Nucleoprotein Diet Ameliorates Arthritis Symptoms in Mice Transgenic for Human T-Cell Leukemia Virus Type I (HTLV-1)

Abstract

Because rheumatoid arthritis (RA), an autoimmune disease, the patients often recognize side-effects due to the medication, alternative therapeutic strategies might potentially offer a clinical advantage. We evaluated the effect of nucleoprotein from salmon soft roe on animal model of arthritis. Mice transgenic for human T-cell leukemia virus type I (HTLV-1 Tg) were divided into three experimental groups and supplemented on either nucleoprotein-free (nonNP), or 0.6% or 1.2% nucleoprotein mixed (NP0.6 or NP1.2) diet for 3 months. The mice were evaluated arthritis by morphology, and measured with rheumatoid factor (RF). Moreover, macrophages and oxidative metabolites were assessed in the ankle and/or serum. Anti-oxidative potentials in nucleoprotein were determined with biological anti-oxidative potential (BAP) test, and electron spin resonance (ESR) analysis. NonNP-diet HTLV-1 Tg mice increased an arthritis symptoms and RF. The symptoms were ameliorated in NP-diet groups. Macrophages detected by F4/80 staining, and oxidative metabolites in the serum and/or joints were clearly decreased in 1.2% NP-diet HTLV-1 Tg mice. Nucleoprotein and DNA-nucleotide, but less protamine, had direct anti-oxidative potency with BAP test and/or ESR in vitro. These observations suggest that dietary nucleoprotein ameliorates arthritis symptoms in HTLV-1 Tg mice and offers hope as an alternative treatment for this debilitating medical condition.

Keywords: alternative medicine; nucleoprotein; radical scavenger; rheumatoid arthritis.

Fig. 1

Schematic diagram of the present study. (A) Diagram of animal study. Artificial experimental chows (see Table 1) are given for 3 months from 6 weeks of age. Body weight and paw swelling are measured every 2 weeks. Blood and histological analysis are carried out 18 weeks of age. (B) Histological criteria of arthritis according to previous paper [24]. (C) Representative images of wave pattern of ¹O₂ in present/absent of nucleoprotein. The highest peak of ¹O₂ generated by rose bengal is recorded without nucleoprotein (0 µg/ml, upper), but the height are decreased with nucleoprotein. (a) is peak of manganese which is internal control. (b) is peak of ¹O₂. The relative ¹O₂ generation was calculated by (b)/(a).

Fig. 2

Changes in body weight and joint swelling in HTLV-1 Tg and wild-type mice. The body weight (A), and thickness of the wrists (B) and ankles (C) are measured for 3 months from 6 to 18 weeks of age. Joint thickness was showed as the paw swelling score (see Materials and Methods). NonNP-fed HTLV-1 Tg mice tend to have low body weights and greater paw swelling scores during the experimental period. NP1.2-fed HTLV-1 Tg mice recorded a significantly improved body weight and paw swelling score at 16 and/or 18 weeks of age. Each value is expressed as mean ± SE. * p<0.05, ** p<0.01, *** p<0.001 compared with nonNP-fed HTLV-1 Tg or wild-type mice (Dunnett’s post hoc test).

Fig. 3

Histopathological score of arthritic degeneration in the wrists and ankles after NP diet. (A) Typical histopathological images (HE staining) of the ankles in the nonNP-fed wild-type (wild), and nonNP-, NP0.6- or NP1.2-fed HTLV-1 Tg (Tg) mice. Ankle in nonNP-fed Tg mice clearly showed proliferation of synovial tissue (asterisks), inflammation and lymph nodes (arrowheads), and destruction of cartilage and bone (arrows). Eighty percentages of nonNP-fed HTLV-1 Tg mice recorded a score of 3 or 4 in wrists (B) and ankles (C). The mice fed nucleoprotein additive chow are ameliorated the score in a content-dependent manner. NP1.2-fed HTLV-1 Tg mice had a significantly reduce the score compared with nonNP-fed animals (p<0.05). Numbers in the columns indicate the incidence (%).

Fig. 4

Dietary effect of nucleoprotein on RF (as IgG) in HTLV-1 Tg (Tg) and wild-type (wild) mice. (A) RF level in serum of nonNP-fed HTLV-1 Tg mice was higher than that of the wild-type mice. NP-fed groups displayed a dose-dependent decrease in serum RF, with NP1.2-fed HTLV-1 Tg mice recording a significant decrease in the level of RF compared with that in the nonNP-fed group (p<0.05). Each value is shown as mean ± SE. (B) Immunohistochemical staining of mouse IgG in the ankles of nonNP-fed wild-type (wild), and nonNP-, NP0.6- or NP1.2-fed HTLV-1 Tg (Tg) mice. Little reactivity was seen in the ankles of wild-type mice. By comparison, intensity of IgG immunoreaction in the ankles was markedly stronger in nonNP-fed HTLV-1 Tg mice, but was decreased in NP0.6- or NP1.2-fed groups. The IgG-positive reactions in the nonNP-fed HTLV-1 Tg mice were observed in the synovial tissue (*) and, to a lesser extent, in the bone (**) and muscles (***).

Fig. 5

Dietary effects of nucleoprotein on inflammatory responses in HTLV-1 Tg mice. Immunohistochemical staining for a macrophage marker, F4/80, in the ankle of nonNP (A)-, NP0.6 (B)- or NP1.2 (C)-fed HTLV-1 Tg mice. A higher magnification image of the boxed regions is shown in (a) to (f). F4/80 immunoreactivity was clearly observed in the ankles of nonNP-fed HTLV-1 Tg mice, especially in the synovial membrane (arrows in a and b). The number of labeled cells and the intensity of F4/80 immunoreactivity decreased in a dose-dependent manner. F4/80 immunoreactivity was reduced or absent in the synovial membrane of the NP1.2-fed HTLV-1 Tg mice.

Fig. 6

Dietary effect of nucleoprotein on oxidative stress in HTLV-1 Tg mice. (A) the mice fed nucleoprotein additive chows decreased the serum level of oxidative metabolite measured with ROM test, being significantly lower in NP1.2-fed HTLV-1 Tg mice than in nonNP-fed group (p<0.05, Dunnett’s post hoc test). Each value is shown as mean ± SEM. (B) Immunohistochemical staining of 3-NT in hyaline cartilage of the ankles in nonNP-fed wild-type, and nonNP- or NP1.2-fed HTLV-1 Tg (Tg) mice. There was marked 3-NT immunoreactivity in the nonNP-fed HTLV-1 Tg group (arrows). However, few positive reactions were observed in the nonNP-fed wild-type or NP1.2-fed HTLV-1 Tg mice.

Fig. 7

Nucleoprotein and DNA-nu, but less protamine exert direct anti-oxidant capacity. (A) Nucleoprotein, DNA-nu, and protamine suspensions were performed BAP test with FREE. Nucleoprotein (close bar) and DNA-nu (gray bar) increase significantly at 5.0 and 2.5 µg/ml or more, respectively, and show strong anti-oxidative potential. In contrast, protamine suspension (open bar) also exerts anti-oxidative potential (75 µg/ml or more), but is obviously less than the others. Each data is shown Mean ± SEM (n = 5). *: p<0.05, **: p<0.01, ***: p<0.001 to compare with 0 µg/ml of each group (Dunnet’s post hoc test). N.D: no data. (B) Quantitative analysis of ¹O₂ with several concentration of nucleoprotein. The height of peak on the ¹O₂ is decreased in a dose-dependent manner. Each data is shown Mean ± SEM (n = 2 – 3). ***: p<0.001 to compare with 0 µg/ml of each group (Dunnet’s post hoc test).