The Role Of COX2/PGE2/PTGER3 Pathway In Regulating The Transcription Of Pro-Inflammatory Mediators In Manganese-Induced Abnormal Spermatogenesis In Mice

Objective:Manganese（Mn）is a necessary trace element of the human body.However,with the long-term occupational and non-occupational manganese exposure,manganese accumulate s in the human body,which causes male testosterone,sperm concentration,sperm count,sperm motility de cline.In addition,excessive manganese exposure can also cause male sexual dysfunction,such as impotence and loss of sexual desire.Although the male reproductive toxicity caused by manganese has been confirmed,its toxicity mechanism has not been fully clarified.Prostaglandin E2（PGE2）synthesized by Cyclooxygenase-2（COX2）plays an important role in the physiological function of normal reproduction,promoting spermatogenesis and androgen secretion.But in pathological conditions,PGE2 is a pro-inflammatory mediator that plays a role in inflammation of the male reproductive system.Therefore,this study explores the role of PGE2 in spermatogenesis in mice after manganese exposure.It also provides a new target for the prevention and treatment of reproductive toxicity caused by manganese exposure and male infertility.Methods:1.In the first phase of animal experiments,24 C57BL/6 mice weighing 20±2 g,all male,were used to construct a mouse manganese exposure model.The 24 mice were randomly divided into control group,low manganese group,medium manganese group and high manganese group,and were given normal saline,12.5 mg/Kg Mn Cl₂,25 mg/Kg Mn Cl₂,and 50 mg/Kg Mn Cl₂,respectively.Manganese is injected intraperitoneally once a day for 5 weeks.2.In the second stage of animal experiments,an intervention model of COX2 inhibitor after manganese exposure in mice was constructed.The 24 mice were randomly divided into control group,high manganese group,COX2 inhibitor-celecoxib（CX）control group,CX+high manganese group.The dose of CX was 22 mg/Kg and the dose of high manganese was 50 mg/Kg.Intraperitoneal injections and gavage are given once daily for 5 weeks.In the above animal experiments,the testes were weighed weekly,while in vivo ultrasound of the testicles was done at 0,2,and 4 weeks.At the end of week5,mice are sacrificed under anesthesia to take testicles and epididymis,weigh the testicles and epididymis separately,and place the epididymis incision in normal saline to determine sperm count,motility,and abnormality.The testicle was fixed,dehydrated,embedded,sectioned and then stained with HE to observe the morphology of testicular tissue.Testicular RNA was extracted and used for the detection of pro-inflammatory mediators HMGB1,IL-6,INOS,TNF-αm RNA.Finally,the ELISA kit was used to measure the PGE2level in the testes of mice.3.Spermatogonia were exposed to different concentrations of Mn Cl₂ and CX for 24 h to evaluate toxicity caused by manganese and CX.Finally,0,100,200,and 400μM Mn Cl₂ were selected as the manganese exposure concentration,and 5μM CX was selected as CX intervention concentration after manganese exposure.After 24h treatment at 0,100,200,400μM Mn Cl₂ and 5μM CX and 5μM CX+400μM Mn Cl₂,cell morphology is observed by microscopy and viability is detected by CCK8.Finally,after24 h of manganese exposure and CX intervention,the extracellular PGE2 level was measured by ELISA kit,and RNA and protein were extracted for the detection of COX2,PTGER3,HMGB1,IL-6,INOS,and TNF-αexpression levels.Results:In the first stage of animal experiments,the testicular ultrasonic volume analysis found that compared with the control group,the testicular volume of the testis of the high manganese group decreased in the 2 and 4 weekends.The organ coefficient shows that the high manganese group decreases compar ed with the testicular and epididymal organs coefficient of the control group.The sperm quality analysis showed that the sperm count and motility of mice in the high manganese group were significantly lower than those in the control group,and the sperm deformity rate was higher than that in the control group.Teste HE staining found that the arrangement of spermatogenic cells in the seminiferous tube in the high manganese group was disordered.In addition,the content of the medium manganese group and the high manganese group increased compared with the control PGE2 in the testis.In the results of the second stage of animal experiments show that CX intervention after manganese exposure can significantly increase the testicular volume of mice,improve the sperm quality of mice,alleviate the seminiferous tube damage,and reduce the PGE2 content in testis.In addition,CX intervention can significantly reduce the levels of INOS,IL-6 and TNF-αm RNA of pro-inflammatory mediators after exposure to manganese.During the cell experiment stage,microscopic observation revealed that the spermatogonia changed in shape after the addition of manganese,swelling occurred,and floating dead cells increased.The CCK8 detection found that the vitality of the high mangane se group’s spermatogonia decreased,while CX could partially restore the vitality of the spermatogonia.PCR and WB showed that the m RNA and protein levels of COX2 and PTGER3 in the high manganese group were increased,and the m RNA levels of pro-inflammator y mediators HMGB1,INOS,IL-6 and TNF-αwere also increased significantly.However,the m RNA and protein of PTGER3 in the high-manganese group after CX intervention were decreased compared with that in the high-manganese group alone,and the above pro-inflammatory mediators were also decreased.Conclusion:Manganese exposure promoted the synthesis of PGE2 by COX2 in mouse spermatogonia,and regulated the transcription of pro-inflammatory mediators through PTGER3 receptor,resulting in damage or dea th of spermatogonia,and ultimately affected the abnormal spermatogenesis of mice.