Cardioprotective Effects of Solidago microglossa DC. in Nicotine-Treated Hypertensive Rats

Solidago microglossa DC. (Asteraceae), “arnica brasileira,” is a Brazilian species popularly used to treat hypertension or renal ailments. This study investigated the cardioprotective effects of standardized S. microglossa extract (EESM) in nicotine-treated spontaneously hypertensive rats (SHRs). Moreover, the molecular mechanisms involved in the cardiovascular effects were also investigated. The acute toxicity was evaluated in female Wistar rats. Afterwards, six-month-old male spontaneously hypertensive rats received the EESM (14, 28, and 56 mg/kg), hydrochlorothiazide (25 mg/kg), and vehicle (filtered water; 0.1 mL/100 g) once daily for 28 days. All treatments were associated with 1.8 mg/kg of nicotine. At the end of the experimental period, the renal function, electrocardiographic profile, blood pressure, ventricular function, biochemical parameter, and mesenteric vascular bed reactivity were evaluated. Relative organ weights and cardiac morphometry were also investigated. Nicotine treatment in 6-month-old SHRs induced a significant reduction in renal function, with reduced urinary volume and lower renal elimination of sodium and creatinine. In addition, serum markers of the redox state and blood pressure levels remained significantly elevated, contributing to changes in vascular reactivity and left ventricular hypertrophy associated with reduced ventricular function. After 28 days of treatment, we found that the highest dose of EESM could mitigate all renal and cardiovascular changes developed by the nicotine-treated hypertensive rats. This study presented EESM as a possible cardioprotective drug that prevents cardiovascular dysfunctions in nicotine-treated hypertensive rats. Our data suggest EESM as a potential adjuvant therapy when cardioprotective effects are required.


Introduction
Hypertension is a multifactorial and highly prevalent disease considered to be the leading risk factor for developing several cardiovascular and renal diseases.Te treatment includes lifestyle changes and pharmacological measures, including renin-angiotensin system inhibitors, betablockers, calcium channel blockers, and diuretics [1].Te drug therapy is lifelong and may cause several adverse effects, contributing to low adherence and inadequate disease control [2].So, natural products emerge as an alternative to complementary and alternative therapy [3].
Due to Brazil's diversity of medicinal plants, public politics has encouraged projects and research to develop new herbal medicines.In 2009, a list of medicinal plants of interest to the Brazilian Unifed Health System (SUS) was published, including Solidago microglossa DC. (Asteraceae) [4].
Terefore, this work evaluated the cardioprotective efects of S. microglossa extract in nicotine-treated spontaneously hypertensive rats (SHRs).Moreover, the molecular mechanisms involved in the cardiovascular efects were also investigated.

Solidago microglossa Extract (EESM) Obtention.
S. microglossa vegan capsules (Brazilian Arnica) were obtained from a Brazilian commercial preparation (Curitiba, Paraná, Brazil).Each capsule contains 650 mg of Brazilian Arnica dry extract.Te extract was produced from the dried aerial parts of S. microglossa using 80-85% (v/v) ethanol.In the drying process, cornstarch was added at a ratio of 0.5-1.5 : 1. Te dry extract was standardized at 1% quercetin-3-rhamnoside.Each capsule (composed of hypromellose and titanium dioxide) has a total weight of 1 g.

Pharmacological Investigations
2.3.1.Animals.Male Wistar-Kyoto (WK) and male SHRs (180 days old), as well as female Wistar rats (90 days old), were obtained from the central vivarium of the Federal University of Grande Dourados (UFGD, Brazil).All animals were kept in a temperature and light-controlled room (22 ± 2 °C; 12 h light/dark cycle) with ad libitum access to water and food.All procedures were previously approved by the Ethics Committee in Animal Experimentation from the UFGD (protocol no.31/2019-1) [10,11] and were following the Brazilian Legal Framework on Scientifc Animals Use.

Safety Evaluation
(1) Acute Toxicity.Te acute toxicity assay was evaluated according to protocols from Palozi et al. [12].Twelve female Wistar rats were equally randomized into two experimental groups.Initially, all animals were subjected to overnight fasting.Ten, the animals were weighed, and a 2,000 mg/kg limit dose of EESM or vehicle (fltered water; 0.2 mL/100 g) was administered orally (by gavage).Te rats were carefully observed for the frst 8 hours and daily for 14 days.Mortality rate, water and food consumption, body weight gain, and any signs of toxicity or behavior changes were registered daily.On the ffteenth morning, all rats were killed by isofurane anesthesia (inhalation) followed by exsanguination.Lungs, liver, kidneys, spleen, and heart were subjected to gross pathology.

Cardioprotective Evaluations
(1) Allometric Extrapolation of the EESM Daily Dose Used in Humans to Rats.Te EESM doses were determined by allometric extrapolation from the daily dose used for humans (1 g/day: 1 capsule).Te average body weight considered for humans was 70 kg.Tus, the daily dose considered was 14, 28 mg/kg.

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Evidence-Based Complementary and Alternative Medicine (3) Renal Function.On days 1 and 28, all rats were treated orally with NaCl 0.9% (5 mL/100 g) to impose hydration uniformity.Ten, the animals were placed individually in metabolic cages.Urine samples were collected over 24 hours, and their volumes were registered.Urine pH and density were determined by a digital pH meter (Q400MT; Quimis Instruments, Brazil) and handheld refractometer (NO107; Nova Instruments, Brazil), respectively.Urinary sodium, potassium, chloride, urea, and creatinine levels were quantifed using an automated biochemical analyzer (Cobas Integra 400 plus, Roche).
( (9) Relative Organ Weight, Histopathology, and Morphometry.At the end of the experiments, the kidneys, liver, and heart were removed and cleaned, and the relative weight was determined (RW% = absolute organ weight × 100/body weight).Ten, heart fragments were fxed in 10% bufered formalin, cleaved, dehydrated with increasing ethanol concentrations, diaphonized in xylol, and embedded in parafn.Sections were then cut at a thickness of 4 μm and stained with hematoxylin and eosin (HE).Te heart area and the right (RV) and left (LV) ventricles and interventricular septum (IS) thickness were measured.All images were obtained and evaluated using Motic Images Plus 2.0 software.

Investigation of the Role of the Endothelin and Renin-
Angiotensin System on the Cardioprotective Efects of S. microglossa Extract.Diferent groups of hypertensive rats (SHRs 28 days nicotine-treated; n � 8) were initially anesthetized and prepared for direct blood pressure measurement, as described above.After cannulation and MAP stabilization, the efects of angiotensin I (Ang I; 10 pmol/kg, i.v.), angiotensin II (Ang II; 10 pmol/kg, i.v.), and ET-1 (10 pmol/kg, i.v.) on the arterial pressure were recorded.
After returning blood pressure levels to baseline, a bolus dose of EESM (56 mg/kg) was administered intraduodenally.After 30 minutes, a new dose of angiotensin I, angiotensin II, and ET-1 was administered.SBP, DBP, and MAP values were registered for 20 min.

Acute Toxicity.
No deaths were detected during the 14day study, allowing to suggest an LD50 as greater than 2,000 mg/kg.We identifed no clinical or behavioral signs indicating EESM toxicity, including body weight and food consumption.Furthermore, no signifcant histopathological changes were observed (data not shown).

Efects on Kidney Function.
Te values that show urinary parameters of all experimental groups on the frst and twentyeighth day of treatment are shown in Tables 1 and 2. On the frst day of treatment, all 6-month-old nicotine-treated hypertensive rats showed a signifcant reduction in urinary volume and renal excretion of sodium and creatinine.On the other hand, after 28 days of treatment, all animals treated with the highest dose of EESM (56 mg/kg) or with HCTZ reversed this change, showing values similar to naïve animals.

Efects on Cardiac Electrical Activity.
Te efects of oral treatment with EESM and HCTZ on electrocardiographic parameters are shown in Table 3.A signifcant increase in the QT and QTc segments and the R wave amplitude was observed in the NC, EESM (14 and 28 mg/kg), and HCTZ groups.On the other hand, the animals in the EESM 56 mg/kg group did not show any signifcant electrocardiographic alterations, with values similar to those found in the naïve rats.Statistical analyses were performed using one-way ANOVA followed by Tukey's test.Values are expressed as mean ± SEM (standard error of the mean) of 8 animals per group.a p < 0.05 when compared to the naïve rats.b p < 0.05 when compared to the NC group.c p < 0.05 when compared to the EESM 14 mg/kg.d p < 0.05 when compared to the EESM 28 mg/kg.EESM: hypertensive rats that received three diferent doses of the Solidago microglossa extract; HCTZ: hypertensive animals that received daily doses of hydrochlorothiazide (25 mg/kg); NC: hypertensive animals that received daily doses of the vehicle; Naïve: normotensive animals that received only the vehicle.DBP: diastolic blood pressure; HR: heart rate; MAP: mean arterial pressure; SBP: systolic blood pressure.Statistical analyses were performed using one-way ANOVA followed by Tukey's test.Values are expressed as mean ± SEM (standard error of the mean) of 8 animals per group.a p < 0.05 when compared to the naïve rats.b p < 0.05 when compared to the NC group.c p < 0.05 when compared to the EESM 14 mg/kg.d p < 0.05 when compared to the EESM 28 mg/kg.e p < 0.05 when compared to the HCTZ.EESM: hypertensive rats that received three diferent doses of the Solidago microglossa extract; HCTZ: hypertensive animals that received daily doses of hydrochlorothiazide (25 mg/kg); NC: hypertensive animals that received daily doses of the vehicle; Naïve: normotensive animals that received only the vehicle.ACE: angiotensin-converting enzyme; MDA: malondialdehyde; NT: nitrotyrosine.Statistical analyses were performed using one-way ANOVA followed by Tukey's test.Values are expressed as mean ± SEM (standard error of the mean) of 8 animals per group.a p < 0.05 when compared to the naïve rats.b p < 0.05 when compared to the NC group.c p < 0.05 when compared to the EESM 14 mg/kg.d p < 0.05 when compared to the EESM 28 mg/kg.EESM: hypertensive rats that received three diferent doses of the Solidago microglossa extract; HCTZ: hypertensive animals that received daily doses of hydrochlorothiazide (25 mg/kg); NC: hypertensive animals that received daily doses of the vehicle; Naïve: normotensive animals that received only the vehicle.ACh: acetylcholine; Phe: phenylephrine; SNP: sodium nitroprusside.

Efects on
Evidence-Based Complementary and Alternative Medicine min).In addition, a signifcant increase in the relative weight of the heart was evidenced, directly infuenced by the thickening of the left ventricular wall.Only hypertensive animals treated with HCTZ or EESM at its highest dose (56 mg/kg) showed normalization of these alterations, showing values similar to those observed in naïve animals.

Efects on Endothelin and Renin-Angiotensin System.
Te efects of intraduodenal administration of EESM (25 mg/kg) on endothelin and renin-angiotensin system are presented in Table 8.Before intraduodenal administration of EESM, intravenous administration of Ang I, Ang II, and ET-1 signifcantly increased SBP, DBP, and MAP levels

Discussion
Smoking is widespread in all societies in the world.Despite addiction, it is agreed that chronic smoking afects the function and structure of the cardiovascular system [17].Among the components of tobacco, nicotine is the most signifcant contributor to the genesis and progression of CVDs, including vascular diseases and cardiomyopathies [18].Nicotine causes endothelial dysfunction and stimulates the oxidative and infammatory cascade that leads to the development of atherosclerosis and hypertension.Furthermore, it can aggravate preexisting hypertension and induce coronary spasms, precipitating a myocardial infarction.Moreover, nicotine increases free radical production, vascular wall adhesion, and plasma reduction in fbrinolytic activity [13].One of the main mechanisms that involve these efects is related to the increase in the release of catecholamines in the sympathetic nerve endings and the channel ion modulations [19].Tus, this work aimed to investigate the efects of a Brazilian pharmacopeial herbal preparation on SHRs treated with nicotine.Terefore, we aim to assess how much the EESM contributed to reducing the renal, cardiac, and vascular dysfunction that may be associated with nicotine-treated hypertensive rats.
Although the hemodynamic and morphological alterations that hypertension induces in rodents are already well known, we used two diferent experimental conditions.First, we used 6-month-old SHRs, stabilizing hypertension and leading to cardiac and vascular morphological changes [20].In addition, we used nicotine to potentialize cardiovascular changes, hypothesizing that EESM could induce cardioprotective efects against these conditions.As expected, nicotine treatment in 6month-old SHRs caused a signifcant reduction in renal function, with lower urinary volume and renal elimination of sodium and creatinine.In addition, serum markers of the redox state and blood pressure levels remained signifcantly elevated, contributing to changes in vascular reactivity and reduced ventricular function.
After 28 days of treatment, the highest dose of EESM could mitigate the renal and cardiovascular changes induced in the nicotine-treated hypertensive rats.Physiopathological alterations observed in the animals that received only the vehicle were completely reversed in the rats treated with the EESM, maintaining cardiovascular conditions similar to those found in the naïve animals.Although the results were interesting, the mechanisms by which the EESM produced these responses remained unclear.Te EESM was standardized with 1% quercetin-3-rhamnoside, a glycosylated favonoid.It is now known that glycosylated favonoids have better solubility and oral absorption than isolated aglycone, e.g., quercetin [21].Nevertheless, it is already widely known that quercetin has signifcant antioxidant, vasodilator, antihypertensive, and cardioprotective activity [22].Data showed that quercetin inhibits vascular superoxide production induced by ET-1 and increases nitric oxide levels [23,24].Furthermore, quercetin induces oxidative and endoplasmic reticulum stress suppression via ET-1/MAPK signalling [25].Although the information about aglycone is promising, its glycosylated form, i.e., quercetin-3-rhamnoside, needs investigation.
We hypothesize that the efects of quercetin-3-rhamnoside and, consequently, of the EESM could be similar to those described for quercetin.Tus, as the extract signifcantly afected lipid peroxidation and tyrosine nitration mediated by reactive nitrogen species, the NO bioavailability was increased, reverberating into a signifcant antioxidant efect.Together, this result directly contributed to a vasodilator response and blood pressure reduction, helping to maintain the integrity of the vascular endothelium [26,27].On the other hand, as vascular reactive oxygen species (ROS) production can be induced by ET-1 and Ang II [28,29], we chose to investigate the role of the reninangiotensin and endothelin system in the hypotensive response of the EESM because it is known that the direct inhibition of the action of ET-1 can prevent the efects of Ang II from stimulating cardiac hypertrophy [28].Te treatment with EESM (at its highest dose) prevented ET1induced hypertension without afecting the renin-angiotensin system, suggesting the direct role of the endothelin signalling pathway.
Endothelins are vasoconstrictor peptides represented by ET-1, ET-2, and ET-3.Only ET-1 is produced by endothelial cells and is related to hemodynamic disorders and endothelial dysfunction.Stimuli such as ischemia, hypoxia, or shear stress induce the transcription of messenger RNA, with consequent synthesis and secretion of ET-1.On the other hand, ET-1 interacts with several hormones, vasoactive peptides, and growth factors, increasing vascular shear stress [30].Smokers have increased ET-1 levels due to nicotine-stimulated production by endothelial cells [31].Furthermore, it was described that the antagonism of endothelin ET(A) receptors attenuated the increase in blood pressure after nicotine injections, suggesting that ET-1 may have a critical role in the efects of nicotine [32].Tus, a limitation of our study was not being able to relate the efects of EESM with the blockade of ETA receptors.
In summary, the data obtained in this study allow us to conjecture that the cardioprotective efects of EESM are likely due to antioxidant efects and ET-1 inhibitors.We cannot claim that the pharmacological efects presented here are only due to quercetin-3-rhamnoside.Still, we believe it is an important agent that acts synchronously with other secondary metabolites in the EESM.

Conclusion
Tis study presented EESM as a possible cardioprotective drug that prevents cardiovascular dysfunctions in nicotinetreated hypertensive rats.Our data suggest EESM as a potential adjuvant therapy when cardioprotective efects are required.

Figure 1 :
Figure 1: Representative images of the left ventricle of nicotine-treated hypertensive rats that received the EESM for 28 days.Te black arrow represents an area of fbrous connective tissue deposition (scar).Te black circle delimits an extensive area of fbrous tissue deposition associated with necrosis of cardiac fbers.EESM: hypertensive rats that received three diferent doses of the Solidago microglossa extract; HCTZ: hypertensive animals that received daily doses of hydrochlorothiazide (25 mg/kg); HE: hematoxylin and eosin stain; NC: hypertensive animals that received daily doses of the vehicle; Naïve: normotensive animals that received only the vehicle.

Table 1 :
Efects of oral treatment with Solidago microglossa extract (EESM) and hydrochlorothiazide (HCTZ) on urinary parameters on the 1 st day of treatment.
a Statistical analyses were performed using one-way ANOVA followed by Tukey's test.Values are expressed as mean ± SEM (standard error of the mean) of 8 animals per group.ap< 0.05 when compared to the naïve rats.EESM: hypertensive rats that received three diferent doses of the Solidago microglossa extract; HCTZ: hypertensive animals that received daily doses of hydrochlorothiazide (25 mg/kg); NC: hypertensive animals that received daily doses of the vehicle; Naïve: normotensive animals that received only the vehicle.4Evidence-BasedComplementary and Alternative Medicine

Table 2 :
Efects of oral treatment with Solidago microglossa extract (EESM) and hydrochlorothiazide (HCTZ) on urinary parameters on the 28 th day of treatment.
bcd Statistical analyses were performed using one-way ANOVA followed by Tukey's test.Values are expressed as mean ± SEM (standard error of the mean) of 8 animals per group.ap < 0.05 when compared to the naïve rats.bp < 0.05 when compared to the NC group.cp < 0.05 when compared to the EESM 14 mg/kg.dp < 0.05 when compared to the EESM 28 mg/kg.EESM: hypertensive rats that received three diferent doses of the Solidago microglossa extract; HCTZ: hypertensive animals that received daily doses of hydrochlorothiazide (25 mg/kg); NC: hypertensive animals that received daily doses of the vehicle; Naïve: normotensive animals that received only the vehicle.Evidence-Based Complementary and Alternative Medicine 2.4.Statistical Analysis.Te results are shown as mean-± standard error of the mean (SEM) of 8 rats or preparations per group.Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Tukey's test.A p value less than 0.05 was considered statistically signifcant.
with HCTZ prevented this change, maintaining blood pressure levels similar to those in the naïve rats.Similarly, treatment with EESM 56 mg/kg or HCTZ also signifcantly reduced the HR of nicotine-treated hypertensive rats.3.5.Efects on BiochemicalParameters.Te efects of oral treatment with EESM and HCTZ on biochemical parameters are shown in Table5.Serum creatinine levels were signifcantly increased in animals from the NC and EESM 14 and 28 mg/kg groups.Treatment with HCTZ and EESM at its highest dose (56 mg/kg) reduced this parameter, maintaining creatinine values similar to those found for naïve animals.NT and MDA serum levels were signifcantly elevated, and nitrite levels were reduced in nicotine-treated hypertensive rats that received vehicle (NC), HCTZ, and EESM at 14 and 28 mg/kg.Treatment with EESM at a dose of 56 mg/kg maintained levels of these oxidative stress markers similar to those found in naïve rats.Serum adrenaline levels were signifcantly increased in nicotine-treated hypertensive rats compared to naïve animals.None of the treatments signifcantly reduced adrenaline serum levels in hypertensive animals.All other evaluated parameters were not altered in any of the experimental groups.
Blood Pressure.Te efects of oral treatment with EESM and HCTZ on blood pressure values are shown in Table4.Rats in the NC group showed a signifcant increase in SBP, DBP, MAP, and HR compared to naïve animals.Treatment with EESM at its highest dose (56 mm/ kg) or 3.7.Efects on Hemodynamics and Cardiac Structure.Te efects of oral treatment with EESM and HCTZ on hemodynamics and cardiac structure are shown in Figure1 and

Table 3 :
Efects of oral treatment with Solidago microglossa extract (EESM) and hydrochlorothiazide (HCTZ) on electrocardiographic parameters.Statistical analyses were performed using one-way ANOVA followed by Tukey's test.Values are expressed as mean ± SEM (standard error of the mean) of 8 animals per group.a p < 0.05 when compared to the naïve rats.b p < 0.05 when compared to the NC group.c p < 0.05 when compared to the EESM 14 mg/kg.d p < 0.05 when compared to the EESM 28 mg/kg.e p < 0.05 when compared to the HCTZ.EESM: hypertensive rats that received three diferent doses of the Solidago microglossa extract; HCTZ: hypertensive animals that received daily doses of hydrochlorothiazide (25 mg/kg); NC: hypertensive animals that received daily doses of the vehicle; Naïve: normotensive animals that received only the vehicle.
6Evidence-Based Complementary and Alternative Medicine Table 7.In the left ventricle myocardium of the animals in the NC group, it was possible to visualize an extensive area of fbrous tissue deposition associated with discreet necrosis and mononuclear cell infammatory infltrate.Hypertensive animals treated with the lowest dose of EESM (14 mg/kg) also showed a discrete focal area of fbrosis in the subendocardial layer of the myocardium.All other experimental groups did not show signifcant histopathological alterations.All hypertensive animals that were treated with the vehicle alone showed a signifcant reduction in ventricular function, including the LVDP (mm Hg), +dp/dt max (mm•Hg/s), −dp/dt min (mm•Hg/s), and RPP (mm•Hg-beats/

Table 4 :
Efects of oral treatment with Solidago microglossa extract (EESM) and hydrochlorothiazide (HCTZ) on blood pressure values.

Table 7 :
Efects of oral treatment with Solidago microglossa extract (EESM) and hydrochlorothiazide (HCTZ) on hemodynamics and cardiac structure.Statistical analyses were performed using one-way ANOVA followed by Tukey's test.Values are expressed as mean ± SEM (standard error of the mean) of 8 animals per group.a p < 0.05when compared to the naïve rats.b p < 0.05 when compared to the NC group.c p < 0.05 when compared to the EESM 14 mg/kg.d p < 0.05 when compared to the EESM 28 mg/kg.EESM: hypertensive rats that received three diferent doses of the Solidago microglossa extract; HCTZ: hypertensive animals that received daily doses of hydrochlorothiazide (25 mg/kg); NC: hypertensive animals that received daily doses of the vehicle; Naïve: normotensive animals that received only the vehicle.IS: interventricular septum; LV: left ventricle; LVDP: left ventricular developed pressure; RPP: rate pressure product; RV: right ventricle; +dp/dt max : peak contraction rate; −dp/dt min : peak relaxation rate.