Fig 1.
Pre-treatment with Peruvioses A and B from P. peruviana ameliorates acute TNBS-colitis.
(A) Wistar rats were treated for 2 days with test compounds (5, 10, and 20 mg/Kg/day, ip) or vehicle. Colitis was induced by the instillation of TNBS. Three days after that, animals were sacrificed, and colitis severity was assessed. (B) Animals weight change was monitored daily. (C) Appearance, damaged area (cm2), and colon weight/length ratio were evaluated as detailed in Materials and Methods. Representative pictures of rat colons are shown. (D) Histologic changes were examined by hematoxylin and eosin (H&E) staining and scored by a blinded pathologist. Representative pictures are shown (10X). (E) Myeloperoxidase (MPO) activity was measured in colon biopsies. Results represent at least two independent experiments and are expressed as the mean ± SEM. (n = 11–20 per group). (****) P<0.0001 vs. control; (+) P<0.05 and (++) P<0.01 vs. TNBS group.
Fig 2.
Peruvioses A and B from P. peruviana diminished the inflammation induced by TNBS.
(A) Colitis was induced in Wistar rats by instillation of TNBS. Animals were treated with the Peruvioses mixture (5 and 10 mg/Kg/day, ip) or vehicle for 15 days. Afterward, rats were sacrificed, and colitis severity was assessed. (B) Survival and (C) body weight changes were monitored daily. (D) Appearance of colon tissue was evaluated, and representative pictures are shown. (E) Macroscopic damage (damaged area (cm2), ulcer index, and colon weight/length) were scored as detailed in Materials and Methods. (F) Myeloperoxidase (MPO) activity was measured in colon biopsies. (G) Histological changes were examined after (1) hematoxylin and eosin (H&E) and (2) Periodic Acid Schiff (PAS) staining. Slides were examined by a blinded pathologist. Representative pictures are shown, magnification 10X (H&E) and 20X (PAS), respectively. Results represent at least two independent experiments and are expressed as the mean ± SEM (n = 10–24 per group). (*) P<0.05, (**) P<0.01, (***) P<0.001, and (****) P<0.0001 vs. control; (+) P<0.05, (++) P<0.01, and (+++) P<0.001 vs. TNBS group.
Fig 3.
Peruvioses A and B pre-treatment reduce the expression of pro-inflammatory enzymes (iNOS, COX-2) and cytokines (TNF-α, IL-10) in acute TNBS-colitis.
Wistar rats were treated with the Peruvioses mixture (5, 10, and 20 mg/Kg/day, ip) or vehicle for 2 days. Afterward, colitis was induced, as described in Materials and Methods. (A) iNOS, COX-2, MUC-2, IL-1β, IL-6, and IL-10 mRNA expression was quantified by RT-PCR. GAPDH was used as a housekeeping gene for normalization. (B) IL-1β, IL-6, IL-10, TNF-α, and IL-4 protein levels were measured by ELISA. Results represent at least two independent experiments and are expressed as the mean ± SEM (n = 9–15 per group). (*) P<0.05, (**) P<0.01, (***) P<0.001, and (****) P<0.0001 vs. control; (+) P<0.05, (++) P<0.01, (+++) P<0.001, and (++++) P<0.0001 vs. TNBS group.
Fig 4.
Treatment with Peruvioses A and B modulates the expression of pro-inflammatory enzymes, cytokines, and NF-κB expression and restores markers of mucus integrity hampered in rats with established TNBS-induced colitis.
Wistar rats instilled with TNBS were treated with the Peruvioses mixture (5 and 10 mg/Kg/day, ip) or vehicle for two weeks, as described in Materials and Methods. (A) Pro-inflammatory enzymes (iNOS, COX-2), (B) Cytokines (IL-1β, IL-6, IL-10, and IL-17A), (C) Mucus integrity markers (MUC-2, MUC-3, and TFF-3), (D) and the transcription factor NF-κB mRNA expression was quantified by RT-PCR. GAPDH was used as a housekeeping gene for normalization. Results represent at least two independent experiments and are expressed as the mean ± SEM (n = 7–10 per group). (*) P<0.05, (**) P<0.01, (***) P<0.001, and (****) P<0.0001 vs. control; (+) P<0.05, (++) P<0.01, (+++) P<0.001, and (++++) P<0.0001 vs. TNBS group.
Fig 5.
Peruvioses A and B reduce NO and cytokines production probably by inhibition of NF-κB pathway.
Wistar rats instilled with TNBS were treated with the Peruvioses mixture (5 and 10 mg/Kg/day, ip) or vehicle for two weeks, as described in Materials and Methods. (A) Cytokines (IL-1β, IL-6, IL-10, TNF-α, IFN-γ, and IL-4) levels were measured by ELISA. (B) NO production was quantified employing the Griess reaction. (C) Expression of iNOS (cytoplasmic) and NF-κB (nuclear) were evaluated by immunoblotting. Results represent at least two independent experiments and are expressed as the mean ± SEM (n = 7–10 per group). (*) P<0.05, (**) P<0.01, and (****) P<0.0001 vs. control; (+) P<0.05, (+++) P<0.001, and (++++) P<0.0001 vs. TNBS group.
Fig 6.
Peruvioses A and B inhibited pro-inflammatory mediators production by LPS-stimulated RAW 264.7 cells.
Macrophages were cultured with the Peruvioses mixture (0–100 μg/mL) for 30 minutes and then stimulated with LPS (10 μg/mL) for 24 hours. (A) Cell viability was measured with the MTT assay, (B) Nitrite production was measured by the Griess assay, and (C) PGE2, IL-6, TNF-α, and MCP-1 production were measured by ELISA. Results represent at least three independent experiments and are expressed as the mean ± SEM (*) P<0.05, (**) P<0.01, (***) P<0.001, and (****) P<0.0001 vs. control.