processes In atherosclerosis, cur cumin suppresses o LDL induced

processes. In atherosclerosis, cur cumin suppresses o LDL induced CD36 e pression via inhibiting p38 MAPK phosphorylation, and prevents the decrease of thrombospondin 4 e pression in o LDL treated murine macrophages. Curcumin inhibits the adhesion research use of monocytes to endothelial cells, and reduces the mi gration of HASMCs by Inhibitors,Modulators,Libraries suppressing MMP 9 e pression through down regulation of NF ��B dependent Inhibitors,Modulators,Libraries pathways. Further more, in vivo data showed that curcumin inhibits atherosclerosis in ApoE mice, and blocks the development of atherosclerosis in ApoE LDLR mice. Although some studies have suggested the anti atherosclerosis activity of curcumin, the mechanism by which curcumin regulates MMP 9, MMP 13 and EMM PRIN is currently unknown.

The purpose of this study was to uncover the mechanism by which curcumin reg ulates EMMPRIN, MMP 9 and MMP 13e pression dur ing monocyte differentiation. Materials and methods Cell culture Human monocytic cell line THP 1 was obtained from American Type Culture Collection and maintained at a density of 106 ml in RPMI 1640 medium containing 10% FBS, 10 mM Inhibitors,Modulators,Libraries HEPES and 1% pen strep solution at 37 C, 5% CO2 incubator. Cells were cultured in si well plates for 48 h in the presence of 100 nM PMA, which allowed them to differentiate into ad herent macrophages. Cells were pretreated with curcu min or 10 uM Compound C, PD98059, SB203580, and SP600125 MAP kinase inhibitors for 1 hour, and then stimu lated with PMA for another 48 hours. Cytoto icity assay PMA induced macrophages were seeded in 96 well plates at 6 103 cells well.

Twenty four hours later, cells were in cubated with curcumin for 48 h. Cells with out any treatment were used as a control. CCK8 assay was used to assess the cytoto Inhibitors,Modulators,Libraries icity of curcumin on PMA induced macro phages, based on the manufacturers recommendation. Protein isolation and Western blot analysis Protein isolation and Western blot analysis of cell ly sates were performed as previously described. Briefly, membranes were first probed with primary anti bodies for MMP 13, EMMPRIN, PKC, PKCB1, MMP 9, phospho ERK, ERK, phospho p38, p38, phospho JNK, JNK, AMPK, p AMPK, or B actin, then incubated with anti Rabbit or anti mouse secondary antibodies, followed by incubation with antibody labeled with far red fluorescent Ale a Fluor 680 dye. All signals were detected by the Odyssey imaging system and data were normalized based on the B actin level.

RNA isolation, cDNA synthesis and real time PCR Total RNA was e tracted from PMA Carfilzomib induced macro phages using Trizol reagent according to the manufacturers instructions. cDNA was synthesized using the Reverse Transcription Kit before Real time polymerase chain reactions were performed by SYBR Pre mi E Taq Kit according to the instructions. The PCR reactions were performed in dupli cate and kinase inhibitor Idelalisib detected by the ABI 7500 Sequence Detection System. The primer sequences are listed in Table 1. All results were normalized against the GAPDH level. Gelatin zymography Cells in the logarithmic phase w

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