The Research Of Relationship Between LOX-1 And P38MAPK Activation And Mechanism Of Simvastatin Anti-atherosclerosis

Objective1. To explore the relationship between LOX -1 and p38MAPK activation in atherosclerosis.2. To explore the mechanisms of simvastatin anti - atherosclerosis.Methods1. The relationship between LOX - 1 and p38MAPK activation and the mechanism of simvastatin anti - atherosclerosis in rabbit aortic atherosclerotic plaques1.1 AS model;30 Japan white rabbits were subjected to two groups randomly: control group(n = 10) fed with common diet, model group(n =20) fed with common diet and 2% cholesterol. After 16 week, model group was randomly divided into hyperlipidemia group ( n = 10, common diet and 2% cholesterol) and simvastatin group( n = 10, common diet and 2% cholesterol + simvastatin). After 8 week, blood and aorta were collected respectively.1.2 Blood lipid assay: Enzymatic colorimetric test was principle. Serum lipid concentrations were detected.1.3 Observation of pathomorphology: Fixed aortic arches were embedded with paraffin, sections were made, and stained with hematoxylin eosin. Pathologic changes were observed by light microscopy.1. 4 Aorta pathological examination: The thickness of aortic intima and media was examine.1. 5 infrastructure observation: infrastructure of aortic arches was observed by transmission electron microscope.1. 6 Reverse transcription - PCR technique;. Total RNA?was isolated using Trizol reagent, following the manufactures instructions, synthesis of single -stranded cDNA according to " First Strand cDNA synthesis Kit". Then Polymer-ase chain was react, p - actin was used as an internal control. The primers were synthesized by Shanghai songong Technologies, Inc. PCR products were elecfro-phoresed in 2% agarose gels in TBE buffer, stained in 0.5 [Ag/ml ethidium bromide and photographed.1.7 Western blotting: tissue lysates were prepared in lysis buffer. Whole lysates were collected and then mixed with sample buffer, each were subjected to SDS - polyacrylamide gel electrophoresis, and the proteins were then transferred to a polyvinylidene difluoride membrane using a Semi - Dry Transblot, and then it was blocked by incubation with skim milk for 2h at room temperature. The blocked membrane was subsequently incubated with first antibody for 12h at 4^. After washing with TTBS, the membrane was incubated for 2h at room temperature with alkaline phosphatase - conjugated secondary antibody IgG, bands of protein on the membrane were visualized with NBT/ BCIP kit.2. The relationship between LOX - 1 and p38MAPK activation and the mechanism of simvastatin anti - atherosclerosis in ECV304 cells2.1 Cell AS model;Human umbilical vein endothelial cell lines(ECV304) were incubated with ox - LDL for 24h.2.2 Observation of cellular morphology: Cells changes in morphology were observed under inverted phase contrast microscope after cultured with ox - LDL for 24 hours.2. 3 Activity of cells: The growing state of cultured endothelial cells were examined by MTT.2.4 Immunocytochemistry examination: All cells were fixed with pure acetone. SP method was used to detect the expression of LOX -1.2.5 Reverse transcription - PCR technique;Total cellular RNA was isolated using Trizol reagent. The experimental process is similar to 1.6.2. 6 Western blotting: Cell lysates were prepared in lysis buffer. The exper-imental process is similar to 1.7.3. Statistical Analysis;All data were expressed as mean ± standard deviation , and SPSS11.5 software was employed to analyze the data. Statistical evaluation was performed using one -way ANOVA and correlation analysis. P <0. 05 was considered significant.Results1. The relationship between LOX - 1 and p38MAPK activation and the mechanism of simvastatin anti - atherosclerosis in rabbit aortic atherosclerotic plaques1.1 Blood lipid changes: Compared with control group, serum TC, LDL -C concentrations in hyperlipidemia group ( HL group) elevated (P<0. 01). Compared with HL group, serum TC, LDL - C concentrations in simvastatin group decreased ( P < 0.01).1. 2 Histological Changes by light microscope: It was shown that aortic inti-ma was smooth, media smooth muscle cells arranged regularly in control group. In HL group, there were lipid sedimentation, foam cells infiltration, plaque in the lesion, collagenous fibers. In simvastatin group, there were a few foam cells in intima.1. 3 Thickness changes: Compared with HL group, thickness of intima and rate of intima and media in simvastatin group decreased(P <0.01).1.4 Effects of simvastatin on aorta LOX - 1 mRNA and protein expression: LOX - lmRNA and protein expression in aorta tissue were reduced more in simvastatin group than that of HL group (P < 0.01).1.5 Aorta p - p38MAPK protein expression;The level of p - p38MAPK protein in aorta tissue was reduced more in simvastatin group than that of HL group(P<0.01).1.6 Correlation analysis;The level of p -p38MAPK protein was related to that of LOX - 1 protein (r = 0. 8082, P <0.001).2. The relationship between LOX - 1 and p38MAPK activation and the mechanism of simvastatin anti - atherosclerosis in ECV304 cells2.1 Changes in cell morphology: In control group, cells were monolayer, and karyons were oval in the center. In ox - LDL groups, there were many black granules and vesicles in the cells. Some became smaller, round, shed and broken. In high concentration ox -LDL group, changes were more obvious.2.2 Activity of cells changes: The OD value in ox - LDL group were reduced remarkably. The effect of ox - LDL was concentration - dependent.2.3 The result of LOX - 1 immunocytochemistry staining: Staining grey levels of positive products of LOX -1 increased in ox - LDL groups. The effect of ox - LDL was concentration - dependent.2.4 Effects of ox - LDL on cell LOX - lmRNA and protein expression;ox - LDL significantly increased LOX - 1 mRNA and protein expression ( P < 0.01), the effect of ox - LDL on LOX - 1 expression was concentration - depend-ent(P<0.05).2. 5 Effects of ox - LDL on p - p38MAPK: ox - LDL activated p38MAPK and increased the expression of phospho - p38MAPK in a concentration - dependent manner. But the expression of phospho - p38MAPK was inhibited by JTX92.2.6 Effects of simvastatin on LOX - lmRNA and protein expression;Pre-treatment of cells with simvastatin markedly decreased LOX - 1 expression (mRNA and protein) induced by ox - LDL( P <0.05 ). High concentration of simvastatin was more potent than the low concentration (P <0.05).Conclusions1. The relationship between LOX - 1 and p38MAPK activation and the mechanism of simvastatin anti - atherosclerosis in rabbit aortic atherosclerotic plaques(1) The expression of LOX - 1 increased in rabbit aortic atherosclerotic plaques. LOX -1 upregulation maybe play a important role in atherosclerosis.(2)p38MAPK activation was related to LOX - 1 upregulation, LOX - 1 maybe mediate p38MAPK activation in atherosclerosis.(3)Simvastatin reduced the expression of LOX - 1 and p38MAPK activa-tion and decelerated the progression of atherosclerosis.2. The relationship between LOX - 1 and p38MAPK activation and the mechanism of simvastatin anti - atherosclerosis in ECV304 cells(1) ox - LDL induced the expression of LOX - 1 and p38MAPK activation in ECV304 ceUs.(2) LOX -1 mediated p38MAPK activation induced by ox - LDL.( 3 ) Inhibition of the expression of LOX -1 was one of mechanisms of simvastatin anti - atherosclerosis, independent of cholesterol - lowering effect. LOX - 1 was a pharmacological potential target of simvastatin.