| It has been well documented that hypertension is one of the most critical risks for the induction of various cardiovascular disorders.Long-term hypertension can lead to both structural and functional impairments of the myocardial tissue,in which the high blood pressure-induced myocardial hypertrophy is regarded as the most common one.Myocardial hypertrophy is characterized by compensatory hypertrophy of cardiomyocytes,proliferation of cardiac interstitial fibroblasts,and massive sedimentation of extracellular matrix,and will eventually cause the occurrence of cardiac arrhythmias,myocardial infarction,heart failure and cardiac death.Besides the overloaded pressure and volume irritation in response to long-term hypertension,it has been shown that the pathological progression of hypertension-myocardial hypertrophy-myocardial fibrosis is also mediated by a large number of neuro-endocrine factors,including prolonged excessive excitation of sympathetic nerves and ?1-adrenergcic receptor,activation of relevant cytokines and growth factors,such as tumor necrosis factor a(TNF-a),transforming growth factor?1(TGF-?1)connective tissue growth factor(CTGF),matrix metalloproteinases(MMPs)and so on.Reversing and improving the left ventricular hypertrophy,myocardial fibrosis and cardiac systolic and diastolic malfunction has been considered as one of the key goals for curing chronic hypertension and been a hot area of research in recent years.Although numerous emerging drugs has been used in clinic to treat high blood pressure,myocardial hypertrophy and cardiac malfunction,only a small proportion patients with hypertension achieved optimal therapeutic effects,which is partially resulted from their low compatibility,high dependency on specific target and multiple side effects.It has been suggested that certain acupoints exert anti-hypertensive effects via the somato-autonomic nervous reflex,e.g.,modulating the output signals of sympathetic nucleus in hindbrain and increasing the activity of vagal nerves to resist the excessive excitation of sympathetic nerves and?1-adrenergcic receptor under chronic hypertensive condition.In addition to the neural mechanisms above,acupuncture can also attenuate hypertension and its complications by regulating the related endocrine networks,including RAS,inflammatory factors and enzymes.Moveover,PC6 is effective to improve cardiac function and benefit for treating arrhythmia,angina and cardiac malfunction clinically,which is attributed to its modulation on the level of Ca2+ and calcium signal-related proteins in cardiomyocytes.Therefore,it is meaningful and prospective to further explore and confirm the therapeutic effects of acupuncture treatment on the pathological progression of hypertension-myocardial hypertrophy-myocardial fibrosis.1 ObjectiveThe effects of 8 weeks-electroacupuncture(EA)treatment at PC6 on dynamic changes of blood pressure and both cardiac structure and function were investigated in 12 w-spontaneously hypertensive rats(SHR)with WKY rats as control by using non-invasive blood pressure recording and echocardiographic analysis.Based on the effectiveness of EA treatment on alleviating the pathological progression of hypertension-myocardial hypertrophy/fibrosis-cardiac malfunction,the underlying biomolecular mechanisms were explored to add a potential approach for hypertension treatment in clinic,by using immunohistochemical staining,Western Blotting,Real-Time PCR and radio-immunoassay.2 MethodsThe present study consists of two sections.The aim of the first section is to verify the effectiveness of EA at PC6 on the pathological progression of hypertension-myocardial hypertrophy-cardiac malfunction.Thirty healthy male SHRs were evenly randomized into model,SHR+EA and SHR+sham EA group with 10 Wistar Kyoto(WKY)rats as normal control.EA treatment(2/15Hz,1mA,30 min)at PC6 was applied once a day in 8 consecutive weeks to SHR+EA group.Sham EA was conducted as shallow insertion at PC6 with no electrical stimulation.Systolic blood pressure(SBP),diastolic blood pressure(DBP)and mean blood pressure(MBP)were non-invasively measured in each group before and after weekly EA treatment.Changes of the myocardial hypertrophic indexes were tested at 2,4,6,8 week after EA treatment using echocardiographic analysis.After 8 week EA,blood sample was collected following decollation,and then heart was harvested via thoracotomy to measure the wet weight of left ventricle,HMI,LVMI.Histopathologic changes and expression of collagen ?,collagen ? in myocardial tissue were measured by HE,VG and immunofluorescence staining assay,as well as Western Blotting.The aim of the second section is to uncover mechanisms underlying the inhibition of myocardial hypertrophy/fibrosis induced by EA at PC6.Eighteen healthy male SHRs were evenly randomized into model,SHR+EA and SHR+sham EA group with 6 Wistar Kyoto(WKY)rats as normal control.EA treatment was identical as described above.By using radio-immunoassay,Elisa assay,Western Blotting and Real-Time PCR,the level of ACE,TNF-uand expression of AT1R?AT2R?1-AR?TGF-?1?CTGF?MMP-2 and MMP-9 were tested in serum or myocardial tissure.3 Results3.1 Effects of EA on hypertension-myocardial hypertrophy-cardiac malfunction3.1.1 Effects of EA treatment at PC6 on blood pressure in SHRNon-invasive blood pressure recording showed that,as compared with basal levels,the diastolic and mean blood pressure of SHR+EA rats were significantly decreased after 5 week-EA treatment(P<0.05).The systolic blood pressure of SHR-EA rats were significantly decreased after 6 week-EA treatment(P<0.05).All three types of blood pressure were markedly increased in SHR group(P<0.05).Moreover,the blood pressure of SHR significantly elevated and remained on an increasing trend as compared with the WKY controls(P<0.05).Following 5 weeks of EA treatment,the systolic,diastolic and mean blood pressure of SHR-EA rats were significantly decreased and maintained stable from the 5th to 8th week,which was markedly lower than that of SHR(P<0.05).However,the blood pressure of SHR+Sham group was stable and similar with that of in SHR group(P>0.05).These results indicated that EA is effective to lower blood pressure of SHR.3.1.2 Effects of EA at PC6 on heart weight(HW)/body weight(BW)ratio and left ventricle weight index(LVW1)As compared with WKY,the HW/BW ratio and LVWI in SHR group were significantly increased(P<0.001),which indicated the formation of myocardial hypertrophy and were reversed by EA at PC6(P<0.001,P<0.01).As compared with SHR,the HW/BW in SHR+Sham rats was slightly lower,but the LVWI showed no difference.These results suggested that EA at PC6 is effective to attenuate the myocardial hypertrophy in SHR.3.1.3 Effects of EA at PC6 on the histopathological changes in SHRAs compared with WKY.the ventricle cavity was reduced and the thickness of myocardial tissue was increased in SHR group,which were significantly improved in Sham+EA group.The morphological features in SHR+Sham group showed no difference with SHR group.By HF staining,the myocyte hypertrophy with inflammatory cell infiltration,dis-arranged and broken myofiber,granular degeneration,irregular shaped and hyperchromatic nuclei were found in SHR,which were also attenuated after 8 week EA treatment.As compared with WKY,the myocyte diameter and cross-sectional area of the cross-sectional tissue were significantly enlarged in SHR(P<0.05,P<0.01).which could be attenuated by EA treatment(P<0.05,p<0.01).There was no difference between SHR and SHR+Sham in terms of these indexes.These results indicated that EA at PC6 improve the pathological changes of cardiac structure and morphology under hypertension-myocardial hypertrophy condition in SHR.3.1.4 Effects of EA treatment at PC6 on cardiac function in SHRThere was no obvious change of LVAWd,LVPWd,LVIDd,LVEF and LVFS in WKY rats during 8 weeks-time course.Compared with WKY controls,both the left ventricular anterior(LVAWDd)and posterior wall thickness(LVPWd)were significantly increased in SHR throughout the course of the study(P<0.05,P<0.01,P<0.001).Meanwhile,LVEF,LVFS LVIDd were significantly decreased,suggesting the myocardial hypertrophic changes in SHR.However,as compared with SHR,the LVAWd was significantly reduced(P<0.01)and the LVEF,LVFS LVIDd were marked increased(P<0.05,P<0.001,P<0.001)in SHR+EA group after 6 week-EA treatment.Additionally,the LVPWd was also increased after 8 week-EA treatment(P<0.05).Pattern of these indexes was similar between SHR+Sham and SHR group.These results showed that EA at PC6 attenuated the symptoms of myocardial hypertrophy.The E/A ratio in WKY rats showed no obvious change throughout the course of the study.As compared with WKY,the E/A ratio was dramatically declined in SHR rats(P<0.001),suggesting the impairment of cardiac function,while markedly restored in SHR + EA group after 6 week-EA treatment(P<0.01,P<0.001).EA ratio of SHR+Sham group was similar with that of in SHR group.These results indicated that EA at PC6 could restore the cardiac malfunction in SHR.3.1.5 Effects of EA at PC6 on the distribution of collagen and the level of hydroxyproline in myocardial tissue of SHRAs compared with WKY,the collagen fiber proliferated obviously in SHR as characterized by enlargement,swell,fracture and disarrange of myocardial fibers,and hypertrophy and hydropic changes of myocardiocyt,as well as hyperchromasia of red cell component.As compared with SHR,these pathological impairments were markedly restored in SHR+EA group,but have no improvement in SHR+Sham group.As compared with WKY,the CA,CVF and level of Hyp in cardiac interstitial tissue were significantly increased in SHR(P<0.01,P<0.001),which were effectively depressed in SHR+EA group(P<0.05,P<0.01),but not in SHR+Sham group.These results suggested that EA at PC6 inhibited the myocardial fibrosis in SHR.3.1.6 Effects of EA at PC6 on the expression of Col I and Col III in myocardial tissue of SHRBased on immunostaining results,as compared with WKY,the expressioin of Col I and Col III in cardiac interstitial tissue of SHR were significantly increased,suggesting the excessive deposition of the collagen.As compared with SHR,the expression of Col I and Col III were reduced in SHR+EA group,but not only Col I was slightly decreased in SHR+Sham group.Quantitative analysis showed that,as compared with WKY,the expressioin of Col ? and Col ? in cardiac interstitial tissue of SHR were significantly increased(P<0.001);as compared with SHR,the expression of Col I and Col III were reduced in SHR+EA group(P<0.001).However,the expression of Col I and Col III showed no difference between,SHR and SHR+Sham group.These results showed that EA at PC6 attenuated myocardial fibrosis in SHR via down-regulating the local expression of Col ? and Col ?.3.2 Mechanisms underlying EA's anti-hypertensive and anti-hypertrophic effects3.2.1 Effects of EA at PC6 on the expression of ?1-AR in SHR myocardial tissueAs compared with WKY,the expression of ?1-AR in SHR myocardial tissue was significantly enhanced(P<0.01),which was significantly attenuated in SHR+EA group(P<0.001),suggesting that EA at PC6 could improve the myocardial hypertrophy in SHR via lowering the expression of ?1-AR.However,the expression of ?1-AR was also reduced in SHR+Sham group compared with that of SHR.These results suggested that down-regulation of ?1-AR contributed to EA's anti-hypertensive and anti-hypertrophic effects.3.2.2 Effects of EA at PC6 on the level of ACE,AT1R and AT2R in SHRAs compared with WKY,the levels of ACE in SHR were significantly higher than WKY,in both serum and heart tissue(P<0.001),which was significantly reduced by EA treatment(P<0.001).The level of ACE showed on difference between SHR and SHR+Sham group.In addition,local expression of AT1R in SHR was significantly higher than WKY(P<0.001),whereas AT2R was markedly decreased(P<0.001).Notably,EA was effective in reversing the expression patterns of AT1R,AT2R receptors in SHR(P<0.001,P<0.01),suggesting that AT1R and AT2R might be involved in EA's anti-hypertrophic effects.In addition,the expression of AT1R was also slightly decreased in SHR+Sham group(P<0.05).These results showed that suppression of RAS mediated EA's anti-hypertensive and anti-hypertrophic effects.3.2.3 Effects of EA at PC6 on the level of TNF-a and TNF-a mRNA in serum or myocardial tissue of SHRAs compared with WKY,the level of serum TNF-? and expression of TNF-a mRNA in myocardial tissue were significantly increased in SHR(P<0.001),both of which were lowered in SHR+EA group(P<0.001).In addition,expression of TNF-a mRNA was also reduced in SHR+Sham group compared with that of in SHR.These results indicated that down-regulation of TNF-a also involved in EA's anti-hypertensive and anti-hypertrophic effects.3.2.4 Effects of EA at PC6 on the expression of TGF-?1 and CTGF in myocardial tissue of SHRAs compared with WKY,the fluorescence intensity,protein and mRNA expression of TGF-? and CTGF were significantly increased in myocardial tissue of SHR group(P<0.001),which were attenuated in SHR+EA group(P<0.01,P<0.001).Additionally,both the fluorescence intensity of TGF-?1 and CTGF,as well as the protein expression of TGF-?1 were also slightly reduced in SHR+Sham group(P<0.01).These results showed that EA attenuated hypertension and myocardial hypertrophy via down-regulating the expression of TGF-?1 and CTGF.3.2.5 Effects of EA at PC6 on the expression of MMP-2 and MMP-9 in myocardial tissue of SHRThe protein and mRNA expression of MMP-2 showed no difference among three groups.As compared with WKY,the protein and mRNA expression of MMP-9 were significantly decreased(P<0.001),which were markedly attenuated in SHR+EA group(P<0.05).The expression of MMP-9 showed no difference between SHR and SHR+Sham group.These results suggested that MMP-9 was involved in EA's anti-hypertensive and anti-hypertrophic effects.4 ConclusionWith the enhancement of blood pressure,the myocardial hypertrophy and cardiac malfunction in SHR are characterized by increased thickness of left ventricular,HW/BW,LVWI,and impaired ejection function,as well as up-regulated expression of CVF,Hyp,collagen I and collagen III,which can be attenuated by EA treatment at PC6.In addition,EA at PC6 improves the pathological changes of myocardial morphology,reduces the increased CVF and diameter and cross sectional area of cardiomyocytes,and inhibits the expression of Hyp,collagen I and collagen III,indicating that EA at PC6 attenuates the pathological progression of hypertension-myocardial hypertrophy-cardiac malfunction.Meanwhile,EA at PC6 suppresses the expression of ACE,?1-AR,AT1R,TNF-?,TGF-?1,CTGF and MMP-9 in serum or myocardial tissue of SHR,suggesting that RAS,?1-AR,relevant cytokines and growth factors are involved in the inhibitory effects of EA on the pathological progression of hypertension-myocardial hypertrophy/fibrosis-cardiac malfunction. |
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