Fig 1.
Effects of different pretreatments on body weight (A) and clinical symptom scores (B) in mice infected with Pythium insidiosum.
Results showed that at the end of preconditioning and infection endpoint, CTX, LPS + CTX, ID + CTX, and ID + LPS groups had significantly decreased body weight and significantly increased clinical symptom scores compared with the control group, with the most pronounced changes in LPS + CTX and ID + CTX groups (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 2.
Effects of different pretreatments on venous blood inflammatory cell counts in mice infected with Pythium insidiosum.
Overall trends showed that compared with the control group, the ID + CTX group had decreased WBC (A), NEUT (B), and LYMPH (C) counts at the end of preconditioning and infection endpoint, suggesting an immunosuppressive state (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 3.
Effects of different pretreatments on BALF NEUT counts in mice infected with Pythium insidiosum.
Compared with the control group, only the ID group showed significantly increased NEUT counts, while other groups showed no statistical differences (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 4.
Effects of different pretreatments on BALF LYMPH counts in mice infected with Pythium insidiosum.
Compared with the control group, LPS, CTX, LPS + CTX, ID + CTX, and ID + LPS groups showed significantly decreased LYMPH counts, while the ID group showed no statistical difference (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 5.
Effects of different pretreatments on lung tissue pathology in mice infected with Pythium insidiosum (HE staining, 300 × , scale bar = 100 μm).
Control group (A) showed clear and intact alveolar structure, slightly septal thickening, and no infiltration in alveolar spaces. LPS group (B) showed alveolar structural disruption, septal thickening (single arrow), and inflammatory cells and erythrocytes infiltration in alveolar spaces (yellow asterisk). CTX group (C) showed alveolar structural disorder, septal destruction, and local hyaline membrane formation (double arrows) in some areas. ID group (D) showed obvious alveolar septal thickening (single arrow), and some alveolar spaces filled with inflammatory cells, erythrocytes, and protein debris (yellow asterisk). LPS + CTX group (E) showed severely disrupted alveolar structure but relatively mild alveolar septal thickening, with slightly inflammatory exudation. ID + CTX group (F) showed local alveolar structural disruption, septal thickening (single arrow), significant inflammatory cells infiltration, and a few erythrocytes and protein debris in alveolar spaces (yellow asterisk). ID + LPS group (G) showed alveolar structural disruption with mild alveolar septal thickening (single arrow), scattered inflammatory cells and erythrocytes in alveolar spaces, and hyaline membrane formation (double arrows) in some areas.
Fig 6.
Effects of different pretreatments on lung injury scores in mice infected with Pythium insidiosum.
Compared with the control group, LPS, ID, LPS + CTX, ID + CTX, and ID + LPS groups showed significantly increased lung injury scores, with relatively higher injury scores and more severe damage in LPS, ID + CTX, and ID groups (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 7.
Effects of different pretreatments on lung tissue hyphal burden in mice infected with with Pythium insidiosum (GMS staining, 300 × , scale bar = 100 μm).
Control group (A), LPS group (B), and ID group (D) showed no black hyphal structures in lung tissue sections. CTX group (C), ID + LPS group (G), and LPS + CTX group (E) showed a little slender, septate black hyphae scattered in alveolar spaces and interstitium (arrow). ID + CTX group (F) showed extensive distribution of numerous black hyphae in lung tissues (arrow) with local hyphal clumping or aggregation (asterisk).
Fig 8.
Effects of different pretreatments on lung hyphal burden scores in mice infected with Pythium insidiosum.
Control, LPS, and ID groups had no detectable hyphae. Compared with the ID + CTX group with the heaviest hyphal burden, CTX, LPS + CTX, and ID + LPS groups showed significantly decreased hyphal burden scores (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 9.
Effects of different pretreatments on serum ferritin levels in mice infected with Pythium insidiosum.
Overall trends showed that at the end of preconditioning and infection endpoint, all groups involving ID pretreatment (ID, ID + CTX, ID + LPS groups) had significantly higher serum ferritin levels compared with the control group, successfully simulating an iron overload state (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 10.
Effects of different pretreatments on serum Th1 cytokine levels in mice infected with Pythium insidiosum.
IL-2 (A) and IL-12p70 (B) showed no statistically significant differences among groups. At the end of preconditioning, compared with the control group, all treatment groups showed significantly suppressed IFN-γ (C) and TNF-α (D) levels, particularly pronounced in ID-related groups. At the infection endpoint, TNF-α showed significant recovery in CTX and ID + LPS groups, but remained suppressed in the ID + CTX group (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 11.
Effects of different pretreatments on serum Th2 cytokine levels in mice infected with Pythium insidiosum.
At the end of preconditioning, IL-4 (A), IL-5 (B), and IL-10 (C) showed no significant differences among groups; LPS, CTX, LPS + CTX, and ID + CTX groups showed significantly decreased IL-13 (D) levels compared with the control group. At the infection endpoint, all Th2 factors showed slight upward trends among groups, but the differences were not statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).