| Objective: The protective effect of Ampelopsin(APS)on the acute lung injury induced by LPS was determined in vivo and in vitro,Elucidated the possible mechanism of action.Method:(1)Acute lung injury animal model studies Healthy KM mice were randomly divided into 9 groups with 6 males and6 females in each group and randomly divided into 3 groups.Orthogonal analysis was used to determine the drug delivery time,drug concentration and poison exposure.The activity,wet/dry weight and lung coefficient of myeloperoxidase(MPO)were measured in lung tissue.(2)The effects of APS on acute lung injury in mice induced by LPS.Health KM of male mice were randomly divided into blank control group,LPS model group,dexamethasone group,the APS high dose group,low dose group,the APS in the APS dose groups,with different concentration of APS continuous dosing 3d to 3d drug delivery immediately after make mould with intraperitoneal injection of lipopolysaccharide method,experiment observed during general status in mice,the weight changes.After 8h,the blood was collected for lung tissue to detect TNF-?,IL-1?level change,and lung tissue myeloperoxidase(MPO)activity detection.Wet/dry weight determination;Lung coefficient determination,pathology observation.(3)The effects of APS on oxidative stress in acute lung injury induced by LPS.Take health KM of male mice were randomly divided into blank control group,LPS model group,vitamin E positive group,the APS high dosegroup,low dose group,the APS in the APS dose groups with different concentration of APS continuous dosing 3d to 3d drug delivery immediately after make mould with intraperitoneal injection of lipopolysaccharide method.During the experiment,the general state of mice was observed and the changes of weight were recorded.T-AOC activity and MDA content were detected after 8h.(4)The effects of APS on autophagy and related pathways of human alveolar epithelial cell A549 induced by LPS.MTS method was used to detect the effect of LPS at different concentrations and time points on the value added rate of A549 cells,and the most timely points and concentrations were selected as the molding conditions.MTS method was used to detect the toxic effects of APS on A549 cells at different concentrations and at different time points,and the time points and concentrations were selected as pretreatment conditions.The effects of APS on the level of TNF-?,IL-1? in alveolar epithelial cell A549 cells were detected.Results:(1)Acute lung injury animal model research results show that the coefficient of the lung: male and female compared with blank group and higher test 8 lung coefficient,aggravating edema degree(P< 0.05).Single females compared with blank,1-9 test does not increase lung coefficient(P> 0.05).Single male compared with blank,rising test 8 lung coefficient,aggravating edema degree(P<0.05);Wet dry than: male and female,together with the blank group comparison,test 3,8,in turn,can reduce dry wet than aggravate the degree of pulmonary edema(P< 0.05 or 0.01).Single females compared with blank,8,9,in turn,can reduce dry wet than aggravate degree of pulmonary edema(P<0.05).Single male compared with blank,test 3,8,in turn,can reduce dry wet than aggravatedegree of pulmonary edema(P<0.05).MPO activity assay: male and female,together with the blank group comparison,test 1,9 is not elevated MPO vigor(P>0.05).Single females compared with blank,test 2,5 can raise MPO vigor,lung damage(P<0.05).Single male compared with blank,8 elevated MPO activity test,lung damage(P<0.05).(2)The APS in LPS induced acute lung injury in mice inflammation results show that the influence of mice general observation:(1)of mice blank eyes,athletic build,good nutrition,back feather soft luster,auricle and limb reddish color,responsive,breath sounds clear,smooth breathing,to eat and drink,normal weight a progressive growth time,no animal died during the experiment.Glassy-eyed,(2)LPS model group mice back feather fluffy upright lacklustre,falls off phenomenon,auricle and limb dark red in colour,unresponsive,don't like activities,common,short of breath,cough asthma,there is a death.(3)positive dexamethasone group mice eyes slightly dull,back feather fluffy upright,weight loss,unresponsive,occasional asthma,etc.,have a cluster phenomenon.(4)the APS eyes slightly dull,high dose group mice back feather fluffy upright,weight gain,occasional short of breath,a cluster phenomenon.(5)in the dose group mice eyes,back feather fluffy but luster,weight gain,sensitive activity.(6)eyes slightly dull,low dose group mice back feather have fall off phenomenon,weight increase slowly,accompanied by a cough,wheeze and asthma,a cluster phenomenon;Pathological form: the alveolar structure of the blank group was normal,the alveolar structure was intact,and the interval was not thickened.The structural integrity of the model group is poor,the alveolar wall is severely thickened,the alveolar cavity is narrow,and there are a large number of neutrophils and pulmonary parenchymal lesions in the pulmonary interstitium.The lowerdose group of APS was less than that of model group.MPO activity assay:APS group can lower the MPO activity in lung tissue of mice,alleviate the acute lung injury(P<0.05),the APS low doses of significant difference(P<0.01);low dose of lung coefficient,the APS can reduce lung coefficient,reduce pulmonary edema(P<0.05),the APS dose group significant difference(P<0.01),the positive group also showed the same effect;Wet dry than: APS high doses can reduce lung coefficient,reduce pulmonary edema(P<0.05),the APS low-dose group significant difference(P<0.01),positive group also showed the same effect;Determination of TNF-?: dose of APS can reduce TNF-? levels,reduce inflammatory lesions(P<0.05);IL-1 ? levels were determined:dose of APS can significantly reduce the IL-1 ? levels,reduce inflammatory lesions(P<0.05).(3)The APS to mouse acute lung injury induced by LPS oxidative stress affect the results showed that mice general observation:(1)of mice blank eyes,athletic build,good nutrition,back feather soft luster,auricle and limb reddish color,responsive,breath sounds clear,smooth breathing,to eat and drink,normal weight a progressive growth time,no animal died during the experiment.Glassy-eyed.(2)LPS model group mice back feather fluffy upright lacklustre,falls off phenomenon,auricle and limb dark red in colour,unresponsive,don't like activities,common cough,asthma,short of breath.(3)vitamin E positive mice eyes slightly dull,back feather fluffy upright,weight is time progressive growth,unresponsive,occasional asthma,etc.,have a cluster phenomenon.(4)the APS eyes slightly dull,high dose group mice back feather fluffy upright,weight gain,occasional short of breath,a cluster phenomenon.(5)in the dose group mice eyes,back feather fluffy but luster,weight gain,sensitiveactivity.(6)eyes slightly dull,low dose group mice back feather have fall off phenomenon,weight increase slowly,accompanied by a cough,wheeze and asthma,a cluster phenomenon;T-AOC activity determination:APS low doses can increase the activity of T-AOC lung tissue,improve the antioxidant capacity(P<0.05),vitamin E positive group also showed the same effect;MDA content determination: APS in low dose could reduce the MDA content in lung tissue,improve the antioxidant capacity(P<0.05),vitamin E positive group also showed the same result.SOD activity assay: APS high dose decreased SOD activity(P<0.05);LC3B protein expression of measurement: model group can enhance LC3 B expression,increase the level of autophagy(P<0.05),the APS the expression of high dose group can decrease LC3 B,reduced autophagy level(P<0.05).(4)The APS to human alveolar epithelial cells A549 cells induced by LPS effect:(1)LPS on alveolar epithelial cells A549 cell increment rate: the influence of LPS treatment 2 h,compared with 0(including g/ml in the control group,LPS(including 150 g/ml cell survival rate rise,statistically significant difference)(P<0.05);LPS treatment 4 h,compared with 0(including g/ml in the control group,LPS(25,50,100,150(including g/ml)4 concentration has no effect on the cell survival rate,there was no statistically significant difference(P>0.05);LPS treatment 8 h,LPS150 including g/ml cell survival rate decreased,statistically significant difference(P<0.05);LPS processing 16 h,compared with 0(including g/ml in the control group,LPS(25,50,100,150(including g/ml)4concentration has no effect on the cell survival rate,there was no statistically significant difference(P>0.05);The effects of APS on lps-induced alveolar epithelial cells A549 cells.cell increment rate: theinfluence of LPS treatment 2h,compared with 0 g/ml in the control group,LPS 150 g/m L cell survival rate rise,statistically significant difference(P<0.05);LPS treatment of 4h was compared with the control group of0g/m L.The 4 concentrations of LPS(25,50,100,150 g/m L)had no effect on cell survival,and the difference was not statistically significant(P>0.05).LPS treatment of 8h,LPS150 g/m L cell survival rate decreased,and the difference was statistically significant(P<0.05).LPS treatment of16 h was compared with the control group of 0 g/ml.The 4 concentrations of LPS(25,50,100,150 g/m L)had no effect on cell survival,and the difference was not statistically significant(P>0.05).(2)the APS on alveolar epithelial cells A549 cell poison: the influence of the APS processing 2 h,compared with 0 ng/m L in the control group,(7.8125,15.625,62.5,500 ng/m L)of alveolar epithelial cells A549 cells have higher effect,hence the statistically significant difference(P<0.05);In the APS treatment of 4h,compared with the 0ng/m L control group,1000ng/m L significantly reduced the appreciation rate of A549 cells in the alveolar epithelial cells,and the difference was statistically significant(P<0.01);APS processing 8h,compared with 0 ng/m L in the control group,(7.8125 and 15.625 ng/m L)of alveolar epithelial cells A549 cells have obvious rise,hence 1000 ng/m L of alveolar epithelial cells A549 cell increment rate is significantly reduced,statistically significant difference(P<0.01);APS processing 16 h,compared with 0 ng/m L in the control group,(500,1000 ng/m L)of alveolar epithelial cells A549 cell increment rate has reduced effect,of which 1000 ng/m L function significantly reduced,statistically significant difference(P<0.05 or 0.01).(3)the APS to the alveolar epithelial cells A549 cells induced by LPS the influence of TNF-? release: compared with pure LPS treatment group,different concentration of APS pretreatment group,inflammatory cytokines TNF-? levels drop,statistically significant difference(P<0.05 or 0.01).(4)The APS to the alveolar epithelial cells A549 cells induced by LPS IL-1? release effect: compared with pure LPS treatment group,3.90625,15.625,31.25 and 62.5 ng/m L.APS pretreatment group,inflammatory cytokines IL-1? levels drop,statistically significant difference(P<0.05 or0.01).Conclusion : APS on LPS induced acute lung injury has a protective effect,its mechanism may be through relieve oxidative stress injury and reduce cell autophagy. |
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