| ObjectiveTo investigate the effectiveness of RARG in ameliorating carotid arterial stiffness and improve endothelial function in essential hypertension patients.MethodsStudy population:grade1and grade2essential arterial hypertension patients aged40-75years old, with qi-asthenia and phlegmatic hygrosis syndrome in Chinese medicine. Study design:patients were randomized into two group:RARG group and basic antihypertensive drug group (basic drug group). All patients should receive antihypertensive drugs to reduce blood pressure <140/90mmHg for general population and<150/90mmHg for aged population. On the top of antihypertensive drugs, RARG group received RARG treatment. Study period is8weeks.Carotid arterial stiffness parameters, including Ep,β,AC,PWV β, AI, YEM and Dist, were evaluated by eTRACKING(ALOKA prosound a7) at baseline and study endpoint, as well as flow mediated vasodilation(FMD).The syndrome was scored according to the level of main and secondary symptoms of qi-asthenia and phlegmatic hygrosis syndrome. Main symptoms score2,4and6for mild,moderate and severe level. Secondary symptoms score1,2and3for mild,moderate and severe level. Symptom scores at baseline and endpoint were compared to evaluate effectiveness of RARG on meliorating symptoms. In addition, number of patients who have achieved ideal blood pressure goal was recorded, as well as antihypertensive drug use.ResultsCarotid arterial stiffness parameters, including Ep,β,AC,PWV β, AI,YEM and Dist, were not significantly different between basic treatment group and RARG group at baseline (P>0.05). After8weeks treatment, Ep, β,AC,PWV β, AI and YEM was not significantly improved (P>0.05) in both groups; carotid distensibility was remarkably improved in RARG group (7.9%vs10.9%, P=0.00) and was not significantly changed in basic treatment group (8.4%比8.9%, P=0.41)。FMD between two groups at baseline was comparable (4.9%vs5.6%, P=0.47) After8weeks treatment, FMD in RARG group was improved from5.6%to8.6%(P=0.01), but not improved in basic treatment group (4.9%vs5.1%, P=0.85)Symptom score between two groups was comparable at baseline (33.66+4.31vs34.82±2.64, P=0.25). RARG treatment for8weeks could significantly reduce symptom score (33.66±4.31vs22.86±3.81, P=0.00). Symptom score was significantly reduced in basic treatment group either (34.82±2.64比29.31±2.64, P=0.00). In addition, symptom score in RARG group was lower than basic treatment group at endpoint (P=0.00)。During follow, number of patients who have achieved ideal blood pressure goal were remarkably increased in both group. It was10at baseline and33at endpoint in basic treatment group,12at baseline and31at endpoint in RARG group. The difference of patients number was not significant (P=0.67),Drug classes used in both group were as follow:CCB was the most common used antihypertensive drug, diuretics,ARB,ACEI and β-blocker rank second to fifth. Number of patients who received only one antihypertensive drug was6in basic treatment group and5in RARG group, and who received two antihypertensive drugs was26in basic treatment group and28in RARG group, and who received three antihypertensive drugs was3in basic treatment group and2in RARG group. The difference of drug use between two groups was not significant (P>0.05)Multivariate nominal logistic stepwise regression analysis demonstrated that hypertension course over10years and RARG were independent predictors for carotid distension increasing, with the OR2.99(95%C.I.1.05-8.52) and3.30(95%C. I.1.16-9.42). RARG was independent predictor for FMD increasing as well, with OR6.30(95%C. I.2.04-19.48).ConelusionIn grade1and grade2essential arterial hypertension patients aged40-75years old and with qi-asthenia and phlegmatic hygrosis syndrome, RARG has additional arterial protection on the top of basic hypertensive drug treatment. RARG could increase carotid distensibility and improve endothelial function. RARG is better than basic hypertensive drug treatment in ameliorating symptoms.Animal researchObjectiveTo investigate arterial protection of RARG per se from extracelluar matrix (ECM) remodeling and endothelial function in spontaneous hypertension rats (SHR)Methodsmale SHRs8-10weeks were randomized into4groups:SHR group(SHR), atorvastatin group(ATV), RARG low-dose group(RARG-L) and RARG high-dose group (RARG-H).8Wistar Kyoto rats was used as normal blood pressure control group (WKY). SHR and WKY group were intragastric administrated of drinking water each day. Atorvastatin(3.6mg/kg·d) was intragastric administrated in ATV group. RARG was intragastric administrated in RARG-L(3.87g/kg·d, herb) and RARG-H(7.74g/kg·d, herb) group.12weeks later, animals were sacrificed and aortas were harvested. Aortic arches were used for histology study. Initial3-5mm segment of descending aortas were cut for vascular rings experiments. Rest aorta tissues were stored at-80℃for total nitric oxide (NO) measurement and gene expression detection.Aortic rings were mounted vertically in a organ bath, which was warmed by circulating water at37℃. One side of the ring was connected to a hook in the organ bath, while the other side was connected to a tension transducer. After lh equilibration, the ring was pre-contracted by10(?)M phenylephrine (PE) and relaxed by acetylcholine (Ach) at10-9-10-4M cumulative concentration. Relaxation percentages in maximum contractile force produced by PE were calculated at each concentration of Ach to evaluate endothelium-dependent vasodilation. Sodium nitroprusside at10-9.5n-10-8M cumulative concentration was used to evaluate endothelium-independent vasodilation.Aortic tissues were lysed by ProteoJETTM Mammalian Cell Lysis Reagent, total NO concentration in supernatant was measured by Total Nitric Oxide Assay Kit(Beyotime).Aortic arches were paraffin-embedded and sectioned into4u m thick slices. H&E staining was use to observe arterial wall thickness. Elastic fiber was stained by Verhoeff's van Gieson staining (VVG). Collagen fiber was stained by Picro-Sirius Red staining (PSR). Image J(NIH, USA) software was used to quantify positive staining area by VVG and PSR staining.eNOS (nitric oxide synthase, endothelial), CTGF (connective tissue growth factor), MMP2(matrix metalloproteinases2) and TIMP4(tissue inhibitor of metalloproteinase4) mRNA expression in aortic tissue were detected by SYBR Green based quantitative PCR.18S was used as internal control.ResultsAorta wall thickness were not increased in all groups. Quantitative analysis of VVG staining showed that elastic fiber content difference in all group were not significant.Collagen contents in SHR group were lower than WKY group (41.02%±2.58%vs46.58%±9.44%, P=0.28). RARG-L could decrease collagen contents of SHRs (33.87%±4.99%vs46.58%±9.44%, P±0.01). Collagen contents in ATV (36.12%±7.51%) and RARG-H (42.99%±12.31%) group were not significantly changed(P=0.21和P=0.53) compared to SHR group.CTGF and MMP2expression were increased in SHR group(P<0.05). Atorvastatin could decrease CTGF but not MMP2expression. RARG-L could significantly reduce CTGF and MMP2expression (P<0.05). CTGF and MMP2expression were not significantly changed after RARG-H administration (P>0.05)TIMP4expression in all groups were not obviously changed (P>0.05) MMP2/TIMP4ratio were greater than1, indicating that ECM degradation exceed accumulation. RARG-L could further increase MMP2/TIMP4ratio. Atorvastatin and RARG-H were not effective in changing MMP2/TIMP4ratio.Endothelium dependent vasodilation percentage in SHR group(45.96%) is lower than WKY group(95.39), P=0.00. Atorvastatin and RARG-L could restore endothelium dependent vasodilation percentage to93.39%and88.93%. The difference between WKY group, ATV group and WKY group was not significant (P>0.05). RARG-H has no effect on endothelium dependent vasodilation percentage. Endothelium independent vasodilation percentages in all group are not significantly different.Aortic tissue total NO concentration in SHR group is lower than WKY group (2.75±1.06μmol/L比5.26±1.13μmol/L, P=0.00). Total NO concentration in RARG-L group (5.31±1.09μmol/L) and atorvastatin group (5.53±1.11μmol/L) were significantly higher than SHR group (P=0.00, P=0.00, respectively) Total NO concentration is both group were nearly the same with WKY group(P=0.93,P=0.64, respectively). RARG-H has no significant effect on elevating NO concentration.Aortic tissue eNOS expression in SHR group, which is17%of WKY group, was significantly down regulated. Atorvastatin could increase eNOS expression to36%of WKY group. eNOS expression in RARG-L group was nearly normal, which is95%of WKY group. RARG-H has no effect on eNOS expression.ConclusionRARG-L has arterial protection per se. It could promote collagen degradation by down regulate CTGF, up regulate MMP2expression and change MMP2/TIMP4balance. RARG-L also restore endothelium dependent vasodilation of SHRs,. The mechanism of endothelial function protection by RARG-L is up regulates eNOS expression and increases NO concentration in SHRs aorta. RARG-H does not have any arterial protection.
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