Effects Of Quercetin On Methamphetamine-induced Oxidative Injury Of MODE-K Cells Based On Nrf2 Pathway

Objective:Methamphetamine(METH)has become the most widely consumed narcotics in China,which seriously threatens social stability and brings heavy medical burden.Enterogenous infection caused by complications such as intestinal bleeding and obstruction is characterized by high incidence and high sudden death rate,which is not only a social problem but also a public health problem.The pathogenesis of intestinal injury complications caused by methamphetamine is extremely complex,and oxidative stress injury is one of the main mechanisms.Quercetin(Que)has been found in a variety of vegetables and fruits,and confirmed having various effects such as anti-inflammatory,anti-oxidation,and anti-proliferation.However,there are few studies on intestinal antioxidant protection.So,we investigated the influences of quercetin on methamphetamine-induced intestinal oxidative damage and the possible mechanism based on Nrf2 signaling pathway and applicated of agonist tertiary butylhydroquinone(TBHQ)and inhibitor all-trans retinoic acid(ATRA),providing new experimental basis and approaches for subsequent animal experiments which can develop quercetin-related new clinical drugs,as well as in order to find out the treatment of methamphetamine intestinal injury complications.Methods:We chose mouse small intestinal epithelial cells(MODE-K cells)and divided into the following five groups,which were:1.control group(METH:0mM,control group),2.methamphetamine group(METH:1.5mM,METH group),3.agonist+methamphetamine group(TBHQ:10μM+METH:1.5mM,TBHQ+METH group),4.inhibitor+methamphetamine group(ATRA:10μM+METH:1.5mM,ATRA+METH group),and 5.quercetin+methamphetamine group(Que:50μM+METH:1.5mM,Que+METH group).After 1.5 hours of pretreatment,methamphetamine intervention was performed for another 24 hours.The MODE-K cells were intervened by methamphetamine and quercetin,and used CCK-8 assay to detect the cell survival rate.So the intervention concentration was determined,the cell morphology was observed by inverted phase contrast microscope,the apoptosis rate was detected by AnnnexinV/PI double staining,the reactive oxygen species(ROS)level was detected by DCFH-DA fluorescent probe,the malondialdehyde(MDA)content was tested by TBA assay,and the expression levels of Nrf2 signaling pathway key genes and their proteins such as Nrf2,HO-1,CAT,NQO1,and SOD1 were detected by qRT-PCR technique and Western Blot technique.Results:1.CCK-8 assay revealed that compared with the control group,there was a significant difference in cell survival rate at four time periods:12h,24h,36h and 48h after methamphetamine intervention in mouse small intestinal epithelial cells,and the survival rate of MODE-K cells gradually decreased with the increase of methamphetamine dose.The result shows a time-dose-response relationship.The 50%inhibitory concentration(IC50)was obtained by computational analysis,and 1.5mM concentration was used in this experiment for 24 hours intervention.To exculde the side effects of quercetin itself on the cells,the survival rate of MODE-K cells was reanalyzed with different concentrations of quercetin,the Que pretreatment dose was 50μM in this experiment.2.Nrf2 signaling agonist TBHQ followed by METH intervention increased the cell survival rate in the same dose group compared with the group without TBHQ,and the difference was statistically significant when the METH concentration was 1.5mM.While Nrf2 signaling inhibitor ATRA followed by METH intervention,the cell survival rate in the same dose group was decreased compared with the group without ATRA,and the difference was statistically significant when the METH concentration was 1.5mM.3.After treatment in each group,the changes of MODE-K cells were observed under inverted phase contrast microscope.In the control group,the cells adhered firmly,grew well in fullness,and the proliferating cells were connected into a network.All the cells process extended,and the cytoplasm spread outwards.In the METH group,the cell morphology was significantly changed which the shape became round.The original synapses turned into shorter or even disappeared,also the number of floating cells and debris were increased significantly.After treatment with agonist TBHQ followed by METH intervention,it was found that the number of condensed and rounded floating cells were reduced.It can be observed synapses in the cells,and the basic morphology of the cells was still apperceived.However,after treatment with the inhibitor ATRA followed by METH intervention,the degree of cell rounding and floating were more observable which produced a large number of cell debris.Whereas after quercrtin pretreatment followed by METH intervention,the basic morphology of MODE-K cells were still observed,and the number of condensed rounded floating cells was reduced compared with the METH group.4.Apoptosis was detected by flow cytometry after intervention in each group.Compared with the control group,METH could make MODE-K cells enter early apoptosis or late apoptosis.The number of apoptosis was reduced compared with the METH group after adding TBHQ,and increased after adding ATRA.While the number of apoptosis was significantly reduced and the level was reduced after querctin pretreatment,compared with the METH group.5.The intracellular ROS level of cells in each group was directly observed under a fluorescence inverted microscope by DCFH-DA fluorescence probe,and then the ROS expression was detected by flow cytometry after collecting the cells.Compared with the control group,the ROS level in the METH group was increased after the methamphetamine intervention.Compared with the METH group,after the agonist TBHQ pretreatment the ROS content was decreased and there was no statistically significant difference compared with the control group.And contrary,the level was increased after pretreatment with the inhibitor ATRA.Furthermore,ROS level was decreased after quercetin intervention,and there was no statistically significant difference compared with the control group.6.The intracellular MDA levels in each group was determinated by TBA assay.Compared with the control group,MDA level was increased in the METH group after methamphetamine intervention.MDA level was decreased after pretreating agonist TBHQ compared with the METH group,and increased in the cells pretreated with inhibitor ATRA.The difference was statistically significant.However,MDA content decreased after Que intervention compared with the METH group,and compared with the control group there was no statistically significant difference.7.Western Blot and RT-qPCR were used to detect the vital proteins and mRNAs expression level of Nrf2 signaling pathway in MODE-K cells,pretreated with agonist and inhibitor followed by methamphetamine treatment.There were no significant statistical differences in Nrf2 and Keapl mRNA,as well as Nrf2 total cellular protein expression in the experimental groups.Compared with the METH group,Nrf2 in nuclear protein expression level was increased with the addition of TBHQ pretreatment,but decreased with the ATRA.The expression levels of HO-1,CAT,NQO1,and SOD1 mRNA and protein were increased in all experimental groups compared with the control group,and the differences were statistically significant.Continue to test the expression levels of HO-1,CAT,NQO1,and SOD1 mRNA and protein were increased in the TBHQ+METH group,while the expression levels of CAT and NQO1 mRNA and protein were decreased in the ATRA+METH group,compared with the METH group.However,the expression levels of HO-1 and SOD1 mRNA and protein in the ATRA+METH group were not statistically different from those in the METH group.Then quercetin intervention up-regulated the expression of critical downstream proteins and mRNAs such as HO-1,CAT,and NQO1,also increased intracellular SOD1 and promoted the clearance of ROS level.Conclusions:Methamphetamine can activate Nrf2 signaling pathway in MODE-K cells.The protective mechanism of Nrf2 signaling pathway in quercetin against methamphetamine-induced MODE-K cell injury may be through activating this pathway and upregulating the expression of genes related to Nrf2 signaling downstream to exert an antioxidant effect on cell injury.