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.
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.
【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 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 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 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.
ObjectiveTo explore the relationship between single nucleotide polymorphisms (SNPs) of the Toll-like receptor 4 (TLR4) gene and the risk of pulmonary tuberculosis (PTB) more comprehensively and objectively through meta-analysis.MethodsWe searched all available articles published before June 13th, 2019 in main Chinese and English databases systematically and comprehensively, including PubMed, Embase, China National Knowledge Infrastructure, Wanfang and CQVIP databases. The literature was screened according to the inclusion and exclusion criteria set in advance. In addition, the basic characteristics and data of the included literature were recorded according to a pre-made data collection form. Statistical analyses were performed using the Stata 15.0 software.ResultsA total of 17 eligible original articles were included in the study eventually. Furthermore, allele and genotype data of the 4 most widely studied SNPs (rs4986790, rs4986791, rs10759932, and rs11536889) in the TLR4 gene were extracted. And their allelic model, dominant model, recessive model, homozygous model, and heterozygous model were separately analyzed by meta-analysis. The results showed that the C allele of rs10759932 increased the risk of PTB [odd ratio (OR)=1.144, 95% confidence interval (CI) (1.043, 1.254), P=0.004]. Compared with the TT genotype, the CC+CT genotype and the CT genotype alone of rs10759932 also increased the risk of PTB [OR=1.218, 95%CI (1.084, 1.369), P=0.001; OR=1.227, 95%CI (1.085, 1.387), P=0.001]. Nevertheless, there was no statistical correlation between the other three SNPs (rs4986790, rs4986791 and rs11536889) and the susceptibility to PTB (P>0.05).ConclusionThe allele model, dominant model (CC+CT vs. TT), and heterozygous model (CT vs. TT) of rs10759932 located on the TLR4 gene are closely related to the risk of PTB.
ObjectiveTo investigate the expression of caspase-3 and Toll-like receptor 4 (TLR4) in the incised rat skin healing process and its relationship with the wound time and to provide an experimental evidence for the prediction of injury time. MethodsAfter the rat incised wound model was established, hematoxylin-eosin dyeing technology and immunohistochemical staining technique were used to observe the expression of caspase-3 and TLR4. Then Image Pro Plus Image analysis software and SPSS statistical analysis software were used to deal with the experimental results. ResultsCaspase-3- and TLR4-positive cells were detected in epidermis, hair follicle and sebaceous gland cells in the control skin. The expression of caspase-3 and TLR4 of the ante mortem groups were significantly different compared with the control group except the 0 h group (P<0.05). Caspase-3- and TLR4-positive cells were detected in neutrophils around the hair follicle half an hour later. Caspase-3- and TLR4-positive cell rate increased with the infiltration of inflammatory cells. Caspase-3- and TLR4-positive cell rate reached the maximum on the 3 rd day, and then it began to decrease, and they were mainly expressed in fibroblasts and mononuclear macrophages. Caspase-3- and TLR4-positive cells were mainly expressed in fibroblasts on the 10th day. There was no significant differences between the postmortem injury groups and the normal control groups (P>0.05). ConclusionCaspase-3- and TLR4-positive cell rate is time dependent and stable in 25℃ temperature environment which makes it possible to determine the time of injury.
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.
Objective To investigate changes of TLR2 mRNA expression in lung of a mouse model of Chlamydia Pneumoniae pneumonitis, and to explore the possible mechanism of signal transduction. Methods Ninety-six male C3H/HeJ mice were randomly divided into four groups as follows: a control group, a model group, a SB203580 intervened group, and a pyrrolidine dithiocarbamate( PDTC) intervened group. Chlamydia Pneumoniae pneumonitis was induced by intranasally inoculated with 4. 0 ×106 IFU/mL of C. Pneumoniae per mouse in the model group and two intervened groups. Then the intervened groups were intraperitoneally injected with the p38MAPK inhibitor SB203580 and nuclear factor kappa B ( NF-κB)inhibitor PDTC, respectively. Six mice in each group were randomly killed in 1st, 4th, 7th and 14th day. The expressional changes of TLR2 mRNA in the mice lung tissue were measured by semi-quantitative RT-PCR. The concentrations of TNF-α in the lung homogenate were measured by ELISA. Results TLR2 mRNA expression in the lung tissue significantly increased after C. Pneumoniae infection, peaking at 4th and 7th days, then decreased after 14th day. Tumor necrosis factor-α( TNF-α) was also elevated in the lung tissue after C. Pneumoniae challenging. Both SB203580 and PDTC treatment effectively inhibited TNF-αand TLR2 mRNA expressions in lung. The inhibitory effect was more obvious by SB203580 treatment. Conclusion C. Pneumoniae can upregulate the expressions of TLR2 and TNF-α in lung, and TLR2/MAPK and TLR2 /NF-κB signal pathways may be involved in Chlamydia Pneumoniae pneumonitis.