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 effects of nitric oxide ( NO) inhalation on lung inflammation of acute lung injury ( ALI) in rats. Methods Twenty-four SD rats were randomly divided into four groups, ie. a normal control group, an ALI group, a 20 ppm NO inhalation group, and a 100 ppm NO inhalation group. ALI model was established by LPS instillation intratracheally and the control group was instilled with normal saline. Then they were ventilated with normal air or NO at different levels, and sacrificed 6 hours later. Pathological changes were evaluated by HE staining. The expression of TLR4 in lung tissues was detected by immunohistochemistry. IL-6 level in lung homogenate was measured by ELISA. Results In the ALI group, the inflammation in bronchus and bronchioles was more apparently, and the expressions of TLR4and IL-6 were elevated significantly compared with the control group. 20 ppm NO inhalation significantly decreased the expression of TLR4 and IL-6, and alleviated the inflammation of ALI. However, 100 ppm NO inhalation did not change TLR4 expression and lung inflammation significantly, and increased IL-6 level.Conclusions Inhalation low level of NO( 20 ppm) can alleviate lung inflammation possibly by reducing theexpression of TLR4 and IL-6.
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.
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 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.
ObjectiveTo investigate the role and mechanism of S100A8/A9 in rat model of chronic obstructive pulmonary disease (COPD). Methods Twelve Wistar rats were randomly divided into a normal control group and a COPD group. The COPD model was established by exposing the rats to cigarette smoke and injected lipopolysaccharide (LPS) in bronchus for 1 month. The pathological changes of the lung tissue were observed under light microscope, and the emphysema indexes of pulmonary mean linear intercept (MLI), mean alveolar numbers (MAN) and pulmonary alveolar area (PAA) were analyzed by image analysis system. The concentrations of S100A8/A9 in serum and bronchoalveolar lavage fluid (BALF) were measured by enzyme linked immunosorbent assay. The mRNA expression levels of S100A8, S100A9, Toll-like receptor-4 (TLR4) and myeloid differentiation factor 88 (MyD88) of lung tissues were measured by real time polymerase chain reaction. The protein expressions of S100A8/A9, TLR4 and MyD88 of lung tissues were detected by immunohistochemistry. Results After cigarette smoking and LPS injection for 1 month, the rat lung tissue appeared in accordance with the typical pathological changes of COPD. The MLI, MAN and PAA had obvious difference compared with the normal control group (P<0.05). The concentrations of S100A8/A9 protein in BALF and serum of the COPD group were obviously higher than those of the normal control group (P<0.05). The levels of S100A8, S100A9, TLR4 and MyD88 mRNA of lung tissues in the COPD group were obviously higher than those in the normal control group (P<0.05), and the expression levels of S100A8 and S100A9 mRNA were positively correlated with the expression levels of TLR4 and MyD88 mRNA respectively (P<0.05). The levels of S100A8/A9, TLR4 and MyD88 protein of lung tissues in COPD group were obviously higher than those in normal control group (P<0.05), and the levels of S100A8/A9 protein were positively correlated with the levels of MyD88 and TLR4 protein (P<0.05). Conclusions As a new inflammatory mediator, S100A8/A9 may be involved in the occurrence and development of COPD. By up-regulating the expression of TLR4 and MyD88, the classical TLR4-MyD88 inflammatory pathway is activated, thus promotes the occurrence and development of COPD.
【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.
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.
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 observe the synthesis of TLR2 protein and its mRNA expression in Kupffer cells (KCs) and sinusoidal endothelial cells(SECs).Methods Thirty-two BALB/c mice divided into two groups (operation group and false operation group) were used to prepare the model of partial hepatic ischemia/reperfusion (I/R) injury. After injury KCs and SECs were isolated with twosteps situ perfusion technique. And these cells were dyed by rat anti-mouse TLR2 IgG and anti-rat IgG2b labeled with flurescein isothiocyanate (FITC). The sysnthesis of TLR2 protein were determined by flow cytometric (FCM) analysis and real time reverse transcription polymerase chain reaction (Real-Time RT-PCR) analysis for gene expression.Results As for KCs: TLR2 expression was significant higher in operation group, compared with false operation group 〔protein expression: （9.19±1.07）% vs （1.52±0.21）%, P＜0.01; gene expression: 0.54±0.77 vs 2.62±2.19, P＜0.05〕. But there were no significant differences with expression in SECs. Conclusion Synthesis of TLR2 protein and its gene expression increased in KCs in the mouse partial hepatic ischemia-reperfusion injury.