Probiotics Prevent Intestinal Barrier Dysfunction in Acute Pancreatitis in Rats via Induction of Ileal Mucosal Glutathione Biosynthesis

Background

During acute pancreatitis (AP), oxidative stress contributes to intestinal barrier failure. We studied actions of multispecies probiotics on barrier dysfunction and oxidative stress in experimental AP.

Methodology/Principal Findings

Fifty-three male Spraque-Dawley rats were randomly allocated into five groups: 1) controls, non-operated, 2) sham-operated, 3) AP, 4) AP and probiotics and 5) AP and placebo. AP was induced by intraductal glycodeoxycholate infusion and intravenous cerulein (6 h). Daily probiotics or placebo were administered intragastrically, starting five days prior to AP. After cerulein infusion, ileal mucosa was collected for measurements of E. coli K12 and ⁵¹Cr-EDTA passage in Ussing chambers. Tight junction proteins were investigated by confocal immunofluorescence imaging. Ileal mucosal apoptosis, lipid peroxidation, and glutathione levels were determined and glutamate-cysteine-ligase activity and expression were quantified. AP-induced barrier dysfunction was characterized by epithelial cell apoptosis and alterations of tight junction proteins (i.e. disruption of occludin and claudin-1 and up-regulation of claudin-2) and correlated with lipid peroxidation (r>0.8). Probiotic pre-treatment diminished the AP-induced increase in E. coli passage (probiotics 57.4±33.5 vs. placebo 223.7±93.7 a.u.; P<0.001), ⁵¹Cr-EDTA flux (16.7±10.1 vs. 32.1±10.0 cm/s10⁻⁶; P<0.005), apoptosis, lipid peroxidation (0.42±0.13 vs. 1.62±0.53 pmol MDA/mg protein; P<0.001), and prevented tight junction protein disruption. AP-induced decline in glutathione was not only prevented (14.33±1.47 vs. 8.82±1.30 nmol/mg protein, P<0.001), but probiotics even increased mucosal glutathione compared with sham rats (14.33±1.47 vs. 10.70±1.74 nmol/mg protein, P<0.001). Glutamate-cysteine-ligase activity, which is rate-limiting in glutathione biosynthesis, was enhanced in probiotic pre-treated animals (probiotics 2.88±1.21 vs. placebo 1.94±0.55 nmol/min/mg protein; P<0.05) coinciding with an increase in mRNA expression of glutamate-cysteine-ligase catalytic (GCLc) and modifier (GCLm) subunits.

Conclusions

Probiotic pre-treatment diminished AP-induced intestinal barrier dysfunction and prevented oxidative stress via mechanisms mainly involving mucosal glutathione biosynthesis.     

Chlorogenic acid,a dietary polyphenol,protects acetaminophen-induced liver injury and its mechanism

Chlorogenic acid (CGA) is one of the most abundant dietary polyphenols, possessing well-known antioxidant capacity. The present study is designed to observe the protection provided by CGA against acetaminophen (AP)-induced liver injury in mice in vivo and the underlying mechanisms engaged in this process. Serum transaminases analysis and liver histological evaluation demonstrated the protection of CGA against AP-induced liver injury. CGA treatment decreased the increased number of liver apoptotic cells induced by AP in a dose-dependent manner. CGA also inhibited AP-induced cleaved activation of caspase-3, 7. Moreover, CGA reversed AP-decreased liver reduced glutathione (GSH) levels, glutamate-cysteine ligase (GCL) and glutathione reductase activity. Further results showed that CGA increased mRNA and protein expression of the catalytic subunit of GCL (GCLC), thioredoxin (Trx) 1/2 and thioredoxin reductase (TrxR) 1. Furthermore, CGA abrogated AP-induced phospholyated activation of ERK1/2, c-Jun N-terminal kinase (JNK), p38 kinases and molecular signals upstream. The results of this study demonstrate that CGA counteracts AP-induced liver injury at various levels by preventing apoptosis and oxidative stress damage, and more specifically, both the GSH and Trx antioxidant systems and the mitogen-activated protein kinase (MAPK) signaling cascade appear to be engaged in this protective mechanism.     

2',4',6'-Tris(methoxymethoxy) chalcone (TMMC) attenuates the severity of cerulein-induced acute pancreatitis and associated lung injury

Acute pancreatitis (AP) is an inflammatory disease involving acinar cell injury and rapid production and release of inflammatory cytokines, which play a dominant role in local pancreatic inflammation and systemic complications. 2',4',6'-Tris (methoxymethoxy) chalcone (TMMC), a synthetic chalcone derivative, displays potent anti-inflammatory effects. Therefore, we aimed to investigate whether TMMC might affect the severity of AP and pancreatitis-associated lung injury in mice. We used the cerulein hyperstimulation model of AP. Severity of pancreatitis was determined in cerulein-injected mice by histological analysis and neutrophil sequestration. The pretreatment of mice with TMMC reduced the severity of AP and pancreatitis-associated lung injury and inhibited several biochemical parameters (activity of amylase, lipase, trypsin, trypsinogen, and myeloperoxidase and production of proinflammatory cytokines). In addition, TMMC inhibited pancreatic acinar cell death and production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 by inhibiting NF-κB and extracellular signal-regulated protein kinase 1/2 (ERK1/2) activation. Neutralizing antibodies for TNF-α, IL-1β, and IL-6 inhibited cerulein-induced cell death in isolated pancreatic acinar cells. Moreover, pharmacological blockade of NF-κB/ERK1/2 reduced acinar cell death and production of TNF-α, IL-1β, and IL-6 in isolated pancreatic acinar cells. In addition, posttreatment of mice with TMMC showed reduced severity of AP and lung injury. Our results suggest that TMMC may reduce the complications associated with pancreatitis.     

Ethanol induces secretion of oxidized proteins by pancreatic acinar cells

The pancreas is vulnerable to ethanol toxicity, but the pathogenesis of alcoholic pancreatitis is not fully defined. The intracellular oxidative balance and the characteristics of the secretion of isolated rat pancreatic acinar cells stimulated with the cholecystokinin analogue cerulein were assayed after acute oral ethanol (4 g/kg) load. Pancreatic acinar cells from ethanol-treated rats showed a significant (p < 0.02) lower content of total glutathione and protein sulfhydryls, and higher levels of oxidized glutathione (p < 0.03), malondialdehyde, and protein carbonyls (p < 0.05). Ethanol-intoxicated acinar cells showed a lower baseline amylase output compared to controls, with the difference being significantly exacerbated by cerulein stimulation. After cerulein, the release of protein carbonyls by ethanol-treated cells was significantly increased, whereas that of protein sulfhydryls was significantly decreased. In conclusion, ethanol oxidatively damages pancreatic acinar cells; cerulein stimulation is followed by a lower output of amylase and by a higher release of oxidized proteins by pancreatic acinar cells from ethanol-treated rats. These findings may account for the decreased exocrine function, intraductular plug formation, and protein precipitation in alcoholic pancreatitis.     

Lipid peroxidation inhibition reduces NF-kappaB activation and attenuates cerulein-induced pancreatitis

Increased lipid peroxidation, enhanced nuclear factor kappa-B (NF-kappaB) activation and augmented tumor necrosis factor-alpha (TNF-alpha) production have been implicated in cerulein-induced pancreatitis. We investigated whether lipid peroxidation inhibition might reduce NF-kappaB activation and the inflammatory response in cerulein-induced pancreatitis. Male Sprague-Dawley rats of 230-250g body weight received administration of cerulein (80 microg/kg s.c. for each of four injections at hourly intervals). A control group received four s.c. injections of 0.9% saline at hourly intervals. Animals were randomized to receive either raxofelast, an inhibitor of lipid peroxidation (20 mg/kg i.p. administered with the first cerulein injection) or its vehicle (1 ml/kg of a 10% DMSO/NaCl solution). All these rats were sacrificed 2 h after the last injection of either cerulein or its vehicle. Raxofelast administration (20 mg/kg i.p. with the first cerulein) significantly reduced malondialdehyde (MDA) levels, an index of lipid peroxidation (CER + DMSO = 3.075 +/- 0.54 micromol/g; CER + raxofelast = 0.693 +/- 0.18 micromol/g; p < 0.001), decreased myeloperoxidase (MPO) activity (CER + DMSO = 22.2 +/- 3.54 mU/g; CER + raxofelast = 9.07 +/- 2.05 mU/g, p < 0.01), increased glutathione levels (GSH) (CER + DMSO = 5.21 +/- 1.79 micromol/g; CER + raxofelast = 15.71 +/- 2.14 micronol/g; p < 0.001), and reduced acinar cell damage evaluated by means of histology and serum levels of both amylase (CER + DMSO = 4063 +/- 707.9 U/l; CER + raxofelast = 1198 +/- 214.4 U/l; p < 0.001), and lipase (CER + DMSO = 1654 +/- 330 U/l; CER + raxofelast = 386 +/- 118.2 U/l; p < 0.001), Furthermore, raxofelast reduced pancreatic NF-kappaB activation and the TNF-alpha mRNA levels and tissue content of mature protein in the pancreas. Indeed, lipid peroxidation inhibition might be considered a potential therapeutic approach to prevent the severe damage in acute pancreatitis.     

N-acetylcysteine prevents intra-acinar oxygen free radical production in pancreatic duct obstruction-induced acute pancreatitis

Although oxygen free radicals (OFR) are considered to be one of the pathophysiological mechanisms involved in acute pancreatitis (AP), the contribution of acinar cells to their production is not well established. The aim of the present study was to determine the effect of N-acetylcysteine (NAC) in the course of AP induced by pancreatic duct obstruction (PDO) in rats, directly analysing by flow cytometry the quantity of OFR generated in acinar cells. NAC (50 mg/kg) was administered 1 h before and 1 h after PDO. Measurements by flow cytometry of OFR generated in acinar cells were taken at different PDO times over 24 h, using dihydrorhodamine-123 as fluorescent dye. Histological studies of pancreas and measurements of neutrophil infiltration in the pancreas, pancreatic glutathione (GSH), malondialdehyde (MDA) levels, plasma amylase activity and hemoconcentration were carried out in order to assess the severity of AP at different stages. NAC effectively blunted GSH depletion at early AP stages and prevented OFR generation found in acinar cells as a consequence of AP induced by PDO. This attenuation of the redox state impairment reduced cellular oxidative damage, as reflected by less severe pancreatic lesions, normal pancreatic MDA levels, as well as diminished neutrophil infiltration in pancreas. Hyperamylasemia and hemoconcentration following AP induction were ameliorated by NAC administration at early stages, when oxidative stress seems to be critical in the development of pancreatitis. In conclusion, NAC reinforces the antioxidant defences in acinar cells, preventing OFR generation therefore attenuating oxidative damage and subsequently reducing the severity of PDO-induced AP at early stages of the disease.     

Fibroblast growth factor 21 alleviates acute pancreatitis via activation of the Sirt1‐autophagy signalling pathway

Fibroblast growth factor 21 (FGF21), a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity, alleviates the process of acute pancreatitis (AP). However, its mechanism remains elusive. The pathological and physiological characteristics of FGF21 are observed in both patients with AP and cerulein‐induced AP models, and the mechanisms of FGF21 in response to AP are investigated by evaluating the impact of autophagy in FGF21‐treated mice and cultured pancreatic cells. Circulating levels of FGF21 significantly increase in both AP patients and cerulein‐induced AP mice, which is accompanied by the change of pathology in pancreatic injury. Replenishment of FGF21 distinctly reverses cerulein‐induced pancreatic injury and improves cerulein‐induced autophagy damage in vivo and in vitro. Mechanically, FGF21 acts on pancreatic acinar cells to up‐regulate Sirtuin‐1 (Sirt1) expression, which in turn repairs impaired autophagy and removes damaged organs. In addition, blockage of Sirt1 accelerates cerulein‐induced pancreatic injury and weakens the regulative effect in FGF21‐activated autophagy in mice. These results showed that FGF21 protects against cerulein‐induced AP by activation of Sirtuin‐1‐autophagy axis.     

Age-Dependent Effects of UCP2 Deficiency on Experimental Acute Pancreatitis in Mice

Reactive oxygen species (ROS) have been implicated in the pathogenesis of acute pancreatitis (AP) for many years but experimental evidence is still limited. Uncoupling protein 2 (UCP2)-deficient mice are an accepted model of age-related oxidative stress. Here, we have analysed how UCP2 deficiency affects the severity of experimental AP in young and older mice (3 and 12 months old, respectively) triggered by up to 7 injections of the secretagogue cerulein (50 μg/kg body weight) at hourly intervals. Disease severity was assessed at time points from 3 hours to 7 days based on pancreatic histopathology, serum levels of alpha-amylase, intrapancreatic trypsin activation and levels of myeloperoxidase (MPO) in lung and pancreatic tissue. Furthermore, in vitro studies with pancreatic acini were performed. At an age of 3 months, UCP2^-/- mice and wild-type (WT) C57BL/6 mice were virtually indistinguishable with respect to disease severity. In contrast, 12 months old UCP2^-/- mice developed a more severe pancreatic damage than WT mice at late time points after the induction of AP (24 h and 7 days, respectively), suggesting retarded regeneration. Furthermore, a higher peak level of alpha-amylase activity and gradually increased MPO levels in pancreatic and lung tissue were observed in UCP2^-/- mice. Interestingly, intrapancreatic trypsin activities (in vivo studies) and intraacinar trypsin and elastase activation in response to cerulein treatment (in vitro studies) were not enhanced but even diminished in the knockout strain. Finally, UCP2^-/- mice displayed a diminished ratio of reduced and oxidized glutathione in serum but no increased ROS levels in pancreatic acini. Together, our data indicate an aggravating effect of UCP2 deficiency on the severity of experimental AP in older but not in young mice. We suggest that increased severity of AP in 12 months old UCP2^-/- is caused by an imbalanced inflammatory response but is unrelated to acinar cell functions.     

The tripeptide analog feG ameliorates severity of acute pancreatitis in a caerulein mouse model

Acute pancreatitis (AP) is associated with significant morbidity and mortality; however, there is no specific treatment for this disease. A novel salivary tripeptide analog, feG, reduces inflammation in several different animal models of inflammation. The aims of this study were to determine whether feG reduced the severity of AP and modifies the expression of pancreatic ICAM-1 mRNA during AP in a mouse model. AP was induced in mice by hourly (x12) intraperitoneal injections of caerulein. A single dose of feG (100 microg/kg) was coadministered with caerulein either at time 0 h (prophylactic) or 3 h after AP induction (therapeutic). Plasma amylase and pancreatic MPO activities and pancreatic ICAM-1 mRNA expression (by RT-PCR) were measured. Pancreatic sections were histologically assessed for abnormal acinar cells and interstitial space. AP induction produced a sevenfold increase in plasma amylase, a tenfold increase in pancreatic MPO activity, and a threefold increase in interstitial space, and 90% of the acinar cells were abnormal. Prophylactic treatment with feG reduced the AP-induced plasma amylase activity by 45%, pancreatic MPO by 80%, the proportion of abnormal acinar cells by 30%, and interstitial space by 40%. Therapeutic treatment with feG significantly reduced the AP-induced abnormal acinar cells by 10% and the interstitial space by 20%. Pancreatic ICAM-1 mRNA expression was upregulated in AP and was reduced by 50% with prophylactic and therapeutic treatment with feG. We conclude that feG ameliorates experimental AP acting at least in part by modulating ICAM-1 expression in the pancreas.     

Differentially expressed proteins in cerulein-stimulated pancreatic acinar cells: implication for acute pancreatitis

The proteins expressed in pancreatic acinar cells during the initiation of acute pancreatitis may determine the severity of the disease. Cerulein pancreatitis is one of the best characterized models for acute pancreatitis. Present study aims to determine the differentially expressed proteins in cerulein-stimulated pancreatic acinar cells as an in vitro model for acute pancreatitis. Rat pancreatic acinar AR42J cells were treated with 10(-8)M cerulein for 12h. The protein patterns separated by two-dimensional electrophoresis using pH gradients of 5-8 were compared between the cells treated without cerulein and those with cerulein. The changed proteins were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. As a result, 10 proteins (Orp150 protein, protein disulfide isomerase related protein, dnaK-type molecular chaperone hsp72-ps1, mitochondrial glutamate dehydrogenase, similar to chaperonin containing TCP-1 beta subunit, RuvB-like protein 1, heterogeneous nuclear ribonucleoprotein H1, aldehyde reductase 1, triosephosphate isomerase 1, peroxiredoxin 2) were up-regulated while four proteins (vasolin-containing protein, 78 kDa glucose-regulated protein precursor, heat shock protein 8, adenosylhomocysteinase) were down-regulated by cerulein in pancreatic acinar AR42J cells. These proteins are related to chaperone, cell defense mechanism against oxidative stress or DNA damage, anti-apoptosis and energy generation. The differentially expressed proteins by ceruein share their functional roles in pancreatic acinar cells, suggesting the possible involvement of oxidative stress, DNA damage, and anti-apoptosis in pathogenesis of acute pancreatitis. Proteins involved in cellular defense mechanism and energy production may protect pancreatic acinar cells during the development of pancreatitis.     

Pro- and anti-inflammatory response of acinar cells during acute pancreatitis. Effect of N-acetyl cysteine

We investigate the ability of acinar cells to produce tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) at different stages of acute pancreatitis (AP). Since oxidative stress is involved in the inflammatory response, the effect of N-acetyl cysteine (NAC) has also been evaluated. AP was induced in rats by bile-pancreatic duct obstruction (BPDO). NAC (50 mg/kg) was administered 1h before and 1h after BPDO. Acinar cells were incubated for 4 h at 37 degrees C in 5% CO2 atmosphere in absence and presence of 24-h BPDO-PAAF (20%, v/v) as stimulant agent. Acinar production of TNF-alpha and IL-10 was analysed by flow cytometry. Plasma amylase activity and histological studies of the pancreas indicated the severity of AP. PAAF significantly stimulated the acinar production of TNF-alpha and IL-10 in control rats. TNF-alpha production was also significantly stimulated in acinar cells of rats with AP, although a decrease in the pro-inflammatory response was found from 6 h after BPDO onwards. However, acinar cells failed to produce IL-10 from 3 h after BPDO. The protective effect of NAC treatment against oxidative cell damage reduced the pancreatic injury and maintained and enhanced the ability of acinar cells to produce IL-10 at early AP stages. As long as acinar cells were not severely damaged in the course of AP, greater ability to produce cytokines in response to PAAF was found in those with higher forward scatter (R2 cells). We suggest that the capability of acinar cells to maintain an appropriate balance between the production of pro- and anti-inflammatory mediators could contribute to determine the degree of severity of AP.     

Role of parathyroid hormone-related protein in the pro-inflammatory and pro-fibrogenic response associated with acute pancreatitis

Pancreatitis is a common and potentially lethal necro-inflammatory disease with both acute and chronic manifestations. Current evidence suggests that the accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic disease, which is associated with an increased risk of pancreatic cancer. While parathyroid hormone-related protein (PTHrP) exerts multiple effects in normal physiology and disease states, its function in pancreatitis has not been previously addressed. Here we show that PTHrP levels are transiently elevated in a mouse model of cerulein-induced AP. Treatment with alcohol, a risk factor for both AP and chronic pancreatitis (CP), also increases PTHrP levels. These effects of cerulein and ethanol are evident in isolated primary acinar and stellate cells, as well as in the immortalized acinar and stellate cell lines AR42J and irPSCc3, respectively. Ethanol sensitizes acinar and stellate cells to the PTHrP-modulating effects of cerulein. Treatment of acinar cells with PTHrP (1-36) increases expression of the inflammatory mediators interleukin-6 (IL-6) and intracellular adhesion protein (ICAM-1), suggesting a potential autocrine loop. PTHrP also increases apoptosis in AR42J cells. Stellate cells mediate the fibrogenic response associated with pancreatitis; PTHrP (1-36) increases procollagen I and fibronectin mRNA levels in both primary and immortalized stellate cells. The effects of cerulein and ethanol on levels of IL-6 and procollagen I are suppressed by the PTH1R antagonist, PTHrP (7-34). Together these studies identify PTHrP as a potential mediator of the inflammatory and fibrogenic responses associated with alcoholic pancreatitis.     

PSP下调miR-345-5p对雨蛙素诱导的AP腺泡细胞氧化应激和炎症因子表达

目的探讨黄精多糖（PSP）对雨蛙素诱导的急性胰腺炎（AP）腺泡细胞氧化应激和炎症因子表达的影响及分子机制。 方法取对数期大鼠胰腺腺泡细胞AR42J,采用100 nmol/L雨蛙素处理细胞6 h,建立AP腺泡细胞损伤模型,并采用不同浓度（1、2、4 mg/mL）PSP处理AP细胞（AP+PSP-L、AP+PSP-M、AP+PSP-H）。试剂盒检测细胞中丙二醛（MDA）含量、超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GPx）活性以及培养液中白细胞介素-6 （IL-6）、肿瘤坏死因子-α （TNF-α）和IL-1β水平,实时荧光定量PCR （RT-qPCR）检测miR-345-5p表达水平。将miR-345-5p抑制物转染AR42J细胞,检测下调miR-345-5p表达对AP细胞氧化应激和炎症因子表达的影响。将miR-345-5p模拟物转染AR42J细胞,检测上调miR-345-5p表达和PSP处理对AP细胞氧化应激和炎症因子表达的影响。两组比较采用t检验,多组间比较采用方差分析,进一步两两比较采用LSD-t检验。 结果与对照比较,AP细胞MDA含量、IL-6、TNF-α、IL-1β水平和miR-345-5p表达水平均升高,SOD和GPx活性降低（P < 0.05）。与AP细胞比较,不同浓度（1、2、4 mg/mL）PSP作用的AP细胞中MDA含量[（1.08± 0.07）比（0.88±0.06）,（0.73±0.06）,（0.60±0.05） nmol/mg]降低,SOD [（43.01±4.37）比（59.60±5.62）,（72.37±6.32）,（94.21±8.70） U/mg]和GPx活性[（29.03±2.51）比（44.11± 4.71）,（58.07±4.20）,（72.67± 6.56） U/mg]均升高,培养液中IL-6 [（310.72±22.27）比（257.01±20.85）,（192.28±17.70）,（146.93±11.90） pg/mL]、TNF-α [（223.82±21.87）比（175.57±15.85）,（137.00±11.31）,（89.26±7.05） pg/mL]、IL-1β表达水平[（41.66±3.85）比（33.82±3.20）,（26.15±2.56）,（20.14±1.71） pg/mL]和miR-345-5p表达水平（2.78±0.24比2.38±0.21,1.91±0.12,1.25±0.13）均降低,且呈浓度依赖性,差异有统计学意义（P均< 0.05）。下调miR-345-5p表达后,AP细胞中MDA含量[（1.13±0.08）比（0.72±0.06） nmol/mg]降低,SOD活性[（41.31±3.98）比（81.73±7.62） U/mg]和GPx [（28.82±2.97）比（61.41±5.81） U/mg]升高,培养液中IL-6 [（314.65±25.02）比159.76±11.93） pg/mL]、TNF-α [（235.18±23.13）比（100.41±8.09） pg/mL]和IL-1β水平[（48.67±4.50）比（27.73±2.54） pg/mL]降低（P均< 0.05）。上调miR-345-5p表达可逆转PSP对AP细胞的影响。其中MDA含量[（0.58±0.03）比（0.95±0.08） nmol/mg]升高、SOD活性[（96.52±9.54）比（54.24±4.15） U/mg]、GPx活性[（79.62±6.23）比（39.81±3.84） U/mg]降低、IL-6 [（145.38±12.49）比（275.38± 21.55） pg/mL]、TNF-α [（84.83±7.81）比（183.73±16.39） pg/mL]和IL-1β [（19.38±1.85）比（36.97±3.62） pg/mL]的表达水平升高（P均< 0.05）。 结论PSP以剂量依赖方式减轻雨蛙素诱导AP细胞炎症反应和氧化应激损伤,其机制与下调miR-345-5p表达有关。     

N-acetylcysteine induces beneficial changes in the acinar cell cycle progression in the course of acute pancreatitis

Oxygen free radicals (OFR) are produced in the course of acute pancreatitis (AP). In addition to injurious oxidative effects, they are also involved in the regulation of cell growth. The aim of the present study was to examine the relationship between the effectiveness of N-acetyl-l-cysteine (NAC) to prevent the generation of OFR and the changes in the cell-cycle pattern of acinar cells in the course of AP induced in rats by pancreatic duct obstruction (PDO). NAC (50 mg/kg) was administered 1 h before and 1 h after PDO. Flow-cytometric measurement of OFR generation in acinar cells was carried out using dihydrorhodamine as fluorescent dye. Plasma amylase activity, pancreatic glutathione (GSH) content and TNF-alpha plasma levels were also measured. The distribution of acinar cells throughout the different cell-cycle phases was analysed at different AP stages by flow cytometry using propidium iodide staining. NAC administration reduced the depletion of pancreatic GSH content and prevented OFR generation in acinar cells of rats with PDO-induced acute pancreatitis. As a result, AP became less severe as reflected by the significant improvement of hyper-amylasaemia and maintenance of plasma TNF-alpha levels at values not significantly different from controls were found. NAC administration inhibited progression of cell-cycle phases, maintaining acinar cells in quiescent state at early PDO times. The protection from oxidative damage by NAC treatment during early AP, allows the pancreatic cell to enter S-phase actively at later stages, thereby allowing acinar cells to proliferate and preventing the pancreatic atrophy provoked by PDO-induced AP. The results provide evidence that OFR play a critical role in the progression of acinar cell-cycle phases. Prevention of OFR generation of acinar cells in rats with PDO-induced AP through NAC treatment, not only protects pancreas from oxidative damage but also promotes beneficial changes in the cell cycle progression which reduce the risk of pancreatic atrophy.     

Cinnamic acid nanoparticles modulate redox signal and inflammatory response in gamma irradiated rats suffering from acute pancreatitis

Acute Pancreatitis (AP) is a multifactorial disease. It was characterized by severe inflammation and acinar cell destruction. Thus, the present study was initiated to evaluate the role the of Cinnamic acid nanoparticles (CA-NPs) as a modulator for the redox signaling pathway involved in the development of pancreatitis. AP in rats was induced by L-arginine and exposure to gamma radiation. The pancreatic injury was evaluated using biochemical and histological parameters. Upon the oral administration of CA-NPs, both the severity of acute pancreatitis and the serum levels of amylase and lipase were decreased. Furthermore, the malondialdehyde (MDA) levels of the pancreatic tissue were significantly reduced and the depletion of glutathione was considerably restored. The injury and apoptosis of pancreatic tissues were markedly improved by the reduction of the caspase-3 levels. Additionally, the alleviation of pancreatic oxidative damage by CA-NPs was accompanied by a down-regulation of the NLRP3, NF-κB, and ASK1/MAPK signaling pathways. Collectively, the current findings showed that CA-NPs could protect the pancreatic acinar cell from injury not only by its antioxidant, anti-inflammatory effect but also by modulation of the redox-sensitive signal transduction pathways contributed to acute pancreatitis severity. Accordingly, cinnamic acid nanoparticles have therapeutic potential for the management of acute pancreatitis.     

Oxidative stress, chronic inflammation, and telomere length in patients with periodontitis

The aim of this study was to determine leukocyte telomere length (LTL) in individuals with periodontitis and controls, exploring its relationship with systemic inflammation and oxidative stress. Five hundred sixty-three participants were recruited for this case-control study: 356 subjects with and 207 subjects without periodontitis. LTL was measured by a qPCR technique from leukocytes' DNA. Global measures of oxidative stress (reactive oxygen metabolites) and biological antioxidant potential in plasma were performed together with high-sensitivity assays for C-reactive protein (CRP). Leukocyte counts and lipid profiles were performed using standard biochemistry. Cases had higher levels of CRP (2.1±3.7mg/L vs 1.3±5.4mg/L, P<0.001) and reactive oxygen metabolites (378.1±121.1 U Carr vs 277.4±108.6 U Carr, P<0.001) compared to controls. Overall, cases had shorter LTL with respect to controls (1.23±0.42 vs 1.12±0.31T/S ratio, P=0.006), independent of age, gender, ethnicity, and smoking habit. When divided by subgroup of periodontal diagnosis (chronic, n=285; aggressive, n=71), only chronic cases displayed shorter LTL (P=0.01). LTL was negatively correlated with age (P=0.001; R=-0.2), oxidative stress (P=0.008; R=-0.2), and severity of periodontitis (P=0.003; R=-0.2) in both the whole population and the subgroups (cases and controls). We conclude that shorter telomere lengths are associated with a diagnosis of periodontitis and their measures correlate with the oxidative stress and severity of disease.     

Antioxidants attenuate myocyte apoptosis and improve cardiac function in CHF: association with changes in MAPK pathways

Antioxidant vitamins reduce cardiac oxidative stress and cardiomyocyte apoptosis produced by exogenous norepinephrine (NE) and attenuate cardiac dysfunction in animals with pacing-induced congestive heart failure (CHF). This study was carried out to determine whether the mitogen-activated protein kinase (MAPK) signal transduction pathways are involved in oxidative stress-induced myocyte apoptosis. Rabbits with rapid pacing-induced CHF and sham operation were randomized to receive either a combination of antioxidant vitamins (beta-carotene, ascorbic acid, and alpha-tocopherol), alpha-tocopherol alone, or placebo for 8 wk. Compared with sham-operated animals, CHF animals exhibited increased oxidative stress as evidenced by decreased myocardial reduced-to-oxidized glutathione (GSH/GSSG) ratio (27 +/- 7 vs. 143 +/- 24, P < 0.05), myocyte apoptosis (77 +/- 18 vs. 17 +/- 4 apoptotic nuclei/10,000 cardiomyocytes, P < 0.05), increased total and phosphorylated c-Jun NH2-terminal protein kinase (p-JNK; 1.95 +/- 0.14 vs. 1.04 +/- 0.04 arbitrary units, P < 0.05) and phosphorylated p38 kinase (p-p38), and decreased phosphorylated extracellular signal-regulated kinase (p-ERK). Administration of antioxidant vitamins and alpha-tocopherol attenuated oxidative stress, myocyte apoptosis, and cardiac dysfunction, with reversal of the changes of total JNK, p-JNK, and p-ERK in CHF. Furthermore, because NE infusion produced changes of JNK, p-p38, and p-ERK similar to those in CHF, we conclude that NE may play an important role in the production of oxidative stress, MAPK activation, and myocyte apoptosis in CHF.