The Effects Of Testosterone On The Hypothalamus Of The Subacute Aging Mice And The Mechanisms

Background:Aging population is a main feature in human development in the 21st century, especially in China.According to newly issued report of National Population Development Research this year,by the end of the 20th century,the aging population more than 60 years old exceeds 10%of the total population in China.By the year 2005,the aging population more than 60 years old approximates 0.144 billion, accounting for 11%of the total population in China.At present,the ageing population is increasing at average annual rate of 3%or so.The aging process is accelerated at the rate of net increasing 0.1 billion ageing population in every 10 years.Along with the increasing aging population,the spectrum of disease in the ageing population in China is also changing from comparatively dispersion to concentration.The former 5 diseases with higher incidence are hypertension,coronary heart disease, cerebrovascular disease,diabetes,and tumor.The aging brain disease has become an important disease for the high incidence of threats to the health of older people.So there was an urgent need to carry out the pathogenesis of brain aging and countermeasures research. The traditional risk factors of brain aging include hypertension,high blood lipid levels,smoking,hyperglycemia,age and gender.Aging belongs to the brain aging has been not considered to be risk factors,therefore,there are less study of it.With the increasing development of aging population,the aging in brain aging mechanism study has become the attention and the new targets.Epidemiological studies show age is the brain aging occurrence and development of independent risk factors,with increasing age,the incidence of brain aging significantly increased.Along with the increase of age,age related changes which happen in brain and partial brain cells ageing.Now,more and more studies have found brain aging in a state of brain cells in aging,cell senescence may be an important reason for the aging brain has been increasingly widely recognized.Research found that,as a senior neuroendocrine central of brain aging process in the hypothalamus is a first appeared in the hypothalamus,aging tips in plays an important role in the development of aging.Geriatrics is a lot of experts suggested that anti-aging treatment as the treatment of brain aging,a new treatment strategy.Why does the aging process take place in brain? What are the cellular and molecular pathologic mechanisms underlying the aging of brain? Are these mechanisms able to give important ideas on amelioration or delaying brain aging? Because the aged population is increasing,these questions need to be answered to prevent and control cardiovascular and cerebrovascular diseases. Whether the Hypothalamus as meuroendocrine regulation of high-level central also occurres aging? At present,there is very little research.In the process of aging,another significant change is the change of balanced sex hormones.Testosterone is the most important in men,belong to a synthetic androgen hormones,the study found that men after age 50,total testosterone and biological activity of testosterone appear gradually decreased.Older men plasma testosterone levels and various age correlation disease incidence negatively.Many testosterone replacement therapy of clinical trials found that testosterone can improve cognitive abilities,adjust the elderly,strengthen muscles fat percentage increase muscular strength,motor skills,inhibit dyslipidemia.Reducing insulin resistance and improve sexual etc,but testosterone can improve the hypothalamus function in older men, delay ageing,senile brain documentary reported.The research shows that,before we testosterone in vitro intervention vascular endothelial cells induced by hydrogen peroxide may inhibit endothelial cells,but the hypothalamus aging urgently.Biological aging changes not only in the macro,more importantly in micro. Aging mechanisms,now from the whole level,organ development to cell and molecular.With the cell biology,molecular biology,the rapid development of the discipline of aging,both at home and abroad,the mechanism of various studies have great progress,and puts forward some high level of aging,have free radicals, mitochondrial DNA damage theory,the theory of telomeres,biological membrane damage theory,theory,chromosome mutation genetic program errors,the theory,the theory of immune theory,endocrine.Generally these theories can be divided into two categories:one kind is to NOS,sugar,hormone disorder,such as a representative of the environmental damage damage theory that aging aging is environmental factors on progressive and cumulative damage cells,another kind is the result,telomere shortening cell cycle regulation factors such as the genetic factors that old procedural aging theory is the orderly gene activity,is genetically programmed in advance or for specific "senescence" gene expression,or for the final depletion.Available genes, These theories from different Angle,the mechanism of aging,deeper exploration of gradually to deeper research direction,and has gradually entered the era of cooperation theories and research focused on environmental factor causing damage to the genetic program changes of aging relevant range.D - galacitiol aging mice caused by subacute model is often used in the aging model,certain time,through continuous subcutaneous injection D - d-galactose,make the body cells galacitiol heighten of chroma,in aldose reductase catalytic reduction, galacitiol and alcohol in cell metabolism and accumulation,influence further normal cellular pressure,causes the cell metabolism disorders,ROS accumulation.D - galacitiol inside can form adanced glycation end products(age-related damage), age-related damage can strengthen ROS damage and cause microtubules related proteins(abnormal) phosphorylation Tau and cytoskeleton filaments was involved in the D - galacitiol mice induced aging process,the enzyme glycosylated function sex is to promote the brain aging ROS.The accumulation of cerebral ROS participation in the brain aging,the mechanism of formation,how? It was unclear.Objective:Therefore,this research uses testosterone to intervent D - galacitiol mice model. Through the observation of the shape and structure of the hypothalamus tissue and the active of ageing related beta galactosidase,we discusse that the hypothalamus whether or not occurre aging through the enzyme activity determination for five months hypodermic D - half lactose 100mg/kg.d.And testosterone whether or not interferes with the hypothalamus aging.Through the determination of expression of cell cycle regulation factor p16^INK4a to explore the mechanism of the hypothalamus aging and the possible ways of testosterone intervention.Methods:1 Subacute aging mice grouping and model establishmentTwenty-four mice were randomly divided into 3 groups:①Testosterone intervention group(11 acid testosterone 5mg/kg,subcutaneous back,1 time per 2 weeks,D-galactose solution 100mg/kg,back subcutaneous injection,1 time per day;②D-galactose group(D-galactose solution 100mg/kg,back subcutaneous injection, 1 time per day;③the normal control group the same dose of normal saline,back subcutaneous injection,1 time per day,8 in each group,drug use 5 months.2 After the establishment of the model,observe the general situation(Tentacles,size, etc)and weight.3 Mice brain separation and making the hypothalamus biopsy.4 Use HE staining to observe the hypothalamus biopsy of dyeing conditions5 Use SA - beta gal dyeing method to determine of the hypothalamus dyeing positive cells percentage6 Use immunohistochemical method to determine of the hypothalamus p16^INK4a protein expression rate of positive cellsStatistical analysis:Experimental data analysis use software SPSS13.0.Income data to^?±s.Data comparison between groups by single factor analysis of variance,pairwise comparison using LSD-t test,P＜0.05 reffers to difference was statistically significant.Results:1.The general situation and weight of the three groups of miceIn the latter period,D galactose mice widespread tentacles off,Camponotus, erect hair,and other signs of slow action,and the normal control group of mice tentacles integrity,quick action.And D-galactose + testosterone treated mice do not appear tentacles off,Camponotus,erect hair,and other signs of slow action.Compared with the normal control group,the mice weights of D - galacitiol group decreased obviously(P＜0.05),And compared with the D - galacitiol group,the mice weight of testosterone intervention group were significantly higher(P＜0.05),but are still lower than the normal control group(P＜0.05).2.The hypothalamus HE dyeing conditionsNormal cellular structure is clear,and the number of neurons are neat, hyperchromatic and nucleoli and nuclear membrane is clear.D - galacitiol group visible organization structure,become pyknotic nuclei,partial neuron cell bodies narrowing gap,neurons deformation around widened.D - galacitiol group + testosterone therapy clearly visible cellular structure,the number of neurons than D galacitiol group,hyperchromatic and clear,nucleoli.3.The hypothalamus SA - beta gal dyeing positive cells percentageCompared with the normal control group,D - galacitiol mice hypothalamus SA - beta Gal dyeing positive cells rate increased significantly(P＜0.05);Compared with D - galacitiol group,testosterone intervention group mice hypothalamus SA - beta Gal dyeing positive cells rate decreased significantly(P＜0.05),but were still higher than normal control group(P＜0.05).4.The hypothalamus p16^INK4a protein expression rate of positive cellsCompared with the normal control group,D - galacitiol mice p16^INK4a hypothalamus protein positive cells expression rate increased significantly(P＜0.05);Compared with D - galacitiol group,the positive cells of testosterone intervention group mice p16^INK4a hypothalamus protein expression rate decreased significantly(P＜0.05),but were still higher than normal control group(P＜0.05).Conclusion:1.After Hypodermic D - half lactose 100mg/kg.d for 5 months,it can be seen that in the hypothalamus HE dyeing visible part of neurons become pyknotic nuclei,SA-β-gal dyeing positive cells percentage increasing,p16^INK4a expression increasing.It fits the aging cell characteristics.It proves that the mice hypothalamus aging model induced by D -galactose succeeds.2.Testosterone intervention can reduce SA - beta gal activity to delay mice hypothalamus aging.3.Testosterone intervents the mice hypothalamus aging partly through reducing p16^INK4a protein expression.