Regulating mitochondrial oxidative phosphorylation and MAPK signaling: wedelolactone as a novel therapeutic for radiation-induced thrombocytopenia

Abstract

Introduction: Radiation-induced thrombocytopenia (RIT) is a serious complication of cancer radiotherapy, for which therapeutic options are limited. This study investigates wedelolactone (WED), a metabolite of a botanical drug, as a potential treatment for RIT.

Methods: In vitro experiments were conducted using Meg-01 and K562 cell lines to evaluate the effects of WED on megakaryocyte differentiation and maturation. Flow cytometry and phalloidin staining were employed to assess the expression of megakaryocyte-specific markers CD41 and CD61, as well as nuclear polyploidization. A mouse model of RIT was established to assess the efficacy of WED in restoring platelet counts and regulating hematopoiesis. RNA sequencing and western blot analyses were performed to explore the underlying molecular mechanisms.

Results: In vitro experiments revealed that WED enhanced megakaryocyte differentiation in a dose-dependent manner, increasing the expression of lineage-specific markers CD41 and CD61, and promoting polyploidization and cytoskeletal reorganization. In vivo, WED significantly restored platelet counts in the mouse model of RIT and promoted the production of hematopoietic stem cells (HSCs), megakaryocytes, and reticulated platelets. RNA sequencing and western blot revealed that WED-induced megakaryocyte differentiation involves the regulation of mitochondrial oxidative phosphorylation mediated by the AMPK signaling pathway and activation of the MAPK signaling pathway. Inhibition of mitochondrial oxidative phosphorylation or MAPK signaling suppressed WED-induced megakaryocyte differentiation, highlighting the central role of these pathways.

Discussion: These findings indicate that WED could be a promising therapeutic candidate for RIT, acting through the modulation of oxidative phosphorylation and MAPK signaling pathways to enhance thrombopoiesis.

Keywords: megakaryocyte differentiation; oxidative phosphorylation; thrombocytopenia; thrombopoiesis; wedelolactone.

Graphical abstract

FIGURE 1

WED induces megakaryocyte differentiation and maturation. (A) CD41 and CD61 expression in the cells after 5 days of PMA and WED treatment. The histograms illustrate the proportion of CD41⁺ CD61⁺ cells in each group. (B) DNA ploidy analysis of cells using flow cytometry after 5 days of PMA and WED intervention. The histograms display the percentage of 2N, 4N, and ≥ 8N cells. (C) Phalloidin staining was conducted on two cell types treated with WED and PMA for 5 days. Scale bar = 100 μm. Data are displayed as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. the control group. Ctrl: Control.

FIGURE 2

WED promotes platelet recovery in RIT mice. (A) Schematic diagram illustrating the study design for the treatment of RIT model mice with WED. (B–E) Changes in platelet parameters, including (B) platelet counts, (C) PDW, (D) MPV, (E) P-LCR, were measured in peripheral blood on days 0, 3, 7, 10, and 12 after RIT mice were treated with WED. Data are displayed as mean ± SD (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001 vs. the model. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. control group. Ctrl: Control.

FIGURE 3

WED stimulates the production of both megakaryocytes and platelets in vivo. (A–C) Flow cytometry analyses of c-Kit⁺CD34⁺ (A), c-Kit⁺CD41⁺ (B), and CD41⁺CD42d⁺ (C) cell percentages in the BM after 10 days of treatment. Histograms represent the proportion of positive cells in each group. (D) Thiazole orange-positive platelets in peripheral blood. Histograms represent the proportion of positive platelets in each group. (E) Immunohistochemical staining of CD41 in the BM and spleen tissue. The CD41⁺ MKs are indicated by arrows. Scale bar = 50 μm. Histograms represent the proportion of CD41⁺ cells in BM and spleen across groups. Data are displayed as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. model group.

FIGURE 4

RNA sequencing analysis. (A) Heatmap displaying gene expression changes. (B) MA plot showing DEGs with upregulated (red) or downregulated (blue). (C) GO enrichment analysis. (D) KEGG enrichment analysis.

FIGURE 5

Mitochondrial oxidative phosphorylation contributes to WED-induced megakaryocyte differentiation. (A) Fluorescence images of JC-1-stained K562 cells showing changes in MMP. (B) Intracellular ATP levels in K562 cells after 5 days of WED treatment. (C–E) Flow cytometry evaluation of MMP (C), ROS levels (D), and mitochondrial mass (E) in K562 cells after 5 days of WED intervention. *p < 0.05, **p < 0.01, ***p < 0.001. vs the control group. (F) Expression of CD41 and CD61. Histograms display the percentage of CD41⁺CD61⁺ cells. Data are displayed as mean ± SD (n = 3). ***p < 0.001 vs. the WED group.

FIGURE 6

MAPK signaling pathway is necessary for WED-induced megakaryocyte maturation. (A-F) Western blot analysis showing the expression levels of RAS, p-MEK, p-ERK, FOS, NF-E2, and GATA1 in cells exposed to WED (2.5, 5, and 10 μM). *p < 0.05, **p < 0.01, ***p < 0.001 vs. the control group. (G) Flow cytometry analysis, with histograms displaying the proportion of CD41⁺CD61⁺ cells. Data are displayed as mean ± SD (n = 3). ***p < 0.001 vs. the WED group.

FIGURE 7

Schematic model showing the role of WED in regulating megakaryocyte differentiation and platelet generation. WED promotes megakaryocyte differentiation and platelet generation by activating the MAPK signaling pathway (RAS/MEK/ERK1/2) and modulating oxidative phosphorylation via the AMPK signaling pathway. Activation of MAPK signaling results in the upregulation of key hematopoietic transcription factors, including FOS, NF-E2, and GATA1, which are essential for megakaryocyte differentiation and maturation. This process ultimately leads to the restoration of platelet counts, as demonstrated in the RIT mice model. MEP: Megakaryocyte-erythroid progenitor.