Protective effects of Ruscus extract in combination with ascorbic acid and hesperidine methylchalcone on increased leukocyte-endothelial interaction and macromolecular permeability induced by ischemia reperfusion injury

Abstract

BACKGROUND:

Despite the well-recognized effectiveness of Ruscus aculetus extract combined or not with ascorbic acid (AA) and hesperidine methyl chalcone (HMC) on ischemia reperfusion (I/R) injury protection, little is known about the contribution of each constituent for this effect.

OBJECTIVE:

To investigate the effects of AA and HMC combined or not with Ruscus extract on increased macromolecular permeability and leukocyte-endothelium interaction induced by I/R injury.

METHODS:

Hamsters were treated daily during two weeks with filtered water (placebo), AA (33, 100 and 300 mg/kg/day) and HMC (50, 150 and 450 mg/kg/day) combined or not with Ruscus extract (50, 150 and 450 mg/kg/day). On the day of experiment, the cheek pouch microcirculation underwent 30 min of ischemia, and the number of rolling and adherent leukocytes and leaky sites were evaluated before ischemia and during 45 min of reperfusion.

RESULTS:

Ruscus extract combined with AA and HMC (Ruscus extract mixture) significantly prevented post-ischemic increase in leukocyte rolling and adhesion and macromolecular permeability compared to placebo and these effects were more prominent than AA and HMC alone on leukocyte adhesion and macromolecular leakage.

CONCLUSION:

Ruscus extract mixture were more effective than its isolated constituents in protect the hamster cheek pouch microcirculation against I/R injury.

Keywords

ischemia/reperfusion injury hamster cheek pouch preparation microcirculation

1 Introduction

Ischemia-reperfusion (I/R) is a condition characterized by an interruption of blood supply to an organ followed by blood flow restoration and tissue reoxygenation [1]. Paradoxically, blood flow restoration and reoxygenation are accompanied by an aggravation of tissue damage due to an enhancement of inflammatory response and reactive oxygen species production (ROS), a phenomenon called reperfusion injury [1 –4].

In clinical setting, an embolic event is responsible to cause arterial blood supply restriction resulting in a mismatch between metabolic supply and demand and consequent tissue hypoxia [1 , 5]. I/R injury occurs in several pathologic conditions including acute myocardial infarction, ischemic stroke and multiple organ failure, the most prevalent cause of death in critically ill patients [1 , 7].

In experimental conditions, the exacerbation of pro-inflammatory response and oxidative stress induced by I/R injury are tightly associated to microvascular dysfunction reflected by impaired endothelial dependent relaxation in arterioles, increased leukocyte-endothelium interactions, elevated macromolecular permeability in post-capillary venules, and capillary hypoperfusion [3 , 9]. One of the most appropriate experimental models devised to study the effects of I/R injury in vivo, is the hamster cheek preparation. This model allows the intravital observation of acute changes in microcirculation, and for this reason is very suitable to study post-ischemic events in microvascular bed [10].

During reperfusion, ROS generated by endothelium elicit P-selectin translocation to endothelial cell membrane, release of leukotriene B₄ (LTB₄) and platelet activating factor (PAF) and activation pro-inflammatory nuclear factors, including nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) [11 –16]. Additionally, LTB₄ and PAF induce expression of β₂-integrins on surface of rolling leukocytes, promoting their firm adhesion to endothelial cells [15, 16].

According to literature, the treatment with antioxidants confers protection against I/R injury by means of neutralization of ROS and downregulation of pro-inflammatory pathways [17]. More recently, a study has shown that acetylcholine (ACh), an agonist of muscarinic receptors, protects cardiomyocytes from I/R injury through inhibition of ROS generation, improvement of mitochondrial biogenesis and function, and reduction of pro-inflammatory factors expression [18].

Ruscus aculeatus (butcher’s broom) extract is a venoactive drug, commonly used in chronic venous disease (CVD) treatment [19]. It is well documented in the literature that Ruscus extract has venotonic [20], anti-inflammatory and antioxidant properties [21]; however, its mechanism of action still need to be fully elucidated [22]. In addition, was demonstrated that Ruscus extract can bind and activate muscarinic receptors both in vitro and in vivo [22]. In this same study, was demonstrated that the protective effects of Ruscus extract on increased leukocyte-endothelial interaction and macromolecular permeability induced by either histamine or I/R injury were mediated by muscarinic receptors activation on microvascular endothelial cells [22]. It was demonstrated that Ruscus extract combined with hesperidine methyl chalcone (HMC) reduced LTB₄, histamine and bradykinin -induced increased in microvascular permeability [23]. More recently, it was shown that this beneficial effect of Ruscus on endothelial barrier function was due to activation of muscarinic receptors on endothelium surface [22].

HMC is a product of methylation of the flavanone hesperidin, a flavonoid found especially in citrus fruits [24]. HMC possesses both anti-inflammatory actions, through inhibition of NF-κB activation [25, 26], and antioxidant properties, by means of activation of nuclear factor erythroid 2-related factor 2 (Nrf2) an important regulator of cells oxidant defense [24].

Ascorbic acid (AA) is an important antioxidant present in plasma and cells and is the first antioxidant to be consumed during oxidative stress [27]. It protects both cytosolic and membrane components of cells from oxidative damage. Ascorbic acid can directly neutralize superoxide anions in aqueous milieu prevent them from attack cell lipids [28]. Moreover, AA can reduce endothelial cell permeability, preserve functional properties of microcirculation maintaining endothelial integrity [29]. It was extensively demonstrated in the literature that ascorbic acid and HMC enhance the beneficial actions of Ruscus extract, improving its anti-inflammatory properties, venous return, lymphatic drainage, and capillary resistance in patients with CVD [30, 31].

Despite the well-recognized effectiveness of Ruscus extract combined or not with AA and HMC on microvascular protection against I/R injury, little is known about the isolated contribution of AA, HMC and Ruscus extract in preventing I/R injury-induced microvascular dysfunction. Thus, this study aims to investigate their effects on increased macromolecular permeability and leukocyte-endothelium interaction induced by I/R injury using the cheek pouch preparation.

2 Materials and methods

2.1 Ruscus extraction

The dry extract of Ruscus titrated in sterolic heterosides was obtained from Ruscus aculeatus L. rhizomes and roots by hydroalcoholic extraction (Pierre Fabre Laboratories, Toulouse, France).

2.2 Animal housing

Animals, 120 male Syrian hamsters (Mesocricetus auratus, Anilab, Paulinia, SP, Brazil), weighing 121–178 g (7 to 10 weeks old) were caged in a light, temperature (20–24°C) and humidity-controlled environment and fed with commercially available chow for small rodents (Nuvilab, Nuvital, Curitiba, Paraná, Brazil) and received tap water ad libitum.

2.3 Animal treatments

Animals were allocated into three major groups according to administered treatment: AA (Pierre Fabre Laboratories, Toulouse, France), HMC (Pierre Fabre Laboratories, Toulouse, France), combined or not with Ruscus. Then each group was divided into three different subgroups according to administered dose of these active compounds as follows: AA at 33, 100 and 300 mg/kg/day; HMC at 50, 150, 450 mg/kg/day and three different Ruscus mixtures. The Ruscus mixtures combined ascorbic acid at 33 mg/kg/day, HMC at 50 mg/kg/day and Ruscus extract at 50 mg/kg/day; AA at 100 mg/kg/day, HMC at 150 mg/kg/day and Ruscus extract at 150 mg/kg/day; ascorbic acid at 300 mg/kg/day, HMC at 450 mg/kg/day and Ruscus extract at 450 mg/kg/day, i.e. at same ratio as they appear in Cyclo 3 Fort (Pierre Fabre Laboratories, Toulouse, France). Another group was separated and received filtered water (placebo group). All solutions were freshly prepared, and animals were treated by oral route, twice a day (at 8 : 00 AM and 5 : 00 PM), for two weeks. The last dose of AA, HMC, Ruscus mixture or filtered water was given two hours before the induction of anesthesia.

2.4 The I/R model in the hamster cheek pouch preparation

On the day of experiment, anesthesia was induced by intraperitoneal injection of 0.1–0.2 ml of xylazine (10 mg/kg, Anasedan, Ceva Saúde Animal Ltda, Paulínia, SP, Brazil) plus ketamine (200 mg/kg, Dopalen, Ceva Saúde Animal Ltda, Paulínia, SP, Brazil) and maintained by α-chloralose (Sigma Chemicals, St. Louis, MO, USA, 100 mg/kg) administered via femoral vein. The temperature was maintained at 37.5°C throughout the surgery and experimental procedures using a heating pad controlled by a rectal thermistor. A tracheal tube was inserted to facilitate spontaneous breathing and the femoral artery was cannulated for mean arterial pressure measurements (PowerLab, AD Instruments, New South Wales, Australia).

The hamster was placed on a microscope stage similar to that described by Duling [33] with minor modifications [34]. The cheek pouch was gently everted and pinned with four to five needles into a circular well filled with silicone rubber to provide a plane bottom layer, thus avoiding stretching of the tissue but preventing shrinkage. In this position, the pouch was submerged in a superfusion solution that continuously flushed the pool of the microscope stage. Before the pouch was pinned, arteries and veins (with approximately 1 mm of diameter) were located with the aid of a Zeiss binocular stereomicroscope (Carl Zeiss Microscopy GmbH, Goettingen, Germany).

Fashioning of a single-layer preparation started with an incision of the upper layer to swing a triangular flap to one side. The exposed area was dissected at X10-16 under the stereomicroscope, and the fibrous, almost avascular, connective tissue covering the vessels was removed with ophthalmic surgical instruments. The dissected part of the pouch was 125–150μm thick. Pouches with petechial formations or those without blood flow in all vessels were discarded (three preparations were discarded due to these problems during the investigation).

The superfusion solution was a HEPES-supported HCO₃-buffered saline solution (composition in mM: NaCl 110.0, KCl 4.7, CaCl₂ 2.0, MgSO₄ 1.2, NaHCO₃ 18.0, HEPES 15.39 and HEPES Na⁺ salt 14.61); temperature of the solution was maintained at 36.5°C, and superfusion rate varied from 4 ml/min (leukocyte-endothelium interactions) to 6 ml/min (macromolecular permeability evaluation). pH was set to 7.40 by bubbling the solutions continuously with 5% CO₂ in 95% N₂.

After 30 minutes of stabilization (resting period), if the preparation presented a brisk blood flow in all parts of the vascular bed including larger veins (where erythrocytes should not be discernible in the image of the blood stream), no spontaneous plasma leakage and few rolling and sticking leukocytes, a local ischemia of 30 minutes was performed. Ischemia in cheek pouch preparations was induced by means of a cuff, made of thin latex tubing, mounted around the proximal part of the everted pouch [35]. The cuff was placed without any visible interference of local blood flow. The intracuff pressure could be quickly increased by air compression using a syringe and rapidly decreased when required. An intracuff pressure of 200 to 220 mmHg resulted in a complete interruption of microvascular blood flow within a few seconds (Fig. 1).

Fig. 1

Photograph of hamster cheek pouch preparation placed on intravital microscope stage: a) Before 30 min of ischemia and; b) During 30 min of ischemia with a cuff positioned around the proximal part of the everted pouch.

Before and after ischemia (reperfusion), changes in number of rolling and adherent leukocytes and number of leaky sites in post-capillary venules were assessed and at the end of all experiments hamsters were euthanized, under anesthesia, by an intravenous injection of potassium chloride (KCl 3M).

2.5 Observation of rolling and adherent leukocytes by intravital microscopy

Circulating leukocytes were labeled by rhodamine 6 G administered by an intravenous injection of 0.4 mL (0.1 mg/mL) immediately prior to observations and followed by a continuous infusion (10μL/min) of the fluorescent dye thereafter (Syringe Pump, model 55-2222, Harvard Apparatus, Hollister, MA, USA). Fluorescent leukocytes were observed in UV-light microscope (Leica DM LS, Leica, Wetzlar, Germany) with a set of filters (Excitation BP 546-12/Emission LP 590, Leica, Wetzlar, Germany) coupled to a closed-circuit TV system (445 X magnification). In each preparation, two venules (with diameters and lengths ranging from 10 to 15μm and from 100 to 400μm, respectively) were selected considering the possibility to return exactly to the same site, (proximity of fat cells and bifurcations), due to the consecutive measurements. Experiments were performed by taking 1 minute videotape recordings of selected microvessel fields in initial control conditions (before ischemia) and subsequently at onset, 15, 30, 45 minutes of reperfusion.

Rolling and sticking leukocytes in post-capillary venules were counted offline, using videotape recordings and frame-by-frame analysis. A leukocyte was considered as rolling when it was in contact with the venular wall and had lower velocity than circulating erythrocytes and as adherent when it was immobilized at one position during at least 30 seconds [36].

2.6 Macromolecular permeability assessment by intravital microscopy

Microvascular permeability for large molecules was quantified as the number of leaky sites (= leaks) per total observed area of the pouch (1 cm²). Briefly, fluorescein isothiocyanate-dextran (FITC-Dextran, MW 150,000, 50 mg/mL) was given intravenously to hamsters at 25 mg/100 mg body weight, just after the resting period. Leaks of labeled dextran were defined as visible extravascular spots (diameter > 40μm) in post-capillary venules (internal diameter ranging from 9 to 16μm) seen under fluorescent light using an UV-light intravital microscope (optical magnification 40X).

The number of leaks in post-capillary venules was manually scanned and counted before ischemia and during reperfusion. Maximal response to I/R occurs at 10 min after the onset of reperfusion and for this reason, this is the value reported for each experiment.

2.7 Statistical analyses

The final sample size was five cases per group and was estimated considering the highest standard deviation (SD) observed in previous I/R induced macromolecular permeability experiments (9.47 leaks/cm²) and the effect size of 10. The calculation of sample size was performed using R software version 3.6.3 (R Project for Statistical Computing).

Results were expressed as mean±SD. The Gaussian distribution of the results was verified by Shapiro-Wilk normality test. Comparisons between groups were performed by Kruskal-Wallis test followed by Dunn’s post-hoc test. All these analyses were performed using Graph Pad Prism 5.0 software (Graph Pad Software Inc., San Diego, CA, USA). A P value of less than 0.05 was considered as significant.

3 Results

3.1 Effects of AA, HMC and their combination with Ruscus extract on leukocyte rolling during I/R

The protective effects of AA at 33 mg/kg/day, HMC at 50 mg/kg/day, Ruscus extract mixture (ascorbic acid 33 mg/kg/day, HMC 50 mg/kg/day, Ruscus extract 50 mg/kg/day) treatments on the number of rolling leukocytes during I/R are represented on Fig. 2.

Fig. 2

Effects of ascorbic acid, HMC and their combination with Ruscus extract on leukocyte rolling during I/R on cheek pouch preparation. Effects of L-ascorbic acid at 33 mg/kg/day, hesperidine methyl chalcone (HMC) at 50 mg/kg/day, Ruscus extract mixture (L-ascorbic acid 33 mg/kg/day, HMC 50 mg/kg/day, Ruscus extract 50 mg/kg/day) treatments on the number of rolling leukocytes: a) before 30 minutes of ischemia; b) At the onset of reperfusion; c) At 15 min of reperfusion; d) At 30 min of reperfusion and; e) At 45 min of reperfusion. Data were expressed as mean±SD. *Significantly different at P level of 0.05. **Significantly at P level of 0.01. ***Significantly different at P level of 0.001.

Before ischemia, and at the onset of reperfusion, no significant differences between treatments were observed (Fig. 2a and 2b). At 15 min of reperfusion, AA 33 mg/kg/day (P < 0.05) and HMC 50 mg/kg/day (P < 0.01), treatments significantly attenuated the increase in leukocyte rolling in comparison to placebo (Fig. 2c). At 30 min of reperfusion, AA 33 mg/kg/day (P < 0.001), HMC 50 mg/kg/day (P < 0.05) and Ruscus extract mixture 50 mg/kg/day (P < 0.001) treatments significantly inhibited the rise in leukocyte rolling when compared to placebo (Fig. 2d). At 45 min of reperfusion, HMC 50 mg/kg/day (P < 0.05) and Ruscus extract mixture 50 mg/kg/day (P < 0.001) treatments significantly inhibited the elevation in the number of rolling leukocytes in comparison to placebo. In addition, Ruscus extract mixture 50 mg/kg/day presented lower number of rolling leukocytes in comparison to AA 33 mg/kg/day treatment (P < 0.05). No other significant differences among groups concerning leukocyte rolling were noticed at all these time points (Fig. 2e).

The beneficial effects of AA at 100 mg/kg/day, HMC at 150 mg/kg/day, Ruscus extract mixture (ascorbic acid 100 mg/kg/day, HMC 150 mg/kg/day, Ruscus extract 150 mg/kg/day) treatments on leukocyte rolling during I/R are depicted on Fig. 3.

Fig. 3

Effects of ascorbic acid, HMC and their combination with Ruscus extract on leukocyte rolling during I/R on cheek pouch preparation. Effects of L-ascorbic acid at 100 mg/kg/day, hesperidine methyl chalcone (HMC) at 150 mg/kg/day, Ruscus extract mixture (L-ascorbic acid 100 mg/kg/day, HMC 150 mg/kg/day, Ruscus extract 150 mg/kg/day) treatments on the number of rolling leukocytes: a) before 30 minutes of ischemia; b) At the onset of reperfusion; c) At 15 min of reperfusion; d) At 30 min of reperfusion and; e) At 45 min of reperfusion. Data were expressed as mean±SD. *Significantly different at P level of 0.05. **Significantly at P level of 0.01. ***Significantly different at P level of 0.001.

Before ischemia, no significant differences between treatments were observed (Fig. 3a). At the onset of reperfusion, AA 100 mg/kg/day (P < 0.05) and HMC 150 mg/kg/day (P < 0.01), treatments significantly prevented the elevation in the number of rolling leukocytes when compared to placebo (Fig. 3b). At 15 min of reperfusion, HMC 150 mg/kg/day and Ruscus extract mixture 150 mg/kg/day treatments significantly inhibited the rise of the number of rolling leukocytes in comparison to placebo (P < 0.001). Moreover, HMC 150 mg/kg/day and Ruscus extract mixture 150 mg/kg/day groups exhibited lower number of rolling leukocytes in comparison to AA 100 mg/kg/day group (P < 0.001) (Fig. 3c). At 30 and 45 min of reperfusion, AA 100 mg/kg/day (P < 0.01), HMC 150 mg/kg/day (P < 0.001) and Ruscus extract mixture 150 mg/kg/day (P < 0.001) treatments significantly inhibited the rise in the number of rolling leukocytes when compared to placebo. We did not notice any other significant differences among groups regarding leukocyte rolling at all these time points. (Fig. 3d and 3e).

The protective changes elicited by AA at 300 mg/kg/day, HMC at 450 mg/kg/day and Ruscus extract mixture (AA 300 mg/kg/day, HMC 450 mg/kg/day, Ruscus extract 450 mg/kg/day) treatments on leukocyte rolling are displayed on Fig. 4.

Fig. 4

Effects of ascorbic acid, HMC and their combination with Ruscus extract on leukocyte rolling during I/R on cheek pouch preparation. Effects of L-ascorbic acid at 300 mg/kg/day, hesperidine methyl chalcone (HMC) at 450 mg/kg/day, Ruscus extract mixture (L-ascorbic acid 300 mg/kg/day, HMC 450 mg/kg/day, Ruscus extract 450 mg/kg/day) treatments on the number of rolling leukocytes: a) before 30 minutes of ischemia; b) At the onset of reperfusion; c) At 15 min of reperfusion; d) At 30 min of reperfusion and; e) At 45 min of reperfusion. Data were expressed as mean±SD. *Significantly different at P level of 0.05. **Significantly at P level of 0.01. ***Significantly different at P level of 0.001.

Before ischemia, no significant differences between treatments were observed (Fig. 4a). At the onset of reperfusion, AA 300 mg/kg/day (P < 0.05) and HMC 450 mg/kg/day (P < 0.01), and Ruscus extract mixture 450 mg/kg/day (P < 0.001) treatments significantly prevented the elevation in the number of rolling leukocytes when compared to placebo (Fig. 4b). At 15 min and 30 min of reperfusion, AA 300 mg/kg/day, HMC 450 mg/kg/day and Ruscus extract mixture 450 mg/kg/day treatments significantly inhibited the rise in the number of rolling leukocytes in comparison to placebo (P < 0.001) (Fig. 4c and 4d). At 45 min of reperfusion, AA 300 mg/kg/day (P < 0.001), HMC 450 mg/kg/day (P < 0.01) and Ruscus extract mixture 450 mg/kg/day (P < 0.001) treatments significantly impeded the leukocytes rolling elevation when compared to placebo. No other significant differences among groups with respect to leukocyte rolling were noticed at all these time points (Fig. 4e).

3.2 Effects of AA, HMC and their combination with Ruscus extract on leukocyte adhesion during I/R

The protective effects of ascorbic acid at 33 mg/kg/day, HMC at 50 mg/kg/day, Ruscus extract mixture (ascorbic acid 33 mg/kg/day, HMC 50 mg/kg/day, Ruscus extract 50 mg/kg/day) treatments on leukocyte adhesion during I/R are represented on Fig. 5.

Fig. 5

Effects of ascorbic acid, HMC and their combination with Ruscus extract on leukocyte adhesion during I/R on cheek pouch preparation. Effects of L-ascorbic acid at 33 mg/kg/day, hesperidine methyl chalcone (HMC) at 50 mg/kg/day, Ruscus extract mixture (L-ascorbic acid 33 mg/kg/day, HMC 50 mg/kg/day, Ruscus extract 50 mg/kg/day) treatments on the number of adherent leukocytes: a) before 30 minutes of ischemia; b) At the onset of reperfusion; c) At 15 min of reperfusion; d) At 30 min of reperfusion and; e) At 45 min of reperfusion. Data were expressed as mean±SD. *Significantly different at P level of 0.05. **Significantly at P level of 0.01. ***Significantly different at P level of 0.001.

Before ischemia, animals treated by Ruscus extract 50 mg/kg/day mixture presented reduced number of adherent leukocytes in comparison to the ones treated by AA 33 mg/kg/day (P < 0.05) (Fig. 5a). At the onset of reperfusion, AA 33 mg/kg/day (P < 0.05) and Ruscus extract mixture 50 mg/kg/day (P < 0.001), treatments significantly prevented the elevation in leukocyte rolling in comparison to placebo. In addition, hamsters treated by Ruscus extract mixture 50 mg/kg/day presented lower number of adherent leukocytes in comparison to the ones treated by AA 33 mg/kg/day (P < 0.05) and HMC (P < 0.001) (Fig. 5b). At 15 min of reperfusion, AA 33 mg/kg/day (P < 0.05) and Ruscus extract mixture 50 mg/kg/day (P < 0.001), treatments significantly prevented the elevation of leukocyte sticking in comparison to placebo. Moreover, animals treated by Ruscus extract mixture 50 mg/kg/day displayed lower number of adherent leukocytes in comparison to the ones treated by AA 33 mg/kg/day (P < 0.05) and HMC 50 mg/kg/day (P < 0.001) (Fig. 5c). At 30 min of reperfusion, AA 33 mg/kg/day (P < 0.01), HMC 50 mg/kg/day (P < 0.05) and Ruscus extract mixture 50 mg/kg/day (P < 0.001) treatments significantly inhibited the rise in the number of adherent leukocytes when compared to placebo (Fig. 5d). At 45 min of reperfusion, AA 33 mg/kg/day (P < 0.01), HMC 50 mg/kg/day (P < 0.01) and Ruscus extract mixture 50 mg/kg/day (P < 0.001) treatments significantly impeded leukocyte sticking elevation in comparison to placebo. No other significant differences among groups concerning leukocyte adhesion were noticed at all these time points (Fig. 5e).

Fig. 6

Effects of ascorbic acid, HMC and their combination with Ruscus extract on leukocyte adhesion during I/R on cheek pouch preparation. Effects of L-ascorbic acid at 100 mg/kg/day, hesperidine methyl chalcone (HMC) at 150 mg/kg/day, Ruscus extract mixture (L-ascorbic acid 100 mg/kg/day, HMC 150 mg/kg/day, Ruscus extract 150 mg/kg/day) treatments on the number of sticking leukocytes: a) before 30 minutes of ischemia; b) At the onset of reperfusion; c) At 15 min of reperfusion; d) At 30 min of reperfusion and; e) At 45 min of reperfusion. Data were expressed as mean±SD. *Significantly different at P level of 0.05. **Significantly at P level of 0.01. ***Significantly different at P level of 0.001.

Before ischemia, hamsters treated by Ruscus extract mixture 150 mg/kg/day displayed significantly reduced number of adherent leukocytes in comparison to the HMC 150 mg/kg/day treated ones (P < 0.05) (Fig. 6a). At the onset of reperfusion, AA 100 mg/kg/day (P < 0.01), HMC 150 mg/kg/day (P < 0.001) and Ruscus extract mixture 150 mg/kg/day (P < 0.001) treatments significantly prevented the elevation in the number of sticking leukocytes when compared to placebo (Fig. 6b). At 15 min of reperfusion, AA 100 mg/kg/day (P < 0.05), HMC 150 mg/kg/day (P < 0.001) and Ruscus extract mixture 150 mg/kg/day (P < 0.001) treatments significantly inhibited the rise of leukocyte adhesion in comparison to placebo (Fig. 6c).

At 30 min of reperfusion, AA 100 mg/kg/day (P < 0.001), HMC 150 mg/kg/day (P < 0.01) and Ruscus extract mixture 150 mg/kg/day (P < 0.001) treatments significantly attenuated the increase of leukocyte adhesion when compared to placebo (Fig. 6d). At 45 min of reperfusion, AA 100 mg/kg/day (P < 0.05), HMC 150 mg/kg/day (P < 0.001) and Ruscus extract mixture 150 mg/kg/day (P < 0.001) treatments significantly prevented the rise in the number of adherent leukocytes when compared to placebo. Furthermore, Ruscus extract mixture 150 mg/kg/day group displayed lower number of sticking leukocytes in comparison to AA 100 mg/kg/day group (P < 0.05). We did not notice any other significant differences among groups regarding leukocyte adhesion at all these time points. (Fig. 6e).

The protective changes elicited by AA at 300 mg/kg/day, HMC at 450 mg/kg/day and Ruscus extract mixture (AA 300 mg/kg/day, HMC 450 mg/kg/day, Ruscus extract 450 mg/kg/day) treatments on leukocyte adhesion are depicted on Fig. 7.

Fig. 7

Effects of ascorbic acid, HMC and their combination with Ruscus extract on leukocyte adhesion during I/R on cheek pouch preparation. Effects of L-ascorbic acid at 300 mg/kg/day, hesperidine methyl chalcone (HMC) at 450 mg/kg/day, Ruscus extract mixture (L-ascorbic acid 300 mg/kg/day, HMC 450 mg/kg/day, Ruscus extract 450 mg/kg/day) treatments on the number of adherent leukocytes: a) before 30 minutes of ischemia; b) At the onset of reperfusion; c) At 15 min of reperfusion; d) At 30 min of reperfusion and; e) At 45 min of reperfusion. Data were expressed as mean±SD. *Significantly different at P level of 0.05. **Significantly at P level of 0.01. ***Significantly different at P level of 0.001.

Before ischemia, Ruscus extract mixture 450 mg/kg/day presented lower number of adherent leukocytes in comparison to placebo (P < 0.01) (Fig. 7a). At the onset of reperfusion, AA 300 mg/kg/day (P < 0.0001) and HMC 450 mg/kg/day (P < 0.01), and Ruscus extract mixture 450 mg/kg/day (P < 0.001) treatments significantly prevented the elevation in the number of sticking leukocytes when compared to placebo (Fig. 7b). At 15 min of reperfusion, AA 300 mg/kg/day, HMC 450 mg/kg/day and Ruscus extract mixture 450 mg/kg/day treatments significantly inhibited the rise of the number of adherent leukocytes in comparison to placebo (P < 0.001) (Fig. 7c). At 30 min of reperfusion, AA 300 mg/kg/day (P < 0.05), HMC 450 mg/kg/day (P < 0.05) and Ruscus extract mixture 450 mg/kg/day (P < 0.001) treatments significantly prevented the elevation leukocyte adhesion in comparison to placebo. In addition, the number of adherent leukocytes were significantly reduced in hamsters treated by Ruscus extract mixture at 450 mg/kg/day in comparison to the ones treated by AA at 300 mg/kg/day and at HMC 450 mg/kg/day (P < 0.05) (Fig. 7d). At 45 min of reperfusion, ascorbic acid 300 mg/kg/day, HMC 450 mg/kg/day and Ruscus extract mixture 450 mg/kg/day treatments significantly inhibited the rise in the number of sticking leukocytes when compared to placebo (P < 0.001). No other significant differences among groups with respect to leukocyte adhesion were noticed at all these time points (Fig. 7e).

3.3 Effects of AA, HMC and their combination with Ruscus extract at different doses on microvascular permeability during I/R

A comparison between the protective effects of AA, HMC, Ruscus extract mixture at different doses on microvascular permeability during I/R are displayed on Fig. 8.

Fig. 8

Effects of ascorbic acid, HMC and their combination with Ruscus extract at different doses on microvascular permeability during I/R, on cheek pouch preparation. a) Effects of L-ascorbic acid at 33 mg/kg/day, hesperidine methyl chalcone (HMC) at 50 mg/kg/day, Ruscus extract mixture (L-ascorbic acid 33 mg/kg/day, HMC 50 mg/kg/day, Ruscus extract 50 mg/kg/day) treatments on macromolecular permeability after 10 minutes of reperfusion; b) Effects of L-ascorbic acid at 100 mg/kg/day, hesperidine methyl chalcone (HMC) at 150 mg/kg/day, Ruscus extract mixture (L-ascorbic acid 100 mg/kg/day, HMC 150 mg/kg/day, Ruscus extract 150 mg/kg/day) treatments on macromolecular permeability after 10 minutes of reperfusion and; c) Effects of L-ascorbic acid at 300 mg/kg/day, hesperidine methyl chalcone (HMC) at 450 mg/kg/day, Ruscus extract mixture (L-ascorbic acid 300 mg/kg/day, HMC 450 mg/kg/day, Ruscus extract 450 mg/kg/day) treatments on macromolecular permeability after 10 minutes of reperfusion. Data were expressed as mean±SD. *Significantly different at P level of 0.05. ***Significantly different at P level of 0.001.

The treatments with AA at 33 mg/kg/day (P < 0.05) and Ruscus extract mixture at 50 mg/kg/day (P < 0.001) significantly inhibited the rise in macromolecular leakage when compared to placebo. The hamsters treated by Ruscus extract mixture at 50 mg/kg/day presented significant lower number of leaks when compared to the HMC 50 mg/kg/day treated ones (P < 0.05) (Fig. 8a). HMC 150 mg/kg/day (P < 0.05) and Ruscus extract mixture 150 mg/kg/day (P < 0.001) treatments significantly inhibited the increase in macromolecular permeability when compared to placebo. Additionally, the number of leaks in animals treated by Ruscus extract mixture at 150 mg/kg/day were significantly reduced in comparison to ones treated with ascorbic acid at 100 mg/kg/day (P < 0.05) (Fig. 8b). The treatments with HMC 450/kg/day (P < 0.05) and Ruscus extract mixture at 450 mg/kg/day (P < 0.001) significantly inhibited the increase in microvascular permeability when compared to placebo. Moreover, the number of leaks in hamsters treated by Ruscus extract mixture at 450 mg/kg/day were significantly lower in comparison to ones treated with AA at 300 mg/kg/day (P < 0.05) (Fig. 8c).

4 Discussion

The major finding of this study was treatment with Ruscus extract mixture at different concentrations (50, 150 and 450 mg/kg/day) for two weeks significantly prevented the increase in leukocyte rolling and adhesion and in macromolecular permeability elicited by I/R injury in hamster cheek pouch post capillary venules. However, the protective effects of Ruscus extract mixture were more prominent on leukocyte adhesion and macromolecular leakage.

NO is the main endogenous inhibitor of leukocyte adhesion on endothelium [37] and its depletion elicited by I/R injury [38] elicits leukocyte activation and chemotaxis and leukocyte-endothelial cell interactions [39] Leukocyte-endothelial cell interactions involves three distinct steps: rolling, firm adhesion and transmigration [40].

Leukocyte rolling is dependent on the expression of a class of adhesion molecules called selectins, which includes L-selectin, constitutively expressed on leukocyte surface, E-selectin, expressed on cytokine-activated endothelium after de novo synthesis, a process that requires 4-6 h, and P-selectin, expressed on surface of activated platelets and endothelial cells [41, 42]. P-selectin is stored in Weibel-Palade bodies of endothelial cells and in α-granules of platelets [43] and is promptly translocated to cell surface when activated by thrombin, histamine, hydrogen peroxide, nitric oxide synthase (NOS) inhibitors and injury, a process that peaks between 10 and 20 min [43] exerting a pivotal role in leukocyte rolling induced by I/R [44] being considered the main selectin involved in I/R injury [42]. L-selectin seems to be important in the early phase of I/R injury, differently from E-selectin that may have a role in leukocyte rolling approximately 4-6 h after reperfusion [42]. In the present study, we have demonstrated that ascorbic acid treatment, in all different daily doses, significantly attenuated the I/R induced increase in leukocyte rolling when compared to placebo group. A study has shown that ascorbate, the ionized form of AA, downregulate the expression of P-selectin in vitro [45]. Moreover, AA prevents endothelial NOS (eNOS) uncoupling, augmenting NO bioavailability [46] and consequently blocking the upregulation of P-selectin [47] (Figs. 1–3).

AA is considered a vitamin only for a few species of vertebrates, including humans, that evolutionary loss the capacity to produce it [48, 49]. Primates, guinea pigs, flying mammals, more evolved birds, fish, insects and other invertebrates, are also examples of species that are not able to synthesize AA [48, 49]. In contrast, AA can be synthesized by plants, amphibians, reptiles, and the remaining species of mammals. Amphibians and reptiles synthesize AA in the kidney, while the mammals that are able to synthesize AA to do so in the liver [49]. The incapacity of humans to synthesize AA is due to the functional loss of l-gulonolactone oxidase encoding gene, the enzyme responsible for the last step of AA biosynthetic pathway in animals [48].

We also observed that treatment with HMC in all studied doses significantly attenuated the post-ischemic increase in the number of rolling leukocytes in comparison to placebo treated hamsters, this effect is probably mediated by its capacity to inhibit NF-κB, downregulating the levels of pro-inflammatory cytokines [50] during reperfusion. Moreover, HMC activates Nrf2, a transcription factor that strengths cell antioxidant defense [24], inducing the expression of detoxifying and antioxidant genes [51, 52] and mitigating the deleterious effect of ROS generated during I/R injury. In addition, Nrf2 acts preventing eNOS uncoupling [52], increasing NO bioavailability and, therefore, inhibiting leukocyte rolling.

This study demonstrated that treatment with crescent daily doses of Ruscus extract combined with ascorbic acid and HMC significantly prevents leukocyte rolling elevation during I/R injury in comparison to placebo group. It has been shown that Ruscus extract per se reduces the number of rolling and leukocytes elicited by I/R injury [22, 53] and that this effect is mediated, at least in part, by muscarinic receptor activation on endothelial cells, since it was blocked by atropine (a muscarinic receptor antagonist) [22].

Firm adhesion of leukocytes to endothelium is mediated by integrins expressed on leukocyte surface and members of immunoglobulin superfamily of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which are expressed on endothelial cells membrane [54]. I/R injury induces the release of LTB₄ and PAF, which in turn upregulate the expression of β₂-integrins (CD11/CD18) on rolling leukocytes surface [11, 14] promoting their firm adhesion to endothelium. In our study, AA, HMC and Ruscus extract mixture treatments in all daily doses significantly prevented the elevation in the number of sticking leukocytes compared to placebo (Figs. 4–6). However, Ruscus extract mixture at lowest daily dose was significantly more effective in preventing post-ischemic elevation in the number of sticking leukocytes than AA and HMC (Fig. 4c–e).

In our study, AA, due to its property to attenuate post-ischemic endothelial release of PAF [55], probably counteracted the PAF-induced upregulation of β₂-integrins on leukocyte surface, which was reflected by a significant reduction of adherent leukocytes during I/R injury.

HMC inhibit cytokine-induced VCAM-1 expression on endothelial cells in vitro [56]. It is well-known in the literature that that HMC acts inhibiting NF-κB activation in endothelial cells [25, 26] resulting in decreased expression of VCAM-1 [57].

Post-ischemic inhibitory effect of Ruscus extract on leukocyte adhesion is similar to that of rolling, which is due in part by the activation of muscarinic receptors on endothelial cells and blocked by atropine [22]. This protective effect on leukocyte adhesion was enhanced by the actions of ascorbic acid and HMC present in the Ruscus extract mixture.

One of hallmarks of microvascular dysfunction induced by I/R is the disruption of endothelial barrier. Once activated, leukocytes release pro-inflammatory soluble factors, such as LTB₄, which elicits rearrangement of endothelial cytoskeleton, gap formation and increased macromolecular leakage [1, 58]. In the present study, only Ruscus extract mixture at all different doses significantly prevented the rise in the number of leaky sites compared to placebo (Fig. 7) being significantly more effective than both AA, at intermediary and highest doses, (Fig. 7b and c) and HMC at lowest dose (Fig. 7a).

During I/R injury, the oxidative stress induced directly by oxidants or indirectly by depletion of antioxidant defenses causes an impairment of endothelial barrier function that is counteracted by AA [59]. This may explain the significant protection against post-ischemic rise on macromolecular permeability in hamsters that received ascorbic acid treatment. Moreover, it has been shown in the literature that Ruscus extract and HMC prevented the elevation of microvascular permeability induced by both LTB₄ and I/R injury [23]. The anti-inflammatory and antioxidant actions of Ruscus extract in our experimental model are in line with studies in vitro [60] and in humans [61]. In addition, other study has demonstrated that the beneficial effect of Ruscus extract on macromolecular leakage, were inhibited by atropine, suggesting that activation of muscarinic receptors was involved in this endothelial barrier function protection [22].

In fact, activation of muscarinic receptor, especially the subtype M₃ that is present in endothelial cells, has been pointed out as an important requirement for maintenance of endothelial barrier function and integrity of adherens junctions [62, 63]. We believe that Ruscus extract exert its protective effects on endothelial cell barrier by means of M₃ receptor activation and this effect was potentialized by the antioxidant property of Ruscus extract [21] and the concomitant antioxidants present in Ruscus extract mixture (AA and HMC).

According to Persson and colleagues [64], 30 min of ischemia in hamster cheek pouch preparation was the time needed to induce reproducible increase in macromolecular permeability, which is fundamental to study postischemic permeability changes in microcirculation. Since they reported these findings, 30 min of ischemia was the time adopted by our group for I/R injury studies using the hamster cheek pouch preparation [36 , 66]. Moreover, 30 min of ischemia in the hamster cheek pouch preparation is also the time used in other studies from other groups [3 , 67] to investigate postischemic alterations in the microvascular bed.

To the best of our knowledge, this is the first study that compared the effectiveness of AA and HMC combined or not with Ruscus extract, in preventing post-ischemic increase in leukocyte rolling and adhesion and macromolecular permeability. Ruscus extract mixture treatment was more effective than its isolated constituents in preventing the elevation of the number of sticking leukocytes and leaky sites induced by I/R injury, an effect that was probably due to the synergism among Ruscus extract mixture constituents.

4.1 Limitations of the study

We did not perform any assay (e.g., evaluation of enzymatic and non-enzymatic antioxidant defenses and/or determination of circulating pro-inflammatory biomarkers levels) to ascertain the specific mechanisms by which AA, HMC and Ruscus extract prevent post-ischemic macromolecular permeability, leukocyte rolling and sticking increase.

5 Conclusion

On the basis of our findings, we may conclude that Ruscus extract in combination with ascorbic acid and hesperidine methyl chalcone significantly inhibited the rise of leukocyte-endothelium interaction and macromolecular permeability induced by ischemia-reperfusion injury in the cheek pouch preparation and this protective effect was more prominent in leukocyte adhesion and microvascular permeability. The superior beneficial effects of Ruscus extract mixture over its isolated constituents, probably can be explained by the synergistic association among them.

Ethics statement

The experimental protocol and all animal procedures were approved by the Ethical Committee of the State University of Rio de Janeiro, Brazil (registered with the number of 021/2017 and approved on March 28, 2017) in accordance to Guide for the Care and Use of Laboratory Animals [32].

Footnotes

Acknowledgments

Authors would like to thank the assistance of Mr. Paulo José Ferreira Lopes and Mr. Claudio Natalino Ribeiro for their care of the animals.

Funding

This work was funded by an Investigator proposed research project to Pierre Fabre’s Laboratory.

Conflict of interest

No conflict of interest to declare.

Author contributions

Conceptualization, E.B; methodology, F.Z.G.A.C. and E.B.; formal analysis, M.G.C.S. and E.B.; investigation, M.G.C.S., F.Z.G.A.C. and E.B.; data curation, M.G.C.S., F.Z.G.A.C. and E.B.; writing—original draft preparation, M.G.C.S.; writing—review and editing, M.G.C.S. and E.B.; supervision, E.B.; project administration, E.B.; funding acquisition, E.B. All authors have read and agreed to the published version of the manuscript.

References

Eltzschig

, Eckle

. Ischemia and reperfusion–from mechanism to translation. Nat Med. 2011;17(11):1391–401.

Abassi

, Armaly

, Heyman

. Glycocalyx Degradation in Ischemia-Reperfusion Injury. Am J Pathol. 2020;190(4):752–767.

Bertuglia

, Giusti

, Del Soldato

. Antioxidant activity of nitro derivative of aspirin against ischemia-reperfusion in hamster cheek pouch microcirculation. Am J Physiol Gastrointest Liver Physiol. 2004;286(3):G437–43.

Kolbenschlag

, Sogorski

, Timmermann

, Harati

, Daigeler

, Hirsch

, Goertz

, Lehnhardt

. Ten minutes of ischemia is superior to shorter intervals for the remote ischemic conditioning of human microcirculation. Clin Hemorheol Microcirc. 2017;66(3):239–248.

Yao

, Lin

, Shi

, Chen

, Wu

, Zhao

, Lin

. Thrombosis density ratio can predict the occurrence of pulmonary embolism and post-thrombotic syndrome in lower-extremity deep vein thrombosis patients. Clin Hemorheol Microcirc. 2023 Dec 7. doi: 10.3233/CH-231778.

Schofield

, Woodruff

, Halai

, Wu

, Cooper

. Neutrophils–a key component of ischemia-reperfusion injury. Shock. 2013;40(6):463–70.

, Ma

, Shi

, Yu

, Gu

, Sun

, Yu

, Pang

. Renal ischaemia-reperfusion injury is promoted by transcription factor NF-kB p65, which inhibits TRPC6 expression by activating miR-150. Clin Hemorheol Microcirc. 2023 Nov 10. doi: 10.3233/CH-231979.

Bertuglia

, Giusti

. Microvascular oxygenation, oxidative stress, NO suppression and superoxide dismutase during postischemic reperfusion. Am J Physiol Heart Circ Physiol. 2003;285(3):H1064–71.

Crimi

, Ignarro

, Napoli

. Microcirculation and oxidative stress. Free Radic Res. 2007;41(12):1364–75.

10.

Bouskela

, Donyo

. Effects of oral administration of purified micronized flavonoid fraction on increased microvascular permeability induced by various agents and on ischemia/reperfusion in diabetic hamsters. Int J Microcirc Clin Ex. 1995;15(6):293–300.

11.

Yoshida

, Granger

, Anderson

, Rothlein

, Lane

, Kvietys

. Anoxia/reoxygenation-induced neutrophil adherence to cultured endothelial cells. Am J Physiol. 1992;262(6 Pt 2):H1891–8.

12.

Arnould

, Michiels

, Remacle

. Increased PMN adherence on endothelial cells after hypoxia: involvement of PAF, CD18/CD11b, and ICAM-1. Am J Physiol. 1993;264(5 Pt 1):C1102–10.

13.

Bandyopadhyay

, Phelan

, Faller

. Hypoxia induces AP-1-regulated genes and AP-1 transcription factor binding in human endothelial and other cell types. Biochim Biophys Acta. 1995;1264(1):72–8.

14.

Ichikawa

, Flores

, Kvietys

, Wolf

, Yoshikawa

, Granger

, et al. Molecular mechanisms of anoxia/reoxygenation-induced neutrophil adherence to cultured endothelial cells. Circ Res. 1997;81(6):922–31.

15.

Bienvenu

, Granger

. Leukocyte adhesion in ischemia/reperfusion. Blood Cells. 1993;19(2):279–88.

16.

Eppihimer

, Granger

. Ischemia/reperfusion-induced leukocyte-endothelial interactions in postcapillary venules. Shock. 1997;8(1):16–25.

17.

Soares

ROS

, Losada

, Jordani

, Évora

, Castro-E-Silva

. Ischemia/Reperfusion Injury Revisited: An Overview of the Latest Pharmacological Strategies. Int J Mol Sci. 2019;20(20):5034.

18.

, Bi

, Lu

, Zhao

, Yu

, Sun

, et al. Reduction of Mitochondria-Endoplasmic Reticulum Interactions by Acetylcholine Protects Human Umbilical Vein Endothelial Cells From Hypoxia/Reoxygenation Injury. Arterioscler Thromb Vasc Biol. 2015;35(7):1623–34.

19.

Allaert

. Combination of Ruscus aculeatus extract, hesperidin methyl chalcone and ascorbic acid: a comprehensive review of their pharmacological and clinical effects and of the pathophysiology of chronic venous disease. Int Angiol. 2016;35(2):111–6.

20.

Cappelli

, Nicora

, Di Perri

. Use of extract of Ruscus aculeatus in venous disease in the lower limbs. Drugs Exp Clin Res. 1988;14(4):277–83.

21.

Lichota

, Gwozdzinski

. Therapeutic potential of natural compounds in inflammation and chronic venous insufficiency. Eur J Med Chem. 2019;176:68–91.

22.

Rauly-Lestienne

, Heusler

, Cussac

, Lantoine-Adam

, de Almeida Cyrino

FZG

, Bouskela

. Contribution of muscarinic receptors to in vitro and in vivo effects of Ruscus extract. Microvasc Res. 2017;114:1–11.

23.

Bouskela

, Cyrino

, Marcelon

. Inhibitory effect of the Ruscus extract and of the flavonoid hesperidine methylchalcone on increased microvascular permeability induced by various agents in the hamster cheek pouch. J Cardiovasc Pharmacol. 1993;22:225–230.

24.

Bussmann

AJC

, Zaninelli

, Saraiva-Santos

, Fattori

, Guazelli

CFS

, Bertozzi

, et al. The Flavonoid Hesperidin Methyl Chalcone Targets Cytokines and Oxidative Stress to Reduce Diclofenac-Induced Acute Renal Injury: Contribution of the Nrf2 Redox-Sensitive Pathway. Antioxidants (Basel). 2022;11(7):1261.

25.

Ruiz-Miyazawa

, Pinho-Ribeiro

, Borghi

, Staurengo-Ferrari

, Fattori

, Amaral

, et al. Hesperidin Methylchalcone Suppresses Experimental Gout Arthritis in Mice by Inhibiting NF-κB Activation. J Agric Food Chem. 2018;66(25):6269–6280.

26.

Rasquel-Oliveira

, Manchope

, Staurengo-Ferrari

, Ferraz

, Saraiva-Santos

, Zaninelli

, et al. Hesperidin methyl chalcone interacts with NFκB Ser276 and inhibits zymosan-induced joint pain and inflammation, and RAW 264. 7 macrophage activation. Inflammopharmacology. 2020;28(4):979–992.

27.

Frei

, England

, Ames

. Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci U S A. 1989;86(16):6377–81.

28.

Molyneux

, Glyn

, Ward

. Oxidative stress and cardiac microvascular structure in ischemia and reperfusion: the protective effect of antioxidant vitamins. Microvasc Res. 2002;64(2):265–77.

29.

Wang

, Song

, Yang

, Wang

, Meng

, Huang

, Li

, Xu

, Xie

, Huang

. Effects of hydrocortisone combined with vitamin C and vitamin B1 versus hydrocortisone alone on microcirculation in septic shock patients: A pilot study. Clin Hemorheol Microcirc. 2023;84(2):111–123.

30.

Jawien

, Bouskela

, Allaert

, Nicolaïdes

. The place of Ruscus extract, hesperidin methyl chalcone, and vitamin C in the management of chronic venous disease. Int Angiol. 2017;36(1):31–41.

31.

Kakkos

, Bouskela

, Jawien

, Nicolaides

. New data on chronic venous disease: a new place for Cyclo 3^® Fort. Int Angiol. 2018;37(1):85–92.

32.

National Research Council. Guide for the Care and Use of Laboratory Animals, The National Academies Press. 2011.

33.

Duling

. The preparation and use of the hamster cheek pouch for studies of the microcirculation. Microvasc Res. 1973;5(3):423–9.

34.

Bouskela

, Grampp

. Spontaneous vasomotion in hamster cheek pouch arterioles in varying experimental conditions. Am J Physiol. 1992;262(2 Pt 2):H478–85.

35.

Svensjö

, Arfors

, Arturson

, Rutili

. The hamster cheek pouch preparation as a model for studies of macromolecular permeability of the microvasculature. Ups J Med Sci. 1978;83(1):71–9.

36.

Simões

, Svensjö

, Bouskela

. Effects of L-NA and sodium nitroprusside on ischemia/reperfusion-induced leukocyte adhesion and macromolecular leakage in hamster cheek pouch venules. Microvasc Res. 2001;62(2):128–35.

37.

Kubes

, Suzuki

, Granger

. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A. 1991;88(11):4651–5.

38.

Tsao

, Aoki

, Lefer

, Johnson

3rd , Lefer

. Time course of endothelial dysfunction and myocardial injury during myocardial ischemia and reperfusion in the cat. Circulation. 1990;82(4):1402–12. doi: 10.1161/01.cir.82.4.1402. PMID: 2401073.

39.

Carden

, Granger

. Pathophysiology of ischaemia-reperfusion injury. J Pathol. 2000;190(3):255–66.

40.

Springer

. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 1994;76(2):301–14.

41.

McEver

, Moore

, Cummings

. Leukocyte trafficking mediated by selectin-carbohydrate interactions. J Biol Chem. 1995;270(19):11025–8.

42.

Lefer

. Pharmacology of selectin inhibitors in ischemia/reperfusion states. Annu Rev Pharmacol Toxicol. 2000;40:283–94.

43.

Lorant

, Patel

, McIntyre

, McEver

, Prescott

, Zimmerman

. Coexpression of GMP-140 and PAF by endothelium stimulated by histamine or thrombin: a juxtacrine system for adhesion and activation of neutrophils. J Cell Biol. 1991;115(1):223–34.

44.

Kanwar

, Smith

, Kubes

. An absolute requirement for P-selectin in ischemia/reperfusion-induced leukocyte recruitment in cremaster muscle. Microcirculation. 1998;5(4):281–7.

45.

Secor

, Swarbreck

, Ellis

, Sharpe

, Feng

, Tyml

. Ascorbate inhibits platelet-endothelial adhesion in an in-vitro model of sepsis via reduced endothelial surface P-selectin expression. Blood Coagul Fibrinolysis. 2017;28(1):28–33.

46.

Okazaki

, Otani

, Shimazu

, Yoshioka

, Fujita

, Iwasaka

. Ascorbic acid and N-acetyl cysteine prevent uncoupling of nitric oxide synthase and increase tolerance to ischemia/reperfusion injury in diabetic rat heart. Free Radic Res. 2011;45(10):1173–83.

47.

Davenpeck

, Gauthier

, Lefer

. Inhibition of endothelial-derived nitric oxide promotes P-selectin expression and actions in the rat microcirculation. Gastroenterology. 1994;107(4):1050–8.

48.

Paciolla

, Fortunato

, Dipierro

, Paradiso

, De Leonardis

, Mastropasqua

, de Pinto

. Vitamin C in Plants: From Functions to Biofortification. Antioxidants (Basel). 2019;8(11):519.

49.

Chatterjee

. Evolution and the biosynthesis of ascorbic acid. Science. 1973;182(4118):1271–2.

50.

Guazelli

CFS

, Fattori

, Ferraz

, Borghi

, Casagrande

, Baracat

, et al. Antioxidant and anti-inflammatory effects of hesperidin methyl chalcone in experimental ulcerative colitis. Chem Biol Interact. 2021;333:109315.

51.

Kopacz

, Kloska

, Forman

, Jozkowicz

, Grochot-Przeczek

. Beyond repression of Nrf2: An update on Keap1. Free Radic Biol Med. 2020;157:63–74.

52.

Yao

, Xie

, He

, Zeng

, Long

, Gong

, et al. Pretreatment with Panaxatriol Saponin Attenuates Mitochondrial Apoptosis and Oxidative Stress to Facilitate Treatment of Myocardial Ischemia-Reperfusion Injury via the Regulation of Keap1/Nrf2 Activity. Oxid Med Cell Longev. 2022;2022:9626703.

53.

de Almeida Cyrino

FZG

, Balthazar

, Sicuro

, Bouskela

. Effects of venotonic drugs on the microcirculation: Comparison between Ruscus extract and micronized diosmine1. Clin Hemorheol Microcirc. 2018;68(4):371–382.

54.

Granger

, Kubes

. The microcirculation and inflammation: modulation of leukocyte-endothelial cell adhesion. J Leukoc Biol. 1994;55(5):662–75.

55.

Lloberas

, Torras

, Herrero-Fresneda

, Cruzado

, Riera

, Hurtado

, et al. Postischemic renal oxidative stress induces inflammatory response through PAF and oxidized phospholipids. Prevention by antioxidant treatment. FASEB J. 2002;16(8):908–10.

56.

Nizamutdinova

, Jeong

, Xu

, Lee

, Kang

, Kim

, Chang

, Kim

. Hesperidin, hesperidin methyl chalone and phellopterin from Poncirus trifoliata (Rutaceae) differentially regulate the expression of adhesion molecules in tumor necrosis factor-alpha-stimulated human umbilical vein endothelial cells. Int Immunopharmacol. 2008;8(5):670–8.

57.

Chen

, Zhang

, Zhu

, Liu

, Chen

, Wang

, et al. Quercetin inhibits TNF-α induced HUVECs apoptosis and inflammation via downregulating NF-kB and AP-1 signaling pathway in vitro. Medicine (Baltimore). 2020;99(38):e22241.

58.

Gautam

, Olofsson

, Herwald

, Iversen

, Lundgren-Akerlund

, Hedqvist

, et al. Heparin-binding protein (HBP/CAP37): a missing link in neutrophil-evoked alteration of vascular permeability. Nat Med. 2001;7(10):1123–7.

59.

May

, Qu

. Ascorbic acid prevents oxidant-induced increases in endothelial permeability. Biofactors. 2011;37(1):46–50.

60.

Rodrigues

JPB

, Fernandes

, Dias

, Pereira

, Pires

TCSP

, C Calhelha

, Carvalho

, Ferreira

ICFR

, Barros

. Phenolic Compounds and Bioactive Properties of Ruscus aculeatus L. (Asparagaceae): The Pharmacological Potential of an Underexploited Subshrub. Molecules. 2021;26(7):1882.

61.

Bihari

, Guex

, Jawien

, Szolnoky

. Clinical Perspectives and Management of Edema in Chronic Venous Disease-What about Ruscus? Medicines (Basel) (2022;9(8):41.

62.

Jiao

, Wu

, Wen

, Zhao

, Du

. M3 muscarinic acetylcholine receptor dysfunction inhibits Rac1 activity and disrupts VE-cadherin/β-catenin and actin cytoskeleton interaction. Biochem Cell Biol. 2014;92(2):137–44.

63.

Jiao

, Wu

, Liu

, Wen

, Zhao

, Du

. Type 3 muscarinic acetylcholine receptor stimulation is a determinant of endothelial barrier function and adherens junctions integrity: role of protein-tyrosine phosphatase 1B. BMB Rep. 2014;47(10):552–7.

64.

Persson

, Erlansson

, Svensjö

, Takolander

, Bergqvist

. The hamster cheek pouch–an experimental model to study postischemic macromolecular permeability. Int J Microcirc Clin Ex. 1985;4(3):257–63.

65.

de Souza

, Conde

, Laflôr

, Sicuro

, Bouskela

. n-3 PUFA induce microvascular protective changes during ischemia/reperfusion. Lipids. 2015;50(1):23–37.

66.

Boa

, Costa

, Souza

, Cyrino

, Paes

, Miranda

, Carvalho

, Bouskela

. Aerobic exercise improves microvascular dysfunction in fructose fed hamsters. Microvasc Res. 2014;93:34–41.

67.

Colantuoni

, Lapi

, Paterni

, Marchiafava

. Protective effects of insulin during ischemia-reperfusion injury in hamster cheek pouch microcirculation. J Vasc Res. 2005;42(1):55–66.