Inhibitory effect of Wendan decoction on atherosclerotic plaque formation in ApoE-/- mice by regulating expression of membrane proteins involved in reverse cholesterol transport

AIM To explore the anti-atherosclerotic mechanism of Wendan decoction based on reverse cholesterol transport. METHODS Eight-week-old apolipoprotein E gene knockout （ApoE^-/-） mice with high-fat diet and daily drug gavage were randomly divided into model group, simvastatin group, and low-, middle- and high-dose Wendan decoction groups, with 15 mice in each group. The C57BL/6 mice of the same age served as control group. The mice were weighed once every week. After 10 weeks, the mice were anesthetized with chloral hydrate. The serum were collected for lipid level examination. The atherosclerotic plaque buildup in aortic root and whole aorta was observed by HE staining and oil red O staining, respectively. The levels of proteins related to cholesterol transport, ATP-binding cassette transporter A1 （ABCA1） and caveolin-1 in the aorta, and scavenger receptor class B type I （SR-BI） and CD36 in the liver, were quantified by Western blot. RESULTS Wendan decoction at middle dose inhibited the increase in the body weight of ApoE^-/- mice fed with high-fat diet （P<0.05）. Wendan decoction at different doses significantly reduced the serum levels of triglyceride, total cholesterol and low-density lipoprotein cholesterol in the ApoE^-/- mice （P<0.05 or P<0.01）, but had no effect on serum high-density lipoprotein cholesterol level （P>0.05）. Wendan decoction at different doses inhibited the formation of atherosclerotic plaques in whole aorta of the ApoE^-/- mice （P<0.05 or P<0.01）. Middle- and high-dose Wendan decoction significantly inhibited the formation of atherosclerotic plaques in the aortic root （P<0.05）. Bedsides, Wendan decoction at different doses increased the protein level of ABCA1 and decreased the protein level of caveolin-1 in the aorta of the ApoE^-/- mice （P<0.01）. Middle- and high-dose Wendan decoction increased the liver protein level of SR-BI in the ApoE^-/- mice （P<0.01）. However, Wendan decoction at different doses had no effect on the liver protein level of CD36 in the ApoE^-/- mice （P>0.05）. CONCLUSION Wendan decoction reduces the body weight, serum lipid levels and formation of atherosclerotic plaques in ApoE^-/- mice fed with high-fat diet, and its mechanism is related to up-regulation of ABCA1 protein level in the aorta and SR-BI protein level in the liver as well as down-regulation of caveolin-1 protein level in the aorta.     

Inhibitory effect of apolipoprotein A-I mimetic peptide D-4F on scavenger receptor A1 in macrophage-derived foam cells

AIM:To investigate the inhibitory effect of apolipoprotein A-I mimetic peptide D-4F on the scavenger receptor A1 (SR-A1) in macrophage-derived foam cells induced by oxidized low-density lipoprotein (ox-LDL). METHODS:RAW264.7 cells were pretreated with different concentrations (12.5, 25 and 50 mg/L) of D-4F or 50 mg/L inactive control peptide scrambled D-4F (sD-4F) for 1 h or endoplasmic reticulum stress (ERS) inhibitor 4-phenylbutyric acid (5 mmol/L) for 30 min, followed by the treatment with 100 mg/L ox-LDL for 12 h. In addition, the cells were pretreated with 50 mg/L D-4F or sD-4F for 1 h, and then stimulated with 2 mg/L tunicamycin (TM; an ERS inducer), for 4 h. The viability of the cells was measured by MTT assay, and the content of intracellular total cholesterol (TC) was measured by a tissue/cell TC assay. The protein and mRNA levels of SR-A1 and glucose-regulated protein 78 (GRP78) were analyzed by Western blotting and quantitative real-time PCR, respectively. The fluorescence intensity of DiI-ox-LDL in the cells was detected by a multifunctional microplate reader. RESULTS:D-4F significantly reduced ox-LDL-induced macrophage injury and intracellular cholesterol accumulation, and attenuated the ox-LDL-induced expression of SRA1 and GRP78 in a dose-dependent manner. Additionally, D-4F significantly inhibited the TM-induced protein expression of SR-A1 and GRP78, and attenuated the uptake of ox-LDL by macrophages. CONCLUSION: D-4F reduces ox-LDL-induced macrophage cholesterol accumulation and injury by inhibiting SR-A1 expression. The mechanism may be related to the inhibition of ERS signaling pathway mediated by GRP78.     

Role of apolipoprotein E in cholesterol efflux mediated by ABCA1 and ABCG1

AIM:To investigate the role of apolipoprotein E(ApoE) in cholesterol efflux mediated by ATP-binding cassette transporter A1(ABCA1) and ATP-binding cassette transporter G1(ABCG1). METHODS:RAW 264.7 cells were seeded in either 6-well or 24-well plates, and then incubated with 20 mg/L low-density lipoprotein receptor gene knockout(LDLr^-/-) mouse lipoprotein 20 mg/L ApoE gene knockout(ApoE^-/-) mouse lipoprotein or culture medium alone. The changes of intracellular lipid content were measured by transmission electron microscopy and enzymatic colorimetric method. The cholesterol efflux was determined by liquid scintillation. The mRNA and protein levels of ABCA1 and ABCG1 were detected by real-time PCR and Western blotting, respectively. RESULTS:The ApoE^-/- mouse lipoprotein increased the content of intracellular cholesterol ester by 60% compared with the control cells. In addition, ApoE^-/- mouse lipoprotein treatment decreased the cholesterol efflux to apolipoprotein A-I(ApoA-I) and high-density lipoprotein(HDL) compared with LDLr^-/- mouse lipoprotein treatment. ApoE^-/- mouse lipoprotein treatment inhibited the mRNA and protein levels of ABCA1 and ABCG1 compared with LDLr^-/- mouse lipoprotein treatment. CONCLUSION:Apolipoprotein E plays an important role in the cholesterol efflux of macrophages, which is associated with its regulatory effect on the expression of ABCA1 and ABCG1.     

Soluble Klotho protein suppresses THP-1-derived foam cell formation

AIMTo investigate the role of soluble Klotho protein in THP-1-derived foam cell formation. METHODSTHP-1 monocytes were induced into macrophages by treatment with 160 nmol/L phorbol myristate acetate for 48 h, and then were divided into 6 groups: negative control group (THP-1-derived macrophages), positive control group [THP-1-derived foam cells induced by oxidized low-density lipoprotein (ox-LDL) for 48 h], and 25, 50, 100 and 200 μg/L soluble Klotho protein groups (THP-1-derived macrophages pretreated with soluble Klotho protein at the indicat?ed concentraions for 2 h and then induced by ox-LDL for 48 h). Lipid droplets in cytoplasm were observed by oil red O staining. The cholesterol outflow rate was detected by scintillation counting technique. The content of intracellular total cholesterol, free cholesterol and cholesterol ester was detected by enzyme fluorescence analysis. The expression of acyl-coenzyme A:cholesterol acyltransferase 1 （ACAT1） and ATP-binding cassette transport?er A1 (ABCA1) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTSOil red O staining and lipid mass quantification showed that THP-1-derived foam cell formation was dose-dependently suppressed by soluble Klotho protein. The cholesterol efflux rate of THP-1-derived foam cells was increased by soluble Klotho protein in a dose-dependent manner (P<0.05). In addition, soluble Klotho protein decreased the expression of ACAT1 and increased the expression of ABCA1 in a dose-dependent manner (P<0.05). CONCLUSION The soluble Klotho protein inhibits THP-1-derived foam cell formation in a dose-dependent manner by down-regulating the expression of ACAT1 and up-regulating the expression of ABCA1.     

Angiotensin-(1-7) increases expression of ABCA1 and decreases content of cholesterol in THP-1-derived foam cells via AC-cAMP pathway

AIM: To establish the THP-1-derived foam cell formation and to evaluate the effects of angiotensin-(1-7) and MDL (an inhibitor of adenylate cyclase) on the expression of ATP-binding cassete transporter A1(ABCA1) and the content of cholesterol. METHODS: THP-1-derived macrophages were treated with oxidized low-density lipoprotein(ox-LDL) to develop into foam cells. The foam cells were divided into 4 groups: control group, MDL group, Ang-(1-7) group and MDL+Ang-(1-7) group. At 24 h after treatment, the content of cAMP was measured by ELISA. The mRNA and protein levels of ABCA1 were determined by real-time RT-PCR and Western blotting, respectively. The content of cholesterol was detected by high performance liquid chromatography. RESULTS: The cAMP, the mRNA and protein levels of ABCA1 in Ang-(1-7) group were significantly higher, and the content of cholesterol was significantly lower than those in control group (P<0.05). On the contrary, the cAMP, the mRNA and protein levels of ABCA1 in MDL group were significantly lower and the content of cholesterol was significantly higher than those in control group (P<0.05). The results in MDL+Ang-(1-7) group were between Ang-(1-7) group and control group. CONCLUSION: Ang-(1-7) inhibits the formation of foam cells by promoting the expression of ABCA1 and decreasing the content of cholesterol. MDL partly antagonizes the effect of Ang-(1-7) by inhibiting the adenylate cyclase and decreasing the content of cAMP.     

Effect of miRNA-33 on ABCA1/ABCG1 expression in RAW264.7 macrophage-derived foam cells after Hcy treatment

AIM:To study whether homocysteine (Hcy) inhibits the expression of ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) by microRNA-33 (miRNA-33) signaling, and reduces the efficiency of reverse cholesterol transport (RCT).METHODS:RAW264.7 macrophages were induced by oxidized low-density lipoprotein (ox-LDL) to establish foam cell model. Oil red O staining was used to determine whether the model was established successfully. miRNA-33 mimics and miRNA-33 inhibitor were transfected into the cells by Lipofectamine 2000, and the cells were exposed to Hcy at concentration of 5 mmol/L for 24 h. The intracellular lipid droplets were observed by Oil red O staining. The expression of ABCA1 and ABCG1 at mRNA and protein levels was determined by real-time PCR and Western blot. The cellular cholesterol content was analyzed by HPLC, and effluent rate of cholesterol was detected by the method of liquid scintillation counting.RESULTS:Compared with blank control group, the lipid content in miRNA-33 mimics group was increased, and the expression of ABCA1 and ABCG1 at mRNA and protein levels was decreased (P<0.05). The intracellular cholesterol content was increased gradually (P<0.05), and the cellular cholesterol efflux rate was gradually decreased (P<0.05) in miRNA-33 mimics group. Compared with blank control group, the testing results in miRNA-33 inhibitor group were the opposition of those in miRNA-33 mimics group (P<0.05). No diffe-rence of the above indexes among blank control group, miRNA-33 mimics-NC group and miRNA-33 inhibitor-NC group was observed.CONCLUSION:Hcy inhibits the mRNA and protein expression of ABCA1 and ABCG1 through miRNA-33 signaling, and reduces the efficiency of RCT in RAW264.7 macrophage-derived foam cells.     

Effects of GW1929 on macrophage TLR expression and inflammation induced by ox-LDL

AIM: To investigate the effects of ox-LDL on TLR2 and TLR4 expression and production of TNF-α, IL-10, IL-12, NO and MDA in macrophages and to observe intervention effect of GW1929 in above procedure. METHODS: The mouse peritoneal macrophages were pretreated with ox-LDL (50 mg/L, 100 mg/L) and GW1929 (20 μmol/L) respectively for 24 h. The concentrations of MDA, NO-2/NO-3, TNF-α, IL-10 and IL-12 in the culture fluid were detected. Flow cytometry was used to observe TLR2 and TLR4 expressions after the mouse peritoneal macrophages were pretreated with ox-LDL (50 mg/L) and GW1929 (20 μmol/L) respectively for 6 h, 12 h, and 24 h. RESULTS: The concentrations of MDA, NO-2/NO-3, TNF-α and IL-10 in ox-LDL (50 mg/L, 100 mg/L) group were higher than those in control and GW1929 group obviously, but the concentrations of above index in ox-LDL (50 mg/L, 100 mg/L)+GW1929 group were lower than those in ox-LDL (50 mg/L, 100 mg/L) group apparently. No IL-12 in every group was detected. Expressions of TLR-2 in ox-LDL+GW1929 (6 h, 12 h, 24 h) group were lower than those in ox-LDL (6 h, 12 h, 24 h) group respectively. TLR-4 expressions in ox-LDL+GW1929 (12 h) were lower than those in ox-LDL (12 h) apparently. CONCLUSION: ox-LDL up-regulates TLR2 and TLR4 expressions and promotes the production of ROX, NO, TNF-α and IL-10 in macrophages. GW1929 is capable of inhibiting the above ox-LDL effects.     

Inflammation disrupts cholesterol efflux in human kidney mesangial cells via PPARγ-LXRα-ABCA1 pathway

AIM: To investigate the perturbative effects of inflammatory stress on cholesterol efflux in human kidney mesangial cells (HMCs) and the relation to peroxisome proliferators activated receptor-γ (PPARγ)-1iver X activated receptor-α (LXRα)-and ATP-binding cassette transporter A1 (ABCA1) pathway. METHODS: HMCs were cultured and divided into control group (incubated with serum free medium), high lipid group , inflammatory stress group or combination treatment group . The mRNA and protein levels of PPARγ, LXRα,ABCA1 were examined by real-time polymerase chain reaction (PCR) and Western blotting. cholesterol assay was performed to evaluate the efflux of cholesterol by liquid scintillation counter. Oil red O staining was used to evaluate lipid droplet accumulation in the cells. Intracellular cholesterol level was measured by enzymic assay. RESULTS: : LDL increased the expression of PPARγ, LXRα and ABCA1 at mRNA and protein levels in HMCs, while TNF-α reduced the expression of these genes at mRNA and protein levels. The cholesterol efflux was increased after LDL loading. However, inflammatory stress inhibited cholesterol efflux in the absence or presence of LDL loading. Oil red O staining and quantitative analysis showed that LDL loading increased the intracellular cholesterol level in HMCs and inflammatory stress further exacerbated the lipid accumulation. CONCLUSION: Inflammatory cytokine reduces cholesterol efflux by inhibiting the expression of PPARγ, LXRα and ABCA1, thereby causing lipid accumulation in HMCs.     

Action of ATP binding cassette transporter A 1 on cholesterol efflux in THP-1 macrophage-derived foam cells

AIM:To study the action of ATP binding cassette transporter(ABC) A 1 on cholesterol efflux in THP-1 macrophage-derived foam cells.METHODS:After exposure of the cultured THP-1 macrophage-derived foam cells to 22(R)-hydroxycholesterol and 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) at different concentration for 24 hours, cholesterol efflux and ABCA1 mRNA level were determined by FJ-2107P type liquid scintillator and reverse trancriptase-polymerase chaim reaction(RT-PCR), respectively.RESULTS:Oxidized LDL promoted cholesterol efflux in THP-1 macrophages and 22(R)-hydroxycholesterol increased cholesterol efflux in THP-1 macrophage-derived foam cells in a dose-dependent manner and DIDS inhibited cholesterol efflux in THP-1 macrophage-derived foam cells in a dose-dependent manner. Exposure of the cultured THP-1 macrophage-derived foam cells to 22(R)-hydroxycholesterol and DIDS at different concentration for 24 hours, resulted in increase and decrease in the expression of ABCA1 mRNA in THP-1 macrophage-derived foam cells in a dose-dependent manner, respectively.CONCLUSION:ABCA1 playes an important role in cholesterol efflux in THP-1 macrophage-derived foam cells.     

Anti-atherosclerosis effect of astragaloside is related to miR-33a/ABCA1 signaling

AIM:To study whether astragaloside affects the expression of ATP binding cassette transporter A1 (ABCA1) by regulating miR-33a and promotes the outflow of cholesterol in macrophages. METHODS:In the in vivo experiments, HE staining was used to detect the pathological damage of the cross section of aorta in the mice. The expression of ABCA1 at mRNA and protein levels in mouse aorta was determined by real-time PCR and Western blot. In the in vitro experiments, THP-1 macrophage-derived foam cells were established and then treated with astragaloside-containing serum. Real-time PCR was used to detect the expression of miR-33a. The cells were randomly divided into blank serum group, astragaloside serum group and astragaloside serum+miR-33a mimic group. The expression of ABCA1 at mRNA and protein levels was determined by real-time PCR and Western blot. Oil red O staining and high-performance liquid chromatography were used to detect intracellular lipid content. The method of[³H] incorporation was used to detect intracellular cholesterol outflow. RESULTS:In vivo experiments showed that the blood vessels of the mice in astragaloside group were structurally normal, with neat arrangement, localized small calcified particles, mild lesions, small plaques, reduced foam cells and li-pid, and basically complete elastic plates, indicating that the pathological changes were significantly lighter than those in model group. Compared with model group, the expression of miR-33a in the aorta of the mice in astragaloside group was decreased and the relative expression of ABCA1 at mRNA and protein levels was increased (P<0.05). In vitro experiments showed that astragaloside significantly up-regulated the expression of ABCA1 at mRNA and protein levels, but this effect was inhibited by the transfection of miR-33 mimic without affecting the cell viability. Astragaloside reduced the lipid accumulation in the cells, but this effect was attenuated by miR-33 mimic. Astragaloside reduced intracellular cholesterol accumulation in relation to its promotion of intracellular cholesterol efflux, and the transfection of miR-33a mimic in the cells inhibited cholesterol efflux. CONCLUSION:Astragaloside inhibits the production of miR-33a to increase the expression of ABCA1 and promote the outflow of cholesterol in macrophages. This may be one of the molecular mechanisms of astragaloside in preventing atherosclerosis.     

Effects of angiotensin Ⅱ on ATP binding cassette transporter A1 in THP-1 derived foam cells

AIM:To study the influence of angiotensin Ⅱ (AngⅡ) on ATP-binding cassette transporter A1 in THP-1 derived foam cells. The variance of the expression of ABCA1, the content and the effluent rate of cholesterol were also investigated. METHODS:The regulatory effect of AngⅡ on the expression of ABCA1 mRNA and protein in THP-1 derived form cells were measured by RT-PCR and Western blotting. The effect of variance of cholesterol content was measured by zymochemistry via-fluorospectrophotometer, cholesterol effluent was measured by liquid scintillator. RESULTS:A positive facilitative effect of Ang Ⅱon form cells was observed. Total cholesterol content were increased significantly by Ang Ⅱ treatment (P<0.05). The mRNA and protein of ABCA1 were down-regulated significantly by Ang Ⅱ stimulation (P<0.05). Irbesartan reduced the total cholesterol content significantly (P<0.05). Meanwhile, the increase in the effluent rate of cholesterol and the expression of ABCA1 were observed (P<0.05). CONCLUSION:The effects of Ang Ⅱ on the formation of foam cells and atherosclerosis may be correlated to the activation of AT1 receptor and down-regulation of ABCA1.     

Effects of different active constituents of Gynostemma pentaphyllum on mitochondrial energy metabolism-related proteins in endothelial cells induced by ox-LDL

AIM To investigate the effects of different components of Gynostemma pentaphyllum ［gypenosides (Gps), gypenoside XLIX (GpXLIX) and ginsenoside Rb3 (GRb3)］ on mitochondrial energy metabolism-related proteins in endothelial cells induced by oxidized low-density lipoprotein (ox-LDL). METHODS EA.hy926 cells were divided into control group, model group, Gps group, GpXLIX group and GRb3 group. The cells in control group were cultured only in DMEM complete medium. The cells in model group were treated with 100 mg/L ox-LDL for 48 h. The cells in Gps group, GpXLIX group and GRb3 group were treated with 100 mg/L ox-LDL for 24 h, and then treated with Gps, GpXLIX and GRb3 at 100 mg/L for another 24 h, respectively. The ATP content in each group was detected by ELISA. The expression levels of mitochondrial energy metabolism-related proteins, cytochrome C oxidase subunit 5a （Cox5a）, NADH:ubiquinone oxidoreductase core subunit S1 （Ndufs1）, ATP synthase F1 subunit alpha (ATP5a) and cytochrome C (Cyt C）, were determined by Wes automatic Western blot quantitative analysis system and Western blot. RESULTS Compared with control group, the ATP content in model group was decreased (P<0.01). After drug intervention, the ATP content increased to different degrees in Gps group, GpXLIX group and GRb3 group (P<0.01). The results of Wes automatic Western blot quantitative analysis system were consistent with those of Western blot. These results showed that compared with control group, the protein expression of Cox5a, Ndufs1 and ATP5a in model group was decreased, and the protein expression of Cyt C was increased (P<0.01). After intervention, the protein expression of Cox5a, Ndufs1 and ATP5a was increased and the protein expression of Cyt C was decreased in Gps group, GpXLIX group and GRb3 group (P<0.05 or P<0.01). Among them, the effect of Gps on the protein expression of Cox5a, Ndufs1 and Cyt C was significantly stronger than those of the 2 monomer components, and the effect of GRb3 was found to be superior in the 2 monomer components. The effect of GpXLIX on ATP5a protein was superior to the other 2 components. CONCLUSION Gynostemma total saponins and related active ingredients protect ox-LDL-induced endothelial cells by affecting mitochondrial energy metabolism-related proteins, thereby preventing and treating atherosclerosis.     

Effects of Danggui Buxue decoction on endothelial progenitor cells,serum VEGF and SDF-1 in atherosclerotic rabbits

AIM: To investigate the effects of Danggui Buxue decoction (DBD) on serum vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1), as well as the activity of circulating endothelial progenitor cells (EPCs) in atherosclerotic rabbits. METHODS: The model of atherosclerosis was established using immune injury and fatty diet for 4 weeks in New Zealand rabbits (n=25). All modeled rabbits were randomized into 5 groups with 5 animals in each group. The rabbits in atherosclerosis group were intragastrically administered with distilled water. The rabbits in simvastatin group were treated with simvastatin at the dose of 1.7 mg/kg. The rabbits in DBD high-dose, middle-dose and low-dose treatment groups were given DBD at the doses of 6 g/kg, 3 g/kg and 1.5 g/kg, respectively. All drugs were given once a day for 2 weeks. After treatment, the levels of serum VEGF and SDF-1 were measured. The mononuclear cells isolated from the rabbit peripheral blood were cultured for 7 days in vitro, and then attached cells were cultured with both DiI-ac-LDL and FITC-UEA-1 for identification. The proliferation was detected by MTT method. The cell migration was observed using Transwell chambers. The adhesion determination and in vitro angiogenesis assay were also performed. RESULTS: Compared with atherosclerosis group, the levels of serum VEGF and SDF-1 were elevated (P<0.05), the proliferation, migration, adhesion and angiogenesis of EPCs were all improved in DBD high-dose, middle-dose treatment groups and simvastatin group (P<0.05). CONCLUSION: DBD elevates the levels of serum VEGF and SDF-1 to improve the activity of EPCs in the process of atherosclerosis.     

Inhibitory effect of cholecystokinin octapeptide on expression of CD14 on rat pulmonary interstitial macrophages induced by lipopolysaccharide in vitro

AIM:To study the effect of cholecystokinin octapeptide(CCK-8) on lipopolysaccharide (LPS)-stimulated pulmonary interstitial macrophage(IM)in vitro.METHODS:Pulmonary IM were isolated and cultured in the presence of LPS, CCK-8, proglumide(the antagonist of CCK receptors) and vehicle alone or together. The expression of mCD14 protein was assayed by flow cytometry, and sCD14 in the supernatant was analyzed semi-quantitatively by Western blot, and TNF-α in the supernatant was detected with ELISA.RESULTS:CCK-8, at concentrations from 10^-7mol/L to 10^-6mol/L inhibited significantly the expression of mCD14, the release of sCD14 and TNF-α to the supernatant up-regulated by LPS(1mg/L). The effect of CCK-8 was inhibited by proglumide. CONCLUSION:CCK-8 modulated negatively several functions of LPS-stimulated pulmonary IM through CCK receptors, which may be one of the mechanisms for CCK-8 to alleviate the inflammation in lung tissues during endotoxemia.     

Effects of Zhenwu decoction on rat kidney injury induced by adriamycin

AIM: To observe the effects of Zhenwu decoction on the expression of podocin and nephrin in podocytes of adriamycin nephropathy (AN) rats, and to clarify the mechanism of Zhenwu decoction in decreasing adriamycin-induced proteinuria in rats. METHODS:Biochemical assay and pathological observation (HE staining, Masson trichrome staining and transmission electron microscopy) were used to evaluate the effects of Zhenwu decoction on renal function, pathological morphology and hydroxyproline (Hyp) content in AN rats with renal fibrosis. Western blotting was used to observe the effect of Zhenwu decoction on the expression of podocin and nephrin which are marker proteins in podocytes. RESULTS:In model group, the levels of urinary total protein (TP), blood urea nitrogen (BUN), serum creatinine (SCr) and renal Hyp were significantly increased, and the clearance of creatinine (CCr) level was decreased (P<0.05). The expression of podocin and nephrin was significantly decreased (P<0.05). Atrophic renal tubules, thickened basement membranes, fusion of foot processes, concentrating renal glomerules, expansion of some renal tubules, degeneration of renal tubular epithelial cells, protein casts, and proliferation of fibroblasts and infiltration of inflammatory cells in renal interstitium were also observed. After treatment with drugs (Zhenwu decoction and valsartan), the above-mentioned parameters were significantly changed. TP, BUN, SCr and Hyp were down-regulated to different levels, and CCr was significantly up-regulated (P<0.05). The expression of podocin and nephrin was up-regulated by treatment with Zhenwu decoction, and renal histological changes were alleviated compared with model group. CONCLUSION: Zhenwu decoction can reduce Hyp content in kidney tissue, alleviate kidney histological changes, and improve renal function in AN rats. It might protect kidney against adriamycin-induced proteinuria via increasing the expression of podocin and nephrin in podocytes.     

IL-17A reduces lipid accumulation by up-regulation of ABCA1 expression in Raw264.7 macrophages

AIM:To investigate the effects of interleukin-17A (IL-17A) on the expression of adenosine triphosphate binding cassette transporter A1 (ABCA1) in RAW264.7 macrophages. METHODS:Mouse RAW264.7 macrophages were treated with IL-17A at different concentrations for 6 or 24 h, or treated with IL-17A at the same concentration for different time. The expression of ABCA1 at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. Cholesterol efflux to apolipoprotein A1 (ApoA-1) was evaluated by NBD-cholesterol method. Lipid accumulation in the cells was evaluated by Oil Red O staining. RESULTS:Compared with control group, IL-17A increased the expression of ABCA1 at protein level in the RAW264.7 cells significantly (P<0.05), but had no effect on the mRNA expression of ABCA1. In addition, cholesterol efflux to ApoA-1 was increased and lipid accumulation in the RAW264.7 cells was decreased obviously after treatment with IL-17A. CONCLUSION:IL-17A increases the protein expression of ABCA1 but not at mRNA level in the RAW264.7 macrophages, which may be correlated with its anti-atherosclerosis effect.     

Inhibitory effect of quercetin on apoptosis of PC12 cells induced by rotenone

AIM: To observe the effects and mechanisms of quercetin on the apoptosis of PC12 cells induced by rotenone. METHODS: PC12 cells were used in the study. Quercetin at the concentration of 300 μmol/L was added into the PC12 cells cultured in DMEM-F12 medium with 10% fetal calf serum. The morphological changes of the cells were observed under fluorescence microscope. The apoptotic rate was determined by flow cytometry assay. The protein levels of Bax and Bcl-2 were determined by Western blotting, and the mitochondrial membrane potential was measured by ratiometric probe JC-1.RESULTS: In the cells treated with rotenone+quercetin, the morphology of the cells was significantly improved, and the apoptotic rate was decreased to 6.7%, significantly lower than that in the cells treated with rotenone alone (P<0.01). The expression of Bcl-2 was up-regulated and Bax was down-regulated in rotenone+quercetin group (P<0.01), while the mitochondrial membrane potential was also increased (P<0.01) as compared to those in rotenone group.CONCLUSION: Pretreatment of quercetin inhibits the development of apoptosis in PC12 cells induced by rotenone. One of the mechanisms may be correlated with up-regulating the expression of Bcl-2 and down-regulating the expression of Bax, thus maintaining mitochondrial membrane potential.     

Effect of Sini decoction on expression of caveolin-1 and eNOS in EAhy926 cells injured by homocysteine

AIM：To investigate the mechanism of Sini decoction in treating human vascular endothelial cell injury and the roles of caveolin-1 and nitric oxide (NO) system in this procedure. METHODS：Model of human umbilical vein endothelial EAhy926 cells injured by homocysteine (Hcy) was established. The protective effect of Sini decoction on the injured EAhy926 cells was observed, and the expression of caveolin-1 and endothelial nitric oxide synthase (eNOS) was detected by real-time fluorescence quantitative PCR and Western blotting. RESULTS：Compared with control group, the Hcy-treated EAhy926 cells showed reduced adherent cell number and NO concentration in culture supernatant, decreased expression of eNOS mRNA and protein, and increased expression of caveolin-1 mRNA and protein (all P<0.05). Compared with Hcy group, better growth of adherent cells, elevated NO concentration in culture supernatant, attenuated expression of caveolin-1 mRNA and protein, and enhanced expression of eNOS mRNA and protein in Sini decoction groups were observed (all P<0.05). CONCLUSION：Homocysteine may injure EAhy926 cells by enhancing the expression of caveolin-1 and suppressing the expression of eNOS, while Sini decoction may protect EAhy926 cells by suppressing the expression of caveolin-1 and enhancing the expression of eNOS.