Upregulation of let-7f-2-3p by long noncoding RNA NEAT1 inhibits XPO1-mediated HAX-1 nuclear export in both in vitro and in vivo rodent models of doxorubicin-induced cardiotoxicity.

Clinical application of doxorubicin (Dox) is limited due to its undesirable side effects, especially cardiotoxicity. Several microRNAs (miRNAs) such as microRNA-140-5p and miR-23a aggravate Dox-induced cardiotoxicity. Here we demonstrate that upregulation of miRNA let-7f-2-3p by long noncoding RNA (lncRNA) NEAT1 inhibits exportin-1 (XPO1)-mediated nuclear export of hematopoietic-substrate-1 associated protein X-1 (HAX-1) in Dox-induced cardiotoxicity. Treatment of the H9c2 cells with the Dox (1 µM) for 6 h inhibited HAX-1 nuclear export and decreased XPO1 expression. Overexpression of XPO1 significantly attenuated the Dox-induced leakage of myocardial enzymes (creatine phosphokinase, creatine kinase-MB and lactate dehydrogenase) and cardiomyocyte apoptosis with the increased HAX-1 nuclear export. Differentially expressed miRNAs including let-7f-2-3p were selected from the Dox or vehicle-treated cardiomyocytes. TargetScan and luciferase assay showed that let-7f-2-3p targeted XPO1 3' UTR. Inhibition of let-7f-2-3p reduced Dox-induced cardiotoxicity and apoptosis by inhibiting XPO1-mediated HAX-1 nuclear export, whereas let-7f-2-3p overexpression aggravated these effects. In addition, lncRNA NEAT1 was identified as an endogenous sponge RNA to repress let-7f-2-3p expression. Overexpression of lncRNA NEAT1 abolished the increased let-7f-2-3p expression by Dox, and thereby attenuated cardiotoxicity. The loss function of let-7f-2-3p increased XPO1-mediated HAX-1 nuclear export and reduced myocardial injury in Dox (20 mg/kg)-treated rats. Importantly, let-7f-2-3p inhibition in mice alleviated Dox-induced cardiotoxicity and preserved the antitumor efficacy. Together, let-7f-2-3p regulated by lncRNA NEAT1 aggravates Dox-induced cardiotoxicity through inhibiting XPO1-mediated HAX-1 nuclear export, and may serve as a potential therapeutic target against Dox-induced cardiotoxicity.

Glabridin Prevents Doxorubicin-Induced Cardiotoxicity Through Gut Microbiota Modulation and Colonic Macrophage Polarization in Mice.

The chemotherapeutic drug doxorubicin (DOX) provokes a dose-related cardiotoxicity. Thus, there is an urgent need to identify the underlying mechanisms and develop strategies to overcome them. Here we demonstrated that glabridin (GLA), an isoflavone from licorice root, prevents DOX-induced cardiotoxicity through gut microbiota modulation and colonic macrophage polarization in mice. GLA reduced DOX-induced leakage of myocardial enzymes including aminotransferase, creatine kinase, lactate dehydrogenase, and creatine kinase-MB. GLA downregulated pro-apoptotic proteins (Bax, cleaved-caspase 9 and cleaved-caspase 3) and upregulated anti-apoptotic proteins (HAX-1 and Bcl-2) in the cardiac tissues. In addition, GLA modulated DOX-induced dysbiosis of gut microbiota and thereby decreased the ratio of M1/M2 colonic macrophage, accompanied by the downregulated lipopolysaccharide (LPS) and upregulated butyrate in the feces and peripheral blood. The leakage of myocardial enzymes induced by the DOX was decreased by antibiotics treatment, but not altered by co-treatment with the GLA and antibiotics. The ratio of M1/M2 colonic macrophage and leakage of myocardial enzymes reduced by the GLA were greatly increased by the Desulfovibrio vulgaris or LPS but decreased by the butyrate. Depletion of the macrophage attenuated DOX-induced cardiotoxicity but failed to further affect the effects of GLA. Importantly, GLA decreased production of M1 cytokines (IL-1ß and TNF-α) but increased production of M2 cytokines (IL-10 and TGF-ß) in the colonic macrophage with the downregulation of NF-κB and the upregulation of STAT6. In summary, GLA prevents DOX-induced cardiotoxicity through gut microbiota modulation and colonic macrophage polarization, and may serve as a potential therapeutic strategy for the DOX-induced cardiotoxicity.

Sulfasalazine alters microglia phenotype by competing endogenous RNA effect of miR-136-5p and long non-coding RNA HOTAIR in cuprizone-induced demyelination.

Sulfasalazine (SF) promotes remyelination and improves the outcome of multiple sclerosis (MS) patients. However, the underlining mechanism remains elusive. Here, we examined whether SF blocks microglia switching to a pro-inflammatory M1-like phenotype through a competing endogenous RNA (ceRNA) effects in cuprizone-induced demyelination. The microglia reprogramming effects of SF in the mice model of cuprizone-induced demyelination was measured by histological, immunohistochemical and molecular biological methods. We also measured the effects of the condition media from SF-treated microglia on the differentiation of OLN-93 cells. Insights of the mechanism of ceRNAs of miR-136-5p and long non-coding RNA (lncRNA) HOTAIR were gained from bioinformatic analysis, luciferase assays and RNA binding protein immunoprecipitation. Microglia switched to a pro-inflammatory M1-like phenotype in cuprizone induced-demyelination. Conversely, SF inhibited the M1-like polarization with the increased remyelination which was attenuated by microglia depletion. SF inhibited production of M1-like factors TNF-α and INF-γ in microglia, and thereby promoted the differentiation of OLN-93 oligodendrocytes. SF down-regulated lncRNA HOTAIR but up-regulated miR-136-5p, and thus inactivated AKT2-NF-κB in cuprizone-treated microglia. Importantly, lncRNA HOTAIR overexpression reversed the increased miR-136-5p expression by SF and thereby attenuated the inhibition of AKT2-mediated NF-κB activation. Mimic of miR-136-5p inhibited cuprizone-induced activation of AKT2-NF-κB in the microglia. In summary, SF blocks microglia switching to a pro-inflammatory M1-like phenotype by ceRNA effect of miR-136-5p and lncRNA HOTAIR in cuprizone-induced demyelination. Our findings show the therapeutic potential of SF for human MS probably by targeting epigenetic regulation in microglia.

Inhibition of COX-2/mPGES-1 and 5-LOX in macrophages by leonurine ameliorates monosodium urate crystal-induced inflammation.

Cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) and microsomal prostaglandin E synthase-1 (mPGES-1)-derived eicosanoids play an essential role in human inflammatory disorders. Here, we investigated whether inhibition of COX-2/mPGES-1 and 5-LOX in macrophages by leonurine ameliorates monosodium urate (MSU) crystal-induced inflammation. Virtual screening assay and in vitro enzyme inhibition assay showed that leonurine was a potential inhibitor of COX-2, mPGES-1 and 5-LOX. Compared with COX-2 inhibitor celecoxib, leonurine (30 mg/kg) significantly decreased ankle perimeter, gait score and neutrophil number in synovial fluid in MSU crystal-treated rats, accompanied with the decreased expression of COX-2, mPGES-1 and 5-LOX and production of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in the synovial fluid macrophages. In addition, leonurine decreased representative M1 marker (iNOS and CD86) expression, NLRP3 inflammasome activation and M1 cytokine (TNF-α and IL-1ß) production. In the in vitro cultured RAW264.7 and human monocyte-derived macrophages (MDMs), blockade of COX-2/mPGES-1 and 5-LOX by leonurine inhibited macrophage M1 polarization and NLRP3 inflammasome activation in response to MSU crystals, and thus down-regulated IL-1ß and TNF-α with STAT1 and NF-κB inactivation. Conversely, these effects were partially abolished by overexpression of COX-2, mPGES-1, 5-LOX or STAT1. Furthermore, leonurine prevented a positive feedback loop between COX-2/mPGES-1/5-LOX and IL-1ß/TNF-α in MSU crystal-induced inflammation. Together, simultaneous down-regulation of COX-2/mPGES-1 and 5-LOX by leonurine ameliorates MSU crystal-induced inflammation through decreasing IL-1ß and TNF-α production. Our study may provide novel multi-target agents toward the arachidonic acid (AA) network for gouty arthritis therapy.

Inhibition of CYP4A by a novel flavonoid FLA-16 prolongs survival and normalizes tumor vasculature in glioma.

Glioblastomas rapidly become refractory to anti-VEGF therapies. We previously showed that cytochrome P450 (CYP) 4A-derived 20-hydroxyeicosatetraenoic acid (20-HETE) promotes angiogenesis. Here, we tested whether a novel flavonoid (FLA-16) prolongs survival and normalizes tumor vasculature in glioma through CYP4A inhibition. FLA-16 improved survival, reduced tumor burden, and normalized vasculature, accompanied with the decreased secretion of 20-HETE, VEGF and TGF-ß in tumor-associated macrophages (TAMs) and endothelial progenitor cells (EPCs) in C6 and U87 gliomas. FLA-16 attenuated vascular abnormalization induced by co-implantation of GL261 glioma cells with CYP4A10high macrophages or EPCs. Mechanistically, the conditional medium from TAMs and EPCs treated with FLA-16 enhanced the migration of pericyte cells, and decreased the proliferation and migration of endothelial cells, which were reversed by CYP4A overexpression or exogenous addition of 20-HETE, VEGF and TGF-ß. Furthermore, FLA-16 prevented crosstalk between TAMs and EPCs during angiogenesis. These results suggest that CYP4A inhibition by FLA-16 prolongs survival and normalizes vasculature in glioma through decreasing production of TAMs and EPCs-derived VEGF and TGF-ß. This may represent a potential therapeutic strategy to overcome resistance to anti-VEGF treatment by effects on vessels and immune cells.

Dopamine induces growth inhibition and vascular normalization through reprogramming M2-polarized macrophages in rat C6 glioma.

Dopamine (DA), a monoamine catecholamine neurotransmitter with antiangiogenic activity, stabilizes tumor vessels in colon, prostate and ovarian cancers, thus increases chemotherapeutic efficacy. Here, in the rat C6 glioma models, we investigated the vascular normalization effects of DA and its mechanisms of action. DA (25, 50mg/kg) inhibited tumor growth, while a precursor of DA (levodopa) prolonged the survival time of rats bearing orthotopic C6 glioma. DA improved tumor perfusion, with significant effects from day 3, and a higher level at days 5 to 7. In addition, DA decreased microvessel density and hypoxia-inducible factor-1α expression in tumor tissues, while increasing the coverage of pericyte. Conversely, an antagonist of dopamine receptor 2 (DR2) (eticlopride) but not DR1 (butaclamol) abrogated DA-induced tumor regression and vascular normalization. Furthermore, DA improved the delivery and efficacy of temozolomide therapy. Importantly, DA increased representative M1 markers (iNOS, CXCL9, etc.), while decreasing M2 markers (CD206, arginase-1, etc.). Depletion of macrophages by clodronate or zoledronic acid attenuated the effects of DA. Notably, DA treatment induced M2-to-M1 polarization in RAW264.7 cells and mouse peritoneal macrophages, and enhanced the migration of pericyte-like cells (10T1/2), which was reversed by eticlopride or DR2-siRNA. Such changes were accompanied by the downregulation of VEGF/VEGFR2 signaling. In summary, DA induces growth inhibition and vascular normalization through reprogramming M2-polarized macrophages. Thus, targeting the tumor microvasculature by DA represents a promising strategy for human glioma therapy.