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.     

Effect of autophagy in SH-SY5Y cells injured by Aβ25-35

AIM: To explore whether the damage of neurons induced by amyloid β-protein (Aβ) is related to the regulation of autophagy and its mechanism based on Akt/mTOR pathway. METHODS: SH-SY5Y cells were incubated with Aβ_25-35 (5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L and 25 μmol/L) for 24 h, and the cell viability was measured by MTT assay. The protein levels of LC3-I, LC3-II, Akt, p-Akt, mTOR and p-mTOR in the SH-SY5Y cells were determined by Western blot. After the SH-5Y5Y cells were incubated with autophagy inducer rapamycin (Rapa) or autophagy inhibitor 3-methyladenine (3-MA) combined with Aβ_25-35 for 24 h, the cell viability and related protein expression were detected by the same methods above mentioned. RESULTS: Each concentration of Aβ_25-35 damaged SH-SY5Y cells and decreased the viability of SH-SY5Y cells. Aβ_25-35 increased the expression of autophagy marker protein LC3-II, increased the level of LC3-II/LC3-I, and down-regulated the phosphorylation level of Akt and mTOR proteins (P<0.05). When combined with autophagy inducer Rapa, the cell viability was not significantly affected, the expression of LC3-II protein was increased, LC3-II/LC3-I was increased significantly, and p-mTOR/mTOR level was decreased (P<0.05). When combined with autophagy inhibitor 3-MA, the protein expression of LC3-II and the level of LC3-II/LC3-I showed a downward trend, while the level of p-Akt/Akt was decreased (P<0.05). CONCLUSION: Aβ_25-35 may induce SH-SY5Y cell autophagy and injury by down-regulating phosphorylation levels of Akt and mTOR proteins.     

Role of P2Y1 receptor in activation of astrocytes induced by Aβ25-35

AIM: To study the role of P2Y₁ receptor in the activation of astrocytes induced by Aβ_25-35.METHODS: Astrocytes were isolated and cultured from newborn Wistar rats and divided into control group, Aβ_25-35 group, MRS2179(P2Y₁receptor inhibitor)+Aβ_25-35 group and MRS2179 group by treating the cells with the corresponding reagents. The expression levels of GFAP and P2Y₁ were determined by the methods of immunohistochemistry, immunofluorescence and Western blotting.RESULTS: No significant change of the astrocyte numbers in all groups was observed. Compared with the control cells, the fluorescence intensity of GFAP significantly increased in Aβ_25-35 group and decreased in both MRS2179+Aβ_25-35 group and MRS2179 group. The expression level of GFAP determined by Western blotting and immunofluorescence showed the similar trend of change in each group. Compared with control group, the expression of P2Y₁ in Aβ_25-35 group was significantly increased (P<0.05), and no significant change between MRS2179+Aβ_25-35 group and MRS2179 group was found (P>0.05).CONCLUSION: Aβ_25-35 activates astrocytes by activation of P2Y₁ receptor.     

Chrysophanol ameliorates spatial memory ability of rats by inhibiting Aβ1-42-induced oxidative stress

AIM: To investigate whether chrysophanol alleviates amyloid β-protein (Aβ)-induced cognitive dysfunction and the underlying antioxidative mechanism.METHODS:Adult male Wistar rats (230~250 g) were randomly divided into control group, Aβ_1-42 group, chrysophanol group, and Aβ_1-42+chrysophanol (1, 10 and 100 mg/kg) groups. Aβ_1-42 was delivered by intracerebroventricular injection under the guidence of a brain stereotaxic apparatus. Y-maze test, open-field test and Morris water maze test were performed 1 week after Aβ_1-42 injection to evaluate the ability of rat spacial learning and memory. Chrysophanol was intraperitoneally injected once daily for 5 consecutive days. After the behavioral tests, the animals were sacrificed immediately by decapitation, and the hippocampus were collected. The malondialdehyde (MDA) content and the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in the hippocampus were measured.RESULTS:Multiple (7 consecutive days, once daily) but not single (once a day) chrysophanol treatment at 1, 10 and 100 mg/kg effectively prevented Aβ_1-42-induced cognitive function deficits in a dose-dependent manner as shown by Y-maze test and Morris water maze test. Moreover, the Aβ_1-42-induced increase in MDA content and decrease in the activity of antioxidant enzymes (SOD, GSH-Px and CAT) in the hippocampus of the rats were also attenuated by multiple chrysophanol treatment.CONCLUSION:Repeated chrysophanol treatment attenuates Aβ_1-42-induced cognitive deficits and synaptic plasticity dysfunction, and the mechanisms underlying the neuroprotective effects are likely due to its antioxidant activity.     

Rapamycin improves circadian rhythm disorder induced by Aβ31-35 in mice

AIM:To observe the effect of rapamycin (Rapa) on the circadian rhythm disorder in C57BL/6 mice and the abnormal expression of Per2 in mouse hippocampal neuronal cell line HT22 induced by amyloid β-protein 31-35 (Aβ31-35).METHODS:The 6~8-week-old male C57BL/6 mice were selected for wheel-running behavior experiment. The effect of Rapa on the circadian rhythm disorder induced by Aβ31-35 was analyzed. The HT22 cells were randomly divided into control group, Aβ31-35 group, Rapa+Aβ31-35 group and Rapa group. The cell viability was measured by CCK-8 assay. Real-time PCR was used to detect the mRNA expression of Per2, and Western blot was applied to examine the expression of Per2 protein at circadian time (CT) 16. RESULTS:Compared with control group, Aβ31-35 disturbed the circadian rhythm which exhibited significantly longer free running period. However, the disruption was significantly relieved by pretreatment with Rapa compared with Aβ31-35 treatment, which manifested significantly decreased free running period (P<0.05). Aβ31-35 decreased the viability of HT22 cells compared with control group, and Rapa pretreatment reduced the toxicity of Aβ31-35 through up-regulating the cell viability. Abnormal mRNA expressions of Per2 was induced by Aβ31-35 in the HT22 cells. Rapa pretreatment reversed the abnormal expression of Per2 at mRNA level induced by Aβ31-35. Aβ31-35 significantly decreased the protein expression of Per2 at CT16. Pretreatment with Rapa significantly improved the protein expression of Per2. CONCLUSION:Rapa attenuates the circadian rhythm disorder induced by Aβ31-35 in C57BL/6 mice, and improves the abnormal expression of Per2 induced by Aβ31-35 in HT22 cells.     

Effect of tenuigenin on tau protein phosphorylation at Ser396 site in neurons of AD rats induced by Aβ1-40

AIM:To investigate the effect of tenuigenin(TEN) on hyperphosphorylation of tau protein in neurons of amyloid β-peptide_1-40(Aβ_1-40) -induced Alzheimer disease(AD) rats. METHODS:Aβ_1-40 was injected into hippocampus CA1 region of the rats to establish AD model. TEN at different doses(18.5 mg/kg, 37.0 mg/kg and 74.0 mg/kg) was intragastrically administered. The protein expression of protein kinase A(PKA),protein phosphatase 2A(PP2A), total tau and p-tau(Ser³⁹⁶) in the neurons was observed by the method of immunohistochemistry. The protein content of total tau and p-tau(Ser³⁹⁶), and the expression level of PKA and PP-2A were detected by Western blotting analysis. RESULTS:In Aβ_1-40 group, the level of total tau, the phosphorylation of tau protein and the expression of PKA were significantly increased compared with those in sham operation group. Meanwhile, the expression of PP2A in Aβ_1-40 group was lower than that in sham operation group. In TEN treatment group, the level of total tau, the phosphorylation of tau protein and the expression of PKA were markedly decreased, and the expression of PP2A was increased as compared with Aβ_1-40 group. CONCLUSION:TEN may protect the neurons from the toxic effect of Aβ_1-40 and reduce the hyperphosphorylation of tau(Ser³⁹⁶) in the neurons of AD rats by activating the expression of PP2A and inhibiting the expression of PKA.     

EZH2-mediated regulation of NF-κB target gene expression in gastric cancer

AIM: To explore the mechanism by which over-expression of enhancer of zeste homolog 2 (EZH2) in a panel of gastric cancer cell lines is involved in tumorigenesis of gastric cancer. METHODS: Real-time PCR and Western blot were employed to examine the mRNA and protein levels of EZH2, respectively. MTS assay, cell migration and soft agar assay were performed to investigate the role of EZH2 in the regulation of stomach cancer behaviors. The effect of EZH2 on NF-κB target gene expression was determined by Luciferase reporter and real-time PCR. Co-immunoprecipitation was used to analyze the interaction of EZH2 and p65 in HEK293T cells. RESULTS: The expression levels of EZH2 were significantly increased in the gastric cancer cells compared with normal gastric epithelial cells. Pharmacological inhibition by DZNep or knockdown of EZH2 significantly compromised AGS and SNU-16 cell activity, cell migration and anchorage-independent cell growth. Moreover, siRNA knockdown of EZH2 impaired NF-κB downstream targets, such as IL-8, CXCL5 and CCL20. In addition, the interaction of EZH2 and p65 was detected. CONCLUSION: EZH2 mediates the growth of gastric cancer cells through the regulation of NF-κB downstream gene expression.     

Expression of MMP-2 and NF-κB in myocardium of mice with viral myocarditis

AIM: To observe the effects of TNF-α/nuclear factor-κB(NF-κB)/matrix metalloproteinase-2(MMP-2) pathway on the expression of MMP-2 in the mice with viral myocarditis. METHODS: Six-week-old inbred male mice were randomly assigned to control and myocarditis group. The mice in myocarditis group and control group were intraperitoneally inoculated with 0.1 mL 10^-5.69 TCID50/mL coxsackievirus B3 and vehicle (PBS), respectively. Ten mice were sacrificed at the 4th and 10th days after injection. The blood and heart specimens were harvested. The serum content of TNF-α was measured by ELISA. The myocardial levels of MMP-2, NF-κB p65 and IκBα were determined by Western blot. RESULTS: Compared with control group, the protein expression of MMP-2 and NF-κB p65 in the myocardium and the serum content of TNF-α were significantly increased in myocarditis group (P<0.05). The protein expression of IκBα was lower in myocarditis group than that in control group (P<0.05).CONCLUSION: TNF-α, NF-κB p65 and MMP-2 were higher in the mice with acute viral myocarditis. The increased expression of them might be involved in the pathogenesis of viral myocarditis.     

Effect of HMGB1 expression knockdown on invasion ability of breast cancer cells induced by TNF-α

AIM:To investigate the effect of high-mobility group box-1 (HMGB1) expression knockdown on the invasion ability of breast cancer cells induced by tumor necrosis factor-α (TNF-α). METHODS:HMGB1 siRNA was used to transfect into the breast cancer MDA-MB-231 cells. The expression of HMGB1 at mRNA and protein levels was determined by RT-qPCR and Western blot. After the MDA-MB-231 cells with HMGB1 expression knockdown were treated with TNF-α, the apoptosis rate was analyzed by flow cytometry, the cell invasion ability was measured by Transwell assay, and the cell migration ability was detected by cell scratch test. The protein expression of E-cadherin, MMP-2, N-cadherin, MMP-9 and Bax was determined by Western blot. RESULTS:The expression of HMGB1 at mRNA and protein levels in the MDA-MB-231 cells transfected with HMGB1 siRNA was significantly lower than that in the non-transfected cells (P<0.05). The apoptosis rate in the cells was increased after TNF-α treatment, and the cell invasion and migration abilities were also increased. The protein level of E-cadherin in the cells was decreased, the protein level of N-cadherin was increased, and the protein levels of MMP-2, MMP-9 and Bax were also increased (P<0.05). After the MDA-MB-231 cells with HMGB1 expression knockdown were induced by TNF-α, the apoptotic rate was increased, the invasion and migration abilities were decreased, the protein levels of E-cadherin and Bax were increased, and the protein levels of N-cadherin, MMP-2 and MMP-9 were decreased, as compared with the cells only induced by TNF-α without knockdown of HMGB1 expression (P<0.05). CONCLUSION:Knockdown of HMGB1 expression enhances the apoptosis of breast cancer cells induced by TNF-α, and inhibited the cell invasion, migration and epithelial-mesenchymal transition induced by TNF-α. The mechanism may be related with the changes of protein expression of MMP-2, MMP-9 and Bax.     

Effect of Yiqi Huayu Huatan decoction on expression of KLF15, HMGB1, NF-κB and its downstream inflammatory factors in rats with UUO-induced renal interstitial fibrosis

AIM: To observe the effect of Yiqi Huayu Huatan decoction (YHHD) on unilaterral ureteral obstruction (UUO)-induced renal interstitial fibrosis in rats, and to investigate the possible mechanism. METHODS: Female SD rats (n=48) were randomly divided into sham group, model group, telmisartan group, and low-, middle-and high-dose YHHD groups, with 8 rats in each group. The UUO model rats was established by ligating left ureter. The rats in sham group and model group were treated with equal volume of normal saline, others were treated with the corresponding drugs daily. After 12 weeks, the rats were sacrificed. The serum samples were collected for determining the concentrations of cystatin C (Cys-C) and uric acid (UA). The morphological changes of the renal tissue were observed by PAS staining. The collagen fiber was observed by Masson staining. The mRNA expression of Krüppel-like factor 15 (KLF15), high-mo-bility group box protein 1 (HMGB1), nuclear factor-κB (NF-κB), IκB, monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), fibronectin (FN), collagen type I (Col I) and Col-Ⅳ was detected by real-time PCR. The protein expression of KLF15, HMGB1 and NF-κB was detected by Western blot. The protein expression of MCP-1 was determined by the method of immunohistochemistry. RESULTS: Compared with sham group, the deposition rate of collagen fibers and the concentration of Cys-C in model group were significantly increased (P<0.05), the mRNA and protein expression of KLF15 was significantly down-regulated (P<0.05), while the mRNA expression of HMGB1, NF-κB, IκB, MCP-1, IL-1β, TNF-α, FN, Col I and Col Ⅳ and the protein expression of HMGB1, NF-κB and MCP-1 were significantly up-regulated (P<0.05). Compared with model group, the deposition rates of collagen fibers in middle-and high-dose YHHD groups and telmisartan group were significantly decreased (P<0.05), with down-regulated protein expression of HMGB1 and NF-κB and mRNA expression of IL-1β and TNF-α (P<0.05). The protein expression of KLF15 was significantly up-regulated in high-dose YHHD group and telmisartan group (P<0.05), while the protein expression of MCP-1 and the mRNA expression of FN were significantly down-regulated (P<0.05). The mRNA expression of KLF15 was significantly up-regulated in high-dose YHHD group (P<0.05), while the mRNA expression of MCP-1, Col I and Col IV was significantly down-regulated (P<0.05). The mRNA expression of NF-κB and IκB was significantly down-regulated and the concentration of Cys-C was significantly decreased in each dose of YHHD groups and telmisartan group (P<0.05). No significant difference of UA level among the groups was observed. CONCLUSION: YHHD alleviates renal interstitial fibrosis in a dose-dependent manner, and YHHD at high dose shows the most obvious effect. The mechanism may be associated with the up-regulation of KLF15 and the down-regulation of HMGB1, NF-κB and its downstream inflammation-related factors in the renal tissue.     

Inhibitory effect of proanthocyanidins B1 and B2 on inflammation of BV-2 cells induced by lipopolysaccharide

AIM: To investigate the different inhibitory effects of proanthocyanidins B1 and B2, which are isomers, on the inflammatory response of BV-2 cells induced by lipopolysaccharide (LPS). METHODS: MTT assay was used to detect the effects of proanthocyanidins B1 and B2 on the viability of BV-2 cells. LPS (1 mg/L) was used to promote BV-2 cells to secrete inflammatory factors. ELISA, chemotaxis assay and Western blot were used to detect the influence of proanthocyanidins B1 and B2 on the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cell chemotaxis and phosphorylation of NF-κB. RESULTS: Proanthocyanidins B1 and B2 did not show cytotoxicity effect on BV-2 cells. Proanthocyanidin B1 and B2 inhibited the cell chemotaxis, phosphorylation of NF-κB, and releases of TNF-α and IL-1β. CONCLUSION: Proanthocyanidins B1 and B2 inhibit the inflammatory response of BV-2 cells induced by LPS, and their action intensity didn't show significant difference.     

Role of TLR4-TRPC6 in LPS-induced NF-κB P65 expression and nuclear translocation in airway epithelial 16HBE cells

AIM: To investigate the role of Toll-like receptor 4 (TLR4) and transient receptor potential channel 6 (TRPC6) signaling pathway in lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) P65 expression and nuclear translocation in airway epithelial cells (16HBE) for supplementing the mechanism for airway inflammation. METHODS: After stimulating the 16HBE cells with LPS at 1 mg/L for 0, 0.5, 2, 6, 12 and 24 h, the expression of NF-κB P65 at mRNA and protein levels in the 16HBE cells were determined by RT-PCR and Western blot respectively, and the nuclear translocation of NF-κB P65 was detected by immunocytochemical staining method. The effects of TLR4 inhibitor CLI-095 at 5 μmol/L and TRPC6 agonist Hyp9 at 10 μmol/L on LPS (1 mg/L)-induced NF-κB P65 expression and nuclear translocation in the 16HBE cells were determined by RT-PCR, Western blot and immunocytochemical staining. RESULTS: LPS increased the mRNA and protein expression of NF-κB P65 and nuclear translocation in the 16HBE cells(P<0.05). TLR4 inhibitor CLI-095 reduced the mRNA and protein expression of NF-κB P65 and nuclear translocation induced by LPS, while Hyp9 enhanced the mRNA and protein expression of NF-κB P65 and nuclear translocation induced by LPS in the 16HBE cells(P<0.05). CONCLUSION: LPS induces the expression and nuclear translocation of NF-κB P65 in the 16HBE cells via TLR4-TRPC6 signaling pathway.     

Effects of Aβ1-42-induced microglial cells on survival of neural stem cells in vitro

AIM: To explore the effects of β-amyloid protein 1-42 (Aβ_1-42)-induced microglia on the survival of cultured neural stem cells (NSCs) in vitro . METHODS: Using the Transwell chambers to build a coculture system of NSCs and microglia, we detected the proliferation, differentiation and apoptosis of the NSCs with the microglia before and after induction by Aβ_1-42. RESULTS: Compared with non-intervention group, the proliferation rate of NSCs in Aβ_1-42 intervention coculture group decreased, as well as the positive expression rates of microtubule-associated protein 2 (MAP-2) and choline acetyltransferase. CONCLUSION: The inflammation mediated by Aβ_1-42 inhib their the proliferation of NSCs and induces their apoptosis. Inflammation also significantly reduces the ratio of NSCs differentiating to neurons, especially to cholinergic neurons.     

Regulation of ectopic trypsin and proinflammatory cytokine expression by NF-κB and AP-1 in influenza A virus induced myocarditis

AIM: To investigate the regulatory effects of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) on the expression of ectopic trypsin and proinflammatory cytokines in influenza A virus (IAV)-induced myocarditis. METHODS: Male BALB/c mice of 8 weeks old (n=40) were randomly divided into 4 groups: normal control group (NC), infection control group (IC), NF-κB inhibitor group (NI) and AP-1 inhibitor group (AI). The mice in NC group and IC group were instilled intranasally with 15 μL saline and 40 plaque forming units (PFU) IAV, respectively. The mice in NI group and AI group were infected intranasally with 40 PFU IAV and injected intraperitoneally with 10 mg/kg NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) or 2.5 mg/kg AP-1 inhibitor nordihydroguaiaretic acid (NDGA) once daily. The mice were euthanized at day 9 after instillation, and the hearts were removed for pathological and biochemical analysis. RESULTS: IAV infection induced significant up-regulation of ectopic trypsin, and proinflammatory cytokines interleukin 6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in the myocardium, and triggered acute myocarditis. PDTC significantly inhibited NF-κB activation and up-regulation of ectopic trypsin and proinflammatory cytokines, and effectively suppressed IAV replication and myocardial inflammatory response (P<0.01). NDGA effectively inhibited AP-1 activity (P<0.01) and mildly suppressed up-regulation of proinflammatory cytokines (P<0.05), but had no effects on the expression of ectopic trypsin, IAV replication and the extent of myocarditis (P>0.05). CONCLUSION: IAV infection induces up-regulation of ectopic trypsin and proinflammatory cytokines in myocardium predominantly by the activation of NF-κB. AP-1 signaling pathway might be only partially involved in the regulation of proinflammatory cytokines.     

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.     

Protective effect of procyanidins on PC12 cells exposed to Aβ25-35

AIM: To investigate the protective effect of procyanidins on the PC12 cells exposed to Aβ_25-35 and the mechanisms.METHODS: Aβ_25-35 at 25 μmol/L was used to treat the PC12 cells for 48 h, and the PC12 cells were pretreated with procyanidins at 25, 50 and 100 mg/L for 24 h. The cell vitality was measured by MTT assay. The content of reactive oxygen species (ROS) was detected by DCFH-DA staining. The change of mitochondrial membrane potential was examined by JC-10 staining. The apoptosis was analyzed by flow cytometry with Annexin V/PI double staining. The protein levels of activated caspase-3 was determined by Western blot.RESULTS: Under the exposure of the PC12 cells to Aβ_25-35, procyanidins increased the cell viability, reduced intracellular ROS level, prevented mitochondrial membrane potential decline, attenuated the caspase-3 activation and inhibited the apoptosis of PC12 cells (P<0.05 or P<0.01).CONCLUSION: Procyanidins have a significant protective effect on the PC12 cells exposed to Aβ_25-35. Its mechanism may be related to removing intracellular ROS induced by Aβ_25-35, relieving the damage to the mitochondrial membrane, and thereby inhibiting cell apoptosis.     

Effect of lysophosphatidic acid on expression of integrin β6 in epithelial cells

AIM: To investigate the effect of lysophosphatidic acid (LPA) on the expression of integrin β₆ (ITGB6) for determining the role of transforming growth factor β (TGF-β) activation induced by LPA in this process. METHODS: Normal human bronchial epithelial (NHBE) cells were primarily cultured in 6 well plate and stimulated with LPA. The mRNA expression of ITGB6 and the level of cell surface ITGB6 protein were detected by RT-PCR and flow cytometry,respectively. The activity of active TGF-β induced by LPA was measured by the method of transformed mink lung epithelial cells (TMLC) transfected with TGF-β responsive plasminogen activator inhibitor 1(PAI-1) promoter fused with firefly luciferase reporter gene. RESULTS: After stimulated with LPA at concentration of 10 μmol/L for 2 h, the mRNA expression of ITGB6 in epithelial cells was significantly increased in a time-dependent manner. The results of flow cytometry showed that the protein level of ITGB6 on cell surface was obviously increased after treated with LPA at concentration of 10 μmol/L for 4 h. The active TGF-β induced by LPA in epithelial cells was blocked by an α_Vβ₆ blocking antibody. However, α_Vβ₆ blocking antibody failed to inhibit the mRNA expression of ITGB6 induced by LPA. CONCLUSION: LPA induces the mRNA and cell surface protein of ITGB6 in epithelial cells. The up-regulated ITGB6 expression by LPA is independent on LPA-induced TGF-β activation.     

Effect of edaravone on expression of Aβ and AQP4 and activity of MMP2 and MMP9 in acute cerebral ischemia/reperfusion rats

AIM: To investigate the effect of edaravone on acute cerebral ischemia/reperfusion rats. METHODS: SD rats were randomly divided into sham operation group (saline), model group (modeling given saline), low dose group (edaravone at 6 mg/kg) and high dose group (edaravone at 10 mg/kg). The rat model was established by Zea Longa suture method. The nerve function scores were evaluated after operation, and the infarct volume was measured by TTC assay. The mRNA expression of aquaporin 4 (AQP4) and amyloid β-protein (Aβ) in the brain tissue was detected by RT-qPCR. The protein levels of APQ4 and Aβ were determined by Western blot. The activity of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9(MMP9) was detected by gelatin zymography. RESULTS: Compared with model group, edaravone administration markedly alleviated neurological deficits, histological damages and brain edema. The mRNA and protein levels of AQP4 and Aβ, and the activity of MMP2 and MMP9 were downregulated (P<0.05). Furthermore, the improvements in high dose group were significantly more effective than those in low dose group. CONCLUSION: Edaravone significantly reduces neurological deficits and brain edema in the rats with acute ischemic stroke, and the mechanisms may be related to the downregulation of AQP4 and Aβ, and the activation of MMP2 and MMP9.