High Insulin To Promote Monocyte/scavenger Source Sex Acat - 1 Foam Cells And Leak Reed On Its Effects

Atherosclerosis is the common pathological basis of cardiovascular and cerebrovascular diseases which is very harmful to human health. The pepole dying of cardiovascular and cerebrovascular diseases in the world reaches one third of total deaths; in China, the pepole dying of cardiovascular and cerebrovascular diseases reaches41%oftotal deaths.In recent years there is a growing number of young people suffering from cardiovascular and cerebrovascular diseases, and therefore early prevention of atherosclerosis has a very important social significance.The hyperinsulinnemia is an independent risk factor of the atherosclerosis. The foam cells from mononuclear-macrophagocyte are the foundation of the atherosclerosis inearly lesion phase.The study about the formation mechanism of the hyperinsulinnemiaand atherosclerosis and looking for the drugs of early prevention and treatment of atherosclerosis will open up a new way to provide prevention and treatment for complications of hyperinsulinemia.The previous studies show that Loulu could reduce the lesion of atherosclerosis and inhibit macrophage uptake of ox-LDL. The foam cells from mononuclear-macrophagocyte could be significantly inhibited by Loulu.Objective:Tn this study, monocyte has been induced to macrophage by PMA, incubating withox-LDL,at the same time,high insulin and Loulu have been added to the cells,finally we can explore the mechanism that the extract of Loulu inhibits macrophage uptake of ox-LDL which is promoted by hyperinsulinemia. We can explore the prevention and treatment effect of the Loulu on early atherosclerosis.lt has an very important meaning to prevention of atherosclerosis.Methods:1. Detecting the optimal PMA concentration of inducing monocytes by MTT assay:After THP-1cells being incubated with PMA, to observe the influence of monocyte from the different concentrations of PMA.2. Determining the concentration of experimental drugs such as insulin, Fluorine simvastatin and Loulu by MTT:After macrophages being incubated with insulin, Fluorine simvastatin and Loulu48h, to observe cytotoxicity of insulin, Fluorine simvastatin and Loulu by detecting cells activity.3. Detecting the rate of foam cell transformation by Oil red O staining, detectingthe cholesterol ester content of foam cells by enzymatic, observing the expression of ACAT-lmRNA by real time PCR and observing the expression of AC AT-1protein by Western blot:after monocyte has been induced to macrophage by PMA,incubating with ox-LDL48h, at the same time, high insulin, Fluorine simvastatin and Loulu have been added to the cells.Results:1. Detecting the optimal PMA concentration of inducing monocytes by MTT assay:After incubating with PMA24h and48h, we could see that the conversion of macrophagesincreases following increasing of PMA concentration between40nmoL/L and160nmoL/L, and the conversion of macrophages reduces following increasing of PMAconcentration between160nmoL/L and480nmoL/L.There is statistically significant difference between160noml/L group and monocyte group (P<0.05). Compared with48h, the conversion of macrophages rate of24h is higher, so we choose the160noml/L PMA to induce THP-1cells24h.2. Determining the concentration of experimental drugs such as insulin, Fluorine simvastatin and Loulu by MTT:After cultivating by different concentrations of insulin48h,compared with the control group,there is no statistically significant difference between0.005U/L and0.1U/L (P>0.05). After cultivating by different concentrations Fluorine simvastatin of48h,compared with the control group,there is no statistically significant difference below10-5mol/L (P>0.05), and there is statistically significant difference exceed10-5mol/L (P<0.05).Therefore, we choose the10-6mol/L as the action concentration of drug. After cultivated by different concentrations of Loulu48h, compared with the control group,there is no statistically significant difference between0.005g/l and0.5g/L (P>0.05),there is statistically significant in lg/L(P<0.05),which appears cytotoxicity.3. Detecting the rate of foam cell transformation by Oil red O staining:There is statistically significant difference between group of ox-LDL and group of ox-LDL and0.02U/L insulin(P<0.05); foam cell transformation rate increases with insulin concentrations. Normal fasting insulin level is0.005-0.015U/L, and definitionof the hyperinsulinemia is insulin level which is higher than the0.015U/L, so we select0.02U/L insulin concentration as the research concentration.There is statistically significant between group of ox-LDL and group of ox-LDL and insulin (P<0.05); foam cell transformation rate increases with insulin concentrations. Compared with the ox-LDL+insulin group by ox-LDL-+insulin+Fluorine simvastatin group,ox-LDL+insulin+high concentrations of Loulu group, ox-LDL+insulin+concentration Loulu group, ox-LDL+insulin+low concentrations of the Loulu group,the difference is statistically significant (P<0.05), foam cell transformation rate reduces following increasing of Loulu concentration.4. Detecting the cholesterol ester content of foam cells by enzymatic:There is statistically significant between group of ox-LDL and macrophage group (P<0.05),the cholesterol ester content of foam cells increased. Compared with ox-LDLgroup,0.02U/L insulin group,0.04U/L insulin group and0.06U/L insulin group have statistical difference(P<0.05), cholesterol ester content increased with insulinconcentrations increasing.There is statistically significant difference between group of ox-LDL and macrophage group (P<0.05), the cholesterol ester content of foam cells increased. Compared with the ox-LDL+insulin group by ox-LDL-+insulin+Fluorine simvastatin group,ox-LDL+insulin+high concentrations of Loulu group, ox-LDL+insulin+concentration Loulu group ox-LDL+insulin+low concentrations of the Loulu group,thedifference is statistically significant (P<0.05), the cholesterol ester content of foam cells reduces following increasing of Loulu concentration.5. Observing the expression of ACAT-1mRNA by real time PCR:There is statistically significant between group of ox-LDL and macrophage group (P<0.05),the ACAT-lmRNA increased. Compared with the ox-LDL+insulin group by ox-LDL-H-insulin+Fluorine simvastatin group,ox-LDL+insulin+high concentrations of Loulu group, ox-LDL+insulin+concentration Loulu group, ox-LDL+insulin+low concentrations of the Loulu group,the differences is statistically significant (P<0.05), the ACAT-1mRNA reduces following increasing of Loulu concentration.6.Observing the expression of ACAT-1protein by Westren blot:There is statistically significant between group of ox-LDL and macrophage group (P<0.05),the ACAT-1protein increased. Compared with the ox-LDL+insulin group by ox-LDL-+insulin+Fluorine simvastatin group,ox-LDL+insulin+high concentrations of Loulu group, ox-LDL+insulin+concentration Loulu group, ox-LDL+insulin+low concentrations of the Loulu group,the differences is statistically significant (P<0.05), the ACAT-1protein reduces following increasing of Loulu concentration.Conclusions:1. Induced by PMA,macrophages were incubated with ox-LDL,which can simulate the formation of foam cells.2. Detecting the rate of foam cell transformation by Oil red O staining and detecting the cholesterol ester content of foam cells by enzymatic:high concentrations of insulin can promote foam cell formation, which can be inhibited by Loulu.3. By Western blot and real time PCR:the function which high concentrations of insulin can promote foam cell formation is related to the exprssion of ACAT-1which is promoted by insulin, which can be inhibited by Loulu.4. So the function that insulin of high concentrations promoting foam cell formation and Loulu inhibiting foam cell formation is close related with the expression of ACAT-1. This study provides preliminary experimental evidence for Loulu preventing atherosclerosis.