Effect And Mechanism Of Resveratrol On Oxidative Injury Of Mouse Testicular Interstitial Cells

Oxidative injury can cause severe spermatogenic disorders in animals,resulting in pathological changes in testicular tissue,decreased testicle index and sperm count,and increased sperm malformation rate,resulting in male infertility.Resveratrol（RES）is a natural antioxidant that plays an important role in the treatment of oxidative injury,inflammation,allergies and cardiovascular disease.At present,the research indicates that RES has protective effect on oxidative damage caused by various factors,but its mechanism is not completely clear.Objective:The oxidative injury model of mouse Leydig cells（TM3）in vitro was established to study the effect of resveratrol（RES）on oxidative-injuried by H₂O₂,and explore its possible mechanism.Methods:Firstly,TM3 cells were treated with different concentrations of H₂O₂to establish the model of cell oxidative injury in vitro.After the model was built,oxidative injury cells were treated with different concentrations of RES.The effects of RES on oxidative injury of TM3 cells induced by H₂O₂ were studied by real-time monitoring of cell growth and proliferation,changes of partial oxidation and antioxidant index content,cell apoptosis and the ability of cells to secrete testosterone.The protective mechanism of RES on oxidative injury of TM3 cells was discussed from the SIRT1/UCP2 signaling pathway,SIRT1/AMPK autophagy pathway and the regulation of major matrix metalloproteinases.Results:1.The establishment of oxidative injury cell model:the results of iCELLigence cell function analyzer showed that 150μM-300μM H₂O₂ treatment of TM3 cells after8 h cell activity decreased significantly,and in a concentration dependent manner,the activity of H₂O₂ decreased to about 75%when it was treated with 150μM H₂O₂.At the same time,the activity of LDH in the supernatant of cell culture,the content of MDA and the activity of SOD in the cell increased significantly,but the activity of GSH-Px decreased significantly.The above results indicated that the cells were in the state of oxidative injury.Therefore,150μM H₂O₂ was used for 8 h to construct the model of cell oxidative injury.2.The effect of RES on the proliferation of normal TM3 cells:the normal TM3cells were treated with different concentrations of RES(2.5μM,5μM and 10μM for24 h,but the cell viability was not significantly affected.3.The effect of RES on oxidative injury cells:when the TM3 cells were treated with the above concentration of RES,the cell viability was significantly increased to a certain extent,and there was no significant difference among the different concentration groups.Therefore,5μM RES was selected to carry out the related experiments in the follow-up experiment.After treated with 5μM RES for oxidative injury of TM3 cells,the changes of related oxidation indexes induced by H₂O₂ were significantly inhibited to some extent,and the activity of LDH,the content of MDA,ROS and the activity of SOD in the supernatant were significantly decreased.The activity of GSH-Px in cells was significantly increased.In addition,RES treatment increased the ability of TM3 cells to secrete testosterone and inhibited apoptosis induced by H₂O₂.4.The mechanism of oxidative injury protection of RES:（1）After oxidative injury induced by H₂O₂,the expression of SIRT1 decreased significantly,but the expression of UCP2,MMP-2/9 and LC3-Ⅱincreased,but the expression of p-AMPK and AMPK did not change significantly.（2）The changes of related factors induced by H₂O₂ were inhibited to some extent when treated with RES,and the levels of p-AMPK and LC3-Ⅱwere significantly up-regulated.（3）In RES treated oxidative injury cells,SIRT1 inhibitor or AMPK inhibitor pretreatment could significantly inhibit LC3-II expression.Moreover,pretreatment with SIRT1 inhibitor could inhibit p-AMPK expression and further decrease the content of MMP-2/9,but AMPK inhibitor pretreatment could not inhibit SIRT1 expression.Conclusions:（1）The mouse testicular interstitial cells TM3 treated with 150μM H₂O₂ for 8 h significantly decreased cell viability and leaved the cells in the state of oxidative injury.When treated with RES,the cells were protected against oxidative injury,which mainly showed that they could improve the viability of oxidative injury cells,protect the integrity of cell membrane,reduce lipid peroxidation,enhance the activity of antioxidant enzymes,and reduce cell apoptosis.It also improves the ability of cells to secrete testosterone.（2）The protective mechanism of RES against oxidative injury of TM3 cells is regulated by the following three pathways:RES may inhibit the expression of UCP2 by activating SIRT1 and inhibit the production of ROS in TM3cells by negative feedback regulation,thereby inhibiting the oxidative injury of TM3cells.RES can reduce the degree of oxidative injury of TM3 cells by reducing the activity of MMP-2/9.SIRT1 may be involved in the regulation of MMP-2/9 in collaboration with RES,but it needs further study.RES may improve oxidative injury of TM3 cells induced by H₂O₂ by mediating SIRT1/AMPK autophagy pathway.