The Effect And Mechanism Of Sterol In Hippocampus Kuda On Mice Reproductive System

Objectives:The aim of this study is to examine the effect and mechanism of sterol extract in Hippocampus Kuda on male mice reproductive system, in order to provide theoretical basis for using hippocampus as traditional Chinese medicine.Methods:1. Three factors associated with extracting effect were determined, including extracting temperature, extracting time and ratio of material to liquid. Sterol in hippocampus were extracted with soxhlet apparatus. Then we determined the absorbance of samples with the vanillin-perchlorate color method after saponification. The content of total sterol in the hippocampus was determined with a standard of cholesterol. Furthermore, we optimized the extracting conditions of total sterol in Hippocampus kuda by Box-Behnken response surface method.2. The purified sample was further identified by high performance liquid chromatography (HPLC) using water, acetonitrile and isopropyl alcohol as eluent.3. Seventy-two ICR health adult male mice were divided into four groups randomly. The control group was treated with0.9%physiological saline intraperitoneally, while the low dose exposure group treated with1mg/mL hippocampus sterol, the middle dose group with3mg/mL, and the high dose group with5mg/mL once a day for10,20and30days respectively. Each group mice were sacrificed in turn by cervical dislocation at the10th,20th and30th day for further analysis of the change of the each viscera coefficient, the sperm concentration and movement parameters and levels of testosterone (T), follicle-stimulating hormone (FSH), luteotrophic hormone (LH), HDL-c and LDL-c of the mice sera. Lactate dehydrogenase (LDH), acidphosphatase (ACP) and alkaline phosphatase (AKP) in testicular homogenate were also detected using the determination kit. The changes of the organizational structure of mice testis and kidney were observed by light microscope. The changes of expression levels of StAR, P450scc, P45017and P450arom genes in mice testis were detected by quantitative RT-PCR technology.Results:1We obtained the data model by the response surface analysis. The amount of total hippocampus sterol extraction (mg/g)=4.85+0.38xA+0.74×B+0.62×C+0.32xAxB+0.17xAxC+0.40xBxC-0.78xA2-0.63xB2-0.10×C2(R2=0.9722).A represents extraction temperature/℃, B represents time/h, C represents ratio of material to liquid/ml-g-1. The results showed that the highest total sterol extraction amount was obtained to5.17mg/g when the extraction temperature was at79℃, extraction time at7.7h, and the ratio of solid to liquid at80ml/g.2By high performance liquid chromatography (HPLC) analysis, sterol material separation conditions were determined. Mobile phase was selected as23%acetonitrile,70%water and7%isopropyl alcohol.3Hippocampus sterol extraction has no significant effect on mice body weight and viscera coefficient.4In certain degree, hippocampal sterol can significantly increase the mice sperm concentration, however, high concentrations of sterol in the early time of gavage had relatively weak effect on the promotion of sperm function.5Hippocampus sterol extract can affect the hormone levels of the mice, which has a promoting effect on T, LH and HDL-c, but little effect on FSH, LDL-c and LDH. The mechanism of impact on the ACP and AKP is complicated which needs to be studied further.6By observation of the kidney and testis of HE staining slice, we found that the hippocampus sterol extract can change mice testicular seminiferous tubule structure, increase more layers of seminoma cells, enhance the numbers of sperm cells, ley dig cells and mature sperm at various levels. It also can promote the formation of sperm. But after high dose of intake hippocampal sterol extract for a long time, the structure of kidney and testis in mice has certainly changed, indicating a chronic toxic effect.7By fluorescence quantitative RT-PCR, we found that low doses of hippocampus sterol extract can increase the expression of the StAR, P450scc and P450c17genes and inhibit P450arom gene synthesis. Also, low doses can increase the testosterone levels in mice, ultimately improve the synthesis and secretion of the sperm.Conclusion:1Hippocampal sterol has no acute toxic effects in mice, but an obvious improved effect on sperm concentration.2High concentrations of hippocampal sterol intake in a long term has some damage effects to mice reproductive system.3Hippocampal sterol can improve the quantity of related gene expression of steroid hormones which affect hormone levels to regulate spermatogenesis in mice.