Fig 1: The effects of STAT3 overexpression on Th17 cell differentiation and the STAT3/RORγt pathway. Naïve CD4+ T cells were transfected with Lenti-STAT3 or Lenti-NC and then induced for Th17 differentiation and treated with 4 μM UA. (A) The protein levels of p-STAT3 and RORγt were analyzed by Western blot. (B) The mRNA levels of STAT3 and RORγt were detected by qRT-PCR. (C) The percentage of Th17 cells was evaluated by flow cytometry assay. (D) The percentage of Treg cells was evaluated by flow cytometry assay. (E) IL-17A concentration was detected by ELISA assay. Lenti-NC: Negtaive control of Lenti-STAT3. *P < 0.05, **P < 0.01.
Fig 2: Bronchoalveolar lavage fluid (BALF) expression of (a) IL-17a and (b) albumin in room air (blue) and DA-exposed rats at Day 12 (yellow) and Day 19 (red). (a) BALF IL-17a expression increased significantly in DA D19 rats compared to room air controls (Kruskal–Wallis, * p < 0.05). (b) BALF albumin increased significantly in both DA D12 and D19 compared to room air controls (Kruskal-Wallis, ** p < 0.01 and * p < 0.05).
Fig 3: The effects of UA on Th17 cell differentiation and the STAT3/RORγt pathway. The naïve CD4+ T cells were induced for Th17 differentiation and treated with UA at different concentrations (1 μM or 4 μM). (A) The proportion of Th17 cells was evaluated by flow cytometry assay. (B) The proportion of Treg cells was evaluated with flow cytometry assay. (C) The mRNA expression of RORγt was quantified with qRT-PCR. (D) The concentration of IL-17A in the cell supernatant was detected by ELISA assay. (E) The protein levels of p-STAT3 and RORγt were analyzed with Western blot. *P < 0.05, **P < 0.01.
Fig 4: Pearson correlations between oxygen saturations, bronchoalveolar lavage fluid (BALF) albumin, BALF IL17a expression, and percent (%) total lung CD4+CD25+ T cells after diacetyl inhalation exposures. (a) Oxygen saturations correlated inversely with % lung CD4+CD25+ T cells (r = −0.7320, p < 0.001). (b) BALF albumin correlated directly with percent lung CD4+CD25+ T cells. (r = 0.8942, p < 0.01). (c) Foxp3 positive T cells correlated directly with CD4+CD25+ T cells. (r = 0.796, p = 0.0005) (d) Day 19 oxygen saturations correlated inversely with BALF IL-17a concentration. (r = 0.5315; p < 0.01). (e) BALF IL-17a correlated directly with percent lung CD4+CD25+ T cells (r = 0.6828; p = 0.0001).
Fig 5: Cytokine levels in BALF and analysis of arterial blood gases. (A) IL-1β, (B) IL-6, (C) IL-17A and (D) TNF-α concentrations in BALF were determined using ELISA. (E) Arterial blood gases, including PaO2 and PaCO2 were analyzed. Data are presented as mean ± standard error of the mean from three independent experiments (n=10 per group). *P<0.05, **P<0.01 vs. Con group; #P<0.05, ##P<0.01 vs. ALI group. BALF, bronchoalveolar lavage fluid; PaO2, partial gas pressure of oxygen; PaCO2, partial gas pressure of carbon dioxide; Con, normal control group; Pue, puerarin control group; ALI, smoke inhalation group; ALI + Pue, puerarin treatment plus smoke inhalation group.
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