Research On The Effect And Mechanism Of Ferulic Acid In Regulating HMGB1 To Interfere With Radiation Intestinal Injury

[Background]Intestine is an important target tissue of ionizing radiation injury due to its high sensitivity to irradiation.Most of the victims in nuclear emergencies including nuclear explosions and nuclear accidents,and those in abdominal pelvic radiotherapy suffered from intestinal injuries.However,there are many difficulties in the research and prevention of radiation enteropathy because of the diversity of the target and the uncertainty of the mechanism.Lacking of effective interventions prompts us to research radiation enteropathy prevention method.Ferulic acid can not only promote the proliferation of injured hematopoietic progenitor cells,but also reduced oxidative and imflammatory damage of vascular endothelial cells induced by radiation.Other research also indicated that ferulic acid can inhibit heat stress enteritis.But its effect on radiation enteropathy and protective mechanism need to be further investigated.Therefore,this work will study the pathogenesis of radiation enteropathy,determine the effect of ferulic acid on radiation enteropathy prevention,and explore the protective mechanism of ferulic acid by HMGB1,a key molecule im inflammatory.[Objective]The aim of this work was to determine the effect of ferulic acid on radiation enteropathy in vivo and in vitro,explore the biological target molecules and effector cells of ferulic acid in radiation enteropathy by bioinformatics and modern biological methods,and illustrate the mechanisms of ferulic acid regulating HMGB1 on radiation enteropathy.This study will provide scientific basis to elucidate the effect and mechanism of ferulic acid in the regulation of HMGB1 on radiation enteropathy,and develop a new potential prevention drug for radiation enteropathy.[Methods]1.Six-week-old male ICR mice were divided into normal control group,irradiation model group,positive drug control group,ferulic acid 10 mg/kg,30 mg/kg,90 mg/kg intervention group.The acute radiation sickness model of ICR mice was established by single whole body irradiation(WBI)with 5.5 Gy 60Co ? ray.The optimal dosing regimen of ferulic acid was determined by peripheral blood test Mouse bone marrow hematopoietic function was analyzed by the hematopoietic progenitor cell colony culture assay,sternal bone marrow polychromatic erythrocyte micronucleus cell rate test,flow cytometry analysis of bone marrow cell cycle and apoptosis,and Western Blot detection of Thbd protein relative expression.The effects of ferulic acid on the levels of HMGB1 and IL-6 in the serum of mice were analyzed by ELISA test.The effects of'ferulic acid on radiation enteropathy were analyzed by the histopathological manifestations of small intestine and the immunohistochemistry changes of vascular endothelial cell density in intestinal villi.The binding sites of ferulic acid and HMGB1 protein interaction were predicted by UCSF Chimera molecular modeling system and compared the sites with HMGB1 positive inhibitors'.The expression and location of HMGB1 protein in the small intestine of the irradiated mice treated with ferulic acid labeled by immunofluorescence to preliminarily confirm the predicted results of molecular fitting2.The radiation sensitivity of the HUVEC and HIEC-6 were analyzed by the cell number of the two cell lines from the first day to the 10th day after different doses of radiation through CCK-8 methods.The effector cell was determined by the viability of HUVEC and HIEC-6 cells at the 5th day after 4 Gy irradiation by CCK-8 method too.Monoclonal count,metrigel culture,flow cytometry detection of cell cycle process,ELISA analysis of HMGB1,IL-6,IL-8 and TNFa.Western Blot analysis of HMGB1,RAGE,TLR2,Thbd protein,and immunofluorescence of HMGB1 were used to study the protective effect and mechanisms of 5,50,500 nM ferulic acid on HUVEC radiation injury.The effect of ferulic acid on Thbd promoter was measured in a recombined vector by dual luciferase assay.[Results]1.Preventive plus treatment regimen of ferulic acid was more beneficial to increase the number of peripheral leukocytes and platelets in ICR mice at the 7th day after 5.5 Gy WBI.The prevention and treatment effect for acute radiation was enhanced with ferulic acid dose increased by this regimen,especially 90 mg/kg treated.Compared with the irradiated model group,90 mg/kg ferulic acid can double the bone marrow hematopoietic progenitor cells(CFU-E,BFU-E,and CFU-GM),reduce the bone marrow macrophage micronucleus by half,decrease the bone marrow cells apoptosis and arrest G2/M cell cycle.Meanwhile,it can also increase the relative expression of Thbd protein in the bone marrow,and then promote the irradiated ICR mice bone marrow hematopoietic function.90 mg/kg ferulic acid can also alleviate the inflammatory response by reducing serum HMGB1 and IL-6 level.Most importantly,compared with the radiation model group,the length of the small intestine villi and the density of vascular endothelial cells were significantly increased.This result indicated that the intestine tissue structure recovered in mice treated with 30 mg/kg and 90 mg/kg ferulic acid.Molecular fitting showed that the interaction binding sites of ferulic acid and HMGB1 were located on the A box domain of methionine and serine,which were completely the same as that of HMGB1 positive inhibitors(ethyl pyruvate and Thbd protein).The molecular fitting result predicted that ferulic acid can inhibit the secretion of HMGB1 and release the inflammatory reaction.Our result of HMGB1 expression in small intestinal of the irradiated mice treated with ferulic acid preliminarily verified this hypothesis.2.HUVEC is more sensitive to 60Co ?-ray than HIEC-6.The number of HUVEC decreased rapidly at the second day after exposured to 8 Gy ?-ray,and cell proliferation ability was completely lost at the 10th day after irradiation.The number of HIEC-6 cells decreased to the lowest at the 4th,5th,6th day after 8 Gy irradiation.At the 10th day after irradiation,the HIEC-6 cell viability was restored up to 15%of the normal control group.HUVEC cell viability was decreased about 65%at 5 days after 4 Gy irradiation compared to normal control group.500 nM ferulic acid can increase HUVEC cell viability by 13%.But ferulic acid had no effect on the cell viability of HIEC-6 dur:ing 5 nM to 500 ?M at the 5th day after sham and 4 Gy irradiation.This suggested that the vascular endothelial cell is the effector cell of ferulic acid in the protection of radiation enteropathy.The HMGB1 expression in the nucleus of HUVEC was decreased over time after 4 Gy irradiation,and its expression in cytoplasm was slightly increased,while the content of HMGB1 in the culture medium was dramatically increased(P<0.05)during the second day to the 5th day.The location of HMGB1 changed with the HUVEC radiation damage progress.Ferulic acid can promote HMGB1 return to the nucleus frorm the extracellular of HUVEC exposed to 4 Gy 60CO y-ray.This effect was enhanced with the increase of ferulic acid doses,especially the 500 nM treated group.At the same time,with HMGB1 return to the nucleus,the expression of inflammatory signal receptor RAGE and TLR2 decreased,and the inflammatory cytokines IL-6,IL-8 in HUVEC culture also reduced.These result indicated ferulic acid can alleviate HUVEC inflammatory response induced by 4 Gy ?-ray.In addition,500 nM f'erulic acid can effectively regulate the cell cycle progression,improve the ability of clone and tube formation.Moreover,ferulic acid can enhance the GLuc luciferase relative expression level of transient transfected cells by activating the Thbd promoter and then increase the expression and activity of Thbd protein.[Conclusions]Vascular endothelial cell is the effector cell of ferulic acid in the protection of radiation enteropathy.Ferulic acid can increase both the expression and activity of Thbd regulate the transfer and secretion of HMGB1 protein by activating Thbd promoter.On the other hand,ferulic acid may also inhibit HMGB1 transfer and secretion by its direct effects on HMGB1.Both two pathways of ferulic acid can regulate HMGB1 transfer and secretion,and then protect the intestinal vascular endothelial cells injury induced by irradiated,supress intestinal bleeding and ischemic inflammation,and alleviate radiation enteropathy.In addition,ferulic acid can also protect the bone marrow cells,improve hematopoietic function,and protect the irradiated mice by multi-system.Our results indicate that ferulic acid can be a potential protection drug for radiation enteropathy.