Study on scavenger receptor A from mouse peritoneal macrophages

AIM: To study the role of scavenger receptor A(SR A) in the uptake of oxidized low density lipoprotein(OxLDL) in mouse peritoneal macrophages(MPM). METHODS: Comparing the difference of the uptake of OxLDL in SR A-deficient and wild-type MPM. RESULTS: The results showed that the binding of OxLDL wasn't apparently reduced in SR A-deficient MPM. The association of OxLDL was reduced by 35.8% and degradation of OxLDL was reduced by 42% in SR A-deficient MPM compared with those in wild-type MPM. CONCLUSION: Studies showed that SR A didn't play an important role in the uptake of OxLDL in MPM. Approximately 70% of the uptake of OxLDL in macrophages was attributable to non-SR A receptor.     

The activative effect of Paecilomyces cicadaeon peritoneal and alveolar macrophages of rats

AIM:To explore the activative effect of water extract from cultured mycelium ofPaecilomyces cicadae (P. cicadae)on peritoneal macrophages (PMΦ) and alveolar macrophages (AMΦ) of rats.METHODS:The rats were randomly divided into four groups: normal control group(Ⅰ), cyclophosphamide (Cy) group (Ⅱ),P. cicadaegroup(Ⅲ), Cy+P.cicadaegroup(Ⅳ).The rats were bred in the same circumstance and were administered with corresponding drugs by subcutaneous injection for 26 days. PMΦ and AMΦ of rats were irrigated by normal saline and collected by centrifuge and incubated in a humidified incubator for 2 h at 37 ℃. The activities of acid phosphatase (ACP) and lactate dehydrogenase (LDH) in the PMΦ and AMΦ were determined by biochemical methods, and the capability of PMΦ and AMΦ in phagocytosis of neutral red were measured by colorimetric method too. RESULTS:After administering P. cicadaeto rats, the activities of ACP and LDH in PMΦ and AMΦ of normal rats were elevated significantly,and the reduction of the activities of ACP and LDH in PMΦ and AMΦ of rats due to Cy was notably antagonized, and the capability of PMΦ and AMΦ phagocytizing the neutral red were strengthened significantly.CONCLUSION:P. cicadaecan activate the PMΦ and AMΦ of rats.     

Effects of HDL from patients with coronary artery disease on lipid deposition and apoptosis in mouse peritoneal macrophages

AIM: To compare the effects of high-density lipoprotein (HDL) from healthy subjects (HDL_heathy) and HDL from the patients with coronary artery disease (HDL_CAD) on the lipid deposition and apoptosis in mouse peritoneal macrophages. METHODS: HDL was isolated from healthy subjects, stable CAD patients (HDL_SCAD) and acute myocar-dial infarction patients (HDL_AMI). The accumulation of intracellular lipids was determined by oil red O staining. The apoptosis of macrophages was measured by fluorescence microscopy with annexin-V/PI staining. DCHF-DA, a redox-sensitive dye, was used to detect intracellular reactive oxygen species (ROS) levels. The protein expression of ATP-binding cassette transporter (ABC) A1, ABCG1, Bcl-2 and Bax was determined by Western blot analysis. RESULTS: Lipid deposition in the macrophages was increased significantly after oxidized low-density lipoprotein (ox-LDL) treatment, and the expression of ABCA1 and ABCG1 was up-regulated (P<0.05). Compared with ox-LDL treatment alone, HDL_healthy decreased lipid deposition in the macrophages and up-regulated the expression of ABCA1 and ABCG1 (P<0.05), while treatment with HDL_SCAD or HDL_AMI further decreased lipid deposition in the macrophages and down-regulated the expression of ABCA1 and ABCG1 (P<0.05). Compared with HDL_SCAD treatment, lipid deposition in the macrophages was further increased after HDL_AMI treatment, and the expression of ABCA1 and ABCG1 was down-regulated (P<0.05). HDL_healthy decreased the levels of intracellular ROS and apoptosis by increasing the level of antiapoptotic protein Bcl-2 and reducing the expression of proapoptotic protein Bax. In contrast, HDL_SCAD and HDL_AMI had opposite effects on the intracellular ROS, the cell apoptosis and the expression of apoptosis-related proteins Bcl-2 and Bax. CONCLUSION: HDL_CAD promotes lipid accumulation in macrophages and induces macrophage apoptosis. These findings provide novel insights into mechanisms leading to altered vascular effects of HDL in CAD.     

Preliminary mechanism of Mcl-1 silencing in regulating apoptosis of mouse peritoneal macrophages infected with MTB

AIM: To investigate the mechanism of myeloid cell leukemia-1 (Mcl-1) silencing in regulating the apoptosis of mouse peritoneal macrophages infected with Mycobacterium tuberculosis (MTB) by observing the changes of Bcl-2 and Bax expression. METHODS: The suspensions of MTB strains with different virulence, BCG, H37Ra, H37Rv and XJ-MTB, were prepared to infect BALB/c mice. The transfection of Mcl-1-shRNA plasmid was used to establish a mouse model, and a corresponding control group at the same time was set up. The mice were executed and their peritoneal macrophages were collected 1 d, 3 d, 5 d and 7 d after the treatment. The apoptosis of the macrophages treated with diffe-rent virulence of MTB strains at different time points was analyzed by flow cytometry. The expression of Bcl-2 and Bax at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: The apoptotic rate of mouse peritoneal macrophages increased to some extent after transfection with Mcl-1-shRNA plasmid compared with control group. The order of apoptotic rates was BCG > H37Ra > H37Rv≈XJ-MTB (P<0.05). The expression of Bcl-2 at mRNA and protein levels was significantly decreased, while the expression of Bax at mRNA and protein levels was significantly increased. The changes in BCG infection group were the most significant, and the negative correlation between the Bcl-2/Bax ratio at mRNA level and the virulence of the MTB strains was observed (P<0.05). CONCLUSION: Inhibition of Mcl-1 expression significantly promotes the apoptosis of peritoneal macrophages in mice infected with different virulence of MTB strains. The regulatory mechanism may be closely related to the protein expression of Bcl-2 and Bax and the virulence of MTB strains.     

Heterogeneity of basal intracellular calcium concentration and its relations to the reactivity in mouse peritoneal macrophages

AIM: To investigate the heterogeneity of basal intracellular free calcium concentration([Ca²⁺]_i) in peritoneal macrophages(PM) and whether it is relative to the reactivity of PM at the single cell level. METHODS:[Ca²⁺]_iimplicated stimulated were measured by fluorescent microscopic imaging system after loading with fluorescent probe fura-2/AM. Superoxide(O₂^-)produced by single PM was determined by modified NBT test. RESULTS: The values of basal[Ca²⁺]_idetermined in 392 PMs of 7 mice showed normal distribution [(54±24) nmol/L, n=392] with wide range(less than 20 nmol/L to more than 100 nmol/L), among which about 50% were in the range of 40-60 nmol/L. When stimulated with PMA or fMLP,[Ca²⁺]_iwas increased, the peak values were positively correlated with the basal[Ca²⁺]_iin one mouse(PMA stimulated cells: r=0.52, P<0.01, n=58; fMLP stimulated cells:r=0.59, P<0.01, n=44. Both of the experiments were repeated in 3 mice, the results in the other 2 mice were similar). The O₂^- in PMA stimulated PMs were also positively correlated with the basal i(r=0.42, P<0.01, n=43, repeated in 4 mice, the results in the other 3 mice were similar). CONCLUSION: Basal[Ca²⁺]_iin murine PM is heterogeneous, and the value of basal[Ca²⁺]_iis tightly correlated to the reactivity of PM stimulated by proinflammatory factors.     

Effects of Mcl-1 silencing on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis

AIM: To investigate the effect of inhibiting Mcl-1 gene expression on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis using a technique of RNA interference. METHODS: The BALB/c mice were infected with prepared bacterium of the virulence strains of Xinjiang, H37Rv, H37Ra and BCG. Mcl-1-shRNA was applied to the mouse model of infection, and the control groups were set up. On 1 d, 3 d, 5 d and 7 d, the mouse peritoneal macrophages were collected. The expression of Mcl-1 at mRNA and protein levels was determined by real-time PCR and Western blot. The apoptotic rate of peritoneal macrophages was analyzed by flow cytometry. RESULTS: The expression of Mcl-1 at mRNA and protein levels was up-regulated in the peritoneal macrophages from the mice infected with different virulence of Mycobacterium tuberculosis, and the cells from the mice infected with virulence strains of Xinjiang and H37Rv expressed higher level of Mcl-1 than the uninfected control cells (P<0.05). The expression of Mcl-1 at mRNA and protein levels was reduced by RNA interference as compared with control group (P<0.05). Inhibition of Mcl-1 expression induced apoptosis of peritoneal macrophages in the mice. CONCLUSION: The Mcl-1 expression at mRNA and protein levels in mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis was effectively suppressed by Mcl-1-shRNA, which can induce macrophage apoptosis.     

Proliferative effect of neonatal mouse brain extracts on neural stem cells in vitro

AIM: To discover specific neural stem cell (NSC) proliferation-promoting factor, which will contribute to study on development of nervous system and treatment of nervous system diseases. METHODS: The extracts of forebrain, midbrain,afterbrain and cerebellum of neonatal mice were prepared, and the NSCs of newborn mice were cultured in vitro. Neurospheres were observed, immunocytochemical staining of characteristic protein, nestin, and MTT assay were performed to identify NSCs and their proliferative properties. RESULTS: A great deal of neurospheres were formed in the presence of the extracts of afterbrain and cerebellum, which were positive for characteristic protein （Nestin） of NSC showed by immunocytochemical staining. CONCLUSION: The afterbrain and cerebellum extracts can increase the total number of NSCs isolated from newborn mice in vitro in a dose-dependent manner.     

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.     

The role of chlamydia pneumoniae in the course of transformation of C57 BL/6J mouse peritoneal macrophages into foam cells

AIM: In order to understand the role of chlamydia pneumoniae in the course of macrophages transformation into foam cells experiments with chlamydia pneumoniae standard strain AR-39 wese made. METHOD: C57 BL/6J Mouse peritoneal macrophages C57 BL/6J wese incubated 24 h, and they were divided into 6 groups to be incubated continually: A₁~DMEM; A₂~DMEM+10 IFUs/L AR-39; B₁~DMEM+10mg/L LDL; B₂~DMEM+10mg/L LDL+10 IFUs/L AR-39; C₁~DMEM+10mg/L OxLDL;C₂~DMEM+10mg/L OxLDL+10 IFUs/L AR-39. 72 h later, morphological changes of the cells were observed and of intracellular cholesterol of content was detected. RESULTS: 1、Morphological studies: there were no lipid particles in A₁, A₂ and B₁ groups, but the lipid particles could be found in B₂、C₁ and C₂ group, Among six groups, the most lipid particles were seen in C₂ groups. 2、Biochemical detection:The ratio of cholesterol ester to total cholesterol was much higher than 50% in B₂、C₁ and C₂ groups, but was less than 50% in A₁、A₂ and B₁ groups. CONCLUSION: Chlamydia pneumoniae may have played a role in promoting C57 BL/6J mouse peritoneal macrophages into foam cells.     

Influence of bifidobacterium on NF-κB and IκB in murine peritoneal macrophages

AIM: To explore the regulatory effect of bifidobacteria adolescence on NF-κB in murine peritoneal macrophages (PMs). METHODS: Macrophages were collected and were divided into tow groups, normal control group and bifidobacterium stimulation groups. The cells were fixed at 60 min after stimulated with bifidobacterium at 106, 107, 108 and 109 cells/L, or fixed respectively at 15, 30, 60, 120 and 180 min after stimulated with bifidobacterium at 108 cells/L, then the total protein and nucleoprotein were extracted. The activities of NF-κB and IκBα were measured with the methods of MSA and Western blotting. RESULTS: NF-κB activity markedly increased and reached the peak at 0.5 h after stimulation, while IκBα markedly decreased at same time. CONCLUSION: NF-κB activity is markedly activated by bifidobacterium. NF-κB pathway participates in the regulation of peritoneal macrophage in this process.     

Effects of fibrous mineral dusts on pulmonary alveolar macrophages in vitro

AIM: To assess the role of surface free radicals and electromotive voltage of fibrous mineral dusts in rabbit pulmonary alveolar macrophage injuries induced by fibrous mineral dusts. METHODS: Changes in cell death ratio, malandialdehyde (MDA) and cellur electrophoresis ratio, lactate dehydrogenate (LDH)and superoxide dismitase(SOD) activities were determined, the technique of cell culture and Scanning electron Microscopy were used to examine the change of membranous permeability, charge and cellular shape. RESULTS: Fibrous wollastonite and tabulate clinoptilolite, which had no OH^-, had no cytotoxicity, while fibrous sepiolite, fibrous palygorskite, fibrous brucite and chrysolite asbestos damaged pulmonary alveolar macrophages in various degrees because of the different OH^- levels. All the six fibrous mineral dusts changed the cellular electrophoresis ratio. CONCLUSION: The surface electromotive voltage of fibrous mineral dusts is not an important factor, and the cytotoxicity of them may be related to OH^- levels on the mineral dust surface.     

Effect of quercetin on ox-LDL-induced lipid accumulation and peroxidation in mouse macrophages

AIM：To investigate the effect of quercetin (QUE) preconditioning on oxidized low-density lipoprotein (ox-LDL)-induced lipid accumulation and peroxidation in mouse RAW264.7 macrophages and the underlying molecular mechanisms. METHODS：RAW264.7 cells were pretreated with different concentrations (20, 40 and 80 μmol/L) of QUE for 30 min and then treated with ox-LDL (100 mg/L) for 24 h. Intracellular lipid droplets were assayed by oil red O staining. Extracellular lactate dehydrogenase (LDH) and malondialdehyde (MDA) and intracellular reactive oxygen species (ROS) were determined to characterize the membrane integrity and the lipid peroxidation, respectively. The mRNA and protein levels of CD36, an important scavenger receptor which mediates ox-LDL uptake, were examined by real-time PCR and Western blotting, respectively. RESULTS：Pretreatment with QUE (20, 40 and 80 mmol/L) significantly attenuated ox-LDL-induced lipid accumulation in RAW264.7 cells and foam cell formation in a dose-dependent manner. Ox-LDL induced LDH release in RAW264.7 cells. This cytotoxic effect was significantly inhibited by QUE pretreatment. Compared with ox-LDL group, the intracellular ROS content and MDA level in culture medium decreased markedly in QUE group. In addition, pretreatment with QUE attenuated ox-LDL-induced up-regulation of CD36 at mRNA and protein levels. CONCLUSION: QUE inhibits ox-LDL-induced lipid accumulation and peroxidation in mouse macrophages and the mechanism may partially involve its ability to down-regulate CD36 expression.     

Effects of anthocyanin on cholesterol efflux from mouse peritoneal macrophage foam cells

AIM: To explore the effect of anthocyanin on cholesterol efflux and elucidate its possible molecular mechanism. METHODS: Mouse Peritoneal macrophages were loaded with 50 mg/L AcLDL to induce macrophage-derived foam cells. Cholesterol efflux from macrophage-derived foam cells induced by anthocyanin was determined by enzymatic methods. PPAR-γ mRNA and protein expression in macrophage-derived foam cells was assayed by quantitative PCR and western blotting. RESULTS: Anthocyanins had the capacity of promoting cholesterol efflux from mouse peritoneal macrophage-derived foam cells and increased PPAR-γ mRNA and protein expression. CONCLUSION: Anthocyain-induced cholesterol efflux may be related to its enhancing PPAR-γ mRNA and protein expression.     

Effect of rapamycin on apoptosis of mouse astrocytes in vitro

AIM: To observe the effect of rapamycin on the apoptosis of mouse astrocytes in vitro.ME-THODS:The astrocytes from C57BL/6J newborn mouse pups were isolated and primarily cultured. The effect of rapamycin on the viability of astrocytes was assessed by MTT assay. The mean fluorescence intensity of SYTOX^® Green stain in the astrocytes was detected by fluorescence microplate reader in order to analyze the effects of rapamycin on the cell death induced by H₂O₂, ionomycin and/or deferorxamin. DiOC₆(3) staining was used to analyze the mitochondrial membrane potential of the astrocytes induced by H₂O₂. Flow cytometry analysis was used to determine the production of ROS in the astrocytes and mitochondria by staining with H₂DCFDA and MitoSOX^TM Red reagent, respectively.RESULTS: Rapamycin at concentration of 0.5 μmol/L protected the astrocytes against cell death induced by H₂O₂ or deferoxamine plus ionomycin. Rapamycin protected the mitochondrial membrane potential of astrocytes from the injury of H₂O₂. It also reduced the production of ROS in the astrocytes and decreased the level of ROS in the mitochondria.CONCLUSION: Rapamycin reduces the ROS overload in the mitochondria, keeps mitochondrial membrane potential safety and protects the astrocytes against apoptosis in vitro.     

Effect of epigallocatechin-3-gallate on LPS-induced p38 MAPK pathway in mouse macrophages

AIM: To investigate the effect of epigallocatechin-3-gallate (EGCG) on lipopolysaccharide (LPS)-induced p38 MAPK activation and tumor necrosis factor-α (TNF-α) secretion in macrophages. METHODS: Western blotting was used to detect the phosphorylation of p38 MAPK in mouse macrophages cultured in vitro. Enzyme linked immunosorbent assay was used to determine the secretion of TNF-α in macrophages. Electron microscopy was used to study the effect of EGCG on the structure of LPS. RESULTS: LPS caused activation of p38 MAPK and more production of TNF-α, EGCG inhibited LPS-induced phosphorylation of p38 MAPK and TNF-α production and had no effect on the structure of LPS. CONCLUSIONS: EGCG has no direct effect on LPS, but blocks cellular signal pathway. The inhibition of EGCG on LPS-induced TNF-α production is mediated, at least in part, through blocking of p38 MAPK pathway.     

Protective effect of astragalus polysaccharide on mitochondria and lysosome in H2O2-stressed skin fibroblasts

AIM: To verify the protection of astragalus polysaccharide (APS) on H2O2-stressed skin fibroblasts. METHODS: A model of acute H2O2 stress in primary skin fibroblast was used at concentration of 0.5 mmol/L by 30 min incubation. Dose responses of APS on cell survival was measured by MTT, cell death was evaluated by DAPI, and effect of APS on mitochondria, mitochondrial membrane potential and lysosome stabilization were measured by confocal microscopy. RESULTS: APS improved cell survival in a dose-dependent manner, starting at 0.5 mg/L and with a maximum at 1 mg/L. Moreover, APS inhibited mitochondrial membrane potential collapse, protected mitochondrial morphology and stablized lysosomal membrane. CONCLUSION: The results suggest the existence, at the mitochondria-lysosome level, of a new pathway of apoptotic regulation by APS. This might constitute a new therapeutic target where oxidative stress and lysosomal impairment are involved.     

Effect of RIP3 expression on apoptosis of mouse macrophages infected with BCG

AIM:To investigate the function of receptor-interacting proteins 3 (RIP3) in regulating Bacillus Calmette-Guérin (BCG)-induced apoptosis of mouse macrophages (RAW264.7 cells). METHODS:The RIP3 adenovirus interference vector was constructed and used to infect the RAW264.7 cells, and then the RAW264.7 cells were infected with BCG. The cell viability was measured by MTT assay. The apoptotic rate, mitochondrial membrane potential and production of reactive oxygen species (ROS) were determined by flow cytometry analysis. The protein levels of RIP3 and apoptosis-associated proteins were examined by Western blot. RESULTS:The viability of RAW264.7 cells was decreased after BCG infection. In the meantime, the expression of RIP3 was up-regulated significantly (P<0.01). Compared with BCG infection group, the apoptotic rate and ROS level in BCG and RIP3 adenovirus interference vector co-infection group were significantly decreased (P<0.01). Importantly, RIP3 was able to further promote apoptosis in BCG-infected RAW264.7 cells in part by increasing mitochondrial membrane potential (P<0.01). In addition, Western blot analysis further demonstrated that RIP3 was involved in BCG-induced apoptosis partly through down-regulation of anti-apoptotic protein Bcl-2, and up-regulation of Bax and cleaved caspase-3 (P<0.01). CONCLUSION:RIP3 is involved in BCG-induced apoptosis of RAW264.7 cells, and this process may be achieved by the mitochondrial pathway.