PCB153 Inhibits Testosterone Production By Inducing Oxidative Stress Via Mark4

Objective:In male reproductive system,Testosterone?T?is one of the most important steroid hormone and plays a crucial role in spermatogenesis and maintenance of male secondary sexual characteristics.Testosterone production is regulated by the hypothalamic-pituitary-gonadal axis and mainly catalyzed by various steroid-generating enzymes in testicular leydig cells.PCBs?Polychlorinated biphenyls?is a kind of widely used environmental pollutants in our daily life.Those permanent pollutants are hard to degradation,can be enriched in the human body in many different ways and bring serious threat to human health,especially to the reproductive system.Previous studies have found that high exposure to PCBs can significantly reduce serum testosterone level,destroy the blood-testis-barrier,reduce testicular relative mass,sperm quality and quantity,and affect male reproductive ability in many ways.However the specific mechanism of its influence on testosterone secretion in testicular leydig cells remains to be further clarified.Mark4?Microtubules regulating kinase 4?is a kind of Ser/Thr protein kinase and it has been shown that it can promote adipose accumulation,induce oxidative stress and inflammation in lipid metabolism,but there is no related report seen in the steroid metabolism.In our previous study,we found that PCB153 can up-regulate the level of Mark4 in testicular leydig cells,so we proposed that Mark4 may be involved in the influence of PCB on the function of leydig cells.In this study,PCB153,a single product of PCBs,is selected as the treatment factor.In vitro and in vivo experiments were conducted to explore the role of Mark4 in affecting the synthesis of testosterone in mesenchymal cells by PCB.Methods:1.Screening dose of PCB153 cells without significant lethal effect.Mouse TM3 cell lines were treated with different concentrations of PCB153,and cell viability was detected at 6 h,12 h,24 h,and 48 h with CCK-8.2.Effects of PCB153 on testosterone secretion in mouse TM3 cells.After TM3 was treated with screened PCB153concentration?5,10 and 20?g/ml?,?1?Detected the level of T in the medium at different time points;?2?Western blot was used to detect the expression of Mark4,steroid-generating enzymes,apoptosis-related proteins and activation of related signaling pathways;?3?Detected ROS and the activity changes of CAT after PCB153 treatment;?4?JC-1 kit was used to detect the changes in mitochondrial membrane potential after PCB153 treatment.3.Verified the effect of PCB153 on testosterone production and spermatogenesis by experiments in vivo.Adult SD rats were intraperitoneally injected with PCB153.Serum and testicular tissues were collected?1?Detect serum T,E₂ and LH levels;?2?Paraffin sections were prepared and HE staining was used to counting the number of?peroid seminiferous tubule;?3?Transmission Electron Microscopy?TEM?was used to observe the structural integrity of mitochondria in testicular leydig cells.4.Effect of Mark4 knockdown on the inhibition of testosterone secretion caused by PCB153 in TM3 cells.Mark4 knockdown was combined with PCB153 to detect the changes of those above indexes,such as T,ROS,CAT.5.Effect of NF-?B inhibitor on oxidative stress and inhibition of testosterone secretion induced by PCB153.The concentration of PDTC was selected to inhibit the activation of NF-?B signaling pathway by PCB153.After treatment with 10?M PDTC for 2 h in advance,the cells were treated with 20?g/ml PCB153 for 24 h.Then the changes of above indicators were detected.6.Effect of PCB153 on testosterone secretion in rats after inhibition of NF-?B activity.Rats were treated with different concentrations of PDTC and PCB153 intraperitoneally.WB was used to detect the activation of NF-?B signaling pathway in testis,and serum T,LH and E2 levels were detected by ELISA.Results:1.PCB153 inhibited TM3 cells survival and testosterone secretion:CCK-8results showed that TM3 cell survival was inhibited after PCB153 treatment.ELISA was used to detect the concentration of testosterone in culture medium,10 and 20?g/ml PCB153 could significantly inhibit the secretion of testosterone in TM3 cells.WB results showed that the expression of steroidogenic enzyme StAR and 3?-HSD were decreased,the expression level of Mark4 increased,and the level of anti-apoptotic protein Bcl-2significantly decreased.2.PCB153 induced oxidative stress and affected mitochondrial function in TM3 cells.The results of WB showed that the p-p65/p65 ratio increased and NF-?B signaling pathway was activated after PCB153 treatment.The kit showed a significant increase of ROS and the CAT activity was decreased.The results of JC-1showed that the intracellular green fluorescence increased significantly and the mitochondrial membrane potential decreased.3.PCB153 inhibits testosterone generating and affect spermatogenesis in mice:Compared with control group,in PCB153 treatment group,the serum level of T was decreased,LH level was elevated;testicular tissue HE was used to cout?period seminiferous tubule and the number was decline;TEM results showed that the leydig cells mitochondria became shallow,cavitation,the mitochondria structure was damaged.4.Mark4 knockdown can weaken the inhibitory effect of PCB153 on testosterone generated:TM3 cells was transfected with Mark4 siRNA to silenced the expression of Mark4,and then combined with PCB153 treatment.The results showed that compared with Ctrl siRNA+PCB153 group,PCB153 could not reduce the StAR level,nor significantly activate the NF-?B signaling pathway in the Mark4 siRNA+PCB153 group,but PCB153 treatment still could reduce the level of Bcl-2.At the same time,after lowering the expression of Mark4,PCB153 could not induce the increase of cellular oxidative stress,the CAT activity and mitochondrial membrane potential reduction caused by PCB153 were alleviated,and the inhibitory effect of PCB153 on the generation of testosterone was partially antagonized.5.Inhibitor of the NF-?B signaling pathway can partially alleviate the inhibitory effect of PCB153on testosterone synthesis:Choosing the concentration of PDTC which can just inhibit the TM3 cells NF-?B signaling pathway activation of PCB153.TM3 cells were pretreated with 10?M PDTC for 2 h,and combined with PCB153 treatment for 24 h,ELISA was used to detect culture supernatant testosterone.The results showed that inhibiting the NF-?B signaling pathway can alleviate the inhibition of TM3 cells testosterone synthesis caused by PCB153;ROS kit results showed that PCB153 still could induce the increase of oxidative stress in PDTC+PCB153 group,but the effect was significantly relieved compared with the PCB153 group.After PDTC inhibiting the activation of NF-?B signaling pathway,PCB153 could not reduce the activity of CAT.It was also found that PCB153 could still reduce mitochondrial membrane potential in the PDTC+PCB153group,but the degree of reduction was lower than that of PCB153 group.In vivo,inhibition of NF-?B signaling pathway significantly alleviated the inhibition of PCB153on serum testosterone synthesis in rats.Conclusion:PCB153 activates the NF-?B signaling pathway and induces oxidative stress in testicular leydig cells by promoting the expression of Mark4 protein,and results in testosterone synthesis disorders.