This study aims to investigate whether the dietary supplement coenzyme Q10(CoQ10)alleviates bisphenol A(BPA)-induced mouse Leydig cell line(TM3)damage through autophagy pathway.Cell activity was measured by CCK-8 assay when treated with different concentrations of BPA for 24 h.TM3 cells were then divided into 5 groups:CON group,BPA group,Torin2 group,CQ group and BPA+CoQ10 group,with three repeats in each group.The morphology of TM3 cells were observed under inverted light microscope.Western blot was used to determine the protein ex-pression of p62 and LC3-Ⅰ/Ⅱ.The autophagy level of TM3 cells was detected by MDC cell auto-phagy staining,the mRNA expression levels of Atg7,Beclin 1,p62 and Atg5 genes were deter-mined by RT-qPCR,and the apoptosis rate of TM3 cells was detected by flow cytometry.The results showed that compared with 0 μmol/L BPA treatment group,the viability of TM3 cells de-creased significantly after 24 h treatment with 60 μmol/L BPA(P＜0.01).Compared with CON group,the number of TM3 cells markedly reduced in the BPA-treated group,the expression of au-tophagy-related proteins(p62,LC3-Ⅱ)significantly increased(P＜0.01),comparable to the CQ group.The MDC fluorescence intensity dramatically enhanced(P＜0.01),the mRNA expression levels of autophagy-related genes(Atg7,Beclin1,p62,Atg5)significantly elevated(P＜0.01),and the apoptosis rate significantly increased(P＜0.01).Compared with BPA group,the expression levels of autophagy-related genes Atg7 and Beclin1 mRNA(P＜0.05),p 62 and Atg5 mRNA(P＜0.01)in TM3 cells treated with BPA+CoQ10 significantly decreased.Moreover,the expres-sion levels of autophagy-related protein p62(P＜0.01)and LC3-Ⅱ(P＜0.05),MDC fluorescence intensity(P＜0.05)and apoptosis rate(P＜0.01)also markedly reduced.In conclusion,CoQ10 could subsequently reduce the apoptosis of TM3 cells by improving the abnormal autophagy flux induced by BPA.

This study aims to investigate whether the dietary supplement coenzyme Q10(CoQ10)alleviates bisphenol A(BPA)-induced mouse Leydig cell line(TM3)damage through autophagy pathway.Cell activity was measured by CCK-8 assay when treated with different concentrations of BPA for 24 h.TM3 cells were then divided into 5 groups:CON group,BPA group,Torin2 group,CQ group and BPA+CoQ10 group,with three repeats in each group.The morphology of TM3 cells were observed under inverted light microscope.Western blot was used to determine the protein ex-pression of p62 and LC3-Ⅰ/Ⅱ.The autophagy level of TM3 cells was detected by MDC cell auto-phagy staining,the mRNA expression levels of Atg7,Beclin 1,p62 and Atg5 genes were deter-mined by RT-qPCR,and the apoptosis rate of TM3 cells was detected by flow cytometry.The results showed that compared with 0 μmol/L BPA treatment group,the viability of TM3 cells de-creased significantly after 24 h treatment with 60 μmol/L BPA(P＜0.01).Compared with CON group,the number of TM3 cells markedly reduced in the BPA-treated group,the expression of au-tophagy-related proteins(p62,LC3-Ⅱ)significantly increased(P＜0.01),comparable to the CQ group.The MDC fluorescence intensity dramatically enhanced(P＜0.01),the mRNA expression levels of autophagy-related genes(Atg7,Beclin1,p62,Atg5)significantly elevated(P＜0.01),and the apoptosis rate significantly increased(P＜0.01).Compared with BPA group,the expression levels of autophagy-related genes Atg7 and Beclin1 mRNA(P＜0.05),p 62 and Atg5 mRNA(P＜0.01)in TM3 cells treated with BPA+CoQ10 significantly decreased.Moreover,the expres-sion levels of autophagy-related protein p62(P＜0.01)and LC3-Ⅱ(P＜0.05),MDC fluorescence intensity(P＜0.05)and apoptosis rate(P＜0.01)also markedly reduced.In conclusion,CoQ10 could subsequently reduce the apoptosis of TM3 cells by improving the abnormal autophagy flux induced by BPA.

Objective To elucidate the underlying mechanisms of sperm-associated antigen 6(SPAG6)in the proliferation and drug resistance of B-cell acute lymphoblastic leukemia(B-ALL).Methods A total of 56 B-ALL patients and 15 iron-deficiency anemia(IDA)patients admitted in the First Affiliated Hospital of Chongqing Medical University from January 2019 to December 2023 were recruited and served as the experimental and control groups,respectively.Bone marrow mononuclear cells(BMMNCs)were derived from the bone marrow tissues of the experimental group.According to the results of qRT-PCR for the expression of SPAG6 in the obtained BMMNCs,the B-ALL patients were stratified into newly-diagnosed(New-diag)group,complete remission(CR)group,minimal residual disease Possitive(MRD+)group,and relapse group.Lentiviral vectors were used to construct B-ALL cells with SPAG6 overexpression and knockdown.CCK-8 assay and methylcellulose-based colony formation experiment were employed to assess the survival,proliferation,and clonogenic potential of the cells with varying SPAG6 expression levels.After f B-ALL cells were treated with the chemotherapeutic drugs,daunorubicin(DNR)and methotrexate(MTX),CCK-8 assay,flow cytometry and Western blot analysis were applied to detect cell viability(drug sensitivity andIC50),cell apoptosis,and protein levels of apoptosis-related molecules Bax,Caspase-3 and Bcl-2,respectively.The mRNA-seq profiles of B-ALL patients were obtained from the TARGET database,and gene set enrichment analysis(GSEA)was conducted to explore SPAG6-associated signaling pathways,which were subsequently validated experimentally.After treatment with the NF-κB agonist Phorbol 12-myristate 13-acetate(PMA)and the antagonist BAY-11-7082,cell viability and sensitivity to chemotherapeutic drugs were assessed with CCK-8 assay,and the expression of pathway-related proteins NF-κB P65,p-NF-κB p65,IK Bα,p-IKBα,TNF-α,and EPO was detected by Western blot analysis.A mouse xenograft tumor model was constructed in SPAG6-/+knockdown mice to observe the effect of DNR on tumor growth and the expression of the NF-κB signaling pathway in the tumor tissues with immunohistochemical assay.Results The mRNA level of SPA G6 was significantly higher in the B-ALL patients(P＜0.05),and in the MRD group(P=0.001)and R group(P=0.003)than the CR group.SPAG6 overexpression and knockdown in B-ALL cells confirmed that SPAG6 promoted the proliferation(P＜0.05)and decreased the sensitivity to DNR and MTX(P＜0.05).SPAG6 resisted chemotherapy-induced apoptosis in B-ALL cells by down-regulating pro-apoptotic proteins Bax and Caspase-3(P＜0.05),and up-regulating the expression of anti-apoptotic protein Bcl-2(P＜0.05).GSEA analysis suggested that the NF-κB pathway was enriched in B-ALL cells,and our experiments confirmed that SPAG6 enhanced the chemoresistance of B-ALL cells to chemotherapy by activating the NF-κB/TNF-α pathway.In in vivo experiments,knocking SPAG6 down significantly reduced the volume of transplanted tumors(P＜0.05),and an additional decrease in tumor growth was observed in the DNR-combined treatment group(P＜0.05),with reduced levels of NF-κB P65 and p-IKBα by immunohistochemical assay.Conclusion SPAG6 promotes the proliferation of B-ALL cells and reduces their chemosensitivity via the NF-κB/TNF-α pathway,suggesting a close association of SPAG6 with the therapeutic outcomes and prognosis in B-ALL patients.