Fig 2: TFL inhibits the release of inflammatory factors by blocking the PD-1/PD-L1 signal pathway. (A) The concentration of IL-6 and TNF-a was determined by enzyme-linked immunosorbent assay. (B) The expression level of SHP-2, PI3K, p-AKT and AKT was detected by western blotting. *P<0.05 vs. control; **P<0.01 vs. control. (C) The expression of PD-1 in CD8+ T cells and the expression of PD-L1 in DC cells were determined by reverse transcription-quantitative PCR assay. **P<0.01 vs. pc-DNA3.1-NC. (D) The expression levels of SHP-2, PI3K, p-AKT and AKT in the co-cultural system of DC cells (wild type or PD-L1 overexpressed) and CD8+ T cells (wild type or PD-1 overexpressed) in the presence of TFL was determined by western blotting. **P<0.01 vs. control; ##P<0.01 vs. TFL. IL-6, interleukin-6; TNF-a, tumor necrosis factor-a; p-, phosphorylated-; DC, dendritic cells; NK cells; natural killer cells; NC, negative control; PD-1, programmed death 1; PD-L1, programmed cell death ligand 1; TFL, Tong-fu-li-fei.

Fig 3: FA alleviates ferroptosis-related inflammation in erastin-exposed HT22 cells. FA treatment (A) improved cell viability, (B) decreased MDA levels, and (C) increased GSH levels in erastin-exposed HT22 cells (n = 8). (D) FA treatment suppressed lipid peroxidation in erastin-exposed HT22 cells (n = 3) as determined using BODIPY 581/591 C11. Red and green fluorescence signals represent non-oxidized and oxidized states, respectively. Scale bar: 100 µm. (E) Ultrastructure of HT22 cells in each group was analyzed using TEM (n = 3). Green, pink, and red arrows indicate disrupted mitochondrial cristae, increased mitochondrial electron density, and rough endoplasmic reticulum expansion. Scale bar: 5 µm for 1000× magnification and 2 µm for 2500× magnification. (F) FA treatment upregulated the expression of p-PI3K, p-AKT, p-GSK-3ß, Nrf2, and NQO1, and downregulated the expression of p-Fyn in erastin-exposed HT22 cells (n = 3). (G) FA treatment upregulated the expression of GPX4, xCT, FTH, FTL, and FPN, and suppressed the expression of TFRC and DMT1 in erastin-exposed HT22 cells (n = 3). (H) FA treatment downregulated the expression of IL-1ß, IL-6, and TNF-a; suppressed the phosphorylation of IKK, I?B, and NF-?B; and upregulated the expression of IL-4 in erastin-exposed HT22 cells (n = 3). The data are presented as mean ± S.E.M. ###p < 0.001 vs. CTRL HT22 cells; ***p < 0.001 vs. erastin-exposed HT22 cells.

Fig 4: Effect of PHC on TNF-a, IL-6 and IL-1ß levels in ALI rat lung tissue. Compared with the sham group, the levels of TNF-a, IL-1ß and IL-6 in lung tissues were significantly increased in the T/HS, PHC1 and PHC2 groups. Compared with the T/HS group, the levels of thee cytokines were significantly decreased in the PHC1 and PHC2 groups. Data are expressed as mean ± standard error of the mean. *P<0.05 vs. the Sham group; #P<0.05 vs. the T/HS group. PHC, penehyclidine hydrochloride; TNF-a, tumor necrosis factor-a; IL, interleukin; ALI, acute lung injury; T/HS, blunt chest trauma and hemorrhagic shock.

Fig 5: Lowly expressed miR-25-3p is observed in the CVEC model treated with ox-LDL. (A) CD31 immunofluorescence staining in CVECs (Scale bar = 25 µm). (B,C) Protein levels and bands of Vcam-1 and Icam-1 in CVECs treated with ox-LDL, as determined by Western blot analysis. (D) Levels of inflammation markers (IL-1ß, IL-6, and TNF-a) in CVECs treated with ox-LDL, as detected by ELISA. (E) MiR-25-3p expression in CVECs treated with ox-LDL, as determined by RT-qPCR. (F) Levels of IL-1ß, IL-6, and TNF-a in CVECs treated with miR-25-3p, as measured by ELISA. *p < 0.05 vs. the 0 group (CVECs without treatment of ox-LDL). #p < 0.05 vs. the NC mimic group (CVECs treated with NC mimic). The measurement data are expressed as mean ± standard deviation, and data between two groups were analyzed using an independent sample t-test while comparisons among multiple groups were conducted using one-way analysis of variance. The experiment was repeated three times independently.