| BackgroundAdvanced oxidation protein products (AOPPs), a new family of oxidized protein compounds, were first identified in the plasma of uremic patients undergoing maintenance dialysis.The AOPPs are a class of dityrosine-containing and cross-linking protein products formed primarily as a consequence of oxidative stress by the reaction of plasma proteins with chlorinated oxidants, such as chloramines or hypochlorous acid (HClO). It contains two distinct molecular weight 60KDa and 600KDa at a special absorption peak in 340nm.AOPPs could also be formed in vitro by incubating serum albumin with hypochlorous acid (HClO), the biological effects of AOPPs prepared by this method are similar with those extracted from uremic patients. It is well known that AOPPs have been considered as a novel marker of oxidant-mediated protein damage. Previous studies showed that AOPPs is the better marker for oxidative stress (OS), compared with others, such as advanced glycation end products (AGEs) and advanced lipoperoxidation end products (ALEs).It may be more appreciate to consider AOPPs as pro-inflammatory factors that play a crucial role in widespread biological events, and not just as markers of oxidative protein damage. AOPPs could cause respiratory burst by stimulating neutrophil and monocyte in vivo or in vitro, and induce the cells to release a large quantity of inflammatory cytokines, such as interleukin (IL) 1 and tumor necrosis factor (TNF)-α, thereby to induce systemic microvascular state. TNF-α not only could induce cells apoptosis that play roles in the progress of acute hepatic failure, but mediate hepatocyte demage by multiple pathways. Inflammation of endothelium induced by AOPPs, and thereby promotes the developments of diabetes mellitus (DM), chronic renal failure, and atherosclerosis. In addition, plasma AOPPs accumulates in inflammatory bowel disease (IBD) patients, and is related to the activity of Crohn’s disease (CD).There is emerging evidence to suggest that the risk of complicating with osteoporosis(OP) is increased in IBD patients. The involvement of oxidative stress-characterized by an oxidant/antioxidant imbalance-has become recognized as a ubiquitous occurrencein the development of IBD and in case of IBD complicated with OP. Previous studies have also illustrated enhanced generation of AOPPs are considered as novel markers of oxidative stress in IBD. Nevertheless, the role of AOPPs in the pathophysiological mechanisms underlying IBD in patients with OP complications is not well understood.Evidence suggests that the reduction of intestinal calcium absorption is an important cause of BMD loss in IBD patients, ultimately leading to OP. The distinct processes by which calcium ions (Ca2+) can be absorbed across the intestinal epithelium include paracellular and transcellular pathways.The calcium transcellular pathway consists of the following three steps:(1) apical calcium entry via transient receptor potential vanilloid (TRPV) 5 and 6 (TRPV5 and TRPV6) Ca2+ channels and the voltage-dependent L-type calcium channel, CaV1.3; (2) cytoplasmic diffusion in a calbindin-D9k (CaBP-D9k)-bound form; and (3) basolateral extrusion via plasma membrane Ca2+-ATPase 1 (PMCA1) and the Na+/Ca2+ exchanger 1 (NCX1). Moreover, Ca2+ balance is regulated by several well-characterized hormones, such as parathyroid hormone (PTH) and vitamin D3 metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3].Although previous studies have revealed that oxidative stress results in plasma accumulation of AOPPs in IBD, the effects of AOPPs in pathophysiological mechanisms underlying IBD in patients with OP complications remain unclear. It is unknown whether AOPPs affect the expressions of calcium transport channels (CTCs) in the small intestinal epithelium. Moreover, there is no information regarding the effects of AOPPs on Ca2+concentrations and its related hormones, such as parathyroid hormone (PTH),25-(OH)D3, and 1,25-(OH)2D3 in the serum. Thus, in the present study, (1) we demonstrated the effects of AOPPs on the expressions of CTCs (including CaV1.3, TRPV6, CaBP-D9k, PMCA1, and NCX) in vitro and in vivo. (2) We investigated the role of AOPPs on the Ca2+concentrations and its related hormones, such as parathyroid hormone (PTH),25-(OH)D3, and 1,25-(OH)2D3 in the serum. (3) We determined cellular pathway underlying the effects of AOPPs.Materials and Methods1. AOPP-albumin preparation and determinationAdvanced oxidation protein product-rat serum albumin (AOPP-RSA) was prepared in vitro by the incubation of RSA (Sigma, St. Louis, MO, USA) with HC10 (Fluke, Buchs, Switzerland), as described previously. In brief,100mg/mL of RSA was exposed to 200 mmol/L of HClO for 30 min at room temperature and dialyzed overnight against phosphate-buffered saline (PBS) to remove the free HClO. The AOPP preparation was passed through a Detoxi-Gel column (Pierce, Rockford, IL, USA) to remove any contaminated endotoxin. The endotoxin levels in the preparation were tested using a Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD, USA) (endotoxin levels were below 0.025EU/mL). The content of the AOPP in the preparations - as determined by the absorbance at 340nm of the reaction mixture of the samples and acetic acid-was 51.6 ± 3.4 nmol/mg protein in AOPP-RSA versus 0.30 ± 0.03 nmol/mg protein in the unmodified RSA.2. Cell cultureCaco-2 human colon adenocarcinoma cells were obtained from the Committee on Type Culture Collection (Chinese Academy of Sciences, Shanghai, China). Cells were cultured and maintained in high-glucose (4500 mg/L D-glucose) Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Grand Island, NY, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Life Technologies, Newcastle, Australia),1000 U/L penicillin,100 mg/L streptomycin, and L-glutamine (2 mmol/L) in a 5% CO2 and 95% air incubator at 37℃.3. Determination in expression of calcium transport channels in Caco-2 cellsCells were plated at 1×105 cells per well in six-well plates (CorningInc., Corning, NY, USA) for experiments. After growing to near confluence, cells were serum-deprived overnight before the addition of AOPPs to the culture medium, as indicated later. To assess dose- and time-dependent changes, cells were incubated either in control medium, RSA (100μg/mL), or AOPPs (100-600μg/mL) for 24h, or with AOPPs 400μg/mL for different periods of time (0,1,3,6,12,24 h).Protein extracts and total RNA of whole cellswere then harvested. The expression in protein levels and RNA levels of calcium transport channels (i.e. CaV1.3, TRPV6, CaBP-D9k, PMCA1, and NCX1) were assessed using Western blotting and Real time-PCR assays, respectively.4. Immunofluorescent stainingCells were plated at 5×104 cells per well in 24-well plates (Corning Inc.) for immunofluorescent staining. The growth medium was changed every other day. After growing to near confluence, cells were serum-deprived overnight before the addition of AOPPs to the culture medium, as indicated later. Cells were stimulated by the control medium, by RSA (100μg/mL), or by AOPP-RSA (400μg/mL) for 24h. Cells were fixed with paraformaldehyde, washed, and permeabilized with 0.1% Triton X-100 for 20 min. After blocking with 5% bovine serum albumin (BSA) in PBS for 1 h, the cells were incubated with primary antibodies (CaBP-D9k、PMCA1) overnight at 4℃.The cells were then incubated with the appropriate fluorescein-labeled immunoglobulin G (IgG) (Alexa Fluor(?) 555 goat anti-rabbit IgG (H+L) antibody; or Alexa Fluor(?) 488 goat anti-rabbit IgG (H+L) antibody) (Invitrogen, Carlsbad, CA, USA). The expressions of calcium transport channels were visualized and images were obtained with an OLYMPUS BX51 fluorescence microscope (Olympus, Tokyo, Japan).5.Phosphorylation of p44/42'JNKFor the detection of the MAPK activation, cells were plated in six-well plates (CorningInc., Corning, NY, USA) and treated with 400μg/mL AOPPs for 0,5,10, 15,20,30,60,120, and 180 min.Protein extracts of whole cells were harvested in RIPA lysis buffer (Beyotime Biotech, Nantong, China) with protease inhibitors (Beyotime Biotech, Nantong, China), according to the manufacturer’s instructions. The expression in protein levels of p-p44/42, p44/42, p-JNK, and JNK were measured using Western blotting assay.6. The effects of p44/42 MAPK and JNK MAPK inhibitors on expression of calcium transport channelsCells were plated in six-well plates (CorningInc., Corning, NY, USA). Inhibition experiments were performed by pre-treatment for 60 min with SP600125 (20μM, a JNK MAPK inhibitor) or U0126 (10μM, a p44/42 MAPK inhibitor). Cells were then stimulated by 400μg/mL AOPPs for 24 h. Protein extracts of whole cells were harvested and the expression in protein levels of calcium transport channels (i.e. TRPV6, CaBP-D9k, PMCA1, and NCX1) were determined using Western blotting assays.7. Animal experimentsExperiments involving male Sprague-Dawley rats (initial weight,180-200 g) were approved by the Animal Experiment Committee of the Southern Medical University (Guangzhou, China). Animals were housed and allowed to acclimate under standardized conditions with free access to water and rodent diet. The rats were randomly divided into three groups (n= 6 for each group). They received the agents by intraperitoneal injections every day, as follows:(1) vehicle (i.e., PBS) for group1; (2) unmodified RSA (50 mg/kg) for group 2; or (3) AOPPs-RSA (50 mg/kg) for group 3. The dosage of AOPPs and the injection interval were determined based on the findings of our preliminary studies.The rats were euthanized at the end of 8 weeks and anesthetized with sevoflurane and exsanguinated.The duodenumwere collected,flushed with ice-cold PBS, andstored for further analyses.8. Collection and biochemical analysis of the serumAfter euthanizing the rats, blood was collected from each rat, transferred to serum separator tubes. Serum biochemical parameters including calcium, sodium, and chloride levels were evaluated using an automatic biochemical analyzer in the Southern Medical University Clinical Laboratory of Nanfang Hospital, Guangzhou, China. The serum levels of PTH,25-(OH)D3, and 1,25-(OH)2D3 were determined using an enzyme-linked immunoabsorbent assay (ELISA) (CUSABIO BIOTECH CO., Ltd, Wuhan, China) method, according to the manufacturer’s instructions.9. Western blotting analysis in ratsAfter euthanizing the rats, protein extracts were collected from frozen rat intestinal tissue samples. The expression in protein levels of calcium transport channels (including CaV1.3, TRPV6, CaBP-D9k, PMCA1, and NCX1), p-p44/42, and p44/42 were assessed by Western blotting assay.10. Reverse transcriptase-polymerase chain reaction in ratsAfter euthanizing the rats, total RNAwere collected from frozen rat intestinal tissue samples. The expression in mRNA levels of calcium transport channels including TRPV6, CaBP-D9k, PMCA1, and NCX1 were evaluated by RT-PCR analysis.11. Immunohistochemical analysisFor immunohistochemical studies, duodenum tissues were fixed in neutral-buffered formalin. Formalin-fixed specimens were embedded in paraffin, and cut into 3-4μm-thick transverse sections. After antigen retrieval, blocking of endogenous peroxidase activity, and normal serum blocking, the sections were incubated with the primary antibody phospho-p44/42 MAPK (rabbit monoclonal, 1:800; Cell Signaling Technology, Danvers, MA, USA) overnight followed by biotinylated secondary antibodies (ZSGB-BIO, Beijing, China). Proteins were visualized as brown pigments via a standard diaminobenzidine protocol (ZSGB-BIO). The sections were lightly counterstained with hematoxylin.12. Statistical analysisAll experimental data are expressed as the mean ± standard deviation (SD), based on experiments performed in triplicate. Statistical differences in the variables between groups were evaluated by one-way ANOVA or by Welch ANOVA (equal variances not assumed). Multiple comparisons were performed using the Bonferroni method or Dunnett’s T3 method. The Dunnett’s procedure was used for comparisons between several treatments and the control. A two-tailed P value<0.05 was defined as statistically significant. Statistical analyses were conducted with SPSS 13.0 software (SPSS Inc, Chicago, IL, USA).Results1. AOPPs reduced the expression of calcium transport-associated channels in Caco-2 cellsWe evaluated the protein levels of CaV1.3 in Caco-2 cells exposed to AOPPs, by Western blotting. The protein and mRNA levels of TRPV6, CaBP-D9k, PMCA1, and NCX1 in Caco-2 cells exposed to AOPPswere analyzed by Western blotting and quantitative real-time PCR assay. There were no significant dose-or time-dependent differences in CaV1.3 protein levels in Caco-2 cells exposed to AOPPs in comparison to the control medium or unmodified RSA groups. However, dose-and time-dependent decreases were observed for the expression of other CTCs, at both protein and mRNA levels, in Caco-2 cells exposed to AOPPs in comparison to the control medium and unmodified RSA. In addition, we next identified and observed the fluorescent strength of the special calcium channels, CaBP-D9k and PMCA, under an immunofluorescence microscope, we found that the expression of CaBP-D9k and PMCA1 was induced down-regulated in the AOPP-challenged group. The expression of CTCs was unchanged in cells cultured with the control medium or unmodified RSA, which suggests that the decreased expression of CTCs was associated with advanced oxidation of RSA (i.e. AOPPs).2. AOPPs induced p44/p42 and JNK MAPK phosphorylation in vitroAOPPs stimulated a time-dependent phosphorylation of p44/42 and JNK MAPK in Caco-2 cells. The phosphorylation of p44/42 was significantly increased from 15 min to 60 min (P<0.05 or P<0.01 at each time point), whereas the phosphorylation of JNK was significantly increased from 10 min to 120 min after the AOPP challenge (P<0.05 or P<0.01 at each time point). These data indicated that AOPPs can induce p44/42 and JNK MAPK activation in Caco-2 cells.3. AOPPs induced the down-regulation of calcium transport channels through p44/42 MAPK phosphorylation in vitroTo further explore whether AOPP-induced down-regulated expression of CTCs is linked to p44/42 and JNK MAPK phosphorylation, Caco-2 cells were pre-treated with U0126 and SP600125, the specific inhibitors for p44/42 and JNK MAPK, respectively. Western blotting was used to detect TRPV6, CaBP-D9k, PMCA1, and NCX1 protein expression. Pre-treatment of Caco-2 cells with U0126 significantly attenuated AOPP-induced decreases in expression of CTCs (i.e. TRPV6, PMCA1, and NCX1) by 100%,145%, and 120%(P<0.01, compared with AOPP). Moreover, the expression of CaBP-D9k was ameliorated by more than 270% after U0126 pre-treatment (P<0.01, compared with AOPP). However, no changes in expression of these proteins were observed after pre-treatment of Caco-2 cells with the JNK MAPK inhibitor, SP600125 (p>0.05, compared with AOPP). These results suggested that activation of the p44/42 MAPK pathway, but not the JNK MAPK pathway, is required for the AOPP-induced down-regulation in expression of CTCs in Caco-2 cells.4. Biochemical characterization of serum from AOPP-challenged ratsThe levels of calcium, sodium, and chloride ions in the serum of each rat were measured at the end of 8 weeks. Neither calcium nor sodium and chloride ions were significantly altered in the vehicle, unmodified RSA, or AOPP-RSA groups. PTH, 25-(OH)D3, and 1,25-(OH)2D3 levels in the serum were also analyzed. The results showed that the levels of PTH,25-(OH)D3, and 1,25-(OH)2D3 were increased in serum of AOPP-challenged rats. However, the variationfor PTH among the three groups did not reach statistical significance (P>0.05).5. AOPPs decreased the expression of calcium transport-associated channels in the duodenum of ratsIn an attempt to examine if the effects of AOPPs on CTCs observed in vitro might also occur in vivo, we measured the expression of protein and genes associated with calcium transporters in the duodenum of rats by Western blotting and RT-PCR.It showed that both the protein and mRNA levels of TRPV6, CaBP-D9k, PMCA1 and NCX1 were decreased significantly in the duodenum after exposure of AOPPs.6. AOPPstriggered p44/42 MAPK activation in vivoImmunohistochemical staining of the duodenum demonstrated increased p44/42 phosphorylation in AOPPs-challenged rats compared with controls. Western blotting confirmed the increases in expression levels. These data show that AOPPs induce p44/42 MAPK activationin vivo as well asin vitro.Conclusions1. AOPPs reduced the expression of calcium transport channels (i.e. TRPV6, CaBP-D9k, PMCA1, and NCX1) in both dose-and time-dependent manner in Caco-2 cells.2. AOPPs decreased the expression of calcium transport channels in Caco-2 cellsvia the p44/42 MAPK signaling pathway.3. AOPPs did not alter the serum calcium ion concentrations in rats, while the levels of the related hormones that regulate calcium absorption including PTH, 25-(OH)D3, and 1,25-(OH)2D3 were increased after exposure of AOPPs. However, the variation for PTH among the three groups did not reach statistical significance.4. AOPP-RSA administration to normal rats induced down-regulation of the calcium transport channels (i.e. TRPV6, CaBP-D9k, PMCA1, and NCX1) in the duodenum. In addition, a significant increase in p44/42 phosphorylation was observed in the basal cell layer of the duodenum tissues following AOPPs stimulation. |
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