Astaxanthin, Compared to Other Carotenoids, Increases the Efficacy of Methotrexate in Rat Adjuvant Arthritis

Abstract

This in vivo study performed in rat adjuvant arthritis aims to advance the understanding of astaxanthin's therapeutic properties for the possible treatment of rheumatoid arthritis (RA) in monotherapy and along with the standard RA treatment, methotrexate (MTX), in combination therapy. The main goal was to elucidate astaxanthin's full therapeutic potential, evaluate its dose dependency, and compare its effects in monotherapy with other carotenoids such as β-carotene and β-cryptoxanthin (KXAN). Moreover, potential differences in therapeutic activity caused by using different sources of astaxanthin, synthetic (ASYN) versus isolated from Blakeslea trispora (ASTAP), were evaluated using one-way ANOVA (Tukey-Kramer post hoc test). KXAN was the most effective in reducing plasma MMP-9 levels in monotherapy, significantly better than MTX, and in reducing hind paw swelling. The differences in the action of ASTAP and ASYN have been observed across various biometric, anti-inflammatory, and antioxidative parameters. In combined therapy with MTX, the ASYN + MTX combination proved to be better. These findings, especially the significant anti-arthritic effect of KXAN and ASYN + MTX, could be the basis for further preclinical studies.

Keywords: adjuvant arthritis; astaxanthin; beta-carotene; beta-cryptoxanthin; carotenoids; inflammation; methotrexate; oxidative stress.

Figure 1

The presented data show the change in animal body weight measured on days 7, 14, 21, and 28 of the pilot experiment. The experimental animals were divided as follows: HC—healthy control group; AA—group of untreated rats with adjuvant arthritis (AA); MTX—rats with AA treated with methotrexate at a dose of 0.3 mg/kg twice weekly (MTX); AS1—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 1 mg/kg daily; AS2—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 5 mg/kg daily. Data are expressed as mean ± SEM. Statistical significance was evaluated by applying ANOVA for independent variables: *** p < 0.001 vs. HC group, +++ p < 0.001 vs. AA group, + p < 0.05 vs. AA group.

Figure 2

The presented data show the change in the hind paw volume measured on days 7, 14, 21, and 28 of the pilot experiment. The experimental animals were divided as follows: HC—healthy control group; AA—group of untreated rats with adjuvant arthritis (AA); MTX—rats with AA treated with methotrexate at a dose of 0.3 mg/kg twice weekly (MTX); AS1—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 1 mg/kg daily; AS2—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 5 mg/kg daily. Data are expressed as mean ± SEM. Statistical significance was evaluated by applying ANOVA for independent variables: *** p < 0.001 vs. HC group, +++ p < 0.001 vs. AA group, + p < 0.05 vs. AA group.

Figure 3

The activity of gamma-glutamyl transferase in the spleen (a), the level of IL-17A in the plasma (b), and the level of MMP-9 in the plasma (c) of the experimental animals measured on day 28 of the pilot experiment. The experimental animals were divided as follows: HC—healthy control group; AA—group of untreated rats with adjuvant arthritis (AA); MTX—rats with AA treated with methotrexate at a dose of 0.3 mg/kg twice weekly (MTX); AS1—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 1 mg/kg daily; AS2—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 5 mg/kg daily. Data are expressed as mean ± SEM. Statistical significance was evaluated by applying ANOVA for independent variables: *** p < 0.001 vs. HC group, +++ p < 0.001 vs. AA group, ++ p < 0.01 vs. AA group, / p < 0.05 vs. AS1 group.

Figure 4

The presented data show the plasmatic level of total CoQ₉ (a) and the level of reduced CoQ₉ in the liver (b) of experimental animals measured on day 28. The experimental animals were divided as follows: HC—healthy control group; AA—group of untreated rats with adjuvant arthritis (AA); MTX—rats with AA treated with methotrexate at a dose of 0.3 mg/kg twice weekly (MTX); AS1—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 1 mg/kg daily; AS2—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 5 mg/kg daily. Data are expressed as mean ± SEM. Statistical significance was evaluated by applying ANOVA to independent variables.

Figure 5

The evaluation of hepatic relative mRNA expressions of heme oxygenase subtype 1 (HO-1) (a), interleukin 1β (IL-1β) (b), and platelet-activating factor-acetylhydrolase (PAF-AH) (c) on day 28. The experimental animals were divided as follows: HC—healthy control group; AA—group of untreated rats with adjuvant arthritis (AA); MTX—rats with AA treated with methotrexate at a dose of 0.3 mg/kg twice weekly (MTX); AS1—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 1 mg/kg daily; AS2—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 5 mg/kg daily. Data are expressed as mean ± SEM. Statistical significance was evaluated by applying ANOVA for independent variables: ** p < 0.01 vs. HC group, *** p < 0.001 vs. HC group, + p < 0.05 vs. AA group, ++ p < 0.01 vs. AA group, ## p < 0.01 vs. MTX group, / p < 0.05 vs. AS1 group.

Figure 6

The effect of two doses of astaxanthin in monotherapy on parameters related to antioxidant status measured in erythrocytes—the superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activity (a–c), and in plasma—the Trolox equivalent antioxidant capacity (TEAC) and lipoperoxides (LPx) (d,e) of experimental animals on day 28. The experimental animals were divided as follows: HC—healthy control group; AA—group of untreated rats with adjuvant arthritis (AA); MTX—rats with AA treated with methotrexate at a dose of 0.3 mg/kg twice weekly (MTX); AS1—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 1 mg/kg daily; AS2—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 5 mg/kg daily. Data are expressed as mean ± SEM. Statistical significance was evaluated by applying ANOVA to independent variables: * p < 0.05 vs. HC group, + p < 0.05 vs. AA group, ++ p < 0.01 vs. AA group, +++ p < 0.001 vs. AA group, ## p < 0.01 vs. MTX group, ### p < 0.001 vs. MTX group, / p < 0.05 vs. AS1 group, // p < 0.01 vs. AS1 group.

Figure 7

The presented data show the change in animal body weight measured on days 7, 14, 21, and 28 of the pivotal experiment. The experimental animals were divided as follows: HC—healthy control group; AA—group of untreated controls with adjuvant arthritis (AA); MTX—rats with AA treated with methotrexate (MTX) at a dose of 0.3 mg/kg twice weekly; ASTAP—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily, ASTAP + MTX—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily and in a combination with MTX at a dose of 0.3 mg/kg twice weekly; ASYN—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily; ASYN + MTX—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily and in combination with MTX at a dose of 0.3 mg/kg twice weekly; BEKA—group of rats with AA receiving β-carotene in the oral dose of 20 mg/kg daily; KXAN—group of rats with AA receiving β-cryptoxanthin in the oral dose of 10 μg/kg daily. Data are expressed as mean ± SEM. Statistical significance was evaluated using ANOVA for independent variables: *** p < 0.001 vs. HC group, ++ p < 0.01 vs. AA group.

Figure 8

The presented data show the percentual change of the hind paw volume of experimental animals measured on 14, 21, and 28 days during the pivotal experiment. HC—healthy control group; AA—group of untreated controls with adjuvant arthritis (AA); MTX—rats with AA treated with methotrexate (MTX) at a dose of 0.3 mg/kg twice weekly; ASTAP—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily; ASTAP + MTX—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily and in a combination with MTX at a dose of 0.3 mg/kg twice weekly; ASYN—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily; ASYN + MTX—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily and in combination with MTX at a dose of 0.3 mg/kg twice weekly; BEKA—group of rats with AA receiving β-carotene in the oral dose of 20 mg/kg daily; KXAN—group of rats with AA receiving β-cryptoxanthin in the oral dose of 10 μg/kg daily. Data are expressed as mean ± SEM. Statistical significance was evaluated using ANOVA for independent variables: *** p < 0.001 vs. HC group, + p < 0.05 vs. AA group, ++ p < 0.01 vs. AA group, +++ p < 0.001 vs. AA group.

Figure 9

The presented data show the arthrogram by using a line graph. The experimental animals were divided as follows: HC—healthy control group; AA—group of untreated controls with adjuvant arthritis (AA); MTX—rats with AA treated with methotrexate (MTX) at a dose of 0.3 mg/kg twice weekly; ASTAP—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily; ASTAP + MTX—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily and in a combination with MTX at a dose of 0.3 mg/kg twice weekly; ASYN—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily; ASYN + MTX—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily and in combination with MTX at a dose of 0.3 mg/kg twice weekly; BEKA—group of rats with AA receiving β-carotene in the oral dose of 20 mg/kg daily; KXAN—group of rats with AA receiving β-cryptoxanthin in the oral dose of 10 μg/kg daily. Data are expressed as mean ± SEM. Statistical significance was evaluated using ANOVA for independent variables: * p < 0.05 vs. HC group, ++ p < 0.01 vs. AA group.

Figure 10

The presented data show the activity of GGT in the spleen of experimental animals measured on day 28 of the pivotal experiment. The experimental animals were divided as follows: HC—healthy control group; AA—group of untreated controls with adjuvant arthritis (AA); MTX—rats with AA treated with methotrexate (MTX) at a dose of 0.3 mg/kg twice weekly; ASTAP—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily; ASTAP + MTX—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily and in a combination with MTX at a dose of 0.3 mg/kg twice weekly; ASYN—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily; ASYN + MTX—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily and in combination with MTX at a dose of 0.3 mg/kg twice weekly; BEKA—group of rats with AA receiving β-carotene in the oral dose of 20 mg/kg daily; KXAN—group of rats with AA receiving β-cryptoxanthin in the oral dose of 10 μg/kg daily. Data are expressed as mean ± SEM. Statistical significance was evaluated using ANOVA for independent variables: ** p < 0.01 vs. HC group.

Figure 11

The presented data show the levels of IL-17A in the plasma of experimental animals measured on days 14 and 28 of the pivotal experiment. The experimental animals were divided as follows: HC—healthy control group; AA—group of untreated controls with adjuvant arthritis (AA); MTX—rats with AA treated with methotrexate (MTX) at a dose of 0.3 mg/kg twice weekly; ASTAP—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily; ASTAP + MTX—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily and in a combination with MTX at a dose of 0.3 mg/kg twice weekly; ASYN—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily; ASYN + MTX—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily and in combination with MTX at a dose of 0.3 mg/kg twice weekly; BEKA—group of rats with AA receiving β-carotene in the oral dose of 20 mg/kg daily; KXAN—group of rats with AA receiving β-cryptoxanthin in the oral dose of 10 μg/kg daily. Data are expressed as mean ± SEM. Statistical significance was evaluated using ANOVA for independent variables: *** p < 0.001 vs. HC group, + p < 0.05 vs. AA group, ++ p < 0.01 vs. AA group.

Figure 12

The presented data show the concentration of MMP-9 in the plasma of experimental animals measured on days 14 and 28 of the pivotal experiment. The experimental animals were divided as follows: HC—healthy control group; AA—group of untreated controls with adjuvant arthritis (AA); MTX—rats with AA treated with methotrexate (MTX) at a dose of 0.3 mg/kg twice weekly; ASTAP—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily; ASTAP + MTX—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily and in combination with MTX at a dose of 0.3 mg/kg twice weekly; ASYN—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily; ASYN + MTX—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily and in combination with MTX at a dose of 0.3 mg/kg twice weekly; BEKA—group of rats with AA receiving β-carotene in the oral dose of 20 mg/kg daily; KXAN—group of rats with AA receiving β-cryptoxanthin in the oral dose of 10 μg/kg daily. Data are expressed as mean ± SEM. Statistical significance was evaluated using ANOVA for independent variables: *** p < 0.001 vs. HC group, + p < 0.05 vs. AA group, ++ p < 0.01 vs. AA group, +++ p < 0.001 vs. AA group, # p < 0.05 vs. MTX, ## p < 0.01 vs. MTX, ### p < 0.001 vs. MTX.

Figure 13

The presented data show the level of the reduced coenzyme Q₉ (CoQ₉red) in the liver of experimental animals measured on day 28 of the pivotal experiment. The experimental animals were divided as follows: HC—healthy control group; AA—group of untreated controls with adjuvant arthritis (AA); MTX—rats with AA treated with methotrexate (MTX) at a dose of 0.3 mg/kg twice weekly; ASTAP—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily; ASTAP + MTX—group of rats with AA receiving astaxanthin of natural origin in the oral dose of 20 mg/kg daily and in a combination with MTX at a dose of 0.3 mg/kg twice weekly; ASYN—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily; ASYN + MTX—group of rats with AA receiving synthetic astaxanthin in the oral dose of 20 mg/kg daily and in combination with MTX at a dose of 0.3 mg/kg twice weekly; BEKA—group of rats with AA receiving β-carotene in the oral dose of 20 mg/kg daily; KXAN—group of rats with AA receiving β-cryptoxanthin in the oral dose of 10 μg/kg daily. Data are expressed as mean ± SEM. Statistical significance was evaluated using ANOVA for independent variables: # p < 0.05 vs. MTX.