| Introduction: BPH(Benign prostate hyperplasia)is a common disease in middle-aged man,what is a kind of geriatrics.Although recent studies on the disease have made great progress,its pathogenesis remains unclear.It is necessary to develop drugs those are effective in prevention and treatment of BPH.Studies have shown that,CGD(Cocoomycxa glocobo-trydisomis)provided by Japan "Nikken" has demonstrated significant anti-aging effects.CGD could enhance the memory of aged rats,lower blood pressure,blood lipids,blood glucose,and increase tissue production of nitric oxide(NO).Mikhon noted,the medicine could cure one kind of geriatrics is likely to play a therapeutic role in a variety of geriatrics.Based on the above information,I think that since CGD has the effect to increase natural aging rats' memory(anti-dementia effect)then it might be effective for the prevention and treatment of the other senile disease,such as BPH.Thus,I design the model of natural aging rats with BPH fed with CGD orally,to observe its effect on the rats' prostates.At the same time,to approach the mechanisms of CGD to prevent the occurrence of BPH.The experimental results show that,natural aging rats can be used as an animal model for BPH,CGD can significantly inhibit the natural aging rat prostatic hyperplasia significantly.This effect is achieved by reducing the expression levels of prostate tissue phosphorylated PDK1 and Phosphorylated Akt protein,increasing the level of phosphorylated PTEN protein;reducing the expression of eNOS and iNOS.Experiments indicate that,as a kind of prevention drugs for senile diseases(including BPH),CGD has a great research and development value and it is promising.In addition,although Coccomyxa gloeobotrydiformis(CGD)is known to offer neuroprotective effects and can improve learning ability in rat model,the involvement of CGD in mediating the protective effects on hypertension-induced cardiovascular and renal injury is not fully explained.Purpose: It has been confirmed that CGD does have some good anti-aging effects,I imagineCGD may have a preventive role in the occurrence and development of BPH.The purpose of this experiment is to detect how CGD effects on BPH in natural aging rats.Based on that,we investigate the mechanism of effect of CGD on BPH in rats.In this study,we also adopted the classic 2-kidney,1-clip(2K1C)hypertension rat model,and examine the protective effects of CGD on cardiovascular system,and discuss the underlying mechanisms.Materials and Methods:1.MaterialsHealthy male rats,weighing 550-650 g,provided by the Experimental Animal Center of China Medical University.CGD provided by Japan"Nikken.Slicer,paraffin,hematoxylin-eosin stain,Masson staining,electronic balance,microscopes.PDKI(ser24).PDK1 and phosphorylated AKt(ser473),AKt and phosphorylated PTEN(ser380)(purchased from Cell Signal company),PTEN,rabbit anti phosphorylated PDK1 polyclonal antibody,rabbit anti phosphorylated AKt polyclonal antibody,rabbit anti phosphorylated PTEN polyclonal antibody(sell Signal),TBS liquid,homogenizer,centrifuge,Westen blot electrophoresis tank;biotinylated goat anti rabbit antibody,SABC complex(Wuhan Boshide biological company).2.MethodsIn BPH study,Feed rats with different doses of CGD,administration according to 50mg/kg/d and 100mg/kg/d.The elderly control group and the youth control group of rats were given normal diet,feeding for three months.When drawn to experiment,elderly control group rats are 24 months old,the youth control group rats are 6 months old.In hypertension study,Male Sprague-Dawley rats were randomly divided as sham-operated control and CGD treatment groups.Low dose(50mg/kg.day)and high dose(100mg/kg.day)concentrations of the drug CGD were intragastrically administrated separately for 12 weeks.At the end of the 12(th)weeks,systolic blood pressure were recorded,cardiac and renal damage were determined,and aortic contents of eNOS,p-eNOS on Ser1177,Akt,p-Akt were assayed.measuring lab rats' prostate wet weightWhen the prostate of the rats was removed,measure its weight immediately,and calculate its prostatic index.Prostatic index=Prostate weight(mg)/body weight(g)X100%Light microscopyUnder the light microscope,observe the prostate tissue HE staining,Masson staining,microscopic photography.SDS-PAGE electrophoresisUltra sonicate the prostate tissue,determinate the protein content.Concentration of separating gel is 10-12%,sample volume is 30 ul,containing 20 ug protein.Electrophoresis,transfer.after chemiluminescence,use Vision Wooks 6,33 image acquisition and analysis software to determinate the expression of phosphorylated PDKI,phosphorylated AKt,phosphorylated PTEN protein in the rats' prostates.Light microscopy the prostate NOS immunohistochemical sliceUse 200 x and 400 x Light microscope to observe the NOS stained particles in slice.Positive for brown or tan,the nuclei are blue or light blue.Statistical analysisMeasurement data is represented by Xt S,and statistical is analysised by SPSS11.5 software package,using ANOVA to analyze between groups,using LSD method to do pairwise comparison,P<0.05 indicates there are significant differences.Results:1.Compared with young rats,the prostate wet weight and prostate index of 24-month-old rats increased significantly.2.The prostate wet weight and prostate index of rats in the CGD(100mg/kg,d)group was significantly lower than that of the age-matched rats in the control group,there was no significant difference compared with the youth group.3.Prostate tissue HE staining of 24 months aging rats showed,glandular epithelial cells arranged densely obviously,which was stratified or pseudostratified,papillary hyperplasia protruding into the gland lumen,in some areas smooth muscle hyperplasia,hierarchical were increased,almost no secretions within the glandular lumens.The prostate tissue HE dyeing of CGD100mg/kg/d group rats could find glandular epithelial cells arranged properly,lumens with discharge.There was only a small amount of smooth muscle cells,and fibroblasts in interstitial.Masson staining showed,the prostate tissue and smooth muscle tissue of natural aging rat was obviously thicken,presented papillary hyperplasia protruding into the gland lumen,fibroblasts increased significantly.Compared to the natural aging group,the smooth muscle cells and collagen components of the prostate tissue of CGD high dose group was significantly reduced.Alveolar walls were thin and smooth,lumens with discharge.4.Compared with the youth control group,the expression of phosphorylated PDK1 protein in prostate tissue of aged control rats was significantly increased.Compared with the aged control group,the expression of phosphorylated PDK1 protein in prostate tissue of CGD(50 and 100mg/kg,d)control group rats was significantly decreased,and in a dose dependent.5.Compared with the youth control group,the expression of phosphorylated AKt(ser473)protein in prostate tissue of aged control rats was significantly increased.The Akt expression levels of two CGD dose groups were obviously lower than the same age groups,and there was no difference with youth group6.The expression levels of phosphorylated PTEN in prostate tissue of natural aging group rats was significantly lower than the youth control group.The phosphorylated PTEN levels of two CDG dose groups of aging rat prostate tissue wereobviously higher than that of natural aging control groups.7.Observations of NOS immunohistochemical staining slice: There was only a small amount of nNOS in rat prostate tissue of aging control group and CGD(100mg/kg,d)group rats,the latter was less than the former,but the difference between the two groups was not obvious.eNOS were hyperchromatic,dense in great measure in epithelial cells and subepithelial tissue of aged control group.eNOS in CGD100mg/kg,d group prostate tissue was significantly reduced than the aged control group.iNOS,in aging control group,the prostate tissue lumens contains a small amount of particles,glandular epithelial cell cytoplasm and nuclei were distributed uneven,piled densely dark brown grain,extremly rarely seen in interstitial.In CGD100mg/kg,d group,lumens of prostate tissue increased,cytoplasm and nucleus of the glandular epithelial cells contain a small amount of yellow grain,scattered all over.rarely seen in interstitial.8.CGD treatment from the time of clipping can not only prevent the development of hypertension,but also prevent renal damage and cardiac impairment in 2K1 C rats.Other evidences show that CGD can improve the NO signaling to prevent the damage of thoracic aortas in 2K1 C hypertension rats.Conclusions:1.24-month-old male SD rats can occur BPH,they can be used to test models of BPH.2.The degree of prostatic hyperplasia of rats fed with CGD100mg/kg,d was reduced significantly.3.The prostatic hyperplastic change of aged rats fed with CGD100mg/kg,d was significantly reduced.4.The occurrence of BPH in aged rats was related to the enhancement of PIK3/AKt pathway activity in prostate tissue and the degression of PTEN activity in prostate tissue.5.The prevention and reduction of prostate hyperplasia in aged rats of CGD was completed by down-regulating the PIK3/AKt activation,up-regulating the PTEN activation.6.eNOS,iNOS were strong positive expressed in aged rats prostate tissue,oral CGD can reduce the expression.NOS(mainly the eNOS,iNOS)may be involved inthe process of BPH occurrence.7.As a drug to prevent BPH,a kind of age-related diseases,CGD has good valuefor development and application prospects.8.Enhancing eNOS protein synthesis and its associated increasing in phosphorylation are a potentially important mechanism by which CGD decrease blood pressure in hypertension. |
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