Release of tumor necrosis factor-α in podocytes and its mechanism by mesangial cell cultured medium with IgA1 from IgA nephropathy

AIM: To explore the effect and mechanism on tumor necrosis factor-α production in podocytes by medium from growth arrested mesangial cells incubated with aIgA1 isolated from IgAN patients and normal control. METHODS: Jacalin affinity chromatography and Sephacryl S-200 molecular sieve chromatography were used to isolate IgA1. Monomeric IgA1 (mIgA1) was transformed to aggregated IgA1 (aIgA1) by heating. IgA-mesangial cell medium was prepared by collecting medium in which growth arrested mesangial cell were incubated with different aIgA1, then the medium with RPMI-1640 containing 0.5% FBS was exposed to podocytes. Real time PCR was used to detect the mRNA expression of Ang and ACE, and TNF-α was measured by ELISA assay. RESULTS: TNF-α level of podocytes by medium from aIgA1 from IgAN cultured with mesangial cells was higher than that in control (6.47±0.45) ng/L vs (1.33±0.65) ng/L, P<0.05. Ang and ACE mRNA in podocytes exposed to the special medium were higher than those in podocytes exposed to control and medium from mesangial cells incubated with aIgA1 from healthy control (P<0.05). Enalaprilat decreased the TNF-α to (7.52±1.12) ng/L (P<0.05), and valsartan decreased TNF-α in the podocytes to (6.64±0.68) ng/L (P<0.05), while enalaprilat and valsartan restored the level of TNF-α in podocytes to normal level (2.72±0.55) ng/L, P>0.05. CONCLUSION: Our findings implicate that medium from mesangial cells cultured with IgA1 from IgA nephropathy stimulates the release of TNF-α in podocytes by the activation of renin-angiotensin system, which might accelerate the progression of IgAN.     

Effect of exogenous hydrogen sulfide on lipophagy in mouse primary hepatocytes

AIM: To evaluate the effect of exogenous hydrogen sulfide (H₂S) from GYY4137 on lipophagy in mouse primary hepatocytes. METHODS: The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion method were divided into 4 groups: the cells in control group were incubated with normal medium; the cells in model group were incubated with 1.2 mmol/L oleic acid (OA) for 48 h; the cells in H₂S group or propargylglycine (PAG) group were incubated with 1.2 mmol/L OA for 48 h followed by serum-free phenol red-free RPMI-1640 medium which contained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h. The cells were collected to conduct immunofluorescence staining of LC3 and photography under fluorescence microscope, phase-contrast microscope or transmission electron microscope. The protein expression of LC3-Ⅰ/Ⅱ in the hepatocytes was determined by Western blot. RESULTS: In contrast with the model group, the fluorescent particles of LC3, the protein expression of LC3, the number of autophagic lysosome and vacuoles in hepatocytes in H₂S group increased. CONCLUSION: In steatosis hepatocytes, exogenous H₂S promotes the lipophagy.     

Effects of aldosterone on expression of angiotensin Ⅱ receptors in cultured rat mesangial cells

AIM: To investigate the effect of aldosterone (ALD) on the mRNA expression of angiotensin Ⅱ (Ang II) type 1 (AT-1a R and AT-1bR) and 2 (AT-2R) receptors in cultured rat mesangial cells (RMCs) treated with high glucose. METHODS: Rat mesangial cells were cultured in high glucose medium containing different concentrations of ALD (10-8-10-6 mol/L). The antagonists of ALD and Ang II receptors including pironolactone (10-7 mol/L, aldosterone receptor antagonist, SPI), losartan (10-7 mol/L, Ang II type 1 receptor blocker, Los) or PD123319 (10-9 mol/L, Ang II type 2 receptor antagonist, PD) were added in the cell culture for 12 h. The control cells were only treated with high (30 mmol/L) or normal (5.6 mmol/L) glucose medium. The viability and proliferation of the RMCs were evaluated by MTT assay. The mRNA expression of AT-1aR, AT-1b R and AT-2R was detected by semi-quantitative RT- PCR. The expression of MCP-1 in cultured RMCs was detected by ELISA. RESULTS: The mRNA expression of AT-1aR, AT-1b R and AT-2R was increased significantly by treatment with ALD in a dose-dependent manner (1.62-1.77, 9.61-9.89 and 7.26-7.35 folds of high glucose control, respectively, P<0.01). SPI significantly reduced the mRNA expression of AT-1aR and AT-1b R (P<0.01) but not affected the mRNA expression of AT-2R. The ratio of AT-1aR/AT-1b R in cultured RMCs treated with high glucose decreased significantly after stimulated with ALD (P<0.01). However, the effect of ALD was inhibited by SPI (P<0.01). Aldosterone treatment induced a significant upregulation of MCP-1 expression in a dose-dependent manner, and previous treatment with spironolactone, losartan or PD123319 abolished this aldosterone-induced MCP-1 expression. CONCLUSION: The results suggest that aldosterone is involved in the inflammatory response by up-regulating the expression of AT-1aR, AT-1bR and AT-2R, changing the proportion of AT-1R subtype, and inducing MCP-1 overproduction in cultured RMCs treated with high glucose.     

Effect of GYY4137 on cytosolic lipid decomposition in mouse primary hepatocytes

AIM: To evaluate the effect of GYY4137, a novel hydrogen sulfide (H₂S) donor, on cytosolic lipid decomposition in mouse primary steatosis hepatocytes. METHODS: Oleic acid (OA) was used to induce hepatic steatosis model in vitro. The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion were divided into 4 groups:the cells in control group were incubated with normal medium for 54 h; the cells in model group were incubated with OA at 1.2 mmol/L for 48 h followed by serum-free phenol red-free RPMI-1640 for 6 h; the cells in H₂S group or DL-propar-gylglycine (PAG; an inhibitor of cystathione γ-lysase, inhibiting H₂S synthesis) group were incubated with OA at 1.2 mmol/L for 48 h followed by serum-free phenol red-free RPMI-1640 which contained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h. The glycerin release and the protein expression of hormone-sensitive lipase (HSL) in the cells were mea-sured. RESULTS: Compared with model group, the glycerin release and the protein expression of phosphorylated HSL (p-HSL) in H₂S group decreased significantly, while those increased significantly in PAG group. CONCLUSION: In steatosis hepatocytes, exogenous H₂S possibly decreases cytosolic lipid decomposition by decreasing the protein level of p-HSL.     

Effect of CHOP/GADD153 on cardiomyocyte apoptosis induced by angiotensin Ⅱ in vitro

AIM: To investigate the relationship between alteration of CHOP/GADD153 protein expression and cardiomyocyte apoptosis induced by angiotensin Ⅱ (AngⅡ),and inhibitory effects of CHOP/GADD153 antisense oligodeoxynucleotide (anti-ODN) on apoptosis in vitro.METHODS: Cultured neonatal cardiomyocytes were exposed to AngⅡ with or without preincubation of CHOP/GADD153 anti-ODN.Variability of myocytes was measured by MTT assay,the lactate dehydrogenase (LDH) release was also detected,the percentage of annexin V positive myocytes was monitored by flow cytometry as apoptosis rate,CHOP/GADD153,Bcl-2 and Bax expressions were determined by Western blotting.RESULTS: Compared with control group,the expression of CHOP/GADD153 was obviously increased from (0.20±0.02 to 0.75±0.06) in AngⅡ group (P＜0.01).The variability of myocytes was obviously decreased from (100.00%±0.00% to 66.32%±7.16%,P＜0.05).The LDH release was significantly increased from (20.23 U/L±2.83 U/L to 79.36 U/L±5.69 U/L,P＜0.05) and apoptosis rate was significantly increased from (3.33%±0.28% to 16.62%±2.09%,P＜0.05).Bcl-2 expression was obviously decreased from (0.73±0.05 to 0.44±0.05,P＜0.01).Bax expression was obviously increased from (0.69±0.08 to 0.90±0.10,P＜0.01) and Bax/Bcl-2 ratio was obviously increased from (0.93±0.09 to 2.00±0.22,P＜0.01).Preincubation of CHOP/GADD153 anti-ODN in the medium significantly reversed above changes except the expression of Bax,but the Bax/Bcl-2 ratio was lower than that in AngⅡ group significantly (P＜0.01).These effects were not observed in mis-ODN group and there was no significant difference between lipofectin and control group.CONCLUSION: The increased expression of CHOP/GADD153 protein may be one of the mechanisms of AngⅡ-induced cardiomyocyte apoptosis,and CHOP/GADD153 anti-ODN can inhibit the apoptosis.     

Effects of panax notoginseng saponins on content of triglyceride and the mRNA expression of LXRα in steatotic hepatocyte L02

AIM: To study the effects of panax notoginseng saponins (PNS) on the content of triglyceride (TG) and the mRNA expression of liver X receptor α (LXRα) in steatotic hepatocyte L02.METHODS: The cells of hepatocyte L02 were cultured with 50% bovine calf serum for 48 h and a model of steatosis of hepatocytes was established. The appropriate concentrations of PNS on steatotic hepatocytes were detected by MTT assay. The cells were divided into 5 groups: normal control group, model group, spontaneous recovery group, low concentration of PNS group (10 mg/L) and high concentration of PNS group (50 mg/L). Meanwhile, hepatocytes in model group were continuously cultured by RPMI-1640 medium containing 50% bovine calf serum while others were cultured by RPMI-1640 medium containing 10% bovine calf serum. After 24 h, the lipid droplets in the cells stained with oil red O were observed under microscope, the intracellular TG levels were determined by automatic biochemical analyzer and the mRNA expression of LXRα in hepatocytes was examined by RT-PCR. RESULTS: Compared to normal control group, Oil red O staining presented numerous orange-red or red lipid droplets in the cytoplasm of human L02 hepatocytes in model group. The content of TG in model group were significantly increased (P<0.01). After treated with PNS for 24 h, the content of TG in PNS treatment groups was significant decreased than that in spontaneous recovery group (P<0.05), especially in low concentration of PNS group (P<0.01). The accumulation of lipid droplets in low concentration of PNS group was decreased significantly. Compared to normal control group, the mRNA expression of LXRα in model group was significantly up-regulated (P<0.01). Compared to spontaneous recovery group, the mRNA expression of LXRα in PNS treatment groups declined in different levels, especially in low concentration group (P<0.05).CONCLUSION: The deposition of lipid in hepatocytes might be related to the high expression of LXRα mRNA. PNS decreases the content of TG in steatotic hepatocytes and promotes the recovery of hepatocyte steatosis by downregulating the mRNA level of LXRα.     

Effects of Chinese medicine WPY on expression of L-selectin and distribution of F-actin in leukocytes from acute necrotizing pancreatitis rats

AIM: To explore the changes of L-selectin expression and F-actin distribution of leukocytes from pancreatic circulation in the experimental acute necrotizing pancreatitis (ANP) rats and impact of Chinese herbs west China pancreatitis yi hao (WPY) on them in vivo. METHODS: Forty-four Wistar rats were randomized into control group, ANP group and ANP WPY treated group. The leukocytes from blood of splenic vein were incubated with PE-anti-CD62L for L-selectin and TRTIC-Phalloidin for F-actin, then the ratio of CD62L positive granulocytes was measured by flow cytometry and leukocytes were taken images with confocal laser scanning microscope. The distribution of F-actin at the border or central part of leukocyte was analyzed by using Mias system. RESULTS: The ratio of CD62L positive granulocytes increased in ANP rats, but those increases at 6 h (31.78%±12.37%) and 12 h (29.83%±13.73%) were significant ［vs control group(11.96%±5.32%), P<0.05］. The ratio of F-actin border/center gray value of leukocytes increased at 1 h, 6 h and 12 h in ANP rats, and those at 1 h (2.52±0.49) and 12 h (2.72±0.48) were significant ［vs control group (1.79±0.26), P<0.05］. The ratio of F-actin border/center gray value of leukocytes (1.54±0.24) declined significantly at 6 h in WPY-treated ANP rats (P<0.05). CONCLUSION: L-selectin expression and F-actin distribution change significantly in leukocytes from pancreatic circulation in ANP rats, suggesting that an increase in adhesive avidity and a decrease in deformability of leukocytes are pathophysiologic factors for progression of ANP, and traditional Chinese medicine WPY may regulate these changes of leukocytes in some degrees.     

Lipopolysaccharide-binding protein inhibitory peptide inhibits the binding of LPS to U937 cells

AIM: To investigate the inhibitory effect of P12, a kind of lipopolysaccharide (LPS)-binding protein (LBP) inhibitory peptide, on the binding of LPS to macrophage in vitro. METHODS: Human monocyte-like cell line (U937 cells) was grown in RPMI-1640 and stimulated with PMA in order to induce their differentiation to macrophage stage. The relative affinity of P12 to LPS was determined by enzyme-linked immunosorbent assay (ELISA). The effects of P12 on the binding of LPS to U937 cells were determined by flow cytometry analysis. The production of tumor necrosis factor-alpha (TNF-α) was measured by ELISA. RESULTS: The relative binding activity of P12 to LPS was higher than that of LBP in the same mass concentration. P12 inhibited the binding of FITC-conjugated LPS (FITC-LPS) to U937 cells. The productions of TNF-α was also significantly suppressed by P12. CONCLUSION: The results suggest that blockage of LBP at the inflammatory sites might attenuate LPS-induced circulatory shock.     

Relationship between Th1/Th2 balance and CD28/CTLA-4 expression on peripheral blood T cells in patients with systemic lupus erythematosus

AIM: To investigate the pattern of Th1/Th2 balance in systemic lupus erythematosus(SLE) patients and the relationship between CD28/CTLA-4(cytotoxic T-lymphocyte antigen-4) molecule expression and Th1/Th2 balance.METHODS: Eighteen SLE patients met the ARA 1997 updated SLE criteria were selected in the study. According to Bombardier's SLEDAI criteria, all patients were classified into two groups: active group(12 cases) and static group(6 cases). Fourteen normal individuals, matched for age and sex of the patients, served as controls. The peripheral blood mononuclear cells(PBMCs) were isolated by density gradient centrifugation and cultured in RPMI-1640 culture medium. After treated with PMA(5 μg/L) and ionomycin(500 μg/L) for 72 h, the PBMCs were collected, the contents of IFN-γ and IL-10 in the supernatant of cultured PBMCs were detected using enzyme linked immunosorbent assay(ELISA). The expression of CD28 and CTLA-4 molecules on T cells were detected by flow cytometric technique with double staining by FITC or PE labeled monoclonal antibodies. RESULTS: The level of IL-10 was higher in the PBMCs of active and static SLE patients(351.29 ng/L±153.31 ng/L and 319.37 ng/L±153.39 ng/L) than that in controls(254.48 ng/L±120.69 ng/L), but the difference did not reach statistical significance(P>0.05). The level of IFN-γ was significantly lower in the PBMCs of active SLE patients(25.76 ng/L±16.09 ng/L) than that in controls(50.71 ng/L±27.92 ng/L, P＜0.05). The ratio of IL-10/IFN-γ was significantly higher in active SLE patients(18.74±13.77) than that in controls(6.66±4.95, P<0.05). Either before or after culture, the expression of CD28 molecule on CD3⁺and CD8⁺ T cells from all SLE patients was not remarkably different from that in the cells of controls. Before culture, the expression of CTLA-4 molecule on CD3⁺T cells of active SLE patients(0.79%+0.37%) was significantly lower than that in the cells of controls(1.31%+0.61%, P<0.05). After culture, the expression of CTLA-4 molecule on CD3⁺ T cells of SLE patients was still lower than that in the cells of normal controls without statistical significance(P＞0.05).The expression level of CD28 molecule on CD3⁺ or CD8⁺ T cells in active SLE patients and controls was not correlated with the levels of IFN-γ and IL-10 in the supernatants(P＞0.05). The level of CTLA-4 molecule expression on CD3⁺ T cells of active SLE patients was positively correlated with IFN-γ level(r=0.681, P<0.05), while was negatively correlated with IL-10 levels(r=-0.624,P＜0.05) and the ratio of IL-10/IFN-γ(r=-0.738, P<0.01). The level of CTLA-4 molecule expression on CD3⁺ or CD8⁺ T cells of controls showed no correlation with IFN-γ levels, while showed negative correlations with IL-10 level(r=-0.587, P<0.05; r=-0.563, P<0.05, respectively).CONCLUSION: There is a bias in the differentiation of Th0 cells towards Th2 in SLE patients. CTLA-4 probably plays an important role in this mechanism through suppressing the signal transmitted by CD28.     

Protective effect of dexmedetomidine-ulinastatin combination on lipopolysaccharide-induced acute lung injury in rats

AIM：To investigate the effects of dexmedetomidine-ulinastatin combination on acute lung injury induced by lipopolysaccharide (LPS) in rats. METHODS：Male Wistar rats were randomly divided into 5 groups: saline control group (NS group) was given saline (5 mL/kg, iv) alone; LPS group (L group) was given LPS (10 mg/kg, over 10 min); dexmedetomidine+LPS group (L+D group) was treated with the additional administration of dexmedetomidine (1 μg·kg -1·h -1) immediately after LPS injection; ulinastatin+LPS group (L+U group) was treated with the addi-tional administration of ulinastatin (50 000 U/kg, ip) immediately after LPS injection; dexmedetomidine+ulinastatin+LPS group (L+D+U group) received dexmedetomidine (1 μg·kg -1·h -1) and ulinastatin (50 000 U/kg) immediately after LPS injection. The animals were sacrificed at 6 h after LPS or NS administration. Partial pressure of arterial oxygen (PaO 2), pH and base excess (BE) were measured, and the lungs were removed for evaluation of histological characteristics and determining the concentrations of TNF-α, IL-1β, macrophage inflammatory protein 2 (MIP-2), malondialdehyde (MDA), nitric oxide (NO), prostaglandin E 2 (PGE 2) and myeloperoxidase (MPO) in lung tissues, lung wet/dry weight ratio (W/D), and albumin in brochoalveolar lavage fluid (BLAF). The pulmonary expression of nuclear factor kappa B (NF-κB) p65 was evaluated by Western blotting. RESULTS：Compared with NS group, PaO 2, pH and BE was lower in L group, which was increased by treatment with dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone. Compared with NS group, LPS induced marked lung histological injury, which was less pronounced in the animals treated with dexmedetomidine-ulinastatin combination but not dexmedetomidine or ulinastatin alone. The levels of IL-1β, IL-6, MIP-2, MDA, NO and PGE 2 in the lung tissues increased in L group compared with NS group, which were reduced by dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone. The MPO activity, MDA level and W/D increased in the lung tissues in L group compared with NS group, which was reduced by dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone. Compared with NS group, the albumin concentration in the BLAF increased, which was reduced by dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone. Compared with NS group, the expression of NF-κB p65 increased in L group, which was reduced by dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone.CONCLUSION：Dexmedetomidine-ulinastatin combination has a protective effect on LPS-induced acute lung injury in the rats.     

中甘21号

AIM: To observe the pathological role of 3-nitrotynosine (3-NT) on Escherichia coli LPS-induced vascular hyporeactivity in rats and the therapeutic effect of antioxidants. METHODS: Forty male SD rats weighting from 200 g to 250 g were randomly divided into four groups: the control group (n=10); LPS shock group (n=10); uric acid-treated group (n=10); melatonin-treated group (n=10). 6 h after LPS shock, phenylephrine (0.5-2.5 μg·kg-1) was applied intravenously to all groups and the percentage increase in MAP was detected, respectively. The concentration-response curve of aorta rings from all groups rats were obtained by cumulative addition of phenylephrine (PE), and PE Emax, EC50 were calculated. The concentrations of plasma malondialdehyde (MDA), nitrate/nitrite and 3-NT were assayed in all groups 6 h after LPS shock. RESULTS: The MAP level induced by PE significantly decreased to 54.60% in LPS shock rats compared with the control (P<0.05). However, PE induced MAP level increased 37.70% and 43.05% in uric acid and melatonin treated rats, respectively, compared with the LPS shock rats (P<0.05). The maximum response and EC50 to PE were significant reduced in LPS shock rats ［Emax, 35.30%±9.80%; EC50, (15.70±4.50)nmol/L］ compared with control group ［Emax, 100%; EC50, (4.71±2.04)nmol/L, P<0.05］; but the reactivity of aorta to PE was improved obviously in uric acid and melatonin treated groups (P<0.05). The plasma concentration of MDA, nitrate/nitrite and 3-NT were much lower in uric acid and melatonin groups than those in LPS shock group (P<0.05). CONCLUSIONS: 3-NT is an important pathological factor on vascular hyporeactivity in LPS shock. Antioxidants effectively improve α-adrenergic receptor-mediated vascular reactivity in LPS shock rats partially by removing lipid peroxidative production, reducing nitric oxide and 3-NT biosynthesis in LPS shock. These results suggest that antioxidants have potential beneficial therapeutic effect for septic shock patients.     

Angiotensin II-induced matrix metalloproteinase-9 expression mediated by NF-κB pathway in human THP-1 cells

AIM: The present study was undertaken to investigate the effect of angiotensin II (AngⅡ) on expression of MMP-9 in THP-1 macrophages. METHODS: Macrophages converted from THP-1 monocytes by incubating with PMA (0.1 μmol/L) for 48 h were divided into PMA group; PMA+AngⅡ group (10-7mol/L, 1 h); PMA+AngⅡ+PDTC group (10 μmol/L, 30 min) and PDTC group. Western blotting was used to detect the MMP-9 and phosphorylation of NF-κB p65, and the expression of MMP-9 mRNA in THP-1 macrophages was measured by RT-PCR.RESULTS: Compared to control group, the expression of MMP-9 (1.06±0.11, P<0.05) and phosphorylation of NF-κB p65 (1.02±0.10, P<0.05) in THP-1 macrophages were expressed when treated with AngⅡ (10-7mol/L); and the expression of MMP-9 mRNA were upregulated (1.22±0.08, P<0.05). However, NF-κB inhibitor PDTC reduced the NF-κB p65 (0.99±0.12, P<0.01) and MMP-9 (1.04±0.14, P<0.01) expressions and decreased the expression of MMP-9 mRNA (0.90±0.06,P<0.01). CONCLUSION: NF-κB signaling pathway contributes to the expression of MMP-9 in THP-1 macrophage induced by AngⅡ.     

Inhibition of HMGB1 expression and release by nicotine in RAW264.7 cells

AIM: To investigate that nicotine inhibits HMGB1 expression and release in RAW264.7 cells.METHODS: (1) RAW264.7 cells were cultured in 6 wells plate, treated with 250 μg/L LPS and 1 μmol/L or 10 μmol/L nicotine, in which the cells treated with or without 250 μg/L LPS were regarded as nicotine 1 group (N1), nicotine 2 group (N2), LPS group (LPS) and control group (C), respectively. HMGB1 protein in the cell culture media and in cell nuclear was examined by Western blotting and the cellular HMGB1 mRNA level was detected by RT-PCR. (2) Transfected with antisense RNA or sense RNA of α7 subunit-containing nicotinic receptor (α7nAChR), RAW264.7 cells were treated with 250 μg/L LPS and 10 μmol/L nicotine, HMGB1 protein in the culture media was also tested by Western blotting.RESULTS: (1) HMGB1 mRNA level in C group was low (1 659.20±121.05) and no significant statistical difference among groups of N1, N2 and LPS was observed (P>0.05). (2) Higher HMGB1 accumulation in the cell culture media was detected in LPS group (445.34±28.52) than that in C group. Compared to LPS group, both N1 and N2 groups distinctly attenuated HMGB1 accumulation in culture media (P<0.05). (3) Nuclear HMGB1 accumulation was lower in LPS group than that in C group, and two different nicotine concentrations markedly increased the nuclear HMGB1 accumulation compared to LPS group (P<0.05). (4) No significant difference of HMGB1 levels in culture media between antisense RNA group and LPS group was observed (P>0.05). In sense RNA group, however, HMGB1 level was observably reduced compared to antisense group (P<0.05).CONCLUSION: The present results suggest that nicotine dramatically inhibits RAW264.7 cell nuclear HMGB1 translocation and extracellular release, and this effect relies on α7nAch receptor expression.     

Effects of inactivated HIV-1 particles on human CD4+T cell activation and Th1/Th2 cytokine secretion in whole blood

AIM: To investigate the effects of AT-2-inactivated HIV-1 particles on human CD4+T cell activation and cytokine secretion in whole blood (WB) in vitro. METHODS: HIV-1ⅢB particles were inactivated by AT-2 chemical and the concentration of p24 antigen was determined by p24 ELISA. AT-2-inactivated HIV-1ⅢB particles were added to human WB culture system in serial concentrations to stimulate the cells. PHA was used as positive control. After 24 h, all the cultural supernatants were harvested and the concentrations of Th1 (IL-2, IFN-γ and TNF-α) and Th2 (IL-4, IL-6 and IL-10) cytokines released to the supernatants were detected by cytometric bead array (CBA). The percentage of CD69 expression on CD4+T cells from WB was detected by immuno-fluorescence staining plus flow cytometry. RESULTS: The concentration of p24 antigen in the AT-2-inactivated specimen was 85.5 μg/L. 24 h later, the percentage of CD69 expression on CD4+T cells from control group was (1.62±0.63) %, whereas it was (38.82±6.00)%, (3.83±1.07)%, (5.94±0.85)% and (9.30±1.22)% in PHA group, HIV-1 (1/500) group, HIV-1 (1/50) group and HIV-1 (1/5) group, respectively. Cytokines secreted by WB in control group were mainly TNF-α and IL-6. However, all the six cytokines tested were strikingly increased in PHA group, as well as in HIV-1ⅢB groups. CONCLUSION: AT-2-inactivated HIV-1ⅢB particles activate CD4+T cells from WB, and up-regulate both Th1 and Th2 cytokine secretion in WB. Besides the effects of viral proteins, other mechanisms may be proposed that HIV-1 particles act as antigen presenting cell (APC) because many host-derived immune molecules are incorporated into HIV-1 envelop when it is released from infected cells by budding, and exert immune modulation.     

Propofol inhibits LPS-induced inflammatory response in microglia via TRPV1 ion channels

AIM:To investigate the effects of propofol (P) on the inflammatory response of microglia induced by lipopolysaccharide (LPS) and the mechanisms. METHODS:Mouse microglia BV2 cells were treated with LPS at 100 μg/L to establish a neuroinflammatory injury model. The BV2 cells were divided into 4 groups:control group (C group), model group (L group), L+P group and LPS+AMG517 group (L+A group). The level of tumor necrosis factor-α (TNF-α) in the cell culture supernatant was measured by ELISA. The mRNA expression of transient receptor potential cation channel subfamily V member 1 (TRPV1) was detected by real-time PCR. The protein levels of TRPV1, TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and phosphorylated calcium/calmodulin-dependent protein kinase Ⅱ (p-CaMKⅡ) were determined by Western blot. The content of free Ca²⁺ in the microglia BV2 cells was detected by Fluo-3 AM assay. RESULTS:Compared with C group, the level of TNF-α was significantly increased in L group (P<0.01), but that in P group was not changed. Compared with L group, the level of TNF-α was significantly lower than that in L+P group within 4 h (P<0.01). Compared with C group, the mRNA expression of TRPV1 was significantly increased in L group (P<0.01). Compared with L group, the mRNA expression of TRPV1 was significantly down-regulated in L+P group (P<0.01).Compared with L group, the protein levels of TNF-α, IL-1β, IL-6 and p-CaMKⅡ and intracellular Ca²⁺ concentration were significantly lower than those in L+P group and L+A group (P<0.01). CONCLUSION:Propofol inhibits the inflammatory response of microglia by reducing the expression of TNF-α, IL-1 and IL-6, which may be related to the down-regulation of TRPV1 and p-CaMKⅡ and the reduction of intracellular Ca²⁺ concentration.     

Inflammation and coagulation activation are associated with gallstone formation in golden hamsters

AIM: To elucidate the relationship of inflammation, coagulation and cholesterol gallstone formation. METHODS: Hamsters were divided into four groups: control group (feeding normal diet), lithogenic diet (LD) 2 weeks group, LD 6 weeks group and LD+aspirin 6 weeks group. Gallstone incidence, antithrombin antigen (AT-Ⅲ:Ag), antithrombin activity (AT-Ⅲ:Ac), thrombin (F-Ⅱa:Ac), plasminogen activator inhibitor activity (PAI:Ac), plasmin activity (Plm:Ac), D-dimer:Ag and C-reactive protein (CRP) in gallbladder bile were observed as read-out parameters. RESULTS: The incidence of gallstones in control group, LD 2 weeks group, LD 6 weeks group and LD+aspirin group were 0%, 20%, 73% and 25%, respectively. AT-Ⅲ:Ag, F-Ⅱa:Ac, D-dimer and CRP in LD 2 weeks group and LD 6 weeks group were significantly higher than those in the control group (P<0.01). AT-Ⅲ:Ag, PAI:Ac, Plm:Ac, D-dimer and CRP in LD 6 weeks group were significantly higher than those in LD 2 weeks group (P<0.01). AT-Ⅲ:Ag, F-Ⅱa:Ac, PAI:Ac, D-dimer and CRP were strikingly decreased in LD+aspirin group compared with those in LD 6 weeks group (P<0.01). The level of CRP was closely correlated with the concentration of D-dimer (γ=0.752, P<0.01). CONCLUSIONS: Coagulation cascade is activated and crosslinked fibrin forms in gallbladder bile before gallstone formation. “Lithogenic bile” might play a central role in the inflammation of gallbladder mucosa, which activates extrinsic coagulation pathway and leads cholesterol gallstone formation.     

Senescence of endothelial cells and gene expression associated with apoptosis induced by angiotensinⅡ

AIM: To study the senescence of human umbilical vein endothelial cells (HUVECs) and Bcl-2, Bax gene expression associated with apoptosis induced by angiotensinⅡ (AngⅡ).METHODS: HUVECs were cultured in vitro and the cell viability was observed by methyl thiazolyl tetrazolium (MTT). HUVECs were intervened by AngⅡ and valsartan (AngⅡ type 1 receptor blocking) and divided into 3 groups: the control group, AngⅡ group (stimulated with AngⅡ10^-6mol/L for 48 h), valsartan group (valsartan was added to cells 1 h before 10^-6mol/L AngⅡ treatment). β-gal staining and cell cycle analysis were used to identify the cell aging status. Morphologic changes and percentage of apoptosis were assayed with Hoechst33258 under fluorescent microscope. The expressions of Bcl-2 and Bax, and the apoptosis-associated genes were detected by immunocytochemical staining, RT-PCR and Western blotting. RESULTS: The cell viability by AngⅡ-induced cells was (81.9%±4.1)%, the positive cell number of β-gal staining was significantly higher in AngⅡ-induced cells (80.10%±6.81)% than that in the control cells. The cell cycle was at G₀-G₁(91.36%±6.45)%, the apoptotic cells significantly increased (31.84±2.86)% under fluorescent microscope. In valsartan group, Bcl-2 mRNA and protein expression increased markedly (P<0.05), but Bax mRNA and protein expression decreased evidently (P<0.05) compared to those in the AngⅡ group.CONCLUSION: Cell apoptosis is possibly an important factor for endothelial cell senescence and vascular aging induced by AngⅡ. One of its molecular mechanisms might be associated with decreasing the expression level of Bcl-2 and increasing that of Bax, which regulate the imbalance between mRNA and protein expression of Bcl-2 and Bax. Valsartan improves endothelial cell aging.     

Effects of survivin inhibitor YM155 on mitochondrial apoptotic pathway of retinoblastoma Y79 cells

AIM: To investigate the effects of survivin inhibitor YM155 {4,9-dihydro-1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(2-pyrazinylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide} on the apoptosis, mitochondrial membrane potential (Δψ_m) and cytochrome C (Cyt C) of retinoblastoma Y79 cells, and to analyze the mitochondrial mechanisms of apoptosis.METHODS: Y79 cells were cultured in vitro and treated with YM155 at concentrations of 0, 0.5, 1, 2, 4 and 8 nmol/L. The cells in control group were treated without YM155. The proliferation of Y79 cells were measured by CCK-8 assay and bromodeoxyuridine (BrdU) labeling assay. Y79 cells were randomly divided into 4 groups:control group (with equal volume of RPMI-1640 nutrient medium), positive control group (10 nmol/L topotecan), low-dose (1 nmol/L) YM155 group and high-dose (2 nmol/L) YM155 group. The effects of YM155 on the apoptosis, the changes of Δψ_m, the mitochondrial distribution and the protein level of Cyt C in the Y79 cells were evaluated by flow cytometry with Annexin V-FITC/PI staining, JC-1 staining, immunofluorescence analysis and Western blot, respectively. RESULTS: Compared with control group, YM155 significantly inhibited the proliferation of Y79 cells and induced apoptosis (P<0.05). YM155 significantly reduced Δψ_m of the Y79 cells, promoted Cyt C which released from mitochondria to the cytosol and reduced the protein level of Cyt C in the mitochondria (P<0.05). CONCLUSION: YM155 inhibits Y79 cell proliferation and induces apoptosis, and the possible mechanisms may be involved in the mitochondrium-mediated apoptotic pathway.     

Relationship between polymorphism of angiotensin I converting enzyme gene insertion/deletion and ACE,PAI-1 activity in patients with myocardial infarction

AIM:To explore the relationship between polymorphism of angiotensin I converting enzyme gene insertion/deletion (I/D) and ACE, PAI-1 activity in patients with myocardial infarction (MI). METHODS:Ninety-three patients with MI and eighty-seven healthy controls were tested. ACE genomic DNA was amplified using the polymerase chain reaction (PCR). Serum ACE activity was measured by colorimetry, plasma level of PAI-1 activity was determined by spectrophotometric assay. RESULTS:① The frequency of ACE DD genotype and D alleles (32.3% and 54.3%) in MI group was significantly higher than those in control group (12.6% and 37.4%, P<0.01, respectively). ② The ACE activity in serum (216.00±58.26)U/L and plasma PAI-1 activity (0.85±0.19)AU/mL in MI group were significantly higher than those in control group (170.19±48.99)U/L, (0.66±0.20)AU/mL, P<0.01, respectively. The serum ACE activity was positively correlated with plasma PAI-1 activity both in MI group and control group (r=0.7108 and r=0.7829;P<0.01, respectively). ③ In MI group, the serum ACE activity and plasma PAI-1 activity showed a significantly higher level in subjects with DD genotype (251.64±57.76)U/L, (0.96±0.16)AU/mL than those with ID (211.47±51.87)U/L, (0.82±0.18) AU/mL and Ⅱ genotypes (179.84±52.65)U/L, (0.71±0.17)AU/mL. The serum ACE activity and plasma PAI-1 activity were significantly higher in subjects with ID genotype than those with II genotype (P<0.05). In control group, the serum ACE activity and plasma PAI-1 activity showed a significantly higher level in subjects with DD genotype (195.53±54.76)U/L, (0.78±0.20)AU/mL than the subjects with Ⅱ genotype (154.98±52.74)U/L, (0.59±0.17)AU/mL (P<0.05). CONCLUSION:The increased ACE activity caused by DD polymorphism may play an important role in elevating the level of plasma PAI-1. The DD genotype of ACE is associated with high PAI-1 level. The genetic variation of ACE contributes to the balance of fibrinolytic pathway, indicating the pathogenesis mechanisms linking to the ACE I/D genotype and MI.     

Hemangioblastic characteristics of human adipose tissue-derived adult stem cells in vivo

AIM: To investigate whether Flk1+CD31-CD34- cells isolated from human adult adipose tissue have characteristics of hemangioblasts in vivo. METHODS: After sublethally irradiated (300cGy) with a caesium source, the female non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice were injected with human adipose tissue-derived Flk1+CD31-CD34- cells (105 cells per mouse) via tail vain with 0.4 mL Roswell Park Memorial Institute medium (RPMI-1640). The control mice received the same volume of RPMI-1640 medium. All mice were killed 2 months after transplantation for further study. The differentiation potential of Flk1+CD31-CD34- cells was assessed in bone marrow and gastrointestinal tract by the methods of flow cytometry, RT-PCR, FISH, and triple-color immunofluorescence. RESULTS: Flk1+CD31-CD34- human adipose tissue-derived adult stem cells differentiated into endothelial cells and hematopoietic cells at the single-cell level in vivo. CONCLUSION: Human adult adipose tissue-derived Flk1+CD31-CD34- cells bear characteristics of hemangioblast in vivo and may have potential application for the treatment of hematopoietic and vascular diseases.