Synergistic Effects of Riluzole and Sodium Butyrate on Barrier Function and Disease Progression of Amyotrophic Lateral Sclerosis in SOD1G93A mice

Synergistic Effects of Riluzole and Sodium Butyrate on Barrier Function and Disease Progression of Amyotrophic Lateral Sclerosis in SOD1G93A mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Synergistic Effects of Riluzole and Sodium Butyrate on Barrier Function and Disease Progression of Amyotrophic Lateral Sclerosis in SOD1 G93A mice Yong-Guo Zhang, KaReisha Robinson, Yinglin Xia, Jun Sun This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5349709/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Emerging evidence has shown that gut-brain barrier dysfunction occurs at the early stages of ALS. Previous studies demonstrated that Sodium butyrate significantly decreased aggregation of the human-SOD1 G93A protein, improved intestinal barriers, delayed the disease onset, and prolonged the life span of ALS mice. Riluzole is the first FDA-approved drug for ALS treatment. We hypothesize that Riluzole and Sodium butyrate combined treatment further decreases aggregation of the h-SOD1 G93A , restores the gut-brain barrier function and delays the ALS progression. SOD1 G93A mice (9-10-week-old) treated with Riluzole (10 mg/kg, I.P. daily), Sodium butyrate (2% in drinking water), or Riluzole and Sodium butyrate combination for 6 weeks. Riluzole and Sodium butyrate combination showed a significantly longer rotarod time, increased grip strength, enhanced intestinal barrier, compared with Riluzole or Sodium butyrate alone treatment. More reduction of h-SOD1 G93A aggregation, an indicator of ALS progression, was observed in the colon, spinal cord lumbar, and brain cortex with Riluzole and Sodium butyrate combination, compared with Riluzole or Sodium butyrate alone treatment. Tight junction proteins (ZO-1 and Claudin-5) significantly increased in the colon, spinal cord lumbar, and brain cortex of mice with Riluzole and Sodium butyrate treatment. In SOD1 G93A mice, Riluzole and Sodium butyrate combination treatment reduced serum lipopolysaccharides (LPS) and inflammatory cytokines (IL-17, IL-6 and IFN-γ) more than that in Riluzole or Sodium butyrate treatment. Our data suggest that Riluzole and Sodium butyrate treatment is more efficient than either Riluzole or Sodium butyrate alone in delaying ALS progress. It provides a potential therapeutic strategy by restoring barrier function through the gut-brain axis for ALS. Biological sciences/Biochemistry Biological sciences/Cell biology Biological sciences/Immunology Biological sciences/Physiology Health sciences/Gastroenterology Health sciences/Neurology Amyotrophic lateral sclerosis treatment barrier function BBB gut-brain axis inflammation tight junctions Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Amyotrophic lateral sclerosis is a fatal neuromuscular disease characterized by progressive motor neuron death and skeletal muscle atrophy and paralysis 1,2 . The lifetime risk of ALS is about 1 in 472 in women and 1 in 350 in men 3 . Because ALS is an age-dependent disease, an increase in the prevalence of ALS can be anticipated as the U.S. population increases and ages. Military veterans, regardless of the branch of service, the era in which they served, or whether they served during a time of peace or war, are at a greater risk of dying from ALS than if they had not served in the military. Most patients with ALS die within 5 years after the disease onset. Despite intensive research efforts, there is no effective cure for ALS. The two available treatments (riluzole and edaravone) are to relieve symptoms and improve the quality of life for patients with ALS. There are significant needs to develop novel treatments for ALS and improve the life quality of ALS patients 4 . Riluzole was the first FDA-approved treatment for ALS and is speculated to reduce glutamatergic neurotransmission by blocking voltage-gated sodium channels on presynaptic neurons. Riluzole extends the patient’s life span for a few months 5 . Increased serum inflammatory cytokines 6 , bacterial lipopolysaccharides (LPS) 6 , and aberrant microbiome have been reported in prior studies of humans ALS 7 . Our study with oral administration of sodium butyrate presents a promising therapeutic strategy 8,9 . Mice with butyrate treatment showed a significantly longer latency to fall in the rotarod test, compared with the G93A non-treatment mice. Bacterial lipopolysaccharides (LPS), glycolipids found in the outer membrane of gram-negative bacteria, are pro-inflammatory and have found to be increased in the serum of ALS patients 6 . Indeed, increased inflammatory cytokine IL-17 and IL-23 have been reported in serum and cerebrospinal fluid of patients with ALS 10 . We have demonstrated increased serum IL-17 in the SOD1 G93A ALS mice, compared to the wild-type mice 11 . Our study showed the dysbiosis was correlated with intestinal inflammation and increased intestinal permeability in ALS 11 . However, the impact and mechanism of Riluzole in the ALS intestine is unknown. Combined role and mechanism of Riluzole and butyrate is also unknown. In the current study, we use the 2% sodium butyrate in the drinking water combining with the Riluzole in the SOD1 G93A mice. The outcomes focused on clinical disease progression (e.g., body weight, ratorad, onset, and neuroprotection (reducing motor neuron loss and inhibiting glial activation, and survival). Moreover, we examined the changes of aggregation of human mutant SOD1 G93A (h-SOD1 G93A ), TJs, barriers, and inflammation. Better understanding of the mechanisms of existing drug and synergistic effects of Riluzole and Sodium butyrate will provide insights into barrier function and ALS progression. Results Riluzole and Sodium butyrate combination showed a significantly protection against disease progression in SOD1 G93A mice. To examine the synergistic effects of Riluzole and Sodium butyrate on disease progression, we set up 4 experimental groups with Riluzole alone, Sodium butyrate alone, and Riluzole and Sodium butyrate combination, and control without treatment in SOD1 G93A mice aged 9 to 10 weeks. Mice were randomly assigned to each group. The treatment was total 6 weeks. The Riluzole-treated group received Riluzole via I.P. at a dose of 10mg per kg body weight daily. The Sodium butyrate-treated group received 2% Sodium butyrate in filtered drinking water ( Fig. 1A ). The date of disease onset is defined as the first date when a mouse fails the 15-rpm rotarod task and start to show muscle tremors in the hind limbs 12,13 . The body weight will be monitored every day during the treatment period. The peak body weight is a reasonable and consistent determinant of onset particularly in combination with a measure of neurological score 14,15 . Body weight changes were shown in Fig. 1B . From the age of 13-14 weeks, Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination treated SOD1 G93A mice groups show slight weight loss without statistical significance, compared to the no-treatment SOD1 G93A mice. From the age of 14-15 weeks, Riluzole/Sodium butyrate combination treatment showed a slight weight loss without statistical significance, compared to the Riluzole alone or Sodium butyrate alone treatment SOD1 G93A mice. At the age of 15-16 weeks, Riluzole and Sodium butyrate combination showed a significant less weight loss, compared to the no-treatment SOD1 G93A mice. The SOD1 G93A mice were tested on an accelerating rotarod 13 to examine the neuromuscular activity performance . The Riluzole and Sodium butyrate combination treated SOD1 G93A mice had a significantly increased rotarod time, compared to Riluzole alone, Sodium butyrate alone treated SOD1 G93A mice ( Fig. 1C ). From the age of 12-13 weeks, Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination treated SOD1 G93A mice all had a significantly increased rotarod time, compared to control mice without treatment. At the age of 15-16 weeks, the combination group had a significantly increased rotarod time, compared to SOD1 G93A mice with only Riluzole or Sodium butyrate . Riluzole and Sodium butyrate combination treated SOD1 G93A mice had a significantly increased strengths of forelimb grip (Fig. 1D ) and hindlimb grip (Fig. 1E ), compared to Riluzole alone, or Sodium butyrate alone treated SOD1 G93A mice. From the age of 12-13 weeks, Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination treated SOD1 G93A mice all had a significantly increased forelimb grip strength and hindlimb grip strength, compared to control mice without treatment. At the age of 15-16 weeks, Riluzole and Sodium butyrate combination treated SOD1 G93A mice had a significantly increased forelimb grip strength and hindlimb grip strength, compared to Riluzole alone, or Sodium butyrate alone treated SOD1 G93A mice. The date of death is defined as the day when the mouse cannot right itself within 30 seconds after being placed on its back 13 . We did not perform the survival curve of the SOD1 G93A mice with and without the treatment in ALS progression due to the restricts of the animal protocol. Overall, our data suggested that Riluzole and Sodium butyrate combination had a significantly protection against disease progression in SOD1 G93A mice. Riluzole and Sodium butyrate combined treatment reserved the permeability and reduced h-SOD1 G93A aggregation in the SOD1 G93A mice. Increased gut permeability was reported in the SOD1 G93A mice 8,9,16 . We then examine whether treatment delays the intestinal dysfunction during ALS progression. Intestinal permeability was decreased in all Riluzole, Sodium butyrate, or Riluzole and Sodium butyrate combination treated SOD1 G93A mice groups, compared to control mice. Meanwhile, Riluzole and Sodium butyrate combination treated SOD1 G93A mice had a significantly decreased intestinal permeability, compared to Riluzole alone or Sodium butyrate alone treated SOD1 G93A mice (Fig. 2A) . The intestinal permeability was regulated by the tight junctions (TJs). We then examined the expression of tight junction proteins. ZO-1 and Claudin-5 detected by Western Blot (WB) were increased in intestines of Riluzole, Sodium butyrate or Riluzole and Sodium butyrate combination, compared to control mice without treatment. Meanwhile, Riluzole and Sodium butyrate combination treated SOD1 G93A mice had the most significantly increase of ZO-1 and Claudin-5 expression in the colon, compared to Riluzole alone or Sodium butyrate alone treated SOD1 G93A mice ( Fig. 2B ). ZO-1 and Claudin-5 expressions increased in the colon of all Riluzole alone, Sodium butyrate alone or Riluzole/Sodium butyrate combination treated SOD1 G93A mice groups compared to control mice. Meanwhile, Riluzole and Sodium butyrate combination treated SOD1 G93A mice had a significantly increased ZO-1 and Claudin-5 expression in the colon, compared to Riluzole alone or Sodium butyrate alone treated SOD1 G93A mice, as determined by immunofluorescence (IF) staining (Fig. 2C &D) . The aggregation of the h-SOD1 mutated protein was tested as an indicator of ALS progression. We also observed decreased aggregation of h-SOD1 G93A in the colon of Riluzole, Sodium butyrate, or Riluzole / Sodium butyrate combination groups, compared to control mice without treatment. As expected, Riluzole and Sodium butyrate combination treated SOD1 G93A mice had the most significantly reduction in aggregation of intestinal h-SOD1 G93A , compared to Riluzole alone or Sodium butyrate alone treated SOD1 G93A mice ( Fig. 2E ). In the spinal cord lumbar, Riluzole and Sodium butyrate combined treatment reduced h-SOD1 G93A aggregation and increased expression of TJ proteins in SOD1 G93A mice. We further examined the TJ proteins in the spinal cord. As shown in Fig. 3A , the expression of TJ proteins, ZO-1 and Claudin-5 were increased in the spinal cord lumbar of all Riluzole, Sodium butyrate, or Riluzole and Sodium butyrate combination groups, compared to control SOD1 G93A mice. Meanwhile, Riluzole and Sodium butyrate combination had the most significant increase of ZO-1 and Claudin-5 expression by WB. The distribution and density of ZO-1 ( Fig. 3B ) and Claudin-5 (Fig. 3C) had the most increase in the spinal cord lumbar of Riluzole and Sodium butyrate treated SOD1 G93A mice, as determined by IF staining. Aggregated h-SOD1 G93A decreased in the spinal cord lumbar of all Riluzole, Sodium butyrate, or combination groups, compared to control mice (Fig. 3D) . Meanwhile, Riluzole and Sodium butyrate combination treated SOD1 G93A mice had the most significant decrease of aggregated h-SOD1 G93A in the spinal cord lumbar, compared to Riluzole alone or Sodium butyrate alone treated SOD1 G93A mice. Riluzole and Sodium butyrate combination treated SOD1 G93A mice had reduced h-SOD1 G93A aggregation and increased ZO-1 and Claudin-5 in the brain cortex. To test the hypothesis that Riluzole and Sodium butyrate combined treatment better decreases aggregation of the h-SOD1 mutated protein, restores the gut-brain barrier function and delays the ALS disease onset, we examined ZO-1 and Claudin-5 in the cortex of brain tissue. Riluzole and Sodium butyrate combination had a significantly increased of ZO-1 and Claudin-5 expression in the brain cortex, compared to Riluzole alone or Sodium butyrate alone treated SOD1 G93A mice by WB ( Fig. 4A ) . As expected, Riluzole and Sodium butyrate combination treated SOD1 G93A mice had a significantly increase of ZO-1 and Claudin-5 expression in the brain cortex, compared to Riluzole alone or Sodium butyrate alone treated SOD1 G93A mice, as determined by IF staining ( Fig. 4B and Fig. 4C ). Claudin-1 expression showed no change in the brain cortex. In the meanwhile, the h-SOD1 G93A protein aggregation was quantified with AggreCount. Riluzole and Sodium butyrate treated SOD1 G93A mice had a significantly decrease in the brain cortex aggregation h-SOD1 G93A , compared to Riluzole alone or Sodium butyrate alone treated SOD1 G93A mice ( Fig. 4D ) . Riluzole and Sodium butyrate combined treatment reduced serum LPS and inflammatory cytokines in SOD1 G93A mice. Intestinal leakage triggers bacterial LPS to blood. Thus, serum LPS could be tested by ELISA 17,18 . As shown in Fig. 5A, LPS were decreased on all mice with treatment. The expression of serum inflammatory cytokine IL-17 was decreased in Riluzole, Sodium butyrate, or Riluzole and Sodium butyrate combination groups, compared to control mice ( Fig. 5B ). Meanwhile, Riluzole and Sodium butyrate combination in SOD1 G93A mice had a significantly decreased serum LPS and IL-17 expression, compared to Riluzole or Sodium butyrate treated SOD1 G93A mice. The levels of serum inflammatory cytokines IL-6 (Fig. 5C) and IFN-γ (Fig. 5D) showed a slight decrease without statistical significance in Riluzole and Sodium butyrate treated groups, compared to SOD1 G93A mice without any treatment. But mice with Riluzole and Sodium butyrate combination had a significantly decreased serum IL-6 and IFN-γ, compared to the no-treatment SOD1 G93A mice. However, serum inflammation cytokines IL-4 and IP-10 didn’t change in all groups ( Fig. 5E & F ), suggesting the role of Riluzole and Sodium butyrate combination might be specific for certain inflammatory cytokines. Discussion In the current study, we investigate the combined roles of Riluzole and Sodium butyrate for the ALS therapy and explore new therapeutic targets for ALS by restoring intestinal homeostasis and barrier functions. Riluzole and Sodium butyrate combination showed a significantly longer rotarod time, more increased grip strength, enhanced intestinal barrier, compared with Riluzole or butyrate alone treatment. The aggregation of the h-SOD1 mutated protein was tested as an indicator of ALS progression. More reduced SOD1 G93A aggregation was observed in the colon, spinal cord lumbar and brain cortex with Riluzole and butyrate combination treatment compared with Riluzole or butyrate alone treatment. The expression of tight junction proteins (ZO-1 and Claudin-5) significantly increased in the colon, spinal cord lumbar and brain cortex with Riluzole and butyrate combination treatment, compared with Riluzole alone or Sodium butyrate alone treatment. The mechanism of Riluzole in protecting intestine in ALS is unknown. Our data not only showed the combined role of Riluzole and Sodium butyrate in slowing down the disease progress through reserving barrier function and inhibiting inflammation, but also demonstrate that Riluzole alone has the beneficial impacts on the intestinal and blood-brain barriers. Combination role of R and butyrate is unknown. A previous study used I.P. treatment of Riluzole and sodium phenlebutyrate (NaPB) in the ALS mice 1 . It showed that Riluzole/NaPB administration increased acetylation at H4 and increased NF-κB p50 translocation to the nucleus in G93A mice. However, the Riluzole and Sodium butyrate has not been tested. In the current study, we did gavage of Sodium butyrate and further checked the combined role on the intestinal functions and TJs in BBB. The results of serum LPS and inflammatory cytokines also explain the influence of epithelial damage on the function of spinal cord motor neurons and neuromuscular structures in the ALS progress. Riluzole and Sodium butyrate combination were able to reduce the serum LPA and inflammatory cytokines. Our data suggestion that ALS mice treatment with Riluzole and Sodium butyrate combination is more efficient than Riluzole alone or Sodium butyrate alone treatment in delay ALS progress. Restoring barrier function through the gut-brain axis provides a potential therapeutic strategy for ALS. Our study opens a new avenue in targeting the gut microbiota-neuron-axis for treating ALS. Materials and Methods Animal model SOD1 G93A strain (B6SJL-Tg (SOD1-G93A) 1Gur/J, stock No.002726) 8,9,21,22 was purchased from Jackson Laboratory (The Jackson Laboratory, Bar Harbor, ME, USA). All mice were housed in specific pathogen-free environments under a controlled condition of 12 h light/12 h dark cycle at 20-22 °C and 45 ± 5% humidity, with free access to the same food and autoclaved water. All materials involved including cage, bedding, water bottles, cage card holder were autoclaved before housing mice. The mice are housed of the same gender and each cage has no more than 5 mice. All experiments were carried out in strict accordance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the IACUC of University of Illinois Chicago Committee on Animal Resources (ACC 21-178 and ACC 23-149). The study is reported in accordance with ARRIVE guidelines. Rotarod test Motor coordination, endurance, and balance were determined in latency by a rotarod test. Mice were trained on a rotarod test three days before performance trials at an accelerating speed from 4 to 40 rpm for 300s using the Rotarod Model LE8205 (Harvard Apparatus, Holliston, MA, USA). Latency to fall was recorded when the mouse fell from the rod. 9,23 Each mouse was tested in 2 trials per day for 2 consecutive days. The mean times for 4 trials of the tests were calculated for each mouse. Assessment of grip strength Forelimb and hindlimb grip measurements were acquired in triplicate with a 25 N Grip strength meter (Harvard Apparatus, Holliston, MA, USA). The mice were lowered onto a triangle bar of the grip strength meter until the animals gripped the bar with their forelimbs or hindlimbs, then the mice were pulled gently backward until they released their grip. The force gauge of the grip meter recorded the maximum force. 9,24 Riluzole and Butyrate treatment in SOD1 G93A mice SOD1 G93A mice aged 9 to 10 weeks were randomly assigned to 4 groups. The Riluzole-treated group intraperitoneally received Riluzole (Sigma-Aldrich, 1604337, St. Louis, MO, USA) at a dose of 10mg per kg body weight daily. The Sodium butyrate-treated group received 2% sodium butyrate (Sigma-Aldrich, 303410, St. Louis, MO, USA) in filtered drinking water. The Riluzole/Sodium butyrate combination-treated group received Riluzole and Sodium butyrate. The Riluzole and Sodium butyrate treatments started at 9 to 10 weeks and finished at 15-16 weeks. All animals were weighed and received a detailed clinical examination, which included assessments of appearance, movement and behavior patterns, skin and hair conditions, eyes and mucous membranes, respiration, and excreta. Restricted outstretching of the hind legs observed on tail holding was considered a symptom of ALS, mice that were unable to turn over within 20 seconds after being laid on their backs were humanely euthanized CO2 inhalation followed by cervical dislocation. Western blot analysis and antibodies Mice colonic epithelial cells were collected by scraping the tissue from the colon of the mouse, including the proximal and distal regions. Mice spinal cord lumbar and brain cortex were harvested and minced into tiny pieces by using scissors. 28,29 The collected tissues were sonicated in lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH 8.0, 1% Triton X-100) with 0.2 mM sodium ortho-vanadate, and protease inhibitor cocktail. The protein concentration was measured using the BioRad Reagent (BioRad, Hercules, CA, USA) and then sonicated. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with primary antibodies. The following antibodies were used: anti-human SOD1 (Abcam, ab52950, Cambridge, MA, USA), anti-ZO-1 (Invitrogen, 33-9100, Carlsbad, CA, USA), anti-Claudin-5 (Invitrogen, 35-2500, Carlsbad, CA, USA), anti-Claudin-1 (Invitrogen, 71-7800, Carlsbad, CA, USA), anti-GAPDH (Cell signaling technology, 5174, Danvers, MA, USA), or anti-β-actin (Sigma-Aldrich, A5316, St. Louis, MO, USA) antibodies and were visualized by ECL (Thermo Fisher Scientific, Waltham, MA, USA). Membranes that were probed with more than one antibody were stripped before re-probing. The software Quantity One has been used for the quantification of the western blot bands. Briefly, the “rectangular tool” was first selected to measure the background and the bands of western blots one by one. All the values of “density” and “volume” after measurement were transferred to an excel file. With the subtraction of background measurement, the “density” values for each band on the western blot were calculated. Immunofluorescence The colonic, spinal cord and brain tissues were freshly isolated and embedded in paraffin wax after fixation with 10% neutral buffered formalin. Immunofluorescence was performed on paraffin-embedded sections (5 μm). After preparation of the slides as described previously, 30,31 tissue samples were incubated with anti-human SOD1 (Abcam, ab52950, Cambridge, MA, USA), anti-ZO-1 (Invitrogen, 33-9100, Carlsbad, CA, USA), anti-Claudin-5 (Invitrogen, 35-2500, Carlsbad, CA, USA) at 4°C overnight. Samples were then incubated with goat anti-rabbit Alexa Flour 488 (Invitrogen, A-11008, Carlsbad, CA, USA), goat anti-mouse Alexa Flour 488 (Invitrogen, A-11001, Carlsbad, CA, USA) or goat anti-mouse Alexa Flour 594 (Invitrogen, A-11032, Carlsbad, CA, USA) and DAPI (Invitrogen, D1306, Carlsbad, CA, USA) for 1 h at room temperature. Tissues were mounted with SlowFade (Invitrogen, s2828, Carlsbad, CA, USA), followed by a coverslip, and the edges were sealed to prevent drying. Specimens were examined with a Zeiss laser scanning microscope LSM 710 (Carl Zeiss Inc., Oberkochen, Germany). The h-SOD1 G93A protein aggregation was quantified with AggreCount software (https://aggrecount.github.io/). Fluorescence intensity was determined by using Image J software. This method determines the corrected total fluorescence by subtracting out background signal, which is useful for comparing the fluorescence intensity between cells or regions. Intestinal permeability Fluorescein isothiocyanate-dextran (average molecular weight 4000, Sigma, 46944, Burbank, CA, USA, diluted in HBSS) was gavaged (25 mg / kg mouse) 4 hours before sample harvest. Mice were anesthetized with avertin; depth of anesthesia was assessed with toe pinch and then blood was collected via cardiac puncture followed by cervical dislocation. Mouse blood samples were collected for intestinal permeability test. 25-27 Serum lipopolysaccharides (LPS) detection LPS in serum samples was measured with limulus amebocyte lysate chromogenic end point assays (Hycult Biotech, HIT302, Plymouth, PA, USA) according to the manufacturer's indications. The samples were diluted 1:4 with endotoxin-free water and then heated at 75°C for 5 minutes on a warm plate to denature the protein before the reaction. A standard curve was generated and used to calculate the concentrations, which were expressed as EU/mL, in the serum samples. Multiplex ELISA assay Mouse blood samples were collected by cardiac puncture and placed in tubes containing EDTA (10 mg/mL). Mouse cytokines were measured using a Cytokine & Chemokine Convenience 26-Plex Mouse ProcartaPlex™ Panel 1 (Invitrogen, EPXR260-26088-901, Carlsbad, CA, USA) according to the manufacturer's instructions. Briefly, beads of defined spectral properties were conjugated to protein-specific capture antibodies and added along with samples (including standards of known protein concentration, control samples, and test samples) into the wells of a filter-bottom microplate, where proteins bound to the capture antibodies over the course of a 2-hour incubation. After washing the beads, protein-specific biotinylated detector antibodies were added and incubated with the beads for 1 hour. After removal of excess biotinylated detector antibodies, the streptavidin-conjugated fluorescent protein R-phycoerythrin was added and allowed to incubate for 30 minutes. After washing to remove unbound streptavidin–R-phycoerythrin, the beads were analyzed with the Luminex detection system (Bio-Rad, Bio-Plex 200 Systems, Hercules, CA, USA). Statistical Analysis All data were expressed as the mean ± SEM or ± SD. All statistical tests were 2-sided. All p-values < 0.05 were considered statistically significant. The differences between samples for more than two groups were analyzed using one-way ANOVA, or two-way ANOVA as appropriate based on data distribution and the number of factors, respectively. The p -values in ANOVA analyses were adjusted for correction of multiple comparisons using the Tukey method to ensure accurate results. Statistical analyses were performed using GraphPad Prism 8 (GraphPad, Inc., San Diego, CA, USA). Declarations Acknowledgements/Funds We would like to acknowledge the VA Merit Award 1 I01BX004824-01, the NIDDK/National Institutes of Health grant R01 DK105118, and R01DK114126 to Jun Sun. The study sponsors play no role in the study design, data collection, analysis, and interpretation of data. Author contributions YZ performed the cellular and animal studies, the detailed analyses of the results; YZ &KR: animal studies; YZ and JS, prepared the figures and the draft text; YX contributed to the statistical analysis of data and the draft text; and JS obtained funds, designed the study, and directed the project. All authors contributed to the writing of the manuscript. Data availability statement Data are available upon request to Jun Sun ( [email protected] ) Competing Interests Statement The authors declare that they have no conflict of interest. References Del Signore, S. J. et al. Combined riluzole and sodium phenylbutyrate therapy in transgenic amyotrophic lateral sclerosis mice. Amyotroph Lateral Scler 10 , 85-94, doi:10.1080/17482960802226148 (2009). Hardiman, O. et al. Amyotrophic lateral sclerosis. Nat Rev Dis Primers 3 , 17071 (2017). https://doi.org:10.1038/nrdp.2017.71 Alonso, A., Logroscino, G., Jick, S. S. & Hernan, M. A. 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Target Metabolites to Slow Down Progression of Amyotrophic Lateral Sclerosis in Mice. Metabolites 12 (2022). https://doi.org:10.3390/metabo12121253 Sun, J. Commentary: Target Intestinal Microbiota to Alleviate Disease Progression in Amyotrophic Lateral Sclerosis. J Neurol Neuromedicine 2 , 13-15 (2017). https://doi.org:10.29245/2572.942x/2017/6.1136 Gilli, F., Royce, D. B. & Pachner, A. R. Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis. J Vis Exp (2016). https://doi.org:10.3791/54616 Huerta Ojeda, A., Fontecilla Diaz, B., Yeomans Cabrera, M. M. & Jerez-Mayorga, D. Grip power test: A new valid and reliable method for assessing muscle power in healthy adolescents. PLoS One 16 , e0258720 (2021). https://doi.org:10.1371/journal.pone.0258720 Zhang, Y. G. et al. Lack of Vitamin D Receptor Leads to Hyperfunction of Claudin-2 in Intestinal Inflammatory Responses. Inflamm Bowel Dis 25 , 97-110 (2019). https://doi.org:10.1093/ibd/izy292 Zhang, Y. G. et al. Vitamin D Receptor Protects Against Dysbiosis and Tumorigenesis via the JAK/STAT Pathway in Intestine. Cell Mol Gastroenterol Hepatol 10 , 729-746 (2020). https://doi.org:10.1016/j.jcmgh.2020.05.010 Zhang, Y. G. et al. Intestinal vitamin D receptor protects against extraintestinal breast cancer tumorigenesis. Gut Microbes 15 , 2202593 (2023). https://doi.org:10.1080/19490976.2023.2202593 Shen, Y. et al. Ultrasound-enhanced brain delivery of edaravone provides additive amelioration on disease progression in an ALS mouse model. Brain Stimul 16 , 628-641 (2023). https://doi.org:10.1016/j.brs.2023.03.006 Lin, Y. S. et al. Neuronal Splicing Regulator RBFOX3 (NeuN) Regulates Adult Hippocampal Neurogenesis and Synaptogenesis. PLoS One 11 , e0164164 (2016). https://doi.org:10.1371/journal.pone.0164164 Zhang, Y., Garrett, S., Carroll, R. E., Xia, Y. & Sun, J. Vitamin D receptor upregulates tight junction protein claudin-5 against colitis-associated tumorigenesis. Mucosal Immunol 15 , 683-697 (2022). https://doi.org:10.1038/s41385-022-00502-1 Zhang, Y., Zhang, J., Xia, Y. & Sun, J. Bacterial translocation and barrier dysfunction enhance colonic tumorigenesis. Neoplasia 35 , 100847 (2023). https://doi.org:10.1016/j.neo.2022.100847 Additional Declarations No competing interests reported. Supplementary Files WBoriginalpictures10.29.2024.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5349709","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":376264333,"identity":"f9f79e73-8c8d-4114-a747-6cf8eaefe0ea","order_by":0,"name":"Yong-Guo Zhang","email":"","orcid":"","institution":"University of Illinois Chicago","correspondingAuthor":false,"prefix":"","firstName":"Yong-Guo","middleName":"","lastName":"Zhang","suffix":""},{"id":376264334,"identity":"9ff63a51-eac6-41f8-beb1-a48788adad5d","order_by":1,"name":"KaReisha Robinson","email":"","orcid":"","institution":"University of Illinois Chicago","correspondingAuthor":false,"prefix":"","firstName":"KaReisha","middleName":"","lastName":"Robinson","suffix":""},{"id":376264335,"identity":"9404618f-b76e-481c-b7c0-2f2deb2525d6","order_by":2,"name":"Yinglin Xia","email":"","orcid":"","institution":"Jesse Brown VA Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yinglin","middleName":"","lastName":"Xia","suffix":""},{"id":376264336,"identity":"15ad3ecd-1c1e-4198-af9b-643a84d36b82","order_by":3,"name":"Jun Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYBACxgYw9V+OgRlEsxGvhdmYeC1QwJwI0UqMFuYZuYc/8+5gS99wnPkBw4eyw0Q4bEZemjTvGZ7cDYfZDBhnnCNKS44ZM2+bBFALDwOQQZwW48+8bQbpBiAtf4nUYiDN25aQANbCSJSWnjdmknPbDhjOBPrlYM+5dMJaDNtzjD+8bTsgz3f+8MMHP8qsidDSgMQ5QFg9EMgTpWoUjIJRMApGNgAAS+A2T6W4i8YAAAAASUVORK5CYII=","orcid":"","institution":"University of Illinois Chicago","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2024-10-28 21:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5349709/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5349709/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68743282,"identity":"c6f3b3bf-b4f8-4e29-b8a4-e64461466b82","added_by":"auto","created_at":"2024-11-11 14:39:14","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":602722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRiluzole and Sodium butyrate combination showed a significantly delayed disease progress, compared with Riluzole or Sodium butyrate alone treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic overview of the SOD1\u003csup\u003eG93A\u003c/sup\u003e mice treated with Riluzole, Sodium butyrate, or Riluzole and Sodium butyrate combination\u003cstrong\u003e \u003c/strong\u003ein\u003cstrong\u003e \u003c/strong\u003eSOD1\u003csup\u003eG93A\u003c/sup\u003e mice (9- to 10-week-old). Riluzole was I.P. at a dose of 10mg / kg body weight daily. 2% Sodium butyrate was given in filtered drinking water. The treatments lasted 6 weeks. \u003cstrong\u003e(B)\u003c/strong\u003e Body weight changes of the SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. At the age of 15-16 weeks, Riluzole and Sodium butyrate combination showed a significant weight loss, compared to the no-treatment SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. Data are expressed as mean ± SEM. N = 8-10, two-way ANOVA test. \u003cstrong\u003e(C)\u003c/strong\u003e Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice had a significantly increased rotarod time, compared to Riluzole alone, Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. Data are expressed as mean ± SD. n = 8-10, two-way ANOVA test. \u003cstrong\u003e(D)\u003c/strong\u003e Riluzole and Sodium butyrate combination led to a significantly increased forelimb grip strength and \u003cstrong\u003e(E\u003c/strong\u003e) hindlimb grip strength, compared to Riluzole alone, or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. From the age of 12-13 weeks, Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice all had a significantly increased forelimb grip strength and hindlimb grip strength compared to control mice. At the age of 15-16 weeks, Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice had a significantly increased forelimb grip strength and hindlimb grip strength, compared to Riluzole alone, or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. Data are expressed as mean ± SD. N = 8-10, two-way ANOVA test. All\u0026nbsp;\u003cem\u003ep\u003c/em\u003evalues are shown in the figures.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5349709/v1/6ec818d063bf9f6dccce6d85.jpeg"},{"id":68743283,"identity":"31abf596-4d0e-4aa7-a1f5-8c1e32b86d63","added_by":"auto","created_at":"2024-11-11 14:39:15","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":981577,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDecreased permeability, reduced h-SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e aggregation and increased expression of tight junction proteins in the colon of SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice with Riluzole/Sodium butyrate combination treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Intestinal permeability was decreased in all Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice groups compared to control mice.\u003cstrong\u003e \u003c/strong\u003eMeanwhile,\u003cstrong\u003e \u003c/strong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice had a significantly decreased intestinal permeability, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. Data are expressed as mean ± SD. N = 6-9, one-way ANOVA test. \u003cstrong\u003e(B)\u003c/strong\u003e The expression of tight junction proteins, ZO-1 and Claudin-5, was increased in the colon of all Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice groups compared to control mice.\u003cstrong\u003e \u003c/strong\u003eMeanwhile,\u003cstrong\u003e \u003c/strong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice had a significantly increased ZO-1 and Claudin-5 expression in the colon compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice by WB. Data are expressed as mean ± SD. n = 4, one-way ANOVA test. \u003cstrong\u003e(C)\u003c/strong\u003e ZO-1 and \u003cstrong\u003e(D)\u003c/strong\u003e Claudin-5 expressions increased in intestines of all of Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice groups compared to control mice, as determined by IF staining. The relative fluorescence intensity was quantified with ImageJ by counting 3 images for each sample. Data are shown as mean ± SD, n\u0026nbsp;= 3 per group, one-way ANOVA test. \u003cstrong\u003e(E)\u003c/strong\u003e Decreased Aggregation of h-SOD1\u003csup\u003eG93A\u003c/sup\u003e in the colon of SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice with Riluzole, Sodium butyrate alone, or combination, compared to control mice without treatment.\u003cstrong\u003e \u003c/strong\u003eMeanwhile,\u003cstrong\u003e \u003c/strong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice had a significantly reduction of aggregated h-SOD1\u003csup\u003eG93A\u003c/sup\u003e, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. The h-SOD1\u003csup\u003eG93A\u003c/sup\u003e protein aggregation was quantified with AggreCount by counting 3 images for each sample. Data are shown as mean ± SD, n\u0026nbsp;= 3 per group, one-way ANOVA test. All\u0026nbsp;\u003cem\u003ep\u003c/em\u003e-values are shown in the figures.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5349709/v1/59d7dd3055cf1e95684cc747.jpeg"},{"id":68743284,"identity":"f1244c9a-7d06-4d55-a6d2-cfcee28210e6","added_by":"auto","created_at":"2024-11-11 14:39:15","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":783088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduced h-SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;aggregation and increased expression of ZO-1 and Claudin-5 in the spinal cord lumbar of Riluzole/Sodium butyrate treated SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The expression of tight junction proteins, ZO-1 and Claudin-5, was increased in the spinal cord lumbar of all Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination groups treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice compared to control mice.\u003cstrong\u003e \u003c/strong\u003eMeanwhile,\u003cstrong\u003e \u003c/strong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice had a significantly increased ZO-1 and Claudin-5 expression in the spinal cord lumbar, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice by WB. Data are expressed as mean ± SD. n = 4, one-way ANOVA test. \u003cstrong\u003e(B)\u003c/strong\u003e ZO-1 and \u003cstrong\u003e(C)\u003c/strong\u003e Claudin-5 expressions increased in the spinal cord lumbar of all Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice compared to control mice.\u003cstrong\u003e \u003c/strong\u003eMeanwhile,\u003cstrong\u003e \u003c/strong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice had a significantly increased ZO-1 and Claudin-5 expression in the spinal cord lumbar, compared to Riluzole alone, and Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, as determined by IF staining. The relative fluorescence intensity was quantified with ImageJ by counting 3 images for each sample. Data are shown as mean ± SD, n\u0026nbsp;= 3 per group, one-way ANOVA test. \u003cstrong\u003e(D)\u003c/strong\u003e Aggregated h-SOD1\u003csup\u003eG93A\u003c/sup\u003e was decreased in the spinal cord lumbar of Riluzole, Sodium butyrate, or Riluzole and Sodium butyrate combination groups, compared to control mice.\u003cstrong\u003e \u003c/strong\u003eThe h-SOD1\u003csup\u003eG93A\u003c/sup\u003e protein aggregation was quantified with AggreCount by counting 3 images for each sample. Data are shown as mean ± SD, n\u0026nbsp;= 3 per group, one-way ANOVA test. All\u0026nbsp;\u003cem\u003ep\u003c/em\u003e-values are shown in the figures.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5349709/v1/53e16a3ffbe1e8be59f2f51e.jpeg"},{"id":68743286,"identity":"0e1e0dff-bf99-435b-9e80-f8fdde389a18","added_by":"auto","created_at":"2024-11-11 14:39:16","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":772951,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduced h-SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;aggregation and increased expression of tight junction proteins (ZO-1 and Claudin-5) in the brain cortex of Riluzole/Sodium butyrate treated SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The expressions of ZO-1 and Claudin-5 by WB were increased in the brain cortex of Riluzole alone, Sodium butyrate, or Riluzole and Sodium butyrate combination treated groups, compared to control mice. Meanwhile,\u003cstrong\u003e \u003c/strong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice had a significantly increased ZO-1 and Claudin-5 expression in the brain cortex, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice by WB. Data are expressed as mean ± SD. N = 4, one-way ANOVA test. \u003cstrong\u003e(B)\u003c/strong\u003e ZO-1 expression and \u003cstrong\u003e(C)\u003c/strong\u003e Claudin-5 increased in the brain cortex of all Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination groups treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice compared to control mice.\u003cstrong\u003e \u003c/strong\u003eMeanwhile,\u003cstrong\u003e \u003c/strong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice had a significantly increase of ZO-1 and Claudin-5 expression in the brain cortex, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, as determined by IF staining. The relative fluorescence intensity was quantified with ImageJ by counting 3 images for each sample. Data are shown as mean ± SD, n\u0026nbsp;= 3 per group, one-way ANOVA test. (\u003cstrong\u003eD\u003c/strong\u003e) Aggregated h-SOD1\u003csup\u003eG93A\u003c/sup\u003e was decreased in the brain cortex of all Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice groups compared to control mice.\u003cstrong\u003e \u003c/strong\u003eMeanwhile,\u003cstrong\u003e \u003c/strong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice had a significantly decrease in the brain cortex of aggregation h-SOD1\u003csup\u003eG93A\u003c/sup\u003e, compared to Riluzole alone, Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. The h-SOD1\u003csup\u003eG93A\u003c/sup\u003e protein aggregation was quantified with AggreCount by counting 3 images for each sample. Data are shown as mean ± SD, n\u0026nbsp;= 3 per group, one-way ANOVA test. All\u0026nbsp;\u003cem\u003ep\u003c/em\u003e-values are shown in the figures.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5349709/v1/d433af3f46a08be9fb00829f.jpeg"},{"id":68743281,"identity":"03e1559d-91ab-48e9-917b-5f03d4f60436","added_by":"auto","created_at":"2024-11-11 14:39:13","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":459664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduced serum\u0026nbsp;LPS, IL-17, IL-6, and IFN-γ\u0026nbsp;in Riluzole/Sodium butyrate treated SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of serum inflammatory cytokines \u003cstrong\u003e(A)\u003c/strong\u003e\u0026nbsp;LPS\u0026nbsp;and\u0026nbsp;\u003cstrong\u003e(B)\u003c/strong\u003e\u0026nbsp;IL-17\u0026nbsp;were\u0026nbsp;decreased in all Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice groups compared to control mice.\u0026nbsp;Meanwhile,\u0026nbsp;Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice had a significantly decreased serum LPS and IL-17 expression, compared to Riluzole or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice. Data are expressed as mean ± SD. N = 6, one-way ANOVA test. The expression of\u0026nbsp;serum inflammatory cytokines \u003cstrong\u003e(C)\u003c/strong\u003e\u0026nbsp;IL-6\u0026nbsp;and\u0026nbsp;\u003cstrong\u003e(D)\u003c/strong\u003e\u0026nbsp;IFN-γ showed a slight decrease without statistical significance in Riluzole alone and Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice groups compared to SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice without any treatment. But Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice had a significantly\u0026nbsp;decreased serum\u0026nbsp;IL-6 and IFN-γ expression, compared to the no-treatment SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice. Meanwhile,\u0026nbsp;Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice had a significant decrease in serum IL-6 and IFN-γ expression, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice. Data are expressed as mean ± SD. N = 6, one-way ANOVA test.\u0026nbsp;The expression of serum inflammation cytokines (\u003cstrong\u003eE)\u003c/strong\u003e\u0026nbsp;IL-4\u0026nbsp;and\u0026nbsp;\u003cstrong\u003e(F)\u003c/strong\u003e\u0026nbsp;IP-10\u0026nbsp;didn’t change in all groups. Data are expressed as mean ± SD. N = 6, one-way ANOVA test. All\u0026nbsp;\u003cem\u003ep\u003c/em\u003e-values are shown in the figures.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5349709/v1/1cb9c0adc713383db77e6444.jpeg"},{"id":71754682,"identity":"7e1aa1e8-e12b-4a8c-a325-94b7dd2fa31a","added_by":"auto","created_at":"2024-12-18 10:02:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4553156,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5349709/v1/5ce8951a-4489-4f23-a5dc-e5a1eebe5512.pdf"},{"id":68743285,"identity":"753c10a4-b861-4497-bf66-2c8fab00d4d0","added_by":"auto","created_at":"2024-11-11 14:39:16","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":540655,"visible":true,"origin":"","legend":"","description":"","filename":"WBoriginalpictures10.29.2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5349709/v1/4649976c64a3c5ec9c7d099f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eSynergistic Effects of Riluzole and Sodium Butyrate on Barrier Function and Disease Progression of Amyotrophic Lateral Sclerosis in SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAmyotrophic lateral sclerosis is a fatal neuromuscular disease characterized by progressive motor neuron death and skeletal muscle atrophy and paralysis \u003csup\u003e1,2\u003c/sup\u003e. The lifetime risk of ALS is about 1 in 472 in women and 1 in 350 in men\u003csup\u003e3\u003c/sup\u003e. Because ALS is an age-dependent disease, an increase in the prevalence of ALS can be anticipated as the U.S. population increases and ages. Military veterans, regardless of the branch of service, the era in which they served, or whether they served during a time of peace or war, are at a greater risk of dying from ALS than if they had not served in the military. \u0026nbsp;Most patients with ALS die within 5 years after the disease onset. Despite intensive research efforts, there is no effective cure for ALS. The two available treatments (riluzole and edaravone) are to relieve symptoms and improve the quality of life for patients with ALS. There are significant needs to develop novel treatments for ALS and improve the life quality of ALS patients \u003csup\u003e4\u003c/sup\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRiluzole was the first FDA-approved treatment for ALS and is speculated to reduce glutamatergic neurotransmission by blocking voltage-gated sodium channels on presynaptic neurons.\u0026nbsp;Riluzole extends the patient\u0026rsquo;s life span for a few months\u003csup\u003e5\u003c/sup\u003e.\u0026nbsp;Increased serum inflammatory cytokines\u003csup\u003e6\u003c/sup\u003e, bacterial lipopolysaccharides (LPS) \u003csup\u003e6\u003c/sup\u003e, and aberrant microbiome have been reported in prior studies of humans ALS \u003csup\u003e7\u003c/sup\u003e. Our study with oral administration of sodium butyrate presents a promising therapeutic strategy \u003csup\u003e8,9\u003c/sup\u003e. Mice with butyrate treatment showed a significantly longer latency to fall in the rotarod test, compared with the G93A non-treatment mice. Bacterial lipopolysaccharides (LPS), glycolipids found in the outer membrane of gram-negative bacteria, are pro-inflammatory and have found to be increased in the serum of ALS patients \u003csup\u003e6\u003c/sup\u003e.\u0026nbsp;Indeed, increased inflammatory cytokine IL-17 and IL-23 have been reported in serum and cerebrospinal fluid of patients with ALS\u003csup\u003e10\u003c/sup\u003e.\u0026nbsp;We have demonstrated increased serum IL-17 in the SOD1\u003csup\u003eG93A\u0026nbsp;\u003c/sup\u003eALS mice, compared to the wild-type mice\u003csup\u003e11\u003c/sup\u003e. Our study showed the\u0026nbsp;dysbiosis was correlated\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewith intestinal inflammation and increased intestinal permeability in ALS\u003csup\u003e11\u003c/sup\u003e.\u0026nbsp;However,\u0026nbsp;the impact\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand mechanism of Riluzole in the ALS intestine is unknown. Combined role and mechanism of Riluzole and butyrate is also unknown. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the current study, we use the 2% sodium butyrate in the drinking water\u0026nbsp;combining with the\u0026nbsp;Riluzole in the SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. The outcomes focused on clinical disease progression (e.g., body weight, ratorad, onset, and neuroprotection (reducing motor neuron loss and inhibiting glial activation, and survival). Moreover, we examined the changes of aggregation of human mutant SOD1\u003csup\u003eG93A\u003c/sup\u003e (h-SOD1\u003csup\u003eG93A\u003c/sup\u003e), TJs, barriers, and inflammation. Better understanding of the mechanisms of existing drug and synergistic effects of Riluzole and Sodium butyrate will provide insights into barrier function and ALS progression.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eRiluzole and Sodium butyrate combination showed a significantly protection against disease progression in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine the synergistic effects of Riluzole and Sodium butyrate on disease progression, we set up 4 experimental groups with Riluzole alone, Sodium butyrate alone, and Riluzole and Sodium butyrate combination, and control without treatment\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ein SOD1\u003csup\u003eG93A\u003c/sup\u003e mice aged 9 to 10 weeks. Mice were randomly assigned to each group. The treatment was total 6 weeks. The Riluzole-treated group received Riluzole via I.P. at a dose of 10mg per kg body weight daily. The Sodium butyrate-treated group received 2% Sodium butyrate in filtered drinking water (\u003cstrong\u003eFig. 1A\u003c/strong\u003e). The date of disease onset is defined as the first date when a mouse fails the 15-rpm rotarod task and start to show muscle tremors in the hind limbs\u003csup\u003e12,13\u003c/sup\u003e. The body weight will be monitored every day during the treatment period. The peak body weight is a reasonable and consistent determinant of onset particularly in combination with a measure of neurological score \u003csup\u003e14,15\u003c/sup\u003e. Body weight changes were shown in \u003cstrong\u003eFig. 1B\u003c/strong\u003e. From the age of 13-14 weeks, Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination\u0026nbsp;treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice groups show slight weight loss without statistical significance, compared to the no-treatment SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. From the age of 14-15 weeks, Riluzole/Sodium butyrate combination treatment showed a slight weight loss without statistical significance, compared to the Riluzole alone or Sodium butyrate alone treatment SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. At the age of 15-16 weeks, Riluzole and Sodium butyrate combination showed a significant less weight loss, compared to the no-treatment SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe SOD1\u003csup\u003eG93A\u003c/sup\u003e mice were tested on an accelerating rotarod\u003csup\u003e13\u003c/sup\u003e to examine the neuromuscular activity performance\u003cem\u003e.\u003c/em\u003e The Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice had a significantly increased rotarod time, compared to Riluzole alone, Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice (\u003cstrong\u003eFig. 1C\u003c/strong\u003e). \u0026nbsp;From the age of 12-13 weeks, Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice all had a significantly increased rotarod time, compared to control mice without treatment. At the age of 15-16 weeks, the combination group had a significantly increased rotarod time, compared to SOD1\u003csup\u003eG93A\u003c/sup\u003e mice with only Riluzole or Sodium butyrate\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice had a significantly increased strengths of forelimb grip \u003cstrong\u003e(Fig. 1D\u003c/strong\u003e) and hindlimb grip \u003cstrong\u003e(Fig. 1E\u003c/strong\u003e), compared to Riluzole alone, or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. From the age of 12-13 weeks, Riluzole alone, Sodium butyrate alone or Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice all had a significantly increased forelimb grip strength and hindlimb grip strength, compared to control mice without treatment. At the age of 15-16 weeks, Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice had a significantly increased forelimb grip strength and hindlimb grip strength, compared to Riluzole alone, or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. The date of death is defined as the day when the mouse cannot right itself within 30 seconds after being placed on its back\u003csup\u003e13\u003c/sup\u003e. We did not perform the\u0026nbsp;survival curve of the SOD1\u003csup\u003eG93A\u003c/sup\u003e mice with and without the treatment in ALS progression due to the restricts of the animal protocol. Overall, our data suggested that Riluzole and Sodium butyrate combination had a significantly protection against disease progression in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRiluzole and Sodium butyrate combined treatment reserved the permeability and reduced h-SOD1\u003csup\u003eG93A\u003c/sup\u003e aggregation in the SOD1\u003csup\u003eG93A\u003c/sup\u003e mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIncreased gut permeability was reported in the\u0026nbsp;SOD1\u003csup\u003eG93A\u003c/sup\u003e mice \u003csup\u003e8,9,16\u003c/sup\u003e. We then examine whether treatment delays the intestinal dysfunction during ALS progression. Intestinal permeability was decreased in all Riluzole, Sodium butyrate, or Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice groups, compared to control mice.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMeanwhile,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice had a significantly decreased intestinal permeability, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice \u003cstrong\u003e(Fig. 2A)\u003c/strong\u003e. The intestinal permeability was regulated by the tight junctions (TJs). We then examined the expression of tight junction proteins. ZO-1 and Claudin-5 detected by Western Blot (WB) were increased in intestines of Riluzole, Sodium butyrate or Riluzole and Sodium butyrate combination, compared to control mice without treatment.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMeanwhile,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice had the most significantly increase of ZO-1 and Claudin-5 expression in the colon, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice (\u003cstrong\u003eFig. 2B\u003c/strong\u003e). ZO-1 and Claudin-5 expressions increased in the colon of all Riluzole alone, Sodium butyrate alone or Riluzole/Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice groups compared to control mice.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMeanwhile,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice had a significantly increased ZO-1 and Claudin-5 expression in the colon, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, as determined by immunofluorescence (IF) staining \u003cstrong\u003e(Fig. 2C \u0026amp;D)\u003c/strong\u003e. The aggregation of the h-SOD1 mutated protein was tested as an indicator of ALS progression. We also observed decreased aggregation of h-SOD1\u003csup\u003eG93A\u003c/sup\u003e in the colon of Riluzole, Sodium butyrate, or Riluzole / Sodium butyrate combination groups, compared to control mice without treatment.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAs expected, Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice had the most significantly reduction in aggregation of intestinal h-SOD1\u003csup\u003eG93A\u003c/sup\u003e, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice (\u003cstrong\u003eFig. 2E\u003c/strong\u003e). \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn the spinal cord lumbar, Riluzole and Sodium butyrate combined treatment reduced h-SOD1\u003csup\u003eG93A\u003c/sup\u003e aggregation and increased expression of TJ proteins in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe further examined the TJ proteins in the spinal cord. As shown in \u003cstrong\u003eFig. 3A\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethe expression of TJ proteins, ZO-1 and Claudin-5 were increased in the spinal cord lumbar of all Riluzole, Sodium butyrate, or Riluzole and Sodium butyrate combination groups, compared to control SOD1\u003csup\u003eG93A\u003c/sup\u003e mice.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMeanwhile,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eRiluzole and Sodium butyrate combination had the most significant increase of ZO-1 and Claudin-5 expression by WB. The distribution and density of ZO-1 (\u003cstrong\u003eFig. 3B\u003c/strong\u003e) and Claudin-5 \u003cstrong\u003e(Fig. 3C)\u003c/strong\u003e had the most increase in the spinal cord lumbar of Riluzole and Sodium butyrate treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, as determined by IF staining.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAggregated h-SOD1\u003csup\u003eG93A\u003c/sup\u003e decreased in the spinal cord lumbar of all Riluzole, Sodium butyrate, or combination groups, compared to control mice \u003cstrong\u003e(Fig. 3D)\u003c/strong\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMeanwhile,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice had the most significant decrease of aggregated h-SOD1\u003csup\u003eG93A\u003c/sup\u003e in the spinal cord lumbar, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRiluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice had reduced h-SOD1\u003csup\u003eG93A\u003c/sup\u003e aggregation and increased ZO-1 and Claudin-5 in the brain cortex.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test the hypothesis\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethat Riluzole and Sodium butyrate combined treatment better decreases aggregation of the h-SOD1 mutated protein, restores the gut-brain barrier function and delays the ALS disease onset, we examined ZO-1 and Claudin-5 in the cortex of brain tissue. \u0026nbsp;Riluzole and Sodium butyrate combination had a significantly increased of ZO-1 and Claudin-5 expression in the brain cortex, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice by WB (\u003cstrong\u003eFig. 4A\u003c/strong\u003e)\u003cstrong\u003e.\u003c/strong\u003e As expected, Riluzole and Sodium butyrate combination treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice had a significantly increase of ZO-1 and Claudin-5 expression in the brain cortex, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, as determined by IF staining (\u003cstrong\u003eFig. 4B and Fig. 4C\u003c/strong\u003e).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eClaudin-1 expression showed no change in the brain cortex. In the meanwhile, the h-SOD1\u003csup\u003eG93A\u003c/sup\u003e protein aggregation was quantified with AggreCount. Riluzole and Sodium butyrate treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice had a significantly decrease in the brain cortex aggregation h-SOD1\u003csup\u003eG93A\u003c/sup\u003e, compared to Riluzole alone or Sodium butyrate alone treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice (\u003cstrong\u003eFig. 4D\u003c/strong\u003e)\u003cstrong\u003e.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRiluzole and Sodium butyrate combined treatment reduced serum LPS and inflammatory\u0026nbsp;cytokines in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntestinal leakage triggers bacterial LPS to blood. Thus, serum\u0026nbsp;LPS could be tested by ELISA\u003csup\u003e17,18\u003c/sup\u003e. As shown in\u003cstrong\u003e\u0026nbsp;Fig. 5A,\u003c/strong\u003e LPS were decreased on all mice with treatment. The expression of serum inflammatory cytokine IL-17 was decreased in Riluzole, Sodium butyrate, or Riluzole and Sodium butyrate combination groups, compared to control mice (\u003cstrong\u003eFig. 5B\u003c/strong\u003e).\u0026nbsp;Meanwhile,\u0026nbsp;Riluzole and Sodium butyrate combination in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice had a significantly decreased serum LPS and IL-17 expression, compared to Riluzole or Sodium butyrate treated SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. The levels of serum inflammatory cytokines IL-6 \u003cstrong\u003e(Fig. 5C)\u003c/strong\u003e and IFN-\u0026gamma; \u003cstrong\u003e(Fig. 5D)\u003c/strong\u003e showed a slight decrease without statistical significance in Riluzole and Sodium butyrate treated groups, compared to SOD1\u003csup\u003eG93A\u003c/sup\u003e mice without any treatment. But mice with Riluzole and Sodium butyrate combination had a significantly decreased serum IL-6 and IFN-\u0026gamma;, compared to the no-treatment SOD1\u003csup\u003eG93A\u003c/sup\u003e mice. However, serum inflammation cytokines IL-4 and IP-10 didn\u0026rsquo;t change in all groups (\u003cstrong\u003eFig. 5E \u0026amp; F\u003c/strong\u003e), suggesting the role of Riluzole and Sodium butyrate combination might be specific for certain inflammatory cytokines.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the current study, we investigate the combined roles of Riluzole and Sodium butyrate for the ALS therapy and explore new therapeutic targets for ALS by restoring intestinal homeostasis and barrier functions. Riluzole and Sodium butyrate combination showed a significantly longer rotarod time, more increased grip strength, enhanced intestinal barrier, compared with Riluzole or butyrate alone treatment. The aggregation of the h-SOD1 mutated protein was tested as an indicator of ALS progression. More reduced SOD1\u003csup\u003eG93A\u003c/sup\u003e aggregation was observed in the colon, spinal cord lumbar and brain cortex with Riluzole and butyrate combination treatment compared with Riluzole or butyrate alone treatment. The expression of tight junction proteins (ZO-1 and Claudin-5) significantly increased in the colon, spinal cord lumbar and brain cortex with Riluzole and butyrate combination treatment, compared with Riluzole alone or Sodium butyrate alone treatment. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe mechanism of Riluzole in protecting intestine in ALS is unknown. Our data not only showed the combined role of\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eRiluzole and Sodium butyrate in slowing down the disease progress through reserving barrier function and inhibiting inflammation, but also demonstrate that Riluzole alone has the beneficial impacts on the intestinal and blood-brain barriers. Combination role of R and butyrate is unknown. A previous study used I.P. treatment of Riluzole and sodium phenlebutyrate (NaPB) \u0026nbsp;in the ALS mice \u0026nbsp;\u003csup\u003e1\u003c/sup\u003e. It showed that Riluzole/NaPB administration increased acetylation at H4 and increased NF-\u0026kappa;B p50 translocation to the nucleus in G93A mice. However, the Riluzole and Sodium butyrate has not been tested. In the current study, we did gavage of Sodium butyrate and further checked the combined role on the intestinal functions and TJs in BBB.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe results of serum LPS and inflammatory cytokines also explain the influence of epithelial damage on the function of spinal cord motor neurons and neuromuscular structures in the ALS progress. Riluzole and Sodium butyrate combination were able to reduce the serum LPA and inflammatory cytokines.\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur data suggestion that ALS mice treatment with Riluzole and Sodium butyrate combination is more efficient than Riluzole alone or Sodium butyrate alone treatment in delay ALS progress. Restoring barrier function through the gut-brain axis provides a potential therapeutic strategy for ALS. Our study opens a new avenue in targeting the gut microbiota-neuron-axis for treating ALS.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSOD1\u003csup\u003eG93A\u003c/sup\u003e strain (B6SJL-Tg (SOD1-G93A) 1Gur/J, stock No.002726)\u003csup\u003e8,9,21,22\u003c/sup\u003e was purchased from Jackson Laboratory (The Jackson Laboratory, Bar Harbor, ME, USA). All mice were housed in specific pathogen-free environments under a controlled condition of 12\u0026thinsp;h light/12\u0026thinsp;h dark cycle at 20-22\u0026thinsp;\u0026deg;C and 45 \u0026plusmn;\u0026thinsp;5% humidity, with free access to the same food and autoclaved water. All materials involved including cage, bedding, water bottles, cage card holder were autoclaved before housing mice. The mice are housed of the same gender and each cage has no more than 5 mice. All experiments were carried out in strict accordance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the IACUC of University of Illinois Chicago Committee on Animal Resources (ACC 21-178 and ACC 23-149).\u0026nbsp;The study is reported in accordance with\u0026nbsp;ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRotarod test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMotor coordination, endurance, and balance were determined in latency by a rotarod test. Mice were trained on a rotarod test three days before performance trials at an accelerating speed from 4 to 40 rpm for 300s using the Rotarod Model LE8205 (Harvard Apparatus, Holliston, MA, USA).\u0026nbsp;Latency to fall was recorded when the mouse fell from the rod.\u003csup\u003e9,23\u003c/sup\u003e Each mouse was tested in 2 trials per day for 2 consecutive days. The mean times for 4 trials of the tests were calculated for each mouse.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of grip strength\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eForelimb and hindlimb grip measurements were acquired in triplicate with a 25 N Grip strength meter (Harvard Apparatus, Holliston, MA, USA). The mice were lowered onto a triangle bar of the grip strength meter until the animals gripped the bar with their forelimbs or hindlimbs, then the mice were pulled gently backward until they released their grip. The force gauge of the grip meter recorded the maximum force.\u003csup\u003e9,24\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRiluzole and Butyrate treatment in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSOD1\u003csup\u003eG93A\u003c/sup\u003e mice aged 9 to 10 weeks were randomly assigned to 4 groups. The Riluzole-treated group intraperitoneally received Riluzole (Sigma-Aldrich, 1604337, St. Louis, MO, USA) at a dose of 10mg per kg body weight daily. The Sodium butyrate-treated group received 2% sodium butyrate (Sigma-Aldrich, 303410, St. Louis, MO, USA) in filtered drinking water. The Riluzole/Sodium butyrate combination-treated group received Riluzole and Sodium butyrate. The Riluzole and Sodium butyrate treatments started at 9 to 10 weeks and finished at 15-16 weeks. All animals were weighed and received a detailed clinical examination, which included assessments of appearance, movement and behavior patterns, skin and hair conditions, eyes and mucous membranes, respiration, and excreta. Restricted outstretching of the hind legs observed on tail holding was considered a symptom of ALS, mice that were unable to turn over within 20 seconds after being laid on their\u0026nbsp;backs were humanely euthanized\u0026nbsp;CO2 inhalation followed by cervical dislocation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis and antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice colonic epithelial cells were collected by scraping the tissue from the colon of the mouse, including the proximal and distal regions. Mice spinal cord lumbar and brain cortex were harvested and minced into tiny pieces by using scissors.\u003csup\u003e28,29\u003c/sup\u003e The collected tissues were sonicated in lysis buffer (10\u0026thinsp;mM Tris, pH 7.4, 150\u0026thinsp;mM NaCl, 1\u0026thinsp;mM EDTA, 1\u0026thinsp;mM EGTA, pH 8.0, 1% Triton X-100) with 0.2\u0026thinsp;mM sodium ortho-vanadate, and protease inhibitor cocktail. The protein concentration was measured using the BioRad Reagent (BioRad, Hercules, CA, USA) and then sonicated. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with primary antibodies. The following antibodies were used: anti-human SOD1 (Abcam, ab52950, Cambridge, MA, USA), anti-ZO-1 (Invitrogen, 33-9100, Carlsbad, CA, USA), anti-Claudin-5 (Invitrogen, 35-2500, Carlsbad, CA, USA), anti-Claudin-1 (Invitrogen, 71-7800, Carlsbad, CA, USA), anti-GAPDH (Cell signaling technology, 5174, Danvers, MA, USA), or anti-\u0026beta;-actin (Sigma-Aldrich, A5316, St. Louis, MO, USA) antibodies and were visualized by ECL (Thermo Fisher Scientific, Waltham, MA, USA). Membranes that were probed with more than one antibody were stripped before re-probing. The software Quantity One has been used for the quantification of the western blot bands. Briefly, the \u0026ldquo;rectangular tool\u0026rdquo; was first selected to measure the background and the bands of western blots one by one. All the values of \u0026ldquo;density\u0026rdquo; and \u0026ldquo;volume\u0026rdquo; after measurement were transferred to an excel file. With the subtraction of background measurement, the \u0026ldquo;density\u0026rdquo; values for each band on the western blot were calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe colonic, spinal cord and brain tissues were freshly isolated and embedded in paraffin wax after fixation with 10% neutral buffered formalin. Immunofluorescence was performed on paraffin-embedded sections (5 \u0026mu;m).\u0026nbsp;After preparation of the slides as described previously,\u003csup\u003e30,31\u003c/sup\u003e tissue samples were incubated with\u0026nbsp;anti-human SOD1 (Abcam, ab52950, Cambridge, MA, USA), anti-ZO-1 (Invitrogen, 33-9100, Carlsbad, CA, USA), anti-Claudin-5 (Invitrogen, 35-2500, Carlsbad, CA, USA) at 4\u0026deg;C overnight. Samples were then incubated with goat anti-rabbit Alexa Flour 488 (Invitrogen, A-11008, Carlsbad, CA, USA), goat anti-mouse Alexa Flour 488 (Invitrogen, A-11001, Carlsbad, CA, USA) or goat anti-mouse Alexa Flour 594 (Invitrogen, A-11032, Carlsbad, CA, USA) and DAPI (Invitrogen, D1306, Carlsbad, CA, USA) for 1 h at room temperature. Tissues were mounted with SlowFade (Invitrogen, s2828, Carlsbad, CA, USA), followed by a coverslip, and the edges were sealed to prevent drying. Specimens were examined with a Zeiss laser scanning microscope LSM 710 (Carl Zeiss Inc., Oberkochen, Germany). The h-SOD1\u003csup\u003eG93A\u003c/sup\u003e protein aggregation was quantified with AggreCount software (https://aggrecount.github.io/). Fluorescence intensity was determined by using Image J software. This method determines the corrected total fluorescence by subtracting out background signal, which is useful for comparing the fluorescence intensity between cells or regions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntestinal permeability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescein isothiocyanate-dextran (average molecular weight\u0026nbsp;4000, Sigma, 46944, Burbank, CA, USA, diluted in HBSS)\u0026nbsp;was gavaged (25 mg / kg mouse) 4 hours before sample harvest.\u0026nbsp;Mice were anesthetized with avertin; depth of anesthesia was assessed with toe pinch and then blood was collected via cardiac puncture followed by cervical dislocation.\u0026nbsp;Mouse blood samples were collected for intestinal permeability test. \u003csup\u003e25-27\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerum lipopolysaccharides\u0026nbsp;(LPS) detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLPS in serum samples was measured with limulus amebocyte lysate chromogenic end point assays\u0026nbsp;(Hycult Biotech, HIT302, Plymouth, PA, USA) according to the manufacturer\u0026apos;s indications. The samples were diluted 1:4 with endotoxin-free water and then heated at 75\u0026deg;C for 5 minutes on a warm plate to denature the protein before the reaction. A standard curve was generated and used to calculate the concentrations, which were expressed as EU/mL, in the serum samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMultiplex ELISA assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse blood samples were collected by cardiac puncture and placed in tubes containing EDTA (10 mg/mL). Mouse cytokines were measured using a Cytokine \u0026amp; Chemokine Convenience 26-Plex Mouse ProcartaPlex\u0026trade; Panel 1 (Invitrogen, EPXR260-26088-901, Carlsbad, CA, USA) according to the manufacturer\u0026apos;s instructions. Briefly, beads of defined spectral properties were conjugated to protein-specific capture antibodies and added along with samples (including standards of known protein concentration, control samples, and test samples) into the wells of a filter-bottom microplate, where proteins bound to the capture antibodies over the course of a 2-hour incubation. After washing the beads, protein-specific biotinylated detector antibodies were added and incubated with the beads for 1 hour. After removal of excess biotinylated detector antibodies, the streptavidin-conjugated fluorescent protein R-phycoerythrin was added and allowed to incubate for 30 minutes. After washing to remove unbound streptavidin\u0026ndash;R-phycoerythrin, the beads were analyzed with the Luminex detection system (Bio-Rad, Bio-Plex 200 Systems, Hercules, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were expressed as the mean \u0026plusmn; SEM or \u0026plusmn; SD. All statistical tests were 2-sided. All p-values \u0026lt; 0.05 were considered statistically significant. The differences between samples for more than two groups were analyzed using one-way ANOVA, or two-way ANOVA as appropriate based on data distribution and the number of factors, respectively. The \u003cem\u003ep\u003c/em\u003e-values in ANOVA analyses were adjusted for correction of multiple comparisons using the Tukey method to ensure accurate results. Statistical analyses were performed using GraphPad Prism 8 (GraphPad, Inc., San Diego, CA, USA).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements/Funds \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge the VA Merit Award 1 I01BX004824-01, the NIDDK/National Institutes of Health grant R01 DK105118, and R01DK114126 to Jun Sun. The study sponsors play no role in the study design, data collection, analysis, and interpretation of data. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYZ performed the cellular and animal studies, the detailed analyses of the results; YZ \u0026amp;KR: animal studies; YZ and JS, prepared the figures and the draft text; YX contributed to the statistical analysis of data and the draft text; and JS obtained funds, designed the study, and directed the project. All authors contributed to the writing of the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available upon request to Jun Sun ([email protected])\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDel Signore, S. 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Bacterial translocation and barrier dysfunction enhance colonic tumorigenesis. \u003cem\u003eNeoplasia\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 100847 (2023). https://doi.org:10.1016/j.neo.2022.100847\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Amyotrophic lateral sclerosis, treatment, barrier function, BBB, gut-brain axis, inflammation, tight junctions","lastPublishedDoi":"10.21203/rs.3.rs-5349709/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5349709/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEmerging evidence has shown that gut-brain barrier dysfunction occurs at the early stages of ALS. Previous studies demonstrated that Sodium butyrate significantly decreased aggregation of the human-SOD1\u003csup\u003eG93A\u003c/sup\u003e protein, improved intestinal barriers, delayed the disease onset, and prolonged the life span of ALS mice. Riluzole is the first FDA-approved drug for ALS treatment. We hypothesize that Riluzole and Sodium butyrate combined treatment further decreases aggregation of the h-SOD1\u003csup\u003eG93A\u003c/sup\u003e, restores the gut-brain barrier function and delays the ALS progression. SOD1\u003csup\u003eG93A\u003c/sup\u003e mice (9-10-week-old) treated with Riluzole (10 mg/kg, I.P. daily), Sodium butyrate (2% in drinking water), or Riluzole and Sodium butyrate combination for 6 weeks. Riluzole and Sodium butyrate combination showed a significantly longer rotarod time, increased grip strength, enhanced intestinal barrier, compared with Riluzole or Sodium butyrate alone treatment. More reduction of h-SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;aggregation, an indicator of ALS progression, was observed in the colon, spinal cord lumbar, and brain cortex with Riluzole and Sodium butyrate combination, compared with Riluzole or Sodium butyrate alone treatment. Tight junction proteins (ZO-1 and Claudin-5) significantly increased in the colon, spinal cord lumbar, and brain cortex of mice with Riluzole and Sodium butyrate treatment. In SOD1\u003csup\u003eG93A\u003c/sup\u003e\u0026nbsp;mice, Riluzole and Sodium butyrate combination treatment reduced serum lipopolysaccharides (LPS) and inflammatory\u0026nbsp;cytokines (IL-17, IL-6 and IFN-γ) more than that in Riluzole or Sodium butyrate treatment. Our data suggest that Riluzole and Sodium butyrate treatment is more efficient than either Riluzole or Sodium butyrate alone in delaying ALS progress. It provides a potential therapeutic strategy by restoring barrier function through the gut-brain axis for ALS.\u003c/p\u003e","manuscriptTitle":"Synergistic Effects of Riluzole and Sodium Butyrate on Barrier Function and Disease Progression of Amyotrophic Lateral Sclerosis in SOD1G93A mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-11 14:38:50","doi":"10.21203/rs.3.rs-5349709/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f7811f56-d922-4d8e-bfec-2fe86fa9f786","owner":[],"postedDate":"November 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40099670,"name":"Biological sciences/Biochemistry"},{"id":40099671,"name":"Biological sciences/Cell biology"},{"id":40099672,"name":"Biological sciences/Immunology"},{"id":40099673,"name":"Biological sciences/Physiology"},{"id":40099674,"name":"Health sciences/Gastroenterology"},{"id":40099675,"name":"Health sciences/Neurology"}],"tags":[],"updatedAt":"2024-12-18T09:54:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-11 14:38:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5349709","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5349709","identity":"rs-5349709","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}