C-reactive protein (CRP) is the inflammation acute phase protein. It was discovered in 1930 by Tillett and Francis. CRP is synthesized by liver cells in response to the production of proinflammatory cytokines (interleukin-1, 6, tumor necrosis factor) [1]. The protein belonging to the family of pentraxins (calcium-dependent ligand-binding plasma proteins) comprises five identical non-glycosylated subunits, 206 amino acid residues each. It became the first laboratory marker of inflammation, tissue damage, and cardiovascular disorders [2–6].

The native pentameric CRP form (nCRP) circulating in blood plasma is capable of local dissociation under the exposure to inflammation or infectious agents. The catalytic factor for such changes may be an alteration in the microenvironment (for example, a temperature increase, calcium ion deficiency, lactic acid concentration increase), resulting in the nCRP transformation into mCRP. In a healthy body, the nCRP concentration < 5 mg/L; with inflammation the value increases 100‒1000-fold. The resulting mCRP exerts a potent proinflammatory effect through interaction with endothelial cells, neutrophils, macrophages, and platelets [7, 8]. The mCRP isoform is insoluble and prone to aggregation and deposition in the sub-endothelial matrix of blood vessels and the synovial membrane, while nCRP is able of partially dissolving in blood plasma. The nCRP function is opsonization of apoptotic cells, due to which activation of phagocytosis occurs [9].

The CRP synthesis rate depends on the disease process intensity [10]. Recently, the hypothesis regarding the role of CRP in the development of neurodegenerative diseases has become widely accepted [11–12]. Obesity is associated with the development of chronic inflammation, during which proinflammatory cytokines and CRP are constantly produced [13–15]. The protein is the main marker of rheumatoid arthritis; it demonstrates inflammation severity in individuals with osteoarthritis [15]. It has been found that CRP levels are higher in patients with radiographic signs of osteoarthritis and that CRP inhibition depends on the anti-inflammatory drug dose [16, 17].

The study aimed to assess plasma concentrations of CRP and its isoforms (nCRP and mCRP) in obese rats with arthrosis depending on administration of different dexamethasone doses throughout 10 days.

METHODS

Arthtosis and obesity modeling, determining the dexamethasone dose

The experiment conducted at the St. Luke Lugansk State Medical University of the Ministry of Health of the Russian Federation involved 180 male white Wistar laboratory rats aged 8–10 weeks with the body weight of 200–250 g. Animals were kept under standard vivarium conditions (22 °C ± 2 °C, 12 h light/dark) with ad libitum access to water and food. Using the "envelope" method, a staff member, who did not take part in the experiment, divided the animals into five groups (n = 36) before the arthrosis and obesity modeling; one group did not take part in the experimental modeling of the disorder. After the arthrosis and obesity modeling 144 animals were randomized into four groups (n = 36) again.

Obesity was simulated by prescribing the high-calorie diet (45% plant and animal fats and 35% carbohydrates) for a period of 8 weeks. The degree of obesity was assessed using the Lee index:

Lee index = 1000 × body weight (g) / naso-anal length (cm).

Animals with the values above 310 and the body weight increase by 25–40% relative to baseline (moderate obesity) were selected for the study [18].

Knee arthrosis was modeled through repeated minimally invasive cartilage damaging. The procedure was performed from the outside in using a thin cable with a frayed tip, introduced into the joint cavity using an injection needle. The cartilage damage was induced by rapid traction through the joint cavity, followed by rigid immobilization of the joint in a physiological position using a plastic splint made from a polymer casting bandage for a period of 4 weeks. The joint area was depilated, then treated with the 70% ethanol solution and anesthetized with 1.0 mL of the 2% lidocaine solution prior to arthrosis modeling. When there were reliable signs of knee osteoarthritis (joint space narrowing, erosions, osteophytes, subchondral sclerosis) on the X-ray images acquired using the Posdion Rextar  X dental system, 70 kV (Posdion, Korea), it was considered a positive result.

After depilation and the injection site treatment with the 70% ethanol solution animals of group I (intact) were intramuscularly administered 1.0 mL of saline (Dalkhimpharm, Russia), group II (osteoarthritis + obesity) was administered 1.0 mL of NaCl; groups III, IV, V (osteoarthritis + obesity) were intramuscularly administered dexamethasone solution with the concentration of 1 ng/mL, 10 ng/mL, 100 ng/mL (1 mL, manufactured by Ellara, Russia); dexamethasone and saline were administered once during the same period. Blood was collected from the tail vein on days 3, 5, and 10. Dexamethasone doses were substantiated as follows: 1 ng/mL is a low and minimally effective concentration allowing one to ensure the minimal antiinflammatory effect without immunosuppression; 10 ng/mL is a therapeutic dose administered to suppress the cytokineinduced inflammation; 100 ng/mL is a high dose used to model a possible “paradoxical" effect upon administration of glucocorticoids. The selected dose range aimed to assess the dose-dependent responses of CRP and its isoforms — from minimal to sub-therapeutic. Such a regimen is fully consistent with the earlier reported dexamethasone anti-inflammatory effect modeling in laboratory animals [19]. The dexamethasone side effects were assessed based on behavioral activity in the open field test [20], body weight and Lee index fluctuations. No apparent side effects were observed upon administration of 1 and 10 ng/mL of dexamethasone; administration of 100 ng/mL resulted in the development of hyperglycemia, decreased activity, which was fully consistent with the known side effects of dexamethasone administration.

Plasma preparation

A total of 1 mL of blood from the lateral tail vein was collected into the test tubes (catalogue number 450531; Greiner BioOne, Austria) with anticoagulant (EDTA) coating. Plasma was obtained by centrifugation at 3000 rpm for 15 min at a temperature of 4 °C, then aliquoted and immediately frozen at –80 °C prior to analysis. Repeated thawing and freezing were avoided in order to avoid the native CRP (nCRP) dissociation into the monomeric form (mCRP).

Determination of plasma total CRP concentrations

CRP concentrations were determined using the commercially available ELISA kits and the diagnostic Rat ELISA Kit (Abcam, UK). Each thawed sample was diluted with buffer solution 1 : 5, then incubated with primary antibodies (100 µL/well) for 2 h at a temperature of 37 °C. After 30 min biotinylated antibodies were added at a temperature of 37 °C. After 30 min streptavidin-HRP was added at a temperature of 37 °C; after 15 min the TMB chromogen was added in complete darkness. Optical density was measured at 450 nm using the BioTek ELx808 microplate reader (BioTek, USA).

Determination of plasma nCRP concentrations

Plasma nCRP concentrations were determined by sandwich ELISA using the commercially available ELISA kit and the diagnostic Rat ELISA Kit (Abcam, UK) in accordance with the manufacturer’s instructions. A total of 100 µL of plasma samples and reference samples were added to the plate wells preliminarily sensibilized using specific antibodies against the rat nCRP. Biotinylated detector antibodies and the avidin– horseradish peroxidase (HRP) conjugate were added after the incubation and washing. Optical density was measured by spectrophotometry at the wavelength of 450 nm using the BioTek ELx808 reader (BioTek, USA).

Determination of plasma mCRP concentrations

mCRP concentrations were determined by the conformation specific sandwich ELISA in accordance with the manufacturer’s instructions. Microplates were adsorbed overnight at 4 °C using a monoclonal antibody specifically recognizing the mCRP cryptic neoepitope (clone 9C9 or 3H12, concentration of 2 µg/mL in the carbonate-bicarbonate buffer, pH 9.6). After blocking free binding sites with the 1% bovine serum albumin (BSA) solution in PBS, plasma samples (diluted 1 : 10) were added to the well and incubated for 2 h at 37 °C. Reference samples of purified native pentameric CRP (catalogue number PHP277; Bio-Rad Laboratories, Inc., UK) were used as a negative control to confirm that there were no cross-reactivity and spontaneous pentamer dissociation during the analysis. Detection was performed using goat polyclonal antibodies against rat nCRP (catalogue number A110-102P; Bethyl Laboratories, Inc., USA) conjugated with the horseradish peroxidase (HRP). The reaction was visualized using the tetramethylbenzidine (TMB) substrate and stopped by adding 2M H₂SO₄. Optical density was measured at 450 nm. The lower limit of mCRP detection was 1.0 ng/mL.

Statistical analysis of the results

Statistical data processing was performed using the Statistica 10.0 software. The normally distributed data (Shapiro–Wilk test) were presented as the mean and standard deviation (М ± SD). Significance of differences between experimental associated with the variance inequality (Levene’s test) was assessed with the Kruskal–Wallis test with subsequent pairwise comparison based on the Student’s t-test using Bonferroni correction. Differences in mean values of the indicators between the  groups and the control group with 95% confidence intervals were used as a measure of the effect. Contributions of the studied factors (effect size) were assessed by calculating the partial eta squared (η²). The differences between groups were considered significant at p < 0.05.

RESULTS

During the study we managed to determine the relationship between obesity associated with arthrosis and adjustment with various dexamethasone doses on days 3, 5, and 10. In group II, CRP concentrations increased due to inflammation; inflammation reduction occurred when using dexamethasone. In group III, CRP concentrations decreased on day 3, while on days 5 and 10 CRP concentrations increased exceeding the values of group II. In groups IV and V, dexamethasone showed the anti-inflammatory effect throughout five days, the effect decreased by day 10, and CRP concentrations were lower compared to group II (tab. 1).

The analysis of CRP values in group II revealed the increase by 2.1% on day 3, η² = 0.005; by 26.4% on day 5, η² = 0.378; by 174.5% on day 10, η² = 0.933 compared to the control group. In group III, there was a decrease in CRP concentrations by 7.9%, η² = 0.089 and by 9.82%, η² = 0.131 on day 3; the increase by 44.2%, η² = 0.632 and by 14.07%, η² = 0.210 was observed on day 5; by 231.6%, η² = 0.919 and by 20.82%, η² = 0.551 on day 10 compared to groups I and II, respectively. In group IV, protein concentrations decreased by 26.1%, η² = 0.498 and by 27.64%, η² = 0.530 on day 3; by 7.9%, η²= 0.069 and by 27.22%, η² = 0.724 on day 5; the increase by 152.3%, η² = 0.951 and the decrease by 8.07%, η² = 0.224 compared to groups I and II, respectively, were reported on day 10. With the dexamethasone dose increase in group V CRP values decreased by 21.4%, η² = 0.425 and by 23.16%, η² = 0.466 on day 3; by 9.52%, η² = 0.091 and by 28.44%, η² = 0.740 on day 5; the increase by 149%, η² = 0.935 and the decrease by 9.10%, η² = 0.274 compared to groups I and II, respectively, were observed on day 10.

The graph clearly demonstrates dynamic changes in CRP concentrations in the studied groups throughout the experiment (fig. 1).

The pentameric form (nCRP) monitoring showed slight changes in the concentration, even under the exposure to dexamethasone. In group II, the CRP concentration increased on day 3 and then decreased, reaching its minimum on day 10.

In group III, the nCRP concentration reached its maximum on day 3 and then decreased to reach its minimum on day 10. In group IV, the maximum concentration was observed on day 3; it decreased on day 5, remaining higher compared to groups II and III, to reach its minimum on day 10. In group V, the nCRP concentration was higher compared to group I on days 3 and 5 and higher compared to group II on day 5; it decreased on day 10, but was higher compared to group II (tab. 2).

The analysis of nCRP levels in group II showed the concentration increase by 11.3%, η² = 0.505 by day 3; it decreased by 5.4%, η² = 0.072 on day 5 and by 25.7%, η² = 0.651 on day 10 compared to the control group. In group III, the levels of this CRP isoform decreased by 3%, η² = 0.039 and by 12.96%, η² = 0.580 on day 3 under the exposure to dexamethasone; the concentration decreased by 7.6%, η² = 0.250 and by 2.33, η² = 0.022 on day 5; by 28.5%, η² = 0.798 and by 3.84, η² = 0.083 on day 10 compared to groups I and II, respectively. In group IV, the protein concentration decreased by 3%, η² = 0.064 and by 12.96, η² = 0.640 on day 3; the concentration decreased by 4.4%, η² = 0.034 and increased by 1.16%, η² = 0.002 on day 5; it decreased by 22.8%, η² = 0.580 and increased by 3.84%, η² = 0.022 on day 10 compared to groups I and II, respectively. In group V, the nCRP concentration increased by 1%, η² = 0.002 and decreased by 9.26%, η² = 0.385 on day 3; the concentration increased by 1.1%, η² = 0.002 and by 6.98%, η² = 0.052 on day 5; it decreased by 18.1%, η² = 0.626 and increased by 10.25, η² = 0.337 on day 10 compared to groups I and II, respectively. Fig. fig. 2 shows dynamic native CRP concentration changes in the studied groups throughout the experiment.

The monomeric CRP analysis showed a significant concentration increase in group II throughout the experiment, with the maximum value reported on day 10. In group III, dexamethasone led to the mCRP concentration decrease on days 3 and 5; the concentration significantly increased on day 10, exceeding the values of groups I and II. In groups IV and V, dexamethasone contributed to the mCRP concentration decrease throughout 5 days; on day 10 the protein levels increased compared to group I, remaining lower compared to group II (tab. 3).

In group II, the mCRP analysis revealed the concentration increase by 91%, η² = 0.977 on day 3; by 161.8%, η² = 0.987 on day 5; by 287.1%, η² = 0.997 on day 10 compared to the control group. In group III, the plasma mCRP concentration decreased by 5.9%, η² = 0.101 and by 50.77%, η² = 0.986 on day 3; the concentration increased by 7.8%, η² = 0.080 and decreased by 58.80, η² = 0.969 on day 5; it increased by 360.2%, η² = 0.999 and by 18.89%, η² = 0,953 on day 10 compared to groups I and II, respectively. In group IV, the concentration decrease by 32.6%, η² = 0.790 and by 64.77% η²= 0.993 was reported on day 3, no changes were reported on day 5 (0% η² = 0.000), as well as the decrease by 61.80%, η² = 0.991; a significant concentration increase by 270.9%, η² = 0.992 and the decrease by 4.17%, η² = 0.274 were reported on day 10 compared to groups I and II, respectively. In group V, mCRP levels decreased by 19.8%, η² = 0.507 and by 58.03%, η² = 0.984 on day 3; the levels increased by 6.7%, η² = 0.126 and decreased by 59.23%, η² = 0.985 on day 5; the concentration increased by 280.6%, η² = 0.994 and decreased by 1.67%, η² = 0.069 on day 10 compared to groups I and II, respectively. 

The dynamic changes in plasma CRP concentrations of obese rats with arthrosis are shown in fig. 3.

DISCUSSION

The blood CRP concentration increases due to pro-inflammatory cytokines released by adipose tissue against the background of obesity and arthrosis [1]. Arthrosis combines with obesity results in the CRP concentration increase throughout the experiment, and the maximum concentration compared to the control group is achieved by day 10. In the experimental rat model of obesity combined with arthrosis plasma CRP concentrations increase, which confirms that there’s inflammation in the body [2–6]. The total CRP levels decrease on day 3 against the background of the administration of 1 ng/mL of dexamethasone, which suggests the inflammation reduction; on days 5 and 10, the concentration increases considerably, even compared with the group having obesity combined with arthrosis. Upon dexamethasone administration throughout 3–7 days against the background of acute ischemia of lower limbs the CRP concentration decreases 2-fold compared to patients, receiving no dexamethasone, which is fully consistent with our research [21].

Such data may suggest low effect of the long-term use of low-dose dexamethasone on the inflammation severity. The use of 10 ng/mL of dexamethasone has a more pronounced effect; the CRP concentration increases on day 10, remaining lower that in the group with obesity and arthrosis. The active substance dose increase to 100 ng/mL results in the more pronounced CRP level decrease: on day 10 the values were higher only compared with the control group. The dexamethasone longterm administration does not have a significant positive effect on the inflammation severity [22]. According to the literature data, the long-term use of glucocorticosteroids can lead to apoptosis of chondrocytes and progression of arthrosis with the risk of developing steroid diabetes, a possible decrease in the quantity of the dexamethasone receptor apparatus [23]. In contrast, when treating COVID-19, dexamethasone showed good results specifically during prolonged administration — over a period of 18–19 days. Efficacy is demonstrated by the reduction in CRP levels as a marker of inflammation from 65.49 ± 71.4 mg/L upon hospital admission to 9.52 ± 12.81 at discharge; the average treatment duration was 19.4 ± 9.0 days [24]. When administering high-dose dexamethasone to treat pneumonia, over the period of 10 days fatal outcomes were reported for nine patients out of 98, and the CRP concentration decreased from 132 mg/L to 7.3 mg/L [25].

In the body, nCRP represents a more stable form consisting of five identical subunits. It performs an anti-inflammatory function and contributes to maintaining homeostasis [9]. In the context of obesity combined with arthrosis, its concentration increases on day 3, which suggests that the protein has an antiinflammatory effect, and then the concentration decreases on days 5 and 10. Since even the low-dose dexamethasone has a pronounced anti-inflammatory effect, the nCRP concentration increases after administration of 1 ng/mL a day, and then decreases. This may suggest suppression of the inflammation caused by arthrosis and obesity; in other groups, the nCRP concentration remains stably high relative to the group having obesity combined with arthrosis, since inflammation suppressed by the active substance shows no pronounced fluctuation depending on the dose.

In the body, mCRP causes the inflammation development and progression, creates the risk of cardiovascular disorders due to local accumulation in the tissues, as well as the risk of neurodegenerative processes [7, 11, 12]. In obesity combined with arthrosis, its concentration increases throughout the experiment, confirming the inflammation progression. In the context of the administration of 1 ng/mL of dexamethasone, the mCRP concentration decreases on days 3 and 5 and rapidly increases on day 10, which suggests ineffectiveness of prolonged dexamethasone administration. The same is observed when administering 10 and 100 ng/mL of dexamethasone, manifesting as a decrease in the monomeric CRP form concentration on days 3 and 5, followed by the increase on day 10. High-dose dexamethasone shows its efficacy upon short-term single administration. Thus, administration of 1 ng/mL of dexamethasone to patients with the body mass index of 26 results in the CRP concentration decrease in the postoperative period following intervention on the abdominal cavity internal organs [26].

High-dose dexamethasone does not lead to significant CRP concentration changes after the total arthroplasty [27]. Such a variable effect it likely to be associated with different effects of dexamethasone on tissue inflammation in the internal organs and joints.

CONCLUSIONS

The combination of experimental obesity and arthrosis results in the persistent systemic inflammatory response characterized by the CRP concentration increase. Such changes occur against the backdrop of the continuous depletion of the native anti-inflammatory pentameric form (nCRP) and the increase in the pro-inflammatory monomeric form (mCRP). Administration of dexamethasone at a dose of 1 ng/mL leads to a short-term anti-inflammatory effect on day 3, and then efficacy decreases. Dexamethasone at a dose of дозе 10 ng/mL remains effective throughout five days, and then efficacy decreases. Efficacy of all dexamethasone doses decreases by day 10, which manifests as an increase in mCRP levels. The prospects of further research include exploration of the more prolonged effects of dexamethasone and other doses on the CRP concentration in inflammation with the assessment of delayed and late effects.