Fig 1: Effect of metformin on bevacizumab-induced apoptosis, vascular endothelial injury markers, and inflammation in HUVECs. Image of flow cytometry of apoptosis (A), cell apoptosis rate (B), CD62E level (C), ET-1 level (D), TM level (E), vWF level (F), TNF-a level (G), and IL-6 level (H) in the control, BEV, MET and MET + BEV groups, respectively. *P<0.05; **P<0.01; ***P<0.001; NS, not significant. HUVECs, human umbilical vein endothelial cells, CD62E, selectin E; ET-1, endothelin-1; TM, thrombomodulin; vWF, von Willebrand factor; TNF-a, tumor necrosis factor-alpha; IL-6, interleukin-6; BEV, bevacizumab; MET, metformin.
Fig 2: IL6 release after PolyIC–WD–TNF in (A) MRC-5 cells and (B) HF-19 cells and the effect of TLR3 and NF-?B inhibitors on its release. Preexposure to Poly (I:C) significantly increased TNFa-induced IL6 release in both MRC-5 (P < 0.005) and HF-19 (P < 0.005) cells. Both inhibitors significantly decreased IL6 release in MRC-5 cells (P < 0.01 and P < 0.0001 for TLR3i and Bay-11-7082, respectively) and HF-19 cells (P < 0.01 and P < 0.05 for TLR3i and Bay-11-7082, respectively). N = 6. Values with * were defined significant when compared to (-)-WD-(-) while *above bar were significant between two variables.
Fig 3: Quercetin inhibits the expression level of XIST and the production of inflammatory cytokines in FLSs induced by TNF-a. (A) Under TNF-a stimulation, the concentrations of inflammatory cytokines (IL-1ß, IL-6 and IL-8) were increased in RAFLSs, which was suppressed by quercetin. (B) Under TNF-a stimulation, the expression level of XIST was significantly upregulated in RAFLSs, which was suppressed by quercetin. *P<0.05 vs. NC group. XIST, X-inactive specific transcript; RAFLSs, rheumatoid arthritis fibroblast-like synoviocytes; NC, negative control.
Fig 4: Gene expressions after PolyIC–WD–TNF at different concentrations of Poly (I:C) and TNFa. MRC-5 cells were initially treated with Poly (I:C) for 24 h and were then washed and rested for 24 h before being secondarily treated with TNFa for another 24 h. (A–E) Effect of different concentrations of Poly (I:C) (0, 1, 10 µg/ml) and TNFa (0, 1, 5, 10 ng/ml) on gene expressions of IL1B, IL6, IL8, MMP8, and MMP9. Secondary treatment to TNFa dose-dependently induced these gene expressions. Preexposure to Poly (I:C) did not affect TNFa-induced IL1B, IL8, MMP8, and MMP9 gene expressions [PolyIC (0 µg/ml)–WD–TNF (10 ng/ml) vs. PolyIC (10 µg/ml)–WD–TNF (10 ng/ml)]. (F) Preexposure to Poly (I:C) (10 µg/ml) and secondary treatment of MRC-5 cells with TNFa (10 ng/mL) did not affect the fibrosis-related genes; ACTA2, COL1A1, POSTN, and TGFB1. N = 4. Values with * were defined significant when compared to (-)-WD-(-) while *above bar were significant between two variables.
Fig 5: Senescent cells induce cell senescence and SASP in adjacent cells. (A) SA-ß-gal staining in HOK and skin fibroblasts at 7 days after radiation (n = 3). (B) mRNA expression levels for p16, p21, MCP1, and IL-6 in HOK and skin fibroblasts at 7 days after radiation (n = 3). (C) Protein expression levels for IL-1a, IL-8, IL-6, IL-1ß, and TNF-a in HOK and skin fibroblast cell supernatant (n = 3). (D) HOK and skin fibroblasts were cultured in Con-CM and SASP-CM for 7 days and assessed by SA-ß-gal staining (n = 3). (E) mRNA expression levels of p16, p21, PAI-1, and SASP genes (IL-1a, IL-10, IL-1ß, TNF-a, IL-6, MMP3, IL-8, MMP12, and MCP1) in HOK and skin fibroblasts (cultured in Con-CM and SASP-CM for 7 days). (F) p-JAK1 and p-JAK2 expression levels in HOK and fibroblasts after radiation. Three independent experiments started with cell plating. (G) Irradiation-induced senescent HOK and fibroblast were treated with JAK inhibitor and vehicle for 72 h. Then RNA was collected, and qRT-PCR was performed (n = 3). (H) Young HOK and skin fibroblasts were treated with SASP-CM and (SASP+JAKi)-CM for 24 h, respectively; the mRNA levels of SASP were analyzed. (I) mRNA levels of SASP in young HOK and skin fibroblasts, which were treated with SASP-CM, followed by addition of JAK inhibitor 1 or vehicle for 24 h. (E) IR+vehicle group compared with IR+JAKi group. (E,G–I) Mean with SD. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001; Student's t-test. (A,D) Scale bar, 100 µm.
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