| Backgrounds and aimsAtherosclerosis(AS)is a complex inflammatory arterial disease caused by lipid deposition and is an important pathogenesis of cardiovascular and cerebrovascular diseases.In the theory of traditional Chinese medicine,"yin deficiency,accumulation of heat and toxin" is the key pathogenesis of AS.Therefore,the Yangyin Qingre Huoxue Prescription(YQHP)used in this experiment has a combination of traditional Chinese medicines that have the functions of nourishing yin,clearing heat and promoting blood circulation,and has achieved remarkable curative effects in the treatment of clinical AS.In AS,the level of activation of T cells,as well as the ratio of regulatory T cells(Treg)to helper T cells(Th)17,play an important role in the progression of the disease.Dendritic cells(DC)serve as a bridge between congenital and acquired immunity,mediate T cell activation and differentiation,control the occurrence and development of AS from the origin of inflammatory response,and the intracellular cholesterol content of DC affects its inflammatory functions.In addition,chronic inflammation caused by infection can also aggravate AS induced by hyperlipidemia.Previous studies have found that YQHP improves AS in apolipoprotein E knockout(ApoE-/-)mice fed with high-fat diet by regulating Treg/Th17 balance,but the effects on the infected environment is unknown.Therefore,in this study,the effects of YQHP on AS in ApoE-/-mice fed with high-fat diet in the background of infection were investigated by bacterial-derived antigen heat shock protein(HSP)65,and whether they are related to the regulation of DC cholesterol metabolism.Furthermore,screening for active ingredients that affect DC cholesterol metabolism and thereby intervene with inflammation.MethodsApoE-/-mice were fed with high-fat diet and injected with HSP65 as an AS disease model in the context of infection.Using simvastatin as a positive drug to study the effects of YQHP on blood lipids,lesions and inflammation,and the correlation with DC cholesterol metabolism after quality testing and hepatotoxicity evaluation:biochemical kits for the measurement of total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C),high density lipoprotein cholesterol(HDL-C),aspartate aminotransferase(AST),alanine aminotransferase(ALT)and alkaline phosphatase(AKP)in serum;HE staining for the evaluation of coronary artery and liver lesion degree;oil red O staining for the determination of aortic plaque area;ELISA for serum IL-6,IL-17,IL-22,IL-2,TGF-β,IL-10,GM-CSF and IL-4 levels;flow cytometry to determine the number of IL-17A+CD4+Th17,Foxp3+CD25+CD4+Treg and CD3+CD4+T cells,and calculate Th17/Treg ratio in anticoagulation blood;realtime quantitative PCR(qPCR)assay for Stat3,Stat5a,Jak2,Socs3,Foxp3,Tgfb1,Cd80,Cd86,Nr1h3,Srebf2 expressed in spleen,and Stat3,Stat5a,Jak2,Socs3,Tgfb1,Cd80,Cd86 mRNA in aorta;Western blotting assay for phosphorylated STAT3(p-STAT3),phosphorylated STAT5(p-STAT5),SOCS3,FOXP3 protein expressed in the spleen,and p-STAT3,SOCS3 expressed in the aorta;immunofluorescence assay for the amount of MHC-II+CD11c+DC and the expression of CD80 in the spleen DC.Bone marrow cells were primarily extracted from ApoE-/-mice and induced to differentiate into bone marrow DC(BMDC).The active components in YQHP affecting cholesterol metabolism of ApoE-/-BMDC were screened out:the CCK-8 method for screening the dose and incubation time of several main components in YQHP in ApoE-/-BMDC;the active constituents and doses influencing sodium taurocholate(NaTC)-mediated cholesterol efflux of ApoE-/-BMDC was screened by NBD cholesterol method;qPCR method for the determination of effects of these active ingredients on cholesterol-metabolizing genes Nr1h3,Srebf2,Abcal and Abcgl in cholesterol-loaded ApoE-/-BMDC in the presence of NaTC.Based on the NaTC-mediated cholesterol efflux model,the effects of selected active ingredients on the inflammatory behavior of ApoE-/-BMDC were investigated:flow cytometry for determination of the number of MHC-Ⅱ+CD11c+ cells in ApoE-/BMDC,and the expression of CD80 and CD86 in MHC-Ⅱ+CD11c+ cells;IL-6,IL-2 and TGF-β1 levels secreted by ApoE-/-BMDC were determined by ELISA;after cocultured with the cholesterol efflux ApoE-/-BMDC model treated with active ingredients,the T cells were determined for the number of CD44hi CD62Llo activated T cells and CD44lo CD62Lhi na?ve T cells by flow cytometry,and the secretion levels of IL-17A,IL-22,TGF-β1 and IL-10 by ELISA.ResultsYQHP can significantly improve the deformation of coronary artery and the formation of aortic plaque in ApoE-/-mice with good quality test results.YQHP can regulate the levels of blood lipids TC,TG,LDL-C,HDL-C and serum inflammatory factors IL-6,IL-22,IL-2,IL-10 and GM-CSF,reduce the increased Th17/Treg ratio and the expression of CD3 in CD4+T cells in peripheral blood,inhibit the transcription of the co-stimulatory molecule CD80 in the aorta,restore the up-regulated p-STAT3 in the spleen and aorta and down-regulate the SOCS3 level in the spleen,up-regulate the spleen p-STAT5 and FOXP3 levels and aortic SOCS3 levels,and can inhibit proliferation and maturation of DC in the spleen,restore the transcription level of Nr1h3.In addition,compared with simvastatin,the liver treated with YQHP had less monocyte infiltration and lower serum AST,ALT and AKP levels.Among the main components of YQHP,40-80μM diphenyl diglycoside and 5-10μM resveratrol can promote NaTC-mediated ApoE-/-BMDC cholesterol efflux.Based on the NaTC-mediated cholesterol efflux model,diphenyl diglycoside down-regulated the expression of Nr1h3,resveratrol down-regulated Nr1h3 and up-regulated Srebf2 in ApoE-/-BMDC.Both diphenyl diglycoside and resveratrol up-regulated expression of Abcal.In the presence of NaTC,diphenyl diglycoside and resveratrol can inhibit the maturation of cholesterol-aggregating ApoE-/-BMDC,attenuating the enhanced antigen presentation and costimulation effects.In addition,diphenyl diglycoside restored the enhanced secretion of IL-6 in ApoE-/-BMDC in the presence of NaTC and up-regulated the secretion of TGF-β1;resveratrol restored the suppressed IL-2 secretion and inhibited excessive release of IL-6 in ApoE-/-BMDC.Under NaTC-mediated cholesterol efflux model,ApoE-/-BMDC treated with diphenyl diglycoside and resveratrol down-regulated the level of CD44hi CD62Llo activated T cells,and restored the depressed IL-10 and increased IL-22 secretion,up-regulated the level of TGF-β1 in co-cultured T cells,and the diphenyl diglycoside-treated ApoE-/-BMDC inhibited the secretion of IL-17A in T cells.Conclusions and significancesYQHP can improve hyperlipidemia and AS lesions in ApoE-/-mice fed with highfat diet under HSP65 attack,and its hepatotoxicity is lower than lipid-lowering drug simvastatin.These effects may be achieved through the inhibition of antigen presenting and co-stimulating effects and maturation of DC via the modification of the cholesterol metabolism,which lead to controlled T cell activation,and the intervention of Th17 and Treg differentiation via the regulation of IL-6-p-STAT3 and IL-2-p-STAT5 signaling.Among the active constituents of YQHP,diphenyl diglycoside and resveratrol can enhance the NaTC-mediated ApoE-/-DC cholesterol efflux by up-regulating Abcal,improving the enhanced antigen presentation.co-stimulation and T cell activation capability of cholesterol-aggregated ApoE-/-DC,inhibiting its maturation,and regulate the inflammatory function of co-cultured T cells.These findings suggest the effectiveness of YQHP on AS in the context of infection with high-fat diet.It also demonstrates the scientificalness of the combination therapy of nourishing yin,clearing away heat and promoting blood circulation on the treatment of internal injuries and miscellaneous diseases,providing a theoretical basis for the clinical use of the prescriptions at home and abroad. |
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