Colchicine Attenuates Microvascular Obstruction after Myocardial Ischemia-Reperfusion Injury by Inhibiting the Proliferation of Neutrophil in Bone Marrow

Abstract

Purpose: Complete and rapid recanalization of blood flow by percutaneous coronary intervention (PCI) is the most effective intervention for patients with ST-segment elevation myocardial infarction (STEMI). However, myocardial ischemia/reperfusion (I/R) injury leads to microvascular obstruction (MVO), limiting its efficacy. Colchicine can reduce myocardial I/R injury, but its effect on MVO is unclear. Hence, this study aimed to assess the role and mechanism of colchicine on MVO.

Methods: Clinical data on STEMI patients with PCI were collected and risk factors related to MVO were analyzed. The rat myocardial I/R model was established to evaluate the MVO by thioflavin S staining. The myocardial I/R model of mice was treated with PBS or colchicine at the reperfusion. The effect of colchicine on cardiomyocyte apoptosis after I/R was evaluated by TUNEL and expression of cleaved caspase-3. ROS levels were detected in H9c2 cells to evaluate the colchicine effect on myocardial oxidative stress. Moreover, the mechanism through which colchicine attenuated MVO was examined using flow cytometry, WB, ELISA, immunohistochemistry, bioinformatics analysis, and immunofluorescence.

Results: Multivariate analysis showed that elevated neutrophils were associated with extensive MVO. Colchicine could attenuate MVO and reduce neutrophil recruitment and NETs formation after myocardial I/R. In addition, colchicine inhibited cardiomyocyte apoptosis in vivo and ROS levels in vitro. Furthermore, colchicine inhibited neutrophil proliferation in the bone marrow (BM) by inhibiting the S100A8/A9 inflammatory signaling pathway.

Conclusions: Colchicine attenuated MVO after myocardial I/R injury by inhibiting the proliferation of neutrophils in BM through the neutrophil-derived S100A8/A9 inflammatory signaling pathway.

Keywords: Colchicine; Microvascular obstruction; Myocardial ischemia/reperfusion injury; NETs; S100A8/A9.

Fig. 1

Colchicine alleviated MVO in rats and inhibits NETs in mice after myocardial I/R injury. (A) Representative image of thioflavin-S-stained hearts of myocardial I/R-injured rats treated with PBS or colchicine for 24 h. The area of the red trace is the MVO (scar bar = 5mm). (B) MVO% (MVO/LV) in the ischemic heart 24 h after myocardial I/R (n = 4 each group) (not normalized to infarct size). (C) Representative immunofluorescence images of NETs in the myocardial infarct area and (D) remote area staining with Ly6G (yellow), CitH4 (citrullinated histone 4, green), MPO (myeloperoxidase, red), and DAPI (blue) (scale bar = 200 μm). (E) Semi-quantification analysis of NETs of infarct and (F) remote areas (n = 4 each group). Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ****P <0.0001. Col, colchicine

Fig. 2

Colchicine inhibited cardiomyocyte apoptosis in mice after myocardial I/R and reduced ROS levels in vitro. (A) Representative immunofluorescence images of cardiac tissues staining with TUNEL (green), α-Actinin (red), and DAPI (blue) in the infarct hearts of the mice at 1 day after myocardial I/R (scar bar = 50 μm). (B) Quantitative analysis of TUNEL (green) positive cells in cardiomyocyte (n = 4 each group). (C) Representative western blot images of total caspase-3 and cleaved caspase-3 in the myocardial infarct area. (D) Statistical analysis of cleaved caspase-3 protein expression in the myocardial infarct area (n = 4 each group). (E) Representative immunofluorescence images of ROS in H9c2 cells treated by H/R with PBS or colchicine pretreatment (scale bars = 50 μm). (F) Statistical analysis of ROS levels in H9c2 cells (n = 3 each group). Data are shown as mean ± SD. ns, not significant. **P < 0.01, ***P < 0.001, ****P < 0.0001. Col, colchicine

Fig. 3

Colchicine reduced the recruitment of neutrophils and inhibited inflammatory factors in mice after myocardial I/R. (A–D) Representative flow cytometry plots and statistical analysis of CD45⁺Ly6G⁺ neutrophils in the blood, BM, spleen, and heart at 12, 12, 12, and 24 h after myocardial I/R separately (n = 4 each group). (E) Quantification of IL-6 protein levels (by ELISA) in the plasma of mice at 12, 24, and 48 h after myocardial I/R (n = 4 each group). (F) Quantification of IL-1β protein levels (by ELISA) in the plasma at 6, 12, and 48 h after myocardial I/R (n = 3 each group). Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Col, colchicine

Fig. 4

Colchicine inhibited neutrophil proliferation and Il-1β receptor expression in the BM of mice after myocardial I/R. (A) Representative flow cytometry plots and (B) statistical analysis of CD45⁺Ly6G⁺ neutrophils (as assessed by EdU incorporation) in the BM of mice at 12 h after myocardial I/R (n = 4 each group). (C) Representative immunohistochemistry images of IL-1RI, IL-1RII, and IL-1Ra in BM of mice at 12 h after myocardial I/R (scale bar = 20 μm). (E–F) Quantification of IL-1RI, IL-1RII, and IL-1Ra positive area in BM of mice at 12 h after I/R (n = 3 each group). Data are shown as mean ± SD. ns, not significant, *P < 0.05, ***P < 0.005, ****P < 0.001. BM, bone marrow. Col, colchicine. ABR-215757, S100A8/A9 inhibitor

Fig. 5

Colchicine inhibited neutrophil proliferation in the BM through the S100A8/A9-NLRP3/IL-1β/IL-1R pathway. (A) The Venn diagram shows the overlap of the four datasets, and colchicine, NETs, MVO, and I/R were selected. (B) Bar chart of enriched items from 74 genes, colored with P-values. (C) Network of enriched items. (D) Interaction network of protein targets. (E) Quantification of S100A8/A9 protein levels (by ELISA) in the plasma of mice at 12 h after myocardial I/R injury (n = 4 each group). (F) Representative western blot images and (G) statistical analysis of NLRP3 protein expression in primary neutrophils (n = 4–6 each group), GAPDH shown as the loading control. (H) Quantification of IL-1β protein levels (by ELISA) in primary neutrophils (n = 3 each group). Data are shown as mean ± SD. *P < 0.05, ***P < 0.005. Col, colchicine

Fig. 6

Schematic representation of the signaling pathway that colchicine inhibited neutrophil proliferation in the BM. In response to myocardial I/R injury, circulating neutrophils and NETs formation are attracted to the areas of MVO, where they are rapidly primed and release S100A8/A9, stimulating the expression of the NLRP3 inflammasome in circulating neutrophils and secretes IL-1β. IL-1β interacts with its receptor (IL-1R) in the bone marrow. The BM is stimulated to produce more neutrophils to the circulation and recruited to the MVO area, thus aggravating myocardial injury. Colchicine can inhibit the recruitment of neutrophils in circulation and inhibit the neutrophils proliferation in BM by inhibiting the S100A8/A9 signaling pathway, thereby attenuating MVO after myocardial I/R injury