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.     

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.     

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.     

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.     

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.     

Effect of puerarin on cholesterol influx and efflux in RAW264.7-treated foam cells

AIM: To study the protective effect of puerarin on the atherosclerosis of RAW264.7-derived foam cells. METHODS: The model of foam cells was established by incubating the RAW264.7 cells with ox-LDL. The cholesterol uptake was evaluated by a DiI-ox-LDL binding assay. The ability of cholesterol efflux of the RAW264.7-derived foam cells was detected by cholesterol efflux assay. The protein levels of LC3Ⅱ, P62, CD36, ABCA1, LAL and p-AMPK were determined by Western blot. RESULTS: Puerarin treatment reduced the cholesterol uptake capacity and enhanced the cholesterol efflux rate. The protein levels of LC3Ⅱ, ABCA1 and LAL in puerarin group were higher than that in ox-LDL group, while the protein levels of P62 and CD36 were obviously decreased, and those in rapamycin treatment group had the same change as puerarin group. The protein levels of LC3Ⅱ, ABCA1 and LAL were obviously decreased and the protein level of p-AMPK was increased after co-treated with 3-MA. CONCLUSION: Puerarin promotes LAL and ABCA1-mediated cholesterol efflux in ox-LDL-treated RAW264.7 macrophages, which might enhance autophagy through AMPK-dependent pathway for cholesterol efflux regulation, and reduce the uptake of lipids by CD36 negative regulation.     

Amiloride slows down calpain-mediated ABCA1 degradation through inhibition of hypoxia-induced NHE1 expression

AIM: To examine the effects of hypoxia on sodium-hydrogen exchange 1(NHE1) expression, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and calpain activity, and to explore the effect of amiloride on adenosine triphosphate-binding cassette transporter A1(ABCA1) degradation and its calpain-related mechanism. METHODS: RAW264.7 cells were exposed to hypoxia for 0 h, 12 h, 24 h and 48 h. The cell viability was measured by MTT assay and the expression of NHE1 at mRNA and protein levels was detected by real-time PCR and Western blot. [Ca₂₊]_i was analyzed by flow cytometry. Calpain activity was assessed by the method of Suc-LLVY-aminoluciferin. Furthermore, the protein levels of ABCA1 in the RAW264.7 cells exposed to hypoxia for 24 h were determined after 6 h or 12 h treatment with NHE1 inhibitor amiloride in the presence of cycloheximide. ABCA1 protein levels and calpain activity were detected after 12 h incubation with calpain inhibitor ALLN or intracellular calcium-chelating agent BAPTA. RESULTS: Hypoxia inhibited the cell viability in a time-dependent manner. Hypoxia up-regulated the mRNA and protein expression of NHE1, and increased [Ca²⁺]_i and calpain activity. Hypoxia increased the degradation of ABCA1 and amiloride slowed down the ABCA1 degradation. ALLN or BAPTA increased ABCA1 protein level and decreased calpain activity. CONCLUSION: NHE1 inhibitor amiloride attenuates the calpain-mediated degradation of ABCA1, indicating that hypoxia-induced NHE1 might, at least in part, participate in the ABCA1 degradation.     

Inhibitory effect of Wendan decoction on formation of foam cells induced by ox-LDL

AIM To explore the anti-atherosclerotic mechanism of Wendan decoction based on formation of foam cells. METHODS The optimal concentrations of Wendan decoction without cytotoxity to cells were selected by MTT assay. After Wendan decoction treatment, the formation of foam cells was examined by oil red O staining. The cholesterol efflux, cholesterol level, free cholesterol level and cholesterol esterification rate were analyzed using cholesterol efflux assay, total cholesterol assay and free cholesterol assay. The expression levels of macrophage membrane proteins, including CD36, scavenger receptor class A （SR-A）, ATP-binding cassette transporter A1 （ABCA1） and scavenger receptor class B type I （SR-BI）, were quantified by Western blot. RESULTS The optimal concentrations of Wendan decoction without cytotoxity to the cells were 0~6 g/L. Wendan decoction at the concentrations of 1.5, 3 and 6 g/L were selected for the experiments. Wendan decoction at these concentrations inhibited the formation of foam cells induced by oxidized low-density lipoprotein （ox-LDL）, and reduced the accumulation of intracellular lipids in a concentration-dependent manner （P<0.05 or P<0.01）. Wendan decoction also reduced intracellular total cholesterol level, cholesterol ester level and cholesterol esterification rate （P<0.05 or P<0.01）, promoted efflux of intracellular cholesterol （P<0.01）, and decreased the protein level of CD36 in THP-1 cell-derived macrophages （P<0.01） in a concentration-dependent manner. Wendan decoction at the concentration of 6 g/L significantly reduced the protein level of SR-A in THP-1 cell-derived macrophages （P<0.05）. At the concentrations of 3 and 6 g/L, Wendan decoction significantly increased the protein levels of ABCA1 and SR-BI in THP-1 cell-derived macrophages （P<0.05 or P<0.01）. CONCLUSION Wendan decoction significantly inhibits ox-LDL-induced formation of foam cells by reducing cholesterol deposition and promoting cholesterol efflux, and its mechanism may be related to the down-regulation of CD36 and SR-A and the up-regulation of ABCA1 and SR-BI.     

Effects of rosiglitazone on expression of SOCS1/SOCS3 and inflammatory reaction in RAW 264.7 cell-derived foam cells

AIM: To investigate whether perioxisome proliferator-activated receptor γ (PPARγ) ligand rosiglitazone regulates suppressor of cytokine signaling 1 (SOCS1) and SOCS3 expression as well as pro-inflammatory/anti-inflammatory responses in RAW 264.7 cell-derived foam cells. METHODS: The concentrations of TNF-α, IL-6 and IL-10 in the cultured supernatant of RAW 264.7 cell-derived foam cells were detected by ELISA, and the ratios of TNF-α/IL-10 and IL-6/IL-10 were calculated. RT-PCR and Western blotting were used to analyze the effects of rosiglitazone on the expression of SOCS1 and SOCS3 at mRNA and protein levels. RESULTS: The concentrations of TNF-α, IL-6 and IL-10, and ratios of TNF-α/IL-10 and IL-6/IL-10 in foam cell group were obviously higher than those in control group, but the concentrations of the above factors in oxidized low-density lipoprotein (ox-LDL) +rosiglitazone group were apparently lower than those in foam cell group. The expression of SOCS1 and SOCS3 at mRNA and protein levels in oxLDL+rosiglitazone group was apparently higher than that in control and foam cell group. CONCLUSION: PPARγ ligand rosiglitazone up-regulates the expression of SOCS1 and SOCS3 at mRNA and protein levels and regulates the balance of pro-inflammatory/anti-inflammatory responses in RAW 264.7 cell-derived foam cells.     

Correlation between expression of ALDH1/ABCG2 and microvessel formation in epithelial ovarian cancer

AIM: To elucidate the correlation between the expression of aldehyde dehydrogenase 1 (ALDH1)/ATP-binding cassette subfaminly G member 2 (ABCG2) and microvessel density (MVD) in epithelial ovarian cancer (EOC).METHODS: In 198 specimens of EOC and 60 specimens of ovarian benign epithelial tumor tissues, the protein expression of ALDH1/ABCG2 and CD105 (microvessel marker) was detected by immunohistochemical staining.RESULTS: The positive rates of ALDH1 and ABCG2 in the EOC were 64.1% and 61.6%, respectively, while the positive rates in benign epithelial tumor tissues were 8.3% and 6.7%, respectively, and there were significant differences between them (P<0.05). In EOC and benign epithelial tumor tissues, the MVD were 22.6±9.7 and 5.03±3.35, respectively, and the difference was also significant (P<0.05). The expression of ALDH1 and ABCG2 in EOC was significantly related to differentiation, FIGO stage,and abdominal organ and lymph node metastasis (P<0.05). MVD had correlation with differentiation, FIGO stage, ascite, and abdominal organ and lymph node metastasis (P<0.05). MVD had positive correlation with the expression of ALDH1 and ABCG2 (P<0.01). There was also a positive correlation between the expression of ALDH1 and ABCG2 (P<0.01). Over-expression of ALDH1/ABCG2 and MVD≥23 were related to the poor prognosis. The survival rates in ALDH1/ABCG2 positive and MVD≥23 groups were significantly lower than those in ALDH1/ABCG2 negative and MVD<23 groups (P<0.05). The FIGO stage, the expression of ALDH1/ABCG2 and MVD were indepen-dent prognosis factors of EOC (P<0.05).CONCLUSION: The results suggest that the expression of ALDH1/ABCG2 and MVD in EOC are related to differentiation, lymph node metastasis, clinical stage and prognosis. Combined detection of these indexes may play an important role in predicting the progression and prognosis of EOC.     

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.     

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.     

Clinical significance of miRNA-326 expression in gastric cancer and effect of up-regulation of its expression on viability and apoptosis of gastric cancer cells

AIM: To investigate the clinical significance of microRNA-326 (miRNA-326) expression in gastric carcinoma and the effect of up-regulation of its expression on the viability and apoptosis of gastric cancer cells. METHODS: The expression of miRNA-326 in 55 tissue samples of gastric cancer was detected by RT-qPCR, and the relationship between the expression and the clinicopathological features was analyzed. The expression of miRNA-326 in gastric cancer BGC-823 cells was detected by RT-qPCR. The BGC-823 cells were transfected by liposome method, and randomly divided into normal control group (untransfected), mimic-NC group (transfected with negative control mimic) and miRNA-326 mimic group (transfected with miRNA-326 mimic). After up-regulation of miRNA-326 expression, the cell viability was measured by CCK-8 assay, and the apoptosis of the cells was analyzed by flow cytometry. The protein levels of matrix metalloprotein 9 (MMP-9), p21, cyclin D1, Bcl-2 and cleaved caspase-3 were determined by Western blot, and the mRNA expression of cyclin D1 was detected by RT-qPCR. Whether CCND1 (the gene of cyclin D1) was the target gene of miRNA-326 was evaluated by dual-luciferase reporter assay. RESULTS: The expression of miRNA-326 in the gastric cancer tissues was significantly lower than that in the adjacent tissues (P<0.05). The miRNA-326 expression had a significant correlation with the tumor size, lymph node metastasis, differentiation, and clinical stages (P<0.05), but it had no correlation with the age and sex of the patients. Moreover, the expression of miRNA-326 was also closely related to the survival rate of the patients (P<0.05). The expression of miRNA-326 in the BGC-823 cells was significantly lower than that in the normal gastric mucosa GES-1 cells (P<0.05). Compared with normal control group, the expression of miRNA-326 in mimic-NC group did not change significantly, while that in miRNA-326 mimic group was increased significantly (P<0.05). Compared with normal control group, the cell viability in miRNA-326 mimic group was significantly decreased, and the apoptosis was increased (P<0.05). In addition, compared with normal control group, the protein levels of MMP-9, cyclin D1 and Bcl-2, and the mRNA expression of cyclin D1 in miRNA-326 mimic group were decreased, while the protein levels of p21 and cleaved caspase-3 were increased (P<0.05). However, no significant difference of above protein and mRNA levels between mimic-NC group and normal control group was observed. Compared with mimic-NC+miR-326 mimic group, the activity of luciferase in the cells transfected with pmiR-CCND1-WT plasmid was significantly decreased (P<0.05), but that in the cells transfected with pmiR-CCND1-Mut plasmid did not change significantly. CONCLUSION: The expression level of miRNA-326 in gastric cancer tissues is low, and it may promote cell viability and inhibit cell apoptosis by targeting CCND1.     

Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

Effect of leonurine on expression of miR-1 in rats with myocardial fibrosis induced by isoproterenol

AIM: To investigate effect of leonurine on the expression of microRNA-1 (miR-1) in rats with myocardial fibrosis induced by isoproterenol (ISO). METHODS: SD rats (n=10) were used as normal control group, and 80 rats were given ISO by intraperitoneal injection daily for 2 weeks to establish the model of myocardial fibrosis. The model rats were randomly divided into 5 groups:model group, low-dose (7.5 mg·kg^-1·d^-1) leonurine group, middle-dose (15 mg·kg^-1·d^-1) leonurine group, high-dose (30 mg·kg^-1·d^-1) leonurine group and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (0.3 mg·kg^-1·d^-1) group. After the treatment for 2 weeks, the ultrastructure of left ventricular myocardial tissues was observed under electron microscope. Masson staining was used to detect collagen fibrosis, and the expression of collagen I and collagen Ⅲ was determined by the method of immunohistochemistry. The contents of endothelin-1 (ET-1) and angiotensin Ⅱ (Ang Ⅱ) were measured by ELISA. The expression of miR-1 and ET-1 mRNA was detected by real-time PCR, and the protein expression of p38 MAPK, β-myosin heavy chain (MHC) and α-MHC was determined by Western blot. RESULTS: Compared with model group, the ultrastructure of left ventricular myocardial tissues in high-dose leonurine group was attenuated, and the expression of miR-1 and the protein expression of α-MHC in left ventricular myocardial tissues of high-dose leonurine group were increased (P<0.05). Collagen volume fraction, collagen I, collagen Ⅲ, the ratio of collagen Ⅰ/collagen Ⅲ, the contents of ET-1 and Ang Ⅱ, the mRNA expression of ET-1, and the protein expression of p38 MAPK and β-MHC in high-dose leonurine group were lower than those in model group (P<0.05). CONCLUSION: Leonurine attenuates myocardial fibrosis in the rats induced by ISO, and it is potentially associated with affecting the expression of miR-1, and inhibiting ET-1/p38 MAPK signaling pathway.     

Effect of ginsenoside Rb1 on proliferation and serotonin transporter and 5-HT1B R expression in hypoxia-induced rat pulmonary artery smooth muscle cells via Rho/Rho-kinase pathway

AIM: To observe the effect of ginsenoside Rb1 on the proliferation and the expression of serotonin transporter (SERT), 5-hydroxytryptamine 1B receptor (5HT_1BR) in rat pulmonary artery smooth muscle cells (PASMCs) under hypoxia condition and the relationship with Rho/Rho-kinase signal pathway.METHODS: PASMCs were isolated from the adult male SD rats and primarily cultured. The subcultured cells from the 4th generation to the 6th generation were harvested and divided into normal group, and hypoxia group, different concentrations of Rb1 incubation groups treated with 50, 100 and 200 mg/L ginsenoside Rb1 under hypoxia (HR50, HR100 and HR200 groups, respectively). The viability of the PASMCs was measured by CCK-8 assay. BrdU positive cells were determined using flow cytometry. The expression of serotonin transporter and 5HT_1BR at mRNA and protein levels was detected by RT-PCR and Western blot, respectively. The PASMCs were randomly divided into normal group, hypoxia group, HR200 group and hypoxia+Y-27632 incubation group (HY group). The mRNA expression of Rho-kinase and phosphorylated myosin phosphatase target subunit 1 (p-MYPT1) protein level were investigated by RT-PCR and Western blot, respectively.RESULTS: Compared with normal group, the proliferation of PASMCs in hypoxia group was significantly increased (P<0.01). The cell viability and the expression of SERT and 5HT_1BR at mRNA and protein levels in all different concentrations of Rb1 groups were obviously decreased compared with hypoxia group (P<0.05). The mRNA expression of Rho-kinase and protein level of p-MYPT1 were markedly decreased in HR200 group, and no significant difference compared with HY group was observed (P<0.01).CONCLUSION: Treatment with ginsenoside Rb1 might prevent hypoxia-induced proliferation of PASMCs and over-expression of SERT and 5HT_1BR through inhibiting the Rho/Rho-kinase pathway.     

Effect of homocysteine on the expression of macrophage inflammatory protein-1α in THP-1 monocytes

AIM: To investigate whether homocysteine (HCY) induce the expression of macrophage inflammatory protein-1α(MIP-1α)in cultured THP-1 monocytes. METHODS: After exposure of THP-1 monocytes to HCY at increasing concentrations (0.05,0.1 and 0.2 mmol/L) for 8 h, or at 0.1 mmol/L of HCY for different incubation times (4, 8 and 16 h), the expressions of MIP-1α mRNA and protein were determined by RT-PCR and immunocytochemistry, respectively. RESULTS: RT-PCR showed that the expression of MIP-1α mRNA increased with the concentrations of HCY compared with the control group. Meanwhile, after the treatment of 0.1 mmol/L HCY to the cells for different times, the MIP-1α mRNA expression increased at 4 h, peaked at 8 h, and then decreased at 16 h. The authenticity of RT-PCR products was confirmed by DNA sequencing. Image analysis of Immunocytochemistry assay showed the expression of MIP-1α protein in experimental groups increased in a dose- and time-dependent manner(P＜0.01). CONCLUSIONS: HCY induced monocytes to express MIP-1α mRNA and protein.