The Role Of Berberine And Its Derivatives Against Atherosclerosis And Underlying Mechanisms

Objective:Endothelial injury and macrophages activation are two key steps in the development of atherosclerosis.Macrophages transferred to foam cells by engulfing oxidized low-density lipoprotein(ox-LDL),then amplifying the inflammatory response and accelerating the progression of atherosclerosis.Endothelial progenitor cells(EPCs),as a special kind of stem cells,play an important role in endothelial repair,and become dysfunctional in patients with atherosclerosis.Berberine(BBR),as an important component of traditional Chinese medicine,Coptis chinensis,can lower lipid profile and exert anti-inflammatory effect.In this study,we will investigate the role and mechanisms of BBR against atherosclerosis in terms of inhibiting macrophage activation and improving EPCs function.We intend to:1)in the first part,to study the role and mechanisms of BBR's inhibition of ox-LDL-induced macrophage activation;2)in the second part,to study the role of BBR's effect on ox-LDL-induced EPCs senescence and underlying mechanisms;3)In the third part,to study the effects of dihydroberberine(dh BBR)on atherosclerosis in Apo E^-/-mice feeding on western diet.Methods:First part:Macrophages were pretreated with BBR,rosuvastatin,BBR combined with rosuvastatin for 1 h,and then induced with ox-LDL for 24 hours.Oil red staining was used to exam lipid droplet aggregation.Real time PCR was used to exam the expressions of IL-6?TNF-?and IL-1?on macrophages.Flow cytometry was used to exam the expressions of CD11b and CD86 on macrophages.Real time PCR,Western Blot and laser confocal were used to exam galectin-3 expression on macrophages.Western Blot was used to detect the expressions of AMPK,p-AMPK,p65 and p-p65on macrophages.Under the condition with or without AMPK signaling pathway inhibitor compound C or NF-?B signaling pathway activator prostratin,the above methods were used to exam macrophage activation and expression of galectin-3.Second part:PBS,ox-LDL,ox-LDL combined with BBR were used to treat EPCs for 24 hours,then colony forming,migration and angiogenic ability of EPCs were examed.?-galactosidase staining was used to exam the senescence of EPCs.Laser confocal was used to exam the expressions of CXCR4 and e NOS on EPCs.Real time-PCR was used to exam the expressions of IL-6,TNF-?,IL-1?,ICAM-1,VCAM-1 and MCP-1.Western blot was used to exam the expressions of Akt,p-Akt,p65 and p-p65.EPCs were pretreated with LY294002(PI3K inhibitor)and PX-316(Akt inhibitor)for 1 hour,and?-galactosidase staining was used to exam the senescence of EPCs.Third part:Apo E^-/-mice were first fed with western diet for 4 weeks,then fed with equal volumes of PBS,BBR,dh BBR,and rosuvastatin in combination with western diet for another 12 weeks.Dairy food intake and weight were recorded.Peripheral blood lipids of each group were examed.The expressions of CD68,i NOS,EMMPRIN and MMP-9 were examed by Western Blot.?-galactosidase staining was used to examed the senescence of born marrow derived EPCs.Real time PCR was used to exam the expressions of IL-6,TNF-?,IL-1?,ICAM-1,VCAM-1 and MCP-1in Apo E^-/-mice aorta.Oil red staining was used to exam the size of aorta atherosclerotic plaque in Apo E^-/-mice.Immunofluorescence was used to exam the expression of CD31 in aorta of Apo E^-/-mice.Western Blot was used to detect the expression of?-SMA in aorta.Mason staining was used to exam the expression of collagen fibers.Results:In the first part,we found that BBR inhibited ox-LDL-induced macrophage activation and galectin-3 expression.Inhibition of galectin-3 abrogated the effects of ox-LDL on macrophage activation,whereas overexpression of galectin-3 enhanced these effects of ox-LDL.Overexpression of galectin-3 intervened the inhibitory effect of BBR on macrophage activation.BBR activated p-AMPK and inhibited p-p65 nuclear translocation.AMPK inhibition and NF-?B activation abolished the inhibitory effects of BBR on galectin-3 expression and macrophage activation.In the second part,we found that BBR can improve the ability of colony formation,migration and angiogenesis of ox-LDL-damaged EPCs.BBR significantly inhibited ox-LDL-induced EPCs senescence and the expressions of IL-6,TNF-?,IL-1?,ICAM-1,VCAM-1 and MCP-1.BBR increased the expressions of CXCR4 and e NOS on EPCs.BBR increased the expression of p-Akt and inhibited the expression of p-p65.Both LY294002(PI3K inhibitor)and PX-316(Akt inhibitor)can abolish the inhibitory effect of BBR on ox-LDL-induced EPCs senescence.In the third part,we found that dh BBR reduced the levels of TC,TG and LDL-C in peripheral blood of Apo E^-/-mice.dh BBR inhibited the expression and activation of macrophages and expressions of IL-6,TNF-?,IL-1?,ICAM-1,VCAM-1 and MCP-1 in the aorta of Apo E(-/-)mice.dh BBR significantly inhibited born marrow derived-EPCs senescence in Apo E(-/-)mice.dh BBR reduced the area of aortic plaques and stabilized aortic plaques by increasing the expression of?-SMA,CD31 and collagen fibers.Conclusion:BBR alleviates ox-LDL-induced macrophage activation by downregulating galectin-3 via the NF-?B and AMPK signaling pathways.BBR inhibits ox-LDL-induced EPCs senescence by activating the PI3K/Akt/e NOS signaling pathway.dh BBR exerts anti-atheroslcerosis ability through reducing the area of aortic,protecting endothelium while increasing the stability of aorta plaque.