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Tongsaimai reverses the hypertension and left ventricular remolding caused by abdominal aortic constriction in rats
Journal Pre-proof Tongsaimai reverses the hypertension and left ventricular remolding caused by abdominal aortic constriction in rats Qinghai Meng, Yao Guo, Dini Zhang, Qichun Zhang, Yu Li, Huimin Bian PII:
S0378-8741(18)34153-9
DOI:
https://doi.org/10.1016/j.jep.2019.112154
Reference:
JEP 112154
To appear in:
Journal of Ethnopharmacology
Received Date: 9 November 2018 Revised Date:
1 August 2019
Accepted Date: 10 August 2019
Please cite this article as: Meng, Q., Guo, Y., Zhang, D., Zhang, Q., Li, Y., Bian, H., Tongsaimai reverses the hypertension and left ventricular remolding caused by abdominal aortic constriction in rats, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.112154. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Tongsaimai reverses the hypertension and left ventricular remolding caused by abdominal aortic constriction in rats Qinghai Meng a, Yao Guo b, Dini Zhang c, Qichun Zhang a, Yu Li d,*, Huimin Bian a,* a School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210023, China b Nanjing TechBoon Biotechnology Company Limited, Nanjing, Jiangsu 211899, China c Department of Environmental Protection, Nanjing Institute of Environmental Sciences, Nanjing, Jiangsu 210042, China d School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210023, China E-mail address: [email protected] (Qinghai Meng) [email protected] (Yao Guo) [email protected] (Dini Zhang) [email protected] (Qichun Zhang) [email protected] (Yu Li) [email protected] (Huimin Bian) * Correspondence should be addressed to Yu Li and Huimin Bian.
Abstract Treating ventricular remodeling continues to be a clinical challenge. Studies have shown that hypertension is one of the most common causes of ventricular remodeling, and is a major cause of cardiovascular risk in adults. Here, we report that Tongsaimai (TSM), a Chinese traditional medicine, could inhibit arterial pressure and left ventricular pressure to improve hemodynamic abnormalities in rats impaired by abdominal aortic constriction (AAC). Administration of TSM significantly reduced the heart mass index and the left ventricular mass index significantly in AAC rats. TSM could also markedly ameliorate cardiac collagen deposition and reduce the concentration of hydroxyproline in the heart of AAC rats. Moreover, TSM alleviated cardiac histomorphology injury resulting from AAC, including reducing cardiomyocyte hypertrophy, fibrous connective tissue hyperplasia, cardiomyocyte apoptosis, replacement fibrosis and the disorders of myocardial myofibrils, intercalated discs, mitochondria and mitochondrial crista. In addition, the levels of transforming growth factor (TGF) - β and inflammation-related molecules including tumor necrosis factor-α (TNF-α), which were over-expressed with AAC, were decreased by STM. In conclusion, STM could reverse the hypertension and left ventricular remolding caused by abdominal aortic constriction in rats Ethnopharmacological relevance: Aim of the study: Materials and methods: Results: Conclusions:
Graphical Abstract: Keywords: Tongsaimai; Abdominal aortic constriction; Hypertension; Left ventricular remolding; Rat
1. Introduction Hypertension with high prevalence, especially arterial pressure changes, is one of the most common causes of ventricular remodeling (Burchfield et al., 2013), and is a major cause of cardiovascular risks (Tomek and Bub, 2017). Result to a deviant augment of left ventricular mass, left ventricular hypertrophy (LVH) has been thought to be adapted to hypertensive cardiac load by minimizing wall stress and normalizing ejection properties (Drazner, 2011). Even without compromising contractile performance due to the compensatory effect of the left ventricle, a lot of preclinical studies have demonstrated that, hypertension induced hypertrophic growth of the left ventricle is possible (Frey et al., 2004; Hill et al., 2000), which has been defined as pathological remodeling (Hill and Olson, 2008). The LVH is maladaptive and predispose to cardiovascular risks. Although there is no dedicated drugs directly act on ventricular hypertrophy, some agents in current use transform the hypertrophic response secondarily, including catechol amines, angiotensin (Ang) II, and aldosterone inhibitors (McKinsey and Kass, 2007). While the experimental researches have demonstrated the role of the renin-angiotensin-aldosterone system (RAAS) in cardiac remodeling, to be more specific, Ang II interacts with Ang II type I (AT1) receptor promotes cardiomyocyte hypertrophy (Sadoshima and Izumo, 1993), fibroblast proliferation and extracellular matrix (ECM) proteins expression (Rosenkranz, 2004), clinical trials released the evidences that angiotensin converting enzyme (ACE) inhibitor and AT1 receptor blocking agents contributed beneficial effects in ventricular hypertrophy (Pfeffer et al., 1995). These findings suggest
that, in addition to treating hypertension, agents that inhibit the RAAS system may also have a beneficial treatment for ventricular remodeling. Ventricular remodeling is featured by collagen and ECM excessive deposition. The ECM constitutes of fibrosis, facilitates dysfunction and arrhythmia of the heart(Spinale, 2007) Under the pathological situation, cardiac fibroblasts proliferate and differentiate into myofibroblasts, gaining the ability to generate collagen and fibronectin, which contributes to collagen and ECM deposition and fibrosis formation(Spinale, 2007). Some evidence has certified that the cardiac fibrosis that long-term maintenance to be irreparable, may be reversed under certain conditions (Berry et al., 2011). Until now, there is no specifically drugs directly target fibrosis in the heart. In spite of the challenge, therapies focusing on cardiac fibrosis may prove benefits in the treatment of ventricular
remodeling. Specifically, Ang II accelerates cardiac fibroblast proliferation, collagen accumulation(Schorb et al., 1993), and cardiac fibrosis(Kim et al., 1995), in hypertensive heart disease patients, losartan reduced cardiac fibrosis and serum collagen markers(Lopez et al., 2001). During the process of ventricular remodeling caused by hypertension, the expression of serum factors is increased, including TGF-β and TNF-α. TGF is known as a pro-fibrosis factor, plays a significant role in the course of cardiac remodeling by the impacts on the inflammatory and refurbishing function (Bujak and Frangogiannis, 2007) Abundant evidence demonstrates a direct crosstalk between the RAAS and TGF-β, suggesting that TGF-β1 acts a downstream factor of AngII (Rosenkranz, 2004). Furthermore, the treatment of hypertrophied (Kim et al., 1996) and myocardial infarction (Sun et al., 1998) with ACE inhibitor or AT1 receptor blocker markedly reduced TGF-β1 level (Yu et al., 2001), suggesting that TGF-β induction in the ventricular remodeling may be mediated via Ang II. As a central, initiative, and sustaining proinflammatory cytokine in the proinflammatory cytokine cascade (Zhang et al., 2006a), TNF-α has been engaged in the pathophysiology of many cardiovascular diseases, including hypertension (Sriramula et al., 2008; Sun et al., 2007; Zhang et al., 2006b). Additionally, the overexpression of TNF-α in mice causes unfavorable cardiac remodeling, characterized by increased ECM and fibrosis (Bryant et al., 1998; Sivasubramanian et al., 2001). In spite of the underlying mechanisms engaged in these diseases have not been illuminated, lots of previous studies have shown that the inhibition of TNF-α was participated in declining Ang II mediated hypertension (Guzik et al., 2007; Sriramula et al., 2013; Sriramula et al., 2008; Sun et al., 2007).
Bursting in the heart, the process of ventricular remodeling is the presentation of many complicated
events,
including
signal pathway,
transcription,
protein
expression,
cell
differentiation, electrophysiology, structural change, and functional change. Due to the highly correlated of these events, treating ventricular remodeling for a single molecule or process may not be sufficient. As a multiple-target treatment, Chinese medicine may reverse ventricular remodeling by targeting multiple pathways. A large amount of clinical and preclinical studies have certified that Chinese medicine have effectiveness in treating ventricular remodeling. For some instance, Tongxinluo Capsules (Chen et al., 2010) and Shexiang Baoxin Pills (Zhang et al., 2014) could reverse ventricular remodeling in clinical trials. Qishen Yiqi Dropping Pills (Wang et al., 2015) could regulate RAAS system to attenuate myocardial fibrosis, and Qili Qiangxin Capsules
(Liang et al., 2016) could rehabilitate ventricular remodeling in rats. As a prescription drug of Chinese medicine formulae, TSM, consists of Astragalus membranaceus (Fisch.) Bunge, Codonopsis pilosula (Franch.) Nannf, Angelica sinensis (Oliv.) Diels, Dendrobium nobile Lindl, Lonicera japonica Thunb, Scrophularia aestivalis Griseb, Achyranthes bidentata Blume, and Glycyrrhiza uralensis Fisch, has a protect effect on focal cerebral ischemia (Zhao et al., 2011) and uses to treat thromboangiitis in clinical practice. A network pharmacology study suggested that multiple components of Tongsaimai may have a protective effect of atherosclerosis (Li et al., 2016). Our previous study demonstrated that Tongsaimai promoted wound recover and tissue regeneration in foot trauma of diabetic foot (Guo et al., 2014). Astragalus membranaceus (Fisch.) Bunge, as a primary component of TSM, can treat hypertension as a folk medicine in traditional applications (Li et al., 2018; Xu et al., 2008). These researches may suggest that TSM has a potential value in treating hypertension and hypertension related diseases. In this study, we investigated the protective effects of TSM in rats with AAC injury, and reported its influences on hypertension and left ventricular remolding.
2. Materials and methods 2.1. Ethics approval All procedures described in this study were performed in accordance with the guidelines of Nanjing University of Chinese Medicine Animal Care and Use Committee.
2.2. Reagents According to the ratio of 15:12:12:12:9:9:9:6, Astragalus membranaceus (Fisch.) Bunge, Codonopsis pilosula (Franch.) Nannf, Angelica sinensis (Oliv.) Diels, Dendrobium nobile Lindl, Lonicera japonica Thunb, Scrophularia aestivalis Griseb, Achyranthes bidentata Blume, and Glycyrrhiza uralensis Fisch are mixed to boil to get the extractum (TSM), and then the prepared extractum were made into sugar-coated tablets. The drug mass was 0.35 g/tablet. This is a standardized production process, and was carried out by Nanxing Pharmaceutical Co., Ltd. The extractum (TSM) used in our study was provided by Nanxing Pharmaceutical Co., Ltd. (lot number: 120713), and its pharmic content was 3.25 g /g (crude herbs / extractum). Captopril (Sino-US Squibb Pharmaceutical Co., Ltd.). Sodium chloride (produced by
Nanjing Chemical Reagent Co., Ltd.). Heparin (Shanghai Ruji Biotechnology Development Co., Ltd.). Urethane (Shanghai Qingfang Chemical Technology Co., Ltd.). Carboxymethyl Cellulose sodium (Sinopharm Chemical Test Co., Ltd.). Plasma renin activity Radioimmunoassay kit, Plasma angiotensin II Radioimmunoassay kit, Plasma aldosterone Radioimmunoassay kit, Plasma TGF-β Radioimmunoassay kit, and Plasma TNF-α Radioimmunoassay kit were purchased from Beijing Huaying Biotechnology Research Institute.
2.3. Animals Adult male Sprague-Dawley rats weighing 180–220g were provided by Shanghai Slack Laboratory Animal Co., Ltd. All animals were housed five per cage under controlled temperature (22 ± 2 °C) and with 12 h light/dark cycle (lights on at 8:00 a.m.). The animals had free access to food and water. After grouping, the rats in different group were intragastrical administered for 8 weeks. The dose of TSM is 2.4g/kg, and the dose of captopril is l0 mg/kg.
2.4. AAC model After diet forbidden and water for free for 12 h, the abdominal aorta was exposed following rat anesthesia (20% Urethane, 12g/kg, intraperitoneally), and the connective tissue surrounding the abdominal aorta were carefully separated. A 4-0 nylon monofilament suture was placed under the abdominal aorta. A six-and-a-half needle with a blunt tip was placed in parallel with abdominal aorta, which was ligated with the abdominal aorta by the nylon monofilament suture. In the
sham group, this same procedure was performed but no abdominal aorta was ligated. After that, the needle was withdrawn followed by the penicillin administration in the abdominal cavity, and then the abdominal cavity was closed layer by layer. 200,000 units of penicillin were intramuscularly injected for 3 consecutive days. Most of the death occurred within 72 hours after the surgery. After modeling for one week, the surviving rats were divided into different group.
2.5. Hemodynamic detection method The RM6240 multi-channel physiological signal acquisition and processing system was used to record and collect data. After diet forbidden and water for free for 12 h, the electro-cardiogram needle electrodes were inserted into the limbs subcutaneously following the anesthesia (20%
Urethane, 12g/kg, intraperitoneally). When the electrocardiogram and temperature (37 ° C) were stabilized, the right common carotid artery was separated and the end of it was ligated for arterial intubation. The pressure transducer was connected with the RM6240 multi-channel physiological signal acquisition and processing system. Systolic blood pressure (SBP), diastolic blood
pressure (DBP) and mean artery pressure (MAP) were recorded for 15 minutes after stabilization. Then, insert the cannula into the left ventricle until the ventricular pressure waveform appears, left ventricular systolic pressure (LVSP), left ventricular end diastolic pressur (LVEDP), the maximal rates that ventricular pressure rise (+dp/dtmax) and the
maximal rates that ventricular pressure fall (-dp/dtmax) were recorded for 15 minutes after stabilization.
2.6. Serologic detection After the hemodynamic detection, blood was collected from the left common carotid artery. After the serum was appeared, centrifugal separated it (3000 rpm, 10 min). The RAAS system was evaluated by the detection of the levels of PRL1, AngII and ALD, and the cardiac collagen deposition was evaluated by the detection of the levels of TGF-β and TNF-α in the serum of AAC rats. All indicators are detected according to the kit instructions
2.7. Evaluation of cardiac hypertrophy After blood collection, opened the chest and removed the heart. The heart was washed in 4 ° C NS to wash off the residual blood, a sheet of filter paper was used to absorb the surface moisture, and the whole heart mass was weighed using an electronic analytical balance. Then, the atrium and right ventricle were removed along the atrioventricular septum, and the left ventricle (including the interventricular septum) was weighed. HMI and LVMI were measured according to the formulas: HMI = whole heart weight / body weight× 100%, LVMI = left ventricle / body weight× 100%.
2.8. Evaluation of cardiac collagen deposition After the evaluation of cardiac hypertrophy, 30 mg of the heart was weighed followed by pyrolysis. The sample was hydrolyzed at 95 ° C for 20 minutes. Adjusted the pH to 6.0~6.8, added
various reagents according to the instruction manual of the kit, then mixed and centrifuged. Measured the absorbance of the supernatant of each tube. The hydroxyproline and collagen concentrations in the hearts of the rats were calculated according to the formulas: Hydroxyproline concentration = (TOD - BOD) / (SOD - BOD) × Cs × Vt × / W, Collagen concentration = Hydroxyproline concentration× 7.46. Abbreviation: TOD, absorbance of the test sample. BOD, absorbance of the blank. SOD, absorbance of the standard substance. Cs, concentration of the standard substance. Vt, volume of the hydrolysate. W, weight of the test sample.
2.9. Hematoxylin and eosin (HE) staining The HE staining method was as reported previously (Fischer et al., 2008). Briefly, after the heart tissue was formed into a block of wax. Bring sections to distilled water, stain nuclei with the hematoxylin for 4 min, then rinse sections in running tap water. After differentiate with 0.3% acid alcohol and wash again, then stain with eosin for 90 s. Dehydrated with different concentration alcohol, made transparent with xylene, and then observed under the microscope.
2.10. Masson's trichrome staining The Masson's trichrome staining method was as reported previously (Yu et al., 2018). Briefly, after heart the tissue was formed into a block of wax. Bring sections to distilled water, stain nuclei with the hematoxylin for 5 min, then rinse sections in running tap water for 10 min. Stain in acid fuchsin solution for 15min. Differentiate in phosphomolybdic acid solution. Transfer sections directly (without rinse) to aniline blue solution and stain for 5 min. Dehydrate very quickly with different concentration of alcohol and clear in xylene, then observed under the microscope.
2.11. Scanning electron microscopy (SEM) detection The SEM detection method was as reported previously (Michelmann et al., 2001). Briefly, 2.5% glutaraldehyde was used to fix the heart sample, osmium tetroxide was used to fix the heart sample again to preserve lipids. Using different concentration of alcohol to remove water from the sample. Sputter coating with gold to make the sample easy to conduct electricity. Adhere the sample to the conductive stage, then find a vision and take a photo.
2.12. Statistical Analysis Statistical analysis was carried out using ANOVA. Results are expressed as mean ± SD. A p value < 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism Version 5.01 software (GraphPad Software Inc., San Diego, CA, USA).
3. Results 3.1. TSM inhibits arterial pressure and left ventricular pressure in AAC rats To evaluate the antihypertensive effects of TSM, we monitored the changes of the arterial blood pressure and left ventricular pressure in different groups. The SBP, DBP, MAP, LVSP, and +dp/dtmax of the AAC rats, were significantly higher (p < 0.01), while the -dp/dtmax was significantly lower (p < 0.01), than that of the sham rats. In the AAC rats that received STM intragastric administration for 8 weeks, the SBP, DBP, MAP, and +dp/dtmax were found to be significantly reduced, and the -dp/dtmax was significantly increased (Fig. 1).
3.2. TSM regulates RAAS system in AAC rats. The RAAS system has the ability to regulate blood pressure, and the hypertension is often accompanied by abnormalities of the RAAS system (Ghazi and Drawz, 2017). Therefore, we next examined the PRA, AngⅡ, and ALD levels in rats, which is believed to indirectly reflect the abnormities of the blood pressure. The results show that the serum levels of PRA, AngⅡ, and ALD in AAC rats were markedly raised compared with normal group (p < 0.01). STM significantly decreased AngⅡ level (p < 0.05) and ALD level (p < 0.01) in AAC rats, but it has no influence on the serum PRA level (Fig. 2).
3.3. TSM suppresses cardiac hypertrophy in AAC rats Many studies have confirmed that cardiac collagen deposition causes cardiac hypertrophy (Isoyama et al., 1992). After 8 weeks of abdominal aortic coarctation， the cardiac hypertrophy occurred in AAC rats due to the increased cardiac after-load, resulting the heart mass index and the left ventricular mass index increased conspicuously than that in sham group (P<0.01). Administration of TSM can suppress ventricular hypertrophy and reduce the heart mass index and
the left ventricular mass index significantly (P<0.01) (Fig. 3).
3.4. TSM reduces cardiac collagen deposition in AAC rats Accounting for about 13% of the total amount of collagen amino acids, hydroxyproline is one of the major components of collagen, and is a specific amino in collagen (Weis et al., 2010). With the high content of hydroxyproline in collagen, the determination of hydroxyproline can be used to evaluate the situation of collagen in the body. Therefore, we examined the effect of TSM on hydroxyproline level of the heart in AAC rats, and calculated the collagen content in cardiac muscle. Higher concentrations of hydroxyproline and collagen were observed in the hearts of AAC rats (p < 0.01), and TSM significantly reduced the concentrations of hydroxyproline and collagen, as shown in Fig. 4.
3.5. TSM restrains the changes of cardiac histomorphology in AAC rats. Next, we examined the effect of TSM on the cardiac histomorphology. The HE staining of the hearts of AAC rats showed cardiomyocyte hypertrophy, fibrous connective tissue hyperplasia, cardiomyocyte apoptosis and replacement fibrosis compared with sham group (Fig. 5A). Masson's trichrome stainin presented that the amount of interstitial and infiltrative fibrosis and collagen deposition in AAC rats’ hearts were significantly increased (Fig. 5B). Furthermore, Scanning electron microscopy detection revealed that the myocardial myofibrils were distorted and discontinuous, the intercalated discs were blurred and widened, the mitochondria were vacuolated, and the mitochondrial crista were broken in the hearts of AAC rats (Fig. 5C). After intragastric administration of TSM in AAC rats, the cardiomyocyte hypertrophy, fibrous connective tissue hyperplasia, cardiomyocyte apoptosis, replacement fibrosis and collagen deposition of the heart were significantly decreased (Fig. 5A-B). And the Myocardial myofibrils and intercalated discs were clear and well-arranged, with complete mitochondria and visible mitochondrial crista (Fig. 5C). These findings suggest that TSM can restrain the changes of cardiac histomorphology in AAC rats.
3.6. TSM decreases the serum factor levels in AAC rats. Evidence shows that TGF-β plays an important role in the process of fibrosis (Rosenkranz,
2004), and the TNF-α involves in the left ventricular remolding following the hypertension (Sriramula et al., 2013; Sun et al., 2007). Therefore, we performed serum factor analysis. As shown in Fig. 6, compared with sham group, the serum levels of TGF-β and TNF-α in model group were increased remarkably (P<0.01). However, the decrease of the TGF-β and TNF-α levels in the serum after captopril treatment were not so significant (P<0.05) compared to other indexes, and there was only a downward trend in the serum TGF-β and TNF-α levels of AAC rats after TSM treatment, but the statistical results of the data were not significant (P>0.05).
4. Discussion Our study demonstrated that the administration of TSM significantly inhibited arterial pressure and left ventricular pressure, decreased AngⅡ and ALD levels, suppressed cardiac hypertrophy and cardiac collagen deposition, restrained the changes of cardiac histomorphology, and suppressed TGF-β and TNF-α levels in AAC rats. These results may enhance our understanding of the protection properties of TSM against left ventricular remolding through reversing the hypertension and provide an alternative option for the future prevention and treatment of hypertension and reconstructive heart disease. In our study, the SBP, DBP, MAP, LVSP, and +dp/dtmax of the AAC rats, were significantly higher (p < 0.01), while the -dp/dtmax was significantly lower (p < 0.01), than that of the sham group. However, the LVEDP did not change significantly compared to the sham group (Fig. 1), these results may indicate that the rat's cardiac afterload was significantly increased after modeling, causing compensatory increasing in systolic pressure. At this stage, the heart function was still in the compensation period, the diastolic function of the heart did not decrease significantly, so there was no significant change in LVEDP. After STM intragastric administration for 8 weeks, the SBP, DBP, MAP, and +dp/dtmax were found to be significantly reduced, and the -dp/dtmax was significantly increased (Fig. 1). Altogether, TSM can inhibit arterial pressure and left ventricular pressure in AAC rats. Previous studies have shown that hypertension is accompanied by abnormalities in the RAAS system (Ghazi and Drawz, 2017). The RAAS system maintains hemodynamic homeostasis by regulating blood pressure, water and electrolyte balance (Ghazi and Drawz, 2017). Pathological stimulations on RAAS causes excessive contraction of vessels, and hypertrophy and fibrosis of the heart (Alves et al., 2010). As the main effector of the RAAS system, AngII plays an important role
in the development of cardiovascular and renal diseases (Romet, 2010). In the kidney, AngII binds to the AT1 receptor, causing the release of ALD, which causes vasoconstriction, water-sodium retention, hypertension, and cardiac contractility increasing (Costerousse et al., 1998; Matsubara, 1998), accompanied by circular blood quantity increasing and cardiovascular remodeling (Savoia and Volpe, 2011). In the heart, AngII can promote fibroblast proliferation, ECM protein expression and collagen synthesis by binding to the AT1 receptor, which directly promoting the hypertrophic growth of neonatal cardiomyocytes (Schultz Jel et al., 2002). In this study, TSM alleviated AngⅡ and ALD levels in AAC rats, but it has no influence on the serum PRA level (Fig. 2). Many animal experiments and clinical studies have shown that, by inhibiting the conversion of AngI to AngII and attenuating the effects of AngII on ALD release, ACE inhibitors can alleviate hypertension and cardiovascular remodeling (Kim et al., 2001; Nakamura et al., 2003). Furthermore, ACE inhibitors reveals the vessels expanding and heart protecting functions, avoids or reverses the LVH (Cohn, 2000). So in this experiment, we used the ACE inhibitor captopril as a positive drug. Previous studies reports that captopril can reduce plasma AngII and ALD levels, but does not reduce PRA level (Sureshkumar, 2008), which is consistent with the results of this experiment. TSM also failed to reduce plasma renin activity, indicating that TSM also does not produce a hypotensive effect by directly inhibiting renin activity. But TSM can reduce the content of AngII and ALD. This finding suggests that the effect of antihypertensive of TSM may be mainly achieved by reducing AngII and ALD. In order to deal with pathological stimuli, like hypertension, the heart increases its weight by rising compensatory cardiomyocytes enlargement, fibroblast proliferation, and ECM deposition, to maintain arterial blood pressure and organ perfusion pressure (Berthelot et al., 2018; Guazzi, 2018). And, LVH is an early manifestation of heart damage in hypertensive patients (Huang and Li, 2018). In this experiment, the heart mass index and the left ventricular mass index were increased in AAC rats, and the administration of TSM can reduce the heart mass index and the left ventricular mass index significantly (P<0.01) (Fig. 3). The results indicating a reduction in cardiac hypertrophy caused by TSM. Hypertension leads to ventricular remodeling, pressure overload-left ventricular remodeling is characterized by cardiomyocytes enlargement, fibroblast proliferation, and ECM deposition (Villar et al., 2009). Furthermore, fibrous collagen is the most common type of collagen in the heart. Inhibition of collagen can significantly reduce ventricular remodeling (Isobe et al., 2010). Therefore, we tested the effect of TSM on hydroxyproline level of the heart in AAC rats, and calculated the collagen content in cardiac muscle. TSM can significantly reduce the concentrations of hydroxyproline and collagen (Fig. 4). This result suggests that TSM can suppress ventricular remodeling by reducing collagen deposition. Cardiac connective tissue plays an important role in maintaining the normal structure of the heart. Excessive ECM collagen deposition increases myocardial stiffness, and subsequently causes cardiac hypertrophy and left ventricular dysfunction (Schultz Jel et al., 2002). In addition, cardiac
hypertrophy and left ventricular dysfunction increase the risk of heart failure and increase the occurrence of arrhythmias (Villar et al., 2009). In this study, our results showed that TSM could inhibit AAC-induced up regulation of cardiomyocyte hypertrophy, fibrous connective tissue hyperplasia, cardiomyocyte apoptosis, replacement fibrosis and collagen deposition in the hearts of rats (Fig. 5A-B). Furthermore, TSM could reverse the disorders of myocardial myofibrils, intercalated discs, mitochondria and mitochondrial crista (Fig. 5C). These findings suggest that TSM can restrain the changes of cardiac histomorphology in AAC rats. TGF-β is a well-defined pro-fibrotic factor, and TNF-α promotes ventricular remodeling by increasing expression and activation of many factors in heart (Creemers et al., 2001). Thus, after confirming the effects of TSM for hypertrophy and left ventricular remodeling, we tested the serum levels of TGF-β and TNF-α in the rats. But, the statistical results of the data were not significant (P>0.05), there was only a downward trend in the serum TGF-β and TNF-α levels of AAC rats after TSM treatment (Fig. 6). Taken together, hypertension with high prevalence, especially arterial pressure changes, is one of the most common causes of ventricular remodeling (Miyoshi et al., 2015; Yao et al., 2016), and is a major cause of cardiovascular risk in adults. Our study found that TSM could significantly improve hemodynamic abnormalities caused by pressure overload, inhibit RAAS by reducing plasma AngII and ALD levels, inhibit cardiac hypertrophy and collagen deposition, restrain the changes of cardiac histomorphology, and reduce the plasma levels of TGF-β and TNF-α in AAR rats. Therefore, STM may potentially be a safe and effective treatment for reverse left ventricular remolding through suppressing hypertension.
Funding sources This study was supported by the Natural Science Foundation of China (No. 81073071) and Jiangsu
Provincial
Science
and
Technology
Department
Social
Development
Fund
(No.BE2011846).
Author contributions Qinghai Meng carried out the experiments, assisted with data analysis, and the manuscript preparation. Yao Guo and Dini Zhang helped with the manuscript preparation and all the experiments. Qichun Zhang helped with the design of the study and conducted the SEM detection. Yu Li performed the kits detection and assisted with the data analysis and manuscript preparation. Huimin Bian helped conduct the experiments and contributed to the writing of the manuscript.
Conflict of Interest Statement
The authors declare that they have no competing interests.
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Figure legends Fig. 1. TSM improves hemodynamics in AAC rats. (A) SBP of the rats. (B) DBP of the rats. (C) MAP of the rats. (D) LVSP of the rats. (E) LVEDP of the rats. (F) +dp/dtmax of the rats. (G) -dp/dtmax of the rats. n = 8.
Fig. 2. TSM regulates RAAS system in AAC rats. (A) Plasma renin activity in rats. (B) Plasma Ang II level in rats. (C) Plasma ALD level in rats.
Fig. 3. TSM reduces cardiac hypertrophy in AAC rats. The heart mass index (A) and left ventricular mass index (B) of the rats.
Fig. 4. TSM reduces cardiac collagen deposition in AAC rats. The hydroxyproline (A) and collagen (B) concentrations in the hearts of the rats.
Fig. 5. TSM improves cardiac histomorphology in AAC rats. (A) HE staining of the hearts of the rats. Scale bar = 50µM. (B) Masson staining of the hearts of the rats. Scale bar = 50µM. (C) SEM dection of the hearts of the rats.
Fig. 6. TSM reduces the serum factor levels in AAC rats. The plasma level of TGF-β (A) and TNF-α (B) in rats.
S0378-8741(18)34153-9
DOI:
https://doi.org/10.1016/j.jep.2019.112154
Reference:
JEP 112154
To appear in:
Journal of Ethnopharmacology
Received Date: 9 November 2018 Revised Date:
1 August 2019
Accepted Date: 10 August 2019
Please cite this article as: Meng, Q., Guo, Y., Zhang, D., Zhang, Q., Li, Y., Bian, H., Tongsaimai reverses the hypertension and left ventricular remolding caused by abdominal aortic constriction in rats, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.112154. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Tongsaimai reverses the hypertension and left ventricular remolding caused by abdominal aortic constriction in rats Qinghai Meng a, Yao Guo b, Dini Zhang c, Qichun Zhang a, Yu Li d,*, Huimin Bian a,* a School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210023, China b Nanjing TechBoon Biotechnology Company Limited, Nanjing, Jiangsu 211899, China c Department of Environmental Protection, Nanjing Institute of Environmental Sciences, Nanjing, Jiangsu 210042, China d School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210023, China E-mail address: [email protected] (Qinghai Meng) [email protected] (Yao Guo) [email protected] (Dini Zhang) [email protected] (Qichun Zhang) [email protected] (Yu Li) [email protected] (Huimin Bian) * Correspondence should be addressed to Yu Li and Huimin Bian.
Abstract Treating ventricular remodeling continues to be a clinical challenge. Studies have shown that hypertension is one of the most common causes of ventricular remodeling, and is a major cause of cardiovascular risk in adults. Here, we report that Tongsaimai (TSM), a Chinese traditional medicine, could inhibit arterial pressure and left ventricular pressure to improve hemodynamic abnormalities in rats impaired by abdominal aortic constriction (AAC). Administration of TSM significantly reduced the heart mass index and the left ventricular mass index significantly in AAC rats. TSM could also markedly ameliorate cardiac collagen deposition and reduce the concentration of hydroxyproline in the heart of AAC rats. Moreover, TSM alleviated cardiac histomorphology injury resulting from AAC, including reducing cardiomyocyte hypertrophy, fibrous connective tissue hyperplasia, cardiomyocyte apoptosis, replacement fibrosis and the disorders of myocardial myofibrils, intercalated discs, mitochondria and mitochondrial crista. In addition, the levels of transforming growth factor (TGF) - β and inflammation-related molecules including tumor necrosis factor-α (TNF-α), which were over-expressed with AAC, were decreased by STM. In conclusion, STM could reverse the hypertension and left ventricular remolding caused by abdominal aortic constriction in rats Ethnopharmacological relevance: Aim of the study: Materials and methods: Results: Conclusions:
Graphical Abstract: Keywords: Tongsaimai; Abdominal aortic constriction; Hypertension; Left ventricular remolding; Rat
1. Introduction Hypertension with high prevalence, especially arterial pressure changes, is one of the most common causes of ventricular remodeling (Burchfield et al., 2013), and is a major cause of cardiovascular risks (Tomek and Bub, 2017). Result to a deviant augment of left ventricular mass, left ventricular hypertrophy (LVH) has been thought to be adapted to hypertensive cardiac load by minimizing wall stress and normalizing ejection properties (Drazner, 2011). Even without compromising contractile performance due to the compensatory effect of the left ventricle, a lot of preclinical studies have demonstrated that, hypertension induced hypertrophic growth of the left ventricle is possible (Frey et al., 2004; Hill et al., 2000), which has been defined as pathological remodeling (Hill and Olson, 2008). The LVH is maladaptive and predispose to cardiovascular risks. Although there is no dedicated drugs directly act on ventricular hypertrophy, some agents in current use transform the hypertrophic response secondarily, including catechol amines, angiotensin (Ang) II, and aldosterone inhibitors (McKinsey and Kass, 2007). While the experimental researches have demonstrated the role of the renin-angiotensin-aldosterone system (RAAS) in cardiac remodeling, to be more specific, Ang II interacts with Ang II type I (AT1) receptor promotes cardiomyocyte hypertrophy (Sadoshima and Izumo, 1993), fibroblast proliferation and extracellular matrix (ECM) proteins expression (Rosenkranz, 2004), clinical trials released the evidences that angiotensin converting enzyme (ACE) inhibitor and AT1 receptor blocking agents contributed beneficial effects in ventricular hypertrophy (Pfeffer et al., 1995). These findings suggest
that, in addition to treating hypertension, agents that inhibit the RAAS system may also have a beneficial treatment for ventricular remodeling. Ventricular remodeling is featured by collagen and ECM excessive deposition. The ECM constitutes of fibrosis, facilitates dysfunction and arrhythmia of the heart(Spinale, 2007) Under the pathological situation, cardiac fibroblasts proliferate and differentiate into myofibroblasts, gaining the ability to generate collagen and fibronectin, which contributes to collagen and ECM deposition and fibrosis formation(Spinale, 2007). Some evidence has certified that the cardiac fibrosis that long-term maintenance to be irreparable, may be reversed under certain conditions (Berry et al., 2011). Until now, there is no specifically drugs directly target fibrosis in the heart. In spite of the challenge, therapies focusing on cardiac fibrosis may prove benefits in the treatment of ventricular
remodeling. Specifically, Ang II accelerates cardiac fibroblast proliferation, collagen accumulation(Schorb et al., 1993), and cardiac fibrosis(Kim et al., 1995), in hypertensive heart disease patients, losartan reduced cardiac fibrosis and serum collagen markers(Lopez et al., 2001). During the process of ventricular remodeling caused by hypertension, the expression of serum factors is increased, including TGF-β and TNF-α. TGF is known as a pro-fibrosis factor, plays a significant role in the course of cardiac remodeling by the impacts on the inflammatory and refurbishing function (Bujak and Frangogiannis, 2007) Abundant evidence demonstrates a direct crosstalk between the RAAS and TGF-β, suggesting that TGF-β1 acts a downstream factor of AngII (Rosenkranz, 2004). Furthermore, the treatment of hypertrophied (Kim et al., 1996) and myocardial infarction (Sun et al., 1998) with ACE inhibitor or AT1 receptor blocker markedly reduced TGF-β1 level (Yu et al., 2001), suggesting that TGF-β induction in the ventricular remodeling may be mediated via Ang II. As a central, initiative, and sustaining proinflammatory cytokine in the proinflammatory cytokine cascade (Zhang et al., 2006a), TNF-α has been engaged in the pathophysiology of many cardiovascular diseases, including hypertension (Sriramula et al., 2008; Sun et al., 2007; Zhang et al., 2006b). Additionally, the overexpression of TNF-α in mice causes unfavorable cardiac remodeling, characterized by increased ECM and fibrosis (Bryant et al., 1998; Sivasubramanian et al., 2001). In spite of the underlying mechanisms engaged in these diseases have not been illuminated, lots of previous studies have shown that the inhibition of TNF-α was participated in declining Ang II mediated hypertension (Guzik et al., 2007; Sriramula et al., 2013; Sriramula et al., 2008; Sun et al., 2007).
Bursting in the heart, the process of ventricular remodeling is the presentation of many complicated
events,
including
signal pathway,
transcription,
protein
expression,
cell
differentiation, electrophysiology, structural change, and functional change. Due to the highly correlated of these events, treating ventricular remodeling for a single molecule or process may not be sufficient. As a multiple-target treatment, Chinese medicine may reverse ventricular remodeling by targeting multiple pathways. A large amount of clinical and preclinical studies have certified that Chinese medicine have effectiveness in treating ventricular remodeling. For some instance, Tongxinluo Capsules (Chen et al., 2010) and Shexiang Baoxin Pills (Zhang et al., 2014) could reverse ventricular remodeling in clinical trials. Qishen Yiqi Dropping Pills (Wang et al., 2015) could regulate RAAS system to attenuate myocardial fibrosis, and Qili Qiangxin Capsules
(Liang et al., 2016) could rehabilitate ventricular remodeling in rats. As a prescription drug of Chinese medicine formulae, TSM, consists of Astragalus membranaceus (Fisch.) Bunge, Codonopsis pilosula (Franch.) Nannf, Angelica sinensis (Oliv.) Diels, Dendrobium nobile Lindl, Lonicera japonica Thunb, Scrophularia aestivalis Griseb, Achyranthes bidentata Blume, and Glycyrrhiza uralensis Fisch, has a protect effect on focal cerebral ischemia (Zhao et al., 2011) and uses to treat thromboangiitis in clinical practice. A network pharmacology study suggested that multiple components of Tongsaimai may have a protective effect of atherosclerosis (Li et al., 2016). Our previous study demonstrated that Tongsaimai promoted wound recover and tissue regeneration in foot trauma of diabetic foot (Guo et al., 2014). Astragalus membranaceus (Fisch.) Bunge, as a primary component of TSM, can treat hypertension as a folk medicine in traditional applications (Li et al., 2018; Xu et al., 2008). These researches may suggest that TSM has a potential value in treating hypertension and hypertension related diseases. In this study, we investigated the protective effects of TSM in rats with AAC injury, and reported its influences on hypertension and left ventricular remolding.
2. Materials and methods 2.1. Ethics approval All procedures described in this study were performed in accordance with the guidelines of Nanjing University of Chinese Medicine Animal Care and Use Committee.
2.2. Reagents According to the ratio of 15:12:12:12:9:9:9:6, Astragalus membranaceus (Fisch.) Bunge, Codonopsis pilosula (Franch.) Nannf, Angelica sinensis (Oliv.) Diels, Dendrobium nobile Lindl, Lonicera japonica Thunb, Scrophularia aestivalis Griseb, Achyranthes bidentata Blume, and Glycyrrhiza uralensis Fisch are mixed to boil to get the extractum (TSM), and then the prepared extractum were made into sugar-coated tablets. The drug mass was 0.35 g/tablet. This is a standardized production process, and was carried out by Nanxing Pharmaceutical Co., Ltd. The extractum (TSM) used in our study was provided by Nanxing Pharmaceutical Co., Ltd. (lot number: 120713), and its pharmic content was 3.25 g /g (crude herbs / extractum). Captopril (Sino-US Squibb Pharmaceutical Co., Ltd.). Sodium chloride (produced by
Nanjing Chemical Reagent Co., Ltd.). Heparin (Shanghai Ruji Biotechnology Development Co., Ltd.). Urethane (Shanghai Qingfang Chemical Technology Co., Ltd.). Carboxymethyl Cellulose sodium (Sinopharm Chemical Test Co., Ltd.). Plasma renin activity Radioimmunoassay kit, Plasma angiotensin II Radioimmunoassay kit, Plasma aldosterone Radioimmunoassay kit, Plasma TGF-β Radioimmunoassay kit, and Plasma TNF-α Radioimmunoassay kit were purchased from Beijing Huaying Biotechnology Research Institute.
2.3. Animals Adult male Sprague-Dawley rats weighing 180–220g were provided by Shanghai Slack Laboratory Animal Co., Ltd. All animals were housed five per cage under controlled temperature (22 ± 2 °C) and with 12 h light/dark cycle (lights on at 8:00 a.m.). The animals had free access to food and water. After grouping, the rats in different group were intragastrical administered for 8 weeks. The dose of TSM is 2.4g/kg, and the dose of captopril is l0 mg/kg.
2.4. AAC model After diet forbidden and water for free for 12 h, the abdominal aorta was exposed following rat anesthesia (20% Urethane, 12g/kg, intraperitoneally), and the connective tissue surrounding the abdominal aorta were carefully separated. A 4-0 nylon monofilament suture was placed under the abdominal aorta. A six-and-a-half needle with a blunt tip was placed in parallel with abdominal aorta, which was ligated with the abdominal aorta by the nylon monofilament suture. In the
sham group, this same procedure was performed but no abdominal aorta was ligated. After that, the needle was withdrawn followed by the penicillin administration in the abdominal cavity, and then the abdominal cavity was closed layer by layer. 200,000 units of penicillin were intramuscularly injected for 3 consecutive days. Most of the death occurred within 72 hours after the surgery. After modeling for one week, the surviving rats were divided into different group.
2.5. Hemodynamic detection method The RM6240 multi-channel physiological signal acquisition and processing system was used to record and collect data. After diet forbidden and water for free for 12 h, the electro-cardiogram needle electrodes were inserted into the limbs subcutaneously following the anesthesia (20%
Urethane, 12g/kg, intraperitoneally). When the electrocardiogram and temperature (37 ° C) were stabilized, the right common carotid artery was separated and the end of it was ligated for arterial intubation. The pressure transducer was connected with the RM6240 multi-channel physiological signal acquisition and processing system. Systolic blood pressure (SBP), diastolic blood
pressure (DBP) and mean artery pressure (MAP) were recorded for 15 minutes after stabilization. Then, insert the cannula into the left ventricle until the ventricular pressure waveform appears, left ventricular systolic pressure (LVSP), left ventricular end diastolic pressur (LVEDP), the maximal rates that ventricular pressure rise (+dp/dtmax) and the
maximal rates that ventricular pressure fall (-dp/dtmax) were recorded for 15 minutes after stabilization.
2.6. Serologic detection After the hemodynamic detection, blood was collected from the left common carotid artery. After the serum was appeared, centrifugal separated it (3000 rpm, 10 min). The RAAS system was evaluated by the detection of the levels of PRL1, AngII and ALD, and the cardiac collagen deposition was evaluated by the detection of the levels of TGF-β and TNF-α in the serum of AAC rats. All indicators are detected according to the kit instructions
2.7. Evaluation of cardiac hypertrophy After blood collection, opened the chest and removed the heart. The heart was washed in 4 ° C NS to wash off the residual blood, a sheet of filter paper was used to absorb the surface moisture, and the whole heart mass was weighed using an electronic analytical balance. Then, the atrium and right ventricle were removed along the atrioventricular septum, and the left ventricle (including the interventricular septum) was weighed. HMI and LVMI were measured according to the formulas: HMI = whole heart weight / body weight× 100%, LVMI = left ventricle / body weight× 100%.
2.8. Evaluation of cardiac collagen deposition After the evaluation of cardiac hypertrophy, 30 mg of the heart was weighed followed by pyrolysis. The sample was hydrolyzed at 95 ° C for 20 minutes. Adjusted the pH to 6.0~6.8, added
various reagents according to the instruction manual of the kit, then mixed and centrifuged. Measured the absorbance of the supernatant of each tube. The hydroxyproline and collagen concentrations in the hearts of the rats were calculated according to the formulas: Hydroxyproline concentration = (TOD - BOD) / (SOD - BOD) × Cs × Vt × / W, Collagen concentration = Hydroxyproline concentration× 7.46. Abbreviation: TOD, absorbance of the test sample. BOD, absorbance of the blank. SOD, absorbance of the standard substance. Cs, concentration of the standard substance. Vt, volume of the hydrolysate. W, weight of the test sample.
2.9. Hematoxylin and eosin (HE) staining The HE staining method was as reported previously (Fischer et al., 2008). Briefly, after the heart tissue was formed into a block of wax. Bring sections to distilled water, stain nuclei with the hematoxylin for 4 min, then rinse sections in running tap water. After differentiate with 0.3% acid alcohol and wash again, then stain with eosin for 90 s. Dehydrated with different concentration alcohol, made transparent with xylene, and then observed under the microscope.
2.10. Masson's trichrome staining The Masson's trichrome staining method was as reported previously (Yu et al., 2018). Briefly, after heart the tissue was formed into a block of wax. Bring sections to distilled water, stain nuclei with the hematoxylin for 5 min, then rinse sections in running tap water for 10 min. Stain in acid fuchsin solution for 15min. Differentiate in phosphomolybdic acid solution. Transfer sections directly (without rinse) to aniline blue solution and stain for 5 min. Dehydrate very quickly with different concentration of alcohol and clear in xylene, then observed under the microscope.
2.11. Scanning electron microscopy (SEM) detection The SEM detection method was as reported previously (Michelmann et al., 2001). Briefly, 2.5% glutaraldehyde was used to fix the heart sample, osmium tetroxide was used to fix the heart sample again to preserve lipids. Using different concentration of alcohol to remove water from the sample. Sputter coating with gold to make the sample easy to conduct electricity. Adhere the sample to the conductive stage, then find a vision and take a photo.
2.12. Statistical Analysis Statistical analysis was carried out using ANOVA. Results are expressed as mean ± SD. A p value < 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism Version 5.01 software (GraphPad Software Inc., San Diego, CA, USA).
3. Results 3.1. TSM inhibits arterial pressure and left ventricular pressure in AAC rats To evaluate the antihypertensive effects of TSM, we monitored the changes of the arterial blood pressure and left ventricular pressure in different groups. The SBP, DBP, MAP, LVSP, and +dp/dtmax of the AAC rats, were significantly higher (p < 0.01), while the -dp/dtmax was significantly lower (p < 0.01), than that of the sham rats. In the AAC rats that received STM intragastric administration for 8 weeks, the SBP, DBP, MAP, and +dp/dtmax were found to be significantly reduced, and the -dp/dtmax was significantly increased (Fig. 1).
3.2. TSM regulates RAAS system in AAC rats. The RAAS system has the ability to regulate blood pressure, and the hypertension is often accompanied by abnormalities of the RAAS system (Ghazi and Drawz, 2017). Therefore, we next examined the PRA, AngⅡ, and ALD levels in rats, which is believed to indirectly reflect the abnormities of the blood pressure. The results show that the serum levels of PRA, AngⅡ, and ALD in AAC rats were markedly raised compared with normal group (p < 0.01). STM significantly decreased AngⅡ level (p < 0.05) and ALD level (p < 0.01) in AAC rats, but it has no influence on the serum PRA level (Fig. 2).
3.3. TSM suppresses cardiac hypertrophy in AAC rats Many studies have confirmed that cardiac collagen deposition causes cardiac hypertrophy (Isoyama et al., 1992). After 8 weeks of abdominal aortic coarctation， the cardiac hypertrophy occurred in AAC rats due to the increased cardiac after-load, resulting the heart mass index and the left ventricular mass index increased conspicuously than that in sham group (P<0.01). Administration of TSM can suppress ventricular hypertrophy and reduce the heart mass index and
the left ventricular mass index significantly (P<0.01) (Fig. 3).
3.4. TSM reduces cardiac collagen deposition in AAC rats Accounting for about 13% of the total amount of collagen amino acids, hydroxyproline is one of the major components of collagen, and is a specific amino in collagen (Weis et al., 2010). With the high content of hydroxyproline in collagen, the determination of hydroxyproline can be used to evaluate the situation of collagen in the body. Therefore, we examined the effect of TSM on hydroxyproline level of the heart in AAC rats, and calculated the collagen content in cardiac muscle. Higher concentrations of hydroxyproline and collagen were observed in the hearts of AAC rats (p < 0.01), and TSM significantly reduced the concentrations of hydroxyproline and collagen, as shown in Fig. 4.
3.5. TSM restrains the changes of cardiac histomorphology in AAC rats. Next, we examined the effect of TSM on the cardiac histomorphology. The HE staining of the hearts of AAC rats showed cardiomyocyte hypertrophy, fibrous connective tissue hyperplasia, cardiomyocyte apoptosis and replacement fibrosis compared with sham group (Fig. 5A). Masson's trichrome stainin presented that the amount of interstitial and infiltrative fibrosis and collagen deposition in AAC rats’ hearts were significantly increased (Fig. 5B). Furthermore, Scanning electron microscopy detection revealed that the myocardial myofibrils were distorted and discontinuous, the intercalated discs were blurred and widened, the mitochondria were vacuolated, and the mitochondrial crista were broken in the hearts of AAC rats (Fig. 5C). After intragastric administration of TSM in AAC rats, the cardiomyocyte hypertrophy, fibrous connective tissue hyperplasia, cardiomyocyte apoptosis, replacement fibrosis and collagen deposition of the heart were significantly decreased (Fig. 5A-B). And the Myocardial myofibrils and intercalated discs were clear and well-arranged, with complete mitochondria and visible mitochondrial crista (Fig. 5C). These findings suggest that TSM can restrain the changes of cardiac histomorphology in AAC rats.
3.6. TSM decreases the serum factor levels in AAC rats. Evidence shows that TGF-β plays an important role in the process of fibrosis (Rosenkranz,
2004), and the TNF-α involves in the left ventricular remolding following the hypertension (Sriramula et al., 2013; Sun et al., 2007). Therefore, we performed serum factor analysis. As shown in Fig. 6, compared with sham group, the serum levels of TGF-β and TNF-α in model group were increased remarkably (P<0.01). However, the decrease of the TGF-β and TNF-α levels in the serum after captopril treatment were not so significant (P<0.05) compared to other indexes, and there was only a downward trend in the serum TGF-β and TNF-α levels of AAC rats after TSM treatment, but the statistical results of the data were not significant (P>0.05).
4. Discussion Our study demonstrated that the administration of TSM significantly inhibited arterial pressure and left ventricular pressure, decreased AngⅡ and ALD levels, suppressed cardiac hypertrophy and cardiac collagen deposition, restrained the changes of cardiac histomorphology, and suppressed TGF-β and TNF-α levels in AAC rats. These results may enhance our understanding of the protection properties of TSM against left ventricular remolding through reversing the hypertension and provide an alternative option for the future prevention and treatment of hypertension and reconstructive heart disease. In our study, the SBP, DBP, MAP, LVSP, and +dp/dtmax of the AAC rats, were significantly higher (p < 0.01), while the -dp/dtmax was significantly lower (p < 0.01), than that of the sham group. However, the LVEDP did not change significantly compared to the sham group (Fig. 1), these results may indicate that the rat's cardiac afterload was significantly increased after modeling, causing compensatory increasing in systolic pressure. At this stage, the heart function was still in the compensation period, the diastolic function of the heart did not decrease significantly, so there was no significant change in LVEDP. After STM intragastric administration for 8 weeks, the SBP, DBP, MAP, and +dp/dtmax were found to be significantly reduced, and the -dp/dtmax was significantly increased (Fig. 1). Altogether, TSM can inhibit arterial pressure and left ventricular pressure in AAC rats. Previous studies have shown that hypertension is accompanied by abnormalities in the RAAS system (Ghazi and Drawz, 2017). The RAAS system maintains hemodynamic homeostasis by regulating blood pressure, water and electrolyte balance (Ghazi and Drawz, 2017). Pathological stimulations on RAAS causes excessive contraction of vessels, and hypertrophy and fibrosis of the heart (Alves et al., 2010). As the main effector of the RAAS system, AngII plays an important role
in the development of cardiovascular and renal diseases (Romet, 2010). In the kidney, AngII binds to the AT1 receptor, causing the release of ALD, which causes vasoconstriction, water-sodium retention, hypertension, and cardiac contractility increasing (Costerousse et al., 1998; Matsubara, 1998), accompanied by circular blood quantity increasing and cardiovascular remodeling (Savoia and Volpe, 2011). In the heart, AngII can promote fibroblast proliferation, ECM protein expression and collagen synthesis by binding to the AT1 receptor, which directly promoting the hypertrophic growth of neonatal cardiomyocytes (Schultz Jel et al., 2002). In this study, TSM alleviated AngⅡ and ALD levels in AAC rats, but it has no influence on the serum PRA level (Fig. 2). Many animal experiments and clinical studies have shown that, by inhibiting the conversion of AngI to AngII and attenuating the effects of AngII on ALD release, ACE inhibitors can alleviate hypertension and cardiovascular remodeling (Kim et al., 2001; Nakamura et al., 2003). Furthermore, ACE inhibitors reveals the vessels expanding and heart protecting functions, avoids or reverses the LVH (Cohn, 2000). So in this experiment, we used the ACE inhibitor captopril as a positive drug. Previous studies reports that captopril can reduce plasma AngII and ALD levels, but does not reduce PRA level (Sureshkumar, 2008), which is consistent with the results of this experiment. TSM also failed to reduce plasma renin activity, indicating that TSM also does not produce a hypotensive effect by directly inhibiting renin activity. But TSM can reduce the content of AngII and ALD. This finding suggests that the effect of antihypertensive of TSM may be mainly achieved by reducing AngII and ALD. In order to deal with pathological stimuli, like hypertension, the heart increases its weight by rising compensatory cardiomyocytes enlargement, fibroblast proliferation, and ECM deposition, to maintain arterial blood pressure and organ perfusion pressure (Berthelot et al., 2018; Guazzi, 2018). And, LVH is an early manifestation of heart damage in hypertensive patients (Huang and Li, 2018). In this experiment, the heart mass index and the left ventricular mass index were increased in AAC rats, and the administration of TSM can reduce the heart mass index and the left ventricular mass index significantly (P<0.01) (Fig. 3). The results indicating a reduction in cardiac hypertrophy caused by TSM. Hypertension leads to ventricular remodeling, pressure overload-left ventricular remodeling is characterized by cardiomyocytes enlargement, fibroblast proliferation, and ECM deposition (Villar et al., 2009). Furthermore, fibrous collagen is the most common type of collagen in the heart. Inhibition of collagen can significantly reduce ventricular remodeling (Isobe et al., 2010). Therefore, we tested the effect of TSM on hydroxyproline level of the heart in AAC rats, and calculated the collagen content in cardiac muscle. TSM can significantly reduce the concentrations of hydroxyproline and collagen (Fig. 4). This result suggests that TSM can suppress ventricular remodeling by reducing collagen deposition. Cardiac connective tissue plays an important role in maintaining the normal structure of the heart. Excessive ECM collagen deposition increases myocardial stiffness, and subsequently causes cardiac hypertrophy and left ventricular dysfunction (Schultz Jel et al., 2002). In addition, cardiac
hypertrophy and left ventricular dysfunction increase the risk of heart failure and increase the occurrence of arrhythmias (Villar et al., 2009). In this study, our results showed that TSM could inhibit AAC-induced up regulation of cardiomyocyte hypertrophy, fibrous connective tissue hyperplasia, cardiomyocyte apoptosis, replacement fibrosis and collagen deposition in the hearts of rats (Fig. 5A-B). Furthermore, TSM could reverse the disorders of myocardial myofibrils, intercalated discs, mitochondria and mitochondrial crista (Fig. 5C). These findings suggest that TSM can restrain the changes of cardiac histomorphology in AAC rats. TGF-β is a well-defined pro-fibrotic factor, and TNF-α promotes ventricular remodeling by increasing expression and activation of many factors in heart (Creemers et al., 2001). Thus, after confirming the effects of TSM for hypertrophy and left ventricular remodeling, we tested the serum levels of TGF-β and TNF-α in the rats. But, the statistical results of the data were not significant (P>0.05), there was only a downward trend in the serum TGF-β and TNF-α levels of AAC rats after TSM treatment (Fig. 6). Taken together, hypertension with high prevalence, especially arterial pressure changes, is one of the most common causes of ventricular remodeling (Miyoshi et al., 2015; Yao et al., 2016), and is a major cause of cardiovascular risk in adults. Our study found that TSM could significantly improve hemodynamic abnormalities caused by pressure overload, inhibit RAAS by reducing plasma AngII and ALD levels, inhibit cardiac hypertrophy and collagen deposition, restrain the changes of cardiac histomorphology, and reduce the plasma levels of TGF-β and TNF-α in AAR rats. Therefore, STM may potentially be a safe and effective treatment for reverse left ventricular remolding through suppressing hypertension.
Funding sources This study was supported by the Natural Science Foundation of China (No. 81073071) and Jiangsu
Provincial
Science
and
Technology
Department
Social
Development
Fund
(No.BE2011846).
Author contributions Qinghai Meng carried out the experiments, assisted with data analysis, and the manuscript preparation. Yao Guo and Dini Zhang helped with the manuscript preparation and all the experiments. Qichun Zhang helped with the design of the study and conducted the SEM detection. Yu Li performed the kits detection and assisted with the data analysis and manuscript preparation. Huimin Bian helped conduct the experiments and contributed to the writing of the manuscript.
Conflict of Interest Statement
The authors declare that they have no competing interests.
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Figure legends Fig. 1. TSM improves hemodynamics in AAC rats. (A) SBP of the rats. (B) DBP of the rats. (C) MAP of the rats. (D) LVSP of the rats. (E) LVEDP of the rats. (F) +dp/dtmax of the rats. (G) -dp/dtmax of the rats. n = 8.
Fig. 2. TSM regulates RAAS system in AAC rats. (A) Plasma renin activity in rats. (B) Plasma Ang II level in rats. (C) Plasma ALD level in rats.
Fig. 3. TSM reduces cardiac hypertrophy in AAC rats. The heart mass index (A) and left ventricular mass index (B) of the rats.
Fig. 4. TSM reduces cardiac collagen deposition in AAC rats. The hydroxyproline (A) and collagen (B) concentrations in the hearts of the rats.
Fig. 5. TSM improves cardiac histomorphology in AAC rats. (A) HE staining of the hearts of the rats. Scale bar = 50µM. (B) Masson staining of the hearts of the rats. Scale bar = 50µM. (C) SEM dection of the hearts of the rats.
Fig. 6. TSM reduces the serum factor levels in AAC rats. The plasma level of TGF-β (A) and TNF-α (B) in rats.