The present study aimed to investigate the protective effect of S-propargyl-cysteine (SPRC) on atherosclerosis progression in mice. A mouse model of vulnerable atherosclerotic plaque was created in ApoE^-/- mice by carotid artery tandem stenosis (TS) combined with a Western diet. Macrophotography, lipid profiles, and inflammatory markers were measured to evaluate the antiatherosclerotic effects of SPRC compared to atorvastatin as a control. Histopathological analysis was performed to assess the plaque stability. To explore the protective mechanism of SPRC, human umbilical vein endothelial cells (HUVECs) were cultured in vitro and challenged with oxidized low-density lipoprotein (ox-LDL). Cell viability was determined with a Cell Counting Kit-8 (CCK-8). Endothelial nitric oxide synthase (eNOS) phosphorylation and mRNA expression were detected by Western blot and RT-qPCR respectively. The results showed that the lesion area quantified by en face photographs of the aortic arch and carotid artery was significantly less, plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) were reduced, plaque collagen content was increased and matrix metalloproteinase-9 (MMP-9) was decreased in 80 mg/kg per day SPRC-treated mice compared with model mice. These findings support the role of SPRC in plaque stabilization. In vitro studies revealed that 100 μmol/L SPRC increased the cell viability and the phosphorylation level of eNOS after ox-LDL challenge. These results suggest that SPRC delays the progression of atherosclerosis and enhances plaque stability. The protective effect may be at least partially related to the increased phosphorylation of eNOS in endothelial cells.
Animals ; Humans ; Mice ; Atherosclerosis ; Cholesterol/metabolism* ; Cysteine/pharmacology* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Lipoproteins, LDL/pharmacology* ; Nitric Oxide Synthase Type III/metabolism* ; Phosphorylation ; Plaque, Atherosclerotic/pathology*

OBJECTIVE: To investigate the effect of microRNA-509-3p (miR-509-3p) on the apoptosis of atherosclerotic vascular endothelial cells. METHODS: Mouse aortic endothelial cells (MAECs) were divided into normal control group, oxidized low-density lipoprotein (ox-LDL) group, miR-509-3p overexpression group, miR-509-3p overexpression control group, miR-509-3p inhibitor + ox-LDL group, and miR-509-3p inhibitor control + ox-LDL group. MAEC were induced with 100 mg/L ox-LDL for 24 hours, and then transfected with miR-509-3p overexpression/inhibitor and corresponding control for 48 hours. The miR-509-3p expression in MAECs exposed to ox-LDL was detected using real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Flow cytometry was used to detect the level of apoptosis, and cell counting kit (CCK-8) was used to detect the proliferation activity of MAECs. The direct gene targets of miR-509-3p were predicted using bioinformatics analyses and confirmed using a dual luciferase reporter assay. The expression of Bcl-2 mRNA and protein was detected by RT-qPCR and Western blotting, respectively. RESULTS: Compared with the normal control group, miR-509-3p was significantly upregulated in ox-LDL-stimulated MAECs (1.68±0.85 vs. 1.00±0.30, t = 2.398, P < 0.05). After transfection of MAECs with miR-509-3p overexpression, the luciferase activity of the BCL2 3'UTR WT reporter gene was significantly lower than that of miR-509-3p overexpression control group (0.83±0.06 vs. 1.00±0.07, t = 4.531, P = 0.001). The luciferase activity of the BCL2 3'-UTR mutant (MUT) reporter gene was not significantly different from that of miR-509-3p overexpression control group (0.94±0.05 vs. 1.00±0.08, t = 1.414, P = 0.188). Compared with the normal control group and miR-509-3p mimics control group, the cell proliferation activity was decreased [(0.60±0.06)% vs. (1.00±0.09)%, (0.89±0.04)%, both P < 0.01], the percentage of apoptotic cells were increased [(23.46±2.02)% vs. (7.66±1.52)%, (10.40±0.78)%, both P < 0.05], and the mRNA and protein expression of Bcl-2 were significantly downregulated (Bcl-2 mRNA: 0.52±0.13 vs. 1.00±0.36, 1.10±0.19, Bcl-2 protein: 0.42±0.07 vs. 1.00±0.11, 0.93±0.10, both P < 0.01) in miR-509-3p overexpression group. Compared with the ox-LDL group, inhibition of miR-509-3p expression could increase the proliferation activity of MAECs induced by ox-LDL [(0.64±0.35)% vs. (0.34±0.20%)%, P < 0.05], and reduce the apoptosis rate [(13.59±2.22)% vs. (29.84±5.19)%, P < 0.01], and up-regulated the expression of Bcl-2 mRNA and protein in MAECs induced by ox-LDL (Bcl-2 mRNA relative expression: 0.82±0.09 vs. 0.52±0.10, Bcl-2 protein relative expression: 0.83±0.17 vs. 0.40±0.07, both P < 0.05). CONCLUSIONS Bcl-2 was one of the target genes of miR-509-3p. miR-509-3p can reduce the proliferation activity of endothelial cells, reduce the expression of Bcl-2, and promote cell apoptosis, thereby promoting the occurrence and development of atherosclerosis. Inhibition of miR-509-3p expression may be a potential therapeutic target for atherosclerosis.
Animals ; Mice ; Humans ; Endothelial Cells ; MicroRNAs/metabolism* ; Signal Transduction ; Lipoproteins, LDL/metabolism* ; Apoptosis ; RNA, Messenger/metabolism* ; Proto-Oncogene Proteins c-bcl-2/pharmacology* ; Atherosclerosis/metabolism* ; Luciferases/pharmacology* ; Cell Proliferation ; Human Umbilical Vein Endothelial Cells

The aim of this paper was to study the protective effect of total flavonoids from Rosa multiflora(TF-RM) on the injury of HUVEC induced by oxidized low density lipoprotein(ox-LDL). SPF male SD rats were randomly divided into blank group, simvastatin group(1.8 mg·kg~(-1)·d~(-1)) and TF-RM group(2.5 g·kg~(-1)·d~(-1)), with 10 rats in each group. They were intragastrically administered with drugs for 7 days, and then blood was collected from the abdominal aorta to prepare drug-containing serum. The HUVEC injury model was established through ox-LDL induction, and added with 15% simvastatin, 5% TF-RM, 10% TF-RM, 15% TF-RM drug-containing serum and blank serum, respectively. Reactive oxygen species(ROS) was determined by flow cytometry. Nitric oxide(NO) content was determined by nitrate reductase method. The contents of ET-1, P-selectin, E-selectin, ICAM-1, VCAM-1, IL-1β, IL-6 and TNF-α were determined by ELISA. The expression of Lox-1 protein was determined by Western blot. Compared with the blank group, ROS level in HUVEC and the contents of ET-1, P-selectin, E-selectin, ICAM-1, VCAM-1 and IL-1β in HUVEC were significantly increased(P<0.05), NO decreased significantly(P<0.01),Lox-1 protein expression increased significantly(P<0.05), and TNF-α and IL-6 showed an increasing trend. Compared with the model group, TF-RM significantly reduced ROS level in HUVEC and ET-1, P-selectin, E-selectin, ICAM-1, TNF-α, IL-1β content in supernatant(P<0.05), significantly increased NO content(P<0.01), and inhibited Lox-1 protein expression(P<0.05). VCAM-1, IL-6 contents showed a decreasing trend. Serum containing TF-RM acts on lectin-like oxidized low-density lipoprotein receptors, and exerts a protective effect on vascular endothelial cells by reducing cell oxidative damage, regulating vasoactive substances, and reducing adhesion molecules and inflammatory cascades.
Animals ; Cells, Cultured ; Endothelial Cells ; Endothelium, Vascular ; Flavonoids/pharmacology* ; Intercellular Adhesion Molecule-1/genetics* ; Lipoproteins, LDL ; Male ; Rats ; Rats, Sprague-Dawley ; Rosa

Objective To investigate the effects of simvastatin on diabetic neuropathic pain and systematic inflammation in diabetic rats and explore their molecular mechanisms.Methods Totally 24 rats were equally randomized into the normal+vehicle(NV)group,diabetic+vehicle(DV)group,and diabetic+simvastatin(DS)group using the random number table.Streptozotocin(STZ)was used to establish the rat models of diabetes.Blood glucose,body mass,paw withdrawal mechanical threshold(PWMT),and paw withdrawal thermal latency(PWTL)in each group were observed on days 7,14,21,and 28 after STZ injection.On day 28 after STZ injection,rats were sacrificed,and the lumbar spinal dorsal horn and serum were collected.Western blotting was used to detect the expression of receptor for advanced glycation end products(RAGE)and the phosphorylation levels of protein kinase B(AKT),extracellular signal-regulated kinase(ERK),p38,and c-Jun N-terminal kinase(JNK)in the spinal dorsal horn of rats in each group.Enzyme-linked immunosorbent assay was performed to determine the serum concentrations of oxidized low density lipoprotein(ox-LDL)and interleukin-1β(IL-1β).Results On days 14,21 and 28 after STZ injection,the PWMT in DV group were(8.6 ± 0.8),(7.1 ± 1.6),and(7.8 ± 0.8)g respectively,which were significantly lower than (12.0 ± 0.9)(=8.482, =0.000),(11.6 ± 1.5)(=11.309, =0.000),and(11.7 ± 1.5)g(=9.801, =0.000)in NV group.The PWMT in DS group on days 21 and 28 were(9.4 ± 1.4)(=5.780, =0.000)and(9.7 ± 0.9)g(=4.775, =0.003),respectively,which were significantly improved comparing with those of DV group.On days 7,14,21,and 28,there were no significant differences in PWTL among these three groups (all <0.05).The expression of RAGE in the spinal dorsal horn of DV group was significantly higher than those of NV group(=6.299, =0.000)and DS group(=2.891, =0.025).The phosphorylation level of AKT in the spinal dorsal horn of DV group was significantly higher than those of NV group(=8.915,=0.000)and DS group(=4.103,=0.003).The phosphorylation levels of ERK( =8.313,=0.000),p38( =2.965, =0.022),and JNK(=7.459, =0.000)in the spinal dorsal horn of DV group were significantly higher than those of NV group;the phosphorylation level of JNK in the spinal dorsal horn of DS group was significant lower than that of DV group(=3.866, =0.004);however,there were no significant differences in the phosphorylation levels of ERK(=1.987,=0.122)and p38(=1.260,=0.375)in the spinal dorsal horn between DS group and DV group.The serum concentrations of ox-LDL and IL-1β in DV group were(41.86 ± 13.40)ng/ml and(108.16 ± 25.88)pg/ml,respectively,which were significantly higher than those in NV group [(24.66 ± 7.87)ng/ml(=3.606,=0.003)and(49.32 ± 28.35)pg/ml(=5.079,=0.000)] and DS group [(18.81 ± 5.62)ng/ml (=4.833, =0.000)and(32.73 ± 11.73)pg/ml(=6.510, =0.000)].Conclusions Simvastatin can relieve the mechanical allodynia of diabetic rats possibly by inhibiting the activation of RAGE/AKT and the phosphorylation of JNK in the spinal dorsal horn.Simvastatin can also decrease the serum concentrations of ox-LDL and IL-1β in diabetic rats,which may contribute to the relief of systematic inflammation.
Animals ; Diabetes Mellitus, Experimental ; complications ; Hyperalgesia ; Inflammation ; drug therapy ; Interleukin-1beta ; blood ; Lipoproteins, LDL ; blood ; Neuralgia ; drug therapy ; Proto-Oncogene Proteins c-akt ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor for Advanced Glycation End Products ; metabolism ; Simvastatin ; pharmacology

BackgroundOxidized low-density lipoprotein (ox-LDL)-induced oxidative stress and endothelial apoptosis are essential for atherosclerosis. Our previous study has shown that ox-LDL-induced apoptosis is mediated by the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2α-subunit (eIF2α)/CCAAT/enhancer-binding protein homologous protein (CHOP) endoplasmic reticulum (ER) stress pathway in endothelial cells. Statins are cholesterol-lowering drugs that exert pleiotropic effects including suppression of oxidative stress. This study aimed to explore the roles of simvastatin on ox-LDL-induced ER stress and apoptosis in endothelial cells.

MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with simvastatin (0.1, 0.5, or 2.5 μmol/L) or DEVD-CHO (selective inhibitor of caspase-3, 100 μmol/L) for 1 h before the addition of ox-LDL (100 μg/ml) and then incubated for 24 h, and untreated cells were used as a control group. Apoptosis, expression of PERK, phosphorylation of eIF2α, CHOP mRNA level, and caspase-3 activity were measured. Comparisons among multiple groups were performed with one-way analysis of variance (ANOVA) followed by post hoc pairwise comparisons using Tukey's tests. A value of P < 0.05 was considered statistically significant.

ResultsExposure of HUVECs to ox-LDL resulted in a significant increase in apoptosis (31.9% vs. 4.9%, P < 0.05). Simvastatin (0.1, 0.5, and 2.5 μmol/L) led to a suppression of ox-LDL-induced apoptosis (28.0%, 24.7%, and 13.8%, F = 15.039, all P < 0.05, compared with control group). Ox-LDL significantly increased the expression of PERK (499.5%, P < 0.05) and phosphorylation of eIF2α (451.6%, P < 0.05), if both of which in the control groups were considered as 100%. Simvastatin treatment (0.1, 0.5, and 2.5 μmol/L) blunted ox-LDL-induced expression of PERK (407.8%, 339.1%, and 187.5%, F = 10.121, all P < 0.05, compared with control group) and phosphorylation of eIF2α (407.8%, 339.1%, 187.5%, F = 11.430, all P < 0.05, compared with control group). In contrast, DEVD-CHO treatment had no significant effect on ox-LDL-induced expression of PERK (486.4%) and phosphorylation of eIF2α (418.8%). Exposure of HUVECs to ox-LDL also markedly induced caspase-3 activity together with increased CHOP mRNA level; these effects were inhibited by simvastatin treatment.

ConclusionsThis study suggested that simvastatin could inhibit ox-LDL-induced ER stress and apoptosis in vascular endothelial cells.

Apoptosis ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; drug effects ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Lipoproteins, LDL ; pharmacology ; Oligopeptides ; pharmacology ; Simvastatin ; pharmacology

OBJECTIVE: : To investigate the effect of chlorogenic acid (CGA) on non-enzymatic glycation and oxidation of low density lipoprotein (LDL). METHODS: : The non-enzymatic glycation incubation system of LDL-glucose was established. The contents of early glycation products (Amodori product) and intermediate products (dicarbonyl compound) were determined by ultraviolet-visible spectrophotometry, and the content of advanced glycation end products (AGEs) was determined by fluorescence spectrophotometry. The LDL oxidation incubation system was established. The contents of thiobarbituric acid reactive substances(TBARS) and conjugated diene were determined by ultraviolet-visible spectrophotometry. The tryptophan fluorescence quenching, and the content of lipofuscin, total fluorescence products, active aldehydes and malondialdehyde were determined by fluorescence spectrophotometry, and further verified by three-dimensional fluorescence spectroscopy. RESULTS: : In the LDL glycation experiment, 150 μg/mL and 300 μg/mL CGA inhibited the formation of Amadori product, dicarbonyl compounds and AGEs. In the LDL oxidation experiment, 15 μg/mL and 25 μg/mL CGA inhibited the formation of TBARS effectively; 5 μg/mL and 10 μg/mL CGA inhibited tryptophan fluorescence quenching, and the formation of active aldehydes, malondialdehyde, total fluorescence products, lipofuscin and conjugated diolefine. And the three-dimensional fluorescence spectroscopy showed the same results. CONCLUSIONS : CGA can inhibit non-enzymatic glycation and oxidation of LDL.
Chlorogenic Acid ; pharmacology ; Glycosylation ; drug effects ; Lipoproteins, LDL ; metabolism ; Oxidation-Reduction ; drug effects ; Thiobarbituric Acid Reactive Substances ; analysis

BACKGROUNDAdipocytes behave like a rich source of pro-inflammatory cytokines including monocyte chemoattractant protein-1 (MCP-1). Oxidized low-density lipoprotein (oxLDL) participates in the local chronic inflammatory response, and high-density lipoprotein could counterbalance the proinflammatory function of oxLDL, but the underlying mechanism is not completely understood. This study aimed to evaluate the effect of apolipoprotein A-I mimetic peptide L-4F on the secretion and expression of MCP-1 in fully differentiated 3T3-L1 adipocytes induced by oxLDL and to elucidate the possible mechanisms.

METHODSFully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentration of L-4F (0-50 μg/ml) with oxLDL (50 μg/ml) stimulated, with/without protein kinase A (PKA) inhibitor H-89 (10 μmol/L) preincubated. The concentrations of MCP-1 in the supernatant, the mRNA expression of MCP-1, the levels of CCAAT/enhancer binding protein α (C/EBPα), and CCAAT/enhancer binding protein β (C/EBPβ) were evaluated. The monocyte chemotaxis assay was performed by micropore filter method using a modified Boyden chamber.

RESULTSOxLDL stimulation induced a significant increase of MCP-1 expression and secretion in 3T3-L1 adipocytes, which were inhibited by L-4F preincubation in a dose-dependent manner. PKA inhibitor H-89 markedly reduced the oxLDL-induced MCP-1 expression, but no further decrease was observed when H-89 was used in combination with L-4F (50 μg/ml) (P > 0.05). OxLDL stimulation showed no significant effect on C/EBPα protein level but increased C/EBPβ protein level in a time-dependent manner. H-89 and L-4F both attenuated C/EBPβ protein level in oxLDL-induced 3T3-L1 adipocytes.

CONCLUSIONSOxLDL induces C/EBPβ protein synthesis in a time-dependent manner and enhances MCP-1 secretion and expression in 3T3-L1 adipocytes. L-4F dose-dependently counterbalances the pro-inflammatory effect of oxLDL, and cyclic AMP/PKA-C/EBPβ signaling pathway may participate in it.

3T3-L1 Cells ; Animals ; CCAAT-Enhancer-Binding Protein-beta ; analysis ; physiology ; Chemokine CCL2 ; genetics ; secretion ; Cyclic AMP ; physiology ; Cyclic AMP-Dependent Protein Kinases ; physiology ; Humans ; Lipoproteins, LDL ; antagonists & inhibitors ; pharmacology ; Mice ; Peptides ; pharmacology ; Signal Transduction ; physiology

OBJECTIVETo investigate the role of aqueous extracts of Tribulus terrestris (TT) against oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) dysfunction in vitro.

METHODSHUVECs were pre-incubated for 60 min with TT (30 and 3 μg/mL respectively) or 10(-5) mol/L valsartan (as positive controls) and then the injured endothelium model was established by applying 100 μg/mL ox-LDL for 24 h. Cell viability of HUVECs was observed by real-time cell electronic sensing assay and apoptosis rate by Annexin V/PI staining. The cell migration assay was performed with a transwell insert system. Cytoskeleton remodeling was observed by immunofluorescence assay. The content of endothelial nitric oxide synthase (eNOS) was measured by enzyme-linked immunosorbent assay. Intracellular reactive oxygen species (ROS) generation was assessed by immunofluorescence and flow cytometer. Key genes associated with the metabolism of ox-LDL were chosen for quantitative real-time polymerase chain reaction to explore the possible mechanism of TT against oxidized LDL-induced endothelial dysfunction.

RESULTSTT suppressed ox-LDL-induced HUVEC proliferation and apoptosis rates significantly (41.1% and 43.5% after treatment for 3 and 38 h, respectively; P<0.05). It also prolonged the HUVEC survival time and postponed the cell's decaying stage (from the 69th h to over 100 h). According to the immunofluorescence and transwell insert system assay, TT improved the endothelial cytoskeletal network, and vinculin expression and increased cell migration. Additionally, TT regulated of the synthesis of endothelial nitric oxide synthase and generation of intracellular reactive oxygen species (P<0.05). Both 30 and 3 μg/mL TT demonstrated similar efficacy to valsartan. TT normalized the increased mRNA expression of PI3Kα and Socs3. It also decreased mRNA expression of Akt1, AMPKα1, JAK2, LepR and STAT3 induced by ox-LDL. The most notable changes were JAK2, LepR, PI3Kα, Socs3 and STAT3.

CONCLUSIONSTT demonstrated potential lowering lipid benefits, anti-hypertension and endothelial protective effects. It also suggested that the JAK2/STAT3 and/or PI3K/AKT pathway might be a very important pathway which was involved in the pharmacological mechanism of TT as the vascular protective agent.

Apoptosis ; drug effects ; Cell Movement ; drug effects ; Cell Survival ; drug effects ; Cytoskeleton ; drug effects ; metabolism ; Endothelium, Vascular ; drug effects ; pathology ; physiopathology ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Gene Expression Regulation ; drug effects ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Lipoproteins, LDL ; adverse effects ; Nitric Oxide Synthase Type III ; metabolism ; Plant Extracts ; pharmacology ; Protective Agents ; pharmacology ; Reactive Oxygen Species ; metabolism ; Tribulus ; chemistry ; Vinculin ; metabolism ; Water ; chemistry

OBJECTIVETo observe the effects of Danlou Tablet (DT) on inflammatory reaction, and expressions of lipoprotein-associated phospholipase A2 (LP-PLA2), secretory phospholipase A2 (sPLA2), and to analyze potential mechanisms.

METHODSForty male Wistar rats were randomly and equally divided into five groups, i.e., the normal control group, the model group, the Western medicine (WM) group, the low dose DT group, the high dose DT group, 8 in each group. Rats in the normal control group were fed with basic forage for 12 successive weeks, while AS rat model was established in rats of the other four groups by feeding high fat and sugar forage plus intraperitoneal injection of vitamin D₃. Normal saline, atorvastatin calcium suspension (at the daily dose of 1.8 mg/kg), low dose DT suspension (at the daily dose of 450 mg/kg), and high dose DT suspension (at the daily dose of 900 mg/kg) were administered to rats in the model group, the WM group, the low dose DT group, the high dose DT group respectively by gastragavage for 8 successive weeks. The general condition of all rats was observed. Rats were sacrificed after gastric administration and their serum collected. Serum levels of lipids (TC, TG, HDL-C, LDL-C) and inflammatory factors [IL-6, TNF-α, monocyte chemoattractant protein 1 (MCP-1), oxidized low-density lipoprotein (ox-LDL), lipoprotein-associated phospholipase A2 (LP-PLA2), secretory phospholipase A2 (sPLA2)] were detected. Pathological changes of thoracic aorta were observed by HE staining. Protein and gene expressions of LP-PLA2 and sPLA2 in thoracic aorta were measured by Western blot and real-time fluorescent quantitative PCR respectively.

RESULTSCompared with the normal control group, rats in the model group were in low spirits and responded poorly. Typical atherosclerotic plaque could be seen in thoracic aorta of rats in the model group. Serum levels of TC, TG, LDL-C, IL-6, TNF-α, MCP-1, ox-LDL, LP-PLA2, and sPLA2 significantly increased (P < 0.05); protein and gene expressions of LP-PLA2 and sPLA2 in rat thoracic aorta increased (P < 0.05) in the model group. After 8 weeks of intervention, rats in 3 medication groups appeared active, and HE staining showed subsidence of plaque in rat thoracic aorta. Compared with the model group, serum levels of TC, TG, LDL-C, IL-6, TNF-α, MCP-1, ox-LDL, and LP-PLA2 decreased in 3 medication groups (P < 0.01, P < 0.05); serum sPLA2 level decreased, protein and mRNA expressions of LP-PLA2 and sPLA2 in rat thoracic aorta decreased in the WM group (P < 0.01, P < 0.05); protein and mRNA expressions of LP-PLA2 in rat thoracic aorta significantly decreased in the low dose DT group (P < 0.01, P < 0.05), and those of LP-PLA2 and sPLA2 decreased in the high dose DT group (P < 0.01, P < 0.05).

CONCLUSIONDT could fight against inflammatory reaction and AS possibly through inhibiting LP-PLA2 expression and reducing ox-LDL production.

1-Alkyl-2-acetylglycerophosphocholine Esterase ; blood ; Animals ; Aorta, Thoracic ; pathology ; Atherosclerosis ; drug therapy ; Chemokine CCL2 ; blood ; Drugs, Chinese Herbal ; pharmacology ; Inflammation ; drug therapy ; Interleukin-6 ; blood ; Lipids ; blood ; Lipoproteins, LDL ; blood ; Male ; Phospholipases A2 ; blood ; Plaque, Atherosclerotic ; Random Allocation ; Rats ; Rats, Wistar ; Tablets ; Tumor Necrosis Factor-alpha ; blood

UNLABELLEDOBJECTIVE : To study the anti-atherosclerotic mechanism of bear bile powder (BBP) in Shexiang Tongxin Dripping Pill (STDP) , and to provide scientific evidence for treating atherosclerosis (AS) by its therapeutic characteristics of cool resuscitation.

METHODSAS model was duplicated using ApoE-/- gene knocked mice fed with high-fat diet. Thirty ApoE-/- deficient male mice were divided into four groups according to body weight using random digit table, i.e., the model group (A, n =9), the STDP group (B, n=E7), the STDP without BBP group (C, n =7), and the BBP group (D, n =9). Besides, another 9 C57BL/6J male mice of the same age were recruited as a normal control group (E). All mice in Group B, C, and D were respectively administered with corresponding drugs (30, 30, and 0. 33 mg/kg) by gastrogavage. Equal volume of normal saline was administered to mice in Group A and E. All medication lasted for 8 successive weeks. Serum levels of inflammatory cytokines such as interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor a (TNF-α), interferon y (IFNγ), and oxidized low-density lipoprotein (ox-LDL) were measured by ELISA. Serum levels of malondialdehyde (MDA), activities of glutathione (GSH) and superoxide dismutase (SOD) were determined using biochemical assay. Contents of reactive oxygen species (ROS) in the aortic root was detected by dihydroethidum (DHE) fluorescent probe. Expression levels of microRNAs (such as miR-20, miR-21, miR-126, and miR-155) were detected by real-time PCR.

RESULTSThe fluorescence intensity of the aorta was obviously enhanced in Group A. But it was obviously attenuated in Group B, C, and D, and the attenuation was the most in Group B. Compared with Group E, serum levels of IL-2, IL-6, TNF-α, IFN-γ, oxLDL, and MDA all increased (P <0. 01), GSH contents and SOD activities decreased (P <0. 01), expression levels of miR-126, miR-21, and miR-155 in aorta increased (P <0. 01), and the expression level of miR-20 decreased in Group A (P<0. 01). Compared with Group A, serum levels of IL-2, IL-6, TNF-α, IFN-γ, oxLDL, and MDA were all down-regulated (P <0. 01), GSH contents and SOD activities were up-regulated (P <0. 01), expression levels of miR-126, miR-21, and miR-155 in aorta were down-regulated in Group B, C, and D (P <0. 01). The expression level of miR20 was up-regulated in Group B and D (P <0. 01). Compared with Group B, serum levels of IL-2, IL-6, TNF-α, IFN-γ increased (P <0.01); GSH contents and SOD activities decreased, levels of MDA and oxLDL increased (P <0. 01) in Group C and D. Expression levels of miR-20 and miR-155 were down-regulated in Group C and D (P <0. 01).

CONCLUSIONSSTDP played roles in significantly regulating inflammatory factors and oxidative stress factors. Its mechanism might be possibly associated with regulating expressions of miR-126, miR-21, miR-155, and miR-20 in aorta. BBP played significant roles in STDP.

Animals ; Aorta ; Apolipoproteins E ; metabolism ; Atherosclerosis ; Bile ; Cytokines ; Diet, High-Fat ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Interleukin-6 ; metabolism ; Lipoproteins, LDL ; metabolism ; Male ; Malondialdehyde ; metabolism ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; Plaque, Atherosclerotic ; drug therapy ; Reactive Oxygen Species ; Superoxide Dismutase ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Ursidae