| As the incidence rate of intestinal cancer increases year by year,the intestinal injury accompanied by radiation therapy in the treatment of intestinal cancer is becoming more frequent.At present,comprehensive therapy is often used for the treatment of acute radiation-induced intestinal injury,but the targeted and specific drugs are almost scare,and the curative effect is barely satisfactory.Accordingly,the discovery of therapeutic targets for acute radiation-induced intestinal injury is extremely important.Studies have shown that apoptosis is an important mechanism for radiation enteritis.Small intestinal stem cells exist at the bottom of the small intestine crypt,and they can differentiate into all other cells of the small intestinal epithelium about 3 to 5 days.The stem cells and epithelial cells are very sensitive to radiation,and they will undergo apoptosis after irradiation soon.Therefore,the prevention or reduction of apoptosis may be an effective treatment target for acute irradiation-induced intestinal injury.It has been reported that glucagon-like peptide-2（GLP-2）can repair acute radiation-induced intestinal injury.However,our preliminary study found that the rats suffered from acute severe irradiation intestinal injury died earlier and more frequent,when administrated with GLP-2immediately after irradiation This result indicated that GLP-2 treatment alone had poor effect.caspase-3 plays a very important role in the process of apoptosis.The chemical structure of Ac-DEVD-CHO contains an aldehyde group.After the addition of an oxidant,the carbon-hydrogen bond in the aldehyde group is broken,and the oxygen atoms in the oxidant are composed of carbon and hydrogen,respectively.Covalent bond of 8 electrons to form a more stable carboxyl group,which plays a better role in the body Therefore,the combination of GLP-2 and caspase-3 inhibitor Ac-DEVD-CHO was used in this study to explore its protective effect and mechanism.Finally,the combination of GLP-2 and caspase agonist PAC-1 provided negative demonstration.1.Establishment of an acute radiation-induced intestinal injury modelAfter anesthesia,the rats were fixed on homemade wooden boards,exposed to the abdomen,and the lead bricks were shielded from other parts.A single abdominal irradiation was performed with 60Coγ-rays at a dose of 15Gy to establish the model of acute radiation-induced intestinal injury.IEC-6 cell line was irradiated with 60Coγ-rays at a dose of 10Gy to establish the model of acute radiation-induced intestinal injury.2.Study on the protective effect of combination of GLP-2 and Ac-DEVD-CHO on acute radiation-induced intestinal injurySeventy-five Wistar rats were randomly divided into five groups:control group（group C）,simple irradiation group（group R）,GLP-2group（group G）,GLP-2 combined agonist PAC-1 group（group P）and GLP-2 combined inhibitor Ac-DEVD-CHO group（group D）,with 15rats in each group.Physiological saline solution,GLP-2 solution,PAC-1 solution and Ac-DEVD-CHO solution were administered immediately after irradiation.The rats’general state,body weight and mortality of each group were recorded in detail during the experiment.The rats were sacrificed at 1d,3d,5d and 14d after irradiation,and the length and weight of the intestine were recorded.After HE staining,the structural changes of jejunum,duodenum and ileum were observed,and the length of villus and the depth of crypt in jejunum were analyzed.Immunohistochemistry was used to detect the expression of Bmi-1 in jejunum tissuesIEC-6 cell line was selected.GLP-2 solution,PAC-1 solution and Ac-DEVD-CHO solution were administered 1h before irradiation,and cell counting kit CCK8 was used to detect proliferation of IEC-6 cells at3h,6h and 24h after irradiation.3.Mechanism of combined application of GLP-2 and Ac-DEVD-CHO for acute radiation-induced intestinal injuryIn situ end labeling and immunoblotting were used to detect apoptosis and expression of cleaved caspase-3 and PARP-1 in jejunum tissues.Finally,flow cytometry was used to detect the apoptosis of IEC-6 cells at 3h after irradiation.Experimental results:1.Study on the protective effect of combination of GLP-2 and Ac-DEVD-CHO on acute radiation-induced intestinal injuryThe combination of GLP-2 and Ac-DEVD-CHO could significantly improve the survival rate of rats after irradiation:The rats of group R began to die on 10 days after irradiation,the mortality rate was 15%;the rats of group G died on 4 days after irradiation;the rats of group P began to die on 1 day after irradiation,and the mortality rate was highest;while there was no death in group D.The combination of GLP-2 and Ac-DEVD-CHO could alleviate the intestinal damage of rats after irradiation:On 1 day after irradiation,group R showed a large number of apoptotic cells.Compared with group R,group G was lighter and group P was heavier,while group D had the least damage in all groups.On 3 days after irradiation,group R had shortened,lodging,and missing villus,and groups G and R had lighter damage of villus compared with group R,and group D had the lightest damage in all groups and recovered best.On 5 days after irradiation,the injury and length of the intestines in group R recovered,the group G was lighter than the group R,the group P was heavier than the group R,and the D group was the lightest in all groups,and the recovery was the best.On 14 days after irradiation,the injury and length of the intestines in the group R recovered normally,the group G recovered better than the group R,the group P returned to normal,and the group D recovered better than the group C,the group R and the group P.The combination of GLP-2 and Ac-DEVD-CHO could alleviate the ultrastructural damage of rat jejunal tissue after irradiation:on the 3rd day after irradiation,group R showed apoptotic cells,missed microvilli,cavitated mitochondria and broken crista.The damage in group G was lighter than that in group R.The injury group P was heavier than that group R,and the damage of group D was the lightest.The combination of GLP-2 and Ac-DEVD-CHO reduced apoptosis of rats jejunal tissue after irradiation:on the 1st day after irradiation,the number of apoptotic cells in group R was significantly higher than that in group C,and the number of apoptotic cells in group G was lower than that in group R.Group P was significantly higher than group R,group D was lower than group R and group P,but higher than group G;on the3rd day after irradiation,the number of apoptotic cells in group R was significantly higher than that in group C,and group G was lower than group R.Group P was close to group R,group D was significantly lower than group R;on the 5th day after irradiation,the number of apoptotic cells in group R was significantly higher than that in group C,group G was lower than group R,group P was higher than group R,group D was significantly lower than the group R;on the 14th day after irradiation,the number of apoptotic cells in the R group was higher than that in the C group,the group G was lower than the R group,P group was lower than the R group,and the D group was significantly lower than group R.The combination of GLP-2 and Ac-DEVD-CHO increased expression of Bmi-1 in intestinal tissues after irradiation:On 1st day after irradiation,the expression of Bmi-1 in group R was significantly lower than that in group C.Compared with group R,group G was significantly elevated,group D was significantly elevated.Group G and group D were significantly lower than group C.On the 3rd day after irradiation,the expression of Bmi-1 in group R was significantly lower than group C.Compared with group R,group G was significantly increased,and group D was significantly lower than group C.On the 5th day after irradiation,group R decreased compared with the C group;group D significantly increased compared with group R,and group P was significantly lower than group C.On the 14th day after irradiation,group R decreased compared with group C;the expression of Bmi-1 in group D was significantly increased compared with group R.The combination of GLP-2 and Ac-DEVD-CHO increased the proliferation activity of IEC-6 cells after irradiation:At 3h after irradiation,the proliferation activity of group R decreased compared with group C;compared with group R,the proliferation of group G increased significantly,the proliferation activity of group P was significantly decreased,the proliferation activity of group D was significantly decreased,and the proliferation of group P and group D was also lower than that of group C.At 6h after irradiation,the cell proliferation activity of group P was significantly lower than that of group C and group R.Compared with the C group,the R group decreased at24h after irradiation.Compared with group R,both group G and group P were significantly decreased,and the cell proliferation activity of group D was significantly increased,which was also significantly higher than that group C.2.Mechanism of combined application of GLP-2 and Ac-DEVD-CHO for acute radiation-induced intestinal injuryThe combination of GLP-2 and Ac-DEVD-CHO affected the expression of cleaved caspase-3 and PARP-1 in jejunum after irradiation:on the 1st day after irradiation,the expression of cleaved caspase-3 in the jejunum of group R was higher than that of group C,and the expression of group D was significantly higher than that of group C.On the 3rd day after irradiation,the expression of group R was decreased,and the expression of group G,group P,and group D was significantly decreased.On the 14th day after the irradiation,there was no difference in the expression of each group.On the 1st day after irradiation,the expression of PARP-1 was increased compared with group C.the expression of groups P and D were both significantly increased compared with group R,On the3rd day after irradiation,the expression of group R was significantly higher than that of group C.On the 14th day after irradiation,the expression of group P was significantly higher than that of group R.The combination of GLP-2 and Ac-DEVD-CHO reduced the percent of apoptosis of IEC-6 cells after irradiation:At 3h after irradiation,the early apoptotic rate of IEC-6 cells in group R was significantly higher than that in group C.Compared with group R,both group G and group D decreased,but there was no statistical difference,and group P was higher than group R.At 3h after irradiation,the late apoptotic rate of IEC-6 cells in group R was significantly higher than that in group C;group G was significantly lower than group R.Conclusion1 The combination of GLP-2 and Ac-DEVD-CHO had a protective effect on acute radiation-induced intestinal injury,and the effect was significantly better than that of GLP-2 alone;2 The combination of GLP-2 and Ac-DEVD-CHO may protect against the expression of cleaved caspase-3 and cleaved PARP-1. |
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