Proteomics Analysis of Lipid Metabolism and Inflammatory Response in the Liver of Rabbits fed on a High Cholesterol Diet.

In this study, we aimed to analyze the proteomics of the liver in rabbits on a high cholesterol diet (HCD). We randomly divided New Zealand white rabbits into the normal diet group and the HCD group. We established the atherosclerosis model and measured plasma cholesterol and triglycerides. The model was successfully established using ultrasound examination and histopathological staining of the intima of aorta and liver of the two groups of rabbits. The differential proteins in the rabbit liver were analyzed using Tandem Mass Tags proteomic analysis technology. Finally, we used western blot to verify the reliability of proteomics. The results showed that compared with the control group, the serum lipid levels of rats in the HCD group was significantly increased, and the pathological sections showed the formation of atherosclerotic plaques in the aorta, inflammation, and adipose lesions in the liver. Proteomic analysis of the liver revealed 149 differences in HCD-expressed protein, which is mainly involved in inflammation and regulation of lipid and sugar metabolism. In addition, we verified differentially expressed liver proteins in the HCD group using western blot. We found that HCD caused lipid accumulation, abnormal glucose metabolism, and inflammatory response in the liver.

Complete mitogenome of the entomopathogenic fungus Orbiocrella petchii.

In this study, the complete mitogenome of an entomopathogenic fungus Orbiocrella petchii (syn. Torrubiella petchii) was assembled and annotated. This circular mitogenome was 23,794 bp in length and consisted of 2 rRNA genes (rnl and rns), 25 tRNA genes, and 14 standard protein-coding genes of the oxidative phosphorylation system. Two group I introns were identified, and they encoded ribosomal protein S3 (in rnl) or a GIY-YIG endonuclease (in nad1). Phylogenetic analysis based on mitochondrial DNA sequences confirms O. petchii in the family of Clavicipitaceae.

SIRT1 Regulates the Inflammatory Response of Vascular Adventitial Fibroblasts through Autophagy and Related Signaling Pathway.

BACKGROUND/AIMS: Autophagy is a lysosomal degradation pathway that is essential for cellular survival, differentiation, and homeostasis. Sirtuin 1 (SIRT1), a NAD+-dependent deacetylase, plays a pivotal role in modulation of autophagy. Recent studies found that autophagy was involved in the regulation of inflammatory response. In this study, we aimed to determine the effect of SIRT1 on autophagy and inflammation, and whether autophagy can regulate the inflammatory response in vascular adventitial fibroblasts (VAFs). METHODS: Cell autophagy was evaluated by fluorescence microscope and transmission electron microscopy. The expression of protein and mRNA were determined by Western blot analysis and real time-PCR. The production of cytokine was detected by ELISA. RESULTS: TNF-α induced autophagy and increased SIRT1 expression in VAFs. SIRT1 activator resveratrol enhanced TNF-α-induced VAF autophagy. In contrast, SIRT1 knockdown attenuated VAF autophagy. Both the Akt inhibitor MK2206 and mTOR inhibitor rapamycin further increased TNF-α-induced VAF autophagy. Furthermore, SIRT1 knockdown increased Akt phosphorylation and inhibited the autophagy in VAFs. However, MK2206 attenuated the effect of SIRT1 knockdown on VAF autophagy. In addition, ingenuity pathway analysis showed that there is a relationship between cell autophagy and inflammation. We found that SIRT1 knockdown increased the expression of NLRP3 and interleukin (IL)-6 and promoted the production of IL-1ß in VAFs. Further study showed that autophagy activation decreased the expression of NLRP3 and IL-6 and inhibited the production of IL-1ß, whereas autophagy inhibition increased the inflammatory response of VAFs. More importantly, our study showed that autophagy was involved in the degradation of NLRP3 through the autophagy-lysosome pathway. CONCLUSION: SIRT1 not only regulates VAF autophagy through the Akt/mTOR signaling pathway but also suppresses the inflammatory response of VAFs through autophagy.

Activation of PPAR alpha by fenofibrate inhibits apoptosis in vascular adventitial fibroblasts partly through SIRT1-mediated deacetylation of FoxO1.

Recent studies demonstrated that the ligand-activated transcription factor peroxisome proliferator-activated receptorα (PPARα) acts in association with histone deacetylase sirtuin 1 (SIRT1) in the regulation of metabolism and inflammation involved in cardiovascular diseases. PPARα activation also participates in the modulation of cell apoptosis. Our previous study found that SIRT1 inhibits the apoptosis of vascular adventitial fibroblasts (VAFs). However, whether the role of PPARα in apoptosis of VAFs is mediated by SIRT1 remains unknown. In this study, we aimed to determine the effect of PPARα agonist fenofibrate on cell apoptosis and SIRT1 expression and related mechanisms in ApoE(-/-) mice and VAFs in vitro. We found that fenofibrate inhibited cell apoptosis in vascular adventitia and up-regulated SIRT1 expression in aorta of ApoE(-/-) mice. Moreover, SIRT1 activator resveratrol (RSV) further enhanced these effects of fenofibrate. In vitro study showed that activation of PPARα by fenofibrate inhibited TNF-α-induced cell apoptosis and cell cycle arrest in VAFs. Meanwhile, fenofibrate up-regulated SIRT1 expression and inhibited SIRT1 translocation from nucleus to cytoplasm in VAFs stimulated with TNF-α. Moreover, the effects of fenofibrate on cell apoptosis and SIRT1 expression in VAFs were reversed by PPARα antagonist GW6471. Importantly, treatment of VAFs with SIRT1 siRNA or pcDNA3.1(+)-SIRT1 showed that the inhibitory effect of fenofibrate on cell apoptosis in VAFs through SIRT1. On the other hand, knockdown of FoxO1 decreased cell apoptosis of VAFs compared with fenofibrate group. Overexpression of FoxO1 increased cell apoptosis of VAFs compared with fenofibrate group. Further study found that fenofibrate decreased the expression of acetylated-FoxO1 in TNF-α-stimulated VAFs, which was abolished by SIRT1 knockdown. Taken together, these findings indicate that activation of PPARα by fenofibrate inhibits cell apoptosis in VAFs partly through the SIRT1-mediated deacetylation of FoxO1.

Increased muscle force production and bone mineral density in ActRIIB-Fc-treated mature rodents.

Myostatin is a highly conserved member of the transforming growth factor-ß ligand family known to regulate muscle growth via activation of activin receptors. A fusion protein consisting of the extracellular ligand-binding domain of activin type IIB receptor with the Fc portion of human immunoglobulin G (ActRIIB-Fc) was used to inhibit signaling through this pathway. Here, we study the effects of this fusion protein in adult, 18-month-old, and orchidectomized mice. Significant muscle growth and enhanced muscle function were observed in adult mice treated for 3 days with ActRIIB-Fc. The ActRIIB-Fc-treated mice had enhanced fast fatigable muscle function, with only minor enhancement of fatigue-resistant fiber function. The ActRIIB-Fc-treated 18-month-old mice and orchidectomized mice showed significantly improved muscle function. Treatment with ActRIIB-Fc also increased bone mineral density and serum levels of a marker of bone formation. These observations highlight the potential of targeting ActRIIB receptor to treat age-related and hypogonadism-associated musculoskeletal degeneration.

SIRT1 regulates TNF-α-induced expression of CD40 in 3T3-L1 adipocytes via NF-κB pathway.

Sirtuin1 (SIRT1), a NAD(+)-dependent deacetylase, not only regulates lipid and glucose homeostasis, but also involves the regulation of proinflammatory cytokine involved in inflammation-associated diseases. The activation of CD40 triggers inflammation that plays a crucial role in the development of many chronic inflammatory diseases including obesity. Growing evidence indicated that SIRT1 exerts anti-inflammatory properties by suppressing proinflammatory cytokines production. However, the effect of SIRT1 on the expression of CD40 in adipocytes has not yet been fully elucidated. The present study showed that SIRT1 expressed both in the nucleus and cytoplasm of 3T3-L1 adipocytes. TNF-α significantly reduced the expression of SIRT1 mRNA and protein and increased the expression of CD40 mRNA and protein in time- and concentration-dependent manners. Overexpression of SIRT1 or SIRT1 activation by resveratrol obviously attenuated the expression of CD40 induced by TNF-α in 3T3-L1 adipocytes, whereas knockdown of SIRT1 or SIRT1 inhibition by nicotinamide and sirtinol significantly enhanced TNF-α-induced expression of CD40. Furthermore, overexpression of SIRT1 or SIRT1 activation by resveratrol diminished TNF-α-induced acetylation of NF-κBp65, while knockdown of SIRT1 or SIRT1 inhibition by nicotinamide and sirtinol augmented TNF-α-induced acetylation of NF-κBp65 in 3T3-L1 adipocytes. NF-κB inhibitor PDTC reduced TNF-α-induced mRNA and protein expression of CD40 in 3T3-L1 adipocytes. The combination treatment of resveratrol and PDTC significantly reduced TNF-α-induced expression of CD40, and the inhibitory effects were higher than that of the single treatment. Taken together, SIRT1 exerts anti-inflammatory property by regulating TNF-α-induced expression of CD40 partially through the NF-κB pathway in 3T3-L1 adipocytes. More importantly, the regulation of SIRT1 on the expression of CD40 provides new insight to understand the anti-inflammatory effects of SIRT1.

The effect of radixin knockdown on the expression and efflux function of MRP2 in SGC-7901 cells.

Multidrug resistance-associated protein 2 (MRP2, ABCC2) is the second member of the MRP transporter family and functions physiologically as an organic anion transporter. Earlier studies have confirmed that radixin, which is a member of the ERM (ezrin/radixin/moesin) family, modulates MRP2 localization at the canalicular membrane in hepatocytes. The relationship between radixin and MRP2 - particularly, the effect of radixin on the expression and function of MRP2 in cells or tissues that co-express all three ERM proteins - has not been well studied. To examine the role of radixin in the expression and function of MRP2 and other MRPs, we chose human gastric carcinoma SGC-7901 cells that express all three ERM proteins rather than hepatocytes, which predominantly express radixin. Radixin stable knockdown SGC-7901 cells, which were constructed by RNAi, exhibited no compensatory up-regulation of ezrin or moesin. The mRNA expression profiles of MRPs in the radixin knockdown cells were primarily evaluated by RT-PCR. Real time quantitative RT-PCR and western blot analysis revealed that the radixin deficiency caused the mRNA and protein expression levels of MRP2 to be reduced by about 50%, respectively. Accordingly, efflux and MTT assays showed that the radixin knockdown cells exhibited lower efflux ability with respect to calcein but no significant change in cell viability. In conclusion, among the MRP1-6 family members, radixin selectively modulates the expression and function of MRP2 in a system co-expressing all three ERM proteins.

The effects of Buyang Huanwu Decoction on hemorheological disorders and energy metabolism in rats with coronary heart disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Buyang Huanwu Decoction (BYHWD), a traditional Chinese medicine (TCM) formula, has been recognized as a clinical treatment for coronary heart disease (CHD) with qi deficiency and blood stasis syndrome. The effects of BYHWD on hemorheological disorders and energy metabolism in CHD with qi deficiency and blood stasis syndrome are still unclear. AIM OF THE STUDY: To investigate whether the ameliorative effects of BYHWD on CHD rats with qi deficiency and blood stasis syndrome are associated with the regulation of hemorheological disorders and energy metabolism. MATERIALS AND METHODS: The rats were lavaged with 25.68, 12.84 and 6.42 g/kg BYHWD (g weight of mixed crude drugs/kg body weight), respectively, once a day for 21 days. The body weight, exhaustive swimming time and tongue characters were observed and recorded. The whole blood viscosity and plasma viscosity were determined by hematology analyzer. The level of fibrinogen (Fbg) in plasma was determined by using Fbg assay kit. The platelet aggregation induced by adenosine diphosphatase was measured by semi-automatic whole blood platelet analyzer. The level of blood glucose (BG) was determined by LifeScan. The activity of Na(+)-K(+)-ATPase in heart tissues was detected by spectrophotometer. RESULTS: BYHWD improved the exterior signs of qi deficiency and blood stasis syndrome in rats with CHD, including the body weight, exhaustive swimming time and tongue quality. The whole blood viscosity in rats treated with 25.68 g/kg BYHWD decreased at the shear rate of 10s(-1) (P<0.05) and the plasma viscosity decreased in rats treated with 25.68 and 12.84 g/kg BYHWD (P<0.05). The plasma Fbg level and the platelet aggregation decreased in rats treated with 25.68 g/kg BYHWD (P<0.01). The results also revealed that the BG level decreased and the Na(+)-K(+)-ATPase activity in heart tissues increased in rats treated with 25.68 and 12.84 g/kg BYHWD (P<0.01). CONCLUSION: The results suggest that the ameliorative effects of BYHWD on CHD rats with qi deficiency and blood stasis syndrome are mediated by the improvement of hemorheological disorders and energy metabolism.

Effect of exercise training on renal function and renal aquaporin-2 expression in rats with chronic heart failure.

1. Chronic heart failure (CHF) is often accompanied by renal dysfunction. Exercise training may relieve the symptomatic burden and improve the overall prognosis of CHF. In the present study, the effects of exercise training on renal function and renal aquaporin (AQP)-2 expression in CHF rats were examined to determine whether exercise training could relieve renal dysfunction in CHF rats. 2. Male Sprague-Dawley rats were divided into three groups: sham, sedentary CHF (Sed-CHF) and exercise training CHF (Ex-CHF) groups. Cardiorenal function was assessed in each group by haemodynamic measurement and ultraviolet spectrophotometry. Pathological changes in cardiac and renal tissues were evaluated histologically and the collagen volume fraction (CVF) was calculated. The expressions of AQP-2 and ß-tubulin were determined by western blotting and immunohistochemistry. 3. The Sed-CHF rats were found to have increased left ventricular end-diastolic pressure (LVEDP) and CVF in the heart compared with sham rats. Exercise training decreased LVEDP and CVF values in Ex-CHF rats. The Sed-CHF rats were found to have increased serum levels of creatinine (sCr), blood urea nitrogen (BUN) and arginine vasopressin (AVP), as well as increased CVF in the kidney, compared with sham rats. Exercise training decreased levels of sCr, BUN, AVP and CVF in Ex-CHF rats. Moreover, exercise training decreased AQP-2 and ß-tubulin protein expression in the kidney of CHF rats. 4. The results suggest that exercise training can significantly improve the renal dysfunction in CHF rats and that the underlying mechanism may be related to water reabsorption and preventing changes to the cytoskeleton.

Buyang Huanwu decoction ameliorates coronary heart disease with Qi deficiency and blood stasis syndrome by reducing CRP and CD40 in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Qi deficiency and blood stasis is traditional Chinese medicine (TCM) syndrome. It leads to many diseases including coronary heart diseases (CHD) and cerebrovascular diseases (CVD). Inflammatory biomarkers and many endothelium-derived vasoactive factors are considered to play pivotal roles in CHD. Buyang Huanwu decoction (BYHWD), a TCM formula, has been recognized as a treatment for CHD with Qi deficiency and blood stasis syndrome and CVD in clinic. The mechanisms of BYHWD effect on CHD with Qi deficiency and blood stasis syndrome are unclear. AIM OF THE STUDY: The aim is to investigate whether the effects of BYHWD on CHD with Qi deficiency and blood stasis syndrome in rats are associated with the inhibition of CRP, CD40 and vascular endothelial regulators. MATERIALS AND METHODS: The treated groups were lavaged with 25.68, 12.84 and 6.42 g/kg BYHWD respectively once a day for 21 days. The level of C-reactive protein (CRP) in serum and the expression of cluster of differentiation 40 (CD40) in the heart and aorta of rats were detected. Moreover, the levels of thromboxaneA(2) (TXA(2)) and prostacyclin (PGI(2)) in plasma were measured and the levels of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in serum were detected. RESULTS: BYHWD (25.68 g/kg) significantly decreased the level of CRP in serum and BYHWD (25.68 and 12.84 g/kg) decreased the expression of CD40 in the heart and aorta (P<0.01). The results also revealed that BYHWD (25.68 g/kg) inhibited the levels of iNOS in serum and TXA(2) in plasma and increased the levels of eNOS in serum and PGI(2) in plasma (P<0.01). CONCLUSION: The study shows that the ameliorative effects of BYHWD on CHD with Qi deficiency and blood stasis syndrome in rats are associated with the inhibition of CRP and CD40 and the regulation of endothelium-derived vasoactive factors.

Down-regulation of CD40 gene expression and inhibition of apoptosis with Danshensu in endothelial cells.

Danshen is commonly used in China for the treatment of atherosclerosis-related disorders such as cardiovascular and cerebrovascular diseases. Research shows that it also has immunostimulation properties. The present study evaluates the protective effect of danshensu, an active water-extractable component isolated from danshen, on an endothelial cell line (CRL-1730) treated with hydrogen peroxide (H(2)O(2)). Danshensu significantly inhibited endothelial cell viability induced by H(2)O(2). The treatment of endothelial cells with danshensu resulted in most cells being arrested in the S and G(2)/M phases of the cell cycle. The fraction of cells in G(0)/G(1) phase was markedly decreased by danshensu treatment compared to the control groups. The apoptosis was also markedly decreased after danshensu treatment. Additionally, danshensu restrains decreased nitric oxide level, increased the release of lactate dehydrogenase and expression of cluster of differentiation 40 (CD40) significantly. These results suggest that danshensu protects endothelial cells from the damage induced by H(2)O(2) through its CD40 anti-inflammatory approach and cell apoptosis inhibition.

Fenofibrate inhibits tumor necrosis factor-alpha-induced expression of CD40 and matrix metalloproteinase in human vascular endothelial cells.

OBJECTIVE: To investigate the regulatory effects of fenofibrate on TNF-alpha-induced CD40 expression and matrix metalloproteinase (MMP) activity in human vascular endothelial cells (HUVECs). METHODS: Quantitative RT-PCR and flow cytometry were employed to evaluate the effect of fenofibrate on TNF-alpha-induced CD40 mRNA and cell surface CD40 expression in HUVECs, and gelatin zymography was used to determine the effect of fenofibrate on the gelatinolytic activities of MMP-2 and MMP-9 in TNF-alpha-stimulated HUVECs. RESULTS: Fenofibrate at the concentrations of 5x10(-5), 1x10(-4) and 2x10(-4) mol/L significantly reduced TNF-alpha-induced increment of CD40 mRNA and cell surface CD40 expressions (P<0.01), with the maximal inhibition achieved at the concentration of 1x10(-4) mol/L. Fenofibrate at 2x10(-4) mol/L did not further decrease CD40 expression induced by TNF-alpha. Fenofibrate significantly inhibited the stimulatory effect of TNF-alpha on MMP-2 and MMP-9 activities in HUVECs. CONCLUSION: Fenofibrate reduces TNF-alpha-induced increment of CD40 expression and MMP-2 and MMP-9 activities in HUVECs.

Protective effect of tanshinone IIA on human umbilical vein endothelial cell injured by hydrogen peroxide and its mechanism.

Tanshinone IIA (Tan IIA) is isolated from Salvia miltiorrhiza, the root of which is widely used as a traditional Chinese medicine to treat atherosclerosis. The aim of the present study was to evaluate the putative protective effect of Tan IIA in a human umbilical vein endothelial cell line (ECV-304) injured by hydrogen peroxide in vitro and the mechanism of its protection. The percentage of cell viability was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The endothelial cell apoptosis and expression of cluster of differentiation 40 (CD40) were detected by flow cytometric analysis. Preincubation with Tan IIA significantly increased the viability of ECV-304 cell injured by hydrogen peroxide, which was accompanied with the increased nitric oxide level and superoxide dismutase activity in a dose-dependent manner. Moreover, cell apoptosis and CD40 expression were decreased in a dose-dependent manner. In conclusion, our data suggests that Tan IIA protects ECV-304 cell damage induced by hydrogen peroxide through its anti-oxidant effect and CD40 anti-inflammatory approach.

[Liposome-mediated human CD40 gene transfection and human umbilical vein endothelial ECV-304 cells].

OBJECTIVE: To construct an eukaryotic expression vector containing human CD40 gene for its efficient, continuous and stable expression in human umbilical vein endothelial ECV-304 cells. METHODS: The recombinant plasmid pUCD40 was digested with endonucleases to obtain human CD40 gene fragment, which was cloned into pCDNA3.1 vector to construct recombinant eukaryotic expression vector pCDNA3.1(+)/CD40. The recombinant vector was identified by enzyme digestion before introduced into ECV-304 cells via liposome, with the positive cell clones selected with G418. The stable transfection and expression of CD40 in ECV-304 cells were identified by reverse transcription (RT)-PCR, Western blotting and flow cytometry, respectively. RESULTS: Enzyme digestion analysis showed that target gene had been cloned into the recombinant vector. The transfected ECV-304 cells successfully expressed human CD40 as determined by RT-PCR and Western-blotting, and 95% of the cells were CD40-positive as shown by flow cytometry. CONCLUSION: The recombinant eukaryotic expression vector pCDNA3.1(+)/CD40 has been successfully constructed, which is capable of stable transfection and expression of CD40 in ECV-304 cells to facilitate further investigation of the roles of CD40 molecule in antiatherosclerotic drug development.

[An experimental study of the effect of structure sealing of infrared CO2 sensor on its accuracy].

OBJECTIVE: To study the effect of structure sealing of infrared CO2 sensor on its accuracy. METHOD: Two experiments were designed. The IR CO2 sensor based on infrared principle and no comparing lighting path with only one beam of light were calibrated in one experiment, and in the other experiment the sensors were placed in an environment simulating the practical condition. The temperature, pressure and standard gas were kept same in the two experiments while CO2 concentrations in the dead volume were varied. RESULT: Readings from the sensors varied with the variation of the CO2 concentration in the dead volume of the sensors. CONCLUSION: The structure sealing of the IR CO2 sensor has great influence on its accuracy. A sealed dead volume in the IR CO2 sensor can decrease or eliminate the effect.

[Effect of ligustrazine on injury of vascular endothelial cells ECV-304 induced by hypoxia and lack of glucose].

OBJECTIVE: To investigate the effects of ligustrazine on nitric oxide (NO), malonaldehyde (MDA) production, release of intracellular lactate dehydrogenase (LDH) and membrane fluidity of the injured human umbilical vein vascular endothelial cell line (ECV-304) with hypoxia and lack of glucose. METHOD: The experiments were performed in culture of ECV-304 injured with hypoxia and lack of glucose in vitro. The released LDH of ECV-304 was measured with automatic biochemistry analyse. NO content of ECV-304 was monitored with colorimetry. Lipid peroxidation of ECV-304 was monitored as MDA with a fluorometric assay. The membrane fluidity of ECV-304 was measured with the fluorescence polarization method. RESULT: After culture ECV-304 in hypoxia and lack of glucose for 24 h, the LDH release, MDA production and the membrane fluidity increased significantly and NO level was decreased. Preincubation of ECV-304 with ligustrazine for 24 h reduced LDH release, MDA production, membrane fluidity increasing and increased the level of NO in ECV-304 due to hypoxia and lack of glucose. CONCLUSION: Ligustrazine has protective effect on injury of ECV-304 induced by hypoxia and lack of glucose.

[The development of a blood pressure monitor based on oscillometric method].

This paper describes a type of blood pressure monitor we have developed using 8031 chip microprocessor based on oscillometric method and the designs of hardware and software. Comparing the measurement results of oscillometric method with that of direct invasive measurement method, we find that the monitor is very useful in clinical applications. Finally in the paper, some improvements that can be made in the monitor are proposed.