Expression of TLR2 and TLR4 on mast cells in human chronic periapical diseases

AIM: To observe the expression of TLR2 and TLR4 on mast cells(MCs) in the periapical tissues from different types of human chronic periapical diseases, and to analyze the role of TLR2 and TLR4 on tryptase-positive MCs in the immunopathogenesis of human chronic periapical diseases. METHODS: A total of 60 donors, including healthy control group, periapical granuloma group and periapical cyst group, were enrolled in the study. The periapical tissue specimens were fixed in 10% buffered formalin and stained with hematoxylin and eosin for histopathology, or stained with double-immunofluorescence for identification of TLR2-tryptase and TLR4-tryptase double-positive MCs in the periapical tissues. RESULTS: Compared with the healthy control, the densities of TLR2-tryptase and TLR4-tryptase double-positive MCs in periapical tissues were significantly increased in human chronic periapical diseases(P<0.01). The densities of TLR2-tryptase and TLR4-tryptase double-positive MCs in periapical cyst group were significantly higher than those in periapical granuloma group(P<0.01). CONCLUSION: TLR2 and TLR4 were expressed on the MCs in the periapical tissues of human chronic periapical diseases. TLR2-tryptase and TLR4-tryptase double-positive MCs may participate in the pathogenesis of chronic periapical diseases.     

Involvement of TLR4/NLRP3 inflammasome in contrast medium-induced inflammation and injury in renal tubular epithelial cells

AIM: To investigate whether Toll-like receptor 4 (TLR4) and Nod-like receptor protein 3 (NLRP3) inflammasome were involved in contrast medium (CM)-induced inflammation and injury in renal tubular epithelial cells. METHODS: Iopromide was used to injure NRK-52E cells in the study. The cell viability was measured by CCK-8 assay. The protein levels of TLR4, NLRP3, apoptosis-associated speckle-like protein (ASC), caspase-1 and cleaved caspase-3 were determined by Western blot. The releases of interleukin (IL)-1β and IL-18 were detected by ELISA. The apoptotic rate was evaluated by Hoechst staining, and mitochondrial membrane potential (MMP) was analyzed by JC-1 staining. siRNA was transfected into the NRK-52E cells to silence NLRP3 expression. RESULTS: CM decreased the viability of NRK-52E cells (P<0.05). CM also elevated the protein levels of cleaved caspase-3, TLR4, NLRP3, IL-1β and IL-18 (P<0.05). Silencing NLRP3 attenuated CM-induced releases of inflammatory cytokines. Moreover, treatment with TLR4 inhibitor TAK-242 or knockdown of NLRP3 by siRNA transfection both attenuated cell apoptosis and loss of MMP caused by CM. CONCLUSION: TLR4/NLRP3 inflammasome takes part in the pathogenesis of CM-induced acute kidney injury, and mediates CM-induced injury and inflammation in renal tubular epithelial cells.     

Effects of panaxadiol saponin on TLR2 and TLR9 mRNA expression in LPS induced shock rats

AIM: To explore the molecular mechanism of panaxadiol saponin (PDS) by observing Toll like receptor (TLR) 2 and TLR9 mRNA expression induced by lipopolysaccharide (LPS).METHODS: Rats were divided into LPS, LPS+PDSL, LPS+PDSM and control group, respectively. Nitric oxide synthase (NOS) activity, nitric oxide (NO) content, LPO content, SOD activity and TLR2 and TLR9 mRNA expression were assayed 4 h after intravenous injection of LPS. RESULTS: NOS activity, NO content, LPO content of LPS+PDSL group and LPS+PDSM group were significantly lower than those in LPS group. TLR2 mRNA expression in the liver tissue of LPS+PDSL group and LPS+PDSM group was decreased compared with LPS group.CONCLUSION: PDS has a protective effect on liver tissues by triggering the down-regulation of TLR2 expression, reducing NOS activity, and NO content.     

Immunological mechanism of long-term stimulation by LPS in macrophages

AIM: To investigate the molecular mechanism and the immunosuppressive phenotype of macrophages under long-term exposure to lipopolysaccharide (LPS). METHODS: We used Ficoll-Hypaque density gradient centri-fugation combined with MicroBeads Separation Kits to separate peripheral blood mononuclear cells from human blood, and then induced the monocytes into macrophages. We observed the morphology of the macrophages by treating the cells with LPS for 48 h, in comparison with a negative control and IFN-γ treatment. ELISA was used to detect the levels of cytokines, such as IL-10, IL-12, IL-6 and TNF-α, and flow cytometry was used to detect the expression of the surface molecules (HLA-DR, CD14, CCR7, HLA-ABC and CD40). To observe the effect of macrophage on T cell proliferation, co-culture experiment was carried out for 6 d. Real-time PCR was used to validate the expression levels of molecules related to MyD88-independent pathway in Toll-like receptor 4 (TLR4) signal pathway. RESULTS: The antigen-presenting ability of the macrophages was reduced and the IL-10 expression level was increased after the cells were treated with LPS for 48 h. We observed a poor proliferative capacity of CD8⁺ T cells after co-culturing of LPS-induced macrophages with CD3⁺ T cells for 6 d. The results of real-time PCR indicated that TRIF, IRF3 and CIITA were down-regulated in LPS-induced macrophages.CONCLUSION: We successfully established a macrophage model in vitro and observed that LPS-induced macrophages into an immunosuppressive phenotype with poor CD8⁺ T cell proliferative capacity, in which MyD88-independent TLR4 signaling pathway was impaired.     

Effect of TLR4 on regulation of autophagy in renal tubular epithelial cells by soluble uric acid

AIM To investigate the role of Toll-like receptor 4 (TLR4) in autophagy induced by soluble uric acid in renal tubular epithelial HK-2 cells. METHODS After HK-2 cells were co-stimulated with soluble uric acid or/and chloroquine (CQ), the protein expression of LC3-Ⅱ and P62 was determined by Western blot. Autophagosomes and autophagolysosomes were observed by transmission electron microscopy. Next, HK-2 cells were co-stimulated with soluble uric acid and TLR4 inhibitor TAK242. The mRNA expression of TLR4 was detected by RT-qPCR, the protein expression of TLR4, LC3-Ⅱ and P62 was determined by Western blot, and the autophagic flux was observed by the method of mRFP-GFP-LC3. RESULTS The expression of LC3-Ⅱand P62 in the HK-2 cells was up-regulated by soluble uric acid. The expression of LC3-Ⅱ and P62 was further increased after co-stimulated with uric acid and CQ,and the number of autophagic body was increased while the number of autophagolyososome was decreased as observed by TEM, indicating that the autophagic flux was blocked. The expression levels of TLR4, LC3-Ⅱ and P62 in soluble uric acid group were higher than those in control group. Meanwhile, TAK242 inhibited the up-regulation of TLR4, LC3-Ⅱ and P62 by soluble uric acid. The results of mRFP-GFP-LC3 experiment showed that the levels of autophagosomes were significantly higher in soluble uric acid group than that in control group, and the levels of autophagolysosomes were lower. Compared with soluble uric acid group, the level of autophagosomes was decreased, and autophagolysosomes was increased in TAK242+soluble uric acid group, indicating that the fusion of autophagosomes and lysosomes was increased, and the process of autophagolysosome formation was smoother. CONCLUSION Soluble uric acid leads blocked autophagic flux in renal tubular epithelial cells. Soluble uric acid mediates abnormal tubular autophagic flux through TLR4.     

Effects of GW1929 on macrophage TLR expression and inflammation induced by ox-LDL

AIM: To investigate the effects of ox-LDL on TLR2 and TLR4 expression and production of TNF-α, IL-10, IL-12, NO and MDA in macrophages and to observe intervention effect of GW1929 in above procedure. METHODS: The mouse peritoneal macrophages were pretreated with ox-LDL (50 mg/L, 100 mg/L) and GW1929 (20 μmol/L) respectively for 24 h. The concentrations of MDA, NO-2/NO-3, TNF-α, IL-10 and IL-12 in the culture fluid were detected. Flow cytometry was used to observe TLR2 and TLR4 expressions after the mouse peritoneal macrophages were pretreated with ox-LDL (50 mg/L) and GW1929 (20 μmol/L) respectively for 6 h, 12 h, and 24 h. RESULTS: The concentrations of MDA, NO-2/NO-3, TNF-α and IL-10 in ox-LDL (50 mg/L, 100 mg/L) group were higher than those in control and GW1929 group obviously, but the concentrations of above index in ox-LDL (50 mg/L, 100 mg/L)+GW1929 group were lower than those in ox-LDL (50 mg/L, 100 mg/L) group apparently. No IL-12 in every group was detected. Expressions of TLR-2 in ox-LDL+GW1929 (6 h, 12 h, 24 h) group were lower than those in ox-LDL (6 h, 12 h, 24 h) group respectively. TLR-4 expressions in ox-LDL+GW1929 (12 h) were lower than those in ox-LDL (12 h) apparently. CONCLUSION: ox-LDL up-regulates TLR2 and TLR4 expressions and promotes the production of ROX, NO, TNF-α and IL-10 in macrophages. GW1929 is capable of inhibiting the above ox-LDL effects.     

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.     

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.     

Postconditioning modulates TLR4 expression in hippocampus of tree shrews with thrombotic cerebral ischemia

AIM: To observe the effects of thrombotic cerebral ischemia and postconditioning on the expression of toll-like receptor 4 (TLR4) in hippocampus of tree shrews.METHODS: The model of thrombotic focal cerebral ischemia was established by photochemical reaction.Four hours after the onset of photochemical reaction, ischemic postconditioning was induced by 3 repeated cycles of carotid artery occlusion for 5 min and reperfusion for 5 min. The histological changes of hippocampus (by HE staining), TLR4 protein level (by Western blotting) and TLR4 mRNA expression (by semiquantitative RT-PCR) were observed.RESULTS: The extensive neuronal degeneration in hippocampus was observed from 4 h to 72 h and peaked at 24 h after cerebral ischemia, but was significantly attenuated after postconditioning. Cerebral ischemia caused a progressive increase in the expression of TLR4 protein at 4 h and 24 h (P<0.05), and decreased at 72 h (P<0.05). In contrast to ischemia groups, postconditioning decreased the expression of TLR4 protein at 4 h and 24 h (P<0.05), but an increase in the expression of TLR4 at 72 h (P<0.05) was observed. Simultaneously, the level of TLR4 mRNA in hippocampus showed the tendency of approximate variation in accordance with the protein expression.CONCLUSION: The expression of TLR4 increases by cerebral ischemia. The protection mechanisms of postconditioning may be associated with modulating TLR4 expression.     

Role of intestinal myofibroblasts in IBD-associated intestinal fibrosis

Intestinal fibrosis is the complications of inflammatory bowel disease (IBD), intestinal myofibroblast cells are the key to intestinal fibrosis. Intestinal myofibroblasts and its interaction with inflammatory cells play an important role in IBD-related intestinal fibrosis.     

Effect of Chinese propolis on PC-PLC activity and TLR4 expression in LPS-treated vascular endothelial cells

AIM: To investigate the effect of Chinese propolis on the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) and the expression of Toll-like receptor 4 (TLR4) in LPS-treated vascular endothelial cells (VECs). METHODS: Confluent VECs were stimulated with LPS at the concentration of 100 μg/L in the presence of 0.5% fetal bovine serum. The cells were treated with Chinese propolis at the concentration of 12.5 mg/L for 12 h and 24 h. The viability of VECs and the level of nitric oxide (NO) were detected by sulforhodamine B (SRB) assay and chemical method, respectively. The activity of PC-PLC was measured using L-α-phosphatidylcholine as substrate. The protein levels of TLR4, nuclear factor-κB p65 (NF-κB p65) and p53 were determined by Western blotting. The level of intracellular reactive oxygen species (ROS) was examined using a fluorescent probe, 2,7-dichlorodihydrofluorescin (DCHF). For the measurement of mitochondrial membrane potential, the fluorescent dye JC-1 was used. RESULTS: Treatment with Chinese propolis for 24 h had no effect on the viability of VECs. However, the levels of NO and ROS were significantly decreased by Chinese propolis. PC-PLC activity and NF-κB p65 expression were significantly depressed by Chinese propolis treated for 12 h, and the expression of TLR4 and p53 were dramatically decreased by Chinese propolis treated for 12 and 24 h. No effect of Chinese propolis on mitochondrial membrane potential was observed. CONCLUSION: Chinese propolis depresses the activity of PC-PLC and the expression of TLR4, and then inhibits the downstream signal molecules such as NF-κB p65, p53, ROS and NO in VECs.     

Expression and significance of TLR4/NF-κB in pulmonary vascular remodeling in smoking rats

ATM: To observe the expression of Toll-like receptor 4 (TLR4), nuclear factor-κB subunit P65 protein (NF-κB P65) and proliferating cell nuclear antigen (PCNA) in the pulmonary vascular tissues of the rats exposed to smoke, and to explore the possible mechanism of TLR4/NF-κB signaling pathway in pulmonary vascular remodeling. METHODS: SPF male healthy rats (n=48) were randomly divided into control group, smoke exposure for 4 weeks group (S4 group), smoke exposure for 8 weeks group (S8 group) and smoke exposure for 12 weeks group (S12 group), with 12 rats in each group. HE staining was used to observe the morphological changes of pulmonary vessels, and then the pulmonary vascular wall area/total vascular area (WA%) and vascular wall thickness/vascular external diameter (WT%) were measured by the medical image analysis system. The expression of TLR4, NF-κB P65 and PCNA in the pulmonary vascular tissues was detected by immunohistochemical staining. The protein content was expressed by the average integral absorbance. The mRNA expression of TLR4 in the pulmonary vessels was detected by RT-qPCR. The relationships between WA%, WT%,TLR4 protein, TLR4 mRNA, P65 protein, PCNA protein and pulmonary vascular remodeling, and another relationships between WA%, WT%, P65 protein, PCNA protein and TLR4 protein were analyzed.RESULTS: The WA% and WT% in smoke exposure groups significantly increased compared with control group, and the ratio was proportional to the time of smoke exposure. The protein expression of TLR4, p65 and PCNA, and the mRNA expression of TLR4 in smoke exposure groups also increased significantly compared with control group. CONCLUSION: The extent of pulmonary vascular remodeling in the rats increases when the protein expression of TLR4 is up-regulated. There is a positive correlation between pulmonary vascular remodeling and the protein expression of TLR4 and NF-κB P65. Pulmonary vascular remodeling may be related to the activation of TLR4/NF-κB signaling pathway.     

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.     

Influence of glycine on LBP mRNA expression induced by LPS in the liver of rats

AIM:To study the influence of glycine(GLY) on lipopolysaccharide-binding protein(LBP) mRNA expression induced by LPS.METHODS:The level of LBP mRNA expression in liver tissues of rats was examined by RT-PCR, and the effects of glycine on LBP mRNA expression in liver tissues of rats induced by LPS were investigated.RESULTS: The level of LBP mRNA expression in hepatic tissue of rats in the LPS group was significantly higher than that in the control group(P＜0.01), the level of LBP mRNA expression in the hepatic tissue of rats in the LPS+GLY group was lower than that in the LPS group(P＜0.01).CONCLUSION:LPS can induce LBP mRNA expression in the hepatic tissue of rats, glycine can inhibit LBP mRNA expression in the hepatic tissue of rats treated by LPS.     

Effects of atorvastatin on cardiac myocyte hypertrophy of neonatal rat induced by angiotensinⅡ in vitro and TLR4 expression

AIM: To assess effects of atorvastatin (Ator) on cardiac myocytes (CM) hypertrophy of neonatal rat induced by angiotensinⅡ (AngⅡ) in vitro and Toll like receptor 4 (TLR4) expression for theoretical bases of preventing and treating myocardial hypertrophy.METHODS: CM of neonatal Sprague-Dawley (SD) rats were isolated with trypsin digestion method and those growth-arrested cells were stimulated with 10^-7 mol/L AngⅡ in the presence of various concentrations of Ator.The method of coomassie brilliant blue was adopted to evaluate the protein contents of CM.The changes in β-MHC,AT₁ receptor and TLR4 mRNA expression were observed by RT-PCR.RESULTS: ① AngⅡ increased the protein content of CM and β-MHC mRNA expression significantly,upregulated AT₁ receptor and TLR4 gene expression.② In a dose-dependent manner,Ator inhibited the increases in the protein contents of CM and β-MHC mRNA expression induced by AngⅡ.③ In a dose-dependent manner,Ator downregulated the AT₁ receptor and TLR4 mRNA expression of CM hypertrophy of neonatal rat induced by AngⅡ.CONCLUSION: Ator inhibits CM hypertrophy,downregulates the AT₁ receptor and TLR4 gene expression.     

Role of purinergic signaling mediated by ATP in Alzheimer's disease-associated colonic motility disorder

AIM: To explore the role of purinergic signaling mediated by ATP in the Alzheimer's disease (AD)-related colon motility disorder and its related molecular mechanisms. METHODS: (1)Clinical trials:AD patients in our hospital were collected and studied. Radioimmunoassay was used for the determination of plasma motilin (MTL), cholecystokinin (CCK), vasoactive intestinal peptide (VIP) and nitric oxide (NO), and high-performance liquid chromatography (HPLC) was applied to test the level of adenosine triphosphate (ATP). The patients were assessed by neuropsychology and scored accordingly. (2)In animal experiments, AD mice received Morris water maze test, and the spatial learning and memory function were evaluated. The plasma levels of MTL, CCK, VIP and NO were examined by radioimmunoassay, and the level of ATP was measured by HPLC. Choline acetyltransferase (ChAT), VIP, nitric oxide synthase (NOS) and ATP synthase were detected by immunohistochemistry. Western blot and immunohistochemistry were used to detect the expression of P2Y receptor. (3)In vitro, organ bath was applied to observe the effect of α,β-methylene ATP (α,β-MeATP), an agonist of P2Y receptor, on both spontaneous and electrically evoked contraction of colonic smooth muscle strip, and the technique of intracellular microelectrode was applied to observe the effect of α,β-MeATP on the membrane potential of colonic smooth muscle cells. RESULTS: Compared with control group, the levels of MTL and CCK were decreased (P<0.01), and the levels of NO and ATP were increased (P < 0.05 or P < 0.01), while the VIP level was not changed. Mini-Mental State Examination (MMSE) score was decreased (P<0.05), Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) score, Neuropsychiatric Inventory (NPI) score and Alzheimer's Disease Cooperative Study-Activities of Daily Living Scale (ADCS-ADL) were all increased as compared with control group (P<0.01). The 4~6 d escape latency of APP/PS1 AD mice was significantly prolonged (P<0.05), and the space exploration ability distinctly reduced (P<0.05). In AD mice, the levels of MTL and CCK were decreased (P<0.01), and the levels of NO and ATP were increased (P<0.05 or P<0.01), while the VIP level was not changed. The protein expression of colonic ATP synthase was significantly increased (P<0.05), but the expression of ChAT, VIP and NOS was not changed. The expression of P2Y receptor was increased (P<0.01). The results of in vitro experiment displayed that α,β-MeATP, from 20 μmol/L to 100 μmol/L, inhibited the spontaneous contraction of colonic smooth muscle strip in the normal mice and AD mice (P < 0.05 or P < 0.01), and this inhibition was reversed by Na⁺ channel inhibitor tetrodotoxin (TTX) (P < 0.05 or P < 0.01). In addition, the effect of α,β-MeATP at 100 μmol/L on the AD mice was more obvious than that on the normal mice (P<0.05), and this inhibition was also antagonized by TTX (P < 0.05 or P < 0.01), pro-minent in AD group as compared with control group (P<0.05). In 10 Hz electrically evoked contraction of colonic smooth muscle strip, α,β-MeATP inhibited both the normal and AD mice (P < 0.05 or P < 0.01), while the inhibition was more obvious in the AD mice at the concentration of 40 μmol/L or 100 μmol/L (P < 0.05 or P < 0.01). CONCLUSION: AD patients and AD mice are accompanied by decreased MTL and CCK levels, and enhanced NO level, thus inducing colonic motor dysfunction along with AD. Meanwhile, ATP in plasma, purinergic neurons, and P2Y receptor expression are increased in the AD mice. Purinergic signaling mediated by ATP inhibits colonic smooth muscle strip contraction and further paralyzes the colonic movement function in AD.     

Toll-like receptor 4 expression on mast cells in human gingival tissues with chronic periodontitis

AIM: To observe the expression of Toll-like receptor 4 (TLR4) on mast cells in human gingival tissues with chronic periodontitis. METHODS: A total of 68 volunteers, including 23 cases of mild chronic periodontitis, 25 cases of severe chronic periodontitis and 20 healthy controls, were involved in this study, and their gingival specimens were taken and fixed in 4% neutral formalin. The histological changes of gingival tissues were observed by HE staining. The expression of TLR4 in gingival tissues was detected by immunohistochemical staining, and TLR4 expression on mast cells was detected by immunofluorescence double staining. RESULTS: The expression of TLR4 in gingival tissues and on mast cells in chronic periodontitis groups was significantly higher than that in normal control group (P<005), and that in severe chronic periodontitis group was significantly higher than that in mild chronic periodontitis group (P<005). CONCLUSION: The expression of TLR4 in gingival tissues and on mast cells is increased with the severity of chronic periodontitis, suggesting that TLR4, especially TLR4 on mast cells, may play an important role in human chronic periodontitis.     

Escherichia coli promotes recovery from dextran sulfate sodium-induced colitis in mice

AIM: To investigate the important role of intestinal microflora in the pathogenesis of inflammatory bowel disease by studying the effects of E.coli on dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: BALB/c mice were given 3.5% DSS in the drinking water for 5 days to induce acute colitis. The control mice (n=6) did not drink DSS water. The mice subject to drinking DSS were randomly divided into 3 groups: (1) simple DSS treatment group; (2) bacteria-depleted mice treated with DSS alone; (3) bacteria-depleted mice treated with DSS+E.coli. Four aspects of the treatment response were evaluated in each group: (1) general conditions, including body weight, disease activity index (DAI) score, colon length and weight; (2) histological score; (3) chemical colorimetric detection of myeloperoxidase (MPO) activity in the diseased tissues; (4) the activation of NF-κB detected by immune histochemistry. RESULTS: The bacteria-depleted mice that did not treat with E.coli were difficult to recover from DSS-induced colitis. Compared with non-treatment group, the general score, histological score and MPO activity in E.coli treatment group improved significantly (P<0.05). The activity of NF-κB in E.coli treatment group was significantly higher than that in non-E.coli treatment group (P<0.05). CONCLUSION: Intestinal microbiota is necessary during the recovery from DSS-induced colitis. E.coli promotes the recovery from DSS-induced colitis in mice. The mechanism may be associated with NF-κB activation.     

Effects of lipopolysaccharide binding protein on activation of p38 signaling pathway induced by LPS in macrophages

AIM:To investigate the regulatory effects of lipopolysaccharide binding protein(LBP)on activation of p38 signaling pathway induced by lipopolysaccharide(LPS)in alveolar macrophages.METHODS:The LBP from actue phase rat serum was purified by ammonium sulphate precipitation, Bio-Rex70 resin and the MonoQ column. Rat alveolar macrophages were exposed to LPS (0.01 mg/L or 1 mg/L) the various concentrations of LBP(0 mg/L, 0.01 mg/L, 0.1 mg/L, 1 mg/L and 10 mg/L).Western blotting were used to detect phospho-p38 in alveolar macrophages. RESULTS:SDS-PAGE analysis indicated that the purified preparation of rat LBP showed homogeneity and the molecu-lar weight was 60 kD.The binding of lipopolysaccharide to mononuclear cells were enhanced by purified rat LBP.Stimu-lation of rat alveolar macrophages with LPS at concentration of 0.01 mg/L was LBP dependent.LBP at concentrations up to 1 mg/L was able to increase the activation of p38.However, when LBP concentrations were further increased to 10mg/L, the phosphorylation levers of p38 were lower as compared with that in the presence of 1 mg/L.Stimulation of ratalveolar macrophages with LPS at concentrations of 1 mg/L was LBP-independent.CONCLUSION:The activation of p38 induced by LPS at lower concentration(0.01 mg/L) was LBP-dependent, meanwhile, LPS at higher concentration(1 mg/L) was LBP-independent.