Atorvastatin inhibits IL-1β and IL-18 releases via lowering the expression of NLRP1 inflammasome

AIM: To explore the role of nucleotide-binding oligomerization domain-like receptor protein 1 (NLRP1) inflammasome in atorvastatin-induced reduction of interleukin-1β (IL-1β) and interleukin-18 (IL-18) releases from the THP-1 macrophages. METHODS: Lipopolysaccharide (LPS, 10 μg/L) was used to trigger the secretion of IL-1β and IL-18 in the THP-1 macrophages. The cells were incubated with different concentrations of atorvastatin (1, 10 and 20 μmol/L) for 24 h, or treated with 10 μmol/L atorvastatin for different time (12 h, 24 h and 48 h). NLRP1 siRNA was transfected into the THP-1 cells. The mRNA expression of NLRP1 inflammasome was detected by RT-PCR. The protein expression of NLRP1 inflammasome was determined by Western blot. The secretion of proinflammatory cytokines IL-1β and IL-18 was quantified by ELISA. RESULTS: Atorvastatin inhibited the mRNA and protein expression of NLRP1 inflammasome in the THP-1 macrophages in a dose- and time-dependent manner. Transfection of NLRP1 siRNA significantly decreased the protein expression of NLRP1 and promoted the suppressive effect of atorvastatin on IL-1β and IL-18 secretion in the THP-1 macrophages. CONCLUSION: Atorvastatin inhibits the production of IL-1β and IL-18 in the macrophages through decreasing NLRP1 inflammasome expression, possibly contributing to the anti-inflammatory effect of atorvastatin on atherosclerosis.     

Relationship between NLRP3 inflammasome-IL-1β axis and End-MT after acute myocardial infarction in rats

AIM: To investigate whether activation of NLRP3 inflammasome-IL-1β axis is consistent with endothelial-mesenchymal transition (End-MT) during the process of myocardial fibrosis after acute myocardial infarction (AMI). METHODS: Adult male SD rats (n=30) were randomly divided into sham operation group (n=15) and AMI group (n=15). After 28 d, Masson staining was used to detect the level of myocardial fibrosis. The activation of NLRP3 inflammasome including NLRP3, ASC, pro-caspase-1 and caspase-1, the endothelial cell markers CD31 and VE-cadherin, and the mesenchymal cell markers α-SMA and FSP1 were analyzed by Western blot. The expression of IL-1β was measured by ELISA. RESULTS: The levels of myocardial fibrosis and End-MT, the activation of NLRP3 inflammasome, and the expression of caspase-1 and IL-1β were significantly increased in AMI group compared with sham operation group (P<0.05). CONCLUSION: The activation of NLRP3 inflammasome-IL-1β axis is significantly consistent with End-MT process, suggesting that NLRP3 inflammasome-IL-1β, as a potential target for the activation of End-MT, will provide a novel theoretical target for the treatment of myocardial fibrosis and heart failure after AMI.     

SO2 derivatives stimulate inflammation of airway epithelial cells through NLRP3 inflammasome

AIM:To investigate the effect of sulfur dioxide (SO₂) derivatives (sodium sulfite and sodium bisulfate) on NLRP3 inflammasome in airway epithelial cells. METHODS:SO₂ derivatives at different concentrations were applied to bronchial epithelial 16HBE cells for 12 h. The production of reactive oxygen species (ROS) was detected by flow cytometry. The protein levels of NLRP3 and caspase-1 p20 were analyzed by Western blot. The level of interleukin-1β(IL-1β) in the cell culture supernatant was measured by ELISA. The cell viability was measued by MTT assay, and the concentration of SO₂ derivatives used in the following experiments was 2 mmol/L. When the NLRP3 gene in 16HBE cells was silenced by RNA interference technique or N-acetyl cysteine (NAC) was used to pretreat 16HBE cells, the intracellular ROS was detected by flow cytometry, and the protein levels of NLRP3 and caspase-1 p20 and the secretion of IL-1β were determined by Western blot and ELISA, respectively. RESULTS:Compared with the control group, the level of intracellular ROS, the protein levels of NLRP3 and caspase-1 p20, and the secretion of IL-1β in cell supernatant were increased significantly in 2 mmol/L and 4 mmol/L SO₂ derivative groups (P<0.05). Compared with the 2 mmol/L group, the protein levels of NLRP3 and caspase-1 p20 were significantly inhibited in NLRP3 siRNA group (P<0.05). The concentration of IL-1β in the cell culture supernatant was significantly decreased (P<0.05). No significant difference of ROS level was observed. Significantly decreased protein levels of NLRP3 and caspase-1 p20, and the concentration of IL-1β in NAC group were found (P<0.05). CONCLUSION:SO₂ derivatives directly promote the production of IL-1β through NLRP3 inflammasome in bronchial epithelial cells.     

Glyburide reduces PFOS-induced lung injury in young rats through inhibiting NLRP3 inflammasome

AIM: To explore the possible mechanism of NLR family Pyrin domain-containing protein 3 (NLRP3) inflammasome involved in perfluorooctane sulfonate (PFOS)-induced lung injury in young rats. METHODS: Twenty-eight SD rats (21-day-old) were randomly divided into control (C) group, PFOS (P) group, glyburide (G) group and glyburide + PFOS (GP) group. PFOS exposure model and glyburide protection model were established. The lung specimens were collected for HE staining. The levels of myeloperoxidase (MPO) in the lung tissues, interleukin-1β (IL-1β) and interleukin-18 (IL-18) in the bronchoalveolar lavage fluid (BALF) were measured by ELISA. The concentration of PFOS in serum was measured by high-performance liquid chromatography (HPLC). The protein expression of NLRP3, caspase-1 and apoptosis-associated speck-like protein containing CARD (ASC) in the lung tissues was determined by Wes-tern blot. RESULTS: HE staining of lung tissues showed that compared with the control rats, there were obvious inflammatory infiltration in trachea and alveolar interstitium of the rats in P group. Glyburide reduced the inflammatory responses significantly. ELISA results showed that the level of MPO in the lung tissues of the rats in P group was higher than those in other 3 groups (P<0.05). The levels of IL-1β and IL-18 in the BALF of the rats in P group were significantly higher than those in control group and GP group (P<0.05). The results of Western blot showed that the protein levels of NLRP3, caspase-1 and ASC in P group were significantly higher than those in control group and GP group (P<0.01). Immunohistochemical staining results showed that compared with the other 3 groups, the expression of NLRP3 in P group was significantly increased (P<0.01). CONCLUSION: PFOS exposure may lead to lung injury in rats by activating NLRP3 inflammasome and then triggering inflammation, releasing inflammatory factors such as IL-1β. Glyburide specifically inhibits the assembly of NLRP3 inflammasome, suppresses the inflammatory responses and reduces the toxicity of PFOS in lung.     

Effects of microglia-derived IL-1β on differentiation of OPCs in corpus callosum of septic neonatal rats

AIM: To explore whether IL-1β inhibits the oligodendrocyte precursor cell (OPCs) differentiation and affects axonal myelination. METHODS: One-day-old SD rats were randomly divided into control group and LPS group (48 rats in each group). The rats in LPS group were intraperitoneally injected with 1 mg/kg LPS. The rats in control group were injected with an equal volume of PBS. The rats in each group were further divided into 3 h, 24 h, 3 d, 7 d, 14 d and 28 d subgroups after injection. The expression of IL-1β and IL-1R₁ in the rat corpus callosum at 3 h, 24 h, 3 d, 7 d was determined by double immunofluorescence and Western blotting. The myelin basic protein(MBP) expression in the rat corpus callosum at 14 d, 28 d after injection was also measured. In vitro, primary OPCs culture was performed and divided into control group, 30 μg/L IL-1β group, 30 μg/L IL-1β+IL-1Ra group and 30 μg/L IL-1Ra group. The expression of MBP in the OPCs induced differentiation for 3 d was observed by double immunofluorescence and Western blotting. RESULTS: The expression of IL-1β and IL-1R₁ in the rat corpus callosum at 3 h, 24 h, 3 d, 7 d after LPS injection was obviously increased and the expression of MBP in the rat corpus callosum at 14 d, 28 d in LPS group was obviously decreased compared with control group in vivo. The level of MBP was significantly decreased after IL-1β treatment for 3 d in vitro. However, IL-1Ra (IL-1R inhibitor) reversed the down-regulation of MBP expression. IL-1β inhibited the expression of p-ERK, ERK over-expression reversed the down-regulation of MBP expression compared with IL-1β group. CONCLUSION: IL-1β inhibits the differentiation of OPCs, which may be involved in ERK pathways, thus leading to axonal hypomyelination in the corpus callosum of septic neonatal rats.     

Sulodexide protects against hyperglycemia-induced retinal vascular injury via suppressing NOX4/NLRP3 pathway

AIM To investigate the effect of sulodexide (SDX) on high glucose-induced damage in retinal microvascular endothelial cells. METHODS (1) High-fat diet combined with intraperitoneal injection of streptozocin were used to induce type 2 diabetes mellitus (DM) followed by injection of saline or SDX in C57BL/6J male mice. Retinal microvascular leakage and density, and the protein levels of NLRP3 inflammasome-related proteins, zonula occludens-1 (ZO-1) and NADPH oxidase 4 (NOX4) were measured. (2) Human retinal microvascular endothelial cells (HRMECs) were treated with normal glucose or high glucose with or without SDX, and were further transfected with siRNA to knock down NOX4, or infected by adenovirus to over-express NOX4. The protein levels of ZO-1, VE-cadherin (VE-Cad), NOX4 and NLRP3 inflammasome-related proteins as well as the level of reactive oxygen species (ROS) were detected. RESULTS Treatment with SDX increased the protein level of ZO-1, attenuated retinal leakage and NLRP3 inflammasome activation, and enhanced the density of microvasculature and the number of ganglion cells in diabetic retinas. The protein levels of ZO-1 and VE-Cad were decreased, while the levels of NOX4, NLRP3 inflammasome-related proteins and ROS generation were increased in high glucose-treated HRMECs. Silencing of NOX4 inhibited high glucose-induced increases in NLRP3 inflammasome and ROS generation, and decreases in the protein levels of ZO-1 and VE-Cad. Over-expression of NOX4 significantly increased the levels of NLRP3 inflammasome-related proteins and ROS generation in HRMECs, and reduced the protein levels of ZO-1 and VE-Cad. Treatment with SDX partly reversed NOX4 over-expression-induced changes. CONCLUSION SDX alleviates hyperglycemia-induced retinal microvascular endothelial injury via inhibiting NOX4/ROS/NLRP3 pathways.     

Cholestane-3β, 5α, 6β-triol induces apoptosis of malignant glioma cells

AIM：To investigate the effect of cholestane-3β, 5α, 6β-triol (Triol) on apoptosis of malignant glioma cells. METHODS：C6 cells and A172 cells were incubated with Triol at different concentrations for different time durations. MTT assay was used to detect the cell viability. Hoechst 3f3342 staining and TUNEL assay were used to analyze the cell apoptosis. The caspase activity was measured. The expression of apoptosis-related proteins, Bcl-2 family members, was determined by Western blotting. RESULTS：Triol decreased the cell viability of C6 and A172 cells in a dose- and time-dependent manner and the IC50 values were (17.8±0.6)μmol/L and (20.6±0.2) μmol/L, respectively. Visible nuclei with apoptotic characteristics, significant increase in TUNEL-positive cells, and the activation of apoptotic execution enzyme caspase-3 indicated that cell apoptosis was induced by Triol in both cell lines. After C6 cells were exposed to Triol for 12 h, 24 h and 48 h, the activity of caspase-8 in extrinsic apoptotic pathway and caspase-9 in intrinsic apoptotic pathway was increased time-dependently. Meanwhile, the levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL, was down-regulated, while pro-apoptotic protein Bak was up-regulated in a time-dependent manner. CONCLUSION：Triol induces apoptosis of malignant glioma cells by activating intrinsic and extrinsic apoptotic pathways, and Bcl-2 family members are involved in Triol-induced apoptosis.     

IL-1β stimulated neuron activation via PI3K/Akt/mTOR pathway

AIM: To study the effect of interleukin-1β (IL-1β) on neuron activation during the process of medial temporal lobe epilepsy (MTLE).METHODS: IL-1β, rapamycin [an inhibitor of mammalian target of rapamycin (mTOR)]and lentiviral transfection to knockdown PI3K-p85 were used to pre-treat the neurons. The protein levels of PI3K-p85, p-Akt, p-p70S6K and MAP2 were detected and the relationship among the tested cytokines was analyzed. The neuron endocytosis was observed in each group. RESULTS: IL-1β increased the protein levels of PI3K-p85, p-Akt and p-p70S6K, up-regulated the expression of PI3K-p85 binding with IL-1RI in the neurons, and increased the neuron endocytosis compared with control group (P<0.05). These processes were inhibited by rapamycin and silence of PI3K-p85 (P<0.05). Inhibition of the PI3K-p85 binding to IL-1RI decreased the protein levels of p-Akt, p-p70S6K and MAP2 which were increased by IL-1β stimulation (P<0.05). CONCLUSION: IL-1β activates PI3K-p85 by binding with IL-1RI to promote the activation and proliferation of neuron synapses via PI3K/Akt/mTOR signaling pathway, which might be one of the mechanisms in MTLE chronic progress.     

Indomethacin induces gastric cancer cell apoptosis through Akt/GSK3β/NAG-1 pathway

AIM: To investigate whether indomethacin induces gastric cancer cell apoptosis through Akt/GSK3β/NAG-1 pathway.METHODS: Gastric cancer cell line MGC-803 was used in the study. Cell viability was measured by MTT method. Hoechst 33258 nuclear staining and flow cytometry analysis were used to determine apoptosis. The protein expression level was examined by Western blotting. RESULTS: Indomethacin induced MGC-803 cell apoptosis via caspase-dependent pathway. Indomethacin inhibited Ser473-Akt and Ser9-GSK3β phosphorylation and up-regulated the expression of non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1). Inhibition of PI3K or Akt alone also increased NAG-1 expression. Moreover, the effect of indomethacin on NAG-1 expression was abolished by pretreatment of the cells with GSK3β inhibitor SB216763. CONCLUSION: Indomethacin induces gastric cancer cell apoptosis through Akt/GSK3β/NAG-1 pathway.     

IL-1β promotes differentiation of naïve T cells into Th22 cells and its expression in non-small cell lung cancer

AIM:To investigate the mechanism of interleukin-1β (IL-1β) promoting the transformation of naïve T cells into Th22 cells and the correlation of its peripheral blood expression in non-small cell lung cancer patients. METHODS:CD4⁺ naïve T cell magnetic bead sorting kit was used to isolate the peripheral blood mononuclear T cells from healthy people. Transforming growth factor-β (TGF-β) and IL-2 were added to promote differentiation and proliferation. IL-1β was used to induce differentiation into Th22 cells. The proportion of CD4⁺ IL-22⁺ T cells was analyzed by flow cytometry, and the expression of IL-22 was detected by ELISA. We selected 60 cases of non-small cell lung cancer patients in our hospital, including 18 in I phase, 20 in Ⅱ phase, 13 in Ⅲ phase and 9 in IV phase, as well as 25 healthy persons. The proportion of Th22 (CD4⁺ IL-22⁺) cells in peripheral blood was detected by flow cytometry, and the serum levels of IL-1β and IL-22 were measured by ELISA. RESULTS:IL-1β induced the transformation of naïve T cells into Th22 cells and promoted the secretion of IL-22 (P<0.05). The proportion of Th22 cells and the IL-22 and IL-1β levels in peripheral blood of the patients with non-small cell lung cancer were higher than those in healthy subjects, and correlated with the clinical stage. CONCLUSION:IL-1β induces the differentiation of Th22 cells and the expression of IL-22. The levels of IL-1β and IL-22 are related to the progression of non-small cell lung cancer, which may be involved in immunosuppression and promote the occurrence of non-small cell lung cancer.     

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.     

Effect of ischemic post-conditioning on Egr-1 and IL-1β expression in ischemia-reperfusion injured lung in rats

AIM: To investigate the protective effects of ischemic post-conditioning on the expression of early growth response factor 1 (Egr-1) and interleukin-1β(IL-1β) in ischemia-reperfusion injured lung in rats. METHODS: The model of lung ischemia-reperfusion injury was established in 24 rats and the rats were randomly allocated to 3 different groups (n=8 in each group): (1) sham group: only sham operation (thoracotomy) and no ischemia for 3 h; (2)ischemia-reperfusion group (I/R group): interruption of pulmonary perfusion and ventilation for 1 h followed by reperfusion for 2 h; (3) ischemic post-conditioning group (IPostC group): ischemic post-conditioning (5 min of reperfusion and 5 min of ischemia for 3 times) between the end of ischemia and the beginning of the reperfusion followed by reperfusion for 1.5 h. The lung tissues (prepared to small pieces of about 20 mg) were collected and homogenized at the end of the experiment. The concentration of myeloperoxidase (MPO) in the homogenate was determined. The wet to dry weight ratio (W/D) of the lung tissues was also measured at the end of reperfusion. The pathological changes of the lung tissues were observed under light microscope after reperfusion. The mRNA expression of Egr-1 and IL-1β in the lung tissues was detected by RT-PCR. RESULTS: Compared with sham group, the mRNA expression of Egr-1 and IL-1β, the levels of MPO and W/D were significantly increased in I/R group (P<0.05). The inflammatory responses of the lungs in I/R group were significantly severer than those in sham group. Compared with I/R group, the mRNA expression of Egr-1 and IL-1β, the levels of MPO and W/D in IPostC group were significantly decreased (P<0.05). The inflammatory responses of the lungs in IPostC group were also significantly attenuated. CONCLUSION: Ischemic post-conditioning significantly reduces ischemic reperfusion injury of the lung by inhibiting the expression of Egr-1 and IL-1β.     

Up-regulation of Snail1 expression in renal tubular epithelial cells through Akt/GSK-3β pathway under high-glucose condition

AIM: To examine the effects of high glucose (HG) on the expression of Snail1 and protein kinase B (Akt)/glycogen synthase kinase 3β (GSK-3β) in primary renal tubular epithelial cells (RTECs). METHODS: The primary RTECs were randomly treated with normal glucose, high glucose or D-mannitol for 30 min~72 h. RT-PCR and Western blotting were used to observe the expression of Snail1, Akt and GSK-3β at mRNA and protein levels in these cells. The primary cultured RTECs were pretreated with LY294002 (a PI3K inhibitor, 25 μmol/L) to observe the specific inhibitory effects of phosphatidylinositol 3-kinase (PI3K) on HG-induced expression of Snail1 protein. RESULTS: Treatment of RTECs with HG resulted in increased mRNA and protein levels of Snail1, Akt1, and phosphorylation of Akt and GSK-3β. LY294002 blocked the HG-induced up-regulation of p-Akt, p-GSK-3β and Snail1 expression at protein level, but no effect of LY294002 was seen on the total protein expression of Akt1 and GSK-3β. HG did not affect the expression of GSK-3β at mRNA and protein levels. CONCLUSION: HG-induced up-regulation of Snail1 may be regulated by Akt/GSK-3β pathway in RTECs.     

Secoisolariciresinol diglucoside attenuates renal inflammatory injury of mice induced by chronic intermittent hypoxia via inhibiting TXNIP and activating NLRP3 inflammasome

AIM To investigate the effectof flax lignan/secoisolariciresinol diglucoside （SDG） on the inflammatory damage of kidney induced by chronic intermittent hypoxia （CIH）. METHODS C57BL/6N mice were divided into normal （control） group, model （CIH） group and treatment （SDG） group. The changes of the body weight was recorded. Hematoxylin-eosin （HE） staining was used to observe the morphological alterations in the renal tissues. The levels of serum creatinine and blood urea nitrogen were measured by a biochemical analyzer. Hydroxylamine and thiobarbituric acid methods were used to detect the activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the renal tissues. The protein levels of thioredoxin-interacting protein （TXNIP） and nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） were detected by immunohistochemical staining, while those of tumor necrosis factor-α （TNF-α）, interleukin-6 （IL-6） and IL-1β were measured by ELISA. The protein levels of TXNIP, NLRP3, apoptosis-associated speck-like protein containing a CARD （ASC）, caspase-1, IL-1β and IL-18 in the renal tissues were also determined by Western blot. RESULTS No significant difference in the body weight and kidney index among the 3 groups was observed （P>0.05）. HE staining showed the swollen epithelial cells of renal tubules with vesicular degeneration, and irregular glomerular morphological change in CIH group, while SDG treatment attenuated the above changes. Compared with control group, the levels of serum creatinine, TNF-α, IL-6 and IL-1β were significantly increased in CIH group （P<0.05）. The significantly increased expression levels of NLRP3 and TXNIP in the cytoplasm of renal tubular epithelial cells in CIH group were detected by immunohistochemical staining. Compared with control group, the activity of SOD was decreased, the content of MDA was increased in CIH group, and the protein expression levels of TXNIP, NLRP3, ASC, caspase-1, IL-1β and IL-18 were up-regulated and then decreased after SDG treatment （P<0.05）. CONCLUSION SDG attenuates the renal inflammatory damage of the mice induced by CIH, and its mechanism may be associated with the inhibition of oxidative stress and activation of NLRP3 inflammasome.     

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.     

Role of ILK siRNA on expression of GSK-3β and β-catenin in human tubular epithelial cells stimulated by high glucose

AIM：To investigate the effects of siRNA targeting integrin-linked kinase (ILK) on the expression of glycogen synthase kinase 3β (GSK-3β) and β-catenin during epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial cell line HKC induced by high glucose. METHODS：HKC cells were divided into 4 groups:normal glucose (NG) group, high glucose (HG) group, HG+HK (a vector containing the non-specific siRNA designed as negative control) group and HG＋ILK siRNA group. The inverted fluorescence microscope was used to examine the expression of green fluorescent protein (GFP). The expression of ILK at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of p-GSK-3β and β-catenin was observed by immunocytochemical staining. The protein expression of total GSK-3β, p-GSK-3β, nuclear β-catenin, total β-catenin, E-cadherin and α-smooth muscle actin (α-SMA) was measured by Western blotting. RESULTS：GFP was observed in HKC cells, indicating that the transfection was successful. Both the protein and mRNA of ILK were down-regulated in HG＋ILK siRNA group compared with HG group and HG+HK group, but still higher than those in NG group. Silencing of ILK down-regulated the expression of p-GSK-3β and nuclear β-catenin. No difference of total GSK-3β or total β-catenin was observed among the 4 groups. CONCLUSION:These data support a functional role of ILK, GSK-3β and β-catenin in tubular EMT induced by high glucose. ILK may promote tubular EMT by regulating the activity of GSK-3β and β-catenin, the downstream effectors of the Wnt/β-catenin pathway.     

17β-estradiol protects cortical neurons from propofol-induced apoptosis by activating PI3K-Akt signaling pathway

AIM: To investigate the protective effects and the mechanisms of 17β-estradiol on the propofol-induced neuroapoptosis in primary cultured cortical neurons. METHODS: The neurons were cultured for 7 d and treated with different concentrations of propofol and/or 17β-estradiol, respectively. The neuron viability, neuroapoptosis and the protein level of p-Akt was determined by MTT assay, Hoechst 33258 staining and Western blot 12 h after different treatments, respectively. RESULTS: Compared with vehicle-control group, propfol inhibited neuron viability in a dose-dependent manner (P<0.05). Compared with propofol treatment group, 17β-estradiol increased the neuron viability in a dose-dependent manner (P<0.05), and IGF increased the neuron viability greatly (P<0.01). Compared with vehicle-control group, the number of apoptotic neurons which was significantly decreased by treatment of 17β-estradiol was markedly increased by propofol (P<0.01). Compared with the 17β-estradiol+propofol group, LY294002 increased the number of apoptotic neurons (P<0.01). Compared with vehicle-control group, propfol decreased the protein level of p-Akt in a dose-dependent manner (P<0.05). Compared with propofol treatment group, 17β-estradiol increased the protein level of p-Akt in a dose-dependent manner (P<0.05). Compared with 17β-estradiol+propofol group, LY294002 significantly decreased the protein level of p-Akt (P<0.01). CONCLUSION: 17β-estradiol exerts the neuroprotective effects against propofol-induced neuroapoptosis by activating the PI3K-Akt signaling pathway.     

Effects of angiotensin II-induced hypertension chemotherapy on the activity of interleukin-1β converting enzyme in transplanted intracerebral rat gliomas

AIM: To investigate the activity of interleukin-1β converting enzyme in transplanted intracerebral rat gliomas under angiotensin II-induced hypertension chemotherapy. METHODS: The brain tumor model was produced in Wistar rats by stereotaxic inoculation of C6 glioma cells (1×10¹² /L). Tumor-bearing rats were treated with carmustine, teniposide and lisplatin (chemotherapy) during angiotensin II-induced hypertension. Then, the survival time of tumor-bearing rats, tumor blood flow, the concentration of drug, volume of gliomas and the activity of interleukin-1β converting enzyme in glioma were examined.RESULTS: The survival time of tumor-bearing rats was significantly longer in chemotherapy with angiotensin II-induced hypertension group than that of chemotherapy alone. In addition, regional tumor blood flow, the concentration of chemotherapeutic drug and the activity of interleukin -1β converting enzyme in transplanted rat gliomas were increased, while the volume of gliomas was decreased in hypertention chemotherapy group compared with chemotherapy alone. CONCLUSION: Chemotherapy with angiotensin II-induced hypertension has a enhancing effect on chemotherapy for improving the drug delivery to tumor tissue by a increased tumor blood flow and enhancing activity of interleukin -1β converting enzyme.     

Effects of JAK-STAT signaling pathway,IL-1β and IL-6 on injury of PC12 cells with X-ray irradiation

AIM: To investigate the role of JAK-STAT pathway, IL-1β and IL-6 in the PC12 cells with X-ray irradiation.METHODS: The PC12 cells were irradiated with X-ray at doses of 2, 4 and 8 Gy. After 24 h, the levels of IL-1β and IL-6 were detected by ELISA. The protein levels of p-JAK1, p-JAK2, p-STAT1, p-STAT3 and p-STAT5 were measured by Western blot.RESULTS: Compared with control group, the levels of IL-1β and IL-6 increased. The protein levels of p-JAK1, p-JAK2, p-STAT1, p-STAT3 and p-STAT5 increased with the doses of X-ray exposed.CONCLUSION: JAK-STAT signaling pathway, IL-1β and IL-6 play a role in the injury of PC12 cells with X-ray irradiation.