Pancreatic acinar-to-ductal metaplasia (ADM) has been identified as an initiating event that can progress to pancreatic intraepithelial neoplasia (PanIN) or pancreatic ductal adenocarcinoma (PDAC). Acini transdifferentiation can be induced by persistent inflammation. Notably, compelling evidence has emerged that chronic alcohol exposure may trigger an inflammatory response of macrophages/monocytes stimulated by endotoxins. In the present study, we aimed to evaluate the role of inflammation induced by chronic alcohol and lipopolysaccharide (LPS) exposure in the progression of pancreatic ADM, as well as to elucidate the possible mechanisms involved. For this purpose, cultured macrophages were exposed to varying doses of alcohol for 1 week prior to stimulation with LPS. Tumor necrosis factor-α (TNF-α) and regulated upon activation, normal T cell expression and secreted (RANTES) expression were upregulated in the intoxicated macrophages with activated nuclear factor-κB (NF-κB). Following treatment with the supernatant of intoxicated macrophages, ADM of primary acinar cells was induced. Furthermore, the expression of TNF-α and RANTES, as well as the phosphatidylinositol-3-kinase (PI3K)/protein kinase B(Akt)/inhibitory κB kinase (IKK) signaling pathway have been proven to be involved in the ADM of acinar cells. Moreover, Sprague-Dawley (SD) rats were employed to further explore the induction of pancreatic ADM by chronic alcohol and LPS exposure
A number of studies have clarified that excessive alcohol consumption is the primary etiological factor in the induction of chronic pancreatitis (CP) or even pancreatic cancer (
The association between alcoholic pancreatitis and susceptibility factors, including genetic polymorphisms (
Alcohol consumption may lead to the enhanced production of pro-inflammatory cytokines and chemokines. Alcoholic hepatitis and pancreatitis, two major clinical complications of chronic alcohol use, have been shown to be intimately associated with increasing circulating levels of pro-inflammatory cytokines that predict poor clinical outcomes (
Chronic inflammation may cause cellular transdifferentiation which can occur in a number of organs, including the pancreas (
In the pancreas, chronic alcohol exposure has been reported to exacerbate the degree of fibrosis induced by LPS through an augmented level of tumor growth factor-β (TGF-β) (
A rat macrophage cell line obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) was cultured in macrophage medium (MaM, Cat. no. 1921) according to the manufacturer’s instructions. The macrophages were stimulated with varying doses (0, 5, 10, 15, 20 and 25 mM) of alcohol [ethanol (EtOH)] for 1 week prior to treatment with
The isolation of primary pancreatic acinar cells was as previously described (
A total of 120 8-week old male SD rats were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The animals were housed under standard conditions with a 12/12-h light/dark cycle at room temperature and fed a common diet with free access to water. To establish chronic alcoholic and LPS-stimulated rat models, the SD rats were randomly divided into 6 groups and intraperitoneally injected with 0, 5, 10, 15, 20 and 25 mmol/kg/day alcohol [ethanol (EtOH)] for 4 weeks. Following alcohol exposure, a dose (1 mg/kg) of LPS was administered by intravenous injection. For TNF-α and RANTES neutralization, the rats were injected with anti-TNF-α (sc-8301; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-RANTES (sc-514019; Santa Cruz Biotechnology) antibodies. The doses of anti-TNF-α or anti-RANTES antibody w as based on the results of preliminary experiments. To inhibit PI3K or IKK activity in rats, LY294002 (a PI3K inhibitor; 100 mg/kg, 10 min before the alcohol injection) was intravenously injected; 25% dimethyl sulfoxide in PBS was used as the vehicle.
All animal experimental procedures were conducted under the guidelines of the National Health and Medical Research Council for the Care and Use of Animals for Experimental Purposes in China. All efforts were made to minimize the suffering of the animals.
Scrambled siRNA and small-interfering RNA (siRNA) targeting NF-κB or the IL-1 receptor-associated kinase (IRAK)-M was purchased from Santa Cruz Biotechnology. The cells were transfected with scrambled or NF-κB/IRAK-M siRNA according to the manufacturer’s instructions. Briefly, the NF-κB/IRAK-M and scrambled siRNA (30 pmol) were diluted in 500
All recombinant adenoviruses were constructed according to a previous report (
The acinar cells were infected with adenovirus-NF-κB-luciferase adenovirus (at 107 IFU/ml), and immediately plated on a 24-well plate and cultured with 6 groups of macrophage supernatants. At 24 and 48 h after infection, the cells were collected and washed with ice-cold PBS, lysed using 250
A Biochemical Analysis kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to measure the plasma alcohol concentration, MDA content, GPx and SOD activity according to the manufacturer’s instructions. Each experiment was performed no less than 3 times.
The levels of TNF-α and RANTES in the serum were analyzed using a commercially available ELISA kit (Yanjin Biotechnology Co., Shanghai, China) according to the manufacturer’s instructions. The absorbance was read at 450 nm using a 680XR microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). All the samples were analyzed in duplicate. The standard curve for the estimation of TNF-α and RANTES expression was created by linear regression analysis.
RNA was extracted from the macrophages or acinar cells using TRIzol RNA extraction reagent (Gibco, Rockville, MD, USA) according to the manufacturer’s instructions. Approximately 5
The cells were homogenized and lysed with RIPA lysis buffer (100 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% Triton X-100, 1 mM EDTA, 10 mM β-glycerophosphate, 2 mM sodium vanadate and protease inhibitor). Protein concentration was assayed using a Micro BCA Protein kit (Pierce, Rockford, IL, USA). Forty micrograms of protein per lane were separated by 12% SDS-PAGE and electroblotted onto nitrocellulose membranes (Amersham Pharmacia, Munich, Germany). Subsequently, non-specific binding was blocked by incubating with 5% non-fat milk in TBST buffer at room temperature for 1 h. Immunodetection of target proteins [TNF-α, RANTES, IκB, phosphorylated (p-)Akt, p-p38 mitogen-activated protein kinase (MAPK), p-c-Jun amino-terminal kinase (JNK), amylase, cytokeratin-19 (CK-19), total caspase-3, cleavage caspase-3 and β-actin] was performed using mouse monoclonal antibody (1:1,000; Santa Cruz Biotechnology) and anti-β-actin antibody (Sigma, St. Louis, MO, USA), respectively. Goat anti-mouse IgG (1:5,000; Sigma) followed by enhanced chemiluminescence (ECL, Amersham Pharmacia, Piscataway, NJ, USA) was used for detection. BandScan 5.0 software was used for the quantification of all the proteins after western blot analysis.
A sequential method for amylase/CK-19 double staining was used according to the immunohistochemistry enzyme double staining protocol described in a previous study (
Data are expressed as the means ± SD. Statistical significance was analyzed with the one-way factorial ANOVA or the Student’s two-tailed t-test. A value of P<0.05 was considered to indicate a statistically significant difference. All analyses were conducted using SPSS software (SPSS, Inc., Chicago, IL, USA).
To explore the effects of chronic alcohol on TNF-α and RANTES expression induced by LPS, we cultured rat macrophages (
To further confirm the role of NF-κB and IκB in the regulation of TNF-α and RANTES expression, cultured rat macrophages were transfected with siRNA targeting NF-κB or adenovirus encoding IκB. These cells were then exposed to alcohol (0 or 25 mM) and LPS (100 ng/ml) as depicted above. The expression of TNF-α and RANTES was analyzed by RT-qPCR. The knockdown of NF-κB or the overexpression of IκB significantly decreased the expression of TNF-α and RANTES (
It has been well established that IRAK-M plays a vital role in activating NF-κB and in the regulation of inflammation induced by alcohol and LPS (
Liou
The progression of ADM has been shown to be implicated in a process of transdifferentiation and the induction of anti-apoptosis (
To further investigate the effects of TNF-α and RANTES on ADM, we employed neutralizing antibodies (NABs) to antagonize TNF-α and RANTES. As a result, neutralizing TNF-α and RANTES markedly reversed the effects on amylase and CK-19 expression (
NF-κB activation and translocation into the nucleus has been proven to be an essential process for initiating ADM (
To explore the effects of chronic alcohol and LPS on the physiological parameters of rats, the animals were injected with a series of doses (0, 5, 10, 15, 20 and 25 mmol/kg/day) of alcohol for 4 weeks and then LPS (1 mg/kg). Following the completion of treatment, all the animals were weighed and then sacrificed by cervical dislocation, with their organs harvested for the calculation of PW, LW, spleen weight (SW) and kidney weight (KW). Compared to the control group (no treatment), increasing the dose of alcohol significantly decreased BW, PW and LW in the rats (
To determine the plasma alcohol concentration in the rats, blood samples of these exposed rats were collected and detected by enzyme-based assays. The plasma alcohol concentrations of the exposed rats were much higher than those of the controls. The results revealed that chronic alcohol exposure induced an increase in the plasma alcohol concentration in the rats (
To determine oxidative stress caused by chronic alcohol and LPS exposure, the MDA level, SOD activity and GPx activity were calculated with the blood samples collected. Exposure to chronic alcohol and LPS induced an increase in the MDA level in serum with a concurrent decrease in SOD and GPx activity (
To investigate the effects of chronic alcohol and LPS on circulating TNF-α and RANTES expression, TNF-α and RANTES expression in serum was analyzed by ELISA. The results revealed that the levels of TNF-α and RANTES in serum were evidently increased by chronic alcohol and LPS exposure (
To further determine whether ADM occurs in pancreatic acinar cells of rats exposed to chronic alcohol and LPS, acinar cells derived from the exposed rats were isolated. Amylase and CK-19 expression in these cells were analyzed by western blot analysis. Chronic alcohol and LPS exposure significantly downregulated amylase expression, but enhanced CK-19 expression in the acinar cells compared with the controls (
We then explored whether ADM is inhibited as TNF-α and RANTES is antagonized
To further examine the role of the PI3K/Akt/IKK pathway in the induction of pancreatic ADM
Several studies have indicated that inflammation of the pancreas may be an important source for the initiation of pancreatic cancer (
It is well established that acute alcoholic exposure inactivates monocyte/macrophage responses to LPS stimulation, while chronic exposure has the opposite effect (
IRAK-M is one of the primary targets in macrophages exposed to alcohol (
The transdifferentiation of acinar cells to duct-like cells may lead to metaplastic duct lesions which are commonly observed in pancreatitis (
PI3K activation has been implicated in the pathogenesis of various pancreatic lesions (
Evidence has indicated that a dedifferentiation process may be a crucial part in ADM. Cultured pancreatic acini will undergo apoptosis under normal conditions. However, once pancreatic ADM has been induced, acinar cells can attain a longer lifespan and proliferative properties (
Our results demonstrated that chronic alcohol exposure and LPS stimulation may have an adverse effect on rats. The body weight, PW and LW of the intoxicated rats were significantly reduced at the end of treatment. Moreover, the injection of alcohol and LPS enhanced the level of alcohol and oxidative stress in the serum. Serum TNF-α and RANTES levels examined by ELISA were distinctly augmented in the exposed rats compared with the controls. Additionally, we found that TNF-α and RANTES expression in monocytes in peripheral blood were evidently upregulated by chronic alcohol exposure.
The monocyte secretion of cytokines, such as TNF-α or RANTES plays a central role in the pathophysiology of pancreatitis (
We observed that the PI3K/Akt/IKK pathway mediated ADM induction by macrophage supernatants in cultured primary acini. Accordingly, the inhibition of PI3K or IKK in the acini of rats also significantly blocked ADM progression, which suggests the role of PI3K/Akt/IKK in the induction of ADM
Collectively, we found that chronic alcohol exposure may promote cytokine secretion in macrophages/monocytes stimulated by LPS both
The present study was supported by grants from the National Natural Science Foundation of China (NSFC) (no. 81172195, 81201824).
acinar-to-ductal metaplasia
protein kinase B
cytokeratin-19
chronic pancreatitis
enzyme-linked immunosorbent assay
glutathione peroxidase
inhibitory κB kinase
interleukin-1
interleukin-6
interleukin-8
interleukin-1 receptor-associated kinase
inhibitory κB
c-Jun amino-terminal kinase
kidney weight
lipopolysaccharide
liver weight
mitogen-activated protein kinase
malondialdehyde
nuclear factor κB
pancreatic intraepithelial neoplasia
pancreatic ductal adenocarcinoma
phosphatidylinositol-3-kinase
pancreatic weight
quantitative reverse transcription-polymerase chain reaction
regulated upon activation, normal T cell expression and secreted
Sprague-Dawley rat
small-interfering RNA
superoxide dismutase
spleen weight
tumor growth factor-β
tumor necrosis factor-α
Chronic alcohol exposure upregulates tumor necrosis factor (TNF)-α and regulated upon activation, normal T cell expression and secreted (RANTES) expression in rat macrophages. (A) Cultured rat macrophages (magnification, ×400, F200). (B) mRNA expression of TNF-α and RANTES in rat macrophages exposed to varying doses (0, 5, 10, 15, 20 and 25 mM) of alcohol [ethanol (EtOH)] and lipopolysaccharide (LPS); *P<0.05 vs. 0 mM EtOH + LPS group. (C) Protein expression of TNF-α and RANTES in rat macrophages exposed to varying doses (0, 5, 10, 15, 20 and 25 mM) of alcohol and LPS. (D) Protein expression was analyzed using BandScan 5.0 software and normalized to β-actin; *P<0.05 vs. 0 mM EtOH + LPS. (E) Western blot analysis of IκB expression in rat macrophages exposed to varying doses (0, 5, 10, 15, 20 and 25 mM) of alcohol and LPS. (F) Protein expression was analyzed using BandScan 5.0 software and normalized to β-actin; *P<0.05 vs. 0 mM EtOH + LPS.
Nuclear factor (NF)-κB is involved in the regulation of tumor necrosis factor (TNF)-α and regulated upon activation, normal T cell expression and secreted (RANTES) expression in rat macrophages. (A) NF-κB expression was targeted by specific siRNA in macrophages prior to exposure to 25 mM alcohol [ethanol (EtOH)] and LPS. *P<0.05 vs. control, **P<0.05 vs. EtOH + LPS group; (B) IκB (inhibitor NF-κB) of overexpression was induced following transfection with adenovirus into macrophages prior to exposure to 25 mM alcohol and LPS. *P<0.05 vs. control, **P<0.05 vs. EtOH + LPS group.
Interleukin-1 receptor-associated kinase (IRAK)-M/p38 mitogen-activated protein kinase (MAPK)/c-Jun amino-terminal kinase (JNK) plays a vital role in the regulation of tumor necrosis factor (TNF)-α and regulated upon activation, normal T cell expression and secreted (RANTES) expression in rat macrophages. (A) Protein expression of IRAK-M in rat macrophages exposed to varying doses (0, 5, 10, 15, 20 and 25 mM) of alcohol [ethanol (EtOH)] and lipopolysaccharide (LPS). (B) Protein expression was analyzed using BandScan 5.0 software and normalized to β-actin; *P<0.05 vs. 0 mM EtOH + LPS. (C) Expression of phosphorylated (p-)p38 MAPK and p-JNK was enhanced in rat macrophages exposed to varying doses (0, 5, 10, 15, 20 and 25 mM) of alcohol and LPS. (D) Protein expression was analyzed using BandScan 5.0 software and normalized to β-actin; *P<0.05 vs. 0 mM EtOH + LPS group. (E) IRAK-M expression was targeted by specific siRNA in macrophages prior to exposure to 25 mM alcohol and LPS. Protein expression of p-p38 MAPK, p-JNK, TNF-α and RANTES was analyzed by western blot analysis. (F) Protein expression was analyzed using BandScan 5.0 software and normalized to β-actin, *P<0.05 vs. control; **P<0.05 vs. 25 mM EtOH + LPS group.
Amylase and cytokeratin-19 (CK-19) expression is altered in acinar cells cultured with macrophage supernatant. (A) Rat macrophages were exposed to varying doses (0, 5, 10, 15, 20 and 25 mM) of alcohol [ethanol (EtOH)]. After exposure for 7 days, lipopolysaccharide (LPS; 100 ng/ml) was added. Supernatants of each group were harvested and added into isolated primary acinar cells. Following culture for 48 h, the protein expression of amylase and cytokeratin-19 (CK-19) in acinar cells was analyzed by western blot analysis. (B) Protein expression was analyzed using BandScan 5.0 software and normalized to β-actin; *P<0.05 vs. 0 mM macrophage supernatant. (C) Expression of total caspase-3 and cleaved caspase-3 in acinar cells was analyzed by western blot analysis. (D) Protein expression was analyzed using BandScan 5.0 software and normalized to β-actin; *P<0.05 vs. 0 mM macrophage supernatant. (E) Tumor necrosis factor (TNF)-α and regulated upon activation, normal T cell expression and secreted (RANTES) expression in supernatant was neutralized by anti-TNF-α and anti-RANTES antibodies before the macrophage supernatants were added into the cultured acinar cells. Following treatment for 48 h, the expression of amylase and CK-19 in acinar cells was analyzed by western blot analysis. (F) Protein expression was analyzed using BandScan 5.0 software and normalized to β-actin; *P<0.05 vs. control, **P<0.05 vs. macrophage supernatant group.
The phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/inhibitory κB kinase(IKK) signaling pathway is involved in nuclear factor (NF)-κB activation induced by macrophage supernatant. (A) NF-κB activity was augmented by macrophage supernatant, *P<0.05 vs. control. (B) p-Akt and p-IKK were reduced by LY294002 (PI3K inhibitor). (C) Protein expression was analyzed using BandScan 5.0 software and normalized to β-actin; *P<0.05 vs. macrophage supernatant group. (D) NF-κB activity was reduced by LY294002; *P<0.05 vs. macrophage supernatant group.
Variation of plasma alcohol and oxidative stress in rats is caused by chronic alcohol and lipopolysaccharide (LPS) exposure. (A) Plasma alcohol was increased in rats exposed to alcohol [ethanol (EtOH)] and LPS; *P<0.05 vs. control (0 mM/kg/day). (B) MDA levels were elevated in rats exposed to alcohol and LPS; *P<0.05 vs. control (0 mM/kg/day). (C) GPx activity was reduced in rats exposed to alcohol and LPS; *P<0.05 vs. control (0 mM/kg/day). (D) SOD activity was reduced in rats exposed to alcohol and LPS, *P<0.05 vs. control (0 mM/kg/day).
Increased expression of tumor necrosis factor (TNF)-α and regulated upon activation, normal T cell expression and secreted (RANTES) is induced by chronic alcohol [ethanol (EtOH)] and lipopolysaccharide (LPS) exposure. (A) Circulating TNF-α and RANTES levels were promoted in rats exposed to alcohol and LPS; *P<0.05 vs. control (0 mM/kg/day). (B) The expression of TNF-α and RANTES was promoted in monocytes of rats exposed to alcohol and LPS; *P<0.05 vs. control (0 mM/kg/day).
Pancreatic acinar-to-ductal metaplasia (ADM) was induced in rats by chronic alcohol [ethanol (EtOH)] and lipopolysaccharide (LPS) exposure. (A) Amylase expression was downregulated in pancreatic acinar cells with an enhancement in cytokeratin-19 (CK-19) expression. (B) Protein expression was analyzed using BandScan 5.0 software and normalized to β-actin; *P<0.05 vs. control (0 mM/kg/day). (C) Restoration of amylase expression was caused by neutralizing antibodies with the decreased expression of CK-19. (D) Protein expression was analyzed using BandScan 5.0 software and normalized to β-actin; *P<0.05 vs. control, **P<0.05 vs. EtOH + LPS group. (E) CK-19 and amylase expression in pancreatic sections was detected by immunohistology. Pancreatic ADM was blocked by LY294002 [phosphatidylinositol-3-kinase (PI3K) inhibitor].
List of primers used for RT-qPCR.
Gene | Primer sequence |
---|---|
TNF-α | F: 5′-ATGAGCACAGAAAGCATGATC-3′ |
RANTES | F: 5′-TCCAATCTTGCAGTCGTGTTTG-3′ |
β-actin | F: 5′-GTG GGG CGC CCC AGG CACCA-3′ |
TNF-α, tumor necrosis factor-α; RANTES, regulated upon activation, normal T cell expression and secreted. F, forward; R, reverse.
Body and organ weight of the animals (n=60).
Groups | n | BW (g) | PW (g) | LW (g) | SW (g) | KW (g) |
---|---|---|---|---|---|---|
0 | 10 | 455.9±29.7 | 1.89±0.27 | 6.19±0.95 | 0.93±0.27 | 3.43±0.91 |
5 | 10 | 437.6±29.4 | 1.61±0.25 | 5.91±0.62 | 1.15±0.33 | 3.37±0.79 |
10 | 10 | 422.7±28.7 |
1.43±0.25 |
5.34±0.81 |
1.26±0.32 | 3.44±0.89 |
15 | 10 | 401.3±27.5 |
1.31±0.26 |
4.68±0.64 |
1.17±0.29 | 3.35±0.88 |
20 | 10 | 392.5±22.8 |
1.20±0.18 |
4.15±0.73 |
1.32±0.41 | 3.47±1.15 |
25 | 10 | 387.8±26.5 |
1.07±0.19 |
3.73±0.71 |
1.24±0.56 | 3.51±1.21 |
BW, body weight; PW, pancreatic weight; LW, liver weight; SW, spleen weight; KW, kidney weight.
P<0.05 vs. 0 mM/kg/day ethanol (EtOH) + LPS group; denotes a significant difference.