| Objective:SARS,H1N1,H5N1 and other major epidemic respiratory system diseases(MERSD)caused great harm,mainly because of its high mortality and strong contagious.The acute lung injury(ALI)and acute respiratory distress syndrome(ARDS)stage are the most dangerous stages,the mortality rate of which is extremely high.The current clinical use of drugs for ALI is controversial,so there is no effective drugs.ALI is the early stage of ARDS,as the control stage,and once into the ARDS,the mortality rate of it is more than 30%.Therefore,the effective drugs for ALI should be studied.In traditional Chinese medicine(TCM),MERSD is a category of Febrile Disease,and ALI causes and clinical manifestations are also attributed to the disease.Chinese medicine had significant effect against the plague and SARS,H1N1,H5N1 of MERSD.Therefore,we intend to find effective drugs from TCM.According to the theory of febrile disease and the clinical experience of TCM in the treatment of ALI,the clearing heat prescriptions on ALI were effective.We decomposed several recipes of the best prescription and we had the different extracts of the best recipe.Finally,we will find the effective components for ALI by the partial least squares regression(PLSR)to guide the clinical use.Method:The research content is divided into five chapters.The first part of the research content(chapter second):First of all,ALI rat model induced by LPS was studied.By intratracheal instillation of LPS,we observed 11 different time point modeling.They were:0h,4h,6h,8h,12h,24h,36h,48h,72h,120 h and 7d after intratracheal instillation of LPS.We focused on inflammatory factors in ALI(total white blood cell count,neutrophil PMNS,TNF-α)and edema index(protein concentration and wet/dry ratio of lung)and pathological changes of lung.Secondly,we studied the effect of four herbs on ALI rats and they were Qinyin decoction(F1),Qingwen Baidu decoction(F2),Qingre Xuanfei decoction(F3)and Qingre Xiefei decoction(F4),respectively.The second part(the third chapter):We selected the best dose of QBD.Further analysis of the QBD recipes,and they were clearing heat and fire group,heat and cooling blood group,clearing heat and expelling dampness group and the fourth recipe,the abbreviations of which were C1,C2,C3,C4.The third part(the fourth chapter):the efficacy and characteristics of six extracts of C2.The 6 extracts were water(S1),10%EtOH(S2),30%EtOH(S3),50%EtOH(S4),70%EtOH(S5),and 90%EtOH(S6)according to the different concentration of ethanol.At the same time,we establish the specific chromatograms of different extracts.The fourth part(the fifth chapter):building the spectrum-effect relationship.There were 21 peaks in HPLC chromatograms of the extracts.Than we set the peak areas as Xn,and the efficacy as Yn,and used the partial least squares regression(PLSR)method to build spectrumeffect relationship.The fifth part(the sixth chapter):Pharmacodynamics validation.We separated X6,and X13.Then,we identified them by HPLC-DAD-ESI-MSn and NMR structures and calculated the content of X6 and X13.Finally,the pharmacodynamics validation was tested.Results:The result of ALI model showed that after 12 h intratracheal instillation of LPS,the typical pathological changes appeared in the ALI:The total cells and PMNs reached the peak at 24h(P<0.01);and the peak time of protein concentration and lung wet dry ratio was at 12h(P<0.01);TNF-α in BALF increased stably at 4h-12h,which had significant difference compared with the control group(P<0.01).Meanwhile,lung tissue pathological changes at 12 h showed that the damaged alveolar structure,increased lung permeability,severe inflammatory cell infiltration and edema appeared in lung tissue.It suggested that the dose of LPS was 5 mg/kg,and ALI model was established after 12 h.The results showed that four decoctions could effectively reduce the number of total cells、PMNs、wet\dry ratio and protein exudation(comparing to model group),and the best decoction was F2(P<0.01).F2 and F3 decoction can effectively reduce the level of TNF-α,(P<0.01),and the best decoction was F2.In other words,Qingwen Baidu decoction can inhibit the inflammatory response and reduce edema in ALI.So we selected Qingwen Baidu decoction(QBD)as the in-depth study decoction.The results showed three doses of QBD could reduce number of the total cells and neutrophils(compared with model group),and the effect was in a dose dependent manner(P<0.01).Meanwhile,the high and middle dose can effectively reduce the protein concentration,TNF-α level and lung wet/dry ratio,and the high dose was the best(P<0.01).Therefore,the high and middle dose of QBD can effectively reduce the ALI damage in a dose-dependent manner.Further analysis of the QBD recipes was as follows:C1,C2 and C3 groups significantly decreased the total cells and PMNs comparing with LPS group(P<0.05),and C3 was best(P<0.01);four recipes of QBD significantly decreased lung wet/dry ratio(P<0.01),especially C3.C1,C2 and C3 significantly decreased the protein concentration in BALF(P<0.05),especially C2(P<0.01).C1,C2 and C4 significantly reduced the levels of TNF-α(P<0.01),especially C1.At the same time,the four recipes were able to reduce the pulmonary interstitial and alveolar congestion,edema,and inflammatory cell infiltration.Than we selected C2(heat and cooling blood group)to get the further research.The results showed six extracts significantly decreased the total cells and PMNs comparing with LPS group(P<0.05),and especially S2(P<0.01).The extracts significantly decreased the total protein concentration and lung wet dry ratio,and especially S4(P<0.01).At the same time,we established the specific chromatograms,which consisted of 21 peaks,and we identified peak P2,P4,P8,P10,P19 and P21 by standerd solution as gallic acid,oxypaeoniflorin,paeoniflorin,verbascoside,paeonol and harpagoside,respectively.According to HPLC-DAD-ESI-MSn,we concluded that 17 compounds which contained the above six compounds and the remaining 11 compounds were P1(6-O-β-glucosylaucubin)、P3(paeoniflorinsulfonate)、P5(apiopaeonoside)、P6(ethyl gallate)、P7(paeonolide)、P9(galloyl paeoniflorin)、P11(quercetin)、P13(1,2,3,4,6-penta-O-galloyl-β-D-glucose)、P14(angoroside C)、P18(benzoyloxypaeoniflorin)、P20(mudanpioside C).The results of the spectrum-effect relationship were as follows:The total cells inhibition was positively related to the 8 peaks:X8,X10,X21,X1,X2,X19,X14,and X16.Among them the contribution rate of X8 was 45.59%,and X2 was 36.32%.The contribution rate of them was 81.91%,so X8 and X2 were the effective compounds of inhibiting total cells.The neutrophil inhibition was positively related to the 7 peaks:X13,X17,X6,X9,X4,X11,X10,and X5.The contribution rate of X8 and X12 was 51.16%,and 30.48%,respectively.The rate of them was 81.64%,so X8 and X12 were the effective compounds of inhibition PMNs.The wet/dry ratio was positively related to 12 peaks,among which the contribution rate of X8,X6,X3,X13,X11,X2 was 22.82%,15.89%,14.9%,9.29%,9.25%and 7.09%,respectively.The contribution rate of them was 80%.So X6,X3,X13,X11 and X2 were the effective components of inhibiting wet/dry ratio.7 peaks positively associated with inhibition of protein concentration:X13,X17,X6,X9,X11,X10,and X5,among which the contribution rate of X13 was 58.11%,and the rate of X6 was 28.8%.The contribution rate of X6 and X13 was 86.91%.So we selected X13 and X6 as the effective components for inhibiting the protein concentration.We separated the compounds X6,X13,X2 and X19 and identified them as X6(ethyl gallate)、X13(1,2,3,4,6-penta-O-galloyl-β-D-glucose)、X2(gallic acid)and X19(paeonol).By linear regression equation,we calculated the content of X6 and X13,and the content of X6(ethyl gallate)and X13(1,2,3,4,6-penta-O-galloyl-β-D-glucose)were 0.11%and 0.26%,respectively.Finally,the results showed that the extract of heat and cooling blood group(S4),P(X13),E(X6),PE(X13,X6)can significantly reduce the total protein concentration(P<0.05),especially extract S4(P<0.01).The order of inhibition of protein concentration was as follows:DEX>F>S(1,2,3,4,6-penta-O-galloyl-β-D-glucose)>PE(Composition)>E(ethyl gallate).Meanwhile,the four groups can decrease alveolar congestion,edema,and the infiltration of inflammatory.The validation results showed that the contribution rate of pharmacopoeia group was 86.9%by PLSR method,and the effective rate was 60.23%.Although the measured value was slightly lower than the predicted value,but basically consistent.Therefore,the results were well verified.Conclusion:X6(ethyl gallate)and X13(1,2,3,4,6-penta-O-galloyl-β-D-glucose)were the main effective compounds of QBD in rat ALI by reducing the total protein concentration. |
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