Experimental Study On Repair Effect And Mechanism Of Phycocyanin On Reproductive Injury Of Male Mice After Smoking

Objective: To study the repair effect of phycocyanin(PC)on the oxidative stress injury of male reproductive system caused by smoking and its mechanism.Methods: Sixty 8-week-old male mice were divided into 6 groups,the smoking group(CON group)was exposed to cigarette smoke for 6 weeks,while the drug treatment group received cigarette smoke exposure.High-dose(500 mg/kg/day,PC-H group),medium-dose(300 mg/kg/day,PC-M group)and low-dose(100 mg/kg/day,PC-L group)phycocyanin were administered by gavage,respectively.The blank control group(CTL group)did not receive cigarette exposure,they were given the same amount of normal saline for 6 weeks only.The PC-only group was given PC(500mg/kg/day,PC group)by gavage for 6 weeks,and was not exposed to cigarette smoke.The mice were sacrificed 1 week after the modeling and the testis and epididymis tissues were collected.Paraffin sections of the right testis and epididymis were prepared for HE staining and TUNEL staining to observe the anatomical structure and apoptosis of testis and epididymis.RNA and total protein were extracted from the left testicle to detect the the expression levels of germ-cell related genes(DDX4,PLZF,Tn P1),antioxidant genes(CAT,SOD,GPX),pro-apoptotic genes(Bax,Casepase3)and anti-apoptotic genes(Bcl2)in testis of mice in each group by Q-PCR.The expression levels of nuclear factor-related factor 2(Nrf2)which is a key transcription factor regulating oxidative stress response,pro-apoptotic protein(Bax,Casepase3)and anti-apoptotic protein(Bcl-2),were detected by Western blot.The expression levels of antioxidant malondialdehyde(MDA),antioxidant superoxide dismutase(SOD),reduced glutathione(GSH)and total antioxidant capacity(T-AOC)in testicular tissues of mice in each group were detected by ELISA.Finally,we made a statistical analysis of the experimental results.Results:1.The testis and epididymis tissues of each group were prepared into paraffin sections,stained with HE,and observed under a microscope: In the blank control group,the basal membrane of the concentric seminiferous tubules of the testis was intact,the spermatocytes at all levels developed normally and were arranged neatly,and the structures of the supporting cells and stroma cells were normal.The lumen surface of the epididymal duct is neat,the lumen is filled with sperm,the epithelial basement membrane of the epididymal duct is complete,and the cells are arranged neatly.In the smoking model group,the distribution of convoluted seminiferous tubules was sparse,the number of interstitial cells was reduced,the basement membrane of the convoluted seminiferous tubules was broken,the number of spermatogenic cells at all levels was reduced and the arrangement was disordered,and the supporting cells were sparse.The epithelial basement membrane of the epididymal duct was incomplete,the number of primary cells and basal cells of the epithelium was reduced,the hierarchy was uneven,some cells were ruptured,the structure of microvilli was abnormal,and the sperms in the epididymal duct were sparse.The physiological structure damage of testis and epididymis in PC-H,PC-M and PC-L groups was alleviated,and the therapeutic effect of PC-H group was the most significant.2.TUNEL staining results showed that compared with the control group,the number of apoptotic cells was increased in the smoking group,mainly located near the basilar layer of the convoluted seminiferous duct.The apoptotic cells were significantly reduced in different doses of cyanobacteria protein.3.Q-PCR results showed that: Smoking decreased the expression of germ-cell related genes PLZF and TNP1,High concentration of cyanobacteria protein could significantly increase the expression of PLZF and TNP1.Smoking reduced the expression of antioxidant enzymes in the testicular tissue of mice,Compared with smoking group,high concentration of phycocyanin could significantly increase the expression of antioxidant enzymes.Smoking increased the expression of pro-apoptotic gene Casepase3 and decreased the expression of anti-apoptotic gene Bcl.Compared with smoking group,high concentration of phycocyanin could decrease the expression of pro-apoptotic genes and increase the expression of anti-apoptotic genes.4.Western blot results: Smoking increased the expression levels of apoptotic proteins Bax and Caspase 3,and decreased the expression levels of anti-apoptotic transcription factors Nrf2 and Bcl2.Different doses of phycocyanin can reduce the expression of Bax and Caspase 3 protein,and increase the expression of Nrf2 and Bcl2 protein,and the effect of H-PC group is better than that of M-PC group and L-PC group.The results showed that smoking could cause apoptosis of testicular tissue cells in mice,and phycocyanin had the repair effect,and the high concentration of phycocyanin had the best effect.The results were consistent with the results of q PCR.5.ELISA results showed that: Compared with the control group,the level of oxide MDA in testicular tissue of mice in smoking group was significantly increased.The levels of SOD GSH and T-AOC decreased significantly,and the differences were statistically significant.The levels of SOD and T-AOC in different doses of phycocyanin treatment group and PC group were higher than those in smoking group,and the difference was statistically significant.The GSH level of H-PC M-PC group and PC group was higher than that of the control group,and the difference was statistically significant,The level of GSH in L-PC group was higher than that in control group,and the difference was not statistically significant.Compared with the control group,the MDA level of H-PC M-PC and PC groups decreased,and the difference was statistically significant,The MDA level of L-PC group was lower than that of the control group,and the difference was not statistically significant.This suggests that smoking can affect the oxidative level of testis in mice;phycocyanin can repair testicular damage caused by smoking through antioxidant pathway.In addition,H-PC group and M-PC group had better effect on the repair of testicular injury caused by smoking than L-PC group.Conclusion:1.The model of male reproductive system injury induced by smoking in mice was successfully established.2.Phycocyanin can effectively repair the damage of male reproductive system functionn caused by smoking in mice,and with the increase of the therapeutic dose of phycocyanin,the therapeutic effect is gradually enhanced,and 500 mg/kg/ day phycocyanin has the most significant therapeutic effect.3.The effect of phycocyanin on male reproductive function injury induced by cigarette smoke in mice may be realized by mediating Nrf2 signaling pathway to regulate oxidative stress response.