Thiolactone DL-homocysteine, tris(hydroxymethyl)aminomethane, o-phenylenediamine, hydrogen peroxide, sodium dodecyl sulfate, Coomassie Blue R-250, gelatin, acrylamide, and N,N'-methylenebisacrylamide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Monoclonal antibodies anti-INFγ, anti-IL-1β, anti-IL4, anti-IL-10, anti-IL-12, and anti-MMP-2 were purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents used in this study were of analytical grade quality and available commercially.

A total of 60 albino non-linear male rats were used in the study. All experiments on animals were performed in compliance with the international principles of the European Convention to protect vertebrate animals used for experimental and other scientific purposes (Strasbourg, 1986). The Ethical Committee approved the study of the Taras Shevchenko National University of Kyiv (protocol №3 approved 25.11.2020). The experiments were started after seven days of animal acclimation in the animal facility of the Taras Shevchenko National University of Kyiv, maintained under constant conditions of temperature (22 ± 3°C), humidity (60 ± 5%), and light (12 h light/12 h dark cycle). Standard rodent food and water were provided ad libitum. Animals of different ages were used in the current study: one-month-old rats (90 - 100 g) corresponded to young animals; six-month-old rats (190 - 230 g) corresponded to adult animals, and twenty-month-old rats (290 - 320 g) corresponded to old animals.

Hyperhomocysteinemia was induced by intragastric administration of DL-homocysteine thiolactone (Sigma-Aldrich, USA) diluted in 1 % starch solution (100 mg·kg^-1 of body weight). DL-homocysteine thiolactone was administered using a nelaton catheter (size 6) once daily for 28 days11. The control rats received an equal volume of 1 % starch. Thus, there were 6 experimental groups with 10 animals in each: 1) Control_young, 2) HC_young, 3) Control_adult, 4) HC_adult, 5) Control_old, and 6) HC_old. HC development was confirmed by the high blood level of homocysteine (above 15 µmol·L^-1). The level of homocysteine in blood plasma was determined by enzyme-linked immunosorbent assay using the kit «Homocysteine EIA» (Axis-Shield, UK). On the 29^th day since the start of the experiment, animals were sacrificed. The animals of the groups Control_young and HC_young were killed by cervical dislocation. The animals of the groups Control_adult, HC_adult, Control_old, and HC_old, were killed by decapitation.

To obtain 10% homogenate of the thyroid gland, 1 g of tissue was homogenized in 9 ml of ice-cold 50 mM Tris-HCl buffer pH 7.4 and centrifuged at 12,000 g for 30 min at +4°C. The supernatant was collected and used for further studies. The Bradford method determined the protein concentration using crystalline bovine serum albumin as a standard12.

The levels of MMP-2 and cytokines were estimated by enzyme-linked immunosorbent assay (ELISA) according to the standard instructions13. ELISA plates were coated overnight at +4°C with samples of thyroid gland previously diluted with Tris–HCl buffer pH 7.4 to a protein concentration of 10 μg·mL^-1. The next day, the plates were washed and blocked with 5% nonfat dry milk for 1 h at 37 °C. After that, plates were incubated for 1 h at +37 °C with the primary antibodies against IFN-g, IL-1β, IL-12, IL-4, IL-10, and MMP-2. Next, plates were washed and incubated for 1 h at +37 °C with the secondary antibodies conjugated to horseradish peroxidase. After washing, substrates (o-phenylenediamine and hydrogen peroxide) were added. The addition of 2.5 N H2SO4 stopped the reaction. The absorbance was read at 492 nm by a microplate reader (mQuant™, BioTek Instruments Inc, USA).

The fraction of LMWS was isolated according to the method described by Nikolajchik et al. (1991)14. First, the supernatant was mixed with 1.2 M HClO₄ at a 1:1 (v/v) ratio to precipitate the proteins. Then, after centrifugation at 10 000 g for 20 min at 4°C, the supernatant was neutralized by 5 M KOH to pH 7.0, and the sample was subjected to the centrifugation step again. After ethanol was added to the final concentration of 80%, the samples were kept at 4°C for 30 min and centrifuged. The optical density of the supernatants was determined with a spectrophotometer Smart SpecTMPlus (BioRad, USA) at 210 nm, 254, and 280 nm. The level of LMWS was expressed as rel. units per g of thyroid gland tissue.

SDS-PAGE was carried out using 4% (w/v) stacking gel and 10% (w/v) separating gel15. SDS-PAGE was performed using Mini-Protean Tetra System (Bio Rad, USA) at 19 mA for stacking and 36 mA for separating gels. Samples were prepared by mixing with sample buffer (0.05 M Tris, pH 8.8, 2% SDS, 5% sucrose, and 0.02% bromophenol blue) at a 1:1 (v/v) ratio. Then, samples were heated at +95 ºC for 1 min before loading into the gel. The total amount of proteins was 20 µg per well. The gels were stained with 2.5% Coomassie brilliant blue R-250 in 10% (v/v) ethanol, 10% (v/v) acetic acid, and 15% (v/v) isopropanol. Furthermore, the molecular weights of proteins were calculated using a protein calibration mixture (Bio Rad, USA).

Zymography was carried out according to the following method16. The separating gel solution (12%) was polymerized in the presence of gelatin (1 mg per mL), and samples were not heated to save enzymatic activity. After electrophoresis, the gels were soaked in 2.5% Triton Х-100 solution with gentle shaking (30 min at +25°C) to remove SDS and renature proteins. Next, the gels were washed with distilled water for 10 min to remove Triton X-100 and then incubated in 50 mM Tris-HCl pH 7.5 at +37 °C for 12 h. The clear areas on the gel indicate the presence of active enzymes. The TotalLab 2.04 program was used to analyze the resultant electropherograms. The represented electropherogram and zymogram are typical for a series of repeated experiments (at least three in each series).

Statistical analysis was performed with Statistica 8.0 software, and figures were plotted using OriginLab 9.1 version software. The data of biochemical estimations were reported as mean ± SEM for each group (n = 10). In addition, the Kolmogorov-Smirnov test was used to verify the normal distribution of results. Statistical analyses were performed using a one-way analysis of variance (ANOVA). Differences were considered to be statistically significant when p < 0.05.

Table 1

Experimental groups	Homocysteine level, µmol·L-1
Control_young	5.45±0.44
HC_young	17.58±1.77*
Control_adult	5.65±0.91
HC_adult	18.15±0.76 **
Control_old	7.35±0.39
HC_old	21.82±2.15***

The data is presented as mean ± SEM (n = 10); * p < 0.05 significantly different from the group “Control_young”; ** p < 0.05 significantly different from the group “Control_adult”; *** p < 0.05 significantly different from the group “Control_old”

Table 2

Experimental groups	Blood plasma LMWS, rel. units·(mL of blood) -1		Thyroid gland LMWS, rel. units·(g of tissue)-1
	Registered at 254 nm	Registered at 280 nm	Registered at 254 nm	Registered at 280 nm
Control_young	0.32 ± 0.03	0.22 ± 0.05	2.87 ± 0.11	3.51 ± 0.16
HC_young	0.62 ± 0.06	0.72 ± 0.07	7.63 ± 0.34*	8.56 ± 0.40*
Control_adult	0.55 ± 0.09	0.29 ± 0.03	3.81 ± 0.17	2.23 ± 0.11
HC_adult	1.45 ± 0.07	0.97 ± 0.10	7.64 ± 0.35**	8.92 ± 0.43**
Control_old	0.70 ± 0.09	0.31 ± 0.04	4.71 ± 0.23	1.71 ± 0.075
HC_old	2.75 ± 0.10	1.01 ± 0.05	9.52 ± 0.46***	9.36 ± 0.45***

The data is presented as mean ± SEM (n = 10); * p < 0.05 significantly different from the group “Control_young”; ** p < 0.05 significantly different from the group “Control_adult”; *** p < 0.05 significantly different from the group “Control_old”

Table 3

Experimental groups	Pro-inflammatory cytokines					Anti-inflammatory cytokines
	TNFα	INFγ	IL-1b	IL-6	IL-8	IL-4	IL-10
	rel. units·(mg of protein)-1
Control_young	29.5 ± 2.3	49.7 ± 2.3	43.5 ± 4.2	50.7 ± 3.8	33.0 ± 6.2	24.5 ± 3.4	55.0 ± 3.4
HC_young	76.5 ± 3.6*	63.2 ± 3.4*	87.2 ± 4.4*	91.5 ± 2.9*	92.0 ± 4.2*	78.2 ± 5.7*	23.5 ± 1.7*
Control_adult	43.2 ± 1.2	56.7 ± 3.7	44.2 ± 1.9	46.5 ± 4.7	36.7 ± 4.9	26.5 ± 1.8	90 ± 4.9
HC_adult	87.7 ± 4.5**	66.7 ± 1.9**	107.5 ± 3.9**	101.2 ± 5.6**	93.5 ± 3.7**	77.5 ± 5.2	42.7 ± 2.7**
Control_old	158.3 ± 2.5	60.0 ± 3.5	46.3 ± 2.7	43.7 ± 2.7	39.1 ± 4.2	27.6 ± 2.3	74.9 ± 5.1
HC_old	115.6 ± 5.3***	120.0 ± 6.7***	110.2 ± 5.6***	109.8 ± 4.5***	107.1 ± 5.5***	65.5 ± 4.7***	31.8 ± 2.7***

The data is presented as mean ± SEM (n = 10); * p < 0.05 significantly different from the group “Control_young”; ** p < 0.05 significantly different from the group “Control_adult”; *** p < 0.05 significantly different from the group “Control_old”

To confirm the state of HC, the plasma level of homocysteine was evaluated. This parameter increased 3.22, 3.21, and 2.96 times in the rats of the HC_young, HC_adult, and HC_old groups compared with the corresponding controls (Table 1), indicating the development of moderate HC17.

It was found that HC causes an increase in the level of LMWS in the blood plasma (Table 2). The concentrations of LMWS registered at 254 nm in the blood of rats in the HC_young, HC_adult, and HC_old groups increased by 1.93, 2.63, and 3.92 times compared with the controls. Further, the concentration of LMWS registered at 280 nm was more than 3-fold higher in the rats with HC than in the rats in the control groups. An elevated level of LMWS in the thyroid gland of HC rats was also observed. The level of LMWS registered at 254 nm was increased 2.7 times in the rats of group HC_young compared to the control value. This parameter for the rats of groups HC_adult and HC_old was found to be 2-fold higher than that in the controls.

It should be noted that the physiological level of LMWS increased with age and was 2.87 ± 0.11 rel. units∙(g of tissue)^-1 in the rats of group Control_young, 3.81 ± 0.17 rel. units∙(g of tissue)^-1 in the rats of group Control_adult, and 4.71 ± 0.23 rel. units∙(g of tissue)^-1 in group Control_old rats. According to the obtained data, the level of LMWS registered at a wavelength of 280 nm changed more significantly. This value increased 2.4-fold in rats of the HC_young group, fourfold in the HC_adult group, and fivefold in the rats of group HC_old.

A significant increase in the contents of all pro-inflammatory cytokines in the thyroid gland of the rats with HC was observed compared with the corresponding controls (Table 3). In most cases, the levels of cytokines were more than twice as high as those in the control. The most pronounced changes were detected for IL-8 as the level of this cytokine increased 3, 2.5, and 2.7 times, respectively, in the thyroid gland of rats of HC_young, HC_adult, and HC_old groups. Furthermore, the level of IL-4 exceeded the control 3.2 times in the rats of the HC_young group, 2.9 times in the HC_adult group, and 2.4 times in the HC_old group. In contrast, the level of IL-10 was reduced in the animals of all experimental groups.

Figure 1

Figure 2

Table 4

Protein fraction, kDa	Experimental groups
	Control_young	HC_young	Control_adult	HC_adult	Control_old	HC_old
> 150	6.92 ± 0.34	11.41 ± 0.56*	6.50 ± 0.23	-	4.71 ± 0.21	-
150 - 100	0.81 ± 0.03	2.38 ± 0.11*	6.42 ± 0.23	6.00 ± 0.27**	8.68 ± 0.41	5.11 ± 2.47***
100 - 70	1.72 ± 0.08	2.10 ± 0.10*	1.61 ± 0.06	1.45 ± 0.06**	2.45 ± 0.11	1.20 ± 0.03***
70 - 50	29.53 ± 1.47	13.54 ± 0.67*	15.84 ± 0.75	8.63 ± 0.41**	14.61 ± 0.71	5.17 ± 2.50***
50 - 30	1.81 ± 0.08	4.79 ± 0.23*	5.35 ± 0.36	7.21 ± 0.33**	4.88 ± 0.22	3.54 ± 0.16***
˂ 30	59.36 ± 2.71	65.5 ± 3.11*	64.1 ± 3.11	76.55 ± 3.78**	64.74 ± 3.21	84.96 ± 4.15***

The data is presented asmean ± SEM (n = 10); *p < 0.05 significantly different from the group “Control_young”; **p < 0.05 significantly different from the group “Control_adult”; ***p < 0.05 significantly different from the group “Control_old”

Figure 3

Figure 4

A significant decrease was observed in the level of peptides in the rats of the HC_young group (2.4 times) compared to the thyroid glands of the control animals. The opposite changes were found in the rats of the HC_adult and HC_old groups, where peptides levels increased 1.7 and 1.2 times, respectively (Figure 1). Findings further revealed some changes in the protein composition, which were more pronounced in aged rats with HC (Figure 2, Table 4). The electropherogram was analyzed using the Total Lab 2.04 software to define the exact molecular weights of proteins. The total amount of proteins in each gel line was set up as 100%, and the level of a particular protein fraction was expressed as a percentage of the total value. According to the obtained data (Table 3), the percentage of proteins with a molecular weight of 50-70 kDa was reduced 2.2 times in the rats of group HC_young. In contrast, the levels of other protein fractions were higher than those in the corresponding controls. Also, a slight decrease in the protein fractions corresponding to proteins with molecular weights of 100-150 kDa (6%) and 70-100 kDa (14%) was found in the thyroid gland of rats in the HC_adult group. On the other hand, the proteins with molecular weights less than 30 kDa were increased in the rats of groups HC_adult and HC_old.

The presence of active enzymes was analyzed using zymography. Following the principle of this method, the appearance of clear areas on the dark background of gel indicates the presence of active enzymes; the more intense the areas, the more active the enzymes are. In addition, this method can be used to estimate the apparent molecular weight of active enzymes. As shown in Figure 3, the samples of thyroid glands of the control rats and rats with HC have the same number of clear areas, but the intensity of these areas differed as there are two bands at the enzyme-electropherogram. One is localized in the region corresponding to 65-80 kDa proteins, and the other is in the region of 25 kDa proteins. Since zymography was performed using gelatin as a substrate protein, clear bands in the region of 65-80 kDa may be associated with the action of MMP-2 and/or MMP-9, the molecular weight of which is 65 kDa and 85 kDa. Therefore, the level of MMP-2 in the thyroid glands of rats with HC was analyzed. As depicted in Figure 4, HC was accompanied by a 2-fold increase in MMP-2 level in the rats of group HC_young. This parameter in the rats of groups HC_adult and HC_old was 2.7 and 3.1 times higher than the corresponding controls.