| BackgroundLate onset hypogonadism(LOH)has recently been defined as the age-related low testosterone syndrome among the middle-aged and elderly men.The main manifestations are decreased libido,erectile dysfunction and decreased frequency of morning erections.The decreased level of testerone is considered to be one of the important causes of LOH.The testosterone is mainly synthesized by leydig cells and regulated by luteinizing hormone(LH).With the age ingcreasing,the synthesis function of leydig cells gradually declines and the level of androgen in the blood circulation decreases.At present,"free-radical theory" is considered to be one of the important mechanisms of aging.It is believed that aging cells are in a state of oxidative imbalance and their antioxidant capacity is not enough to exclude excessive reactive oxygen species(ROS).Finally ROS causes oxidative damage,apoptosis and so on.During the aging process,accumulation of ROS in leydig cells can damage mitochondrial function.It is said that ROS may reduce the gene expression of testosterone synthesis by inhibiting the transcription of Nur77,resulted in a decrease of testosterone level.Amount of studies have shown that excessive ROS can interrupt the synthesis of testosterone in leydig cells.The Nrf2-ARE signaling pathway plays a key role in anti-oxidative stress of the body.Nuclear factor-like 2(Nrf2)and anti-oxidation response element(ARE)regulates the expression of downstream II phase metabolizing enzymes,anti-oxidation proteins and antioxidant enzymes.more and more research shows that Nrf2 plays a key role in antioxidation function of the body.Recent studies have shown that vitamin D3 receptors and their metabolic enzymes are also widely distributed in the reproductive system,indicating that VitD3 plays an important role in the reproductive system.Studies have shown that 25-hydroxy vitamin D3 levels are negatively correlated with age and positively correlated with circulating testosterone levels.In addition,active forms of vitamin D3 can delay the progression of diabetic nephropathy by inhibiting oxidative stress and regulating the Nrf2 pathway.Active vitamin D3 can attenuate LPS-induced macrophage HMGB1 secretion via the Nrf2/HO-1 pathway.In this experiment,we established an oxidative damage model induced by hydrogen peroxide of leydig cells to explore the effect of calcitriol on the damage of leydig cells and the mechanism whether it played a role through Nrf2/ARE signaling pathway.PurposeTo explore the effect of calcitriol on testis cells and its possible molecular mechanism.MethodsTM3 cells were divided into 8 groups according to different concentrations of vitamin D3 and with or without hydrogen peroxide intervention,respectively:(1)blank control group(CON);(2)1nM calcitriol group(C1);(3)10nM calcitriol group(C10);(4)100nM calcitriol group(C100);(5)hydrogen peroxide group(HI);(6)Hydrogen peroxide + 1 nM calcitriol group(HI1);(7)10 nM calcitriol group + hydrogen peroxide group(HI10);(8)100 nM calcitriol + hydrogen peroxide group(HI100).Total protein was extracted and Western blot was used to detect the expression levels of VDR,Nrf2,GCLC,and SOD2.Real-time fluorescence quantitative PCR was used to detect the expression of Nrf2,GCLC and SOD2 mRNA.The cell viability was dected with the CCK-8 assay.The testosterone levels was dected by ELISA.Homogenates were prepared for the detection of SOD activity.The ROS level in each group was detected.Results1.Effect of calcitriol on the viability of leydig cellsCompared with CON group,the activity of the cells added with calcitriol increased,and the activity is highest in the C1 group.The activity was decreased in the HI group(P<0.05).Compared with the HI group,the activity of HI1 and HI10 group is increased and the HI10 group is the highest one(P<0.05).2.Effect of calcitriol on Antioxidant Activity of leydig cellsCompared with the CON group,SOD activity is increased(P<0.05)and ROS level is decreasd(P<0.05)in the C1 and C10 group.SOD activity is decreased in the C100group(P>0.05).And the level of ROS is increased(P<0.05).Compared with the group HI,SOD activity is increased(P<0.05)and ROS levels are decreased(P<0.05)in group H1 and H10.However,the ROS level is increased when VitD3 concentration was 100nM(P<0.05).3.The effect of vitamin D3 on the testosterone secretion of leydig cellsCompared with the CON group,testosterone level is increased in the group C1.In the HI group the testosterone level is decreased(P<0.05).Compared to the HI group,the testerone level of HI1 is decreased(P<0.05).4.Effect of calcitriol on relative NRF2,GCLC,and SOD2 mRNA in leydig cellsCompared with the CON group,the levels of NRF2,GCLC and SOD2 mRNA are increased in C1?C10 and C100 group(P<0.05).Compared with the HI group,the levels of NRF2,GCLC,and SOD2 mRNA in the vitamin D3 group are increased(P<0.05).5.Effect of Vitamin D on the relative Expression of VDR,NRF2,SOD2,and GCLC Proteins in leydig cellsCompared with the CON group,the levels of VDR,NRF2,GCLC and SOD2 are increased in C1 and C10 group(1 nM,10 n M vitamin D3)(P<0.05),but the level of NRF2 and GCLC are decreased when the vitamin D3 concentration is 100 nM.Compared with the HI group,the expression levels of VDR,SOD2,Nrf2,and GCLC are increased in the group of adding calcitriol(P<0.05).6.The effect of vitamin D3 on the expression of nuclear Nrf2 of leydig cellsCompared with the CON group,the expression of cytoplasmic Nrf2 protein is decreased in C1 and C10groups(P<0.05).But there is no significant difference between the 100 nM group and CON group(P<0.05).The expression level of nuclear Nrf2 protein in the 10 and 100 nM groups was increased.Compared with the HI group,the expression of Nrf2 protein in the cytoplasm of 100 nM vitamin D3 group is increased(P<0.05).There is no significant difference between C1 and C10 group.The level of Nrf2 protein in the nuclei increased with the concentration of 1,10 nM vitamin D3(P<0.05).ConclusionVitamin D3 has a protective effect on oxidative damage of mouse Leydig cells,probably through NRF2 pathway. |
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