Research On The Expression Of Klotho Gene In The Brain Tissue And Blood Of The Hypertensive Patient

With the development of economy and improvement of people's standard of living,hypertension has become a commonly and frequently encountered disease.It is rooted from the persistent and strong arterial systole of the whole body,which causes the blood pressure to rise to result in the dysfunction of many bodily organs such as the heart,brain, liver and so on.And the impairment of the cerebral vessel is among the complications of hypertension.In spite of the breakthrough in the research of the pathogenesis of cerebral damage caused by hypertension in the recent years,the precise incidence mechanism of it remains to be clarified.It is known to all that persistent hypertension can cause pathological change of systemic anteriola,which is represented by the hyperplasy and fibrosis of vessel intelmedial smooth muscles,the thickening vessel wall,and stenosis of lumens.These changes conduce to atherosclerosis,cerebral thrombosis and result in the emergence of microaneurysm which often ruptures and leads to hematencephalon.The eventual results of the above mentioned pathogenesis are brain atrophy and functional deterioration,like the insenecence process of human organs.In order to make a research on the incidence mechanism of cerebral damage caused by hypertension and to probe into its relationship with Klotho(an aging-associated gene)and some hypertension-associated risk factors,we make our research through the animal studies and clinical observation.The First Chapter(Animal Experiment)Expression of Klotho Gene in the Brain Tissue of the Spontaneously Hypertensive Rat and Object of the Experiment of Drug Interference with Fosinopril and ValsartanObjectsTo observe the expression of the anti-aging gene Klotho, ICAM-1(intercellular adhesion molecule-1)and VCAM-1(vascular cell adhesion molecule-1)in the brain tissue of the SHR(the Spontaneously Hypertensive Rat)model rats interfered by Fosinopri,Valsartan or Fosinopri and Valsartan synergistically,and to research the effect and role of Klotho gene and ICAM-1 and VCAM-lon brain harm caused by hypertention,and to explore the effective treatment of it in the early stage.Methods20 SHR male rats with 22 weeks' ages were divided averagely into four groups at random:the spontaneously hypertensive rats group(SHR; lavaged with water),the Fosinopril treatment group(Fos;lavaged with Fosinopril 10mg/kg/d),the Valsartan treatment group(Val;lavaged with Valsartan 50mg/kg/d),the Fosinopril and Valsartan treatment group (Fos+Val;lavaged with Fosinopril 10 mg/kg/d and Valsartan 50mg/kg/d). The homogenous wky rats were 5 Wistar-kyoto rats,served as the normal control group(WKY).All the rats had been fed for 8 weeks,drinking and eating freely,until they were 30 weeks old.During the experiment:①the changes of hypertension and weight of the SHR model rats were observed before and after drugs treatment;②the electron microscope was used to detect the neuron structure and apoptosis of the brain tissue when harmed by hypertension,and the effect of the drug interference of Fosinopril and Valsartan on the structure of the brain nerve cell of the SHR model rats;③the modern biological methods RT-PCR,Western bolt and immunohistochemistry were used respectively to detect the changes of the mRNA and protein expression of Klotho,ICAM-1 and VCAM-1 before and after the drug interference of Fosinopril and Valsartan in the brain tissue of the SHR model rats.Results1.Effect of Fosinopril and Vaisartan on the Blood Pressure and Weight of the Spontaneously Hypertensive RatBefore drugs(Fos,Val and Fos+Val)treatment,the blood pressure in the SHR,Fos,Val and Fos+Val groups was obviously higher than the WKY group(p＜0.01);after drugs interference with Fosinopril, Valsartan,Fosinopril and Valsartan for 8 weeks,the blood pressure in the SHR group was obviously reduced,compared with that before drugs treatment.There were no obvious changes in the weight of the 5 groups before and after drugs treatment(p＞0.05).This suggests that Fosinopril, Valsartan,and Fosinopril and Valsartan combined treatments could reduce the blood pressure of the spontaneously hypertensive rat,and inhibit hypertension progress(p＜0.05)2.Changes of the Ultrastructure in the Brain Tissue of the Spontaneously Hypertensive Rat and Effect of Drug Interference on itAfter the rats had been treated with Fosinopril,Valsartan,and Fosinopril and Valsartan combined treatment for 8 weeks,the brain tissues of the rats were detected by electron microscope scanning.There were the findings:(1)the WKY group:the modality and structure of the nerve cells were normal;rough surfaced endoplasmic reticulum and episomal ribosome were rich and their configurations were clear;the cell nucleus were large and round,the karyothecas were clear,the nucleoli were obvious and the chromatins were light;(2)the SHR group:different apoptosis changes were found in about 50%of the nerve cells;the nerve cells were karyopyknosis,the chromatins centralized around,the karyothecas began to disintegrate,and apoptotic-bodies were formed;(3) the Fos group:a few nerve cells were karyopyknosis and the chromatins centralized around;and a large number of the nerves cells were normal; (4)the Val group:a part of the nerve cells were karyopyknosis,the karyothecas began to disintegrate,and there were apoptotic-bodies in a number of the nerve cells;and apoptotic-bodies in the early stage were formed(5)the Fos+Val group:a large number of the nerves cells were normal,rough surfaced endoplasmic reticulums and episomal ribosomes were rich and their configurations were clear;and a few nerve cells were karyopyknosis,chromatins centralized around,and karyothecas began to disintegrate.Apoptosis changes were compared among the 5 groups and the conclusion was that the sequence of the apoptosis degree was the SHR group→Val group→Fos group→Fos+Val group→WKY normal group.The SHR group ranks the highest,and the WKY normal group ranks the lowest.The above data demonstrate that hypertension could harm the configuration of the brain nerve cells,but Fosinopril,Valsartan, and Fosinopril and Valsartan combined treatments could resume or slow the harm of the brain nerve cells in different degrees.This shows that Fosinopril and Valsartan could protect the function of brain cells.3.Expression of Klotho,ICAM-1 and VCAM-1 in the Brain Tissue of the Spontaneously Hypertensive Rat and Effect of Drug Interference on it(1)Changes of the mRNA and protein expression of Klotho,ICAM-1 and VCAM-1 in the brain tissue of the SHR model rats before and after drug interference,detected by RT-PCR and Western boltA.Changes of the expression of Klotho:①the expression of Klotho in the SHR group was reduced,compared with the WKY nomal control group;②after 8 weeks' drug treatmtent,compared with the WKY group,there was no obvious difference in the expression of Klotho in the Fosinopril,Valsartan,Fosinopril and Valsartan combined treatment groups(p＞0.05);but compared with the SHR group,it was obviously up-regulated(p＜0.05)B.Changes of the expression of ICAM-1 and VCAM-1:①the expression of ICAM-1 and VCAM-1 in the brain tissue of the SHR model rats was up-regulated(p＜0.01),compared with the WKY nomal control group;②after 8 weeks' drug treatmtent,there was no obvious difference in the mRNA expression of ICAM-1 and VCAM-1 in the Fosinopril,Valsartan,Fosinopril and Valsartan combined treatment groups(p＞0.05),compared with the WKY groups;but compared with the SHR group,there was obvious difference in it in these groups (p＜0.01).This shows that the mRNA expression of ICAM-1 and VCAM-1 in the brain tissue of the SHR group is at a high level,and the drugs treatments with Fosinopril or Valsartan and Fosinopril and Valsartan combined treatment can down-regulate the mRNA expression of ICAM-1 and VCAM-1 in the brain tissue of the SHR group(p＜0.05 and p＜0.01)(2)Changes of the protein expression of Klotho,ICAM-1 and VCAM-1 in the brain tissue of the SHR model rats before and after drug interference,detected by immunohistochemistry. A.Klotho protein was mainly expressed in the nerve cell plamid:①it was little expressed in the WKY group;②the expression of Klotho protein was obviously decreased in the nerve cell of the SHR group (p＜0.05),compared with the WKY group;③after 8 weeks' drugs treatment,compared with the WKY group,there was no statistic meaning in the changes of the expression of Klotho protein in the Fosinopril,Valsartan,Fosinopril and Valsartan combined treatment groups(p＞0.05);however,compared with the SHR group,the expression of Klotho protein was obviously enhanced(p＜0.05).B.ICAM-1 and VCAM-1 protein was mainly expressed in haemangioendothelium and adventitia,and it was little expressed in the nerve cell,and glio-cell andinterstitial cell:①the ICAM-1 and VCAM-1 protein was little expressed in the WKY group;②compared with the WKY group,it was obviously up-regulated in the nerve cell of the SHR group(p＜0.05).③after the rats had been treated for 8 weeks with Fosinopril,Valsartan,Fosinopril and Valsartan combined treatment, compared with the WKY group,there was no statistic meaning in the changes of the expression of ICAM-1 and VCAM-1 protein in the Fosinopril,Valsartan,Fosinopril and Valsartan combined treatment groups(p＞0.05);compared with the SHR group,the expression of ICAM-1 and VCAM-1 protein was obviously down-regulated(p＜0.05).Summary1.Hypertension can result in brain harm;2.In brain harm caused by hypertention,the expression of Klotho in the brain tissue is low,and the expression of ICAM-1 and VCAM-1 is high;3.Besides depressing hypertension,Fosinopril or / and Valsartan can protect the cell function by regulating the expression of Klotho gene and ICAM-1 and VCAM- 1.4.Inflammation genes play a part in the harm of the brain caused by hypertention,and might be partly relative to the anti-aging gene Klotho.5.Fosinopril and Valsartan might prevent brain harm caused by hypertention at the early stage. The Second Chapter(Clinical Research)Test of the Klotho Protein Expression of Blood Serum in the Hypertensive Patient with Brain Harm and Object of the Analysis of the Associated Risk FactorsObjectsTo test the changes of the Klotho protein expression of blood serum in the hypertensive patient with brain harm from the angle of clinical study,based on the above findings of the animal experiment;to make comprehensive analysis of the hypertension-associcated risk factors such as the age,blood pressure,blood sugar,cholesterin and hypertension medical record of the patient;to further probe into the incidence mechanism of brain harm caused by hypertension and the associated risk factors,so as to provide a basis for early prevention of brain harm caused by hypertension.MethodsThe research objects were the hypertensive patients without syndrome(56 cases),hypertensive patients with brain harm(114 cases), and normal control humans(no hypertension,30 cases),who were emergency patients in Xiangya Hospital from September,2007 to March, 2008.Compared with the normal control group of 30 humans with normal blood pressure,the Klotho protein concentration of blood serum, cholesterin,creatinine,blood sugar and blood pressure of these three groups of patients were tested.And correlation analysis of the above factors and the patients' ages and hypertension medical records was made.ResultsThe Klotho protein concentration of blood serum in the normal control group is obviously higher than that of the hypertensive patients without syndrome and the hypertensive patients with brain harm(p＜0.05); the Klotho protein concentration of blood serum of the hypertensive patients without syndrome is obviously higher than that of the hypertensive patients with brain harm(p＜0.05).The Klotho protein concentration of blood serum is negatively pertinent with hypertension and the cholesterol,creatinine,blood sugar,blood pressure,age,and hypertension medical record of the hypertensive patient with brain harm.Summary1.The Klotho protein concentration of blood serum reduces gradually with the patient's hypertension progress.2.The Klotho protein concentration of blood serum is negatively pertinent with hypertension and the cholesterol,creatinine,blood sugar, blood pressure,age,and hypertension medical record of the hypertiensive patient with brain harm.The higher the cholesterol,creatinine,blood sugar and blood pressure is,the older the age is,and the longer hypertension medical record is,the lower the Klotho protein concentration of blood serum is.3.The increase of the Klotho protein concentration might be a protective factor of hypertension and brain harm caused by hypertension.4.In order to monitor and prevent brain harm caused by hypertension at the early stage,it is beneficial for the hypertensive patient to have a test of the Klotho protein concentration of blood serum regularly.Conclusion1.There is brain harm in the SHR model rat in the progress of hypertension.It is showed by the form of apoptotic-bodies in the brain tissue of the SHR model rat,observed from the electron microscope.2.The up-regulation of the mRNA and protein expression of ICAM-1 and VCAM-1 in the brain tissue of the SHR model rat demonstrates that there is change of vascellum inflammation in brain harm caused by hypertension.3.Besides the change of vascellum inflammation in the SHR model rat, the down-regulation of the expression of Klotho gene in the brain tissue suggests that brain harm caused by hypertension might be associated with aging.4.After the experiment of interference with Fosinopri or/and Valsartan, the expression of Klotho gene up-reguates with the improvement of inflammation in the brain tissue(the down-regulation of the mRNA and protein expression of ICAM-1 and VCAM-1).This shows that besides depressing hypertension,Fosinopri or / and Valsartan might be associated with the change of the expression of Klotho gene.5.The clinical research demonstrates that the Klotho protein concentration of blood serum of the hypertensive patient is lower than the normal control person,and it is negatively pertinent with hypertension and the cholesterol,creatinine,blood sugar,blood pressure,age,and hypertension medical record of the hypertensive patient with brain harm. The higher the cholesterol,creatinine,blood sugar and blood pressure is, the older the age is,and the longer hypertension medical record is,the lower the Klotho protein concentration of blood serum is.This shows that in order to monitor and prevent brain harm caused by hypertension at the early stage,it is beneficial for the hypertensive patient to have a test of the Klotho protein concentration of blood serum regularly.