The Effects Of Shexiang Baoxin Pill On The Development Of Atherosclerosis In Apolipoprotein E-deficient Mice Fed HFD

Epidemiological studies have established that cardiovascular diseases（CVDs）are the predominant cause of death in the world.Atherosclerosis,featured by progressive plaque lesion formation leading to lumen stenosis,has been demonstrated to be the underlying cause of several CVDs,such as coronary artery disease and ischemic stroke.Recently basic studies have demonstrated that the onset and progression of atherosclerosis is associated with complex pathophysiological events including lipid metabolism dysfunction,vascular endothelial injury,inflammatory response,thrombosis and immune regulation disorder.Shexiang Baoxin Pill（SBP）is a traditional Chinese medicine formulation and has been broadly applied for the treatment of CVDs in East Asia.However,up to now,the effects of SBP on atherosclerosis development are still unclear.In this study,we investigated whether SBP administration exerted beneficial roles in delaying the progression of atherosclerotic plaques,and then analyzed the relevant underlying molecular mechanisms.Part I The effect of SBP on the atherosclerotic plaque burden of apoE^-/- mice and regulatory roles of SBP in lipid metabolismAim:To ascertain whether SBP gavage affects the extent and degree of atheromatous lesions in apoE^-/-mice and investigating the roles of SBP treatment in regulating the lipid profiles and specific molecules responsible for lipid metabolism.Methods:8-week-old apoE^-/-mice with the C57BL/6 background were acclimatized to the environment for 2 weeks.Then,apoE^-/-mice at 10 weeks old were randomly divided into two groups.The mice were fed HFD（21%fat and 0.15%cholesterol）supplemented with oral gavage of SBP（25 mg/kg/day）or normal saline（Control）for 20 weeks.At the end of the experiment,all animals were fasted overnight and sacrificed.Then the blood samples and heart,liver and aortic tissues were collected for further study.En face lesions stained with Oil Red O in the entire aorta were quantified to analyze the burden and range of atherosclerosis.Cross-sections of the aortic sinus were stained with specific markers for analyzing the characteristics of plaque lesions.Then,Plasma levels of triglyceride（TG）,total cholesterol（TC）,low density lipoprotein cholesterol（LDL-C）and high density lipoprotein（HDL-C）were determined using corresponding commercially available reagents according to the manufacturer’s protocols.The expression of LDLR,HMRCR,p-HMGCR,SREBP1c,ACC,FAS,PPARα,CPT-1A,AMPK and p-AMPK in the liver were measured via western blot.Results:Oil Red O staining showed that the model of atherosclerosis were developed after HFD feeding for 20 weeks.We observed that the extent of plaque lesions in the en face prepared aorta in the SBP group was markedly decreased as compared to the control group.Biochemical analysis indicated that treatment obviously lowered the contents of plasma TG,TC and LDL-C,while elevating the level of plasma HDL-C when compared with normal saline management.The data of western blot showed that SBP administration induced the elevation of LDLR,PPARα,CPT-1A,p-HMGCR and p-AMPK and reduced the level of SREBP1c,ACC and FAS.Conclusion:SBP gavage displayed inhibitory roles in the development of atherosclerosis and regulated the level of blood lipid via mediating proteins involved in lipid metabolism in the liver.Part II The effects of SBP on the inflammatory reactions andvascular endothelial damage of apoE^-/-miceAim:Investigating the roles of SBP in systemic inflammation and vascular inflammatory response and analyzing whether SBP treatment alleviates the impairments of vascular endothelium in apoE^-/-mice.Methods:After 20 weeks of SBP intervention,the mice plasma,heart and aortic tissues were collected.Cross-sections of the aortic sinus were used for immuno-staining.The contents of plasma TNF-α,CRP,IL-1β,IL-6,MCP-1,IL-10 and TGF-β1 were detected by ELISA kits.In addition,levels of MDA,MPO,H2O2,SOD,GSH and NO were quantified using biochemical assays.Western blot was performed to detect the proteins involved in inflammation response and apoptotic signal pathway.Results:When compared with the normal saline group,there was decreased level in plasma TNF-α,CRP,IL-1β,IL-6 and MCP-1and increased content in IL-10 and TGF-β1 of the SBP group.Moreover,circulating oxidative injury-related markers including MDA,MPO and H2O2 were reduced and anti-oxidative indicators including SOD and GSH were elevated separately in the SBP group.Findings of immunohistochemistry showed that SBP administration significantly lower the level of cleaved caspase-3 in the vascular wall and increased NO content in the plasma.In addition,SBP treatment reduced the expression of VCAM-1,ICAM-1,IL-6 and IL-2 and diminished the activities of p38,JNK and NF-κB and increased the level of Mfn2 in the aorta.Meanwhile,SBP gavage inhibited the signal transduction of mitochondria-dependent apoptotic pathway,as seen by decrease of Bax/Bcl-2,cleaved caspase-9,cleaved caspase-3 and cleaved PARP.Conclusion:SBP administration was capable of attenuating the systemic and vascular inflammation response and improving vascular endothelial damage,thereby inhibiting the development of atherosclerosis.