Objective To evaluate the role of Toll-like receptor 2/nuclear factor-κB(TLR2/NF-κB)signaling pathway pretreatment in ventilator-induced lung injury(VILI). Methods Thirty male Sprague-Dawley(SD)rats were randomly divided into three groups by using random number scale,with 10 rats in each group. Group A:rats were given 200μL of TLR2 monoclonal antibodies(TLR2mAb,10μg/kg)by slow instillation through tracheal catheter, and then ventilated with a high tidal volume(VT)of 40 mL/kg. Group B:ventilated with a normal VT of 8 mL/kg. Group C:rats were tracheally instilled with 10 μg/kg of TLR2mAb devoid of biologic activity,and then ventilated with a high VT of 40 mL/kg. The rats were mechanically ventilated for 4 hours,the lung wet to dry weight ratio(W/D)was calculated. The changes in pathology and ultrastructure in lung tissue were observed with microscope. Enzyme linked immunosorbent assay(ELISA)was performed to determine the concentration of interleukins(IL-1β,IL-6)and tumor necrosis factor-α(TNF-α)in serum and brconchoalveolar lavage fluid(BALF). Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR)was used to assess the mRNA expressions of TLR2, NF-κB and myeloid differentiation factor 88(MyD88)in lung tissue. Results No obvious pathological changes in lungs were found in group A and group B,and no obvious damages to ultra-microstructure were found in lung macrophages, typeⅠepithelial cell and typeⅡepithelial cell. In group C,pathological changes were observed,including pulmonary alveoli fusion,alveoli septum thickening,inflammatory cells infiltration,and damages to ultrastructure of lung macrophage,damage to cell membrane of typeⅠepithelial cells and typeⅡepithelial cells,vacuoles in cytoplasm, damage to organelle,and even pyknosis and perinuclear cistern thickening. Compared with group C,W/D ratio and mean concentration of inflammatory cytokines in serum and BALF showed a significant decrease in group A and B〔W/D ratio:1.151±0.026,1.128±0.048 vs. 1.403±0.062;concentration of IL-1βin serum(ng/L):37.05±5.61, 34.52±4.31 vs. 51.45±8.18;concentration of IL-6 in serum(ng/L):53.65±5.16,55.77±5.62 vs. 89.96±7.08;concentration of TNF-αin serum(ng/L):71.93±13.29,67.36±11.42 vs. 96.20±11.60;concentration of IL-1βin BALF(ng/L):56.48±6.16,54.44±7.26 vs. 99.77±8.41;concentration of IL-6 in BALF(ng/L):172.44±21.26, 163.47±18.70 vs. 216.22±23.90;concentration of TNF-α in BALF(ng/L):235.81±42.75,231.72±40.38 vs. 374.85±69.61,all P<0.01〕,but there were no significant differences between group A and group B(all P>0.05). The mRNA expressions of TLR2,MyD88,and NF-κB were significantly decreased in group A and group B compared with those in group C〔TLR2 mRNA(2-ΔΔCt):1.021±0.287,0.938±0.196 vs. 3.862±0.871;MyD88 mRNA (2-ΔΔCt):1.235±0.277,1.300±0.306 vs. 3.618±1.107;NF-κB mRNA(2-ΔΔCt):0.519±0.036,1.043±0.170 vs. 20.280±9.466,P<0.05 or P<0.01〕,but there was no significant difference among the parameters mentioned above between group A and B(all P>0.05). Conclusion To some extent,pre-intervention with TLR2mAb to block the TLR2/NF-κB signal pathway can inhibit the release of pro-inflammatory factors,and regulate the VILI.

To establish the amplification-refractory mutation system quantitative real-time PCR (ARMS-qPCR) method based on qPCR technique for detecting the c.2T>C mutation of SLC25A13 gene and validate its diagnostic performance. According to the principle of ARMS-qPCR primer design, the specific primers were designed for the conserved sequence of SLC25A13. The c.2T>C mutation ARMS-qPCR detection assay of SLC25A13 gene and the corresponding Sanger sequencing system were established through the use of the synthetic plasmids of homozygous mutation and 200 human peripheral blood specimens which were verified by Sanger sequencing as templates, and the diagnostic efficacy of the qPCR assay was validated by using nucleic acid extracted from another 200 human peripheral blood specimens and the results obtained were compared with the Sanger sequencing results as the gold standard, and the consistency of the two detection methods was analyzed. The results showed that the qPCR assay could accurately identify artificial plasmids carrying different mutations of SLC25A13 gene, and distinguish between wild type SLC25A13 gene and the c.2T>C mutation. This method was used to detect the mutation status of SLC25A13 c.2T>C in human peripheral blood, and the detection results were 100% consistent with the Sanger sequencing results. Among the 200 blood samples, 8 samples (4%) carried the c.2T>C mutation of SLC25A13 gene and 192 samples (96%) did not carry it. In conclusion, the ARMS-qPCR test established in this study can quickly, simply and accurately detect the c.2T>C mutation of SLC25A13 gene, which is helpful for the diagnosis of citrin deficiency (CD).

To establish the amplification-refractory mutation system quantitative real-time PCR (ARMS-qPCR) method based on qPCR technique for detecting the c.2T>C mutation of SLC25A13 gene and validate its diagnostic performance. According to the principle of ARMS-qPCR primer design, the specific primers were designed for the conserved sequence of SLC25A13. The c.2T>C mutation ARMS-qPCR detection assay of SLC25A13 gene and the corresponding Sanger sequencing system were established through the use of the synthetic plasmids of homozygous mutation and 200 human peripheral blood specimens which were verified by Sanger sequencing as templates, and the diagnostic efficacy of the qPCR assay was validated by using nucleic acid extracted from another 200 human peripheral blood specimens and the results obtained were compared with the Sanger sequencing results as the gold standard, and the consistency of the two detection methods was analyzed. The results showed that the qPCR assay could accurately identify artificial plasmids carrying different mutations of SLC25A13 gene, and distinguish between wild type SLC25A13 gene and the c.2T>C mutation. This method was used to detect the mutation status of SLC25A13 c.2T>C in human peripheral blood, and the detection results were 100% consistent with the Sanger sequencing results. Among the 200 blood samples, 8 samples (4%) carried the c.2T>C mutation of SLC25A13 gene and 192 samples (96%) did not carry it. In conclusion, the ARMS-qPCR test established in this study can quickly, simply and accurately detect the c.2T>C mutation of SLC25A13 gene, which is helpful for the diagnosis of citrin deficiency (CD).