| Fritillaria cirrhosa D.Don,a traditional Chinese medicine belonging to Fritillaria L.of Liliaceae.It is mainly used for the treatment of acute bronchitis,recurrent asthma and other respiratory diseases.The anti-inflammatory effect of Fritillaria cirrhosa D.Don can cooperate with the treatment of respiratory tract infection,which is the pharmacological basis of Fritillaria cirrhosa D.Don to relieve cough and phlegm.In this study,CCK-8 method was used to determine the effects of the positive drugs DXMS,ethanol extract of Fritillaria cirrhosa D.Don 1,ethanol extract of Fritillaria cirrhosa D.Don 2,Fritillaria cirrhosa D.Don fluid extract,lipid-soluble components of Fritillaria cirrhosa D.Don,verticine,verticinone,peimisine and sipeimine on RAW 264.7 cell viability under different concentrations;The in vitro inflammatory model of RAW 264.7 cells was successfully established by LPS induction,and the in vitro anti-inflammatory activities of four Fritillaria cirrhosa D.Don extracts and four alkaloids were studied.The interaction mechanism between active components and anti-inflammatory targets of Fritillaria cirrhosa D.Don was studied by network pharmacology.The binding ability between the active components and the target proteins was verified by molecular docking,and the binding mode with the highest matching degree was screened,which provided theoretical basis and reference value for the development and utilization of lead compounds and innovative drugs of Fritillaria cirrhosa D.Don in the future.The main results of this study are as follows:1.The effects of the DXMS,4 kinds of Fritillaria cirrhosa D.Don extracts and 4kinds of alkaloids on the vitality of RAW 264.7 cells at different concentrations were determined by CCK-8 method.The results showed that DXMS concentration of 1～20μg/m L had no obvious toxicity to cells(P>0.05).The four kinds of Fritillaria cirrhosa D.Don extracts at concentrations of 25 μg/m L,50 μg/m L and 100 μg/m L had no obvious toxicity to cells(P>0.05).The four alkaloids at concentrations of 25ng/m L,50 ng/m L and 100 ng/m L had no obvious toxicity to cells(P>0.05).2.The inflammatory model of RAW 264.7 cells was established by LPS stimulation in vitro to study the effect of Fritillaria cirrhosa D.Don effective components on inflammatory cytokines of RAW 264.7 cells.The results showed that the four kinds of extracts and four kinds of alkaloids at different concentrations could reduce the production of inflammatory cytokines IL-1β,IL-6 and TNF-α in RAW264.7 cells modeled successfully(P<0.01).The anti-inflammatory effects of the 4kinds of Fritillaria cirrhosa D.Don extracts were ethanol extract of Fritillaria cirrhosa D.Don 2 > Fritillaria cirrhosa D.Don fluid extract > ethanol extract of Fritillaria cirrhosa D.Don 1 > lipid-soluble components of Fritillaria cirrhosa D.Don.The anti-inflammatory effects of 4 kinds of alkaloids were peimisine >verticine > verticinone > sipeimine.3.Network pharmacology was used to screen the active ingredients of Fritillaria cirrhosa D.Don,and their related anti-inflammatory targets were enriched.GO enrichment analysis and KEGG enrichment analysis were used to explore the anti-inflammatory mechanism of Fritillaria cirrhosa D.Don.The results showed that ten active ingredients were obtained by screening.Ten core target proteins related to anti-inflammatory;By GO enrichment analysis,746 biological processes(BP),30 cell components(CC)and 107 molecular functions(MF)were obtained.Seventy-three signal pathways were obtained by KEGG enrichment analysis.4.Molecular docking technology was used to verify the above obtained active ingredients and core targets related to inflammation,and the binding mode with optimal binding ability was screened.The results showed that peimisine had the best binding ability to the target protein,followed by beta-sitosterol,Korseverinine and ZINC03860434,finally,cyclopamine and sinpemine A.The binding force of peimisine was in good agreement with the results of alkaloids in vitro. |
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