| Research Background and Purpose:Because the wounds that are difficult to heal have a great negative impact on the body and mind of patients how to promote the healing of wounds is a hot issue in modern medical research.Wound healing is a complex process consisting of inflammation,proliferation and remodeling,which is accomplished through epithelialization of epidermal cell regeneration.When the skin is damaged,a hypoxic microenvironment is formed on the wound,leading to the secretion of a variety of factors that promote tissue repair.A large number of studies have shown that the initiation of wound healing is related to hypoxic microenvironment,among which the effect of hypoxia on epidermal cell migration has been widely confirmed,but its specific mechanism remains unclear.Clarifying the mechanism of hypoxia promoting cell migration is crucial for understanding the physiological process of wound healing,and studying the molecular mechanism of clinically applied therapeutic means(hypoxia environment caused by short-term application of semi-permeable dressing in the early stage of acute wound formation can promote wound healing),so as to provide a theoretical basis for more accurate and efficient treatment.Therefore,this study continued our previous exploration of the possible mechanism of short-term hypoxia promoting cell migration through high-throughput gene detection,and was verified in vitro and in vivo experiments.Research methods:1.Cell experiments:(1)The cell scratch experiment was used to observe the migration ability of Ha Ca T cells after short-term hypoxia at different time points(0,3,6,12,24h).(2)The proliferation ability of Ha Ca T cells after short-term hypoxia at different time points(0,3,6,12,24h)was observed by CCK8 experiment.(3)Enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of TNF-α and CCL2 in the supernatant of Ha Ca T cells at different time points of short-term hypoxia(0,3,6,12,24h).(4)Western blotting was used to detect the expressions of TNF-α signaling pathway-related proteins(p-NF-κB,p-P38 and p-ERK1/2)and migration-related proteins(N-cadherin and E-cadherin)in Ha Ca T cells after short-term hypoxia at different time points(0,3,6,12,24h).(5)Construct interference and overexpression CCL2 cells,verify the construction of lentivirus cells at the transcription level and protein expression level,and observe the changes of cell migration ability under short-term hypoxia conditions(0,3,6,12,24h)using stable cells.(6)The changes of ERK protein in Ha Ca T cells were observed by TNF-α stimulation.(7)The migration ability of Ha Ca T cells and Ha Ca T cells with overexpression of CCL2 was observed under short-term hypoxia conditions(0,3,6,12,24h)after the addition of ERK inhibitor by cell scratch experiment.2.Animal experiments(1)BALB/c male mice were randomly divided into blank control group and ERK inhibitor group.A full-layer skin injury wound with a diameter of 18 mm was formed on the back of Balb/C male mice.In the ERK inhibitor group,FR180204 was injected around the wound,and wound healing was recorded immediately after injury(0),3,6,9,12,15 days.(2)HE staining was used to observe neovasculum formation,inflammatory cell infiltration and epidermis formation on day 1,3,6 and 15 of blank control group and ERK inhibitor group,and Masson staining was used to observe collagen deposition on day 1,3,6and 15 of blank group and inhibitor group.(3)Western blotting was used to observe the protein expression of p-NF-κB,p-P38,p-ERK1/2,N-cadherin and E-cadherin in the wound tissue of blank control group and inhibitor group at 1,3,6 and 15 days.(4)Immunohistochemical staining was performed to detect the number of positive cells of Ki-67 and the average optical density value of VEGF in the wound tissue of blank control group and ERK inhibitor group at 1,3,6,and 15 days,and to observe the cell proliferation and angiogenesis at different time points in wound healing.(5)ELISA was used to detect the expressions of cytokines(IL-10,IL-1β,IL-6)and chemokines(CCL20)in the wound tissue of blank control group and ERK inhibitor group at1,3,6 and 15 days.Result:1.Cell experiments:(1)ELISA results showed that the expression of CCL2 and TNF-α increased significantly after 24 h hypoxia.(2)The cell scratch test results showed that the migration ability of Ha Ca T cells was gradually enhanced in a time-dependent manner after hypoxia treatment,but the migration ability of Hacat cells was decreased after hypoxia for more than 24 h.(3)CCK8 experiment showed that hypoxia had little effect on cell proliferation;(4)Western blotting showed that p-NF-κB,p-P38,p-ERK1/2 and N-cadherin increased with the increase of time,while E-cadherin decreased significantly.(5)By constructing Ha Ca T cells with interference/overexpression of CCL2,it was found that under short-term hypoxia conditions,the migration ability of cells with overexpression of CCL2 was stronger than that of cells with interference of CCL2,but this enhanced migration ability was reduced by ERK inhibitors.At the same time,the enhanced migration ability of normal Ha Ca T cells under short-term hypoxia condition was also reduced by ERK inhibitor,and ERK protein could be activated by TNF-α.2.Animal experiments:(1)The results of animal experiments showed that the inhibitor group significantly inhibited wound healing compared with the blank control group;(2)HE and Masson staining showed that,compared with blank control group,inflammatory cell infiltration increased and collagen deposition decreased in inhibitor group.(3)Western blotting showed that compared with the blank control group,the expressions of p-NF-κB,p-P38,p-ERK1/2 and N-cadherin in the wound tissue of the inhibitor group were decreased,while the expression of E-cadherin was increased.(4)Immunohistochemical staining showed that the expressions of Ki-67 and VEGF were decreased in the inhibitor group;(5)ELISA results showed that the expressions of IL-6 and IL-1β were increased and the expressions of IL-10 and CCL20 were decreased in the inhibitor group compared with the blank control group.Conclusions:1.Under short-term hypoxia conditions,the expressions of CCL2 and TNF-α in Ha Ca T cells were increased,and the migration ability of Hacat cells was enhanced,but there was no significant change in cell proliferation.Meanwhile,the downstream protein ERK of the TNF-α signaling pathway was significantly increased.2.CCL2 promoted the migration of Ha Ca T cells in the condition of short-term hypoxia,and TNF-α activated ERK protein in Ha Ca T cells.However,the migration ability of overexpressed CCL2 cells and normal Ha Ca T cells in the condition of short-term hypoxia was weakened by ERK inhibitors,suggesting that in the condition of short-term hypoxia,CCL2 and TNF-α signaling pathways are involved in the regulation of Ha Ca T cell migration.3.The ERK signaling pathway can affect the migration of wound cells,regulate the healing of full-thickness skin defects in mice,and affect the expression of inflammatory factors and chemokines in the wound of mice. |
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