Combined Enzymatic and Antioxidative Treatment Reduces Ischemia-Reperfusion Injury in Rabbit Skeletal Muscle

Background

Ischemia/reperfusion (I/R) injury is characterized by the production of oxygen-free radicals leading to disturbances in vasomotility (microvascular constriction) and microvascular permeability (interstitial edema formation). The objective was to evaluate the effect of the combined antioxidative and enzymatic preparation Phlogenzym® on I/R injury of skeletal muscle.

Materials and methods

A rabbit hindlimb model of I/R (2.5/2 h) was used (IR group). Phlogenzym®, containing rutin, trypsin, and bromelain, was applied enterally (60 mg/kg body weight) as a bolus 30 min prior to ischemia (Ph group). Sham-operated animals served as controls (CO group). Plasma malondialdehyde, potassium, and microvascular perfusion (monitored by laser flowmetry) were assessed. Histomorphometry and electron microscopy were performed from major adductor muscles.

Results

Two hours after reperfusion, potassium levels were significantly elevated in IR compared to Ph group (6.7 ± 1.2 versus 4.9 ± 0.9 mmol/l, P < 0.006). Enhanced lipid peroxidation, apparent by increased plasma malondialdehyde levels, was ameliorated in the Ph group (1.0 ± 0.1 versus 0.7 ± 0.1 nmol/ml, P < 0.0001). No-reflow (reduction of blood flow by 62% in IR group) was not observed in the Ph group (P < 0.004). Phlogenzym® treatment prevented microvascular constriction (17.6 ± 2.3 versus 12.6 ± 1.1 μm², P < 0.0001) and mollified interstitial edema (21.5 ± 2.0 versus 26.0 ± 3.7%, P < 0.017), resulting in mild ultrastructural alterations in contrast to pronounced sarcolemmal and mitochondrial damage in untreated rabbits.

Conclusions

Phlogenzym® had a protective effect on skeletal muscle during I/R injury expressed by prevention of no-reflow and preservation of muscle tissue.

Introduction

Various mechanisms have been implicated to explain the development of ischemia/reperfusion (I/R) injury in skeletal muscle [1]. Stimulated generation of superoxide (O₂⁻) and reduction of nitric oxide (NO) production are believed to play a key role in this process [2, 3].

There are many sources of O₂⁻ during I/R injury, one major source being disarranged constitutive nitric oxide synthase (cNOS). This enzyme, which produces NO, is one of the most important vasodilators. At the onset of ischemia, intracellular calcium influx activates cNOS, leading to the consumption and relative local depletion of intracellular l-arginine. In l-arginine-starved environments, cNOS uses molecular oxygen as substrate to produce O₂⁻ [4]. Superoxide induces generation of other extremely reactive oxygen-derived free radicals (OFR) [5]. Excessive OFR formation overwhelms the endogenous antioxidative defense capacity, resulting in cell damage through increased lipid peroxidation in cell membranes [6]. The consequences of such injury are remote and local tissue destruction.

I/R injury is accompanied by disturbances in vasomotility and microvascular permeability. Deficiency of vasodilator NO due to its consumption by vasoconstrictor OFR results in microvascular constriction and prominent reduction of blood flow in reperfused tissue. OFR also promote the formation of inflammatory responses causing leukocyte “rolling and sticking” [7]. In addition, altered rheological conditions lead to thrombus formation in microvessels. These mechanisms contribute to the development of no-reflow phenomenon [1, 8, 9] Concurrently, increased OFR damage endothelial cells, enhance microvascular protein efflux, and potentiate endothelial permeability, causing interstitial reperfusion edema [10].

Different treatment strategies are viable for reducing I/R injury. Supplementation of NO or its precursor l-arginine aims at prevention of derangement of cNOS and excessive O₂⁻ production [4, 11], whereas radical scavengers such as antioxidative vitamins [12, 13] and bioflavonoids [14, 15] annihilate OFR. A combination of both strategies was reported to have an additional beneficial effect on I/R injury [16].

Hydrolytic enzymes, such as bromelain and trypsin, may represent another, new policy to reduce I/R injury. Although trypsin also has antioxidative properties [17], other specific actions of these hydrolytic enzymes have been described. These include (1) reduction of platelet aggregation and lymphocyte adherence [18, 19] and activation of protease-activated receptor (PAR)-2 causing vasodilatation [20] by trypsin; and (2) prevention of platelet, leukocyte, and erythrocyte adherence [18, 21, 22, 23], and fibrinolytic activities dissolving fibrin clots [23, 24] by bromelain. These mechanisms might prevent plugging of microvessels, thereby ameliorating no-reflow phenomenon.

The current investigation was undertaken to evaluate the effect of Phlogenzym® (MUCOS Pharma GmbH & Co., Geretsried, Germany), a commercially available preparation combining antioxidative and enzymatic properties, in a rabbit model of skeletal muscle I/R injury. Phlogenzym® consists of three active compounds, the bioflavonoid rutin with well-known antioxidative effect, and the hydrolytic enzymes bromelain and trypsin. Rutin was reported to ameliorate I/R injury in heart [25, 26] and brain [27]. Bromelain, a cysteine proteinase extracted from pineapples, was described to have antiedematous, antiinflammatory, antithrombotic, and fibrinolytic activities in vitro and in vivo [24]. Trypsin was reported to scavenge OFR and to reduce edema formation and tissue destruction [17].

Treatment with Phlogenzym® was reported as safe and effective in clinical use. It has been shown that such treatment reduces hemodynamic support in septic children [28]. Pathogenesis in sepsis has similarities with I/R injury. In both situations stimulated overproduction of OFR is playing a key role [29] with subsequent disturbances of microcirculation and increased endothelial permeability [30, 31].

Therefore, we tested if enzymatic treatment in combination with antioxidants using Phlogenzym® might positively influence I/R injury in skeletal muscle.