Knockdown MD-2 Gene In Alveolar Macrophages With RNA Interference And Its Effect On Rat Acute Lung Injury Induced By Lipopolysaccharide

Knockdown MD-2 gene in alveolar macrophages with RNA interference and its effect on rat acute lung injury induced by lipopolysaccharideBackground Excessive amount of proinflammation cytokines and effector cells as the core of pulmonary inflammation lead to acute lung injury (ALI). There is no effective anti-flammation therapy because of the complex courses of inflammation. The mortality of ALI is still very high. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, has been recognized as an important cause of ALI. Toll like receptor 4 (TLR4) and myeloid differentiation protein 2 (MD-2) are the main LPS receptor complex. MD-2 is required in the course of LPS recognization and signal transmembrane transduction through TLR4. These signal transduction intermediary molecules in turn up-regulate inflammatory mediators and cytokines synthesis and also trigger preformed mediator release. Here we explore the expreeions of MD-2 in lung tissues of ALI rat induced by LPS, and the effect of knockdown MD-2 gene of alveolar macrophages with RNA interference on the upper-stream course of LPS/TLR4- MD-2 /MyD88 signal transduction and proinflammation cytokines secretion during ALI.Methods 1) A rat model of ALI was established by vena caudalis injection of LPS. 20 SD rats were randomly divided into control group and LPS group. The wet/dry ratios of lung tissue were measured and the histology changes of lung tissue were observed under microscope. Rat alveolar macrophages were collected form bronchial alveolar lavage fluid (BALF). The MD-2 mRNA and protein expressions were detected by semi-quantitative revels transcription polymerase (RT-PCR),Western Blot and immunohistochemistry, respectively. The levels of TNF-a in serum were detected by ELISA.2) Rat alveolar macrophage cell NR8383 was cultured with F-12K medium and stimulated with LPS. The expressions of TLR4 mRNA and MD-2 mRNA in cells were detected by RT-PCR. The contents of TNF-α,IL-6 and IL-1βin the cell cultured supernatant were tested by ELISA.3) Five MD-2 siRNA oligos were transfected into NR8383 by Lipofectamine 2000. The gene expression of MD-2 was detected by realtime PCR. The synthesis of MD-2 protein was analyzed by Western blot analyze. The MD-2 siRNA with highest gene knockdown effect was screened and been used in the following experiments. The cells treated with MD-2 siRNA were stimulated with LPS by two different methods, LPS+1 and LPS+2. In method LPS+1, the cells were stimulated by LPS and then treated with MD-2 siRNA. In method LPS+2, the cells were treated with MD-2 siRNA and then stimulated by LPS. The siRNA untreated cells were used as control group. The gene expressions of MD-2,TLR4 and MyD88 were detected by realtime PCR. The synthesis of MD-2 protein was analyzed by Western blot. The levels of TNF-αI1-1βand IL-6 in cell supernatants were measured by ELISA.Results 1) Compared with the control group, W/D ratio and the expressions of MD-2 mRNA and protein of both lung tissue and alveolar macrophages in LPS group increased significantly. The serum level of TNF-αin LPS group was also higher than that in control group (P<0.05).2) Both the TLR4 and MD-2 mRNA expressed in rat alveolar macrophages cell NR8383. The mRNA expression of TLR4 and MD-2 in control group were 0.52±0.05 and 0.44±0.09, respectively. There was no obviously change after O.01μg/ml LPS stimulation. The increase occurred in a dose dependent manner from 0.1μg/ml to 10μg/ml. The highest TLR4 and MD-2 mRNA expression were 0.72±0.06 and 0.65±0.10 (P＜0.01). The changes of TNF-αIL-6 and IL-1βcontents in cell cultured supernatant were similar with that of TLR4 and MD-2 gene expression (P<0.01). When stimulated with 1μg/ml LPS, the mRNA expressions of TLR4 and MD-2 in NR8383 cell were increased from hour 2. The highest mRNA expressions of TLR4 and MD-2 occurred at hour 6, and then decreased slowly from hour 8. The mRNA expressions at hour 24 were still higher than those in cells without LPS stimulation (P＜0.01). The changes of TNF-α,IL-6 and IL-1βin cell cultured supernatant were also similar with that of gene expression (P<0.01). The secretion peaks of TNF-αand IL-1βoccurred from hour 6 to hour 8, and the secretion peak of IL-6 occurred from hour 8 to hour12. The mRNA expression of MD-2 was related with that of TLR4 positively (r=0.513, P＜0.01).3) A pair of MD-2 siRNA (Sense: 5'- CCA UAU UUA CUG AAU CUG ATT-3'; antisense:5'- UCA GAU UCA GUA AAU AUG GGA-3') was affirmed to be effective by detecting the expressions of MD-2 mRNA and protein in NR8383 cell. The best work time was 24-hour and the gene knockdown effect was 67% in mRNA level. The expression of MD-2 mRNA and MD-2 protein increased obviosly after LPS stimulation in MD-2 siRNA untreated cells, as well as the expressions of TLR4 mRNA and MyD88 mRNA. After simulated with LPS in LPS+1 style, the expressions of MD-2 mRNA and MD-2 protein in MD-2 siRNA treated groups didn't increase (P＞0.05). The expressions of TLR4 mRNA and MyD88 mRNA increased obviously(0.69±0.10 and 0.82±0.09 vs 0.44±0.12 and 0.62±0.06,P<0.05), as well as the levels of TNF-αIL-1βand IL-6 in cell supernatants. After simulated with LPS in LPS+2 style, the expressions of MD-2 mRNA,MyD88 mRNA and TLR4 mRNA in MD-2 siRNA treated cells didn't increase, as well as the levels of TNF-αI1-1βand IL-6 in cell supernatants (P>0.05).Conclusion MD-2 may play an important role in rat acute lung injury induced by LPS. Stimulation by LPS can upregulate the expressions of MD-2 and TLR4 mRNA in rat alveolar macrophage cell NR8383 and increase pro-inflammation cytokines secretion. Knockdown MD-2 gene in NR8383 by RNAi can blockade LPS/TLR4-MD-2/MyD88 signal transduction, and then reduce the secretion of pro-inflammation cytokines.