| Background: Ischemic diseases can lead to structural and functional damage in various ischemic tissues and organs,and to a substantial increase in mortality.Angiogenesis plays a key role in the pathological progression of tissue repair.Several pro-angiogenic drugs have achieved encouraging success,and some of them have even been licensed for clinical application in tissue repair engineering.Here,we synthesized a novel pro-angiogenic drug,strontium-based nanoparticles(Sr NPs)using melanin encapsulation,and explored its function and mechanism in mice ischemic model,with the expectation of providing a new approach for the treatment of ischemic diseases.Aims: To validate the role of the novel Sr NPs in HLI and AMI models of mice,and to investigate the mechanisms through vitro/vivo studies.Methods: In vivo experiments: HLI and AMI models were established and randomly divided into control and treatment groups,which received saline/Sr NPs injection treatment,respectively.The changes in recovery of blood flow in the hind limbs of mice and the cardiac function were continuously detected by Laser Doppler imaging and Ultrasound Cardiogram at different time points.HE staining and electron microscopy were used to evaluate the morphology of muscle tissues and blood vessels.Immunofluorescence staining of tissues(including CD31,α-SMA and CD206)was performed 7 days after surgery to clarify the role of Sr NPs in the regulation of angiogenesis and vascular inflammation.The drug distribution and tissue targeting of Sr NPs were examined by electron microscopy and ICP.The safety of Sr NPs was clarified by HE staining of important organs and blood biochemistry.RNA-sequencing revealed the potential functions and signalling pathways of Sr NPs in post-ischemic tissue repair.In vitro experiments: Electron microscopy,XPS and SOD-like enzyme activity assay were used to clarify the basic characterization of Sr NPs.The role of Sr NPs in regulating angiogenesis was examined by HUVECs functional assay(proliferation and tube formation).LPS was used to induce M1 polarization in RAW264.7 cells,cellular immunofluorescence(COX2)and Western Blot(COX2 and CD206)were used to detect the effect of Sr NPs blocking LPS-induced M1 polarization.Results: 1.Laser Doppler imaging and Ultrasound Cardiogram results showed that Sr NPs effectively promoted the restoration of blood flow in the hind limbs and the recovery of cardiac function;2.Immunofluorescence results showed that Sr NPs could increase the number of CD31 and α-SMA positive small vessels and promote angiogenesis,as well as effectively increase the number of perivascular M2 polarized macrophages and regulate inflammation;3.Morphological and electron microscopy results showed that Sr NPs could significantly reduce the damage of tissue,and maintain the structural integrity of muscle and mitochondrial,and the morphology and structure of endothelial cells;4.Endothelial cell assay results showed that Sr NPs could promote the ability of endothelial cells to proliferate and become tubes,and RAW264.7 cell assay showed that Sr NPs can inhibit the expression of COX2 and promote the expression of CD206;5.Morphological assay and blood biochemical results showed that Sr NPs did not damage important organs and liver and kidney functions,and ICP assay showed that strontium content of injured tissues was significantly increased in Sr NPs group,suggesting that Sr NPs successfully localized injured tissues;6.Sr NPs may be related to the regulation of angiogenesis and vascular inflammatory,and the RNA-sequencing results suggested that VEGF \ PI3K-Akt \ MAPK \ TNF \ JAK-STAT \ Fox O signaling pathways may be the potential action pathway of Sr NPs.Conclusion: 1.Novel Sr NPs showed good therapeutic effects in promoting tissue repair in two ischemic models.2.Novel Sr NPs have the ability to promote angiogenesis and regulate vascular inflammation.3.Novel Sr NPs showed good drug safety and targeting properties. |
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