Low-Dose Piperlongumine Rescues Impaired Function of Endothelial Progenitor Cells and Reduces Cerebral Ischemic Injury in High-Fat Diet-Fed Mice

Abstract

It is of great clinical significance to develop potential novel strategies to prevent cardio-cerebrovascular complications in patients with hyperlipidemia. Vascular Endothelial integrity and function play a key role in the prevention of cardio-cerebrovascular diseases. Endothelial progenitor cells (EPCs) can home to sites of ischemic injury and promote endothelial regeneration and neovascularization. Hypercholesterolemia impairs the function of EPC. The present study attempted to identify the effect of piperlongumine on EPCs' angiogenic potential and cerebral ischemic injury in high-fat diet-fed (HFD-fed) mice. Here, we showed that treatment with low-does piperlongumine (0.25 mg/kg/day) for 8 weeks significantly improved EPCs function and reduced the cerebral ischemic injury (both infarct volumes and neurobehavioral outcomes) in HFD-fed mice. In addition, low-dose piperlongumine administration increased intracellular NO level and reduced intracellular O₂ ^- level in EPCs of HFD-fed mice. Moreover, incubation with piperlongumine (1.0 μM, 24 h) reduced thrombospondin-1/2 (TSP-1/2, a potent angiogenesis inhibitor) expression levels in EPCs from HFD-fed mice, increased the therapeutic effect of EPC from HFD-fed mice on cerebral ischemic injury reduction and angiogenesis promotion in HFD-fed mice, and the donor derived EPCs homed to the recipient ischemic brain. In conclusion, low-dose piperlongumine can enhance EPCs' angiogenic potential and protect against cerebral ischemic injury in HFD-fed mice. It is implied that treatment with low-dose piperlongumine might be a potential option to prevent ischemic diseases (including stroke) in patients with hyperlipidemia, and priming with piperlongumine might be a feasible way to improve the efficacy of EPC-based therapy for ischemic diseases.

Keywords: angiogenesis; cerebral ischemic injury; endothelial progenitor cells; hyperlipidemia; piperlongumine.

FIGURE 1

Effects of low-dose piperlongumine administration on food intake, body weight, fasting blood glucose and lipid levels in HFD-fed mice. Male C57BL/6J mice at 8 weeks of age were randomly allocated to three groups and fed with high-fat rodent chow with or without receiving an intraperitoneal injection of low-does piperlongumine (0.25 mg/kg/d) for 8 weeks. After 8 weeks treatment, the mice were subjected to various analyses. (A), Food intake during the 8 weeks experiments (n = 8–11). (B), Body weight during the 8 weeks experiments (n = 7–11). (C), Effect of piperlongumine on fasting blood glucose (n = 7–9). (D–F), Blood total cholesterol (TC) levels, triglyceride (TG) levels, low-density lipoprotein cholesterol (LDL-c) levels, and high-density lipoprotein cholesterol (HDL-c) levels after 8 weeks treatment (n = 5–9). Throughout, bars represent means; error bars represent SEM. Values were normalized to Con. *p < 0.05, **p < 0.01. Con, control mice; HF, HFD-fed mice; PIP, HFD-fed mice treated with piperlongumine.

FIGURE 2

Low-dose Piperlongumine administration rescued EPC dysfunctions in HFD-fed mice. (A), Experimental protocols: Male C57BL/6 mice (8 weeks) were randomized into three groups and fed with high-fat rodent chow with or without receiving an intraperitoneal injection of low-does piperlongumine (0.25 mg/kg/d) for 8 weeks. Then, the EPCs were isolated, cultured and examined in HFD-fed mice. (B), EPC migration assay (n = 15–16). (C), EPC tube formation assay (n = 20 per group). (D), EPC adhesion assay (n = 20–26). (E), Intracellular superoxide level of EPCs assessed by DHE staining flow cytometry (n = 4–5). (F), Intracellular NO level of EPCs assessed by DAF-FM staining flow cytometry (n = 4–5). Throughout, bars represent means; error bars represent SEM. Values were normalized to Con. *p < 0.05, **p < 0.01. Con, control mice; HF, HFD-fed mice; PIP, HFD-fed mice treated with piperlongumine. Scale bar: 200 μm.

FIGURE 3

Low-dose Piperlongumine administration protected against cerebral ischemic injury in HFD-fed mice. (A), Surgical protocols: Male C57BL/6 mice (8 weeks) were randomized into three groups and fed with high-fat rodent chow with or without receiving an intraperitoneal injection of low-does piperlongumine (0.25 mg/kg/d) for 8 weeks. Then the mice were subjected to focal cerebral ischemia by permanent. On day 3 after cerebral ischemia, behavioral test (including Body Asymmetry Test and Beam Test) was performed, and then the cerebral infarct volumes were determined. (B,C), Images are representative of TTC-stained brain sections (B) and cerebral infarct volumes (C). (D,E), Neurobehavioral outcomes: Body Asymmetry Test (D) and Beam Test (E). Throughout, bars represent means; error bars represent SEM. Values were normalized to Con. *p < 0.05, **p < 0.01. n = 8 per group. Con, control mice; HF, HFD-fed mice; PIP, HFD-fed mice treated with piperlongumine.

FIGURE 4

Incubation with piperlongumine rescued the impaired functions of EPCs from HFD-fed mice. (A), Surgical protocols: Male C57BL/6 mice (8 weeks) were fed with high-fat rodent chow for 8 weeks. Then, the EPCs were isolated from the HFD-fed mice. After 6 days, piperlongumine was added to EPCs. Then, EPC functions were measurement at the 7^th day. (B), Effects of piperlongumine (0.1 and 1.0µM, 24 h) on migration function of EPCs from HFD-fed mice (n = 15 per group). (C), EPC migration assay (n = 13 per group). (D), Tube formation assay of EPCs (n = 15–20). (E), Adhesion assay of EPCs (n = 17–20). (F), the representative images and the protein expression levels of TSP-1 (n = 7) and TSP-2 (n = 6). Throughout, bars represent means; error bars represent SEM. Values were normalized to Con or HF. *p < 0.05, **p < 0.01. Con, control EPC; HF, EPC from HFD-fed mice; HF+0.1, piperlongumine (0.1µM, 24 h)-incubated EPC from HFD-fed mice; HF+1.0, piperlongumine (1.0µM, 24 h)-incubated EPC from HFD-fed mice . HF + PIP, piperlongumine (1.0µM, 24 h)-incubated EPC from HFD-fed mice. Scale bar: 200 μm.

FIGURE 5

Incubation with piperlongumine increased the therapeutic effect of EPC from HFD-fed mice on cerebral ischemic injury reduction in HFD-fed mice. (A), Surgical protocols: Male C57BL/6 mice (8 weeks) were fed with high-fat rodent chow for 8 weeks. Then, the EPCs were isolated from the HFD-fed mice. After 6 days, piperlongumine (1.0 μM) was added to EPCs for 24 h. EPCs were harvested at the 7^th day and injected into the HFD-fed mice via tail vein just after the cerebral ischemia (1×10⁶ EPCs per mice). Behavioral test and the infarct volumes were assessed on day 3 after middle cerebral artery occlusion. (B,C), Images are representative of 2,3,5-triphenyltetrazolium chloride-stained brain sections (B) and cerebral infarct volumes (C). (D,E), Neurobehavioral outcomes: Body Asymmetry Test (D) and Beam Test (E). Throughout, bars represent means; error bars represent SEM. Values were normalized to HF. *p < 0.05, **p < 0.01. n = 8-9 per group. HF, HFD-fed mice; HF + HF, HFD-fed mice treated with EPCs; HF + PIP, HFD-fed mice treated with piperlongumine (1.0µM, 24 h)-incubated EPCs.

FIGURE 6

Incubation with piperlongumine increased the therapeutic effect of EPC from HFD-fed mice on angiogenesis promotion in HFD-fed mice. (A), Surgical protocols: Male C57BL/6 mice (8 weeks) were fed with high-fat rodent chow for 8 weeks. Then, the EPCs were isolated from the HFD-fed mice. On day five of culture, BrdU were added to the media, and new medium supplemented with BrdU was replenished daily until day 7. Piperlongumine (1.0 μM) was added to EPCs on day 6 for 24 h. EPCs were harvested at the 7^th day and injected into the HFD-fed mice via tail vein just after the middle cerebral artery occlusion (1×10⁶ EPCs per mice). On day 3 after middle cerebral artery occlusion, the local angiogenesis in ischemic brain were determined. (B), immunostaining for CD31 shows typical microvessels in the ischemic boundary area of ischemic brains. The bar graph shows increased capillary density in the two groups of EPCs-treated mice. Moreover, piperlongumine-incubated EPCs exerted a more positive effect on angiogenesis promotion compared to the EPCs without piperlongumine incubation (n = 9–12). Scale bars: 200 μm (top); 100 μm (bottom). (C), immunostaining for vWF shows typical microvessels in the ischemic boundary area of ischemic brains. The bar graph shows increased capillary density in the two groups of EPCs-treated mice. Moreover, piperlongumine-incubated EPCs exerted a more positive effect on angiogenesis promotion compared to the EPCs without piperlongumine incubation (n = 8–9). Throughout, bars represent means; error bars represent SEM. Values were normalized to HF. **p < 0.01. Scale bars: 200 μm (top); 100 μm (bottom). (D), The ischemic area and the ischemic boundary area of the ischemic brain, the red squares indicate the fields that were examined in this study. (E), Photographs of ischemic boundary area indicate that the donor derived BrdU-positive EPCs (red fluorescence) were incorporated into CD31-positive microvessels (green fluorescence). Some BrdU-positive cells were found surrounding the microvessels. The nucleus was stained with DAPI (blue fluorescence). Scale bar: 50 μm. HF, HFD-fed mice; HF + HF, HFD-fed mice treated with EPCs; HF + PIP, HFD-fed mice treated with piperlongumine (1.0µM, 24 h)-incubated EPCs.

FIGURE 7

Putative mechanisms underlying low-dose piperlongumine reducing cerebral ischemic injury in HFD-fed mice. High-fat diet impaired endothelial progenitor cell function, decreased the ischemic angiogenesis, increased cerebral infarct volumes, and reduced the corresponding neurobehavioral outcomes in mice. Low-dose piperlongumine reduces cerebral ischemic injury via rescuing impaired EPC-mediated angiogenesis in HFD-fed mice.