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Objective: To investigate the effects of bisphenol A(BPA) on the proliferation and apoptosis of mouse testicular sertoli cells(TM4 cells) and the role of PERK-eIF2α-ATF4-CHOP pathway., Methods: TM4 cells were treated with different concentrations of BPA(0, 25, 50, 100 μmol/L) and 100 μmol/L BPA combined with protein kinase R-like ER kinase(PERK) inhibitor GSK2656157 for 24 h, and the apoptosis of TM4 cells was observed by TUNEL staining. The expression levels of Bax, Bcl-2, cleaved Caspase-3, GRP78 and PERK-eIF2α-ATF4-CHOP pathway-related proteins were detected by Western blot., Results: The apoptosis rate of TM4 cells in 25, 50 and 100 μmol/L BPA exposed groups was increased to 3.31%±0.34%, 7.51%±1.10% and 14.58%±0.91%, respectively, which was significantly higher than that in control group(0.73%±0.03%, P<0.05). Compared with the control group(1.00), cleaved Caspase-3 protein expression of TM4 cells in the 25, 50 and 100 μmol/L BPA exposed groups increased to 1.49±0.11, 1.59±0.12, 2.42±0.24, respectively; the ratio of Bax/Bcl-2 increased to 2.06±0.19, 3.94±0.034, 6.14±0.71, respectively; the protein expression of GRP78 increased to 1.29±0.06, 1.39±0.06, 1.92±0.17, respectively; the expression of p-PERK protein was increased to 1.64±0.03, 2.52±0.09, 2.80±0.11, respectively; the expression of p-eIF2α protein was increased to 1.79±0.05, 2.48±0.10, 4.77±0.32, respectively; ATF4 protein expression was increased to 2.51±0.03, 3.24±0.14 and 7.45±0.51, respectively; CHOP protein expression was increased to 1.44±0.01, 3.20±0.11 and 3.80±0.11, respectively, and all the differences were statistically significant(P<0.05). Compared to 100 μmol/L BPA group, the expression level of p-PERK, p-eIF2α, ATF4, CHOP, cleaved Caspase-3 protein and the ratio of Bax/Bcl-2 in 100 μmol/L BPA+10 μmol/L GSK2656157 group were decreased to 2.17±0.11, 1.81±0.13, 1.71±0.23, 2.18±0.22, 1.43±0.03, 2.22±0.13, respectively; the apoptosis rate of TM4 cells was also decreased to 7.28%±0.47%, all the differences were statistically significant(P<0.05)., Conclusion: BPA can induce apoptosis of TM4 cells by activating endoplasmic reticulum stress and regulating PERK-eIF2α-ATF4-CHOP pathway.

Objective: To investigate the effect of icariin (ICA) on the bisphenol A (BPA)-enhanced proliferation function of thyroid carcinoma cell B-CPAP and underlying mechanism. Methods: The proliferation of Gastric B-CPAP cell line was evaluated by cell counting kit-8 (CCK-8). Apoptosis and ROS expression in B-CPAP cells were detected by flow cytometry. The expression of superoxide dismutase (SOD) and malondialdehyde (MDA) in B-CPAP cells were measured by individual assay kits. The expressions of Bcl-2 and γ-HA2X were detected by Western blot. SPSS 18.0 software was used to analyze the data. Results: B-CPAP cell activity was promoted by treatment with 3×10(-7)mol/L BPA for 48 h, with significant difference in absorbance between BPA and control groups (1.089±0.053 vs 0.935±0.010, P <0.05). The cell activities of BPA+ ICA(25), BPA+ ICA(50), BPA+ ICA(100) and BPA+ ICA(200) groups was 0.780±0.036, 1.007±0.050, 0.958±0.033 and 0.625±0.064, respectively (all P <0.01). The proliferation of B-CPAP cells treated with BPA for 72 hours showed a similar trend to 48 hours. There was no significant difference between all treatment groups in 24 hours. The apoptosis rate was (19.272±0.186)% in BPA-treated cells, and was (22.412±0.238)% in control cells ( P <0.05). The apoptosis rates of BPA+ ICA(50) and BPA+ ICA(200) groups were (23.688±0.412)% and (30.270±0.696)%, respectively ( P <0.01). The intracellular accumulation of ROS in BPA, BPA+ ICA(50), and BPA+ ICA(200) groups were 806±21, 1 772±37, 2 041±16, respectively ( P <0.01). The expressions of anti-apoptotic protein Bcl-2 in control, BPA, BPA+ ICA(50), BPA+ ICA(200) groups were 7 120±151, 9 801±286, 5 902±171 and 4 203±216, respectively ( P <0.01). Conclusion: BPA can promote the proliferation of thyroid carcinoma B-CPAP cells and decrease the apoptosis of cells, and this effect can be inhibited by ICA. The possible mechanism is to induce high expression of intracellular ROS and inhibit the expression of antioxidase system, leading to cell oxidative damage, thereby inducing apoptosis.

Objective: To discuss the effect of Sirtuins（Sirt) protein family on the male reproductive system damage cell and animal models induced by bisphenol A (BPA), and to clarify its effect on the male reproductive system damage induced by BPA. Methods: Forty Kunming mice were randomly divided into control group, low dose of BPA group (given 3 mg·kg-1·d-1 BPA), medium dose of BPA group (given 30 mg·kg-1·d-1 BPA), and high dose of BPA group (given 300 mg · kg-1·d-1 BPA), and there were 10 mice in each group. The mice in low, medium, and high doses of BPA groups were gavaged with corn oil (0.01 mL·g-1) mixed with corresponding concentrations of BPA, while the mice in control group were given 0.01 mL·g-1 corn oil. After 5 weeks, the body weights, testis indexes, and epididymal indexes of the mice in various groups were detected； the sperm qualities of the mice in various groups were detected by using computer assisted semen analysis (CASA) system； the morphology of testis tissue of the mice in various groups was observed by HE staining； the expression levels of Sirt1-Sirt7 proteins in testis tissue of the mice in various groups were detected by Western blotting method. The GC-2 cells were divided into 0, 20, 40, and 80 μmol·L-1 BPA groups (treated with 0, 20, 40, and 80 μmol·L-1 BPA).The proliferation rates of the GC-2 cells in various groups were detected by EdU staining； the percentages of the GC-2 cells at different cell cycles and the apoptotic rates of GC-2 cells in various groups were detected by flow cytometry；the expression levels of Sirt1-Sirt7 proteins in the GC-2 cells in various groups were detected by Western blotting method. Results: Compared with control group, the testis index of the mice in high dose of BPA group was decreased (P<0.05); compared with control group, the percentage of immobile sperm of the mice in high dose of BPA group was increased (P<0.01), the percentage of rapid progressive sperm was decreased (P<0.01), the percentage of medium progressive sperm was decreased (P<0.05), and the deformity rate of the sperm was increased (P<0.01).The HE staining results showed that the number of spermatogenic cells at all levels in the seminiferous tubules of the mice in different doses of BPA groups showed a decreasing and loosely arranged trend. Compared with control group, the expression level of Sirt6 protein in testis tissue of the mice in low dose of BPA group was decreased (P<0.01), while the expression levels of Sirt1, Sirt2, and Sirt6 proteins in testis tissue of the mice in medium dose of BPA group mice were decreased (P<0.01), the expression levels of Sirt1, Sirt2, Sirt3, Sirt4, Sirt5, Sirt6, and Sirt7 proteins in testis tissue of the mice in high dose of BPA group were decreased (P<0.05 or P<0.01). The EdU staining results showed that compared with 0 μ mol·L-1 BPA group, the proliferation rates of the GC-2 cells in 20, 40, and 80 μmol·L-1 BPA groups were decreased (P<0.01). The flow cytometry results showed that after treated for 48 h, compared with 0 μmol·L-1 BPA group, the apoptotic rates of the GC-2 cells in 20, 40, and 80 μmol·L-1 BPA groups were increased (P<0.01). The Western blotting assay results showed that after treated for 24 h, compared with 0 μmol·L-1 BPA group, the expression levels of Sirt4 and Sirt7 proteins in GC-2 cells of the mice in 20 μmol·L-1 BPA group were decreased (P<0.05 or P<0.01), while the expression levels of Sirt4 and Sirt7 proteins in GC-2 cells of the mice in 80 μmol·L-1 BPA group decreased (P<0.05 or P<0.01).After treated for 48 h, compared with 0 μmol·L-1 BPA group, the expression levels of Sirt2, Sirt3, Sirt4, and Sirt6 proteins in GC-2 cells of the mice in 20 μmol·L-1 BPA group were decreased (P<0.05 or P<0.01), while the expression levels of Sirt1, Sirt2, Sirt3, Sirt4, and Sirt6 proteins in GC-2 cells of the mice in 40 μmol·L-1 BPA group were significantly decreased (P<0.05 or P<0.01), and the expression levels of Sirt 5 and Sirt 6 proteins were decreased (P<0.05 or P<0.01). Conclusion: The expression levels of Sirt1-Sirt7 proteins in the male reproductive injury cells and animal models induced by BPA are decreased, which exacerbates the male reproductive system damage induced by BPA. [ABSTRACT FROM AUTHOR]

Objective: To explore the protective effect and its mechanism of the protein and peptide extracts from the muscle of Chinese giant salamanders (SP) on the protection of bisphenol A (BPA)-induced sperm disorder in mice. Methods: Forty male C57BL/6 mice were divided into four groups, Control group, BPA group, BPA+CS group and BPA+SP group. BPA group was injected with 50 mg/kg/d of BPA, which dissolved in corn oil. Control group was injected with corn oil only. The BPA+CS group was given compound substances, which have been proved to be beneficial for spermatogenesis, and were used as controls. The BPA+SP group was given the above mentioned compounds plus SP dissolved in physiological saline. Mice received the compounds plus SP by gavage. The doses were 4 g/kg/d for both groups. The model was established continuously for 28 days, during which the body weight of mice was measured weekly. Testicular volume and testosterone-body ratio were measured after establishment of animal model. Epididymal sperms were collected to measure sperm number and sperm motility by using the computer aided analysis system (CASA). The amount of testosterone in the serum was determined by ELISA. HE staining and TUNEL staining were used to analyze testicular histopathology. Finally, immunofluorescence staining and Western blotting were used to analyze the expression of metastasis associated protein 2 (MTA2), and Oil Red O staining was used to observe the phagocytosis of Sertoli cells in vitro and in vivo. Results: Compared with the control group, the testicular volume (P<0.05), testicular weight (P<0.05), testosterone-body ratio (P<0.05), sperm number (P<0.05), sperm motility (P<0.05), and the amount of testosterone in the serum of mice (P<0.01) in BPA group were significantly decreased. Testicular morphology was damaged and the number of apoptotic spermatogenic cells in BPA group was increased compared with control group(P<0.01). At the same time, the expression of MTA2 and the phagocytosis of Sertoli cells in BPA group were decreased compared with control group (P<0.01). There was no difference between BPA + CS group and BPA group. Surprisingly, the above changes in BPA group were significant difference by using SP(P<0.05). Conclusion: SP has obvious protective effect on BPA-induced sperm disorder in mice, and the mechanism of action may be related to enhanced phagocytosis of Sertoli cells by improving the expression of MTA2. [ABSTRACT FROM AUTHOR]

Objective To explore the possible key pathways in the male reproductive toxicity induced by exposure to low-dose bisphenol A (BPA). Methods BPA-related genes were screened based on Comparative Toxicogenomics Database (CTD). Functional enrichment analysis was used for these genes to explore the possible pathways and identify the key genes in related pathways. Mouse spermatogonia cell line GC-2 cells were treated with BPA at a final concentration of 0 (DMSO control group), 20, 40 and 80 μmol/L for 72 h. Then the changes of cell morphology and apoptotic rate were observed. The mRNA and protein expression levels of the obtained key genes were detected by RT-qPCR and Western blotting, respectively. Results A total of 1 460 BPA-related genes were screened out from the CTD database. Gene Ontology (GO) enrichment analysis showed that these genes were significantly enriched in molecular biological processes, such as reproductive system development and reproductive structure development, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that they were significantly enriched in HIF-1 and PI3K-Akt signaling pathways (P < 0.01). Visual analysis showed that the key genes in the pathway were HIF-1α, PKC and P300/CBP, and PI3K, JAK, P53 and BCL-2. The apoptotic rate of GC-2 cells exposed to BPA was significantly increased in a dose-dependent manner (P < 0.05). Compared with the control cells, the mRNA expression levels of PI3K and JAK were significantly down-regulated, while those of P53 and BCL-2 were up-regulated (P < 0.05). The protein levels of PI3K and BCL-2 were obviously down-regulated, while that of P53 up-regulated (P < 0.05). Conclusion The PI3K-Akt signaling pathway may play an important role in the process of GC-2 cell apoptosis induced by low-dose BPA exposure.