The Study Of Ziqin And Kuqin Quality Comparison And Their Effects And Mechanism On Treating Acute Lung Injury

1 OBJECTIVEScutellaria baicalensis,the dry root of Scutellaria baicalensis Georgi,has the functions of heating-cleaning and damp-drying,purging fire for removing toxin,hemostasis and tocolysis a plant of the Lamiaceae family.The medical monographs in ancient time recorded that Scutellaria baicalensis G.can form Ziqin and Kuqin due to different harvesting years.Kuqin(over 3years),an old perennial root,is hollow and withered,is the upper part of the root of Ziqin;Ziqin(under 3 years),the lower part of Kuqin,is solid and firm.Accorading to medical documentations,Ziqin and Kuqin are often used as medicines respectively.Ziqin is used to relieve the heat in the lower coke in the large intestine,and Kuqin is good at clearing the heat in the part of the lung and stomach,which are located in the upper part of viscera,but this operation is about to be discarded at contemporary society.Since the 1990s,Scutellaria baicalensis G.has not been no longer divided into Ziqin and Kuqin clinically,and the name of the doctor’s prescription is Scutellaria baicalensis G..There are few related studies on Kuqin in modern literatures.In order to verify the reasonableness and scientificity that the ancients dividing Scutellaria baicalensis G.into Ziqin and Kuqin further so as to use them as decoction pieces,this paper studies the differences in characteristic and chemical components of Ziqin and Kuqin,as decoction pieces,and their intervention effects on acute lung injury.To clear the reasonableness of the different application of Ziqin and Kuqin;on this basis,the material basis and differences of the anti-inflammatory effects of Ziqin and Kuqin are analyzed through the spectrum-effect relationship,and the protection of Kuqin alcohol extract on mice with acute lung injury was discussed.The feasiblity of researches above-mentioned provides a modern theoretical research basis for the clinical application of Ziqin and Kuqin.2 Methods and results2.1 Comparative study on characteristic and chemical components of Ziqin and Kuqin with different harvesting years2-year-old Ziqin and 3-,5-,7-,and 10-year-old Kuqin were harvested from the same production area,and the differences in charactersitic between Ziqin and Kuqin with pieces of different ages were compared when they were decoction pieces.Matlab software was used to calculate the proportion of hollow decay area,and a colorimeter was used to measure the color difference as the two medicinal materials were shaped into powder.The results showed that with the increase of harvesting years,the diameter of all batch of decoction pieces continue to increase,Ziqin were no hollow decay,and the hollow decay area of Kuqin gradually increased.The hollow and putrescent proportions of 3-,5-,7-,and 10-year-old Kuqin were 26.65 %,32.52 %,43.90 % and 52.80 % in turn,and the 10-year-old Kuqin grew endogenous roots inside,and then formed inner layers and hollow areas;the green-red numercial value a* of the two medicinal material’ spowder color increases,the numercial brightness value L*,the yellow-blue value b* and the total color difference numercial value ΔE* decreased.Ziqin and Kuqin with different ages were tested contents of main chemical components by ultra-high liquid chromatography.The results showed that the contents of flavonoid glycosides such as baicalin and Wogonoside decrease year by year,the contents of baicalein,wogonin,oroxylin A increased,and eventually the total content of the five chemical components decreased.Pearson correlation analysis showes that the harvesting period of Scutellaria baicalensis G.was significantly correlated with the decay area,color difference and chemical composition content.2.2 Comparative study on the therapeutic effects of Ziqin and Kuqin ethanol extracts on acute lung injuryTaking SPF grade male C57BL/6 mice as experimental subjects,the acute lung injury model was acuted by intranasal lipopolysaccharide instillation,myeloperoxidase(MPO)activity and malondialdehyde(MDA)content in lung tissue,and the changes of inflammatory factors IL-6,IL-1β and TNF-α content in bronchoalveolar lavage fluid were tested as indicators.The effects of Ziqin and 70% ethanol extracts from Kuqin in the prevention and treatment of acute lung injury in mice were compared.The results showed that the lung tissue structure was disordered in the model group,the alveolar wall was widened and thickened,the pulmonary interstitium appeared edema,and was accompanied by a large number of inflammatory cell infiltration,each administration group could improve the tissue morphology of the model group,the lung tissue in the high dose Kuqin group was closest to the normal form.Compared to the model group,the high and low dose Kuqin groups and the high dose Ziqin group could reduce the white blood cell count of mice,and the difference was statistically significant(P < 0.01 or P < 0.05);The high dose Kuqin and Ziqin groups could reduce the wet-dry specific gravity of lung tissue in the model group to varying degrees(P < 0.01 or P < 0.05);Both high and low dose Kuqin and Ziqin groups could reduce the MDA content in the model group(P < 0.01);The high dose Kuqin group and the low dose Ziqin group could reduce the MPO in the model group(P < 0.01 or P < 0.05);the high and low dose Kuqin and Ziqin groups could reduce the content of inflammatory factors IL-1β and TNF-α in the bronchoalveolar lavage fluid of the mice in the model group(P < 0.01);The high dose Kuqin and Ziqin groups could reduce the content of inflammatory factor IL-6 in the model group(P <0.01 or P < 0.05);The high dose Kuqin group metrics improved the best.The effects of baicalein,the highest component of kuqin transformation increment,and baicalein ethanol extract on ALI were further compared.The results showed that the baicalein group(the content in the corresponding dose of kuqin was the same)and the kuqin group could improve the structure of lung tissue lesions,reduce the The m RNA expression of inflammatory factors IL-1β,TNF-α,and IL-6(P < 0.01),the efficacy of the two was effective and there was no conspicuous difference.2.3 Study on the Spectrum-effect relationship of the anti-inflammatory effects of Ziqin and Kuqin ethanol extractsThe fingerprints of different batches of Ziqin(S1-S10)and Kuqin(S11-S20)were established individually by ultra-high performance liquid chromatography(UPLC);macrophages were stimulated with lipopolysaccharide to release inflammatory factors IL-1β,TNF-α.level were the efficacy index;the Spectrum-effect relationship between Ziqin and Kuqin was analyzed by orthogonal partial least squares regression(OPLS),and the Spectrum-effect relationship of anti-inflammatory effects of 20 batches of Scutellaria baicalensis G.was further analyzed comprehensively.The results showed that 17 common peaks were showed by the fingerprints of 20 batches of samples.The results of the orthogonal partial least squares regression analysis showed that the main related components of the anti-inflammatory effect of Ziqin were peaks 4,9,and 13,and the main anti-inflammatory effect of Kuqin were peaks 4,13,14,and 2.The main related components of Ziqin and Kuqin has two common components,has three different components;The main related components of the anti-inflammatory effects of Scutellaria baicalensis in 20 batches were peaks 4,9 and 13.After the comparasion of control article,the three components were baicalin,Wogonoside and baicalein.2.4 Study on the protective mechanism of Kuqin ethanol extract on acute lung injuryUsing the network pharmacology method,with the help of the Swiss Target Prediction database,String Website database and Gene Cards database,to analyze the potential targets and ALI-related genes of the five major chemical components(baicalin,wogonoside,baicalein,wogonin,oroxylin A)of ethanol extract from Kuqin and the results showed that the anti-inflammatory mechanism of alcohol extract from Kuqin was main related to NF-κB and NLRP3 pathways.Two possible molecular mechanisms were further verified in vitro and in vivo.Taking LPS-induced ALI mice as a model,to compare with the model group,the results showed that each dose Kuqin group decreased the fluorescence expression of macrophage marker F4/80 in the lung tissue of the model group;inhibited the production of inflammatory cytokines such as IL-1β,IL-6 and TNF-α in the model group(P < 0.01),and reduced the key enzymes i NOS in the progression of inflammation.and the m RNA expression(P < 0.01)of the PTGS2,F4/80 and p65 proteins,F4/80 and NLRP3 in the lung tissue of mice were detected by double immunofluorescence(IF)double labeling,compared with the model group,the fluorescence expression of F4/80 and p65,F4/80 and NLRP3 in the high dose Kuqin group decreased significantly;Western blotting(WB)was used to detect NF-κB and NLRP3pathway-related proteins The expression of p-IκB,p-p65,NLRP3,ASC,cleaved-caspase-1,and cleaved-IL-1β protein in the model group was significantly inhibited in the high dose Kuqin group(P < 0.01 or P < 0.05).Taking LPS-stimulated THP-1 macrophages as a model,the each dose Kuqin groups inhibited the m RNA expressions of inflammatory factors IL-1β,TNF-α,IL-6,i NOS and PTGS2 in a concentration-dependent manner(P <0.01).The expression of NF-κB pathway-related proteins was detected by WB.The results showed that the high dose Kuqin group could significantly inhibit the expression of p-IκBα and p-p65 proteins in the cell model(P < 0.01);the expression of NLRP3 pathway-related proteins was detected by WB.,there was no significant difference in the expressions of NLRP3 and ASC among the groups,and the high dose Kuqin group could significantly inhibit the expression of cleaved-caspase-1 and cleaved-IL-1β proteins in the cell model(P < 0.01).3 Conclusion3.1 Comparative study on characteristic and chemical components of Ziqin and Kuqin with different harvest yearsWith the increase of the harvesting period of Scutellaria baicalensis G.,the hollow decay area increases,the powder brightness value,yellowness value,and chromaticity decrease.The transformation amount increased,and the total flavonoid content decreased.Based on the change of chemical composition content and the change of harvesting year,decayed area and powder color,the roots of 10-year-old Kuqin was too hollow and rotten,and it was easy to wrap the sediment and dust,the ash content exceeds the standard,and the total flavonoid content was too low.it was suggested that Kuqin should be harvested within 7 years.3.2 Comparative study on the therapeutic effects of Ziqin and Kuqin ethanol extracts on acute lung injuryBoth Ziqin and Kuqin alcohol extracts could improve the inflammatory symptoms of lung tissue on ALI mice.The efficacy indicators suggest that the Kuqin groups was better than the Ziqin groups,which verifies that the Kuqin has a better effect on improving lung inflammation than Ziqin.The effects of baicalein,the highest component of Kuqin transformation increment,and Kuqin on ALI were further compared.The results showed that both Kuqin group and baicalein group had significant medicinal effects,which suggested that baicalein may be the main contributing component of Kuqin’s efficacy over Ziqin’s.3.3 Study on the Spectrum-effect relationship of the anti-inflammatory effects of Ziqin and Kuqin ethanol extractsThere were significant differences in the fingerprints and in vitro anti-inflammatory efficacy between Ziqin and Kuqin.The fingerprints of Kuqin and Ziqin both identified 17 common peaks.The spectrum-effect relationship showed that peaks 4,9,and 13 were mainly related to the anti-inflammatory effect of Ziqin,and peaks 4,13,14,and 2 were mainly related to the anti-inflammatory effect of Kuqin.They have 2 common components and 3 different components,which indicated that the material basis of anti-inflammatory effect of the two were similar,and there were also certain differences;the spectrum-effect relationship results of 20 batches of Scutellaria baicalensis G.suggest that the main anti-inflammatory related components were peak 4,9 and 13,compared with the reference substances,the three components that contributed the most to the anti-inflammatory effect of Scutellaria baicalensis G.were baicalin,Wogonoside and baicalein.3.4 Study on the protective mechanism of Kuqin ethanol extract on acute lung injuryNetwork pharmacology analysis revealed that the possible mechanism of Kuqin ethanol extract in the treatment of lung injury was related to NF-κB and NLRP3 pathways.In an animal model of LPS-induced ALI,the Kuqin ethanol extract could inhibit the expression of NF-κB/NLRP3 pathway-related proteins and the release of inflammatory factors,thereby inhibiting the activation of macrophages.Similar results were obtained in in vitro experiments on LPS-stimulated macrophages.This study clarified the potential of Kuqin in the treatment of ALI and revealed its possible mechanism,that is,by inhibiting the NF-κB/NLRP3 inflammatory pathway,thereby inhibiting the pro-inflammatory phenotype activation of lung tissue macrophages.To sum up,this study clarified the differences between Ziqin and Kuqin with different harvesting years through the comparison of characters and chemical components,and provided a basis for the best harvesting period of Kuqin for 7 years;it was clear that Kuqin has more advantages than Ziqin,and preliminarily explore that the advantage was not the contribution of the highest incremental ingredient baicalein,which may be caused by the different proportions of main components in Kuqin and Ziqin.The three components that contribute to the anti-inflammatory effect of Ziqin and Kuqin were:baicalin,Wogonoside and baicalein,which provide the basis for further research on the basis of pharmacodynamic substances;the research on the pharmacodynamic mechanism preliminarily verified that inhibition of macrophage activation via the NF-κB/NLRP3 inflammatory pathwaythe was the potential mechanism for Kuqin to exert its medicinal effect.The results of this study verified the rationality of the different application of Ziqin and Kuqin,excavated the potential efficacy of Kuqin,provides a basis for clinical precision treatment,and provided a reference research model for the regulation of Chinese herbal medicine for rescue protection.