The protective effects of polysaccharide extract from Xin-Ji-Er-Kang formula on Ang II-induced HUVECs injury, L-NAME-induced hypertension and cardiovascular remodeling in mice

Hypertension is a significant health threat in the USA due to its mortality rate and treatment costs. Approximately 31% of Americans have been diagnosed with hypertension [1]. In addition, it is a major risk factor for fatal diseases, including stroke, atherosclerosis and congestive heart failure. Evidently, effective control of blood pressure reduces the morbidity and mortality rates of these diseases [2, 3]. Recently, the curative effects of traditional Chinese medicine (TCM) have received growing attention both in China and abroad. The therapeutic effect of a typical Chinese herbal formula (Table 1) Xin-Ji-Er-Kang (XJEK) in hypertension-induced coronary heart disease, toxic myocarditis and virus myocarditis has been confirmed in both clinical and basic studies [4, 5]. It has been demonstrated that the underlying mechanism of XJEK may involve decreasing oxidative stress (OS) and improving endothelium function [6, 7].

Furthermore, an increasing amount of evidence has revealed various beneficial medicinal effects of polysaccharides in TCM, including antitumor [8], antivirus [9, 10] and anti-oxidative effects, and regulation of the immune system and cytokine secretion. Notably, several main components of XJEK, such as Polygonatum odoratum (Mill.) Druce, Astragalusmongholicus Bunge and Ophiopogon japonicus (Thunb.) Ker Gawl exert protective effects in cardiovascular diseases [11‐14]. In our previous study, it was implied that polysaccharides from XJEK decoction exhibited an anti-cardiac remodeling effect in an isoprinosine-treated mouse model [15]. Recent research demonstrated that XJEK formula has a protective effect against Nω-nitro-L-arginine methyl ester (L-NAME)-induced hypertension and angiotensin II (Ang II)-induced Human umbilical vascular endothelial cells (HUVECs) injury [16, 17], but the effect of polysaccharides from XJEK decoction on these two models has not been elucidated.

Chronic administration of nitric oxide (NO) synthase inhibitors, including L-NAME, can lead to severe chronic hypertension, which is a method commonly used for the establishment of animal models with pharmacological hypertension. In addition, administration of NO synthase inhibitors has been associated with decreased NO bioactivity and endothelial dysfunction (ED). Furthermore, as suggested by recent studies, NO deficiency is directly associated with the generation of inflammatory cytokines, including tumor necrosis factor-α [18, 19]. Therefore, the anti-hypertensive effects of polysaccharide extract from XJEK were investigated in the present study in L-NAME-induced hypertensive mice, and the underlying molecular mechanism of cardiovascular protection was assessed by examining ED, anti-oxidative stress and anti-inflammatory activities.

Long-term administration of L-NAME in drinking water is a commonly used method for the establishment of chronic hypertension models, in which endothelial dysfunction, increased ventricular hypertrophy, elevated oxidative stress, decreased eNOS expression levels, increased plasma ADMA expression levels and an increased inflammatory status are typical characteristics, as summarized in Fig. 9. The findings in the present study validated the effects of L-NAME administration on BP, ED, cardiac hypertrophy, vascular remodeling and inflammatory status, as previously reported. Furthermore, it was demonstrated that treatment with AqE reduces these pathophysiological changes, as does treatment with the positive control drug fosinopril.

In recent years there has been a novel but increasingly important trend of studies investigating the modernization of TCM to achieve a lower dose requirement with the same efficacy, which has predominantly been attempted by the elimination of impurities and the guarantee of active ingredients in compound preparations of TCM. Notably, both clinical and basic studies have demonstrated the efficacy of polysaccharides extracted from Panax ginseng C.A.Mey. [24], Polygonatum odoratum (Mill.)Druce [25], Astragalusmongholicus Bunge [26] and Ophiopogon japonicus (Thunb.) Ker Gawl [27] in the prevention of cardiovascular diseases and associated disorders. Specifically, a randomized controlled trial demonstrated that Panax ginseng C.A.Mey. polysaccharide may be an effective natural alternative for enhancing the immune system [28]. Zhang et al [20] reported that Panax ginseng C.A.Mey polysaccharide can improve pressure overload-induced cardiac remodeling by protecting mitochondria function and reducing energy metabolism dysfunction. The anti-hypertensive function of Astragalusmongholicus Bunge polysaccharide has been identified to be associated with an enhancement of endothelial function and anti-inflammation [29‐31]. Similarly, in clinical reports byJiang [32] and Luan [33], Astragalusmongholicus Bunge polysaccharide was associated with a reduction of the haemodynamic index and cardiac fibrosis in spontaneous hypertension rats (SHR) and in isoproterenol-induced cardiac hypertrophy. Shenmai injection, also known as a traditional Chinese herbal medicine extraction, has gained popularity in China in therapies targeting chronic heart failure, hypertension, and angina pectoris [34, 35]. Furthermore, Ophiopogon japonicus (Thunb.) Ker Gawl polysaccharide has been demonstrated to exhibit anti-myocardial ischemic activities in studies by of the Ph. Xu D. S (Shanghai University of Traditional Chinese Medicine) group [36, 37]. Recent studies by Chen [38] and Gu [39] indicateda strong anti-oxidative effect of polysaccharide from Polygonatum odoratum (Mill.) Druce for the treatment of metabolic disorders. Polygonatum odoratum (Mill.) Drucehas been used for nutritional and medicinal purposes for over 2000 years in China, however, a limited number of studies have investigated its role and pharmacological mechanisms. In accordance with the aforementioned studies, the present study demonstrated an expected effect of AqE and this effect was observed at a lower dose compared with that of XJEK (2.47 g/kg vs. 7.5 g/kg). The pathological characteristics of the animal models used in the current study may explain the similar effect of AqE.

eNOS inhibition induces thickening of the aorta in hypertensive mice, which impacts the elasticity of the aorta vessel wall [40, 41]. A recent study reported an early 50% increase in fibrosis surrounding the aortas of L-NAME-treated mice compared with those of untreated mice [42]. ADMA regulates eNOS activity, which results in decreased levels of NO synthesis and increased generation of superoxide [43]. ADMA activity is also susceptible to reactive oxygen and nitrogen species [44]. Therefore, increased systemic ADMA levels may contribute to the pathogenesis and progression of cardiovascular diseases that are associated with endothelial dysfunction. These effects were detected in the current study by the observation of impaired NO mediated responses to ACh in aorta, in addition to decreased NO content and eNOS activity, and increased ADMA consent in plasma and cardiac remodeling. Treatment with AqE for 4 weeks improved cardiovascular remodeling, which was indicated by the restoration of cardiac haemodynamics and an improved HW/BW index, cardiomyocyte CSA, aorta wall thickness, TAA and M. Furthermore, 4-week experimental therapy with AqE alleviated L-NAME-induced ED, as indicated by the enhancement of NO-dependent artery relaxation and the restoration of NO content, ADMA content and eNOS activity in plasma. In addition, the present in vitro study demonstrated marked protective effects of AqE on AngII-induced injury of HUVECs, which was indicated by increased NO content and eNOS activity in a dose-dependent manner.

TNF-α interacts with its receptors and in turn activates multiple signals, including the activation of NADPH oxidase. Subsequently, NADPH oxidase induces superoxide production, which reacts with NO and produces the strong oxidant peroxynitrite. In addition, TNF-α inhibits the eNOS promoter and induces the destabilization of eNOS mRNA, which ultimately downregulates the levels of eNOS protein and the ability of the endothelium to produce NO [45, 46]. The aorta ring experiment in the current study demonstrated that TNF-α reduces endothelium-dependent vasodilatation. TNF-α knockout mice may inhibit BP elevation and left ventricular hypertrophy [47]. Furthermore, Mauno et al [48] reported that baseline levels of IL-1β are markedly increased in hypertensive individuals, which suggests a critical role of IL-1β in hypertension. Cytokinesare understood to exhibit anti-inflammatory and anti-hypertensive effects. Didion et al demonstrated an impairment of endothelial dependent vasodilatation in IL-10 knockout mice treated with Ang II. In addition, increased function of Tregs decreases the expression of Ang II and improves aldosterone-induced hypertension, cardiac fibrosis, coronary inflammation, electric remodeling and endothelial-dependent vasodilation, which are likely partly mediated by the release of IL-10 from Tregs [49‐52].

Furthermore, NO regulatesthe endogenous production of IL-10, which maintains homeostasis in healthy individuals. It has been suggested that NO may sustain the endogenous levels of IL-10 by promoting the proliferation and survival of Treg cells [53]. Therefore, a balance between pro-inflammatory and anti-inflammatory cytokines is important for cardiac remodeling, hypertension-associated ED and myocardial infarction injury, and the restoration of this balance may prevent cardiovascular damage [54]. It has been reported that polysaccharides perform potent immune-modulatory activities, as demonstrated by the induction of pro-inflammatory and anti-inflammatory cytokines [55]. In the present study an immunological imbalance was revealed in an L-NAME-induced hypertensive mouse model, indicated by increased expression levels of TNF-α and IL-1β, and decreased expression of IL-10. Furthermore, the balance between pro-inflammatory and anti-inflammatory status could be restored by treatment with polysaccharide from XJEK (Fig. 9).