Role Of Cx43 In Lycopene Against DEHP-induced Testicular Toxicity Damage In Mice

Di(2-ethylhexyl)phthalate(DEHP)is a common endocrine disruptor commonly used as a plasticizer in the manufacture of various polyvinyl chloride(PVC)products,which are widely distributed in the environment.Exposure to DEHP is a serious public health problem,and its environmental accumulative effects can cause damage and dysfunction of male reproductive organs,interferes with spermatogenesis,and leads to adverse pregnancy outcomes.Lycopene(LYC)is an internationally recognized foodborne nutrient,known as “plant gold” and “the new favorite of health care product in the 21 st century”,and has become a potential therapeutic agent for male infertility due to its natural antioxidant properties.In order to explore the role and potential mechanism of LYC in DEHP-induced testicular injury in mice,the 21-day-old male ICR mice were used as subjects in the vivo experimental part of this experiment,and they were randomly divided into 7 groups,which were the blank control group(Con),vehicle control group(Vcon),LYC control group(5 mg/kg LYC),DEHP low-dose group(50 mg/kg DEHP),DEHP medium-dose group(200 mg/kg DEHP),DEHP high-dose group(500 mg/kg DEHP),LYC intervention group(5 mg/kg LYC+500 mg/kg DEHP).After 28 days by gavage,the clinical status,genital morphology,serum sex hormone levels,DEHP and its metabolite contents,sperm-related parameters,alterations of oxidative stress and antioxidant function,and related indicators of spermatogenesis,testosterone synthesis and blood-testis barrier(BTB)of mice were observed.To further investigate the role and potential mechanism of LYC in mouse testicular injury induced by DEHP and its main metabolite(mono-(2-ethylhexyl)phthalate)MEHP,mouse testicular spermatogonia line(GC-1),mouse Leydig cell line(TM3),mouse Sertoli cell line(TM4)and mouse primary Sertoli cells were used as research objects in the vitro part of this experiment.Firstly,the three cell lines of GC-1,TM3 and TM4 were divided into 6 groups,blank control group(Con),LYC control group(1 μM LYC),MEHP low-dose group(50 μM MEHP),MEHP medium-dose group(100 μM MEHP),MEHP high-dose group(200 μM MEHP),and LYC intervention group(1 μM LYC+200 μM MEHP).Subsequently,the three cell lines(GC-1,TM3 and TM4)were respectively detect the changes in related factors of spermatogenesis,testosterone synthesis and secretion function.The ultrastructure,cell viability,apoptosis levels,oxidative stress levels,antioxidant function,mitochondrial membrane potential,mitochondrial membrane permeability transport channels,changes in cell junctions and cell migration capacity were also observed in these three cell lines.Finally,plasmid Cx43 overexpression vector and Cx43 si RNA(si Cx43)were transfected in the culture system,TM4 cells and primary Sertoli cells were respectively divided into 8 groups,empty vector control group(NC+Con),Cx43 overexpression group(Cx43+Con),MEHP group(NC+200 μM MEHP),Cx43 overexpression and MEHP group(Cx43+200 μM MEHP);si RNA control group(si NC+Con),si Cx43 group(si Cx43+Con),LYC intervention group(si NC+1 μM LYC+200 μM MEHP),si Cx43 and LYC intervention group(si Cx43+1 μM LYC+200 μM MEHP),the changes of cell scratch and cell resistance were detected respectively.Research indicates:(1)LYC alleviated DEHP-induced reduction of body weight,food intake,anogenital distance and testicular organ coefficient in mice.It is indicated that LYC could antagonize DEHP-induced growing developmental disorders in mice.(2)LYC reduced the content of the main metabolite(MEHP)and secondary metabolites(MEHHP,MEHOP and 2cx MMHP)of DEHP in the mouse testis,and the content of MEHP in all metabolites in the DEHP-treated groups was much higher than that in other metabolites.It is indicated that LYC could reduce the accumulation of DEHP metabolites in mouse testicular tissue,thereby reducing the toxic effect of DEHP.(3)LYC alleviated DEHP-induced structural damage of spermatogenic cells in mouse testicular tissue,damages of sperm DNA and protein,decreased motility,increased deformity rate,decreased motility and ability to penetrate cumulus granulosa cells,and decreased spermatogenesis-related proteins(DDX25,CRM1,HMGB2 and PGK2),acrosome formation-related protein(DPY19L2)and flagellar structure-related protein(AKAP4).It is indicated that LYC could antagonize DEHP-induced spermatogenic dysfunction in mouse testis.(4)LYC inhibited DEHP-induced damage to the Leydig cell structure in mouse testicular tissue,disorders of steroid hormone biosynthesis and metabolic,and changes of serum sex hormone levels,alterations of testosterone synthases(STAR,P45017α,P450 scc,3β-HSD and 17β-HSD)and orphan nuclear receptors(SF1,TR4,Nur77,DAX1 and SHP)proteins levels.It is indicated that LYC could antagonize DEHP-induced decrease of testosterone synthesis ability in mouse testis.(5)LYC alleviated DEHP-induced damage to the Sertoli cell structure in mouse testicular tissue,disorder of secretion function-related proteins(AR,ABP,FSHR and Tf)levels,the structural and functional impairment of BTB,and alterations of ectoplasmic specialization-related proteins(Integrinβ1,N-cadherin,E-cadherin,α-catenin,β-catenin and γ-catenin),tight junction-related proteins(Claudin5,Claudin 11,ZO-1 and Occludin),gap junction-related proteins(Cx43 and P-Cx43)levels.It is indicated that LYC could antagonize DEHP-induced destruction of the BTB in mouse testis.(6)LYC attenuated DEHP-induced increase of oxidative stress products,decrease of antioxidant enzyme activity and disorder of glutathione metabolism in mouse testis.It is indicated that LYC could antagonize DEHP-induced oxidative stress in mouse testicular tissue by activating antioxidant enzymes and glutathione system,which is also a guarantee for the prevention for mouse testicular spermatogenesis disorder.(7)LYC inhibited MEHP-induced decreased cell viability,ultrastructural damage,increased levels of apoptosis,increased ROS levels in cell and mitochondria,lipid peroxide production,decreased antioxidant enzyme activity,decreased mitochondrial membrane potential,mitochondrial permeability transition pore damage,and decrease of spermatogenesis-related proteins(TDRD1,DDX25,CRM1,HMGB2,and PGK2),acrosome-related protein(DPY19L2)and flagellar structure-related proteins(AKAP4 and CFAP44)levels,the disorder and decrease of cytoskeleton morphological structure,decreased cell migration capacity,changes of cell junctions-related proteins levels in GC-1 cells.It is indicated that LYC could alleviate MEHP-induced the structure and function damage of GC-1 cells,further suggesting that LYC could antagonize DEHP-induced spermatogenic dysfunction in mouse testis.(8)LYC inhibited MEHP-induced increase in apoptosis,decreased cell viability,ultrastructural damage,increased intracellular ROS levels,lipid peroxide production,decreased antioxidant function,decreased mitochondrial membrane potential,mitochondrial permeability transition pore damage,changed testosterone synthases(STAR,P45017α,P450 scc,3β-HSD and 17β-HSD)and orphan nuclear receptors(SF1,TR4,Nur77,DAX1,and SHP)proteins levels,the disorder and decrease of cytoskeleton morphological structure,decreased cell migration capacity,decreased cell junctions-related proteins levels in TM3 cells.It is indicated that LYC could protect MEHP-induced structure and function of damage in TM3 cells,further suggesting that LYC could antagonize DEHP-induced testosterone synthesis decline in mouse testis.(9)LYC alleviated MEHP-induced ultrastructural damage,apoptosis level in increase,increased intracellular ROS and lipid peroxides,decreased antioxidant enzyme activity,decreased mitochondrial membrane potential,impaired mitochondrial permeability transition pore,decreased secretion function-related proteins(AR,ABP,FSHR,and Tf),the disorder and decrease of cytoskeleton morphological structure,decreased cell migration capacity,disorders of cell junction-related pathways,disorder of ectoplasmic specialization-related proteins(N-cadherin,E-cadherin,α-catenin,β-catenin andγ-catenin),tight junctions-related proteins(Claudin 5,ZO-1 and Occludin)and gap junctions-related proteins(Cx43 and P-Cx43)levels in TM4 cells.It is indicated that LYC could inhibit MEHP-induced the damage of structure and function in TM4 cells,further indicating that LYC could antagonize DEHP-induced damage of the BTB in mouse testis.(10)Overexpression of Cx43 alleviated MEHP-induced the decline of cell migration ability in TM4 cells and the impairment of BTB function in primary Sertoli cells.Knockdown of Cx43 could inhibit the protective effect of LYC on MEHP-induced the decline of cell migration ability in TM4 cells and the impairment of BTB function in primary Sertoli cells.It indicated that LYC could antagonize DEHP-induced destruction of the BTB in mice by regulating Cx43.Conclusion: DEHP induces spermatogenesis dysfunction,decreased testosterone synthesis and BTB damage in testicular tissue.LYC reduces the accumulation of DEHP metabolites in testicular tissue,antagonizes the testicular toxicity caused by DEHP,and alleviates DEHP-induced BTB destruction by regulating Cx43.This experiment provides new evidence for the molecular mechanism of LYC antagonizing DEHP-induced male reproductive toxicity.This study proved that LYC could effectively antagonize the toxic effects of environmental persistent organic pollutants on male reproduction,providing a new target for the prevention and treatment of male reproductive toxicity in medical and veterinary clinic,and providing a new idea for the effective guarantee of livestock production and reproduction.