Application of PPARalpha (peroxisome proliferator activated receptor alpha) agonists Fenofibrate, Wy14643 and PEA (peroxisome proliferator activated receptor alpha)

Abstract

The invention relates to the technical field of biological medicines, and provides application of a PPARalpha (peroxisome proliferator activated receptor alpha) agonist, namely fenofibrate Feyfibrate, Wy14643 and PEA (peroxisome proliferator activated receptor alpha). The PPARalpha agonists Fenofirate, Wy14643 and PEA provided by the invention can be directly or indirectly combined with PPARalpha protein through specificity and activate the activity of the PPARalpha protein, so that a PPARalpha signal channel is regulated, and diarrhea reaction after stimulation of new coronavirus is relieved. The Fenofibrate, the Wy14643 and the PEA disclosed by the invention are high in treatment specificity, small in side effect and good in effect when being used for treating new coronavirus infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the application of PPARα agonists fenofibrate, Wy14643 and PEA. Background Art

[0002] SARS-CoV-2 is a novel coronavirus. In addition to primary respiratory symptoms, SARS-CoV-2 infection is often accompanied by gastrointestinal symptoms, such as diarrhea. Analysis of clinical data and epidemiological statistics of COVID-19 patients revealed that patients with diarrhea-associated COVID-19 had significantly higher rates of fecal occult blood, critical illness, mortality, ICU admission rates, and length of hospital stay than those without diarrhea. COVID-19 patients with diarrhea also experience increased inflammation and multi-organ damage, and their prognosis is far worse than that of COVID-19 patients without diarrhea.

[0003] Current drug development for the novel coronavirus SARS-CoV-2 (SARS-CoV-2) primarily focuses on antiviral therapy and immune regulation, but a specific treatment for SARS-CoV-2-induced diarrhea remains lacking. Among direct-acting antivirals, 3CL protease inhibitors (such as Paxlovid and the domestically produced sennotevir) and RNA polymerase inhibitors (such as remdesivir and monolavir) are core options. Paxlovid, by blocking a key enzyme in viral replication, can reduce the risk of hospitalization or death in high-risk patients by approximately 89%, but it must be taken within five days of onset and is associated with drug-drug interactions. Remdesivir requires intravenous administration and is effective for hospitalized patients, but is costly. Neutralizing antibodies were previously used in early treatment, but their use has largely been discontinued due to immune evasion by the Omicron variant. New broad-spectrum antibodies (such as the Chinese SA55 / SA58) are in clinical trials. Regarding immune regulation, glucocorticoids (dexamethasone) and IL-6 / JAK inhibitors (tocilizumab and baricitinib) can suppress the excessive inflammatory response in critically ill patients, but the risk of secondary infection should be considered.

[0004] The occurrence of diarrhea is mainly due to changes in the transport status of electrolytes in the body. When the gastrointestinal tract is infected by viruses, bacteria, etc., the absorption function of the intestinal mucosa is impaired, resulting in abnormal expression of diarrhea-related ion transport proteins, which in turn affects the absorption and secretion of nutrients in the gastrointestinal tract, resulting in diarrhea. Currently, there are no drugs on the market for diarrhea symptoms caused by SARS-CoV-2. Currently, the treatment methods for diarrhea are limited and the efficacy varies. Therefore, there is an urgent need to find a drug that can prevent or treat diarrhea. Summary of the Invention

[0005] The present invention provides an application of PPARα agonists Fenofibrate, Wy14643, and PEA to solve the problems in the existing technology, such as the lack of targeted treatment and prevention drugs for COVID-19 diarrhea, and the large side effects and insufficient efficacy of existing drugs.

[0006] The present invention provides use of a regulator capable of regulating PPARα activity in preparing a medicament for treating or preventing diarrhea after SARS-CoV-2 infection.

[0007] Alternatively, the modulator comprises an agonist, an antagonist, a coactivator or a co-repressor.

[0008] Alternatively, the modulator comprises the PPARα agonist Fenofibrate, Wy14643 or PEA.

[0009] As an alternative, the regulator includes a PPARα agonist, and the drug includes a PPARα agonist and a pharmaceutically acceptable excipient.

[0010] As an option, the dosage form of the drug includes an oral dosage form or a non-oral dosage form.

[0011] As an optional solution, the oral dosage form includes tablets, capsules, granules or microcapsules; the non-oral dosage form includes injection or lyophilized powder injection.

[0012] As an alternative, the fenofibrate, Wy14643 or PEA can specifically bind to the PPARα protein and activate its activity, thereby regulating the PPARα signaling pathway.

[0013] Alternatively, the fenofibrate, Wy14643 or PEA can reduce lung inflammation caused by SARS-CoV-2 infection.

[0014] Alternatively, the lung inflammation is caused by the SARS-CoV-2 E protein inhibiting PPARα activity in lung epithelial cells, resulting in reduced CFTR expression.

[0015] Alternatively, the diarrhea is induced by the SARS-CoV-2 E protein inhibiting PPARα activity in colon epithelial cells.

[0016] This study, screening, and confirmation demonstrate that the PPARα agonists fenofibrate, Wy14643, or PEA can specifically bind directly or indirectly to the PPARα protein and activate its activity, thereby regulating the PPARα signaling pathway. These agonists can be used to treat COVID-19 diarrhea as active ingredients in preventive or therapeutic drugs. These three agonists offer significant advantages as therapeutic agents. First, they have high specificity, specifically binding to and activating the PPARA (i.e., PPARα) protein, demonstrating high selectivity for the PPARα signaling pathway. Second, they have minimal side effects, as these agonists act only on the PPARα protein in the PPARα signaling pathway and have no effect on other cellular signaling pathways. Third, they are highly effective, with fenofibrate, Wy14643, or PEA effectively alleviating ion disturbances and improving diarrheal symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a GSEA enrichment analysis diagram, which shows the diarrhea-related gene set enriched in the epithelial cells of the COVID-19 group and the healthy group through the GSEA algorithm. The volcano plot presents the distribution of differentially expressed genes in the epithelial cells of the COVID-19 group and the healthy group in the single-cell sequencing data.

[0018] Figure 2 This is a Venn diagram showing the gene intersection of three sets of data: single-cell sequencing differentially expressed genes (DEGs), proteomic differentially expressed genes in SARS-CoV-2 infected Caco-2 cells, and known diarrhea-related genes.

[0019] Figure 3 This is a protein interaction network diagram constructed based on the STRING database, showing the interaction relationship between the above-mentioned intersection genes.

[0020] Figure 4 The violin plots compare the expression level distribution of SPINT2, SLC9A3, and SLC26A3 genes in epithelial cells of the COVID-19 group and the healthy group.

[0021] Figure 5 This is a Venn diagram showing the intersection of transcription factors that regulate SPINT2, SLC9A3, and SLC26A3 with M3 cluster transcription factors that showed high activity scores in colonic epithelial cells of healthy individuals and COVID-19 patients and were significantly reduced after infection. Five potential master regulators were found, namely PPARA, NR2F6, HNF4A, ESRRA, and NR1I2.

[0022] Figure 6This is a VlnPlot analysis diagram, which shows that PPARA activity is significantly reduced in epithelial cells, suggesting that PPARA is the main regulatory factor for absorption-related proteins in colon epithelial cells.

[0023] Figure 7 This is a correlation analysis of single-cell transcriptome data, showing that PPARA is significantly positively correlated with SLC26A3, SLC9A3, and SPINT2. The UMAP plot further demonstrates that PPARA activity is primarily enriched in epithelial cells and significantly decreased after infection. Taken together, these findings indicate that PPARA is a key master regulator of protein absorption in colonic epithelial cells following SARS-CoV-2 infection.

[0024] Figure 8 RT-qPCR results show the RNA expression levels of SPINT2, SLC9A3, and SLC26A3 in Caco-2 (AC) and T84 (EG) cells after stimulation with 0, 100, 500, and 1000 ng / ml E protein.

[0025] Figure 9 Western blot results show the protein expression levels of SPINT2, SLC9A3, SLC26A3, PPARA and phosphorylated PPARA (p-PPARA) in Caco-2 and T84 cells after stimulation with 0, 100, 500 and 1000 ng / ml E protein.

[0026] Figure 10 Western blot results show the protein expression levels of SPINT2, SLC9A3, SLC26A3 and PPARA in Caco-2 and T84 cells after transfection of siPPARA.

[0027] Figure 11 PPARA ChIP-seq data were downloaded from the ENCODE database to predict potential PPARA motifs. The results showed that there were significant PPARA peaks in the promoter regions of SLC 26 A3, SLC 9A 3, and SPINT 2, which were consistent with the predicted PPARA motifs.

[0028] Figure 12 This is a ChIP-seq data visualization diagram, which uses IGV software to display the binding signal of PPARA in the promoter region of the CFTR gene.

[0029] Figure 13Western blot results show the protein expression levels of CFTR, PPARA and phosphorylated PPARA (p-PPARA) in A549 and Beas-2B cells after stimulation with 0, 100, 500 and 1000 ng / ml E protein.

[0030] Figure 14 RT-qPCR results show the detection of RNA expression levels of SPINT2, SLC9A3, and SLC26A3 in the control group (Ctrl), E protein group, E protein + Fenofibrate, Wy14643, PEA group, E protein + Wy14643 group, and E protein + PEA group in Caco-2 and T84 cells.

[0031] Figure 15 Western blot results show that the protein expression levels of SPINT2, SLC9A3, SLC26A3, PPARA and phosphorylated PPARA (p-PPARA) in the above groups were detected in Caco-2 and T84 cells.

[0032] Figure 16 RT-qPCR results show the detection of CFTR RNA expression levels in A549 and Beas-2B cells in the control group (Ctrl), E protein group, E protein + Fenofibrate, Wy14643, PEA group, E protein + Wy14643 group, and E protein + PEA group.

[0033] Figure 17 Western blot results show the protein expression levels of CFTR, PPARA and phosphorylated PPARA (p-PPARA) in A549 and Beas-2B cells in the above groups. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] The "new crown" mentioned in the present invention refers to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus.

[0036] Peroxisome proliferator-activated receptors (PPARs) include three subtypes: PPARα, PPARγ, and PPARβ / δ. These subtypes are encoded by distinct genes on different chromosomes and share a high degree of homology. PPARs function as transcription factors, regulating gene expression. The PPARα gene is strongly implicated in the development of human diseases such as diabetes, arteriosclerosis, and obesity. It plays a crucial role in lipid metabolism, adipogenesis, and maintaining metabolic homeostasis, and influences the transcription of various inflammatory mediator genes in vascular endothelial cells, smooth muscle cells, and monocytes / macrophages.

[0037] The present invention has shown that PPARα activity plays a key role in COVID-19 diarrhea, and discloses the use of a modulator capable of regulating PPARα activity in the preparation of a medicament for treating or preventing diarrhea following SARS-CoV-2 infection. Furthermore, the diarrhea is induced by the SARS-CoV-2 E protein inhibiting PPARα activity in colonic epithelial cells.

[0038] More specifically, the present invention discloses the use of three PPARα agonists—fenofibrate, Wy14643, and PEA—in the preparation of medicaments for treating or preventing diarrhea following SARS-CoV-2 infection. Fenofibrate is a widely used fibric acid lipid-lowering drug in clinical practice. As a PPARα agonist, it can significantly increase the β-oxidation rate of fatty acids, reduce plasma triglyceride levels, increase high-density lipoprotein cholesterol, and exhibit anti-inflammatory and antioxidant effects. Wy14643 is a specific PPARα agonist that can activate the PPARα signaling pathway, thereby promoting fatty acid oxidation and energy metabolism and alleviating inflammatory responses. PEA (palmitoylethanolamine) is an endogenous lipid mediator that also has PPARα agonist effects and can regulate processes such as inflammatory responses and cell apoptosis.

[0039] Fenofibrate, Wy14643, or PEA in the present invention regulate the PPARα signaling pathway by specifically binding to and activating the PPARα protein. These compounds can also alleviate pulmonary inflammation caused by SARS-CoV-2 infection, and are therefore useful in the preparation of medicaments for treating or preventing diarrhea following SARS-CoV-2 infection. In some embodiments, the pulmonary inflammation is caused by the SARS-CoV-2 E protein inhibiting PPARα activity in lung epithelial cells, leading to decreased CFTR expression. In the preparation of these medicaments, in addition to the three PPARα agonists, fenofibrate, Wy14643, and PEA, as active ingredients, the pharmaceutical composition also includes pharmaceutically acceptable excipients, such as excipients and carriers. Specific dosage forms include oral dosage forms (e.g., tablets, capsules, granules, or microcapsules) or parenteral dosage forms (e.g., injections or lyophilized powders).

[0040] More specifically, the present invention discloses that PPARA (i.e., PPARα) is the primary transcription factor causing SARS-CoV-2 diarrhea. The PPARA agonists screened in this study, fenofibrate, Wy14643, and PEA, are effective in preventing SARS-CoV-2 diarrhea and lung inflammation. Furthermore, the PPARA agonists of the present invention have a salvage effect on pulmonary inflammation caused by SARS-CoV-2 infection. Specifically, the present invention discovered and confirmed that fenofibrate, Wy14643, and PEA can directly or indirectly increase the expression of phosphorylated PPARA in colonic epithelial cells, activating the PPARA pathway and the activity of downstream target genes. SPINT2, SLC26A3, and SLC9A3 are membrane protein genes involved in ion absorption in colonic epithelial cells. Their expression can be inhibited in a concentration-dependent manner by the SARS-CoV-2 E protein, triggering ion homeostasis in the colon and causing diarrheal symptoms. Fenofibrate, Wy14643, and PEA rescued the downregulation of SPINT2, SLC26A3, and SLC9A3 protein expression caused by E protein, maintained the ion exchange balance in the intestine, and thus prevented the occurrence of diarrhea symptoms.

[0041] In addition, in lung epithelial cells, CFTR is a chloride ion channel protein located on the cell membrane that mediates the - ) and bicarbonate (HCO3 -) transport across the cell membrane, regulating intracellular and extracellular ion balance. The SARS-CoV-2 E protein can cause abnormal CFTR expression or activity, impairing lung mucus clearance and exacerbating the inflammatory response following viral infection. Fenofibrate, Wy14643, and PEA can also activate PPARA activity in the lungs, increasing the level of phosphorylated PPARA protein and upregulating CFTR expression, thereby alleviating lung inflammation caused by the SARS-CoV-2 E protein.

[0042] The main transmission route of the new coronavirus SARS-CoV-2 is droplet transmission. It can also be transmitted through aerosols in a relatively closed environment. SARS-CoV-2 can also be detected in the gastrointestinal tract, urine, saliva and tears of patients, and can cause lung inflammation and gastrointestinal discomfort. Therefore, the relevant scheme of the present invention uses colon epithelial cells Caco-2, T84 and lung epithelial cells A549 and Beas-2B cells.

[0043] 1. Experimental Materials Human intestinal epithelial cells (Caco-2, FH0029) were purchased from Fuheng Biotechnology (Shanghai) and cultured in Caco-2 medium supplemented with NEAA, 20% FBS, and 1% P / S. Human colorectal cancer lung metastasis cells (T84, LH-H327) were obtained from Leibaiha Biotechnology (Shanghai) and cultured in T84 medium consisting of DMEM / F12 supplemented with 5% FBS and 1% P / S. Adenocarcinoma human alveolar basal epithelial cells (A549), maintained in our laboratory, were cultured in RPMI 1640 supplemented with 10% FBS and 1% P / S. Human bronchial epithelial cells immortalized with adenovirus 12-SV 40 hybrid virus (Beas-2B, LH-H118) were purchased from Leibaiha (Shanghai) Biotechnology and cultured in Beas-2B medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 1% P / S. All these cell lines were cultured under standard conditions in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37°C in a 5% CO2 incubator and used within 6 months.

[0044] 2. Experimental Reagents

[0045] 3. Experimental Instruments Cell culture: culture dishes, culture flasks, 6-well plates, 12-well plates, 1.5 ml centrifuge tubes, centrifuges, pipettes, etc.

[0046] Western blotting experiments: 1.5 ml centrifuge tubes, electrophoresis apparatus, vortexer, microcentrifuge, vertical electrophoresis tank, glass plates, combs, transfer tanks, clamps, shakers, incubation boxes, fully automatic chemical exposure analysis system, and pipettes.

[0047] qPCR experiment: real-time fluorescence quantitative PCR instrument, vortex oscillator, microcentrifuge, eight-tube strips, and pipette.

[0048] 4. Experimental content and results Example 1: PPARA (also written as PPARα) is the main regulator of diarrhea symptoms after COVID-19 infection, while SLC26A3, SLC9A3, SPINT2, and CFTR are its downstream proteins and are regulated by it.

[0049] 1.1 Experimental methods 1.1.1 Single-cell RNA sequencing (scRNA-seq) data analysis scRNA-seq data from COVID-19 and normal lung tissue samples were downloaded from the GEO database (GSE171524) (PMID: 33915568). These datasets were analyzed using Seurat v4.3.0. Raw expression matrices from these datasets were read into R and assembled into a list of Seurat objects. Each dataset was normalized, and the variance stabilizing transformation (VST) method was used to identify highly variable features. Dataset integration was performed using the Seurat integration pipeline. Anchors were identified between datasets, and the data were integrated to eliminate individual batch effects. The integrated dataset was then scaled for downstream analysis. Principal component analysis (PCA) was performed on the integrated dataset using the highly variable features. Clustering was performed using the Louvain algorithm at a resolution of 0.8 to identify distinct cell clusters. Unified Mapping (UMA) dimensionality reduction was then applied to visualize the clusters. Cell types were annotated based on known marker genes and expression patterns. Differential expression analysis was performed using the FindAllMarkers function to identify markers specific to each cluster. Select the top markers for each cluster and visualize them using dot plots, VlnPlot, and FeaturePlot to highlight expression patterns across different cell types and conditions. Use stacked bar charts to compare the proportions of different cell types in healthy and COVID-19 conditions.

[0050] 1.1.2 Differential analysis, enrichment analysis, and GSEA analysis Epithelial cells from healthy and COVID-19 colon or lung tissue were extracted using the subset function and differential expression analysis was performed using FindMarkers. Diarrhea-related genes were collected from the literature, and functional enrichment analysis, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and GSEA analysis were performed using the clusterProfiler package to explore the biological significance of the identified genes. GSEA results were visualized using gseaplot 2, volcano plots were generated using ggplot 2, and Venn diagrams were drawn using the jvenn website. Protein-protein interactions were analyzed using STRING. The expression levels of diarrhea-related genes in epithelial cells were visualized using VlnPlot.

[0051] 1.1.3 Gene regulator network analysis and master regulator screening The SCENIC pipeline was initialized using the required databases (500 bp and 10 kb cisTarget databases) and parameters. Meta-cell matrices were normalized using a log2 (CPM / 100 + 1) transformation, and lowly expressed genes were filtered out. Genes were correlated using Spearman correlation, and co-expression networks were constructed using GENIE 3. This step identified potential regulatory relationships between genes. The co-expression network was converted into a transcriptional regulatory network by identifying enriched motifs and linking them to transcription factors (TFs). This step generated regulons, which are groups of genes regulated by specific TFs. TF activity scores were calculated for each cell using the AUCell package. This step generated a TF activity score matrix, which was integrated into a Seurat object for downstream analysis. Regulons were clustered based on their activity scores using hierarchical clustering and the connectivity specificity index (CSI). This step identified distinct TF modules driving transcriptional changes. The relationship between TF activity and downstream gene expression was analyzed using scatter plots and correlation coefficients. Heatmaps and UMAP were used to visualize the activity of TF modules and their correlation with cell type and condition. Visualize the activity profiles of master regulators using violin plots and feature plots.

[0052] 1.1.4 Drug stimulation Caco-2 and T84 cells were stimulated with SARS-CoV-2 S, E, and N proteins at a concentration of 1000 ng / mL for transcriptome sequencing. Additionally, four cell types (Caco-2, T84, A549, and Beas-2B) were stimulated with a gradient of E protein (0, 100 ng / mL, 500 ng / mL, and 1000 ng / mL). RNA and protein were extracted after 24 hours of culture. Caco-2 and T84 cells were transfected with siRNA targeting PPARα (siPPARA) and control siRNA (siCtrl) using Lipofectamine 3000 reagent. RNA and protein were extracted 48 hours after transfection.

[0053] 1.2 Experimental Results GSEA analysis revealed significant repression of many genes in the Arabidopsis genome ( Figure 1 We identified 1416 differentially expressed genes in colon epithelial cells between patients infected with the novel coronavirus and healthy subjects ( Figure 1 To ensure accuracy, we crossed these differentially expressed genes with genes in the proteomic data of Caco-2 cells infected with SARS-CoV-2 (PMID: 32408336) and diarrhea-related genes, generating 15 potential target genes ( Figure 2 ). Enrichment analysis in the STRING database identified absorption-related proteins closely associated with diarrhea in the disease database: SLC26A3, SLC9A3, and SPINT2 ( Figure 3 ) (PMID: 35348648). Visualization of this gene in our scRNA-seq data showed that expression levels were significantly reduced in patients compared to healthy individuals ( Figure 4 Taken together, these results suggest that SARS-CoV-2 induces the inhibition of absorption-related proteins in colonic epithelial cells, leading to diarrhea.

[0054] To identify the master regulators of SARS-CoV-2-induced diarrhea in colonic epithelial cells, we used the SCENIC algorithm to analyze regulator activity scores (RAS) and transcriptional regulatory networks in single-cell data. RAS heatmaps for each cell cluster revealed significant differences between healthy and COVID-19 colonic epithelial cells. Hierarchical clustering grouped transcription factors with similar activity into 11 clusters. Visualization of the average activity score of each module on the UMAP plot showed that module 3 (M3) transcription factors exhibited high activity scores in colonic epithelial cells of both healthy individuals and COVID-19 patients, which were significantly reduced after infection. To identify the master regulators responsible for inhibiting absorption-related proteins, we aligned the transcription factors that regulate SLC26A3, SLC9A3, and SPINT2 with those in M3 and identified five potential candidates: PPARA, ESRRA, NR2F6, HNF4A, and NR1I2 ( Figure 5 ). VInPlot analysis showed that only PPARA activity was significantly decreased in epithelial cells, while the other four transcription factors were also decreased in endothelial cells ( Figure 6 Given that PPARA agonists have been shown to affect clinical outcomes in COVID-19 patients, we hypothesized that PPARA is a major regulator of absorption-related proteins in colonic epithelial cells. Correlation analysis in our scRNA-seq data confirmed significant positive correlations between PPARA and SLC26A3, SLC9A3, and SPINT2 ( Figure 7 The UMAP map further showed that PPARA activity was mainly enriched in epithelial cells and was significantly reduced after infection ( Figure 7 Collectively, these findings indicate that PPARA is a major regulator of absorption-related proteins in colonic epithelial cells following SARS-CoV-2 infection.

[0055] To elucidate which components of SARS-CoV-2 are responsible for the reduction in PPARA activity in colonic epithelial cells, we stimulated Caco-2 and T84 cells with SARS-CoV-2 S, E, and N proteins and performed bulk RNA-seq. PCA analysis revealed that Caco-2 cells exhibited the most significant differences after E protein stimulation, with S protein stimulation also showing significant differences. In contrast, T84 cells showed significant differences only upon E protein stimulation, suggesting that the SARS-CoV-2 E protein plays a key role in colonic epithelial cell infection. GSEA analysis demonstrated that E protein-related gene sets were significantly downregulated in E protein-stimulated colonic epithelial cells, consistent with the scRNA-seq findings, suggesting that the E protein is causal for the diarrheal phenotype. To further validate the relationship between E protein and PPARA activity, we constructed a gene set representing PPARA activity based on the results of the SCENIC algorithm. GSEA analysis showed a significant decrease in PPARA activity after E protein stimulation, confirming that the SARS-CoV-2 E protein inhibits PPARA activity in colonic epithelial cells. Additional GSEA analysis of all KEGG pathways revealed that the E protein primarily activated immune-related signaling pathways in colonic epithelial cells, such as TNF and MAPK signaling pathways. Given that PPARA negatively regulates immune inflammatory responses, activation of this pathway further supports the idea that the E protein inhibits PPARA activity. Collectively, these results suggest that the SARS-CoV-2 E protein induces diarrhea by inhibiting PPARA activity in colonic epithelial cells.

[0056] To experimentally verify the above findings, we stimulated Caco-2 and T84 cells with E protein and examined the RNA and protein levels of absorption-related proteins (SLC26A3, SLC9A3, and SPINT2) and PPARA protein activity. The results showed that the RNA and protein levels of SLC26A3, SLC9A3, and SPINT2 were significantly reduced in an E protein concentration-dependent manner, indicating that these proteins are regulated by E protein at the transcriptional level ( Figure 8 Similarly, PPARA protein activity decreased with increasing E protein concentration, suggesting that PPARA may be a master regulator of SLC26A3, SLC9A3, and SPINT2 ( Figure 9 To confirm the causal relationship between PPARA and these absorption-related proteins, we used siRNA to knock down PPARA expression in Caco-2 and T84 cells. The RNA and protein levels of SLC26A3, SLC9A3, and SPINT2 were significantly reduced after PPARA knockdown, confirming that PPARA is the main regulator of these proteins ( Figure 10To determine the regulatory mechanism of PPARA, we downloaded PPARA ChIP-seq data from the ENCODE database and used MEME to predict potential PPARA motifs. The results showed significant peaks of PPARA in the promoter regions of SLC26A3, SLC9A3, and SPINT2, which were consistent with the predicted PPARA motifs ( Figure 11 Collectively, these findings suggest that E protein stimulation significantly reduces PPARA activity in colonic epithelial cells, leading to decreased transcription of absorption-related proteins and ultimately causing a diarrheal phenotype.

[0057] Given that E protein stimulation primarily reduces PPARA activity in colonic epithelial cells, and previous studies have shown that E protein stimulation of lung epithelial cells leads to decreased CFTR expression and worsens pneumonia, we investigated the regulatory relationship between PPARA and CFTR. Visualization of PPARA ChIP-seq data revealed a significant peak in the CFTR promoter region, consistent with the PPARA motif ( Figure 12 Stimulation of lung epithelial cells A549 and Beas-2B with E protein also showed that PPARA activity and CFTR protein levels were significantly reduced in an E protein concentration-dependent manner ( Figure 13 Collectively, these results suggest that SARS-CoV-2 infection of lung AT2 cells inhibits PPARA activity, reduces CFTR expression, and worsens pneumonia.

[0058] Example 2: PPARα agonists rescued the diarrhea and pneumonia phenotypes induced by E protein in in vitro experiments.

[0059] 2.1 Experimental methods Three PPARA agonists: palmitoylethanolamide (PEA), fenofibric acid, and Wy 14643 (pyrinic acid) were dissolved in DMSO to prepare 0.1 mM stock solutions and stored at -20°C. During stimulation with 1000 ng / mL E protein, these agonists were added to the culture medium at a final concentration of 0.1 µM. Cells were cultured for 24 hours before further experiments. RNA was extracted and converted to cDNA using a reverse transcription kit. RT-qPCR quantification was performed using target gene primers. Protein was extracted and analyzed by western blotting.

[0060] 2.2 Experimental Results To investigate whether PPARA agonists could rescue the diarrhea and pneumonia phenotypes caused by E protein, we treated E protein-stimulated colon and lung epithelial cells with three PPARA agonists: palmitoylethanolamide (PEA), fenofibric acid, and Wy 14643 (pyrinic acid) and measured the RNA and protein levels of downstream target genes. In colon epithelial Caco-2 and T84 cells, all three PPARA agonists enhanced PPARA activity, thereby increasing the RNA and protein levels of SLC26A3, SLC9A3, and SPINT2 to rescue the diarrhea phenotype caused by E protein ( Figure 14-15 Similarly, in lung epithelial cells A549 and Beas-2B, three PPARA agonists enhanced PPARA activity, thereby increasing CFTR RNA and protein levels and rescuing the pneumonia phenotype caused by E protein ( Figure 16-17 Collectively, these findings demonstrate that PPARA agonists can enhance PPARA activity in cells and promote the expression of downstream target genes to rescue the diarrhea and pneumonia phenotypes caused by E protein.

[0061] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions are not to be construed as limiting the present invention. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications, or improvements may be made to the technical solutions and implementations of the present invention, all of which fall within the scope of the present invention.

Claims

1. Use of a modulator capable of regulating PPARα activity in the preparation of a drug for treating or preventing diarrhea after SARS-CoV-2 infection.

2. The use according to claim 1, wherein the regulator comprises an agonist, an antagonist, a coactivator or a co-repressor.

3. The use according to claim 1, wherein the regulator comprises PPARα agonist fenofibrate, Wy14643 or PEA.

4. The use according to claim 1, characterized in that The regulator includes a PPARα agonist, and the drug includes a PPARα agonist and pharmaceutically acceptable excipients.

5. The use according to claim 1, characterized in that The dosage form of the drug includes an oral dosage form or a non-oral dosage form.

6. The use according to claim 5, characterized in that The oral dosage forms include tablets, capsules, granules or microcapsules; the non-oral dosage forms include injections or freeze-dried powder injections.

7. The use according to claim 3, characterized in that The fenofibrate, Wy14643 or PEA can specifically bind to the PPARα protein and activate its activity, thereby regulating the PPARα signaling pathway.

8. The use according to claim 3, characterized in that The fenofibrate, Wy14643 or PEA can reduce lung inflammation caused by SARS-CoV-2 infection.

9. The use according to claim 8, characterized in that The lung inflammation is caused by the SARS-CoV-2 E protein inhibiting PPARα activity in lung epithelial cells, resulting in reduced CFTR expression.

10. The use according to claim 1, characterized in that The diarrhea is induced by the SARS-CoV-2 E protein inhibiting PPARα activity in colon epithelial cells.

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