Main Research Findings

1) Co-administration of coenzyme Q and selenium was shown to reduce metabolic dysfunction associated with Steatohepatitis, induced by a methionine choline deficient diet.

2) Combined treatment of vitamin C, choline chloride, and selenium was found to reduce allergic effects demonstrated in an animal model of airway disease.

 

Selected Data

1) The study performed by the research team of Choi et al was designed to investigate the ameliorative effects of Coenzyme Q (CoQ) and Selenium (Se) co-supplementation on methionine choline-deficient (MCD) diet-induced metabolic dysfunction-associated steatohepatitis (MASH) in mice. All animal experiments were meticulously conducted under protocols approved by the Kyung Hee University Institutional Animal Care and Use Committee, ensuring ethical treatment and adherence to animal welfare guidelines. For the experimental setup, male C57BL/6J mice, aged 4 weeks, were maintained under controlled environmental conditions, including a temperature of 22 ± 2 °C, 50 ± 10% humidity, and a 12-hour light/dark cycle [1].

Following an adaptation period, the mice were randomly assigned to one of eight experimental groups, with 10 mice per group. Four groups received a methionine choline-deficient (MCD) diet to induce MASH, while the other four groups were fed a methionine choline-sufficient (MCS) diet as controls. Within both the MCD and MCS diet cohorts, treatment groups included vehicle (corn oil), 100 mg/kg CoQ, 158 µg/kg Se, or a combination of both (CoQ + Se). The respective treatments were administered via oral gavage once daily for a duration of 4 weeks, with dosages selected based on previous research findings.

At the conclusion of the 4-week feeding period, mice were humanely euthanized using carbon dioxide (CO2) anesthesia. Liver tissue samples were promptly collected; a portion was fixed in 10% neutral buffered formalin for histological analysis, while another portion was snap-frozen in liquid nitrogen and stored at –80 °C for subsequent biochemical and molecular analyses. Various serum and hepatic biochemical parameters were measured to assess liver injury and metabolic dysfunction. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, key indicators of liver damage, were quantified [1].

Hepatic triglyceride (TG) and total cholesterol (TC) concentrations were also measured using specific assay kits from the same supplier to evaluate lipid accumulation. Oxidative stress markers included hepatic malondialdehyde (MDA) content, measured with a Sigma-Aldrich assay kit, and the ratio of reduced to oxidized glutathione (GSH/GSSG). Antioxidant enzyme activities, including hepatic catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX), were determined using commercially available kits, all performed according to manufacturer protocols. Hepatic non-heme iron content, a marker for iron accumulation, was measured using a previously described method [1].

For histological analysis, fixed liver tissues were embedded in paraffin, sectioned into 5 µm thick slices, and stained with hematoxylin and eosin (H&E) to evaluate steatosis, lobular inflammation, and hepatocellular ballooning, which together comprise the MASLD activity score (MAS score). Liver fibrosis was assessed by Sirius Red staining, with the fibrotic area quantified as a percentage of the total area. All stained sections were observed and imaged under an optical microscope at 200× magnification. Molecular analyses included Quantitative Real-Time RT-PCR (qRT-PCR) to quantify mRNA expression levels of various genes. Total RNA was extracted from 25 mg of liver tissue using RNAiso Plus reagent, and 100 ng of total RNA was reverse-transcribed into cDNA using the PrimeScript™ RT reagent Kit. Gene-specific mRNA expression levels were then amplified and quantified relative to control groups.

Western blot analysis was performed to evaluate protein expression of ferroptosis-related markers. 50 mg liver tissue samples were lysed, and protein concentrations were determined. Lysates were separated by SDS-PAGE, transferred to a PVDF membrane, and incubated overnight with primary antibodies against GPX4, 4-hydroxynonenal (4-HNE), ferritin, acyl-CoA synthetase long-chain family member 4 (ACSL4), and 12-lipoxygenase (LOX), with GAPDH serving as a loading control. Membranes were then incubated with secondary antibodies and visualized using an enhanced chemiluminescence solution. All data underwent statistical analysis, employing one-way Analysis of Variance (ANOVA) followed by Duncan’s multiple range test to determine significant differences between groups [1].

2) This study conducted by Bansal et al employed a mouse model of allergic airway disease to investigate the therapeutic potential of intranasally administered choline chloride (ChCl) in combination with vitamin C (Vit C) and selenium (Se). Male Balb/c mice, aged 4–6 weeks and weighing 20–25 g, were obtained and acclimatized before being randomly allocated into eight groups of six animals each. Standard housing conditions were maintained, including controlled temperature, humidity, and a 12-hour light/dark cycle with ad libitum access to food and water [2].

To induce allergic airway disease, mice were sensitized and challenged with cockroach extract (CE), a common allergen. Sensitization involved intraperitoneal (ip) injections of CE (10 µg/100 µl PBS) on days 0, 7, and 14. A control group received PBS ip injections. Following sensitization, mice were challenged with 1% CE nebulized in a Plexiglas chamber for 30 minutes daily from day 25 to 30. The control group received a PBS challenge. A booster challenge was also administered on day 38 to ensure a sustained allergic response.

Therapeutic interventions were initiated after the initial challenge phase. On days 31, 33, 35, 37, and 39, mice received intranasal treatments after being anesthetized with 3% isoflurane. Treatment groups included 1 mg/kg ChCl, 308.33 mg/kg Vit C, 1 mg/kg Se, or a combination of ChCl + Vit C + Se. A vehicle group received PBS intranasally. For comparative purposes, additional groups were treated with standard anti-inflammatory therapies: 1 mg/kg dexamethasone and 100 mg/kg α-lipoic acid [2].

At the culmination of the study on day 40, all mice were euthanized. Blood, bronchoalveolar lavage fluid (BALF), lungs, and spleens were meticulously collected for various analyses to assess the effects of the treatments on allergic airway disease parameters. Airway hyperresponsiveness (AHR), a hallmark of asthma, was measured using a computer-controlled FlexiVent ventilator. Mice were anesthetized, and a precalibrated cannula was inserted into the trachea. Respiratory resistance was then recorded in response to increasing doses of methacholine, ranging from 2 to 20 mg/ml PBS.

BALF samples were processed to evaluate cellular infiltration into the lungs. Briefly, 1.5 ml of chilled PBS was used for lavage, and the collected fluid was centrifuged. The supernatant was stored for biochemical assays, while the cell pellet was resuspended in PBS for total cell counting. Differential cell counts of eosinophils and neutrophils were performed on Leishman’s-stained BALF cell smears, and absolute numbers were calculated [2].

Blood was collected, and serum was separated and stored for subsequent immunological analyses, specifically for the determination of allergen-specific antibodies. Lung tissue was divided for histopathology and molecular assays. For histopathology, lung sections were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin (H&E). Tissue slides were observed under a microscope by two blinded observers to quantify cell infiltration and measure the thickness of the airway-lumen wall, generating an inflammation score.

Splenocyte cultures were prepared to assess immune cell responses. Spleens were excised, minced, and processed to obtain single-cell suspensions. Splenocytes were cultured in RPMI 1640 medium supplemented with heat-inactivated fetal bovine serum, HEPES buffer, L-glutamine, penicillin, and streptomycin. Cells were stimulated with CE or phytohemagglutinin (PHA) for 72 hours. For specific treatment effects on cellular responses, CE-stimulated splenocytes were co-incubated with the ChCl + Vit C + Se combination. Intracellular staining was performed using FITC-labeled IL-10 and PE-labeled FOXP3 antibodies, and cells were analyzed by flow cytometry to quantify IL-10-producing FOXP3+ cells [2].

Oxidative stress markers were comprehensively assessed. Intracellular reactive oxygen species (iROS) in BALF cell pellets were measured by incubating cells with 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and analyzing mean fluorescence intensity using a fluorescence-activated cell sorter. Eosinophil peroxidase (EPO) activity in BALF supernatant was determined using a substrate solution containing o-phenylenediamine dihydrochloride, Triton X-100, and hydrogen peroxide, with absorbance read at 492 nm. Lung homogenates were used to measure glutathione peroxidase (GPx) activity using a commercial kit. Levels of 8-isoprostanes, an indicator of lipid peroxidation, were quantified in BALF samples using enzyme immunoassay kits, with absorbance read at 420 nm and a detection limit of 0.8 pg/ml.

Molecular analyses focused on inflammatory transcription factors and cytokines. Nuclear fractions of lung homogenates were extracted to estimate NF-κB (p65) protein levels using an immunoassay kit, where samples were added to microtiter plates precoated with NF-κB response element DNA, followed by primary and secondary antibody incubations and absorbance readings at 450 nm. Cytokine levels including IL-4, IL-5, IFN-γ, and IL-10, in BALF and splenocyte culture supernatants were determined by ELISA (BD Pharmingen), with specific detection limits for each cytokine. Serum levels of CE-specific immunoglobulins including IgE, IgG1, and IgG2a, were also quantified [2].

 

Discussion

1) The findings of the study performed by Choi et al demonstrate that co-supplementation with CoQ and Se effectively alleviates MCD diet-induced MASH in mice, primarily by reducing oxidative stress, lipid peroxidation, and suppressing ferroptosis. Histopathological analysis of liver sections revealed that the MCD diet significantly induced severe hepatic steatosis, characterized by a marked increase in fat droplet size and number, along with pronounced inflammatory cell infiltration and hepatocellular ballooning, compared to the MCS control group. However, treatment with CoQ, Se, or their combination significantly ameliorated these pathological features, with the CoQ + Se co-supplementation group exhibiting the most substantial reductions in hepatic steatosis, inflammatory foci, and ballooning degeneration. This was further corroborated by significantly lower MAS scores in the CoQ, Se, and CoQ + Se groups compared to the MCD group, with the combined treatment yielding the lowest scores [1].

Liver injury, as indicated by serum markers, was significantly mitigated by the treatments. Serum levels of ALT and AST, which were markedly elevated in the MCD group, were notably reduced by individual CoQ and Se treatments, and even more so by their combination. Furthermore, the MCD diet led to significantly higher hepatic TG and TC concentrations. While individual CoQ or Se treatments showed a trend towards reducing these lipid levels, the combined CoQ + Se supplementation significantly lowered both hepatic TG and TC concentrations to near-normal levels compared to the MCD vehicle group.

The study also revealed the anti-inflammatory and anti-fibrotic effects of the co-supplementation. The degree of liver fibrosis was markedly increased in the MCD group but significantly reduced in all treatment groups, with the CoQ + Se combination showing the greatest reduction in collagen staining. Molecular analysis confirmed these findings, as mRNA expression levels of key fibrotic indicators such as Collagen 1α1, Collagen 3α1, and TGF-β were significantly upregulated in the MCD group but substantially downregulated by CoQ, Se, and particularly their combination. Moreover, pro-inflammatory cytokine-related genes, including IL-1β and IL-6, showed significantly increased mRNA levels in the MCD group, which were markedly downregulated by all treatments. COX2 protein expression, a mediator of inflammatory responses, was also significantly elevated in the MCD vehicle group, and its expression was significantly reduced by the CoQ + Se combination, but not by CoQ or Se alone [1].

A crucial aspect of the study was the assessment of hepatic oxidative stress and lipid peroxidation. The MCD diet significantly compromised antioxidant defenses, evidenced by reduced CAT and SOD activities and a decreased GSH/GSSG ratio compared to MCS controls. While individual CoQ or Se treatments showed trends of improvement, their combination significantly enhanced both CAT and SOD activities, demonstrating a synergistic effect against oxidative stress. CoQ + Se also substantially improved the GSH/GSSG ratio to levels comparable to the MCS group, highlighting its capacity to restore redox balance. Correspondingly, hepatic lipid peroxidation markers, MDA and 4-HNE, were significantly elevated in the MCD group. All treatments, especially the combination, significantly decreased hepatic MDA levels, and the CoQ + Se combination notably reduced 4-HNE levels, underscoring their effectiveness in mitigating lipid peroxidation [1].

 

Figure 1: Changes in A) hepatic MDA and B) 4-HNE levels across experimental treatment groups included in either the MCD or MCS diet groups.

Finally, the investigation into ferroptosis-related markers provided deeper mechanistic insights. The MCD group exhibited significant increases in the protein expression of ACSL4 and LOX, both critical drivers of ferroptosis. CoQ, Se, or their combination significantly decreased ACSL4 protein expression, and only the combined CoQ + Se treatment significantly reduced LOX protein expression. Furthermore, GPX4 protein levels, which are crucial for ferroptosis suppression, were increased in the MCD group as a compensatory response and further elevated by individual Se and combined CoQ + Se treatments. Hepatic non-heme iron content, a pro-ferroptotic factor, was significantly higher in the MCD group but significantly reduced by CoQ + Se supplementation. These comprehensive results indicate that CoQ and Se co-supplementation synergistically targets multiple pathways implicated in MASH pathogenesis, including oxidative stress, inflammation, fibrosis, and ferroptosis, presenting a promising therapeutic strategy [1].

 

Figure 2: Changes in the expression of A) ACSL4, B) LOX, and C) GPX4 across experimental treatment groups included in either the MCD or MCS diet groups.

2) The results of the study by Bansal et al reported that intranasal administration of a combination of ChCl, Vit C, and Se effectively attenuated allergic airway disease in a mouse model, addressing key pathological features such as airway hyperresponsiveness (AHR), inflammation, oxidative stress, and immune dysregulation. AHR, a critical physiological characteristic of allergic asthma, was significantly increased in CE-immunized mice compared to PBS controls. While individual treatments with ChCl, Vit C, or Se all significantly reduced AHR, the combination of ChCl + Vit C + Se proved to be the most effective, bringing AHR levels back to normal, comparable to the PBS control group. This highlights a synergistic effect of the combined therapy [2] .

Airway inflammation was comprehensively assessed through various parameters. CE exposure led to a substantial increase in total cell count in BALF, indicative of significant leukocyte recruitment into the lungs. This was accompanied by marked increases in eosinophil and neutrophil infiltration, key inflammatory cells in allergic responses. All treatments, including individual components and the combination, significantly reduced these cellular infiltrates. The ChCl + Vit C + Se combination exhibited the highest reduction in total leukocyte count, eosinophil and neutrophil numbers.

Furthermore, EPO activity, a marker of eosinophilic inflammation, was significantly reduced by the combination treatment. Histopathological analysis of lung sections corroborated these findings, showing a high inflammation score in CE-challenged mice which was drastically reduced by all treatments, with the ChCl + Vit C + Se combination producing the most significant amelioration of inflammation. Notably, standard therapies like dexamethasone and α-lipoic acid also reduced inflammation but to a lesser extent than the combined ChCl + Vit C + Se treatment [2].

Oxidative stress, a major contributor to allergic pathology, was significantly elevated in CE-immunized mice. iROS and 8-isoprostanes in BALF were markedly increased, while GPx activity in lung homogenates was significantly decreased. All individual and combination treatments effectively mitigated these oxidative stress markers. The ChCl + Vit C + Se combination led to the highest reductions in both iROS and 8-isoprostanes and restored GPx activity to normal levels, demonstrating its potent antioxidant capacity.

The NF-κB p65 transcription factor, a central regulator of inflammatory gene expression, was significantly elevated in the nuclear fraction of lung tissue from CE-immunized mice. All treatments resulted in marked reductions in NF-κB p65 levels, indicating an inhibition of its nuclear translocation. The ChCl + Vit C + Se combination exhibited the highest reduction in NF-κB p65, suggesting a strong anti-inflammatory effect through this pathway [2].

Immune responses were further characterized by cytokine and immunoglobulin analyses. Pro-inflammatory cytokines, IL-4 and IL-5, were significantly increased in both BALF and splenocyte culture supernatants of CE-immunized mice. All treatments significantly reduced the levels of these cytokines. The ChCl + Vit C + Se combination produced the greatest reduction in IL-4 and IL-5 levels, making them comparable to the PBS control group. Conversely, IL-10, an important immunoregulatory cytokine, was significantly decreased in CE-immunized mice. All treatments led to an increase in IL-10 levels, with the combination treatment restoring IL-10 to normal, thereby suggesting a restoration of immune balance. IFN-γ levels remained unchanged across all groups.

Serum immunoglobulin levels also reflected the allergic response. CE-immunized mice showed significant increases in allergen-specific IgE and IgG1. All treatments significantly reduced these immunoglobulin levels. The ChCl + Vit C + Se combination again achieved the highest reduction in IgE and IgG1. Serum IgG2a levels remained unaffected by both sensitization and treatments. Lymphocyte proliferation, which was significantly increased in CE-immunized mice, was also reduced by all treatments. The ChCl + Vit C + Se combination showed the highest decrease in lymphocyte proliferation, further indicating its immunomodulatory effects. Delving deeper into the mechanism of immune regulation, splenocyte cultures revealed that IL-10-producing FOXP3+ cells, crucial for immune tolerance, were present in CE-stimulated splenocytes. The ChCl + Vit C + Se combination treatment significantly increased the percentage of these IL-10+FOXP3+ cells, suggesting that the combined therapy enhances regulatory T cell activity, which in turn contributes to the increased IL-10 production and subsequent attenuation of the allergic response [2].

In conclusion, the results unequivocally demonstrate that the intranasal administration of ChCl + Vit C + Se exerts a potent therapeutic effect in a mouse model of allergic airway disease. This combined therapy effectively alleviates AHR, reduces airway inflammation and oxidative stress, suppresses NF-κB activation, modulates pro- and anti-inflammatory cytokine profiles, lowers allergen-specific immunoglobulin levels, and enhances regulatory T cell activity. These findings highlight the synergistic potential of ChCl, Vit C, and Se as a promising therapeutic strategy for managing allergic airway diseases, possibly through a multifaceted mechanism involving antioxidant, anti-inflammatory, and immunoregulatory actions [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Choi H, Choi J, Go Y, Chung J. Coenzyme Q and Selenium Co-Supplementation Alleviate Methionine Choline-Deficient Diet-Induced Metabolic Dysfunction-Associated Steatohepatitis in Mice. Nutrients. 2025;17(2):229. Published 2025 Jan 9. doi:10.3390/nu17020229

[2] Bansal P, Saw S, Govindaraj D, Arora N. Intranasal administration of a combination of choline chloride, vitamin C, and selenium attenuates the allergic effect in a mouse model of airway disease. Free Radic Biol Med. 2014;73:358-365. doi:10.1016/j.freeradbiomed.2014.05.018

 

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