Protective Effect Of C-phycocyanin On Reproductive System Of Male Mice By Regulating RIPK1-RIPK3-MLKL Signal Pathway

Infertility,cardiovascular disease and tumor are the three major diseases affecting human life and health,in which male infertility has attracted more and more attention of scholars at home and abroad because of its rising incidence.Male infertility is usually caused by a variety of causes,and the treatment is limited.At present,it mainly depends on drug treatment,which has poor clinical effect and high toxicity,so the search for low-toxic and efficient therapeutic drugs has become the focus of current research.As a treasure house of natural medicines,the ocean is rich in exploitable resources.C-phycocyanin(C-PC)is a protein complex extracted from spirulina with multiple pharmacological effects,such as anti-oxidant,anti-inflammatory,anti-tumor and immunosuppressive activities.Many studies have shown that C-PC can improve the fertilization ability of porcine oocytes and the developmental potential of embryos.During the development of reproductive system,in addition to apoptosis and necrosis,there is a kind of programmed necrosis,it’s necroptosis,which is regulated by activating receptor interacting protein kinase-1(RIPK1),receptor interacting protein kinase-3(RIPK3)and mixed lineage kinase domain-like protein(MLKL).Necroptosis has negative effects on the development and evolution of reproductive system.Through the study of the RIPK1-RIPK3-MLKL signal pathway,the necroptosis inhibitors can effectively maintain the vitality of germ cells to protect the male reproductive system.The purpose of this study was to explore whether C-PC can be used as an inhibitor of necroptosis,so as to protect the male reproductive system,provide a new direction and theoretical basis for the development and utilization of C-PC,and has important research value.Objective:To explore the specific mechanism of the protective effect of C-phycocyanin(C-PC)on the reproductive system of male mice by regulating RIPK1-RIPK3-MLKL signal pathway.Methods:1.Construction of GC-1 spg mouse spermatogonia necroptosis model: GC-1 spg cells were treated with necroptosis inducing reagent(TSZ)to construct reproductive injury cell model.The experiment was divided into four groups: Control,TSZ,TSZ+C-PC,TSZ+Nec-1,Necrostain-1(Nec-1)is a kind of RIPK1 inhibitor,which can inhibit RIPK1 phosphorylation.CCK-8 assay and flow cytometry were used to detect the effect of C-PC on the proliferation and necrosis rate of GC-1 spg cells induced by TSZ.Immunofluorescence,q RT-PCR and Westernblot were used to detect the expression of necroptosis-related genes and proteins(TNF-α,RIPK1,RIPK3,MLKL,p-MLKL)induced by TSZ to explore the regulatory effect of C-PC on RIPK1-RIPK3-MLKL signal pathway.2.Construction of reproductive injury model of ICR male mice: the mouse model of reproductive injury was established by intraperitoneal injection of cyclophosphamide(Cy).The mice were divided into four groups: Control,Cy Model,Cy+C-PC(low concentration)and Cy+C-PC(high concentration).The effects of C-PC on the reproductive organs and sperm quality of mice were evaluated by calculating the coefficient of reproductive organs,counting the number of sperm,calculating the rate of deformity and mortality.3.The contents of superoxide dismutase(SOD)and malondialdehyde(MDA)in testicular tissue was detected to explore the protective effect of C-PC on testicular oxidative damage.The levels of testosterone(T),follicle stimulating hormone(FSH)and luteinizing hormone(LH)in serum of mice were detected by ELISA to study the effect of C-PC on spermatogenesis in mice.4.The testicular tissue was stained with HE,the pathology of testicular tissue was observed,and the spermatogenic kinetics of mice was analyzed to explore the protective effect of C-PC on testicular spermatogenesis.5.The expression of RIPK1,RIPK3,MLKL and p-MLKL in testicular tissue of male mice induced by Cy was detected by immunohistochemistry and westernblot to explore the specific mechanism of protective effect of C-PC on testicular tissue.Results:1.Low concentration of C-PC can promote the proliferation of GC-1spg cells,while high concentration of C-PC can inhibit the proliferation of GC-1spg cells.TSZ can inhibit the proliferation of GC-1 spg cells in a time-and dose-dependent manner.With the increase of the treatment time of TSZ,the necrosis rate of GC-1 spg cells increased gradually,and the expression of RIPK1,RIPK3,p-MLKL,TNF-α and other proteins also increased with the increase of TSZ concentration.Compared with TSZ treatment group,the viability of GC-1spg cells increased significantly and the necrosis rate decreased significantly in TSZ+C-PC and TSZ+Nec-1 treatment group.In addition,the expression of RIPK1,RIPK3,p-MLKL and TNF-α decreased in TSZ+C-PC and TSZ+Nec-1 treated group.2.Compared with the control group,the reproductive organ coefficient and sperm quality of the Cy model group decreased,the sperm number decreased,the sperm deformity rate and mortality increased,and the fecundity of the mice decreased.Compared with the Cy model group,the reproductive organ coefficient and sperm quality of the C-PC group were improved,the sperm number increased,the deformity rate and mortality decreased,and the fecundity increased.And the effect of high concentration of C-PC is more significant.3.Compared with the control group,the activity of SOD decreased and the content of MDA increased in the testicular tissue of mice treated with Cy,while the activity of SOD increased and the content of MDA decreased in the group treated with C-PC,the effect of high-concentration C-PC was more significant.Compared with the control group,the release levels of T,FSH and LH in the serum of Cy-treated mice decreased,while the release levels of these three hormones in C-PC-treated mice increased compared with Cy model group.4.Through the analysis of the spermatogenic kinetics of mice,compared with the control group,the number of spermatogenic cells in testicular tissue of Cy model group decreased,the volume of Leydig cells and nucleus decreased,the epithelial cells of seminiferous tubules became thinner,the number of spermatogenic cells decreased,and a cavity was formed in the lumen.Compared with Cy model group,the number of spermatogenic cells in testicular tissue of C-PC group increased.The volume of Leydig cells and nucleus increased,the injury degree of seminiferous tubules improved,and the spermatogenic cells increased and arranged orderly.The effect of high concentration of CPC was more significant.5.Compared with the control group,the expression levels of RIPK1,RIPK3,p-MLKL and MLKL in the Cy model group increased,but after C-PC treatment,the expression of these proteins decreased.Conclusion:C-PC could reduce the damage of GC-1 spg cells induced by TSZ,improve cell viability,reduce the rate of cell necrosis,inhibit the signal pathway of necroptosis,and play a protective role in GC-1 spg cells.In addition,C-PC could also improve the decrease of sperm density caused by Cy,reduce the rate of sperm deformity and mortality,improve the fecundity of mice,alleviate the damage of testis caused by Cy,and protect the reproductive system of mice.The protective mechanism may be that C-PC inhibited Cy-induced oxidative stress and necroptosis signal pathway.