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Cadmium chloride (CdCl2), tauroursodeoxycholic acid (TUDCA), and bacitracin were purchased from Sigma-Aldrich (USA). Antibodies for Nrf2, Bip, PERK, and β-actin were purchased from Proteintech Group (USA). Peroxidase-conjugated goat anti-mouse IgG and peroxidase-conjugated goat anti-rabbit IgG were purchased from Proteintech Group (USA). Prestained Protein Marker was purchased from Thermo Fisher Scientific (USA). Reverse transcription reagents were purchased from TaKaRa (Japan). SYBR green RT-PCR kit was purchased from Applied Biosystems (USA). RIPA lysis buffer was purchased from Beyotime (China). All other chemicals were of reagent grade.
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Healthy male and female SD rats, weighing 180-220 g, were obtained from Guangdong Provincial Medical Laboratory Animal Center, China. The laboratory animal production license number was SCXK(Yue)2013-0002, and the experimental animal license was SYXK(Yue)2014-0137. All animal treatment procedures were approved by the Ethical Committee of Guangzhou Quality Supervision and Testing Institute (serial number: 2016-03-08). The animals were kept under standard laboratory conditions and were supplied with standard pelleted diet and water. The SD rats were quarantined for 7 days before the experiment, and healthy rats were selected for the experiment.
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The experiment was designed to include the control group, CdCl2-alone groups, ERS activator bacitracin pretreatment groups, and ERS inhibitor TUDCA pretreatment groups. All reagents were injected into the animals by the intraperitoneal route at a dose of 10 mL/kg body weight for 48 h.
The rats were divided into 12 groups of six animals each. Treatments were done as follows:
Group 1: Control group. Rats were injected with 0.9% physiological saline.
Groups 2-4: CdCl2 groups. Rats were injected at doses of 5, 10, or 20 μmol/kg body weight (bw) CdCl2 for 48 h.
Group 5: Bacitracin group. Rats were injected at a dose of 50.0 mg/kg body weight bacitracin for 48 h.
Groups 6-8: Bacitracin+CdCl2 groups. Rats were pre-administered with bacitracin (50.0 mg/kg·bw) for 120 min before they were injected with CdCl2 (5, 10, or 20 μmol/kg·bw) for 48 h.
Group 9: TUDCA group. Rats were injected at a dose of 50.0 mg/kg·bw TUDCA for 48 h.
Groups 10-12: TUDCA+CdCl2 groups. Rats were pre-administered with TUDCA (50.0 mg/kg·bw) for 120 min before they were injected with CdCl2 (5, 10, or 20 μmol/kg·bw) for 48 h.
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Total RNA was extracted from the rat kidney tissues using TRIzol. The total RNA was reverse-transcribed into cDNA using TaKaRa reverse transcription reagents. The expression level of target genes was detected by RT-PCR using Applied Biosystems SYBR green RT-PCR kit on Applied Biosystems 7500HT Fast Real-Time PCR System. The PCR conditions were as follows: initial denaturation at 95 ℃ for 2 min, followed by 40 cycles at 95 ℃ for 15 s, and 60 ℃ for 1 min. The differences between samples were determined using the comparative Ct method. The levels of Bip, PERK, ATF4, Nrf2, GCLC, GST-P1, HO-1, and NQO1 mRNA were measured. The sequences of the primers used in this study are listed in Table 1.
cDNA Primer Sequences Bip F 5'-CATCACGCCGTCCTATGTCG-3' R 5'-CGTCAAAGACCGTGTTCTCG-3' PERK F 5'-GGAAACGAGAGCCGGATTTATT-3' R 5'-ACTATGTCCATTATGGCAGCTTC-3' ATF4 F 5'-ATGACCGAAATGAGCTTCCTG-3' R 5'-GCTGGAGAACCCATGAGGT-3' Nrf2 F 5'-TCAGCGACGGAAAGAGTATGA-3' R 5'-CCACTGGTTTCTGACTGGATGT-3' GST-P1 F 5'-GGCCCACCTAGCCATCAATG-3' R 5'-CGCTGAATGCAGTTGAAGATGT-3' GCLC F 5'-GGAGACCAGAGTATGGGAGTT-3' R 5'-CCGGCGTTTTCGCATGTTG-3' HO-1 F 5'-GAGCAGAACCAGCCTGAACTA-3' R 5'-GGTACAAGGAAGCCATCACCA-3' NQO1 F 5'-ACCCCACTCTATTTTGCTCC-3' R 5'-ACTTACTCCTTTTCCCATCCTC-3' GAPDH F 5'-CTGGGCTACACTGAGCACC-3' R 5'-AAGTGGTCGTTGAGGGCAATG-3' Table 1. Primer Sequences of Bip, PERK, ATF4, Nrf2, GST-P1, GCLC, HO-1, NQO1, and GAPDH
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The kidney tissues were lysed with RIPA lysis buffer containing 1 mmol/L PMSF. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a PVDF transfer membrane, and blocked with 5% skim milk in TBST buffer for 1 h. The membranes were incubated overnight with anti-Nrf2, anti-Bip, anti-PERK, and anti-β-actin antibodies at 4 ℃. Then, the membranes were washed three times in TBST and incubated with peroxidase-conjugated secondary antibody for 1 h at room temperature. The membranes were again washed three times in TBST, and the blots were developed using ECL chemiluminescence solutions. Band intensities were quantified by the Tanon 5200 Multi fluorescence image analysis system (Tanon, Shanghai, China).
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Data were expressed as mean ± standard deviation. Data analysis was conducted using one-way ANOVA, and comparisons between groups were performed using Student-Newman-Keuls (SNK) post hoc test. SPSS 17.0 was used for all statistical analyses. Values were considered to be statistically significant when P < 0.05.