Abstract:Aim To explore the effect of TanshinoneⅡA(TanⅡA) on macrophage polarization by inhibiting histone deacetylase 3(HDAC3). Methods Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) was applied to screen drug targets of TanⅡA and therapeutic targets of atherosclerosis(As). The intersection genes were enriched and visualized by KEGG pathway analysis and Cytoscape 3.7.1 software, respectively. THP-1 monocytes were induced as adherent macrophages by phorbol 12-myristate- 13-acetate(PMA), and divided into four groups:M0 group, oxidized low density lipoprotein(ox-LDL) group, TanⅡA＋ox-LDL group and ox-LDL＋HDAC3 siRNA group. Flow cytometry, immunofluorescence and qRT-PCR were used to detect the change of polarization and expression of HDAC3 mRNA in macrophage. Results Twenty-three intersection genes were obtained of TanⅡA drug targets and As therapeutic targets by Bioinformatics analysis. The top twenty signal pathways were selected for visual analysis and displayed that these genes concentrated in signaling pathways such as atherosclerosis, fluid shear force and TNF signaling pathway, among which JUN, FOS, RELA and NFKBIA, were involved in the regulation of macrophage polarization. Results of Flow cytometry, immuno fluorescence and qRT-PCR showed that ox-LDL could significantly increased the expression of CCR7 and CCL2 in M0 macrophages. Compared with ox-LDL group, HDAC3, CCR7 and CCL2 expression decreased in TanⅡA＋ox-LDL group, consistent with ox-LDL＋HDAC3 siRNA group. Conclusion TanⅡA could effectively prevent ox-LDL-induced macrophage polarization toward M1 direction, the mechanism may be mediated by regulating HDAC3 expression.

Abstract:Aim With the application of two-dimensional electrophoresis and mass spectrometry to study the effect of tanshinoneⅡA on ox-LDL-induced RAW264.7 mouse macrophage-derived foam cells proteome and explore the molecular mechanism of lipid and anti-atherosclerotic effect of tanshinoneⅡA. Methods The isolated purified human serum LDL, oxidized with CuSO4 to give ox-LDL, co-incubated with RAW264.7 cells, the formation of foam cells containing tanshinoneⅡA 20 mg/L to continue for 24 hours was used as the tanshinoneⅡA group; and the control group excluding tanshinoneⅡA solution was incubated for 24 hours; cells were disrupted by ultrasonic and 4℃ 12000 g centrifugal 30 min, to get supernatant for protein quantitation; protein of the control group and the tanshinone ⅡA group was loaded with the same amount and two-dimensional electrophoresis (IEF and SDS-PAGE); after the completion of electrophoresis, they were stained with silver nitrate to get proteome map with different samples. Through Labscan difference proteomic analysis software, select the protein with expression difference of more than 2-fold. After gel digestion and mass spectrometry analysis, peptide mass fingerprinting was obtained, by Mascot database searching, combined with the 2-D map of protein molecular weight and isoelectric point information protein identification was realized. Results In TanshinonⅡA treated group calreticulin, vimentin, peroxidase-2, CuZn-SOD, zipper protein, stabilin-1, hematopoietic cell specific protein were up-regulated, while GTP protein, ATP synthesis, mimitin, IL-5, HSP70, translationally controlled tumor protein, chloride ion channel protein were down-regulated. Conclusions TanshinonⅡA improved the lipid regulation of foam cells by decreasing the expression of GTP protein and HSP70 and increasing the expression of CRT. TanshinonⅡA regulated the ability of endocytosis by increasing the expression of stabilin-1 and decreasing the expression of ATP synthesis. It also stimulated the metabolism of lipid and lipoprotein by improving the expression of leucine zipper protein and vimentin. Meanwhile, tanshinonⅡA eliminated ROS and lipid peroxidation by increasing the expressions of peroxidase-2 and CuZn-SOD. It also had the effects of anti-inflammation and anti-apoptosis by reducing the expressions of mimitin and IL-5. TanshinonⅡA played a role of anti-tumor by subsiding the expressions of CLIC1 and TCTP. In conclusion, tanshinonⅡA may have function of lipid regulation and anti-atherosclerosis, and can be used to treat cardiovascular diseases in clinic.

Abstract:Aim To explore the expression of matrixmetallop roteinase-9 （MMP-9） and tissue inhibitor of metallop roteinase-1 （TIMP-1） in different treatment for patients with Kawasaki disease （KD）, and discuss the protective effect of Tanshinone ⅡA （TanⅡA） on the coronary artery 1esion （CAL） with KD. Methods Forty-eight Kawasaki patients including twenty-eight no coronary artery 1esion （NCAL） and twenty CAL, and twenty-four healthy children as controls were recruited into the study. The patients with Kawasaki disease were randomly divided into two groups: TanⅡA treatment group and contrast treatment group （including fourteen NCAL and ten CAL patients in each group）. The serum MMP-9 and TIMP-1 levels were detected by ELISE. The MMP-9 and TIMP-1 mRNA expression were detected by real-time fluorescence quantification PCR （qRT-PCR） in the peripheral blood mononuclear cells （PBMC）. Results Compared with the normal group, the expression of MMP-9 and TIMP-1 were increased before treatment, and decreased after treatment of five to seven days both at the mRNA and protien in the KD group （P<0.05）. However, the KD patients

Abstract:AimThe effect of tanshinoneⅡA on cholesterol homeostasis in foam cells was studied in vitro.MethodsThe foam cell induced from RAW264.7 macrophage with oxidized low density lipoprotein (ox-LDL) were treated with different concentration of tanshinoneⅡA.The relative cell viability were detected by MTT methods, and the change of total cholesterol and cholesterol ester in the cells were detected with the enzyme colorimetric quantifies.The lipid accumulation in cell was observed by oil red O dyeing, then the expressions of CD36, ATP binding cassette transporter A1(ABCA1), liver X receptor α (LXRα) and peroxisome proliferator activated receptors γ (PPARγ), PPARα were detected with the methods of fluorescent quantitation PCR and Western Blot.ResultsThe cell viability was not affected when tanshinone ⅡA concentrations were in the range of 0～20 mg/L.The ratio of cholesterol ester and total cholesterol decreased and lipid accumulation decreased when the foam cells were treated with 20 mg/L tanshinone ⅡA.Meanwhile, the expression of ABCA1, LXRα, PPARα were increased，but the expression of CD36 had no obvious variance.ConclusionTanshinoneⅡA can inhibit the formation of foam cells induced from RAW264.7 by ox-LDL，the mechanism is possibly related to inducing the expressions of PPARα, LXRα and ABCA1 and promoting the excretion of cholesterol.