Effects of PTEN on apoptosis,oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation

AIM: To investigate the effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) on the apoptosis, oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation (A/R). METHODS: The H9c2 cells were used to establish a model of A/R. The H9c2 cells were transfected with PTEN small interfering RNA (siRNA) and negative control. After A/R, the expression of PTEN at mRNA and protein levels was determined by RT-PCR and Western blot, respectively. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Xanthine oxidase method was used to determine superoxide dismutase (SOD) activity. The content of malondialdehyde (MDA) was detected by thiobarbituric acid method. The lactate dehydrogenase (LDH) activity in the supernatant was evaluated by 4-dinitrophenylhydrazine method. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 in culture supernatant were examined by ELISA. The protein levels of cleaved caspase-3, Bax and FasL in the cells were determined by Western blot. RESULTS: After A/R, the expression of PTEN at mRNA and protein levels was significantly increased in the H9c2 cells (P<0.05). The mRNA and protein levels of PTEN were decreased significantly after transfection with PTEN siRNA (P<0.05). The viability of H9c2 cells was decreased after A/R, while the apoptotic rate was increased. The protein levels of cleaved caspase-3, Bax and FasL were increased in the cells. The MDA level was elevated, the activity of SOD was decreased, and the levels of LDH, TNF-α, IL-1β and IL-6 in the culture supernatant were increased (P<0.05). Down-regulation of PTEN partly antagonized the effects of A/R on the viability, apoptotic rate, MDA content, SOD activity, and the levels of LDH, TNF-α, IL-1β and IL-6 in culture supernatant. CONCLUSION: Down-regulation of PTEN attenuates oxidative damage induced by A/R, reduces apoptosis and secretion levels of TNF-α, IL-1β and IL-6 in the H9c2 cells.     

Effects of procyanidins on oxidative damage of osteocytes caused by TCP wear particles

AIM: To investigate the effects of procyanidins (PC) on oxidative damage of osteocytes caused by tricalcium phosphate (TCP) wear particles, and to explore the underling mechanism. METHODS: Mouse long bone osteocyte MLO-Y4 cells were treated with TCP wear particles (0.1 g/L) for 48 h to establish the model of osteocyte injuries. The MLO-Y4 cells were divided into 4 groups:control group, TCP group, PC (10 μmol/L) group and PC (50 μmol/L) group. Calcein-AM staining and MTT assay were used to observe the viability of MLO-Y4 cells. The levels of dentin matrix protein 1 (DMP-1), sclerostin (SOST) and interleukin-1β (IL-1β) in the culture media were examined by ELISA. The apoptosis of MLO-Y4 cells was analyzed by flow cytometry with Annexin V/PI double staining. The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity of MLO-Y4 cells, and lactate dehydrogenase (LDH) release in the culture media were measured by chemical colorimetry. The protein levels of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), cleaved caspase-1 and IL-1β in the MLO-Y4 cells were determined by Western blot. RESULTS: Compared with control group, MLO-Y4 cell injuries, apoptosis rate and MDA level were obviously increased in TCP group, while SOD activity was significantly decreased (P<0.05) The protein levels of NLRP3, ASC, cleaved caspase-1 and IL-1β were remarkably up-regulated (P<0.05) in the MLO-Y4 cells, and the level of IL-1β and LDH release were increased in the culture media (P<0.05). Compared with TCP group, the injuries of MLO-Y4 cells, apoptosis rate and MDA level were decreased obviously (P<0.05) in PC groups, whereas SOD activity was increased (P<0.05). The protein levels of NLRP3, ASC, cleaved caspase-1 and IL-1β were down-regulated remarkably in the MLO-Y4 cells (P<0.05), and the level of IL-1β and LDH release were significantly decreased in the culture media (P<0.05). CONCLUSION: PC obviously inhibit oxidative damage of osteocytes caused by TCP wear particles, which may be related to alleviating NLRP3 inflammasome activation and pyroptosis.     

Effect of berberine on endoplasmic reticulum stress-autophagy pathway in human ovarian cancer SKOV3 cells

AIM: To investigate the regulatory effect of berberine on the endoplasmic reticulum stress-auto-phagy pathway in human ovarian cancer SKOV3 cells. METHODS: Human ovarian cancer SKOV3 cells were cultured in vitro, and berberine at doses of 12.5, 25, 50, 100, 200 and 400 μmol/L were added. After exposure for 12 h, 24 h and 48 h, the viability of the SKOV3 cells was measured by MTT assay. The cells were divided into control group, berberine (50 μmol/L) group, berberine (100 μmol/L) group, and berberine (200 μmol/L) group. After treatment with berberine for 24 h, the effects of berberine on the morphological changes of SKOV3 cells were observed under inverted phase-contrast microscope. The protein expression of microtubule-associated protein 1 light chain 3 (LC3) and ubiquitin-binding protein p62 was observed by indirect immunofluorescence method under laser confocal microscope. The protein expression of beclin-1,LC3,p62, CCAAT/lenhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) was determined by Western blot. RESULTS: Berberine at 12.5, 25, 50, 100, 200 and 400 μmol/L significantly decreased the viability of SKOV3 cells at 12 h, 24 h and 48 h, and the IC₅₀ values of 12 h, 24 h and 48 h were (764.7±0.3) μmol/L, (231.6±0.1) μmol/L and (96.2±0.1) μmol/L, respectively. Laser confocal microscopy showed that the LC3 and p62 proteins were scattered and the fluorescence intensity was increased, while the point-like aggregation was also observed. Berberine at 200 μmol/L obviously enhanced the co-localization of LC3 and p62 proteins. Compared with control group, the expression of endoplasmic reticulum stress-related proteins GRP78 and CHOP, and autophagy-related proteins beclin-1, LC3 and p62 in berberine (200 μmol/L) group was increased significantly (P<0.05). CONCLUSION: Berberine may promote endoplasmic reticulum stress in SKOV3 cells by regulating autophagy.     

Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via ERK-mediated autophagy inhibition

AIM: To explore the effect of Pycnogenol on transforming growth factor-β1 (TGF-β1)-induced hepatic stellate cell activation. METHODS: Cultured LX-2 cells were treated with 5 μg/L TGF-β1 and different concentrations (0, 10, 25 and 50 mg/L) of Pycnogenol. The viability of the LX-2 cells under the conditions with or without autophagy inhibitor 3-MA and ERK inhibitor PD98059 was determined by MTT assay. The protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2 and ERK1/2 were detected by Western blot. RESULTS: Compared with control group, 5 μg/L TGF-β1 treatment elevated the cell viability, and increased the protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2, and ERK1/2 in the LX-2 cells (P<0.05). However, these effects were reversed by Pycnogenol pretreatment in a dose-dependent manner and the inhibitory effect of 50 mg/L Pycnogenol was the most significant in the LX-2 cells (P<0.05). Furthermore, compared with TGF-β1 group, pretreatment with 50 mg/L Pycnogenol, 5 mmol/L 3-MA or 20 μmol/L PD98059 downregulated TGF-β1-induced cell viability and the protein levels of α-SMA and LC3-Ⅱ/Ⅰ in the LX-2 cells (P<0.05). CONCLUSION: Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via p-ERK and autophagy inhibition.     

USP14 regulates H2O2 induced oxidative stress in H9c2 cells

AIM:To evaluate the effect of inhibiting ubiquitin-specific protease 14(USPl4) activity on oxidative stress induced by H₂O₂ of H9c2 cells.METHODS:The H9c2 cells were incubated with H₂O₂ at 25 μmol/L for 2 h to establish the oxidative stress injury model.The cells were divided into control group,H₂O₂ group,IU1 group (25 μmol/L or 50 μmol/L) and IU1+H₂O₂ group.The H9c2 cells activity was measured by MTS assay.The level of intracellular reactive oxygen species (ROS) and cell survival rate were analyzed by flow cytometry assay.The changes of the mitogen-activated protein kinase (MAPK) family related proteins were detected by Western blot.RESULTS:Compared with control group,the cell activity and the viability rate in H₂O₂ group were decreased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were increased (P<0.05).Compared with H₂O₂ group,the cell activity and the viability rate of the H9c2 cells in IU1+H₂O₂ group were increased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were decreased (P<0.05).CONCLUSION:Inhibition of USPl4 activity reduces the oxidative stress injury of the H9c2 cells.The mechanism may be related to inhibition of the MAPK signaling and down-regulation of apoptosis related proteins.     

Angiotensin-(1-7) protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting the interaction between TLR4 activation and necroptosis

AIM: To investigate whether angiotensin-(1-7)[Ang-(1-7)] protects H9c2 cardiac cells against high glucose (HG)-induced injury and inflammation by inhibiting the interaction between Toll-like receptor 4 (TLR4) activation and necroptosis. METHODS: The expression levels of receptor-interacting protein 3 (RIP3; an indicator of necroptosis) and TLR4 were determined by Western blot. Cell viability was measured by CCK-8 assay. The activity of lactate dehydrogenase (LDH) in the culture medium was measured with a commercial kit. The releases of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by ELISA. The intracellular level of reactive oxygen species (ROS) was analyzed by 2', 7'-dichlorfluorescein-diacetate (DCFH-DA) stating followed by photofluorography. Mitochondrial membrane potential (MMP) was examined by rhodamine 123 staining followed by photofluorography. RESULTS: After the H9c2 cardiac cells were treated with HG (35 mmol/L glucose) for 24 h, the expression of RIP3 was obviously increased. Co-treatment of the cells with 30 μmol/L TAK-242 (an inhibitor of TLR4) attenuated the up-regulation of RIP3 induced by HG. Furthermore, the expression of TLR4 was significantly increased after the cells were exposed to HG for 24 h, and co-treatment of the cells with 100 μmol/L necrostatin-1 (Nec-1; a specific inhibitor of necroptosis) and HG for 24 h attenuated the up-regulation of TLR4 expression induced by HG. Moreover, 1 μmol/L Ang-(1-7) simultaneously blocked the up-regulation of the RIP3 and TLR4 induced by HG. On the other hand, co-treatment of the cells with 1 μmol/L Ang-(1-7), 30 μmol/L TAK-242 or 100 μmol/L Nec-1 and HG for 24 h attenuated HG-induced injuries and inflammatory response, leading to the increase in the cell viability, and the decreases in the activity of LDH, ROS generation, MMP loss as well as the releases of IL-1β and TNF-α. CONCLUSION: Ang-(1-7) protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the interaction between TLR4 activation and necroptosis.     

Berberine promotes apoptosis of human breast cancer cells by regulating miRNA-146a

ATM: To observe the effect of berberine on apoptosis of MCF-7 cells and its potential mechanism. METHODS: The MCF-7 cells were divided into control group and the groups with 3 different doses of berberine. The cell viability was detected by MTT assay, while the cell apoptosis was measured by Hoechst 33258 staining and flow cytometry assay. The protein levels of p-P65, Bax and Bcl-2 were Western blot. The levels of microRNA-146a(miRNA-146a) in the MCF-7 cells were detected by RT-qPCR. The miRNA-146a siRNA was transfected to the MCF-7 cells after an evaluation of transfection efficacy, which was co-incubated with berberine to observe its effects on the mRNA levels of Bax and Bcl-2. RESULTS: Compared with control group, the cell viabilities were decreased significantly in medium and high doses of berberine treatment groups with a dose-dependent manner (P<0.01). The cell apoptosis was increased significantly in medium and high doses of berberine treatment groups dose-dependently (P<0.05). The protein levels of Bax were up-regulated, while those of Bcl-2 and p-P65 were down-regulated significantly by the treatment of berberine (P<0.05). In addition, the miRNA-146a levels were increased significantly in medium and high doses of berberine treatment groups (P<0.05) and showed a dose-dependent manner. The mRNA levels of Bax were decreased, while the mRNA levels of Bcl-2 were increased after transfection with miRNA-146a siRNA and co-incubated with berberine.CONCLUSION: Berberine promotes apoptosis of MCF-7 cells. The mechanism may be related to inhibit the activity of NF-κB by incresing the levels of miRNA-146a.     

Anticancer function of Shp2 in lung adenocarcinoma A549 cells and rela-ted molecular mechanisms

AIM: To explore the anticancer function of Shp2 in lung adenocarcinoma A549 cells and the related molecular mechanisms. METHODS: The viability and proliferation of A549 cells treated with Shp2 specific inhibitor Phps-1 or cisplatin (DDP) were measured by CCK-8 assay and EdU assay. Annexin V-FITC/PI double staining was applied to detect apoptotic rate of A549 cells with different interventions. The protein levels of caspase-3-17p, Bcl-2, Bax, p-STAT3/STAT3 and p-ERK/ERK were determined by Western blot. RESULTS: Compared with control group, Phps-1 at the concentration of 20 μmol/L significantly increased the viability of A549 cells after 24 h of treatment (P<0.05). Meanwhile, the proliferation rate of A549 cells in Phps-1 20 μmol/L group was significant increased compared with control group (P<0.05). The apoptotic rate of A549 cells in DDP treatment group decreased from 13.01%±2.62% to 3.67%±0.93% after adding Phps-1 (P<0.05). Phps-1 down-regulated the protein levels of caspase-3-17p, Bax and p-ERK, but up-regulated p-STAT3.CONCLUSION: Shp2 is a tumor suppressor in A549 cells, which may be associated with the activation of STAT3 signal pathway.     

Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.     

Role of ATP-sensitive potassium channels in inhibitory effect of hydrogen sulfide on high glucose-induced inflammation mediated by necroptosis in H9c2 cardiac cells

AIM: To investigate the role of ATP-sensitive potassium (K_ATP) channels in the inhibitory effect of hydrogen sulfide (H₂S) on high glucose(HG)-induced inflammation mediated by necroptosis in H9c2 cardiac cells.METHODS: The expression levels of receptor-interacting protein 3 (RIP3; an indicator of necroptosis) and cyclooxyge-nase-2 (COX-2) were determined by Western blot. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected by ELISA.RESULTS: After H9c2 cardiac cells were treated with 35 mmol/L glucose (HG) for 24 h, the expression of RIP3 was significantly increased. Pre-treatment of the cells with 100 μmol/L diazoxide (DZ; a K_ATP channel opener) or 400 μmol/L NaHS (a donor of H₂S) for 30 min considerably blocked the up-regulation of RIP3 induced by HG. Moreover, pre-treatment of the cells with 100 μmol/L 5-hydroxydecanoic acid (5-HD; a K_ATP channel blocker) attenuated the inhibitory effect of NaHS on HG-induced up-regulation of RIP3. On the other hand, co-treatment of the cells with 100 μmol/L necrostatin-1 (a specific inhibitor of necroptosis) or pre-treatment of the cells with 100 μmol/L DZ or 400 μmol/L NaHS attenuated HG-induced inflammatory responses, evidenced by decreases in the expression of COX-2 and secretion levels of IL-1β and TNF-α. However, pre-treatment of the cells with 100 μmol/L 5-HD significantly attenuated the above anti-inflammatory effects of NaHS.CONCLUSION: K_ATP channels play an important role in the inhibitory effect of H₂S on HG-induced inflammation mediated by necroptosis in H9c2 cardiac cells.     

Effect of netrin-1 on high glucose induced injury of renal tubular epithe-lial cells

AIM:To study the effect of netrin-1 on the damage of renal tubular epithelial cells induced by high glucose. METHODS:Human renal tubular epithelial HK-2 cells were treated with high glucose. Real-time PCR and Western blot were used to detect the expression level of netrin-1 in the cells. HK-2 cells were infected with netrin-1-over-expressing lentivirus, and the effect of netrin-1 over-expression on the HK-2 cells treated with high glucose was observed. The apoptosis rate was analyzed by flow cytometry. The protein level of cleaved caspase-3 was determined by Western blot. lactate dehydrogenase (LDH) activity in the culture medium was measured by 2,4-binitrobenzene hydrazine method. The content of malondialdehyde (MDA) in the culture medium was detected by thiobarbituric acid method. The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the culture medium were measured by ELISA. RESULTS:The expression of netrin-1 at mRNA and protein levels in the HK-2 cells after high glucose treatment was significantly lower than that in the control cells (P<0.05). Infection with netrin-1-over-expressing lentivirus up-regulated the expression of netrin-1 in the HK-2 cells treated with high glucose. High glucose promoted the secretion of IL-1β and TNF-α, decreased the levels of LDH and MDA in the cell culture supernatant, and induced apoptosis and activation of caspase-3 in renal tubular epithelial cells (P<0.05). After the HK-2 cells with up-regulation of netrin-1 were induced by high glucose, the IL-1β and TNF-α secretion, the levels of LDH and MDA in the culture medium, the apoptosis, and the level of activated caspase-3 protein in the cells were all decreased, as compared with the control cells (P<0.05). CONCLUSION:Up-regulation of netrin-1 expression attenuates oxidative damage and inflammatory injury, and reduces apoptosis induced by high glucose in renal tubular epithelial cells.     

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.     

Urotensin Ⅱinduces pulmonary arterial smooth muscle cell proliferation and up-regulates Egr-1 expression through ERK1/2 pathway in rats

AIM: To investigate the effect of urotensinⅡ (UⅡ) on the proliferation of cultured rat pulmonary arterial smooth muscle cells (PASMCs), and to explore whether mitogen-activated protein kinase (MAPK) signaling pathways and early growth response factor-1 (Egr-1) involved in the regulation of the PASMCs proliferation stimulated by UⅡ. METHODS: The rat PASMCs were isolated and cultured in vitrowith explant culture technique. The proliferation of cultured PASMCs stimulated by different doses of UⅡwas detected by BrdU incorporation. The mRNA expression of extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase (SAPK), p38 MAPK and Egr-1 in cultured PASMCs treated with UⅡ, UⅡ-specific antagonist urantide, and ERK1/2 inhibitor PD98059 was detected by real-time PCR. The protein levels of phosphorylated ERK1/2 (p-ERK1/2), p-SAPK, p-p38 and Egr-1 in cultured PASMCs were determined by Western blotting. RESULTS: UⅡ at concentrations of 1 μmol/L, 0.1 μmol/L and 0.01 μmol/L increased the proliferation of cultured PASMCs in a dose-dependent manner (P<0.01 or P<0.05), with the maximal effect at a concentration of 1 μmol/L. However, urantide inhibited the promotion effect of UⅡ on PASMC proliferation (P<0.05). UⅡ up-regulated the mRNA expression of ERK1/2, SAPK and Egr-1 (P<0.01 or P<0.05), but not the p38 MAPK. However, the up-regulatory effect of UⅡ on ERK1/2 and Egr-1 expression was inhibited by PD98059 and/or urantide (P<0.01 or P<0.05). UⅡ also increased the protein levels of p-ERK1/2, p-SAPK and Egr-1 (P<0.01 or P<0.05), but the promotion effect was also inhibited by PD98059 and/or urantide (P<0.01 or P<0.05).CONCLUSION: UⅡ increases the proliferation of PASMCs, and U Ⅱand Egr-1 participates in UⅡ-mediated proliferation of cultured PASMCs through activation of ERK1/2 signal pathway.     

Effect of CIP2A over-expression on proliferation and apoptosis of gastric epithelial cells

AIM: To investigate the effects of cancerous inhibitor of protein phosphatase 2A (CIP2A) over-expression on the proliferation and apoptosis of gastric epithelial cells.METHODS: The mRNA expression of CIP2A and cyclin D1 in the tissues of normal gastric mucosa and gastric polyps was detected by RT-qPCR. The GES-1 cells were divided into control group, Ad-emp group and Ad-CIP2A group. The cell proliferation ability was detected by MTT assay and BrdU assay, and the cell apoptosis was analyzed by flow cytometry. The expression of apoptosis-related molecules was determined by Western blot and RT-qPCR. The levels of inflammatory cytokines were measured by ELISA after GES-1 cells were infected with Ad-emp and Ad-CIP2A. Furthermore, the protein levels of p-Rb, E2F1 and cyclin D1 in the GES-1 cells was determined by Western blot after transfected with CIP2A siRNA.RESULTS: The expression of CIP2A and cyclin D1 in adenomatous gastric polyps tissues was significantly higher than that in normal gastric mucosa tissues, and no significant change of that between hyperplastic gastric polyps tissues and normal gastric mucosa was observed. After transfected with CIP2A, the proliferation ability of GES-1 cells was increased, the cell apoptosis was inhibited, the concentrations of IL-1β and IL-10 was up-regulated and the protein levels of p-Rb, E2F1 and cyclin D1 were increased, while the protein levels of p-Rb, E2F1 and cyclin D1 were significantly decreased after transfected with CIP2A siRNA.CONCLUSION: CIP2A promotes the proliferation and inhibits the apoptosis of GES-1 cells by activating Rb/E2F1.     

Minocycline inhibits BV-2 cell activation by regulating P2X7 receptor

AIM：To explore the role of P2X7 receptor in inhibition of lipopolysaccharide (LPS)-stimulated BV-2 cell activation by minocycline. METHODS：BV-2 cells were divided into 5 groups: control group, LPS group, LPS+0.1 μmol/L Mino group, LPS+1 μmol/L Mino group and LPS+10 μmol/L Mino group. The expression of P2X7 receptor was determined by real-time PCR and Western blotting. The levels of TNF-α and IL-1β in the microglia culture supernatants were measured by ELISA. The morphological changes of the cells were also observed. RESULTS：After exposed to LPS, the expression of P2X7 receptor increased in BV-2 cells at mRNA and protein levels. The concentrations of TNF-α and IL-1β in the microglia culture supernatants also increased. Meanwhile, 0.1~10 μmol/L minocycline inhibited those changes in a dose-dependent manner. CONCLUSION：Minocycline inhibits the activation of microglia. The mechanism may be related to the P2X7 receptor.     

Adipophilin induces expression of inflammatory factors through ERK1/2-AP-1 signaling pathway

AIM: To observe the effects of adipose differentiation-related protein (adipophilin) on the expression of inflammatory factors in RAW264.7 macrophage and to clarify the related mechanism. METHODS: The cell models with high expression and low expression of adipophilin were constructed by transfecting PA317 packaging cells with stable high or low expression adipophilin retroviral vectors into the RAW264.7 cells. The concentrations of IL-6, MCP-1 and TNF-α in the cell culture medium were detected by ELISA. The protein levels of AP-1, p-AP-1, ERK1/2 and p-ERK1/2 were measured by Western blot. The protein levels of adipophilin, p-ERK1/2 and p-AP-1 and the releases of the inflammatory factors in the RAW264.7 cells treated with or without ERK1/2 inhibitor PD98059 or AP-1 inhibitor curcumin were determined. RESULTS: The RAW264.7 cells with high expression of adipophilin had higher levels of IL-6, MCP-1 and TNF-α, and higher protein levels of p-AP-1 and p-ERK1/2 than those in the cells with low expression of adipophilin. ERK1/2 inhibitor had no significant effect on the expression of adipophilin, but the protein expression of ERK1/2 and AP-1 was significantly inhibited (P<0.05). The administration of AP-1 inhibitor curcumin had no significant effect on the protein expression of adipophilin and ERK1/2, but the protein expression of AP-1 was significantly inhibited (P<0.05). At the same time, the releases of inflammatory factors IL-6, MCP-1 and TNF-α were significantly decreased. CONCLUSION: Adipophilin may regulate the expression of inflammatory factors through ERK1/2-AP-1 pathway in RAW264.7 macrophages.     

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.     

Opening of ATP-sensitive K+ channels protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting TLR4/NF-κB pathway

AIM: To investigate whether the opening of ATP-sensitive K⁺(K_ATP) channels protects H9c2 cardiac cells against high glucose(HG)-induced injury and inflammation by inhibiting the Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) pathway. METHODS: The protein levels of TLR4 and NF-κB p65 were determined by Western blot. The levels of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) were detected by ELISA. The cell viability was measured by CCK-8 assay. Mitochondrial membrane potential(MMP) was examined by rhodamine 123(Rh 123) staining followed by photofluorography. The intracellular levels of reactive oxygen species(ROS) were detected by 2', 7'-dichlorfluorescein- diacetate(DCFH-DA) staining followed by photofluorography. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography. RESULTS: After the H9c2 cardiac cells were treated with HG(35 mmol/L glucose) for 24 h, the protein levels of TLR4 and phosphorylated NF-κB p65(p-NF-κB p65) were significantly increased. Pretreatment of the cells with 100 μmol/L diazoxide(DZ, a K_ATP channel opener) for 30 min before exposure to HG considerably blocked the up-regulation of the TLR4 and p-NF-κB protein levels induced by HG. Moreover, co-treatment of the cells with 30 μmol/L TAK-242(an inhibitor of TLR4) obviously inhibited the HG-induced up-regulation of the p-NF-κB p65 protein level. On the other hand, pretreatment of the cells with 100 μmol/L DZ had a clear myocardial protection effect, which attenuated the HG-induced cytotoxicity, inflammatory response, mitochondrial damage, oxidative stress and apoptosis, evidenced by an increase in the cell viability, and decreases in the levels of IL-1β and TNF-α, MMP loss, ROS generation and the number of apoptotic cells. Similarly, co-treatment of H9c2 cardiac cells with 30 μmol/L TAK-242 or 100 μmol/L PDTC(an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries and inflammation induced by HG.CONCLUSION: The opening of K_ATP channels protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the TLR4/NF-κB pathway.     

Knock-down of TLR4 expression reduces secretion of inflammatory factors in LPS-induced pancreatic acinar AR42J cells

AIM:To study the effect of Toll-like receptor 4 (TLR4) on the secretion of inflammatory factors in the pancreatic acinar AR42J cells induced by lipopolysaccharides (LPS). METHODS:The rat pancreatic acinar AR42J cells were treated with LPS. The expression of TLR4 at mRNA and protein levels was determined by real-time PCR and Western blot. The lentivirus carrying TLR4 small interfering RNA (siRNA) was used to infect the AR42J cells. Under LPS stimulation, the interference efficacy was measured by real-time PCR and Western blot. The cell viability was measured by MTT assay, and the leakage rate of lactate dehydrogenase (LDH) was examined by dinitrophenylhydrazine method. The releases of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell culture medium were detected by ELISA, and the malondialdehyed (MDA) content in supernatant was measured by thiobarbituric acid method. The activity of superoxide dismutase (SOD) in the supernatant was determined by xanthine oxidation, and the activity of glutathione peroxidase (GSH-Px) and catalase (CAT) was detected by colorimetry. RESULTS:The expression of TLR4 at mRNA and protein levels in LPS-treated AR42J cells was significantly increased (P<0.05). Infection with TLR4 siRNA-carrying lentivirus significantly inhibited the expression of TLR4 at mRNA and protein levels in the AR42J cells under LPS stimulation(P<0.05). The viability of AR42J cells was decreased after LPS treatment. The leakage rate of LDH was increased, the levels of IL-1β and TNF-α secreted by the AR42J cells were increased, the content of MDA was increased in the supernatant, and the activity of SOD, GSH-Px and CAT was reduced (P<0.05). After knock-down of TLR4 expression, the viability of AR42J cells was increased under LPS stimulation, the LDH leakage rate, secreted levels of IL-1β and TNF-α, and the content of MDA in cell culture medium were decreased, and the SOD, GSH-Px and CAT levels were increased (P<0.05). CONCLUSION:LPS induces the expression of TLR4 in the pancreatic acinar AR42J cells. Knock-down of TLR4 expression reduces the secretion of inflammatory factors IL-1β and TNF-α, and attenuates the oxidative damage in pancreatic acinar AR42J cells induced by LPS.