated, and EA group, which received EA at ST36 at a frequency of 1-2 Hz and amplitude of 1 mA for 30 min twice a day. Fourteen rats were also included as the control group. After EA, the intestinal propulsion was measured. In the distal ileum myenteric plexus, the density of HuC/D and the proportion of cholinergic neurons were measured using immunohistochemistry. Compared to the SAP model group, the EA group demonstrated significant improvements in intestinal propulsion rates. Furthermore, after EA, the density of myenteric neurons in the ileum returned to normal levels and the proportion of cholinergic neurons was increased compared to the SAP model group. And finally, EA alleviated the damage to the pancreas. Thus, our results suggest that EA stimulation at ST36 can partly restore the enteric neuron function and improve intestinal motility dysfunction, therefore could ameliorate SAP. The enteric nervous system can participate in changes in intestinal motility by affecting cholinergic neurons.

Restoration of Intestinal Mucosa in Euphorbia kansui-treated Severe Acute Pancreatitis Rats based on HMGB1/MFG-E8 Expression.

The pathogenesis of Severe Acute Pancreatitis (SAP) is mediated substantially by dysfunctions in the intestinal barrier. Euphorbia kansui (EK) is a medicinal plant used widely in traditional Chinese medicine to treat inflammation; however, its efficacy and mechanism of action in SAP treatment are not yet well understood. To investigate the role of EK in intestinal barrier tissue repair and in the pathogenesis and development of SAP. The rat SAP model was established by a retrograde injection of sodium taurocholate into the pancreatic bile duct. The SAP model group and the SAP + EK treatment groups were divided into 6 subgroups according to timing: 2, 6, 12, 24, 48, or 72h after inducing SAP. The progression of the SAP rats and of the rats receiving the EK treatment was evaluated using the ascites volume, serum amylase and plasma endotoxin levels, and histological grading of intestinal mucosal damage. In addition, serum inflammatory factor contents were measured using Enzyme-Linked Immunosorbent Assay (ELISA) tests and apoptotic cells in damaged ileum tissue were detected using TUNEL staining. Apoptosis markers and other signaling proteins in intestinal mucosal cells were detected by immunohistochemical assays and then validated by combining these data with quantitative polymerase chain reactions and western blotting. Compared with the results of the SAP model rats, the results of the rats that received EK treatment demonstrated that EK could effectively reduce the ascites volume and serum amylase and plasma endotoxin levels. EK treatment also greatly reduced the abnormal intestinal morphological alterations in the rat SAP model and significantly downregulated the serum contents of Interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. EK treatment inhibited the elevation of capapse-3, inhibited the decrease of the Bcl-2 protein, and decreased the number of apoptotic cells in rat ileum tissue. Finally, EK treatment abrogated the increase of HMGB1 and the suppression of MFG-E8 protein expression in the SAP + EK rat ileum tissue. EK suppresses SAP pathogenesis by restoring the intestinal barrier function and modulating the HMGB1/MFG-E8 signaling axis.

Da-Huang-Fu-Zi-Tang Ameliorates Severe Acute Pancreatitis by Elevation of M2 Kupffer Cells in Rats.

Introduction Severe acute pancreatitis (SAP) is a clinical emergency often accompanied by inflammatory response syndrome (SIRS), which eventually leads to acute lung injury and failure of other organs. The activation of liver Kupffer cells (KCs) plays a major role in the process of SIRS and multiorgan damage caused by SAP. Da-Huang-Fu-Zi-Tang (DHFZT), a traditional Chinese prescription, has been widely used for SAP in the clinic. The present study investigated the activation state of KCs in SAP and the potential mechanism of DHFZT. Methods A total of 24 Sprague Dawley rats were randomly assigned to four groups: SH (sham operation group + saline enema), SH-DHFZT (sham operation group + DHFZT enema), SAP (model group + saline enema), and SAP-DHFZT (model group + DHFZT enema). Blood samples were drawn from the abdominal aorta for measuring serum endotoxin, amylase, calcium ion, IL-1β, TNF-α, iNOS, and IL-10. Then, the pancreas, lung, liver, and ileum were harvested for histological observation, and the liver was used to detect the level of F4/80, CD86, and CD163 in KCs with immunohistochemistry and western blot. Results In the SAP group, the CD86+ KCs were significantly increased with a high level of IL-1β, TNF-α, and iNOS, and the organs were impaired. In the SAP-DHFZT group, CD163+ KCs were significantly increased with the high level of IL-10, and the damage to organs was alleviated. Conclusion These phenomena suggested that the SIRS and multiple organ damage in SAP might be related to the excessive activation of M1 KCs, and DHFZT might alleviate the SIRS by inducing the differentiation of KCs into the M2-type and promote the expression of anti-inflammatory factor IL-10.

Mechanism of gasdermin D on intestinal injury in severe acute pancreatitis by mediating pyroptosis

To investigate the function of gasdermin D (GSDMD) in intestinal damage of mice with severe acute pancreatitis (SAP). The healthy C57BL/6 mice were divided into four groups randomly, including normal saline (NS) group, small interfering RNA (siRNA)-NS group, SAP model group and siRNA-SAP group, with 6 mice in each group. The SAP mouse model was reproduced by intraperitoneal injection of caerulein 50 μg/kg combined with lipopolysaccharide (LPS) 10 mg/kg; the NS group was given the same amount of NS; in the siRNA-SAP group and siRNA-NS group, siRNA 50 mg/kg was injected through the tail vein three times before modeling or injection of NS. The blood of mice eyeball in each group was taken 12 hours after modeling, and serum interleukins (IL-1β, IL-18) levels were detected by enzyme linked immunosorbent assay (ELISA). The mice were sacrificed to observe the general changes in abdominal cavity, the pancreas and ileum tissues were taken to observe the pathological changes under a light microscope. The expression of long-chain non-coding RNA uc.173 (lnc uc.173) was detected by reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical method was used to detect the expression of tight junction proteins zonula occluden-1 (ZO-1) and Occludin in intestinal mucosal epithelial cells. Western blotting was used to detect the GSDMD protein expression level in the intestinal tissue. The serum levels of IL-1β and IL-18 in the SAP model group were significantly higher than those in the NS group and the siRNA-NS group [IL-1β (ng/L): 146.66±1.40 vs. 44.48±5.76, 81.49±10.75, IL-18 (ng/L): 950.47±177.09 vs. 115.43±16.40, 84.84±21.90, all P < 0.05]; and the levels of IL-1β and IL-18 in the siRNA-SAP group were significantly lower than those in the SAP model group [IL-1β (ng/L): 116.26±15.54 vs. 146.66±1.40, IL-18 (ng/L): 689.96±126.08 vs. 950.47±177.09, both P < 0.05]. General observation showed that there were no obvious abnormalities in the abdominal cavity of the mice in the NS and siRNA-NS groups; the mice in the SAP model group and the siRNA-SAP group had different degrees of edema and congestion in the intestine; compared with the SAP model group, the abnormalities in the siRNA-SAP group was significantly reduced. Under light microscope, there were no obvious changes in the pancreas and intestinal mucosa in the NS group and the siRNA-NS group; the pancreatic tissue of the SAP model group and the siRNA-SAP group had different degrees of edema, inflammatory cell infiltration, and lobular structure damage, and the intestinal mucosa was damaged to a certain degree, and the villi were broken to varying degrees, but the damage in the siRNA-SAP group was lighter. The results of RT-PCR showed that the expression of lnc uc.173 in the intestinal tissues of the model SAP group was significantly lower than that of the NS group and the siRNA-NS group (2-ΔΔCt: 0.26±0.12 vs. 1.01±0.37, 0.67±0.32, both P < 0.05), while the expression of lnc uc.173 in the siRNA-SAP group was significantly higher than that in the SAP model group (2-ΔΔCt: 0.60±0.39 vs. 0.26±0.12, P < 0.05). Immunohistochemistry showed that ZO-1 and Occludin proteins in the NS group were distributed along the epithelial cells of the intestinal mucosa, showing a strong expression; ZO-1 and Occludin expressions were significantly reduced in the SAP model group and siRNA-SAP group, but the expressions in the siRNA-SAP group was higher than that in the SAP model group. Western blotting showed that the expression level of GSDMD protein in the intestinal tissues of the SAP model group was significantly higher than that of the NS group and the siRNA-NS group [GSDMD protein (GSDMD-N/β-actin): 1.99±0.46 vs. 1, 1.00±0.78, both P < 0.05]. Compared with the SAP model group, the expression of GSDMD protein in the siRNA-SAP group was significantly decreased [GSDMD protein (GSDMD-N/β-actin): 1.42±0.42 vs. 1.99±0.46, P < 0.05]. The systemic inflammatory response and intestinal mucosal barrier damage of SAP mice may be related to the increase of GSDMD expression in intestinal tissues. GSDMD mediates cell pyrolysis to promote the release of inflammatory factors, cause intestinal injury, and down-regulate the expression of intestinal epithelial cell tight junction proteins such as ZO-1 and Occludin, resulting in intestinal mucosal damage.

Protective Mechanism of MIF Inhibitor ISO-1 on Intrahepatic Bile Duct Cells in Rats with Severe Acute Pancreatitis.

This study aimed to explore the protection mechanism of ISO-1 on severe acute pancreatitis-associated intrahepatic bile duct (IBD) injury in rats. Forty-eight specific-pathogen-free male Wistar rats were randomly divided into four groups (N = 12): a sham operation group (SO group), a severe acute pancreatitis model group (SAP group), a ISO-1 treatment group (ISO-1 + SAP group), and a ISO-1 control group (ISO-1 + SO group). All rats were killed after 12h of being made models. Immunohistochemistry was used to detect the expression of MIF and P38 in IBD cells. MIF mRNA expression in IBD cells was observed using real-time fluorescent quantitative polymerase chain reaction (real-time PCR). In addition, Western blotting was performed to detect the protein expression of P38, phosphorylated P38 (P-P38), nuclear factor-κB (NF-κB p65), and tumor necrosis factor alpha (TNF-α). Enzyme-linked immunosorbent assays were used to analyze the levels of TNF-α, IL-1β, and IL-6 in the IBD of rats. Compared with SAP, after treatment with ISO-1, the pathological injuries of pancreas, liver, and IBD cells in ISO-1 treatment group remarkably relieved. The expression of MIF in the IBD cells was significantly downregulated both at mRNA and at protein levels in ISO-1 treatment group. Besides, the protein expression levels of P38, P-P38, NF-κBp65, TNF-α, IL-1β, and IL-6 in the IBD in rats were also significantly decreased in ISO-1 treatment group (all P < 0.05). ISO-1 may protect the IBD cells, reduce pathological injuries, and reduce the inflammatory response in SAP rats. Its mechanisms may be via inhibiting the expression of MIF and then blocking the activation of p38-MAPK and NF-κB signaling pathway.

Effect of SOCS3 on lung injury in rats with severe acute pancreatitis through regulating JAK2/STAT3 signaling pathway.

To explore the effect of suppressor of cytokine signaling 3 (SOCS3) on the lung injury in rats with severe acute pancreatitis (SAP) by regulating the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway. Sprague-Dawley rats were divided into control group (n=20) and SAP model group (established via injection of 5% sodium taurocholate, n=40). Then, SOCS3 was overexpressed using the adenovirus in 20 rats in SAP model group. The serum amylase (AMY) was detected, whether the transfection was successful was verified via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), the hepatic function indexes were detected, the pathological changes were observed using hematoxylin-eosin (HE) staining, and the wet/dry weight ratio (W/D) was calculated. Moreover, the content of serum inflammatory factors was detected via enzyme-linked immunosorbent assay (ELISA) and the expression levels of JAK2/STAT3 signaling pathway genes and proteins were detected through RT-PCR and Western blotting. The content of AMY in SAP model group was significantly increased, indicating the successful modeling. SOCS3 was significantly increased in transfection group, suggesting that the transfection efficiency was significant. The content of alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in SOCS3 transfection group was significantly lower than in model group. According to the histopathological observation, there were lung injury, pulmonary edema, hemorrhage, severe inflammatory response, and alveolar congestion in SAP model group. There were almost no pathological changes in SOCS3 transfection group. In SOCS3 transfection group, the content of serum interleukin-6 (IL-6), IL-18 and tumor necrosis factor-α (TNF-α), the mRNA and protein expressions of IL-6, JAK2, and STAT3 were all remarkably declined. SOCS3 inhibits the activation of the JAK2/STAT3 pathway and the increase of inflammatory factors, promoting the repair of lung injury in SAP rats.

4-Phenylbutyric Acid Attenuates Endoplasmic Reticulum Stress-Mediated Intestinal Epithelial Cell Apoptosis in Rats with Severe Acute Pancreatitis.

The present study aimed to determine whether intestinal epithelial cell (IECs) apoptosis could be induced by endoplasmic reticulum stress (ERS) in severe acute pancreatitis (SAP), and the role of chemical chaperone 4-phenylbutyric acid (4-PBA) in SAP-associated intestinal barrier injury. Twenty-four male Sprague Dawley rats were randomly divided into three groups: the sham operation group, the SAP group, and the SAP model plus 4-PBA treatment group (4-PBA group). A rat model of SAP was induced by retrograde injection of 5% sodium taurocholate (STC) into the biliopancreatic duct; in the 4-PBA group, 4-PBA was injected intraperitoneally at a dose of 50mg/kg body weight for 3days before modeling. The results indicated that 4-PBA attenuated the following: (1) pancreas and intestinal pathological injuries, (2) serum TNF-α, IL-1β, and IL-6, (3) serum DAO level, serum endotoxin level, (4) the apoptosis of IECs, (5) ER stress markers (caspase-12, CHOP, GRP78, PERK, IRE1α, ATF6) and caspase-3 expression in intestinal. However, the serum AMY, LIPA levels, and the expression of caspase-9, caspase-8 were just slightly decreased. ERS may be considered a predominant pathway, which is involved in the apoptosis of IECs during SAP. Furthermore, 4-PBA protects IECs against apoptosis in STC-induced SAP by attenuating the severity of ERS.

Emodin attenuated severe acute pancreatitis via the P2X ligand‑gated ion channel7/NOD‑like receptor protein3 signaling pathway.

Acute pancreatitis (AP) is an aseptic inflammation characterized with an annual incidence rate, and ~20% patients progressing to severe AP (SAP) with a high mortality rate. Although Qingyi decoction has been frequently used for SAP treatment over the past 3 decades in clinic, the actual mechanism of its protective effects remains unknown. As the major active ingredient of Qingyi decoction, emodin was selected in the present study to investigate the effect of emodin against severe acute pancreatitis (SAP) in rats through NOD-like receptor protein 3 (NLRP3) inflammasomes. The rats were randomly divided into a sham operation group, an SAP model group induced by a standard retrograde infusion of 5.0% sodium taurocholate into the biliopancreatic duct, and low-dose (30 mg/kg) and high-dose (60 mg/kg) emodin-treated groups. At 12 h after the event, the plasma amylase, lipase, interleukin (IL)-1β, IL-18 and myeloperoxidase (MPO) activities were examined. Furthermore, the pathological scores of pancreases were evaluated by hematoxylin and eosin staining. The expression levels of P2X ligand-gated ion channel 7 (P2X7), NLRP3, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain and caspase-1 were also analyzed by western blot analysis. The data demonstrated that, compared with the SAP group, emodin could significantly relieve the pancreatic histopathology and acinar cellular structure injury, and notably downregulate the plasma amylase and lipase levels, as well as the MPO activities in pancreatic tissues, in a dose-dependent manner. Furthermore, emodin inhibited the P2X7/NLRP3 signaling pathway followed by the decrease of pro-inflammatory factors, and the latter is beneficial for the recovery of SAP. Collectively, the data indicated that emodin may be an efficient candidate natural product for SAP treatment.

Therapeutic effects of Saussurea involucrata injection against severe acute pancreatitis- induced brain injury in rats

ObjectivesTo observe the therapeutic effects of Saussurea involucrata (Sau) injection against severe acute pancreatitis (SAP)-induced brain injury. MethodsSodium taurocholate-induced SAP-modeled rats were equally randomized into an SAP model group (SAP group) and a Sau treated group (Sau + S group). Healthy rats were equally randomized into a Sau treated group (Sau + H group) and a sham operation group (SO group). Serum amylase levels, endothelin-1 (ET-1) and nitric oxide (NO) contents were determined by optical turbidimetry, ELISA and nitrate reductase method respectively. Western blot was used to detect protein expression levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), ET-1, inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) while mRNA levels of these biomarkers in brain tissue were measured by quantitative real-time PCR. Furthermore, pathological changes, as well as all above indexes of pancreas and brain, were observed at 6, 24 and 48 h after administration. ResultsThere was a significant difference in mortality between SAP and Sau + S groups (P < 0.05). Serum amylase levels, ET-1 and NO contents, ET-1/NO ratio, relative expression levels of ET-1 and iNOS protein/mRNA of brain tissue in Sau + S group were lower than those in SAP group at 24 and 48 h post-operation (P < 0.05 or 0.01), meanwhile, pancreas and brain pathological scores showed similar tendency (P < 0.01). However, both protein and mRNA levels of PI3K, Akt and eNOS of brain tissue in Sau + S group were higher than those in SAP group (P < 0.05 or P < 0.01). There were no significant differences in all indexes between Sau + H and SO groups at all designated time points (P > 0.05). ConclusionsSau injection has therapeutic effects on SAP-induced brain injury in rats.

Emodin diminishes immune stress in rats with severe acute pancreatitis

Objectives: The aim was to observe the immune stress in rats with Severe Acute Pancreatitis (SAP) and diminish by emodin. Methods: Rats were divided into four groups: sham operation group (SO), SAP model group (SAP), emodin treatment group (ET), and two times dose emodin treatment group (HT). Samples of blood, terminal ileum and lung were harvested on 6 h, 12 h, 18 h and 24 h for examination. Serum amylase, and high-mobility group box 1 (HMGB 1), cyclooxygenase-2 (COX-2), and peroxisome proliferator activated receptor gamma-γ (PPAR-γ) in terminal ileum and lung was determined. Results: The values for emodin treated showed distinctly treatment effect, with lower levels of serum amylase, HMGB 1 and COX-2 on 12, 18 and 24 h and higher levels of PPAR-γ in the ET and HT groups compared with the SAP group on four time-points. The levels of detection indicators in the ET group were quite different from the HT group, with higher levels of serum amylase, HMGB 1 and COX-2 on 12, 18 and 24 h and lower expression of PPAR-γ on each time points. Conclusions: Emodin can diminish immune stress in rats with SAP. The strategy of two times may be a better therapeutic effect.

Therapeutic effect of hesperidin on severe acute pancreatitis in rats and its mechanism

To investigate the protective effect of hesperidin on severe acute pancreatitis (SAP) in rats and its related mechanism. Sixty male Sprague-Dawley (SD) rats were randomly divided into five groups (n = 12 in each group): sham group, SAP model group, dexamethasone group (5 mg/kg), low and high dose of hesperidin groups (10 mg/kg and 20 mg/kg). SAP rats were administered a retrograde infusion of 3.5% sodium taurocholate solution into the biliopancreatic duct after laparotomy. Sham rats were administered with equivalent saline. The treatment was intravenously injected 5 minutes after operation through femoral vein. After 24 hours, the survival of animals was observed, the level of serum amylase, the volume of ascites and the relative specific gravity of the pancreas were measured; the pathological changes of pancreatic tissue were observed by Hematoxylin-eosin (HE) staining; the levels of serum and pancreatic tissue interleukin (IL-1β, IL-6) and tumor necrosis factor-α (TNF-α) were detected by enzyme linked immunosorbent assay (ELISA); the expression of Toll-like receptor 4 (TLR4), the phosphorylation of IL-1 receptor associated kinase (IRAK1) and nuclear factor-κB (NF-κB) were detected by Western Blot. Compared with SAP model group, the 24-hour survival rate were increased in low and high dose of hesperidin groups (83.3%, 100% vs. 58.3%), the volume of ascites were reduced (mL: 7.36±0.91, 6.10±1.02 vs. 13.82±2.06), the levels of serum amylase were reduced (U/L: 1 081.48±78.23, 1 048.58±49.97 vs. 1 990.37±127.27), the relative specific gravity of the pancreas were reduced [(7.52±1.02)%, (5.59±0.96)% vs. (11.22±0.96)%], and the pathological damage of pancreatic tissue were reduced; the levels of serum and pancreatic tissue inflammatory factors were reduced in high dose hesperidin group [serum IL-1β (ng/L): 68.08±10.49 vs. 130.30±23.35, IL-6 (ng/L): 63.88±10.47 vs. 158.41±21.38, TNF-α (ng/L): 10.42±1.49 vs. 18.16±2.01; pancreas IL-1β (pg/μg): 13.87±1.84 vs. 20.08±1.66, IL-6 (pg/μg): 21.90±3.12 vs. 38.13±3.57, TNF-α (pg/μg): 1.88±0.20 vs. 4.26±0.58]; the expression of TLR4, and the phosphorylation levels of IRAK1 and NF-κB were decreased in low and high dose of hesperidin groups (the sham operation group was 100, TLR4/β-actin: 91.9±15.6, 83.7±11.2 vs. 168.5±9.0, p-IRAK1/IRAK1: 117.4±7.6, 104.7±11.5 vs. 173.5±15.8, p-NF-κB p65/NF-κB p65: 119.9±9.3, 105.8±12.6 vs. 174.1±13.0), with statistically significant differences (all P < 0.05). The effects of dexamethasone were similar to that of high dose of hesperidin. Hesperidin could significantly protect SAP rats, and this protection was related to the inhibition of TLR4/IRAK1/NF-κB signaling pathway, and to the reduction of pro-inflammatory cytokine expressions. The effect of high dose hesperidin (20 mg/kg) was more significant.

Exogenous leptin protects rat models of sodium taurocholate‑induced severe acute pancreatitis through endocrinal and immunological pathways.

Acute pancreatitis (AP) is a common non‑bacterial disease compromising pancreatic tissues. Adipocyte‑derived leptin is closely associated with the severity and clinical outcome of pancreatitis. The potential protective effects of exogenous leptin administration on a rat model of severe AP (SAP) remain to be elucidated, and were examined in the present study. Male Wistar rats were divided into a sham operation group (SO), SAP model group (SAP) and leptin group (LEP). Each group was divided into three sub‑groups by observation time (24, 48 and 72h). The SAP models were prepared by retrograde injection of 6% sodium taurocholate into the pancreatic‑bile duct. Following model establishment, exogenous leptin was intraperitoneally injected into mice at 50mg/kg in the LEP group. Subsequently, serum amylase, lipase and glucose levels at particular time‑points were analyzed using a fully‑automatic biochemical analyzer, and serum levels of tumor necrosis factor (TNF)‑α and interleukin (IL)‑10 were detected using an enzyme‑linked immunosorbent assay. The pathological changes in pancreatic tissues were observed using hematoxylin and eosin staining, and the pancreatic expression of the long form of the leptin receptor (OB‑Rb) was detected and evaluated using Nest‑polymerase chain reaction analysis. The mortality rates of the model rats were compared between the groups. Following the administration of exogenous leptin, the serum level of amylase in the LEP group was significantly decreased at 48h, compared with that in the SAP group, with serum lipase levels decreased at 48 and 72h, and blood glucose levels decreased at 72h. Regarding the serum inflammatory factors, the level of TNF‑α in the LEP group was significantly lower, compared with that in the SAP group at 24h; whereas no significant difference was observed in the serum level of IL‑10 between the two groups. Regarding the pathological changes in the pancreas, the tissues in the LEP group showed significantly alleviated pancreatic inflammation. In addition, the pancreatic expression of OB‑Rb in the LEP group was significantly higher, compared with that in the SAP group at 24 and 48h. No significant difference in 3‑day mortality rates were observed between the SAP group and the LEP group. Taken together, exogenous leptin administration regulated inflammatory factors and the expression of OB‑Rb at the early stage of AP, which exerted protective effects by through the immunological and endocrinal pathways.

4-Phenylbutyric Acid Attenuates Pancreatic Beta-Cell Injury in Rats with Experimental Severe Acute Pancreatitis.

Endoplasmic reticulum (ER) stress is a particular process with an imbalance of homeostasis, which plays an important role in pancreatitis, but little is known about how ER stress is implicated in severe acute pancreatitis (SAP) induced pancreatic beta-cell injury. To investigate the effect of 4-phenylbutyric acid (4-PBA) on the beta-cell injury following SAP and the underlying mechanism, twenty-four Sprague-Dawley rats were randomly divided into sham-operation (SO) group, SAP model group, and 4-PBA treatment group. SAP model was induced by infusion of 5% sodium taurocholate into the biliopancreatic duct. 4-PBA or normal saline was injected intraperitoneally for 3 days in respective group before successful modeling. Results showed that 4-PBA attenuated the following: (1) pancreas and islet pathological injuries, (2) serum TNF-α and IL-1β, (3) serum insulin and glucose, (4) beta-cell ultrastructural changes, (5) ER stress markers (BiP, ORP150, and CHOP), Caspase-3, and insulin expression in islet. These results suggested that 4-PBA mitigates pancreatic beta-cell injury and endocrine disorder in SAP, presumably because of its role in inhibiting excessive endoplasmic reticulum stress. This may serve as a new therapeutic target for reducing pancreatic beta-cell injury and endocrine disorder in SAP upon 4-PBA treatment.

The protective effect of hydrogen-rich saline on lung injury associated with severe acute pancreatitis and its impact on P38MAPK and NF-κB expression

Objective To investigate the protective effect of hydrogen-rich saline on lung injury associated with severe acute pancreatitis and its impact on P38MAPK and NF-κB expressions. Methods Fifty-four male Wistar rats were randomly (random number) divided into three groups: (1) hydrogen-rich saline treatment group (HRS group, n=18 ) , in which the rats were treated with hydrogen-rich saline (6 mL/kg) administered intravenously via tail vein and HRS (20 mL/kg) administered subcutaneously at 5 min after successful modeling. (2) Severe acute pancreatitis model group (SAP group, n=18) , in which rats received equivalent volume of normal saline instead of hydrogen-rich saline both intravenously and subcutaneously as in HRS group. (3) Sham operation group (SO group, n=18) , in which rats were treated with sham surgery, and received equivalent volume of normal saline as in SAP group. The model of severe acute pancreatitis (SAP) was made by retrograde injection of 5% sodium taurocholate (1 mL/kg) into cholepancreatic duct. All rats were sacrificed at 3 h, 12 h, and 24 h separately after the operation (n=6 at a time). The levels of serum amylase, lipase were measured. The ratio of wet and dry lung tissues was measured. The histopathological changes of lung tissues were observed under optic microscope. The expressions of P38MAPK, p-P38MAPK and NF-κB were measured by using immunohistochemistry method. Results Compared with SAP group, there were no significant differences in levels of serum amylase [12 h (5 306.7±909) vs. (5 435.0±441.2) ]and lipase [12 h (1 897.8±149.4) vs. (1 917.9±106.8) ]in HRS group (P >0.05) , but there were significant differences in the ratio of wet and dry lung tissues [12 h (3.12±0.58) vs. (1.87±0.25) ]and histopathology scores [12 h (2.14±0.38) vs. (3.58±0.32) ] (P <0.05). There was no significant difference in expression of P38MAPK in lung tissues among three groups at 12 h. Compared with SO group, the expressions of p-P38MAPK and NF-κB were significant increased in SAP group at 12 h, however, they were lower significantly in HRS group than those in SAP group. Conclusions Hydrogen-rich saline has a protective effect on lung injury associated with severe acute pancreatitis, and its mechanism may be likely related to the antioxidant effect and inhibiting the activation of P38MAPK and NF-κB. Key words: Hydrogen-rich saline; Pancreatitis; Lung injury; Protective effect; P38MAPK; NF-κB

Ulinastatin Reduces T Cell Apoptosis in Rats with Severe Acute Pancreatitis

Purpose: To investigate the immunoregulative effects of ulinastatin (UTI) on T lymphocytes apoptosis in rats with severe acute pancreatitis (SAP) and to elucidate its underlying molecular mechanism.Methods: Thirty six Wistar rats were randomly divided into 3 groups (n =12): sham, SAP model and UTI-treated group. SAP model was established by intrapancreatobiliary duct injection of 5% sodium taurocholate. A bolus of 10000 U/kg UTI was intravenously injected after SAP establishment. T cell apoptosis was determined by Annexin-V/PI double-staining. Oxidative stress was evaluated by examining changes in the levels of reactive oxygen species (ROS). Total superoxide dismutase (SOD) in serum was tested by hydroxylamine colorimetric assay, and malondialdehyde levels were examined by thiobarbituric acid assay. Mitochondrial function was evaluated by analyzing mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (MPTP).Results: We found CD4 + T cells (32.10±2.87% vs. 45.22±4.38%, P&lt;0.01) and CD4 +/CD8 + T cells in SAP rats significantly decreased compared with sham group (1.15±0.12 vs. 2.23±0.12%, P&lt;0.01), while the percent of the apoptotic CD4 + and (17.70±2.10 vs. 3.82±0.50%, P&lt;0.01) CD8+ T lymphocytes was highly increased (2.78±0.45 vs. 1.97±0.36%, P&lt;0.01 compared with sham group). After UTI treatment, the apoptosis of CD4+ T lymphocytes significantly decreased compared with SAP group (8.58±1.09 vs. 17.70±2.10%, P&lt;0.01), while the percent of CD4 + T and CD4 +/CD8 + lymphocytes significantly enhanced (P&lt;0.01). ROS (mean fluorescence intensity): 5107±430 vs. 12904±840, P&lt;0.01) and MDA levels(4.41±0.32 vs. 7.25±0.57nmol/ml, P&lt;0.01) in serum in UTI-treated group were decreased compared with SAP group. SOD activity was enhanced after UTI treatment (59.72±5.45 vs. 48.32±3.81nmol/ml, P&lt;0.01). Mitochondrial function assays showed that MMP (17.30±1.60 vs. 46.94±3.49%, P&lt;0.01) and MPT (30.14±2.46 vs. 51.31±3.23%, P&lt;0.01) were inhibited by UTI.Conclusion: UTI reduces T lymphocytes apoptosis and improves immunological function in SAP rats, possibly via enhancing the scavenging capacity of oxygen free radical and attenuating the influence of oxidative stress.Key words: Ulinastatin, T cell, Apoptosis, Severe acute pancreatitis, Mitochondrion

Effects and mechanisms of alveolar type II epithelial cell apoptosis in severe pancreatitis-induced acute lung injury

This study aimed to examine the role of alveolar type II epithelial cell (AEC II) apoptosis in severe pancreatitis-induced acute lung injury (ALI) and the intervening role of Qingyi decoction (QYT). An SAP model was established in male Sprague-Dawley rats. Immunohistochemical analysis was conducted to observe the pathological changes in the pancreas and lung tissue. AEC II apoptosis was detected by flow cytometry and the free Ca2+ concentration in AECs II was determined by laser scanning confocal microscopy. A radioimmunoassay was performed to determine serum TNF-α content. Quantitative polymerase chain reaction (qPCR) and immunohistochemical analysis were performed to detect the mRNA and protein expression levels of Bax and caspase-8 in the lung tissue. Hematoxylin and eosin staining of lung tissue sections in the severe acute pancreatitis (SAP) group showed pathological changes from control tissue, consistent with acute lung injury (ALI). Flow cytometry showed that the level of AEC II apoptosis in the SAP group was significantly increased compared with that in the control group (P<0.01). Laser scanning confocal microscopy indicated that the free Ca2+ concentration in the AECs II of the SAP group was also significantly increased compared with that in the control (P<0.01). Radioimmunoassay demonstrated that the TNF-α levels were significantly increased in the SAP group compared with those in the control group (P<0.01), and qPCR results showed that the levels of Bax and caspase-8 apoptotic gene expression in the AECs II of the SAP group were significantly elevated (P<0.01). The aforementioned indicators were significantly lower following drug treatment compared with the levels observed in the SAP model group. These results suggest that AEC II apoptosis is involved in the ALI procedure associated with SAP. The mitochondrial pathway and death receptor pathway may have key regulatory roles in AEC II apoptosis. The use of QYT may significantly reduce the extent of lung injury.

Da-Huang-Fu-Zi-Tang attenuates liver injury in rats with severe acute pancreatitis

Ethnopharmacological relevanceDa-Huang-Fu-Zi-Tang (DHFZT) is a famous traditional Chinese prescription with strong anti-inflammatory effects. Our previous work found that DHFZT could act against pancreatic injury in rats with severe acute pancreatitis (SAP) via inhibiting the Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) signaling pathway in pancreatic tissues. Aim of the studyTo investigate the therapeutic effects of DHFZT on liver injury in SAP rats, and the effects on JAK2/STAT3 signaling in liver tissue and Kupffer cells (KCs). Materials and methodsFifty SD male rats were randomly divided into five groups: sham operation group (SO), SAP model group, DHFZT treatment groups (12, 24, and 48mg/kg body weight). The model of SAP was constructed by injecting sodium taurocholate (3.5%) into pancreatic and biliary ducts. One hour before constructing the model, DHFZT was perfused into the stomach. All rats were sacrificed after 24h following the operation; livers were examined with hematoxylin and eosin staining. The protein expression of pJAK2 and pSTAT3 in liver tissue was detected by immunohistochemical staining. The activity of ALT, IL-6 and TNF-α in serum was detected. KCs of each group were isolated. After culture for 4h, the protein expression of JAK2, pJAK2, STAT3 and pSTAT3, the mRNA expression of IL-6 and TNF-α in KCs were examined. ResultsSodium taurocholate induced liver injury concomitant with increased expression of pJAK2 and pSTAT3 in liver tissue and KCs. Pretreatment with DHFZT significantly attenuated liver injury induced by SAP, and concurrently, effectively lowered the serum ALT level. Furthermore, our studies showed that DHFZT obviously decreased the expression of pJAK2 and pSTAT3 in liver tissue and KCs. ConclusionsDHFZT could ameliorate liver injury in rats with SAP.

Therapeutic effects of electroacupuncture at ST36 acupoint on sodium-taurocholate-induced severe acute pancreatitis

To explore the protective effects and mechanisms of electroacupuncture (EA) at Zusanli (ST36) acupoint in rats with severe acute pancreatitis (SAP). Sixty-six male Sprague-Dawley rats were randomly assigned to three groups of 22 each: a SAP model group (SAP group), a shamoperated group (sham group) and a EA at ST36 acupoint group (EA group). A rat model of SAP was induced by pancreatic duct injection with 3.5% sodium taurocholate. EA was performed at ST36 acupoint for 30 min after induction of SAP and 30 min before sacrificed. The rats were killed at 3 h (n=7), 6 h (n=7) and 12 h (n=8) after operation, and blood samples were taken for the measurement of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and acetylcholine (Ach) by enzyme-linked immunosorbnent assay. The pathological changes of pancreatic tissue, volume of ascites and pancreatic weight/body weight ratio were measured. The serum concentrations of TNF-α and IL-6 in the EA group were significantly lower than in the SAP group at 3, 6 and 12 h after the operation (p<0.05). Serum Ach in the EA group was significantly higher than in the SAP group at various time points after operation (p<0.05). The other parameters were clearly improved after treatment with EA. EA at ST36 acupoint might have a therapeutic effect in rats with SAP through activating the cholinergic anti-inflammatory pathway.

Effect of Emodin on Endoplasmic Reticulum Stress in Rats with Severe Acute Pancreatitis

This study aimed to investigate the protective effect of emodin on endoplasmic reticulum (ER) stress in rats with severe acute pancreatitis (SAP) and the underlying molecular mechanism. Sprague-Dawley male rats were randomly divided into sham operation group, SAP model group, and emodin treatment group. SAP was constructed through injecting sodium taurocholate into pancreatic and biliary duct in rats. Half an hour before establishing the animal model, emodin or sodium carboxymethylcellulose was intragastrically administrated to the rats in respective group. Rats were killed at 3, 6, and 12 h postdisease induction. The amylase, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) levels in serum, pancreatic histopathology, acinar ER ultrastructure, protein expression of Bip, IRE1α,TRAF2, ASK1, p-JNK, and p-p38 MAPK in pancreas were examined. Sodium taurocholate induced pancreatic injury and ER lumen dilated in exocrine pancreas in rats at 3-, 6-, and 12-h time points. ER stress transducers Bip, IRE1α, and their downstream molecules TRAF2, ASK1 in pancreatitis were upregulated. Furthermore, phosphorylation of JNK and p38MAPK in pancreas was increased, which induced high expression level of inflammatory cytokines such as TNF-α and IL-6. Treatment with emodin obviously ameliorated pancreatic injury and decreased the release of amylase and inflammatory cytokines. Further studies showed that emodin significantly decreased the expression of Bip, IRE1α, TRAF2, and ASK1, inhibited phosphorylation of JNK and p38 MAPK in pancreas in rats at all time points. Emodin could reduce pancreatic injury and restrain inflammatory reaction in SAP rats partly via inhibiting ER stress transducers IRE1α and its downstream molecules.

Effects of Shenmai injection on the expression of intercellular adhesion molecule-1 in rats with severe acute panereatitis

Objective To explore the impact of Shenmai injection on the levels of plasma soluble intercellular adhesion molecule-1 ( sICAM-1 ) and ICAM-1 mRNA in the pancreas tissue of rats with severe acute pancreatitis (SAP).Methods One hundred and eight SD male rats were randomly divided into three groups:sham operation group (group A),SAP model group (group B),Shenmai injection group ( group C).The rats in groups B and C were further divided into four subgroups based on the time points of constructing model:4,8,16 and 24 h subgroups (n =12 each).The levels of plasma sICAM-1 and the ICAM-1 mRNA in the pancreas tissue of rats were compared among the three groups.Results The levels of plasma sICAM-1 were significantly higher in group B than in 4,8,16 and 24 h subgroups of group A (128.07±15.86 vs 87.31 ±13.16,142.33±22.61 vs 98.82 ±11.39,187.45±17.39 vs 129.51 ±15.61,and 176.18 ± 19.36 vs 123.57 ± 17.03) ( all P ＜0.05).The levels of plasma sICAM-1 were significantly lower in group C than those in 4,8,16 and 24 h subgroups of group B (92.55 ± 8.09 vs 128.07 ± 15.86,105.28 ± 14.29 vs 142.33 ± 22.61,142.36 ± 18.25 vs 187.45 ± 17.39,131.32 ± 21.83 vs 176.18 ±19.36) ( all P ＜ 0.05 ).The expression of 1CAM-1 mRNA in the pancreatic tissue was significantly higher in group B than that in4,8,16 and 24 h subgroups of group A (0.97 ±0.12 vs 0.52 ±0.11,1.08 ±0.18 vs 0.61±0.13,1.32±0.18 vs0.82±0.14,1.23 ±0.09vs0.74±0.13) (allP＜0.05).The expression of ICAM-1 mRNA in the pancreatic tissue was significantly lower in group C than that in 4,8,16 and 24 h subgroups of group B ( 0.59 ± 0.13 vs 0.97 ± 0.12,0.68 ± 0.09 vs 1.08 ± 0.18,0.96 ±0.20vs1.32±0.18,0.88±0.09vs1.23±0.09) (allP＜0.05).Conclusion Shenmai injection could improve microcirculation and protect rats with SAP through decreasing the levels of plasma sICAM-1and the ICAM-1 mRNA of the pancreas tissue. Key words: Severe acute pancreatitis ; Intercellular adhesion molecule-1 ; Shenmai injection