| Purpose:The topic takes Huangqin Shegan Decoction as the research object.Based on the literature research and serum medicinal chemistry research,the effective component group of Huangqin Shegan Decoction is screened.The molecular docking technology is used to predict the action mode and strength of active components and related targets.To simulate the possible mechanism of action of Huangqin Shegan Decoction.Established cough model of rat induced by LPS to detect related indicators,verify molecular docking results from protein expression and biomarker changes in vivo,and explore its mechanism of action.The HPLC method was used to study the quality control of the active ingredients of Huangqin Shegan Decoction.The above research will lay the foundation for the clinical application of Huangqin Shegan Decoction.Material and method:1 The molecular structures of 16 flavonoids were summarized through the literature retrieval and TCMSP databases.By consulting relevant domestic and foreign literatures and TCMSP database,16 reported flavonoids with clear pharmacological activity were screened,and 10 protein structures related to airway inflammation pathway were summarized.Find protein-ligand complexes from PDB website.The structure of the crystal was analyzed by using the Autodock 4.02 docking software to study the molecular docking of the compound and the protein.Cocrystallization ligand was extracted from crystal structure as control,while the effect strength of compounds and targets was compared by the relative score(R= compound score/controlled molecular score).Stronger proteins were used for subsequent validation experiments.2 According to the conclusion of the R value of the molecular docking,an in vivo verification experiment was performed.Based on the cough model of rat,the morphology of trachea,bronchi and alveoli in lung tissue of model animals were observed by hematoxylin-eosin(HE)staining method.The expression levels of related proteins in lung tissues were detected by western-blot method.The levels of cytokines in serum and alveolar lavage fluid of rats were detected by ELISA.3 The HPLC-VWD method was used to determine the simultaneous determination of eight active ingredients such as baicalin in Huangqin Shegan Decoction,which provided a basis for the quality control of Huangqin Shegan Decoction.Results:1 Through molecular docking technology,using the relevant database and molecular docking software,the molecular weight,hydrogen bond donor,hydrogen bond acceptor and CLog P of16 flavonoids contained in Huangqin Shegan Decoction were summarized.The results showed that flavonoids of Huangqin Shegan Decoction were accorded with Class rules of medicinal of Lipinski,which indicated its drug-likeness.The molecular docking energy relative scores showed that the combination of flavonoids and COX-2 and 5-LOX was better than other targets,R>0.8,and P38 MAPK,PLA2,JNK 3 and CYP450 2C9 subtype molecular docking relative scores are also better,R>0.7,the relative scores of individual components and JNK 1,LTA4 H,CYP450 2C8 and IKK-β molecules docking is slightly worse,but greater than 0.6,From the mode of action of the compound and the protein,16 flavonoids can bind to10 proteins.It indicates that the flavonoids contained in Huangqin Shegan Decoction may exert anti-airway inflammation through multi-component,multi-channel and multi-target,and the anti-inflammatory effect of Huangqin Shegan Decoction maybe probably relevant to the COX-2 and 5-LOX activity.2 Based on the results of molecular docking experiments,the cough model of rat induced by LPS was selected as a model for preliminary drug model study.The cough time within 5 min of the control group were increased obviously,while the cough times within 5 min of Huangqin Shegan Decoctionn group were decreased compared to the control group,and reduced the bronchial and peripheral inflammation in the lung tissue.The expression levels of COX-2 and 5-LOX in lung tissue of control group were assayed by Western-blot method.The results of Western-blot indicated that the levels of COX-2 and 5-LOX were increased in the control group(P<0.05),indicated that the established cough model of rat induced by LPS can activate COX-2 and 5-LOX in the arachidonic acid metabolic pathway.The level of COX-2in Huangqin Shegan Decoction group was significantly decreased(P<0.05),while the level of5-LOX showed no significant difference(P>0.05),suggested that Huangqin Shegan Decoction may play a role by inhibiting the COX metabolic pathway of arachidonic acid.The metabolites of prostaglandins E2(PGE2)and leukotriene C4(LTC4)of arachidonic acid were determined by ELISA.The results of the content of arachidonic acid metabolites showed that the metabolite contents of PGE2 metabolic pathways in the control group were statistically increased compared to the blank group(P<0.05),while the content of PGE2 was significantly decreased(P<0.05)in Huangqin Shegan Decoction group.Combined with the results of Western blotting,it is speculated that Huangqin Shegan Decoction may slow down the inflammatory response by inhibiting the expression of COX-2 and reducing the release of the metabolite PGE2.The results of the content of arachidonic acid metabolites showed that the metabolite contents of LTC4 metabolic pathways in the control group were statistically increased compared to the blank group(P<0.05),while the content of LTC4 showed no significant difference in Huangqin Shegan Decoction group.Combined with the results of western blotting,it was speculated that the inhibitory effect of Huangqin Shegan Decoction on LOX and its metabolites was not obvious.3 HPLC-VWD method was used to detect the content of tectoridin,tectorigenin,iridin,irisflorentin,baicalin,wogonoside,baicalein and oroxylin A.The method validation experiments showed that in the range of 0.05625~0.3375μg、0.008656~0.05194μg、0.01613~0.09677μg、0.01134~0.06802μg、0.3146~1.888μg、0.1199~0.7194μg、0.05632~0.3379μg1 and 0.01035~0.06211μg,the linear relationship with peak area is good,r value is 0.9999,0.9999,1.000,1.000,1.000,1.000,0.9997 and 0.9998,respectively.Conclusion:1 The results of molecular docking experiments of Huangqin Shegan Decoction showed that the 16 flavonoids in Huangqin Shegan Decoction had the best binding strength to COX-2 and5-LOX among the ten target proteins in the inflammatory pathways,followed by P38 MAPK,PLA2,JNK 3 and CYP450 2C9.It is slightly inferior to JNK 1,LTA4 H,CYP450 2C8 and IKK-β.It is speculated that the pathway of anti-inflammatory effect may be related to COX-2and 5-LOX target proteins in the arachidonic acid inflammatory pathways.2 Verification experiment selected the cough model of rat induced by LPS.Huangqin Shegan Decoction can significantly inhibit the cough frequency of model animals,reduce lung tissue inflammation,inhibit the expression of COX-2 in lung tissue,and reduce the content of cytokine PGE2 in alveolar lavage fluid,indicating that Huangqin Shegan Decoction can intervene COX-2 in the arachidonic acid inflammatory pathways.3 The HPLC-VWD method as a quality control method for Huangqin Shegan Decoction is simple,sensitive and reproducible for the determination of the content of tectoridin,tectorigenin,iridin,irisflorentin,baicalin,wogonoside,baicalein and oroxylin A. |
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