| 1 ObjectiveSome satistics show that there are 16.7 million pepole dying of cardiovascular and cerebrovascular diseases in the world, which reachs one third,29.2% of total deaths; In China, there are 2.6 million pepole dying of cardiovascular and cerebrovascular diseases,41% of total deaths.50 years of the 20th century, the studies of medical autopsy form soldiers of American from 18 years to 22 years who were died in the United States invasion of the Korean war have found that 60-70% of people had different degrees of atherosclerosis; Recently we see form studies that not only the incidence of stroke,cornary heart disease and hypertension is increasing,but also incidence trends show younger, the studies of medical autopsy of 18 years to 22 years in BeiJing find that 74% of people have different degrees of atherosclerosis. So experts call at the 8th south international congress of cardiology that we must foucs on prevention,early intervention cardiovascular and cerebrovascular diseases which is considered to be number one killer of hazardous to human life and health, even preventive health care should begin from the embryo.The traditional view is that hyperinsulinnemia can result in As by high blood suger,but studies suggest in recent years that hyperinsulinnemia is an independent risk fator of As,which can result in As by:insulin resistanceâ†'hyperinsulinnemiaâ†'monocyte/macrophage-derived foam cellsâ†'lipid stripeâ†'As. So it is important that we explore the mechanism of insulin-induced As for preventing occurrence or delaying development of As earlier, which will reduce the incidence of As.There is no corresponding name of disease and atherosclerosis in the classical theory of traditional chinese medicine,the reason is that pulse is an odd constant organs,by which gas and blood can yun, attacheding to the heart and without independent physiological function and pathological changes. Obesity can result in phlegm which stain in blood and vessels, it induces the stagnation of gas and blood, and cause the obstruction of blood and vessels. The main methods to treat this diease is removing phlegm, promoting blood circulation and detoxification from the aspects of phlegm, damp and toxin.The herb Loulu can detoxify,abscess swelling and through milk. It has been proved that Loulu can inhibit the formation of foam cells by reducing the expression of CD36 which result in reducing intake of ox-LDL by monocyte. In this study, monocyte has been induced to macrophage by PMA, incubating with ox-LDL, at the same time, high insulin and Loulu have been added to the cells, finally we can explore the mechanism that high insulin/Loulu promote/inhibit formation of foam cell from macrophage. It is very important of this study for preventing As.2 Methods2.1 Detecting the adhersion rate of foam cells by MTT, so that which concentration of PMA will be used. After monocyte from mouse being incubated with PMA, to observe the influence for monocyte from the different concentrations of PMA.2.2 Determining the concentration of experimental drugs such as insulin and Loulu by MTT:After monocyte from mouse being incubated with insulin and Loulu 24h and 48h, to observe cytotoxicity of insulin and Louou by detecting cells activity.2.3 Detecting the rate of foam cell transformation by Oi 1 red 0 staining:monocyte has been induced to macrophage by PMA, incubating with ox-LDL 24h,at the same time, high insulin and Loulu have been added to the cells.2.4 Observing the expression of CD36 mRNA by real time PCR:monocyte has been induced to macrophage by PMA, incubating with ox-LDL 24h,at the same time, high insulin and Loulu have been added to the cells.2.5 Observing the expression of CD36 protein by Westren blot:monocyte has been induced to macrophage by PMA, incubating with ox-LDL 24h, at the same time, high insulin and Loulu have been added to the cells.3 Results3.1 Detecting the adhersion rate of foam cells by MTT:monocyte are round, less cytoplasm, lucent cytoplasm, small cell processes, large nucleus and round and suspended growth. After monocyte incubating with PMA from 6 hours to 7 hours, cell morphology began to change from suspension into adherent type; Incubated with PMA 24h, most of cells were adherent and amoeba-like growth,cell bodies became large and extend pseudopodia and rich particles,which showed the formation of macrophage. After incubating with PMA 24h and 48h, we see that the adherence rate which is named the conversion of macrophages increases following increasing of PMA concentration between 40nmoL/L and 160nmoL/L. But there is no statistically significant 320nmoL/L,and there is significant cytotoxicity 640nmoL/L. Compared with 48h, the adherence rate of 24h is higher.3.2 Detecting cytotoxicity of insulin and Loulu by MTT:After cultivating by different concentrations of insulin 24h,compared with the control group, there is no statistically significant between 40uU/ml and 120uU/ml (P> 0.05), there is statistically significant between 150uU/ml and 200uU/ml (P<0.05). After cultivated by different concentrations of Loulu 24h, compared with the control group, there is no statistically significant between 0.005mg/ml and 0.5mg/mL(P >0.05), there is statistically significant in 5mg/mL(P<0.05), which appears cytotoxicity. there is statistically significant in 50mg/mL(P<0.01), which appears significant cytotoxicity.3.3 Detecting the rate of foam cell transformation by Oil red 0 staining:after cultivating with ox-LDL 48h, there is a lot of red lipid granules in cytoplasm by Oil red staining, which show the morphological characteristics of foam cells. Compared with group A which is also named group of ox-LDL, there is statistically significant group B which is also named group of ox-LDL and insulin (P<0.01);Compared with group B, there is statistically significant positive control group and group of high concentration loulu (P< 0.01), and group of moderate,low concentration also have statistically significant (P< 0.05)3.4 Observing the expression of CD36 mRNA by real time PCR:Copmared with group ox-LDL, there is statistically significant(P<0.01) group ox-LDL and insulin; Compared with group ox-LDL and insulin, there is statistically significant group positive control and group of high,low concentration Loulu(P<0.05),but there is no statistically significant group of moderate concentration Loulu (P>0.05).3.5 Observing the expression of CD36 protein by Westren blot:Compared with group A, there is statistically significant (P<0.01) group B; Compared with group B, there is statistically significant (P<0.01)group C and D;but there is no statistically significant (P>0.05).4 ConclusionsInduced by PMA, macrophages were incubated with ox-LDL, which can simulate the formation of foam cells. By Oil red 0 staining:high concentrations of insulin can promote foam cell formation, which can be inhibited by Loulu. 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 CD36 which is promoted by insulin, which can be inhibited by Loulu. So the function that high concentrations of insulin promote foam cell formation or Loulu inhibit foam cell formation is very close relationship with the expression of CD36. |
PDF Full Text Request