OBJECTIVES: To investigate the effects and molecular mechanisms of asiatic acid on β-cell function in type 2 diabetes mellitus (T2DM). METHODS: The T2DM model was established by high fat diet and streptozotocin injection in ICR mice, and the effects of asiatic acid on glucose regulation were investigated in model mice. The islets were isolated from palmitic acid-treated diabetic mice. ELISA was used to detect the glucose-stimulated insulin secretion, tumor necrosis factor (TNF)-α and interleukin (IL)-6. ATP assay was applied to measure ATP production, and Western blotting was used to detect protein expression of mature β cell marker urocortin (Ucn) 3 and mitofusin (Mfn) 2. The regulatory effects of asiatic acid on glucose-stimulated insulin secretion (GSIS) and Ucn3 expression were also investigated after siRNA interference with Mfn2 or treatment with TNF-α. RESULTS: Asiatic acid with the dose of 25 mg·kg^－1·d^－1 had the best glycemic control in T2DM mice and improved the homeostasis model assessment β index. Asiatic acid increased the expression of Mfn2 and Ucn3 protein and improved the GSIS function of diabetic β cells in vitro and in vivo (both P<0.05). Moreover, it improved the ATP production of islets of T2DM mice in vitro (P<0.05). Interfering Mfn2 with siRNA blocked the up-regulation of Ucn3 and GSIS induced by asiatic acid. Asiatic acid inhibited islet TNF-α content and increased Mfn2 and Ucn3 protein expression inhibited by TNF-α. CONCLUSIONS Asiatic acid improves β cell insulin secretion function in T2DM mice by maintaining the β cell maturity, which may be related to the TNF-α/Mfn2 pathway.
Mice ; Animals ; Insulin Secretion ; Diabetes Mellitus, Type 2/drug therapy* ; Islets of Langerhans/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Insulin/therapeutic use* ; Diabetes Mellitus, Experimental ; Mice, Inbred ICR ; Glucose/therapeutic use* ; Interleukin-6/metabolism* ; RNA, Small Interfering/pharmacology* ; Adenosine Triphosphate ; GTP Phosphohydrolases/therapeutic use*

OBJECTIVETo investigate the change in the expression of tight junction protein ZO-1 in intestinal epithelial cells (Caco-2 cells) and the protective effect of eicosapentaenoic acid (EPA) after adherent-invasive Escherichia coli (E.coli) LF82 infection.

METHODSThe Caco-2 cell line was used to establish an in vitro model of tight junction of intestinal epithelial cells. Caco-2 cells were divided into EPA treatment groups (0, 25, 50, 100, and 200 μmol/L EPA) and EPA (0, 25, 50, 100, and 200 μmol/L EPA)+E.coli LF82 treatment (0, 6, and 12 hours) groups. A microscope was used to observe the morphological characteristics of the cells. MTT assay was used to determine the cell growth curve. The activity of alkaline phosphatase (ALP) at both sides of the cell membrane was compared to evaluate the Caco-2 cell model. MTT assay and flow cytometry were used to investigate the effects of different concentrations of EPA on the survival rate and apoptosis rate of Caco-2 cells. RT-qPCR was used to measure the mRNA expression of ZO-1 in Caco-2 cells after EPA and/or E.coli LF82 treatment. ELISA was used to measure the change in the level of tumor necrosis factor-α (TNF-α) in culture supernatant.

RESULTSAfter EPA treatment (25 and 50 μmol/L), the proliferation of Caco-2 cells was induced in a dose-dependent manner. The survival rates of the cells were significantly higher than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant inhibitory effect on the proliferation of Caco-2 cells in a dose-dependent manner. The survival rates of the cells were significantly lower than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant increase in cell apoptosis rate compared with the control group (P<0.05). The 6- and 12-hour E.coli LF82 treatment groups had decreasing mRNA expression of ZO-1 in Caco-2 cells over the time of treatment and had significantly lower mRNA expression of ZO-1 than the untreated group (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed an increase in the mRNA expression of ZO-1 with the increasing concentration of EPA, as well as significantly higher mRNA expression of ZO-1 than the Caco-2 cells treated with E.coli LF82 alone (P<0.05). The Caco-2 cells treated with E.coli LF82 alone for 6 or 12 hours had increasing secretion of TNF-α over the time of treatment and had significantly higher secretion than the untreated Caco-2 cells (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed a reduction in the secretion of TNF-α with the increasing concentration of EPA and had significantly lower secretion than the Caco-2 cells treated with E.coli LF82 alone (P<0.05).

CONCLUSIONSEPA can effectively prevent the destruction of tight junction of intestinal epithelial cells induced by E.coli LF82 infection and inhibit the secretion of inflammatory factors. Therefore, it has a certain protective effect on intestinal mucosal barrier.

Apoptosis ; drug effects ; Caco-2 Cells ; Eicosapentaenoic Acid ; pharmacology ; Escherichia coli ; pathogenicity ; Humans ; Intestinal Mucosa ; metabolism ; microbiology ; RNA, Messenger ; analysis ; Tight Junctions ; drug effects ; Tumor Necrosis Factor-alpha ; secretion ; Zonula Occludens-1 Protein ; genetics

OBJECTIVETo investigate the role of non-receptor tyrosine kinase Tec in the production of TNF-α and IL-1β from macrophages induced by LPS and its related mechanism.

METHODSRAW264.7 mononuclear-macrophages cultured in 6-well plates were divided into 4 groups according to the random number table, with 24 wells in each group. Cells in blank group were routinely cultured (cultured with DMEM medium containing 10% FBS) for 2 hours. Cells in LFM-A13 group were pretreated with 75 µmol/L Tec specific inhibitor LFM-A13 for 1 hour and then routinely cultured for 1 hour. Cells in LPS group were routinely cultured for 1 hour and then treated with 0.1 µg/mL LPS for 1 hour. Cells in LPS+LFM-A13 group were pretreated with 75 µmol/L LFM-A13 for 1 hour and then treated with 0.1 µg/mL LPS for 1 hour. The content of TNF-α and IL-1β in culture supernatant of cells was determined with ELISA. The mRNA expressions of TNF-α and IL-1β in cells were assayed with real-time fluorescent quantitative RT-PCR. The activity of intracellular Tec, p38 MAPK, and transforming growth factor activated kinase 1 (TAK1) was determined with Western blotting. Data were processed with one-way analysis of variance and LSD test.

RESULTSThe content of TNF-α and IL-1β in culture supernatant of cells in LFM-A13 group was close to that in blank group (with P values above 0.05). The mRNA expressions of TNF-α and IL-1β in the cells of LFM-A13 group were close to those of blank group (with P values above 0.05). The content of TNF-α and IL-1β in culture supernatant of cells in LPS group was respectively (1 213 ± 154) and (636 ± 90) pg/mL, which was higher than that in blank group [(330 ± 44) and (211 ± 31) pg/mL, with P values below 0.01]. The mRNA expressions of TNF-α and IL-1β in the cells of LPS group were respectively 1.57 ± 0.22 and 1.44 ± 0.24, which were significantly higher than those of blank group (1.00 ± 0.18 and 1.00 ± 0.19, with P values below 0.01). The content of TNF-α and IL-1β in culture supernatant of cells in LPS+LFM-A13 group was respectively (787 ± 109) and (453 ± 64) pg/mL, which was significantly lower than that in LPS group (with P values below 0.05). The mRNA expressions of TNF-α and IL-1β in the cells of LPS+LFM-A13 group were respectively 1.21 ± 0.15 and 1.21 ± 0.22, and they were significantly lower than those of LPS group (with P values below 0.05). The activity of intracellular Tec, TAK1, and p38 MAPK of cells in LPS+LFM-A13 group was close to that in blank group (with P values above 0.05). The activity of intracellular Tec, TAK1, and p38 MAPK of cells in LPS group was respectively 2.69 ± 0.41, 3.99 ± 0.65, and 2.07 ± 0.31, which was significantly higher than that in blank group (1.00 ± 0.17, 1.00 ± 0.16, and 1.00 ± 0.18, with P values below 0.01) and LPS+LFM-A13 group (1.02 ± 0.17, 1.18 ± 0.20, and 1.58 ± 0.28, P < 0.05 or P < 0.01).

CONCLUSIONSTec promotes the production and release of pro-inflammatory cytokines TNF-α and IL-1β from macrophages induced by LPS via TAK1-p38 MAPK signaling pathway.

Amides ; metabolism ; Cell Line ; Cytokines ; Interleukin-1beta ; metabolism ; secretion ; Lipopolysaccharides ; Macrophages ; metabolism ; Nitriles ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism ; secretion

OBJECTIVETo investigate the effects of zinc deficiency on the relevant immune function in rats with LPS-induced sepsis.

METHODSSixty rats were divided into low zinc group (LZ), normal zinc pair-fed group (NP), and normal zinc control group (NC) according to the random number table, with 20 rats in each group. The rats in group LZ were fed with low zinc diet, and the rats in group NP were fed with normal zinc diet, with the same intake as that of group LZ by manual control, and the rats in group NC were fed with normal zinc diet freely. After being fed for 7 d, the rats all fasted and were further divide into the below subgroups named LZ-LPS, LZ-normal saline (NS), NP-LPS, NP-NS, NC-LPS, and NC-NS according to the random number table, with 10 rats in each subgroup. Rats in the LPS subgroups were intraperitoneally injected with 1 mg/mL LPS solution with the dosage of 5 mg/kg, rats in the corresponding NS subgroups were intraperitoneally injected with equivalent NS. The rats were sacrificed at post injection hour 6 to collect blood, spleen, and thymus. The serum level of zinc was detected by inductively coupled plasma mass spectrometry, and the serum alkaline phosphatase (ALP) activity was detected by automatic blood biochemical analyzer. The body weight and weight of spleen and thymus of rats were weighed, and the indices of spleen and thymus were calculated. Six routine blood indices were examined by automatic blood cell analyzer. The serum levels of interferon gamma (IFN-γ), TNF-α, IL-4, and IL-10 were determined with ELISA, and the ratio of IFN-γ to IL-4 was calculated. Data were processed with one-way analysis of variance and SNK test.

RESULTS(1) Serum levels of zinc and ALP activity in the LPS subgroups were significantly lower than those in the corresponding NS subgroups (with P values below 0.05). The two former indices in subgroups NP-NS and NC-NS were significantly higher than those in subgroup LZ-NS (with P values below 0.05). The two former indices in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS (with P values below 0.05). (2) Body weight, spleen and thymus weight, indices of spleen and thymus in the LPS subgroups were similar with those in the corresponding NS subgroups (with P values above 0.05). The 4 former indices, except for body weight, in subgroups NP-NS and NC-NS were significantly higher than those in subgroup LZ-NS (with P values below 0.05). The 4 former indices, except for body weight, in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS (with P values below 0.05). (3) Levels of leucocyte count in subgroups LZ-LPS and NP-LPS were significantly higher than those in the corresponding NS subgroups (with P values below 0.05). Level of leucocyte count in subgroup NC-NS was significantly higher than that in subgroup LZ-NS (P<0.05). Level of leucocyte count in subgroup NC-LPS was significantly lower than that in subgroup LZ-LPS (P<0.05). Levels of neutrophilic granulocyte count (NGC) and NG in the LPS subgroups were significantly higher than those in the corresponding NS subgroups (with P values below 0.05). The two former indices in subgroup NC-LPS were significantly lower than those in subgroup LZ-LPS (with P values below 0.05). Level of NG in subgroup NC-NS was significantly lower than that in subgroup LZ-NS (P<0.05). Levels of lymphocyte count and lymphocyte in subgroups LZ-NS, LZ-LPS, NP-NS, NP-LPS, NC-NS, and NC-LPS were respectively (1.8 ± 0.4) × 10⁹/L, (1.0 ± 0.3)× 10⁹/L, (2.6 ± 0.7) × 10⁹/L, (1.4 ± 0.4) × 10⁹/L, (3.3 ± 0.6) × 10⁹/L, (1.5 ± 0.5) × 10⁹/L, and 0.39 ± 0.10, 0.11 ± 0.03, 0.47 ± 0.12, 0.14 ± 0.04, 0.50 ± 0.09, 0.24 ± 0.07. The two former indices in the LPS subgroups were significantly lower than those in the corresponding NS subgroups (with P values below 0.05). The two former indices in subgroup NC-NS were significantly higher than those in subgroup LZ-NS (with P values below 0.05). The two former indices in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS (with P values below 0.05). Level of lymphocyte count in subgroup NP-NS was significantly higher than that in subgroup LZ-NS (P<0.05). Levels of platelet count (PC) in subgroups NP-LPS and NC-LPS were significantly lower than those in the corresponding NS subgroups (with P values below 0.05). Levels of PC in subgroups NP-NS and NC-NS were significantly higher than those in subgroup LZ-NS (with P values below 0.05). Level of PC in subgroup NC-LPS was significantly higher than that in subgroup LZ-LPS (P<0.05). (4) Serum levels of TNF-α, IL-4, and IL-10 in each subgroup showed no significant differences (with P values above 0.05). Serum levels of IFN-γ and ratios of IFN-γ to IL-4 in subgroups LZ-NS, LZ-LPS, NP-NS, NP-LPS, NC-NS, and NC-LPS were respectively (75 ± 21), (233 ± 40), (80 ± 14), (345 ± 74), (66 ± 7), (821 ± 189) pg/mL, and 3.1 ± 1.0, 6.6 ± 1.7, 3.9 ± 1.7, 20.2 ± 8.3, 3.4 ± 1.5, 45.7 ± 7.6. The two former indices in the LPS subgroups were significantly higher than those in the corresponding NS subgroups (with P values below 0.05). The two former indices in subgroups NP-NS and NC-NS were similar with those in subgroup LZ-NS (with P values above 0.05). The two former indices in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS (with P values below 0.05).

CONCLUSIONSZinc deficiency can induce the atrophy of spleen and thymus, and reduction of peripheral blood lymphocyte. In sepsis, zinc deficiency can further decrease the production of IFN-γ, thus making the cytokines of Th1/Th2 shift to Th2 and the immune imbalance worse.

Animals ; Cytokines ; Interferon-gamma ; Interleukin-10 ; Interleukin-4 ; Lipopolysaccharides ; pharmacology ; Rats ; Sepsis ; chemically induced ; Tumor Necrosis Factor-alpha ; metabolism ; secretion ; Zinc ; deficiency

OBJECTIVETo investigate the expression of ASK1 and PRKCD in the process of monocyte differentiation, and explore their role in functional changes of hypersplenism spleen macrophages (Mφ) in portal hypertension (PH).

METHODSU937 cells were stimulated to differentiate into monocyte/macrophage-like cells by cultivation in PMA and the mRNA expressions of ASK1 and PRKCD were detected by q-PCR and the changes of protein expression were identified by western blot analysis. The secretion of phagocytose related cytokines such as IL-10 and TNF-α were tested by ELISA, and the function of the macrophage-like cells were studied by chicken red blood cell phagocytose test.

RESULTSThe expressions of PRKCD and ASK1 mRNA were gradually decreased along with the cell differentiation, while the secretion of TNF-α was increased, IL-10 secretion reached a maximum at 24 h after PAM stimulation, and then gradually fell. The expression of ASK1 and p-ASK1 were rapidly increased compared with the non-stimulated U937 cells, while the expression of PRKCD and p-PRKCD were sightly declined. The phagocytose test show that U937 cells induced with PMA were able to swallow the chicken red blood cell.

CONCLUSIONUp-regulated protein expression of ASK1 and p-ASK1 and down-regulated protein expression of PRKCD and p-PRKCD in the process of PMA induced monocyte differentiation, are consist with the expression changes of splenic macrophage phagocytosis in hypersplenism, which leads to increased activity of Mφ.

Cell Differentiation ; Down-Regulation ; Humans ; Hypersplenism ; Hypertension, Portal ; Interleukin-10 ; secretion ; MAP Kinase Kinase Kinase 5 ; physiology ; Macrophages ; cytology ; Phagocytosis ; Protein Kinase C-delta ; physiology ; RNA, Messenger ; Tumor Necrosis Factor-alpha ; secretion ; U937 Cells

OBJECTIVETo investigate extracellular splitting pattern of mitochondria and the depressant effects of CsA on the process and explore the mechanism of post-traumatic SIRS and its therapeutic strategy.

METHODSTen male SD rats with 60 to 70 days age and 240 to 280 g weight were used for mitochondrial isolation. Freshly isolated mitochondria were randomly divided into two groups, which were cultured in blood plasma with or without CsA respectively for 8 h. COX and MDH were assayed by ELISA every 30 min. Meanwhile, Rat macrophage cell line NR8383 were treated as follows, control (group A): cultivation with normal medium; NR8383+CsA co-culture group (group B): culture medium was supplemented with CsA of 10 mmol/L; NR8383+intact mitochondria co-culture group (group C): culture medium was supplemented with intact mitochondria (mtDNA=5 g/ml); NR8383+intact mitochondria+CsA co-culture group (group D): culture medium was supplemented with intact mitochondria (mtDNA=5 μg/ml)and CsA of 10 mmol/L; NR8383+disrupted mitochondria co-culture group (group E): culture medium was supplemented with disrupted mitochondria (mtDNA=5 μg/ml); NR8383+disrupted mitochondria+CsA co-culture group (group F): culture medium was supplemented with disrupted mitochondria (mtDNA=5 μg/ml)and CsA of 10 mmol/L. TNF-α and IL-6 concentrations in supernatant were assessed at 1, 3, 5 h after culture.

RESULTSIn the mitochondria plasma cultures, MDH and COX levels were increased with the time and peaked at about 3 h and 3.5 h; CsA can delay the appearance of peak to 4.5 h. Among different treated groups,there was no significant difference in TNF-α and IL-6 between group A and group B; there was significant difference in TNF-α and IL-6 other groups. After 1 h culture, compared with group C, no significant difference of TNF-α and IL-6 was observed in group D, while TNF-α and IL-6 were significant higher in group E; after 3 h culture, compared with group C, TNF-α and IL-6 were significantly lower in group D, while TNF-α and IL-6 were significantly higher in group E; after 5 h culture, compared with group C, TNF-α and IL-6 were significantly lower in group D, while no significant difference of TNF-α and IL-6 were observed in group E. At each time point, there was no significant difference in TNF-α and IL-6 between group F and group E.

CONCLUSIONMitochondria can split in serum with time, which will further activate macrophages. CsA has depressant effect to mitochondrial splitting on the process and will therefore inhibit the activation of macrophages.

Animals ; Cells, Cultured ; Cyclosporine ; pharmacology ; Interleukin-6 ; secretion ; Male ; Mitochondria ; drug effects ; Prostaglandin-Endoperoxide Synthases ; analysis ; Rats ; Rats, Sprague-Dawley ; Systemic Inflammatory Response Syndrome ; drug therapy ; etiology ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo observe the effect of formula of removing both phlegm and blood stasis (TYTZ) in inhibiting the inflammatory reaction in Chinese mini-swine with coronary atherosclerosis.

METHODTotally 36 Chinese mini-swine were randomly divided to six groups: the normal control group, the model group, the Shujiangzhi group and TYTZ groups with does of 2.0, 1.0 and 0.5 g x kg(-1), and six each in every group. Except for the normal control group, all of other groups were fed with high-fat diet for 2 weeks. Interventional balloons are adopted to injure their left anterior descending artery endothelium. After the operation, they were fed with high-fat diet for 8 weeks to prepare the coronary atherosclerosis model. In the 8th week after the operation and administration, the intravascular ultrasound was adopted to observe the coronary artery plaque burden of each group and the pathological morphology of coronary artery. Such inflammatory factors as high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 were detected by ELISA. The expression of NF-kappaB p65 nuclear translocation was observed by the immunohistochemical method.

RESULTCompared with the normal control group, the model group showed significant increase in the coronary artery plaque burden at the end of the experiment (P < 0.01), notably abnormal structural changes in atherosclerotic vascular tissues, luminal stenosis, a large number of foam cells and inflammatory cell infiltration, remarkable growth of hs-CRP, TNF-alpha and IL-6 levels (P < 0.01). The immunohistochemical staining also showed the significant increase in the NF-kappaB p65 nuclear translocation of coronary artery of Chinese mini-swine in the model group. Compared with the model group, TYTZ could significantly attenuate atherosclerotic plaque burden (P < 0.01), inhibit the coronary luminal stenosis, reduce inflammatory cell infiltration, decrease such inflammatory cell factors as hs-CRP, TNF-alpha and IL-6 in serum, and inhibit the NF-kappaB p65 nuclear translocation of coronary artery (P < 0.05 or P < 0.01).

CONCLUSIONTYTZ can reduce the downstream inflammatory reaction by controlling NF-kappaB p65 nuclear translocation, so as to inhibit the occurrence and development of coronary atherosclerotic plaque in Chinese mini-swine.

Animals ; C-Reactive Protein ; metabolism ; Coronary Artery Disease ; blood ; complications ; drug therapy ; pathology ; Female ; Inflammation ; complications ; Interleukin-6 ; blood ; Male ; Medicine, Chinese Traditional ; methods ; Mucous Membrane ; drug effects ; secretion ; Swine ; Swine, Miniature ; Tumor Necrosis Factor-alpha ; blood

This study is to evaluate the effects of the metformin (Met) on β cell function of diabetic KKAy mice. Female diabetic KKAy mice selected by insulin tolerance test (ITT) were divided randomly into two groups. Con group was orally administered by gavage with water, Met group with metformin hydrochloride at a dose of 0.2 g x kg(-1) for about 12 weeks. ITT and glucose tolerance tests (OGTT) were determined. Beta cell function was assessed by hyperglycemic clamp. Pancreatic biochemical indicators were tested. The changes of gene and protein expression in the pancreas and islets were also analyzed by Real-Time-PCR and immunostaining. Met significantly improved glucose intolerance and insulin resistance in KKAy mice. Fasting plasma glucose and insulin levels were also decreased. In addition, Met markedly increased glucose infusion rate (GIR) and elevated the Ist phase and maximum insulin secretion during clamp. It showed that Met decreased TG content and iNOS activities and increased Ca(2+) -Mg(2+)-ATPase activity in pancreas. Islets periphery was improved, and down-regulation of glucagon and up-regulated insulin protein expressions were found after Met treatment. Pancreatic mRNA expressions of inflammation factors including TLR4, NF-κB, JNK, IL-6 and TNF-α were down-regulated, p-NF-κB p65 protein levels also down-regulated by Met. And mRNA expressions of ion homeostasis involved in insulin secretion including SERCA2 and Kir6.2 were up-regulated by Met. Met increased SIRT5 expression level in pancreas of KKAy mice under the hyperglycemic clamp. These results indicated that chronic administration of Met regulated pancreatic inflammation generation, ion and hormone homeostasis and improved β cell function of diabetic KKAy mice.
Animals ; Blood Glucose ; Diabetes Mellitus, Experimental ; drug therapy ; Down-Regulation ; Female ; Glucose Tolerance Test ; Homeostasis ; Inflammation ; drug therapy ; Insulin ; secretion ; Insulin Resistance ; Insulin-Secreting Cells ; drug effects ; Interleukin-6 ; metabolism ; Metformin ; pharmacology ; Mice ; NF-kappa B ; metabolism ; Pancreas ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the effect of bone marrow mesenchymal stem cells (BMSCs) on secretion of inflammatory cytokines in macrophages stimulated by lipopolysacharide (LPS).

METHODSRat BMSCs and macrophages were isolated, cultured, and identified. The BMSCs and macrophages, cultured alone or in co-culture, were treated with LPS or PBS or without treatment and tested for interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) concentrations in the supernatants at 1, 3, 5, 7, 12, and 24 h after the treatment using enzyme-linked immunosorbent assay.

RESULTSExposure to LPS caused significantly increased IL-10 and TNF-α concentrations in the supernatant of cultured macrophages but not in BMSC culture. Macrophages co-cultured with BMSCs showed significantly lowered IL-10 and TNF-α secretions in response to LPS exposure as compared with the macrophages cultured alone.

CONCLUSIONBMSCs can reduce LPS-induced secretion of inflammatory cytokines by the macrophages to ameliorate inflammatory reactions.

Animals ; Cells, Cultured ; Coculture Techniques ; Enzyme-Linked Immunosorbent Assay ; Inflammation ; Interleukin-10 ; secretion ; Lipopolysaccharides ; Macrophages ; secretion ; Mesenchymal Stromal Cells ; cytology ; Rats ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo explore the role of HIV-1 tat gene variations in AIDS dementia complex (ADC) pathogenesis.

METHODSHIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC and an AIDS patient without ADC were cloned for sequence analysis. HIV-1 tat gene sequence alignment was performed by using CLUSTAL W and the phylogentic analysis was conducted by using Neighbor-joining with MEGA4 software. All tat genes were used to construct recombinant retroviral expressing vector MSCV-IRES-GFP/tat. The MSCV-IRES-GFP/tat was cotransfected into 293T cells with pCMV-VSV-G and pUMVC vectors to assemble the recombinant retrovirus. After infection of gliomas U87 cells with equal amount of the recombinant retrovirus, TNF-α, and IL-1β concentrations in the supernatant of U87 cells were determined with ELISA.

RESULTSHIV-1 tat genes derived from peripheral spleen and central basal ganglia of the AIDS patient with ADC and the other one without ADC exhibited genetic variations. Tat variations and amino acid mutation sites existed mainly at Tat protein core functional area (38-47aa). All Tat proteins could induce U87 cells to produce TNF-α and IL-1β, but the level of IL-1β production was different among Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen. The level of Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen were obviously higher than that from the non-ADC patient's basal ganglia.

CONCLUSIONTat protein core functional area (38-47aa) may serve as the key area of enhancing the secretion of IL-1β. This may be related with the neurotoxicity of HIV-1 Tat.

AIDS Dementia Complex ; metabolism ; pathology ; virology ; Adult ; Amino Acid Sequence ; Basal Ganglia ; virology ; Cell Line, Tumor ; Gene Expression Regulation, Viral ; Genes, tat ; HIV-1 ; genetics ; pathogenicity ; Humans ; Interleukin-1beta ; biosynthesis ; genetics ; secretion ; Middle Aged ; Molecular Sequence Data ; Neuroglia ; pathology ; secretion ; Spleen ; virology ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics ; secretion ; tat Gene Products, Human Immunodeficiency Virus ; genetics ; physiology