| Spontaneous hypertensive(HP),namely hypertension,the "cardiovascular syndrome"with features of arterial blood pressure rising,is one of the most common chronic disease,with long duration,complex etiology,health damage and serious social harm etc.Hypertension is the most significant risk factor of cardio-cerebrovascular disease in China and a major public health problem worldwide.Hypertension is a major risk factor for cardiovascular mortality through its effect on the target organs including heart,brain,kidney and vessels.Therefore,mechanism of hypertension is a hot topic in the field of cardiovascular.Vascular remodeling is not only the pathological origin leading to hypertension,but also the damage result on its residing organs.Thus,intervention to vascular remodeling is the main strategy of primary and secondary prevention of hypertension.Vascular remodeling is a dynamic processes containing cell proliferation,migration,apoptosis,and synthesis,degradation and rearrange of extracellular matrix;vascular remodeling is a complicated dynamic response process of incentive,including receiving of the signal,conduction of the signal,as well as the synthesis and release of regulate factors and finally produce structural changes.The most typical features include media thickening,inner diameter narrowing and substrate increasing,but different vascular have different remodeling.It is considered that the performance of the large arteries such as aorta is smooth muscle cell hypertrophy;the performance of mesenteric small arteries such as in the third stage arterial is hyperplasia.Currently,we lack systematic studies on hypertensive vascular remodeling mechanisms and the intervention of small artery.Many researches about vascular remodeling focused on the endothelial cells of tunicae intima vasorum and smooth muscle cells(VSMC).Recently it was found that adventitia fibroblast(AF)of under inflammatory cytokines stimulation,adventitia fibroblast participate in and promote the vascular remodeling by activating,intraluminal migration and phenotypic transformation then make excessive collagen synthesis and deposition in the vessel wall,which differ from the process of vascular remodeling about large artery;the runaway about the balance between cell proliferation and apoptosis on vascular wall promote vascular remodeling process.Grape seed proanthocyanidin extract(GSPE),by most accounts,is proved a effective natural antioxidant protective in cardiovascular system,including atherosclerosis,myocardial ischemic-reperfusion injury,hypertension and target organs damage caused by hypertension,due to not only its anti-oxidative but also its anti-inflammation and endothelial cell protective effect.GPSE is well known in protecting ischemia heart from reperfusion injury;however,its role in vascular remodeling,especially in small vascular remodeling is not clear.This study is the oabservation and research of the different characteristics of vascular remodeling about the aorta artery and the mesenteric small artery of SHR,(Spontaneous hypertensive rat,SHR)-forty-three week old rats.This study started from collagen hyperplasia of small artery differed from the characteristics of large artery,then observed fibroblast proliferation,migration and transformation of phenotypic about fibroblast and the apoptosis and proliferation of blood vessel walls about vascular remodeling.From tissue,cell and molecular level to discuss the mechanism of vascular remodeling and to explore the protection mechanism of GSPE on small artery remodeling by interfering in different dose of GSPE,we aimed to offer new thoughts and novel drug targets for small vascular remodeling in hypertension.Part IDifferentiation of vascular remodeling between small arteries and aorta in Spontaneous Hypertensive RatsBackgroundEpidemiological studies confirmed that rapid economic development led to a fundamental change in lifestyle,then the incidence of "lifestyle disease" increased rapidly,in which hypertension is one of the most common representative diseases.The hypertension-related cardiovascular diseases in China have become more dangerous than cancer.The damage of its target organs and poor prognosis induce inestimable burden and loss for our economy and life.So hypertension is a major challenge for us in the field of cardiovascular and cerebrovascular diseases.Studies of hypertension have made significant progress and the ratio of hypertension awareness,treatment and control have reached unprecedented levels.But clinical studies show that even if patients' blood pressure reduces to the normal level,target organ of hypertension trending further damage.That is to say the relevance between the damage of target organs and blood pressure levels is different from the early theories.Vascular remodeling is a pathogen of the initiation and development of hypertension,and in turn is the pathological presence of multi-organs damage caused by hypertension.Prevention and alleviation to vascular remodeling have extremely clinical significance for protecting target organs,improving the prognosis value,and controlling many vascular diseases.Vascular remodeling is the pathological basis of various risk factors and diseases so as to target organ damage.Recent researches indicate that the vascular remodeling,especially in small arteries,is an independently predictable factor for cardiac vascular disease.Characterized by hyperplasia and hypertrophy of the small vascular remodeling of hypertension,its vascular wall thickened obviously and inner diameter(ID)decreased significantly under circumstance of morphological features of the blood vessels outside diameter(OD)almost without distinction,which has the distinct morphological performance of the vascular remodeling of large arteries.The increased thickness of vascular wall and the decreased lumen are major pathologic presences in hypertension,in which the proliferation of vascular muscle cells and the accumulation of collagens in ECM are involved.At this stage,there are lack research about intervention of vascular remodeling.The vascular remodeling includes not only the change of diameter of vascular,but also the change of wall composition:such as the proliferation and activity change of cells,the unbalance of cell's proliferation and apoptosis,the imbalanced synthesis and degradation of ECM and its wrong distribution.The collagen synthetized by fibroblast participated in vascular remolding is a kind of fibrin which is not soluble and the main component of the extracellular matrix and plays a decisive role in the maintenance of tissue and organ.The vascular wall mainly contains collagen type ? and type ?,and the synthesis of collagen is more than degradation.The abnormal changes of the characteristic configuration and arrangement are the basic reasons for the remodeling of hypertension.In our experiment,we used Spontaneous hypertensive rats as the experimental group and wistar Kyoto rats as the control group to observe characteristic changes of SHR small mesenteric arterial remodeling and the aorta vascular remodeling.We found the vascular remodeling of large arteries are vessel wall cell hyperplasia mainly,especially VSMC's hyperplasia and hypertrophy.However,the vascular remodeling of small artery is mainly stromal hyperplasia,especially vascular adventitia collagen hyperplasia significantly.We also observed the more apoptosis of wall cells may involve in the balance about the proliferation and apoptosis of the vessel wall and observe the fiber cell of vascular adventitia proliferation,migration and phenotype transformation.In the end,a new theoretical basis for prevention and treatment of small vascular remodeling is provided.Purposes1 Compare blood pressure,body weight,blood lipid,blood sugar and renal function between SHR and WKY rats2 Observe the different characteristics of SHR mesenteric arteriolar vascular remodeling and aortic vascular remodeling in morphology,tissue and cell several aspects3 Demonstrate hypertensive small vessel remodeling characteristics,from the perspective of collagen hyperplastic.4.Evaluate the role of vessel wall cell apoptosis and proliferation 's position in different vascular remodeling.MethodsThirty twenty-week-old male spontaneous hypertensive rats were randomly divided into three groups:ten spontaneous hypertensive rats(SHR-C),ten small doses GSPE of intervention group(SHR-L)and ten large doses GSPE of intervention group(SHR-H).The ten same weeks of male WKY rats(WKY)group C(WKY-C)went into the experiment after 1 week's adaptive feeding.Twenty-week-old male spontaneous hypertensive rats(SHR,n=10)and Wistar-Kyoto rats(WKY)(body weight 369.25±12.11 g,n=10)were fed with standard rat chow and free accessible tap water for 1 week.Rats in WKY-C and SHR-C groups were orally administered with 1.0 ml 0.9%saline per day.Rats in GSPE intervention groups were respectively given 100 ug/kg,250 ug/kg GSPE lavage 1.0 ml.At the end of week 22,rats were sacrificed after 10%chloral hydrate intraperitoneal injection.mesenteric small artery and thoracic aorta tissue of four groups rats were separated quickly after taking blood.Peripheral blood serum from abdominal aorta and related vessels were collected and stored at-80?.The part I compares the difference of mesenteric small artery and thoracic aorta vascular remodeling in the WKY-C group and the SHR-C group.The part II will explore the protective effect and mechanism of GSPE on small vessel remodeling.1.Measure the systolic blood pressures and body weight initially once a week before experiments until the end of the experiment.Systolic blood pressures was placed by its tail systolic blood pressures.2.Draw blood from the abdominal aorta,and measure the serum's total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),triglycerides(TG),blood sugar(BS),creatinine(Cr)and blood urea nitrogen(BUN)after centrifugation.3.Collect and slice Mesenteric small artery and thoracic aorta tissue.Vascular tissue slices were stained with HE and Sirius red-victoria blue to respectively observe the change of morphology and collagen.The distribution and expression of type I and III collagen were detected by indirect immunofluorescence histochemical staining with confocal laser scanning microscope.Arterial ultrastructure was imaged using transmission electron microscopy.The appoptosis and proliferation of arterial vascular wall were examined with TUNEL and PCNA.Samples tissue were stored at-80?,in order to conduct Western Blot and RT-PCR in the second part of the experiment.Results1.Systolic blood pressure and body weightDuring the course of the experiment,the systolic pressure of SHR-C group was significantly increased,the difference was statistically significant(P<0.01),but no significant difference of body weight was found between the two groups.2.Serum lipids(TC,TG,LDL-C)and BS,Cr,BUNThe serum total cholesterol,low density lipoprotein cholesterol,triglycerides and blood sugar,creatinine and blood urea nitrogen were no significant difference between WKY-C group and SHR-C group(P>0.05).3.Morphological changes of artery tissue in two groups rats3.1 Stained with HEThe rats's mesenteric small artery and aorta of WKY-C group were normal and each layer was clear and orderly,while the rats's mesenteric small artery wall in the SHR-C group was thickened,with lumen stenosis and with medial VSMCs disordered.More specifically,adventitia was thickened and cell proliferation.However,the aortic wall in the SHR-C group was also thickened,especially in media.Further,the VSMCs were hypertrophy and proliferation,and the medial elastic fibers arranged at random,loose,uneven thickness and breaks,at the same time,part of endothelial cyte was sheded.3.2 Stained with Sirius red-Vitoria blueThe Sirius red-Vitoria blue staining bright field shows that the mesenteric small artery and aorta in the WKY-C group rats were normal,with blue-green elastic layer structure clear and with red collagen distribution normal,whereas mesenteric small artery and aortic wall of SHR-C group were thicker.At the same time,red collagen fiber was severe hyperplasia,especially in adventitia of mesenteric small artery,it was significantly more proliferous than the aorta.However,there is no obvious change in the blue-green elastic plate in one layer.But the hyperplastic collagen of aorta was mainly in medial,with destroyed and structure disordered elastic fibers.Morphological detection analysis shows that the ID and LCSA of mesenteric small artery in SHR-C group was significantly decreased(P<0.05)compared with WKY-C group,whereas the WT,WCSA,WT/ID,WCSA/LCSA was significantly increased the difference was statistically significant(P<0.01),but the ID,LCSA and WT/ID of aorta was significantly increased(P<0.05),WT,WCSA,WCSA/LCSA was also significantly increased(P<0.01),the difference was statistically significant.4.Comparison of the collagen fibers in the two groups ratsImage Pro-Plus analysis system for quantitative analysis found that the total collagen content per area of mesenteric small artery in the SHR-C group was significantly increased than in the WKY-C group(P<0.01);the total collagen content per area of aortic artery in the SHR-C group was all significantly increased than in the WKY-C group(P<0.01),especially in small artery.5.The distribution of type ?/? collagen in the two groups ratsSirius red-Vitoria blue staining dark field using polarization microscopy shows that red-yellow type ? collagen in the WKY-C group rats was mainly located in adventitia and green type ? collagen was mainly appeared in adventitia and few in media.However,in SHR-C mesenteric small arteries and aortic,collagen was obvious hyperplasia and observed overload disarranged,overload type ? collagen was found reached to media and in adventitia,type ? collagen fibers are distributed in the outer membrane and beyond.Compared with the aorta with increased type ? collagen in media,mesenteric small arteries's collagen increased more significantly,especially adventitia ? collagen.6.Indirect immunofluorescence histochemical staining with confocal laser scanning microscope to detect the expression of type ?/? collagenArterial structure of WKY-C group in rats was normal,the density of green collagen fibers and red nuclei was also normal.Compared with WKY-C group,arterial wall of SHR-C group was thick obviously,with the number of red nuclei increased significantly.At the same time,green type ?/? collagen was overloaded,it was found in media and in adventitia.Compared with the aorta,collagen of mesenteric small artery was increased significantly,especially in the adventitia.Large artery blood wall nuclei increased significantly,especially the mieda.The results of fluorescence quantitative analysis(IOD)showed that:compared with WKY-C group,the expression of type ?/? collagen in SHR-C group was increased,but the mesenteric small artery was more significantly(P<0.01),especially in type ?collagen.The difference was statistically significant.Aorta increased of collagen was mainly in type I,the difference was statistically significant(P<0.05).7.Ultrastructural changes of the artery in two groups ratsUnder the transmission electron microscope,the collagen of WKY-C group in rats was distributed mostly in adventitia and a minor portion in media.Meanwhile,the endothelial cells,VSMCs and AF were normal.The inner membrane of artery in SHR-C group was broken obviously and the apoptotic cells can be even detected.Meanwhile,VSMCs of mesenteric small artery were hypertrophy,proliferation and disordered in the media and adventitia collagen was obvious hyperplasia.AF with expansion of the endoplasmic reticulum was migrated to the media,even appeared phenotypic transformation:dense patch was detected under the cytomembrane;In aorta of SHR-C group,the endothelial cells were damaged seriously and with parts of it shed.Meanwhile,the basement membrane had a worm-eaten proliferation for repairing,and the internal elastic membrane was split and structure disturbance.At the same time,the VSMCs were hypertrophy and proliferation and the organelles in cytoplasmic were significantly increased.The hypertrophy of collagen was mainly in media,adventitia AF did not change significantly.8.TUNEL to detect of apoptosisTerminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL)detected the vessel wall apoptosis cells and apoptotic cell's nucleus were stained brown.Compared with WKY-C group,the apoptotic cells of the mesenteric small artery in SHR-C group were significantly increased,meanwhile,the AI was significantly increased(P<0.01).The aortic apoptosis was significantly increased and AI was increased(P<0.05)too.9.Immunohistochemical staining to detect of proliferating cell nuclear antigen(PCNA)Immunohistochemical of PCNA staining revealed that the proliferation of vascular wall cell's nucleus were stained brown.Compared with the WKY-C group,proliferation cells of artery tissue in SHR-C group were significantly increased.The PI of mesenteric small artery was increased(P<0.05).In turn,the proliferation of vascular wall cells of aorta was more obvious than mesenteric small artery(P<0.01).The results shows that cell proliferation of aortic wall in SHR-C was more significant.10.TUNEL-PCNA double-stainingAfter TUNEL detecting the apoptosis cells,Immunohistochemical of PCNA detected the positive PNCA cells.The positive cells stained by the TUNEL express brown,meanwhile the positive cells stained by the PNCA expressed purple.The image sharply shows that apoptosis positive cells were increased obviously in hypertension of the small artery,but in the large artery blood wall cell hyperplasia is bigger than the trend of apoptosis.Conclusions1.The vascular remodeling in hypertensive small artery was significant collagen hyperplasia,especially in adventitia,which was accompanied with AF migration and phenotypic transformation.2.Aortic remodeling of hypertension was mainly vascular wall cell hyperplasia especially smooth muscle proliferation in the media,it fitted with the traditional research.3.Proliferation and apoptosis were both existed in hypertensive artery remodeling,but the non-cellular components-collagen hyperplasia was a predominant status in small artery remodeling.Vascular remodeling in hypertensive artery was a dynamic pathological process with multifactor,multi cell and multi Pathology.Small artery remodeling is different from the characteristic change of aortic remodeling.Part ?Protective mechanism of GSPE on small artery vascular remodeling in Spontaneous Hypertensive RatsBackgroundHypertension(HE)and its complications development is closely correlated to vascular remodeling(VR).Simple " up to standard blood pressure " cannot fully control target organ damage and its complications,however,target organ damages can even occur before the blood pressure increased,indicating that the correlation between hypertension target-organ damage and blood pressure levels is not as strong as early theory predicted.There is still significant need to explore the mechanism of the vascular remodeling thus to better understand how to prevent and treat VR for ensureing patient safty,and improve the quality of human survival and reducing the pressure of the national economy.The research shows that the oxidative stress induced by reactive oxygen species(reactive oxygen species,ROS)is involved in every link of hypertensive vascular remodeling.The oxidative stress's level enhancement is involved in the occurrence and development of hypertension.Oxidative stress is implicated in endothelial dysfunction,inflammation,hypertrophy,hyperplasia,apoptosis,migration and fibrosis,which are importantly triggering and promoting factor involved in vascular remodeling in hypertension.Reactive oxygen species(ROS),such as H2O2,ONOO-and HOCO3-,play important roles in vascular remodeling.Grape seed procyanidins(GSPE)is mainly derived from grape seeds,it is a polyphenol compound possessed strong antioxidant activity.A large number of research materials confirmed that GSPE is a potent antioxidant proved to be protective in cardiovascular system:not only due to its anti-atherosclerosis,blood press control,lipid lowering,anti-myocardial ischemia-reperfusion injury but also anti-inflammation and endothelial cell's protective effect.Recent studies have found that it has anti-tumor activity,effective lethal effect to a variety of tumor cells.However,the role of GSPE in hypertensive vascular remodeling is not clear,especially in small vascular remodeling.The experiment in Part ? has detected collagen hyperplasia's important position in small artery's vascular remodeling and small blood vessel wall apoptosis cells increase involved in the imbalance between blood wall cell's proliferation and apoptosis participates in the development of small vascular remodeling.The research in this Part ? adopted the SHR rats model and given different doses of GSPE to intervention,then observed the change of blood pressure,blood lipid,blood sugar and the level of oxidative stress after applying GSPE,and observed the GSPE's effect to small artery vascular remodeling 's morphology,histology and cytology by light and electron microscope.TUNEL and PCNA detected respectively the influence on apoptosis and proliferation of blood vessel cell.Discuss different doses GSPE whether can play an important role in improving the hypertensive small artery vascular remodeling and finally detect collagen ?/?,TGF-beta 1 and related apoptosis proteins-BAX,Bcl-2 expression using quantitative PCR and Western bloting.The effect of GSPE on vascular remodeling and the underlying mechanism may be evaluated about the clinical utilization of GSPE in small vascular remodeling.Purposes1.Research the relationship between oxidative stress's level and hypertensive small vessel collagen hyperplasia remodeling.2.Confirm different doses of GSPE's protective effect on hypertensive small vascular collagen hyperplasia remodeling.3.Observe the relationship between GSPE and cell proliferation,apoptosis of the vessel wall cell.4.Explore the GSPE'S mechanism for improving hypertensive small artery remodeling from the molecular level.MethodsThe experimental groups,the experimental animals feeding,dosing and specimens to return are the same as those in Part ?.1.Measure the systolic blood pressures and body weight once a week before experiments until the end of the experiment.Systolic blood pressures is placed by its tail systolic blood pressures.2.Draw blood sample to test total cholesterol(TC),low density lipoprotein cholesterol(LDL-c),triglyceride(TG),and blood sugar(BS),creatinine(Cr)and urea nitrogen(BUN)using serum after centrifugal in part ?.3.Measure the the four group's content of Malondialdehyde(MDA),H2O2,ROS,superoxide dismutase(SOD),and catalase(CAT)of mesenteric artery tissues,using the Multilabel Counter following the manufacturer's instructions.4.Dissect and slice the Three group's mesenteric small arteries.The morphology of mesenteric artery was observed by HE staining.The change of collagen fiber and elastic fiber was observed by Sirius red-Victoria blue dyeing,The expression of type ? and ? collagen were detected by indirect immunofluorescence histochemical staining with confocal laser scanning microscope.The three group's ultrastructure change of mesenteric small artery was observed using transmission electron microscope.The TUNEL and PCNA respectively observed cell proliferation and apoptosis of the vessel wall using.Samples tissue were stored at-80?,in order to conduct RT-PCR and Western Blot summarizing the part ?'s experimental tissue samples.Results1.Systolic blood pressure and body weightAlthough the systolic pressure rising gradually as the experimental process,compared SHR-group C with two GSPE intervention group,the systolic pressure difference between groups have no statistical significance(P>0.05).No significant weight alternation was observed in the three groups during the course of the experiment.2.Serum lipids(TC,TG,LDL-C)and BS,Cr,BUNThe serum total cholesterol,low density lipoprotein cholesterol,triglycerides and blood sugar,creatinine and blood urea nitrogen were no significant difference between three groups(P>0.05).3.Oxidative stress index of mesenteric arteriolar in the four groupsCompared with WKY-C group,significant high level of ROS,H2O2 and MDA was measured in SHR-C hypertensive rats,nevertheless low activity of SOD,CAT was measured in SHR-C hypertensive rats.The difference was statistically significant(P<0.01).After the GSPE intervention,ROS(P<0.05),H2O2 and MDA(P<0.01)decreased in SHR-H group meanwhile SOD(P<0.01),CAT(P<0.05)increased,the difference was statistically significant.However,in SHR-L group,merely ROS,H2O2 content decreased obviously(P<0.05),and the H2O2 decline in the two intervention groups was statistically significant(P<0.01),which shows that GSPE inhibiting oxidative stress damage has dose-dependent trend.4.Morphological changes of mesenteric small artery4.1 HE staining showed SHR-group C shows obvious signs of vascular remodeling:wall thickened,lumen stenosis,VSMCs hypertrophy and proliferation and arranged at random.In the SHR-L and SHR-H group,mesenteric vascular remodeling signs have improved,especially in the SHR-H,degree of the wall thickness and luminal stenosis significantly alleviated:vascular intima smooth,VSMCs also hyperplasia with irregular arrangement?4.2 The Sirius red-Vitoria blue staining bright field shows that in the SHR-C group,mesenteric arteriolar wall was markedly thickened,luminal stenosis.The blue-green single layer elastic plate has no obvious thickening.The red collagen fiber was severe hyperplasia,mainly in the adventitia and media;GSPE intervention group improved:wall thinner,lumen enlargement,the collagen fiber hyperplasia alleviating,especially in the SHR-H group.After conducting image capture by Olympus DP72 light microscope and quantitative detection analysis by Image pro-plus image analysis system,the results shows that compared with the SHR-C group,in the SHR-H,ID?LCSA increased obviously(P<0.05),whereas WT?WT/ID?WCSA?WCSA/LCSA decreased obviously(P<0.01),the difference was statistically significant.In the SHR-L,WT?WT/ID?WCSA?WCSA/LCSA obviously reduced(P<0.01),Which shows that GSPE's improvement to small vascular remodeling morphology has a dose-dependent trend.5.Comparison of the collagen and elastic fibers in the three groups ratsSirius red-Vitoria blue staining bright field shows that in SHR-C group,mesenteric arteriolar wall was markedly thickened,luminal stenosis;blue-green single elastic plate thickened to some extent;Red collagen was severe hyperplasia in the adventitia and media and the collagen arranged at random and even ruptured.After GSPE treatment,the wall thickness and luminal stenosis of small mesenteric arteries were alleviated significantly as a dose-dependent manner:wall thickness alleviated,lumen diameter enlarged.Red collagen fibers was dramatically reduced,irregular arrangement,particularly in SHR-H,blue-green single elastic plate has no significant change.Image Pro-Plus quantitative analysis system detected that the total collagen content per area of mesenteric small artery in the GSPE treatment group was significantly decreased than in the SHR-C group(P<0.05),however,there was no significant difference in the elastic fiber per area(P>0.05).Further,the ratio of collagen/elastin decreased observably in the GSPE treatment group(SHR-L,P<0.05;SHR-H,P<0.01)compared with the SHR-C group.6.The distribution of type ?/? collagen in mesenteric small arteryType ?/? collagen and elastine were stained with Sirius red-victoria blue and were observed in the dark field using polarization microscopy.In the SHR-C group,mesenteric arteriolar wall collagen fibers was severe hyperplasia,serious thickening and arranged at random.Overload yellow-red type ? collagen was found in adventitia and extended to media,green type ?collagen fibers were distributed in the middle and adventitia margin.However,after GSPE treatment,wall collagen fiber hyperplasia alleviated,especially type?collagen fibers in the media and adventitia.Meanwhile,type ? collagen decreased significantly too,their distribution was still irregular.7.Indirect immunofluorescence histochemical staining with confocal laser scanning microscope to detect the expression of type ?/? collagenThe confocal laser scanning microscope shows that in the SHR-C group,mesenteric arteriolar wall thickened obviously,green collagen and red cell nuclei increased significantly,the luminal narrowed.The type ? collagen increased and distributed in media and adventitia.The type ? collagen also increased and mainly distributed in adventitia and margin.However,after GSPE treatment,the blood vessel wall thickness alleviated and numbers of cell nuclei reduced,type ? and type ? collagen all decreased.Fluorescence quantitative analysis results(IOD)shows that compared with the SHR-C group,in the GSPE treatment group,type ? and type ?collagen expression decreased significantly(P<0.01),the difference had statistical significance.especially in the SHR-H group,the difference between groups was statistically significant(P<0.01).8.Ultrastructural changes of mesenteric small artery in the three group under the transmission electron microscope.Under the transmission electron microscope,the endangium of SHR-C group was severely broken and part of them fell off and even detected the apoptotic cells.Meanwhile,AF with expansion of the endoplasmic reticulum was migrated to the media,even phenotypic transformation:dense patch was detected under the cytomembrane accompanied by a large number of collagen irregularity hyperplasia and distribution disorder in adventitia and margin,AF was observed mitochondrial "vacuolization" changes;After GSPE treatment,inner membrance damage alleviated,collagen hyperplasia relieved as a dose-dependent manner.AF 'S migration was detected in the SHR-L group.The AF with abundant endoplasmic reticulum was also detected in the adventitia.9.TUNEL to detect of apoptosis cellsThe TUNEL's results shows that the apoptotic cell nucleus of blood vessel wall was stained brown.Compared with SHR-C group,the apoptotic cells of the mesenteric small artery in SHR-H group decreased significantly;meanwhile,AI decreased significantly(P<0.01).The results was statistically significant compared with the SHR-L group(P<0.01).10.Immunohistochemical staining to detect of PCNAImmunohistochemical of PCNA staining revealed that the proliferation state of vascular wall cell's nucleus were stained brown.Compared with the SHR-C group,proliferation cells of small artery tissue in SHR-H group decreased significantly and PI of mesenteric small artery in SHR-H group decreased(P<0.05),the difference was statistically significant.11.TGF-?1 and type ?/? collagen s mRNA and protein expression of mesenteric arteriolar in four group ratsThe results shows that compared with the WKY-C,in the SHR-C group,the mRNA levels of type ?/? collagen,TGF-?1 all increased(P<0.05),the results has remarkably statistical significance.But low dose and high dose of GSPE can all antagonize these effects.Compared with the SHR-C group,the mRNA levels of type ?/? collagen,TGF-?1 all decreased in the SHR-H and SHR-L group were decreased,the results have remarkably statistical significance(P<0.05).Western Blot confirmed this results.In the SHR-H and SHR-L group,the proteins levels of type ? collagen(P<0.05),type ? collagen(SHR-L,P<0.05;SHR-H,P<0.01),TGF-?1(SHR-L,P<0.05;SHR-H,P<0.01)were decreased.12.Bcl-2 and Bax 's mRNA and protein expression of mesenteric arteriolar in four group ratsThe results shows that compared with the WKY-C,the mRNA levels of mesenteric arteriolar Bcl-2 significantly decreased(P<0.01)and Bax increased(P<0.05)significantly in the SHR-C group,the statistics has a significant meaning.Therefore,the ratio of Bcl-2/Bax significantly decreased.(P<0.01);But low dose and high dose of GSPE can all antagonize these effects.Compaed with the SHR-C,Bcl-2's mRNA expression in the SHR-L(P<0.05)and SHR-H(P<0.01)increased,Bax's expression in the SHR-H decreased(P<0.05),and the ratio of Bcl-2/Bax increased(SHR-L,P<0.05;SHR-H,P<0.01),the statistics has a significant meaning.Western Blot confirmed that the protein expression of Bcl-2 and Bax are consistent with them.Compared with the SHR-C,Bcl-2's protein expression increased(P<0.05)in two treat groups,Bax's expression in the SHR-H decreased(P<0.05),and the ratio of Bcl-2/Bax increased(P<0.05)in two treat groups,the statistics has a... |
PDF Full Text Request