| BackgroundAtherosclerosis (atherosclerosis, AS) is a kind of chronic vascular disease that the cause is not entirely clear. Its main features include lipid depositing in the vessel wall, the formation of atherosclerotic plaque and fibrous plaque, severe plaque rupturing and even forming thrombosis. Plaque can cause blood vessels be narrowed or even completely blocked, lead some corresponding organs being ischemic and even infarcted. Currently its etiology and pathogenesis is not fully understood. The clear and effective clinical treatment targets are lack and it brings great difficulties with the AS treatment.Although there is no word in ancient books directly with AS corresponding disease name, but according to their clinical symptoms, AS can be expressed as dizziness, headache, Chest, stroke in the traditional Chinese medicine (TCM). TCM thinks the pathogenesis of AS is that the vacuity is the basic, phlegm and blood stasis infested. On behalf of the drug under the theoretical guidance from phlegm and blood, Dan Lou Tablet (DLT) can promote stasis and eliminate phlegm and supply Qi and dredge paralysis. Previous studies found that DLT has a good effect against the AS, but its mechanism of effect has not been elucidated. The formation of the macrophage foam cell plays a key role in the development and transfer process of AS. There is no study on the relationship of DLT and macrophage foam cell against the AS. To further explore the effect of the intervention with DLT on the AS, we designed this study relying on National Natural Science Foundation Youth Project (No.81202782)ObjectiveIn this study, we hope to define the precise role of DLT protecting against AS as inhibiting the forming of macrophages foam cell. We focused on how the macrophage scavenger receptor CD36, the hemagglutinin-like oxidized LDL receptor 1 (LOX-1) and the ATP-binding cassette transporter Al (ABCA1) work in forming macrophage foam cells in the AS and the effect and mechanism of DLT protect against AS in macrophage foam cells and inflammation. This study of DLT protect against AS in order to provide a theoretical basis for its clinical application.Methods1.The effects of DLT on AS in ApoE-/- mice modelGrouping:The ApoE-/- mice were assigned randomized to six groups:Control group (Control), Model group (Model), DLT low-dose group (DLT-L), DLT median-dose group (DLT-M), DLT high-dose group (DLT-H), A atorvastatin group (LPT). The ApoE-/- mice fed with normal diet were as the control group, and the atorvastatin group (LPT) as a positive control. Each group had 10 ApoE-/- mice.Approach:The classical AS model-simply fed high fat diet for long time to set up the AS mouse model.8 weeks old male ApoE-/- mice were selected to set up an atherosclerosis model by feeding high-fat diet for 16weeks. The intervention groups were given the appropriate treatment at the beginning of high-fat diet. The DLT-L, DLT-M, DLT-H three groups were treated with DLT ultrafine solution 1,2,4g/kg/d according to dose equivalent conversion between animals and humans formula. The LPT group was given 2mg/kg/d atorvastatin according to the literature. The Control group and the Model group were received 2ml/kg/d normal saline. Every mouse was gavaged once daily for 16 weeks.Observations:the status and body weight of six groups; the number and area of plaque in carotid artery by carotid ultrasound; the level of serum HDL-C, LDL-C, TC and TG; the vascular tissue, especially the aorta were stained by hematoxylin-eosin and immunohistochemistry with anti-CD68 antibody to evaluate the endometrial or the macrophage infiltration in plaques.Statistical analysis:All values are expressed as the mean±SD. Repeated measures analysis was used for repeated measures data, and ANOVA analysis was used to compare means between three or more groups, and Post Hoc multiple comparisons was used for any two groups. Choose LSD when the variance is homogeneous, and choose Tamhane T2 when the variance is not homogeneous. All data analyses were conducted using SPSS17.0 software. Statistical significance was determined when P-values were less than 0.05.2. The effect and mechanism of DLT on macrophage foam cellGrouping:(1) THP-1 was cultured and grouped as Model group, DLT low-dose group (DLT20μg/ml), DLT median-dose group (DLT1000μg/ml), DLT high-dose group (DLT2000μg/ml). (2) RAW264.7 cell was also cultured and grouped as Control group, Model group, and DLT20, DLT100, DLT500, DLT1000, DLT1500, DLT1750, DLT2000, DLT2250, DLT2500 groups.Approach:(1) We used PMA to stimulate the THP-1 cells transformed into macrophages, and plus oxLDL 50 μg/ml to form macrophage-derived foam cells. The Model group was without drug intervention; the DLT intervention groups plus the corresponding ultrafine solution to make the final concentrations as DLT20 μg/ml, DLT1000 μg/ml, DLT2000μg/ml. Both of the ox-LDL and the DLT solution were simultaneously added to the cell culture wells and the cells with them were incubated for 24h. (2) The RAW264.7 cells were grouped. The Control group was without intervention oxLDL nor DLT. The Model group plus oxLDL or Dil-oxLDL but not DLT. The DLT intervention groups plus oxLDL or Dil-oxLDL and the corresponding ultrafine solution to make the final concentrations as DLT20, DLT100, DLT500, DLT1000, DLT1500, DLT1750, DLT2000, DLT2250, DLT2500 (μg/ml). The ox-LDL or Dil-oxLDL and DLT were simultaneously added to the cell culture wells and incubated for 24h plus ox-LDL, add Dil-oxLDL were incubated for 2h,4h,6h and 24h.Observations:Oil Red 0 staining of THP-1-derived macrophages foam cells in each group were observed. Using the flow cytometry to analysis the mean fluorescence intensity (MFI) of RAW264.7 derived foam cells which plus Dil-oxLDL 10 μg/ml in each group. We used fluorescent quatititive PCR (RT-PCR) to test the effect of DLT on expression level of related components in RAW264.7 derived foam cells, that was the mRNA of expression of CD36, Lectin like oxidized low density lipoprotein receptor 1 (LOX-1), ATP binding cassette transporter Al (ABCA1).Statistical analysis:All values are expressed as the mean+SD. Repeated measures analysis was used for repeated measures data, and ANOVA analysis was used to compare means between three or more groups, and Post Hoc multiple comparisons was used for any two groups. Choose LSD when the variance is homogeneous, and choose Tamhane T2 when the variance is not homogeneous. All data analyses were conducted using SPSS17.0 software. Statistical significance was determined when p-values were less than 0.05.3. The effects of DLT on the inflammation of AS in ApoE-/- miceGrouping:The ApoE-/- mice were assigned randomized to six groups:Control group (Control), Model group (Model), DLT low-dose group (DLT-L), DLT median-dose group (DLT-M), DLT high-dose group (DLT-H), A atorvastatin group (LPT). The ApoE-/- mice fed with normal diet were as the control group, and the atorvastatin group (LPT) as a positive control. Each group had 10 ApoE-/- mice.Approach:The classical AS model-simply fed high fat diet for long time to set up the AS mouse model.8 weeks old male ApoE-/- mice were selected to set up an atherosclerosis model by feeding high-fat diet for 16weeks. The intervention groups were given the appropriate treatment at the beginning of high-fat diet. The DLT-L, DLT-M, DLT-H three groups were treated with DLT ultrafine solution 1,2,4g/kg/d according to dose equivalent conversion between animals and humans formula. The LPT group was given 2mg/kg/d atorvastatin according to the literature. The Control group and the Model group were received 2ml/kg/d normal saline. Every mouse was gavaged once daily for 16 weeks.Observations:FACS analysised the percentage of Th1-IFNγ in spleen cell suspension in each group. RT-PCR and Protein chip tested the mRNA and protein of expression of Tumor necrosis factor-a, Interferon-y.Statistical analysis:All values are expressed as the mean±SD. Repeated measures analysis was used for repeated measures data, and ANOVA analysis was used to compare means between three or more groups, and Post Hoc multiple comparisons was used for any two groups. Choose LSD when the variance is homogeneous, and choose Tamhane T2 when the variance is not homogeneous. All data analyses were conducted using SPSS17.0 software. Statistical significance was determined when P-values were less than 0.05. 4. The effect of DLT on the inflammation of macrophage foam cellGrouping:RAW264.7 cell was also cultured and grouped as Control group, Model group, DLT20 group, and DLT2000 group.Approach:The RAW264.7 cells were grouped. The Control group was without intervention oxLDL nor DLT. The Model group plus oxLDL but not DLT. The DLT intervention groups plus oxLDL and the corresponding ultrafine solution to make the final concentrations as DLT20μg/ml, DLT2000 μg/ml. The ox-LDL and DLT were simultaneously added to the cell culture wells and all of them were incubated for 24h.Observations:RT-PCR and Protein chiptested the mRNA expression of Tumor necrosis factor-α, Interferon-γ and TLR4 and the protein expression of Tumor necrosis factor-α, Interferon-γ.Statistical analysis:All values are expressed as the mean±SD. Repeated measures analysis was used for repeated measures data, and ANOVA analysis was used to compare means between three or more groups, and Post Hoc multiple comparisons was used for any two groups. Choose LSD when the variance is homogeneous, and choose Tamhane T2 when the variance is not homogeneous. All data analyses were conducted using SPSS17.0 software. Statistical significance was determined when P-values were less than 0.05.Results1.The effects of DLT on AS in ApoE-/- mice modelThe status and body weight of six groups:The status of the mice from every group were not different. The time fed with high-fat diet and the changes of body weight of each group were positively correlated, feeding longer, more severe body weight, while there is no interaction between time and group. Before the start of the experiment, the body weight of each group, the difference was not statistically significant (P>0.05), indicating a consistent baseline body weight. After fed 8 weeks to 16 weeks, the body weight of DLT-H group was decreased (P<0.05 and P<0.01), the difference was statistically significant; Since experiment 12 weeks, compared with the model group, the body weight of DLT-H group was decrease (P<0.01), were statistically significant; In The 16th weeks of the experiment, compared with the model group, the weight of DLT-M group was decreased (P<0.05), were significant difference. Experimental results show that compound DanLou tablet control ApoE"A mice increased weight in a dose-dependent manner.The overall assessment of mouse carotid ultrasound:the carotid artery of a uniform continuous control group, no see abnormal luminal. The carotid artery acoustic shadowing of model group mice were most uneven continuous compared with the surrounding tissue (thyroid gland as the reference), part of their blood vessel wall showed hyperechoic light, indicating significant thickening of their blood vessel. The carotid artery of DLT-L can also see a continuous strong echo uneven lighting, the DLT-M group, DLT-H group and atorvastatin group situation gradually improved mitigation.The serum lipid:After 16 weeks, compared with control group, the level of LDL-C, TC, TG of model group were increased (P<0.01), the difference were statistically significant. Compared with the model group, DLT-L group can decrease the level of TG (P<0.05), DLT-M could decrease the level of LDL-C (P<0.05) and TG (P<0.01), DLT-H group can decrease the level of LDL-C (P<0.01), TC (P<0.05) and TG (P<0.01), the difference were statistically significant. The level of LDL-C, TC and TG of Atorvastatin (LPT) group were statistically decreased compared with model group.HE staining:The model group cortex was off, the vascular with a large wall plaque, internal elastic lamina was yet completed, intimal Proliferated seriously, a large number of foam cells in plaque, irregular thickening of the middle and outer membrane, visible foam cell infiltration, indicating that we set up successfully a mice AS model. The foam cell infiltration in mice aorta AS lesions of the DLT-L, DLT-M, DLT-H and Atorvastatin group were gradually reduced. The results show that DLT can reduce the plaque formation and inhibit the vascular smooth muscle cell proliferation remodeling in a dose-dependent manner.CD68 immunohistochemical staining:The aortic AS lesion in each group of mice was brown particles. The results showed that DLT could suppress the macrophage infiltration in the plaque from the high-fat diet fed ApoE-/- mice.2.The effect and mechanism of DLT on macrophage foam cellOil red 0 staining:Compared with the model group, the lipid droplets of DLT (2000μg/ml) were reduced, juse left scattered small lipid droplets. Compared with the model group, the red area of lipid droplets from DLT was significantly reduced.The MFI values with the foam cells:We used flow cytometry to analysis the MFI values with the foam cells which were stimulated with Dil-oxLDL. The results suggested that compared with the model group, DLT were able to significantly reduce the MFI form incubating for 2h, the difference was statistically significant (P<0.05).The expression levels of the mRNA of CD36:The results of RT-PCR showed that compared with control group, the expression levels of the mRNA of CD36 of model group were increased, the difference was statistically significant (P<0.01). And compared with the model group, the DLT2000 group could reduce the expression levels of the mRNA of CD36, the difference was statistically significant (P<0.01)The expression levels of the mRNA of LOX-1:The results of RT-PCR showed that compared with control group, the expression levels of the mRNA of LOX-1 of model group were increased, the difference was statistically significant (P<0.01). And compared with the model group, the DLT2000 group could reduce the expression levels of the mRNA of LOX-1, the difference was statistically significant (P<0.01).The expression levels of the mRNA of ABCA1:The results of RT-PCR showed that compared with control group, the expression levels of the mRNA of ABCA1 of model group was reduced, the difference was statistically significant (P <0.01).And compared with the model group, the DLT group could increase the expression levels of the mRNA of ABCA1, the difference was statistically significant (P<0.05).3. The effects of DLT on the inflammation of AS in ApoE-/- miceThe percentage of Thl-IFNγ in spleen cell suspension:Compared with the control group, the model group could increase the percentage of Thl-IFNγ in spleen cell suspension, the difference was statistically significant (P <0.01). Compared with the model group, the DLT-M, DLT-H group and atorvastatin group all could reduce the percentage of Thl-IFNγ in spleen cell suspension, the difference was statistically significant (P<0.05, P<0.05, P<0.01)The protein expression of inflammatory cytokines TNF-α:We used protein chip method to detect the expression of inflammatory cytokines TNF-a The fold change of TNF-α from the model group compared with DLT-H group was 1.496, more than 1.2, made it significant.The protein expression of inflammatory cytokines IFN-γ:We used protein chip method to detect the expression of inflammatory cytokines IFN-y. The fold change of IFN-γfrom the model group compared with DLT-H group was 1.662, more than 1.2. The IFN-γwas expressed differently and made it significant.The expression levels of the mRNA of TNF-a: The results of RT-PCR showed that compared with control group, the expression levels of the mRNA of TNF-a of model group were increase, the difference was statistically significant (P<0.01).And compared with the model group, the DLT group could reduce the expression levels of the mRNA of TNF-α, the difference was statistically significant (P<0.01).The expression levels of the mRNA of IFN-γ:The results of RT-PCR showed that compared with control group, the expression levels of the mRNA of IFN-y of model group were increased, the difference was statistically significant (P<0.01). And compared with the model group, the DLT group could reduce the expression levels of the mRNA of IFN-y, the difference was statistically significant (P<0.01).4. The effect of DLT on the inflammation of macrophage foam cellThe protein expression of inflammatory cytokines TNF-a:We used protein chip method to detect the expression of inflammatory cytokines TNF-a from foam cells. The fold change of TNF-a from the model group compared with DLT-L group was 1.214, and compared with DLT-H group was 1.311, both more than 1.2. The TNF-a was expressed differently and made it significant.The protein expression of inflammatory cytokines IFN-y:We used protein chip method to detect the expression of inflammatory cytokines IFN-y from foam cells. The fold change of IFN-y from the model group compared with DLT-H group was 1.380, more than 1.2. The inflammatory cytokines IFN-y was expressed differently and made it significant.The expression levels of the mRNA of TNF-a:The results of RT-PCR showed that compared with control group, the expression levels of the mRNA of TNF-a of model group were increase, the difference was statistically significant (P<0.05). And compared with the model group, the DLT group could reduce the expression levels of the mRNA of TNF-a, the difference was statistically significant (P<0.01).The expression levels of the mRNA of IFN-γ:The results of RT-PCR showed that compared with control group, the expression levels of the mRNA of IFN-γ of model group were increase, the difference was statistically significant (P<0.01). And compared with the model group, the DLT group could reduce the expression levels of the mRNA of IFN-γ, the difference was statistically significant (P<0.01)The expression levels of the mRNA of TLR4:The results of RT-PCR showed that compared with control group, the expression levels of the mRNA of TLR4 of model group were increase, the difference was statistically significant (P<0.01).And compared with the model group, the DLT group could reduce the expression levels of the mRNA of TLR4, the difference was statistically significant (P<0.01).ConclusionDan Lou tablet could reduce the phagocytosis of oxLDL with macrophage, increase lipid efflux, reduce the plasma levels of the LDL-C, TG and TC, inhibit macrophage foaming, and reduce the formation of atherosclerotic plaque in the ApoE-/- mice AS model via reducing the expression levels of the mRNA of CD36, LOX-1 and increasing the expression levels of the mRNA of ABCA1.Dan Lou tablet also could relieve the chronic inflammation of blood vessels, and thus play a role in the prevention and treatment of AS since DLT can reduce the macrophages infiltration in plaques, reduce the expression of inflammatory cytokines TNF-α and IFN-γ, reduce the expression levels of the mRNA of TNF-α, IFN-γ and TLR4. |
PDF Full Text Request