Ferulic Acid Interferes With Radioactive Intestinal Injury Through The DJ1-Nrf2 And Sirt1-NF-κB-NLRP3 Pathways

Objective:Radiation-induced intestinal injury mainly occurs in nuclear accidents or radiotherapy for abdominal and pelvic malignant tumors.Acute injuries that occur early of radiation are mainly related to oxidative stress,changes in gene expression,and inflammatory damage.Because of radiation damage to the intestine,there is no exact pathogenesis and its protective drugs.The preliminary study of our group showed that Ferulic acid（FA）,as an effective component of Siwu Decoction of Traditional Chinese medicine,can improve the survival rate of irradiated mice,improve the hematopoietic function of bone marrow,and has anti-radiation effects such as anti-oxidation and anti-inflammation,and can intervene the radiation intestinal injury by regulating HMGB1.Therefore,the potential of ferulic acid in the prevention and treatment of radiation-induced intestinal injury remains to be further developed.In this study,cells and animal models of radioactive intestinal injury were firstly established to further explore the new pathway of ferulic acid in the treatment of radioactive intestinal injury,providing data support for the treatment of radioactive intestinal injury.Methods:（1）HIEC6 cells were irradiated with different doses of ⁶⁰Coγ-rays at a single time（the dose rate was 64.46c Gy/min）,the morphology of HIEC6 cells was observed under a microscope,and the cell viability was detected by CCK-8 method;ELISA was used to detect the level of IL-1βin the cell supernatant,and the fluorescent probe DCFH-DA was used to detect the intracellular reactive oxygen species（ROS）,so as to establish a radiation-induced intestinal injury cell model.（2）Different concentrations of ferulic acid were used to pretreat HIEC6cells.Cell viability was determined by CCK-8 method and IL-1βlevel in cell supernatant was determined by ELISA method,so as to select the optimal concentration of ferulic acid;（3）The protein expression level of NLRP3 in HIEC6 cells was detected by immunofluorescence method,the mRNA levels of Sirt1,NLRP3,Csapase-1 and IL-1βwere detected by Q-PCR,and DJ1,Nrf2 and Sirt1,NF-κB,p-NF-κB,NLRP3,Caspase-1,Cleaved-Caspase-1,IL-1βprotein expression levels were detected by Western Blot;（4）The abdominal cavity of mice was shields with ⁶⁰Coγ-ray with a dose rate of 96.72c Gy/min of 11Gy,and the mouse intestinal tissue was collected after irradiation.The thiobarbituric acid（TBA）method was used to detect malondialdehyde（MDA）in the intestinal tissue.)content,water-soluble tetrazolium（WST-8）method to detect superoxide dismutase（SOD）activity,ELISA method to detect the expression of TNF-α,IL-6 inflammatory factors,HE staining to observe the small intestinal tissue damage,in order to establish an animal model of radiation-induced intestinal injury;（5）The expression level of NLRP3protein in the intestinal tissue of mice after irradiation was detected by immunofluorescence method,and the expression levels of NLRP3,IL-1βand IL-18 were detected by ELISA method;（6）The mRNA and protein expression levels of Sirt1,NF-κB,NLRP3,Caspase-1,IL-1β,IL-18 were detected by Q-PCR and Western Blot.Results:（1）Through the observation of cell morphology, CCK-8,IL-1βand ROS detection results,10Gy ⁶⁰Coγ-ray was established as the best model of radiation oxidation and inflammatory injury in HIEC6 cells;（2）The CCK-8 method was used to detect the viability of HIEC6 cells pretreated with ferulic acid and the secretion of IL-1βin the supernatant of the cells,and the optimal dosage of ferulic acid was established to be 0.01,0.1,and 1μmol/L;（3）The results of immunofluorescence experiments showed that 0.1 and 1μmol/L ferulic acid could significantly reduce the expression level of NLRP3 protein in HIEC6 cells after irradiation;Q-PCR results showed that ferulic acid could significantly improve radiation-induced Sirt1 mRNA.The level of NLRP3,Caspase-1,IL-1βmRNA increased.Western Blot results showed that ferulic acid could significantly increase the protein expression levels of DJ1 and Nrf2,increase the protein expression level of Sirt1,and decrease the protein expression levels of p-NF-κB,NLRP3,Cleaved-Caspase-1 and IL-1β;（4）Ferulic acid can improve the levels of MDA and SOD antioxidant indexes and inflammatory factors TNF-αand IL-1βin small intestine tissues of mice after irradiation,and improve the pathological changes of small intestine in mice caused by irradiation.Alleviate radiation-induced oxidative and inflammatory damage to the small intestine;（5）Ferulic acid can play an antioxidant role by increasing the mRNA and protein expression levels of DJ1 and Nrf2;（6）Ferulic acid can reduce NLRP3 fluorescence intensity,increase Sirt1 mRNA and protein expression levels,reduce NF-κB,NLRP3,Caspase-1,IL-1β,IL-18 mRNA levels and protein expression level and exert an anti-inflammatory effect.Conclusion:This study preliminarily verified the protective effect of ferulic acid on radiation-induced HIEC6 cells and mouse small intestinal tissue oxidative and inflammatory damage from in vitro and in vivo experiments,which may be through the DJ1-Nrf2 and Sirt1-NF-κB-NLRP3 pathways realized.