| Testicular torsion is an acute urologic emergency occurring in male newborns,children or adolescents,and it can cause blood circulation in the testis to be impaired.It is necessary to adopt reduction and surgical treatment timely to restore blood supply and prevent impaired spermatogenesis.Testicular torsion/detorsion（T/D）is considered to be the primary pathophysiological event of testicular ischemia-reperfusion injury（IRI）.Even in patients with manipulative reduction,there is still a possibility of testicular atrophy,decreased spermatogenesis,and even male infertility after surgery.Therefore,treatment and prevention of testicular IRI after surgical reduction has become a hot topic in the field of andrology in recent years.With the continuous development of research technology,as a new type of gas signal molecule discovered in recent years,hydrogen sulfide（H2S）has been proved to be involved in the functional regulation of various organs in the human body.GYY4137 is one of the novel H2S donors that,compared with the traditional donor sodium hydrogen sulfide（Na HS）,GYY4137 can release H2S stably under the conditions of the temperature and p H of physiological environment,which is closer to the synthesis of endogenous hydrogen sulfide in the body.According to research reports,GYY4137 can play an important role in the regulation of ischemia-reperfusion injury in various organs,but its function and mechanism in testicular IRI have not been reported in the literature.Therefore,in this experiment,we respectively established rat testis IRI model and spermatogenic cell hypoxia/reoxygenation model on animal and cell models,to explore the role of GYY4137 in the rat testicular IRI and its possible mechanism.Our experimental results show that GYY4137 can play an important role in inhibiting and reducing the level of oxidative stress in cells,reducing apoptosis and inflammation damage and thus to play a certain protective function in the rat testicular IRI model and spermatogenic cell hypoxia/reoxygenation model.On this basis,we found the therapeutic effect of GYY4137 injected through the scrotum was better than traditional intraperitoneal injection by comparing two different modes of administration.Built on the above studies,we investigated the effect and mechanism of GYY4137 in testicular IRI,and provided new ideas for future clinical treatment of testicular torsion.Part one:The effect of GYY4137 on testicular ischemia-reperfusion injury in ratsObjective:To investigate the protective effect of GYY4137 on testicular ischemia-reperfusion injury in rats.Methods:Forty healthy adult male SD rats were randomly divided into 5 groups of 8 rats each:control group（group A）,testicular ischemia-reperfusion injury（IRI）model group（group B）,and IRI+50μmol/kg GYY4137 group（group C）,IRI+100μmol/kg GYY4137 group（group D）,and IRI+150μmol/kg GYY4137 group（group E）.Group A was a sham operation group.Rats in groups B,C,D,and E established unilateral testicular torsion reduction models.GYY4137 was injected intraperitoneally,and 4 hours after reduction,rats were sacrificed to collect specimens.Histopathological changes were detected by HE staining;the contents of malondialdehyde（MDA）and superoxide dismutase（SOD）were measured by thiobarbituric acid method and xanthine oxidase method;real-time fluorescent quantitative PCR（q RT-PCR）was used to detect the expression levels of Bax,Caspase-3,TNF-αand IL-1β.Results:Compared with group A,spermatogenic cells were widely exfoliated and necrotic and the epithelium was disordered in group B.Compared with group B,the changes caused by testicular ischemia-reperfusion injury were significantly improved in groups C,D and E.Compared with group A,the content of MDA in group B increased observably,and the content of SOD decreased observably.Compared with group B,the content of MDA in groups C,D and E decreased observably,and the content of SDA increased observably,among which the degree of change in group D was the most significant.PCR results showed that the expression of Bax and Caspase-3 in group B were significantly increased compared with group A,while the expression of TNF-αand IL-1βwas also significantly increased.Compared with group B,the expression of Bax,Caspase-3,TNF-αand IL-1βwere all reduced in group C,D and E,and the degree of decrease was the most significant in group D.Conclusion:GYY4137 has a certain degree of protection against ischemia-reperfusion injury in testis of rats,which can effectively reduce the level of oxidative stress in cells and reduce the damage of apoptosis and inflammation,and this effect is related to the concentration of administration,the protective effect is most obvious at100μmol/kg.Part two:The effect of GYY4137 on hypoxia/reoxygenation injury of spermatogoniaObjective:To investigate the protective effect of GYY4137 on hypoxia/reoxygenated spermatogonia.Methods:A hypoxia/reoxygenation injury model of spermatogonia was established.Under these conditions,we gave different concentrations（10,50,and 100μM）of GYY4137 for intervention.MTT assay was used to detect the viability of isolated cells,and flow cytometry was used to detect the apoptosis rate,q RT-PCR was used to detect the expression of Bax and Caspase-3.Results:Compared with the control group,the cell viability of the model group was markedly decreased,and the cell viability of each group in the GYY4137intervention group was significantly higher than that of the model group,of which the50μM group had the most significant increase.Compared with the control group,the apoptosis rate of the model group increased markedly,and the apoptosis rate of the cells in each group intervened with GYY4137 decreased compared with the model group,and the 50μM group showed the most markedly decrease.The expression levels of Bax and Caspase-3 in the model group were markedly increased compared with the control group,while the expression of Bax and Caspase-3 in the GYY4137 intervention group was lower than those in the model group.The decrease was most obvious at a concentration of 50μM.Conclusion:GYY4137 has a certain protective effect on hypoxia/reoxygenation injury of spermatogonia cells,which can effectively decrease the level of oxidative stress,alleviate apoptosis,and increase the level of cell proliferation,and this effect is related to the concentration of administration,the protective effect is most obvious at50μM.Part three:Comparison of intraperitoneal and intratesticular GYY4137 therapy for the treatment of testicular ischemia reperfusion injury in ratsObjective:To compare the efficacy of intraperitoneal injection and intratesticular injection of GYY4137 on ischemia-reperfusion injury of testis in rats.Methods:Thirty-two healthy adult male SD rats were randomly divided into 4groups,8 in each group:control group（group A）,testicular ischemia-reperfusion injury group（group B）,testicular ischemia-reperfusion injury+GYY4137 intraperitoneal administration group（group C）,testis ischemia-reperfusion injury+GYY4137intratesticular administration group（group D）.Group A was a sham operation group,rats in group B,group C,and group D established unilateral testicular torsion/detorsion models.Group C and group D were injected intraperitoneally and intratesticularly with GYY4137（100μmol/kg）during reduction.4 hours after reduction,took the left testis for measurement.HE staining was used to observe the pathological changes of testis tissues,and measured the contents of malondialdehyde（MDA）and superoxide dismutase（SOD）;terminal d UTP transferase nick end labeling（TUNEL）was used to detect the spermatogenic cell apoptosis index.Immunohistochemistry,Western Blot and q RT-PCR were used to detect the expression level of Bax,Bcl-2 and Caspase-3expression in testis.Results:A large number of spermatogenic cells atrophy and necrosis were seen in group B.Compared with group B,group C and group D significantly improved the change caused by testicular ischemia-reperfusion injury.Compared with group A,the content of MDA in group B increased markedly and the content of SOD decreased markedly.Compared with group B,the content of MDA in group C and group D was significantly decreased,and the content of SDA was significantly increased,the degree of change in group D was more pronounced;the apoptosis rate in group B was significantly higher than that in group A.Compared with group B,the apoptosis rates in group C and group D were significantly reduced,of which the degree of decline in group D was more pronounced.The expression levels of Bax and Caspase-3 in group B were significantly increased compared with group A,while the expression levels of Bcl-2 were significantly reduced.Compared with group B,the expression of Bax and Caspase-3 in group C and group D both decreased,and the expression of Bcl-2increased,among them,the improvement degree of group D is more significant.Conclusion:GYY4137 has obvious protective effect on testicular ischemia-reperfusion injury in rats.The efficacy of intracordal administration is greater than that of intraperitoneal administration,which may provide a new idea for the treatment of testicular torsion in the future. |
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