THE EFFECT OF N-ACETYLCYSTEINE ON LIPID PEROXIDATION AND ANTIOXIDANT SYSTEM PARAMETERS IN THE EYE TISSUES OF RABBITS WITH EXPERIMENTAL UVEITIS

ABSTRACT

Uveitis is one of the leading causes of permanent visual impairment in the world. Recent data indicates that oxidative stress plays an important role in the pathogenesis of uveitis. A key role in the antioxidant defense of an eye is played by the glutathione system. N-acetylcysteine is potent antioxidant, but its application in uveitis was never investigated. An experimental study on rabbits with immunogenic uveitis was conducted, showing that N-acetylcysteine has prominent antioxidant action on the eye tissues in uveitis and allows replenishing contents of glutathione.

Keywords: uveitis, oxidative stress, glutathione, n-acetylcysteine.

Relevance. The relevance, social, and medical significance of the problem of uveitis today are determined by its high prevalence, commonly occurring severe complications resulting in disability, predominant involvement of young and working-age individuals, as well as the insufficiently studied and complex etiopathogenesis of the disease [1, 2]. By now, substantial experimental and clinical evidence has accumulated showing that oxidative stress plays an important role in the onset and progression of uveitis [3, 4].

A key role in the antioxidant defense of the eye is played by the glutathione system. Glutathione is the main endogenous antioxidant, occupying a central position in the cell’s anti-radical and anti-peroxide defense [5]. The rate-limiting factor in glutathione synthesis is L-cysteine [6].

Currently, N-acetylcysteine (NAC) is considered one of the most promising pharmacological antioxidant agents capable of repleneshing intracellular glutathione. However, the literature lacks data on parenteral use of NAC for correcting oxidative stress in ocular tissues during uveitis, and there is no evidence regarding the ability of NAC to cross the blood–ocular barrier [7].

Objective. To investigate the effect of parenteral administration of NAC on indicators of lipid peroxidation and antioxidant defense during experimental immunogenic uveitis (EIU) in the tissues of the anterior segment of the rabbit eye.

Methods. An experimental study was conducted on male rabbits in accordance with the Helsinki Declaration on the humane treatment of animals. All rabbits (25 animals – 50 eyes) were divided into 5 groups (5 animals each). Control-1 included intact animals. The remaining rabbits underwent induction of EIU using a standardized method [8]. Rabbits with EIU were divided into 4 groups: Control-2 – received daily intramuscular placebo (saline) for 3 days; Experimental-2 – received daily intramuscular NAC injections (40 mg/kg) for 3 days; Control-3 – received daily intramuscular placebo for 7 days; Experimental-3 – received daily intramuscular NAC injections (40 mg/kg) for 7 days.

After rabbits were withdrawn from the experiment eyeballs were enucleated. Oxidative stress intensity was assessed by determining the levels of lipid peroxidation products (LPO): diene conjugates (DC), triene conjugates (TC), malondialdehyde (MDA). Antioxidant system status was analyzed by measuring levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and their ratio (GSH/GSSG). Statistical analysis was performed using the Mann–Whitney test.

Results and Discussion. The obtained data (table 1) indicate that the development of EIU was accompanied by a significant progressive elevation of LPO products (DC, TC, MDA), GSSG in ocular tissues, as well as progressive depletion of GSH and decrease in GSH/GSSG ratio on days 3 and 7.

In the groups treated with NAC, LPO levels were significantly lower there was a significant decrease in LPO products in ocular tissues and a significant increase in antioxidant defense markers compared to untreated animals. In particular, by day 7 the level of DC was 2.5 times lower, TC was 2.19 times lower, and MDA was 4.64 times lower. It is especially worth noting the effect of NAC on the state of the glutathione system in eye tissues. By day 7, level of of GSH increased 1.85 times, while level of GSSG decreased 1.32 times, which was reflected in the ratio between them (GSH/GSSG), which increased 2.44 times.

The experimental results objectively show that NAC parenteral administration reliably replenishes the intracellular pool of reduced glutathione – the main intracellular antioxidant – both by participating in its synthesis via intracellular cysteine delivery and by reducing its consumption and conversion to the oxidized form due to the direct neutralization of free radicals and reactive oxygen species in the eye tissues.

Table 1.

Content of LPO products, indicators of the glutathione system in the eye tissues of rabbits in different groups

*** - p≤0,001

Conclusions.

1. NAC parenteral administration to rabbits with EIU enabled an effective reduction in oxidative stress and an increase in antioxidant potential in ocular tissues both at early stages (day 3) of the inflammatory process and throughout the subsequent 7 days.

2. NAC administration to animals with EIU restored the depleted reserves of glutathione – the key intracellular antioxidant – in the tissues of the anterior segment of the eye.

3. The results obtained confirm the ability of NAC, under conditions of oxidative stress, to cross the blood–ocular barrier, providing a rationale for its use in the form of parenteral injections.

4. Considering that oxidative stress is a major pathogenetic factor initiating ocular tissue damage and inflammation in uveitis the inclusion of NAC in the comprehensive therapy of uveitis may significantly improve the effectiveness of treatment for this pathology.

Reference:

1. Prevalence and Incidence of Uveitis: A Systematic Review and Meta-analysis / Á. García-Aparicio [et al.] // Ophthalmic Epidemiol. – 2021. – Vol. 28, № 6. – P. 461-468.
2. Bottner, K. Social Medical conseguences of Uveitis in childhood and Adolescence: Results of a Pilot Study / K. Bottner, P. Mussinghoff, M. Meyer Zur Horste, C. Heinz, A. Heiligenhaus // Klin Monbl Augenhtilkd. – 2017. – Vol. 234, № 9. – P. 1 – 5.
3. Ung, L. Oxidative stress and reactive oxygen species: a review of their role in ocular disease / L. Ung, U. Pattamatta, N. Carnt, J. L. Wilkinson Berka, G. Liew, A. J. R. White // Clinical Sci. – 2017. – Vol. 131. – P. 2865 – 2883.
4. Кравчук, Е.А. Роль свободнорадикального окисления в патогенезе увеита / Е. А. Кравчук // Офтальмология. – 2005. – № 4. – С. 5 – 9.
5. Калинина, Е. В. Роль глутатиона, глутатионтрансферазы и глутаредоксина в регуляции редокс-зависимых процессов / Е. В. Калинина, Н. Н. Чернов, М. Д. Новичкова // Успехи биологической химии. – 2014. – Т. 54. – С. 299 – 348.
6. Liu, Y. Emerging regulatory paradigms in glutathione metabolism / Y. Liu, A. S. Hyde, M. A. Simpson, J. J. Barycki // Adv Cancer Res. – 2014. – Vol. 122. – P. 69 – 101.
7. Rushworth, G. F. Existing and potential therapeutic uses for N-acetylcysteine: The need for conversion to intracellular glutathione for antioxidant benefits / G. F. Rushworth, I. L. Megson // Pharmacol Ther. – 2014. – Vol. 141, № 2. – P. 150 – 159.
8. Нероев, В.В. Моделирование иммуногенного увеита у кроликов / В. В. Нероев, Г. А. Давыдова, Т. С. Перова // Бюл. Эксп. Биол. Мед. – 2006. – Т. 142, № 11. – С. 598–600.