Intervention Effect And Mechanism Of Wuzi Yanzong Pill On Heat Shock In Rats

Objective:Male infertility is a clinical common disease, recently, it has become a global medical and social problems which influence human health and social development by the rising of proportion of male infertility. A variety of reasons, such as spermatogenic or sperm maturation dysfunction, vas deferens obstruction, sexual dysfunction, abnormal accessory sex gland, reproductive tract infection and immune factors can lead to male infertility, of which the spermatogenic dysfunction is the main reason. Local high temperature exposure of the testis is recognized as one of the strongest destructive determinant on male reproductive ability by world health organization (WHO).From the viewpoint of traditional Chinese medicine (TCM), the kidneys store the essence and dominate reproduction. The exuberance and debilitation of kidney essence decides the male reproductive ability, and kidney essence deficiency is the etiology of male infertility. Wuzi Yanzong Pill (WYP) is known as the "the first seed party in ancient and modern", and has been used as a classical prescription for male infertility with kidney essence insufficiency. Although WYP can significantly improve male reproductive ability, in current clinical application and animal experimental studies, the mechanism is unclear. To investigate the potential mechanisms of WYP treating spermatogenic dysfunction can provide new targets and thinking for clinical treatment of male infertility.In our study, spermatogenic dysfunction models were established by rat testicular heat stress at 43℃ for 20 min, to investigate the function and potential mechanisms of WYP preventing spermatogenic dysfunction induced by heat stress, and could provide new targets for clinical treatment of male infertility.Methods:1. Thirty-five 60-day-old male Wistar rats were randomly allotted into five experimental groups:control, model, low-dose WYP, medium-dose WYP, and high-dose WYP. The rats in the low-dose WYP, medium-dose WYP and high-dose WYP groups were given intragastrically medicated with WYP granules at 418 mg/kg,836 mg/kg, and 1672 mg/kg, respectively. The model and control groups were given intragastrically distilled water at equal volume, once daily for 15 days. After anaesthesia with an intraperitoneal injection of 10% chloral hydrate (0.3 ml/100g body weight), the scrotum containing testes were then immersed in a thermostatically controlled water bath at 43℃ for 20 min. Control rats were treated in the same way, except the testes were immersed in a water bath maintained at 22℃. Then we sacrificed these animals and weighed testis at 30 min after heat stress.2. The serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were measured by radioimmunoassay (RIA).3. The expression and distribution of Ki67 of rats testicular tissue were detected with immunohistochemistry.4. Germ cell apoptosis was evaluated by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay.5. The ultrastructural changes in rat seminiferous tubules were examined with the transmission electron microscope.6. The expression of Akt, p-Akt, CK-18, AR, EPO and EPOR proteins of testicular tissue were detected with Western Blot.Results:Experiment one:No significant changes were noted in serum FSH and LH levels in different groups. After heat stress, serum T levels decreased in the model group, but it had no statistically significance (P>0.05).This elevated serum T levels were measured in the WYP groups, and the serum T levels of Low-dose WYP group had significantly increased comparing with the model group (P<0.05), the serum T levels of Medium-dose WYP group nearly returned to the levels of control group.In the control group, TUNEL positive apoptotic germ cells were rarely observed in seminiferous tubules, the morphology and structure of cells were intact, the junctions between cells were tightness, and there were many Ki67-positive germ cells in seminiferous tubules. After heat stress, the number of TUNEL-positive germ cells of seminiferous tubules were increased, the structure of the seminiferous tubules became loose because of many vacuoles, and the number of Ki67-positive germ cells were sharply decrease, leading to testicular weight significant decrease (P<0.01). In the WYP groups, compared with the model group, the number of TUNEL-positive germ cells were decreased(In particular, TUNEL-positive germ cells of Medium-dose WYP group were significantly decreased), the morphology and structure of cells in the seminiferous tubules were nearly intact, and the number of Ki67-positive germ cells were increased(In particular, Ki67-positive germ cells of Low-dose and Medium-dose WYP groups were significantly increased), the testicular weight significantly increased (P<0.01).Experiment two:The representative results of Western blot analysis show that the expression of p-Akt protein was dramatically decreased (P<0.01) in the model group, but the expression of p-Akt protein increased (P<0.05) in the Medium-dose WYP group compared with the model group.The results of CK-18 protein show that the expression was rarely detected in the control group, the expression of CK-18 protein increased significantly (P<0.01) in the model group. However, the expression of CK-18 protein was decreased significantly (P<0.01) in the WYP groups compared with the model group. The results of AR protein show that the expression was strongly detected in the control group, after heat stress, the expression of AR protein levels decreased significantly (P<0.01) in the model group. However, the expression of AR protein was significant increased (P<0.01) in the Low-dose and Medium-dose WYP groups compared with the model group. The results of EPO protein show that the expression of EPO protein levels decreased significantly (P<0.01) in the model group after heat stress. However, the expression of EPO proteins were significant increased (P<0.01) in the WYP groups compared with the model group. The results of EPOR protein show that the expression of EPOR protein was almost no change. However, the expression of EPOR proteins were significant increased (P<0.01) in the WYP groups compared with the model group.Conclusions:1. WYP can effectively prevent from germ cells apoptosis and ultrastructural changes of seminiferous tubules induced by heat stress, and promote the activity of germ cells cell proliferation.2. WYP could regulate the expression of AR and T through stimulating the expression of EPO and its receptor increased, to promote Sertoli cells differentiation and germ cells proliferation, and effectively prevent from germ cells apoptosis and ultrastructural changes of seminiferous tubules induced by heat stress, in order to protect the testicular function and prevent from spermatogenic dysfunction, and this process may be closely related with Akt signaling pathways.