Systemically Silencing Long Non-coding RNAs Maclpil With Short Interfering RNA Nanoparticles Alleviates Experimental Ischemic Stroke by Promoting Macrophage Apoptosis and Anti-inflammatory Activation

Abstract

Background: Maclpil is a proinflammatory long non-coding RNA highly expressed on monocyte-derived macrophages in the ischemic brain. This study investigated the impact and the mechanisms of systemically delivering nanoparticle Maclpil short interfering RNA (siRNA) on experimental ischemic stroke in a mouse model.

Methods: Ischemic stroke (focal cerebral ischemia) was induced in male C57BL/6 mice through the middle cerebral artery occlusion. Three hours thereafter, mice were intravenously injected with Maclpil siRNA or scramble siRNA nanoparticles. Bone marrow cell-derived macrophages were transfected with Maclpil or scramble siRNA and subjected to oxygen glucose deprivation culture. The influence of silencing Maclpil on stroke outcomes, neuroinflammation, and macrophage fates was assessed via histology, flow cytometry, Western blotting, and quantitative PCR analysis.

Results: Three days following stroke induction, siRNA silencing Maclpil substantially reduced ischemic infarction size and improved neurological behaviors. Silencing Maclpil also markedly attenuated the accumulation of monocyte-derived macrophages, CD4⁺ T cells, and CD8⁺ T cells in the ischemic hemisphere without affecting microglia cellularity. Reciprocally, myeloid cells and both subsets of T cells were elevated in mouse peripheral blood following Maclpil siRNA treatment. Under oxygen glucose deprivation conditions that mimicked hypoxia and hypoglycemia in vitro, Maclpil siRNA silencing augmented macrophage apoptosis in conjunction with upregulation of proapoptotic Bax and caspase 3 expressions. siRNA knocking down Maclpil skewed macrophages from proinflammatory classical toward anti-inflammatory alternative activation as evidenced by increased arginase 1, Ym1, and Fizz1 and reduced inducible nitric oxide synthase, IL-1β, and TNF-α mRNA levels. Consistent with macrophage phenotype switching, silencing Maclpil by siRNA enhanced fatty acid oxidation as indicated by increased mRNA levels of 3 key metabolic enzymes (ACADM, ACADVL, and HADHA).

Conclusion: Systemically silencing Maclpil by siRNA nanoparticles attenuated experimental ischemic stroke by promoting macrophage apoptosis and anti-inflammatory alternative activation. Identifying and targeting Maclpil human homolog(s) may help develop a novel therapy for stroke clinical management.

Keywords: SiRNA nanoparticles; ischemic stroke; lncRNA maclpil; macrophages; neuroinflammation.

FIGURE 1

siRNA silencing Maclpil reduces ischemic infraction size and improves neurological behaviors. (A) Three hours after MACO surgery, mice were intravenously injected with nanoparticle conjugated Maclpil or scramble siRNA. Neurological behaviors and infarction size were assessed 3 days after the MCAO surgery. (B) Images for infarction area identified by TTC staining and quantification of infarction size. (C) Quantification of neurological score. All data are mean and SD from 6 (B) and 7–8 (C) mice in each group. Student’s t-test, **p < 0.01 compared to scramble siRNA treatment.

FIGURE 2

siRNA silencing Maclpil alleviates leukocyte accumulation in experimental ischemic stroke. (A) Flow cytometric analysis strategies for identifying individual subsets of leukocytes. Leukocytes: CD45⁺; Microglia: CD11b⁺CD45^low; monocyte-derived macrophages (MoDMs): CD11b⁺CD45^high; CD4⁺ cells: CD4⁺; and CD8⁺ cells: CD8⁺. (B,C) Quantification of relative and absolute numbers of leukocytes, MoDM, microglia, CD4⁺ cells and CD8⁺ cells. All data are given as mean and SD from 5 mice per group. Student’s t-test, *p < 0.05 compared to scramble siRNA treatment.

FIGURE 3

siRNA silencing Maclpil reduces cells expressing CD68 and iNOS in ischemic infraction area. Brain sections were stained with antibodies against CD68 and iNOS, respectively, counterstained with DAPI for nuclei, and imaged on a confocal microscope. (A,B) Representative staining images for CD68 (A) and CD68⁺ cell density estimated as positively stained cells/0.1 μm² (B). (C,D) Representative staining images for iNOS (C) and iNOS⁺ cell density quantified as positively stained cells/0.1 μm² (D). All data are presented as mean and SD from 3 mice in each group. Student’s t-test, ^**p < 0.01 compared to scramble siRNA treatment.

FIGURE 4

siRNA silencing Maclpil increases circulating leukocytes in experimental ischemic stroke. (A) Flow cytometric analysis strategies for identifying individual subsets of leukocytes. Leukocytes: CD45⁺; myeloid cells: CD45⁺CD11b⁺CD45^low; CD4⁺ cells: CD4⁺; and CD8⁺ cells: CD8⁺. (B,C) Quantification of relative (B) and absolute (C) numbers of leukocytes, myeloid cells, CD4 + cells and CD8 cells. All data are given as mean and SD from 5 mice per group. Student’s t-test, *p < 0.05 compared to scramble siRNA treatment.

FIGURE 5

siRNA silencing Maclpil increases apoptosis and proapoptotic protein expression in macrophages undergoing oxygen glucose deprivation treatment. (A) Representative flow cytometric pseudocolor plots showing Annexin⁺PI⁺ apoptotic cells in Maclpil or scramble siRNA-treated bone marrow-derived macrophages (BMDCs). (B) Quantification of apoptotic cells in Maclpil and scramble siRNA-treated BMDCs. (C) Representative Western blots for proapoptotic proteins (Bax and caspase 3) and house-keeping gene (GAPDH) in Maclpil- and scramble siRNA-treated BMDCs. (D) Quantification of the expression of Bax and caspase 3 in Maclpil and scramble siRNA-treated BMDCs as the ratio to GAPDH. All data are mean and SD from triplicate experiments. Student’s t-test, ^**p < 0.01 compared to scramble siRNA treatment.

FIGURE 6

siRNA silencing Maclpil promotes anti-inflammatory gene expression in macrophages undergoing oxygen glucose deprivation treatment. Following oxygen glucose deprivation treatment, total RNA was extracted from marrow-derived macrophages (BMDCs) and subjected to reverse transcript and digital PCR amplification sequentially. (A) Representative 2-D fluorescence amplitude (FAM) plots acquired via digital PCR for individual genes in each treatment group. (B,C) Quantification of anti-inflammatory (Arg1, Fizz1, Ym1) gene (B) and pro-inflammatory (iNOS, IL-1β and TNFα) gene (C) expression in BMDCs from different treatment groups. All data are mean and SD from triplicate experiments. Student’s t-test, *p < 0.05 and ^**p < 0.01 compared to scramble siRNA treatment.

FIGURE 7

siRNA silencing Maclpil increases the expression of key fatty acid oxidation enzymes in macrophages undergoing oxygen glucose deprivation treatment. Following oxygen glucose deprivation treatment, marrow-derived macrophages (BMDCs) were stained with unlabeled primary antibodies against ACADM, ACADVL, and HADHA followed by incubation with fluorochrome-conjugated secondary antibodies and analyzed on a flow cytometer. (A) Representative flow cytometric overlay histograms showing the staining for ACADM, ACADVL, and HADHA in Maclpil and scramble siRNA-treated BMDCs after oxygen glucose deprivation. (B) Quantification of the expression levels (mean fluorescence intensity, mean and SD, n = 3) of ACADM, ACADVL, and HADHA in differentially treated BMDCs. Student’s t-test, ^**p < 0.01 compared to scramble siRNA treatment.