The Role Of TSP-1 In The Healing Process Of Combined Radiation-wound Injury

Background:Combined Radiation-Wound Injury（CRWI）is one of the main injury types in activities such as nuclear accident and nuclear war,and can also be seen in postoperative radiotherapy for tumors.In the case of public health emergency radiation exposure,CRWI account for approximately 65%of nuclear explosion injuries and significantly increase morbidity and mortality compared to radiation injuries alone.Wound healing is a complex and coordinated process,which can be divided into three distinct but interrelated stages:inflammatory response,proliferative repair and tissue remodeling.Under the influence of radiation,after CRWI is formed,the inflammatory response in wound healing is weakened,proliferation and repair are slowed down,and tissue remodeling process is inhibited,wound healing is very difficult.The mechanism of the difficult healing is complex and has not been fully clarified,which requires us to continue to explore.Thrombospondin-1（TSP-1）is a multifunctional extracellular matrix protein that is involved in physiological and pathological processes such as inflammation regulation and angiogenesis,and plays an important role in wound repair.The present study shows that TSP-1 plays a different role in different wound models.TSP-1 is also sensitive to radiation.However,its current role in the healing process of CRWI remains unclear.Therefore,this study investigated the role of TSP-1 in the healing process of CRWI,in order to provide experimental basis for the treatment of CRWI.Method:In this study,a mouse skin CRWI model of 5 Gy ⁶⁰Coγwhole body irradiation combined with a single wound diameter of 1 cm was constructed to investigate the healing state and inflammatory response of mouse skin CRWI.The expression level of TSP-1 in mouse skin CRWI was detected by RT-q PCR and immunofluorescence staining.Secondly,taking TSP-1Knock out（KO）mice as the research object,the CRWI healing of TSP-1KO mice and Wild type（WT）mice was compared.On the 7th day after the formation of CRWI in the skin of mice,the wound tissues of TSP-1KO mice and WT mice were selected for F4/80immunohistochemical staining.The expression level of NF-кB protein was analyzed by WB,and the expression levels of cytokines IL-1β,IL-6 and TNF-αwere detected by RT-q PCR.To determine the inflammatory status of CRWI wound site in the skin of mice in both groups.On the 7th day after the formation of skin CRWI in mice,CD31 immunohistochemical staining andα-SMA immunofluorescence staining were performed on the wound tissues of TSP-1KO mice and WT mice to determine the angiogenesis status of skin CRWI in the two groups.In order to further understand the role of TSP-1 in different stages of skin CRWI healing in mice,a mouse model treated with TSP-1 inhibitor was constructed to inhibit the expression of TSP-1 in the early stage of skin CRWI healing（day 0-3）and in the middle and late stages of skin CRWI healing（day 6-10）,wound healing in TSP-1 inhibitor group and control group was recorded.HE staining,F4/80 immunohistochemical staining and NF-кB immunofluorescence staining were performed on the wound tissues of mice in TSP-1inhibitor group and control group in the early healing stage on the 3rd day.Elisa kit was used to detect the expression levels of cytokines IL-1β,IL-6 and TNF-αat the wound site of the two groups.The purpose of this study was to evaluate the wound healing status and the inflammatory response status of skin CRWI in mice after inhibiting the expression of TSP-1in the early stage of healing;HE staining,CD31 immunohistochemical staining andα-SMA immunofluorescence staining were performed on the wound tissues of mice in the TSP-1inhibitor group and control group in the middle and late healing stages on the 10th day.The expression levels of VEGF,PDGF andβ-FGF in the wound sites of the two groups were detected by RT-q PCR.The purpose of this study was to evaluate the wound healing status and angiogenesis of skin CRWI in mice after inhibition the expression of TSP-1 in the middle and late stages of healing.Results:In this study,TSP-1KO mouse model and TSP-1 inhibitor treated mouse model were used to verify the role of TSP-1 in the healing process of skin CRWI in mice.The results showed that:1.Compared with simple skin wounds of mice,skin CRWI of mice treated with 5 Gy⁶⁰Coγwhole body irradiation combined with a single wound diameter of 1 cm was more difficult to heal,and the inflammatory response of mice was weakened.The results of RT-q PCR and immunofluorescence staining showed that the expression of TSP-1 in the wound site of mouse skin CRWI was lower in the early stage and higher in the middle and late stages than in the simple wound site of mouse skin.2.CRWI healing was delayed in TSP-1KO mice compared with WT mice.On the 7th day after the formation of CRWI in the skin of mice,there was less infiltration of F4/80macrophages at the wound site of TSP-1KO mice,and the expression levels of NF-кB protein and inflammatory cytokines IL-1βand IL-6 were also relatively low.Moreover,the number of CD31 positive cells andα-SMA positive cells decreased at the wound site.3.The expression of TSP-1 was inhibited in the early stage of skin CRWI healing,and the wound healing was delayed in the TSP-1 inhibitor group,and the infiltration of F4/80macrophages at the wound site was less,the expression of NF-кB protein was lower,and the expression levels of inflammatory cytokines IL-1βand IL-6 were also decreased.The expression of TSP-1 was inhibited in the middle and late stages of skin CRWI healing in mice.The wound healing was accelerated in the TSP-1 inhibitor treatment group,and the number of CD31 andα-SMA positive cells at the wound site increased,as well as the expression levels of angiogenesis related cytokines VEGF,PDGF andβ-FGF were also increased.Conclusion:1.The expression level of TSP-1 in CRWI healing was different from that in simple wounds.Weakened inflammatory response is an important reason for the difficulty of CRWI wound healing.Knockout of TSP-1 can reduce the level of inflammatory response in the early stage of CRWI healing and delay the wound healing,suggesting that TSP-1 plays an important role in the healing process of CRWI.2.TSP-1 plays different roles in the early and the middle and late stages of skin CRWI healing in mice.In the early stage of skin CRWI healing,TSP-1 can induce inflammatory cell formation,promote cytokine release,and accelerate wound healing.TSP-1 can inhibit angiogenesis and delay wound healing in the middle and late stages of skin CRWI healing in mice.