Fig 2: Blockage of ERK/VEGF axis can alleviate SARS-CoV-2 spike RBD-induced hyperpermeability and intestinal inflammation AELISA analysis of VEGF concentration in intestinal tissues of mice treated with ERK inhibitor or anti-VEGF antibody. SCH772984, ERK inhibitor; Bevacizumab, anti-VEGF antibody. For each group, n = 4. n, biologically independent samples (mice).BEvaluation of intestinal permeability in animals treated with SCH772984 or Bevacizumab by Evans Blue dye extravasation assay. For each group, n = 6. n, biologically independent samples (mice).C, DRepresentative H&E images (C) and the degrees of inflammation (D) of intestinal tissues from animals treated with SCH772984 or Bevacizumab. The inflammatory infiltrates were indicated by a yellow arrow. The edema area was indicated by a red star. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (mice).EModel for the SARS-CoV-2 spike RBD-mediated vascular hyperpermeability and intestinal inflammation. ? Binding of spike RBD and receptors on enterocytes; ? Activation of the Ras-Raf-MEK-ERK pathway; ? VEGF overproduction; ? VEGF-triggered phosphorylation of Y731 and internalization of VE-cadherin; ? Increase of endothelial permeability accompanied by interstitial edema and inflammation in the intestinal tissue. Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA. Source data are available online for this figure.

Fig 3: SARS-CoV-2 spike RBD promotes VEGF production via the Ras-Raf-MEK-ERK pathway AqRT–PCR analysis of levels of VEGF mRNA in HUVEC treated with Control-Fc or Spike RBD-Fc. For each group, n = 3. Data are from three independent biological experiments.BELISA analysis of VEGF concentration in the supernatant from Caco-2 cells treated with Control-Fc or Spike-Fc. For each group, n = 4. Data are from four independent biological experiments.CImmunofluorescence analysis of VEGF, cytokeratin (CK) and CD34 protein in the intestinal tissues of COVID-19 patients. CK and CD34 staining in green, VEGF staining in red, and nuclear staining in blue. Scale bars, 50 µm.DLevels of ERK and pERK in Caco-2 cells treated with Control-Fc or Spike RBD-Fc by Western blot. Protein expression was normalized to ß-actin. Data are from three independent biological experiments.EELISA analysis of VEGF concentration in the supernatant from Caco-2 cells treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984. For each group, n = 4. Data are from four independent biological experiments.F, GImmunohistochemical staining shows ERK (E) and pERK (F) expression in the duodenum of COVID-19 patients and healthy controls. Scale bars, 100 µm. For each group, n = 7. n, biologically independent samples (human specimen).HImmunohistochemical staining shows ERK and pERK expression in the intestinal tissues of mice treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (human specimen).IThe levels of Ras, c-Raf, MEK, pMEK, ERK, pERK, P90RSK, and p-P90RSK in Caco-2 cells treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 by Western blot. Protein expression was normalized to ß-actin. Data are from three independent biological experiments. Data information: All data are shown as mean ± SD. For (A), (B), (D), P values are determined by Paired Student’s t-test; (F) and (G), P values are determined by Student’s t-test; for (E), P values are determined by Mann–Whitney test; for (H) and (I), P values are determined by one-way ANOVA. Source data are available online for this figure.

Fig 4: SARS-CoV-2 spike promotes VEGF production in enterocytes via the ERK pathway qRT–PCR analysis of the transcription level of VEGF isoforms in Caco-2 cells treated with the Control-Fc or Spike RBD-Fc. Data are from three technical replicates with similar results from three biological replicates.Immunofluorescence analysis of VEGF, CK, and CD34 protein in the duodenum of hACE2-B6J mouse. CK and CD34 staining in green, VEGF staining in red and nuclear staining in blue. Scale bars, 50 µm. Representative images of two to three biological replicates.The expression of the pERK and pAKT in Caco-2 cells were detected by Western blot. Caco-2 cells were treated with Control-Fc or Spike RBD-Fc. ß-actin was used as a reference gene.The expression of the ERK and pERK in HUVECs were detected by Western blot. HUVECs were treated with Control-Fc or Spike RBD-Fc. ß-actin was used as a reference gene.qRT–PCR analysis of the transcription level of ERK pathway in Caco-2 cells treated with the Control-Fc or Spike RBD-Fc. Data are from three technical replicates with similar results from three biological replicate experiments.The levels of Ras, pERK, ERK, and p-P90RSK in NCM460 cells treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984 by Western blot. ß-actin was used as a reference gene.The levels of ACE2 and pERK in Caco-2 and HUVEC cells treated with Control-Fc or Spike RBD-Fc by Western blot. ß-actin was used as a reference gene.The levels of Ras, c-Raf, pMEK, pERK, ERK, and p-P90RSK in Caco-2 with ACE2 knockdown by Western blot. ß-actin was used as a reference gene.The levels of ACE2, pERK, and ERK in Caco-2 cells treated with Control-Fc, Spike RBD-Fc with ACE2 knockdown by Western blot. ß-actin was used as a reference gene. Data information: All data are shown as mean ± SD. For (A), P values are determined by Student’s t-test; for (E), P values are determined by one-way ANOVA.