The cardioprotective effect of S. africana caerulea/Blue Sage in ischaemia and reperfusion induced oxidative stress

Background: Since antiquity, alternative herbal remedies, such as S. africana caerulea/Blue Sage (BLS) water infusion extract (WIE) has been used by traditional healers, for the effective treatment of various chronic inflammatory disorders associated with reduced cellular antioxidant defense mechanisms and free radical cellular damage. In the heart, ischaemia—reperfusion (I/R) induced oxidative stress becomes an early crucial event in the pathogenesis of ischaemia—reperfusion injury (I/RI) and subsequent heart failure. Purpose/Aim: To investigate whether BLS WIE treatment during ischaemia and/or reperfusion may be cardioprotective. Study design: Isolated perfused rat hearts were exposed to 35 min regional ischaemia (RI) and 60 min reperfusion. The BLS WIE was applied: i) for the last 10 min of RI (PerT) or ii) from onset of reperfusion (PostT) or iii) both (PerT) + (PostT). Methods: Endpoints were functional recovery and infarct size (IS). In another set of experiments, left ventricles were freeze-clamped after RI and 10 min reperfusion for detection of total and phosphorylated p-ERK p44/p42, p-Akt, p-p38-MAPK, p-JNK, Nrf-2, NF-kB, Bax, Bcl-2, Caspase-3, and PGC-1α by Western blot analysis. Results: BLS (PostT) significantly increased ERK p44, p-Akt, Nrf-2, and Bcl-2 levels; significantly decreased p-p38-MAPK as well as p-JNK p46 phosphorylation; did not affect Bax levels and significantly decreased Bax/Bcl-2 ratios. This was associated with significantly reduced Caspase-3 levels and increased PGC-1α phosphorylation, particlarly when BLS WIE was administered as PostT. Conclusion: The administration of polyphenol-rich BLS WIE at different stages of ischaemia and/or reperfusion, activate/inhibit several signaling events simultaneously and mediate cardioprotection in a multitarget manner.


Introduction
In the South African context, the self-care system of any chronic disease, including diabetes, can be physically, emotionally and especially financially very difficult.Even though these illnesses can be treated and managed clinically, first-line treatments are often associated with numerous side effects.Consequently, the application of alternative herbal remedies, has been reported to be effective and simple for the treatment of many chronic ailments (Arendse, 2013;Department of Biodiversity, 2013).
The use of traditional medicine/alternative herbal remedies was endorsed by the World Health Organization (WHO) Traditional Medicine Strategy (2005) with future possibilities of plant extracts as substitute therapy.It is common knowledge since antiquity that the application of alternative herbal remedies, has been simple effective treatments of various chronic ailments.One such alternative herbal remedy, is S. africana caerulea/Blue Sage (BLS).There is no lack in the validation of its use but there is a lack of scientific information regarding the pharmacological/physiological properties of BLS for example, to corroborate its traditional use and to extend the use of this plant into new therapeutic applications.This necessitates further research into BLS, which exhibits promising in vitro activity in various research models (Kamatoua et al., 2008).BLS has significant antioxidant capacity that has the potential to effectively ameliorate inflammatory related disorders such as diabetes mellitus, neurodegenerative diseases, cancer and heart disease, all associated with reduced cellular antioxidant defense mechanisms and ensuing free radical cellular damage (Hess and Manson, 1984).
Low levels of reactive oxygen species (ROS) are constantly being generated in cardiac myocytes and serve as vital intracellular regulators, whereas excessive ROS is neutralized by endogenous antioxidant mechanisms.However, I/R can induce alterations in the redox balance and oxidative stress becomes an early crucial event in the pathogenesis of several cardiac disorders, I/RI and subsequent heart failure.Notably, it is not always possible to completely counteract the damaging effects of oxidative stress, mainly due to the many sources of ROS.However, it may be possible to regulate ROS signaling pathways in such a manner to allow essential cell function and simultaneously prevent oxidative stress induced I/R cardiac damage.This may be achieved by means of a multitarget cardioprotective approach using BLS WIE, which contains several polyphenolic compounds applied at different stages of I/R.In this manner several signaling cascades can be simultaneously targeted to ensure an improved cardioprotective effect.Consequently, this study investigated the antioxidant capacity of BLS in ischaemia and reperfusion induced oxidative stress in the ex vivo working Wistar rat heart model.

Animal ethics and regulations
Animal ethics was obtained from the Ethics Committee for Research on Animals (ECRA): ref [13][14][15][16][17][18]South African Medical Research Council (SAMRC).Experimental animals were used in accordance to ethical guidelines as set out by the ECRA, SAMRC Guideline Book 3: 1990-Use of Animals in Research and the South African National Standard for Care and Use of Animals for Scientific Purpose (SANS 10386: 2008).The rats had free access to food and water before experimentation.Rats were anaesthetised with sodium pentobarbital (120 mg/kg) by intraperitoneal injection before surgical removal of the hearts.
Preparation of BLS WIE: 1 L of boiling water was added onto 200 g of BLS plant material and extracted for 3 days at room temperature.The plant material was separated from the BLS WIE, which was subjected to Liquid chromatography-mass spectrometry (LC-MS) analysis (Figures 2A-C).
Experimental design: BLS WIE dose response (n = 6 per dosage) Three different concentrations of BLS WIE (5 μL, 100 µL or 500 µL in 100 mL (v/v) Krebs-Henseleit buffer) were tested by administration for 10 min at the onset of reperfusion, as a post-treatment (PostT) after RI to determine the most appropriate cardioprotective dosage using Infarct size (IS) and Functional recovery (FR) as end-points (Figure 1A).The concentrations of active ingredients (Figures 2A-C) in 5 μL, 100 µL or 500 µL BLS WIE in 100 mL (v/v) Krebs-Henseleit buffer were calculated, for example Rosmarinic acid equated to 28.37, 567.40, and 2837.00 ng/mL, respectively.
Experimental groups for RI Untreated (UT) group (n = 6) In the untreated group (UT), isolated rat hearts were stabilized for 45 min, followed by 35 min RI and 60 min reperfusion.

Per-treatment group (PerT) (n = 6)
Per-treated (PerT) hearts were exposed to a 45 min stabilization period, 35 min RI, subjected to a BLS WIE treatment during the last 10 min of RI and followed by 60 min reperfusion.

Post-treatment group (PostT) (n = 6)
Post-treated (PostT) hearts were exposed to a 45 min stabilization period, 35 min RI and BLS WIE treatment for 10 min at the onset of the 60 min reperfusion after RI.
Per-and post-treatment group (PerT + PostT) (n = 6) Per-and post-treatment (PerT + PostT) hearts were exposed to a 45 min stabilization period (Arendse, 2013), 35 min RI and subjected to a BLS WIE treatment during the last 10 min of RI as well as for 10 min at the onset of the 60 min reperfusion after RI.

Perfusion technique and IS determination
Hearts were perfused as previously described by Lochner et al., 1999.The percentage functional recovery (% FR) of hearts was determined by expressing the post-ischaemic cardiac output (CO), calculated as Qc + Qa rates in mL/min, as a percentage of the pre-ischaemic CO.
At the completion of regional ischaemia and reperfusion, the silk suture around the LAD was permanently tied and a 0.25% Evan's blue solution infused into the heart to outline viable tissues.Hearts were removed, frozen, cut into 2 mm thick transverse tissue segments and incubated in 1% triphenyl tetrazolium chloride (TTC) in phosphate buffer, pH 7.4 for 10 min.Damaged tissues take on a deep red coloration.Infarcted tissue areas are not stained and have a white color.The reaction with TTC was stopped by placing the tissue segments in 10% formalin.Tissue segments were placed between two glass plates and ImageJ software was used to outline and analyze the left ventricle area at risk (AR) (red colored area), area of infarct tissue (I) (unstained white area) and area of viable tissue (blue outlined area).The infarct size (IS) was determined and expressed as a percentage of the area at risk (% I/AR) (Figure 3).

Experimental groups for western blot analysis
After each BLS WIE treatment regiments (PerT, PostT, and PerT + PostT) and RI, the left ventricles were freeze-clamped at 10 min of reperfusion (Figure 1B).Freeze-clamped left ventricular tissue was used for subsequent detection and measurement of total and phosphorylated ERK p44/p42, p-Akt, p38-MAPK, JNK, Nrf-2, NF-kB, Bax, Bcl-2, Caspase-3, and PGC-1α by Western blot analysis (Gel electrophoresis).Samples were subjected to electrophoresis on a 12% or 7.5% polyacrylamide gel (SDS-PAGE), depending on the size of the protein of interest, using the standard BIO-RAD Mini Protean III system.The separated proteins were transferred to an Immobilon membrane (Millipore) (Billerica, MA, United States: Polyvinylidenedifluoride (PVDF) membrane), using the Trans-Blot ® Turbo ™ Transfer system.Non-specific binding sites on the membrane were blocked with 5% fat free milk (5 g) in TBS-Tween (Tris-buffered saline +0.1% Tween 20) for 1-2 h at room temperature and incubated overnight at 4 °C with the primary antibodies (Cell Signaling Technology, Massachusetts, United States) that recognize total or phosphorylated proteins: ERK p44/p42, Akt, p38-MAPK, JNK, Nrf-2, NF-κB, Bax, Bcl-2, Caspase-3, and PGC-1α.The membranes were washed with TBS-T (3 × 5 min) and then incubated at room temperature with a diluted horseradish peroxidase-labelled secondary antibody (Cell Signaling Technology).After thorough washing with TBS-T, membranes were covered with Enhanced chemiluminescence ECL detective reagent for 1 minute and briefly exposed with the Chemidoc MP Imager System with Image lab 5. Stain-Free membranes and the Chemidoc MP Imager System with Image lab 5 were used to validate protein transfer and equal loading of samples.Note that, densitometry measurements were normalized to those of beta tubulin and the fold change is presented as a comparison to the Untreated group.

Negative control group (-ve C) (n = 6)
In the negative control group (-ve C), isolated rat hearts were stabilized for 45 min and left ventricles freeze-clamped.

Untreated group (UT) (n = 6)
In the untreated group (UT), isolated rat hearts were stabilized for 45 min, followed by 35 min RI, after which the left ventricles were freeze-clamped at 10 min of reperfusion.Post-treatment group (PostT) (n = 6) BLS WIE was administered for 10 min at the onset of reperfusion after RI and freeze-clamped at the end of this period.
Per-and post-treatment group (PerT + PostT) (n = 6) BLS WIE was administered during the last 10 min of RI as well as for 10 min at the start of reperfusion after RI and freeze-clamped at the end of this period.

Statistical analysis
Results were expressed as mean ± standard error of the mean (SEM).For multiple comparison one-way analysis of variance (ANOVA) was utilised (Graph Pad Prism Plus Version 8.0.1 software).Post-hoc testing for differences between selected groups was done using Bonferroni's method.A p-value of <0.05 was considered significant.2A-C).

The area at risk zone
The AAR represents the entire myocardial perfusion bed distal to an occluded coronary artery and is a major determinant of final IS and prognosis (Califf et al., 1985).However, the variability of both the branching pattern and myocardial vascular regions supplied by the LAD makes consistent reproducibility in the size of the AAR difficult, which necessitates consistent positioning of the ligature around the LAD (Kanno et al., 2002).Subsequently, a similar AAR among experimental groups represents a consistent positioning of the ligature around the LAD and in this study the area at risk zone (49.85% ± 1.29%), expressed as a percentage of the left ventricular area was similar in untreated and all BLS WIE treated groups, implying that results are comparable.

p-JNK
BLS WIE as a PerT, PostT and PerT + PostT had no effect on the phosphorylation of p-JNK p54.Similarly, p-JNK p46 activation was not affected by BLS PerT or PostT.However, BLS PerT + PostT Representative images of heart slices for untreated and BLS WIE treated hearts.The infarcted area, calculated as % IS outlined as unstained area, area at risk (red color) and the viable area (dark blue).The BLS WIE treated heart shows significantly reduced % IS when compared to untreated heart, illustrating the cardioprotective effects of the BLS WIE.

Bax/Bcl-2 ratio
Comparing the relative Bax to the Bcl-2 expression level (Bax/ Bcl-2 ratio), it was found that the Bax/Bcl-2 ratio was significantly decreased with BLS (PerT), whereas the Bax/Bcl-2 ratio of BLS PostT as well as BLS (PerT + PostT) showed marginal but not significant decreases (Figure 11).

NF-kB
BLS PerT, BLS PostT or BLS PerT + PostT had no effect on NF-kB phosphorylation when compared to the untreated group (Figure 12).

Discussion
It is well recognized that diminished antioxidant mechanisms and ROS-induced oxidative stress has been linked with cardiovascular injury (Kilgore et al., 1999).However, low levels of ROS are crucial in physiological processes and feature prominently as key cardioprotective elements (Matsushima et al., 2014).Importantly, I/R contributes to ROS production and subsequent myocardial I/RI (Matsushima et al., 2013), which affects a plethora of cell signaling cascades, some of which may be cardioprotective.Notably, cardiovascular risk factors and comorbidities such as sex, age, hypertension, and metabolic diseases such as hyperlipidemia and diabetes (Ferdinandy et al., 2014), will ultimately impact the outcome of any treatment schedule.Consequently, it may be suitable to consider a multitarget cardioprotective therapy, such as BLS WIE which contain several polyphenolic compounds (Figures 2A-C).In this setting, BLS WIE may target multiple cardioprotective cascades, especially when applied at 3 different time periods of acute myocardial I/R; during ischemia as PerT, at reperfusion as PostT or late ischemia into early reperfusion as a PerT + PostT.Subsequently, the prospect of applying more than one cardioprotective therapy using one therapeutic agent at 3 phases of acute myocardial I/R may result in the activation and cohesion of multiple cardioprotective pathways.Frontiers in Pharmacology frontiersin.org08 Salie et al. 10.3389/fphar.2023.1254561Ischaemia/reperfusion and the impact of BLS WIE on infarct size and functional recovery The application of 100 µL BLS as BLS PerT; BLS PostT or as a BLS PerT + PostT, resulted in a significant infarct-sparring effect and improved functional recovery after I/R (Figures 4A, B).
In this model of I/R induced oxidative stress, only BLS PostT significantly increased p-ERK p44 activation.Even though BLS PerT and BLS PerT + PostT caused p-ERK p44 activation, this found not to be significant.A similar pattern was found with p-ERK p42 (Figure 5).However, this form of p-ERK p44/p42 activation was associated with significantly reduced IS (Figure 4A) at these specific time periods when BLS was applied as a PerT, PostT as well as BLS PerT + PostT.BLS PerT and BLS PostT had no effect onp-p38-MAPK or p-JNK p54 phosphorylation.However, p-p38-MAPK as well as p-JNK p46 phosphorylation was significantly decreased when the extract was applied as PerT at the end of ischaemia and continued as a PostT treatment at the onset of reperfusion (Figures 7, 8).These findings, highlight the effectiveness of BLS WIE when applied particularly at the end of ischaemia and continued as a PostT treatment at the onset of reperfusion, reducing I/R induced oxidative stress and curtailing cardiac damaging.

Nrf-2 and p-Akt
Nrf-2 controls transcriptional regulation of antioxidant enzymes and cytoprotective proteins, such as catalase (CAT), superoxide dismutase (SOD), heme oxygenase-1 (HO-1), and glutathione peroxidase (GPx) (Ruiz et al., 2013).Nrf-2 activity is enhanced by kinases, such as p-ERK, p-p38 (MAPK), PI3K and PKC, through the phosphorylation and inhibition of GSK-3β (Kaidanovich-Beilin and Woodgett, 2011).Notably, the application of BLS WIE during these critical time periods of I/R was shown to have beneficial effects, since BLS PerT, PostT as well as BLS PerT + PostT caused significant activation of Akt (Figure 6), which was associated with significantly increased Nrf-2 levels particularly at the onset of reperfusion with BLS PostT (Figure 13) and enhanced cardioprotective effects (Figure 3A), possibly due to a reduction of oxidative stress.

NF-kB
Recent evidence showed that NF-kB represses Nrf-2 signaling at transcription level (Li et al., 2008).In addition, Rosmarinic acid, a known inducer of Nrf-2, was also found to be cardioprotective against myocardial I/RI through suppression of the NF-kB inflammatory signaling pathway and ROS production in mice (Quana et al., 2021).Caffeic acid can reduce cardiac remodeling through downregulation of the MEK/ERK signaling pathway in vivo and in vitro (Ren et al., 2017).Caffeic acid also suppresses the NF-kB pathway by reduction of iNOS, COX-2 enzymes and the release of pro-inflammatory cytokines such as TNF-α, leading to the decrease of ROS production (Natarajan et al., 1996).Like Carnosic acid, Caffeic acid induces phosphorylation of Akt and AMP-activated protein kinase (AMPK), resulting in an increased   GLUT4 expression, reduced pro-apoptotic factors and improving cell survival (Lee et al., 2015a).In the current study it was found that BLS PerT, BLS PostT or BLS PerT + PostT had no effect on NF-kB phosphorylation (Figure 12).This finding together with elevated Akt and Nrf-2 levels (Figure 13) as well as significantly reduced IS (Figure 4A), validating the inference of augmented antioxidant enzymes, the suppression of the NF-kB inflammatory signaling pathways and oxidative stress.
Bax, Bcl-2, and Caspase-3 activation The Bcl-2 family of proteins, Bax and Bid are pro-apoptotic proteins, located mainly in the cytoplasm induces opening of the mitochondrial permeability transition pore (mPTP), whereas the anti-apoptotic proteins Bcl-2 and Bcl-xl, primarily located in the nuclear, mitochondrial and endoplasmic reticulum membranes.Opening of the mPTP subsequently, causes cytochrome-c (Cyt-c) release from the mitochondrial intermembrane into the cytoplasm (Yoo et al., 2019), where it binds to adenosine triphosphate, apoptotic protease-activating factor 1 (Apaf-1) and pro-caspase 9, which subsequently activates caspase-3 and results in DNA fragmentation (Pollack and Leeuwenburgh, 2001).A crucial role of RA in apoptosis was shown when RA pretreatment inhibited caspase-1 downstream signaling cascade, namely activation of caspase-3 and 9, release of Cyt-c and translocation of apoptosisinducing factor (Jeong et al., 2011).It was also reported that carnosic acid enhanced the nuclear translocation of Nrf-2, upregulated the phase II/antioxidant enzyme activities as well as Bcl-2 levels and reduced myocardial expression of cleaved caspase-3, caspase-9, p53 and Bax (Sahu et al., 2014).
In this study, BLS WIE treatment, particularly during BLS PostT as well as BLS PerT + PostT, significantly minimized cellular damage of myocardial I/RI (Figure 4A).This cardioprotective response was shown to be triggered by activation of Akt (Figure 6) and increased Nrf-2 (Figure 13) as well as Bcl-2 levels (Figure 10).The activation of these cytoprotective proteins, unchanged Bax levels (Figure 9) and significantly reduced Caspase-3 levels (Figure 14), mediate increased antioxidant enzymes production, minimises oxidative stress and enhanced the cardioprotective effects of BLS WIE.

Bax/Bcl-2 ratio
Bcl-2 and Bcl-XL dimerize with pro-apoptotic proteins, such as Bax, to suppress apoptosis (Basu and Haldar, 1998).Since many cell types trigger Bcl-2 and Bax expression to suppress or enable apoptosis, the ratio of Bax/Bcl-2 has been considered as the independent regulator to determine apoptotic threshold (Harnois et al., 1997).Accordingly, cells with a high Bax/Bcl-2 ratio will be more sensitive to a given apoptotic stimulus when compared to a similar cell type with a comparatively low Bax/Bcl-2 ratio.It follows then, that the Bax/Bcl-2 ratio can be regarded as a valuable means to gauge of oxidative stress.
Salvigenin, a known polyphenol flavonoid, also shown to be present in BLS WIE, inhibits hydrogen peroxide-induced apoptosis, reduces the generation of ROS and reduces caspase-3 levels as well as Bax/Bcl-2 ratio in SH-Sy5Y neuroblastoma cells (Rafatian et al.,

FIGURE 16
Schematic representation of the administration of polyphenol-rich BLS WIE at different stages of ischaemia and/or reperfusion; activate/inhibit several signaling events simultaneously and mediate cardioprotection in a multitarget manner.
Frontiers in Pharmacology frontiersin.org13 Salie et al. 10.3389/fphar.2023.1254561 FIGURE 1 (A) Schematic representation of BLS WIE administration to the PerT, PostT, and PerT + PostT groups, followed by infarct size and functional recovery measurements as endpoints.(B) Schematic representation of BLS WIE PerT, PostT, and PerT + PostT, showing time points of freeze clamping for Western blot analysis.
FIGURE 2 (A) Schematic representation of LC-MS Spectrum, (B) Table showing the LC-MC retention times, fragments ions and compound identification, (C) Table showing the relative concentrations Polyphenolic compounds in BLS WIE in mg/L.

FIGURE 3
FIGURE 3Representative images of heart slices for untreated and BLS WIE treated hearts.The infarcted area, calculated as % IS outlined as unstained area, area at risk (red color) and the viable area (dark blue).The BLS WIE treated heart shows significantly reduced % IS when compared to untreated heart, illustrating the cardioprotective effects of the BLS WIE.