The Protective Effect Of MA-5 Activating Mfn2-related Mitophagy On Microglial Cells Wiht LPS-induced Injury

Objectives: To study the protective effect and the mechanism of mitochonic acid-5(MA-5)on microglial's damage induced by LPS.Methods:1.MTT assay,caspase-3 activity assay and TUNEL assay were used to investigate cell apoptosis.2.We detected the mitochondrial membrane potential via JC1 staining,mPTP opening rate and intracellular calcium.3.Mfn2 was silenced by siRNA interference.Western Blotting was used to detect the expressions of Mfn2 and caspase-3,caspase-9,and kit detection was used to detected the activities of caspase-3 and caspase-9.The mitochondrial lysosome fluorescene co-localization was used to observe mitophgagy.4.We dectecting ATP productions by ATP assay kit,intracellular ROS and myocardial phospholipid expression by immunofluorescence.5.The effects of MA-5 mediated mitophacy on cell migration was determined by Transwell and F-actin immunostaining.Results:1.MA-5 protects microglial BV-2 cells against LPS-induced apoptosis.Compared with the control group,LPS significantly decreased cell viability,increased caspase-3 activity,and increased the number of apoptosis cells.After MA-5 treatment,it increased the viability of LPS-induced BV-2 cells and reduced apoptosis.It was shown that MA-5 can protects microglial BV-2 cells against LPS-induced apoptosis.2.LPS induces mitochondrial damage,which is inhibited by MA-5.MA-5 increased the mitochondrial membrane potential of LPS-treated BV-2 cells,relieved the intracellular calcium overload,and decreased the activities and expressions of caspase-9 and caspase-3,indicating that MA-5 inhibits LPS-induced mitochondrial damage.3.MA-5 treatment increases Mfn2-related mitophagy activity.The mitophagy level(mitochondria fused with lysosome)was decreased in response to LPS treatment but was increased in MA-5-treated cells in a Mfn2-dependent manner,and the activity of caspase-9 also increased.Silencing Mfn-2 with siRNA to reduce the expression of Mfn2,the mitophagy level(mitochondria fused with lysosome)and the activity of caspase-9 was decreased,indicating that MA-5 treatment increases mitophagy activity in a Mfn2-dependent manner.4.MA-5-activated mitophagy reverses cellular energy production.MA-5 treatment significantly increased cellular ATP production,but reduced ROS production and cardiolipin oxidation in the LPS-treated cells.This protective effect was inhibited by Mfn2 knockdown.5.Mitophagy promotes the migration of BV-2 cells following LPS-induced inflammatory injury.MA-5 treatment increases the number of migrating cells and maintained F-actin expression in the LPS-treated cells.Notably,once Mfn2 was silenced,the reversal effect of MA-5 was disappeared.These data indicated that following LPS-induced inflammatory injury,MA-5 improved the BV-2 migratoy response via promoting mitophagy.Conclusion: MA-5 activates mitophagy to protect LPS-injured microglial cells BV-2,and its mechanism may be mediated by Mfn2.