| Objective With the development of medical imaging technology,the radiotherapy of tumors has developed rapidly,and many advanced radiotherapy techniques are widely used in clinical practice.While killing the tumor,the normal tissues in the irradiated area also appear acute and chronic injuries.Intestinal mucosal tissue proliferation faster,so,the intestinal become the most sensitive organ to prone to radioactive injury.Ubiquitin is a small molecule protein widely existed in eukaryotes with 76 amino acid residues.Its amino acid sequence is extremely conservative and its primary structure is almost the same in different species of higher organisms.Studies have shown that extracellular ubiquitin has anti-inflammatory properties,which can be used as an immunomodulatory.Ubiquitin also has been shown to suppress inflammation and reduce tissue damage in vivo and tissue damage models.However,the relationship between extracellular free Ub and ionizing radiation,and the effect of ionizing radiation on intestinal cells under the action of free Ub have not been reported in the literature.Thus,we envisage whether free Ub can reduce the radiation-induced intestinal damage and repair the damage.Methods Cell viability was by MTT assay.Cell membrane integrity and cell proliferation were detected by LDH and EDU.Cell number and cell migrate were detected by hand held cell counter and wound healing assay.The Intestinal cell mitochondrial function after irradiation were detected by JC-1kit.Apoptosis was detected by flow cytometry.The differences signal pathin intestinal tissue of c57 mice between free Ub intraperitoneal injection group and control group was detected by bioinformatics analysis;Luminex method for high-throughput detection of cytokine changes in serum of free Ub pretreated and control mice.Western blot was used to detect the expression of active factor-related protein after intestinal cell irradiation;the effect of supernatant of intestinal cell culture medium on cell viability was detected by MTT assay and the effect of free Ub pretreatment on the activity of active factors in the supernatant of intestinal cell culture medium was detected by ELISA kit.LDH kit was used to detect the release of lactate dehydrogenase from free Ub combined SDF-1α after irradiation,and the effect of free Ub combined with SDF-1α on the proliferation of intestinal cells after irradiation.Results Compared with the control group,IEC-6 cells pretreated with free Ub increased the viability of IEC-6 cells,decreased the content of extracellular lactate dehydrogenase,and also increased the cell proliferation ability and the number of cells.After pretreatment with free Ub,the migration of intestinal cells accelerated,mitochondrial function was more complete,but the inhibitory effect on intestinal apoptosis was not obvious.By bioinformatics analysis,secretory pathway were detected in small intestine of free Ub intraperitoneal injection group and control group of c57 mice,and the most significant differences cytokines FGF2 were found in blood of mice by high-throughput analysis,and the protein was detected by Western blot in free Ub pre-irradiated intestinal cells.MTT assay showed that the cell viability of gut cells pretreated with supernatant of intestinal cell culture increased after irradiation,and ELISA detected that free Ub promoted the extracellular secretion of extracellular FGF2 by irradiated intestinal cells.SDF-1α treatment of intestinal epithelial cells alone can promote cell viability,and combined with free Ub can effectively reduce cell LDH release after irradiation,and also reduce irradiation-induced cell necrosis.The combination of free Ub with SDF-1α or free Ub alone both could improve the proliferation of intestinal cells,but non-irradiation free Ub combined with SDF-1α could promote cell proliferation compared with free Ub alone.The effect of free Ub alone treatment group on the proliferation of intestinal cells was more obvious,and the expression of intracellular free Ub protein was more.Conclusion Free Ub preconditioning can effectively enhance the cell viability,cell proliferation and the cell migration after ionizing radiation.AT the same time free Ub preconditionin also reduced the damage of cell membrane and protects the loss of mitochondrial function after irradiation.The mechanism may be related with free Ubinduced protein expression of intracellular fibroblast growth factor FGF2 and extracellular secretion.This study revealed the role of free Ub activation of FGF2 of intestinal cells on radiation injury,but also provided a new method for the treatment of radiation-induced intestinal injury. |
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