Qing Xia Jie Yi Formula granules alleviated acute pancreatitis through inhibition of M1 macrophage polarization by suppressing glycolysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbal formulas from Traditional Chinese Medicine are common and well-established practice for treating acute pancreatitis (AP) patients. However, little is known about their bioactive ingredients and mechanisms, such as their targets and pathways to inhibit inflammation. AIM OF THE STUDY: This study aimed to evaluate the effect of Qing Xia Jie Yi Formula (QXJYF) granules on AP and discuss the molecular mechanisms involved. MATERIALS AND METHODS: Major compounds in QXJYF granules were identified using UPLC-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap MS). The effect of QXJYF granules on experimental AP models both in vitro and in vivo, and detailed mechanisms were clarified. Two AP models were induced in mice by intraperitoneally injections of caerulein or L-arginine, and QXJYF granules were used to treat AP mice in vivo. Histological evaluation of pancreas and lung, serum amylase and lipase levels, serum inflammatory cytokines, inflammatory cell infiltration and macrophage phenotype were assessed. Bone marrow derived macrophages (BMDMs) were cultured and treated with QXJYF granules in vitro. BMDM phenotype and glycolysis levels were measured. Lastly, clinical effect of QXJYF granules on AP patients was verified. Predicted severe AP (pSAP) patients eligible for inclusion were assessed for enrollment. RESULTS: Nine major compounds were identified in QXJYF granules. Data showed that QXJYF granules significantly alleviated AP severity both in caerulein and L-arginine-induced AP models in vivo, pancreatic injury and inflammatory cell infiltration, systematic inflammation, lung injury and inflammatory cell infiltration were all improved after QXJYF treatment. QXJYF granules significantly reduced M1 macrophages during AP both in vivo and in vitro; besides, the mRNA expression levels of M1 genes such as inos, Tnfα, Il1ß and Il6 were significantly lower after QXJYF treatment in M1 macrophages. Mechanistically, we found that HK2, PFKFB3, PKM, LDHα levels were increased in M1 macrophages, but significantly decreased after QXJYF treatment. Clinical data indicated that QXJYF granules could significantly reduce CRP levels and shorten the duration of organ failure, thereby reducing the incidence of SAP and preventing pSAP patients from progressing to SAP. CONCLUSION: QXJYF granules alleviated AP through the inhibition of M1 macrophage polarization by suppressing glycolysis.

Luteolin protects mice from severe acute pancreatitis by exerting HO-1-mediated anti-inflammatory and antioxidant effects.

Reseda odorata L. has long been used in traditional Asian medicine for the treatment of diseases associated with oxidative injury and acute inflammation, such as endotoxemia, acute lung injury, acute myocardial infarction and hepatitis. Luteolin, the main component of Reseda odorata L., which is also widely found in many natural herbs and vege-tables, has been shown to induce heme oxygenase-1 (HO-1) expression to exert anti-inflammatory and antioxidant effects. In this study, we aimed to examine the effects of luteolin on mice with severe acute pancreatitis (SAP), and to explore the underlying mechanisms. Cerulein and lipopolysaccharide were used to induce SAP in male Institute of Cancer Research (ICR) mice in the SAP group. The SAP group was divided into 4 subgroups, as follows: the vehicle, luteolin, zinc protoporphyrin (ZnPP) only, and luteolin (Lut) + ZnPP (luteolin plus zinc protoporphyrin treatment) groups. The wet/dry weight ratios, hematoxylin and eosin staining and pathological scores of pancreatic tissues were assessed and compared to those of the control mice. Amylase, lipase, nuclear factor-κB (NF-κB) and myeloperoxidase activities, and malondialdehyde, tumor necrosis factor α (TNFα), interleukin (IL)-6, IL-10 and HO-1 levels, as well as the expression of HO-1 were determined in serum and/or pancreatic tissue samples. SAP was successfully induced in male mice compared to normal control mice. The wet/dry weight ratios, pathological scores, and amylase and lipase activity, as well as the levels of TNFα and IL-6 were significantly reduced in the pancreatic tissues of the mice in the Lut group compared with those of the mice in the vehicle group. The Lut group exhibited a significant increase in HO-1 expression in the pancreas and enhanced serum HO-1 and IL-10 levels compared with the vehicle group. The suppression of HO-1 activity in the ZnPP group significantly abolished the protective effects of luteolin. NF-κB expression in the pancreatic tissues from the mice in the Lut + ZnPP group was significantly increased following the suppression of HO-1 activity. On the whole, our findings demonstrate that luteolin protects mice from SAP by inducing HO-1-mediated anti-inflammatory and antioxidant activities, in association with the suppression of the activation of the NF-κB pathway.

Sex differences in colonization of gut microbiota from a man with short-term vegetarian and inulin-supplemented diet in germ-free mice.

Gnotobiotic mouse model is generally used to evaluate the efficacy of gut microbiota. Sex differences of gut microbiota are acknowledged, yet the effect of recipient's gender on the bacterial colonization remains unclear. Here we inoculated male and female germ-free C57BL/6J mice with fecal bacteria from a man with short-term vegetarian and inulin-supplemented diet. We sequenced bacterial 16S rRNA genes V3-V4 region from donor's feces and recipient's colonic content. Shannon diversity index showed female recipients have higher bacteria diversity than males. Weighted UniFrac principal coordinates analysis revealed the overall structures of male recipient's gut microbiota were significantly separated from those of females, and closer to the donor. Redundancy analysis identified 46 operational taxonomic units (OTUs) differed between the sexes. The relative abundance of 13 OTUs were higher in males, such as Parabacteroides distasonis and Blautia faecis, while 33 OTUs were overrepresented in females, including Clostridium groups and Escherichia fergusonii/Shigella sonnei. Moreover, the interactions of these differential OTUs were sexually distinct. These findings demonstrated that the intestine of male and female mice preferred to accommodate microbiota differently. Therefore, it is necessary to designate the gender of gnotobiotic mice for complete evaluation of modulatory effects of gut microbiota from human feces upon diseases.

Retinoic Acid Ameliorates Pancreatic Fibrosis and Inhibits the Activation of Pancreatic Stellate Cells in Mice with Experimental Chronic Pancreatitis via Suppressing the Wnt/ß-Catenin Signaling Pathway.

Pancreatic fibrosis, a prominent feature of chronic pancreatitis (CP), induces persistent and permanent damage in the pancreas. Pancreatic stellate cells (PSCs) provide a major source of extracellular matrix (ECM) deposition during pancreatic injury, and persistent activation of PSCs plays a vital role in the progression of pancreatic fibrosis. Retinoic acid (RA), a retinoid, has a broad range of biological functions, including regulation of cell differentiation and proliferation, attenuating progressive fibrosis of multiple organs. In the present study, we investigated the effects of RA on fibrosis in experimental CP and cultured PSCs. CP was induced in mice by repetitive cerulein injection in vivo, and mouse PSCs were isolated and activated in vitro. Suppression of pancreatic fibrosis upon administration of RA was confirmed based on reduction of histological damage, α-smooth muscle actin (α-SMA) expression and mRNA levels of ß-catenin, platelet-derived growth factor (PDGF)-Rß transforming growth factor (TGF)-ßRII and collagen 1α1 in vivo. Wnt 2 and ß-catenin protein levels were markedly down-regulated, while Axin 2 expression level was up-regulated in the presence of RA, both in vivo and in vitro. Nuclear translation of ß-catenin was significantly decreased following RA treatment, compared with cerulein-induced CP in mice and activated PSCs. Furthermore, RA induced significant PSC apoptosis, inhibited proliferation, suppressed TCF/LEF-dependent transcriptional activity and ECM production of PSC via down-regulation of TGFßRII, PDGFRß and collagen 1α1 in vitro. These results indicate a critical role of the Wnt/ß-catenin signaling pathway in RA-induced effects on CP and PSC regulation and support the potential of RA as a suppressor of pancreatic fibrosis in mice.

Rosmarinic Acid Attenuates Sodium Taurocholate-Induced Acute Pancreatitis in Rats by Inhibiting Nuclear Factor-κB Activation.

Rosmarinic Acid (RA), a caffeic acid ester, has been shown to exert anti-inflammation, anti-oxidant and antiallergic effects. Our study aimed to investigate the effect of RA in sodium taurocholate ( NaTC )-induced acute pancreatitis, both in vivo and in vitro. In vivo, RA (50 mg/kg) was administered intraperitoneally 2 h before sodium taurocholate injection. Rats were sacrificed 12 h, 24 h or 48 h after sodium taurocholate injection. Pretreatment with RA significantly ameliorated pancreas histopathological changes, decreased amylase and lipase activities in serum, lowered myeloperoxidase activity in the pancreas, reduced systematic and pancreatic interleukin-1 ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) levels, and inhibited NF-κB translocation in pancreas. In vitro, pretreating the fresh rat pancreatic acinar cells with 80 µ mol/L RA 2 h before 3750 nmol/L sodium taurocholate or 10 ng/L TNF-α administration significantly attenuated the reduction of isolated pancreatic acinar cell viability and inhibited the nuclear activation and translocation of NF-κB. Based on our findings, RA appears to attenuate damage in sodium taurocholate-induced acute pancreatitis and reduce the release of inflammatory cytokines by inhibiting the activation of NF-κB. These findings might provide a basis for investigating the therapeutic role of RA in managing acute pancreatits.

Shikonin ameliorates cerulein-induced acute pancreatitis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Shikonin, a highly liposoluble naphthoquinone pigment isolated from the traditional medical herbs Lithospermum erythrorhizon (LE), was considered to exhibit an anti-inflammatory property. While the potential of shikonin to ameliorate acute pancreatitis (AP) is unknown. Our aim was to investigate the effects of shikonin in a murine model of cerulein-induced pancreatitis. MATERIALS AND METHODS: AP was induced in mice by six intraperitoneal injection of cerulein (50 µg/kg) at hourly intervals. Vehicle or shikonin (50 mg/kg) was pretreated 2 h before the first cerulein injection. After 6 h, 9 h and 12 h of the first cerulein injection, the severity of acute pancreatitis was assessed by biochemistry, myeloperoxidase activity, histological grading, proinflammatory cytokines levels and nuclear factor kappa B (NF-κB) activity. RESULTS: Shikonin administration significantly reduced serum amylase and lipase activities, pancreatic histological scores, TNF-α, IL-1ß, IL-6 levels, MPO activity and NF-κB activity. CONCLUSION: Taken together, these results suggest that shikonin might protect against experimental pancreatitis by reducing release of inflammatory cytokines via inhibition of NF-κB activity. The therapeutic role of shikonin in AP needs further investigation.

Reg4 protects against acinar cell necrosis in experimental pancreatitis.

Background and aims Reg4 is a recently discovered member of the regenerating gene family with distinctive expression profiles in primary cancers. To date, the physiological function of Reg4 is poorly understood. Previously, the authors found that Reg4 was markedly upregulated during acute pancreatitis (AP). The aim of this study was to investigate the role of Reg4 in experimental pancreatitis. Methods AP was induced in C57BL/6 mice by administration of either l-arginine or caerulein, and Reg4 expression was assessed by immunofluorescence, reverse transcriptase (RT)-PCR and western blot analyses. Recombinant human Reg4 protein (rReg4), heat-inactivated Reg4, neutralising antibody and vehicle were also administered to mice by subcutaneous injection. The severity of AP was determined by measuring amylase and lipase activities in the serum and histological grading. The effect of rReg4 on cell death was examined and epidermal growth factor receptor (EGFR), p-EGFR, Akt, p-Akt, Bcl-2 and Bcl-xL expression were assessed by western blot analysis of isolated murine acinar cells treated with l-arginine. Results Reg4 mRNA and protein were markedly upregulated during arginine-induced pancreatitis. Reg4 was widely expressed in residual acinar cells around the islets and regenerating metaplastic epithelium. rReg4 could protect against arginine-induced necrosis of acinar cells both in vivo and in vitro. This protective effect was also confirmed in the caerulein-induced murine model of AP. It was shown that arginine induced expression of Bcl-2 and Bcl-xL, while rReg4 upregulated Bcl-2 and Bcl-xL expression by activating the EGFR/Akt pathway. The upregulation of Bcl-xL correlated inversely with cell necrosis in isolated pancreatic acinar cells. Conclusions The data suggest that Reg4 may protect against acinar cell necrosis in experimental pancreatitis by enhancing the expression of Bcl-2 and Bcl-xL via activation of the EGFR/Akt signalling pathway.

Effect of Lianshu preparation on lipopolysaccharide-induced diarrhea in rats.

AIM: To investigate the effect of Lianshu preparation on lipopolysaccharide (LPS)-induced diarrhea in rats. METHODS: A diarrhea model was established in Sprague Dawley rats via injection of 1 mL of 30 mg/kg LPS. A total of 40 rats were randomly divided into normal group, LPS group, LPS + Lianshu group, LPS + berberine group (n = 10 in each group). Their intestinal mucosal barrier and frequency of diarrhea were observed. Levels of glucose, serum Na(+), K(+), Cl(-) and hematocrit, plasma nitrogen monoxide (NO), diamine oxidase (DAO), and D (-)-lactate were measured. The number of IgA+ plasma cells in small intestine was detected and SIgA levels in the intestinal fluid were measured. The antipyretic activity of Lianshu preparation in rats was evaluated using Brewer's yeast-induced pyrexia (10 mL/kg of 20% aqueous suspension). Acetaminophen (250 mg/kg, intragastric administration, bid) was used as a standard drug for comparison. Temperature was recorded 1 h before and 6 h after Brewer's yeast injection. Finally, small intestinal transmission in mice treated with Lianshu was detected after intraperitoneal injection of methyl prostigmin (2 mg/kg). Atropine (10 g/kg) was used as a control. The ink content in intestine was determined and the total length of intestine was measured. RESULTS: The frequency of diarrhea was higher in LPS group than in LPS + Lianshu group and LPS + berberine group (36.70 +/- 5.23 vs 28.50 +/- 4.06 and 32.70 +/- 9.30 respectively, P < 0.01), and lower in LPS + Lianshu group than in LPS + berberine group (P = 0.03). The levels of Na(+), glucose, Cl(-), K(+) were significantly lower in LPS + Lianshu group than in LPS + berberine group (140.35 +/- 3.19 mmol/L vs 131.99 +/- 4.86 mmol/L, 8.49 +/- 1.84 mmol/L vs 6.54 +/- 2.30 mmol/L, 106.29 +/- 4.41 mmol/L vs 102.5 +/- 1.39 mmol/L, 5.08 +/- 0.66 mmol/L vs 4.32 +/- 0.62 mmol/L respectively, P < 0.05). The level of hematocrit was lower in LPS + Lianshu group than in LPS + berberine group (0.50% +/- 0.07% vs 0.59% +/- 0.10% respectively, P < 0.05). The plasma levels of NO, DAO and D (-)-lactate were higher in LPS group than in normal group (79.74 +/- 7.39 micromol/L vs 24.94 +/- 3.38 micromol/L, 2.48 +/- 0.42 micro/mL vs 0.82 +/- 0.33 micro/mL, 5.63 +/- 0.85 microg/mL vs 2.01 +/- 0.32 microg/mL respectively, P < 0.01), and lower in LPS + Lianshu group than in LPS + berberine group (48.59 +/- 4.70 micromol/L vs 51.56 +/- 8.38 micromol/L, 1.43 +/- 0.53 micromol/mL vs 1.81 +/- 0.42 micromol/mL, 4.00 +/- 0.54 microg/mL vs 4.88 +/- 0.77 microg/mL respectively, P < 0.05). The morphology of the intestinal mucosa showed destroyed villi in LPS group and atrophied intestinal mucosa in other groups. The pathological intestinal mucosal changes were less in LPS + Lianshu group than in LPS group. The number of IgA+ plasma cells and amount of SIgA were higher in LPS + Lianshu group than in LPS group (1.16 +/- 0.19/microm(2) vs 1.09 +/- 0.28/microm(2), P = 0.026; 0.59 +/- 0.12 mg/L vs 0.15 +/- 0.19 mg/L respectively, P = 0.000). Lianshu had counteractive effects on yeast-induced pyrexia and enterokinesia in rats. CONCLUSION: Lianshu preparation has therapeutic effects on LPS-induced diarrhea and enterokinesia in rats.

[Effects of enteral nutrition supplemented with glutamine and arginine on gut barrier in patients with severe acute pancreatitis: a prospective randomized controlled trial].

OBJECTIVE: To investigate the effects of continuous early enteral nutrition (EEN) supplemented with glutamine and arginine on gut barrier function in patients with severe acute pancreatitis (SAP). METHODS: Thirty two patients with a diagnosis of acute pancreatitis predicted to develop severe disease were randomized into 2 groups: EEN group (n = 18) and EEN + glutamine and arginine group (enteral immunonutrition group, n = 14). EEN was initiated when homeostasis was achieved within 72 hours after attack, and both group received isocaloric isonitrogenous nutrition. Glutamine and arginine were administered into jejunum in the enteral immunonutrition group. Serum amylase, plasma diamine oxidase (DAO), C-reactive protein (CRP), plasma endotoxin, urinary excretion of lactulose (L), and mannitol (M) were measured, and APACHE-II scores were recorded on days 1, 7, and 14. Complications, and length and cost of hospitalization were recorded as well. RESULTS: EEN and enteral immunonutrition were both tolerated well. There was no difference in APACHE-IIscore between the two groups (P > 0.05). The DAO, CRP, plasma endotoxin, and urinary L/M levels decreased with the course of SAP. However, the plasma endotoxin and urinary L/M on day 7 of the enteral immunonutrition group were (10.0 +/- 3.8) EU/ml and 0.29 +/- 0.15 respectively, both significantly higher than those of the EEN group [(7.9 +/- 2.8) EU/ml and 0.16 +/- 0.08 respectively, both P < 0.05]. The length of hospital stay and cost showed no differences between the two groups. CONCLUSION: EEN is safe and feasible in treatment of SAP. Enteral immunonutrition containing glutamine and arginine improves the gut barrier function by reducing the gut permeability and decreasing plasma endotoxin level in the early stage of SAP.

The therapeutic effect of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine in rodents with acute necrotizing pancreatitis and its mechanism.

OBJECTIVES: This study aims to investigate the therapeutic effect of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OXPAPC) in rodents with acute necrotizing pancreatitis (ANP) and its mechanism. METHODS: The ANP model was induced by cerulein challenged by lipopolysaccharide in mice and taurocholic acid in Sprague-Dawley rats. Both ANP models were treated with OXPAPC. Twenty animals of each group were separated to investigate mortality. Detection included serum levels of amylase and lactate dehydrogenase, histological changes of pancreas, activity of myeloperoxidase in pancreas, mRNA expression of inflammatory factors, expression of signal transduction factor proteins, and binding activity of transcriptional factors. RESULTS: After treatment with OXPAPC, survival rate was improved in the rat model. In both models, OXPAPC significantly decreased serum amylase and lactate dehydrogenase levels. Histologically, OXPAPC reduced the severity of pancreatic injury. There was a significant decline of myeloperoxidase activity. The mRNA levels of intrapancreatic inflammatory factors were depressed. Activated p38, C-jun N-terminal kinase 1, and inhibitor of kappa-B kinase beta proteins were down-regulated. Electrophoretic mobility shift assay showed that the binding activity of nuclear factor-kappaB and activator protein 1 to DNA was inhibited. CONCLUSIONS: The OXPAPC decreased the severity of experimental ANP in rodents. The protective effect of OXPAPC was mediated, at least in part, through blocking the lipopolysaccharide signal pathway.