Allicin alleviates lead-induced hematopoietic stem cell aging by up-regulating PKM2

Abstract

Hematopoietic stem cells (HSCs) aging is associated with hematopoietic dysfunction and diseases. Our previous study showed that lead exposure induced a functional decline in HSCs. Allicin, a chemical extracted from the garlic (Allium sativum L.), has been reported to have antioxidative and anti-inflammatory effects. However, the biological activities of allicin on lead-induced toxicity, especially in the hematopoietic system, remain unclear. Here, we found that lead exposure elicited aging phenotypes in HSCs, including perturbed cell quiescence, disabled self-renewal function and colony-forming ability, and myeloid-biased differentiation, all of which contributed to significant hematopoietic disorders in mice. Intragastric administration of allicin substantially ameliorated lead-induced HSCs aging phenotypes in vivo Lead exposure induced a peroxide condition in HSCs leading to DNA damage, which reduced expression of the glycolytic enzyme pyruvate kinase M2 isoform (PKM2), a phenotype which was significantly ameliorated by allicin treatment. These findings suggested that allicin alleviated lead-induced HSCs aging by up-regulating PKM2 expression; thus, it could be a natural herb for preventing lead toxicity.

Keywords: Aging; Allicin; Hematopoietic stem cell; Lead; PKM2.

Figure 1. Lead-exposed mice exhibited a perturbed hematopoiesis and aging phenotype

C57BL/6 mice were exposed with lead acetate for 8 weeks. Quantification of the WBC level (A), the RBC level (B), the hemoglobin level (C), the inflammatory factors IL-6 (D) and TNF-α (E) in lead exposed mice. All data are shown as mean (M) ± SD, n=6, *P<0.05, **P<0.01.

Figure 2. HSCs from lead-exposed mice exhibited premature aging

(A) LSK cells population in vehicle and lead exposed mice, (B) the cell ratio of lymphoid cells and myeloid cells in vehicle and lead exposed mice. (C) Colony forming ability, (D) self-renewal function of HSCs that isolated from vehicle and lead-exposed mice by serial competitive transplantation. All data are shown as mean (M) ± SD, n=6, *P<0.05, **P<0.01, ***P<0.001, scale bar = 50 μm.

Figure 3. Allicin alleviated the aging features induced by lead exposure

(A) Schematic chart of mouse exposure to lead and allicin in different groups. (B) the ratio of LSK cells in BM cells from different groups; (C) the ration of lymphoid cells to myeloid cells in whole BM cells from different groups; (D-E) the inflammatory factors IL-6 (D) and TNF-α (E) in peripheral blood in different groups. All data are shown as mean (M) ± SD,n=6, *P<0.05, **P<0.01.

Figure 4. Allicin attenuated lead-induced self-renewal function of HSCs

LSK cells isolated from mice (n=6) in different groups (vehicle, allicin, lead, lead/allicin) were collected: (A) colony forming ability of LSK cells; LSK cells were isolated from C57BL/6 mice and followed by lead or lead/allicin exposure for 72 h. (B) colony forming ability of LSK cells, (C) self-renewal function of HSCs that isolated from different group mice (n=6) performed by serial competitive transplantation. All data are shown as mean (M)±SD, *p<0.05, **P<0.01, scale bar=50 μm. The experiments were performed in triplicate.

Figure 5. Allicin attenuated lead-induced increased cellular ROS and DNA damage

(A) intracellular ROS levels were determined in LSK cells that isolated from mice (n=6) in different groups. (B) intracellular ROS levels were determined in LSK cells pretreated by lead or lead/allicin. (C) DNA damage level (γ-H2XA) were determined in LSK cells isolated from mice (n=6) in different groups. (D) DNA damage level (γ-H2XA) were determined in LSK cells pretreated with lead or lead/allicin. All data are shown as mean (M)±SD, **P<0.01. The experiments were performed in triplicate.

Figure 6. Allicin alleviated lead-induced HSCs aging by upregulating PKM2

(A,B) mRNA and protein expression of PKM2 on LSK cells that isolated from mice (n=6) in different groups were determined; (C) colony forming ability of LSK after stimulation by the PKM 2 activator DASA10; (D) cellular ROS accumulation in LSK after stimulation by the PKM2 activator DASA10; (E) colony forming ability of LSK treated by PKM2 inhibitor shikonin; (F) cellular ROS accumulation in LSK treated by PKM2 inhibitor shikonin. All data are shown as mean (M)±SD, **p<0.01, ***p<0.001. The experiments were performed in triplicate.