Colchicine prevents oxidative stress-induced endothelial cell senescence via blocking NF-κB and MAPKs: implications in vascular diseases

Abstract

Background: Smoking, alcohol abuse, and hypertension are - among others, potential risk factors for cardiovascular diseases. These risk factors generate oxidative stress and cause oxidative stress-induced DNA damage, resulting in cellular senescence and senescence-associated secretory phenotype (SASP). The SASP factors in feed-forward response exacerbate inflammation and cause tissue remodeling, resulting in atherosclerotic plaque formation and rupture.

Results: Colchicine inhibited ROS generation and mitigated oxidative stress-induced DNA damage. It dampened oxidative stress-induced endothelial cell senescence and improved the expression of DNA repair protein KU80 and aging marker Lamin B1. The drug attenuated the expression of senescence marker P21 at mRNA and protein levels. The pathway analysis showed that colchicine inhibited NF-κB and MAPKs pathways and subdued mTOR activation. Colchicine also attenuated mRNA expression of interleukin (IL)-1β, IL-6, IL-8, MCP-1, ICAM-1, and E-selectin. Furthermore, colchicine reduced the mRNA and protein expression of matrix metalloproteinase (MMP-2).

Conclusion: In summary, colchicine blocked oxidative stress-induced senescence and SASP by inhibiting the activation of NF-κB and MAPKs pathways.

Keywords: Cellular senescence; Colchicine; Endothelial cells; Inflammation; MAPKs; NFκ-B; Oxidative stress; mTOR.

Fig. 1

Colchicine mitigates oxidative stress and oxidative stress-induced senescence in endothelial cells. (A) Immunofluorescence staining for oxidative stress-induced DNA damage marker 8-OHDG, (B) β-gal staining. Quantification of (C) ROS generation, (D) 8-OHDG positive cells and (E) β-gal staining. (F) Western blot showing protein expression of senescence markers. Relative protein expression of (G) KU70, (H) KU80, (I) Lamin B1, (J) P21 and relative mRNA expression of (K) P21. β-actin was used as a loading control. The experiment was performed with biological triplicates. The data was analyzed by applying One-way ANOVA followed by Tukey’s test. Error bars represent SD, (****<0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05), scale bar = 100 μm

Fig. 2

Colchicine inhibits NF-κB activation: (A) Western blot showing protein expression of NF-κB subunits. Relative protein expression of (B) P65 and (C) p-P65. (D) Ratio of p-P65/P65. β-actin was used as a loading control. The experiment was performed with biological triplicates. The data was analyzed by applying One-way ANOVA followed by Tukey’s test. Error bars represent the SD (*** p < 0.001, ** p < 0.01, * p < 0.05)

Fig. 3

Colchicine inhibits oxidative stress-induced P38 and ERK activation: (A) Western blot showing protein expression of MAPKs. Relative protein expression of (B) p-P38, (C) p-ERK and (D) p-JNK. β-actin was used as a loading control. The experiment was performed with biological triplicates. The data was analyzed by applying One-way ANOVA followed by Tukey’s test. Error bars represent the SD, ****p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05

Fig. 4

Colchicine modulates mTOR pathway activation: (A). Western blot showing protein expression of p-AKT, p-mTOR, p-4EBP1, and P-S6. Relative protein expression of (B) p-AKT, (C) p-mTOR, (D) p-S6 and (E) p-4EBP1. β-actin was used as a loading control. The experiment was performed with biological triplicates. The data was analyzed by applying One-way ANOVA followed by Tukey’s test. Error bars represent the SD, (*** p < 0.001, ** p < 0.01, and * p < 0.05)

Fig. 5

Colchicine mitigates the relative mRNA expression of SASP factors. Relative mRNA expression of (A) IL-1β, (B) IL-6, (C) IL-8, (D) MCP-1, (E) ICAM-1, (F) VCAM-1 (G) E-Selectin, (H) MMP-1, (I) MMP-2 (J) MMP-8, (K) MMP-11, (L) TIMP-1 and (M) TIMP-2. (N) Western blot showing protein expression of MMP-2. (O) Relative protein expression of MMP-2. β-actin was used as a loading control. qPCR data are the mean of three independent technical replicates and WB data are the mean of the biological triplicates. The data was analyzed by applying One-way ANOVA followed by Tukey’s test. Error bars represent the SD, *** p < 0.001, ** p < 0.01, and * p < 0.05)