| Objectives:Skin damage caused by radiation therapy for oncology and nuclear accidents is still a serious problem and there is a lack of effective prevention and treatment methods.The aim of this study is to observe the protective effect and the underlying mechanism of 1,2-Propanediol(PPD)combined with exosomes from human dental pulp stem cells(DPSCs)modified with human hepatocyte growth factor(HGF)gene on radiation skin cell injury.Methods:1.Radiation damage to human keratinocyte(Ha Ca T)cells was induced by X-ray irradiation.The Ha Ca T cells were pretreated with different concentration of PPD two hours before irradiated and then after irradiation the radioprotective effect of PPD was evaluated by measuring cell proliferation viability using CCK-8 assay,apoptosis by flow cytometry,and cell migration by cell scratch assay.The m RNA expression levels of inflammatory factors of TNF-α,IL-1β and IL-6 were detected using real time PCR.The cellular reactive oxygen species(ROS)was detected using fluorescence microscopy,and the cellular oxidative stress damage response was evaluated by the cellular content of malondialdehyde(MDA),glutathione(GSH)and superoxide dismutase(SOD).Furthermore,the expression of nuclear factor erythroid 2 related factor-2(NRF2)and NAD(P)H quinone oxidoreductase 1(NQO1),the key proteins in the oxidative stress pathway,was detected with western blot method.2.After infection of human DPSCs using Ad.HGF virus,the cultures were collected and Ad.HGF DPSC-Exo were isolated by ultracentrifugation.The number and size of Ad.HGF DPSC-Exo particles were detected by nanoparticle tracking analysis(NTA)and the surface markers were identified by western blotting.Furthermore,the expression of HGF in Exo and DPSCs infected with Ad.HGF was determined by PCR and western blotting respectively.3.After pretreatment with PPD for 2 hours,Ha Ca T cells were irradiated 10 Gy with X-ray,and then the cultures immediately were changed into fresh complete media.Thereafter,the irradiation protection of PPD combined with exosomes derived from DPSCs modified with HGF was evaluated through cellular proliferation,apoptosis and migration,as well as the expression of P53 and P21.Results:1.The irradiation protection of PPD: the results showed that pretreatment of Ha Ca T cells with PPD before X-ray irradiation yielded a good radiation protection,with a significant improvement of cell proliferation and migration as well as a decrease of apoptosis.In addition,PPD significantly reduced the expression of intracellular inflammatory factors,and the level of ROS and MDA,but promoted the production of intracellular antioxidant GSH and antioxidant enzyme SOD.Moreover,PPD also promoted the expression of NRF2 as well as NQO1,a downstream antioxidant protein.In conclusion,pretreatment with PPD produced a significant radiation protective effect on radiation-induced Ha Ca T cell damage by activating the antioxidant signal pathway and the antioxidant system to eliminate the redundant radicals,and it may be used to prevent radiation-induced skin damage.2.The separation and determination of exosomes: the results analyzed by NTA showed that the diameter distribution of the particles separated from the cell cultures was concentrated between 30 and 150 nm;in addition,the expression of exosome markers CD9、CD63 and TSG101 was detected in the obtained Exo.Moreover,the high expression of HGF in the exosome and DPSCs infected with Ad.HGF was confirmed with PCR and western blot methods,indication that HGF could be expressed through exosomes.3.The irradiation protection of PPD combined with Ad.HGF DPSC-Exo: the results demonstrated that both PPD pretreatment and Ad.HGF DPSC-Exo treatment generated a good irradiation protection to Ha Ca T cells,displaying by the improvement of cell proliferation and migration ability,as well as the decrease of apoptosis.And the protection effect was more significant in the group of PPD pretreatment combined with Ad.HGF DPSC-Exo treatment.In addition,a significant alleviation of the cellular G2/M phase block and the expression of cell cycle gene P53 and P21 was detected in the group of PPD pretreatment combined with Exo treatment.Conclusion : The results of this study demonstrated that PPD pretreatment effectively protected the radiation injury of Ha Ca T cells by activating the antioxygen signal pathway and antioxygen system to alleviate the redundant free radicals;gene modified stem cells increased the expression of HGF in exosomes;both PPD pretreatment and Ad.HGF DPSC-Exo treatment had significant protective effects on radiation-induced skin cell injury,and the protective effects was the best in the group of PPD combined with Ad.HGF DPSC-Exo.Therefore,this study provided a novel ideas and potential methods for the prevention and treatment of radiation skin injury. |
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