| Objective:Acute lung injury(ALI)and acute resratory distress syndrome(ARDS)are clinical server diseases with high morbidity and mortality while the effective agent is lacked clinical.The fever,cough,asthma,dyspnea and other clinical manifestations are similar to "lung heat syndrome","storm surge","chest" and "take off"caused by "heat evil"and "heat","phlegm heat"and "blood stasis"in TCM theory,belong to the epidemic febrile disease of Chinese medicine category.As a representative of the drug with the effect of eliminating pathogenic heat from the blood,Scrophulariae Radix often appeared in the "Qingying Decoction"and"Qingwenbaidu Decoction"and other classic prescription in the treatment of heat-toxin syndrome for febrile disease.Modern pharmacological research shows that Radix Scrophulariae has anti-inflammatory and analgesic,antibacterial,antioxidant,immune enhancement,anti-fatigue,anti-platelet aggregation and other pharmacological effects,and which were related to the pathogenesis of ALI,suggesting that Scrophularia Radix have possibility for the treatment of ALI.There were no reports of Scrophularia Radix on the prevention and treatment of ALI,this study intends to establish lipopolysaccharide(lipopolysaccharides,LPS)in a rat model of acute lung injury induced by the exploration of the material for ALI prevention and treatment effect of Scrophulariae Radix and its possible mechanism,basis,provide experimental basis for clinical treatment and Scrophulariae Radix quality standard establishment of acute lung injury.Methods:1.Study on dynamic model of acute lung injury in ratsALI model was established by tracheal instillation of LPS(5 mg/kg),and the lung were harvested immediately,the model group respectively in the model 4 h,6 h,8 h,12 h,24 h,36 h,48 h,72 h and 120 h after the animal were sacrificed.Observation of bronchoalveolar lavage fluid(BALF)in polymorphonuclear leukocytes(PMN),tumor necrosis factor(TNF-α)concentration,total protein concentration and lung wet to dry ratio(W/D)of lung tissue and pathology,to evaluate and determine the tracheal instillation of LPS induced acute lung injury rat model and the best time.2.Preventive and therapeutic effect of Scrophulariae Radix on LPS induced acute lung injury in ratsScrophulariae Radix extract were Prepared by extraction,and the high dose,middle dose,low dose were 9 g/kg,4.5 g/kg,1.5 g/kg,respectively.After 5 days of continuous intragastric administration,the model group was induced by intratracheal instillation of LPS,and the normal control group(ALI)rats were injected with the same volume of saline.Positive drug group:intraperitoneal injection of dexamethasone 2 mg/kg before intratracheal instillation of LPS.To observe the BALF PMN number and total protein concentration,lung wet to dry the pathological changes of lung tissue and to study the protective effect of Scrophulariae Radix on ALI rats induced by LPS.3.Analysis of the relationship of HPLC chromatogram of Scrophulariae Radix with its preventive and therapeutic effect on LPS induced acute lung injury in ratsFirstly,HPLC characteristic of Scrophularia Radix 6 extraction(water extraction,30%ethanol extract,50%ethanol extract,70%ethanol extract,90%ethanol extract,water extract alcohol parts)HPLC characteristic chromatogram was established,and the chemical components of Scrophulariae Radix were preliminary recognized by HPLC-MS/MS.90 male SD rats were randomly divided into 9 groups(n=10 each groups):normal control group(NS),model group(LPS),positive drug group and 6 experimental groups.gavage for 5 days,ALI model was established by intratracheal instillation of LPS method to the number of BALF in PMN and lung wet dry ratio spectrum as pharmacodynamic index effect analysis.Finally,using partial least squares regression analysis method to study the relationship between different parts of the HPLC characteristic chromatograms,to filter out the main active components of Scrophulariae Radix in resisting acute lung injury.4.Isolation and structure identification of active ingredients of Radix Scrophulariae5 kg pieces of Scrophularia Radix were extracted by 50%ethanol extraction,the extract was separated by D101 macroporous resin,Sephadex LH-20,Preparative liquid chromatography,successively,and with TLC and HPLC with tracing.Finally,the pure compound were identified by LC-MS/MS,1H NMR,13C NMR,and finally the structure were confirmed.Results:1 the best time to establish ALI model by intratracheal instillation of LPS was 12h.Based on analysis of the observed index,the model of acute lung injury in rates was established according to the comprehensive evaluation for each time point,the results showed that there was significant difference in each index compared with the normal control group(P<0.05)at 12 h point,and at this time,the pathological section of rat lungs showed lung structure was severely damaged,alveolar wall thickening,alveolar cavity the narrow,alveolar and interstitial inflammatory cell infiltration and tissue fluid,which were meet the acute lung injury evaluation criteria,and the model was established successfully at 12h point.2.Scrophulariae Radix has certain preventive and therapeutic effect on LPS induced acute lung injury in ratsThe middle dose of Scrophularia Radix extract can significantly reduce the number of PMN,total protein concentration and lung wet dry ratio in BALF(P<0.01),In the middle dose group,the alveolar structure was relatively complete and clear.In the high dose and low dose group,the degree of alveolar destruction in the model group was decreased,but the effect was not significant.3 The results of spectrum-activity relationship analysisThis experiment adopts the HPLC-MS/MS technology to analyze the constituents of Scrophularia Radix extraction,18 compounds were quickly identified of Scrophulariae Radix according to the molecular ion peaks provided by MS,MS2,MS3 provided by ion fragmentation information combined with the literature data.The spectrum-activity relationship analysis showed that 10 compounds of Scrophulariae Radix associated with inhibition of neutrophil activity positively,13 compounds in Scrophulariae Radix associated with inhibition of pulmonary edema positively,and these compounds have been shown to play a synergistic role in the prevention and treatment of acute lung injury,Possible.Coefficient Ni in the regression equation reflects the influence of peak area of independent variable(Xi)on the efficacy of Y,that is,when the regression coefficient is larger,the small change of the peak area of the independent variable will cause the change of the dependent variable Y to be larger,and have obvious difference in the efficacy.The contents of 6 extracts were different because of the different extraction methods,and Content factor is also crucial to the efficacy.Therefore,the contribution rate of a compound was evaluated by considering the contribution rate of a compound,and the contribution rate means that the percentage of all positive correlation coefficient and peak area product is the sum of all positive correlation coefficient and peak area product.Statistical results show that the contribution rate of No.6 and No.16 peak to Y1 is 89.22%,and that of No.6 and No.14 peak to Y2 is 83.83%.the No.6,No.14,No.16 peak were Harpagide,Verbascoside and Angoroside C,respectively,which were confirmed by LC-MS/MS and the standards.4 the results of active ingredient separationFive pure compounds were isolated In this study,and Compound 1,2,3 are effective components of Scrophulariae Radix confirmed by spectrum-activity relationship.In addition,2 other compounds were obtained in the separation process,compound 4 and compound 5,respectively.Conclusion:Scrophulariae Radix can inhibit PMN aggregation,activation in lung,and Reduce the release of pro-inflammatory mediators,and then inhibit the inflammatory cascade reaction of ALI,effectively.It also has a certain role in the repair of damaged lung tissue,reduce the degree of pulmonary edema.It has a good protective effect on lung tissue.The main active ingredient of Scrophulariae Radix in prevention and treatment of acute lung injury might be Harpagide,Verbascoside and Angoroside C. |
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