Role and mechanism of mitochondrial calcium uniporter in Caco-2 cell hypoxia and reoxygenation injury
Objective:To investigate the role of mitochondrial calcium uniporter(MCU)in hypoxia/reoxygen-ation(H/R)injury in Caco-2 cells.Methods:Human intestinal epithelial cells Caco-2 cells grown at nor-mal logarithmic phase were randomly divided into four groups:control group(CTL group)(n=6),con-trol+Ru360 group(CTL+Ru360)(n=6),hypoxia/reoxygenation group(H/R group)(n=6),and H/R+Ru360 group(H/R+Ru360 group)(n=6).Caco-2 cells were cultured,and the cell model of intesti-nal ischemia-reperfusion injury was established by hypoxia for 12 h and reoxygenation for 2 h.The MCU inhibitor Ru360 was administered 1 h before the H/R stimulation.Cell viability was detected by the CCK-8 method;apoptosis was assayed by the TUNEL method;lactate dehydrogenase(LDH)was detected for cell damage;mitochondrial ROS levels were measured by ROS detection kit,and mi-tochondrial membrane potential level was assayed by JC-1labelingg method;labeled fluorescent probe ATP assay kit was adopted to detect ATP content;mitochondrial calcium content was detected with Rhod-2 AM labeling method,and MCU was detected by Western Blot.Results:Compared with the CTL group,the H/R group showed decreased cell activity,increased LDH levels,increased apopto-sis,decreased mitochondrial membrane potential,decreased ATP content,increased mitochondrial ROS production,increased MCU,and increased mitochondrial calcium ion levels(P＜0.05);after Ru360 treatment,compared with the H/R group,the H/R+Ru360 group showed increased cell activi-ty,decreased LDH levels,decreased apoptosis,increased mitochondrial membrane potential,and in-creased ATP content,decreased mitochondrial ROS production,decreased MCU and decreased mito-chondrial calcium ion levels(P＜0.05);there was no statistical difference in the above indexes between the CTL group and the CTL+Ru360 group(P＞0.05).Conclusion:Ru360 may protect Caco-2 cells from H/R-induced injury by inhibiting MCU activation,maintaining mitochondrial calcium homeosta-sis,and reducing mitochondrial dysfunction.
Ischemia ReperfusionMitochondrial Calcium UniporterHypoxia/Reoxygen-ationMitochondriaMitochondrial Function
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