The Effects And Mechanism Of A20Protein On The Inflammatory Response In Acute Lung Injury

Part I:Change and significance of A20protein in acute lung injury induced by intravenous injection of LPS in ratsAbstract Objective To build the model of acute lung injury (ALI) induced by intravenous injection of LPS in vivo and to investigate the A20Protein Expression in ALI.Methods54specified-pathogens free male Sprague-Dawley rats were randomized into two groups:Group control (intravenous injection of PBS,1ml per rat, defined as Oh); group ALI (intravenous injection of LPS,10mg/kg, and divided into eight subgroups according to the different time points1h,2h,4h,6h,8h,12h,24h,48h after injection of LPS). All animals were sacrificed at the corresponding time. The pathological change of lung was observed under light microscope. The wet/dry weight ratio of lung(W/D), the levels of total proteins and the total cell count in bronchoalveolar lavage fluid (BALF) were examined. TNF-alpha, IL-1β levels in BALF and NF-κB DNA binding activity in lung tissue were tested by ELISA. The levels of nuclear protein p65and A20protein were examined by Western blot. A20mRNA levels in lung tissue were analysis by RT-PCT.Results Compared with controls,24hours after LPS injection, the W/D ratio of lung, the levels of total proteins, the number of total cells in BALF were significantly higher (p <0.05). There were widespread alveolar walls thickening, massive neutrophils infiltration and hemorrhage under light microscope in the ALI group. In addition, after injection of LPS, the levels of TNF-aand IL-1(3in BALF, the DNA binding activity of NF-kappa B and the nuclear protein p65content increased with time, reached a peak at2h, then decreased gradually, but still higher than group control (p<0.05). As well as NF-kappa B and cytokines, A20protein and mRNA levels in lung tissue also gradually increased and reached a peak at2h, then decreased, but still higher than group control (p<0.05). Furthermore, immunohistochemistry showed that2hours after LPS injection, the A20was mainly expressed in alveolar macrophages cytoplasm.Conclusions In acute lung injury, the expression of A20was elevated, with a peak as high as early cytokines and NF-κB. In early phase of ALI, A20protein was mainly expressed in alveolar macrophages cytoplasm, which indicated that A20may regulate inflammatory responses in ALI through alveolar macrophage. Part II:The effects and mechanisms of A20on the inflammation responses of alveolar macrophageAbstract Objective To establish the alveolar macrophage A20gene silencing and over-expression in vitro and to investigate the effects and mechanisms of A20on LPS induced lung injury.Methods Constructed lentivirus-mediated iRNA vector and expression vector of A20gene, then transfected into rat alveolar macrophage cell lines (NR8383) respectively. The cells were treated with LPS (lug LPS per ml medium), and the culture supernatants and cells were collected at different time points:0.5,1,2,4hours after simulation of LPS. The levels of TNF-a and IL-1(3in supernatant levels, the DNA binding activity of NF-κB were analysis by ELISA. The levels of A20protein and nuclear p65were detected by Western Blot. The mRNA levels of A20, TNF-alpha and IL-1(3were detected by RT-PCR.Results Compared with control, the levels of A20protein and mRNA in A20gene silencing group (A20-SH group) were significantly lower (p<0.05), with an interference efficiency of80%, while the levels in A20gene overexpression cells (A20group) were dramatically increased (p<0.05), by more than10times. After stimulation of LPS, the A20protein and mRNA content in control groups increased and reached a peak at1h, and then gradually decreased, while the levels in A20-SH group significantly lower (p<0.05), and remained at a low level all the time. On the contrary, compared with control group, the A20protein and mRNA content in A20group were significantly increased (p<0.05). In addition, after LPS simulation, in the control groups, the levels of TNF-alpha and IL-1β in supernatants and mRNA in cells increased with time, and reached a peak at1h, and then gradually decreased, as well as DNA binding activity NF-κB and nuclear p65content. Compared with control, the levels of TNF-alpha and IL-1β protein in supernatants and mRNA in cells, the DNA binding activity NF-κB and nuclear p65content in A20-SH group significantly increased (p<0.05), while A20group decreased (p<0.05).Conclusions A20gene silencing led to increase in NF-κB activity, the promotion of TNF-alpha, IL-1β expression and secretion. On the contrary, A20gene overexpression can inhibit NF-κB activity and TNF-alpha, IL-1β expression and secretion of alveolar macrophage. A20protein can inhibit the inflammation activity of alveolar macrophages, thereby reducing the lung injury.