Curcumin as a Potential Therapeutic Agent for Mitigating Carbon Monoxide Poisoning: Evidence from an Experimental Rat Study

Introduction

Carbon monoxide (CO) is a non-irritating, tasteless, odorless, and colorless poisonous gas. It results from the incomplete combustion of molecules containing carbon. Asphyxia and cellular hypoxia, binding to hemoglobin, myoglobin, and cytochrome p450, and ischemia and reperfusion injury are all mechanisms by which carbon monoxide causes tissue damage. CO poisoning is believed to account for more than half of all fatal poisonings worldwide [1,2].

Although the carboxyhemoglobin (COHb) level is helpful in diagnosis, there is a weak correlation between the measured COHb level and clinical symptoms and findings [3]. Thus, clinicians can frequently face dilemmas in the treatment of patients considered to have CO poisoning [4]. The conventional treatment for CO poisoning is the injection of a large quantity of oxygen as soon as possible. Furthermore, hyperbaric oxygen therapy appears to be the preferred treatment for individuals with considerable CO exposure. The decision to commence hyperbaric oxygen treatment needs careful consideration of a number of parameters, such as the blood COHb level, comorbid disorders (including pregnancy), the patient’s stability, and the proximity of the nearest facility with emergency hyperbaric capabilities [1].

Oxidative stress is characterized by an imbalance between reactive oxygen species (ROS), such as hydroxyl radicals (OH), ROS, and hydrogen peroxide (H2O2), which are produced during normal cellular metabolism, and antioxidants, which scavenge free radicals [5]. ROS, free radicals, and neuronal nitric oxide are thought to cause oxidative damage, which is one of the current hypotheses on the molecular mechanism of CO poisoning [6]. The natural compound curcumin, which is widely present in the Curcuma longa plant, is specifically suggested to serve as an anti-apoptotic enhancer or moderator against oxidative shocks. Its action may include its anti-oxidative effect and changes in signal targets [7].

The hypothesis of the study is that curcumin can prevent/reduce oxidative stress damage caused by CO poisoning. The objective of this study is to investigate the neuroprotective role of curcumin by measuring levels of superoxide dismutase (SOD), nitric oxide (NO), malondialdehyde (MDA), catalase (CAT), dimethylarginine (ADMA), and Bcl-2 in rats with CO poisoning.

Material and Methods

Establishment of Experimental Groups

ESTABLISHMENT OF EXPERIMENTAL GROUPS:

The study included 24 female rats (Sprague Dawley) with an average weight of 250 g. They were kept under standard conditions (at 21–23°C with 12 h/12 h light/dark cycles) and had free access to food and water. Carbon-monoxide gas of 3000 ppm concentration was obtained from the HABAS Industrial and Medical Gas Industry Company (İzmit, Turkey). Then, the rats were divided into 4 subgroups as follows (Figure 1).

CONTROL GROUP (GROUP 1, N=6):

Rats in this group were exposed to room air for 30 min. In this group, only room air was administered to the rats. CO gas and curcumin treatment were not administered. Subsequently, they were killed, and blood/tissue samples were collected.

CURCUMIN GROUP (GROUP 2, N=6):

Rats in this group were administered curcumin (50 mg/kg) intraperitoneally. The rats in this group were treated only with curcumin; CO inhalation was not administered. After 30 min of room air, they were killed, and blood/tissue samples were taken.

CO GROUP (GROUP 3, N=6):

Rats in this group were exposed to 3 L/min of CO gas at 3000 ppm concentration for 30 min. The rats in this group were exposed only to carbon monoxide inhalation. Subsequently, they were killed, and blood/tissue samples were collected.

CURCUMIN+CO GROUP (GROUP 4, N=6):

Rats in this group were exposed to 3 L/min of CO gas at a concentration of 3000 ppm for 30 min. Following CO exposure, they were intraperitoneally administered curcumin at a dosage of 50 mg/kg. After an additional 30 min, the rats were killed, and blood/tissue samples were collected.

All rats were decapitated after intraperitoneal administration of ketamine (75 mg/kg) and xylazine (10 mg/kg). Intracardiac puncture was used to collect blood samples from the deceased rats for biochemistry analysis. Thereafter, parts of the hippocampus CA1 were surgically removed. One part of the tissue samples was used for biochemical analysis, while the other part was used for histopathological inspection. Although the tissues reserved for histological investigation were immersed in a 10% formaldehyde solution, processed through a series of graded alcohols and xylene, and ultimately embedded in paraffin blocks, the tissues reserved for biochemical analysis were kept at 80°C until analysis.

TISSUE HOMOGENIZATION AND BIOCHEMICAL ANALYSIS:

The tissues were homogenized using a glass-distilled water homogenizer. The supernatants of blood were centrifuged at 2500 rpm after a 10-min resting period. Hippocampus tissues were rinsed with physiological saline after removal, and the right hemispheres were weighed, promptly frozen in liquid nitrogen, and stored at −80°C. For analysis, all tissue samples underwent homogenization using a blade homogenizer in a 1/6 phosphate buffer (pH: 7.4), followed by centrifugation at 20 000×g to isolate the supernatants. Total protein levels in all homogenates and supernatants were determined through spectrophotometric analysis, using the Bradford method (Shimadzu UV 1800 Spectrophotometer) [8].

Supernatant CAT activities were measured using Aebi’s technique, and the maximal absorbance of hydrogen peroxide (H2O2) was at 240 nm. Catalase reduces ultraviolet absorption by converting H2O2 in the experimental environment into water and oxygen. The activity of the CAT enzyme is directly linked to a reduction in absorbance [9].

Supernatant SOD activities were measured by the method of Sun et al, based on the changes adapted by Durak et al [10]. This technique depends on the reduction of nitroblue tetrazolium by oxygen radicals produced by the xanthine oxidase system. Its reduction terminates with the formation of a blue color at 560 nm of maximum absorption.

Homogenate MDA levels were assayed by the method proposed by Placer et al and modified by Matkovics et al to measure lipid peroxidation [11]. This method is based on the reaction between butyric acid and MDA, an aldehyde made by lipid peroxidation that reacts with thiobarbituric acid to make a pink chromogen that can be measured at 532–535 nm on a spectrophotometer.

The NO production was assessed by the accumulation of nitrites by the modified technique of Griess, as described previously [12]. The optical density of the assay samples was measured spectrophotometrically at 540 nm.

Supernatant CAT, SOD, NO, and homogenate MDA results were expressed in proportion to gram protein. Serum ADMA levels were measured by the ELISA method using commercially available kits, according to the manufacturer’s instructions (Thermo Scientific Multiscan GO, Finland).

IMMUNOHISTOCHEMICAL STUDY:

Bcl-2 is one of many important apoptotic regulators that are necessary for healthy development, tissue homeostasis, and defense against external pathogens. Many sets of genes control this pathway, but the Bcl-2 family is the best known [13]. The Bcl-2 family includes members that are proapoptotic (Bax, Bak, Bid, and Bad) and antiapoptotic (Bcl-2, Bcl-xL, and Bcl-w) [14].

The paraffin blocks were cut into 5-m-thick sections using a microtome before being put on the slide. These sections were incubated for a whole night at 54°C before being deparaffinized twice for a total of 20 min. The sections were then rehydrated by allowing them to soak for 5 min in a series of alcohols with gradually lower concentrations (100%, 90%, 80%, and 70%). The sections were subjected to microwave irradiation (7+5+5 min) in 250 mL of freshly produced citric acid (pH 6.0) buffer, washed twice with phosphate-buffered saline (PBS), and then cooled at room temperature for 20 min to show antigenic epitopes. They were washed 3 times with PBS for 5 min at room temperature. The endogenous peroxidase activity in the tissues was then saturated by incubating them for 20 min at room temperature in a 3% H₂O₂ solution. The sections were held for 7 min at room temperature in blocking solution (Invitrogen, Carlsbad, CA, USA) to prevent non-specific binding. Following the removal of the blocking solution, bcl-2 primary antibodies (mouse monoclonal anti-Bcl-2, Dako) were added to the samples, which were then kept overnight at 4°C in a humid environment.

The sections were then incubated for 45 min at room temperature with biotin-labeled (Invitrogen) secondary antibodies. After the application of the secondary antibody, the streptavidin-peroxidase complex was introduced to the tissue samples and incubated for 30 min at room temperature. Following the PBS washing steps, dripping diaminobenzidine (DAB; Invitrogen) solution made antigen-antibody complexes visible. After DAB application, sections were rinsed with water and stained with Mayer hematoxylin. The samples underwent dehydration through a series of increasing alcohol concentrations, were cleared by passing through xylol, and were finally covered with a closure solution. Under a microscope, the resulting chromogenic reaction was examined.

Using the procedure described in the prior report, immunopositive cells were counted [15]. Hippocampal sections were examined under a 200×-magnification light microscope for immunostaining, and immunopositive cells per 1000 cells were counted. The evaluation was performed using a light microscope (Nikon Eclipse Ni), a digital camera (Nikon DS-R12), and software (NIS-Elements 4.50).

STATISTICAL ANALYSES:

The biochemical study was statistically analyzed using MedCalc (version 15.8), and the results were presented as the median and 25^th percentile and 75^th percentile. Kruskal-Wallis variance analysis was used to examine the median comparisons of more than 2 groups. Following the analysis of variance, post-hoc tests were used to determine the various groups, and the Bonferroni test was used for paired comparisons. The level of statistical significance accepted was 0.05.

SPSS was used to perform statistical analyses for immunohistochemistry (version 22.0, IBM Corp, Armonk, NY, USA). Descriptive statistics were reported as the mean±standard deviation. Shapiro-Wilk tests were used to analyze the group distributions. Since the data met the assumptions of normality, the differences between 3 or more groups were assessed using the ANOVA test. Post-hoc tests with Bonferroni correction or Games-Howell were applied for pairwise comparisons to identify specific group differences. A significance level of P<0.05 was considered statistically significant.

ETHICS STATEMENT:

The study obtained approval from the Local Ethics Committee of Ankara Training and Research Hospital Animal Experiments (537; 13.08.2018).

Results

Our findings revealed that CAT values in the control, curcumin, and curcumin+CO groups were significantly higher than those in the CO group (P<0.001, P<0.001, P=0.007, respectively). Notably, the CAT value in the curcumin group was significantly elevated compared to the curcumin+CO group (P=0.042) (Figure 2). SOD values demonstrated a similar pattern, with the control and curcumin groups exhibiting significantly higher values than the CO group (P<0.001, P=0.002, respectively) (Figure 3). Regarding MDA levels, the control, curcumin, and curcumin+CO groups all showed significantly lower values than the CO group (P<0.001). Furthermore, MDA values in the curcumin+CO group were significantly higher than those in the control group (P=0.023) (Figure 4). ADMA values in the control, curcumin, and curcumin+CO groups were all significantly lower than those in the CO group (P=0.001, P=0.001, P=0.041, respectively). Notably, in the control and curcumin groups, ADMA values were significantly lower than in the curcumin+CO group (P=0.003, P=0.004, respectively) (Figure 5). Additionally, NO values in the control and curcumin groups were higher than those in the CO group (P<0.001, P=0.010, respectively). Moreover, the NO value in the curcumin group was significantly higher than in both the CO and curcumin+CO groups (P=0.001, P=0.023, respectively) (Figure 6, Table 1).

The Bcl-2 value showed a significant increase in the control, curcumin, and curcumin+CO groups, compared with the CO group (P<0.001, P<0.001, P=0.017, respectively). Specifically, in the control and curcumin groups, the Bcl-2 immunopositive cell values were significantly higher than those in the curcumin+CO group (P<0.001, P<0.001, respectively). Moreover, within the curcumin+CO group, the Bcl-2 value was higher than that in the CO group (P=0.017) (Table 2).

In the immunostaining conducted with the anti-Bcl-2 antibody in the hippocampus, widespread expression (indicated by arrows) is evident in the control group (Figure 7A). Contrastingly, the presence of immunopositive cells notably decreased in the CO group (Figure 7C). In the Curcumin+CO group (Figure 7D), there was a significant increase in the presence of immunopositive cells, compared with the CO group. Notably, in the curcumin-treated group (Figure 7B), immune expression slightly increased when compared with that of the control group (scale bar: 100 μm) (Figure 4).

Discussion

LIMITATIONS OF THE STUDY:

First, no measurements were conducted regarding the levels of curcumin in the plasma of the animals. Due to curcumin’s poor bioavailability stemming from inadequate absorption, rapid metabolism, and quick systemic elimination, further studies are required to assess proper monitoring of both the administered dose and its effects. Second, this study exclusively focused on the short-term effects and single dose of curcumin at the tissue level. However, additional investigations exploring the long-term effects of curcumin at various doses may provide a better understanding of its potential therapeutic properties.

Conclusions

Curcumin can protect oxygen-dependent tissues from the toxic effects of CO gas. The positive effect on tissue CAT activities and serum ADMA levels, together with the suppression of MDA, are important mediators of the exaggerated inflammatory process that causes cell destruction. These findings support the hypothesis that the destructive effect of CO can be limited by curcumin. In addition, curcumin-mediated increased expression of Bcl-2 may limit the process leading to apoptosis and cell death.