Study On The Interventing Mechanism Of Yerba Mate Polyphenols In The Early Stage Of Therosclerosis Formation

BackgroundAtherosclerosis (AS) is the common pathological basis of many cardiovascular, cerebrovascular and peripheral vascular diseases. Lipid dysregulation and lipid deposition are the key step in the occurrence and development of atherosclerosis. However,in the early stages of atherosclerosis, elevated blood lipids play a crucial role. Blood lipids have long been thought to be a potential predisposing factor of AS. A large number of studies have shown that dyslipidemia can lead to blood hyperviscosity, microcirculatory disorders and endothelial dysfunction. Therefore, reducing blood lipids, blood viscosity and accelerating microcirculation have become the treatment of atherosclerosis concerning a therapeutic target.Yerba Mate is derived from the leaves of Ilex Paraguariensis in South American countries. Many pharmacological studies have shown the effects of Yerba Mate, including anti-obesity, anti-diabetic, anti-hypertension, anti-oxidant and lipid-lowering properties. Previous work demonstrated that aqueous Yerba Mate extract has a protective effect against hyperlipidemia in animals. The clinical effects of Yerba mate on the reduction of blood lipids have not been verified. Our team will further observe the effect of Yerba mate on blood lipids, blood viscosity and blood flow in humans, to investigate the impact on the AS.ObjectiveThis study aims to investigate the effect of Yerba mate on blood lipids, blood viscosity and microcirculation of volunteers with high blood lipids.Methods1. Subjects and MethodsFrom November 2012 to December 2013, volunteers with high LDL-C、TC and abnormal nail fold circulation pattern in microscopic images were recruited through physical examination at the Medical Examination Center of Shandong Hospital of Integrated Traditional Chinese and Western Medicine.142 eligible subjects with high blood lipids were randomized at a 1:1 ratio into either the Yerba mate tea-receiving group or placebo treatment. After 6 weeks treatment, results of indexes were compared at Baseline and after 6 Weeks of Yerba Mate or Placebo Intake.2. Blood sampleA fasting-state blood sample of venous blood (14 mL) was collected before and after the 6-week treatment. Eight milliliters was used for Lipid Profile and hemorheological tests. The other blood sample was centrifuged at 4℃ and 4000 rpm for 6 min to isolate the serum phase and immediately cooled to -20℃ for 30 min, and then stored at -80℃ until the time of analysis.3. Lipid Profile AssessmentsFasting blood samples of venous blood were taken in the morning and were transported directly to the Medical Examination Center at the hospital for analysis. TC, HDL-C, LDL-C and TG were observed and compared before and after treatment in the two groups.4. Nailfold Microcirculation AssessmentsEvery subject was maintained indoors for at least 15 min at a room temperature between 23℃ and 25℃. The right ring finger was observed under a microscope base plate with a drop of immersion oil on the nail-fold bed to maximize the translucency of the keratin layer. Nail-fold microcirculation was examined by XW880 Color Microscopic Observation Instrument. Images were captured were taken for all subjects. Images were stored for analysis with image processing software.5. Hemorheology AssessmentsThe blood samples (between 20 min and 4 h) were immediately measured by a MEN-C100 automatic blood rheology dynamic analyzer.6.6-keto-PGF1α and TXB2 AssessmentsDeterminations of 6-keto-PGFlaand TXB2 were expressed by ABC-ELISA technology.7. Statistical AnalysisStatistical analyses were performed with SPSS version 19.0. Differences between two group descriptive characteristics were compared by paired t-test’s, if data was distributed normally. Comparison among multiple groups was performed using LSD analysis. The level of significance was defined as p-value<0.05.Results1. Analysis of blood lipid resultsAfter Yerba Mate and placebo intervention for 6 weeks, HDL-C was increased from basal levels in the Mate-tea population after treatment completion while TC> LDL-C and TG levels were decreased in this group. In fact, no significant changes were observed in any of the lipid parameters within the placebo-tea receiving group. Yerba Mate consumption improved blood lipid profiles to levels expressed by normal un-treated controls.2. Analysis of Nailfold MicrocirculationAfter treatment, the diameter of the afferent limb capillary, diameter of the efferent limb capillary, the ratio between the diameter of the efferent limb and afferent limb capillary, the apical diameter of the capillary loop and weighted integral values were significantly decreased from baseline levels in the Mate tea-receiving group. The blood flow rate was significantly increased in the Mate tea group. At the termination of the treatment, Mate tea drinkers displayed values no different than normal, untreated controls. Conversely, no significant differences were detected after placebo treatment in any of the microcirculatory parameters.3. Analysis of hemorheologic valuesAfter treatment, the values of high shear whole blood viscosity, middle shear whole blood viscosity, low shear whole blood viscosity, plasma viscosity and equation K value of erythrocyte sedimentation rate were significantly decreased in the Mate tea group compared to baseline levels. These values were unchanged in the placebo group. Mate tea group participant hemorheologic values after treatment were statistically similar to normal, untreated group values4. Analysis of TXB2 and 6-keto-PGF1αAfter treatment, TXB2 levels dropped to nearly normal values while 6-keto-PGF1α values rose to nearly normal. Placebo-group serum TXB2 and 6-keto-PGF1α levels, on the other hand, remained unchanged at the end of the treatment periodConclusions1. Yerba mate played a role in the regulation of blood lipids of volunteers with high blood lipids, Yerba Mate consumption decreased TC, LDL-C, TG and increased HDL-C levels2. Yerba mate tea played a role in the regulation of various indexes of nailfold microcirculation, hemorheology, and the platelet aggregating factors 6-keto-PGF1a and TXB2 of volunteers with high blood lipids.3.The clinical study demonstrated that daily consumption of Yerba mate normalized high blood lipids, hyperviscosity and promoted healthy blood flow in patients at risk for AS.BackgroundThe formation of macrophage derived foam cells is a key link in early stage of atherosclerosis. Oxidized low-density lipoprotein has been regarded as the primary villain initiating early atherogenesis by inducing the transfer of monocyte-derived macrophages into foam cells. In the early stage of foam cell formation, Intracellular scavenger receptors of macrophages combines with OX-LDL specifically, and unlimited uptake of OX-LDL leads to intracellular cholesterol and Cholesterol ester accumulation, and the cells then transform into foam cells, followed by the formation of fatty streak. Blood lipid metabolism and inflammatory response play an important role in this process. Inflammation promotes lipid accumulation, and lipid enhances inflammatory response in turn. On one hand, OX-LDL promotes the infiltration and activation of inflammatory cells as well as the release of multiple inflammatory factors. On the other hand, phagocytosis and degradation of OX-LDL by activated microphages lead to intracellular cholesterol accumulation and formation of foam cells, and thus inflammatory factors are further released, which accelerate the intracellular cholesterol accumulation in turn. Although our previous clinical studies have demonstrated that Yerba Mate tea can lower the blood lipid level in sub-healthy people with abnormal blood lipid, the effect of Yerba mate tea on the inhibiting the foam cell formation of AS has not been verified. According to the unsolved problem aboved,we designed the cell Experimental study to investigate the inhibitory effect of Yerba mate on formation of macrophage derived foam cells. The cell Experimental study was divided into two parts. The first Part study aims to investigate the effects of Yerba mate Polyphenols on intracellular lipid accumulation and inflammatory response in the foam cell formation. And then we found that LOX-1 is involved in the anti-AS function of Yerba Mate. Second Part, We first established the LOX-1 gene lentivirus interference vector, and observed the influence of LOX-1 gene lentivirus interference vector and Yerba mate Polyphenols on the lipid metabolism of macrophages and the expression of inflammation-related genes.Part 1. Effects of Yerba mate Polyphenols on intracellular lipid accumulation and inflammatory response in the foam cellObjectiveThis study aims to investigate the effects of Yerba mate Polyphenols on endothelial injury, inflammatory response and blood lipid metabolism in the foam cell formation process of AS.Methods1. Preparation of Yerba Mate PolyphenolsCircumfluence extraction was performed at 80℃ for 60 min using 1000g Yerba Mate tea and 70% ethanol at a solid-liquid ratio of 1:10. Ethanol was recovered by vacuum concentration to obtain the extract. The extract was dissolved in distilled water to pass AB-8 macroporous resin column, and dried by vacuum concentration to obtain 25.3 g Yerba Mate Polyphenols (MP).2. Foam cell cultureThe 100 nmol/L PMA was added into human THP-1 cell culture medium for 48 h to induce the formation of macrophages. Macrophages were then incubated with 50μg/ml OX-LDL for 24 h to generate foam cells. Lipid droplets in foam cells were observed with oil red O staining.3. Cell grouping and treatmentMacrophages were randomly divided into five subgroups:OX-LDL group, OX-LDL+Simvastatin group, OX-LDL+20ug/ml MP group, OX-LDL+100ug/ml MP group, OX-LDL+200ug/ml MP group4.Measured in each group LOX-1mRNA,ACATl mRNA,PCSK9 mRNA,CD36 mRNA,SR—AmRNA Using real-time quantitative RT-PCR technique.5. The intracellular TC、FC、CE、TNF-α、IL-6、IL-1β、VCAM1 levels were measured using a commercially available ELISA kit6.Determination of LOX-1、AKT、NF-κB、ERK1/2 protein expression as well as phosphorylation changes by western blot.7. Statistical analysisAll the experiments were repeated three times. Statistical analyses were performed with SPSS version 19.0. Comparison among multiple groups was performed using LSD analysis. The level of significance was defined as p-value<0.05.Results1. Establishment of foam cell model by THP-1 mononuclear macrophagesThe macrophages were induced with 50μg/ml OX-LDL, followed by oil red O staining after 24 h. Observation under microscope found that intracellular lipid content exceeded 80% in most of cells; cell plasma within a large number of red dye, thus the foam cell model was successfully established.2. Analysis of foam cells interferenceThe foam cells were treated with Simvastatin and MP treatment respectively for 24h, and both significantly reduced intracellular TC,FC and CE content. The result showed that they had the effect of inhibiting foam cell formation.3. Analysis of Scavenger receptorCD36,SR—A,LOX-1 expression were measured by Real-time PCR in THP-1 derived foam cells that had been incubated with OX-LDL alone or in combination with Simvastatin or MP. Data showed that CD36,SR—A,LOX-1 expression in Simvastatin were decreased, and MP only reduced the level of LOX-1 (P<0.01) CD36 and SR—A level was slightly down-regulate,but there was no Statistical significant difference compared to OX-LDL group.4. Analysis of inflammatory factors and adhesion moleculesELISA was used to determine the level of TNF-α,IL-6,IL-1β and VCAM1 in each cell experiments, and the results showed that simvastatin and MP intervention can reduce the level of TNF-α,IL-6,IL-1β and VCAM1. 5.Analysis of AKT、NF-kb、ERK1/2western blot was used to determine the activation rates of AKT、NF-kb、ERK1/2 in each cell experiments, and the results showed that simvastatin and MP intervention can reduce the activation rate of PAKT/AKT、PNF-kb/NF-kb、PERK1/2/NF-kb. 6. Analysis of ACAT1Real-time PCR was used to determine the level of ACAT1 in each cell experiments, and the results showed that simvastatin and MP intervention can reduce the level of ACAT1.Conclusions1. Yerba mate Polyphenols inhibit the formation of macrophage derived foam cells, it can decreased intracellular lipid accumulation and reduced intracellular TC,FC and CE content.2. Yerba mate Polyphenols played a role in the regulation of Scavenger receptor, inflammatory factors, adhesion molecules and blood lipid metabolism. The cell study showed that Yerba mate Polyphenols can inhibit the expression of Scavenger receptor such as LOX-1, inflammatory factors such as TNF-α, IL-1β, IL-6, adhesion molecules VCAM-1 and blood lipid metabolism gene such as ACAT1, and it have no interference effect of PCSK9 and some Scavenger receptor,such as CD36 and SR-A.3. Yerba Mate Polyphenols has a regulatory effect on ERK1/2、AKT、NF-KB,and reduce the intracellular lipid accumulation and inflammatory response in the foam cell formation.Part 2. Construction of LOX-1-targeting RNAi lentivirus and investigation of the effect of Yerba mate Polyphenols on intracellular lipid metabolism and inflammatory response in the foam cellObjective1. Construct RNA interference lentivirial vectors targeting LOX-1 gene and prepare mature virus, and screening the best target sequence.2. Research the effects of Yerba mate Polyphenols on the lipid metabolism and inflammation-related genes in the foam cell induced by OX-LDL with the silencing expression of LOX-1 gene.Methods1. Construction of LOX-1 gene lentivirus interference vectorThree target sequences of LOX-1 gene were designed. The synthesized single-stranded DNA formed double-stranded DNA after annealing.DNA oligo nucleotides of target sequence were synthesized and cloned into lentivirial vector. Lentiviral vector LOX-1 shRNA was co-transfected into 293T cells with packaging plasmid. Determination of LOX-1 protein expression in 293 T cells by western blot2. Cell grouping and treatmentThe foam cells were divided into 5 groups:Blank vector group,Simvastatin group,200μg/ml MP group,LOX-1 interference group:200μg/ml MP+LOX-1 interference group.3. Determination of TNF-a, VCAM-1, MCP-1,eNOS and ACAT expression in OX-LDL induced foam cells after LOX-1 lentivirus interference by ELISA4. Determination of LOX-1 protein expression and NF-κB protein expression as well as phosphorylation changes in OX-LDL induced foam cells after LOX-1 lentivirus interference by western blot.5. Statistical analysisAll the experiments were repeated three times. Statistical analyses were performed with SPSS version 19.0. Comparison among multiple groups was performed using LSD analysis. The level of significance was defined as p-value<0.05.Results1. Construction of LOX-1 gene lentivirus interference vectorDNA sequencing demonstrated that the inserted sequences were successfully constructed. The results of Westen Blotting showed that the relative expression level of LOX-1 protein in 3 RNAi groups was significantly reduced compared with that in blank control group and negative control group, and the effect on the interference site 2 of LOX-1 was the best inhibition efficiency.2. Effect of LOX-1 lentivirus vector on NF-κB in the process of inducing foam cellsWestern blot showed that LOX-1 gene silencing can lead to activation of NF-κB, and can promote activation of NF-κB if combined with MP.3. Effect of LOX-1 lentivirus vector on TNF-α, VCAM-1, MCP-1, eNOS and ACAT expression in the process of inducing foam cellsELISA showed that LOX-1 gene lentivirus interference vector can significantly reduce the expression of TNF-α, VCAM-1, MCP-1 and ACAT, and increase the expression of eNOS; it can further promote this effect if combined with MP.Conclusions1. RNAi lentivirial vectors targeting LOX-1 were constructed successfully. The expression of LOX-1 can be inhibited effectively.2.The inhibition of LOX-1 can inhibit the expression of NF-κB、TNF-α、IL-6、 VCAM-1、ICAM-1、MCP-1、ACAT1, up-regulating the expression of eNOS. MP intervention can enhance the regulatory effects on above molecules, prevent the formation of foam cells.3. LOX-1 gene silencing can inhibit activation of NF-κB pathway to reduced the inflammatory factors, adhesion molecules and intracellular lipid accumulation. MP may interfere with the LOX-1/NF-κB pathway to inhibit the formation of macrophage derived foam cells, which played the role of anti-atherosclerosis.BackgroundMacrophage derived foam cells,as one of early event of atherosclerosis, is a Lipid metabolism disorder in vascular, characterized by blood lipid deposition, monocyte aggregation, vascular endothelial dysfunction, intracellular and extracellular lipid accumulation, foam cell formation and smooth muscle cells proliferation, which leading to the formation of fatty streak and Atherosclerotic Plaque. Therefore, foam cells play a key role in the formation and development of Atherosclerotic Plaque. Our previous clinical studies have demonstrated that Yerba Mate tea can lower the blood lipid level in sub-healthy people with abnormal blood lipid in the first part. Then, in the second part we observed the role of Yerba mate Polyphenols in macrophage derived foam cells Scavenger receptor,inflammatory factors,adhesion molecules and chemokine expression, intracellular lipid content and lipid accumulation. Next, we observed the effect of LOX-1 RNAi lentivirus on intracellular lipid accumulation and inflammatory response in the foam cell formation.and Yerba mate Polyphenols intervention can enhance above regulatory effects. Although Previous researches suggest that Yerba mate Polyphenols inhibit the formation of macrophage derived foam cells, but the effect of Yerba mate Polyphenols on the AS plaques has not yet been verified. And now there is no report about the relationship between Yerba mate Polyphenols and the atherosclerotic plaques. Therefore we choose to explore it. According to the unsolved problem above, we designed this animal’s experimental study to investigate the inhibitory influence of Yerba mate on formation of atherosclerotic plaques and its mechanisms in Apolipoprotein E-deficient Mice. Our findings provide new insights for revealing the potential roles, mechanisms and therapeutic value of Yerba mate in atherosclerosis.ObjectiveThis study aims to observe the effect of MP on lipid metabolism and inflammatory response in atherosclerotic plaque of Apolipoprotein E-deficient Mice, to investigate the part of the role of anti-atherosclerosis mechanism.Methods1. Animal ProtocolSixty Six-week-old male apoE-/- mice were fed with a high fat high cholesterol diet including 0.15% cholesterol and 21% fat. Twelve eight-week-old male C57 mice were fed a normal diet. After 12 weeks, apoE-/-mice were randomly divided into five groups:Model group, Simvastatin group, MP low-dose group(50 mg/kg MP), MP medium-dose group (100 mg/kg MP), MP high-dose group (200 mg/kg MP) (n=12 for each group). C57 mice were the normal control group. MP or Simvastatin were given orally, once a day for 12 weeks respectively. Mice in model and normal control groups were received the same volume of water at the same time.2. Blood sampleAt the end of the study, the blood samples were collected from eye of mice that fasting for 12 hours, after injected with excessive pentobarbital sodium. And the serums of mice with 3000rpm for 10min were used to measure the level of serum lipid and serum other indexes respectively by automatic biochemical analyzer and ELISA assay.3. Histopathological analysis:At the end of the experiment, the aortic root were removed to produce specimens which stained with hematoxylin and eosin and oil red O.4. Lipid Profile AssessmentsThe blood samples of Fasting were transported directly to the Medical Examination Center at Hospital for analysis. TC, HDL-C, LDL-C and TG were observed in the six groups.5. The aortas of mice were used for analyzing the expression of TNF-α、IL-6、ICAM-1、 VCAM-1 and MCP-1 by ELISA.6. The aortas of mice were used for measuring the phosphorylated expression of NF-KB and LOX-1 protein expression by western blot.7. Statistical analysisStatistical analyses were performed with SPSS version 19.0. Comparison among multiple groups was performed using LSD analysis. The level of significance was defined as p-value<0.05.Results1. Histological and Morphology analysisAfter the MP intervention for 12 weeks, the structures of the aortic arches by HE stain and observed showed that the normal group, Simvastatin group and MP high-dose group are normal aorta wall. There was no lipid deposition in normal aortic wall. Atherosclerotic lipid plaques were observed in other groups (all apoE-/- mice) and the lumens were narrowed in a different degree. Compared with model group, lipid infiltration in plaques were lower in each treatment group (There was no lipid deposition in normal aortic wall. There were a lot of red dye plaque lipid deposition in model group. Compared with model group,lipid infiltration in plaques were lower in each treatment group)2. Serum lipid concentration analysisTC, TG and LDL-C concentration was significantly higher in the Model group than that in normal group. TC, TG and LDL-C levels in each treatment group were significmitly lower compared with that in Model group. So MP can regulate lipid metabolism by reducing the levels of TC、TG、LDL-C.3. Inflammatory response analysisELISA was used to determine the level of TNF-α、IL-6、VCAM-1、ICAM-1 and MCP-1 in each group, and western blot was used to determine the activation rates of NF-kb. The results showed that MP suppress inflammatory response by reducing the levels of NF-kb、TNF-α、IL-6、VCAM-1、ICAM-1 and MCP-1.4. Protective effect of vascular endothelium analysis The ELISA results showed that MP improves the aortas endothelial function with high level of eNOS.5. Analysis of AC AT1ELISA was used to determine the level of ACAT1 in each groups, and the results showed that simvastatin and MP intervention can reduce the level of AC AT1.Conclusions1.MP can reducing the levels of TC、TG、LDL-C、LOX-1、ACAT-1 in the ApoE mice, which could significantly inhibit OX-LDL-induced macrophage foam cell formation and the growth of lipid plague of atherosclerosis. MP can regulate lipid metabolism, which may be one of the mechanisms of their anti-atherosclerosis effect.2.MP can inhibit activation of NF-κB pathway to reduced the inflammatory factors, adhesion molecules and chemokine in lipid plague of ApoE mice, such as TNF-α、 IL-6、ICAM-1、VCAM-1、MCP-1. MP played the role in the regulation of inflammatory response in the ApoE mice, which may be another important mechanisms of inhibiting the foam cell formation and their anti-atherosclerosis effect.3. MP can inhibit the progress of existing atherosclerotic lesions in ApoE-/- mice. The possible mechanisms may be associated with lipid metabolism and inflammatory response. We conclude that MP might be a potential diet for the development of antiatherosclerotic therapeutics.