Beneficial Effect Of Berberine On Atherosclerosis Based On Attenuating Vascular Inflammation And Calcification

Background and aimAtherosclerosis is a chronic inflammatory disease associated with cardiovascular dysfunction including myocardial infarction,unstable angina,sudden cardiac death,stroke and peripheral thromboses.It has been predicted that atherosclerosis will be the primary cause of death in the world.Atherogenesis is initiated by endothelial injury,oxidative stress,inflammation and so on.It is represented by the development of vascular lesions or plaques in the blood vessels following inflammatory/oxidant response to endothelial damage.Those events do not work independently but interplay each other.They finally result in plaque rupture and thrombosis leading to serious cardiovascular and cerebrovascular disease.Clinically,the commonly used drugs for atherosclerosis are statins.Long-term use of statins may cause liver damage cause by statins,so it is essential to developnew therapeutic drugs.Berberine(BBR)is an alkaloid originally isolated from Huanglian(Coptis chinensis).In China,BBR has been extensively used as a nonprescription drug to treat diarrhea caused by bacteria.Recently berberine is identified as a novel cholesterol-lowering drug working through a unique mechanism distinct from statins.It is reported that the main mechanism of BBR is elevating LDLR expression.The other mechanisms have not been fully understood yet.To investigate the beneficial effect of berberine(BBR)on atherosclerosis in Apo-/-E mice and explore the underlying mechanisms based on attenuating vascular inflammation and modulating calcification in human umbilical vein endothelial cells(HUVECs)and smooth muscle cells(HASMCs).METHODS48 Apo-/-E mice,at 6-8 weeks old,were randomly allocated into 4 groups:Control,Model,BBR and Atorvastatin(Positive Control)groups with 12 mice in each group.They were fed with high-fat diet for 4 weeks except those in Normal group and then treated with indicated drugs or solvent for another 4 weeks.The morphology and inflammation infiltration of aortic were examined with HE staining.Calcium deposition were examined with Von Kossa staining.The expression of BMP-2,OPG,OCN and RUNX2 in aortic was examined by immumohistochemical staining.Blood lipid levels were examined by automatic biochemical analyzer.The expression of IL-6,TNF-? and BMP-2 in serum and tissues was detected by ELISA method.The expression of ALP and the content of calcium were detected by commercially-available kits.HUVEC cells were stimulated with TNF-? and incubated with various concentrations(7.5,10,15,20 mg/L)of BBR for 24h.The contents of intercellular cell adhesion molecule-1(IC AM-1),vascular cell adhesion molecule(VCAM-1),matrix metalloprotein-9(MMP-9)in the culture supernatant were detected by ELISA method.Calcification was induced with?-glycerophosphatein HASMC cells and the effect of BBR on the content of calcium was examined.HASMC cells were stimulated with TNF-a and incubated with various concentrations(10,15,20 mg/L)of BBR for 4 days.BMP-2 and RUNX2 in HASMC were detected by immunofluorescence.RESULTS1.The mouse model of atherosclerosis can be successfully established in Apo-/-E mice by high-fat feeding for 4 weeks.Serum lipids(TC,TG,LDL-C and HDL-C)levels in Apo-/-E model mice were significantly increased compared with that in control group.The aortic vessel wall was thickened,the plaque in the vessel was obvious,and even ruptured plaquescan be observed in model group.2.Serum biochemical data showed that 4-week berberine treatment markedly reduced serum TC and LDL-c levels and improved the plaque stability in Apo-/-E mice fed with a high-fat diet(P<0.05)which was comparable with the effect of atorvastatin.In the pathological section,there was no plaque formation in blood vessels of berberine-treated mice of,the vascular wall was smooth without thickening,and there was no obvious inflammatory infiltration.3.After a four-week high-fat diet,the expression of inflammatory factors in serum and aortic tissues of model mice increased significantly.In the pathological section of aortic tissue,the inflammatory infiltration of blood vessels in model group was obvious,especially in the plaques.Berberine significantly decreased the levels of IL-6 and TNF-a in mice serum and aortic tissues(P<0.05).4.The expressions of ICAM-1,VCAM-1,and MMP-9 in TNF-?-stimulated HUVECs were obviously increased compared with that in control group.Berberine significantly reduced the levels of ICAM-1,VCAM-1,and MMP-9(P<0.05).5.Von Kossa staining results showed that calcium deposits in model group were obvious,especially in plaque.Immunohistochemistry results showed that the expression of calcification-associated proteins ALP,BMP-2,OPG,OCN and RUNX2 in vascular tissues of model mice were increased(P<0.05).Berberine tended to decrease ALP,BMP-2,OPG,OCN and RUNX2 levels and the content of calcium in mice serum and aortic tissues(P<0.05)which were not observed in atorvastatin group.6.Compared with the control group,the calcium content in HASMC was significantly increased in odium phosphatide-induced model group s(P<0.05).Berberine can inhibit the increase of calcium content.7.Compared with the control group,the BMP-2 and RUNX2 contents in HASMC were significantly increased in model group stimulated with TNF-a.Berberine can inhibit the increase of BMP-2 and RUNX2.CONCLUSIONBerberine has a good therapeutic effect on Apo-/-E atherosclerosis mice model established by high-fat feeding.Berberine can profitably regulate the levels of blood lipid in mice fed with a high-fat diet,decrease the injury caused by inflammation through reducing secretion of inflammatory factors and adhesion molecules,and attenuate vascular calcification evidenced by reduced calcification-associated proteins.In addition,some inflammatory cytokines such as TNF-? can activate BMP-2 and RUNX2.Berberine might indirectly block calcification by inhibiting TNF-?-mediated response.Hence,berberine may attenuate atherosclerotic plaque development in an experimental model of atherosclerosis and play a role in cardiovascular protection.