Effects of Ubiquinol with Fluid Resuscitation following Hemorrhagic Shock

Abstract

Abstract Hemorrhagic shock (HS) and fluid resuscitation triggers ischemia-reperfusion injury in cells and increases the production of reactive oxygen species (ROS) which are known to activate the intrinsic pathway of apoptosis and contribute to organ dysfunction.1 Ubiquinol is a potent free radical scavenger which is produced endogenously and functions as part of the mitochondrial respiratory chain.2 No study has been conducted to investigate the effects of ubiquinol related to HS. The overall aim of this study was to examine the effects of ubiquinol on leukocyte mitochondria and in the lungs, diaphragm, heart and kidneys as a supplemental treatment for HS. A randomized experimental design was used for this study. Adult male Sprague-Dawley rats (n = 20) were anesthetized and HS was induced by withdrawing 40% of the rat's blood volume to maintain a mean arterial pressure of 45-55 mmHg for 60 minutes. Following HS the rats were resuscitated with blood and lactated Ringer's (LR) with or without ubiquinol (1 mg per 100 g of body weight). The rats were monitored for 120 minutes, the animals were euthanized and the organs harvested. Leukocyte mitochondria superoxide (O2*⁻) was measured by flow cytometry using MitoSOX Red, a mitochondrial-targeted variant of the fluorescent probe hydroethidine. Superoxide levels were measured at baseline, end of HS and 120 minutes following fluid resuscitation. Arterial blood values were also recorded at these times. At the end of experiment, diaphragms were evaluated for hydrogen peroxide (H2O2) using the fluorescent probe dihydrofluorescein-diacetate (Hfluor). The lungs, diaphragm, heart, and kidneys were examined for percent of apoptotic nuclear membrane damage using a differential dye uptake method with acridine orange and ethidium bromide. No significant differences were found between groups with regard to the volume of blood removed, hemodynamic status or arterial blood values (p 0.05). Ubiquinol decreased leukocyte mitochondrial production of O2*⁻ at the end of the experiment by 35% compared to the control group (4687.2 ± 265.4 versus 7227.9 ± 534.5, p ˂ 0.001). Similarly, the mean fluorescence intensity (MFI) of diaphragm H2O2 was significantly lower in the ubiquinol group compared to control (4193 ± 333 versus 23513 ± 5098, p ˂ 0.001). The percent of apoptosis in the lungs, diaphragm, heart, and kidneys was significantly reduced in the animals treated with ubiquinol compared to the control group (6.0 ± 0.7% versus 39.2 ± 1.1%, 4.7 ± 0.5% versus 30.6 ± 2.4%, 2.9 ± 0.6% versus 23.6 ± 1.2%, 2.4 ± 0.3% versus 42.1± 1.9%, respectively, p ˂ 0.001). Ubiquinol was effective in decreasing leukocyte mitochondrial O2*⁻ formation, which suggests that ubiquinol scavenged O2*⁻ within the mitochondria. Since ubiquinol is a potent antioxidant, it also probably scavenged other free radicals outside the mitochondria. The increased concentration of ubiquinol within the mitochondria would assist in maintaining the activities within the electron transport chain during HS. In addition, the decreased mitochondrial O2*⁻ would result in lower H2O2 production. The significant reduction in the percent of apoptosis in lungs, diaphragm, heart and kidneys between the control and treatment rats, suggests that decreased ROS production attenuated the activation of the intrinsic (mitochondrial) apoptosis pathway.3 The findings could also be attributed to the stabilization of the mitochondrial membrane by ubiquinol, which has been demonstrated in a previous study.4 In conclusion, ubiquinol may have application as a supplemental treatment to reduce free radical damage and apoptosis- related injury following HS and fluid resuscitation.