Regulatory Mechanism Of Squid Ink Polysaccharide Against Autophagy And Apoptosis Of Testicular Cells In Mice Exposed To Cyclophosphamide

Cyclophosphamide(CP), one of the most widely used alkylating agent, has no anti-tumor activity in vitro, unless it is metabolized in the body. Acrolein(ACR) is a major metabolite of CP in vivo with strong oxidative capacity. As a main toxic factor of CP, ACR has been found to damage normal organs. Numerous researches show that CP can obviously lead to damage of the male reproductive system, but its mechanism of injury is still unclear. This study revealed that, it may be an important reason that CP-induced toxicity of male reproductive system was resulted from CP’s metabolite-mediated oxidative stress and apoptosis. After administration of CP mice were observed a mass of abnormal features in testes, such as weight loss, hollowed seminiferous tubules, damaged Leydig cells, decreased spermatogenic cells, added abnormal sperms, increased ROS and MDA, declined activities of SOD and CAT. Moreover, in spermatogonia and Sertoli cells, nuclei were broken or pyknosis and cristae disappeared in swelled mitochondria. Many autophgosomes and autophagic vacuoles were presented in Leydig cells. Apoptotic index significantly increased and the positive cells of apoptosis occured mainly in spermatogonia. Increased contents of LC3 and Beclin-1 proteins were mainly distributed in Leydig cells. While Leydig cells were exposed to ACR in vitro, activities of the cells were reduced on a dose-dependent manner, intracellular activity of SOD and T-AOC were decreased and content of MDA was elevated. Cells were damaged by oxidative stress ACR caused, which resulted in apoptosis and autophagy of Leydig cells and reduction of secreted testosterone. ACR-caused autophagy occurred before apoptosis, apoptosis should be triggered while autophagy was inhibited, which suggested that an antagonistic association must exist between autophagy and apoptosis.Autophagy and apoptosis share mutual regulatory mechanisms, such as p38 MAPK and PI3KI/Akt signaling pathway. In the process of CP-induced autophagy and apoptosis of testicular germs cells, p38 MAPK and PI3K/Akt signaling pathway were activated. It was also found that p38 MAPK signaling pathway promoted autophagy and apoptosis in the ACR-treated Leydig cell. While the autophagic capacity of Leydig cells overloaded, p38 MAPK pathway triggered proapoptotic program to induce apoptosis.Squid ink polysaccharide(SIP), a marine active substance with chemoprophylactic activity, is able to inhibit toxic injury of male reproductive system caused by CP. In this study, SIP-1, a main component of SIP, was used to antagonize CP-induced apoptosis and autophagy of testicular germ cells. The results show that SIP-1 could effectively alleviate CP-mediated testicular oxidative damage through improving the activities of SOD, CAT and reducing the content of MDA, removing excess intracellular reactive oxygen species(ROS), preventing structural damage of seminiferous tubules, increasing the number of spermatogenic cells, reducing sperm deformity rate and suppressing the decrease of body weight in mice. In vitro, SIP-1 has significant protective effects on Leydig cells under the stimulation of ACR. SIP-1 improved cell viability, inhibited oxidative stress, promoted testosterone secretion and protected secretory function of Leydig cell. It was because SIP-1 eliminated the excess ROS in germ cells which was helpful for avoiding damage of mitochondria structure, prevented activation of apoptotic factor Caspase-3 and reduced Bax/Bcl-2 ratio which resulted in inhibition on apoptosis of germ cells. Meanwhile, SIP-1 also reduced the over-expression of autophagy-related proteins, Beclin-1 and LC3, and avoided apoptosis caused by autophagy that surpassed cellular tolerance. However, in this process, moderate autophagy was still remained to resist apoptosis and to maintain cellular homeostasis and prolong cell life. These results imply that SIP-1 mainly employs antioxidative function to play cytoprotective roles. In addition, SIP-1 was able to impair autophagy and apoptosis regulated by p38 MAPK pathways. Therefore, SIP-1 antagonized CP- or ACR-induced apoptosis and autophagy, improved the tolerance of germ cells to chemotherapy drug, and resultantly relieved reproductive toxicity of CP.