Sulindac Derivative Inhibit Macrophage Apoptosis Of Vulnerable Plaque Through Activating Protective Autophagy Pathway

Objective: To investigate the effects and mechanisms of sulindac derivatives K80003 on macrophage apoptosis in vulnerable atherosclerotic plaque.Methods: Using the method of partial ligation of left carotid and left renal arteries,we generated an ApoE-/-mouse model of vulnerable carotid atherosclerotic plaque.The mice were random divided into 2 groups: control group and K80003 group.The necrotic cores of atherosclerotic plaques were evaluated using HE staining.The apoptosis in atherosclerotic plaques was assessed by TUNEL staining and immunofluorescence staining.The level of ROS in plaque was examined by using DHE fluorescent probe.Macrophages(murine RAW 264.7 cells)were cultured and stimulated with 7-ketocholesterol(7-KC)to indue apoptosis.The protein expression of cleaved caspase-3 and cleaved PARP was detected by Western blotting.The number of apoptotic cells and proportion were assessed by Annexin V/PI staining and TUNEL staining.And intracellular ROS was detected with the fluorescent probe DCF-DA by laser confocal microscopy.The level of macrophage autophagy was examined by Western blotting,immunofluorescence staining and GFP-LC3II/ GFP-RFP-LC3 II.Results: 1)Atherosclerotic plaques in K80003 group displayed dramatically decreased necrotic core area,necrotic core ratio and apoptotic cell numbers compared to control.In vitro,K80003 significantly inhibits 7-KC induced apoptotic cell numbers and protein expression of apoptosis markers(cleaved caspase-3 and cleaved PARP)in murine RAW 264.7 cells.2)Lesional ROS level was significantly decreased in K80003 group compared to control.In vitro experiments showed that K80003 inhibits 7-KC induced apoptosis by downregulating ROS.3)K80003 significantly restores 7-KC induced impairment of autophagic flux in murine RAW 264.7 cells.K80003 inhibits macrophage apoptosis by alleviating oxidative stress through activating protective autophagy pathway.Conclusion: Sulindac derivative K80003 significantly inhibits macrophage apoptosis of atherosclerotic plaque and reduces necrotic cores formation in ApoE-/-mice by alleviating oxidative stress through activating protective autophagy pathway.