Effects And Mechanisms Of Xuezhikang On The Formation Of Vulnerable Carotid Atherosclerotic Plaque In Apoe-/-Mice

Objective:To investigate the effects and mechanisms of Xuezhikang(XZK)on the formation of vulnerable carotid atherosclerotic plaque in ApoE-/-mice.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 4 groups:control group(n=16),atorvastatin group(n=16),low-dose XZK group(n=19)and high-dose XZK group(n=21).The pathological changes of atherosclerotic plaques were evaluated using HE staining.The lipid deposition and collagen content were assessed by Oil Red O and Picric Sirius Red staining.The level of ROS in plaque was examined by using DHE fluorescent probe.The expression of MMP-8,MMP-13,P-IRE1,P-PERK,P-eIF2a,CHOP and cleaved caspase-3 was detected by laser confocal microscopy after immunofluorescence staining.We also detected apoptosis in atherosclerotic plaques by TUNEL staining.Macrophages(murine RAW 264.7 cells)were cultured and stimulated with 7-ketocholesterol(7-KC)to indue endoplasmic reticulum stress(ER stress).The protein expression of XBP-1s,P-PERK,CHOP,cleaved caspase-3 and cleaved PARP was detected by Western blotting.The morphological change of nucleus was observed by DAPI staining.And intracellular ROS was detected with the fluorescent probe DCF-DA by laser confocal microscopy.Results:1)The incidence of vulnerable plaque and plaque rupture with thrombus were significantly reduced in atorvastatin group,low-dose XZK group and high-dose XZK group when compared with control.There was no difference in lesion area in atorvastatin group and low-dose XZK group compared to control,but lesion area in high-dose XZK group was markedly reduced,accompanied with significantly decreased macrophage infiltration,lipid deposition and expression of MMPs,elevated vascular smooth muscle cell numbers and intimal collagen content.2)Atherosclerotic plaques in high-dose XZK group displayed dramatically decreased necrotic core area,necrotic core ratio and apoptotic cell numbers compared to control.In vitro,xuezhikang 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.3)Lesional ROS level,protein expression of P-IRE1,P-PERK,P-eIF2a and CHOP were significantly decreased in high-dose XZK group compared to control.In vitro experiments showed that 7-KC induced upregulation of ROS,XBP-1s,P-PERK and CHOP in murine RAW 264.7 cells were significantly inhibited by xuezhikang.Conclusion:Xuezhikang significantly improves the stability of carotid atherosclerotic plaque and inhibits vulnerable carotid plaque formation in ApoE-/-mice by alleviates oxidative stress,inhibits ER stress activation and reduces cell apoptosis.