Fig 2: Schematic illustration of neuroprotection of C+P through the PKC-d/NOX/MnSOD pathway. After I/R, PKC-d translocated to the cell membrane is phosphorylated and activated. PKC-d interacted with p47phox which facilitates the activation of NOX. Moreover, MCAO also induced the elevated expression of NOX subunits gp91phox, p47phox, p67phox, and p22phox, resulting in NOX activation. NOX activation further increased ROS production and oxidative stress. Additionally, MnSOD expression is repressed after stroke. C+P suppressed both PKC-d and NOX activation and promoted MnSOD expression, resulting in the reduced ROS production and oxidative stress.

Fig 3: E2 inhibits TNF-a-induced proliferation and oxidative stress in VSMC. (A–H) VSMCs were stimulated with TNF-a for indicated doses and times. The cell viability was determined by MTS assay (A,B). *p < 0.05 and **p < 0.01 vs. untreated group, respectively. Cell counting was carried out using a Countess automated counter (C,D). *p < 0.05 and **p < 0.01 vs. untreated group, respectively. qRT-PCR detected the mRNA expression of PCNA, cyclin D1, NOX1, NOX4 and p47phox (E,F). *p < 0.05 and **p < 0.01 vs. untreated group, respectively. Western blotting detected PCNA, cyclin D1, NOX1, NOX4 and p47phox protein expression (G,H). Statistic of band intensities is shown on the right. *p < 0.05 and **p < 0.01 vs. untreated group, respectively. (I,J) VSMCs were pretreated with 25, 50 and 100 nM of E2 for 6 h and then were stimulated with TNF-a (10 ng/mL) for the indicated times. The cell viability was determined by MTS assay (I), and cell counting was carried out using a Countess automated counter (J). *p < 0.05 and **p < 0.01 vs. DMSO group, #p < 0.05 and ## p < 0.01 vs. TNF-a group. (K–O) VSMCs were pretreated with E2 (100 nM) for 6 h and then were stimulated with TNF-a (10 ng/mL) for 24 h. PCNA, cyclin D1, NOX1, NOX4 and p47phox expression was determined by Western blotting (K) and qRT-PCR (L). Statistic of band intensities is shown on the right. *p < 0.05 and **p < 0.01 vs. DMSO group, #p < 0.05 and ##p < 0.01 vs. TNF-a group. Cell proliferation was detected by EdU staining (M). Scale bar = 100 µm. Analysis of the percentage of EdU positive cells (N). **p < 0.01 vs. DMSO group, #p < 0.05 vs. TNF-a group. ROS levels were detected by DCFH-DA staining (O). Scale bar = 25 µm.

Fig 4: Downregulation of BHLHE40 promotes TNF-a-induced proliferation and oxidative stress in VSMC. (A) VSMCs were transfected with si-BHLHE40, and the expression of BHLHE40 was detected by Western blotting and qRT-PCR. **p < 0.01 vs. si-Ctrl group. (B–H) VSMCs were transfected with si-BHLHE40 and then treated or not with TNF-a for 24 h. The expression of PCNA, cyclin D1, NOX1, NOX4 and p47phox was determined by qRT-PCR (B) and Western blotting (C). Statistic of band intensities is shown on the bottom. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. si-Ctrl group. The cell viability was determined by MTS assay (D), and cell counting was carried out using a Countess automated counter (E). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. si-Ctrl group. Cell proliferation was detected by EdU staining (F). Scale bar = 100 µm. Analysis of the percentage of EdU positive cells (G). *p < 0.05, and ** p < 0.01 vs. si-Ctrl group. ROS levels were detected by DCFH-DA staining (H). Scale bar = 25 µm.

Fig 5: IL-27 enhances phosphorylation of p47phox in macrophages.IL-27 treated and untreated macrophages were stimulated with 100 ng/ml PMA for 20 min at 37 °C and then whole cell lysates were prepared in the presence of a phosphatase inhibitor and proteinase inhibitor. Western blot was performed with anti-phosphorylated p47phox, anti-p47phox, anti-gp91phox, and anti-ß-actin was used for detecting a loading control.