| The incidence of acute lung injury(ALI) is high. Its mechanism is complex. When ALI developed to acute respiratory distress syndrome(ARDS), its mortality rate as high as 40%. Studies found the imbalance of proinflammatory cytokines and anti-inflammatory cytokines may be the main pathogenesis of ALI, The common pathogenic factors of ALI/ARDS are Gram-negative bacterial infection and endotoxemia. Cholinergic anti-inflammatory pathway(CAP) was found recently years, It also can regulate the systemic inflammatory response, inhibit the inflammatory response synthesis by suppresing vagus nerve to release proinflammatory cytokines. The CAP is constituted of the vagus nerve and its neurotransmitter acetylcholine with cholinergic receptors. The α7 nicotinic acetylcholine receptor(α7nAChR) plays an important role in cholinergic anti-inflammatory pathway. The results of a large number of animal experiments showed that using α7nAChR agonists can inhibit the release of inflammatory cytokines in trauma, sepsis and rheumatoid arthritis, and other systemic inflammation. GTS-21 is the agonist of α7nAChR,it whether playing a role in anti-inflammatory effects in acute lung injury and the mechanism, there is reported less at present.Objective To observe the dynamic change of expression α7nAChR and inflammatory cytokines IL-1β, IL-18 and HMGB1 in LPS-induced acute lung injury in different periods. To explore the intervention of α7nAChR agonist GTS-21 on LPS-induced acute lung injury in mice model, to study its anti-inflammatory mechanism, and provide new therapeutic strategies for the treatment of acute lung injury.Methods The study includes two parts. The first part: To study the mechanism of α7nAChR in acute lung injury. The 50 C57BL/6 mice were randomly divided into five groups: normal control group, ALI 3 hours group, 6 hours group, 12 hours group, 24 hours group, each group taken eight mice. Normal group were injected with normal saline, the mice were sacrificed after one hour, the other groups were intraperitonealled of 30mg/kg LPS to model, after LPS injection corresponding time mice were sacrificed. Detection of mouse lung tissue specimens from the left lung wet/dry(W/D) gravity; HE staining, light microscopy of lung pathological changes; used ELISA to assay of IL-1β,IL-18 and HMGB1 the level of lung tissue expression; used Real-Time PCR to test the α7nAChR m RNA level of the lung tissue.The second part, observation of GTS-21 targeted Intervention. 63 mice were randomly divided into four groups: normal control group(NC group,n=9), acute lung injury model group(LPS group, n=9), the drug group(GTS-21 group, n=9), GTS-21 intervention group(LPS +GTS-21 group, n=36). NC group were given intraperitoneal injection of phosphate buffered saline(PBS); LPS group received intraperitoneal injection of 30mg/kg of LPS modeling; GTS-21 group injected PBS before 30 minutes injected 3 mg/kg of GTS-21; LPS+GTS-21 group injected PBS before 30 minutes injected 3 mg/kg of GTS-21. Normal control group, ALI model group, drug group were injected with PBS or LPS after 6 hours, all mice were sacrificed. GTS-21 intervention group were intraperitoneal injection LPS after 3 hours, 6 hours, 12 hours, 24 hours,take 8 mice sacrificed from each group. To observe the lung wet/dry(W/D) proportion, pathological changes, used ELISA to assay of expression of IL-1β, IL-18 and HMGB1 the level in lung tissue; used Real-Time PCR to test the α7nAChR m RNA level of the lung tissue. Results Part I(1) Compared with the normal control group, the ALI model group, the lung W/D ratio was increased significantly(3h:4.49±0.16 vs 4.06±0.11,6h:5.57±0.18 vs 4.06±0.11,12h: 5.32±0.14 vs 4.06±0.11,24h:5.25±0.20 vs 4.06±0.11 s,p<0.01)lung pathology score increased and IL-1β levels increased(3h:2875.28±169.95 vs 555.73±132.89,6h:3136.51±130.56 vs 555.73±132.89, 12h:2475.36±190.50 vs 555.73±132.89,24h:2256.83 ± 413.40 vs 555.73±132.89,p<0.01);IL-18 levels increased(3h:176.72±20.86 vs 4.92±21.43, 6h:200.96±21.16 vs 144.92±21.43, 12h: 183.82±7.76 vs 144.92±21.43, 24h:159.59±16.09 vs 144.92±21.43,p<0.05); HMGB1 levels increased(3h: 3.99±0.82 vs 1.88±0.73, 6h: 5.9±1.33 vs 1.88±0.73,12h: 7.85±0.65 vs 1.88±0.73, 24h:6.49±0.51 vs 1.88±0.73, p<0.01)wherein the lung W/D proportion of lung pathology score, IL-1β, IL-18 levels peaked at 6 hours, and HMGB1 levels peaked at 12 hours, the difference was statistically significant between the two groups(p<0.05).(2)Compared with normal control group, the expression of the α7nAChR m RNA were gradually decreased, the differences were statistically significant.(3h:1.57±0.16 vs 2.06±0.29, 6h: 0.79±0.13 vs 2.06±0.29, 12h: 0.65±0.12 vs 2.06±0.29, 24h: 0.19±0.04 vs 2.06±0.29, p<0.01).Part II(1)Compared with normal control group, the lung W/D ratio,the lung pathology score,IL-1β,IL-18, HMGB1 levels and α7nAChR m RNA of GTS-21 group had no obvious statistics significance(p>0.05).(2) Compared with the model group, α7nAChR m RNA expression of GTS-21 intervention group was increased significantly.(6h: 1.23±0.41 vs 0.81±0.13, p<0.05).(3)Compared with ALI model group, lung W/D ratio of the GTS-21 interventiongroup decreased significantly(3h: 4.20±0.13 vs 4.49 ± 0.16, 6h: 5.21±0.11 vs 5.57 ± 0.18, 12h: 5.17±0.13 vs 5.32±1.14, 24h: 5.06 ±0.13 vs 5.25 ± 0.20, p<0.05); The lung histopathology score decreased significantly.(3h: 5.25±0.46 vs 6.00±0.75, 6h: 10.5±0.92 vs 12.37±1.18, 12h: 7.25 ±0.71 vs 8.50 ±1.19, 24h: 5.75 ± 1.03 vs 7.25 ±1.58, p<0.05); IL-1β levels decreased significantly(3h:2585.10±143.27 vs 2875.28 ±169.95,6h : 1899.01 ± 121.74 vs 3136.51 ± 130.56, 12h: 1640.89 ± 211.90 vs 2475.36 ± 190.50, 24h: 1624.06 ± 130.13 vs 2256.83 ± 413.40, p<0.01); IL-18 levels decreased significantly(3h: 155.73 ± 13.90 vs 176.72 ± 20.86, 6h: 164.05 ± 15.2 vs 200.96 ± 21.16, 12h: 151.83 ± 22.06 vs 183.82 ± 7.76, 24h: 132.45 ± 13.05 vs 159.59 ± 16.09, p<0.05);HMGB1 levels were decreased significantly(3h:3.16 ± 0.61 vs 3.99 ± 0.82, 6h: 4.07 ± 1.40 vs 5.9 ± 1.33, 12h: 5.37±1.03 vs 7.85±0.65, 24h: 5.12±1.31 vs 6.49±0.51, p<0.05). Conclusions(1)α7nAChR plays an important role in LPS-induced acute lung injury in mice.(2)GTS-21 has a protective effect on LPS-induced acute lung injury. |
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