Effects of interferon-γ, tumor necrosis factor-α and interleukin-1β on apoptosis of human airway smooth muscle cellsin vitro

AIM:To clarify if interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)can induce apoptosis of human airway smooth muscle cells (ASMCs) in vitro.METHODS:Human ASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Passage 4-6 cell was used in the experiment. IFN-γ,TNF-α and IL-1β, were used separately or together in the treatment of human ASMCs. The effects of IFN-γ,TNF-α and IL-1β on the growth of the cells was detected by MTT method at the hour 0,24,48 and 72. Light microscopy and electron microscopy were used to examine the morphological change. DNA fragmentation was analyzed by agarose gel electrophoresis. SP immunohistological staing method was performed to detect the change of expressions of p 53, bcl- 2 and bax gene. The apoptosis cell percentage were detected by in situ end labeling technique (TUNEL)of fragmental DNA. RESULTS:(1)IFN-γ or IFN-γ together with TNF-α and IL-1β decreased the number of viable cells in a time dependent manner. (2) Light and electron microscopic examination showed cell shrinkage, membrane blebbing, nuclear contraction, chromatin condensation and nuclear fragmentation in human ASMCs. (3) Agarose gel electrophoresis showed a characteristic"ladder"of DNA bands representing integer multiples of the internucleosomal fragments (about 180-200 bp) in cytokine cotreated human ASMCs. (4)The expression of p 53 and bax gene in cytokine cotreated group was significantly higher than in control group, but the expression of bcl-2 gene was lower than in control group. (5)Stimultaneous treatment with IFN-γ(4×10⁵ U/L),TNF-α(4×10⁵ U/L)and /or IL-1β (10×10⁴ U/L) induced apoptosis of human ASMCs. Apoptotic index of human ASMCs in cytokine co-treated group was significantly higher than in control group (P＜0.01).CONCLUSION:Stimultaneous treatment with IFN-γ,TNF-α and /or IL-1β induced apoptosis of human ASMCs. These immune cytokines may play an important role in airway remodeling of asthma and of chronic obstructive pulmonary disease.     

CB2 receptor agonist JWH133 exerts protective effects on rat model of paraquat-induced acute lung injury

AIM: To study the protective effects of cannabinoid CB2 receptor agonist JWH133 on rat acute lung injury induced by paraquat (PQ).METHODS: Male Sprague-Dawley rats (n=72) were randomly divided into 4 groups. PQ group: PQ was administered intraperitoneally at the dose of 20 mg/kg; Low-dose JWH133 pretreatment group (L-JWH133 group): JWH133 (5 mg/kg, ip) was administered 1 h before PQ exposure; high-dose JWH133 pretreatment group (H-JWH133 group): JWH133 (20 mg/kg, ip) was administered 1 h before PQ exposure; control group: 1 mL saline was administered intraperitoneally. Arterial blood, bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected at 8 h, 1 d and 3 d after PQ exposure. PaO2 and the levels of TNF-α and IL-1β in BALF were measured via blood gas analyzer and ELISA, respectively. The pathological changes and lung injury scores were assessed at 3 d after PQ exposure. NF-κB and AP-1 protein levels were also determined by Western blotting.RESULTS: The decrease in PaO2, structural injury of the lung tissues, interstitial pulmonary edema, and the increase in IL-1β and TNF-α in BALF were observed in PQ-treated rats compared with control group. JWH133 pretreatment reduced the degree of lung tissue injury, decreased the levels of IL-1β and TNF-α in BALF and the NF-κB and AP-1 protein expression in the lung tissue compared with PQ group, especially in H-JWH133 group. CONCLUSION: CB2 receptor agonist JWH133 inhibits NF-κB and AP-1 protein expression in the lung tissues, and reduces the secretion of IL-1β and TNF-α in BALF after paraquat exposure, thus attenuating paraquat-induced acute lung injury.     

Poly(adenosine diphosphate ribose) glycohydrolase regulates apoptosis-inducing factor and inflammatory factors in rat hippocampus after seizures

AIM: To investigate the effects and molecular mechanisms of poly(adenosine diphosphate ribose) glycohydrolase (PARG) on rat hippocampus neurons after seizures and to study the effects of gallotannin on the expression of apoptosis-inducing factor (AIF), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in rat hippocampus after seizures. METHODS: Seizures were induced by kainic acid (KA). The damage of hippocampus neurons was evaluated by Nissl staining. The protein expression levels of poly(adenosine diphosphate ribose) (PAR), AIF, IL-1β and TNF-α were detected by Western blotting analysis. RESULTS: The number of damaged hippocampal pyramidal neurons in gallotannin-treated group was significantly lower than that in KA-treated group (P<0.05). The expression of PAR was increased in gallotannin-treated group compared with KA-treated group(P<0.05).AIF in mitochondrial fraction increased and its accumulation in nucleus fraction decreased in gallotannin-treated group compared with KA-treated group (P<0.05). In addition, gallotannin significantly decreased the protein expression of IL-1β and TNF-α, which were obviously increased in hippocampus after seizures (P<0.05). CONCLUSION: PARG inhibition by gallotannin has the neuroprotective effect on the damage of hippocampal neurons induced by seizures. In addition, gallotannin suppresses the translocation of AIF from mitochondria to nucleus and the expression of IL-1β and TNF-α in rat hippocampus after seizures.     

Pretreatment with external trigeminal nerve electrostimulation inhibits seizures and decreases expression of IL-1β and TNF-α in hippocampus with status epilepticus induced by pentylenetetrazol in rats

AIM:To observe the effect of pretreatment with external trigeminal nerve electrostimulation (eTNS) on behavioral changes and the expression of interleukin-1β (IL-1β) and 　tumor necrosis factor α (TNF-α) in hippocampus of pentylenetetrazol (PTZ)-treated rats. METHODS:The rats were randomly divided into control group, PTZ group and eTNS group, and kindled by PTZ after administered 7 d, 14 d and 28 d of consecutive fake electrostimulation or eTNS. Subsequently, the severity and duration of seizure were quantitatively evaluated. The concentrations of IL-1β and TNF-α in hippocampus were detected by the methods of ELISA and immunohistochemisty. RESULTS:Compared with PTZ group, treatment with eTNS significantly inhibited the severity and duration of seizure (P<0.05), and significantly reduced the content of IL-1β and TNF-α in the hippocampus after status epilepticus (P<0.05 or P<0.01). CONCLUSION:Pretreatment with eTNS may provide a new approach for prevention and treatment of epileptogenesis.     

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.     

Effects of atorvastatin on expression of PAPP-A induced by TNF-α and IL-1β in rat aortic endothelial cells

AIM: To investigate the effects of atorvastatin on the expression of pregnancy-associated plasma protein A(PAPP-A)induced by TNF-α and IL-1β in endothelial cells. METHODS: The rat aortic endothelial cells were isolated from thoracic aortas and cultured by the tissue explant method. The cells in passage 3-4 were used in the experiment and were randomly divided into 4 groups: blank control group: the cells were treated without any intervention; atorvastatin concentration groups: the cells were incubated with atorvastatin at the concentrations of 0.1, 1 and 10 μmol/L for 24 h; atorvastatin time groups: the cells were incubated with atorvastatin at the concentration of 10 μmol/L for 6 h,12 h and 24 h; atorvastatin+inflammatory factors groups: the cells were pre-incubated with 60 μg/L TNF-α or 20 μg/L IL-1β for 1 h, then different concentrations of atorvastatin (0.1, 1.0, 10 μmol/L) were added for 6 h,12 h and 24 h. MTT reduction assay was used to observe the cell proliferation. The mRNA expression of PAPP-A was detected by RT-PCR. The protein level of PAPP-A in the supernatants of cultured cells was measured by ELISA. RESULTS: Compared with blank control group, no significant change of cell proliferation was observed after the intervention of atorvastatin and TNF-α/IL-1β for 3 h, 6 h, 12 h, 24 h and 48 h, indicating that the drugs had no toxic effects on the cells. No significant difference of PAPP-A expression between atorvastatin groups and blank control groups was found. Compared with TNF-α groups and IL-1β groups, PAPP-A expressions in atorvastatin intervention groups significantly decreased. The protein level of PAPP-A was gradually decreased with the raised concentration of atorvastatin and the prolonged time in a concentration- and time-dependent manner. CONCLUSION: Atorvastatin doesn't influence the PAPP-A expression, but inhibits the expression of PAPP-A activated by inflammatory factors in a concentration- and time-dependent manner in primary cultured rat aortic endothelial cells.     

Experimental study on anti-endotoxin activity of a tetrahydropyrimidine derivative,ZL-5015

AIM: To investigate the protective effect of 1, 3-dicyclopentyl-1, 2, 3, 6-tetrahydropyrimidine-4, 5-dicarboxylic acid diethyl ester (ZL-5015) on lethal endotoxin-challenged mice and to explore the underlying mechanism. METHODS: Mouse model of lethal endotoxin challenge and endotoxemia were established by intraperitoneal administration of lipopolysaccharide (LPS) at a dose of 70 mg/kg to the C57BL/6J mice. Mouse peritoneal macrophages stimulated with LPS (10 mg/L) were used as an in vitro inflammatory model. The levels of interleukin-1β (IL-1β), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was used to evaluate the mRNA expression of the cytokines. RESULTS: Prophylactic treatment of the mice with ZL-5015 (100 and 200 mg/kg, ig) slightly increased the survival rate, extended the survival time, decreased the serum levels of IL-1β and TNF-α, and increased the serum level of IL-10 in the early stage of endotoxemia as compared with model group. The results of in vitro study demonstrated that treatment of the endotoxin-stimulated mouse peritoneal macrophages with ZL-5015 (10, 20 and 40 μmol/L) inhibited the expression of IL-1β and TNF-α at both mRNA and protein levels but promoted the expression of IL-10 at both mRNA and protein levels. CONCLUSION: The tetrahydropyrimidine derivative ZL-5015 shows a moderate anti-endotoxin effect by increasing the survival rate and extending the survival time of the mice challenged by endotoxin, which may result from inhibition of the expression of pro-inflammatory cytokines such as IL-1β and TNF-α, and promotion of the expression of anti-inflammatory cytokine IL-10.     

Role of NOX1 in TNF-α-induced oxidative damage and inflammation in alveolar epithelial cells

AIM: To explore the role of NADPH oxidase 1 (NOX1) in tumor necrosis factor-α (TNF-α)-induced oxidative damage and inflammation in alveolar epithelial cells.METHODS: The mRNA and protein expression levels of NOX1 in alveolar epithelial cells after TNF-α treatment were determined by real-time PCR and Western blot. NOX1 siRNA and its negative control were transfected into the alveolar epithelial cells. After the induction of TNF-α, NOX1 levels in the cells were measured by real-time PCR and Western blot, and the content of malondialdehyde (MDA) in the cells was detected by thiobarbituric acid method. Xanthine oxidation assay was used to detect the activity of superoxide dismutase (SOD) in the cells. The contents of interleukin-4 (IL-4), IL-6 and IL-1β in cell culture medium were examined by ELISA. The rate of apoptosis was analyzed by flow cytometry. Western blot was used to detect the level of apoptotic protein cleaved caspase-3.RESULTS: The expression of NOX1 at mRNA and protein levels in TNF-α-induced cells was increased after induction (P<0.05). After transfection of NOX1 siRNA, the expression of NOX1 at mRNA and protein levels in the cell was downregulated (P<0.05). Transfection of siRNA negative control had no effect on the expression level of NOX1 in the cells. The content of MDA in the cells after TNF-α treatment was increased, the activity of SOD was reduced, the releases of IL-4, IL-6 and IL-1β by the cells were increased, and the apoptotic rate and the level of apoptotic protein cleaved caspase-3 were increased as compared with the cells that were not treated with TNF-α (P<0.05). The content of MDA in the cells with NOX1 knockdown induced by TNF-α was reduced, the activity of SOD elevated, and the releases IL-4, IL-6 and IL-1β, the apoptotic rate and the level of apoptotic protein cleaved caspase-3 decreased, as compared with the cells only treated with TNF-α induction (P<0.05).CONCLUSION: TNF-α induces the expression of NOX1 in the alveolar epithelial cells. Knockdown of NOX1 expression reduces cellular oxidative damage, releases of inflammatory factors, and cell apoptosis.     

Effect of NOD8 on production of nitric oxide,IL-1β and TNF-α in LPS-treated macrophages

AIM: To investigate the effect of NOD8 on lipopolysaccharide (LPS)-induced releases of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 cells. METHODS: The plasmids of pEGFP-C2 and pEGFP-NOD8 were transfected into RAW264.7 cells respectively. The transfected and non-transfected cells were stimulated by LPS for 0, 6, 12 and 24 h. NO production was evaluated by Griess reagent assay, and the levels of IL-1β and TNF-α were measured by ELISA. The protein expression of NOD8 and the nuclear translocation of nuclear factor κB (NF-κB) p65 subunit were detected by Western blotting. The level of activated caspase-1 was determined by fluorimetric method. RESULTS: Compared with pEGFP-C2 group, the protein expression of NOD8 was significantly elevated in pEGFP-NOD8+LPS group. The releases of NO, IL-1β and TNF-α were obviously increased after RAW264.7 cells were treated with LPS for 6 h, 12 h and 24 h, and while the secretion of NO was significantly reduced in the cells transfected with pEGFP-NOD8 and induced by LPS for 12 h and 24 h, and the release of IL-1β was also significantly reduced at 6 h, 12 h and 24 h. However, no significant difference of TNF-α release was observed between pEGFP-C2+LPS group and pEGFP-NOD8+LPS group. The activation of caspase-1 in RAW264.7 cells stimulated with LPS for 6 h, 12 h and 24 h was markedly increased, and the expression of NF-κB p65 subunit in the cytoplasm was significantly decreased, indicating that p65 nuclear translocation was increased. In addition, the activation of caspase-1 and the nuclear translocation of p65 were significantly inhibited in pEGFP-NOD8+LPS group. CONCLUSION: NOD8 suppresses the releases of LPS-induced NO and IL-1β in RAW264.7 cells by inhibiting the activation of caspase-1 and NF-κB.     

Artemisinin attenuates intestinal epithelial barrier damage induced by LPS

AIM: To investigate the effect of artemisinin on lipopolysaccharide(LPS)-induced intestinal epithelial barrier damage in IEC-6 cells and its molecular mechanism. METHODS: Cultured IEC-6 cells were divided to 5 groups: control group, LPS(100 mg/L) group and LPS+Artemisinin(30, 50 and 100 μmol/L) groups. The cytotoxicity was detected by MTT assay. The releases of TNF-α, IL-1β and IL-6 in the IEC-6 cells were measured by ELISA. The transepithelial electrical resistance(TER) was detected by electrical resistance tester, and the horseradish peroxidase(HRP) flux permeability were analyzed by a microplate reader. The expression of tight junction proteins, ZO-1, claudin-1 and occludin, and the expression of TLR4/MyD88/NF-κB at mRNA and protein levels were determined by RT-qPCR and Western blot. RESULTS: Artemisinin alone(up to 100 μmol/L) or in combination with LPS(100 mg/L) was not toxic to IEC-6 cells. Compared with control group, the releases of TNF-α, IL-1β and IL-6 in the culture supernatant of IEC-6 cells significantly increased after treatment with LPS. The expression of TLR4/MyD88/NF-κB was activated by LPS. LPS down-regulated the protein expression of ZO-1, claudin-1 and occludin. However, artemisinin treatment decreased the releases of TNF-α, IL-1β and IL-6 in the culture supernatant of IEC-6 cells. The expression of TLR4/MyD88/NF-κB at mRNA and protein levels was gradually reduced after treatment with artemisinin. In addition, artemisinin upregulated the protein expression of ZO-1, claudin-1 and occludin significantly(P<0.01) in a dose-dependent manner. CONCLUSION: Artemisinin attenuates LPS-induced intestinal epithelial barrier damage by inhibiting TLR4/MyD88/NF-κB activation in the IEC-6 cells.     

Role of p38MAPK in production of pro-inflammatory cytokines in Kupffer cells from severe acute pancreatitis rats

AIM:To investigate the role of p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in the Kupffer cells (KCs) production of pro-inflammatory cytokines, tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β), in severe acute pancreatitis (SAP) rats.METHODS:Sprague-Dwaley rats were randomized into three groups:①sham operation rats, ②SAP rats, ③SAP rats given the p38 MAPK inhibitor CNI-1493(10 mg/kg, iv). The SAP model was induced by the bili-pancreatic duct infusion with 5% sterile soduim taurocholate solution. Rats from each group were killed at 12 h after sham operation or SAP and Kupffer cells (KCs) were isolated. The mRNA expressions of TNF-α and IL-1β (by quantitative real-time RT-PCR) and p38 MAPK activity (by Western blot analysis) in KCs were examined. The levels of TNF-α and IL-1β in plasma were determined by ELISA.RESULTS:There was a significant acvitation of p38 MAPK in KCs harvested from SAP rats than those from sham operation rats. SAP also promoted the mRNA expressions of TNF-α and IL-1β in KCs and the plasma levels of TNF-α and IL-1β. These events were significantly inhibited by treatment with CNI-1493.CONCLUSIONS:p38 MAPK activation is one important aspect of the signaling events that may mediate the KCs production of pro-inflammatory cytokines, TNF-α and IL-1β, in SAP rats. The inhibition of the p38 MAPK may be a potential target in the prevention and treatment of SAP.     

Effects of icariin preconditioning on inflammatory responses in myocar-dial ischemia-reperfusion injury

AIM:To observe the effects of icariin on myocardial ischemia-reperfusion injury. METHODS:The left anterior descending coronary artery was ligated for 30 min and then loosened for 2 h to establish the rat model of myocardial ischemia-reperfusion injury. Forty-eight healthy adult male SD rats weighing 250~300 g were randomly divided into sham group, model group, low-, middle-and high-dose icariin groups, and aspirin group. The morphological changes of the myocardium were observed by HE staining. The protein expression of NF-κB p65 in the myocardial nucleus was determined by the method of immunohistochemistry. The content of tumor necrosis factor α (TNF-α) in the myocardial tissues was detected by Western blotting. The level of interleukin 1β (IL-1β) in the serum was measured by ELISA. The activity of myeloperoxidase (MPO) in the myocardial tissues was assayed by colorimetry. RESULTS:Compared with sham group, TNF-α content, IL-1β concentration, NF-κB expression and MPO activity in all other groups increased. Compared with model group, TNF-α content, IL-1β concentration, NF-κB expression and MPO activity in low-, middle- and high-dose icariin groups and aspirin group all decreased. No significant difference of the above parameters between high-dose icariin group and aspirin group was observed. CONCLUSION: Icariin preconditioning reduces inflammatory responses in the process of myocardial ischemia-reperfusion injury in a dose-dependent manner.     

Analysis of cytokines derived from murine yolk sac endothelial cells cultured in vitro

AIM:To purify murine yolk sac endothelial cells (mYS-EC) and investigate the cytokines mRNA expression in mYS-EC. METHODS:The murine yolk sacs were digested with 0.1% collagenase, resuspended in DMEM and counted after digestion and centrifugation. The yolk sac adherent cells were cultured in DMEM containing 15% FBS with 10% mBMEC-CM or 5μg/L VEGF, ECGF and bFGF. The phagocytose function and expression of vWF were evaluated via particle phagocytosis and immunohistochemistry method. Atlas cDNA expression array was used for analysis of cytokine expression in mYS-EC. RESULTS:Colonies consisting of pure yolk sac endothelial cells were obtained in liquid culture system containing 15% FBS and 10% mBMEC-CM or 5μg/L VEGF, ECGF and bFGF. For complete purification of the endothelial cells, subsequent passage was also necessary. Cellular cord formed during passage culture. The endothelial cells were round or oval sharp in morphology, positive in phagocytosis and factor VIII related antigen (von Willebrand's Factor, vWF). The mRNA expressions of cytokines, such as TGF-β2, TNF-α, IFN-γ, FL, BMP-4, MIP-1β, BMP-2A, FLT2, endothelin 2, thymosin β10, IL-6, IL-13, IL-9, SCYA5 and ACBP were detected in mYS-ECs. CONCLUSION:mYS-EC was purified and expanded in vitro. The mRNA expression of 15 kinds of cytokines was detected in mYS-ECs by Atlas arrays.     

Heme oxygenase-1 protects renal function in septic rats via influencing expression of thrombomodulin

AIM: To investigate the protective effect of heme oxygenase-1 (HO-1) on the kidney of septic rats and the influence of HO-1 on the expression of thrombomodulin (TM) in the kidney. METHODS: Sepsis in rats was developed with cecal ligation and puncture (CLP). The septic rats were randomly divided into sham group, CLP group, CLP+HO-1 inducer group and CLP+HO-1 inhibitor group (n=18). The plasma levels of creatinine (Cr), cystatin-C (Cys-C), carboxyhemoglobin (COHb), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and TM, and the changes of prothrombin time (PT) and activated partial thromboplastin time (APTT) in each group were measured. Histopathological examination was performed in the kidney. The expression of TM in the kidney tissue was detected by Western blot. RESULTS: Compared with sham group, significantly elevated plasma levels of Cr, Cys-C, TNF-α, IL-1β and TM (P<0.05), shortened PT and APTT (P<0.05), significantly increased microthrombus formation, and lowered TM expression in the kidney (P<0.05) of CLP group were observed. The administration of hemin lowered the plasma levels of Cr, Cys-C, TNF-α, IL-1β and TM (P<0.05), prolonged PT and APTT (P<0.05), attenuated microthrombus formation, and up-regulated the expression of TM in the kidney (P<0.05). In contrast, ZnPP had the opposite effects. CONCLUSION: HO-1 increases the expression of TM in the kidney and exerts anticoagulatory and antiinflammatory effects, thereby improving renal function in the septic rats.     

miR-451 inhibits inflammatory responses in glomerular mesangial cells by targeting Psmb8 in diabetic nephropathy mice

AIM: To investigate the effect of microRNA (miR)-451 by targeting proteasome subunit β type 8 (Psmb8) on the inflammatory responses in mouse glomerular mesangial cells (MCs) under high-and low-glucose conditions. METHODS: The expression levels of miR-451, IL-18 mRNA and TNF-α mRNA were detected by qPCR. The protein expression levels of IL-18, TNF-α and Psmb8 were determined by Western blot when miR-451 was over-expressed and down-expressed in the MCs. Moreover, the expression of IL-18 and TNF-α was detected when Psmb8 was silenced by si-Psmb8 in MCs. RESULTS: The expression of miR-451 was significantly decreased in the MCs treated with high glucose compared with low glucose group (P<0.01). However, the expression of Psmb8 was increased in high glucose group as compared with low glucose group (P<0.01). Moreover, the expression levels of Psmb8, IL-18 and TNF-α were significantly decreased when miR-451 was over-expressed in high glucose group (P<0.01). Additionally, the expression levels of IL-18 and TNF-α were significantly reduced when Psmb8 was silenced in the MCs under high glucose condition. CONCLUSION: miR-451 reduces the expression of inflammatory factors via targeting Psmb8 in the MCs under high glucose condition. Therefore, miR-451 may play a role in inflammation of diabetic nephropathy.     

Suppressive effect of interferon γ on IL-13-induced fibrosis in fibroblasts

AIM：To investigate the suppressive effect of interferon γ (IFN-γ) on fibrosis induced by interleukin 13 (IL-13) in fibroblasts. METHODS：The fibroblasts were divided into IFN-γ (4×105U/L) group, IL-13 (100 μg/L) group, IFN-γ+IL-13 group and blank control group. At 24 h, 48 h and 72 h, the secreted collagen from fibroblasts was measured by hydroxyproline release assay. The mRNA expression of collagen type I α1 (Col1A1) in fibroblasts was examined by RT-PCR. The protein level of collagen type I synthesized in fibroblasts was analyzed by Western blotting. RESULTS：IFN-γ at 4×105U/ L significantly inhibited the proliferation of fibroblasts and down-regulated Col1A1 mRNA and cellular collagen. The mRNA expression of Col1A1 and the protein level of collagen type I in IFN-γ group were lower than those in blank control group at 48 h and 72 h. At 72 h, the mRNA expression of Col1A1 and the protein level of collagen type I in IL-13 group were substantially higher than those in blank control group, those in IFN-γ + IL-13 group were remarkable lower than those in blank control group, and those in IFN-γ group were also lower than those in blank control group. CONCLUSION：IFN-γ inhibits the fibrotic effect of IL-13 in fibroblasts.     

Therapeutic effect of Jiawei Danzhi Xiaoyao powder combined with intravenous human umbilical cord mesenchymal stem cells on post-stroke depression

AIM: To observe the clinical therapeutic effect of Jiawei Danzhi Xiaoyao powder combined with transplantation of human umbilical cord mesenchymal stem cells (HUC-MSCs) in the treatment of post-stroke depression (PSD) patients and to detect the changes of the serum cytokine levels of the tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, norepinephrine (NE), 5-hydroxytry-ptamine (5-HT) and brain derived neurotrophic factor (BDNF). METHODS: The patients with PSD (n=60) were randomly divided into observation group and control group. The patients in observation group was given Jiawei Danzhi Xiaoyao powder combined with HUC-MSCs, and the patients in control group was given Jiawei Danzhi Xiaoyao powder combined with fluoxetine. The course of treatment was 8 weeks. The effects of the treatments on the patients were evaluated with Hamilton Depression Scale (HAMD). The serum levels of IL-1β, IL-6, TNF-α, 5-HT, NE and BDNF were measured by ELISA. RESULTS: After 8 weeks of treatment, the total effective rate in observation group was significantly higher than that in control group (P<0.05). Compared with the same group before treatment, the HAMD scores and the serum levels of TNF-α, IL-1β, IL-6 of the 2 groups were significantly decreased after treatment (P<0.05), while 5-HT, NE and BDNF were significantly increased (P<0.05). After 8 weeks of treatment, the HAMD scores in observation group was significantly lower than that in control group (P<0.05), and the serum levels of TNF-α, IL-1β, IL-6 were significantly decreased in observation group (P<0.05), while 5-HT, NE and BDNF were significantly increased (P<0.05). CONCLUSION: Jiawei Danzhi Xiaoyao powder combined with human umbilical cord mesenchymal stem cells is effective in the treatment of post-stroke depression. The mechanism may be related to the effects of HUCMSCs such as anti-inflammatory effect, increased release of monoamine neurotransmitters, and stimulation of secretion of neurotrophic factors in the brain.     

Curcumin reduces neuroinflammation stimulated by Aβ25-35 in primary rat microglial cells

AIM: To investigate the effects of curcumin (Cur) on the expression of High mobility group box 1 protein (HMGB1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) in amyloid-β (Aβ)-induced primary rat microglial cells. METHODS: Microglia were derived from the cerebral cortices of postnatal rat brains. The cells were identified by immunocytochemistry using mouse anti rat Iba-1 monoclonal antibody. A cell model using primary rat microglial cells incubated with Aβ_25-35 as an inflammation model of Alzheimer's disease (AD) was set up. The morphological characters of primary rat microglial cells were observed. The concentration of Aβ_25-35 and the treatment concentration of curcumin were selected by CCK-8 assay. Cultured primary rat microglial cells were divided into 5 groups:normal cell group, Aβ_25-35 group, Cur group, Aβ_25-35+Cur group and Aβ_25-35+DMSO group. The expression of HMGB1, NF-κB, and receptor for advanced glycation end products (RAGE) was detected by Western blot. The levels of HMGB1, IL-1β, and TNF-α in the culture supernatant were measured by ELISA. RESULTS: The purity of primary microglias determined by Iba-1 immunofluorescence was more than 95%. The protein levels of HMGB1, RAGE and NF-κB were significantly increased after Aβ_25-35 stimulation. After treatment with Cur, the protein levels of HMGB1, RAGE and NF-κB were significantly decreased (P<0.05). The levels of HMGB1, IL-1β and TNF-α in the supernatant were significantly increased after Aβ_25-35 stimulation. Cur significantly decreased the level of HMGB1, IL-1β and TNF-α in the supernatant. CONCLUSION: Curcumin significantly inhibits neuroinflammation stimulated by Aβ_25-35 in primary rat microglial cells.