| Objective To study the expression level of TLR2, TLR4mRNA andprotein in rat lung tissue, and the expression level of IL-6, TNF-α inbronchoalveolar lavage fluid after smoke inhalation. Discuss the expressionlevel and the utility of Toll-like receptors and downstream inflammatorycytokines after smoke inhalation injury.Methods Thirty SD rats, Each half of the male and female, Weightat200-300g were randomly devided into five groups(n=6).Define A group ascontrol, and B,C,D,E group as0.5h,2h,4h,6h after smoke inhalation. Mixture ofsawdust and kerosene were heated in a self-made Smoke Generator. Rats wereplaced in the injury room for5min and fresh air out for another5min. The Injuryprocess were repeated3times. Rats were anesthetized by chloral hydrate at eachtime point after smoke inhalation. The chest of rats were opened, and the heartblood, alveolar lavage fluid and lung tissue were collected for the subsequent experiment.1. The general changes of the lung tissue were observed.2. Wet anddry ratio (W/D) of the lung tissue were detected to assess the extent of edema.3.Polymerase chain reaction (PCR) were set to detect the TLR2and TLR4mRNAexpression in the lung homogenates.4. TLR2and TLR4protein expression levelwere detected by Western blot(WB).5. Inflammatory cytokines IL-6and theexpression of TNF alpha level were detected by enzyme-linked immunosorbentassay (ELISA).6. Observe the lung tissue structure and morphology changesunder optical microscope.7. The immunohistochemical methods were used todetect the TLR2and TLR4protein expression. The change of histological oflung tissue, TLR2and TLR4mRNA and protein expression level, the level of IL-6and TNF alpha before and after smoke inhalation were analysed by statisticalanalysis.Results (1). The lung volume increased slightly after smokeinhalation injury. Subpleural lung surface is a large number of scattered andpatchy bleeding. Some mucus and foamy fluid were in the tracheal and bronchus.Mucosal were in hyperemia and swelling. Scattered and patchy bleeding andfoamy fluid were observed when cutting the lung tissue.(2). Lung tissue werestoved into constant weight to calculate the W/D ratio. The results showed thatall of the four groups had a higher W/D ratio than that of the control group(P<0.05). The2h group had a highest W/D ratio of5.225±0.263. There are nodifferences in W/D ratio between the four groups after smoke inhalation injury(P>0.05).(3).PCR results showed higher TLR2mRNA level in the four groupsafter inhalation injury than the control group (P<0.05). The4h group had thehighest TLR2mRNA expression in all the five groups (P<0.05), which showedthat TLR2mRNA reached top and then had a down-regulation after6h. The0.5h, 2h and6h groups had no statistical differences (P>0.05). TLR4mRNA continueto rise after smoke inhalation injury, and the four experiment groups all had ahigher expression level than the control group (P<0.05), and the2h,4h, and6hgroups had no statistical differences between (P>0.05),but they all had a higherexpression level than the0.5h group (P<0.05).(4). Western blot showed asimilar results with PCR. TLR2protein had a higher expression in the fourgroups after inhalation injury than the control group (P<0.05). The4h grouphad the highest TLR2protein expression in all the five groups with37.52±3.29ng/ml (P<0.05), which showed that TLR2protein expression reachedtop and then had a down-regulation after6h, but it had no statistical differenceswith the2h and6h groups (P>0.05). TLR4protein continue to rise after smokeinhalation injury, and the four experiment groups all had a higher expressionlevel than the control group (P<0.05), and the6h group had a higher expressionlevel than the other four groups (P<0.05).(5). ELISA showed that the IL-6had ahigher expression in the four groups after inhalation injury than the controlgroup (P<0.05). The0.5h group had no statistical differences with the control.The4h group had the highest IL-6expression in all the five groups (P<0.05),and had a statistical difference with the0.5h group (P>0.05). TNF-α continue torise after smoke inhalation injury, and the four experiment groups all had ahigher expression level than the control group (P<0.05). No statisticaldifferences were found between the0.5h、2h and4h groups (P>0.05). The6hgroup had a higher expression level than the other four groups (P<0.05).(6). HEstaining of the lung tissue showed the four groups after smoke inhalation hadDifferent levels of Inflammatory infiltration and edema.0.5h group had alightest inflammatory injury of the five groups.(7). Immunohistochemical detection showed a positive expression of TLR2and protein in all the fivegroups. The expression level constantly rised at each time point. The overalltrend were similar with PCR and WB results. TLR2and TLR4expressed in bothcell surface and cytoplasm.Conclusions (1.)In condition of smoke inhalation injury, theup-regulated TLR2and TLR4mRNA and protein may play a role in the lunginflammation.(2). The inflammation of lung tissue of rats gradually aggravate astime past after smoke inhalation, and the same with IL-6and TNF alpha.Smoke inhalation can lead to a damage of lung tissue and inflammation.(3). Theexpression level of TLR2and TLR4has a similar trend with that of IL-6andTNF alpha, which indicate that the signal pathway of Toll-like receptors has acritical role in smoke inhalation injury. |
PDF Full Text Request