Sasanquasaponin-induced Cardioprotection Involves Inhibition Of MPTP Opening Via Attenuating Intracellular Chloride Accumulation

Objective:This study aims to investigate whether the cardioprotection afforded by SQS may be mediated by preventing mPTP opening via inhibition of H/R-induced elevation of[Cl^-]_i and subsequent improving mitochondrial function.Methods:1.To examine the effect of SQS on the extent of mPTP opening in H9c2 cells undergoing H/R.H9c2 cells were preincubated with SQS?10?M?for 24 h prior to H/R.The extent of mPTP opening was qualitatively observed via a phase-contrast fluorescence microscope and quantitatively analyzed by flow cytometry with the calcein-AM and CoCl₂ co-loading method.2.To investigate whether the inhibitory effect of SQS on mPTP opening is relevant for inhibition of H/R-induced elevation of[Cl^-]_i,we further tested the effect of external Cl^--free condition,as a positive control for the[Cl^-]_i inhibition,on mPTP opening.H9c2 cells were incubated in Cl^--free Tyrode solution,in which Cl^-was replaced with equimolar gluconate,during the entire experimental period.The[Cl^-]_i was analyzed by flow cytometry.The extent of mPTP opening was qualitatively observed via a phase-contrast fluorescence microscope and quantitatively analyzed by flow cytometry.3.In order to explore whether the inhibition of the increased[Cl^-]_i is an upstream event of the mPTP closing by SQS in H9c2 cells undergoing H/R.H9c2 cells were pretreated with SQS or external Cl^--free condition and then atractyloside?ATR,as a specific mPTP opener?for 20 min from the beginning of reoxygenation.The[Cl^-]_i was analyzed by flow cytometry.The extent of mPTP opening was qualitatively observed via a phase-contrast fluorescence microscope and quantitatively analyzed by flow cytometry.4.To further analyze whether SQS-induced mPTP inhibition contributes to its cardioprotective effect against H/R.H9c2 cells were pretreated with SQS or external Cl^--free condition and then atractyloside for 20 min from the beginning of reoxygenation.Mitochondrial membrane potential???_m?and mitochondrial ROS generation was qualitatively observed via a phase-contrast fluorescence microscope and quantitatively analyzed by flow cytometry.LDH and the activity of mitochondrial complex I,II,III,and IV were detected by spectrophotometry.The intracellular ATP content was detected by bioluminescence assay.Cell viability was detected by Methyl thiazolyl tetrazolium?MTT?method.Results:1.SQS suppressed mPTP opening,and protected mitochondria,as indicated by preserved mitochondrial membrane potential and respiratory chain complex activities,decreased mitochondrial reactive oxygen species generation,and increased ATP content.Moreover,SQS attenuated H/R-induced the elevation of[Cl^-]_i,accompanied by reduction of lactate dehydrogenase release and increase of cell viability.2.Cl^--free inhibited H/R-induced elevation of[Cl^-]_i,accompanied by inhibition of mPTP opening similar to SQS preconditioning.3.ATR significantly attenuated the inhibitory effect of SQS and Cl^--free on H/R-induced mPTP opening but to not[Cl^-]_i elevation.4.ATR abolished all the protective effects induced by SQS or Cl^--free,including suppression of mPTP opening,maintenance of mitochondrial membrane potential,and subsequent improvement of mitochondrial function.Conclusion:The above results allow us to conclude that SQS-induced cardioprotection may be mediated by preserving the mitochondrial function through preventing mPTP opening via inhibition of H/R-induced elevation of[Cl^-]_i.