A Comparison Between Paeonol To Kaempferol On The Supressive Mechanism Of Macrophage-derived Foam Cell Formation

Aim Atherosclerosis is the major death cause of human diseases,whichcould be found in critical organs such as heart,brain and kidney. Andatherosclerosis is responsible for coronary heart disease, myocardialinfarction，cerebral stroke.In abroad,there are some reports about theantiatherogenic effects of kaempferol.For instance, kaempferol inhibitsfoam cell formation by reducing the uptake of ox-LDL in macrophages;kaempferol also suppresses the development of atherosclerosis inApoE-/-mice through regulating OPN-CD44pathway.These data suggestthat the antiatherogenic effect of kaempferol is recognized. Our previousstudy showed that compared to model group, progression of atherosclerosisin rabbits was significantly inhibited after ten weeks treatment ofkaempferol(30mg/kg,150mg/kg). And the mechanism possibly involvedupregulation of antioxidative ability and downregulation of inflammatorycytokines such as TNF-α,IL-1β,and vascular adhision molecular includingE-sel,ICAM-1and VCAM-1. Paeonol, a potent antioxidant isolated from cortex moutan, exerts protective effects on cardiovascular disease. Thoughpaeonol could retards the development of atherosclerosis, theantiatherosclerotic effect of paeonol is rarely reported abroad. Both paeonoland kaempferol have beneficial effects on atherosclerosis, however, reportsof kaempferol are more than paeonol, and the antiatheroscleroticmechanism of kaempferol is more detailed than paeonol. Therefore, wecompared the mechanism of paeonol to kaempferol whoseantiatherosclerotic effects are identified. On one hand, we provide moreevidences for the differences between paeonol and kaempferol onantiatherosclerotic mechanism; on another hand, we complement theantiatherosclerotic mechanism of kaempferol. It is reported thataccumulation of foam cells in atherosclerotic lesions is a hallmark ofearly-stage atherosclerosis. Therefore, we compared the differencesbetween kaempferol and paeonol on the mechanism of inhibitingmacrophage-derived foam cell formation.Methods MTT assay was used to select non-cytotoxic concentrations ofPaeonol and Kaempferol on macrophages; Effects of paeonol andKaempferol on ox-LDL-induced macrophage foam cell formation and theability of paeonol to reduce arterial plaque formation in ApoE-/-micewere detected by Oil red O staining. Western blot and real time PCR wereused to detect protein and mRNA expression of ABCA1, ABCG1, SR-BI, SR-A and CD36in macrophages respectively. After the blockage of HO-1gene expression by HO-1shRNA transfection in macrophages, theprotection effects of paeonol and Kaempferol on macrophage-derived foamcell formation were further investigated.Results （一） Treatment with kaempferol markedly suppressedoxLDL-induced macrophage foam cell formation, a consequence that wasdue to a decrease in lipid accumulation and an increase in cholesterol effluxin THP-1derived macrophages. Additionally, kaempferol treatment ofmacrophages showed down-regulation of cluster of differentiation36(CD36) protein levels, up-regulation of ATP-binding cassette (ABC)transporter A1(ABCA1), scavenger receptor class B type I(SR-BI), andABCG1protein level, while having no effect on scavenger receptorA(SR-A) expression. The mRNA expression of ABCA1, SR-BI, SR-A,CD36, ABCG1modulated by kaempferol was similar to that observed inprotein levels. The reduced CD36expression by kaempferol resulted frominhibition of c-Jun-activator protein-1(AP-1) nuclear translocation, and thiswas confirmed by the augmented reduction of CD36by kaempferol afterAP-1inhibition using SP600125. Accordingly, kaempferol-mediatedsuppression of lipid accumulation in macrophages was also augmented bySP600125. The increased expression of ABCA1, SR-BI, ABCG1bykaempferol was accompanied by enhanced protein expression of haem oxygenase-1(HO-1) as revealed by the abolition of the increase after thegene knockdown of HO-1using small hairpin RNA(shRNA). Moreover,the kaempferol-mediated attenuation on lipid accumulation and promotingof cholesterol efflux was also inhibited by HO-1shRNA. In conclusion,c-Jun-AP-1-dependent down-regulation of CD36and HO-1-dependentup-regulation of ABCG1, SR-BI, ABCA1may mediate the beneficial effectof kaempferol on foam cell formation.(二)Paeonol treatment significantly attenuated intracellular lipidaccumulation in macrophages, which may be results of decreased oxidizedlow-density lipoprotein (ox-LDL) uptake and increased cholesterol efflux.Additionally, paeonol markedly inhibited the mRNA and protein expressionof cluster of differentiation36(CD36) by decreasing nuclear translocationof c-Jun(one subunit of activator protein-1). Moreover, paeonolup-regulated the protein stability of ATP-binding cassette transporter A1(ABCA1) by inhibiting calpain activity, while ABCA1mRNA expressionwas not altered. Furthermore, small hairpin RNA (shRNA) targeting haemoxygenase-1(HO-1) inhibited the paeonol-mediated beneficial effects onthe expression of c-Jun, CD36, ABCA1, calpain activity and lipidaccumulation in macrophages. Accordingly, paeonol retarded the progressof atherosclerosis in ApoE/mice and modulated the expression of CD36and ABCA1in aortas similarly to that observed in macrophages. These results indicate that Paeonol provides protective effects on foam cellformation by a novel HO-1-dependent mediation of cholesterol efflux andlipid accumulation in macrophages.（三）（1）Both Paeonol and Kaempferol treatment significantly attenuatedintracellular lipid accumulation in macrophages, which may be results ofdecreased oxidized low-density lipoprotein (ox-LDL) uptake and increasedcholesterol efflux.（2） Additionally, Both paeonol and Kaempferolmarkedly inhibited the mRNA and protein expression of cluster ofdifferentiation36(CD36) by decreasing nuclear translocation of c-Jun(onesubunit of activator protein-1).（3）Moreover, paeonol up-regulated theprotein stability of ATP-binding cassette transporter A1(ABCA1) byinhibiting calpain activity, while ABCA1mRNA, ABCG1,SR-BI mRNAand protein expression was not altered. However, kaempferol treatment ofmacrophages showed up-regulation of ATP-binding cassette (ABC)transporter A1(ABCA1), scavenger receptor class B type I(SR-BI), andABCG1protein level. The mRNA expression of ABCA1, SR-BI, ABCG1modulated by kaempferol was similar to that observed in protein levels.And HO-1is responsible for the increased expression of ABCA1,SR-BI,ABCG1by kaempferol in macrophages.(4) Furthermore, small hairpinRNA (shRNA) targeting haem oxygenase-1(HO-1) inhibited thepaeonol-mediated beneficial effects on the expression of c-Jun, CD36, ABCA1, calpain activity and lipid accumulation in macrophages. As forKaempferol, the increased expression of ABCA1, SR-BI, ABCG1bykaempferol was accompanied by enhanced protein expression of haemoxygenase-1(HO-1) as revealed by the abolition of the increase after thegene knockdown of HO-1using small hairpin RNA(shRNA). Moreover,the kaempferol-mediated attenuation on lipid accumulation and promotingof cholesterol efflux was also inhibited by HO-1shRNA.(5) Accordingly,paeonol retarded the progress of atherosclerosis in ApoE/mice andmodulated the expression of CD36and ABCA1in aortas similarly to thatobserved in macrophages. Because the antiatherosclerotic effect ofkaempferol in ApoE-/-mice has been reported and our previous report alsodemonstrated that kaempferol inhibits atheroslcrosis development in rabbits,so we did not do similar study again.Conclusion These results indicate that though both Paeonol andKaempferol inhibits macrophage-derived foam cell formation. Theirmechanisms are different. Paeonol provides protective effects on foam cellformation by a novel HO-1-dependent mediation of ABCA1proteinstability and HO-1-c-Jun-AP-1-dependent inhibition of CD36expression.While for Kaempferol, c-Jun-AP-1-dependent down-regulation of CD36and HO-1-dependent up-regulation of ABCG1, SR-BI, ABCA1maymediate the beneficial effect of kaempferol on foam cell formation. These differences in antiatherosclerotic mechanism between kaempferol andpaeonol may be results of differences in their chemical structure.Through compare the differences of kaempferol and paeonol in inhibitingmacrophage-derived foam cell formation, not only provide more efficientevidences for the differences between kaempferol and paeonol onantiatherosclerotic mechanism, but also enrich the antiatheroscleroticmechanism of kaempferol, and supply some new insights in identifyingnew drugs for the treatment of atherosclerosis.