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 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.
【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 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 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.
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 effect and mechanism of microRNA (miR)-146a-3p on acute lung injury (ALI) and inflammation induced by lipopolysaccharide (LPS) in mice.MethodsThirty-two BALB/c mice were randomly divided into sham group, ALI group, ALI+agomiR-negative control (NC) group, ALI+miR-146a-3p agonist (agomiR-146a-3p) group, with 8 mice in each group. The ALI model was established by instilling 5 mg/kg LPS into the lungs through the trachea, and the same amount of saline was instilled slowly in the sham group. The mice in the ALI+agomiR-146a-3p group/NC group were injected with 8 mg/kg agomiR-146a-3p or agomiR-NC respectively through the tail vein, once a day, for 3 days. The sham group and the model group were given the same amount of normal saline injection through the tail vein. After 24 hours, they were sacrificed and lung tissues were collected. The expressions of miR-146a-3p and toll-like receptor 4 (TLR4) mRNA in lung tissue were detected by RT-qPCR, the expression levels of TLR4, cleaved caspase-3, Bcl-2 related X protein (Bax), B cell lymphoma-2 (Bcl-2) protein in lung tissue were detected by Western blot. The changes of lung pathology were observed by hematoxylin-eosin staining. The apoptosis of lung tissue was detected by TdT-mediated dUTP nick-end labeling. The expression levels of IL-1β, IL-6 and TNF-α in lung tissue were detected by enzyme-linked immunosorbent assay (ELISA). The dual luciferase reporting system verified the targeting relationship between miR-146a-3p and TLR4 in MRC-5 cells. MRC-5 cells were divided into control group, LPS group, LPS+miR-146a-3p mimic group, LPS+pcDNA3.1(pc)-TLR4 group, LPS+miR-146a-3p mimic+pc-TLR4 group. 100 nmol/L miR-146a-3p mimic and pc-TLR4 plasmids were transfected into MRC-5 cells separately or jointly for 24 hours, and then treated with 1000 ng/mL LPS or normal saline for 72 hours. The apoptosis rate was detected by flow cytometry. The expression levels of TLR4, cleaved caspase-3, Bax, and Bcl-2 proteins were detected by Western blot. The levels of IL-1β, IL-6 and TNF-α were detected by ELISA.ResultsCompared with the ALI group, the expression of miR-146a-3p was up-regulated, the expressions of TLR4 mRNA and protein were down-regulated, the apoptotic rate was decreased, the expressions of cleaved caspase-3 and Bax protein was down-regulated, the expression of Bcl-2 protein was up-regulated, and the levels of TNF-α, IL-6 and IL-1β in lung tissue were decreased in the lung tissues of the ALI+agomiR-146a-3p group (P<0.05). Dual-luciferase reporter assay confirmed that miR-146a-3p regulates transcription by targeting TLR4 3’UTR sequence (P<0.05). Compared with the LPS group, the expression of TLR4 protein in MRC-5 cells of the LPS+miR-146a-3p mimic group was down-regulated, the apoptosis was reduced, the expressions of cleaved caspase-3 and Bax protein were down-regulated, and the levels of TNF-α, IL-6 and IL-1β in lung tissue were decreased (P<0.05). Overexpression of TLR4 reversed the effect of miR-146a-3p mimic overexpression on LPS-induced apoptosis and inflammation of MRC-5 cells (P<0.05).ConclusionmiR-146a-3p alleviates LPS-induced ALI in mice by down-regulating TLR4.
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 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.