Use of ELMO1 and its downstream pathway genes in the diagnosis, prevention and treatment of intestinal fibrosis

Abstract

This invention discloses the use of ELMO1 and its downstream pathway genes in the diagnosis, prevention, and treatment of intestinal fibrosis. This study demonstrates that ELMO1 plays an important role in epithelial cell aging and intestinal fibrosis. Furthermore, it was discovered that irisin treatment, which targets the AMPK-SIRT1 pathway, can alleviate cellular aging and reduce intestinal fibrosis by enhancing ELMO1 activity. ELMO1 can serve as an anti-aging marker, providing a theoretical basis for how intestinal epithelial cell aging promotes the adverse progression of intestinal inflammation. It also offers new insights into the treatment of IBD fibrosis, potentially reducing the recurrence and progression of intestinal fibrosis.

Description

Technical Field

[0001] The present invention relates to the technical field of diagnosis and treatment of inflammatory bowel disease, and more specifically, to the use of ELMO1 and its downstream pathway genes in the diagnosis, prevention and treatment of intestinal fibrosis. Background Art

[0002] Intestinal fibrosis is a long-term complication of inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD). While typically seen in ileal CD, intestinal fibrosis is also a common complication of chronic, progressive UC and colonic CD. Due to the lack of specific antifibrotic therapies, approximately 50% of CD patients require surgical intervention within 10 years of diagnosis, and these therapies do not prevent disease recurrence or fibrosis.

[0003] Therefore, there is an urgent need to study the mechanisms of IBD-related fibrosis, fibrotic stenosis, and possible treatment options. Summary of the Invention

[0004] The present invention aims to overcome at least one of the shortcomings of the above-mentioned prior art and provides a use of ELMO1 and its downstream pathway genes in the diagnosis, prevention and treatment of intestinal fibrosis. By activating ELMO1 and its downstream pathways, it is possible to reduce intestinal cell senescence caused by ELMO1 downregulation, further preventing intestinal fibrosis caused by intestinal cell senescence, and providing a new treatment strategy for intestinal fibrosis. By detecting ELMO1 levels and comparing them with normal intestinal tissue, the risk of intestinal fibrosis can be promptly diagnosed.

[0005] One object of the present invention is to provide a method for preparing a medicament for treating intestinal fibrosis using an activator of the FNDC5-AMPK-SIRT1 pathway. In one embodiment of the present invention, it was found that knockdown of ELMO1 leads to downregulation of the related pathway, FNDC5-AMPK-SIRT1, which in turn increases the accumulation of senescent cells and exacerbates intestinal fibrosis. However, the use of an activator of the FNDC5-AMPK-SIRT1 pathway can eliminate cellular senescence-related signs, such as p65 acetylation, and further alleviate and mitigate intestinal fibrosis. In particular, the purified FNDC5 protein, irisin, can mitigate senescence induced by ELMO1 depletion through AMPKα-SIRT1 signaling and further improve fibrosis in chronic intestinal inflammation. Irisin, a hormone produced by the hydrolysis of fibronectin type III domain-containing protein 5 (FNDC5), is considered an anti-aging agent and has been found to inhibit chronic diseases such as Alzheimer's disease, cardiac dysfunction, and intervertebral disc degeneration.

[0006] Furthermore, the FNDC5-AMPK-SIRT1 pathway activator includes a FNDC5 activator, an AMPK activator, a SIRT1 activator and / or an ELMO1 activator.

[0007] Further, the FNDC5 activator includes irisin; and / or the AMPK activator includes metformin; and / or the SIRT1 activator includes quercetin.

[0008] Furthermore, intestinal fibrosis includes intestinal fibrosis and intestinal fibrosis stricture in inflammatory bowel disease. In one or more embodiments of the present invention, it is found that the aforementioned pathway activator can effectively treat intestinal fibrosis and intestinal fibrosis stricture in inflammatory bowel disease including chronic colitis.

[0009] Another object of the present invention is to provide an anti-aging drug and / or a p65 acetylation inhibitor for use in the preparation of a medicament for treating intestinal fibrosis. In one or more embodiments of the present invention, it was found that reducing intestinal epithelial cell senescence can effectively alleviate intestinal fibrosis. In one or more embodiments of the present invention, p65 acetylation appears to be crucial in the senescence process induced by ELMO1 depletion. Therefore, when the goal is to inhibit senescence, drugs that inhibit its acetylation should also be considered as medicaments for treating intestinal fibrosis.

[0010] Furthermore, the anti-aging drugs include intestinal epithelial tissue cell anti-aging drugs.

[0011] Another object of the present invention is to provide a use of an ELMO1 detection reagent in the preparation of a kit for diagnosing intestinal fibrosis. In one or more embodiments of the present invention, it was found that inflammatory intestinal tissue with fibrosis showed lower ELMO1 expression levels compared to normal intestinal tissue.

[0012] Furthermore, intestinal fibrosis includes intestinal fibrosis in inflammatory bowel disease.

[0013] Another object of the present invention is to provide an intestinal fibrosis marker, including ELMO1, which has a relatively low expression level in the intestine.

[0014] Another object of the present invention is to provide a drug for treating inflammatory bowel disease fibrosis, comprising a FNDC5 activator, an AMPK activator, a SIRT1 activator and / or an Elmo1 activator.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The research of this application shows that ELMO1 plays an important role in epithelial cell aging and intestinal fibrosis, and on this basis, it was found that irisin treatment targeting the AMPK-SIRT1 pathway can reduce cell aging and reduce intestinal fibrosis by enhancing ELMO1 activity. ELMO1 can be used as an anti-aging marker, providing a theoretical basis for intestinal epithelial cell aging to promote the adverse progression of intestinal inflammation, and at the same time providing new ideas for the treatment of IBD fibrosis to reduce the recurrence and development of intestinal fibrosis. ELMO1, as an anti-aging marker, plays an important role in preventing intestinal fibrosis. Based on the research of this application, a new treatment strategy for intestinal fibrosis in inflammatory bowel disease is provided, which is beneficial for application in clinical treatment to reduce the recurrence and development of intestinal fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Increased intestinal senescence is observed in a mouse model of chronic colitis and patients with stricturing IBD. [A] Representative H&E-stained colon sections from uninflamed controls and a chronic colitis model. [B] Absolute mucosal thickness was measured in controls (n=5) and mice with chronic colitis (n=5) (**P<0.015, ***P<0.001). [C] Masson's trichrome staining of colon sections from mice and animals subjected to the chronic DSS or chronic TNBS model; fibrosis levels are depicted (n=5, **P<0.01, ***P<0.001). [D] Relative expression of fibronectin, type I collagen, and α-SMA mRNA in the chronic DSS or chronic TNBS model (n=5, **P<0.01, ***P<0.001). [E] Volcano plot of differentially expressed genes (DEGs) between normal colon tissue and DSS-induced colitis tissue. [F] GO analysis of DEGs in DSS-induced colitis tissue. [G] Expression of p21, p16, and SASP genes in normal colon tissue and DSS-induced colitis tissue. [H] Representative immunohistological staining images of SA-β-gal activity (scale bar, 100 μm). [I] Representative immunofluorescence staining images of LAMNB1 (scale bar, 200 μm). [J] Relative expression of SASP gene mRNA in chronic DSS or chronic TNBS models. (n=5, **P<0.01, ***P<0.001). [K] Relative mRNA expression of p53, p16, and p21 in chronic DSS or chronic TNBS models. (n=5, **P<0.019 ***P<0.001)

[0017] Figure 2ELMO1 deficiency leads to severe fibrosis and is associated with epithelial cell senescence in a chronic TNBS-induced colitis model. [A] ELMO1 expression in acute colitis, chronic colitis, and normal colon tissues. (n=3, ***P<0.001). [B] Correlation between ELMO1 and p16, IL-6, PDGFα, and α-SMA in animals with chronic colitis. [C] - / - Chronic TNBS colitis was established in mice or control mice, and colon tissue was analyzed by microendoscopy [C] and H&E staining [D]. [E] In Elmol - / - Absolute thickness of the mucosa was measured in mice with colitis (n=5) and wild-type mice (n=5). - / - Immunohistochemical images of fibronectin, collagen I, and α-SMA in colitis mice (n=5) and wild-type mice (n=5, **P<0.01, ***P<0.001). [GH] In Elmol - / - SA-β-gal activity and LAMINB1 staining were detected in colitis mice and wild-type mice (scale bar, 100 pm). - / - Relative mRNA expression of p21, p16, p53, and SASP genes in colitis mice and wild-type mice (n=5, **P<0.01, ***P<0.001)

[0018] Figure 3 ELMO1 depletion promotes IEC6 senescence and activates 3T3-L1 fibrosis. IEC6 cells were transfected with the sh-Elmo1 plasmid to exclude the effects of ELMO1 on senescence. Senescence activity was detected in silenced and perturbed cells by QR-PCR [A-B] and LAMINB1 staining [C]. 3T3-L1 cells were treated with conditioned medium from Elmo1-KD IEC6 cells (SCM), conditioned medium from normal IEC6 cells (CCM), normal medium (Ctrl), and 2 ng / ml TGF-β1. [D] Relative mRNA expression of fibronectin, collagen I, and α-SMA. [E] Representative immunofluorescence images of fibronectin. [F] Representative, quantitative immunoblot analysis of α-SMA.

[0019] Figure 4ELMO1 targets the FNDC5-AMPK-SIRT1 pathway. [AB] Transcriptome sequencing was performed, and pathways were assessed for differentially expressed genes (fold change > 1.0, Benjamini-Hochberg corrected P < 0.05) using the Molecular Signature Database signature gene set. [C] Western blotting was used to examine the expression of p65, p65-K310 acetylation, p53, p53-K382 acetylation, IL-6, and IL-1β in Elmo1-silenced and control cells. [D] Quantitative PCR was used to examine the expression of Prkaa1, Sirt1, Ppargc1, and Fndc5. [E] Western blotting was used to examine the expression of AMPK, AMPK-T172 phosphorylation, SIRT1, and FNDC5 in Elmo1-silenced and control cells. Cells were treated with DMSO, 5 mM metformin, 5 pM quercetin, or 100 ng / ml irisin for 24 hours. [F] Expression of p65-K310, IL-6, IL-1β, AMPK-T172, SIRT1, and FNDC5 was also assessed by Western blotting, with β-actin used as a loading control. [GH] Relative mRNA expression of p21, p16, and SASP genes was examined in shElmo1 and scramble cells under the indicated treatments. [I] TGF-β protein concentration in supernatants of Elmo1-KD cells co-cultured with DMSO, 5 mM metformin, 5 pM quercetin, or 100 ng / ml irisin. (**P < 0.01, ***P < 0.001). [J] Protein levels of p65-K310, IL-6, IL-1β, AMPK-T172, SIRT1, and LAMINB1 in Elmo1-KD cells treated with Compound C. [K] Relative mRNA expression of p21, p16, and SASP genes in Elmo1-KD cells treated with Compound C. (***P<0.001, Student's t-test)

[0020] Figure 5FNDC5 / irisin treatment ameliorates ELMO1 depletion-induced senescence and fibrosis. IEC6 cells were treated with the indicated concentrations of irisin. [A] Combining ELMO1 downregulation and irisin treatment selectively reduces the viability of senescent IEC6 cells. [B] Relative mRNA expression of Bcl-2 and BAX. [C] Representative immunoblot images of Bcl-2, BAX, and cleaved caspase-3, with tubulin used as a loading control. [D] Representative immunofluorescence images of LAMINB1 (Scale bar: 100 pM). [E] 3T3-L1 cells were treated with normal culture medium (Ctrl), CM from negative control IEC6 cells (CCM), CM from Elmo1 knockdown IEC6 cells (SCM), CM from DMSO-treated Elmo1 knockdown IEC6 cells (DCM), CM from irisin-treated Elmo1 knockdown IEC6 cells (ICM), 2 ng / ml TGF-β, and 100 ng / ml irisin. The relative mRNA expression levels of fibronectin, type I collagen, and a-SMA were detected by QPCR (***P<0.001, Student's t-test).

[0021] Figure 6 .Irisin treatment improves interstitial fibrosis in a mouse model of chronic colitis. Animals were injected with the same volume of irisin or saline daily for 3 weeks (starting 6 weeks after the development of chronic TNBS colitis). Senescence activity was detected by microendoscopy [A], SA-β-gal activity [B], and QPCR assay [C]. [D] IHC analysis of immune cell marker expression on the colonic mucosa of chronic TNBS animals. [E] H&E staining of colon samples from experiment A. [F] Masson trichrome staining analysis of colon samples from experiment A. [G) Analysis of fibronectin and α-SMA in colon tissue from the chronic TNBS model by IHC staining.

[0022] Figure 7 ELMO1 deficiency leads to severe fibrosis and is associated with epithelial cell senescence in a chronic DSS-induced colitis model.

[0023] Figure 8 . Short hairpin RNA (shRNA) was used to knock down Elmo1 in intestinal epithelial cells (IEC6) and fibroblasts (3T3-LI).

[0024] Figure 9 .The mRNA levels of p21, p16, and SASP factors, as well as the relative mRNA expression levels of fibronectin, type I collagen, and a-SMA in Elmo1-knockout 3T3-L1 cells.

[0025] Figure 10.The expression levels of AMPKα-T172 phosphorylation, SIRT1, p65-lys310 acetylation and IL-1β in IEC6 cells under irisin treatment. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0027] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0028] The present invention will now be further described in conjunction with specific examples. The following examples are only intended to explain the present invention but do not limit the present invention. The test samples and test procedures used in the following examples include the following (if the specific experimental conditions are not specified in the examples, they are usually based on conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources).

[0029] The mice used in this example include C57BL / 6, Elmo1 - / - (B6 / JGpt-Elmo1 em1Cd / Gpt) mice; chronic colitis mouse models, including DSS- and TNBS-induced models, were established using conventional techniques. The mouse intestinal epithelial cell line (IEC6) used was purchased from the American Type Culture Collection. In this example, Elmo1 silencing was performed using at least the following sequences: shElmol#1: 5'-GCAGCTCCATGAACGAATACA-3'; shElmol#2: 5'-GGAGATCACCATTGGCCAACT-3'. Scramble shRNA was purchased from Gene Copoeia. SA-β-gal staining was performed according to the manufacturer's instructions (Beyotime, Hairnen, China). IBD: inflammatory bowel disease; ELMO1: phagocytic and cellular motility protein 1; IEC: intestinal epithelial cell; SASP: senescence-associated secretory phenotype; AMPK: AMP-activated protein kinase; SIRT1: silent information regulator 2-related enzyme 1; α-SMA: α-smooth muscle actin.

[0030] Example 1

[0031] 1. The number of senescent intestinal epithelial cells increased in both chronic colitis models.

[0032] To determine whether cellular senescence is involved in IBD-associated fibrosis, we compared tissues from mice with DSS- or TNBS-induced chronic colitis with tissues from uninflamed control individuals. For both DSS- and TNBS-induced models, histological changes of chronic inflammation and fibrosis were confirmed by quantitative polymerase chain reaction (QPCR) assays, H&E, and Masson's trichrome staining, making them suitable for further study.

[0033] H&E staining showed that the mucosa and submucosa of chronic colitis mice were significantly widened compared with normal control individuals, and the inner layer of the muscularis propria tended to be widened (e.g. Figure 1 Masson's trichrome staining showed that there was a lot of collagen deposition in the submucosa and mucosa of mice with chronic colitis ( Figure 1 C). QPCR results from colitis samples in mice showed increased expression of fibrosis markers, including α-SMA, fibronectin, and collagen I ( Figure 1 D).

[0034] To verify the hypothesized profibrotic mechanism, the present inventors collected colon tissue samples from the chronic DSS model for RNA transcriptome sequencing to compare their mRNA expression profiles with those of control mice. A total of 2,035 differentially expressed genes (DEGs) were detected in chronic DSS-induced colitis tissues compared with the control group (P < 0.05, as shown in Table 1). Figure 1 E). Gene ontology (GO) analysis of these genes showed that processes such as “wound healing,” “cell cycle arrest,” “DNA damage response,” “immune response,” “extracellular matrix disorganization,” and “cellular senescence and aging” were upregulated in chronic colitis tissues ( Figure 1 F). In addition, the expression of p21 and senescence-associated secretory phenotype (SASP) genes (IL-1β and TGF-β1) were upregulated in the chronic DSS model, indicating the onset of senescence ( Figure 1 G).

[0035] To determine the role of senescence in IBD-associated fibrosis, we analyzed the hallmarks of senescence in a mouse model of colitis induced by DSS and TNBS. To detect senescent cells, colon sections were stained for a panel of senescence biomarkers, including senescence-associated β-galactosidase (SA-β-gal) and LAMNB1. A decrease in LAMINB1 protein in the nuclear membrane is considered a hallmark of senescence.

[0036] The results showed that SA-β-gal activity increased ( Figure 1 H), LAMNB1 decreased ( Figure 1 I). p16 and p21 mRNA levels are upregulated in stricture colitis tissues ( Figure 1 J). Furthermore, a significant increase in SASP genes, including IL-1β, IL-6, IL-8, PDGFα, TGF-β, and MCP-1, was observed in colitis tissues by QPCR detection. Figure 1 These results suggest a correlation between intestinal aging and intestinal fibrosis in a mouse model of chronic colitis.

[0037] 2. ELMO1 deficiency accelerates epithelial tissue aging and aggravates intestinal fibrosis.

[0038] Previous studies by the present inventors have identified ELMO1 as a key regulator of wound healing in IBD, but the function of ELMO1 in cellular senescence and intestinal fibrosis is unclear. Based on this, the present inventors first examined ELMO1 expression in normal, acute colitis, and chronic colon tissues. They found that ELMO1 expression was significantly reduced in stenotic tissues and correlated with high expression of p16, IL-6, PDGFα, and α-SMA in stenotic tissues. Figure 2 A and B).

[0039] To analyze the potential role of ELMO1 in fibrosis and aging in vivo, we - / - Furthermore, the researchers investigated senescence and fibrosis in two established IBD models.

[0040] In the chronic TNBS model, microendoscopy revealed that mice lacking Elmo1 had exacerbated mucosal inflammation compared with control mice ( Figure 2 C) In Elmo1 - / - In mice, the number of activated colonic fibroblasts increased, the submucosal layer was wider, and the expression of fibrotic markers (α-SMA, fibronectin, and collagen I) was increased ( Figure 2 DF). Elmo1 was observed using SA-β-gal staining and LAMNB1 staining and light-sheet fluorescence microscopy. - / - The accumulation of senescent cells in confined areas of animals ( Figure 2 G and H). In addition, senescent cells were observed to be located in intestinal epithelial cells ( Figure 2 H).

[0041] Similar assays were performed on chronic DSS and showed similar intestinal inflammation, aging and fibrosis, as well as gene expression (e.g. Figure 7To further understand this process, the present inventors used RNA sequencing (RNA-seq) to compare the Elmo1 cells treated with chronic TNBS. - / - Transcriptome of mice and wild-type control mice. - / - Compared with wild-type controls, p21, p16, and approximately 30 SASP genes were differentially expressed in mice ( Figure 2 I). QPCR was used to further confirm the increase in the levels of these central SASP components (IL-1β, IL-6 and IL-8) and cell cycle regulators p21 and p16 (e.g. Figure 2 I~2J; Figure 7 H~7I).

[0042] These data indicate that ELMO1 inactivation leads to a significant increase in senescence and intestinal fibrosis in two different chronic colitis models by regulating the SASP pathway.

[0043] 3. ELMO1 depletion-induced senescence activates intestinal fibroblasts.

[0044] Next, to investigate whether ELMO1 plays a key role in cellular senescence, two independent short hairpin RNAs (shRNAs) were used to stably knock down Elmo1 in intestinal epithelial cells (IEC6) and fibroblasts (3T3-LI). Figure 8 shown).

[0045] We found that knockout of Elmo1 induced higher levels of p21, p16, and SASP factors in IEC6 cells, and reduced LAMINB1 abundance ( Figure 3 AC). However, compared with their controls, the mRNA levels of p21, p16, and SASP factors remained unchanged in Elmo1-knockout 3T3-L1 cells (e.g. Figure 9 A and 9B).

[0046] Furthermore, previous studies have shown that senescent cells can activate fibroblasts. However, whether senescent Elmo1-silenced IEC6 cells can activate intestinal fibroblasts is still unknown. On this basis, the inventors launched a related study, treating 3T3-L1 cells with conditioned medium (CM) from senescent IEC6 cells depleted of Elmo1. The stimulation of 3T3-L1 fibroblasts was divided into groups according to the treatment mode, including normal culture medium (Ctrl), CM from negative control IEC6 cells (CCM), CM from Elmo1 knockout IEC6 cells (SCM) and 2ng / ml TGF-β (TGF-β). TGF-β was used as a positive control for activating fibroblasts.

[0047] The results showed that elevated levels of type I collagen, α-SMA, and fibronectin were detected in the SCM group ( Figure 3 D). To exclude the effect of Elmo1 ablation on fibroblast activation, we also tested whether knockdown of Elmo1 could activate fibroblasts. The results showed that knockdown of Elmo1 could not activate fibroblasts ( Figure 9 C). In addition, immunofluorescence staining of fibronectin and immunoblotting of α-SMA also confirmed the activation of 3T3-L1 cells by SCM ( Figure 3 E and F).

[0048] The above experimental results show that Elmo1 depletion-induced senescence can stimulate the transformation of fibroblasts to myofibroblasts (the transformation of fibroblasts to myofibroblasts is a hallmark of intestinal fibrosis).

[0049] 4. Decreased FNDC5-AMPK-SIRT1 signaling activity is the cause of ELMO1 depletion-induced aging.

[0050] Previous studies have shown that upregulation of p21 and SASP genes indicates activation of the p53 and NF-KB pathways. Transcriptome sequencing data showed that PI3K-AKT signaling, MAPK signaling, p53 signaling, NF-KB signaling, and longevity regulatory pathways were associated with ELMO1 deficiency and chronic colitis disease ( Figure 4 A and B). Based on transcriptome sequencing results and previous studies by others, we examined the expression of p53 and RelA / p65 (key transcription factors in the NF-κB pathway) in IEC6 cells depleted of Elmo1. The results showed that acetylation of p65 at Lys310 was significantly increased in three independent experiments, while the levels of p53-Lys382, total p53, and p65 proteins did not change ( Figure 4 C).

[0051] Consistent with the acetylation of p65, the expression of IL-1β and IL-6 was upregulated in Elmo1-knockout IEC6 cells, suggesting that activation of p65 through increased acetylation is responsible for the cell cycle arrest and SASP induced by ELMO1 depletion. Figure 4 C) QPCR assays showed that key genes in the longevity regulatory pathway, including Prkaa1 (also known as Ampka), Sirtl, Ppargc1 (also known as Pgc-1, and Fndc5), were expressed in Elmo1 - / - Down-regulated in colitis colon tissue ( Figure 4 D) Studies have shown that SIRT1, a deacetylase, improves inflammation in aged COPD mice by regulating p65-lys310 acetylation and AMPKa-SIRT1 signaling.

[0052] Next, the inventors attempted to investigate the enzyme responsible for regulating p65 acetylation downstream of ELMO1. To determine whether AMPKa-SIRT1 signaling is involved in the effects of ELMO1 on intestinal aging and inflammation, the expression of SIRT1 and AMPKα in Elmo1-depleted cells and their controls was measured by immunoblotting. The results showed that the protein level of AMPKα did not change significantly in cells with Elmo1 downregulation, while the phosphorylation of AMPKα at Thr172 was significantly inhibited, and SIRT1 activity was significantly decreased ( Figure 4 E), FNDC5 (a novel upstream substrate of AMPK) had its protein level significantly decreased ( Figure 4 E). Further examination showed that the elevated p65-lys310 acetylation was abolished in the presence of an AMPKα activator (metformin), a SIRT1 activator (quercetin), or purified FNDC5 protein (irisin) ( Figure 4 F). These treatments also abolished the induction of SASP genes, p21, and p16, and reduced the secretion of the cytokine TGF-β ( Figure 4 GI).

[0053] More importantly, immunoblotting showed that the AMPKα inhibitor Compound C alone could induce significant senescence in IEC6 cells, and SIRT1 activity was significantly inhibited ( Figure 4 Compound C did not further increase p65 acetylation levels, and there were no significant changes in p21, p16, and SASP factors, indicating that Elmo1-KD-mediated senescence is entirely achieved through AMPK-SIRT1 signaling ( Figure 4 J and K). Irisin treatment alone promoted AMPKα-T172 phosphorylation and SIRT1 levels, while decreasing p65-lys310 acetylation and IL-1β expression (Fig. Figure 10 A and 10B).

[0054] These observations indicate that the FNDC5-AMPK-SIRT1 pathway, which mediates p65 acetylation and activation, is essential for ELMO1 depletion-induced senescence.

[0055] 5. Irisin treatment can eliminate senescence caused by ELMO1 depletion.

[0056] Combined with the above findings, we found that irisin treatment induced the phosphorylation of AMPK-T172 and promoted the nuclear translocation of SIRT1 ( Figure 4 F).

[0057] To determine whether irisin can eliminate senescent cells by enhancing ELMO1 function, IEC6 cells with downregulated ELMO1 were treated with irisin and DMSO. CCK-8 assay showed that under irisin treatment, the viability of the downregulated cells was significantly decreased compared with the control cells ( Figure 5 A). Irisin reduced the mRNA expression of the anti-apoptotic gene Bcl-2 and increased the expression of the pro-apoptotic gene BAX ( Figure 5 B). The results of immunoblotting were similar to those of QPCR ( Figure 5 C). In the ELMO1 knockout group, the protein level of cleaved caspase 3 was upregulated after treatment with irisin ( Figure 5 C). Consistent with the activation of apoptosis, the accumulation of senescent cells was reduced in irisin-treated Elmo1-silenced cells as assessed by LAMINB1 staining ( Figure 5 D).

[0058] Based on in vitro data, to explore whether irisin treatment can improve intestinal fibrosis, the inventors next analyzed the effect of irisin treatment on 3T3-L1 cells. Culture medium was collected to stimulate 3T3-L1 fibroblasts. It was found that in the ISCM group (culture medium from Elmo1-knockdown IEC6 cells under irisin treatment), Figure 5 In E), the mRNA levels of α-SMA, fibronectin, and collagen I were downregulated.

[0059] These results indicate that irisin treatment can alleviate the burden of cellular senescence caused by ELMO1 depletion and reduce aging-induced intestinal fibrosis.

[0060] Irisin treatment reduces senescent cells and intestinal fibrosis in a TNBS-induced colitis mouse model

[0061] Since irisin treatment could abrogate ELMO1 depletion-induced senescence in vitro, we further tested whether irisin could reduce intestinal senescence and intestinal fibrosis in the chronic TNBS model.

[0062] We have studied wild-type, Elmo1 - / - Mice were injected with the same volume of irisin or saline once a week for 3 weeks (starting 6 weeks after the development of chronic TNBS colitis). Irisin treatment was found to reduce mucosal inflammation and the positive area of SA-β-gal staining in wild-type mice ( Figure 6 A and B). With the decrease of senescent cells under irisin treatment, the mRNA expression of p21, p16, and SASP factors (including IL-1β, IL-6, MCP-1, PDGFa, and MMP3) was downregulated ( Figure 6 C). Reduced inflammatory cells and mucosal inflammation ( Figure 6 D). Then, we analyzed the fibrosis markers in the chronic TNBS model under irisin treatment. We detected reduced distortion of crypt structure, reduced submucosal thickness, and decreased collagen accumulation ( Figure 6 E and F). ( Figure 6 E and F). Irisin reduced the levels of α-SMA and fibronectin ( Figure 6 G).

[0063] These data suggest that irisin's important effects on intestinal aging and fibrosis are required for ELMO1 function in the chronic TNBS model.

[0064] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Use of irisin in preparing a medicament for preventing and / or treating intestinal fibrosis, characterized in that: The intestinal fibrosis is intestinal fibrosis in inflammatory bowel disease.

2. Use of ELMO1 detection reagent in the preparation of a kit for diagnosing intestinal fibrosis in inflammatory bowel disease.

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