The Study Of The Mechanism Of Nervilia Fordii Total Flavonoids In Treating Acute Lung Injury

Part Ⅰ Extraction and Compar ison of Nervilia Fordii Total FlavonoidsObjectiveWe wanted to extract the total flavoids from the Nervilia Fordii,and identi fy components of the Nervilia Fordii total flavoids.Methods"percolation method" was used to extract the components of Nervilia Fordi i and the alcohol solution was collected.AB-8 macroporous and polyamide were used to extract the Nervilia Fordii Total Flavonoids.High performance liquid chromatography(HPLC)and liquid phase mass spectrometry(LMS)were used to identify the total flavonoids.ResultsThe total flavonoids were identified as complanatuoside and nervilifordizin A(rhamnol-3-O-β-D-xylose-(1→4)-β-d-glucoside).ConclusionThe total flavonoids were identified as complanatuoside and nervili fordizin A(rhamnol-3-O-β-D-xylose-(1→4)-β-d-glucoside).Part Ⅱ Effects of different doses of LPS and different time of LPS intervention on ALI model of BALB/c miceObjectiveThrough intratracheal instillation of LPS,the effects of different concentrations of LPSand different time intervention of LPS on BALB/c mice.the BALB/c mice ALI model state,body weight,dry weight and wet weight of lung,effects of lung tissue active transport protein and cytokines,fresh lung tissue appearance changes were explored.Methods66 BALB/c mice were randomly divided into 10 groups:Blank group,6h Sham group,6h 100ug LPS group,6h 200ug LPS group,24h Sham group,24h 100ug LPS group,24h 200ug LPS group and 72h Sham group,72h 100ug LPS group,72h 200ug LPS group.The ALI animal model was established by intratracheal instillation of LPS.Blank group was not treated.Sham group was given 50ul normal saline(NS).During the modeling period,the mental state,activity,diet,fur and body weight of the mice were recorded,and the left lung tissue was taken to weigh the wet or dry weight.The appearance of fresh lung tissue was observed and recorded during the sampling period.The expression of AQP-1,AQP-5,a ENaC,β ENaC,y ENaC,TNF-a,IL-1β,IL-6,CXCL-1,GM-CSF,CCL-2,C XCL-15,MMP-9 and MMP-12 in lung tissue was detected by RT-PCR.In addition,the airway lavatory fluid cells of the same treatment mice were counted.Results1.Mice in Blank group had good mental state,uniform respiration,sensitive reaction,smooth wool color and normal di et.6 hours after operation,Sham group mice were still in good condition,even breath,good reaction,poor diet and disordered hair color.24 hours after operation,the mice in Sham group were in good mental state,breathing evenly,eating well,and hair color was normal.72h after operation,the mice in Sham group had good mental state,uniform breathing,sensitive response,hair color restored to normal color,smooth oil and normal diet.At 6h after LPS intratracheal instillation,the mice in 100ug LPS group and 200ug LPS group had poor state,shortness of breath and slow reaction,Poor diet,hair disorderly;After 24 hours of LPS intratracheal instillation,the mice in 100ug LPS group and 200ug LPS group were in poor condition,shortness of breath and cough,slow reaction,reduced diet and yellow hair color.After 72 hours of modeling,the mice of 100ug LPS group and 200ug LPS group were very poor,the reaction was very slow,the diet was further reduced,the hair color was disorderly yellow and lustrous.2.The mice weight changing:24 hours after LPS intratracheal instillation,the weight of Sham group,100ug LPS group and 200ug LPS group were all reduced.48h later,the weight of Sham group began to recover,while those of 100ug LPS group and 200ug LPS group continued to lose weight,and 72h later,the Sham group’s weight had increased,but the weight of the 100ug LPS group and the 200ug LPS group was still reduced.3.Comparison of wet lung dry lung weight in mice:In the wet lung comparison,there was no significant difference in 6h Sham group,6h 200ug LPS group compared with Blank group,there was a difference in 6h between 100ug LPS group and blank group.6h 100ug LPS group,6 h 200ug LPS group had no difference compared with 6 h Sham group;there was significant difference between 24h 100ug LPS group,24 h 200ug LPS group and Blank group(P<0.01).There was significant difference between 24 h 100ug LPS group and 24h 200ug LPS group compared with 24h Sham group {P<0.05),there was significant difference between 72h 100ug LPS group,72h 200ug LPS group and Blank group(P<0.01).72h 100ug LPS group and 72h 200ug LPS group had significant difference compared with 72h Sham group(p<0.01).In the dry lung comparison,there was no significant difference in 6h Sham group,6h 100ug LPS group,6h 200ug LPS group compared with Blank group,6h 100ug LPS group,6h 200ug LPS group had no difference compared with 6h Sham group;There was significant difference between 24h 100ug LPS group,24h 200ug LPS group and 24h Sham group(P<0.05),24h 100ug LPS group was significantly different from 24h Sham group(P<0.05),24h 200ug LPS group was significantly differerit from 24h Sham group(P<0.01).There was significant dilffercnce between 72h 100ug LPS group,72h 200ug LPS group and Blank group(P<0.01).72h 100ug LPS group and 72h 200ug LPS group had significant difference compared with 72h Sham group(P<0.01).In the aspect of wet-dry weight ratio of lung,there were significant differences between 72 h LPS group and 72 h Sham group {P<0.05),6 h LPS group and Blank group(P<0.06),24 h 100ug LPS group and Blank group(P<0.05).In the comparison of excess lung water in mice,there were significant differences in 6h 100ug LPS group compared with Blank group(P<0.01),6h 100ug LPS group compared with 6h Sham group(P<0.05).There was no difference between 6h 200ug LPS group and 6h Sham group.There was significant difference between 24h 100ug LPS group,24h 200ug LPS group and Blank group(P<0.01).There was significant difference between 24h 100ug LPS group and 24h 200ug LPS group compared with 24h Sham group(P<0.05).There was significant difference between 72h 100ug LPS group,72h 200ug LPS group and Blank group(P<0.01).72h 100ug LPS group and 72h 200ug LPS group had significant difference compared with 72h Sham group(P<0.01).4.RT-PCR in lung tissue:AQP-1:There were significant differences between 6h 100ug LPS group,6h 200ug LPS group and Blank group,6h Sham group,and there was significant difference between 6h Sham group and Blank group(P<0.05).There was significant difference between 24 h 100ug LPS group,24h 200ug LPS group and Blank group and 24h Sham group(P<0.01).There was significant difference between 72h 100ug LPS group,72h 200ug LPS group and Blank group(P<0.01).There was significant difference between 72h 200ug LPS group and 72h Sham group(P<0.01).AQP-5:There was significant difference between 6h 100ug LPS group,6h 200ug LPS group and Blank group(P<0.01),and there was significant difference between 6h 200ug LPS group and 6h Sham group(Pp<0.01).There was significant difference between 24h 100ug LPS group and Blank group,24h Sham group(P<0.01),there was difference between 24h 200ug LPS group and Blank group,24h Sham group(P<0.05),and there was significant difference between 72h 200ug LPS group and Blank group,72h Sham group(P<0.01).a ENaC:There was no difference in 6h LPS group compared with Blank control group and 6h Sham group.There was no difference between 24h Sham group,24h 100ug LPS group,24h 200ug LPS group and Blank group.There was no difference between 24h LPS groups and 24h Sham group.There was no difference between 72h LPS groups and Blank group,72h Sham group.β ENaC:There was no difference between 6h 100ug LPS group and Blank group,but there was significant difference compared with 6h Sham group(P<0.05).There was significant difference between 6h 200ug LPS group and Blank group(P<0.05),and significant difference between 6h Sham group and 6h Sham group(P<0.01).There was difference between 24h 100ug LPS group,24h 200ug LPS group and Blank group,24h Sham group.There was significant difference between 72h 200ug LPS group and Blank group,72h 200ug LPS group and 72h Sham group(P<0.05).There was no difference between 72h 100ug LPS group and Blank group and 72h Sham group.γ ENaC:There were significant differences between 6h Sham group compared with Blank control group(P<0.01),6h 200ug LPS group compared with 6h Sham group(p<0.05).24h 100ug LPS group,24h 200ug LPS group and Blank control group,24h Sham group had no difference.There was no difference between 72h 100ug LPS group,72h 200ug LPS group and Blank control group,72h Sham group.TNF-α:There was significant difference between 6h 100ug LPS group,6h 200ug LPS group and Blank group(P<0.01),and there was no difference between 6h LPS group and 6h Sham group.There was significant difference between 24h 100ug LPS group,24h 200ug LPS group and Blank group and 24h Sham group(P<0.01),and there was significant difference between 24h Sham group and Blank group(P<0.01).There was significant difference between 72h 100ug LPS group,72h 200ug LPS group and Blank group,72h Sham group(P<0.01).There was significant difference between 6h Sham group,24h Sham group and Blank group P<0.01).IL-1β:There were significant differences between 6h 100ug LPS group,6h 200ug LPS group and Blank group(P<0.01),6h 100ug LPS group and 6h Sham group(P<0.05),6h Sham group and Blank group(p<0.05).There were significant differences between 24h 200ug LPS group and Blank group(P<0.01),24h 100ug LPS group,24h 200ug LPS group compared with 24h Sham group(P<0.01).There was significant difference between Sham group and Blank group at 24h(P<0.05).There was significant difference between 100ug LPS group,200ug LPS group and Blank group,72hours Sham group(P<0.01).IL-6:There was significant difference between 6h 100ug LPS group,6h 200ug LPS group and Blank group(P<0.01).There was no significant difference between 6h 100ug LPS group,6h 200ug LPS group and 6h Sham group.There was signi ficant difference between 6h Sham group and Blank group(P<0.01).There was significant difference between 24h 100ug LPS group,24h 200ug LPS group and Blank group,24h Sham group(P<0.05),and there was significant difference between 72h 100ug LPS group,72h 200ug LPS group and Blank group(P<0.01),there was significant difference between 72h 200ug LPS and 72h Sham group(P<0.01),there was difference between 72h 100ug LPS and 72h Sham group(P<0.05)·CXCL-1:There was significant difference between 6h groups and Blank group(p<0.01).There was no difference between 6h 100ug LPS group,6h 200ug LPS group and 6h Sham group.There was significant difference between 24 h 100ug LPS group,24 h 200ug LPS group and Blank group,24 h Sham group had significant difference(P<0.01).There was significant difference between 72h 100ug LPS group and Blank group and 72h Sham group(p<0.01).There was significant difference between 72h 200ug LPS group,24h Sham group and72h Sham group(P<0.01).GM-CSF:There were differences between 6h 100ug LPS group,6h 200ug LPS group and Blank group(P<0.01),6h 100ug LPS group and 6h Sham group(P<0.01),6h 200ug LPS group and 6h Sham group(P<0.01),and there was significant difference between 6h 200ug LPS group and 6h Sham group(P<0.01).There was significant difference between 6h Sham group and Blank group(P<0.05).There was significant difference between 24h 100ug LPS group,24h 200ug LPS group and Blank group,24h 200ug LPS group and 24h Sham group(P<0.01),24h 100ug LPS group and 24h Sham group(P<0.01),and there was significant difference between 24h 100ug LPS group and 24h Sham group(P<0.05).There was difference between 24h Sham group and Blank group(P<0.05).There was significant difference between 72h 100ug LPS group,72h 200ug LPS group and Blank group,72h Sham group(P<0.05).CCL-2:There was significant difference between 6h 100ug LPS group,6h 200ug LPS group and Blank group(P<0.01),and there was difference between 6h Sham group and Blank group(P<0.05).There were differences between 24h 100ug LPS group,24h 200ug LPS group and Blank group(P<0.01),24h 100ug LPS group,24h 200ug LPS group and 24h Sham group(p<0.05),24h 200ug LPS group and 24h Sham group(P<0.01),and there was significant difference between 24h Sham group and Blank group(P<0.05).There was difference between 72h 100ug LPS group,72h 200ug LPS group and Blank group,72h 100ug LPS group and 72h Sham group(P<0.05),72h 100ug LPS group and 72h Sham group(P<0.01).MMP-9:There was significant difference between 6h 100ug LPS group,6h 200ug LPS group and Blank group(P<0.01),and there was no difference between 6h Sham group and 6h Sham group,but there was significant difference between 6h Sham group and Blank group(P<0.01),There was significant difference between 24h 100ug LPS group,24h 200ug LPS group and Blank group(P<0.01).There was significant difference between 24h 200ug LPS group and 24h Sham group(P<0.05).There was no significant difference between 24h 100ug LPS group and 24h Sham group.There was significant difference between 24h Sham group and Blank group(P<0.05).There was significant difference between 72h 100ug LPS group,72h 200ug LPS group and Blank group,72hours Sham group(P<0.01).CXCL-15 and MMP-12:Their changes do not seem to have much with LPS-induced ALT.5.The appearance of fresh lung tissue:In 6h group,the appearance of lung tissue in 6h sham group could be seen as a large area of capillary hemorrhage point,accompanied by scattered silt spots.Pulmonary capillary hemorrhage spots were seen in the lung tissue of mice in 6h 100ug LPS group,accompanied by stasis points and even fusing into plaques.In 6h 200ug LPS group,a large area of pulmonary capillary hemorrhage spots were found in the lung tissue,accompanied by large patches of congestion.In the 24-hour group,the lung tissue of 24-hour sham group was fresh and very few scattered in the silt spots.In the 24 100ug LPS group,pulmonary capillary congestion and edema could be seen,accompanied by hemorrhage of capillary rupture,silt points,and even fusing into plaques,In the 24h 200ug LPS group,large areas of pulmonary capillary hemorrhage were observed in the lung tissue,accompanied by large patches of ecchymosis.In 72h group,the appearance of lung tissue in 72h sham group was normal,there was no obvious pulmonary capillary congestion edema or hemorrhage,and there was no obvious congestion and edema in lung tissue.The pulmonary tissue congestion and edema were obvious in the 100ug LPS group at 72h,the pulmonary capillaries ruptured and the pulmonary capillaries ruptured in the naked eye,and the pulmonary tissue congestion and edema were very obvious,in the 200ug LPS group at 72h,and the ruptured pulmonary blood vessels could be seen in the naked eye,and fused to form large patches of bruise.6.Total cell count in bronchoalveolar lavage fluid:There was no significant difference between 6h 100ug LPS group and Blank group(P<0.05),but there was no difference in 6h 100ug LPS group,6h 200ug LPS group and 6h Sham group.There was difference between 24h Sham group and Blank group(P<0.05),24h 100ug LPS group,24h 200ug LPS group compared with Blank group,24h Sham group(P<0.01).There was significant difference between 72h groups and Blank group(P<0.01).There was significant difference between 72h 100ug LPS group,72h 200ug LPS group and 72Sham group(P<0.01).Neutrophil count in bronchoalveolar lavage fluid:There was no difference between 6h LPS groups and 6h Sham group,24h 100ug LPS group,24h 200ug LPS group.There were significantly different from Blank group and 24 Sham group(P<0.01).There was significant difference in 72h 100ug LPS group,72h 200ug LPS group compared with Blank group,72h Sham group(P<0.01).Count of Monocytes and Macrophages in bronchoalveolar lavage fluid:There was no difference between 6h groups and Blank group,there was no difference 6h model group and 6h Sham group,24h groups and Blank group,24h model group and 24h Sham group.There was significant difference between 72h Sham group,72h 100ug LPS group,72h 200ug group and Blank group(P<0.01).There was significant difference between 72h 200ug group and 72h Sham group(P<0.05).ConclusionLPS could induce the changes of lung wet weight,lung dry weight and excess lung water content in mice.With the prolongation of time,the lung water content gradually increased,and the lung water content was the most at 72 h time point,and this change was not obvious at 6 h time point.However,the changes were most obvious at 24 h and 72 h,and the increase of lung water content suggested that pulmonary edema was more serious.For the effect of wet-dry weight ratio of lung,there was little difference in the ratio between LPS and lung wet weight and dry lung weight of mice due to its effect on lung wet weight and lung dry weight.LPS could inhibit the gene expression of AQP-1,AQP-5,β ENaC,and there was no significant positive correlation between the degree of inhibition and the dosage of 100ug or 200ug in LPS.At 24 h point,the concentration of 100ug LPS and 200ug LPS inhibited the expression of AQP-1,AQP-5,and β ENaC gene most stably,while when LPS reached 200ug,the expression of a AQP-1,AQP-5,β ENaC gene was strongly inhibited at three time points.LPS can promote the gene expression of TNF-α,IL-1β,IL-6,CXCL-1,CCL-2,GM-CSF,MMP-9 and other inflammatory factors,chemokine and metal.loproteinases,this promotion was strongest at 6h point,and the gene expression of these factors decreased gradually with the advance of time.Compared with the appearance of fresh lung tissue,with the passage of time,the congestion area and edema degree of lung injury would deepergradually;this kind of injury would show a more serious trend due to the increase of the dosage of LPS.Different concentrations of LPS could induce the change of total cell number and cell classification count in bronchoalveolar lavage fluid of BALB/c mice at different time.We can find that the total number of BALF cells,neutrophil count and macrophage count increase with the passage of time,which also indicates that the lung injury becomes deep gradually with time.The analysis of Sham group showed that intratracheal instillation of surgical trauma or NS intratracheal stimulation could lead to abnormal gene expression in sham group,also the appearance of fresh lung tissue was changed especially at 6h time point.However,at 72 h,there was no difference between some indexes and blank group or corresponding sham group,so the sampling time could be selected at 24 h time point.Part Ⅲ the Mechanism of Nervilia Fordii Total Flavonoids in treating ALI BALB/c mice Model(I)ObjectiveWe wanted to explore the mechanism of Nervilia Fordii Total Flavonoids in treating Acute Lung Injury.The dosage of Total Flavonoids were divided into three groups:40 mg/kg,180 mg/kg,320 mg/kg.The model of ALI was established by intratracheal infusion of 10mg/kg LPS for 24 hours.From the point of view of the lung wet/dry weight ratio,excess lung water volume,lung water transporter protein,tight junctional protein,inflammatory factor,chemokine and metalloproteinases,the mechanism of Nervilia Fordii Total Flavonoids on acute lung injury was studied.Methods64 BALB/c mice were randomly divided into 7 groups:Blank group(No.1-6),Sham group,LPS Model group,40mg/kg administration group(40mg/kg TCM)group.180mg/kg(180mg/kg TCM)group,320mg/kg administration(320mg/kg TCM)group and Dexamethasone(5mg/kg DEX)group.Once a day for 7 days,the Sham group and LPS Model group were given the same dose of solvent,that is 0.2mlNS.Dexamethasone(5mg/kg DEX)group was injected intraperitoneally with 5mg/kg DEX,that is,each mouse was injected intraperitoneally with 0.lmg DEX/0.1mINS 30min after LPS intratracheal instillation.The model was established by intratracheal instillation of 10mg/kg LPS,and the mice were sacrificed 24 hours after intervention.The wet weight and dry weight of left lung were compared.The other lung tissues were frozen at-80 ℃.AQP-1,AQP-5,α ENaC,β ENaC,γ ENaC,Claudin-3,Claudin-4,Claudin-18,Occludin,ZO-1,TNF-α,IL-1β,IL-6,CXCL-1 GM-CSF and MMP-9 were detected by RT-PCR.Results1.Comparison of dry weight and wet weight of lung:In wet weight comparison,’there was difference between LPS group and Sham group(P<0.01),there was difference between 180mg/kg TCM group and LPS group(P<0.01),and there was no difference between the other drug groups and LPS group(P<0.01).In the dry weight comparison,there were significant differences between LPS group and Sham group(P<0.01),but there was no difference between drug groups and LPS group.In W/D comparison,there was difference between LPS group and Sham group(p<0.01),there was difference between 180mg/kg TCM group and LPS group(P<0.01),and there was no difference between the other drug groups and LPS group(P<0.01).In Excess Lung Water comparison,there was difference between LPS group and Sham group(P<0.01),there was difference between 180mg/kg TCM group and LPS group(P<0.01),and there was no difference between the other drug groups and LPS group(P<0.01).2.Lung tissue RT-PCR comparison:Occludin:There was significant difference between LPS group and Sham group(P<0.01),there was significant difference between 40mg/kg TCM group,180mg/kg TCM group and LPS group(P<0.05).and there was significant difference between 5mg/kg DEX group and LPS group(P<0.01).INF-α and IL-1 β:There was significant difference between LPS group and Sham group(P<0.01),there was significant difference between 180mg/kg TCM group and LPS group(P<0.05).There was no fit the results of the experiment in the expression of AQP-1,AQP-5,α ENaC,β ENaC,γ ENaC,Claudin-3,C1 audin-4,Claudin-18,ZO-1,IL-6,CXCL-1 GM-CSF and MMP-9 gene between the TCM groups and the LPS group.ConclusionThe Nervilia Fordii Total Flavonoids can reduce the lung tissue wet weight,then reduce the ratio of lung dry to wet weight and excess lung water,thus alleviate the LPS-induced acute lung injury in BALB/c mice with pulmonary edema.The mechanism may be achieved by increasing the gene expression of tight junction protein occludin and decreasing the gene expression of TNF-α and IL-1β.Part Ⅳ the Mechanism of Nervilia Fordi i Total Flavonoids in treating ALI BALB/c mice Model(Ⅱ)ObjectiveWe wanted to to investigate the mechanism of Nervilia Fordii Total Flavonoids on lung histopathology,bronchoalveolar lavage fluid count and protein expression of occludin in ALI model of BALB/c mice with ALI.Methods32 SPF grade BALB/c mice were randomly divided into 4 groups:Sham group(n=8),Sham+180mg/kg TCM group(n=8),LPS model group(n=8)and LPS+180mg/kg TCM group(n=8),Sham+180mg/kg TCM group(n=8)and LPS+180mg/kg TCM group(n=8)were given 3.6mg Nervilia Fordii Total Flavonoids/0.2ml ns,Sham group(n=8)and LPS model group(n=8)were given 0.2ml NS.These were given once a day for 7 days.The model was established by intratracheal instillation of 10mg/kg LPS,and the mice were taken 24 hours after intervention.Paraformaldehyde was fixed in the middle lobe of the right lung,and HE slices were performed.The total cell count and classification cell count of mouse BALF were retained.The remaining lung tissue was frozen at-80℃.The expression of occludin protein was examined.Results1.Comparison of cell count in BALF:comparison of total cell count in BALF:There was significant difference between LPS group and Sham group(P<0.01).There was significant di fference between LPS+TCM group and LPS group(P<0.05).Comparison of neutrophil count in BALF:there was significant difference between LPS group and Sham group(P<0.01).There was significant difference between),LPS+TCM group and LPS group(P<0.01).Comparison of macrophage count in BALF:there was no difference between LPS group and Sham group(P>0.05).There was no difference between LPS+TCM group and LPS group(P>0.05).2.L.ung histopathology and ALI score:in Sham group and Sham+TCM group,lung tissue structure was intact,no pulmonary edema was found in HE section,macrophages and a small number of neutrophils were observed.In LPS group,lung tissue structure was destroyed and pulmonary interstitial edema was obvious in HE section.With a large number of neutrophil infiltration and alveolar collapse,the degree of injury in,LPS+TCM group was lower than that in LPS group.The results showed that there was significant difference between LPS group and Sham group(P<0.01).There was significant difference between LPS+TCM group and LPS group(P<0.01).3.Western Blot:The expression of occludin protein in LPS group was lower than that in Sham group,these existed difference between LPS group and Sham group(P<0.01).The expression of occludin protein in LPS+TCM group was higher than that in LPS group,these existed difference between them(P<0.01).