Remdesivir inhibits the progression of experimental colitis stimulated by dextran sodium sulfate

doi:10.21203/rs.3.rs-4976035/v1

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Remdesivir inhibits the progression of experimental colitis stimulated by dextran sodium sulfate

https://doi.org/10.21203/rs.3.rs-4976035/v1

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Remdesivir, a broad-spectrum antiviral prodrug, has been investigated for its potential effects on inflammatory bowel disease (IBD). Using a mouse model with acute colitis induced by 3% dextran sulfate sodium (DSS), we administered remdesivir at doses of 12.5 and 25 mg/kg from day 1 to 7. Our research demonstrated that remdesivir treatment notably decreased disease activity scores and improved colon tissue damage under the microscope. It also boosted the levels of tight junction proteins such as occludin and claudin-1, while reducing the production of inflammatory cytokines like IL-1β, IL-6, and TNF-α, as well as the adhesion molecule ICAM-1. Further analysis showed that remdesivir significantly reduced the expression of inflammatory markers CD3, EMR, and MPO in the mice's colorectal tissues. Additionally, it was found to regulate the gut microbiota and restore bile acid levels. Remdesivir was also observed to stabilize AnxA5, modulating the NF-κB pathway and thereby reducing inflammation, which was confirmed by its ability to counteract the effects of Si-AnxA5 suppression in LPS-treated Caco-2 cells. These findings indicate that remdesivir may activate the AnxA5 signaling pathway, offering a new perspective for treating experimental colitis. This suggests that remdesivir could be a valuable candidate for further development and therapeutic refinement in the context of IBD.

Remdesivir

Gut microbiota

Bile acid

AnxA5

IBD

In the realm of modern medicine, the quest to understand and combat a variety of diseases is an ongoing endeavor. Inflammatory bowel disease (IBD) represents a group of chronic conditions characterized by inflammation that affects the digestive tract, with Crohn's disease and ulcerative colitis being the most common forms[1, 2]. These conditions can be debilitating, with symptoms ranging from mild to severe, and often require lifelong management. Traditional treatments for IBD have focused on managing symptoms and preventing complications[3], but there is a growing interest in exploring novel therapeutic agents that can target the underlying causes of these conditions.

The etiology and pathogenesis of IBD are highly complex, involving the joint action of multiple factors, and currently, no single factor or mechanism can fully account for the etiology of IBD. It is widely believed that genetic factors, environmental influences, intestinal barrier damage, and microbial infections interact with one another, leading to a dysregulation of the mucosal immune system, which in turn results in chronic inflammation and irreversible damage to the intestinal mucosa[4].

The intestinal epithelium is composed of a tightly continuous single layer of epithelial cells, and abnormal regulation of epithelial cell proliferation and cell death can exacerbate intestinal damage[5]. The NF-κB signaling pathway in intestinal epithelial cells plays a crucial role in maintaining mucosal homeostasis and mediating pathogen-specific responses[6]. The integrity of the intestinal barrier is primarily supported by the tight junction proteins of the epithelial cells, which effectively prevent harmful substances (such as pathogens and endotoxins) from passing through the intestinal mucosa into the bloodstream[7]. Tight junctions are essential structures that constitute the intestinal mucosal barrier, mainly composed of transmembrane proteins (such as Occludin, Claudins, etc.) and peripheral tight junction proteins (such as ZO-1, ZO-2, etc.)[8, 9]. These components tightly link adjacent cells together, preventing harmful substances from entering the bloodstream through the intestinal mucosa. Abnormal downregulation of tight junctions can lead to increased intestinal permeability, allowing bacteria to infiltrate from the lumen into the intestinal wall, triggering a cascade of inflammatory responses[10, 11]. These bacteria induce the production of pro-inflammatory factors (such as TNF-α and IFN-γ), thereby inducing apoptosis in intestinal epithelial cells and further causing a loss of intestinal barrier function[12, 13].

Annexin A5 (AnxA5) can be activated by high concentrations of calcium ions or phosphatidylserine (PS), which leads to its spontaneous binding to the cell membrane and the formation of a trimer[14]. This structure allows it to play a key role in processes such as cell differentiation, apoptosis, and cell signaling. Additionally, AnxA5 also plays a significant role in inflammation, anti-apoptosis, tumorigenesis, thrombosis, heart failure, and autoimmune diseases[15]. It is particularly noteworthy that the research by Tschirhart and Zhang is directly related to the pathogenesis of IBD. Tschirhart's research has revealed that Annexin A5 (AnxA5) can effectively suppress the expression of inflammatory factors and adhesion molecules in microvascular endothelial cells activated by lipopolysaccharides (LPS) through the inhibition of platelets or microvesicles[16]. This mechanism has significant implications for the control of inflammatory responses in the microvasculature of the intestine in IBD. Zhang's research, on the other hand, has delved into how ANXA5, by binding to phosphatidylserine on the surface of colon vascular endothelial cells, induces the internalization of Toll-like receptor 4 (TLR4), thereby inhibiting the activation of the NF-κB pathway triggered by LPS[17]. This is manifested by the inhibition of P65 phosphorylation and IκB-α degradation, thus alleviating experimental colitis induced by trinitrobenzene sulfonic acid (TNBS) in mice.

One such agent that has garnered attention in recent years is Remdesivir, a broad-spectrum antiviral prodrug initially developed to combat viral infections[18]. Its unique mechanism of action, which involves the inhibition of viral replication through the targeting of specific viral enzymes, has led to its successful use in treating various viral diseases[19]. However, the potential of Remdesivir extends beyond its antiviral capabilities, as recent studies have suggested its possible therapeutic effects on IBD.

In conclusion, the findings from our study suggest that Remdesivir may offer a novel approach to the treatment of experimental colitis by activating the AnxA5 signaling pathway and modulating key aspects of the inflammatory response. These results open up new avenues for the development of therapeutic strategies for IBD and highlight the potential of repurposing existing drugs to address unmet medical needs. As we continue to delve into the complexities of IBD, the potential of Remdesivir as a therapeutic candidate warrants further investigation and optimization to harness its full potential in the management of this challenging condition.

Animal and ethical approval

In this experiment, male C57BL/6J mice at 8 weeks of age were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. These mice were bred in a Specific Pathogen Free environment and were received by the Animal Center of Nankai University for subsequent experimentation. All animal experimental procedures were conducted in accordance with standardized protocols and approved by the Ethics Committee.

Experimental colitis induced by DSS

Forty male C57BL/6J mice, aged 7–8 weeks and weighing approximately 22–23 grams, were pre-acclimated in the animal facility for one week to adapt to the environment. The 40 experimental mice were randomly divided into 5 groups, with each group consisting of 8 mice (2 cages, 4 mice per cage). During the initial 7 days of the experiment, the control group mice were provided with pure drinking water; the DSS model group, SASP positive drug group, and both low and high dose Remdesivir groups were all given 3% DSS water to drink. The body weight of the mice was recorded daily, their general condition was monitored, and a Disease Activity Index (DAI) score was assessed[20]. Starting from the 8th day of the experiment, all groups of mice were switched to drinking pure water. The SASP group mice were administered the drug daily via gavage at a dose of 200 mg/kg. The low dose Remdesivir group mice were given the drug via intraperitoneal injection at a dose of 25 mg/kg, and the high dose Remdesivir group mice were administered the drug at a dose of 50 mg/kg through the same route. During the drug administration period, the body weight of the mice was recorded daily, their general condition was observed, and a DAI score was assigned based on symptoms such as weight loss, stool consistency, and the presence of fecal blood. On the last day, fresh fecal samples were collected from the mice, and colon tissue was excised from the anal end to the cecum. The tissue samples were then stored in a freezer at -80°C for subsequent biological analysis.

Histological assessment

A section of colon tissue approximately 1 cm close to the anus was taken, washed in PBS buffer, drained of excess liquid, and then placed into an ample amount of 4% paraformaldehyde solution for fixation on a shaking bed for 24 hours. The fixed tissues were then sequentially processed for paraffin embedding and sectioning. Subsequently, the tissue sections underwent deparaffinization, staining with hematoxylin and eosin (H&E), dehydration, and other necessary steps. Randomly, three H&E stained sections from each group were selected and the colon pathological damage was evaluated using a blind method, referring to the scoring criteria published in the literature[21].

ELISA

Experiment according to the kit instructions (Shanghai Jianglai Biotechnology Co., Ltd.)

Alcin Blue - Nuclear Fast Red Staining

After the preparation of paraffin sections, the first step is to perform deparaffinization. Following this, 50 µL of Alcian blue staining solution is applied to each section. The sections are then placed in a humid chamber for staining for 1 hour, after which they are rinsed with distilled water for 10 seconds, repeated three times. Next, 50 µL of nuclear fast red staining solution is applied to each section and allowed to stain for 10 minutes. The sections are then washed with tap water for 5 minutes. Finally, the sections undergo dehydration and mounting procedures. Once air-dried, the sections are imaged using a microscope.

Immunohistochemistry, IHC

The tissue sections are placed in a drying oven at 60°C for 1 hour, followed by the processes of deparaffinization and antigen retrieval. A non-specific binding blocker is applied for 10 minutes to block any non-specific binding sites.The corresponding primary antibody solution is prepared and the tissue sections are incubated with it in a humid chamber at 4°C overnight. After the incubation, the tissue sections are taken out and left at room temperature for 30 minutes. They are then washed with PBS for 3 minutes, repeated three times.Biotin-labeled secondary antibodies are applied for 10 minutes, followed by streptavidin-horseradish peroxidase (HRP) for 10 minutes. The sections are then subjected to DAB (3,3'-diaminobenzidine) chromogen development for visualization of the bound antibodies. After DAB staining, the sections are counterstained with hematoxylin, followed by dehydration and mounting. Once the sections are dry, they are imaged using a microscope. Using a blind method, three randomly selected tissue sections from each group are scored according to the criteria published in the literature[22] to evaluate the tissue.

Immunofluorescence assessment

Cells are plated onto glass slides in culture dishes, and allowed to grow into a monolayer before being treated with drugs. For tissue sections, the steps of slide baking, dewaxing, and antigen retrieval are the same as those in the aforementioned immunohistochemistry experiment. Cover the cells or tissues with a 0.2% Triton solution for 20 minutes. Block the cells or tissues with a 5% BSA solution for 1 hour. Prepare the corresponding antibody solution according to experimental requirements, ensuring it fully covers the cells or tissues. Add the fluorescent secondary antibody in the dark, covering the cells or tissues completely, and incubate at room temperature for 1 hour. Finally, add the DAPI solution in the dark, and use an anti-fluorescence quenching sealing agent to seal the slide. Capture fluorescence images using a confocal microscope.

Western blot

Prepare the lower separating gel and the concentrated gel. Depending on the molecular weight of the protein, select a gel with a different concentration for the lower layer to achieve the best separation effect. Then proceed with electrophoresis to separate the proteins. After electrophoresis is complete, trim off the unnecessary separating gel, place the gel and PVDF membrane together on a piece of filter paper soaked with transfer buffer, assemble and clamp into a "sandwich" structure, and then transfer it into the transfer tank for blotting. Soak the PVDF membrane in 50 mL of 5% non-fat milk and gently rock on a shaker at room temperature for 1 hour to block non-specific binding sites. Dilute the primary antibody with TBST to the appropriate concentration, place the membrane into the primary antibody, and incubate overnight on a shaker at 4°C. Place the membrane in the diluted secondary antibody and incubate on a shaker at room temperature for 2 hours. Mix the ECL reagent A and B in a 1:1 ratio, and finally, capture the image in the chemiluminescence imaging system.

Quantitative real-time polymerase chain reaction (PCR) assay

Total RNA was extracted from cellular and colorectal tissue samples via the Trizol method. Subsequently, reverse transcription was performed following the protocol outlined in the Hifair® III 1st Strand cDNA Synthesis Kit (gDNA digester plus) manual. Primers for the gene of interest were designed and procured from TsingkeBiotechnologyCo., Ltd., with their sequences detailed in Table 1. The qPCR reaction mixture was assembled in an eight-well strip on ice, adhering to the guidelines of the Hieff UNICON® Power qPCR SYBR Green Master Mix (antibody method, No Rox) manual. Upon completion of the reaction, the amplification and melting curves were verified, and the data were subsequently processed to generate the corresponding graphs.

Microbial community analysis

To explore the impact of Remdesivir on the gut microbiota of mice with acute colitis, the following experimental scheme was designed. In this study, fecal samples from three groups of mice were collected for high-throughput sequencing of the 16S rRNA to analyze the composition of the mice's gut microbiota. The experiment was commissioned by Beijing Microread Technology Co., LTD

Bile acid quantitative analysis

Weigh an appropriate amount of the sample. Following a ratio of 6 µL of precooled ice-cold methanol for every 1 mg of sample, grind and disrupt the sample, then thoroughly agitate for 1 minute. Allow the mixture to stand at 4°C for 30 minutes, and then centrifuge at 12000 rpm for 10 minutes. After the first centrifugation, to the pellet, add a second portion of ice-cold methanol solution in a 1:6 ratio. Agitate the mixture thoroughly for 1 minute, let it stand at 4°C for another 30 minutes, and centrifuge again at 12000 rpm for 10 minutes. Combine the extracts from both rounds, and then evaporate the solvent to dryness. Finally, reconstitute the dried residue with 120 µL of methanol containing an internal standard at a concentration of 50 ng/mL. After centrifugation for 15 minutes, transfer the supernatant for analysis using liquid chromatography-mass spectrometry (LC-MS). The above experiments were commissioned by Beijing Abace Biotechnology Co., LTD.

Drug Affinity Responsive Target Stability (DARTS)

Drug Affinity Responsive Target Stability (DARTS) has emerged as an effective approach in the identification of small molecule target proteins, demonstrating its unique advantages as referenced[13]. The fundamental principle of DARTS lies in the ability of small molecule drugs to alter the conformation of the target protein upon binding, which in turn endows the protein with resistance to proteolytic degradation. The specific experimental workflow adheres to the research methodologies outlined by Xiang[23]and Zhu [24]

Materials

Mouse monocytic macrophage leukemia cells (RAW 264.7) and human colorectal adenocarcinoma cells (Caco-2) were both purchased from Procell Life Technology Co., Ltd. Sulfasalazine (SASP) and Dextran Sodium Sulfate (DSS) were purchased from Sigma-Aldrich. Lipo8000 and an Alcian Blue-Periodic Acid Schiff (AB-PAS) staining kit (pH 1.0) were acquired from Beyotime Biotech. Inc. A BCA protein concentration detection kit was purchased from Beijing Solarbio Science & Technology Co.,Ltd. The Hifair® III 1st Strand cDNA Synthesis Kit (gDNA digester plus) and the Hieff UNICON® Power qPCR SYBR Green Master Mix (antibody method, No Rox) were purchased from Yeasen Biotechnology (Shanghai) Co., Ltd. A Mouse Calprotectin (CALP) ELISA detection kit was acquired from Shanghai Jianglai Biotechnology Co., Ltd.

Statistical analysis

Graphpad Prism 8 software was used for the statistical analysis of experimental data. Two independent sample T-tests were used for comparison between two groups, and one-way ANOVA was used for comparison between multiple groups. p < 0.05 was considered statistically significant. Each experiment was performed at least three times independently, and data are expressed as the mean ± standard deviation (SD).

1. Remdesivir alleviates the progression of DSS-induced colitis in mice

This study induced colitis in mice using a 3% Dextran Sulfate Sodium DSS aqueous solution (Fig. 1A). As shown in Fig. 1B, the high-dose Remdesivir group exhibited a significantly superior rate of body weight recovery compared to the other groups, while the low-dose group and the positive control group treated with SASP showed similar trends in weight recovery, with the model group experiencing the slowest recovery. Post DSS-induced modeling, both the model and treated groups of mice demonstrated an increasing trend in their DAI scores over time. However, in comparison to the model group, the Remdesivir-treated groups showed a marked improvement in DAI scores in the later stages (Fig. 1C). As shown in Fig. 1D, calprotectin (CALP), a key biomarker for inflammatory bowel disease, was significantly elevated in the model group compared to the normal control group, while a notable decrease in calprotectin levels was observed in mice treated with both low and high doses of Remdesivir. As illustrated in Figs. 1E-F, the colon length of mice in the model group was significantly shorter than that of the normal control group, with some degree of restoration observed in the groups treated with the positive drug SASP and a low dose of Remdesivir. The high-dose Remdesivir group demonstrated the most pronounced restoration effect on colon length. Histological differences between the various treatment groups were clearly observable through H&E staining of the mice's colon tissue. The colon tissue of mice in the model group showed evident damage, characterized by severe damage to the colonic surface mucosa, deformation or disappearance of the crypt structure, substantial infiltration of inflammatory cells in the mucosal lamina propria, and destruction of goblet cells. Upon treatment with the positive drug SASP and a low dose of Remdesivir, the damage to the mice's colon tissue was significantly ameliorated. The high-dose Remdesivir group showed even more pronounced therapeutic effects.

2. Remdesivir alleviates intestinal inflammation in mice with DSS induced colitis

This study employed qRT-PCR technology to examine the expression of inflammatory cytokines in the colons of mice. The experimental results showed that, compared to the model group, there was a significant decrease in the mRNA expression levels of IL-1β, IL-6, TNF-α, and ICAM-1 in the colon tissues of mice treated with SASP and Remdesivir. To further explore the anti-inflammatory mechanism of Remdesivir in IBD, this study used immunohistochemistry to assess the expression levels of CD3, MPO, and EMR1 in the colon tissues of mice with DSS-induced colitis, in order to evaluate the inhibitory effect of Remdesivir on the aggregation of inflammatory cells. The experimental results indicated that, compared to the control group, the protein expression levels of these three markers significantly increased in the colon tissues of mice in the model group. In the groups treated with the positive drug SASP and Remdesivir, the levels of these proteins all decreased, with the high-dose Remdesivir group showing a more pronounced downward trend in protein expression. These results further suggest that high-dose Remdesivir has a more significant effect in reducing the colonic inflammatory response in mice.

3. Remdesivir improves intestinal barrier damage in mice with DSS induced colitis

The colonic epithelial barrier, which includes the mucus layer and tight junction proteins, serves as the first line of defense against the invasion of external pathogens and harmful substances into the gut. The integrity of its function is crucial for maintaining intestinal health. As shown in Figs. 3A-B, Alcian Blue staining results indicated that the colonic epithelial tissue of mice in the DSS model group was damaged, with blurred boundaries of goblet cells, thinning and partial loss of the mucus layer, and a significant reduction or even disappearance of mucin expression. However, after treatment with Remdesivir, the damage to the colonic epithelial tissue in mice was restored, the number of goblet cells increased, and both the thickness and integrity of the mucus layer were improved, comparable to the therapeutic effect of the positive control drug, Sulfasalazine. Additionally, the mRNA expression levels of MUC2 in the colonic tissue of mice, as shown in Fig. 3C, also confirmed this observation. Western Blot results (Figs. 3D-E) showed that, compared to the control group, the expression levels of tight junction proteins (ZO-1, Occludin, and Claudin-1) in the colon of mice in the model group were significantly downregulated. However, in the Remdesivir treatment group, the expression levels of tight junction proteins were significantly increased. Immunohistochemistry and immunofluorescence experiments (Supplementary Figs. 1A-C) also confirmed the upregulation of tight junction proteins in the Remdesivir treatment group.

4. Remdesivir improves LPS-induced cellular inflammation and epithelial barrier damage

In the LPS-induced Caco-2 cell model, Remdesivir significantly inhibited the downregulation of tight junction proteins ZO-1, Occludin, and Claudin-1 induced by LPS (Fig. 4A-B). Additionally, immunofluorescence also confirmed the upregulating effect of Remdesivir on the ZO-1 protein (Fig. 4C). As shown in Fig. 5D, in LPS-induced RAW264.7 cells, stimulation with LPS significantly increased the mRNA expression levels of pro-inflammatory cytokines IL-1β, TNF-α, TNF-R1, and the adhesion molecule ICAM-1, while pretreatment with Remdesivir significantly reduced the mRNA expression levels of these inflammatory factors, showing a clear dose-dependent inhibitory effect.

5. Effects of remdesivir on gut microbiota and bile acid metabolism in colitis mice

In order to study the impact of Remdesivir on the gut microbiota composition of UC mice, we conducted 16S rRNA gene sequencing on fecal samples from three groups of mice. The alpha diversity of the microbial community showed that Remdesivir did not improve the fecal microbial abundance induced by DSS in mice (Supplementary Fig. 2A). The species accumulation curve tended to flatten (Supplementary Fig. 2B). Principal component analysis was used to analyze the similarity of the microbial community composition. The results indicated that there were significant differences in the gut microbiota composition between the DSS group and the control group. However, Remdesivir treatment did not significantly modulate the changes in the gut microbiota of mice with DSS-induced colitis (Fig. 5A). A Venn diagram was created using the ASV/OTU abundance table (Fig. 5B). To further explore the species classification distribution and composition in each region of the Venn diagram, the number and relative abundance of each ASV/OTU in the corresponding region were statistically analyzed at the phylum and genus levels, and displayed using bar charts (Fig. 5C and Supplementary Fig. 3A). Figures 5D and 5E describe the top 20 gut bacteria at phylum and genus levels, respectively. To further compare the differences in species composition among samples and to illustrate the trends in species abundance distribution across various samples, a heatmap was used for the analysis of species composition at the genus level. The results revealed that Remdesivir can significantly improve and suppress the abundance of 12 types of bacterial communities induced by DSS, including Lawsonia, Staphylococcus, Clostridium, and Anaeroplasma (Supplementary Fig. 3B).

To identify robust differential species between groups, we performed Linear Discriminant Analysis Effect Size (LEfSe) analysis. The characteristic microbial communities in the Remdesivir group were identified as Parabacteroides and Porphyromonadaceae (with an LDA score greater than 2) (Supplementary Figs. 4A-B). Additionally, Supplementary Figs. 5A-D display the relative abundance distribution of signature species at the class level across different groups.

Network inference analysis based on the interrelationships among microbial members is also a common method for analyzing microbial communities. By using associative analysis methods, it is possible to search for temporal and spatial changes in specific microbial communities. The associative analysis between different grouped microbial members is shown in the supplementary Fig. 6A-B.

Additionally, we analyzed the MetaCyc and KEGG secondary pathway enrichment for all samples (Supplementary Fig. 7A-B). After obtaining the abundance data of metabolic pathways, we attempted to identify metabolic pathways with significant inter-group differences. The significantly different MetaCyc and KEGG metabolic pathways between the control group and the remdesivir group are shown in Fig. 6A and Supplementary Fig. 8A. The significantly different MetaCyc and KEGG metabolic pathways between the model group and the remdesivir group are depicted in Fig. 6B and Supplementary Fig. 8B. Finally, based on the significantly different metabolic pathways, an analysis of the species composition of the differential pathways was conducted (Figs. 6C-F).

Bile acids are small molecules synthesized from cholesterol through a multi-enzyme process in the liver, and 95% of bile acids are reabsorbed when they reach the distal ileum, entering the enterohepatic circulation (Fig. 7A). In addition, bile acids and the gut microbiota have a bidirectional influence on each other. Therefore, we conducted a quantitative analysis of 43 types of bile acids in the feces of mice from different groups (Supplementary Fig. 9). As shown in Fig. 7B, the content of 12 types of bile acids was significantly increased in the model group, while the content of these 12 types of bile acids significantly decreased after administration of remdesivir. In summary, remdesivir can regulate some aspects of the gut microbiota and bile acid metabolism.

6. Remdesivir inhibits activation of the p65 signaling pathway in vivo and in vitro

The abnormal activation of the NF-κB signaling pathway plays a pivotal role in the pathogenesis of IBD. This pathway controls the expression of multiple inflammatory-related factors, impacting intestinal barrier functions such as mucin expression, intestinal epithelial cell apoptosis, and the reduction of tight junction proteins, thereby intensifying the progression of IBD. As depicted in Figs. 8A-B, there was a notable increase in the phosphorylation levels of P65 and IKBα in the colon samples of mice in the model group. Treatment with Remdesivir significantly reduced the phosphorylation levels of both P65 and IKBα, indicating that Remdesivir may function by inhibiting the activation of the NF-κB signaling pathway. To further validate and delve into the mechanisms of Remdesivir's action, this study constructed an LPS-induced inflammatory model in RAW264.7 macrophages. As illustrated in Figs. 8C-E, Remdesivir administration mitigated the elevation of LPS-induced phosphorylation levels of P65 and IKBα in a dose-dependent manner, a result that further substantiates the potential role of Remdesivir in modulating the NF-κB signaling pathway.

7. Remdesivir inhibits p65 signaling pathway activation by interacting with AnxA5

To delve into the mechanisms of action of remdesivir, we employed DARTS technology to identify potential targets of remdesivir. To characterize these protein fragments, protein bands at the marked sites were precisely excised and subjected to gel recovery. After drying, the recovered protein samples were analyzed by mass spectrometry (Fig. 9A). The mass spectrometry analysis identified the top ten scoring proteins (Table 2). Annexin A5 is a protein with multiple functions in the body that can reduce the infiltration of inflammatory cells in TNBS-induced colitis. To verify that the binding of remdesivir indeed enhances the stability of AnxA5, we adopted two experimental approaches: Western Blot analysis of DARTS samples and cellular thermal shift assay. Firstly, in the DARTS-treated samples, the binding of remdesivir to AnxA5 was confirmed by immunoblotting, which enhances the resistance of AnxA5 to proteases, thereby preventing its degradation (Fig. 9B). In the cellular thermal shift assay, as the heating temperature increased, the Anxa5 protein gradually degraded. Compared to the DMSO control group, the Anxa5 protein content in the remdesivir group was significantly higher at 68°C, and this trend was also observed at 72°C and 76°C. This finding indicates that remdesivir indeed forms a specific complex with the Anxa5 protein, enhancing its thermal stability and making it less prone to degradation upon heating (Fig. 9C). Molecular docking showed that the binding energy of remdesivir to Anxa5 reached − 7.172 kcal/mol, a value that reflects their high affinity (Fig. 9D).

To further explore the role of AnxA5 in the NF-κB pathway and the impact of remdesivir on it, we designed three siRNA sequences targeting AnxA5. By comparing the silencing effects of different sequences, we found that one siRNA sequence (Sequence 3) could significantly and specifically reduce the expression level of AnxA5 (Fig. 9E), and thus was chosen for subsequent experiments. As shown in Figs. 9F-G, under LPS stimulation, the phosphorylation levels of P65 and IKB were significantly increased. However, in cells where AnxA5 was silenced, the phosphorylation levels induced by LPS stimulation further increased, suggesting that AnxA5 may play a role in negatively regulating the activation process of the NF-κB pathway. Upon addition of remdesivir and subsequent LPS stimulation, the phosphorylation levels of P65 and IKB were somewhat reduced, indicating that remdesivir could inhibit the activation of the NF-κB pathway induced by LPS. Nevertheless, in cells with silenced AnxA5, the effect of remdesivir in reducing phosphorylation was markedly suppressed, suggesting that AnxA5 may be one of the key targets through which remdesivir exerts its pharmacological effects.

Inflammatory bowel disease (IBD), as a complex digestive tract disorder, has seen a year-on-year increase in its incidence rate globally in recent years, imposing a heavy burden on patients and society. Although existing chemical drugs and biological agents can alleviate the symptoms of IBD to some extent, curative treatment remains a significant challenge. Therefore, finding new therapeutic strategies and drug targets is crucial for the clinical treatment of IBD.

In this study, we delved into the potential role of remdesivir in the treatment of inflammatory bowel disease (IBD). Remdesivir, a medication originally designed to target RNA viruses, has garnered significant attention in recent years due to its notable efficacy against coronavirus disease 2019 (COVID-19)[25]. Numerous studies have demonstrated that remdesivir exhibits a robust antiviral effect in the treatment of COVID-19 and has been considered by the U.S. FDA as one of the candidate drugs for this disease [19, 26, 27]. Research in SARS-CoV-infected mouse models has shown that remdesivir not only mitigates pulmonary lesions and bronchiolitis but also provides protection against acute lung injury [28]. This finding has been further corroborated by clinical trials, where remdesivir has shown positive effects in enhancing the rate of clinical improvement in hospitalized patients, reducing the risk of disease progression, and decreasing the hospitalization rate for mild cases, with its safety widely recognized [29]. Beyond its prominent performance in the field of antiviral therapy, remdesivir has also shown potential applications in the treatment of inflammation-related diseases. Studies indicate that remdesivir can improve various disease conditions, including metabolic dysfunction-related diseases [30], acute kidney injury [31], and tumors [32], by modulating inflammatory cytokines and immune responses. These studies suggest that remdesivir may have broad potential in treating inflammation-related diseases. Although COVID-19 and IBD differ significantly in their pathogenesis and clinical manifestations, both involve the infiltration of inflammatory cells and the release of a large number of inflammatory cytokines, leading to tissue damage. Based on remdesivir's dual roles in antiviral and anti-inflammatory actions, we hypothesize that it may have similar therapeutic effects in the treatment of IBD.

Inflammation plays a central role in the pathogenesis of enteritis. An increasing body of evidence indicates that an overactivation of the inflammatory response within the gut leads to intestinal tissue damage and the manifestation of a range of clinical symptoms. To treat IBD, researchers have developed drugs targeting inflammatory factors, mitigating intestinal inflammation by disrupting cytokine signaling and reducing their expression levels within the gut[33]. Additionally, studies have shown that some natural plant extracts, such as Artemisia capillaris, can improve IBD symptoms by upregulating anti-inflammatory factors and downregulating pro-inflammatory factors [34]. Initially, we used a mouse model of acute colitis induced by DSS to preliminarily confirm the therapeutic effect of remdesivir on IBD. The experimental results showed that remdesivir could significantly improve indicators such as weight loss, Disease Activity Index (DAI) scores, colon length, and fecal calprotectin content in mice, demonstrating efficacy comparable to or even superior to that of positive control drugs. Further research found that remdesivir could reduce the infiltration of inflammatory cells, restore the number of goblet cells, and thus maintain the integrity of the intestinal epithelial mucosa. At the same time, remdesivir was also able to decrease the expression levels of inflammatory cytokines in animal and RAW264.7 cell models, demonstrating its potent anti-inflammatory effects. These results provide strong evidence for our deeper understanding of the role of remdesivir in the treatment of IBD.

When delving into the pathogenesis of IBD, we must pay close attention to the importance of intestinal barrier function. The intestinal barrier, a complex structure composed of the mucus layer, epithelial cells, and intercellular tight junctions, plays a crucial role in maintaining intestinal health and defending against harmful substances. From a physiological perspective, the permeability of tight junctions has a decisive impact on the function of the intestinal barrier. However, inflammatory and injurious factors can often disrupt these tight junctions, leading to increased permeability [10]. The mucus layer, as the first line of defense of the intestinal barrier, is essential for intestinal health. Mucin secreted by goblet cells, particularly MUC2, plays a key role in the physical structure of the intestinal mucosal barrier [35]. In the pathological process of IBD, especially ulcerative colitis (UC), the thickness of the mucus layer often thins, which is closely related to the abnormal glycosylation of the MUC2 protein [36]. Tight junction proteins, as an important component of the intestinal barrier, are crucial for preventing harmful substances from passing through the intestinal mucosa into the bloodstream [1, 37, 38]. Researchers, through exploring different mechanisms, have found that mannose can protect the intestinal epithelial barrier function and maintain the integrity of the epithelial barrier, thereby effectively alleviating intestinal inflammation[1]. In addition, compounds such as lithospermate B and wedelolactone have also been found to have the effect of repairing damaged epithelial barriers, exerting therapeutic effects on enteritis by inhibiting related signaling pathways and reducing the infiltration and activation of neutrophils[39, 40]. Our research found that remdesivir could restore the reduction of intestinal mucosal protein MUC2 content induced by DSS in IBD mice and increase the expression levels of tight junction proteins ZO-1, Occludin, and Claudin-1 in the intestines of IBD mice and Caco-2 cell models, thereby improving intestinal barrier function. This discovery has revealed a new mechanism of remdesivir in the treatment of IBD for us.

Bile acids, as important substances in the human metabolic process, play a crucial role in the gut-liver axis. They are not only key factors in the digestion and absorption of fats but also act as signaling molecules that convey information between the intestine and the liver, coordinating their functions. In the pathogenesis of inflammatory bowel disease (IBD), abnormalities in bile acid metabolism often affect the balance of the gut microbiota through the gut-liver axis, thereby exacerbating intestinal inflammation. This abnormal bile acid metabolism forms a vicious cycle with intestinal inflammation, making the condition of IBD difficult to control[41]. Intestinal bacteria also play an important role in bile acid metabolism; they produce secondary bile acids by metabolizing bile acids, which regulate the number and function of RORγ + regulatory T cells in the intestine, maintaining intestinal immune homeostasis[42]. In this study, we conducted an in-depth sequencing analysis of fecal samples from mice in the normal group, the model group, and the high-dose remdesivir treatment group. The results showed that high-dose remdesivir treatment could significantly improve the abnormal secretion of bile acids in the IBD mouse model, helping to restore their normal levels. This finding provides strong experimental evidence for the potential of remdesivir in the treatment of IBD.

The activation of the NF-κB pathway is closely related to the pathogenesis of IBD[43]. Studies have shown that mutations in the NFκB1 gene exacerbate the pathological manifestations of IBD, and the persistent activation of the NF-κB signaling pathway further intensifies the inflammatory response. Additionally, mice with mutated NFκB1 lacking p105 expression exhibit spontaneous intestinal inflammation similar to IBD, highlighting the key role of NF-κB in IBD. Annexin A5 (AnxA5), as an important calcium and phospholipid-binding protein, has garnered widespread attention for its biological functions in the bio-medical field. In the study of inflammatory bowel disease, the anti-inflammatory effects of AnxA5 are particularly prominent. It can effectively inhibit the expression of inflammatory cytokines and adhesion molecules induced by LPS-activated platelets or microvesicles, thereby effectively controlling the inflammatory response in IBD[16]. Furthermore, AnxA5 can also suppress the activation of the NF-κB pathway by binding to phosphatidylserine on the surface of colonic vascular endothelial cells, inducing the internalization of TLR4[17]. As a candidate drug, remdesivir may play a significant role in the treatment of IBD by regulating AnxA5 and affecting the activation state of the NF-κB pathway. By inhibiting the overactivation of the NF-κB pathway, remdesivir is expected to alleviate intestinal inflammation and improve the clinical symptoms of IBD patients.

In summary, this study not only reveals the potential application value of remdesivir in the treatment of IBD but also elucidates its mechanism of action in inhibiting the progression of inflammatory bowel disease by targeting Annexin A5 to regulate the NF-κB pathway. These findings provide new ideas and methods for the development of novel, safe, and effective drugs for the treatment of IBD. However, there are still some limitations and deficiencies in this study. Firstly, our research is mainly based on mouse models, and although certain experimental results have been obtained, further verification of its efficacy and safety in clinical practice is still required. In addition, the number of samples within the gut microbiota and bile acid sequencing groups is relatively small, and we will collect more mouse fecal samples in the future. Secondly, the mechanism of action of remdesivir still needs to be further explored to fully understand its role in the treatment of IBD. Moreover, we also need to pay attention to the combined application of remdesivir with other drugs in order to achieve better therapeutic effects.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This work was financially supported by the Fundamental Research Funds for the Central Universities“, Nankai University [Grant 735-63241345], The National Natural Science Foundation of China [Grant 82270069], The National Natural Science Foundation of China [Grant 82370073] and the 111 Project [Grant B20016].

Author Contribution

Xiaoting Gu, Honggang Zhou, Hailong Cao, and Cheng Yang supervised the project, designed experiments, and wrote the manuscript. Hailong Li, Ying Yang and Jinhe Li performed experiments and analyzed data. YaYue Hu, Ruiqi Mao, Xiaoman Yang, Xi Wu , Zherui Li and Liqing Han performed experimental colitis modeling experiments in mice. Ying Yang provided technical assistance in pharmacology. All authors read and approved the final manuscript.

Data availability

Data will be made available on request.

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Tables 1 and 2 are available in the Supplementary Files section.

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Remdesivir inhibits the progression of experimental colitis stimulated by dextran sodium sulfate

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Version 1

Abstract

Figures

Introduction

Materials and methods

Animal and ethical approval

Experimental colitis induced by DSS

Histological assessment

ELISA

Alcin Blue - Nuclear Fast Red Staining

Immunohistochemistry, IHC

Immunofluorescence assessment

Western blot

Quantitative real-time polymerase chain reaction (PCR) assay

Microbial community analysis

Bile acid quantitative analysis

Drug Affinity Responsive Target Stability (DARTS)

Materials

Statistical analysis

Results

1. Remdesivir alleviates the progression of DSS-induced colitis in mice

2. Remdesivir alleviates intestinal inflammation in mice with DSS induced colitis

3. Remdesivir improves intestinal barrier damage in mice with DSS induced colitis

4. Remdesivir improves LPS-induced cellular inflammation and epithelial barrier damage

5. Effects of remdesivir on gut microbiota and bile acid metabolism in colitis mice

6. Remdesivir inhibits activation of the p65 signaling pathway in vivo and in vitro

7. Remdesivir inhibits p65 signaling pathway activation by interacting with AnxA5

Discussion

Declarations

Declaration of Competing Interest

Funding

Author Contribution

Data availability

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1