| Farrerol, a major flavanone-type compound present in plants, is the main active substance of “Man-shan-hong”(Rhododendron dauricum L.). It has obvious effect for the treatment of chronic bronchitis and cough expectorant. Moreover, modern pharmacological studies suggest that farrerol also has antibacterial, anti-inflammatory, immune suppression, etc. The previous studies by our group have demonstrated that farrerol exhibited a significant cytoprotective activity against hydrogen peroxide induced injury in human umbilical vein endothelial cells and also has markedly inhibitory effects against the proliferation of rat vascular smooth muscle cells(VSMCs). These results suggest that farrerol has important significance in the treatment of the cardiovascular system diseases. However, the effect of farrerol for hypertension in vivo is limited. Herein, the present study was performed to observe the vasoactive effects of farrerol on rat aorta, to investigate its role of farrerol as a potential anti-hypertension agent as well as its resistance to aortic injury, and futher explor its effect on aorta gene expression.Part 1 The vasodilation effect of farrerol on the isolated rat aorta and its mechanismObjectives To observe the vasoactive effect of farrerol on rat aorta and investigate the possible underlying mechanisms.Methods 1. Isolated aortic rings of rat were mounted in an organ bath system and the myogenic effects stimulated by farrerol were studied.2. Intracellular Ca2+([Ca2+]in) was measured by molecular probe Fluo-4-AM using Laser scanning confocal microscopy method.3. The activities of L-type voltage-gated Ca2+ channels(LVGC) were studied with whole-cell patch clamp in cultured VSMCs.Results 1. Farrerol induced relaxation on aortic rings with KCl- or PE-induced contraction in a dose-dependent manner. At a concentration of 100 μM, farrerol induced the maximal relaxations of 66.97% for KCl and 65.13% for PE, respectively. The experiment indicated that there was a slight or no difference in the potency or sensitivity of the farrerol-induced relaxation effect in rings contracted by KCl or PE. The respective EC50 values were 14.02 μM for KCl and 35.94 μM for PE. This vasorelaxation was not affected by NG-nitro-L-arginine methylester ester or endothelium denudation.The endothelium-denuded aorta rings contraction induced by KCl(60 m M) was mainly due to the depolarization of VSMCs and the influx of extracellular Ca2+ through LVGC. In high-K+, Ca2+-free Krebs’ solution, cumulative addition of Ca Cl2(1–100 μM) induced a gradually increased tension of aortic rings. Pretreatment with farrerol at 14.02 μM noticeably depressed the maximal contraction to 53.90% ± 6.67%. The contraction induced by PE was mainly caused by the influx of extracellular Ca2+ through the receptor-operative Ca2+ channel(ROC). The effect of farrerol on the ROC was studied in Ca2+-free Krebs’ solution; PE(1 μM) pre-stimulated endothelium-denuded aortic rings were used. Farrerol(35.94 μM) pre-incubation significantly inhibited the contraction induced by extracellular Ca Cl2(0.01–3 m M), and the maximum contraction was decreased to 51.43% ± 7.87%.In this experiment, endothelium-denuded rings were pretreated for 30 min with 1 μM Verapamil, an LVGC inhibitor, and subsequently contracted by PE. Except in the control(vehicle) group, farrerol(35.94 μM) was added in another three experimental groups, and then the curves of relaxation were clearly shown. In addition, this effect of farrerol was enhanced by 10 μM ruthenium red(RR), a ryanodine receptor inhibitor(p < 0.01 compared to farrerol only), but not by 50 mg/L heparin(HP), an IP3 receptor inhibitor. The result demonstrated that the effect of farrerol on vasorelaxation was related to the ryanodine receptor.2. Our preliminary experiments showed that 60 m M KCl was optimal for measuring [Ca2+]in. The fluorescence intensity increased by 100.6% after addition of KCl which means that KCl could significantly increase [Ca2+]in. Farrerol(14.02 μM) was then applied to the cells and we observed a decrease of 72.9% in the intracellular fluorescence intensity. The VSMCs were then washed with 60 m M KCl in order to remove farrerol while keeping the cells constantly exposed to 60 m M KCl. KCl induced again an increase of [Ca2+]in, gradually restoring the calcium levels to the basal level measured before the application of farrerol. The above results indicate that farrerol could decrease [Ca2+]in in cultured VSMCs.3. The effects of farrerol on [Ca2+]ex influx currents were measured using whole-cell patch clamp technique. The peak currents of LVGC appeared at 0 m V. The maximal current density was-10.72 ± 0.45 p A/p F. At a test potential of 0 m V, 14.02 μM farrerol reduced LVGC currents by 59.50%.Part 2 Antihypertensive activity of farrerol and its attenuation action against the aortic lesion in SHRObjective The aim of this study was to explore the antihypertensive activity of farrerol and its attenuation action against the aortic lesion in SHR. Materials and methods1. Blood pressure, heart rate and body weight measurement.WKY(n = 8) and SHR(n = 24) aged 12 weeks were divided into four groups of eight animals each:(1) WKY+C(control group),(2) SHR+C(untreated),(3) SHR+F(treated with farrerol), and(4) SHR+V(treated with verapamil). The rats were orally administrated farrerol or verapmil(50 mg/kg body wt) dissolved in 0.5% CMC-Na solution during 8 weeks, respectively. The control group received an equal volume 0.5% CMC-Na solution. Systolic blood pressure, the heart and body weight were measured in conscious animals before the start of treatment and weekly during treatment.2. Determination of plasma biochemical indicesPlasma superoxide dismutase(SOD), Malonaldehyde(MDA), Nitric oxide(NO), Nitricoxide synthase(NOS), Endothelin(ET-1), Angiotensin II(Ang II), Lnterleukin-6 (IL-6) and Tumor necrosis factor-α(TNF-α) were measured in plasma using a quantitative sandwich enzyme immunoassay.3. Determination of aortic homogenates biochemical indicesAortic homogenates were prepared on ice in lysis buffer. e NOS, Nitrite, NAD(P)H oxidase and O2- were measured in accordance with the Kit instructions and the literatures.4. Aorta tissue staining with Hematoxylin-Eosin and MassonPortions of the aorta were fixed for 24 hours in buffered 10% neutral buffered formalin, routinely processed and embedded in paraffin, cut into 5 μm thick slices and fixed on glass slides for staining with Hematoxylin-eosin and Masson.5. Immune histochemical method to determine the protein expression of aortaCollagen I, Collagen III, MMP-2, MMP-9 and ROS were measured using Immune histochemical method in the aorta of SHR and WKY.6. Determinate the m RNA and protein expression of rat aortae NOS, p22 phox, NF-k B and SM-α-actin were measured in aorta using RT-PCR quantification and Western blot analysis.Results 1. Effect of farrerol treatments on systolic blood pressure, heart rate and body weightBody weight, blood pressure and heart rate were weekly observed to represent the growth for all rats. Systolic blood pressure levels of SHR were higher than those of WKY(119 ± 5 mm Hg in WKY+C vs 190 ± 6 mm Hg in SHR+C, p < 0.01), while administration with verapamil or farrerol for 8 weeks significantly decreased systolic blood pressure levels of SHR(190 ± 6 mm Hg in SHR+C vs 128 ± 5 mm Hg in SHR+V, p < 0.01 or 162 ± 5 mm Hg in SHR+F, p < 0.01). After 8 weeks of treatment(from 12-20 weeks of age), the heart rate of SHR+V was significantly lower(p < 0.01) than that of SHR+F and SHR+C. The body weights administrated in treated animals over the 8 weeks treatment period were not significantly different from that of untreated animals.2. Plasma biochemical indicesOur experiments were observed the relationship of oxidant stress, inflammatory factor and endothelial function occurs with hypertension through testing the changes of SOD, MDA, NO, NOS, ET-1, Ang II, IL-6 and TNF-α from plasma of SHR after application of farrerol. Plasma levels of MDA, ET-1, Ang II, IL-6 and TNF-α, were significantly higher in SHR than in WKY+C, SHR+F, and SHR+V; while plasma levels of SOD, NO and NOS, were significantly lower in SHR+C than in WKY+C, SHR+F and SHR+V.3. Effect of farrerol on nitrite and O2- levels along with the activity of e NOS and NAD(P)H oxidase in WKY and SHRThe e NOS activity was found to be significantly lower than that in WKY+C aortic homogenates. Meanwhile, the levels of O2-, nitrite and the activity of NAD(P)H oxidase were higher in SHR+C aortic homogenates than those in WKY+C aortic homogenates. After treatments with farrerol NAD(P)H oxidase activity were markedly decreased(NADH: by 54.9 ± 4.1%, NADPH: by 48.6 ± 5.4%), the levels of nitrite and O2- were also decreased by 6.8 ± 0.8%, 33.5 ± 5.7%, respectively. However, e NOS activity was increased by 84.7 ± 6.2%.4. Effect of farrerol treatment on aorta morphology and histologyThere were no differences in the outer diameter and lumen diameter between WKY+C and SHR+C. However, media thickness(+20%),wall area(+43%) and the media-lumen ratio(+20%) were greater(p < 0.05) in SHR+C than those in WKY+C. Moreover, nuclei size was significantly larger in SHR+C aortic rings than that in WKY+C, while nuclei number was notably lower. After administration with farrerol for 8 weeks, we found that farrerol partially reversed the morphological remodeling of the SHR aorta, resulting in a notable reduction in media thickness, wall area, media-lumen ratio, and nuclei size. About Masson staining, compared with WKY+C, SHR+C of the relative collagen contents was significantly increased(p < 0.05); Compared with SHR+C, SHR+V and SHR+F of the relative collagen contents were significantly decreased(p < 0.05).5. Effect of farrerol on Collagen I, Collagen III, MMP-2, MMP-9 and ROS production in the aortaCompared to the WKY+C,Collagen I, Collagen III, MMP-2, MMP-9 and ROS levels were markedly increased in the aorta of SHR+C(p < 0.05). Farrerol markedly reduced them in the SHR aortas(p < 0.05).6. Aortic e NOS, p22 phox, NF-k B and SM-α-actin m RNA expressionAortic e NOS m RNA expression was lower in SHR+C than that in WKY+C(p < 0.05), while treatment with farrerol increased e NOS expression(p< 0.05). In contrast, aortic p22 phox, NF-k B and SM-α-actin m RNA expression was higher in SHR+C than that in WKY+C(p < 0.05), while treatment with farrerol or verapamil increased its expression(p < 0.05).7. Aortic e NOS, p22 phox, NF-k B and SM-α-actin protein expressionProtein expression of e NOS was higher in the aortas of WKY+C than that in the SHR+C. After treatment with farrerol for 8 weeks, e NOS expression was significantly increased in the aorta of SHR. We also studied the protein expression of p22 phox, NF-k B and SM-α-actin, we found higher protein levels of p22 phox, NF-k B and SM-α-actin in SHR+C aortas than that in WKY+C aortas. However, treatment with farrerol decreased p22 phox protein levels in SHR aortas. Part 3 Effect of farrerol on aorta gene expression in SHRObjective The aim of this study was to investigate the effect of farrerol on aorta gene expression in SHR, find maximum of differential expressed genes and signaling pathways associated with hypertension, further study the mechanism of its anti-hypertension providing new ideas. Materials and methods1. Digital gene expression library were prepared and sequenced by a commercial service(Genomics institution, Beijing) using Illumina Hi Seq? 2000 technology according to the manufacturers’ protocols, the sequencing quality was rigorously evaluated.2. The p value corresponds to the differential gene expression test. We obtained the significance of the gene expression difference through an FDR(False Discovery Rate) of ≤ 0.001 and the absolute value of log2 ratio ≥1. More stringent criteria with smaller FDR and bigger fold-change values can be used to identify DEGs. The gene ontology(GO) classification system was used to determine the possible functions of all differentially expressed genes. The p value was calculated by GO and Bonferoni corrected. A corrected p value of ≤0.05 was selected as a threshold for significant enrichment of the gene sets. For pathway enrichment analysis, we mapped all differentially expressed genes to terms in KEGG database and further searched for significantly enriched metabolic pathways or signal transduction pathways.The genes selected according to the DGE-tag copy number were evaluated, and some of these genes were investigated by RT-PCR. A total of 1 μg of total RNA from each sample was used to synthesize the first strand c DNA using the Prime Script Reverse Transcriptase kit according to the protocol of the manufacturer. GAPDH was used as a reference gene. RT-PCR was carried out in an ABI PRISM® 7300 Sequence Detection System according to the instructions of the manufacturer.Results 1. We used gene express profile to identify differentially expressed genes in the aorta from WKY+C, SHR+C, SHR+F and SHR+V. Compared with WKY+C, SHR+C showed 444 upregulated genes. Compared with SHR+C, SHR+F increased the expression of 2329 genes in the aorta. Similarly, SHR+V increased the expression of 1037 genes in the aorta.In the converse analysis, Compared with WKY+C, SHR+C showed 1547 downregulated genes. Compared with SHR +C, SHR+F decreased the expression of 575 genes in the aorta. Similarly, SHR+V decreased the expression of 629 genes in the aorta.2. To understand the functions of differentially expressed genes, we mapped all the genes to terms in KEGG database, and compared this with the complete reference gene background to search for genes involved in signal-transduction pathways that were significantly enriched. Among all the genes with KEGG pathway annotation, 1991 differentially expressed genes were identified between WKY+C and SHR+C,1666 differentially expressed genes between SHR+C and SHR+F, and 2904 differentially expressed genes between SHR+C and SHR+V of libraries. The seven differentially expressed signaling pathways related with hypertension from every comparison group have been listed.3. Hypertension involves many aspects, such as vascular smooth muscle contraction, MAPK signaling pathway, regulation of actin cytoskeleton, VEGF signaling pathway, calcium signaling pathway and renin angiotensin system. In vascular smooth muscle contraction, this pathway plays a central, obligatory role in determining myogenic responsiveness but aslo plays a major role in the maintenance and regulation of blood pressure.4. In total, 453 genes were found to be differentially expressed in different groups,with expression profiles clustering into three different groups using the K-means algorithm. 18 genes showing more than a 4-fold increase or less than a 4-fold decrease in expression were isolated and candidate genes were selected as significantly enriched genes. To validate the DGE data, the expression levels of 10 genes were measured using real-time PCR. These results correlate with those from our DGE data. The fold changes in expression among samples were completely consistent within the RT-PCR in several cases; however this difference is not surprising given the difference in sensitivity of gene detection in the two methods.5. By analyzing the gene expression profiling of aorta tissues, we found that most of the genes from the vascular smooth muscle contraction signaling pathways were upregulated by aorta in SHR+C. Furthermore, the expression of ADRA1 was significantly lower in the aorta of SHR+C than WKY+C, and elevated after oral administration farrerol.Conclusions: 1. Farrerol has significant relaxation effect on rat aorta, its mechanism of action is inhibition of L-type voltage-gated calcium channel(LVGC), receptor-operated calcium channel(ROC) and sarcoplasmic ryanodine receptor, thereby descrease the calcium content of rat VSMCs.2. Farrerol significantly lower blood pressure and protect aortic lesion in SHR, the mechanism may be related to the regulation of oxidative stress, immune factors and diastolic contraction of vascular factors.3. Preliminary studies of gene expression profiling indicate that farrerol reduce blood pressure and protect aortic lesion in SHR,which may be related to upregulate Adra1 d genes in the vascular smooth muscle contraction pathway. |
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