ObjectiveTo observe the expression of Toll-like receptor 4 (TLR4) and inflammatory cytokines, leucocytic density and permeability in retina of diabetic rat. MethodsA total of 106 Brown Norway rats were randomly divided into experimental group and control group with 53 rats in each group. Diabetic model was established in experimental group by intraperitoneal injection of streptozotocin, and control rats received intraperitoneal injection of an equal volume of citric acid-sodium citrate buffer. Four weeks later, the retinas were collected for further analysis. TLR4 RNA and protein expression were measured by quantitative polymerase chain reaction and Western blot. Inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemo-attractant protein-1 (MCP-1), were measured by enzyme-linked immunosorbent assay in rat retina homogenate. Leukocyte density in the retina was measured by acridine orange fundus angiography. The retinal permeability was evaluated by Evans blue (EB) staining. ResultsTLR4 expression was significantly increased in diabetic rats of experimental group compared with non-diabetic rats of control group (F=1.606, 0.789; P < 0.05). Inflammatory cytokines (TNF-α, IL-1β and MCP-1) were significantly increased in retina of diabetic rats of experimental group versus non-diabetic rat of control group (F=24.622, 5.758, 4.829; P < 0.05). The retinal leukocyte density was (6.2±0.5)×10-5, (2.2±0.3)×10-5 cells/pixel2 in experimental and control group respectively, the difference was statistically significant (F=2.025, P < 0.05). The amount of retinal EB leakage was (23.41±4.47), (13.22±3.59) ng/mg in experimental and control group respectively, the difference was statistically significant (F=21.08, P < 0.05). ConclusionTLR4 and inflammatory cytokines expression, leucocytic density and permeability increased significantly in retina of diabetic rat.
Objective To observe the expression of GdCl3 on Toll-like receptors （TLRs） of RAW264.7 from murine macrophage cell line induced by lipopolysaccharide (LPS) stimulation. Methods Cells were divided into 3 groups: blank group, LPS group and GdCl3 group. And these cells dyed by goat anti-mouse TLR2/4 poly-antibody and anti-goat IgG labelled with fluorescein isothiocyanate (FITC). The synthesis of TLR2/4 protein were determined by flow cytometry (FCM) analysis and reverse transcription polymerase chain reaction (RT-PCR) analyzed their gene expression. Cell supernatants were taken to measure TNF-α production following the ELISA (enzyme-linked immunosorbent assay) protocol. Results The expressions of TLR2/4 protein and mRNA in GdCl3 group under action of different concentration of GdCl3〔TLR2/4 protein, 200 μmol/L: (70.2±1.28）％/(66.7±2.59)％, 400 μmol/L: (64.9±1.43)％/(60.4±1.25)％, 2 000 μmol/L: (47.4±0.98)％/(32.1±0.74)％; TLR2/4 mRNA (the value of absorbance), 200 μmol/L: (76.42±2.76)/(101.72±3.14), 400 μmol/L: (75.60±3.76)/(89.65±5.17), 2 000 μmol/L: (64.22±4.67)/(78.44±4.88)〕 were significantly lower than those of in LPS group 〔TLR2/4 protein: (94.4±1.76)％/(95.7±0.87)％, P＜0.01; TLR2/4 mRNA: (127.64±3.25)/(119.82±5.59), P＜0.05, P＜0.01〕. The expression of TNF-α in GdCl3 group under action of different concentration of GdCl3〔200 μmol/L: (2 540±77) pg/ml, 400 μmol/L: (2 041±106) pg/ml, 2 000 μmol/L: (1 020±220) pg/ml〕 was also significantly lower that that of in LPS group 〔(4 688±127) pg/ml, P＜0.01)〕. Conclusion GdCl3 significantly inhibits TLR expression and secretion of TNF-α under the condition of LPS stimulation in vivo.
Objective To investigate the effect of peptidoglycan (PGN) on the secretion of pro-inflammatory cytokines by dendritic cells (DCs) and the regulation of T helper 17 (Th17) responses in experimental autoimmune uveitis. Methods Bone marrow cells from naive mice were cultured with granulocyte macrophage-colony-stimulating factor and interleukin (IL)-4 to induce DCs. DCs cultured for six days were randomly divided into two groups: PGNtreated group and control group. The DCs in PGNtreated group were stimulated with PGN and the same volume of phosphate buffered saline was added to the DCs as control group. The relative mRNA expression levels of IL-23, tumor necrotic factor alpha; (TNF-alpha;), IL-6,IL-1beta;were measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Peptide fragment of interphotoreceptor retinoidbinding protein (IRBP1-20)specific T cells, which were isolated from the spleen and draining lymph nodes of C57BL/6 mice immunized with IRBP1-20 peptide fragments 13 days earlier, were co-cultured with PGN-treated or untreated DCs, respectively. Total RNA from T cells cocultured for two days were isolated and the relative expression of retinoic acid receptor-related orphan receptor gamma;t (ROR-gamma;t), IL-17, T-box expression in T cells (T-bet), interferon gamma; (IFN-gamma;) mRNA were detected by realtime RT-PCR. On the second, the fifth and the seventh day, the cocultured T cells were analyzed by flow cytometry to detect the percentages of IFN-gamma;, IL-17 positive cells. Results The real-time RT-PCR results revealed that the level of IL-23, IL-1beta;, IL-6, TNF-alpha; mRNA from PGNstimulated DCs were significantly increased compared to the control group (t=-14.363, -5.627, -3.85, -28.151; P＜0.05). The level of RORgamma;t, IL-17 mRNA from the T cells cocultured with PGN-stimulated DCs were greatly increased compared with the control group (t=-5.601, -19.76；P＜0.05). However, the level of T-bet, IFN-gamma; mRNA from the T cells cocultured with PGNstimulated DCs were significantly decreased compared with the control group (t=4.717, 11.207; P＜0.05). Data of flow cytometry showed that at two days, five days, seven days after cocultured with PGN-treated DCs, the percentages of IL-17 positive T cells were increased compared to the control group (t=-2.944, -3.03, -4.81; P＜0.05), and the percentages of IFN-gamma; positive T cells had no remarkable change (t=-1.25, -0.18, -2.16; P＞0.05). Conclusion PGN can promote the secretion of Th17-related cytokines by DCs, which favors proliferation and differentiation of Th17 in experimental autoimmune uveitis.
Objective To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis (OA). Methods Human cartilage cell line C28/I2 was cultured in vitro and treated with 10 ng/mL interleukin 1β (IL-1β) for 24 hours to construct an in vitro OA model. C28/I2 cells were transfected with miR mimics, mimics negative control (NC), over expression (oe)-NC, and oe-Toll-like receptor 4 (TLR4), respectively, and then treated with 10 ng/mL IL-1β for 24 hours to establish OA model. Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2’-deoxyuridine, cell apoptosis and cell cycle were detected by flow cytometry, and B-cell lymphoma 2 protion (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-Caspase-3, TLR4, myeloid differentiation primary response gene 88 (MyD88), p65 and phosphorylated p65 (p-p65) protein expression levels were detected by Western blot. Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4, and ELISA was used to detect pro-inflammatory factor prostaglandin E2 (PGE2), tumor necrosis factor α (TNF -α), and IL-6 levels in cell supernatant. The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database, and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay. Results After the treatment of C28/I2 cells with IL-1β, the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced. The expression levels of Bax and cleaved-Caspase-3 protein, the levels of pro-inflammatory factors (PGE2, TNF-α, IL-6) in the supernatant of C28/I2 cells, and the apoptosis of C28/I2 cells significantly increased. In addition, the proportion of the cells at S phase and G2 phase decreased significantly, and the proportion of cells at G1 phase increased significantly, suggesting that the cell cycle was blocked after IL-1β treatment. After transfection with miR mimics, the expression level of miR-515-5p in the cells significantly up-regulated, partially reversing the apoptosis of OA chondrocytes induced by IL-1β, and alleviating the cycle arrest and inflammatory response of OA chondrocytes. After treating C28/I2 cells with IL-1β, the mRNA and protein levels of TLR4 significantly increased. Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factor κB (NF-κB) pathway. Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1β-induced apoptosis and inflammation of OA chondrocytes. ConclusionmiR-515-5p negatively regulates the expression of TLR4, inhibits the activation of MyD88/NF-κB pathway and apoptosis of OA chondrocytes, and effectively alleviates the inflammatory response of the cells.
Objective To review the research progress of Toll-like receptors （TLRs） signaling and its effects in organ transplantation. Methods The structural and functional features of TLRs and their ligands were summarized，the literatures in recent years about the research progress of TLRs signaling in animal experiment and clinical organ transplantation were reviewed. Results TRLs played an important role in the organ transplantation，the activation of TLRs could activate the specific immune system，and contribute to ischemic reperfusion injury，acute and chronic allograft rejections，and induce the immune tolerance. Early treatment intervention could reduce the activation of TRLs through ischemic reperfusion injury in the organ transplantation，and improve the allograft survival. The efficient immunosuppressive drugs which aimed at the related immunosuppressive target in immune and its signal transduction pathway could reduce ischemic reperfusion injury in the organ transplantation and immune rejection. Conclusions TRLs signaling plays an important role in ischemic reperfusion injury，immune rejection，and immune regulation.
【Abstract】Objective To explore Toll-like receptor 4 (TLR4) expression and distribution in rat pancreas.Methods Reverse transcriptase-polymerase chain reaction (RTPCR) and immunohistochemistry (IHC) were applied to detect expression of TLR4-mRNA and TLR4 protein respectively. Results RT-PCR of RNA isolated from rat pancreatic tissue yielded the predicted amplicon for the TLR4. IHC/immunofluorescence revealed TLR4 protein mainly distributed in the epithelium of the pancreatic duct, vascular endothelium of the exocrine section, endocrine islet also had some signs of distribution. No TLR4 protein signal could be detected in the acinar cells. Conclusion TLR4 could be detected in rat pancreas. Its distribution is consistent with its roles in immune surveillance, mainly in tissues exposed to the external environment such as pancreatic duct as well as in immunologically important settings such as pancreatic vascular endothelium. Islet also has some signs of distribution. No TLR4 expression in acinar cells, suggesting TLR4 immunological involvement in the pathophysiology of pancreas.
Objective To verify tissue factor (TF)-bearing microparticle (TF-MP) could be released from Kupffer cells (KCs) stimulated by lipopolysaccharide (LPS) and TF controlled by Toll-like receptor 4 (TLR4) could induce acute pancreatitis. Methods After the acute pancreatitis model completed, the wild type C57/BL6 mouse (WT group) and the TLR4-/- mouse (TLR4-/- group) received intraperitioneal injections of 10 mg/kg LPS. The degree of pathological lesion and the TF expression were detected in the pancreas tissue. The TF and TLR4 protein and mRNA expressions in the KCs were detected at 6, 12, and 24 h after the last injection of LPS. The survival rates were campared in these estabilshed acute pancreatitis model mice. The TF and TLR4 protein and mRNA expressions in the KCs stimulated with LPS (300 μg/L) were also detected at 0, 15, 30, 60, and 120 min. The TF and TF-MP levels were detected in the supernatants of the KCs at these time point. Results The injury of the pancreas in the TLR4-/- group was slighter than that in the WT group. The TF proteins in the liver and pancreas tissues of the TLR4-/- group were significantly lower than those of the WT group (P<0.05). The survival rate of the TLR4-/- group was significantly higher than that of the WT group under the situation of the acute pancreatitis (P<0.05). The TLR4 and TF protien and mRNA expressions of the KCs were significantly decreased in the TLR4-/- group as compared with the WT group at 30, 60, and 120 min (P<0.05). The levels of TF and TF-MP in the supernatant of the TLR4-/- group were significantly lower than those of the WT group at 30, 60, and 120 min (P<0.05). Conclusion Acute pancretitis can be induced by TF and TF-MP expressions in KCs which could be regulated by TLR4 pathway.
【Abstract】 Objective To study the role of house dust mite ( HDM) induced airway epithelium TLR4 expression and T lymphocyte activation in asthmatic inflammation. Methods Thirty BALB/ c mice were randomly divided into an ovalbumin ( OVA) group, a HDMgroup, and a control group. The mice in the OVA group were sensitized with OVA and Al( OH) 3 , and repeatedly exposed to aerosolized OVA. The mice in the HDMgroup and the control group were sensitized and challenged with HDMand saline, respectively.Histopathology changes of pulmonary tissue and airway were observed under light microscope. Levels of IL-4, IL-5, IL-13, IL-17, and IFN-γin BALF were measured by ELISA. Total and differential cell counts in bronchoalveolar lavage fluid ( BALF) were also measured. The mRNA and protein expressions of TLR4 weredetected by quantitative real-time PCR and Western blot, respectively. Th1, Th2, and cells in the peripheral blood were detected by flow cytometry. Results Light microscope revealed eosinophil specific inflammatory cells infiltration around the peribronchovascular region,mucus gland hyperplasia, and airway mucous plug inthe OVA group. The HDM group showed more severe alveolar and intersititial congestion and neutrophils infiltration. The control group showed intact alveolus with few mucous plug and inflammatory cells.Compared with the OVA group, significant increases in the number of total cells and neutrophils, as well as significantly higher expression of IL-4, IL-5, IL-13, and IL-17 were detected in the HDMgroup ( P lt;0. 05) ,while IFN-γexpression had no significant change ( P gt;0. 05) . The expression of TLR4 mRNA and protein significantly increased in the HDMgroup( P lt; 0. 05) , and did not change significantly in the other two groups ( P gt;0. 05) . The percentages of Th2 and Th17 cells in peripheral blood in the HDMgroup were significantly higher than the OVA group ( P lt;0. 05) . Conclusion HDM may induce inflammatory cells infiltration andactivation of Th2 and Th17 lymphocyte cells via up-regulation of TLR4 expression in airway epithelium,which might play an important role in asthmatic inflammation.
ObjectiveTo investigate the correlation between TLR5 rs5744174 gene polymorphism and Streptococcus pneumoniae pneumonia.MethodsOne-hundred and six patients with Streptococcus pneumoniae pneumonia admitted to this hospital from January 2014 to October 2018 were selected as an observation group, and 85 healthy subjects were selected as a control group during the same period. The clinical and pathological data of the subjects were collected, polymorphism of TLR5 rs5744174 gene was analyzed by PCR and sequencing, and the relationships between cell classification count, C-reactive protein (CRP) level in bronchoalveolar lavage fluid (BALF) and TLR5 rs5744174 gene polymorphism in the patients with Streptococcus pneumoniae pneumonia were analyzed.ResultsThere were significant differences in age, smoking, alcoholism, diabetes and the other general data between the observation group and the control group (P<0.05). The distribution of TLR5 rs5744174 genotype in the observation group and the control group was in accordance with Hardy-Weinberg equilibrium test level (χ2=16.89 for the observation group, χ2=10.76 for the control group, both P>0.05). There was no significant difference in the distribution frequency of TLR5 rs5744174 (C < T) genotype and allele between the two groups (P>0.05). There were significant differences in the proportion of diabetes mellitus among the three genotypes (CC, CT, TT) of the patients with Streptococcus pneumoniae pneumonia (P<0.05). The percentage of neutrophils and CRP levels in BALF were significantly different (P<0.05).ConclusionThe polymorphism of TLR5 rs5744174 gene may not be related to the occurrence of Streptococcus pneumoniae pneumonia, but is related to the proportion of complicated diabetes mellitus, the percentage of neutrophils and the level of CRP in patients with Streptococcus pneumoniae pneumonia, which may affect the degree of inflammation.
ObjectiveTo investigate the protective effect and mechanism of curcumin on lipopolysaccharide (LPS)-induced acute lung injury.MethodsTotally 24 SD rats were randomly divided into a control group, a LPS group and a LPS+curcumin group (n=8 in each group). The degree of lung injury (oxygen partial pressure, wet/dry ratio, pathological scores) and inflammatory levels [tumor necrosis factor (TNF)-α, interleukin (IL)-6, monocyte chemotactic protein (MCP)-1, Toll-like receptor 4 (TLR4), mobility group box 1 protein (HMGB1) expression] of the lung were detected in different groups.ResultsOxygen partial pressure was significantly lower in the LPS group than that in the control group (P<0.05), while wet/dry ratio, pathological scores and expression levels of TNF-α, IL-6, MCP-1, TLR4 and HMGB1 were significantly higher in the LPS group than those in the control group (P<0.05). Compared with the LPS group, curcumin significantly reduced wet/dry ratio, pathological scores and expression levels of TNF-α, IL-6, MCP-1, TLR4 and HMGB1 in the LPS+curcumin group (P<0.05), while it significantly improved oxygen partial pressure (P<0.05).ConclusionCurcumin might protect LPS-induced acute lung injury through inhibition of TLR4-HMGB1-inflammation pathway.