MitoQ Protects Intestinal Barrier From Ischemia/Reperfusion Damage Through Suppression Of Mitochondrial DNA Oxidative Injury

Objective:Ischemia-reperfusion(I/R)injury is a clinical challenging problem,especially those involving the gastrointestinal tract.Actually,the small intestine is believed to be themost vulnerable tissue induced by I/R.I/R injury to intestinal epithelium occurs in trauma,shock,especially infections that may induce hypoperfusion.Intestinal I/R injury can not only cause local damage of intestinal tissue,but also has been related to loss of gut barrier function and the ensuing translocation of bacteria and endotoxin from the gut to the portal and systemic circulations,leading to sepsis and shock,further damaging the liver,lungs,kidneys and other organs,and ultimately leading to death.Methods:1.The mice intestine was suffered from regional ischemia for 30 minutes,followed by 6 hours' reperfusion to establish intestinal I/R-injury models in vivo.The IEC-6 cells were subjected to hypoxia for 6 hours and followed by reoxygenation for 12 hours to establish intesitinal I/R-injury models in vitro.To found out wheather I/R-injury related to mtDNA damage,the 8-hydroxyguanine(8-OHdG)was detected through immunostaining,and the mtDNA content and mt DNA transcription levels were detected through real time PCR both in vivo and in vitro.2.We detected the mitochondrial membrane potential(??m)and ATP level of cardiomyocytes both in vivo and in vitro to ensure if mitochondrial dysfunction related to I/R-injury in our models.3.To find out wheather MitoQ can protect enterocyte from I/R by inhibiting mtDNA damage.HE staining was used to detect the injury level of intestine.We analysed the levels of apoptosis through terminal deoxynucleotidyl transferase d UTP nick end labeling(Tunel)staining.Apoptosis was also examined with FITC-labeled Annexin V/propidiumiodide(PI)Apoptosis Detection kit(BD Biosciences,New Jersey,USA)following the manufacturer's instructions.At the same,the 8-OHdG,mtDNA content,mtDNA transcription levels,??m and ATP levels of enterocyte were also detected at the same time to ensure the protective effect of lycopene on mtDNA and mitochondrial function.4.To o further explore the protective mechanism of lycopene on mtDNA,malondialdehyde(MDA),reactive oxygen species(ROS)and mitochondrial superoxide levels were determined.Result:1.The number of 8-OHdG-positive cells significantly increased in the I/R-injured IEC-6 cells.Most of the 8-OHdG co-localized with mitochondria.PCR results indicated that both I/R-treatment in vivo and in vitro decreased mtDNA copy number and mtDNA transcripts significantly as compared with control.These results suggested that I/R-injury is related to mt DNA damage.2.ATP concentrations were significantly decreased in the I/R groups both in vivo and in vitro compared with controls.In I/R-treated IEC-6 cells the ??m also decreased.These results proved that I/R caused enterocyte mitochondrial dysfunction.3.MitoQ pretreatment can help to rescue Intestinal mucosal damage cause by I/R injury.We also found lycopene pre-treatment significantly reduced Tunel-positive enterocyte both in vivo and in vitro.Pre-treatment with MitoQ aslo reduced cleaved caspase-3 expression as compared to I/R group.The results of the Annexin V/PI analysis with flow cytometry confirmed that MitoQ reduced H/R-induced cell death.At the same time,we noticed lycopene signicantly prevented I/R indued 8-OHdG-positive cells increase and mtDNA copy number and mt DNA transcripts decrease.MitoQ also prevented I/R indued ??m and ATP decrease both in vivo and in vitro.MtDNA is very susceptible to oxidative stress.In our experiments,we found that pre-treatment with MitoQ significantly reduced the elevated MDA production and ROS generation induced by I/R both in vivo;and in vitro.Conclusions:Inconclusion,this study suggested that mtDNA oxidative damage is involved in intestinal I/R-injury.MitoQ has a pharmacological potential in the treatment intestinal I/R-injury by protecting enterocyte mt DNA from oxidative damage.