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Abstract：
OBJECTIVE: To investigate the cell membrane-penetrating capacity of human cell-penetrating peptide hPP10 carrying human antioxidant protein Cu-Zn superoxide dismutase (Cu, Zn-SOD) and assess the antioxidant and anti-inflammatory activity of these fusion proteins.
METHODS: The fusion protein hPP10-Cu, Zn-SOD was obtained by genetic engineering and identified by Western blotting. The membrane-penetrating ability of the fusion protein was evaluated by immunofluorescence assay, fluorescence colocalization assay and Western blotting, its SOD enzyme activity was detected using a commercial kit, and its effect on cell viability was assessed with MTT assay. In a HEK293 cell model of H2O2-induced oxidative stress, the effect of hPP10-Cu, Zn-SOD on cell apoptosis was analyzed with flow cytometry and RT-qPCR, and its antioxidant effect was assessed using reactive oxygen species (ROS) assay; its anti-inflammatory effect was evaluated in mouse model of TPA-induced ear inflammation by detecting expression of the inflammatory factors using RT-qPCR, Western blotting and immunohistochemistry.
RESULTS: The fusion protein hPP10-Cu, Zn-SOD was successfully obtained. Immunofluorescence assay confirmed obvious membrane penetration of this fusion protein in HEK293 cells, localized both in the cell membrane and the cell nuclei after cell entry. hPP10-Cu, Zn-SOD at the concentration of 5 μmol/L exhibited strong antioxidant activity with minimal impact on cell viability at the concentration up to 10 μmol/L. The fusion protein obviously inhibited apoptosis and decreased intracellular ROS level in the oxidative stress cell model and significantly reduced mRNA and protein expression of the inflammatory factors in the mouse model of ear inflammation.
CONCLUSIONS: The fusion protein hPP10-Cu, Zn-SOD capable of penetrating the cell membrane possesses strong antioxidant and anti-inflammatory activities with only minimal cytotoxicity, demonstrating the value of hPP10 as an efficient drug delivery vector and the potential of hPP10-Cu, Zn-SOD in the development of skincare products.
METHODS: The fusion protein hPP10-Cu, Zn-SOD was obtained by genetic engineering and identified by Western blotting. The membrane-penetrating ability of the fusion protein was evaluated by immunofluorescence assay, fluorescence colocalization assay and Western blotting, its SOD enzyme activity was detected using a commercial kit, and its effect on cell viability was assessed with MTT assay. In a HEK293 cell model of H2O2-induced oxidative stress, the effect of hPP10-Cu, Zn-SOD on cell apoptosis was analyzed with flow cytometry and RT-qPCR, and its antioxidant effect was assessed using reactive oxygen species (ROS) assay; its anti-inflammatory effect was evaluated in mouse model of TPA-induced ear inflammation by detecting expression of the inflammatory factors using RT-qPCR, Western blotting and immunohistochemistry.
RESULTS: The fusion protein hPP10-Cu, Zn-SOD was successfully obtained. Immunofluorescence assay confirmed obvious membrane penetration of this fusion protein in HEK293 cells, localized both in the cell membrane and the cell nuclei after cell entry. hPP10-Cu, Zn-SOD at the concentration of 5 μmol/L exhibited strong antioxidant activity with minimal impact on cell viability at the concentration up to 10 μmol/L. The fusion protein obviously inhibited apoptosis and decreased intracellular ROS level in the oxidative stress cell model and significantly reduced mRNA and protein expression of the inflammatory factors in the mouse model of ear inflammation.
CONCLUSIONS: The fusion protein hPP10-Cu, Zn-SOD capable of penetrating the cell membrane possesses strong antioxidant and anti-inflammatory activities with only minimal cytotoxicity, demonstrating the value of hPP10 as an efficient drug delivery vector and the potential of hPP10-Cu, Zn-SOD in the development of skincare products.
摘要：
目的：研究携带人抗氧化蛋白Cu-Zn超氧化物歧化酶（Cu，Zn-SOD）并评估这些融合蛋白的抗氧化和抗炎活性。
方法：融合蛋白hPP10-Cu，Zn-SOD是通过基因工程获得的，并通过Western印迹进行鉴定。用免疫荧光法评价融合蛋白的膜穿透性,荧光共定位测定和蛋白质印迹，使用商业试剂盒检测其SOD酶活性，用MTT法评估其对细胞活力的影响。在H2O2诱导的氧化应激的HEK293细胞模型中，HPP10-Cu的作用，用流式细胞术和RT-qPCR分析Zn-SOD对细胞凋亡的影响,并采用活性氧（ROS）法评估其抗氧化作用；通过RT-qPCR检测炎症因子的表达，在TPA诱导的小鼠耳部炎症模型中评估其抗炎作用。蛋白质印迹和免疫组织化学。
结果：融合蛋白hPP10-Cu，成功获得Zn-SOD。免疫荧光实验证实了这种融合蛋白在HEK293细胞中的明显膜渗透，定位在细胞膜和细胞进入后的细胞核。hPP10-Cu,浓度为5μmol/L的Zn-SOD表现出较强的抗氧化活性，但浓度高达10μmol/L时对细胞活力的影响最小。在小鼠耳部炎症模型中，融合蛋白明显抑制细胞凋亡，降低细胞内ROS水平，显著降低炎症因子的mRNA和蛋白表达。
结论：融合蛋白hPP10-Cu，能够穿透细胞膜的Zn-SOD具有很强的抗氧化和抗炎活性，细胞毒性很小，证明了hPP10作为有效药物递送载体的价值和hPP10-Cu的潜力，Zn-SOD在护肤品开发中的应用
方法：融合蛋白hPP10-Cu，Zn-SOD是通过基因工程获得的，并通过Western印迹进行鉴定。用免疫荧光法评价融合蛋白的膜穿透性,荧光共定位测定和蛋白质印迹，使用商业试剂盒检测其SOD酶活性，用MTT法评估其对细胞活力的影响。在H2O2诱导的氧化应激的HEK293细胞模型中，HPP10-Cu的作用，用流式细胞术和RT-qPCR分析Zn-SOD对细胞凋亡的影响,并采用活性氧（ROS）法评估其抗氧化作用；通过RT-qPCR检测炎症因子的表达，在TPA诱导的小鼠耳部炎症模型中评估其抗炎作用。蛋白质印迹和免疫组织化学。
结果：融合蛋白hPP10-Cu，成功获得Zn-SOD。免疫荧光实验证实了这种融合蛋白在HEK293细胞中的明显膜渗透，定位在细胞膜和细胞进入后的细胞核。hPP10-Cu,浓度为5μmol/L的Zn-SOD表现出较强的抗氧化活性，但浓度高达10μmol/L时对细胞活力的影响最小。在小鼠耳部炎症模型中，融合蛋白明显抑制细胞凋亡，降低细胞内ROS水平，显著降低炎症因子的mRNA和蛋白表达。
结论：融合蛋白hPP10-Cu，能够穿透细胞膜的Zn-SOD具有很强的抗氧化和抗炎活性，细胞毒性很小，证明了hPP10作为有效药物递送载体的价值和hPP10-Cu的潜力，Zn-SOD在护肤品开发中的应用
