| Background and Objectives:With the development of science and technology, humans have longer life span. The world has entered the aging era. The elderly’s health become the current social problems of common concern. In the male science, male climacteric syndrome is one of the hot research direction. Although the diagnosis and treatment of male climacteric syndrome are still controversy, partial androgen deficiency is regarded as one of the important causes. With the increase of age, the body organs and systems including the reproductive organs are gradually going senile degeneration. The males showed testicular atrophy, Leydig cells apoptosis, synthetic androgen secretion function decay. Androgen supplement therapy (AST) by chemical synthesis is now the main treatment of the male menopause syndrome. Long term application of human androgen synthesis can cause a variety of adverse reactions. Drug safety become a bottleneck of the popularity of the use of androgen replacement therapy. Therefore, to explore the development of biological androgen replacement therapy, promote the Leydig cell regeneration and so on, opens up a new way for the study of new treatment method.Leydig cells are distributed in groups in the seminiferous tubules in loose connective tissue. From adolescence onwards, Leydig cells functioned by the anterior pituitary basophil interstitial cell stimulating hormone (luteinizing hormone, LH), can synthesize and secrete male hormone (testosterone, T). Testosterone is transported to target organs throughout the body. It plays an important physiological function by binding to the androgen receptor, such as the promotion of spermatogenesis and male reproductive development, and maintain the secondary sexual characteristics and sexual function.Basic fibroblast growth factor (bFGF) widely exists in the brain, pituitary, liver, kidney, bone, cartilage, skin cells, vascular smooth muscle cells, myoblast, stellate cells and other tissues. It can promote cell proliferation, differentiation and regulation of cell function in the mesoderm and neural ectoderm. Research showed that, this factor can promote angiogenesis, wound healing, tissue repair and regeneration, and the embryo development and differentiation. Insulin-like growth factor1(IGF1) is mainly from the human liver cell synthesis and secretion. It plays an important regulating role in embryo differentiation, ontogeny, sugar and fat metabolism. The male reproductive system derived from the mesoderm. Research aspects of bFGF and IGF1on Leydig cell differentiation, proliferation and regeneration of the adjustment effect is seldom reported.This paper described is to use adult Leydig cell specific inhibitor (Ethane Dimefhane sulfonaft, EDS) on the rat adult Leydig cells (ALC) apoptosis, construction of vivo animal model, and the seminiferous tubules in in-vitro cultured model. And to systematically study the regeneration and proliferation of the bFGF and IGF1on LC. It aims to clarify the molecular mechanism in order to carry out induced differentiation in the Leydig stem cells, and to promote regeneration method of the LC for the treatment of male climacteric syndrome, and provide theoretical and experimental basis. According to the study objectives, the experiment is divided into3parts:(1) Construction of rat ALC knock-out model. From two aspects which are rats in vivo and in vitro cultured seminiferous tubules construction.(2) Investigate the effects of bFGF and IGF1on Leydig cell regeneration and secretion of testosterone in vivo rats(3) Investigate the effects of bFGF and IGF1on Leydig cell regeneration of seminiferous tubules in vitro cultured rats, and effects of secretion of testosterone.Objective:First, we induced Leydig cell of adult male rats apoptosis with intraperitoneal injection of ethane dimethane sulfonate (EDS). Secondly, we used the intraperitoneal injection of bFGF and IGF1and discussed the influence and mechanism of testosterone secretion on rats. At the same time, Q-PCR and Western Blot were employed to detect the changes of bFGF and IGF1during LC regeneration after EDS treatment in rats. Moreover, we performed studies with in vitro model to investigate bFGF and IGF1effects on proliferation, differentiation and androgen secretion of Leydig cells.Methods:The adult male SD rats were randomly divided into normal group, solvent control group, EDS-treated group. EDS-treated groups were divided into physiological saline group, bFGF group, FGFR-inhibitor (PD166866) group, IGF1group, IGFR-inhibitor (PPP) group.7d later, we begain to use bFGF and IGF1by intraperitoneal injection accordance with100μg/kg/day for1week after single intraperitoneal injection of EDS to the rats belong to bFGF group and IGF1group. Simultaneously, we begain to use PD166866and PPP by intraperitoneal injection accordance with100μg/(kg·d) to the rats belong to FGFR-inhibitor group and IGFR-inhibitor group for one week. Physiological saline are used by intraperitoneal injection accordance with0.6mL/d to the rats belong to hysiological saline group for one week. And then, we killed the rats on the14th,21st,35th,56th day after the injection of EDS based on the Leydig cell proliferation and differentiation of reresentatives at different time points. We weighed the weight of the liver, bilateral kidneys and bilateral testis. We determined the FSH and testosterone content with RIA method at all time poins. We collected total testicular tissue protein at all time points,detect the espression of StAR,one of the testosterone synthesis key speed limited enzymes. Finally, we analysised and compared the data in each group by SPSS software.We established the organ culture model of rat seminiferous tubules, which were isolated from90-day-old rat testes. After treatment with different concentrations of bFGF and IGF1, examined cell proliferation with EdU labeling; Investigating bFGF and IGF1effects on SLC differentiation by radioimmunoassay, Q-PCR and western blot.Results:2to7d after apoptosis of Leydig cells by treated with EDS, the testosterone content of adult male rats has a very low level in serum. There is a Leydig cell regeneration process in EDS-treated rats, we found that the LHR can expressed in testis interstitial with the passage of times. The liver weight of rat at the14th,21st,35th,56th days after EDS-treated has the significant difference compared with the control, in addition the kidney weight of rat in the56th days has the significant difference compared with the control. Afer the injection of bFGF, the testis weight of rat at14th,21st,35th,56th days were compared with FGFR-inhibitors group and physiological saline group they have significant difference; and the serum testosterone levels at35th,56th days were compared with FGFR inhibitors group and physiological saline group they have significant difference, but the serum FSH levels have no significant difference at all time points; after the injection of bFGF, the expression of StAR protein in testicular tissue at14th,21st,35th days have significant difference, compared with FGFR inhibitors group and physiological saline group. Afer the injection of IGF1, the testis weight of rat at56th days were compared with IGFR-inhibitors1group and physiological saline group they have significant difference; and the serum testosterone levels at56th days were compared with IGFR inhibitors group and physiological saline group they have significant difference, but the serum FSH levels have no significant difference at all time points.bFGF and IGF1promoted stem Leydig cell to proliferate significantly in a dose-dependent manner. bFGF at100ng/ml induced not only high testesterone production, but also high expression of steroidogenic enzyme significantly. The western blot showed that High phosphorylation of Erk1/2may be involved in bFGF and IGF1effects on SLC.Conclusion:Acording to75mg/kg, after the single intraperitoneal injection of EDS, in a certain period, there will be a reduction of the number of liver, kidney cells in rats, we guess it may be exist the same failure mechanism with EDS causing the ledig cell apoptosis. After the injection of EDS in rats, it can promote the secretion of testosterone with intraperitoneal injection of bFGF and IGF1active protein factor in rats. This effects could be associated with bFGF and IGF1, which can promote the proliferation and differentiation of stem Leydig cells and increased the StAR protein. Our present studies have revealed that bFGF and IGF1induce SLC, PLC and ILC to proliferate. Moreover, bFGF and IGF1induced SLCs to differentiate into PLC and PLC into ILC. Based on described above, bFGF and IGF1may be a potential drug candidates for the treatment of PADAM. |
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