Tetraiodothyronine (T4) and triiodothyronine (T3) ELISA kits were purchased from Nanjing Jiancheng Bioengineering Institute. Expression levels of Bax, Bcl-2, ERK1, ERK2, MEK1, eIf2α, p-eIf2α, GRP78 and GRP94 genes were determined using transcription-polymerase chain reaction technique (Shanghai bioengineering Limited by Share Ltd). Thyroid tablets were purchased from Biochemical Pharmaceutical Factory (Laiyang city, shandong province, China). Analytical grade CdCl, fucoxanthin and other chemicals were purchased from Solarbio Science & Technology Co., Ltd (Beijing, China).
The specific pathogen-free (SPF) adult male Kunming mice (22-26g) were obtained from the institute of drug inspection of Qingdao. The animals were fed with a experimental mice diet (moisture ≤10%, crude protein ≥18%, crude fat ≥4%, crude fiber ≤5%, crude ash ≤8%) and pure water (Jiangshan wahaha hongzhen drinking water co., LTD., Quzhou City, zhejiang province, China). The animals were kept in laboratory animal houses, under a 12h/12h light-dark cycle conditions (lights on at 7: 00, off at 19: 00) with temperature 22°C-25°C and 50% relative humidity. The animals were adapted to laboratory animal houses conditions for 1 week prior to experiments.
All experimental procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Ethical Committee and the protocols were approved by the Committee on the Ethics of Animal Experiments of the Institute of Oceanology, Chinese Academy of Sciences, Shandong, China. All efforts were made to minimise suffering of the animals. The animals were anesthetised using ether before blood sampling. The laboratory animal quality certificate code is scxk20140001.
Animals (N = 120) were divided into control group (N=20) and CdCl-exposed group (N=100). The blank control group (BCG) were given pure water only, whereas animals of the CdCl-exposed group were given CdCl orally at a dose of 30 mg/kg body weight (bw)/day for 30 days (21). Fucoxanthin was previously administered orally at doses of 1-100 mg/kg bw/day and significant antioxidant effect and health benefits were demonstrated in various organisms (22-25). Herein, fucoxanthin was administered at doses of 10, 25 and 50 mg/kg bw/day. The CdCl-exposed group were divided into following 5 groups (N=20) to evaluate the intervention effect of fucoxanthin supplementation on cadmium-induced thyroid injury in mice: 1) negative control group (NCG, animals were supplied with pure water); 2) positive control group (PCG, animals were supplied with 50 mg/kg bw/day thyroid tablets); 3) low fucoxanthin concentration group (F1, animals were supplied with 10 mg/kg bw/day fucoxanthin); 4)medium fucoxanthin concentration group (F2, animals were supplied with 25 mg/kg bw/day fucoxanthin); 5) high fucoxanthin concentration groups (F3, animals were supplied with 50 mg/kg bw/day fucoxanthin). A 14-day treatment was conducted for these animals.
Animal Sample Collection
After 14-day treatment, these animals were sacrificed and thyroid tissue and blood samples were collected immediately. These samples were kept in 10% neutral-buffered formalin, 2.5% neutral-buffered glutaraldehyde and ultra low temperature refrigerator for further analysis.
Estimation of Malondialdehyde (MDA), Ascorbate Peroxidase (APX), Catalase (CAT), T4 and T3 Levels
Immediately before sacrifice, blood samples were collected from animals under ether anaesthesia. MDA, CAT and APX levels were measured spectrophotometrically. T4 and T3 levels were assayed using enzyme-linked immuno assays. T4, T3, MDA, APX and CAT levels were expressed in ng/ml, pg/ml, ng/ml, μmol/min/g and nmol/min/mL, respectively.
Total mRNA Extraction and Real-Time Quantitative PCR
Total RNA from thyroid tissues was prepared using Trizol reagent. Subsequently, mRNA was reverse-transcribed to cDNA using oligonucleotide dT primers according to the manufacturer’s instructions. RT-PCR analysis were then performed using Platinum Taq polymerase and 4S Red Plus nucleic acid stain. Primer sequences were designed using Primer Premier 5.0 software (Table 1).
Microstructures and Ultramicrostructures of Thyroid Tissues
After sacrifice, mice thyroid tissues were removed immediately and fixed in 10% neutral-buffered formalin or 2.5% neutral-buffered glutaraldehyde at 4℃. Tissues that were fixed in formalin were made into paraffin sections, stained with haematoxylin and eosin, and tissue histopathology were recorded and analyzed with a optical microscope(Olympus, BX63). Tissues that were fixed in glutaraldehyde were used to analyze the ultrastructure under an electron microscope (Hitachi, H-9500).
Statistical analysis were performed using SPSS 22.0 software. Data were expressed as means ± standard deviations (SD). Differences between two groups were tested by Student’s t-test, while multiple groups were examined by one-way analysis of variance (ANOVA)P < 0.05 was considered as a significant difference.