The Role Of Lycopene In Hypoxia-induced Testicular Injury And Its Molecular Mechanism Study

The First Part Lycopene plays an inhibitory role in testicular injury in varicocele rats.Background:Varicocele is a common disease of male reproductive system,which may be related to male infertility,pain and discomfort of ipsilateral scrotum and its contents,abnormal synthesis and secretion of testosterone,and it is one of the main causes of male infertility.For the treatment of male infertility caused by varicocele,there is no particularly effective drug presently,and most of the drugs used in clinic are empirical and nonspecific drugs.Therefore,it is an urgent problem in the research of varicocele infertility to clarify the exact pathological mechanism of varicocele,which leading to low fertility,and find new and more effective therapeutic drugs in order to improve the low fertility of patients with varicocele.Lycopene is a natural functional pigment and a phytochemical in the carotene family.Studies have found that lycopene has strong anti-inflammatory and antioxidant effects,and it can be used as an adjuvant drug for chronic prostatitis,chronic pelvic pain syndrome,benign prostatic hyperplasia and prostate cancer.However,it is not clear whether lycopene can play a role in varicocele infertility.Objective:To explore the role of lycopene in testicular injury in rats with varicocele.Methods:In this part of the study,we first established a rat model of varicocele,and verified the testicular injury of varicocele by weighing the testis and epididymis tissues of rats,and detecting the sperm quality and hormone secretion of rats.Subsequently,in order to explore whether lycopene has therapeutic effect on testicular injury in varicocele rats,we first divided the selected rats into the following three groups:sham operation group(NG),varicocele group(VG),varicocele+lycopene 60 mg/kg administration group(VLG);After 4 weeks,the samples were collected and detected by ELISA,HE staining,immunohistochemistry and TUNEL.Results:In varicocele group,the weight of testis and epididymis decreased,the sperm density and motility decreased,and the sperm deformity rate increased.Serum testosterone content decreased.However,the sperm quality and testosterone secretion of rats in lycopene treatment group were significantly improved compared with those in varicocele group.Subsequently,the HE staining found that the damage of spermatogenic epithelium in the testis of VLG group was significantly better than that of VG group.TUNEL staining found that the apoptosis of spermatogenic cells in VG rats was significantly aggravated,while that in VLG group was reduced.Immunohistochemical staining showed that PCNA in VG rats decreased significantly,while PCNA in VLG group increased significantly compared with VG group.The results showed that lycopene can reduce the apoptosis of spermatogenic cells in testis caused by varicocele and reduce the damage of varicocele to spermatogenic epithelium in rats.The ELISA.results showed that the expression of inflammatory factors in testis of varicocele rats increased,but the expression of inflammatory factors IL-1β and IL-2 decreased significantly after lycopene was applied.By detecting the levels of ROS and SOD in the testis of three groups of rats,it was found that the level of ROS in lycopene treatment group decreased significantly and the level of SOD increased.It shows that lycopene plays an anti-inflammatory and antioxidant role in varicocele.Conclusion:lycopene can inhibit the apoptosis of spermatogenic cells in varicocele rats,reduce the damage of spermatogenic epithelium and improve the spermatogenic function of rats’ testis.The second part studies the mechanism of lycopene inhibiting testicular injury induced by varicocele hypoxia.Background:The possible causes of infertility caused by varicocele include scrotal temperature rise,oxidative stress,testicular hypoxia,metabolite reflux from adrenal gland,accumulation of carbon dioxide and nitric oxide,and autoimmunity,but the exact pathological mechanism of infertility caused by varicocele is still unclear.Testicular hypoxia is recognized as an important pathophysiological feature of varicocele.Studies have shown that the hypoxic environment of testis induced by varicocele can cause oxidative stress and inflammatory reaction,which will cause extensive damage to cells and tissues,affect cell proliferation and apoptosis,and finally cause testicular spermatogenic function damage.MicroRNA(miRNA)is a highly conserved small non-coding RNA.Under pathological conditions,the abnormal expression of miRNA can participate in the regulation of many diseases,including varicocele.Differentially expressed lncRNA and miRNA may be potential biomarkers to predict the risk of spermatogenic dysfunction and the effect of surgical repair in varicocele.PROK2 is a secreted protein with many biological functions.PROK2 can participate in many pathological processes,such as inflammation,abnormal angiogenesis and organ development disorders.The researchers found that PROK2 expression increased in rats with varicocele,and it can also participate in the occurrence of varicocele in rats by inducing apoptosis of spermatocytes.It is suggested that PROK2 is closely related to the occurrence of varicocele.Methods:According to the results of previous animal experiments,we found that lycopene significantly improved the damage of spermatocytes in VC rats,so we selected mouse spermatocytes GC-2 to construct a hypoxia-induced cell model in vitro,and divided GC-2 cells into the following three groups:normoxic treatment group,hypoxia+DMSO treatment group,hypoxia+lycopene 10μM treatment group.After 24 hours culture,Wetsern blot experiment and flow cytometry technology were adopted.Then,the expressions of miR-23a-3p and miR-23b-3p in rat testis and mouse GC-2 spermatocytes were detected by bioinformatics analysis and qRT-PCR experiment.Subsequently,we verified whether PROK2 interacted with miR-23a-3p and miR-23b-3p through STARBASE database and Luciferase reporter experiment.In the further study,we stimulated the mouse GC-2 spermatocytes with hypoxia,treated them with lycopene,transfected miR-23a-3p inhibitor and miR-23b-3p inhibitor respectively,and detected the changes of PROK2 expression in the cells by qRT-PCR and Western blot experiment.Finally,Wetsern blot was used to detect the protein level of HIF-1α,NF-κB,VEGF,AKT and p-AKT in three groups.Results:The expression of BCL-2 and Cleaved Caspase-3 in three groups of GC-2 spermatocytes was detected by Western blot in vitro.The results showed that the ratio of bcl-2/bax in GC-2 spermatocytes decreased while the ratio of C-/T-caspase-3 increased under hypoxia,which indicated that apoptosis increased under hypoxia,and lycopene could alleviate the apoptosis of spermatocytes induced by hypoxia.The same result was obtained by flow cytometry.Through cck-8 and EdU experiments,we found that lycopene can alleviate the inhibition of hypoxia on the proliferation of GC-2 spermatocytes.This shows that lycopene can promote the proliferation of spermatocytes and inhibit apoptosis.Then,we detected the expression profiles of miRNA in the testis of three groups of rats by sequencer,in which the expression of miR-23a-3p and miR-23b-3p decreased in the varicocele group,but increased in the lycopene treatment group.Subsequently,we detected the expression of miR-23a-3p and miR-23b-3p in testicular tissue and GC-2 spermatocytes of three groups by qRT-PCR experiment,and found that the expression of miR-23a-3p and miR-23b-3p in testicular tissue and GC-2 spermatocytes of rats in lycopene treatment group was significantly higher than that in varicocele hypoxia-induced group.Then,we transfected the GC-2 spermatocytes with miR-23a-3p and miR-23b-3p mimics,and detected the apoptosis of GC-2 spermatocytes by flow cytometry.The results showed that overexpression of miR-23a-3p and miR-23b-3p could significantly inhibit the apoptosis of GC-2 spermatocytes caused by hypoxia.In order to further verify the anti-apoptosis effects of miR-23a-3p and miR-23b-3p,we detected the expression of apoptosis-related proteins by Western blot.The results showed that overexpression of miR-23a-3p and miR-23b-3p significantly up-regulated the expression of bcl-2/bax in GC-2 spermatocytes and down-regulated the expression of C-/T-caspase-3.In addition,CCK8 and EdU experiments show that overexpression of miR-23a-3p and miR-23b-3p can improve the proliferation activity of GC-2 spermatocytes induced by hypoxia.These results indicate that miR-23a-3p and miR-23b-3p can inhibit the apoptosis of GC-2 spermatocytes induced by hypoxia and promote cell proliferation.Then,the downstream potential targets of miR-23a-3p and miR-23b-3p were predicted by STARBASE database,and it was found that PROK2 was the common target of miR-23a-3p and miR-23b-3p.Luciferase reporter experiment confirmed that PROK2 is the target gene of downstream action of miR-23a-3p and miR-23b-3p.Next,we detected the expression of PROK2 at mRNA and protein levels in the testis of three groups of rats.The results showed that compared with VG group,the expression of PROK2 at mRNA and protein levels in the testis of VLG group was significantly reduced.In the cell experiment,compared with the hypoxia treatment group,the expression of PROK2 in GC-2 spermatocytes in lycopene treatment group was also significantly reduced.However,the inhibitory effect can be reversed by adding miR-23a-3p inhibitor or miR-23b-3p inhibitor to GC-2 spermatocytes in lycopene treatment group.Finally,we detected the changes of HIF-1α,NF-κB,VEGF and p-AKT in the testis of three groups of rats by Western blot experiment.The results showed that compared with varicocele group,the expression of HIF-1α,NF-κB and VEGF in the testis of rats in lycopene treatment group decreased,while the expression of p-AKT increased.The results showed that lycopene inhibited the expression of HIF-1α,NF-κB and VEGF in rat testis under the condition of varicocele hypoxia and promoted the expression of p-AKT.Conclusion:Lycopene can reduce the apoptosis of spermatocytes induced by hypoxia by activating the miR-23a-3p/23b-3p/PROK2 axis,thus improving the testicular spermatogenic function damage induced by varicocele.In addition,HIF-1α/NF-κB/VEGF/AKT axis also plays an important role in the process of lycopene inhibiting testicular injury induced by hypoxia.Our research results provide a new therapeutic drug for the treatment of varicocele infertility in the future,and have important clinical significance for improving the fertility of patients with varicocele infertility.