Mechanism of Renshentang in Treatment of Atherosclerosis Based on Autophagic Effect of TRPV1 on Arterial Smooth Muscle / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Renshentang， recorded in Synopsis of Golden Chamber， in the treatment of atherosclerosis （AS） based on the autophagic effect of transient receptor potential vanilloid subtype 1 （TRPV1） on arterial smooth muscle. MethodFourteen SPF-grade 8-week-old male C57BL/6J mice were assigned to the normal group and 70 8-week-old apolipoprotein E knockout （ApoE-/-） mice were assigned to the experimental group. The AS model was induced by a high-fat diet in the mice in the experimental group for eight weeks. The model mice were then randomly divided into model group， low-， medium-， and high-dose Renshentang groups （2.715， 5.43， and 10.68 g·kg-1·d-1）， and simvastatin group （0.02 g·kg-1·d-1）. Drug treatment lasted eight weeks. Serum was taken and serum total cholesterol （CHO）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， and high-density lipoprotein cholesterol （HDL-C） levels were measured by assay kits to observe the changes in lipid levels in mice. The aorta was stained with hematoxylin-eosin （HE） to observe the overall pathology of the aortic root and oil red O staining was used to detect the lipid deposition in the aortic plaque and calculate the percentage of the aortic root area to the lumen area. The protein expression of TRPV1， adenylate-activated protein kinase （AMPK）， phosphorylated AMPK （p-AMPK）， autophagy effector-1 （Beclin-1）， and microtubule-associated protein 1 light chain 3 （LC3Ⅱ） in mouse aortic tissues was determined by Western blot. ResultCompared with the normal group， the model group showed increased serum CHO， TG， and LDL-C levels， decreased HDL-C， and increased aortic root plaque area （P<0.01）. Compared with the model group， the Renshentang groups showed decreased levels of CHO， TG， and LDL-C in serum （P<0.05， P<0.01）， especially in the low- and medium-dose Renshentang groups （P<0.01）. Compared with the normal group， the simvastatin group and the Renshentang groups showed reduced aortic root plaque area （P<0.05）， especially in the high-dose Renshentang group （P<0.01）. Compared with the normal group， the model group showed decreased relative expression levels of TRPV1， p-AMPK/AMPK， Beclin-1， and LC3Ⅱ/LC3Ⅰ（P<0.05， P<0.01）. Compared with the model group， the medium- and high-dose Renshentang groups showed increased relative expression levels of TRPV1， p-AMPK/AMPK， Beclin-1， and LC3Ⅱ/LC3Ⅰ（P<0.05，P<0.01）. ConclusionThe anti-AS effect of Renshentang recorded in Synopsis of Golden Chamber may be achieved by up-regulating TRPV1 expression to restore the level of autophagy mediated by AMPK.

Intervention of Huanglian Jiedutang on Atherosclerosis in ApoE-/- Mice and Its Mechanism / 中国实验方剂学杂志

ObjectiveTo study the intervention of Huanglian Jiedutang on atherosclerosis （AS） in apolipoprotein E knockout （ApoE-/-） mice induced by the high-fat diet. MethodThe ApoE-/- mouse model of AS was induced by the high-fat diet， and Huanglian Jiedutang was used to intervene in the AS in the ApoE-/- mice. The pathological changes of aorta were observed by hematoxylin-eosin （HE） staining. The levels of serum total cholesterol （TC）， triglyceride （TG）， high-density lipoprotein cholesterol （HDL-C）， and low-density lipoprotein cholesterol （LDL-C） were detected by an automatic biochemical analyzer. The protein expression levels of sirtuin-1 （SIRT1） and nuclear factor-kappa B （NF-κB） were determined by Western blot assay， and the mRNA expression levels of adenosine 5'-monophosphate-activated protein kinase （AMPK）， peroxisome proliferators-activated receptors α （PPARα）， tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， interleukin-1β （IL-1β）， and NOD-like receptor pyrin domain-containing 3 （NLRP3） were determined by real-time quantitative polymerase chain reaction （Real-time PCR）. ResultAs compared with the normal group， there was a large amount of lipid accumulation in the blood vessels of the model group. In the model group， the levels of serum TG， TC， and LDL-C were increased （P<0.01）， and the level of HDL-C was decreased （P<0.01）. The protein expression level of SIRT1 in the aorta was decreased， while that of NF-κB was increased in the model group （P<0.01）. The mRNA expression levels of IL-6， TNF-α， and IL-1β were higher （P<0.01）， while those of AMPK in the liver were lower in the model group （P<0.01）. Compared with the model group， the Huanglian Jiedutang group reduced the lipid accumulation and inflammatory reaction in the aorta of mice with AS， reduced the levels of TC， TG， and LDL-C （P<0.01）， and increased the level of HDL-C （P<0.01）. Huanglian Jiedutang significantly increased the protein expression level of SIRT1 in the aorta of ApoE-/- mice （P<0.01） and decreased the protein expression levels of NF-κB in the aorta （P<0.05， P<0.01）. Huanglian Jiedutang down-regulated the mRNA expression levels of TNF-α， IL-6， IL-1β， and NLRP3 in the aorta （P<0.05， P<0.01）， and up-regulated the mRNA expression levels of AMPK and PPARα in the liver of ApoE-/- mice （P<0.05， P<0.01）. ConclusionHuanglian Jiedutang has a certain intervention effect on the formation of atherosclerotic aortic plaque in ApoE-/- mice. Its mechanism may be related to the decrease of serum TC， TG， and LDL-C levels， the increase of HDL-C levels， thus playing a role in lowering blood lipid， the increase of SIRT1 protein， the decrease of NF-κB protein， the decrease of inflammatory factors such as TNF-α and IL-6， which protects blood vessels from inflammatory injury， and the improvement of AMPK and PPARα levels to participate in autophagy and apoptosis.

Effect of Ethyl Acetate Extract of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis on Intestinal Flora in ApoE-/- Mice with Atherosclerosis / 中国实验方剂学杂志

ObjectiveTo observe the effect of ethyl acetate extract of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis on high-fat diet-induced apolipoprotein E gene knockout （ApoE-/-） mice， and explore its mechanism of treating atherosclerosis by regulating intestinal flora. MethodThirty-two 8-week-old male ApoE-/- mice were randomly divided into model group， rosuvastatin group （10 mg·kg-1）， high-， low-dose groups of ethyl acetate extract of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis （75， 25 mg·kg-1）， with 8 mice in each group. Eight C57BL/6 mice were used as blank group. After 8 weeks of continuous administration， blood was taken to determine the blood lipid level. Enzyme-linked immunosorbent assay （ELISA） was used to detect the contents of related indexes in serum of mice. Hematoxylin-eosin （HE） staining was used to observe the formation of aortic plaque in mice. Cecal contents were collected and 16S rRNA amplicon sequencing was used to detect intestinal flora. ResultCompared with the blank group， the plaque area of the model group was significantly increased with inflammatory infiltration， the contents of triglyceride （TG）， total cholesterol （TC）， low-density lipoprotein cholesterol （LDL-C）， inflammatory factors and inducible nitric oxide synthase （iNOS） were increased， while the content of high-density lipoprotein cholesterol （HDL-C） was decreased. Compared with the model group， rosuvastatin group and high- and low-dose groups of ethyl acetate extract of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis could improve the deposition of aortic plaque， reduce the contents of TG， TC， LDL-C， inflammatory factors and iNOS， and increase the content of HDL-C. Compared with the blank group， the relative abundances of Firmicutes and Proteobacteria in the model group increased， while the relative abundance of Bacteroidetes decreased. Alpha and Beta diversity analysis showed that samples of each group could be significantly isolated， and the total number and abundance of intestinal flora species in the model group were low. Compared with the model group， ethyl acetate extract of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis could increase the relative abundance of beneficial bacteria and decrease the relative abundance of pathogenic bacteria. ConclusionEthyl acetate extract of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis was mainly composed of flavonoids， which can treat atherosclerosis by regulating the intestinal flora and improve the pathological changes in the aorta of ApoE-/- mice induced by high-fat diet. The mechanism may be related to its ability to reduce the level of inflammatory factors， improve antioxidant capacity and repair the disorder of intestinal flora structure.

Effect and Mechanism of Longshengzhi Capsule on Atherosclerosis in ApoE-/- Mice / 中国实验方剂学杂志

ObjectiveTo study the effect of Longshengzhi capsule （LSZC） on high fat diet （HFD）-induced atherosclerosis （AS） in apolipoprotein E knockout （ApoE-/-） mice. MethodApoE-/- mice were fed with HFD for 8 weeks to induce AS. Then the mice were randomized into model group， simvastatin group （4 mg·kg-1）， high-dose LSZC group （1.6 g·kg-1）， medium-dose LSZC group （0.8 g·kg-1）， and low-dose LSZC group （0.4 g·kg-1）. C57BL/6J Mice with normal diet were used as the blank control. After 10 weeks， serum levels of triglyceride （TG）， total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， malondialdehyde （MDA）， superoxide dismutase （SOD）， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） were detected. Hematoxylin-eosin （HE） and oil red O were used to detect aortic plaque in each group. The levels of CD34 and F4/80 in aorta were determined by immunohistochemistry （IHC）. ResultCompared with the blank control， the model group demonstrated obvious aortic plaque， a large amount of lipid accumulation， serious damage of aortic intima， increase in serum levels of TC， TG， LDL-C， HDL-C， MDA， IL-1β， and IL-6 （P<0.01）， decrease in SOD level （P<0.01）， and rise of the expression of CD34 and F4/80 （P<0.01）. Compared with the model group， LSZC of the three doses all decreased the serum levels of TG and LDL-C （P<0.05）， and the levels of IL-1β and IL-6 （P<0.05， P<0.01）， and the high-dose and medium-dose LSZC improved SOD level， decreased MDA content （P<0.05， P<0.01）， and reduced the expression of the CD34 and F4/80 in blood vessels （P<0.05， P<0.01）. ConclusionLSZC has certain intervention effect on the formation of aortic plaque in atherosclerosis ApoE-/- mice. The mechanism is that it reduces the levels of serum TG and LDL-C to lower blood lipid， decreases MDA level and improves SOD activity to inhibit lipid peroxidation， lowers the levels of IL-1β and IL-6 and down-regulates the expression of CD34 and F4/80 to protect blood vessels from inflammatory damage.

Effects of Total Flavonoids from Chamomile on Lipid Metabolism of Hyperlipidemia Model Mice and Its Mechanism / 中国药房

OBJECTIVE：To st udy t he effects of total flavonoids from chamomile on lipid metabolism of hyperlipidemia model mice and its potential mechanism. METHODS ：Thirty male C 57BL/6J-ApoE－/－ mice were randomly divided into model group ， positive control group（fenofibrate 30 mg/kg）and chamomile total flavonoids low-dose ，medium-dose and high-dose groups （88， 176，352 mg/kg），with 6 mice in each group. In addition ，6 male C 57BL/6J mice were used as normal control group. Mice in normal control group were fed with ordinary diet ，and mice in other groups were fed with high-fat diet for 8 weeks to replicate hyperlipidemia model. At the time of making model ，administration groups were given relevant liquid （using 1％ sodium carboxymethyl cellulose as solvent ）；normal control group and model group were given 1％ sodium carboxymethyl cellulose intragastrically，200 mL per gavage ，once a day ，for consecutive 8 weeks. The body weight of mice in each group was weighed before medication and 8 weeks after medication. The serum contents of total cholesterol （TC），triacylglycerol（TG），low-density lipoprotein cholesterol （LDL-C），high-density lipoprotein cholesterol （HDL-C），aspartate aminotransferase （AST）and alanine aminotransferase （ALT） in mice were detected after last administration ；the contents of superoxide dismutase （SOD） and malondialdehyde（MDA）as well as the protein expressions of peroxisome proliferator-activated receptor α（PPARα），carnitine palmityl transferase 1A（CPT1A）and peroxase acyl-CoA oxidase 1（ACOX1）in liver tissue were determined. The pathological changes i n liver tissue were observed. RESULTS：Compared w ith before medication ，the body weight of each group showed an increasing trend after 8 weeks of medication. Compared with normal control group ，body weight ，the contents of TC ，TG， LDL-C，AST and ALT in serum and MDA content in live r lan- tissue of mice in model group were significantly increased wei516@sina.com after 8 weeks of medication （P＜0.05 or P＜0.01）. The ·2706· China Pharmacy content of HDL-C in serum and the cont ent of SOD in liver tissue ，as well as the protein expressions of PPARα，CPT1A and ACOX1 were significantly decreased （P＜0.05 or P＜0.01），and the structure of liver tissue was disorganized ，with circular fat vacuoles of different sizes and lipid droplets of different sizes in the cytoplasm. Compared with model group ，body weight （except for chamomile total flavonoids low-dose group ）of mice ，serum contents of TC ，TG，LDL-C，AST and ALT ，content of MDA in liver tissue （except for chamomile total flavonoids low-dose and medium-dose groups ）were significantly decreased （P＜0.05 or P＜ 0.01）. Serum content of HDL-C ，content of SOD in liver tissue ，protein expressions of PPARα，CPT1A（except for chamomile total flavonoids low-dose and medium-dose groups ） and ACOX 1 were significantly increased （P＜0.05 or P＜0.01）；liver tissue structure was clear ，and liver fat vacuoles were improved to varying degrees ，and less lipid droplets. The improvement effect of the above indexes was the best in the chamomile total flavonoids high-dose group. CONCLUSIONS ：Chamomile total flavonoids can prevent the occurrence of hyperlipidemia in C57BL/6J-ApoE －/－ mice，the mechanism of which may be associated with up-regulation of PPARα expression，the improvement of liver injury and oxidant stress injury.

Study on effect of "Trichosanthis Fructus-Allii Macrostemonis Bulbus" on atherosclerosis in ApoE~(-/-) mice based on liver metabonomics / 中国中药杂志

In this study, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS)-based liver metabolomics approach was used to explore the mechanism of "Trichosanthis Fructus-Allii Macrostemonis Bulbus" in improving atherosclerosis(AS) of mice with apolipoprotein E gene knockout(ApoE~(-/-)). AS mouse model was induced by high-fat diet. The pathological and biochemical indexes such as the histopathological changes, body weight, liver weight, blood lipid level and inflammatory factors in the liver of mice were determined. The metabolic profiling of mice liver samples was performed with UPLC-Q-TOF-MS. Multiple statistical analysis methods including partial least squares discriminant analysis(PLS-DA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were employed to screen and identify biomarkers. The levels of related enzymes including LCAT, sPLA2, EPT1 and ACER1 were detected. The results showed that "Trichosanthis Fructus-Allii Macrostemonis Bulbus" significantly reduced the areas of aortic plaque and fat vacuoles of liver in AS mice and decreased the accumulation of lipid droplets and liver coefficient. "Trichosanthis Fructus-Allii Macrostemonis Bulbus" also regulated the levels of blood lipid and inflammatory injury in the liver. The metabolites of the control group, the model group and the "Trichosanthis Fructus-Allii Macrostemonis Bulbus" group could be distinguished significantly. Fifteen potential biomarkers related to AS were discovered and preliminarily identified, seven of which could be regulated by "Trichosanthis Fructus-Allii Macrostemonis Bulbus" in a trend of returning to normal. Metabolic pathway analysis screened out two major metabolic pathways. "Trichosanthis Fructus-Allii Macrostemonis Bulbus" obviously regulated the levels of LCAT, sPLA2, EPT1 and ACER1. It was inferred that "Trichosanthis Fructus-Allii Macrostemonis Bulbus" could play a major role in AS treatment by regulating glycerophospholipid and sphingolipid metabolism disorders in the liver, with the mechanism probably relating to the intervention of the expression of LCAT, sPLA2, EPT1 and ACER1.

Schisandrin C alleviates atherosclerosis and inflammation in ApoE-/-mice fed with arterial high-fat diet / 中国药理学通报

Aim To investigate the effects of schisandrin C on arterial inflammation and atherosclerosis of ApoE-/-mice fed with high-fat diet. Methods ApoE-/-mice were divided into high fat diet group (HFD) and high fat diet group + schisandrin group (HFD + Sch C). High fat diet and Sch C were given at the same time. After 12 weeks, mice were executed and arterial tissues were collected. RT-q PCR and Western blot were respectively used to detect the expressions of IL-6, TNF-α, ICAM-1, β-actin and the phosphorylation and expression of IκB-α in arteries. Oil red O staining and biochemical kit were respectively used to detect the lipid changes in aortic root and the blood lipid levels. Results Compared with HFD group, the area of atherosclerotic plaque in HFD + Sch C was reduced (P < 0. 05), but Sch C did not affect blood lipid levels. Compared with HFD group, the mRNA expression of TNF-α, IL-6 and ICAM-1 and the phosphorylation of IκB-α of arterial tissue in HFD + Sch Cgroup decreased (P < 0. 05). Conclusions Sch C could effectively alleviate atherosclerosis induced by high-fat diet in ApoE-/-mice, accompanied by alleviation of arterial inflammation.

Based on "intestinal flora-inflammation" pathway to investigate effect and mechanism of Danlou Tablet on atherosclerosis of ApoE-/- mice / 中草药

Objective: To investigate the efficacy and mechanism of Danlou Tablet against atherosclerosis model of ApoE-/- mice fed with high fat diet. Methods: C57BL/6J mice were used as controls and ApoE-/- mice were randomly divided into two groups after 24 weeks of high fat feeding, including the model group received saline and the treatment group received Danlou Tablet. Animals were executed after 8 weeks of treatment and serum was collected to measure blood lipids; Plaque formation in the aorta was observed by red O and HE staining; 16 S rRNA sequencing was used to analyze changes in intestinal flora, and GC-MS test for detection fecal SCFAs content, ELISA for the determination of serum LPS, and real time PCR for detection of mRNA expression. Results: Compared with the control group, the blood lipid levels were increased; intestinal flora was imbalance with increased harmful bacteria and reduced beneficial bacteria; The level of serum LPS and inflammation around the aorta were increased in the model group. Compared with the model group, the contents of TG, TC, LDL-C and the plaque area of Danlou Tablet group were decreased (P < 0.01); Danlou Tablet group can regulate intestinal flora, thus effectively reducing serum LPS and inflammatory factors TNF-α, ICAM-1 and IL-1β levels around the aorta (P < 0.01). Conclusion: Danlou Tablet exerts an anti-atherosclerosis action with favorable efficacy through restructing the intestinal flora stucture, inhibiting endotoxin releasing and constraining the inflammatory response induced by dysbacteriosis.

Effects of Yangyin Huoxue Prescription on the imbalance of Treg/Th17 and atherosclerosis in ApoE-/- mice induced by high-fat diet / 中国药科大学学报

@#Atherosclerosis(AS), characterized with the accumulation of lipids on the vessel wall, is an immune-related inflammatory disease which promotes the progression of cardiovascular diseases(CVD). The imbalance of Treg/Th17 accelerates the progression of AS. Yangyin Huoxue Prescription(YHF)is an efficient traditional Chinese medicine used in the treatment of AS, but the effects of YHF on the balance of immunity have still not been clarified. This project was designed to investigate the effects of YHF on the imbalance of Treg/Th17 and AS in ApoE-/- mice induced by high-fat diet(HFD). ApoE-/- mice were given HFD to induce AS and administered low-dose YHF(18 g/kg)or high-dose YHF(36 g/kg)for 20 weeks. Atherosclerotic plaque area was analyzed by oil red O staining. Serum lipids were measured by biochemical kits. Treg or Th17 cells in peripheral blood were detected by flow cytometry. mRNA and protein expression of Foxp3 and RORγt of aortas were determined by qRT-PCR, Western blot and immunohistochemistry. Splenic CD4+T cells of mice were isolated and activated by anti-CD3/CD28, and then treated with lipopolysaccharide(LPS)and YHF. The expression of mRNA and protein of Foxp3 and RORγt were detected by qRT-PCR and immunofluorescence. It was found that YHF reduced the plaque area, decreased lipid level and increased the ratio of Treg cells in peripheral blood. Moreover, YHF increased mRNA or protein expression of Foxp3 in aortas in vivo or CD4+T cells in vitro while decreasing mRNA or protein expression of RORγt. These results suggested that YHF can regulate the imbalance of Treg/Th17 in ApoE-/- mice induced by HFD, and reduce the inflammatory stimulation of LPS on CD4+T cells, thereby improving AS.

MiR-590 Inhibits Endothelial Cell Apoptosis by Inactivating the TLR4/NF-κB Pathway in Atherosclerosis

PURPOSE: Previous study has well documented the anti-apoptotic effects of miR-590 on oxidized low-density lipoprotein (ox-LDL)-treated endothelial cells (ECs). However, the mechanism underlying the anti-apoptotic effects of miR-590 in ox-LDL-treated ECs remains to be further addressed. MATERIALS AND METHODS: ApoE(−/−) mice fed with a high-fat diet (HFD) and human aortic endothelial cells (HAECs) treated with ox-LDL were used as in vivo and in vitro models of atherosclerosis. The expressions of miR-590 and toll-like receptor 4 (TLR4) were detected by quantitative real-time PCR and Western blot, respectively. Atherosclerotic lesion analysis was performed using Evans blue and hematoxylin-eosin staining. Cell proliferation was assessed by MTT assay. Apoptosis was examined using flow cytometry analysis and Western blot analysis of Cleaved poly (ADP-ribose) polymerase (PARP) and Cleaved Caspase-3 levels. The effect of miR-590 on TLR4/nuclear factor kappa B (NF-κB) pathway was evaluated by Western blot. Binding between miR-590 and TLR4 was confirmed by luciferase reporter assay and Western blot. RESULTS: miR-590 was downregulated in the aorta tissues from HFD-fed apoE(−/−) mice and ox-LDL-treated HAECs. miR-590 overexpression inhibited atherosclerotic lesion in HFD-induced apoE(−/−) mice and promoted proliferation and inhibited apoptosis of ox-LDL-treated HAECs. Additionally, TLR4 was identified as a direct target of miR-590 in ox-LDL-treated HAECs. Moreover, anti-miR-590 reversed TLR4 knockdown-mediated promotion of cell proliferation and suppression of apoptosis in ox-LDL-treated HAECs. miR-590 overexpression suppressed the TLR4/NF-κB pathway, and inhibition of the TLR4/NF-κB pathway promoted cell proliferation and impeded apoptosis in ox-LDL-treated HAECs. CONCLUSION: miR-590 promoted proliferation and blocked ox-LDL-induced apoptosis in HAECs through inhibition of the TLR4/NF-κB pathway.

Effect of Huayu Qutan Recipe in Regulating Aortic MAPK Pathway-related Gene Expression in Atherosclerosis Mice / 中国实验方剂学杂志

Objective: To investigate the expression changes of mitogen activated protein kinase(MAPK) signaling pathway-related genes in ApoE-/- mice and the intervention effect of Huayu Qutan recipe on them, in order to explore the anti-atherosclerotic(AS) effect and possible mechanism of Huayu Qutan recipe. Method: Fifty ApoE-/- mice were randomly divided into model group, tanshinone ⅡA group (30 mg·kg-1·d-1), and high-dose phlegm group (20 g·kg-1·d-1), the middle-dose group (10 g·kg-1·d-1), and the low-dose group (5 g·kg-1·d-1), and 10 C57BL/6/J mice were included in the blank controls group. Automated biochemical analyzer was used to detect serum triglyceride(TG), cholesterol(TC), high-density lipoprotein cholesterol(HDL-C), low-density lipoprotein cholesterol(LDL-C) content; hematoxylin-eosin(HE) was used to detect aortic plaque in each group; oil red O was used to detect liver lipid deposition in each group; enzyme-linked immuno sorbent assay(ELISA) was used to determine vascular cell adhesion protein-1(VCAM-1), intercellular adhesion molecule-1(ICAM-1), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α) in serum of each group; Western blot was performed to detect c-Jun N-terminal kinase (JNK), phosphorylated c-Jun N-terminal kinase(p-JNK), c-Jun N-terminal kinase(JNK), p38 MAPK, p-p38 MAPK, extracellular regulated protein kinases (ERK), and expression of p-ERK. Result: Compared with the blank control group, serum TC, TG, LDL-C levels in the model group were significantly increased, while HDL-C levels were significantly decreased; aortic plaques were observed in the aortic lumen, and lots of lipid deposition were observed in the liver cells. Serum inflammatory factors TNF-α, IL-6, VCAM-1, ICAM-1 increased(PPα, IL-6, VCAM-1 and ICAM-1 were decreased. The expressions of p-p38 MAPK and p-JNK genes in intravascular associated MAPK signaling pathway were significantly decreased. p-ERK expression trend was not obvious(PConclusion: Huayu Recipe can inhibit the formation of aortic plaque in ApoE-/- mice, and its mechanism may be related to the regulation of vascular MAPK signaling pathway gene expression.

Anti-atherosclerotic effect of extract of traditional Chinese medicine formula Dan-yi-Iian in ApoE(-/-)mice / 中国药理学与毒理学杂志

OBJECTIVE To explore the anti-atherosclerotic effect of the extract of traditional Chinese medicine formula Dan-yi-lian(DYL)and the related mechanism.METHODS Atherosclerosis(AS)mod-el was established in ApoE(-/-)mice with a western diet. The mice were orally administered with differ-ent doses of DYL or vehicle daily for 28 d.The anti-atherosclerotic effect was evaluated by measuring the aortic atherosclerotic lesion area and media thickness with ultrasound imaging and histological sec-tions staining method. The effect on blood lipid was investigated by determining TC, TG, LDL, HDL, Apo-A1, Apo-B, etc. The anti-oxidative activity as assessed by determining the level of SOD, CAT, GSH,GSH-Px and MDA.Western blot analysis was used to determine the effect on ICAM-1,VCAM-1, MMP-2 and TNF-α. RESULTS In Dan-yi-lian administered ApoE(-/-)mice,the plaque area and media thickness were significantly reduced. Meanwhile, serum TC, TG, LDL and Apo-B were decreased, in contrast to the increased level of HDL and Apo-A1.On the other hand,SOD,CAT,GSH and GSH-Px were increased, while MDA was reduced in liver homogenate. In addition, the expression of ICAM-1, VCAM-1,MMP-2 and TNF-α was obviously inhibited by Dan-yi-lian.CONCLUSION Dan-yi-lian exhibit-ed potent anti-athero-sclerotic efficacy,in which the lipid-regulating,anti-oxidative and anti-inflammato-ry mechanism might be involved.

Protective effect and mechanism of dihydromyricetin on atherosclerosis in ApoE-/-mice / 中成药

AIM To explore the protective effect and mechanism of dihydromyricetin (DHM) on atherosclerosis (AS) in ApoE-/-mice induced by high fat diet (HFD).METHODS Thirty healthy 6-week-old male ApoE-/-mice were randomly divided into model group,DHM group [50 mg/(kg · d)] and Atorvastatin group [5 mg/(kg · d),i.g.],and another 10 male C57BL/6J mice were taken for control group.The mice in the control and model group were administered with 0.5％ CMC-Na solution,while the other two groups were given DHM and atorvastatin suspension (0.2 mL/10 g).All mice went on with a 10-week HFD diet (0.3％ cholesterol,20％ fat),after which the orbit blood was procured for serum isolation and subsequent determination of levels of SOD,GSH-Px,CAT and MDA by the biochemical analyzer and test of lipid accumulation in the aotic root by oil red O.The detection of macrophages accumulation and Caspase-3 expression level in the aortic root were achieved by immunofluorescence,and the macrophages apoptosis by TUNEL assay.RESULTS Significant post-treatment reduction in levels of TG,TC,and remarkable amelioration of LDL-C and plaque area in the aortic root were observed in DHM group when compared with model group.Meanwhile,DHM contributed to marked decrease of MDA level and improvement in activities of CAT,GSH-Px and SOD,the obvious macrophages accumulation prevention in atherosclerotic plaque.Additionally,its significant inhibition on macrophage apoptosis in aortic root was verified by the TUNEL assay.CONCLUSION DHM reduces the lipid accumulation in HFD-induced ApoE-/-mice,mechanically a resultant of its inhibition on macrophage apoptosis.

Effects of Aerobic Exercise on the Expression of Hepatic Hepcidin, and the Level of Serum IL-6 and MCP-1 in ApoE-/-Mice / 中国运动医学杂志

Objective To observe the preventive effects of the aerobic exercise on the expression of hepatic hepcidin,and the level of serum IL-6 and MCP-1 in ApoE-/-rats with atherosclerosis (AS) and to explore the mechanism.Methods Twenty 8-week male ApoE-/-mice were randomly divided into a control group and an exercise group,each of 10.The AS model was established in both groups through feeding high-fat diet.The control group was not given exercise intervention,while the exercise group underwent 14-week progressive intensity treadmill training,5 times a week,starting at 10 n/min for 30 min for the first 5 weeks and increasing to 13 m/min for 60 min at the end.At the end of the experiment,aortic samples were fixed and treated using the hematoxylin-eosin staining and Oil Red O.Then the pathological changes of the aorta were analyzed.The hepcidin expression of liver and serum hepcidin was assessed using Western blotting,and the level of the,interleukelin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) were detected using ABC-ELISA.Results The pathological slice showed that the vascular fiber plate and internal membrane of the aorta in the exercise group were better than that of the control group,with less ruptured vascular fibreboards,foam cell infiltration and fiber plaques.The hepcidin expression of liver and the level of serum hepcidin,the level of the serum IL-6 and MCP-1 in the exercise group were significantly lower than those of the control group (P＜0.01 and P＜0.01).Conclusion The long-term aerobic exercise can prevent AS for ApoE /-mice through lowering the expression of the hepatic hepcidin and serum hepcidin,as well as the level of serum IL-6 and MCP-1.

Suppression effect of MaiShu on formation of atherosclerotic plaque of apolipoprotein E knock-out mice / 药学学报

The research aimed to investigate the suppression effect of MaiShu which contains hawthorn, hippophae, medlar, phytosterols (β-sitosterol, stigmasterol and campesterol), β-glucan and lycopeneon formation of atherosclerotic plaque in apolipoprotein E knock-out (ApoE-/-) mice. Liquid chromatography-ultraviolet-mass spectrometry (LC-UV-MC) methods were used to analyze the main chemical composition of MaiShu.Atherosclerotic mice models were established by high-fat diet. The mice were administrated with MaiShu (1, 2, 4 g·kg-1·d-1) or other contrast materials by intragastric route for 10 weeks continuously. At the end of administration, the blood of mice was collected for tests of the serum total cholesterol (TC), total triglyceride (TG) and high density lipoprotein cholesterol (HDL-C) level. Atherosclerotic lesions in aorta and aortic root were assessed by calculating the relative area of lesions (oil red O stained). Intravital fluorescence microscopic system was used to evaluate the leukocyte-endothelial adhesion in mesenteric artery of mice by detecting the rolling velocity of white blood cells (WBC). Collagenous fibers and macrophages in lesions were detected by sirius red staining and immunological histological chemistry to evaluate the atherosclerotic plaque stability. Results showed that MaiShu contains various flavonoids (9.5%), phytosterols (23.8%) and polysaccharides (8.9%). The serum lipid level of model animals was significantly higher than the control animals. Serum TC level was decreased by MaiShu (4 g·kg-1, P-1) compared to model (P-1, P-1, P-1, P-1, P-1, P-1) and macrophages were decreased (2, 4 g·kg-1) compared to model. These results demonstrate that MaiShu can obviously decrease the serum lipid levels and the risk of leukocyte-endothelial adhesion in ApoE-/- mice. The effect of MaiShu may be associated with the decrease of macrophages in plaque.

Mechanism of the effect of rAd-APN gene on atherosclerosis in ApoE-/-mice / 中国实验动物学报

Objective Atherosclerosis ( AS) is a common pathological basis of cardiovascular diseases.Adiponec-tin ( APN) has been shown to have an anti-AS effect, and the underlying mechanisms, however, are largely unknown.Nu-clear transcription factorκB ( NF-κB) has also been regarded as a proatherogenic factor, mainly because of its regulation of a variety of the proinflammatory genes linked to AS.It is hypothesized that the inhibitory effects of APN on AS is through the inhibition of NF-κB signaling pathway.The aim of this study was to test the hypothesis via investigation and validation of the inhibitory effect of APN on AS in ApoE-/-mice, and to delineate the roles of NF-κB signaling pathway in modulating the APN effect on AS in vivo.Methods APN overexpression in ApoE-/-mice were mediated by transfecting adenovirus bearing a vector encoding for APN and enhanced green fluorescent protein ( Ad-APN-eGFP) .The AS in ApoE-/-mice was induced by feeding a high-fat diet.To validate the inhibitory effect of the adenovirus mediated APN overexpression on AS in the ApoE-/-mice.120 male ApoE-/-mice aged 12 weeks were randomly and evenly assigned into two groups (60 mice per group), and were fed with a high-fat diet to induce AS.At 0 day, 2, 4, and 6 weeks of high-fat diet feeding.The 2 groups of mice were injected intravenously in the tail with either 100 μL (3.0 ×108 p.f.u) of Ad-eGFP virus ( control group) or the same amount of Ad-APN-eGFP virus ( APN group) .Blood samples and aortic tissues were taken at 0 day, 4, and 8 weeks of high-fat diet feeding.For the blood samples, FABA was used to analyze the concentrations of blood lipids and ELIZA was used to test the concentrations of serum APN.For the aortic tissues, oil red O staining was used to detect the surface lesion percentage.Masson staining was used to evaluate the collagen content and fibrous cap thickness of the plaque area.Immunofluorescence method was used to detect APN and NF-κB p65 expression.Western blot was used to de-tect the expressions of APN,nuclear NF-κB p65 and the downstream factors of NF-κB pathway.Results APN inhibited the formation of atherosclerotic plaque in ApoE-/-mice.The lesion formation in aortic sinus was significantly inhibited ( P<0.01).Compared with the control group, the oil red O staining showed that the surface area ratio of atherosclerotic le-sions was decreased significantly in the Ad-APN group ( P<0.001 ): the percentage of surface lesions in the 4 weeks groups was 27.78 ±8.64 vs.33.02 ±5.18 (%);the 8 weeks groups was 31.58 ±5.87 vs.52.16 ±5.79 (%) .As the serum APN was increased,the concentration of TC, TG and LDL-C were significantly decreased( P<0.001 for all) , and the growth of body weight was slowed down(P<0.05).APN effectively inhibited the expression of NF-κB nuclear protein p65 and inflammatory factors.Conclusions Adiponectin reduces the inflammatory reactions in atherosclerosis through in-hibiting the NF-κB pathway.

Effect of atorvastatin on adventitial fibroblast phenotype differentiation in atherosclerosis of apolipoprotein E-knockout mice / 中国病理生理杂志

AIM: To explore the effect of atorvastatin on the expression of α-SMA and TGF-β1 in the adventi-tia of ApoE-/-mice with atherosclerosis, and to investigate the underlying mechanism of atorvastatin therapy.METHODS:Male ApoE-/-mice (n =40) at 6-weeks of age were used to establish the atherosclerosis model by feeding with high fat diet. The mice were randomly divided into model group and atorvastatin group.In atorvastatin group, the mice were lavaged with atorvastatin at dose of 20 mg? kg-1? d-1 .The mice in model group were given normal saline.C57BL/6 mice of the same age served as control group, feeding with ordinary food.The mice were respectively sacrificed at the time points of 10 and 15 weeks after feeding with different diets.The ascending aorta was removed for serial sectioning.Some sections were per-formed with Movat staining in order to observe the morphological changes of the tissues, and to measure the relative athero-sclerotic plaque area and the thickness of the adventitia.Some sections were stained with Sirius red to identify the collagen synthesis.Immunohistochemistry assay was prepared to observe the expression of α-SMA and TGF-β1 in the adventitia at different time points.The expression of TGF-β1 at mRNA and protein levels in the thoracoabdominal aorta was measured by RT-qPCR and Western blot.RESULTS: Compared with model group, the formation of plaque in atorvastatin group signifi-cantly descended.Meanwhile the adventitial thickness and collagen synthesis also decreased.The results of immunohisto- chemical staining showed that compared with 10 weeks-model group, α-SMA and TGF-β1 in 15 weeks-model group was in-creased.The expression of α-SMA and TGF-β1 in atorvastatin group decreased significantly compared with model group. The expression of TGF-β1 at mRNA and protein levels in model group were higher than those in control group.They de-creased in atorvastatin group compared with model group.Compared with 10 weeks-model group, the mRNA and protein of TGF-β1 in 15 weeks-model group were increased.CONCLUSION: Atorvastatin modulates adventitial fibroblast phenotype differentiation by suppressing expression of TGF-β1 and intervenes atherosclerotic development in ApoE.

Effects of Moxibustion and Moxa Smoke on Blood Lipids, and Hepatic Pathologic Morphology and CD36 and ABCA1 Expressions in ApoE-/-mice / 上海针灸杂志

ObjectiveTo investigate the intervention effects of moxibustion and moxa smoke on blood lipids,hepatic pathological changes and intrahepatocytic molecules related to cholesterol metabolism and analyze the regulating effects of moxibustion and moxa smoke on cholesterol metabolism and explore the mechanisms of actions of moxibustion and moxa smoke. MethodFifty-one 8-week-old ApoE-/-mice were randomized into model, moxa smoke and moxibustion groups, 17 mice each. Twenty C57BL/6 mice comprised a blank control group. The normal and model groups of mice were routinely grabbed and fastened. The moxa smoke group of mice was exposed to 10-15 mg/m3moxa smoke circumstances. The moxibustion group of mice was given moxibustion on point Guanyuan(CV4). All interventions were made 20 min daily, 6 times a week, for 12 consecutive weeks. Total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) were measured using an automatic biochemical analyzer. Hepatic pathologic morphology was observed by HE staining. Hepatic CD36 and ABCA1 expressions were determined by immunohistochemical method.ResultIn the model group of mice, serum TG and LDL-C contents were significantly higher than in the normal group (P=0.003;P=0.001);HDL-C content was significantly lower than in the normal group (P=0.007); TC content had no significant difference compared with the normal group (P>0.05). In the moxibustion group of mice, serum TG and LDL-C contents were significantly lower than in the model group (P=0.03;P=0.001) and HDL-C content had no significant difference compared with the model group (P=0.11). In the moxa smoke group of mice, serum TG and LDL contents were significantly lower than in the model group (P=0.01;P=0.008) and HDL content had no significant difference compared with the model group (P=0.11). There were no significant differences in various blood lipid indicators between the moxibustion and moxa smoke groups (P>0.05). There were hepatic cell cord and sinusoid derangement and obvious hepatocytic swelling in the model group of mice. In the moxa smoke and moxibustion groups, hepatocytic swelling subsided significantly, and inflammatory cell infiltration reduced compared with the model group. In the model group,CD36 expression was significantly higher than in the normal group (P=0.004) and ABCA1 expression was significantly lower than in the normal group (P=0.001). In the moxibustion group, CD36 expression had no significant difference compared with the model group (P=0.09) and ABCA1 expression was significantly higher than in the model group (P=0.03). In the moxa smoke group, CD36 expression was significantly lower than in the normal group (P=0.02) and ABCA1 expression was significantly higher than in the model group (P=0.002). There were no significant differences in CD36 and ABCA1 expressions between the moxibustion and moxa smoke groups (P>0.05).ConclusionEarly moxibustion on point Guanyuan can regulate disorders of blood lipid metabolism, delay the occurrence of hepatic lesions and reduce intrahepatic accumulation of cholesterol to a certain extent in an ApoE-/-mouse model of atherosclerosis. That may be one of the mechanisms by which moxibustion therapy prevents atherosclerosis. Moxa smoke as the product of moxibustion is an effective factor in moxibustion producing a therapeutic effect.

CFTR suppressing hyperhomocysteinemia-induced hepatocyte damage in ApoE-/-mice / 实用医学杂志

Objective To investigate the function of CFTR in ApoE-/- mice with HHcy-induced hepato-cellular injury. Methods Thirty six 5-week old ApoE-/- mice were divided into three groups , including the ApoE-/- group, the HHcy group and the intervention group, (n = 12). Twelve normal C57BL/6J mice were fed with regular mouse diet as the normal control (SPF grade). HL-7702 human liver cells were intervened by Hcy (100 μmol/L) and 100 μmol/L Hcy + folic acid (100 μmol/L Hcy + F). The changes of Hcy, ALT and AST in the serum and the expression of CFTR mRNA and protein in liver and liver cells were detected. The concen-trations of ALT and AST in the liver cell intervened by VX-770 agonist and CFTR(inh)-172 inhibitor were mea-sured by ELISA. Results Compared with the control group , the levels of Hcy , ALT and AST were higher and the levels of CFTR mRNA and protein were lower in the Meth group (P < 0. 05 ) , while the reverse result in the Meth + F group (P < 0.05). Compared with the control group, the levels of CFTR mRNA and protein were de-creased and the levels of ALT and AST were increased in the 100 μmol/L Hcy group (P < 0.05). Compared with the 100 μmol/L Hcy group , the levels of CFTR mRNA and protein were increased and the levels of ALT and AST were decreased in the 100 μmol/L Hcy + F group (P < 0.05). Stimulated with VX-770 can reduce the concentrations of ALT and AST and the vice versa in the CFTR (inh)-17 group the concentration was increased in liver cells. Conclusion CFTR plays an important role in the regulation of hepatocellular injury by HHcy.

Effects of Moxa Smoke Versus Tobacco Smoke on Autonomous Behaviors and Hippocampal GFAP Expression in Apolipoprotein E Gene Knockout Mice / 上海针灸杂志

ObjectiveTo investigate the effects of moxa smoke versus tobaccosmoke on autonomous behaviors and hippocampal expression of glial fibrillary acidic protein (GFAP) in apolipoprotein E gene knockout (ApoE-/-) mice.Method Thirteen 8-week-old C57BL/6 mice constituted a blank control group. Twenty-seven ApoE-/-mice of the same age were randomized into ApoE-/-model, moxa smoke and tobacco smoke groups. The tobacco smoke and moxa smoke groups of mice were exposed to smoke 5-15 mg/m3circumstances. Every group of mice was intervened in 20 min. daily, six days a week, for atotal of 12 weeks. A behavioral test was conducted in week 13. The animals were then sacrificed to take the materials. Hippocampal GFAP in the brain was measured by an immunohistochemical method.ResultAutonomous activities were significantly more in theblank group than in the model group (P0.05). Moving distance was longer in the moxa smoke group than in the model group (P<0.05). Standing-up number was smaller in the moxa smoke group than in the blank group (P<0.05). Integral optical density of GFAP immune reaction products in the hippocampus was significantlyhigher in the model group of mice than in the blank and moxa smoke groups (P<0.05). Hippocampal GFAP expression was significantly higher in the group of mice than in the moxa smoke and blank groups (P<0.05).Conclusion Moxa smoke can increase the excitability of central nervous system in mice and reduce hippocampal GFAP expression in a mice model of Alzheimer disease.