OBJECTIVE: To investigate the effect of zedoarondiol on neovascularization of atherosclerotic (AS) plaque by exosomes experiment. METHODS: ApoE^-/- mice were fed with high-fat diet to establish AS model and treated with high- and low-dose (10, 5 mg/kg daily) of zedoarondiol, respectively. After 14 weeks, the expressions of anti-angiogenic protein thrombospondin 1 (THBS-1) and its receptor CD36 in plaques, as well as platelet activation rate and exosome-derived miR-let-7a were detected. Then, zedoarondiol was used to intervene in platelets in vitro, and miR-let-7a was detected in platelet-derived exosomes (Pexo). Finally, human umbilical vein endothelial cells (HUVECs) were transfected with miR-let-7a mimics and treated with Pexo to observe the effect of miR-let-7a in Pexo on tube formation. RESULTS: Animal experiments showed that after treating with zedoarondiol, the neovascularization density in plaques of AS mice was significantly reduced, THBS-1 and CD36 increased, the platelet activation rate was markedly reduced, and the miR-let-7a level in Pexo was reduced (P<0.01). In vitro experiments, the platelet activation rate and miR-let-7a levels in Pexo were significantly reduced after zedoarondiol's intervention. Cell experiments showed that after Pexo's intervention, the tube length increased, and the transfection of miR-let-7a minics further increased the tube length of cells, while reducing the expressions of THBS-1 and CD36. CONCLUSION Zedoarondiol has the effect of inhibiting neovascularization within plaque in AS mice, and its mechanism may be potentially related to inhibiting platelet activation and reducing the Pexo-derived miRNA-let-7a level.
Animals ; MicroRNAs/genetics* ; Exosomes/drug effects* ; Plaque, Atherosclerotic/genetics* ; Neovascularization, Pathologic/genetics* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Blood Platelets/drug effects* ; Apolipoproteins E/deficiency* ; Thrombospondin 1/metabolism* ; CD36 Antigens/metabolism* ; Platelet Activation/drug effects* ; Male ; Mice ; Mice, Inbred C57BL

OBJECTIVETo investigate the effects of the DPP4 inhibitor sitagliptin on the expressions of early growth response-1 (Egr-1) and fibronectin in the kidney of ApoE gene knockout mice.

METHODSEight-week-old male ApoE gene knockout mice were randomly divided into sitagliptin + apoE(-/-) group and apoE(-/-) group (n=6), with 6 C57BL mice as the normal control group. After feeding with high-fat diet and drug treatment for 16 weeks, the mice underwent intraperitoneal glucose tolerance test (IPGTT) and were measured for 24-h urinary albumin using ELISA. All the mice were then sacrificed to examine the changes of blood lipid profile and for detection of Egr-1 and fibronectin mRNA and proteins in the renal tissue using real-time PCR and Western blotting.

RESULTSThe mice in both apoE(-/-) group and sitagliptin+apoE(-/-) group all showed prominently increased blood lipids as compared with the control group (P<0.05) without significant differences between the two apoE(-/-) groups. The level of HDL was significantly higher in sitagliptin +apoE(-/-) group than in apoE(-/-) group (P<0.001) and control group (P<0.001). IPGTT showed no significant differences in the levels of blood glucose among the 3 groups. The excretion of urinary albumin was increased in apoE(-/-) group compared with the control group (P<0.01), but was significantly lower in sitagliptin+ apoE(-/-) group than in apoE(-/-) group (P<0.01). Real-time PCR and Western blotting showed significantly decreased mRNA and protein expressions of renal cortical Egr-1 and fibronectin in sitagliptin+apoE(-/-) group compared with apoE(-/-) group.

CONCLUSIONSitagliptin can reduce the renal expression of fibronectin by regulating the expression of Egr-1 to achieve renal protection.

Animals ; Apolipoproteins E ; genetics ; Diet, High-Fat ; Dipeptidyl-Peptidase IV Inhibitors ; pharmacology ; Early Growth Response Protein 1 ; metabolism ; Fibronectins ; metabolism ; Gene Knockout Techniques ; Kidney ; metabolism ; Lipids ; blood ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Real-Time Polymerase Chain Reaction ; Sitagliptin Phosphate ; pharmacology

OBJECTIVETo study the effect of polydatin on the expression level of miR-214 and liver function in atherosclerotic mice.

METHODSForty male ApoE(-/-) mice were randomly allocated into 4 groups (n=10), namely the model group, low- and high-dose polydatin groups, and simvastin group, with 10 male C57BL/6J mice serving as the normal control group. Mouse models of atherosclerosis were established by feeding the ApoE(-/-) mice with a high-fat diet. After 12 weeks of treatment, blood levels of glucose, lipids, AST, and ALT and the contents of T-SOD and MDA in the liver tissue were detected. The pathologies of the liver were examined with HE staining, and miR-214 expression in the liver was detected using quantitative real-time PCR.

RESULTSCompared with the normal control mice, the mice in the model group showed significantly increased blood glucose, serum TC, TG, LDL-C, ALT, and AST levels, and MDA contents in the liver (P<0.01), with significantly decreased serum HDL-C level and SOD and miR-214 levels in liver (P<0.01). Polydatin treatment significantly ameliorated such changes in blood glucose, serum ALT, AST, TC, TG, LDL-C, and HDL-C levels, and MDA, SOD, and miR-214 contents in liver tissue (P<0.05).

CONCLUSIONs Polydatin can reduce blood glucose and lipid levels and protect the liver function in atherosclerotic mice possibly by up-regulating the expression of miR-214 and T-SOD and down-regulating MDA in the liver.

Animals ; Apolipoproteins E ; genetics ; Atherosclerosis ; drug therapy ; Blood Glucose ; analysis ; Diet, High-Fat ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Glucosides ; pharmacology ; Lipids ; blood ; Liver ; drug effects ; Male ; Malondialdehyde ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; MicroRNAs ; metabolism ; Stilbenes ; pharmacology ; Superoxide Dismutase ; metabolism

OBJECTIVETo study the effect of Jinlida on changes in expression of skeletal muscle lipid transport enzymes in fat-induced insulin resistance ApoE -/- mice.

METHODEight male C57BL/6J mice were selected in the normal group (NF), 40 male ApoE -/- mice were fed for 16 weeks, divided into the model group (HF), the rosiglitazone group ( LGLT), the Jinlida low-dose group (JLDL), the Jinlida medium-dose group (JLDM), the Jinlida high-dose group (JLDH) and then orally given drugs for 8 weeks. The organization free fatty acids, BCA protein concentration determination methods were used to determine the skeletal muscle FFA content. The Real-time fluorescent quantitative reverse transcription PCR ( RT-PCR) and Western blot method were adopted to determine mRNA and protein expressions of mice fatty acids transposition enzyme (FAT/CD36), carnitine palm acyltransferase 1 (CPT1), peroxide proliferators-activated receptor α( PPAR α).

RESULTJinlida could decrease fasting blood glucose (FBG), cholesterol (TC), triglyceride (TG), free fatty acid (FFA) and fasting insulin (FIns) and raise insulin sensitive index (ISI) in mice to varying degrees. It could also up-regulate mRNA and protein expressions of CPT1 and PPARα, and down-regulate mRNA and protein levels of FAT/CD36.

CONCLUSIONJinlida can improve fat-induced insulin resistance ApoE -/- in mice by adjusting the changes in expression of skeletal muscle lipid transport enzymes.

Animals ; Apolipoproteins E ; deficiency ; genetics ; Blood Glucose ; metabolism ; CD36 Antigens ; genetics ; metabolism ; Carnitine O-Palmitoyltransferase ; genetics ; metabolism ; Dietary Fats ; adverse effects ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Hypoglycemic Agents ; administration & dosage ; Insulin ; metabolism ; Insulin Resistance ; Lipid Metabolism ; drug effects ; Male ; Metabolic Diseases ; drug therapy ; enzymology ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Muscle, Skeletal ; drug effects ; metabolism

We evaluated the association of the APOE polymorphism with serum C-reactive protein levels and white blood cell count in two large population-based studies in Korean. The datasets included the Dong-gu study (n = 8,893) and the Namwon Study (n = 10,032). APOE genotypes were identified by polymerase chain reaction-restriction fragment length polymorphism. Multivariable linear regression analysis was performed to evaluate the relationship of APOE genotypes with C-reactive protein levels and white blood cell count with adjustments for age, sex, body mass index, smoking, diabetes, hypertension, and serum lipids. In the multivariate model, carriers of E3E4 or E4E4 genotype had significantly lower C-reactive protein levels compared with carriers of E3E3 genotype group (0.50 mg/L vs. 0.67 mg/L; 0.37 mg/L vs. 0.67 mg/L, respectively, for the Dong-gu Study and 0.47 mg/L vs. 0.66 mg/L; 0.45 mg/L vs. 0.66 mg/L, respectively, for the Namwon Study). However, there was no difference in white blood cell count among APOE genotypes. We found that the APOE E4 allele is associated with lower C-reactive protein levels, but not white blood cell count. Our results suggest that APOE genotype may influence C-reactive protein levels through non-inflammatory pathway.
Aged ; Apolipoproteins E/*genetics ; C-Reactive Protein/*metabolism ; Female ; Genetic Association Studies ; Genotype ; Humans ; Inflammation/*blood/immunology ; Leukocyte Count ; Male ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide/genetics ; Prospective Studies ; Republic of Korea

OBJECTIVETo investigate the effects of carbon disulfide (CS(2)) inhalation on the lipid levels of ApoE knockout gene mice and C57BL/6J mice.

METHODSFifty-one male ApoE gene knockout mice were randomly divided into four groups: CS(2)-exposed normal diet group, CS(2)-unexposed normal diet group, CS(2)-exposed high-fat diet group, and CS(2)-unexposed high-fat diet group. Fifty male C57BL/6J mice were divided into four groups in the same way. The exposed groups received 1000 mg/m3 CS(2) by static inhalation (5h/d, 5d/w) for four weeks. The weight of each mouse was determined and recorded once a week. On the 14th day of exposure, six mice in each group were randomly selected to measure serum total cholesterol (TC) levels. On the 28th day of exposure, the serum levels of TC and low-density lipoprotein (LDL) in the remaining mice were measured.

RESULTSThe mean weight gain of exposed groups was less than that of the unexposed groups. On the 14th and 28th days of experiment, the TC levels of the CS2-exposed high-fat diet group were significantly higher than those of the CS(2)-unexposed high-fat diet group among ApoE knockout gene mice (P < 0.01 for both). On the 14th day of experiment, the TC levels of the CS(2)-unexposed high-fat diet group were significantly higher than those of the CS(2)-unexposed normal-diet group among C57BL/6J mice group (P < 0.05). On the 28th day of experiment, the LDL levels of the CS(2)-exposed high-fat diet group were significantly higher than those of the CS(2)-unexposed high-fat diet group among ApoE knockout gene mice (P = 0.003).

CONCLUSIONCS(2) exposure, high-fat diet, and ApoE gene knockout can elevate blood lipids in mice, thus increasing the risk of atherosclerosis.

Administration, Inhalation ; Animals ; Apolipoproteins E ; genetics ; Atherosclerosis ; Body Weight ; Carbon Disulfide ; toxicity ; Diet, High-Fat ; adverse effects ; Gene Knockout Techniques ; Lipid Metabolism ; drug effects ; Lipids ; blood ; Lipoproteins, LDL ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout

The role of methyl-CpG binding domain protein 2 (MBD2) in an ApoE-deficient mouse model of age-related macular degeneration (AMD) was investigated. Eight-week-old Mbd2/ApoE double deficient (Mbd2(-/-) ApoE(-/-)) mice (n=12, 24 eyes, experimental group) and MBD2 (wt) ApoE(-/-) mice (n=12, 24 eyes, control group) were fed on Western-type diet for 4 months. The mice were sacrificed, and total serum cholesterol levels were analyzed and Bruch's membrane (BM) of the eyes was removed for ultrastructural observation by transmission electron microscopy. Moreover, intercellular adhesion molecule 1 (ICAM-1) immunoreactivities were evaluated by fluorescence microscopy in sections of the eyes in both groups for further understanding the function mechanism of MBD2. There was no significant difference in the total serum cholesterol levels between control group and experimental group (P>0.05). Transmission electron microscopy revealed that AMD-like lesions, various vacuoles accumulated on BM, notable outer collagenous layer deposits and dilated basal infoldings of retinal pigment epithelium (RPE) were seen in both groups, and the BM in control group was significantly thickened as compared with experimental group (P<0.05). Fluorescence micrographs exhibited the expression of ICAM-1 in choroid was higher in control group than in experimental group. We are led to conclude that MBD2 gene knockout may lead to accumulation of more deposits on the BM and influence the pathogenesis of AMD via triggering endothelial activation and inflammatory response in choroid, improving microcirculation, and reducing lipid deposition so as to inhibit the development of AMD-like lesions. Our study helps to provide a new therapeutic approach for the clinical treatment of AMD.
Animals ; Apolipoproteins E ; genetics ; metabolism ; Bruch Membrane ; metabolism ; ultrastructure ; Cholesterol ; blood ; Choroid ; metabolism ; ultrastructure ; DNA-Binding Proteins ; genetics ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Macular Degeneration ; blood ; genetics ; metabolism ; Male ; Mice ; Mice, Knockout ; Microscopy, Electron, Transmission ; Microscopy, Fluorescence ; Retinal Pigment Epithelium ; metabolism ; ultrastructure

OBJECTIVETo study the effect of Dangua Recipe (DGR) on glycolipid metabolism, vascular cell adhesion molecule-1 (VCAM-1) and its mRNA expression level of transgenic Apo E(-/-) mouse with spontaneous atherosclerosis, thus revealing its partial mechanism for curing diabetes mellitus (DM) with angiopathy.

METHODSDiabetic model was prepared by peritoneally injecting streptozotocin (STZ) to Apo E(-/-) mouse. Totally 32 modeled mice were stratified by body weight, and then divided into 4 groups referring to blood glucose levels from low to high by random digit table, i.e., the model group (MOD, fed with sterile water, at the daily dose of 15 mL/kg), the DGR group (fed with DGR at the daily dose of 15 mL/kg), the combination group (COM, fed with DGR at the daily dose of 15 mL/kg and pioglitazone at the daily dose of 4.3 mg/kg), and the pioglitazone group (PIO, at the daily dose of 4.3 mg/kg), 8 in each group. Another 8 normal glucose C57 mouse of the same age and strain were recruited as the control group. All interventions lasted for 12 weeks by gastrogavage. The fasting blood glucose (FBG), body weight, food intake, water intake, skin temperature, the length of tail, and the degree of fatty liver were monitored. The hemoglobin A1c (HbA1c), total cholesterol (TC), and LDL-C were determined. Endothelin-1 (ET-1) was determined by radioimmunoassay. Nitrogen monoxidum (NO) was determined by nitrate reductase. The kidney tissue VCAM-1 level was analyzed with ELISA. The expression of VCAM-1 mRNA in the kidney tissue was detected with real time quantitative PCR.

RESULTSCompared with the control group, the body weight and food intake decreased, water intake increased in all the other model groups (P < 0.05). Besides, the curve of blood glucose was higher in all the other model groups than in the control group (P < 0.01). Compared with the model group, the body weight increased; levels of HbAlc, TC, LDL-C, ET-1, and VCAM-1 were significantly lower; and skin temperature was higher in the DGR group (P < 0.05, P < 0.01). Compared with the PIO group, body weight, the increment of body weight, FBG, TC, and LDL-C were lower (P < 0.05, P < 0.01); food intake and water intake increased more and the tail length was longer in the DRG group (P < 0.01). There was no statistical difference in the level of NO among groups. The degree of fatty liver in the model group was significantly severer than that in the control group (P < 0.05). It was obviously alleviated in the DGR group (P < 0.05) when compared with the model group and the PIO group (P < 0.05, P < 0.01). But it was severer in the PIO group than in the model group (P < 0.01). The degree of fatty liver in the combination group ranged between that of the DGR group and the PIO group (P < 0.05). The level of VCAM-1 mRNA expression was significantly lower in the DGR group than in the model group, the PIO group, and the combination group (P < 0.05).

CONCLUSIONSDGR had effect in lowering blood glucose and blood lipids, and fighting against fatty liver of transgenic Apo E(-/-) mouse with spontaneous atherosclerosis. DGR played an effective role in preventing and treating DM with angiopathy by comprehensively regulating glycolipid metabolism and promoting the vascular function.

Animals ; Apolipoproteins E ; genetics ; Blood Glucose ; metabolism ; Diabetes Mellitus, Experimental ; blood ; drug therapy ; Diabetic Angiopathies ; drug therapy ; Drugs, Chinese Herbal ; pharmacology ; Lipids ; blood ; Male ; Mice ; Mice, Knockout ; RNA, Messenger ; genetics ; Random Allocation ; Thiazolidinediones ; pharmacology ; Vascular Cell Adhesion Molecule-1 ; genetics ; metabolism

OBJECTIVETo observe the effect of Huanglian Jiedu IJecoction (HJU) on systemic and vascular immune responses of high fat diet fed apoE deficient (apoE(-/-)) mice.

METHODSEight wild type C57BL6 mice were recruited as the wild type common food group. Totally 24 apoE(-/-) mice were randomly divided into the ApoE'common food group, the ApoE(-/-) hyperlipidemia group, and the ApoE(-/-) hyperlipidemia plus HJD group, 8 in each group. In the present study, the common food mice and high fat fed mice were fed with a chow diet or a high cholesterol diet for 4 weeks. HJD was given to mice in the ApoE(-/-) hyperlipidemia plus HJD group at the daily dose of 5 g/kg by gastrogavage, while equal volume of pure water was given to mice in the rest groups by gastrogavage. Four weeks later, the plasma levels of blood lipids, the ratio of peripheral blood mononuclear cells, and expressions of Toll-like receptor 4 (TLR-4) and CD36 on the monocytes were detected. The pathological changes and expressions of cytokines in local aorta were detected. The plasma cytokine levels in response to lipopolysaccharide (LPS) were analyzed. Results (1) Compared with the wild type common food group, TO, TG, and LDL-O significantly increased in the ApoE(-/-) common food group (P < 0. 05, P < 0.01). Compared with the ApoE(-/-) common food group, TC and LDL-C significantly increased in the hyperlipidemia group (P < 0. 05). There was no statistical difference in each index between the ApoE(-/-) hyperlipidemia group and the ApoE(-/-) hyperlipidemia plus HJD group (P > 0.05). (2) Compared with the wild type common food group, no obvious change of the ratio of peripheral blood mononuclear cells happened, the TLR4 expression level significantly increased in the ApoE'common food group (P < 0. 05). Compared with the ApoE common food group, the ratio of peripheral blood mononuclear cells and the TLR4 expression level significantly increased in the ApoE' hyperlipidemia group (P < 0.05). Compared with the ApoE(-/-) hyperlipidemia group, the ratio of peripheral blood mononuclear cells and the TLR4 expression level significantly decreased. Besides, the CD36 expression level also significantly decreased (P<0.05). (3) After stimulated by LPS for 3 h, compared with the wild type common food group, plasma TNF-ct and IL-b expressions significantly increased in the ApoE(-/-) common food group (P < 0.05). Compared with the ApoE(-/-) common food group, plasma expressions of IL-12, TNF-alpha, MCP-1, and IL-10 increased, but with no statistical difference in the ApoE(-/-) hyperlipidemia group (P > 0.05). After 4-week intervention of HJD, compared with the ApoE(-/-) hyperlipidemia group, the MCP-1 expression was significantly down-regulated, while the IL-10 expression significantly increased, showing statistical difference (P < 0.05). Compared with the wild type common food group, mRNA expression levels of IFN-gamma, MCP-1 , TNF-alpha, IL-10, and IL-1beta significantly increased (P < 0. 05, P < 0.01). Compared with the ApoE(-/-) common food group, not only mRNA expression levels of IFN-gamma, MCP-1, TNF-alpha, and IL-1beta, further significantly increased, but also IL-12, IL-10, and TGF-beta significantly increased (P < 0. 05, P < 0. 01). After 4-week intervention of HJD, compared with the ApoE(-/-) hyperlipidemia group, mRNA expression levels of MCP-1, TNF-alpha, IL-1beta, and IL-12 significantly decreased in the ApoE(-/-) hyperlipidemia plus HJD group (P < 0.05, P < 0.01).

CONCLUSIONSHigh fat diet induced systemic reaction and inflammatory reactions of local vessels. The local inflammatory response of vessels exceeded systemic inflammatory response. Intervention of HJD could attenuate inflammatory response, especially in local arteries. Meanwhile, it enhanced systemic anti-inflammatory reactions.

Animals ; Aorta ; pathology ; Apolipoproteins E ; genetics ; CD36 Antigens ; metabolism ; Chemokine CCL2 ; metabolism ; Dietary Fats ; adverse effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hyperlipidemias ; blood ; etiology ; immunology ; Inflammation ; Interleukin-10 ; blood ; Interleukin-12 ; blood ; Interleukin-1beta ; blood ; Leukocytes, Mononuclear ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Systemic Inflammatory Response Syndrome ; blood ; etiology ; immunology ; Toll-Like Receptor 4 ; metabolism ; Transforming Growth Factor beta ; blood ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo study the effects of ApoE gene polymorphism on anti-inflammatory action of Xuezhikang Capsule.

METHODSOne hundred and two patients with hyperlipidemia (as the treated group) and one hundred healthy volunteers (as the control group) were enrolled in the case-control study. Total DNA of the peripheral blood was extracted and ApoE genotypes were determined by PCR sequence analysis. The serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and high sensitivity C reactive protein (hs-CRP)were measured in all subjects. The changes of TNF-alpha, IL-6, and hs-CRP were detected before and after 6-week Xuezhikang Capsule treatment, thus analyzing the correlation between ApoE gene polymorphism and changes of each inflammatory factor.

RESULTSThe frequency of E3/3 genotype was 86% (86/100 cases)in the control group, significantly higher than that of the treated group (62.7%, 64/102 cases). The frequency of E3/4 genotype was 6% (6/100 cases) in the control group, significantly lower than that of the treated group (21.6%, 22/102 cases; both P < 0.05). Compared with the control group, the serum levels of TNF-alpha, IL-6, and hs-CPR were higher in the treated group before treatment (P < 0.05). In hyperlipidemia patients with E3/4 + E4/4 genotype, the serum level of TNF-alpha was higher than that of E3/3 genotype (P < 0.05); the serum level of IL-6 was higher than that of E2/E2 + E2/E3 genotype (P < 0.05); the serum level of hs-CRP was higher than that of E2/E2 + E2/E3 and E3/E3 genotype (P < 0.05). But there was no statistical difference in the serum levels of TNF-alpha, IL-6, or hs-CPR between E3/3 and E2/E2 + E2/E3 genotype. After 6-week intervention of Xuezhikang Capsule, the serum levels of TNF-alpha, IL-6, and hs-CRP were lower in the treated group (P < 0.05), but the serum levels of TNF-alpha and IL-6 were still higher than those of the control group (P < 0.05). But there was no statistical difference in the decrement of TNF-alpha, IL-6, or hsCRP among E2/E2 + E2/E3, E3/E3, or E3/4 + E4/4 genotypes (P > 0.05).

CONCLUSIONSThe distribution of ApoE gene polymorphism is different between the hyperlipidemia patients and the healthy people. Chronic inflammatory reactions exist in hyperlipidemia patients, especially in those with e4 allele. Xuezhikang Capsule showed anti-inflammatory effects, but ApoE gene polymorphism did not affect its effects.

Adult ; Aged ; Anti-Inflammatory Agents ; therapeutic use ; Apolipoproteins E ; genetics ; C-Reactive Protein ; metabolism ; Case-Control Studies ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Genotype ; Humans ; Hyperlipidemias ; drug therapy ; genetics ; Interleukin-6 ; blood ; Male ; Middle Aged ; Phytotherapy ; Polymorphism, Genetic ; Tumor Necrosis Factor-alpha ; blood