Immunological Mechanisms Of Squid Ink Polysaccharide Relieves Premature Ovarian Failure Induced By Cyclophosphamide In Mice

Objective: At present, cyclophosphamide(CP)is an commonly used as nitrogen mustard chemotherapy drug in clinic, not only could killing tumor cells, but also having strong side effects to the body normal tissues, which makes it is limited to widely used in clinic. Premature ovarian failure(POF)is a kind of ovarian function failure syndrome induced by various diseases, however, abnormal autoimmune is an important factor which causes premature ovarian failure. Squid ink polysaccharide is the marine active substances separated and extracted from squid ink, which composition, activity and structure of squid ink polysaccharide has been widely reported, but the immunological efficacy and mechanism of squid ink polysaccharide alleviate premature ovarian failure induced by chemotherapy is very few study and report.Therefore, the study establish a model of premature ovarian failure induced by chemotherapy drug cyclophosphamide, at the same time polysaccharide oral gavage, so that could trying to observe the changes of viscera tissue physiological indexes, blood indexes and ovarian tissue pathological slices. Thus to discuss the alleviation efficacy and the possible immunological mechanisms of squid ink polysaccharide to premature ovarian failure induced by cyclophosphamide, which provides a scientific theory basis for the development and clinical application.Method: According to the weight, 60 SPF female KM mice are randomly divided into 6 groups: normal group, model group, treatment group1(SIP :50 mg/kg),treatment group2(SIP :65 mg/kg),treatment group3(SIP :80 mg/kg)and treatment group4(SIP :110 mg/kg). After 15 days of the trial period, detecting the index changes of weight, ovary index, spleen index and thymus index, ovary SOD activity and MDA content, so that could trying to explore and determine the best dose of squid ink polysaccharides.According to the weight, 40 SPF female KM mice are randomly divided into 4 groups: normal group(CON), model group(CP), the polysaccharide group(SIP) and treatment group(CP+SIP). After 15 days of the trial period, detecting the changes of ovary index and serum sex hormone levels, and observing ovarian tissue pathological slices.According to the weight, 40 SPF female KM mice are randomly divided into 4 groups: normal group(CON), model group(CP), the polysaccharide group(SIP) and treatment group(CP+SIP). After 15 days of the trial period, detecting the changes of ovary index, spleen index and thymus index, serum immune level and ovarian immune factor content.According to the weight, 40 SPF female KM mice are randomly divided into 4 groups: normal group(CON), model group(CP), the polysaccharide group(SIP) and treatment group(CP+SIP). After 15 days of the trial period, detecting the changes of Nrf2/ARE signaling pathways related expression amount of proteins and enzymes, so that could trying to explore the antioxidant pathways and mechanism of squid ink polysaccharide activation female POF mice mediated by cyclophosphamide Nrf2/ARE signaling pathways.Result: Squid ink polysaccharide can alleviate or inhibit ovarian oxidative damage caused by chemotherapy, suppressing the reducing of ovary index, spleen index and thymus index, Significantly improving SOD activity(P<0.05)and reduce MDA content P<0.05)in ovary, in order to reduce the level of lipid ovary, maintain normal growth performance in mice, which shows strong antioxidant power. Finally, it is concluded that each with 80 mg/kg squid ink polysaccharide(CP: 120mg/kg) is the best dose to alleviate the damage of POF induced by cyclophosphamide.After intraperitoneal injection of cyclophosphamide in mice, ovary index decreased significantly(P<0.05), FSH levels and LH levels in serum significantly increased(P<0.01), E2 levels in serum significantly lower(P<0.01), the number of ovarian follicles at different levels decreased(P<0.01), ovarian reserve capacity is reduced(P<0.01), and ovarian tissue mesenchymal transition. Compared with model group, treatment group of squid ink polysaccharide can change the changes of the above-mentioned indexes(P<0.05 or P<0.01) in different degrees, which caused by POF mice induced in cyclophosphamide.Cyclophosphamide can significantly decrease ovary index,spleen index and thymus index of POF mice(P<0.05, P<0.01), serum levels of CD4+T%, CD4+/CD8+ratio, IL-2and TNF alpha level significantly decreased(P<0.05, P<0.01), and CD8+T%, NK% content increased significantly(P<0.05, P<0.01), IL-2 and TNF alpha in ovarian tissue levels were significantly lower(P<0.05, P<0.01); Compared with model group, the indicators above-mentioned in treatment group have been changed indicators in different degrees(P<0.05, P<0.01), so the squid ink polysaccharide could protect the mice immunity from the damage of POF induced by cyclophosphamide.Cyclophosphamide could obviously decrease(P<0.05, P<0.01) expression quantity of Nrf2/ARE signaling pathways related protein including Nrf2、HO-1、NQO1、HDAC2 in POF mice ovary, while the PKC and Keap1 expression quantity increased significantly(P<0.05, P<0.01); Compared with model group, treatment group squid ink polysaccharide can obviously(P<0.05, P<0.01) improve the expression quantity of Nrf2, HO-1, NQO1, HDAC2 and reduce the amount of PKC and Keap1 expression. From the above results, it can be concluded that squid ink polysaccharide by regulating the expression quantity of Nrf2/ARE signaling pathways related protein and II detoxification enzymes, so that alleviate the oxidative damage of ovary in POF mice induced cyclophosphamide.Conclusions: Squid ink polysaccharide has some of degree alleviation and protective effect to the damage of POF mice induced by cyclophosphamide immune system, reproductive system and viscera, and its mechanism maybe through adjusting mice body’s immunity in order to reduce the damage in ovarian and related viscera, at the same time, stimulating the ovaries Nrf2/ARE signaling pathways, alleviating the oxidative damage of POF induced by cyclophosphamide in mice ovary.