GT1-7 cell line was provided by Pediatrics Laboratory of Ruijin Hospital in Shanghai. All interventions were conducted as the cells were at the logarithmic growth phase.
First, a series of concentration of BPA solutions (500ng/l, 50ug/l, 500ug/l, 5mg/l and 10mg/l) were prepared by dissolving different dose of BPA in an equal volume of phosphate buffered saline (PBS). Then, BPA-containing PBS diluted in cell medium, depending on the group designation, was used for an additional culture period of 24 hours.
The NYPF herbs are an original prescription from TCM Department, Children's Hospital of Fudan University. In this study, particle prescription of NYPF herbs was used. It is composed of nine herbal concentrate-granules, namely, Rehmannia glutinosa libosch root (Sheng-Di-Huang 5g), Scrophularia buergeriana (Xuan-Shen 3g), Anemarrhena asphodeloides (Zhi-Mu 3g), Cortex phellodendri (Huang-Bai 3g), Alisma plantago-aquatica L. var. orientale Sam (Ze-Xie 3g), Carapax et Plastrum Testudinis (Zhi-Gui-Ban 2g), Hordeum vulgare L (Mai-Ya 6g), Asparagus cochinchinensis (Tian-Dong 3g) and Roast Radix Glycyrrhizae (Zhi-Gan-Cao 2g), provided by PuraPharm Corporation (Nanning, Guangxi, China). The particle mixture was dissolved in distilled water at a final concentration of 0.4g/ml. The dose for animal administration was 1.0g/100g.d, which was the equivalent dosage for the treatment of precocious puberty in clinic17.
Pregnant SD rats were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd (Certification number: SCXK (Zhe) 2019-0001) and raised in the Laboratory Animal Center at Fudan University Shanghai Medical College, with controlled photoperiod (12/12 h light/dark cycle) and temperature (23–25 °C), meeting Specified Pathogen Free standards. All the animals had ad libitum access to tap water and pelleted chow. All the animals and experimental procedures were approved by the research ethics board in accordance with established ethical guidelines.
The preparation methods of Pharmaceutic Serum were based on previous research 18 with a little modification. The brief protocol was as follows. Female rats at postnatal 21 (P21) were randomly divided into TCM group or normal saline (NS) group (N=10 each). They were continuously given TCM for NYPF or equal volume of NS as designated by gavage twice a day, respectively, from P21 to P25. On the morning of P25, one hour after the last administration, rats were sacrificed and blood samples were drawn from abdominal aorta. The serum was then separated by centrifugation and inactivated through heating at 56℃ for 30 min followed by aseptic filtration. Finally, the samples were stored at -80℃ for later use.
GT1-7 cells were intervened first by BPA, followed by serum containing TCM or NS at the final concentration of 10% (vol/vol). Then the cells were proceeded to culture for an additional 24 hours before harvesting.
Real-Time PCR (RT-PCR) Analysis
The mRNA levels of Kiss1 and GnRH1 were detected by RT-PCR. Total RNA was extracted using RNAiso Plus reagent (9109, TAKARA, Japan) and quantified by ultraviolet spectrophotometry (Nanodrop2000, Thermo-Scientific). Then 1ug of RNA per sample was transcribed into cDNA using reverse transcriptase kit (RR036A, Takara, Japan). Each tube for RT-PCR contained 2ul of cDNA, 1.6ul of primers (0.8ul each), and TB Green® Premix Ex Taq™ II reagent (RR820B, TAKARA, Japan) in a final volume of 20ul. And PCR amplified conditions were as follows: a cycle of 30s at 95°C for pre-denaturation and followed by 40 cycles for denaturation at 95°C for 15s and annealing at 60°C for 60s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference, using the 2−ΔΔCt method to calculate the relative mRNA levels. All the primers used in the assay (shown in Table 1) were designed by Primer3 software and synthesized by Shanghai Sangon Biotech Inc. (Shanghai, China).
Western Blot Analysis
Kiss1 and GnRH1 proteins were quantitatively analyzed by WB. Total protein was obtained using RIPA lysis buffer (Medium 20115ES60, YEASEN, Shanghai, China). BCA protein detection kit (20201ES76, YEASEN, Shanghai, China) was used to test protein concentration. Afterward, 40–60ug of total protein were separated by 15% SDS-PAGE, and it then was transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were locked with 5% (wt./vol) skimmed milk for 2 hours and probed with primary antibody overnight at 4 ℃. Next, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:4,000; CST, New York) for 2 hours. Subsequently, the greyscale images were obtained and quantified using the Image J software (NIH). The primary antibodies used in the assay contained rabbit polyclonal anti-Kisspeptin antibody (ab19028; 1:200 dilution; Abcam, Los Angeles, CA), anti-GnRH1 antibody (abs138073, 1:1,000 dilution, Absin, Shanghai, China), mouse monoclonal anti-GAPDH (T0004, Affinity, Jiangsu, China) or rat polyclonal anti-Tubulin antibody (30302ES20, YEASEN, Shanghai, China).
Bisulfite DNA Sequencing PCR (BSP)
The DNA methylation levels at Kiss1 promoter were tested by BSP. Genomic DNA was extracted using a genomic DNA extraction kit (DP304, Tiangen, Beijing, China). First, the genomic DNA was modified using Methylamp DNA Modification Kit (P-1001-1, EPIGENTEK, Farmingdale, USA). Afterward, a total of 20ul mixture, including 2ul of sulfite-modified DNA, 2ul of primers (1ul each), 2ul of 10X buffer, 2.7ul of Mg+, 9.8ul of ddH2O, 1.7ul of dNTP and 0.1ul of enzyme, was prepared for PCR reaction. PCR fragments were confirmed by agarose gel electrophoresis and they were then cloned to pGEM-T Vector (A1360, PROMEGA, USA). Subsequently their identity was veriﬁed by Sanger sequencing by Shanghai BioSune Co., LTD (Shanghai, China). The sequence was blasted online using PubMed comparison software (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Finally, methylation rate was determined using the formula: methylated CpG/ (methylated + unmethylated CpG) *100%. BSP primers were designed using MethPrimer software (http://www.urogene.org/cgi-bin/methprimer2/MethPrimer.cgi) and shown in Table1.
Chromatin Immunoprecipitation Assay
To assess the content of MLL1 and H3K4me3 at the promoter region of Kiss1, we performed Chip assays using chromatin extracted from each group according to the instructions of Magna ChIP® A/G Chromatin Immunoprecipitation Kit (17-10085, Millipore, USA). The brief protocols were as follows: cell samples counting 2*106 each group were washed once in ice-cold phosphate-buffered saline (PBS) containing a protease inhibitor cocktail II. Thereafter, cells were crosslinked by exposing to 1% formaldehyde for 10 min at room temperature. After two additional washing steps the samples were lysed with cell and nuclear lysis buffer, and sonicated for 30 s by 10 times to yield chromatin fragments of 200~500 base pairs (bp) using the Ultrasonic Processor (Bioruptor Pico, Diagenode, Belgium) at 4℃. Size fragmentation was confirmed by agarose gel electrophoresis. The sonicated chromatin was clarified by centrifugation at 12,000g for 10 min at 4℃, then take 50ul of supernatant into a clean EP tube, brought up to 500ul in chip dilution buffer for each reaction. Input sample with 50ul was removed from each tube of chromatin in advance and the remains were incubated with 5ug of MLL1(05-765, Millpore, USA) or H3K4me3(9727, CST, USA) antibodies and with 25μl of protein A or G beads solution (Dynabeads) for 4 hours with rotation at 4°C. Four hours later the beads were washed first with 0.5 ml low-salt wash buffer, followed by high-salt wash buffer, LiCl buffer and finally with TE buffer. Thereafter, the complexes were eluted with 100μl of chip elution buffer containing proteinase K at 62°C for 2 hours with rocking. To reverse the crosslinking reaction the samples were incubated at 95°C for 10 min. Then DNA for qPCR analysis was recovered using ChIP DNA Clean & Concentrator columns. All the reagents mentioned above were included in the Magna ChIP Kit.
Real-time PCR Detection of Chromatin Immunoprecipitated DNA
The 5’-flanking regions of Kiss1 gene between nt -2000 and nt +1, using transcriptional start site (TSS) as reference point, were supposed as promoter region. The putative promoter fragments were predicted online using the website (https://www.fruitfly.org/cgi-bin/seq_tools/promoter.pl) and qualified by RT-PCR using Chromatin Immunoprecipitated (IP) samples. Each reaction system was comprised of 2uL of IP or input sample, 2ul of primers (1ul each) and TB Green® Premix Ex Taq™ II reagent (Tli RNaseH Plus) (RR820B, Takara, Japan) in a final volume 20 ul. Data are expressed as % of IP signal/input signal.
All the data were presented as mean ± standard error (SEM) and statistical analyses were performed using SPSS 17.0 (IBM Corporation, Armonk, NY, USA). One-way ANOVA was used to make comparisons among multiple groups. The data were first subjected to a normality and an equal variance test. If the variance was homogeneous, the LSD tests were used. If not, the Dunnett T3 tests were conducted. In addition, comparison of proportion analysis was evaluated by Chi-square test. Values were considered to be significantly different as P < 0.05.
Table1.Primers used in the study