WO2022121110A1 - Mécanisme basé sur des symptômes gastro-intestinaux provoqués par le sras-cov-2 et son application - Google Patents

Mécanisme basé sur des symptômes gastro-intestinaux provoqués par le sras-cov-2 et son application Download PDF

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WO2022121110A1
WO2022121110A1 PCT/CN2021/075675 CN2021075675W WO2022121110A1 WO 2022121110 A1 WO2022121110 A1 WO 2022121110A1 CN 2021075675 W CN2021075675 W CN 2021075675W WO 2022121110 A1 WO2022121110 A1 WO 2022121110A1
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cov
sars
vegf
gastrointestinal
mice
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Chinese (zh)
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何欢欢
单鸿
曾发敏
何建忠
邓昭华
李颍雯
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中山大学附属第五医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
    • A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals

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  • the present invention relates to the field of medical technology, and more particularly, to the mechanism and application of gastrointestinal symptoms caused by SARS-CoV-2.
  • COVID-19 is a severe acute respiratory illness caused by the SARS-CoV-2 virus that has caused a worldwide pandemic since its outbreak in late 2019.
  • SARS-CoV-2 infection the common feature is typical respiratory symptoms, and the airway is also identified as a target organ.
  • SARS-CoV-2 infection has been further studied, clinical reports of cases with gastrointestinal symptoms, which typically manifest as diarrhea, anorexia, nausea, and vomiting, continue to increase.
  • some patients infected with SARS-CoV-2 exhibited only digestive symptoms at the time of onset or even during the course of the disease, indicating that the gastrointestinal tract is highly susceptible to SARS-CoV-2.
  • the technical problem to be solved by the present invention is to overcome the defect that the mechanism of gastrointestinal symptoms caused by SARS-CoV-2 in the prior art is not clear. Gastrointestinal symptoms in patients with -19 provide an explanatory mechanism and a therapeutic strategy.
  • Angiotensin-converting enzyme maintains circulatory homeostasis by regulating angiogenesis, thrombosis, and vascular remodeling, and it has been reported that ACE2 is the major binding receptor for SARS-CoV-2.
  • ACE2 is expressed in many organs, including the digestive and vascular systems. Notably, human ACE2 is most highly expressed in the small intestine.
  • SARS-CoV-2 replicates in intestinal epithelial cells using ACE2 as an entry receptor using human intestinal organoids and a colon epithelial cancer cell line (Caco-2), respectively.
  • SARS-CoV-2 can also bind to ACE2 in other vertebrates, such as mice and rats, without being infected, raising the question of whether SARS-CoV-2 binding to ACE2 is possible in these organisms cause any signaling.
  • the present invention first provides a method of inhibiting SARS-CoV-2-induced damage to the vascular barrier in gastrointestinal tissue, comprising administering to the gastrointestinal tissue a vascular rejection targeting signal.
  • VEGF vascular permeability
  • SARS-CoV- 2 Spike The protein can regulate the phosphorylation of downstream ERK by binding to intestinal epithelial ACE2, thereby promoting the secretion of VEGF.
  • Secreted VEGF stimulated lower VE-cad expression or higher VE-cad phosphorylation in vascular endothelial cells. Increase vascular permeability.
  • ERK inhibitors can reduce the phosphorylation of pERK and the production of VEGF, thereby restoring the expression of VE-cad in vascular endothelial cells, indicating that ERK inhibitors can be used as reference drugs.
  • VEGF antagonists inhibit the binding of VEGF to VEGF receptors, thereby inhibiting the increase of vascular endothelial permeability.
  • the repulsive targeting signal includes an ERK inhibitor and/or a VEGF antagonist
  • the ERK inhibitor refers to a substance that can bind to ERK and block the biological activity of ERK
  • the VEGF antagonist refers to Substances that bind to VEGF and block the biological activity of VEGF.
  • the ERK inhibitors include clinical stage inhibitors SCH772984, GDC-0994, Ulixertinib, KO-947, LY3214996, MK-8353, CC-90003, LTT462, etc., and those in preclinical stage or biological activity evaluation stage
  • the VEGF antagonists include bevacizumab, ramucirumab, ranibizumab, aflibercept, conbercept and the like.
  • the above-mentioned ERK inhibitor is SCH772984, and the VEGF antagonist is bevacizumab.
  • the duodenum is the most sensitive to the SARS-CoV-2 Spike protein, so preferably, the above-mentioned gastrointestinal tract tissue is the duodenum.
  • the above study of the present invention also highlights the sensitivity of the duodenum to SARS-CoV-2 Spike-induced increased vascular permeability and its potential pathways, providing a potential therapeutic target for gastrointestinal symptoms in COVID-19 patients.
  • the present invention also provides a method for screening or evaluating an agent that inhibits SARS-CoV-2-induced damage to the vascular barrier in the gastrointestinal tract, specifically: determining the effect of the agent on the VEGF-mediated increase in the vascular permeability of the gastrointestinal tract. ability.
  • the present invention studies the mechanism of gastrointestinal symptoms caused by SARS-CoV-2, namely SARS-CoV-2 Spike protein directly binds to ACE2 on intestinal epithelial cells, activates the downstream ERK/VEGF signaling pathway, and induces increased vascular permeability.
  • SARS-CoV-2 Spike protein directly binds to ACE2 on intestinal epithelial cells, activates the upstream molecules of ERK Ras, Raf and MEK in intestinal epithelial cells, further activates ERK, promotes the secretion of VEGF from intestinal epithelial cells, and makes intestinal endothelial cells VE-cad Decreased expression or increased phosphorylation results in increased intestinal endothelial vascular permeability.
  • the ability to increase vascular permeability refers to determining the ability of the agent to inhibit ERK, MEK, Ras, Raf, VEGF.
  • the effect of the agent on the increase in vascular permeability of the gastrointestinal tract mediated by VEGF is determined by the following steps:
  • the endothelial cells in the candidate drug group can detect higher VE-cad expression or lower VE-cad phosphorylation level than the endothelial cells in the placebo group, indicating that the candidate drug can Inhibits VEGF-mediated increase in gastrointestinal vascular permeability.
  • the placebo described in step (2) is selected from PBS or DMEM.
  • said agent when in vivo, affects VEGF-mediated increase in gastrointestinal vascular permeability as determined by the following steps:
  • a lower expression level of VEGF can be detected in the gastrointestinal tract tissue of the animals after (3) treatment than in the gastrointestinal tract tissue of the animals after treatment (2), indicating that the agent can Inhibits VEGF-mediated increase in gastrointestinal vascular permeability.
  • the placebo described in step (2) is selected from PBS or DMEM.
  • the gastrointestinal tract tissue in step (4) is the duodenum.
  • the construction method of the SARS-CoV-2 gastrointestinal inflammation animal model in step (1) includes the following steps:
  • mice were intraperitoneally injected with SARS-CoV-2 Spike protein;
  • SARS-CoV-2 Spike protein in the present invention Before intraperitoneal injection of SARS-CoV-2 Spike protein in the present invention, acid enema treatment is performed on the epithelium of the gastrointestinal tract, and then SARS-CoV-2 Spike protein stimulation is more conducive to the binding of SARS-CoV-2 Spike protein; the animal model obtained in this way is consistent with the clinical symptoms of COVID-19 patients reported in the present, so the animal model constructed by the method of the present invention can replicate clinical symptoms Gastrointestinal manifestations in patients with COVID-19.
  • the SARS-CoV-2 described in step S3 The dosage of Spike protein was 5.5 nM/kg mouse body weight.
  • step S4 refers to 6-24 hours.
  • mice were fed normally. After 16-24 hours, the candidate drugs were injected into the mice through the abdominal cavity respectively to obtain the mice in the candidate drug group; the placebo was injected into the mice through the abdominal cavity respectively to obtain the mice in the placebo group. ;
  • mice in the candidate drug group and the placebo group were injected with SARS-CoV-2 by intraperitoneal injection.
  • Spike protein mice in the control group were intraperitoneally injected with the same amount of IgG-Fc protein;
  • mice in the control group were taken respectively.
  • the gastrointestinal tissues of the mice in the placebo group were able to detect higher
  • the gastrointestinal tissue of the candidate drug group could detect a lower VEGF expression level, indicating that the candidate drug can inhibit the VEGF-mediated gastrointestinal vascular permeability Increase.
  • the concentration of acetic acid in the above step (2) is 1-2% v/v, and the volume is 500 ⁇ L to 1000 ⁇ L.
  • the SARS-CoV-2 described in the above step (4) The dosage of Spike protein was 5.5nM/kg mouse body weight.
  • the present invention also provides a method for treating or preventing gastrointestinal symptoms caused by SARS-CoV-2, characterized by comprising the following steps:
  • the repulsive guidance signal includes an ERK inhibitor and/or a VEGF antagonist
  • the ERK inhibitor refers to a substance that can bind to ERK and block the biological activity of ERK
  • the VEGF antagonist refers to a substance that can interact with VEGF Substances that bind to and block the biological activity of VEGF.
  • the ERK inhibitors include clinical stage inhibitors SCH772984, GDC-0994, Ulixertinib, KO-947, LY3214996, MK-8353, CC-90003, LTT462, etc., which are in the preclinical stage or the biological activity evaluation stage.
  • Inhibitors FR180204, VTX-11e, BL-EI-001, etc.; the VEGF antagonists include bevacizumab, ramucirumab, ranibizumab, aflibercept, conbercept and the like.
  • the above-mentioned ERK inhibitor is SCH772984
  • the VEGF antagonist is the anti-VEGF drug bevacizumab.
  • the present invention also provides a medicament for treating or preventing or alleviating gastrointestinal symptoms caused by SARS-CoV-2, the medicament having at least one of the following functions:
  • the drugs include, but are not limited to: ERK inhibitors and/or VEGF antagonists.
  • the ERK inhibitors include clinical stage inhibitors SCH772984, GDC-0994, Ulixertinib, KO-947, LY3214996, MK-8353, CC-90003, LTT462, etc., which are in the preclinical stage or the biological activity evaluation stage.
  • Inhibitors FR180204, VTX-11e, BL-EI-001, etc.; the VEGF antagonists include bevacizumab, ramucirumab, ranibizumab, aflibercept, conbercept and the like.
  • the present invention has the following beneficial effects:
  • the present study found that the SARS-CoV-2 Spike protein can combine with human and mouse ACE2 to induce increased vascular permeability.
  • Spike protein activates the Ras-Raf-MEK-ERK pathway by binding to ACE2 of intestinal epithelial cells, and promotes the secretion of VEGF from epithelial cells, which does not exist in endothelial cells.
  • ERK or VEGF blockade rescued Spike protein-enhanced vascular permeability in vivo and alleviated gastrointestinal symptoms.
  • Figure 1a shows the co-localization of ACE2 and SARS-CoV-2 Spike protein observed in the gastrointestinal tissue of COVID-19 patients;
  • Figure 1b shows the local inflammation in H&E stained gastrointestinal tissue;
  • Figure 2a shows the construction process of the animal model
  • Figure 2b shows the HE staining images of different parts of the experimental group (Spike-Fc) mice and the control group (Control-Fc) mice
  • Figure 2c and Figure 2d show the experimental group (Spike-Fc) mice.
  • Figure 3c shows that the HUVECs of experimental group (Spike-Fc) and control group (Control-Fc) were determined by WB
  • Figure 4a shows the pull-down results of Spike-Fc protein and human ACE2 in Caco-2 cells and 239T cells, respectively, where Input represents total cell lysate, and IP:Fc represents the protein pulled down by Fc peptide;
  • Figure 4b shows Spike-Fc Fluorescent staining results of Fc protein and human ACE2 in 239T cells;
  • Figure 5a shows the detection of cohesin ZO-1, VE-cad, pVE-cad (Y658) and pVE-cad (Y731) in the supernatant of the co-culture system of Caco-2-HUVECs incubated with Spike-Fc or Control-Fc by WB.
  • Figure 5b and Figure 5c show VE-cad and IHC plot of pVE-cad(Y731), the scale bar is 100 ⁇ m, and the scatter plot shows the expression levels of VE-cad and pVE-cad(Y731) in the duodenum, jejunum, colon and rectum (data are presented as mean ⁇ SD represents, p value is by t test, ns represents no significance, * represents p ⁇ 0.05, ** represents p ⁇ 0.01); Figure 5d and Figure 5e show VE-cad in duodenum of healthy and COVID-19 patients and the detection of pVE-cad (Y731) (data are represented by mean ⁇ SD,
  • Figure 6a shows the RT-PCR analysis of VEGF transcript levels in the duodenum, jejunum, colon and rectum of experimental (Spike-Fc) and control (Control-Fc) mice (data are expressed as mean ⁇ SD, The p value was tested by t-test, * represents p ⁇ 0.05);
  • Figure 6b shows the ELISA analysis of VEGF in the duodenum, jejunum, colon and rectum of experimental (Spike-Fc) and control (Control-Fc) mice The scatter plot shows the protein concentration of VEGF in intestinal tissue (data are represented by mean ⁇ SD, p value is by t test, ns represents no significance); The amount of VEGF secreted by Cntrol-Fc-treated Caco-2 cells;
  • Figure 6d shows the amount of VEGF in plasma of mice treated with Spike-Fc or Control-Fc was analyzed by ELISA; scatter plot shows the protein of VEGF in plasma Concentration (data are represented by mean ⁇ SD,
  • Figure 7 shows immunoblot analysis of ERK or pERK in Caco-2 cells treated with Spike-Fc or Control-Fc; histograms show quantification of protein expression relative to the amount of ⁇ -actin by densitometric scanning. Data are represented by mean ⁇ SD, p value is by paired t test, * represents p ⁇ 0.05;
  • Figure 8a shows the detection of Caco-2 siRNA (with three siRNA knockdown sequences 01, 02 and 03) by western blot. Compared with the control sequence (NC), all three sequences showed obvious knockdown effect, and the endogenous ACE2 After knockdown, the expressions of Ras, C-Raf, pMEK, pERK and p-P90RSK were significantly increased;
  • Figure 8b shows that after stimulating HUVCE cells with Control-Fc or Spike-Fc protein for 1 h, the expression of ERK and pERK proteins was detected by western blotting happening;
  • Figure 9a shows the expression levels of each protein in the Ras-Raf-MEK-ERK signaling pathway after Spike-Fc, Control-Fc and SCH772984 treatment of Caco-2 cells, the histogram shows the protein expression relative to ⁇ -actin was quantified by protein grayscale The experimental data were repeated three times, the data were represented by the mean ⁇ SD, the p value was tested by paired t test, * represents p ⁇ 0.05, ns represents no significance;
  • Figure 9b shows that Spike-Fc, Control-Fc and SCH772984 were compared with Caco After the -2 cells were co-cultured, the amount of VEGF was determined by ELISA, the data were expressed as mean ⁇ SD, the p value was expressed by t test, * represents p ⁇ 0.05;
  • Figure 9c shows the experimental group (Spike-Fc) and the control group (Control-Fc) Fc) IHC map of ERK and pERK in intestinal tissues (duodenum, jejun
  • Figure 10a shows the expression and localization of VE-cad in Caco-2 cells treated with Spike-Fc or Control-Fc or SCH772984 or Bevacizumab
  • Figure 10c shows the experimental group (Spike-Fc) and the control group (Control-Fc) intraperitoneal injection of SCH772984 or Bevacizumab model mice intestinal tissue (12 IHC plots of VE-cad and pVE-cad (Y731) in the denum, jejunum, ileum, colon and
  • Figure 11a shows the use of ELISA to analyze the expression of VEGF in the intestinal tissues (duodenum, jejunum, colon and rectum) of model mice injected with SCH772984 or Bevacizumab in the experimental group (Spike-Fc) and the control group (Control-Fc) by intraperitoneal injection.
  • FIG. 11b shows the experimental group (Spike-Fc) and the control group (Control-Fc) ) or immunoblot analysis of ERK or pERK in intestinal tissues (duodenum, jejunum, colon, and rectum) of model mice injected with SCH772984 or Bevacizumab intraperitoneally;
  • the amount of ⁇ -actin, the experimental data was repeated three times, the data were expressed as mean ⁇ SD, and the p value was expressed by paired t test, *represents p ⁇ 0.05, **represents p ⁇ 0.01, and ***represents p ⁇ 0.0001;
  • Figure 12b shows the evens permeated per unit weight (g) of intestinal tissue (duodenum, jejunum, colon and rectum) The OD value of blue dye;
  • Figure 12c shows the experimental group (Spike-Fc) and the control group (Control-Fc) or the intestinal tissue (duodenum, jejunum, colon and rectum) of model mice injected with SCH772984 or Bevacizumab intraperitoneally ) of H&E staining; yellow arrows represent inflammatory infiltrates; red stars represent edema, and no inflammation, moderate inflammation, and severe inflammation were assessed in the intestinal tissue; his
  • Figure 13 shows the model mechanism of SARS-CoV-2 Spike protein-mediated vascular hyperpermeability and intestinal tissue inflammation.
  • the clinical data of 17 patients are shown in Supplementary Table 1. Ethically approved by the Ethics Committee of the Fifth affiliated Hospital of Sun Yat-Sen University (No. 2020L029-1), all patients signed informed consent.
  • Human umbilical vein endothelial cells were purchased from ScienCell (Cat. No. 8000, Cat. No. 5000) in ECM medium (ScienCell, Cat. No. 5000) supplemented with 10% fetal bovine serum. 1001) in culture.
  • Human colorectal adenocarcinoma cells (Caco-2) were purchased from (Guangzhou IGE Biotechnology Company, Guangzhou) and cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum, 50 U /mL penicillin and 50 mg/mL streptomycin (Gibco, catalog number 15140-122).
  • a murine endothelial cell line (C166) was obtained from the American Biological Resource Center (ATCC, Manassas, USA), supplemented with 10% fetal bovine serum, 50 U/mL penicillin, and 50 mg of Dulbecco's modified Eagle's medium ( Gibco). Cells were cultured in a humidified 37°C incubator with 5% CO . The following studies were approved by the Fifth affiliated Hospital of Sun Yat-Sen University. For all animal experiments, the permission of the Experimental Animal Ethics Committee of the Fifth affiliated Hospital of Sun Yat-sen University was obtained. Statistical analysis: Statistical analysis was performed using SPSS v13.0 software.
  • SARS-CoV-2 Spike Protein preferentially induces duodenal interstitial edema
  • Tissue sections Clinical specimens from COVID-19 patients were formalin-fixed, paraffin-embedded and sectioned (4 ⁇ m).
  • Sections need to be placed in 10% goat serum prepared in PBST, incubated at room temperature for 1 hour, and then added with primary antibodies (anti-ACE2, Santa Cruz, sc390851, 1:100; anti-SARS-Cov-2 Spike, Sino Biological, 40150-R007, 1:500), the sections were incubated at 4°C overnight. The next day, wash 3 times with PBST and add fluorescent secondary antibodies (AlexaFluor® 647-conjugated goat anti-rabbit IgG, bs-0296G-AF647, Bioss, 1:100; Dylight-550 goat anti-rabbit IgG secondary antibody BA1135, 1:200 ) at room temperature for 1 h.
  • primary antibodies anti-ACE2, Santa Cruz, sc390851, 1:100; anti-SARS-Cov-2 Spike, Sino Biological, 40150-R007, 1:500
  • RESULTS Using double immunofluorescence staining, co-localization of ACE2 and SARS-CoV-2 Spike protein was observed in the duodenum in gastrointestinal tissues obtained by endoscopy from patients with COVID-19, whereas in the duodenum Spike proteins were detectable in the denum (Fig. 1a). H&E staining further showed marked edema of the mucosal lamina limba interstitium, local inflammation with plasma cell and lymphocyte infiltration (Fig. 1b).
  • interstitial edema was significantly associated with disease type, acid reflux, total bilirubin, glutamate-pyruvate aminotransferase (ALT) and aspartate aminotransferase (AST) (Table 2), suggesting progression of the disease prediction features.
  • mice 8-9 weeks old C57BL/6J mice were randomly divided into two groups (experimental group and control group); (2) all mice were fasted for 24 hours; (3) after fasting for 24 hours, the abdominal cavity of each mouse was Inject 100 ⁇ L of 1% pentobarbital for anesthesia; (4) After 5 minutes of anesthesia, apply petroleum jelly on the surface of the hose, and then gently insert it from the anus to a depth of 4 cm; (5) Connect a syringe to one end of the hose and inject 500 ⁇ L of 1% acetic acid, and the control group was injected with an equal volume of PBS; (6) the mice were inverted for 1 min, and then lavaged with 500 ⁇ L of PBS twice to wash off the injected acetic acid; (7) The mice were put back into the cage and supplemented with food; (8) 16-24 hours later, the S protein (SARS-CoV-2 Spike protein) 5 ⁇ g/mice (or 5.5 nmol/kg) was intraperitoneally injected, and the control
  • SARS-CoV-2 gastrointestinal inflammation model mice and control groups (Mouse IgG1-Fc protein) was injected into the tail vein of TRITC-dextran, and after half an hour, the peritoneal cavity was washed with 2.5 mL of PBS; after taking the ascites, the ascites was centrifuged at 1500 rpm for 10 min, and the excitation and emission wavelengths were 540 nm and 590 using a microplate reader, respectively. Fluorescence was measured at nm; duodenal, jejunum, colon and rectal tissues were taken after the mice were sacrificed, embedded and dehydrated, and then sliced and stained with H&E.
  • mice were intravenously injected with TRITC-dextran, followed by quantitative detection of dextran leaking into the abdominal cavity. Greater leakage was observed in mice treated with Spike-Fc compared to controls (Control-Fc) (Fig. 3a), suggesting that Spike proteins can cause impairment of the intestinal vascular barrier.
  • ACE2 has been reported to be expressed in various cell types including endothelial cells, so here we first investigated whether Spike proteins could mediate permeability by directly affecting the endothelium.
  • HAVEC human umbilical vein endothelial cells
  • Spike protein did not significantly affect the permeability of HUVEC cells (Fig. 3b).
  • ACE2 is the main binding receptor of SARS-CoV-2.
  • ACE2 is expressed in a variety of organs, including the digestive and vascular systems.
  • Caco-2 colon epithelial cancer cell line
  • SARS-CoV-2 replicates in intestinal epithelial cells using ACE2 as an entry receptor
  • the in vivo permeability experiments described above give Therefore, it is reasonable to speculate that Spike protein induces endothelial permeability by affecting intestinal epithelial cells.
  • HUVEC cells were incubated with conditioned medium of Spike-Fc-treated intestinal epithelial cells Caco-2, as determined by permeability, using Spike-Fc (0.25 mg/mL) for Caco-2 (5 ⁇ 10 5 ), the control group was treated with IgG-Fc (0.25mg/mL) for 24h, and then the cultured Caco-2 cell supernatant was filtered through a 0.22 ⁇ m filter.
  • HUVEC cells (1 x 105 ) were plated in a monolayer in the upper chamber of a 24-well plate overnight.
  • the wells were pipetted into the same 24-well plate, medium with 1% FBS was added until the HUVECs reached confluency for 6 h, and then the filtered Caco-2 supernatant was replaced to the bottom of the Transwell chamber and co-cultured with HUVEC for 12 h.
  • the upper chamber medium was removed, and tetramethylrhodamine isothiocyanate-Dextran (T1162, Sigma) (2 mg/mL) was added to the upper wells. After 3 hours, the Dextran-added cells were collected. culture medium, and fluorescence was measured at excitation and emission wavelengths of 540 nm and 590 nm, respectively, using a microplate reader.
  • Recombinant SARS-CoV-2 Spike protein (RBD, Fc tag) was purchased from Sino Bioloical.
  • Caco-2 cells and cells were treated with Spike-Fc (0.25 mg/mL) or control, respectively IgG-Fc (0.25 mg/mL) was incubated at 37 °C for 1 h, and then the protein in the lysate was pulled down with Protein G Sepharose for immunoblotting.
  • Cell lysates were analyzed by western blot and quantitative PCR, and cell culture supernatants (24 h) were filtered through 0.22 ⁇ m filters (MILLEX GP) and stored at -80°C for Elisa and co-culture experiments.
  • ERK, pERK, VE-cad and pVE-cad(Y731) proteins were assessed for tissue samples.
  • HE staining was also performed on human/mouse tissue sections to assess tissue morphological characteristics and distribution of target proteins.
  • Tight junctions and adherent junctions are the basic components of the intestinal vascular barrier. Changes in tight junction proteins such as ZO-1 were first analyzed, but no significant changes were found. However, the expression of a key adhesion protein, VE-cadherin (VE-cad), was reduced, accompanied by an increase in its phosphorylation at Tyr731 but not at 658 (pVE-cad) (Fig. 5a). Then, we measured the expression and phosphorylation levels of VE-cad in vivo. Different parts of the gastrointestinal tract of mice treated with Spike protein were analyzed by western blot and immunohistochemistry (IHC). The results showed a persistent decrease in VE-cad in the duodenum (Fig.
  • SARS-CoV-2 Spike protein through ERK/VEGF pathway mediates vascular permeability
  • RNA from tissues was isolated using TRIzol by conventional RNA extraction protocols.
  • Total RNA from cells was isolated with Total RNA Kit I.
  • the isolated RNA was reverse transcribed into cDNA (Vazyme, Nanjing, China).
  • qRT-PCR was performed using a real-time PCR system (Bio-Rad, America) and ChamQ Universal SYBR qPCR master mix (Vazyme, Nanjing, China). Primers were designed and synthesized by Guangzhou IGE Biotechnology Company. GAPDH was used as an internal control and all reactions were repeated three times. Relative RNA expression was calculated using the 2 - ⁇ Ct method.
  • VEGF concentration of VEGF produced from cells, tissues and serum was detected by ELISA.
  • Quantikine ELISA human VEGF immunoassay catalog. No. DVE00, R&D
  • Determination of VEGF concentration for mouse samples, use mouse VEGF according to the manufacturer's instructions Simplestep ELISA kit (cat. No. ab209882, Abcam) to measure VEGF levels in tissue homogenates (prepared in cold PBS using an electric homogenizer) and serum.
  • VEGF known as a potent vascular permeability factor
  • SARS-CoV-2 infection significantly increases VEGF expression in human lung epithelial cells.
  • enterocytes which may be responsible for gastrointestinal symptoms in COVID-19 patients. The above experimental data verified our expectation.
  • Caco-2 cells were attached to a six-well plate; (2) Lipofectamine was used LTX (Invitrogen, ThermoFisher Scientific Corporation) transfection reagent, and the negative control siRNA and ACE2 siRNA were transfected into Caco-2 cells; (3) After 48h, use RIPA buffer containing protease/phosphatase inhibitor mixture to lyse Caco-2, and extract the protein; (4) Western blot was used to detect the knockdown efficiency and the expression of related proteins.
  • LTX Invitrogen, ThermoFisher Scientific Corporation
  • Caco-2 was transfected with negative control siRNA and ACE2 siRNA for 48 hours using Lipofectamine LTX (Invitrogen, ThermoFisher Scientific), then Caco-2 was lysed using RIPA buffer containing a protease/phosphatase inhibitor cocktail in preparation for a western blot.
  • Caco-2 (5 ⁇ 10 5 ) was treated with Spike-Fc (0.25mg/mL), control group was treated with IgG-Fc (0.25mg/mL), Spike-Fc with SCH772984 (1uM), bevacizumab Bevacizumab (25ug/ml) cells were treated for 24h, and then the cultured Caco-2 cell supernatant was filtered through a 0.22 ⁇ m filter. A monolayer of HUVEC ( 1 x 105) was plated in the upper chamber of a 24-well plate overnight.
  • the wells were pipetted into the same 24-well plate, medium with 1% FBS was added until the HUVEC cells reached confluency for 6 h, and then the filtered Caco-2 cell supernatant was added. The solution was changed to the bottom of the Transwell chamber and co-cultured with HUVEC cells for 12 h.
  • the upper chamber medium was removed, and tetramethylrhodamine isothiocyanate-Dextran (T1162, Sigma) (2 mg/mL) was added to the upper well. After 3 hours, the collection was added with Dextran of medium, and measured fluorescence using a microplate reader at excitation and emission wavelengths of 540 nm and 590 nm, respectively.
  • mice 8-9 weeks old C57BL/6J mice were randomly divided into six groups: control group, placebo group I, placebo group II, Spike protein group, SCH772984 group and bevacizumab group; (2) All mice were fasted for 24 hours; (3) After fasting for 24 hours, each mouse was anesthetized by intraperitoneal injection of 100 ⁇ L of 1% pentobarbital; (4) After 5 minutes of anesthesia, the surface of the hose was smeared with Vaseline, and then injected from the anus.
  • the insertion depth is 4cm; (5) Connect a syringe to one end of the hose, inject 500 ⁇ L of 1% acetic acid, and the control group is injected with an equal volume of PBS; (6) Invert the mouse for 1 min, and then use 500 ⁇ L of PBS for Lavage, lavage twice, in order to wash off the injected acetic acid; (7) Put the mice back in the cage and supplement with food; (8) After 16-24 hours, the mice in the SCH772984 group were injected intraperitoneally at a dose of 50 mg/kg, The bevacizumab group was injected intraperitoneally at a dose of 5 mg/kg, the placebo group I mice were intraperitoneally injected with the solvent of SCH772984, the placebo group II mice were intraperitoneally injected with the bevacizumab solvent, and the control group did not.
  • the SCH772984 group, the bevacizumab group, the placebo group I, and the placebo group II were given intraperitoneal injection of S protein (SARS-CoV-2 Spike protein) 5 ⁇ g / animal (or 5.5 ⁇ g) respectively.
  • the control group was injected with the same type control Mouse IgG1-Fc protein; (10) 1 ⁇ 2h, the mice in the SCH772984 group were again injected intraperitoneally at a dose of 50 mg/kg, the bevacizumab group was injected at a dose of 5 mg/kg, and the placebo group I was injected with SCH772984 (11) TRITC-dextran was injected into the tail vein after 4-6 hours, and 2.5ml of PBS was washed in the abdominal cavity after half an hour; (12) after taking the ascites, the ascites was rotated at 1500rpm.
  • mice were sacrificed, and the duodenum, colon, jejunum, ileum, and rectum were taken, and some tissues were embedded, dehydrated, and sliced. After HE staining; and immunofluorescence staining experiments were performed to investigate the co-localization of ACE2 and SARS-CoV-2. The content of VEGF in some fresh tissues was detected by Elisa kit.
  • HUVECs were seeded into 15 mm glass bottom cell culture dishes (2.5 ⁇ 10 5 ) and co-cultured with the supernatant of Caco-2 (treated with Spike-Fc and IgG-Fc, respectively) for 24 h. It was then fixed with 4% paraformaldehyde. Samples were stained sequentially with the following antibodies or fluorescent dyes: VE-Cadherin (Cat.No.44-1145G, ThermoFisher, 1:1,000), Dylight-488 Goat Anti-mouse IgG secondary antibody (Cat.No. BA1126, BOSTER, 1:200), Antifade Mounting Medium with DAPI (Cat. No. Ab104139, Abcam). The results were acquired with a confocal microscope, and the sample pictures were taken by a Zeiss 880 with a 60x objective lens, and image processing was performed using ImageJ software.
  • VE-Cadherin Cat.No.44-1145G, ThermoFisher, 1:1,000
  • mice were fasted for 24 hours in advance; (2) 100ul of 1% pentobarbital was injected intraperitoneally into the mice; (3) After the mice were anesthetized for about 5 minutes, apply Vaseline to the surface of the tube, and the tube was removed from the anus.
  • mice in the SCH772984 group were injected intraperitoneally according to the dosage of SCH772984: 50 mg/kg, and the mice in the bevacizumab group were intraperitoneally injected with the dosage of 5 mg/kg , the mice in the placebo group I were intraperitoneally injected with the solvent of SCH772984, and the mice in the placebo group II were injected with the solvent of bevacizumab, and the control group was not treated; (8) SCH772984 group, bevacizumab group and Placebo group I and placebo group II were intraperitoneally injected with S protein (SARS)

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Abstract

La présente invention se rapporte au domaine de la technologie médicale. Des études mécanistes ont découvert que la protéine de spicule du SARS-CoV active une voie Ras-Raf-MEK-ERK par liaison aux récepteurs ACE2 des cellules épithéliales intestinales, ce qui favorise la sécrétion de VEGF par les cellules épithéliales intestinales. Cependant, la voie n'est pas altérée dans les cellules endothéliales. Un inhibiteur d'ERK ou de VEGF peut remédier à la perméabilité vasculaire amplifiée par la protéine de spicule et atténuer les symptômes gastro-intestinaux chez les souris. Les résultats indiquent que dans le tractus gastro-intestinal, notamment dans le duodénum dans lequel ACE2 est fortement exprimée, la protéine de spicule du SARS-CoV-2 peut se lier directement à ACE2 dans les cellules épithéliales intestinales, activer un trajet de signal ERK/VEGF en aval et induire une augmentation de la perméabilité vasculaire, ce qui conduit à des symptômes gastro-intestinaux liés à l'inflammation. La présente étude fournit un mécanisme explicatif et une stratégie de traitement pour des symptômes gastro-intestinaux chez des patients atteints de la COVID-19.
PCT/CN2021/075675 2020-12-11 2021-02-06 Mécanisme basé sur des symptômes gastro-intestinaux provoqués par le sras-cov-2 et son application WO2022121110A1 (fr)

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