| BackgroundAs a clinical common disease and frequently occurring disease, essential hypertension is an important risk factor to vascular diseases of heart, brain and kidney. Vascular remodeling (VR) is the pathological basis of target organ damage during high blood pressure. It is of be great significance to discover and study the antihypertensive drugs that can fight and reverse VR.Vascular adventitia is an important participant in VR. Recent studies have shown that the adventitia is the most sensitive layer to respond to blood pressure, and is the earliest vascular structure in which pathological changes occur. Adventitial fibroblasts (AFs), the main adventitial cells, play a key role in controlling VR through regulatory systems. Under the action of damage, AFs can change phenotype, increase activity of proliferation and migration, release the extracellular matrix, promote new adventitia formation, and participate in the occurrence of vascular adventitia remodeling. The role of vascular adventitia in the vascular remodeling during hypertension is increasingly arousing attention in clinically. Therefore, the research on medicine to intervene VR has theoretical basis and application value.The main components of extracellular matrix (ECM) is collagen, with type I and III as the main types in vascular wall. The abnormal synthesis and expression in collagen is regarded as the important pathological changes of vascular lesions in hypertension. Transforming growth factor β1(TGF β1) is powerful initiating factor to stimulate the synthesis and deposition of collagen in AFs. The classical signal transduction pathway of the TGF-P family is based on Smad signal pathway. Smad3belongs to receptor activated Smad (R-Smad) and plays a core role in the TGF-β1intracellular signal transduction pathways. Our previous study showed that Smad3can respond to collagen deposition in AFs induced by TGF-β1.Qindan capsule (QC), a prepared compound used in traditional Chinese medicine, has been used as an anti-hypertensive agent in clinical settings for years to treat patients with essential hypertension manifests as "liver fire and blood stagnation" symptoms. Our previous clinical study showed that QC can decrease blood pressure and plasma TGF-β1levels so as to help in clearing up clinical symptoms and the prothrombotic state of of this kind of patients.Additionally, our previous animal studies showed that QC has the actions in decreasing blood pressure and angiotensin-Ⅱ level, inhibiting or reversing aorta damage by improving the morphological index of the artery, down-regulating the collagen volume fraction in the media and inhibiting the expression of osteopontin and the transformation of smooth muscle cells. The latest in vitro research has confirmed that QC can reduce the high expression of collagen and inhibit the activity of Smad3excited by TGF-β1in AFs.This research is based on the past studies, further study and discuss the effect and mechanism of QC on vascular remodeling in hypertension by observing the pathological changes and detecting Ⅰ,Ⅲcollagen and TGF-β1and Smad3expression in adventitia of spontaneously hypertensive rats (SHRs). Our study will provide scientific reason for the clinical application of QC.Aims1. To investigate the effect of QC on blood pressure and vascular adventitial morphology change in SHRs.2. To investigate the effect of QC on collagen synthesis and the mechanism underlying the process in SHRs.Methods1. Object of study(1) Experimental rats:Twenty-four male SHRs aged14weeks and weighing 250-290g and8male WKY rats aged14weeks and weighing240-280g were used in the study.(2) Experimental groups:Twenty-four SHRs were divided into three groups: the positive control (PC) group, the QC treatment (QD) group, and the losartan treatment (LSD) group. Eight male WKY rats were included in the negative control (NC) group.(3) Experimental treatment:Rats in the QD group were administered QC at a dose of750mg/kg/day through irrigation of the stomach. Rats in the LSD group were administered losartan at a dose of30mg/kg/day through irrigation of the stomach. Rats in the PC group and the NC group were administered an equal volume of distilled water for three months.(4) Obtain adventitia:Rats were fast for24h at the end of medical administration of whole-day drugs and anesthetized with10%chloral hydrate by intraperitoneal injection. The thoracic aorta adventitia of the experimental rats was segregated.2. Contents of study(1) The systolic blood pressure (SBP) of the rats was monitored by tail-cuff plethysmographic (TCP) method.(2) The thoracic aorta adventitia was observed by Van Gieson (V-G) histological staining method.(3) The mRNA expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ was measured by reverse transcription PCR method.(4) The protein expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ was measured by immunohistochemical staining method.3. Statistical analysisData are expressed as mean±SD. All of the experiments data were analyzed by SPSS20.0software. An independent-sample t-test was applied when only two groups were compared, whereas comparisons among groups were analyzed by two-way ANOVA. Differences were considered to be statistically significant when the P values were less than0.05. Results1. Comparison of SBP before and after experimental treatments.Before experimental treatment, compared with WKY rats (aged14weeks) in NC group, the SBP of SHR rats (aged14weeks) in PC, QD and LSD groups were significantly increased, the difference was statistically significant (p<0.01), the compares among PC, QD and LSD groups were no statistically significance (p>0.05). During experimental treatment, the SBP of SHR rats in PC group was keeping high level, the SBP of SHR rats in QD and LSD groups was significantly continue decreased, and the SBP of WKY rats in PC group was keeping normal level. By the end of experimental treatment, compared with WKY rats (aged26weeks) in NC group, the SBP of SHR rats (aged26weeks) in QD and LSD groups were significantly decreased, the difference was statistically significant (p<0.01). From the third week after the treatments, the compares between PC, QD and LSD groups have statistical difference (p<0.05or p<0.01). From the fifth week after the treatments to the end of treatments, the compares between PC, QD and LSD groups both had significantly statistical difference (p<0.01) and the compare between QD and LSD groups was no statistically significance (p>0.05)2. Morphological observation of thoracic aorta adventitia histological after experimental treatments.The Van Gieson stain showed that the vascular adventitia in the PC group was thicker than that in the NC group, and QC-and losartan-treated rats showed lower adventitial thicknesses than the control rats in the PC group. Compared with WKY rats, the collagen fibers of the adventitia in SHRs showed significant hyperplasia and were intensely dyed. However, a significant reverse effect was observed in the QD group and the LSD group at the end of treatment. The QD group was not significantly different from the LSD group.3. Comparison of the mRNA expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ after experimental treatments.The expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ in the PC group was significantly increased compared with that in the NC group (p<0.01). However, the expression was significantly decreased in the QD group and the LSD group compared with the PC group (p<0.05or p<0.01). There was no significant difference in TGF-β1, Smad3, or collagen Ⅰ and Ⅲ mRNA expression between the QD group and the LSD group (p>0.05).4. Comparison of the protein expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ after experimental treatments.TGF-β1, Smad3, and collagen Ⅰ and Ⅲ expression in the PC group was significantly increased compared with that in the NC group (p<0.01). However, the expression of TGF-β1, Smad3, and collagens Ⅰ and Ⅲ was significantly decreased in the QD group and the LSD group compared with the PC group (p<0.05or p<0.01). There was no significant difference in TGF-β1, Smad3, or collagen Ⅰ and Ⅲ protein expression between the QD group and the LSD group (p>0.05).Conclusions1. The significantly antihypertensive effect of QC on SHR rats was verified again, our study suggested that its antihypertensive mechanism through down-regulating the expression of TGF-β1, Smad3, and collagen Ⅰ and Ⅲ in thoracic aorta adventitia.2. QC could reduce collagen content of SHR rats in vascular adventitia, improve the adventitia morphology of vascular and reverse the vascular remodeling. The possible mechanism was related to inhibit the TGF-β1/Smad3signal pathway. BackgroundHarmful vascular remodeling (VR) caused by vascular hyperplastic lesions is the common pathological basis on a variety of cardiovascular diseases. Fibroblasts are the most abundant cell type in the adventitia, which lead to vascular harmful reconstruction and restenosis after damage. Hypertention could prompt the adventitial fibroblasts (AFs) turn into myofibroblasts (MFs), MFs migrate to the tunica media or the intima, and prompt the composition or fibrosis of extracellular matrix (ECM) at the same time, and lead to coarctation or VR. More and more evidence to support the AFs is "the central part" of vascular hyperplastic lesions, and become a new target to the prevention and control of harmful VR.The studies have found that the TGF-β1is the most important and the most direct cell factor to promote AFs to proliferation, migration, phenotypic switching and synthetic function, and has most close relations to vascular adventitia reconstruction. Smad signal path is a TGF-β1downstream signaling pathways found in recent years and closely related with many tissue fibrosis. Our previous studies have confirmed that Smad2and Smad3mediated the AFs to TGF-β1reaction.And the biological function of TGF-β1is also controlled by the mitogen activated protein kinase (MAPK). This effect can be blocked by specific MAPK inhibitor. MAPK signaling pathways (mainly including ERK, JNK and p38) is one of the most important intracellular signals in the network. It is involved in many kinds of pathophysiological process such as mediating cell proliferation, differentiation and so on, and closely related with the happening of fibrosis diseases. The receptor tyrosine kinase activated MAPK may be participate in cross-talk with the Smad pathway by phosphorylation and change the TGF-β1signal. But there has little research for specific signal path TGF-β1/ERK on vascular remodeling in hypertension so far.The process of VR is along with synthesis and degradation of ECM. Matrix metalloproteinase (MMP) is in the main enzyme system in degradation of ECM, MMP2and MMP9are the main composition. Matrix metalloproteinase synthesis (MMPs) participated in the process of hypertensive heart and vascular reconstruction.Connective tissue growth factor (CTGF) is a kind of ecretory polypeptide with rich cysteine. The main biological activities are to promote cell mitosis, chemotactic cytokines, inducing adhesion, apoptosis regulation and the formation of blood vessels, it also can promote cell proliferation and ECM synthesis. CTGF can be secreted by fibroblast under the induction of TGF-β1. The studies found that the CTGF excessive expression is closely related with the happening of some hyperplastic or fibrosis disease s. However, the relation between CTGF and vascular function has been less reported.Hypertension disease belongs to the "vertigo" category in traditional Chinese medicine,"live fire and blood stagnation"s the common symptoms. Qindan capsule (QC) is a clinical empirical formula by the tutor to treat hypertension patients with "liver fire and blood stagnation" symptoms. Our previous clinical study showed that QC could influent TGF-β1/Smad signaling pathways, restrain AFs proliferation and collagen synthesis and play an important role in reversing vascular adventitia remodeling of SHRs.But the influence on the AFs biological activity. MMP2, MMP9, CTGF, and TGF-β1/ERK signaling pathway is still unknown. This research is based on our previous studies to continue to explore the influence of QC on AFs proliferation, migration, cycle and apoptosis regulation and MMP2and MMP9, CTGF synthesis induced by TGF-β1, and the role of ERK signal transduction pathway during the process. Our study would provide more scientific basis for the clinical application of QCAims 1. To investigate the effect of QC medicated serum on the biological activity of AFs induced by TGF-(31by observing the proliferation, migration, cycle and apoptosis regulation.2. To investigate the role of TGF-β1/ERK signaling pathway by observing the expression of extracellular signal-regulated kinase1/2(ERK1/2)、phospho-ERK1/2(p-ERK1/2)、CTGF、MMP2and MMP9in AFs induced by TGF-β1and affected by QC medicated serum.Methods1. Preparation of QC-containing serumNinety male WKY rats (weight:240-280g) were divided into three groups randomly:the high dosage QC group (QCH,750mg/kg/d), the low dosage QC group (QCL,150mg/kg/d) and Losartan group (30mg/kg/d)). Medicines were given2times a day and four consecutive days. Rats were starved for24h and were anaesthetised for1.5h after the final medicine treatment. Blood was drawn from the abdominal aorta. Serum was separated and mixed in the same group, filtered bacteria, at56℃inactivated for30min. Serum freeze dry powder was made in low temperature vacuum drying machine and was stored at-20℃for use.2. Acquisition and intervention of AFs(1) Vascular adventitia was stripped from thoracic aorta of WKY rat. Paste tissue pieces method was used to cultivate original generation fibroblasts. AFs were frozen storage, carried anabiosis and subculture.(2) Medicated serum preliminary stimulus:The subcultured AFs were continue to be cul tured in serum free medium for4h, then isovolumetric serum freeze-dried powder was added and continue to be cultured for48h. And the negative control group was not given any processing factors.(3) TGF-β1induced AFs:AFs were cultured by common nutrient solution with TGF-β1(20ng/ml) for24h.3. Experimental groupsThe AFs were divided into5groups as follows:(1) The negative control group (NC). (2) The positive control group (PC).(3) The QC high dose group (QCH).(4) The QC low dose group (QCL).(5) The Losartan group (Losartan).4. Contents of study(1) The cell proliferation rate was measured by MTT colorimetry assay.(2) The cell migration ability was measured by transwell assay.(3) The cell cycle and apoptosis regulation was measured by flow cytometry analysis.(4) The mRNA expression of MMP2, MMP9and CTGF was measured by reverse transcription-polymerase chain reaction (RT-PCR).(5) The protein expression of MMP2, MMP9and ERK1/2, p-ERK1/2and CTGF was measured by western blot.5. Statistical analysisData are expressed as mean±SD. All of the experiments data were analyzed by SPSS20.0software. An independent-sample t-test was applied when only two groups were compared, whereas comparisons among groups were analyzed by ANOVA. Differences were considered to be statistically significant when the P values were less than0.05.Results1. Comparison of proliferation activity of AFs among experimental groupsThe proliferation activity of AFs in the PC group was significantly increased compared with that in NC group (P<0.01). After treated by medicine, the proliferation activity of AFs in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05or P<0.01). The compare between QCH and Losartan groups has no statistically significance (P>0.05).2. Comparison of migration number of AFs among experimental groupsThe migration number of AFs in the PC group was significantly increased compared with that in NC group (P<0.01). After treated by medicine, the cell migration number of AFs in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).3. Comparison of cell cycle of AFs among experimental groupsThe cell percentage of G0/G1and G2/M in the PC group was increased compared with that in NC group (.P<0.05or P<0.01). After treated by medicine, the cell percentage of G0/G1and G2/M in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).4. Comparison of cell apoptisis of AFs among experimental groupsThe cell percentage of early apoptosis in the PC group was significantly decreased compared with that in NC group (P<0.01). After treated by medicine, the cell percentage of early apoptosis in QCH, QCL and Losartan group was increased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).5. Comparison of the mRNA expression of MMP2, MMP9and CTGF in AFs among experimental groupsThe mRNA expression of MMP2, MMP9and CTGF in the PC group was significantly increased compared with that in NC group (P<0.01). After treated by medicine, the mRNA expression of MMP2, MMP9and CTGF in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).6. Comparison of protein expression of MMP2, MMP9and ERK1/2, p-ERK1/2and CTGF in AFs among experimental groupsThe protein expression of MMP2, MMP9and p-ERK1/2and CTGF in the PC group was significantly increased compared with that in NC group (P<0.01). After treated by medicine, the protein expression of MMP2, MMP9and p-ERK1/2and CTGF in QCH, QCL and Losartan group was decreased compared with that in PC group (P<0.05). The compare between QCH and Losartan groups has no statistically significance (P>0.05).The protein expression of ERK1/2among five experimental groups has no statistically significance (P>0.05).Conclusion1. QC could inhibit or reverse vascular adventitia remodeling by inhibiting the biological activity of AFs and the synthesis of MMP2, MMP9and CTGF induced by TGF-β1.2. The mechanism of QC to inhibit or reverse vascular adventitia remodeling maybe related to the inhibiting effect of TGF-β1/ERK signaling pathway. |
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