| Objective:Epidemiologic studies show that the levels of air pollutants and particulate matters are positively associated with the morbidity and mortality of cardiovascular diseases.Here we demonstrate whether the inhalational exposure to multi-walled carbon nanotubes(MWCNTs),a fine particle in air pollutants,exacerbate doxorubicin(DOX)-induced cardiotoxicity in mice through altering gut microbiota and pulmonary and colonic macrophage phenotype,and may identify a novel mechanism of action of inhaled particle-driven cardiotoxicity,and may serve as a preventive strategy against air pollution-exacerbated cardiotoxicity.Methods:(1)MWCNTs(25 μg/kg per day,5 days a week for 3 weeks)promoted cardiotoxicity and apoptosis in the DOX(2 mg/kg,twice a week for 5 weeks)-treated C57BL/6 mice.The peripheral blood was obtained for detecting of myocardial enzymes;pro-apoptotic proteins(Bax,cleaved-caspase 9 and cleaved-caspase 3)and anti-apoptotic proteins(HAX-1 and Bcl-2)expression in the cardiac tissues were detected by western blot.The cardiomyocyte apoptosis was evaluated by the TUNEL assay.The histological alterations of cardiac tissues or interstitial fibrosis was evaluated by H&E or Masson’s Trichrome;The gut microbiota was analyzed by 16 s r RNA sequencing;Macrophage phenotype was detected by flow cytometry;q PCR was used to detect M1 markers(i NOS and CXCL9)and M2 markers(CD206 and arginase-1)expression;ELISA was used to detect Th1 cytokine(TNF-α and CCL2)and Th2 cytokine(TGF-β and IL-10);Chromo-LAL was performed to detect LPS level.(2)Pulmonary and colonic macrophages were depletion by the anti-CSF-1,and the cardiomyocyte apoptosis and gut microbiota of DOX,MWCNTs,DOX plus MWCNTs or vehicle-treated mice was evaluated.(3)The gut microbiota of DOX or DOX plus MWCNTs-treated mice was depleted by antibiotics,and the microbial products(LPS),leakage of myocardial enzymes and macrophage phenotypes were detected.(4)The fecal microbiota from the DOX,MWCNTs or MWCNTs plus DOX-treated mice were transplanted into the normal mice,and the microbial products(LPS),leakage of myocardial enzymes and macrophage phenotypes were detected.(5)IL-1β and TNF-αwere neutralized by the antibodies in MWCNTs plus DOX-treated mice,and the leakage of myocardial enzymes and macrophage phenotypes were detected.(4)The colonic macrophages from the DOX,MWCNTs,MWCNTs plus DOX or vehicle-treated mice were isolated and cultured,and then the expressions of TLR4,p-IκBα,p-p65 and HMGB1 were detected by Western blot.Result:(1)MWCNTs induced cardiomyocyte apoptosis in the DOX-treated mice.MWCNTs exaggerated DOX-induced gut microbiota dysbiosis characterized by the increased abundances of Helicobacteraceae and Coriobacteriaceae.In addition,MWCNTs promoted M1-like polarization of the colonic macrophages in the DOX-treated mice with an increase in TNF-α,IL-1β and CC chemokine ligand 2 in peripheral blood.(2)Macrophage depletion by the anti-CSF-1 reduced the DOX plus MWCNTs-exaggerated cardiotoxicity and gut microbiota dysbiosis.The conditioned medium from MWCNTs-treated pulmonary macrophages promoted gut microbiota dysbiosis and colonic macrophage polarization in DOX-induced cardiotoxicity.(3)Importantly,treatment with the antibiotics attenuated MWCNTs plus DOX-induced apoptosis of cardiomyocytes and M1-like polarization of the colonic macrophages.(4)The fecal microbiota transplantation from the MWCNTs plus DOX-treated mice into the normal mice induced the cardiotoxicity with M1 polarization of the colonic macrophages compared with the DOX or MWCNTs alone.(5)Furthermore,the combination or single blockade of IL-1β and TNF-α attenuated MWCNTs-exacerbated cardiotoxicity.(6)The combination of DOX with MWCNTs upregulated HMGB1 and NF-κB in the colonic macrophages.Conclusions: MWCNTs exacerbate DOX-induced cardiotoxicity in mice through gut microbiota and pulmonary and colonic macrophage interaction.Our findings identify a novel mechanism of action of inhaled particle-driven cardiotoxicity. |
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