Rat Model Of Severe Acute Pancreatitis Research Articles

Dexmedetomidine attenuates inflammation and pancreatic injury in a rat model of experimental severe acute pancreatitis via cholinergic anti-inflammatory pathway.

BackgroundExcessive inflammatory responses play a critical role in the development of severe acute pancreatitis (SAP), and controlling such inflammation is vital for managing this often fatal disease. Dexmedetomidine has been reported to possess protective properties in inflammatory diseases. Therefore, this study aimed to investigate whether dexmedetomidine pre-treatment exerts an anti-inflammatory effect in rats with SAP induced by sodium taurocholate, and if so, to determine the potential mechanism.MethodsSAP was induced with sodium taurocholate. Rats received an intraperitoneal injection of dexmedetomidine 30 min before sodium taurocholate administration. α-bungarotoxin, a selective alpha-7 nicotinic acetylcholine receptor (α7nAchR) antagonist, was injected intra-peritoneally 30 min before dexmedetomidine administration. The role of the vagus nerve was evaluated by performing unilateral cervical vagotomy before the administration of dexmedetomidine. Efferent discharge of the vagal nerve was recorded by the BL-420F Data Acquisition & Analysis System. Six hours after onset, serum pro-inflammatory cytokine (tumor necrosis factor α [TNF-α] and interleukin 6 [IL-6]) levels and amylase levels were determined using an enzyme-linked immunosorbent assay and an automated biochemical analyzer, respectively. Histopathological changes in the pancreas were observed after hematoxylin and eosin staining and scored according to Schmidt criteria.ResultsPre-treatment with dexmedetomidine significantly decreased serum levels of TNF-α, IL-6, and amylase, strongly alleviating pathological pancreatic injury in the rat model of SAP (TNF-α: 174.2 ± 30.2 vs. 256.1±42.4 pg/ml; IL-6: 293.3 ± 46.8 vs. 421.7 ± 48.3 pg/ml; amylase: 2102.3 ± 165.3 vs. 3186.4 ± 245.2 U/L). However, the anti-inflammatory and pancreatic protective effects were abolished after vagotomy or pre-administration of α-bungarotoxin. Dexmedetomidine also significantly increased the discharge frequency and amplitude of the cervical vagus nerve in the SAP rat model (discharge frequency: 456.8 ± 50.3 vs. 332.4 ± 25.1 Hz; discharge amplitude: 33.4 ± 5.3 vs. 20.5 ± 2.9 μV).ConclusionsDexmedetomidine administration attenuated the systemic inflammatory response and local pancreatic injury caused by SAP in rats through the cholinergic anti-inflammatory pathway involving vagus- and α7nAChR-dependent mechanisms.

Prophylactic low-molecular-weight heparin administration protected against severe acute pancreatitis partially by VEGF/Flt-1 signaling in a rat model.

The present study was aimed to explore the effects and the underlying mechanism of prophylactic low-molecular-weight heparin (LMWH) treatment on taurocholate-induced severe acute pancreatitis (SAP) in a rat model. Rat SAP model was induced by injection of 4% sodium taurocholate into the pancreatic duct. LMWH was applied half an hour before the induction of pancreatitis at the dose of 200 IU/kg subcutaneous injection. The rats were euthanized at 1 h, 6 h, and 12 h after taurocholate-induced SAP. The inflammatory and oxidative response markers were assessed. And the vascular endothelial growth factor (VEGF) and Fms-related tyrosine kinase 1 (Flt-1) expression were evaluated by immunohistochemistry (IHC) and western blot methods. The expression of inflammatory and oxidative response markers increased after induction of SAP. IHC and western blot results showed the VEGF and Flt-1 expression were increased in SAP group. Prophylactic LMWH administration reduced the inflammatory and oxidative response markers expression and decreased the expression of VEGF and Flt-1. This study suggested that prophylactic LMWH treatment mitigated the severity of pancreatitis in rat SAP model by anti-inflammation and oxidative response. The underlying mechanism may result from downregulating VEGF/Flt-1 signaling of LMWH in SAP rat model.

Inhibition of Ferroptosis Attenuates Acute Kidney Injury in Rats with Severe Acute Pancreatitis

Acute kidney injury (AKI) is a frequent complication of severe acute pancreatitis (SAP). Ferroptosis is involved in a range of diseases. However, the role of ferroptosis in SAP-induced AKI has yet to be elucidated. We aimed to investigate whether ferroptosis is induced in the kidney after SAP and whether inhibition of ferroptosis ameliorates AKI in a rat model of SAP. Sodium taurocholate (5%) was retrogradely perfused into the biliopancreatic duct to establish a model of SAP with AKI in rats. The levels of serum amylase, lipase, tumor necrosis factor (TNF)-α, interleukin (IL)-6, creatinine (Cr) and blood urea nitrogen (BUN) in rats were measured. We also determined the biochemical and morphological changes associated with ferroptosis in renal tissue, including iron accumulation, lipid peroxidation assays, and mitochondrial shrinkage. H&E staining was used to assess pancreatic and renal histological changes. Western blot analysis, RT-PCR, and immunofluorescence staining were performed to analyze the expression of ferroptosis-related proteins and genes. SAP-induced AKI was followed by iron accumulation, increased lipid peroxidation, and upregulation of ferroptosis-related proteins and genes. Twenty-four hours after SAP, TEM confirmed the presence of typical shrunken mitochondria. Furthermore, treatment with liproxstatin-1 lowered the levels of serum amylase, TNF-α, IL-6, Cr and BUN, decreased kidney lipid peroxidation and alleviated pancreatic and renal histopathology injury in SAP rats. Our findings are the first to demonstrate the involvement of ferroptosis in SAP-associated renal damage and present ferroptosis as a therapeutic target for effective treatment of SAP-induced AKI.

Effects of macrophage migration inhibitory factor on early acute pancreatitis

To investigate the value of macrophage migration inhibitor factor (MIF) in early severe acute pancreatitis (SAP). (1) Animal experiment: according to the random number table method, 24 male Sprague-Dawley (SD) rats were divided into Sham group and SAP 3, 6 and 12 hours groups, with 6 rats in each group. SAP rat model was prepared by injecting 5% sodium taurocholate via the retrograde cholangiopancreatic duct. Liver, kidney, lung, pancreas and serum samples were harvested after 3, 6 and 12 hours. In the Sham group, tissue and serum were harvested immediately after pancreas was turned over. The histopathological changes of the pancreas were observed microscopically by hematoxylin-eosin (HE) staining. The MIF levels of serum, liver, kidney, lung and pancreas were measured by enzyme linked immunosorbent assay (ELISA). (2) Clinical study: an observational study was conducted. Seventy-two adult patients within 24 hours of the onset of abdominal pain (blood amylase was 3 times the normal level), and the clinical diagnosis met the criteria of acute pancreatitis (AP) admitted to the emergency department of the First Affiliated Hospital of Zhengzhou University from December 2018 to October 2019 were enrolled. Venous blood was extracted and serum MIF level was determined by ELISA. Acute physiology and chronic health evaluation II (APACHE II) was recorded for 24 hours. Patients were divided into SAP group (17 cases), moderate severe acute pancreatitis (MSAP) group (25 cases), and mild acute pancreatitis (MAP) group (30 cases) according to the revised Atlanta criteria for comparison between groups. (1) The results of animal experiments showed that the serum, liver, and pancreatic MIF levels of rats in the SAP group all reached the peak at 6 hours after modeling, and the differences were statistically significant compared with the Sham group [serum MIF (ng/L): 2 862.79±238.33 vs. 1 728.32±197.59, liver MIF (ng/L): 2 141.39±328.07 vs. 1 372.70±163.41, pancreas MIF (ng/L): 4 468.00±1 324.31 vs. 1 572.06±108.40, all P < 0.01]; although the levels of MIF in serum, liver and pancreas decreased at 12 hours after modeling, they were still significantly higher than Sham group. However, there was no statistically significant difference in MIF levels of lung and kidney in SAP rats compared with Sham group at 3, 6 and 12 hours after molding. (2) Clinical observation showed that early serum MIF levels of SAP, MSAP and MAP patients decreased in order, (14.83±2.99), (10.17±2.64), and (7.21±2.47) μg/L, respectively; APACHE II scores also decreased in order, 10.41±3.74, 7.60±3.18 and 4.00±2.41 respectively. Correlation analysis showed that serum MIF levels in patients with SAP, MSAP, and MAP had a good correlation with APACHE II scores of the respective groups, showing that MIF levels was positively correlated with disease severity (SAP: r = 0.51, P = 0.03; MSAP: r = 0.45, P = 0.02; MAP: r = 0.45, P = 0.01). MIF can predict the occurrence of early SAP, and it is related to the severity of early AP.

Protective Effects of Bone Marrow–Derived Mesenchymal Stem Cells on Insulin Secretion and Inflammation in the Treatment of Severe Acute Pancreatitis in Rats

ObjectiveThe present study aims to demonstrate the protective effect of bone marrow mesenchymal stem cells (bmMSCs) on transplanted islets and its potential therapeutic role of severe acute pancreatitis (SAP) in rat model. Materials and MethodsMesenchymal stem cells (MSCs) were isolated from 6 male SD rats, and were identified. The Islets isolated from 20 SD rats were evenly and randomly divided into co-culture group, and basic culture group (control group), in which the islets were cultured in DMEM/F12 medium, so as to compare the insulin secretion and stimulation index. Severe AP was induced in SD rats by retrograde injection of sodium taurocholate. Ninety rats were randomly and evenly assigned into 5 groups: control group (healthy rats), SAP group, tail vein injection group, intraperitoneal injection group and combined injection (tail vein + intraperitoneal) group. Rats were sacrificed on day 1, 2, and 3. The pancreatic tissues and blood were collected. The plasma levels of IL-10, IL-1β, TNF-α, IL-6 were determined using ELISA. Pathologic changes of the pancreas were observed using HE staining, and the positioning of DAPI labeled bmMSCs in vivo were detected. ResultsInsulin secretion and the stimulation index of co-culture group were significantly higher than those of basic culture group (P < .05), after 7 and 14 days of culture. Inflammation, edema, hemorrhage and necrosis in each model of pancreatitis were reduced significantly in BMMSCs injection group as compared to SAP group (P < .05). Infused BMMSCs through combined injection indicated improved outcome than that of tail-vein injection or intraperitoneal injection alone. ConclusionCo-culture of BMMSCs with transplanted islets prolongs the survival time of islets and maintains in vitro activity. In the rat model of SAP, combined injection of BMMSCs through tail vein and intraperitoneal significantly suppresses the inflammatory reaction and alleviates pancreatic injury in rat SAP model.

Berberine hydrochloride mitigates acute pancreatitis by suppressing the TLR4/IκBα/NFκB pathway

Severe acute pancreatitis (SAP) usually causes multiple-organ dysfunction syndrome and exhibits a high mortality rate. It has been previously reported that berberine, as a dominant alkaloid, can be effectively used against inflammation. However, there is limited data on the effects of berberine hydrochloride (BBRH) on inflammation in SAP rat models. Male Sprague–Dawley rats were given preliminary intraperitoneal injections of BBRH 30 min prior to retrograde infusion of 5% sodium taurocholate in their biliopancreatic ducts. The rats were sacrificed 12 h after taurocholate administration, and tissues and blood samples were then acquired for histological, chemical, and molecular analyses. The SAP models exhibited enhanced edema, ascitic fluid volume, neutrophil infiltration, serum amylase and lipase levels, and interleukin (IL)-6, tumor necrosis factor-α, and IL-1β levels, and reduced IL-10 concentration, while BBRH administration reversed these changes. Further study showed that in the SAP model, protein and mRNA levels of nuclear factor kappa-light-chain-enhancer of activated B (NFκB) p65 and toll-like receptor 4 (TLR4) were increased and inhibitor of nuclear factor kappa B (IκBα) expression was decreased, while pretreatment with BBRH significantly decreased the activation of the TLR4/IκBα/NFκB pathway. The data obtained displayed that BBRH mitigated acute pancreatitis by suppressing the TLR4/IκB/NFκB pathway.

A New Role for the Spleen: Aggravation of the Systemic Inflammatory Response in Rats with Severe Acute Pancreatitis

Little is known about the role of the spleen in mediating systemic inflammatory responses in severe acute pancreatitis (SAP). We investigated the role played by the spleen in rats after SAP induction. Splenectomy was performed at designated time points after SAP induction. Pancreatic tissue and serum samples were collected and subjected to histologic, immunohistochemical, and immunologic analyses. After SAP induction, the splenic immune response was enhanced during SAP progression, as shown by the increased diameter of the splenic periarterial lymphatic sheath and the thickness of the splenic marginal zone. Rats with splenectomy developed acute pancreatitis more slowly than rats without splenectomy. In addition, pancreatic tissues of rats with splenectomy contained lower levels of serum amylase, tumor necrosis factor-α, and IL-6 and exhibited less acinar cell death, leukocyte infiltration, and interstitial edema than those of rats without splenectomy. Compared with splenectomy alone, cotreatment with splenectomy and the administration of splenic cells originating from a rat with SAP 12 hours after induction increased systemic inflammation in SAP rats. Splenic factors exacerbated SAP-associated liver and lung injury and accentuated intestinal mucosal barrier dysfunction. Splenectomy altered the serum cytokine profile in rats with SAP. In a rat model of SAP, the spleen exacerbated the systematic inflammatory responses and injury to multiple organs, indicating a new role for the spleen in SAP.

Improvement of Gut Microbiota by Inhibition of P38 Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Rats with Severe Acute Pancreatitis.

BackgroundGut microbiota dysbiosis plays a key role in pathogenesis of severe acute pancreatitis (SAP). In this study, we explored the protective effects of the p38 MAPK inhibitor, SB203580, against gut inflammation and microbiota dysbiosis induced by pancreatic duct injection with 3.5% sodium taurocholate in an SAP rat model.Material/MethodsNinety male Sprague-Dawley rats were randomly assigned to sham-operated, SAP model, and SAP plus SB203580 groups (n=30/group). Histological examination was conducted to assess gut and pancreatitis injury. The levels of amylase, D-lactate, diamine oxidase, tumor necrosis factor α, IL-6, IL-1β, and phospho-p38MAPK in the plasma and intestine were evaluated at 3, 6, or 12 h after SAP induction. The gut microbiome was investigated based on16S rDNA gene sequencing at 12 h after SAP induction.ResultsHistological examination revealed edema and inflammatory infiltrations in the pancreas and distal ileum. The expression of tumor necrosis factor α, IL-1β, and IL-6 in plasma and distal ileum was increased in the SAP group, which were restored after treatment with SB203580. Significantly lower bacterial diversity and richness was found in the SAP group. In the SAP group, the abundance of Bacteroidetes and Firmicutes was decreased, and there was a higher proportion of Proteobacteria at the phylum level. The SAP plus SB203580 group exhibited significantly less damage to the gut microbiota, with higher bacterial diversity and a more normal proportion of intestinal microbiota.ConclusionsSB203580 mediated suppression of the p38 MAPK signaling pathway via reduced gut inflammatory response and microbiota dysbiosis.

Qingyi decoction attenuates severe acute pancreatitis in rats via inhibition of inflammation and protection of the intestinal barrier.

ObjectiveQingyi decoction (QYD) has beneficial effects in severe acute pancreatitis (SAP). We assessed the therapeutic effect and mechanisms of QYD in SAP.MethodsA rat model of SAP was induced by pancreatic ductal injection of sodium taurocholate. QYD was administered intragastrically immediately postoperatively and once every 12 hours. Serum amylase, endotoxin, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and D-lactate levels were measured at 12, 24, and 48 hours. Histological changes in the pancreas and ileum were analyzed. Expression of nuclear factor kappa-light-chain-enhancer of activated B cells p65 (NF-κB p65), Toll-like receptor 4 (TLR4), and zonula occludens-1 (ZO-1) in the small intestinal mucosa was also assessed.ResultsPancreatic tissue showed extracellular space expansion, inflammatory infiltration, vessels with necrotic walls, and hemorrhage. Ileal tissue showed hemorrhage, inflammatory infiltration, and ileal mucosa destruction. These histological features were dramatically improved by QYD. Increased serum levels of amylase, endotoxin, TNF-α, IL-6, and D-lactic acid were significantly decreased by QYD administration. Increased expression of NF-κB p65 and TLR4 and decreased expression of ZO-1 in the ileal mucosa were also restored to normal levels by QYD treatment.ConclusionQYD alleviates SAP by reducing intestinal barrier dysfunction, inhibiting intestinal bacteria and endotoxin translocation, and preventing NF-κB activation.

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.

Bidirectional Effects of Pyrrolidine Dithiocarbamate on Severe Acute Pancreatitis in a Rat Model.

Introduction:The mechanism by which intestinal mucosal barrier is damaged in severe acute pancreatitis (SAP)-associated impairment is not fully understood.Methods:We established an l-arginine-induced SAP rat model, pretreated with or without pyrrolidine dithiocarbamate (PDTC). Hematoxylin and eosin staining was performed to evaluate the pathological alterations. Western blotting was conducted to detect the expression of autophagy-related proteins. Oxidative stress was assessed by the levels of malondialdehyde and superoxide dismutase.Results:We found significant injury of the intestinal mucosal barrier in SAP rats, with overexpression of Beclin-1, LC3, and p65. Pyrrolidine dithiocarbamate showed a bidirectional effect in protecting SAP rats. A high dose of PDTC aggravated disease in rats, while a low or medium dose of PDTC pretreatment, was able to alleviate tissue damage. Pyrrolidine dithiocarbamate changed the expression of Beclin-1, LC3, and p65 in the intestines. The fatty acid-binding protein level was increased in SAP rats with high-dose PDTC or without PDTC pretreatment and was reduced in SAP rats with low- or medium-dose PDTC exposure.Conclusions:Autophagy is involved in the impairment of intestinal mucosal barrier during SAP. A suitable dose of PDTC (1 or 10 mg/kg) may decrease the severity of SAP by inhibiting autophagy in intestinal mucosal cells.

Correlation between the liver injury and the expression of interleukin-10 in severe acute pancreatitis at high altitude: a rat experimental result

To observe the indexes of liver injury and the expression of inflammation-related factor interleukin-10 (IL-10) in rats with severe acute pancreatitis (SAP), and to discuss the correlation between the expression of IL-10 and the related factors of liver injury in SAP rats at different altitudes. 280 male Wistar rats with SPF grade aged 5 to 6 months were divided into four groups according to random number table with 70 rats in each group, and the rats were placed in different altitudes such as Xi'an (at an altitude of 1 027 m), Xining (at an altitude of 2 260 m), Xinghai (at an altitude of 3 300 m) and Wenquan (at an altitude of 3 950 m). The rats in each altitude were randomly divided into sham operation group (Sham group, n = 10) and SAP 1, 6, 12, 24 hours groups (all n = 15). SAP rat model was reproduced by injecting sodium cholate into the posterior membrane of pancreas, and the rats of Sham group were only turned pancreas over several times after opening the abdomen and then closed the abdomen. The rats were sacrificed at the corresponding time points after model reproduction in SAP groups, and rats in Sham group were sacrificed at 6 hours after sham operation. At the same time, the abdominal aorta blood was harvested, and the contents of serum amylase (AMY), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by automatic biochemical analyzer. Liver tissues were harvested, and the expression level of IL-10 was determined by immunohistochemistry. Pancreatic tissues were harvested, and hematoxylin-eosin (HE) staining was performed to observe the pathological changes under light microscopy. The correlations among the indicators were analyzed by Pearson correlation. At different altitudes, no significant abnormality was found in the pancreas of Sham group, but significant pathological changes were found in the pancreas of all SAP groups, mainly manifested as pancreatic acinar swelling, inflammatory cell infiltration, vascular congestion and hemorrhage, acinar cell degeneration and dissolution, changes in glandular lobule structure, peri-pancreatic fat necrosis, and continuous aggravation with the increasing of time and altitude. At the same altitude, the pancreatic pathology score, the serum AMY, ALT and AST levels, and the hepatic IL-10 expression were all significantly increased in all the SAP groups as compared with those in Sham group, and they were continuously increased with time. In Sham group, there was no statistically significant difference in pancreatic pathology score, AMY, ALT, AST, or IL-10 level among different altitudes. At the corresponding time point after model reproduction, the pancreatic pathology score, AMY, ALT, AST and IL-10 levels in the SAP groups were also shown a continuous rising tendency with altitude increase, and the differences in above parameters of SAP 24 hours group in Wenquan area were statistically significant as compared with those of Sham group [pathology score: 11.06±0.94 vs. 0.23±0.15, AMY (mmol/L): 2 706.6±208.3 vs. 336.5±94.3, ALT (U/L): 267.00±5.37 vs. 52.00±4.84, AST (U/L): 465.88±11.02 vs. 139.00±11.61, IL-10 (A value): 0.579±0.006 vs. 0.281±0.006, all P < 0.05]. It was shown by correlation analysis that IL-10 of SAP rats at different altitudes was positively correlated with pancreatic pathology score, AMY, ALT and AST, the correlation coefficient (r value) between IL-10 and the above indicators in the Wenquan area with the highest altitude was 0.959, 0.928, 0.977, 0.983, respectively (all P < 0.01). The severity of SAP rats was positively correlated with altitude. IL-10 was involved in the pathological expression process of SAP liver damage, and its expression level was positively correlated with altitude and the degree of SAP liver damage.

SRT1720 ameliorates sodium taurocholate-induced severe acute pancreatitis in rats by suppressing NF-κB signalling.

Severe acute pancreatitis (SAP) is a medical emergency that is often associated with multiple organ failure and high mortality. Although an SAP diagnosis requires prompt treatment, therapeutic options remain limited. SRT1720 is a newly formulatedSIRT1 activator that exerts multiple pharmacological activities with beneficial health effects. However, its potential as an SAP treatment has not been explored. The current study assessed the effect of SRT1720 on a rat model of sodium taurocholate-induced SAP and explored the underlying mechanism. SAP was induced in rats by retrograde injection of a 3.5% sodium taurocholate solution (1 ml/kg) in the biliopancreatic duct. SRT1720 (5 mg/kg) was administered intraperitoneally after sodium taurocholate exposure. Serum samples were analysed for inflammatory cytokine levels and select enzymatic activities using the enzyme-linked immunosorbent assay and commercial enzyme activity assay kits, respectively; protein expression levels were evaluated by western blotting; mRNA levels of biomarkers were determined by quantitative real-time PCR; histopathological changes were analysed by haematoxylin and eosin staining and immunohistochemistry.SRT1720 treatment significantly reduced serum amylase, lipase, pancreatic histological scores, proinflammatory cytokine (TNF-α and IL-6) levels, and expression of NF-κB and p65 in sodium taurocholate-induced SAP rats. Importantly, the treatment stimulated SIRT1 and IκBα levels in pancreatic tissue. Our data suggest that SRT1720 protects rats from sodium taurocholate-induced SAP by suppressing the NF-κB signalling pathway.

Antithrombin III Attenuates AKI Following Acute Severe Pancreatitis.

Antithrombin III (ATIII), the predominant coagulation factor inhibitor, possesses anti-inflammatory properties and exerts renoprotective effects on renal ischemia-reperfusion injury in animal models. However, the ATIII's protective effects of ATIII on acute kidney injury (AKI) following severe acute pancreatitis (SAP) need to be confirmed. We assessed the association between ATIII activities and the incidence of AKI in patients with SAP, and explored therapeutic effects and potential mechanisms of ATIII on kidney injury in sodium taurocholate induced SAP rat model. Rats were intravenously injected with ATIII (500 μg/kg) before or after the induction of SAP. The results demonstrated ATIII did not attenuate pancreatic injury, but significantly ameliorate renal dysfunction and renal histological injury. ATIII administration alleviated renal inflammation response, oxidative stress, and cell apoptosis. Moreover, ATIII attenuated tumor necrosis factor α (TNFα)-stimulated intercellular cell adhesion molecule 1(ICAM-1) and monocyte chemotactic protein 1 (MCP-1) upregulation in cultured renal tubular epithelial cells. ATIII appears to ameliorate SAP-induced kidney injury by inhibiting inflammation, oxidative stress, and apoptosis. ATIII supplementation may have a potential prophylactic and therapeutic effect on SAP induced AKI.

Effects of inhibiting peritoneal macrophage caspase recruitment domain-containing protein 9 on inflammatory reaction of severe acute pancreatitis in rats

Objective To investigate the effects of caspase recruitment domain-containing protein 9 (CARD9) expression in peritoneal macrophages on severe acute pancreatitis (SAP) in rats and its mechanism. Methods A total of 60 male Sprague Dawley rats were divided into control group (n=6), SAP group (n=18), small interfering RNA (siRNA) control group (n=18) and siRNA CARD9 group (n=18). SAP rat models were established. At three, six and twelve hours after the models were established, ascites was collected, peritoneum was lavaged and peritoneal macrophages were isolated and cultured. The expressions of CARD9, nuclear factor-kappaB (NF-κB), p38 mitogen-activated protein kinase (p38MAPK) at mRNA level in peritoneal macrophages was measured by real-time polymerase chain reaction (RT-PCR). The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in peripheral blood were detected by enzyme-linked immunosorbent assay (ELISA). LSD or Tamhane′s T2 methods were performed for statistical analysis. Results At three, six and twelve hours after the models were established, CARD9 mRNA levels of peritoneal macrophages in SAP group were 1.63±0.05, 1.68±0.24 and 2.61±0.02, respectively, which were all higher than that of control group (1.01±0.23), and the differences were statistically significant (t=25.97, 6.86 and 131.59; all P<0.05); the levels of CARD9 mRNA of siRNA CARD9 group were 1.45±0.02, 1.24±0.03 and 1.63±0.03, respectively, which were lower than that of SAP group at the same time points, and the differences were statistically significant (t=-7.81, -4.46 and -62.13; all P<0.05). At three, six and twelve hours after the models were established, the mRNA levels of NF-κB and p38MAPK of peritoneal macrophages of rats in SAP group were 1.51±0.08, 1.81±0.10, 2.30±0.05 and 1.37±0.13, 1.69±0.18, 2.42±0.23, respectively, which were higher than those of control group (1.00±0.01, 1.03±0.08), and the differences were statistically significant (tNF-κB=15.10, 19.95 and 60.36; tp38MAPK=5.37, 8.34 and 14.11; all P<0.05); the levels of NF-κB mRNA in siRNA CARD9 group were 1.38±0.05, 1.57±0.06 and 1.76±0.09, respectively, which were lower than that of SAP group at the same time points, and the differences were statistically significant (t=-3.32, -5.07 and -12.70; all P<0.05). At six and twelve hours after the models were established, the p38MAPK mRNA levels of siRNA CARD9 group were 1.50±0.10 and 2.00±0.09, respectively, which were lower than that of SAP group, and the differences were statistically significantly (t=-2.30 and -4.17, both P<0.05). At three, six and twelve hours after the models were established, the levels of TNF-α, IL-1β and IL-6 in peripheral blood of SAP group were (53.49±21.64) pg/mL, (108.62±22.76) pg/mL and (139.00±15.35) pg/mL; (43.86±18.30) pg/mL, (87.51±17.10) pg/mL and (117.27±14.57) pg/mL; (78.38±32.70) pg/mL, (156.39±30.56) pg/mL and (209.56±26.09) pg/mL, respectively, which were higher than those of control group ((2.79±1.17), (7.13±4.52), (12.73±8.08) pg/mL), and the differences were statistically significant (tTNF-α=5.73, 11.37 and 21.69; tIL-1β=4.77, 11.13 and 17.68; tIL-6=4.77, 11.32 and 17.68; all P<0.05). At six and twelve hours after the models were established, the levels of TNF-α, IL-1β and IL-6 of siRNA CARD9 group were (75.73±16.93) pg/mL, (108.23±14.02) pg/mL; (63.05±11.98) pg/mL, (91.56±14.28) pg/mL and (112.67±21.40) pg/mL, (163.62±25.51) pg/mL, respectively, which were lower than those of SAP group, and the differences were statistically significant (tTNF-α=-2.84, -3.63; tIL-1β=-2.88, -3.09; tIL-6=-2.88, -3.09; all P<0.05). Conclusions There are CARD9-related NF-κB and p38MAPK pathways in peritoneal macrophages of SAP rats. Intervention of the expression of CARD9 in peritoneal macrophages, especially at early stage of SAP may relieve the inflammation reaction and pancreatic injury, which may provide a new method for SAP treatment. Key words: Macrophages, abdominal cavity; NF-kappaB; p38 mitogen-activated protein kinases; RNA, small interfering; Rats; Pancreatitis, severe acute; Caspase recruitment domain-containing protein 9

Interleukin-10 attenuates impairment of the blood-brain barrier in a severe acute pancreatitis rat model

BackgroundImpairment of the blood-brain barrier (BBB) in severe acute pancreatitis (SAP) could result in life-threatening pancreatic encephalopathy. Interleukin-10 (IL-10) is a classical cytokine that is well-known for its strong immunoregulatory and anti-inflammatory abilities. However, whether and how IL-10 protects the BBB in SAP are still unclear.MethodsThis study includes in vivo experiments using a SAP rat model and in vitro experiments using an in vitro BBB model consisting of a monolayer of brain microvascular endothelial cells (BMECs). The study groups are divided into the control, SAP (in vivo)/TNF-α (in vitro), IL-10 treatment, IL-10 + signal transducer and activator of transcription 3 (STAT3) inhibitor S3I-201 treatment groups. Pancreatic pathological scores, serum amylase, serum TNF-α levels and BBB permeability by Evan’s blue assay in SAP rat models were evaluated. BMEC apoptosis in SAP rats or induced by TNF-αin vitro was detected by terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and flow cytometry, separately. Expression levels of claudin-5 and proteins involved in the STAT3 signaling pathway were measured by Western blotting. Location and changes of junctional structure of claudin-5 on BMECs were assessed by immunohistochemistry and immunofluorescence.ResultsIn vivo, IL-10 alleviated the severity of inflammation, attenuated the increased BBB permeability in SAP rat models by reducing BMEC apoptosis via the STAT3 pathway and ameliorated the down-regulation of claudin-5 expression in BMECs; in vitro, IL-10 improved BBB integrity against TNF-α by attenuating BMEC apoptosis via the STAT3 pathway, the impairment of tight junction structure and the down-regulation of claudin-5 expression in BMECs.ConclusionsIL-10 improves BBB properties in SAP by attenuating the down-regulation of claudin-5 expression and the impairment of tight junctions and by STAT3 pathway-mediated anti-apoptotic effects on BMECs.

Free Total Rhubarb Anthraquinones Protect Intestinal Injury via Regulation of the Intestinal Immune Response in a Rat Model of Severe Acute Pancreatitis.

Intestinal mucosal immune barrier dysfunction plays a key role in the pathogenesis of severe acute pancreatitis (SAP). Rhubarb is a commonly used traditional Chinese medicine as a laxative in China. It markedly protects pancreatic acinar cells from trypsin-induced injury in rats. Free total rhubarb anthraquinones (FTRAs) isolated and extracted from rhubarb display the beneficial effects of antibacteria, anti-inflammation, antivirus, and anticancer. The principal aim of the present study was to investigate the effects of FTRAs on the protection of intestinal injury and modification of the intestinal barrier function through regulation of intestinal immune function in rats with SAP. We established a rat model of SAP by injecting 3.5% sodium taurocholate (STC, 350 mg/kg) into the biliopancreatic duct via retrograde injection and treated the rats with FTRAs (36 or 72 mg/kg) or normal saline (control) immediately and 12 h after STC injection. Then, we evaluated the protective effect of FTRAs on intestinal injury by pathological analysis and determined the levels of endotoxin (ET), interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), nitric oxide (NO), myeloperoxidase (MPO), capillary permeability, nucleotide-binding oligomerization domain-like receptors 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD domain (ASC), casepase-1, secretary immunoglobulin A (SIgA), regulatory T cells (Tregs), and the ratio of Th1/Th2 in the blood and/or small intestinal tissues or mesenteric lymph node (MLN) cells. Moreover, the chemical profile of FTRAs was analyzed by HPLC-UV chromatogram. The results showed that FTRAs significantly protected intestinal damage and decreased the levels of ET, IL-1β, TNF-α, and NO in the blood and TNF-α, IL-1β, and protein extravasation in the intestinal tissues in SAP rats. Furthermore, FTRAs significantly decreased the expressions of NLRP3, ASC, and caspase-1, the number of Tregs and the ratio of Th1/Th2, while significantly increased the expression of SIgA in the intestinal tissues and/or MLN cells in SAP rats. Our results indicate that FTRAs could protect intestinal injury and improve intestinal mucosal barrier function through regulating immune function of SAP rats. Therefore, FTRAs may have the potential to be developed as the novel agent for the treatment of SAP clinically.

Autophagy Strengthens Intestinal Mucosal Barrier by Attenuating Oxidative Stress in Severe Acute Pancreatitis.

Intestinal mucosal barrier dysfunction can be caused by severe acute pancreatitis (SAP). It is normally associated with changes to mucosal autophagy and oxidative stress. The aim of this study was to investigate the correlation between autophagy and oxidative stress on the intestinal mucosal barrier of SAP rat model. SAP was induced by retrograde injection of sodium taurocholate (5%) into the biliopancreatic duct. Bacterial translocation (BT) was detected by 16S rDNA sequencing analysis. Morphological alterations in the pancreas and gut were determined by hematoxylin-eosin staining. Oxidative stress status was determined by measuring the level of intestinal malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx). Western blot, RT-PCR, and immunofluorescent staining were preformed to analyze the expression of tight junction and autophagy proteins. According to the sequencing analysis, rats in SAP group were divided into BT (+) group (n=9) and BT (-) group (n=8). Pancreatic and intestinal injuries in SAP group were significantly higher than sham operation group. The content of MDA was clearly elevated, and SOD as well as GPx activities were decreased in BT (+) group as compared with BT (-) group. The expression of LC3II and Beclin1 in BT (-) group was higher than that observed in BT (+). In contrast, BT (+) group had a higher level of claudin-2 and a lower level of zonula occluden-1, occludin, and claudin-1. These results suggest that activated autophagy may attenuate intestinal mucosal barrier dysfunction by preventing and reducing the oxidative stress in SAP.

Curcumin protects against acute renal injury by suppressing JAK2/STAT3 pathway in severe acute pancreatitis in rats.

The aim of the present study was to investigate the effect of curcumin on acute renal injury in a rat model of severe acute pancreatitis (SAP). A SAP model with acute kidney injury was established in rats by retrograde injection of 5% sodium taurocholate into the pancreatic duct. The serum amylase, creatinine (Cr) and blood urea nitrogen (BUN) levels in rats were measured. Hematoxylin and eosin staining was used to assess pancreatic and renal histological changes. Serum tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were measured using ELISA kits. Renal protein levels of Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 pathway components were determined by western blot assay. The results showed that curcumin significantly decreased serum amylase, Cr and BUN levels, and alleviated pancreatic and renal histological changes in SAP rats. Furthermore, curcumin markedly decreased serum TNF-α and IL-6 levels and downregulated renal protein levels of JAK2/STAT3 pathway components. These results proved that curcumin ameliorates acute renal injury in a rat model of SAP. The molecular mechanism of its effect may be associated with the suppression of the JAK2/STAT3 pathway to reduce TNF-α and IL-6 levels in SAP-induced acute renal injury. Therefore, the findings of the present study revealed the potential use of curcumin for the prevention and treatment of SAP and the associated renal injury.

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.