- Email: [email protected]
The toll-like receptor 4 antagonist transforming growth factor-β-activated kinase(TAK)-242 attenuates taurocholate-induced oxidative stress through regulating mitochondrial function in mice pancreatic acinar cells
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The toll-like receptor 4 antagonist transforming growth factor-b-activated kinase(TAK)-242 attenuates taurocholate-induced oxidative stress through regulating mitochondrial function in mice pancreatic acinar cells Long-Fei Pan, MD,a Lei Yu, MD,b Li-Ming Wang, MD,a Jun-Tao He, MD,c Jiang-Li Sun, MD,a Xiao-Bo Wang, MD,a Zheng-Hai Bai, MD,a Li-Juan Su, MD,a and Hong-Hong Pei, MDa,* a
Department of Emergency Medicine, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an Jiaotong University, Xi’an, Shaanxi, China b Department of Clinical Laboratory, Xi’an Medical College, Xi’an, Shaanxi, China c Department of Clinical Laboratory, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an Jiaotong University, Xi’an, Shaanxi, China
article info
abstract
Article history:
Background: Acute pancreatitis (AP) is a commonly occurring and potentially life-
Received 12 March 2016
threatening disease. Recently, toll-like receptor 4 (TLR4) has been considered as a new
Received in revised form
clue for studying the pathogenesis of AP due to its important role in inflammatory response
27 June 2016
cascade.
Accepted 2 August 2016
Materials and methods: The aim of this study was to investigate the potential protective
Available online 9 August 2016
effect of transforming growth factor-b-activated kinase (TAK)-242, a novel TLR4 antagonist, in taurocholate-treated mice pancreatic acinar cells. The protective effects were measured
Keywords:
by cell viability, lactate dehydrogenase release and apoptosis, and oxidative stress was
Acute pancreatitis
assayed by lipid peroxidation and oxidative enzyme activities. To determine the potential
Oxidative stress
underlying mechanisms, mitochondrial cytochrome c release, swelling, and calcium
Mitochondrial dysfunction
buffering capacity were measured in isolated mitochondria, and mitochondrial biogenesis
TLR4
and expression of mitochondrial dynamic proteins were detected by reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blot. Results: Treatment with 6-mM taurocholate significantly increased the expression of TLR4 at both mRNA and protein levels. TAK-242 markedly increased cell viability, decreased lactate dehydrogenase release, and inhibited apoptotic cell death as measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining in pancreatic acinar cells. These protective effects were accompanied by the suppressed lipid peroxidation and enhanced endogenous antioxidative enzyme activity. Using isolated and purified mitochondria from pancreatic acinar cells, we found that TAK-242 treatment also inhibited cytochrome c release into the cytoplasm, mitochondrial swelling, and decrease in
* Corresponding author. Department of Emergency Medicine, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an Jiaotong University, 157 Xiwu Road, Xi’an, Shaanxi 710004, China. Tel.: þ86 29 87679412; fax: þ86 29 87679413. E-mail address: [email protected] (H.-H. Pei). 0022-4804/$ e see front matter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2016.08.011
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mitochondrial Ca2þ buffering capacity after taurocholate exposure. In addition, TAK-242 significantly promoted mitochondrial biogenesis, as evidenced by increased mtDNA and upregulated mitochondrial transcription factors. The results of Western blot analysis showed that TAK-242 also differently regulated the expression of mitochondrial fusion and fission proteins. Conclusions: All these data strongly indicated that blocking TLR4 activity via TAK-242 exerts protective effects in an in vitro AP model, and it could be a possible strategy to improve clinical outcome in AP patients. ª 2016 Elsevier Inc. All rights reserved.
Introduction Acute pancreatitis (AP) is a commonly occurring and potentially life-threatening disease. In most cases, it follows a selflimiting course and can be treated conservatively. Unfortunately, approximately 20%-30% of AP patients eventually progress to develop severe AP with a high morbidity and mortality due to the lack of specific treatment.1 AP involves a complicated cascade of events initiated by the injury of pancreatic acinar cells, followed by local and systemic inflammatory responses.2,3 Although the pathogenic mechanisms of acinar cell injury remains elusive, there are many clinical and experimental findings supporting the involvement of oxidative stress and mitochondrial dysfunction.4,5 The toll-like receptors (TLRs) are a large family of pattern recognition receptors that play a central role in the regulation of the host immunity and inflammatory responses.6 To date, 13 members of these mammalian receptors and 11 human TLRs have been described, among which TLR4 is identified as the signaling receptor for lipopolysaccharide, an outer membrane component of gram-negative bacteria.7 Accumulating evidence has shown that the activation or suppression of TLR4 is involved in the development and progression of a number of inflammatory diseases, including AP.8 TLR4 is mainly expressed extracellularly in human and rodent pancreas with an extracellular region, a transmembrane segment and a cytoplasmic tail.9 Results of animals’ experiments showed that TLR4 mRNA is increased in the pancreas of rats in the early stage of pancreatitis,10 and anti-TLR4 antibody inhibited pancreatic elastase-induced TNF-a release in a human leukemic cell line (THP-1) cells.11 In addition, in AP patients, the upregulation of plasma TNF-a levels was found to be positively correlated with the expression of TLR4.12 Transforming growth factor-b-activated kinase (TAK)-242, also known as resatorvid, is a novel small-molecule compound that binds to the intracellular domain of TLR4, and selectively inhibits TLR4 signal transduction and its downstream signaling cascades.13 In the present study, we investigated the potential protective effect of TAK-242 in an in vitro AP model induced by taurocholate in mice pancreatic acinar cells.
Materials and methods Primary culture of pancreatic acinar cells Pancreatic acinar cells were isolated from adult male (to avoid the effects of estrogen levels) C57BL/6J mice (weighing 25-30 g)
as previously described.14 Briefly, the animals were sacrificed, and the pancreas was removed quickly and incubated in buffer solution (containing 130-mM NaCl, 4.7-mM KCl, 1.3-mM CaCl2, 1-mM MgCl2, 1.2-mM KH2PO4, and 0.2% bovine serum albumin (BSA) at 37 C for 10 min. Then, the cell suspension was centrifugated at 30 g for 5 min at 4 C, and the acinar cell clumps were resuspended in Na-Hepes buffer without collagenase. To mimic AP in vitro, cultured pancreatic acinar cells were treated with taurocholate (Gibco, MD) at the concentration of 6 mM under the conditions detailed in the following sentences. To investigate the potential protective effect of TAK-242, pancreatic acinar cells were treated with TAK-242 (1 mM) immediately after exposure to taurocholate, and the concentrations of taurocholate and TAK-242 were selected according to previous published data.15,16 Twenty C57BL/6J mice were used, and all experimental protocols and animal handling procedures were performed in accordance with the Institutional Animal Care and Use Committee guidelines for the use of experimental animals and approved by the Institutional Animal Care and Use Committee of the Xi’an Jiaotong University.
Cell viability assay Cell viability was determined by the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay according to the instruction from the company (Sigma, MO). Briefly, pancreatic acinar cells were cultured in 96-well plates at the concentration of 1 104 cells/well and exposed to various treatments. At 24 h after taurocholate treatment, 5 g/L MTT reagent was added into each well before incubation for 4 h at 37 C. Then, the blue colored formazan product was dissolved in dimethyl sulfoxide (DMSO), and the spectrophotometric absorbance was subsequently measured at 570 nm using a microplate reader. All results were compared to the untreated control group and expressed as the percentage of the control values.
Lactate dehydrogenase release assay The cytotoxicity in pancreatic acinar cells was determined by measuring lactate dehydrogenase (LDH) release as a function of membrane integrity using a diagnostic kit according to the manufacturer’s instructions (Institute Jianchen Biological Engineering, Nanjing, China). Briefly, 50 mL of supernatants were collected and incubated with nicotinamide adenine dinucleotide diaphorase (NADH) and 0.1% sodium pyruvate for 15 min at 37 C. Absorbance of the sample was monitored
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Quantification of cytochrome c release
at 440 nm, and the activity of LDH was represented as the fold of control values.
Cytochrome c release into the cytoplasm was determined by Western blot analysis using specific antibodies after mitochondrial isolation and purification. Briefly, pancreatic acinar cells were lysed by the protease inhibitor mixed lysis buffer, and the cell lysate was centrifuged for 10 min at 600 g at 4 C. After discarding the pellets containing the nucleus, the supernatant was centrifuged for 10 min at 14,000 g at 4 C. The resulting pellet and supernatant were separated and used as mitochondrial fraction and cytosolic fraction, respectively.
TUNEL staining Apoptosis in pancreatic acinar cells subjected to taurocholate and/or TAK-242 treatment was detected by TUNEL staining, the method to observe DNA strand breaks in nucleus. Briefly, cells seeded on glass slides were fixed by 4% methanol-free formaldehyde solution in PBS for 20 min and labeled with the fluorescein TUNEL reagent mixture (Promega, Madison, WI) for 1 h at 37 C in the dark. Then, 2-(4-Amidinophenyl)-6indolecarbamidine dihydrochloride (DAPI) (Cell Signaling Technology, MA) was added to stain the nucleus before washing with phosphate buffered saline (PBS) three times. Fluorescence evaluation was performed under a confocal microscope (FV10i, Olympus, Tokyo, Japan), and the number of TUNEL-positive (green) cells was counted.
Measurement of mitochondria swelling After mitochondrial isolation and purification, effects of TAK242 on mitochondria swelling after taurocholate treatment were measured by a previously published method.18 The results were represented as the decrease in absorbance at 540 nm with 200-mM CaCl2.
Measurement of lipid peroxidation Determination of mitochondrial calcium buffering capacity To determine the levels of lipid peroxidation after taurocholate and/or TAK-242 treatment, the content of malonaldehyde (MDA) and 4-hydroxynonena (4-HNE) in pancreatic acinar cells were measured using assay kits according to the manufacturer’s instructions (Cell Biolabs, MA). All values were compared with the control group, and the results were expressed as the percentage of control values.
To determine mitochondrial Ca2þ metabolism in pancreatic acinar cells, mitochondrial Ca2þ buffering capacity was measured using the Ca2þ sensitive Calcium Green 5N fluorescent dye (Molecular Probes, OR). After taurocholate and/or TAK-242 treatment, pancreatic acinar cells were incubated with the prepared working liquid at 37 C, and bolus additions of CaCl2 were performed. The change in green fluorescence was monitored at 532 nm with a QuantaMaster spectrofluorometer (Invitrogen, Sacramento, CA), and the results were expressed as the percentage of control values.
Measurement of SOD, CAT, and GPX activity To investigate the effect of TAK-242 on endogenous antioxidative system, the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) was measured using assay kits according to the manufacturer’s instructions (Cayman Chemical, MI). All values were compared with the control group, and the results were expressed as the percentage of control values.
Real-time RT-PCR Total RNA was isolated from pancreatic acinar cells using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). After extraction, 500 ng of total RNA were used to synthesize the first strand of cDNA using a reverse transcription kit (Takara, Tokyo, Japan). The mRNA levels of TLR4, peroxisome proliferator activated receptor coactivator-1 (PGC1), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (TFAM) was determined by real-time RT-PCR using the primers as summarized in Table 1, and the results were normalized to the value of GAPDH. In addition, mtDNA was determined using primers used for the amplification of mitochondrial genomes: 50 -ATA TTT TCA CTG CTG AGT CCC GTG G-30 ; reverse, 50 -AAT TTC GGT TGG GGT GAC
Determination of ROS generation To determine the intracellular reactive oxygen species (ROS) generation after various treatments, pancreatic acinar cells were incubated with the indicator 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA, Cell Signaling Technology, MA) in the dark at room temperature.17 After washing with PBS for three times, the fluorescence was detected with a fluorescence plate reader (excitation/emission 480/530 nm).
Table 1 e Primers sequences used in real-time PCR. Gene
Forward sequences
Reverse sequences
TLR4
50 -AGAAAATGCCAGGATGATGC-30
50 -AGGGATTCAAGCTTCCTGGT-30
PGC-1
50 -GTGCAGCCAAGACTCTGTATGG-30
50 -GTCCAGGTCATTCACATCAAGTTC-30
NRF-1 TFAM GAPDH
0
5 -GAGTGACCCAAACCGAACA-3 0
5 -GGTGTATGAAGCGGATTT-3 0
0
50 -GGAGTTGAGTATGTCCGAGT-30
0
5 -ATGTATCCGTTGTGGATCTGAC-3
50 -CTTTCTTCTTTAGGCGTTT-30 0
50 -CCTGCTTCACCACCTTCTTG-30
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CTC GGA G-30 . The results were represented as the fold of control group.
Results
Western blot analysis
Blocking TLR4 via TAK-242 attenuates taurocholateinduced cytotoxicity
The pancreatic acinar cells were harvested using ice-cold lysis buffer containing protease inhibitors (Sigma, MO), and the protein concentrations were determined using the bicinchoninic acid (BCA) method (Institute Jianchen Biological Engineering, Nanjing, China). A total of 40-mg lysates was separated by 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes using standard techniques. The nonspecific binding of antibodies was blocked with 5% nonfat milk solution for 1 h, and the following primary antibodies purchase from Sigma (St. Louis, MO) were used: TLR4 (1:600), cytochrome c (1:500), Tubulin (1:1000), COX V (1:500), Opa-1 (1:800), Mfn-1 (1:600), Drp-1 (1:800), Fis-1 (1:800), and bactin (1:1000). The blots were performed using chemiluminescence, and the optical density of each band was quantified using Image J software (Scion Corporation, MO).
Statistical analysis The statistical analysis of results was performed using GraphPad Prism 6.0 (GraphPad, San Diego, CA). Statistical evaluation of the data between groups was performed by oneway analysis of variance followed by Bonferroni post hoc test or Student’s t-test when appropriate. A value of P < 0.05 was considered statistically significant.
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To investigate the effect of AP on TLR4 expression, pancreatic acinar cells were treated with 6-mM taurocholate, and the expression of TLR4 at mRNA and protein levels were detected by RT-PCR and Western blot, respectively. The results showed that TLR4 mRNA was significantly increased from 3 h to 12 h after injury and then decreased to normal level (Fig. 1A). At the same time, the protein level of TLR4 was elevated from 3 h and continued to 48 h and then decreased to normal level at 72 h after taurocholate treatment (Fig. 1B). To block the activation of TLR4, pancreatic acinar cells were treated with 1-mM TAK242, an exogenous TLR4 antagonist. We found that TAK-242 protects against taurocholate-induced cytotoxicity, as evidenced by increased cell viability (Fig. 1C) and decreased LDH release (Fig. 1D). In addition, we also detected apoptosis using TUNEL staining, the results of which showed that TAK-242 significantly decreased the number of TUNEL-positive cells as compared to the taurocholate-treated group.
TAK-242 reduces taurocholate-induced oxidative stress To determine whether TAK-242 protects pancreatic acinar cells via antioxidative activity, we investigated the effect of TAK-242 treatment on lipid peroxidation by measuring MDA
Fig. 1 e Blocking TLR4 via TAK-242 attenuates taurocholate-induced cytotoxicity. Pancreatic acinar cells were treated with 6-mM taurocholate (Tauro), and the expression of TLR4 at mRNA and protein levels were detected by RT-PCR (A) or Western blot (B). Pancreatic acinar cells were treated with 1-mM TAK-242 (TAK) in the presence or absence of 6-mM Tauro. Cell viability (C) and LDH release (D) were measured at 24 h after Tauro exposure. The apoptotic cell death was detected by TUNEL staining (E). Scale bar: 100 mm. Data are shown as mean ± standard error of mean (n [ 6). #P < 0.05 versus control. *P < 0.05 versus Tauro. (Color version of figure is available online.)
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Fig. 2 e TAK-242 reduces taurocholate-induced oxidative stress. Pancreatic acinar cells were treated with 1-mM TAK in the presence or absence of 6-mM Tauro. The expression levels of MDA (A), 4-HNE (B), and the activities of endogenous antioxidant enzymes (C) were measured. Data are shown as mean ± standard error of mean (n [ 6). #P < 0.05 versus control. *P < 0.05 versus Tauro. and 4-HNE levels. As shown in Figure 2A and B, taurocholate significantly increased the expression of MDA and 4-HNE, while there was no difference between control and TAK-242 groups. Blocking TLR4 activation via TAK-242 markedly attenuated taurocholate-induced increases in both MDA and 4-HNE levels. Furthermore, we also detected the enzymatic activity of SOD, CAT, and GPX after TAK-242 and/or taurocholate treatment to test the effects of TLR4 blocking on the endogenous antioxidant system (Fig. 2C). The results showed that TAK-242 partially prevented the taurocholate-induced decreases in SOD, CAT, and GPX activities, indicating the antioxidative activity of TAK-242.
TAK-242 inhibits taurocholate-induced mitochondrial dysfunction Intracellular ROS generation was assayed to investigate the effect of TAK-242 on mitochondrial function, and we found that taurocholate-induced increase in ROS generation was significantly inhibited by TAK-242 treatment (Fig. 3A). We also detected cytochrome c release by Western blot analysis (Fig. 3B). The results showed that TAK-242 inhibited
cytochrome c release after taurocholate exposure, as evidenced by decreased cytosolic cytochrome c (Fig. 3C) and increased mitochondrial cytochrome c content (Fig. 3D). After isolation of mitochondria from pancreatic acinar cells, we determined the effect of TAK-242 on mitochondria swelling, which was induced by 200-mM Ca2þ (Fig. 3E). The results showed that TAK-242 treatment significantly prevented mitochondria swelling induced by taurocholate exposure. As shown in Figure 3F and G, a similar result in mitochondrial Ca2þ buffering capacity was also observed.
TAK-242 differently regulates mitochondrial biogenesis and dynamics To determine whether TAK-242 protects pancreatic acinar cells via regulating mitochondrial biogenesis, long-fragment PCR was performed to quantify the amount of intact mtDNA (Fig. 4A). The results showed that taurocholate treatment significantly decreased mtDNA content, which was partly prevented by TAK-242. We also used real-time RT-PCR to detect the expression of three transcription factors considered essential for mitochondrial gene expression and increased
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Fig. 3 e TAK-242 inhibits taurocholate-induced mitochondrial dysfunction. Pancreatic acinar cells were treated with 1-mM TAK in the presence or absence of 6-mM Tauro. The intracellular ROS production was detected by 2,7-dichlorofluorescein diacetate (DCF) fluorescence staining (A). Mitochondria in each group were isolated and purified. The expression of cytochrome c was detected by Western blot analysis (B) in cytosolic fraction (C) and mitochondrial fraction (D). Mitochondrial swelling was examined by monitoring the absorbance at 540 nm induced by 200-mM Ca2D (E), and the baseline absorbance was measured without Ca2D. Relative Ca2D uptake capacity of isolated mitochondria were determined (F) and calculated (G). Data are shown as mean ± standard error of mean (n [ 6). #P < 0.05 versus control. *P < 0.05 versus Tauro.
expression of PGC-1, NRF-1, and TFAM mRNA was observed in Tauro þ TAK group compared with that in taurocholatetreated cells (Fig. 4B). In addition, the expression of Opa-1, Mfn-1, Drp-1, and Fis-1 at protein levels was detected by Western blot to investigate the effect of TAK-242 on mitochondrial dynamics (Fig. 4C). As shown in Figure 4D, TAK-242 increased the expression of opa-1 and Mfn-1 but decreased the expression of Drp-1 after taurocholate treatment, with no effect on Fis-1 expression.
Discussion Significant findings on TLR4-mediated signaling cascades in inflammatory diseases have been reported, such as nuclear factor-kB (NF-kB) and members of the interferon (IFN)-regulatory factor (IRF) family.19 Moreover, the upregulation of plasma TNF-a levels in AP patients was found to be positively correlated with the expression of TLR4.12 All these data suggest a critical role of TLR4 in AP. However, the direct link between TLR4 and pancreatic acinar cell injury are still elusive. In the present study, we first observed that the expression of TLR4 was increased after taurocholate treatment at both mRNA and protein levels. Then, we used TAK-242 to demonstrate that blocking of TLR4 exert protective effects against taurocholate-induced toxicity in mice pancreatic acinar cells. After that, we found that TAK-242-induced protection was associated with preserved mitochondrial function and the
regulation of mitochondrial biogenesis and dynamics. All these results indicate that TLR4 plays an important role in AP. TLR4 was originally identified to generate innate immune responses to pathogens by activating a cascade of proinflammatory events.20 Through recognizing its ligands, such as heat shock proteins, extracellular domain A, CD14 and MD-2, TLR4 recruits the adaptor molecules TIR (Toll/IL-1 receptor) domain-containing adaptor protein, myeloid differentiation factor 88 (MyD88), IL (interleukin)-1-receptor-associated kinase, and TNF-receptor-associated factor-6, and this crosstalk between these proteins enables downstream signaling via the IKK-NF-kB complex and the mitogen-activated protein kinase cascades.21e23 TLR4 is highly expressed in the rodent and human pancreas, which providing the molecular basis for its involvement in the initiation of inflammatory response during AP.8 Results of animals’ experiments showed that TLR4 mRNA is increased in the pancreas of rats in the early stage of pancreatitis,10 and anti-TLR4 antibody inhibited pancreatic elastase-induced TNF-a release in THP-1 cells.11 In addition, in AP patients, the upregulation of plasma TNF-a levels was found to be positively correlated with the expression of TLR4.12 However, the time expression profiles of TLR4 after AP in pancreatic acinar cells have not been reported. In the present study, we demonstrated, for the first time, that TLR4 mRNA was significantly increased from 3 h to 12 h after injury and then decreased to normal level. Meanwhile, the protein level of TLR4 was elevated from 3 h and continued to 48 h and then decreased to normal level at 72 h after taurocholate
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Fig. 4 e TAK-242 differently regulates mitochondrial biogenesis and dynamics. Pancreatic acinar cells were treated with 1mM TAK in the presence or absence of 6-mM Tauro. The relative mitochondrial DNA content was quantified by longfragment PCR (A), and the mRNA expression of mitochondrial biogenesis factors was measured by real-time RT-PCR (B). The expression of mitochondrial dynamic proteins, including Opa-1, Mfn-1, Drp-1, and Fis-1, were detected by Western blot (C) and calculated (D). Data are shown as mean ± standard error of mean (n [ 6). #P < 0.05 versus control. *P < 0.05 versus Tauro.
treatment. Our results strongly supports that the expression of TLR4 was significantly increased at both transcription and translation levels in our in vitro AP model. Ductal infusion or treatment with taurocholate is widely used to mimic AP because it reproduces pancreatic edema, hemorrhage, and necrosis in in vitro and in vivo experimental conditions.24 Previous studies showed that taurocholate treatment leads to the loss of adenosine triphosphate (ATP) synthesis and excessive generation of ROS, which together, could induce a marked inhibition of endogenous antioxidative system.25 Based on these findings, antioxidative agents have been suggested to be effective treatment for pancreatitis. In this study, taurocholate treatment significantly increased lipid peroxidation and decreased antioxidative enzyme activities, and all these effects were partially reversed by TAK-242, findings that support the involvement of antioxidative activity in TAK-242-induced actions in pancreatic acinar cells. However, previous studies demonstrated that the use of antioxidative compounds in AP is not sufficient to prevent cell death, and it can even increase cell death by necrosis.26,27 Using isolated mitochondria from pancreatic acinar cells, we further investigated whether TAK242-induced protection was associated with mitochondrial function, which plays an important role under oxidative stress conditions. Our results showed that TAK-242 treatment
significantly prevented mitochondria swelling and decreases in mitochondrial Ca2þ buffering capacity induced by taurocholate exposure, which was accompanied by reduced mitochondrial cytochrome c release and ROS generation. It was shown that the Ca2þ overload in mitochondria responsible for cell death is increased by mitochondrial dysfunction.28e30 Therefore, it appears that TAK-242, through maintaining mitochondrial membrane and mitochondrial Ca2þ buffering, preserves mitochondrial integrity and function. All these data strongly support the hypothesis that TLR4 could be a downstream signaling activated under stress conditions to regulate cell oxidative state in AP, which needs to be further determined. Stimulation of secretion increases the energy demand of pancreatic acinar cells, and enhanced mitochondrial function to synthesis more ATP is required to adjust the cellular metabolism.31 Previous studies characterized novel mechanisms of toxins that induce pancreatitis, among which the direct impairment of mitochondrial ATP generation is the key event.32,33 It is well known that the preservation of mitochondrial ATP production could be achieved by increasing mitochondrial biogenesis, which is defined as the growth and division of mitochondria.34 In the present study, an increase in mtDNA content in TAK-242 treated pancreatic acinar cells was found, indicating a stimulation of mitochondrial
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biogenesis. In consistent, we also observed the significantly increased expression of PGC-1, NRF-1, and TFAM, three transcriptional factors required for mtDNA replication and transcription. To adapt energy metabolism to physiological and pathologic conditions, mitochondria became a highly dynamic organelle, remodeling by fusion and fission events.35 The balance of mitochondrial fission and/or fusion is commonly regulated by high-molecular weight GTPases, such as Opa-1 and Mfn-1 as fusion proteins, as well as Drp-1 and Fis-1, two fission proteins.36 Our results showed decreased expression of Opa-1 and Mfn-1, and increased expression of Drp-1 after taurocholate treatment, indicating decreased mitochondrial fragmentation and mitochondrial mass.37 Treatment with TAK-242 significantly prevented the changes of these mitochondrial dynamic proteins expression, which was consistent with preserved mitochondrial biogenesis. All these data strongly suggested that TAK-242 exerted protective effects against taurocholate-induced toxicity through regulating mitochondrial biogenesis and dynamics. A limitation of our experiments is that we used an in vitro model to examine the protective effect of TAK-242 against AP. The samples for assays on mitochondrial function and the expression of mitochondrial dynamic factors were harvested from primary cultured mice pancreatic acinar cells. If the experiments were performed in isolated pancreatic tissues or in in vivo conditions, more detailed information on TAK-242induced anti-inflammatory activity and related mechanisms would be provided. In summary, blocking TLR4 activity using TAK-242 was effective in ameliorating cytotoxicity and apoptosis induced by taurocholate exposure in our cellular model of AP. These effects are likely to be associated with inhibited oxidative stress and preserved mitochondrial function.
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Acknowledgment This work was financially supported by the project fund from Science and Technology Project of Shaanxi Province (no. 2010K15-03-02). The authors would like to thank Dr. Terry Chen for his technical support for the experiments and the preparation of the manuscript. Authors’ contribution: H.H.P. and L.F.P. conceived and designed the experiments. L.Y., L.M.W., J.T.H., J.L.S., and X.B.W. performed the experiments. Z.H.B. and L.J.S. analyzed the data.
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Disclosure The authors declare that there are no conflicts of interest.
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association with insulin receptor substrate-1 and phosphorylation of Ser(307). J Biol Chem. 2000;275:9047e9054. Hemi R, Yochananov Y, Barhod E, et al. p38 mitogen-activated protein kinase-dependent transactivation of ErbB receptor family: a novel common mechanism for stress-induced IRS-1 serine phosphorylation and insulin resistance. Diabetes. 2011;60:1134e1145. Wan MH, Huang W, Latawiec D, et al. Review of experimental animal models of biliary acute pancreatitis and recent advances in basic research. HPB (Oxford). 2012;14:73e81. Voronina SG, Barrow SL, Simpson AW, et al. Dynamic changes in cytosolic and mitochondrial ATP levels in pancreatic acinar cells. Gastroenterology. 2010;138:1976e1987. Siriwardena AK, Mason JM, Balachandra S, et al. Randomised, double blind, placebo controlled trial of intravenous antioxidant (n-acetylcysteine, selenium, vitamin C) therapy in severe acute pancreatitis. Gut. 2007;56:1439e1444. Leung PS, Chan YC. Role of oxidative stress in pancreatic inflammation. Antioxid Redox Signal. 2009;11:135e165. Lahlou S, Magalhaes PJ, de Siqueira RJ, et al. Cardiovascular effects of the essential oil of Aniba canelilla bark in normotensive rats. J Cardiovasc Pharmacol. 2005;46:412e421. Chen T, Yang YF, Luo P, et al. Homer1 knockdown protects dopamine neurons through regulating calcium homeostasis in an in vitro model of Parkinson’s disease. Cell Signal. 2013;25:2863e2870.
30. Dai SH, Chen T, Wang YH, et al. Sirt3 protects cortical neurons against oxidative stress via regulating mitochondrial Ca2þ and mitochondrial biogenesis. Int J Mol Sci. 2014;15:14591e14609. 31. Korc M, Williams JA, Goldfine ID. Stimulation of the glucose transport system in isolated mouse pancreatic acini by cholecystokinin and analogues. J Biol Chem. 1979;254:7624e7629. 32. Maleth J, Rakonczay Jr Z, Venglovecz V, Dolman NJ, Hegyi P. Central role of mitochondrial injury in the pathogenesis of acute pancreatitis. Acta Physiol (Oxf). 2013;207:226e235. 33. Mukherjee R, Criddle DN, Gukovskaya A, et al. Mitochondrial injury in pancreatitis. Cell Calcium. 2008;44:14e23. 34. Ren J, Pulakat L, Whaley-Connell A, Sowers JR. Mitochondrial biogenesis in the metabolic syndrome and cardiovascular disease. J Mol Med (Berl). 2010;88:993e1001. 35. Szabadkai G, Simoni AM, Chami M, et al. Drp-1-dependent division of the mitochondrial network blocks intraorganellar Ca2þ waves and protects against Ca2þ-mediated apoptosis. Mol Cel. 2004;16:59e68. 36. Martinou JC, Youle RJ. Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. Developmental Cel. 2011;21:92e101. 37. Zhu J, Wang KZ, Chu CT. After the banquet: mitochondrial biogenesis, mitophagy, and cell survival. Autophagy. 2013;9:1663e1676.
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The toll-like receptor 4 antagonist transforming growth factor-b-activated kinase(TAK)-242 attenuates taurocholate-induced oxidative stress through regulating mitochondrial function in mice pancreatic acinar cells Long-Fei Pan, MD,a Lei Yu, MD,b Li-Ming Wang, MD,a Jun-Tao He, MD,c Jiang-Li Sun, MD,a Xiao-Bo Wang, MD,a Zheng-Hai Bai, MD,a Li-Juan Su, MD,a and Hong-Hong Pei, MDa,* a
Department of Emergency Medicine, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an Jiaotong University, Xi’an, Shaanxi, China b Department of Clinical Laboratory, Xi’an Medical College, Xi’an, Shaanxi, China c Department of Clinical Laboratory, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an Jiaotong University, Xi’an, Shaanxi, China
article info
abstract
Article history:
Background: Acute pancreatitis (AP) is a commonly occurring and potentially life-
Received 12 March 2016
threatening disease. Recently, toll-like receptor 4 (TLR4) has been considered as a new
Received in revised form
clue for studying the pathogenesis of AP due to its important role in inflammatory response
27 June 2016
cascade.
Accepted 2 August 2016
Materials and methods: The aim of this study was to investigate the potential protective
Available online 9 August 2016
effect of transforming growth factor-b-activated kinase (TAK)-242, a novel TLR4 antagonist, in taurocholate-treated mice pancreatic acinar cells. The protective effects were measured
Keywords:
by cell viability, lactate dehydrogenase release and apoptosis, and oxidative stress was
Acute pancreatitis
assayed by lipid peroxidation and oxidative enzyme activities. To determine the potential
Oxidative stress
underlying mechanisms, mitochondrial cytochrome c release, swelling, and calcium
Mitochondrial dysfunction
buffering capacity were measured in isolated mitochondria, and mitochondrial biogenesis
TLR4
and expression of mitochondrial dynamic proteins were detected by reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blot. Results: Treatment with 6-mM taurocholate significantly increased the expression of TLR4 at both mRNA and protein levels. TAK-242 markedly increased cell viability, decreased lactate dehydrogenase release, and inhibited apoptotic cell death as measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining in pancreatic acinar cells. These protective effects were accompanied by the suppressed lipid peroxidation and enhanced endogenous antioxidative enzyme activity. Using isolated and purified mitochondria from pancreatic acinar cells, we found that TAK-242 treatment also inhibited cytochrome c release into the cytoplasm, mitochondrial swelling, and decrease in
* Corresponding author. Department of Emergency Medicine, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an Jiaotong University, 157 Xiwu Road, Xi’an, Shaanxi 710004, China. Tel.: þ86 29 87679412; fax: þ86 29 87679413. E-mail address: [email protected] (H.-H. Pei). 0022-4804/$ e see front matter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2016.08.011
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mitochondrial Ca2þ buffering capacity after taurocholate exposure. In addition, TAK-242 significantly promoted mitochondrial biogenesis, as evidenced by increased mtDNA and upregulated mitochondrial transcription factors. The results of Western blot analysis showed that TAK-242 also differently regulated the expression of mitochondrial fusion and fission proteins. Conclusions: All these data strongly indicated that blocking TLR4 activity via TAK-242 exerts protective effects in an in vitro AP model, and it could be a possible strategy to improve clinical outcome in AP patients. ª 2016 Elsevier Inc. All rights reserved.
Introduction Acute pancreatitis (AP) is a commonly occurring and potentially life-threatening disease. In most cases, it follows a selflimiting course and can be treated conservatively. Unfortunately, approximately 20%-30% of AP patients eventually progress to develop severe AP with a high morbidity and mortality due to the lack of specific treatment.1 AP involves a complicated cascade of events initiated by the injury of pancreatic acinar cells, followed by local and systemic inflammatory responses.2,3 Although the pathogenic mechanisms of acinar cell injury remains elusive, there are many clinical and experimental findings supporting the involvement of oxidative stress and mitochondrial dysfunction.4,5 The toll-like receptors (TLRs) are a large family of pattern recognition receptors that play a central role in the regulation of the host immunity and inflammatory responses.6 To date, 13 members of these mammalian receptors and 11 human TLRs have been described, among which TLR4 is identified as the signaling receptor for lipopolysaccharide, an outer membrane component of gram-negative bacteria.7 Accumulating evidence has shown that the activation or suppression of TLR4 is involved in the development and progression of a number of inflammatory diseases, including AP.8 TLR4 is mainly expressed extracellularly in human and rodent pancreas with an extracellular region, a transmembrane segment and a cytoplasmic tail.9 Results of animals’ experiments showed that TLR4 mRNA is increased in the pancreas of rats in the early stage of pancreatitis,10 and anti-TLR4 antibody inhibited pancreatic elastase-induced TNF-a release in a human leukemic cell line (THP-1) cells.11 In addition, in AP patients, the upregulation of plasma TNF-a levels was found to be positively correlated with the expression of TLR4.12 Transforming growth factor-b-activated kinase (TAK)-242, also known as resatorvid, is a novel small-molecule compound that binds to the intracellular domain of TLR4, and selectively inhibits TLR4 signal transduction and its downstream signaling cascades.13 In the present study, we investigated the potential protective effect of TAK-242 in an in vitro AP model induced by taurocholate in mice pancreatic acinar cells.
Materials and methods Primary culture of pancreatic acinar cells Pancreatic acinar cells were isolated from adult male (to avoid the effects of estrogen levels) C57BL/6J mice (weighing 25-30 g)
as previously described.14 Briefly, the animals were sacrificed, and the pancreas was removed quickly and incubated in buffer solution (containing 130-mM NaCl, 4.7-mM KCl, 1.3-mM CaCl2, 1-mM MgCl2, 1.2-mM KH2PO4, and 0.2% bovine serum albumin (BSA) at 37 C for 10 min. Then, the cell suspension was centrifugated at 30 g for 5 min at 4 C, and the acinar cell clumps were resuspended in Na-Hepes buffer without collagenase. To mimic AP in vitro, cultured pancreatic acinar cells were treated with taurocholate (Gibco, MD) at the concentration of 6 mM under the conditions detailed in the following sentences. To investigate the potential protective effect of TAK-242, pancreatic acinar cells were treated with TAK-242 (1 mM) immediately after exposure to taurocholate, and the concentrations of taurocholate and TAK-242 were selected according to previous published data.15,16 Twenty C57BL/6J mice were used, and all experimental protocols and animal handling procedures were performed in accordance with the Institutional Animal Care and Use Committee guidelines for the use of experimental animals and approved by the Institutional Animal Care and Use Committee of the Xi’an Jiaotong University.
Cell viability assay Cell viability was determined by the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay according to the instruction from the company (Sigma, MO). Briefly, pancreatic acinar cells were cultured in 96-well plates at the concentration of 1 104 cells/well and exposed to various treatments. At 24 h after taurocholate treatment, 5 g/L MTT reagent was added into each well before incubation for 4 h at 37 C. Then, the blue colored formazan product was dissolved in dimethyl sulfoxide (DMSO), and the spectrophotometric absorbance was subsequently measured at 570 nm using a microplate reader. All results were compared to the untreated control group and expressed as the percentage of the control values.
Lactate dehydrogenase release assay The cytotoxicity in pancreatic acinar cells was determined by measuring lactate dehydrogenase (LDH) release as a function of membrane integrity using a diagnostic kit according to the manufacturer’s instructions (Institute Jianchen Biological Engineering, Nanjing, China). Briefly, 50 mL of supernatants were collected and incubated with nicotinamide adenine dinucleotide diaphorase (NADH) and 0.1% sodium pyruvate for 15 min at 37 C. Absorbance of the sample was monitored
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Quantification of cytochrome c release
at 440 nm, and the activity of LDH was represented as the fold of control values.
Cytochrome c release into the cytoplasm was determined by Western blot analysis using specific antibodies after mitochondrial isolation and purification. Briefly, pancreatic acinar cells were lysed by the protease inhibitor mixed lysis buffer, and the cell lysate was centrifuged for 10 min at 600 g at 4 C. After discarding the pellets containing the nucleus, the supernatant was centrifuged for 10 min at 14,000 g at 4 C. The resulting pellet and supernatant were separated and used as mitochondrial fraction and cytosolic fraction, respectively.
TUNEL staining Apoptosis in pancreatic acinar cells subjected to taurocholate and/or TAK-242 treatment was detected by TUNEL staining, the method to observe DNA strand breaks in nucleus. Briefly, cells seeded on glass slides were fixed by 4% methanol-free formaldehyde solution in PBS for 20 min and labeled with the fluorescein TUNEL reagent mixture (Promega, Madison, WI) for 1 h at 37 C in the dark. Then, 2-(4-Amidinophenyl)-6indolecarbamidine dihydrochloride (DAPI) (Cell Signaling Technology, MA) was added to stain the nucleus before washing with phosphate buffered saline (PBS) three times. Fluorescence evaluation was performed under a confocal microscope (FV10i, Olympus, Tokyo, Japan), and the number of TUNEL-positive (green) cells was counted.
Measurement of mitochondria swelling After mitochondrial isolation and purification, effects of TAK242 on mitochondria swelling after taurocholate treatment were measured by a previously published method.18 The results were represented as the decrease in absorbance at 540 nm with 200-mM CaCl2.
Measurement of lipid peroxidation Determination of mitochondrial calcium buffering capacity To determine the levels of lipid peroxidation after taurocholate and/or TAK-242 treatment, the content of malonaldehyde (MDA) and 4-hydroxynonena (4-HNE) in pancreatic acinar cells were measured using assay kits according to the manufacturer’s instructions (Cell Biolabs, MA). All values were compared with the control group, and the results were expressed as the percentage of control values.
To determine mitochondrial Ca2þ metabolism in pancreatic acinar cells, mitochondrial Ca2þ buffering capacity was measured using the Ca2þ sensitive Calcium Green 5N fluorescent dye (Molecular Probes, OR). After taurocholate and/or TAK-242 treatment, pancreatic acinar cells were incubated with the prepared working liquid at 37 C, and bolus additions of CaCl2 were performed. The change in green fluorescence was monitored at 532 nm with a QuantaMaster spectrofluorometer (Invitrogen, Sacramento, CA), and the results were expressed as the percentage of control values.
Measurement of SOD, CAT, and GPX activity To investigate the effect of TAK-242 on endogenous antioxidative system, the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) was measured using assay kits according to the manufacturer’s instructions (Cayman Chemical, MI). All values were compared with the control group, and the results were expressed as the percentage of control values.
Real-time RT-PCR Total RNA was isolated from pancreatic acinar cells using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). After extraction, 500 ng of total RNA were used to synthesize the first strand of cDNA using a reverse transcription kit (Takara, Tokyo, Japan). The mRNA levels of TLR4, peroxisome proliferator activated receptor coactivator-1 (PGC1), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (TFAM) was determined by real-time RT-PCR using the primers as summarized in Table 1, and the results were normalized to the value of GAPDH. In addition, mtDNA was determined using primers used for the amplification of mitochondrial genomes: 50 -ATA TTT TCA CTG CTG AGT CCC GTG G-30 ; reverse, 50 -AAT TTC GGT TGG GGT GAC
Determination of ROS generation To determine the intracellular reactive oxygen species (ROS) generation after various treatments, pancreatic acinar cells were incubated with the indicator 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA, Cell Signaling Technology, MA) in the dark at room temperature.17 After washing with PBS for three times, the fluorescence was detected with a fluorescence plate reader (excitation/emission 480/530 nm).
Table 1 e Primers sequences used in real-time PCR. Gene
Forward sequences
Reverse sequences
TLR4
50 -AGAAAATGCCAGGATGATGC-30
50 -AGGGATTCAAGCTTCCTGGT-30
PGC-1
50 -GTGCAGCCAAGACTCTGTATGG-30
50 -GTCCAGGTCATTCACATCAAGTTC-30
NRF-1 TFAM GAPDH
0
5 -GAGTGACCCAAACCGAACA-3 0
5 -GGTGTATGAAGCGGATTT-3 0
0
50 -GGAGTTGAGTATGTCCGAGT-30
0
5 -ATGTATCCGTTGTGGATCTGAC-3
50 -CTTTCTTCTTTAGGCGTTT-30 0
50 -CCTGCTTCACCACCTTCTTG-30
pan et al resatorvid and pancreatitis in vitro
CTC GGA G-30 . The results were represented as the fold of control group.
Results
Western blot analysis
Blocking TLR4 via TAK-242 attenuates taurocholateinduced cytotoxicity
The pancreatic acinar cells were harvested using ice-cold lysis buffer containing protease inhibitors (Sigma, MO), and the protein concentrations were determined using the bicinchoninic acid (BCA) method (Institute Jianchen Biological Engineering, Nanjing, China). A total of 40-mg lysates was separated by 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes using standard techniques. The nonspecific binding of antibodies was blocked with 5% nonfat milk solution for 1 h, and the following primary antibodies purchase from Sigma (St. Louis, MO) were used: TLR4 (1:600), cytochrome c (1:500), Tubulin (1:1000), COX V (1:500), Opa-1 (1:800), Mfn-1 (1:600), Drp-1 (1:800), Fis-1 (1:800), and bactin (1:1000). The blots were performed using chemiluminescence, and the optical density of each band was quantified using Image J software (Scion Corporation, MO).
Statistical analysis The statistical analysis of results was performed using GraphPad Prism 6.0 (GraphPad, San Diego, CA). Statistical evaluation of the data between groups was performed by oneway analysis of variance followed by Bonferroni post hoc test or Student’s t-test when appropriate. A value of P < 0.05 was considered statistically significant.
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To investigate the effect of AP on TLR4 expression, pancreatic acinar cells were treated with 6-mM taurocholate, and the expression of TLR4 at mRNA and protein levels were detected by RT-PCR and Western blot, respectively. The results showed that TLR4 mRNA was significantly increased from 3 h to 12 h after injury and then decreased to normal level (Fig. 1A). At the same time, the protein level of TLR4 was elevated from 3 h and continued to 48 h and then decreased to normal level at 72 h after taurocholate treatment (Fig. 1B). To block the activation of TLR4, pancreatic acinar cells were treated with 1-mM TAK242, an exogenous TLR4 antagonist. We found that TAK-242 protects against taurocholate-induced cytotoxicity, as evidenced by increased cell viability (Fig. 1C) and decreased LDH release (Fig. 1D). In addition, we also detected apoptosis using TUNEL staining, the results of which showed that TAK-242 significantly decreased the number of TUNEL-positive cells as compared to the taurocholate-treated group.
TAK-242 reduces taurocholate-induced oxidative stress To determine whether TAK-242 protects pancreatic acinar cells via antioxidative activity, we investigated the effect of TAK-242 treatment on lipid peroxidation by measuring MDA
Fig. 1 e Blocking TLR4 via TAK-242 attenuates taurocholate-induced cytotoxicity. Pancreatic acinar cells were treated with 6-mM taurocholate (Tauro), and the expression of TLR4 at mRNA and protein levels were detected by RT-PCR (A) or Western blot (B). Pancreatic acinar cells were treated with 1-mM TAK-242 (TAK) in the presence or absence of 6-mM Tauro. Cell viability (C) and LDH release (D) were measured at 24 h after Tauro exposure. The apoptotic cell death was detected by TUNEL staining (E). Scale bar: 100 mm. Data are shown as mean ± standard error of mean (n [ 6). #P < 0.05 versus control. *P < 0.05 versus Tauro. (Color version of figure is available online.)
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Fig. 2 e TAK-242 reduces taurocholate-induced oxidative stress. Pancreatic acinar cells were treated with 1-mM TAK in the presence or absence of 6-mM Tauro. The expression levels of MDA (A), 4-HNE (B), and the activities of endogenous antioxidant enzymes (C) were measured. Data are shown as mean ± standard error of mean (n [ 6). #P < 0.05 versus control. *P < 0.05 versus Tauro. and 4-HNE levels. As shown in Figure 2A and B, taurocholate significantly increased the expression of MDA and 4-HNE, while there was no difference between control and TAK-242 groups. Blocking TLR4 activation via TAK-242 markedly attenuated taurocholate-induced increases in both MDA and 4-HNE levels. Furthermore, we also detected the enzymatic activity of SOD, CAT, and GPX after TAK-242 and/or taurocholate treatment to test the effects of TLR4 blocking on the endogenous antioxidant system (Fig. 2C). The results showed that TAK-242 partially prevented the taurocholate-induced decreases in SOD, CAT, and GPX activities, indicating the antioxidative activity of TAK-242.
TAK-242 inhibits taurocholate-induced mitochondrial dysfunction Intracellular ROS generation was assayed to investigate the effect of TAK-242 on mitochondrial function, and we found that taurocholate-induced increase in ROS generation was significantly inhibited by TAK-242 treatment (Fig. 3A). We also detected cytochrome c release by Western blot analysis (Fig. 3B). The results showed that TAK-242 inhibited
cytochrome c release after taurocholate exposure, as evidenced by decreased cytosolic cytochrome c (Fig. 3C) and increased mitochondrial cytochrome c content (Fig. 3D). After isolation of mitochondria from pancreatic acinar cells, we determined the effect of TAK-242 on mitochondria swelling, which was induced by 200-mM Ca2þ (Fig. 3E). The results showed that TAK-242 treatment significantly prevented mitochondria swelling induced by taurocholate exposure. As shown in Figure 3F and G, a similar result in mitochondrial Ca2þ buffering capacity was also observed.
TAK-242 differently regulates mitochondrial biogenesis and dynamics To determine whether TAK-242 protects pancreatic acinar cells via regulating mitochondrial biogenesis, long-fragment PCR was performed to quantify the amount of intact mtDNA (Fig. 4A). The results showed that taurocholate treatment significantly decreased mtDNA content, which was partly prevented by TAK-242. We also used real-time RT-PCR to detect the expression of three transcription factors considered essential for mitochondrial gene expression and increased
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Fig. 3 e TAK-242 inhibits taurocholate-induced mitochondrial dysfunction. Pancreatic acinar cells were treated with 1-mM TAK in the presence or absence of 6-mM Tauro. The intracellular ROS production was detected by 2,7-dichlorofluorescein diacetate (DCF) fluorescence staining (A). Mitochondria in each group were isolated and purified. The expression of cytochrome c was detected by Western blot analysis (B) in cytosolic fraction (C) and mitochondrial fraction (D). Mitochondrial swelling was examined by monitoring the absorbance at 540 nm induced by 200-mM Ca2D (E), and the baseline absorbance was measured without Ca2D. Relative Ca2D uptake capacity of isolated mitochondria were determined (F) and calculated (G). Data are shown as mean ± standard error of mean (n [ 6). #P < 0.05 versus control. *P < 0.05 versus Tauro.
expression of PGC-1, NRF-1, and TFAM mRNA was observed in Tauro þ TAK group compared with that in taurocholatetreated cells (Fig. 4B). In addition, the expression of Opa-1, Mfn-1, Drp-1, and Fis-1 at protein levels was detected by Western blot to investigate the effect of TAK-242 on mitochondrial dynamics (Fig. 4C). As shown in Figure 4D, TAK-242 increased the expression of opa-1 and Mfn-1 but decreased the expression of Drp-1 after taurocholate treatment, with no effect on Fis-1 expression.
Discussion Significant findings on TLR4-mediated signaling cascades in inflammatory diseases have been reported, such as nuclear factor-kB (NF-kB) and members of the interferon (IFN)-regulatory factor (IRF) family.19 Moreover, the upregulation of plasma TNF-a levels in AP patients was found to be positively correlated with the expression of TLR4.12 All these data suggest a critical role of TLR4 in AP. However, the direct link between TLR4 and pancreatic acinar cell injury are still elusive. In the present study, we first observed that the expression of TLR4 was increased after taurocholate treatment at both mRNA and protein levels. Then, we used TAK-242 to demonstrate that blocking of TLR4 exert protective effects against taurocholate-induced toxicity in mice pancreatic acinar cells. After that, we found that TAK-242-induced protection was associated with preserved mitochondrial function and the
regulation of mitochondrial biogenesis and dynamics. All these results indicate that TLR4 plays an important role in AP. TLR4 was originally identified to generate innate immune responses to pathogens by activating a cascade of proinflammatory events.20 Through recognizing its ligands, such as heat shock proteins, extracellular domain A, CD14 and MD-2, TLR4 recruits the adaptor molecules TIR (Toll/IL-1 receptor) domain-containing adaptor protein, myeloid differentiation factor 88 (MyD88), IL (interleukin)-1-receptor-associated kinase, and TNF-receptor-associated factor-6, and this crosstalk between these proteins enables downstream signaling via the IKK-NF-kB complex and the mitogen-activated protein kinase cascades.21e23 TLR4 is highly expressed in the rodent and human pancreas, which providing the molecular basis for its involvement in the initiation of inflammatory response during AP.8 Results of animals’ experiments showed that TLR4 mRNA is increased in the pancreas of rats in the early stage of pancreatitis,10 and anti-TLR4 antibody inhibited pancreatic elastase-induced TNF-a release in THP-1 cells.11 In addition, in AP patients, the upregulation of plasma TNF-a levels was found to be positively correlated with the expression of TLR4.12 However, the time expression profiles of TLR4 after AP in pancreatic acinar cells have not been reported. In the present study, we demonstrated, for the first time, that TLR4 mRNA was significantly increased from 3 h to 12 h after injury and then decreased to normal level. Meanwhile, the protein level of TLR4 was elevated from 3 h and continued to 48 h and then decreased to normal level at 72 h after taurocholate
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Fig. 4 e TAK-242 differently regulates mitochondrial biogenesis and dynamics. Pancreatic acinar cells were treated with 1mM TAK in the presence or absence of 6-mM Tauro. The relative mitochondrial DNA content was quantified by longfragment PCR (A), and the mRNA expression of mitochondrial biogenesis factors was measured by real-time RT-PCR (B). The expression of mitochondrial dynamic proteins, including Opa-1, Mfn-1, Drp-1, and Fis-1, were detected by Western blot (C) and calculated (D). Data are shown as mean ± standard error of mean (n [ 6). #P < 0.05 versus control. *P < 0.05 versus Tauro.
treatment. Our results strongly supports that the expression of TLR4 was significantly increased at both transcription and translation levels in our in vitro AP model. Ductal infusion or treatment with taurocholate is widely used to mimic AP because it reproduces pancreatic edema, hemorrhage, and necrosis in in vitro and in vivo experimental conditions.24 Previous studies showed that taurocholate treatment leads to the loss of adenosine triphosphate (ATP) synthesis and excessive generation of ROS, which together, could induce a marked inhibition of endogenous antioxidative system.25 Based on these findings, antioxidative agents have been suggested to be effective treatment for pancreatitis. In this study, taurocholate treatment significantly increased lipid peroxidation and decreased antioxidative enzyme activities, and all these effects were partially reversed by TAK-242, findings that support the involvement of antioxidative activity in TAK-242-induced actions in pancreatic acinar cells. However, previous studies demonstrated that the use of antioxidative compounds in AP is not sufficient to prevent cell death, and it can even increase cell death by necrosis.26,27 Using isolated mitochondria from pancreatic acinar cells, we further investigated whether TAK242-induced protection was associated with mitochondrial function, which plays an important role under oxidative stress conditions. Our results showed that TAK-242 treatment
significantly prevented mitochondria swelling and decreases in mitochondrial Ca2þ buffering capacity induced by taurocholate exposure, which was accompanied by reduced mitochondrial cytochrome c release and ROS generation. It was shown that the Ca2þ overload in mitochondria responsible for cell death is increased by mitochondrial dysfunction.28e30 Therefore, it appears that TAK-242, through maintaining mitochondrial membrane and mitochondrial Ca2þ buffering, preserves mitochondrial integrity and function. All these data strongly support the hypothesis that TLR4 could be a downstream signaling activated under stress conditions to regulate cell oxidative state in AP, which needs to be further determined. Stimulation of secretion increases the energy demand of pancreatic acinar cells, and enhanced mitochondrial function to synthesis more ATP is required to adjust the cellular metabolism.31 Previous studies characterized novel mechanisms of toxins that induce pancreatitis, among which the direct impairment of mitochondrial ATP generation is the key event.32,33 It is well known that the preservation of mitochondrial ATP production could be achieved by increasing mitochondrial biogenesis, which is defined as the growth and division of mitochondria.34 In the present study, an increase in mtDNA content in TAK-242 treated pancreatic acinar cells was found, indicating a stimulation of mitochondrial
pan et al resatorvid and pancreatitis in vitro
biogenesis. In consistent, we also observed the significantly increased expression of PGC-1, NRF-1, and TFAM, three transcriptional factors required for mtDNA replication and transcription. To adapt energy metabolism to physiological and pathologic conditions, mitochondria became a highly dynamic organelle, remodeling by fusion and fission events.35 The balance of mitochondrial fission and/or fusion is commonly regulated by high-molecular weight GTPases, such as Opa-1 and Mfn-1 as fusion proteins, as well as Drp-1 and Fis-1, two fission proteins.36 Our results showed decreased expression of Opa-1 and Mfn-1, and increased expression of Drp-1 after taurocholate treatment, indicating decreased mitochondrial fragmentation and mitochondrial mass.37 Treatment with TAK-242 significantly prevented the changes of these mitochondrial dynamic proteins expression, which was consistent with preserved mitochondrial biogenesis. All these data strongly suggested that TAK-242 exerted protective effects against taurocholate-induced toxicity through regulating mitochondrial biogenesis and dynamics. A limitation of our experiments is that we used an in vitro model to examine the protective effect of TAK-242 against AP. The samples for assays on mitochondrial function and the expression of mitochondrial dynamic factors were harvested from primary cultured mice pancreatic acinar cells. If the experiments were performed in isolated pancreatic tissues or in in vivo conditions, more detailed information on TAK-242induced anti-inflammatory activity and related mechanisms would be provided. In summary, blocking TLR4 activity using TAK-242 was effective in ameliorating cytotoxicity and apoptosis induced by taurocholate exposure in our cellular model of AP. These effects are likely to be associated with inhibited oxidative stress and preserved mitochondrial function.
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15.
Acknowledgment This work was financially supported by the project fund from Science and Technology Project of Shaanxi Province (no. 2010K15-03-02). The authors would like to thank Dr. Terry Chen for his technical support for the experiments and the preparation of the manuscript. Authors’ contribution: H.H.P. and L.F.P. conceived and designed the experiments. L.Y., L.M.W., J.T.H., J.L.S., and X.B.W. performed the experiments. Z.H.B. and L.J.S. analyzed the data.
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Disclosure The authors declare that there are no conflicts of interest.
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