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. 2024 Mar 6;25(5):3064.
doi: 10.3390/ijms25053064.

The Potential of Twendee X® as a Safe Antioxidant Treatment for Systemic Sclerosis

Fukka You  1   2 , Carole Nicco  3 , Yoshiaki Harakawa  1 , Toshikazu Yoshikawa  4   5 , Haruhiko Inufusa  1   2
Affiliations

The Potential of Twendee X® as a Safe Antioxidant Treatment for Systemic Sclerosis

Fukka You et al. Int J Mol Sci. .

Abstract

Systemic sclerosis (SSc) is an autoimmune disease characterized by systemic skin hardening, which combines Raynaud's phenomenon and other vascular disorders, skin and internal organ fibrosis, immune disorders, and a variety of other abnormalities. Symptoms vary widely among individuals, and personalized treatment is sought for each patient. Since there is no fundamental cure for SSc, it is designated as an intractable disease with patients receiving government subsidies for medical expenses in Japan. Oxidative stress (OS) has been reported to play an important role in the cause and symptoms of SSc. HOCl-induced SSc mouse models are known to exhibit skin and visceral fibrosis, vascular damage, and autoimmune-like symptoms observed in human SSc. The antioxidant combination Twendee X® (TwX) is a dietary supplement consisting of vitamins, amino acids, and CoQ10. TwX has been proven to prevent dementia in humans with mild cognitive impairment and significantly improve cognitive impairment in an Alzheimer's disease mouse model by regulating OS through a strong antioxidant capacity that cannot be achieved with a single antioxidant ingredient. We evaluated the effectiveness of TwX on various symptoms of HOCl-induced SSc mice. TwX-treated HOCl-induced SSc mice showed significantly reduced lung and skin fibrosis compared to untreated HOCl-induced SSc mice. TwX also significantly reduced highly oxidized protein products (AOPP) in serum and suppressed Col-1 gene expression and activation of B cells involved in autoimmunity. These findings suggest that TwX has the potential to be a new antioxidant treatment for SSc without side effects.

Keywords: Twendee X®; antioxidant; autoimmunity; oxidative stress; systemic sclerosis.

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Conflict of interest statement

F.Y., Y.H., and H.I. are employees of Gifu University. The Division of Antioxidant Research is a laboratory that has been established at the Life Science Research Center at Gifu University, based on a research fund from the TIMA Establishment (Liechtenstein). T.Y. is an advisor to TIMA Establishment (Liechtenstein). The sponsor had no control over the interpretation, writing, or publication of this work.

Figures

Figure 1
Figure 1
Effect of Twendee X® on sera redox status. Concentrations of advanced oxidation protein products (AOPP) in the sera from mice (mM of chloramine T equivalent). Each box represents mean ± SD from n = 10 individual mice. Kruskal–Wallis test with Dunn’s multiple comparison test was used for statistical analysis). * p < 0.05.
Figure 2
Figure 2
Effect of Twendee X® on fibrosis parameters. (A) Change in skin fold thickness in millimeters from day 1 to day 36, measured weekly (n = 10). **: p < 0.01, ***: p < 0.001 (PBS vs. HOCl), †: p < 0.05 (HOCl vs. HOCl + TwX). (B) Collagen type I levels in skin (mg/punch biopsy) and (C) in lung (mg/lobe biopsy) were evaluated by hydroxyproline dosage. Each box represents mean ± SD from n = 10 individual mice. * p < 0.05; ** p < 0.01; *** p < 0.001. (D) Relative mRNA level of Collagen-1. Results were standardized by GAPDH. Each box represents mean ± SD from n = 10 individual mice. ** p < 0.01; *** p < 0.001. (E) Representative H&E dyed skin sections of 6 μm, showing enhanced fibrosis in mice. Photographs were taken with a Nikon Eclipse 80i microscope (Nikon Instruments, Inc., Melville, NY, USA). Original magnification ×20. The scale represents 500 μm. All data are expressed as the mean ± SD. Kruskal–Wallis test with Dunn’s multiple comparison test was used for all statistical analysis.
Figure 3
Figure 3
Effect of Twendee X® on fibroblast differentiation. (A) α-SMA and (B) H-Ras in skin were evaluated by Western blot. Results were normalized to tubulin. Each box represents mean ± SD from n = 10 individual mice. Kruskal–Wallis test with Dunn’s multiple comparison test was used for all statistical analysis. * p < 0.05.
Figure 4
Figure 4
Effect of Twendee X® on Collagen and cytokines expression. Relative mRNA level of (A) Il-6, (B) Il-33, and (C) Il-17 mRNA levels in skin evaluated by qRT-PCR. Results were normalized to GAPDH. Each box represents mean ± SD from n = 10 individual mice. Kruskal–Wallis test with Dunn’s multiple comparison test was used for all statistical analysis. No significant difference was observed.
Figure 5
Figure 5
Effects of Twendee X® on B and T CD4+ cells activation assessed by flow cytometry in SSc mice. The side-scatter (SSC) and the forward-scatter channels (FSC) were used to gate the leukocytes. A total of 100,000 events were accumulated for each sample. Doublets were excluded with FSC-A and FSC-H channels. (A,B) Activation of B220+ cells assessed by CD40 expression (A) and MHC II expression (B). (C,D) Activation of CD4+ T cells assessed by CD69 (C) and CD44 (D) expression. Each box represents mean ± SD from n = 10 individual mice. Kruskal–Wallis test with Dunn’s multiple comparison test was used for all statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 6
Figure 6
Flow cytometric analysis of the effects of Twendee X® on splenic macrophage phenotype in SSc mice. Splenic macrophages were gated on CD11b- and F4/80-positive cells among CD45-positive. A total of 100,000 events were accumulated for each sample. Doublets were excluded with FSC-A and FSC-H channels. (A) Percentage of splenic macrophages among total splenic cells. Data represent the percentage and SD. (B–D) Flow cytometric analysis of CD86, Ly6C, and CD206 expression on splenic macrophages. (E) Ratio of M1/M2 macrophages’ frequency. M1 macrophages were defined as B220-F4/80+CD11b+Ly6CHighCD206- and M2 macrophages as B220-F4/80+CD11b+Ly6cLowCD206+. Each box represents mean ± SD from n = 10 individual mice. Kruskal–Wallis test with Dunn’s multiple comparison test was used for all statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001.
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