Dexmedetomidine Reverses Methotrexate-induced Neurotoxicity And Inflammation In Hippocampal HT22 Cell Lines Via HuR/NCOA4-mediated Ferritinophagy

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Methotrexate-induced cognitive dysfunction and inhibition of hippocampal ferritinophagy in miceObjective:To investigate the effects of methotrexate on cognitive function and hippocampal ferritinophagy in mice.Methods:Chemotherapeutic drug-induced cognitive dysfunction in mice model was established by intraperitoneal injection of methotrexate.SPF male 4-month-old C57BL/6 mice,weighing 20-30 g,were divided into 2 groups(n=12 per group):Control group and methotrexate treatment group(MTX group)using the random number table method.Mice in the MTX group received methotrexate treatment(100 mg/kg,three times)every 7 days,and mice in the Control group received the same volume of PBS.7 days after the last treatment,behavioral training and testing(open field,new object recognition and fear condition tests)were performed.After the tests,the mice were executed under deep anesthesia,and the expression levels of ferritinophagy-related proteins(HuR,NCOA4,FTH1,and LC3?/LC3?)and mRNA(HuR,NCOA4,and FTH1)in hippocampus were detected by Western blot and qRT-PCR,respectively.The total iron concentration in hippocampal tissues was measured by the iron assay kit.Results:1.Open field test:there was no statistical difference between the total exploration distance of mice in Control and MTX groups(P>0.05);compared with group control,the time spent in center of mice in MTX group was significantly decreased(P<0.01).New object recognition test:there was no statistical difference between the two groups of mice in the training phase for the exploration time of the two objects(P>0.05);there was a significant increase in the exploration time of the control group mice for the new object compared with the familiar object in the testing phase(P<0.01),while there was no statistical difference between the MTX group mice for the exploration time of the familiar and new objects(P>0.05).Fear condition test:in the context test,the freezing time of mice in the MTX group was significantly decreased compared with the control group(P<0.001).In tone test,there was no significantly difference in the freezing time of mice between MTX group and control group(P>0.05).2.Compared with the control group,protein(P<0.001)and mRNA(P<0.001)levels of HuR,protein(P<0.001)and mRNA(P<0.01)levels of NCOA4,and LC3?/LC3? protein(P<0.05)levels were significantly decreased,while protein(P<0.05)and mRNA(P<0.01)levels of FTH1 and total iron concentration(P<0.001)were significantly increased in the hippocampus of mice in the MTX group.Conclusions:Methotrexate-induced cognitive dysfunction and inhibition of hippocampal ferritinophagy in mice.Part ? Dexmedetomidine reverses methotrexate-induced neurotoxicity and inflammation in hippocampal HT22 cell lines via NCOA4-mediated ferritinophagyObjective:To investigate the mechanism of NCOA4 in the improvement of methotrexate-induced hippocampal HT22 neuronal injury by dexmedetomidine.Methods:In vitro model was constructed by stimulating mouse hippocampal HT22 cells with 10 ?M methotrexate for 24 h.HT22 cells were divided into five groups in the first phase:Control group,methotrexate treatment group(MTX group),methotrexate+1 ng/mL dexmedetomidine group(MTX+DEX1 group),methotrexate+10 ng/mL dexmedetomidine group(MTX+DEX10 group)and methotrexate+50 ng/mL dexmedetomidine group(MTX+DEX50 group).Methotrexate stimulated HT22 cells for 24 h and then treated with different concentrations of dexmedetomidine for 1 h.HT22 cells were divided into four groups in the second phase:Control group,methotrexate treated group(MTX group),methotrexate+50 ng/mL dexmedetomidine+NC-siRNA group(MTX+DEX+NC-siRNA group)and methotrexate+50 ng/mL dexmedetomidine+NCOA4-siRNA group(MTX+DEX+NCOA4-siRNA group).HT22 cells were first transfected with NC-siRNA or NCOA4-siRNA for 48 h,followed by methotrexate stimulation for 24 h,and finally treated with 50 ng/mL dexmedetomidine for 1 h.HT22 cell viability was measured by CCK-8 assay,and the kit measured LDH activity,TNF-? and IL-1 ? concentration,ROS level,intracellular labile iron pool and total iron level,flow cytometry for apoptotic changes in HT22 cells,western blot for NF-?B,NCOA4,FTH1 and LC3?/LC3? changes,and immunofluorescence for NCOA4 and LC3II co-labeling.Results:Phase ?:10 M methotrexate treatment for 24 h was able to significantly decreased the HT22 cells viability(P<0.05);increased LDH activity(P<0.05);promoted apoptosis(P<0.05);upregulated inflammation-related proteins NF-? B(P<0.05),TNF-?(P<0.05),IL-1?(P<0.05)and FTH1(P<0.05)levels;increased ROS(P<0.05)and total iron concentration(P<0.05);decreased intracellular labile iron pool concentration(P<0.05)and ferritinophagy-associated protein NCOA4(P<0.05)and LC3?/LC3?(P<0.05)expression.10 and 50 ng/mL dexmedetomidine significantly attenuated these changes(P<0.05),but 1 ng/mL dexmedetomidine treatment did not(P<0.05).Phase ?:transfection of NCOA4-siRNA was able to significantly down-regulated NCOA4 levels(P<0.05).Compared with the MTX+DEX+NC-siRNA group,the HT22 cells viability was reduced(P<0.05),LDH activity(P<0.05)and apoptosis were increased(P<0.05),and TNF-?(P<0.05),IL-1?(P<0.05),FTH1(P<0.05),ROS(P<0.05)and total iron concentration(P<0.05)increased,and the levels of NCOA4(P<0.05),LC3?/LC3?(P<0.05)and intracellular labile iron pool concentration(P<0.05)decreased in the MTX+DEX+NCOA4-siRNA group.Conclusions:Dexmedetomidine alleviates MTX-induced neurotoxicity and inflammation in hippocampal HT22 cell lines via NCOA4-mediated ferritinophagy.Part ? Dexmedetomidine reverses methotrexate-induced neurotoxicity and inflammation in hippocampal HT22 cell lines via HuR/NCOA4-mediated ferritinophagyObjective:To investigate the role of HuR/NCOA4 pathway in the improvement of methotrexate-induced hippocampal HT22 neuronal injury by dexmedetomidine.Methods:In vitro model was constructed using 10 M methotrexate to stimulate mouse hippocampal HT22 cells for 24 h.HT22 cells were divided into four groups:control group,methotrexate treatment group(MTX group),methotrexate+50 ng/mL dexmedetomidine+NC-siRNA group(MTX+DEX+NC-siRNA group)and methotrexate+50 ng/mL dexmedetomidine+HuR-siRNA group(MTX+DEX+HuR-siRNA group).HT22 cells were transfected with NC-siRNA or HuR-siRNA for 48 h,then methotrexate was stimulated for 24 h,and finally treated with 50 ng/mL dexmedetomidine for 1 h.The viability of HT22 cells was measured by the CCK-8 assay,and the LDH activity,TNF-? and IL-1? concentration,ROS level,intracellular labile iron pool and total iron level were measured by kit,respectively.Flow cytometry was performed to detect apoptotic changes in HT22 cells,western blot was used to detect HuR,NCOA4,FTH1 and LC3?/LC3? changes,and NCOA4 and HuR co-labeling was detected by immunofluorescence.Results:Transfection of HuR-siRNA was able to significantly down-regulated NCOA4 levels(P<0.05).Compared with MTX+DEX+NC-siRNA group,HT22 cells in MTX+DEX+HuR-siRNA group showed decreased viability(P<0.05);increased LDH activity(P<0.05)and apoptosis(P<0.05);increased TNF-?(P<0.05),IL-1?(P<0.05),FTH1(P<0.05),ROS(P<0.05)and total iron concentration(P<0.05);decreased levels of HuR(P<0.05),NCOA4(P<0.05),LC3?/LC3?(P<0.05)and intracellular labile iron pool concentrations(P<0.05).Conclusions:Dexmedetomidine alleviates MTX-induced neurotoxicity and inflammation in hippocampal HT22 cell lines via HuR/NCOA4-mediated ferritinophagy.