Apolipoprotein A-I mimetic peptide D4F protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting caspase-12

AIM: To investigate the effect of D4F, an apolipoprotein A-I mimetic peptide, on oxidized low-density lipoprotein (ox-LDL)-induced macrophage apoptosis and activation of caspase-12, a key molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, and to elucidate the underlying molecular mechanisms. METHODS: RAW264.7 macrophages were pretreated with D4F (12.5, 25 and 50 mg/L), 4-phenylbutyric acid (PBA, 5 mmol/L) or diphenyleneiodonium (DPI, 5 μmol/L) for 1 h and then treated with ox-LDL (100 mg/L) or tunicamycin (TM, 4 mg/L) for 24 h. The cell viability and apoptosis were determined by MTT assay and TUNEL detection, respectively. The levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in the cells and the activities of superoxide dismutase (SOD) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were determined. The protein level of caspase-12 was examined by Western blot analysis. RESULTS: Similar to the ERS inhibitor PBA, D4F protected RAW264.7 macrophages from ox-LDL or TM (an ERS inducer)-induced decrease in the viability and increase in apoptotic rate in a dose-dependent manner. Like DPI (an oxidative stress inhibitor), D4F significantly inhibited ox-LDL-induced oxidative stress, as expressed by the decreased generation of ROS and MDA (P <0.01), the increased activity of SOD and the decreased activity of NADPH oxidase (P <0.05). Moreover, similar to PBA and DPI, D4F significantly suppressed ox-LDL-induced activation of caspase-12 in a concentration-dependent manner (P <0.05). Furthermore, D4F also inhibited the caspase-12 activation induced by TM (P <0.05). CONCLUSION: D4F inhibits macrophage apoptosis induced by ox-LDL, and the mechanism is at least partially by reducing oxidative stress and inhibiting the activation of caspase-12.     

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.     

L-4F attenuates the injury of lupus nephritis in a lupus mouse model

AIM: To investigate the effects of apolipoprotein A-I mimetic peptide L-4F on the process of nephropathia in apoE-/-Fas-/-C57BL/6 lupus mice. METHODS: The apoE-/-Fas-/-C57BL/6 lupus mice (8~9 weeks old, female) were treated with L-4F by peritoneal injection for 25 weeks. RESULTS: Compared with the vehicle controls, the mice treated with L-4F presented smaller lymph nodes and glomerular tufts (P<0.05), lower serum levels of IgG antibodies to double-stranded DNA (P<0.05) and oxidized phospholipids, as well as lower levels of inflammatory factors including IL-6 and TNF-α (P<0.05). Furthermore, serum adiponectin level in apoE-/-Fas-/-C57BL/6 mice was significantly increased after L-4F treatment for 25 weeks. CONCLUSION: L-4F treatment significantly attenuates the development of lupus nephritis in apoE-/-Fas-/-C57BL/6 lupus mice, indicating a potential clinical value of L-4F in the treatment of lupus nephritis.     

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.     

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.     

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.     

Inhibitory effect of IGF-1 overexpression on oxidative damage in umbilical cord mesenchymal stem cells

AIM: To analyze the inhibitory effect of insulin-like growth factor-1 (IGF-1) overexpression in umbilical cord mesenchymal stem cells (UC-MSCs) on oxidative damage and to develop new application model for UC-MSCs. METHODS: UC-MSCs were isolated from human umbilical cord with enzymatic digestion, and further characte-rized with flow cytometry. IGF-1-overexpressing UC-MSCs (UC-MSCs-IGF-1) were established by retrovirus infection. IGF-1 expression of UC-MSCs-IGF-1 was evaluated by real-time quantitative PCR and flow cytometry, and its surface markers, as well as osteogenic and adipogenic differentiation ability, were further analyzed. The proliferation, anti-oxidative damage and anti-apoptosis abilities of UC-MSCs-IGF-1 were evaluated when treated with H₂O₂ at different concentrations (0 μmol/L, 10 μmol/L, 50 μmol/L and 100 μmol/L). RESULTS: UC-MSCs showed positive expression of CD29, CD90 and CD105, but negative expression of CD34, which coincided with the normal phenotype of mesenchymal stem cells. UC-MSCs-IGF-1 established with retrovirus infection showed much higher expression of IGF-1 compared with normal UC-MSCs, and expressed the same surface markers as UC-MSCs.The osteogenic and adipogenic differentiation abilities were also observed. With the oxidative damage by H₂O₂ treatment, UC-MSCs-IGF-1 showed more strong proliferation, anti-oxidative damage and anti-apoptosis abilities as compared with normal UC-MSCs. In addition, the activity of SOD in UC-MSCs-IGF-1 was a little higher than that in control group. CONCLUSION: IGF-1 overexpression in UC-MSCs inhibits oxidative damage and cell apoptosis. UC-MSCs-IGF-1 may have more advantagies in clinical application.     

H IF-1αm ediates effect of interm ittent hypoxia on biological behavior of A 549 cells in vitro

ATM: To investigate whether hypoxia-inducible factor 1α (HIF-1α) mediates the effect of intermittent hypoxia on A549 cell viability, apoptosis and invasive ability METHODS: A549 cells were transfected with HIF-1α-siRNA and cultured under intermittent hypoxia. The expression of HIF-1α and its downstream genes, such as Bcl-2, Bax, P53, P21 and VEGF at mRNA and protein levels was determined by real-time PCR and Western blot. The viability of the A549 cells was measured by MTT assay. The apoptosis and cell cycle distribution of the A549 cells were examined by flow cytometry. The invasive ability of the A549 cells was detected by transwell test. RESULTS: The expression levels of HIF-1α, Bcl-2 and VEGF in non-HIF-1α-siRNA transfected A549 cells cultured in intermittent hypoxia environment[blank controlgroup(IH C),empty vector control group (IH E) and negative control group (IH N)] were higher than those in the A549 cells in normoxia group (RA), but the expression levels of Bax and P21 were lower than those in RA group (P<0.05). The siRNA-mediated HIF-1α gene silencing[intermittent hypoxia silenced group (IHS)] resulted in obvious down-regulation of HIF-1α, Bcl-2 and VEGF, and significant increase in the protein expression of P21 and Bax(P<0.05). The expression level of P53 in intermittent hypoxia groups was significantly higher than that in RA group, and no significant difference of P53 expression in different intermittent hypoxia groups was observed. Compared with normoxia, intermittent hypoxia resulted in significantly enhanced cell viability, decreased apoptosis, and enhanced invasive ability of non-HIF-1α-siRNA transfected A549 cells (P<0.05). The siRNA-mediated HIF-1α gene silencing resulted in significant cell viability inhibition, elevated apoptotic rate and decreased invasive ability under hypoxic condition (P<0.05).CONCLUSION: Intermittent hypoxia promotes the viability and invasion of A549 cells by HIF-1α-mediated downstream gene expression. HIF-1α gene silencing inhibits A549 cell growth and invasion under intermittent hypoxia by inhibition of HIF-1α signal pathways in vitro.     

Effect of 5-HT1A receptor in hippocampal CA1 region on spatial memory of PTSD rats

AIM: To investigate the change of long-term potentiation (LTP), and the expression of 5-hydroxytryptamine 1A receptor (5-HT_1A receptor) and postsynaptic density protein 95 (PSD-95) in the hippocampus of the rats with posttraumatic stress disorder (PTSD), and to explore the mechanism of 5-HT_1A receptor in the regulation of spatial memory in the PTSD rats.METHODS: Healthy adult SD rats (n=36) were randomly divided into control group and model group, with 18 rats in each group. The rats in model group were treated with single prolonged stress to construct the model of PTSD. Morris water maze (MWM) was used to test the learning and memory ability. The LTP induced by high-frequency stimulation (HFS) was detected by electrophysiological method. The protein expression of 5-HT_1A receptor and PSD-95 in the hippocampus was determined by Western blot and immunofluorescence. RESULTS: The MWM analysis showed that the latency of the rats searching for the underwater platform in model group was significantly longer than that in control group (P<0.01). The results of electrophysiological analysis showed that the amplitude of the evoked potential in both groups were significantly increased after HFS in the hippocampus, but that in model group was significantly lower than that in control group (P<0.01). The results of Western blot and immunofluorescence analysis showed that compared with control group, the protein expression of 5-HT_1A receptor was obviously increased (P<0.05), while the expression of PSD-95 was obviously decreased in model group (P<0.05).CONCLUSION: The spatial memory impairment in the PTSD rats may be associated with the increase in the expression of 5-HT_1A receptor and the decrease in the expression of PSD-95 in the CA1 region of hippocampus.     

Regulatory effect of astragaloside IV on SDF-1α/CXCR4 in EPCs

AIM: To investigate the effects of astragaloside IV (AS-IV) on chemokine receptor 4 (CXCR4) and stromal cell-derived factor 1α (SDF-1α) in endothelial progenitor cells (EPCs) and its mechanism. METHODS: Rat bone marrow-derived EPCs were cultured in vitro. The proliferation, adhesion, migration, apoptosis and tube formation capacity of EPCs treated with AS-IV and AMD3100, a specific blocker of CXCR4, were observed. The effects of AS-IV on the expression of SDF-1α/CXCR4 at mRNA and protein levels and the protein level of p-CXCR4 in the EPCs were determined. RESULTS: AS-IV significantly enhanced the proliferation, adhesion, migration and tube formation abilities of EPCs, reduced the apoptosis of EPCs, and up-regulated the mRNA and protein expression of SDF-1α and CXCR4 and the p-CXCR4 protein level in the EPCs. On the other hand, AMD3100 blocked the up-regulating effect of AS-IV on the mRNA and protein expression of CXCR4 and the p-CXCR4 protein level in the EPCs, but did not affect the effect of AS-IV on the expression of SDF-1α. CONCLUSION: AS-IV might enhance the biological function of EPCs by regulating the expression of SDF-1α/CXCR in EPCs.     

Effects of erythropoietin on expression of SDF-1 and its receptor in peripheral blood endothelial progenitor cells from rats with chronic renal failure

AIM:To investigate the effects of erythropoietin (EPO) on the expression of homing factors in peripheral blood endothelial progenitor cells (EPCs) from rats with chronic renal failure (CRF). METHODS:The CRF model was established by a two-stage 5/6 nephrectomy procedure in rats. Experimental rats were randomly divided into three groups: sham operation group, CRF model group and EPO treatment group. From the third week after the second stage of 5/6 nephrectomy procedure, rats in EPO treatment group received subcutaneous injection of human recombinant EPO at 50 U/kg every time and three times a week for 6 weeks, and then all the rats were sacrificed. EPCs were isolated from rat peripheral blood and primarily cultured. The mobilization, angiogenesis and functional activity of EPCs in vitro were detected. The mRNA and protein expression of EPO, EPO receptor (EPOR), stromal cell-derived factor 1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) in EPCs was also detected by the methods of real-time PCR and Western blotting. RESULTS:Compared with CRF model group, the expression of EPO and EPOR in EPCs in EPO treatment group was significantly up-regulated (P<0.05). Moreover, the expression of SDF-1 and its receptor CXCR4 in EPCs was also up-regulated by administration of EPO (P<0.05). CONCLUSION: EPO can mobilize EPCs from CRF rat peripheral blood, which may be associated with the increased expression of SDF-1 and its receptor CXCR4.     

Effect of peroxisome proliferator activated receptor δ on mRNA expression of MCP-1 induced by homocysteine in cultured human umbilical vein endothelial cells

AIM: To investigate the effects of peroxisome proliferator activated receptor δ (PPARδ) on the mRNA expression of monocyte chemoattractant protein 1 (MCP-1) induced by homocysteine (Hcy) in human umbilical vein endothelial cells (HUVECs). METHODS: Collagenase was used to isolate endothelial cells from human umbilical vein, and the cells were cultured in vitro . The HUVECs were divided into blank control group, Hcy group, GW0742 (a specific agonist of PPARδ) group and diphenyleneiodonium (DPI,a specific inhibitor of NADPH oxidase) group. RT-PCR was used to examine the mRNA expression of MCP-1 and PPARδ. The protein level of PPARδ was detected by Western blotting.2',7'-Dichlorofluorescin diacetate(DCFH-DA) was added to monitor intracellular production of reactive oxygen species (ROS). RESULTS: Compared with control group, Hcy promoted the mRNA expression of MCP-1 in a concentration-dependent manner, and decreased the mRNA expression of PPARδ in HUVECs. The mRNA expression of MCP-1 was significantly elevated by Hcy at the concentration of 10^-5 mol/L, and the mRNA expression of PPARδ was decreased remarkably (P<0.01). GW0742 decreased the mRNA expression of MCP-1 compared with Hcy group (P<0.01). Hcy remarkably increased the production of ROS compared with control group. Hcy-induced production of ROS was also significantly attenuated by GW0742. CONCLUSION: The activation of PPARδ decreases the Hcy-induced mRNA expression of MCP-1 by suppressing Hcy-stimulated production of ROS.