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Minocycline abrogates lung oxidative damage and haematological perturbations in mice exposed to hypoxia

Article Info:

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Article History:

Received 17 December 2021

Reviewed 29 January 2022

Accepted 06 February 2022

Published 15 February 2022

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Cite this article as:

Eduviere A, Otomewo L, Minocycline abrogates lung oxidative damage and haematological perturbations in mice exposed to hypoxia, Journal of Drug Delivery and Therapeutics. 2022; 12(1-s):82-90

DOI: http://dx.doi.org/10.22270/jddt.v12i1-s.5342

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*Address for Correspondence:

Dr. Anthony Eduviere, Department of Pharmacology, Delta State University, Abraka, Nigeria

ORCID ID: https://orcid.org/0000-0001-6822-7672

Abstract

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Objectives: Hypoxia results from the unavailability of sufficient oxygen to tissues. Since previous studies have shown that hypoxia results in inflammation, this research has found it necessary to investigate the impact of chronic hypoxia on the lungs and blood of mice and also the potential benefit of minocycline which is a well-known antibiotic.

Design: Thirty mice were split into 5 groups of 6 animals each. A physical model of hypoxia was used. Here, mice were locked in a 250 mL airtight container for 20 mins each day for seven consecutive days.

Interventions: One hour before exposure to hypoxia, mice in all groups except the vehicle group, were treated with varying doses of minocycline (10, 20, 40 mg/kg, p.o.).

Main Outcome Measures: The mice in the positive control group expressed a sign of lung inflammation with an increase in myeloperoxidase activity, as well as lung oxidative stress with an increase of prooxidants over antioxidants. While in the blood, white blood cells were significantly increased whereas red blood cell count depreciated as blood volume did too.

Results: The various effects of hypoxia on the blood and lungs of the mice were significantly prevented and attenuated to an acceptable degree by minocycline

Conclusion: In conclusion, minocycline expresses an acceptable benefit in the respiratory and circulatory systems of mice in hypoxic conditions.

Keywords: Hypoxia, Lungs, Blood, Haematology, Oxidative stress, Minocycline

Hypoxia is a condition that results when the cellular supply of oxygen does not meet the demand. Most commonly, previous research have focused on investigating this condition and its effect on the brain since the brain is considered the organ with the greatest need for oxygen.¹ Other studies had also investigated the potential benefit of minocycline on such hypoxic conditions in the brain (such as hypoxia-ischemia) due to the fact that the drug is highly lipophilic and crosses the blood-brain barrier.² In those studies, the benefit of minocycline was linked to its intrinsic anti-inflammatory activity.

Minocycline is a tetracycline antibiotic with highly profitable pharmacokinetic properties (good tolerance, safety, broad spectrum of action, and high efficacy). Apart from its known antibiotic activity, various studies have elucidated a potential role of minocycline in oxidative stress attenuation which has been linked to its anti-inflammatory ability.^1-4

From the foregoing, the main objectives of the current study were to elucidate the potential benefit of minocycline on lung tissue damage and blood dyscrasia induced by chronic hypoxia in mice.

Thirty (30) healthy albino mice (male; weight=24.0±2.0 g) which were used in this research were procured from the animal house of the Basic Medical Sciences faculty of the institution. The NIH protocols for animal experimentation (under the Guide for Care and Use of Laboratory Animals; publication 85-23) were followed with minute modifications in housing and feeding the mice.

Minocycline (100 mg) was dissolved in an appropriate volume of distilled water to obtain the stock solution. Further serial dilutions were carried out to acquire the three dose levels used in this study (10, 20, 40 mg/kg). Therefore, the mice were allotted into five (5) treatment groups of six animals each (n = 6) based on the treatment they received:

The animals were treated for 7 consecutive days and all administrations were oral.

Hypoxia-induced stress was initiated in mice using a physical model. One hour after mice were orally treated with either distilled water or minocycline, mice in groups 2–5 only were subjected to the hypoxic stress design by locking them individually in an airtight cylindrical container of ~250 mL capacity for 20 min daily for the total duration of treatment i.e., seven (7) days.⁵ On day 8, haematological assays and biochemical assays of the lungs were carried out. Histology of the lungs was also done.

Following euthanization, each mouse lung was harvested, weighed and homogenized with phosphate buffer and cold centrifuged for 15 min. Aliquots of the resulting supernatant were then used for each of the biochemical assays.

Reduced glutathione (GSH) concentration and glutathione peroxidase (GPx) activity

GSH concentration and GPx activity were determined using an established method.⁶

MPO activity in the lungs was determined according to the method previously described.^11,12

Lung tissue obtained for histological studies from the mice in each group was prepared and subjected to H&E staining following the method of Akinluyi et al.¹³ with slight modifications. Micrographs were then obtained digitally.

Subsequent to euthanasia, blood was obtained from mice via cardiac puncture into EDTA tubes. A full blood analysis was carried out according to previously described methods.¹⁴

All data are presented as means + SEM. Statistical comparisons were made using one way-ANOVA followed by a Bonferroni’s post-test using the Graph Pad Biostatistics software. The level of significance for all tests was set at p <0.05.

Figures 1 and 2 reveal that hypoxia-induced stress resulted in a significant hike in prooxidant levels in the lungs. The former represented MDA content and the latter depicted nitrites concentration. In both figures also, minocycline-treated groups showed signs of attenuation of this effect.

On the other hand, the antioxidant molecules of the mice exposed to hypoxia alone were significantly diminished with reduced activity when compared to the vehicle group. Figures 3-6 showed reduced SOD activity, reduced CAT activity, reduced GSH concentration, and reduced GPx activity, respectively. However, minocycline administration significantly attenuated these effects.

Myeloperoxidase (MPO) activity in lung of mice exposed to hypoxia-induced stress

Mice in the positive control group showed a significant increase in MPO activity during the biochemical assays (Figure 7). However, minocycline groups responded oppositely by presenting with significant reductions in MPO activity.

Lungs of mice in the positive control group (depicted as slide G) showed the presence of fewer pneumocytes and the shrinking of capillaries more than the mice of the vehicle group (depicted as slide N). However, the tables turned when the mice which received any of the doses of minocycline showed an improved architecture of the alveoli (slides A-C; see Figure 8).

Figure 9 revealed the significant increase in number of white blood cells of mice in the positive control when compared to the vehicle group. Minocycline treatment caused a significant suppression in this increase.

Figure 10 revealed the significant depletion in white blood cell count of mice in the positive control when compared to the vehicle group. Minocycline treatment caused a significant attenuation in this decrease.

Haemoglobin content of mice exposed to hypoxia-induced stress was significantly reduced when compared to mice in the vehicle group. However, minocycline administration caused a significant increase in haemoglobin content in a manner resembling dose dependence (Figure 11).

From Table 1, it is easy to deduce that hypoxic stress caused a significant reduction in blood volume markers – PCV, MCV, and MCHC – when compared to the vehicle only group. However, minocycline caused a significant boost in blood volume towards normal.

Table 1: Effect of minocycline on blood volume markers of mice exposed to hypoxic stress

Treatment	PCV (%)	MCV (fl)	MCHC (pg/dL)
VEH 10 mL/kg	54.10+3.12	67.33+5.16	40.72+2.57
VEH 10 mL/kg + HS	26.33+2.20^#	29.80+3.77^#	15.20+2.13^#
MCL 10 mg/kg + HS	34.50+3.35	43.20+3.81	19.83+1.62
MCL 20 mg/kg + HS	40.97+2.87^*	52.63+3.15^*	24.67+1.41^*
MCL 40 mg/kg + HS	45.50+2.64^*	55.47+4.39^*	27.50+1.80^*