Nrf2/ARE Signal Pathway Participates In The Interventional Effects Of Squid Ink Polysaccharides On Cyclophosphamide-associated Oxidative Damage In Testis

At present, many studies have confirmed that Squid ink polysaccharide（SIP）was amarine bioactive substance which has broad-spectrum biological functions, it can be usedin the fields of food&health protect&medicine. The researchers in our lab had spent along-term commitment to study the protective effect of SIP on chemotherapy. SIP couldsignificantly protect heart, lung, liver and spleen, and also could reduce the toxic effect oftestes induced by cyclophosphamide（CP）. Studing the chemotherapy protective effect ofSIP on reproductive injure induced by CP is the primary goal of this study.First of all, SIP was obtained from squid ink by enzyme extraction and ethanolprecipitate, and purified by column chromatography. Then the purity and molecularweight of it were detected by ultraviolet spectroscope method and high performanceliquid chromatography. The basic structure of SIP was verified by infrared. The resultsshow that a single elution sample was obtained by isolated and purified. The averagemolecular weight of the sample was37068Da. UV scanning result showed that therewere no absorption peak detected at260nm and280nm, this explained that the sampledid not contain any protein and nucleic acid. High performance liquid chromatographyfingerprint had a good symmetrical peak, which implied the sample was pure. Thecharacteristic absorption peak of polysaccharides and sulfuric acid group were observedby infrared spectrum.60healthy male kunming rats were randomly divided into6groups,10rats pergroup. The index of reproductive organ, sperm parameter, testis pathological section, andmale reproductive capacity were observed after10weeks. CP could lead to malereproductive damage, consistent with the result of previous study. it could significantly（p<0.01or p<0.05）decreased the mice weight, the weight of testis and epididymis, andthe rate of testis, but had no obvious effect（p>0.05）on the rate of epididymis. Spermcounts and sperm viability were also significantly (p<0.01or p<0.05）declined, and allkind of teratospermia and sperm abnormality rate were increased significantly（p<0.05）. Microscopic analysis of testis displayed that the testicular tissue was seriously damaged,the decreasing of different stages of spermatogenic cells, especially primary sperm cells.The gap between the seminiferous increased, leydig cells damaged, and the demeter ofseminiferous tubule also significantly(p<0.01) lowered by CP. Male reproductive abilitywas reduced by CP, fecundation index lowered, the number of litter size and liver fetusesrate sharply(p<0.01) dropped and fetal deaths rate increased, but there was no difference（p>0.05）on fetal malformations rate. Compared with the model group, SIP couldimprove all kinds of target significantly(p<0.01or p<0.05), but had no obvious effects（p>0.05） on sperm counts, the number of midpiece broken sperm and fetalmalformations rate.40healthy male kunming rats were randomly assigned to4groups (control group,model group, SIP group and treated group). Iconic enzyme activity, sex hormone levelsand oxidative stress injury were observed. Compared with the model group, SIPsignificantly (p<0.01or p<0.05）improved the abnormal effect of the characteristicenzyme in the testis, for instance, the activity of LDH increased remarkablely(p<0.05),the activity of AKP, ACP and γ-GT significantly(p<0.01or p<0.05) reduced, but theactivity of LDH、ACP and γ-GT were no difference（p>0.05）with the blank group. SIPperfected the abnormal changes of hormones levels in the serum and testis, the content ofFSH、LH and E reduced significantly(p<0.01or p<0.05), testosterone content increasedsignificantly (p<0.01) in the serum, and testosterone content rose in the testis. SIPimproved oxidative stress damage of mice testis induced by CP. Compared with the blankgroup, CP could remarkablely（p<0.01）increase the content of MDA and NO, sharply（p<0.01）reduced the activity of SOD, CAT, GR, GPX and GST, and significantlydropped the content of GSH and Vc, significantly（p<0.01）dropped T-AOC. Whencompared with the model group, SIP significantly(p<0.01or p<0.05) improved theactivity of antioxidant enzyme and the content of antioxygen, dropped MDA and NOcontent, and remitted the redox damage of mouse testes.The way of grouping was the same with above. Total protein were extracted frompyrolysised testicular tissue and by high-speed refrigerated centrifuge extraction. Thecontrolling genes and target genes related with Nrf2/ARE signaling partway in testiculartissue were examined by western blot. Compared with the blank group, CP significantly(p<0.01or p<0.05)dropped Nrf2, Keap1and NQO1protein expression, but had noobviou（sp>0.05）effect on HO-1, HDAC and PKC protein expression. When compared inthe model group, Nrf2, NQO1, HO-1, Keap1, HDAC2and PKC protein expression significantly increased by SIP pretreatment, which display that SIP remitted the decreaseof HO-1, HDAC and PKC protein expression by CP.SIP could effectively improve male reproductive damage induced by CP. Itsprotective effect is associated with that it improved the activity of the charateristicenzyme and redox homeostasis in testis, and maintain the balance of sex hormone level inthe serum and testis. The protective effect of SIP on male reproductive damage related toits antioxidant actions. It is a powerful antioxidants, could directly eliminate excess ROS,and maintain redox homeostasis. It, is a inducer, could indirectly activate Nrf2/AREsignaling pathway and promote the expression of antioxidant enzyme gene andphaseⅡdetoxifying enzyme gene to resist the adverse factor. Nrf2/ARE signalingpathway was activited through the sulfydryl of Keap1was oxidized by SIP. the stabilityof Nrf2was maintained through SIP increased the expression of HDAC2and p-PKC,anddeacetylation of HDAC2and phosphorylation of p-PKC.