CN116531508A - ELMO1 and use of downstream pathway genes thereof in diagnosis, prevention and treatment of intestinal fibrosis - Google Patents
ELMO1 and use of downstream pathway genes thereof in diagnosis, prevention and treatment of intestinal fibrosis Download PDFInfo
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- CN116531508A CN116531508A CN202310115891.4A CN202310115891A CN116531508A CN 116531508 A CN116531508 A CN 116531508A CN 202310115891 A CN202310115891 A CN 202310115891A CN 116531508 A CN116531508 A CN 116531508A
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Abstract
The invention discloses an ELMO1 and application of a downstream pathway gene thereof in diagnosis, prevention and treatment of intestinal fibrosis. Studies in this application have shown that ELMO1 plays an important role in epithelial cell aging and intestinal fibrosis, and on this basis, it has been 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, provides theoretical basis for aging of intestinal epithelial cells and promotion of poor progress of intestinal inflammation, and simultaneously provides a new thought for IBD fibrosis treatment so as to reduce recurrence and development of intestinal fibrosis.
Description
Technical Field
The invention relates to the technical field of diagnosis and treatment of inflammatory bowel disease, in particular to application of ELMO1 and a downstream pathway gene thereof in diagnosis, prevention and treatment of intestinal fibrosis.
Background
Intestinal fibrosis is a long-term complication of Inflammatory Bowel Disease (IBD), including Ulcerative Colitis (UC) and Crohn's Disease (CD). Intestinal fibrosis is typically seen in ileal CD, and is also a common complication of chronic progressive UC and colonic CD. Due to the lack of specific anti-fibrotic therapies, about 50% of CD patients require surgical intervention within 10 years after diagnosis and are unable to prevent disease recurrence or fibrosis.
Thus, there is an urgent need to investigate the mechanisms of IBD-related fibrosis, fibrotic stenosis, and possible therapeutic regimens.
Disclosure of Invention
The present invention aims to overcome at least one of the above-mentioned shortcomings of the prior art, and provides a use of ELMO1 and its downstream pathway genes in diagnosing, preventing and treating intestinal fibrosis, which can reduce intestinal cell aging caused by ELMO1 down-regulation, further prevent intestinal fibrosis caused by intestinal cell aging, provide a new therapeutic strategy for intestinal fibrosis, and can diagnose whether there is a risk of intestinal fibrosis in time by detecting ELMO1 levels and comparing with normal intestinal tissues.
An object of the present invention is to provide a use of an FNDC5-AMPK-SIRT1 pathway activator in the preparation of a medicament for treating intestinal fibrosis. In one embodiment of the invention, ELMO1 knockdown was found to cause downregulation of the relevant pathway FNDC5-AMPK-SIRT1, thereby increasing the accumulation of senescent cells, exacerbating intestinal fibrosis, while using FNDC5-AMPK-SIRT1 pathway activators, the characterization of cellular senescence by p65 acetylation, etc. was eliminated, and intestinal fibrosis was further alleviated, alleviated. In particular the FNDC5 purified protein irisin. Iris can reduce ELMO1 depletion-induced aging by AMPK alpha-SIRT 1 signaling, and further improve fibrosis in chronic intestinal inflammation. Among them, irisin ((Irisin)) is a hormone hydrolyzed from fibronectin type III domain-containing protein 5 (FNDC 5), and is considered as an anti-aging agent, and has been found to inhibit chronic diseases such as alzheimer's disease, cardiac dysfunction and disc degeneration.
Further, the FNDC5-AMPK-SIRT1 pathway activator comprises a FNDC5 activator, an AMPK activator, an SIRT1 activator, and/or an ELMO1 activator.
Further, the FNDC5 activator comprises irisin; and/or, AMPK activators include metformin; and/or, the SIRT1 activator comprises quercetin.
Further, intestinal fibrosis includes intestinal fibrosis and intestinal fibrosis stenosis in inflammatory bowel disease. In one or more embodiments of the invention, the aforementioned pathway activators have been found to be effective in treating intestinal fibrosis, intestinal fibrosis stenosis, in inflammatory bowel disease, including chronic colitis.
It is still another object of the present invention to provide a use of an anti-aging agent and/or a p65 acetylation-inhibiting agent in the preparation of a medicament for treating intestinal fibrosis. In one or more embodiments of the present invention, it has been found that intestinal fibrosis can be effectively reduced by reducing intestinal epithelial cell senescence. In one or more embodiments of the invention, p65 acetylation appears to be important during aging induced by ELMO1 depletion, and where the aim is to inhibit aging, the pharmacologic inhibition of its acetylation should also be considered as a drug for the treatment of intestinal fibrosis.
Further, the anti-aging medicine comprises an anti-aging medicine of intestinal epithelial tissue cells.
It is still another object of the present invention 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 invention, inflammatory bowel tissue with fibrosis is found to exhibit lower levels of ELMO1 expression compared to normal bowel tissue.
Further, intestinal fibrosis includes intestinal fibrosis in inflammatory bowel disease.
It is a further object of the present invention to provide a marker for intestinal fibrosis, comprising ELMO1 with low relative intestinal expression levels.
It is still another object of the present invention to provide a therapeutic agent for inflammatory bowel disease fibrosis, including an FNDC5 activator, an AMPK activator, a SIRT1 activator, and/or an Elmo1 activator.
Compared with the prior art, the invention has the beneficial effects that: studies in this application have shown that ELMO1 plays an important role in epithelial cell aging and intestinal fibrosis, and on this basis, it has been 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, provides theoretical basis for aging of intestinal epithelial cells and promotion of poor progress of intestinal inflammation, and simultaneously provides a new thought for IBD fibrosis treatment so as to reduce recurrence and development of intestinal fibrosis. ELMO1 as an anti-aging marker has an important role in preventing intestinal fibrosis. On the basis of the research of the application, a novel inflammatory bowel disease intestinal fibrosis treatment strategy is provided, and is beneficial to being applied to clinical treatment so as to reduce recurrence and development of intestinal fibrosis.
Drawings
Figure 1. Mice model of chronic colitis and patients with stenotic IBD have increased intestinal aging. [A] Representative H & E stained colon sections showed non-inflamed control individuals and a model of chronic colitis. [B] Absolute thickness of mucosa (< P <0.015 (< P < 0.001)) was measured in control individuals (n=5) and chronic colitis mice (n=5). [C] Trichromatic staining results of Masson, derived from colon sections of mice, animals, respectively, of the chronic DSS or chronic TNBS model; the fibrosis level is depicted. (n=5, ×p <0.01, ×p < 0.001). [D] Relative expression of fibronectin, type I collagen, and α -SMA mRNA in chronic DSS or chronic TNBS models (n=5, P <0.01, P < 0.001). [E] Volcanic map of Differentially Expressed Genes (DEG) between normal colon tissue and DSS-induced colitis tissue. [F] DSS induced GO analysis of DEG of colitis tissue. [G] Expression of the 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 immunofluorescent staining image of LAMNB1 (scale bar, 200 μm). [J] Relative expression of the 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)
Fig. 2.Elmo1 deficiency resulted in severe fibrosis and was associated with epithelial cell senescence in a chronic TNBS-induced colitis model. [ A ]]ELMO1 expression in acute colitis, chronic colitis and normal colon tissue. (n=3,/P)<0.001)。[B]Correlation of ELMO1 with p16, IL-6, PDGF alpha and alpha SMA in chronic colitis animals. [ C-D ]]In Elmo1 -/- Construction of chronic TNBS colitis in mice or control mice and analysis of colon tissue by micro-endoscopy [ C]And H&E staining [ D]。[E]In Elmol -/- The absolute thickness of the mucosa was measured in (n=5) and wild-type colitis mice (n=5). [ F]Elmo1 -/- (n=5) and wild-type colitis mice (n=5, P<0.01,***P<0.001 Immunohistochemical images of fibronectin, collagen I, and a-SMA). [ G-H ]]In Elmol -/- And wild-type colitis mice, SA- β -gal activity and LAMINB1 staining (scale bar, 100 pm) were examined. [ I-J]Elmo1 -/- And relative mRNA expression of p21, pl6, p53 and SASP genes in wild-type colitis mice. (n=5,/P)<0.01,***P<0.001)
FIG. 3 ELMO1 depletion promotes IEC6 aging and activates 3T3-L1 fibrosis. IEC6 cells were transfected with sh-Elmo1 plasmid to exclude the effect of ELMO1 on senescence. Senescence activity was detected in silencing and disrupting cells by QRCR [ a-B ] and lamnb1 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 2ng/ml TGF-beta 1. [D] Relative mRNA expression of fibronectin, collagen I, and α -SMA. [E] Representative immunofluorescence image of fibronectin. [F] Representative, quantitative immunoblot analysis of α -SMA.
FIG. 4.ELMO1 targets the FNDC5-AMPK-SIRT1 pathway. [ A-B ] transcriptome sequencing was performed and the pathway of differentially expressed genes (fold change >1.0, benjamini-Hochberg corrected P <, 05) was assessed using molecular signature database marker gene sets. [C] Western blot detection of Elmo1 silencing and control cells, p65-K310 acetylation, p53-K382 acetylation, IL-6 and IL-1β expression. [D] QPCR detects expression of Prkaa1, sirt1, ppargc1 and Fndc 5. [E] Western blot analysis of Elmo1 silencing and control cells, AMPK-T172 phosphorylation, SIRT1 and FNDC5 expression. Wherein the cells were treated with DMSO, 5mM metformin, 5pM quercetin or 100ng/ml irisin, respectively, for 24 hours. [F] Expression of p65-K310, IL-6, IL-1β, AMPK-T172, SIRT1 and FNDC5 was also assessed by Western blotting, wherein β -actin was used as a loading control. [ G-H ] relative mRNA expression of p21, p16 and SASP genes was detected in shElmo1 and scramble cells under the indicated treatments. [I] Concentration of TGF-beta protein in Elmo1-KD cell supernatants co-cultured with DMSO, 5mM metformin, 5pM quercetin or 100ng/ml irisin. P <0.01, P < 0.001). [J] Protein levels of p65-K310, IL-6, IL-1β, AMPK-T172, SIRT1 and LAMINB1 in Compound C treated Elmo1-KD cells. [K] Compound C-treated Elmo1-KD cells, relative mRNA expression of p21, pl6 and SASP genes. P <0.001,Student's t-test
FIG. 5. FNDC5/irisin treatment improved ELMO1 depletion-induced aging and fibrosis. Wherein IEC6 was treated with a specified concentration of irisin. [A] Binding ELMO1 down-regulation and tectorigenin treatment selectively reduced viability of senescent IEC6 cells. [B] Relative mRNA expression of Bcl-2 and BAX. [C] Representative immunoblots of Bcl-2, BAX and cleaved caspase-3, tubulin was used as loading control. [D] Representative immunofluorescence image of lamINB1 (scale: 100 pM). [E]3T3-L1 cells were treated with normal medium (Ctrl), CM (CCM) from negative control IEC6 cells, CM (SCM) from Elmo1 knockdown IEC6 cells, CM (DCM) from DMSO-treated Elmo1 knockdown IEC6 cells, elmo1 knockdown IEC6 cells (ICM) from tectoridin treatment, 2ng/ml TGF-. Beta.and 100ng/ml tectoridin, respectively. QPCR detects the relative amounts of mRNA expression of fibronectin, type I collagen, and a-SMA. P <0.001,Student's t-test).
Figure 6. Iris treatment improves interstitial fibrosis in a mouse model of chronic colitis. Animals were injected daily with the same volume of tectorigenin or saline for 3 weeks (starting 6 weeks after chronic TNBS colitis formation). Aging activity was detected by miniature endoscope [ A ], SA- β -gal activity [ B ] and QPCR assay [ C ]. [D] IHC analysis of immune cell marker expression on colonic mucosa of chronic TNBS animals. [E] H & E staining from a colon sample from a experiment. [F] Masson trichromatic staining analysis of colon samples from the a experiment. [ G) analysis of fibronectin and alpha-SMA in colon tissue from the chronic TNBS model by IHC staining.
Fig. 7.Elmo1 deficiency resulted in severe fibrosis and was associated with epithelial cell senescence in a chronic DSS-induced colitis model.
FIG. 8 Elmo1 in intestinal epithelial cells (IEC 6) and fibroblasts (3T 3-LI) were knocked down using short hairpin RNA (shRNA).
FIG. 9 shows mRNA levels of p21, p16 and SASP factors, and relative expression levels of fibronectin, type I collagen and a-SMA in Elmo1 knockdown 3T3-L1 cells.
FIG. 10 expression levels of AMPK alpha-T172 phosphorylation, SIRT1, p65-lys310 acetylation, IL-1β, and the like in IEC6 cells under tectorigne treatment.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The invention will now be further illustrated with reference to specific examples, which are given solely for the purpose of illustration and are not to be construed as limiting the invention. The test specimens and test procedures used in the following examples include those (if the specific conditions of the experiment are not specified in the examples, generally according to conventional conditions or according to the recommended conditions of the reagent company; the reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified).
The mice used in this example include C57BL/6, elmo1 -/- (B6/JGpt-Elmo1 em1Cd Gpt) mice; the chronic colitis mouse model, including DSS and TNBS induction model, is constructed by conventional technique. The mouse intestinal epithelial cell line (IEC 6) used was purchased from the american type culture collection. In this example, elmo1 silencing is 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-. Beta. -gal staining was performed according to the manufacturer's instructions (Beyotime, hairnen, china). IBD: inflammatory bowel disease; ELMO1: phagocytic and cell motor protein 1; IEC: intestinal epithelial cells; SASP: a senescence-associated secretory phenotype; AMPK: AMP-activated protein kinase; SIRT1: silencing information regulator 2-related enzyme 1; alpha-SMA: alpha-smooth muscle actin.
Example 1
1. Aged intestinal epithelial cells were increased in both chronic colitis models.
To determine whether cellular senescence is involved in IBD-associated fibrosis, we compared tissues from DSS or TNBS-induced chronic colitis mice to that of non-inflamed control individuals. And for DSS and TNBS induction models, histological changes in chronic inflammation and fibrosis were confirmed by Quantitative Polymerase Chain Reaction (QPCR) assays, H & E and Masson trichromatic staining, making them suitable for further study.
H & E staining showed significant broadening of the mucosa and submucosa in chronic colitis mice compared to normal control individuals, with a tendency for broadening of the intrinsic myolayer (as shown in figures 1A and 1B). Trichromatic staining of Masson showed significant collagen deposition in submucosa and mucosa of chronic colitis mice (fig. 1C). QPCR assay results from samples of mouse colitis showed increased expression of fibrotic markers, including α -SMA, fibronectin and collagen I (fig. 1D).
To verify the putative pro-fibrotic mechanism, the inventors collected colon tissue samples from the chronic DSS model for RNA transcriptome sequencing to compare their mRNA expression profile with that of control mice. As a result, 2035 Differentially Expressed Genes (DEG) were found in the chronic DSS-induced colitis tissue in total, compared to the control group; (P <0.05, as shown in FIG. 1E). Analysis of the Gene Ontology (GO) for these genes indicated that the processes of "wound healing", "cell cycle arrest", "DNA damage response", "immune response", "extracellular matrix disintegration" and "cell senescence and aging" were up-regulated in chronic colitis tissues (fig. 1F). Furthermore, expression of p21 and senescence-associated secretory phenotype (SASP) genes (IL-1β and TGF-. Beta.1) was up-regulated in the chronic DSS model, indicating initiation of senescence (FIG. 1G).
To determine the role of aging in IBD-associated fibrosis, the characteristics of aging in DSS and TNBS-induced colitis mouse models were analyzed. To detect senescent cells, colon sections were stained for a series of senescence biomarkers, including senescence-associated beta-galactosidase (SA-beta-gal) and LAMNB1. Among them, the decrease of lamnb1 protein in the nuclear membrane is considered as a feature of aging.
The results showed an increase in SA- β -gal activity (FIG. 1H) and a decrease in LAMNB1 (FIG. 1I). mRNA levels of p16 and p21 were up-regulated in stenotic colitis tissues (FIG. 1J). Further, significant increases in SASP gene in colitis tissues were also observed by QPCR detection, including IL-1β, IL-6, IL-8, PDGF α, TGF- β and MCP-1 (as shown in FIG. 1K). These results indicate that there is a correlation between intestinal aging and intestinal fibrosis in the mouse model of chronic colitis.
2.ELMO1 deficiency accelerates aging of epithelial tissues and aggravates intestinal fibrosis.
In previous studies by the inventors, ELMO1 was identified as a key regulator of IBD wound healing, but it was not clear that ELMO1 functions in cell senescence and intestinal fibrosis. On this basis, the inventors first examined ELMO1 expression in normal, acute colitis and chronic colon tissues. It was found that the expression of ELMO1 was significantly reduced in stenotic tissue and correlated with high expression of p16, IL-6, pdgfα and α -SMA in stenotic tissue (fig. 2A and B).
To analyze the potential role of ELMO1 in fibrosis and aging in vivo, ELMO1 was tested -/- Mice were subjected to experiments. Further, aging and fibrosis were also studied in two established IBD models.
In the chronic TNBS model, it was found by micro-endoscopy that the mucosal inflammation of mice lacking Elmo1 was worsened compared to control mice (fig. 2C). In Elmo1 -/- In mice, the number of activated colon fibroblasts was increased, with a wider submucosal layer thickness, and the expression of fibrosis markers (α -SMA, fibronectin, and collagen I) was increased (fig. 2D-F). Using SA- β -gal staining and LAMNB1 staining, and observation by light sheet fluorescence microscopy, elmo1 was observed -/- Accumulation of senescent cells in the stenotic area of animals (FIGS. 2G and H). Furthermore, senescent cells were also observed to be located in intestinal epithelial cells (fig. 2H).
Similar assays were performed on chronic DSS, and the results showed similar intestinal inflammation, aging and fibrosis, as well as gene expression (as shown in fig. 7A-7I). To further understand this process, the inventors compared chronically TNBS treated Elmo1 using RNA sequencing (RNA-seq) -/- Transcriptome of mice and wild-type control mice. Discovery of Elmol -/- Mice had differential expression of p21, pl6 and approximately 30 SASP genes compared to wild-type controls (fig. 2I). And the increase in the levels of these central SASP components (IL-1. Beta., IL-6 and IL-8) and cell cycle regulatory factors p21, p16 was further confirmed using QPCR (see FIGS. 2I-2J; FIGS. 7H-7I).
These data indicate that ELMO1 gene inactivation leads to significant increases in aging and intestinal fibrosis in two different models of chronic colitis by modulating the SASP pathway.
3.ELMO1 depletion-induced aging activates intestinal fibroblasts.
Next to investigate whether ELMO1 plays a key role in cell senescence, two independent short hairpin RNAs (shRNA) were used to stably knock down ELMO1 in intestinal epithelial cells (IEC 6) and fibroblasts (3T 3-LI) (as shown in fig. 8).
The knockout of Elmo1 was found to induce higher levels of IEC6 cell p21, pl6 and SASP factors, and reduced lamnb1 abundance (fig. 3A-C). However, the mRNA levels of p21, p16 and SASP factors remained unchanged in the Elmo1 knocked-out 3T3-L1 cells compared to their control group (as shown in fig. 9A and 9B).
Further, previous studies have shown that senescent cells can activate fibroblasts. While it is currently unknown whether senescent Elmo1 silences IEC6 cells can activate intestinal fibroblasts. Based on this, the present inventors have developed a related study to treat 3T3-L1 cells with Conditioned Medium (CM) from aged IEC6 cells depleted of Elmo1. Stimulation of 3T3-L1 fibroblasts was grouped in a treatment regimen, including normal medium (Ctrl), CM (CCM) from negative control IEC6 cells, CM (SCM) from Elmo1 knockout IEC6 cells, and 2ng/ml TGF- β (TGF- β). TGF-beta was used as a positive control for activating fibroblasts.
As a result, elevated levels of type I collagen, α -SMA, and fibronectin were found to be detected in SCM groups (fig. 3D). To rule out the effect of Elmo1 ablation on activation of fibroblasts, it was also tested whether down-regulation of Elmo1 could activate fibroblasts. The results showed that knocking down Elmo1 failed to activate fibroblasts (fig. 9C). In addition, immunofluorescent staining of fibronectin and immunoblotting of α -SMA also confirmed activation of 3T3-L1 cells by SCM (fig. 3E and F).
The above experimental results indicate that aging induced by Elmo1 depletion can stimulate the conversion of fibroblasts to myofibroblasts (the conversion of fibroblasts to myofibroblasts is a marker of intestinal fibrosis).
4. The reduced FNDC5-AMPK-SIRT1 signaling activity is responsible for the aging induced by ELMO1 depletion.
Previous studies have shown that upregulation of the p21 and SASP genes indicates activation of the p53 and NF-KB pathways. Transcriptional sequencing data showed that PI3K-AKT signaling, MAPK signaling, p53 signaling, NF-KB signaling and long-lived regulatory pathways were associated with ELMO1 deficiency and chronic colitis disease (fig. 4A and B). Based on transcriptome sequencing results and other previous studies, we examined the removal of p53 and RelA/p65 (key transcription factors in the NF-KB pathway) expression in IEC6 cells of Elmo1. The results show that in three independent experiments, lys310 site acetylation of p65 was significantly increased, while p53-Lys382, total p53, p65 protein levels were unchanged (fig. 4C).
Consistent with the acetylation of p65, the expression of IL-1 β and IL-6 was up-regulated in Elmo1 knockdown IEC6 cells, suggesting that activation of p65 by increasing acetylation is responsible for induction of cell cycle arrest and SASP by Elmo1 depletion (fig. 4C). QPCR assays revealed that key genes for long-lived regulatory pathways, including Prkaa1 (also known as Ampka), sirtl, ppargc1 (also known as Pgc-1 and Fndc 5), were found in Elmo1 -/- Downregulation in colonic tissue of colitis (fig. 4D). Studies have shown that SIRT1 is a deacetylase that improves inflammation in aged COPD mice by modulating p65-lys310 acetylation, AMPKa-SIRT1 signaling.
Next, the inventors sought to investigate the enzymes responsible for regulating p65 acetylation downstream of ELMO1. To determine whether AMPKa-SIRT1 signaling is associated with the effects of ELMO1 on intestinal aging and inflammation, expression of SIRT1 and AMPK alpha in ELMO1 depleted cells and their controls was measured by immunoblotting. As a result, it was found that there was no significant change in the protein level of AMPK.alpha.in cells downregulated by Elmo1, while the phosphorylation of the Thr172 site of AMPK.alpha.was significantly inhibited, and the SIRT1 activity was significantly decreased (FIG. 4E), and that the protein level of FNDC5, a novel upstream substrate of AMPK, was significantly decreased (FIG. 4E). Further examination showed that the elevation of p65-lys310 acetylation was eliminated in the case of purified proteins (irisin) using AMPK alpha activator (metformin), SIRT1 activator (quercetin) or FNDC5 (fig. 4F). By these treatments, induction of the SASP gene, p21 and p16 was also eliminated and secretion of the cytokine TGF- β was reduced (FIG. 4G-I).
More importantly, immunoblots showed that AMPK alpha inhibitor Compound C alone induced significant aging of IEC6 cells and SIRT1 activity was significantly inhibited (fig. 4J). The use of Compound C did not further increase the level of p65 acetylation, and there was no significant change in p21, p16 and SASP factors, indicating that Elmo1-KD mediated senescence was achieved entirely through AMPK-SIRT1 signaling (FIGS. 4J and K). The treatment with irisin alone promoted AMPK alpha-T172 phosphorylation and SIRT1 levels, while reducing p65-lys310 acetylation and IL-1β expression (as shown in fig. 10A and 10B).
These observations indicate that the FNDC5-AMPK-SIRT1 pathway mediates acetylation and activation of p65, which is critical for aging induced by ELMO1 depletion.
5. Tectorigenin treatment can eliminate aging caused by ELMO1 depletion.
In combination with the above findings, we found that tectorigenin treatment induced phosphorylation of AMPK-T172 and promoted SIRT1 nuclear translocation (fig. 4F).
To determine whether irisin could eliminate senescent cells by enhancing ELMO1 function, ELMO1 down-regulated IEC6 cells were treated with irisin and DMSO. CCK-8 assays showed a significant decrease in viability of the deleted cells compared to control cells under tectorigenin treatment (fig. 5A). Irisin reduced mRNA expression of the anti-apoptotic gene Bcl-2, while increased expression of the pro-apoptotic gene BAX (fig. 5B). The results of immunoblotting were similar to those of QPCR (fig. 5C). In the ELMO1 knockout group, protein levels of cleaved caspase 3 were up-regulated following treatment with irisin (fig. 5C). Consistent with activation of apoptosis, it was observed by lamnb1 staining evaluation that senescent cell accumulation was reduced in tectorigenin-treated Elmo 1-silenced cells (fig. 5D).
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. The medium was collected to stimulate 3T3-L1 fibroblasts. It was found that in ISCM group (medium from Elmo1-knockdown IEC6 cells under tectoridin treatment, fig. 5E), mRNA levels of α -SMA, fibronectin and collagen I were down-regulated.
The above results indicate that irisin treatment can reduce the burden of cellular aging caused by ELMO1 depletion and reduce aging-induced intestinal fibrosis.
6. Irisin treatment reduces senescent cells and intestinal fibrosis in TNBS-induced colitis mice models
Since irisin treatment can eliminate ELMO 1-depletion induced aging in vitro, we further tested whether irisin could reduce intestinal aging and intestinal fibrosis in a chronic TNBS model.
We refer to wild type, elmo1 -/- Mice were injected once a week with the same volume of irisin or saline for 3 weeks (starting 6 weeks after chronic TNBS colitis formation). Iris treatment was found to reduce mucosal inflammation and reduce the positive areas of SA- β -gal staining in wild type mice (FIGS. 6A and B). mRNA expression of p21, p16 and SASP factors (including IL-1β, IL-6, MCP-1, PDGFa and MMP 3) was down-regulated with decreasing senescent cells under tectorigenin treatment (FIG. 6C). Inflammatory cells and mucosal inflammation decreased (fig. 6D). Then, fibrosis markers of the chronic TNBS model under tectorigenin treatment were analyzed. Reduced crypt distortion was detected, reduced submucosal thickness, and reduced collagen accumulation (fig. 6E and F). (FIGS. 6E and F). Irisin reduced the levels of α -SMA and fibronectin (fig. 6G).
These data indicate that an important role for irisin in intestinal aging and fibrosis is necessary for the realization of ELMO1 function in the chronic TNBS model.
It should be understood that the foregoing examples of the present invention are merely illustrative of the present invention and are not intended to limit the present invention to the specific embodiments thereof. Any modification, equivalent replacement, improvement, etc. that comes within the spirit and principle of the claims of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
- Use of an fndc5-AMPK-SIRT1 pathway activator in the manufacture of a medicament for the prevention and treatment of intestinal fibrosis.
- 2. The use of claim 1, wherein the FNDC5-AMPK-SIRT1 pathway activator comprises a FNDC5 activator, an AMPK activator, a SIRT1 activator, and/or an ELMO1 activator.
- 3. The use according to claim 2, wherein the FNDC5 activator comprises irisin; and/or, AMPK activators include metformin; and/or, the SIRT1 activator comprises quercetin.
- 4. Use according to any one of claims 1 to 3, wherein intestinal fibrosis comprises intestinal fibrosis and intestinal fibrosis stenosis in inflammatory bowel disease.
- 5. Use of an anti-aging agent and/or a p65 acetylation-inhibiting agent in the manufacture of a medicament for the treatment of intestinal fibrosis.
- 6. The use according to claim 5, wherein the anti-aging agent comprises an intestinal epithelial tissue cell anti-aging agent.
- Use of an elmo1 detection reagent for the preparation of a kit for diagnosing intestinal fibrosis.
- 8. The use according to claim 7, wherein intestinal fibrosis comprises intestinal fibrosis in inflammatory bowel disease.
- 9. A marker for intestinal fibrosis, comprising ELMO1 at a low level of intestinal relative expression.
- 10. A therapeutic agent for inflammatory bowel disease fibrosis, comprising a FNDC5 activator, an AMPK activator, a SIRT1 activator, and/or an ELMO1 activator.
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