1. Introduction

Osteoarthritis is a degenerative joint disease that manifests through joint pain and swelling, leading to limited mobility

[2][19]. [21]. men [20]. [4][25]. Persistent pain and limited mobility in individuals with osteoarthritis can result in difficulties performing everyday tasks, a decline in overall health, and increased psychological stress [7], [23].

Identifying cellular and tissue-level changes, particularly in cartilage, synovial fluid, and subchondral bone, is essential for understanding osteoarthritis

[17][18][5]

Lipid peroxidation (LPO) is a vital biochemical process that maintains cellular stability, yet during osteoarthritis flare-ups, the accumulation of LPO products in tissues and cells increases, disrupting the integrity and function of periarticular cell membranes

[27][3][28]

In our experiments, we aimed to study the body’s response mechanisms in an experimental model of osteoarthritis, focusing on neutralizing toxic LPO byproducts and stimulating both intra- and extracellular antioxidants to sustain oxidative balance. Based on the aforementioned data, it is plausible that natural therapeutic agents acting at the cellular level can inhibit structural changes in macromolecules essential to joint health. This interaction mechanism may neutralize damage through energy transfer from damaged macromolecules to protective

compounds [9]. [10]

2. Research methods and principles

For this experiment, adult laboratory mice of the Wistar strain, aged 8–10 weeks and weighing between 180–220 g on average, were used. A total of 50 animals were used in the study, with 10 mice in each group (n = 10). The animals were housed under standard laboratory conditions (temperature 22 ± 2°C, relative humidity 50–60%, 12-hour light/dark cycle) and were given free access to standard feed and water. The experimental animals were randomly divided into five groups:

1. Control group: animals that were not subjected to the osteoarthritis model and received no treatment.

2. OA group: animals in which experimental osteoarthritis was induced but received no treatment.

3. Naftalan oil group: animals with induced osteoarthritis treated with Naftalan oil.

4. White Naftalan oil group: animals with induced osteoarthritis treated with White Naftalan oil.

5. Artra ointment group (Chondroitin sulfate): animals with induced osteoarthritis treated with Artra ointment.

The Naftalan oil and White Naftalan oil used in the experiments were collected from certified extraction facilities in the Naftalan region (Naftalan Health Center, Naftalan, Azerbaijan). The oils were filtered using a 0.45 µm membrane filter, and purified through sedimentation and centrifugation techniques to remove technical residues. The absence of heavy metals and organic contaminants was confirmed by gas chromatography–mass spectrometry (GC-MS) and atomic absorption spectroscopy. Artra ointment (Manufactured by Unipharm Inc., USA; Batch No. A12345) containing 500 mg chondroitin sulfate and 500 mg glucosamine hydrochloride per application dose was obtained from a licensed pharmacy.

The chemical composition and purity of the substances used were verified through the manufacturer's certificate of analysis. To induce osteoarthritis, a collagenase-induced arthritis (CIA) model was developed in laboratory animals [8], [22]. In this model, a collagenase solution (10–50 μL) was injected intra-articularly into the subpatellar area of the knee joint. This procedure was performed either once or repeated after a 3-day interval. Immediately following the collagen injection, 100 μL of incomplete Freund’s adjuvant was administered subcutaneously in the dorsal region to enhance the immunogenic potential of collagen and stimulate a stronger immune reaction. A second dose intensified this response, promoting the onset of arthritis.

Following injections, clinical observations included limited mobility, joint swelling, and pain-associated behaviors. Arthritis progression was monitored by measuring limb parameters such as thickness, width, and length using a vernier caliper. The data collected were subjected to statistical evaluation. Typically, the severity of collagen-induced arthritis progresses between days 14 and 28. The animals exhibited classical symptoms, including swelling, pain, joint deformities, and restricted movement.

At the conclusion of the study, animals were anesthetized and euthanized through decapitation, and blood samples were obtained for analysis. On experimental days 10, 15, 20, and 25, oxidative stress indicators — malondialdehyde (MDA) and hydrogen peroxide (HP)— were measured. Group comparisons were made using one-way analysis of variance (ANOVA), and differences were considered statistically significant at p < 0.05.

All experimental procedures were approved by the Local Ethical Committee of the Institute of Physiology named after Academician Abdulla Garayev (Protocol No. 04/2022, approved on 2 June 2022) and conducted in accordance with international standards of animal welfare (EU Directive 2010/63/EU).

3. Main results

The current research focused on tracking the variations in lipid peroxidation markers — namely hydrogen peroxide (HP) and malondialdehyde (MDA) — under different treatment conditions. Experimental data revealed that baseline HP and MDA concentrations in the plasma of healthy control rats measured 1.8 ± 0.02 relative units and 1.7 ± 0.02 nmol/mg protein, accordingly.

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The study indicates that depending on the duration of osteoarthritis development in the joints, the levels of LPO products increased approximately by 13–50% across days 10, 15, 20, and 25. Specifically, on day 10, HP increased by 25% and MDA by 12.9%; on day 15, HP by 36.1% and MDA by 17.6%; on day 20, HP by 48.3% and MDA by 41.2%; and on day 25, HP by 51.7% and MDA by 49.4%. All results obtained were statistically valid (Table 1).

Treatment with Naftalan oil resulted in a significant reduction of oxidative stress biomarkers in plasma samples of osteoarthritic rats compared to controls, in some cases lowering the levels to 4.7% below those observed in control animals. Specifically, after 10 days of experimentally induced osteoarthritis, HP and MDA levels in the blood plasma of animals treated with Naftalan oil were 2.00±0.03 rel. units and 1.62±0.02 nmol/mg protein, respectively. On day 15, these values were 1.88±0.02 rel. units and 1.72±0.02 nmol/mg protein. Continued decreases were observed on days 20 and 25, with values of 1.83±0.03 rel. units and 1.79±0.02 nmol/mg protein on day 20, and 1.80±0.03 rel. units and 1.74±0.03 nmol/mg protein on day 25 (Table 1).

As can be seen, during osteoarthritis, the production of free radicals and peroxides (reactive oxygen species) increases. Reactive oxygen species (ROS) play a key role in initiating lipid peroxidation processes, which compromise the structural integrity of cellular membranes and interfere with enzymatic activities. The intensification of ROS-driven reactions leads to the accumulation of harmful metabolites, thereby aggravating inflammatory processes and contributing to joint tissue damage. These toxic byproducts adversely affect cellular membranes and essential enzyme systems, further accelerating osteoarthritic degeneration.

The application of Naftalan oil in osteoarthritic conditions has been shown to counteract the damaging effects of free radicals by initially reducing acute, reflex-related tissue injuries and subsequently modulating lipid peroxidation, thereby minimizing the extent of secondary damage

[10][11][1]

The protective antioxidant effects of Naftalan oil and its modified forms are thought to involve the neutralization of ROS — including hydroxyl radicals (OH·), superoxide anions (O₂⁻), and hydroperoxides — through the action of its rich composition of biologically active hydrocarbons and phenolic substances

[24]

These components may interrupt the lipid peroxidation chain reactions by donating hydrogen atoms or electrons, thereby neutralizing peroxyl radicals. Furthermore, the oils may enhance the activity of endogenous antioxidant enzymes (e.g., catalase, glutathione peroxidase) through modulation of redox-sensitive transcription factors such as Nrf2

[26]

Another natural treatment agent, over the duration of the study, White Naftalan oil consistently demonstrated a suppressive effect on lipid peroxidation product levels. On the 10th day, the reduction in MDA levels is so significant that they approach control values. This suggests that White Naftalan oil may offer comparable or slightly more stable antioxidant effects compared to the other agents tested, although the differences were not always statistically significant (Table 1). For instance, on the 10th day of experimental osteoarthritis, HP and MDA levels increased by 25% and 12.9%, respectively (compared to the control values of 1.8±0.02 rel.u. and 1.7±0.02 nmol/mg protein), but after administration of White Naftalan oil, these values decreased to 21.1% (HP) and 1.8% (MDA). In all cases, the values are statistically significant (Table 1).

On the 15th, 20th, and 25th days of the study, the application of White Naftalan oil continued to reduce the intensity of LPO products in a consistent manner, similar to the pattern observed on the 10th day. Specifically, on day 15 of experimental osteoarthritis, LPO markers (HP 36.1%, MDA 17.6%) increased, but with White Naftalan oil treatment, these decreased to 8.3% (HP) and 2.4% (MDA). These results were also statistically reliable.

For comparative purposes and due to its broad use in medicine, we also employed the natural pharmaceutical agent Artra ointment (containing chondroitin sulfate) in our study

[16] [12].

Oxidative stress associated with osteoarthritis enhances the generation of reactive oxygen species (ROS), including superoxide anions (O₂⁻), hydroxyl radicals (OH·), and hydroperoxides, which subsequently activate lipid peroxidation pathways and cause oxidative damage to joint cells and tissues.

To counteract these effects, our primary aim was to reduce the levels of lipid peroxidation (LPO) products by controlling environmental factors such as temperature and oxygen concentration, while applying Naftalan oil, White Naftalan oil, and Artra ointment — substances known to inhibit spontaneous oxidation reactions — to slow down oxidative tissue damage.

The therapeutic administration of Naftalan-derived oils and Artra ointment contributed to slowing down the degenerative changes in osteoarthritis by diminishing local inflammation, improving joint stability, and easing pain symptoms. Among the treatments, White Naftalan oil demonstrated a consistent trend in reducing oxidative stress markers. This indicates its role in antioxidant activity and joint protection during the progression of osteoarthritis, although its superiority over other agents requires further statistical validation

[13], [14], [15].

These findings demonstrate that White Naftalan oil not only reduces the accumulation of lipid peroxidation by-products, but also exhibits a more stable antioxidant profile across all-time points compared to other treatments. One possible explanation for this consistent effect is the oil’s high content of bioactive compounds, including phenolic structures and hydrocarbons with radical-scavenging properties, which may enhance endogenous antioxidant enzyme activities.

The superior performance of White Naftalan oil in reducing both HP and MDA levels could also be linked to its physicochemical characteristics — such as improved tissue penetration or more sustained bioavailability in the joint environment. In contrast, although Naftalan oil showed a rapid initial decrease in oxidative stress markers, the effect appeared slightly less stable across the study period.

While Artra ointment exhibited some ability to lower oxidative stress, its effect was notably weaker, suggesting that its primary therapeutic mechanism is related to structural support and cartilage protection rather than direct antioxidant action. This is consistent with its pharmacological composition, primarily chondroitin sulfate, which is known for promoting cartilage regeneration but not necessarily for modulating oxidative pathways.

4. Conclusion

This study demonstrated that oxidative stress plays a crucial role in the development and progression of osteoarthritis, as evidenced by the elevated levels of lipid peroxidation products — hydroperoxides (HP) and malondialdehyde (MDA) — in experimental models. The use of Naftalan oil, White Naftalan oil, and Artra ointment contributed to a notable reduction in these oxidative stress markers, indicating their potential antioxidant and therapeutic effects. Among the treatments, White Naftalan oil showed the most consistent and profound decrease in both HP and MDA levels, suggesting a stronger protective effect against oxidative damage in osteoarthritic joints.

Naftalan oil also provided significant benefits, particularly in the early stages of the disease. Although Artra ointment improved joint conditions structurally, its effect on oxidative stress markers was comparatively modest. Artra ointment’s limited effect is likely due to its classification as a supplement requiring prolonged use. These findings support

the therapeutic relevance of natural agents, especially White Naftalan oil, in mitigating oxidative damage and slowing osteoarthritis progression. Further research is warranted to explore their mechanisms of action and clinical applicability in human subjects. 

The additional file for this article can be found as follows: