Study On The Material Basis And Molecular Mechanism Of The Glycoside Components Of Picrorhizae Rhizoma Against Colitis

Objective:Picrorhizae Rhizoma is a well-known bitter Chinese medicine recorded in the Chinese Pharmacopoeia.It is mainly used clinically for damp-heat diseases such as colitis and hepatitis.Among them,the glycoside components are the main substances through which they exert the efficacy of clearing heat and dampness.At present,the material basis and mechanism of its pharmacological effects in treating colitis are still unclear.The purpose of this study is to use UPLC-QTOF-MS technology combined with network pharmacology and experimental verification methods to explore the material basis and molecular mechanism of the glycoside extract of P.scrophulariiflora(GPS)in the treatment of colitis.Methods:Crude herb Picrorhizae Rhizoma is extracted by reflux of 70% ethanol,and then enriched by macroporous adsorption resin D-101 to obtain GPS.Its chemical composition was identified and analyzed by UPLC-QTOF-MS.The anti-inflammatory material basis and molecular mechanism of its treatment of colitis were first analyzed by using network pharmacology methods and enriched with the Kyoto Encyclopedia of genes and genomes(KEGG)approach to predict its potential active ingredients and the signaling pathways it affects.Then,the predicted active substances and molecular mechanisms were verified by using dextran sodium sulfate(Dextran Sulfate Sodium,DSS)-induced colitis mouse model and LPS-induced RAW264.7 cell model and molecular docking methods.In vitro verification experiment: establishment of LPS-induced RAW264.7macrophage inflammation model.The experimental groups are as follows: Control group(Control),LPS group(1μg/m L LPS),GPS-L group(1μg/m L LPS + 50μg/m L GPS),GPS-M group(1μg/m L LPS + 100μg/m L GPS),GPS-H group(1μg/m L LPS)+ 150μg/m L GPS).After the cells were added with GPS intervention and LPS stimulation for 24 h,the NO release was detected(Greiss);the m RNA level of inflammatory factor IL-6,IL-1β,and i NOS were measured(RT-q PCR);the protein level of i NOS was determined(Western Blot);the levels of inflammatory cytokines IL-6 and IL-1β were determined(Elisa);the protein expressions of p-Akt,p-p38,p-ERK and p-JNK were detected(Western Blot).In vivo validation experiment: replicating a DSS-induced experimental colitis mouse model.The experimental groups are as follows: Control group(Control),DSS group(2%DSS),GPS-L group(2%DSS + 200mg/m L/day GPS,ig),GPS-H group(2%DSS + 400mg/m L/day GPS,ig)and Cs A group(2%DSS + 25mg/m L/day Cs A,ig).During the experiment,the body weight,fecal occult blood and traits of the mice were recorded every day,and the Disease activity index(DAI)was evaluated.After the experiment,the mice were sacrificed,and the colon length,colon pathological changes(HE)were measured or observed;the m RNA levels of the cytokines IL-6,IL-1β,i NOS and IL-10 in the colon were determined(RT-q PCR);the contents of inflammatory factors IL-6,IL-1β and MPO in colon were detected(Elisa);and the protein expressions of p-Akt,p-p38,p-ERK and p-JNK were detected(Western Blot).Results:The results of in vitro experiments showed that compared with the Control group,LPS stimulation could lead to a significant increase in the release of NO,the transcription and protein levels of inflammatory factors(IL-1β,IL-6)and inflammatory mediators(i NOS)in RAW264.7;GPS pretreatment can significantly reverse the above changes in RAW264.7 cells.The results of in vivo experiments also showed that compared with the Control group,DSS could lead to a significant decrease in body weight,a significant increase in DAI,a significant shortening of the colon length,significant damage to the colon tissue,and inflammatory factors in the colon(IL-1β,IL-6),the levels of inflammatory mediators(i NOS)and MPO were significantly increased while the levels of anti-inflammatory cytokines(IL-10)were significantly decreased;and the intervention of GPS and positive drug Cs A can significantly improve the above clinical symptoms and Inflammatory conditions.A total of 30 potential active ingredients were identified from GPS by UPLC-QTOF-MS analysis,and the structural types were mainly iridoid glycosides,organic acids,phenylethanol glycosides and ketones.Based on these components,related targets were collected through the database,and intersected with anti-inflammatory and IBD related targets,a total of 361 potential targets were obtained;After further analysis of the PPI network topology,a total of 77 hub targets were screened out;Subsequently,through KEGG pathway enrichment,the Top20 signaling pathways for GPS treatment of colitis were predicted;Among them,PI3K/Akt and MAPK signaling pathways are the main signaling pathways mediating the regulation of GPS.In vivo and in vitro verification experiments found that GPS intervention treatment could significantly reduce the protein expressions of p-Akt,p-p38,p-ERK and p-JNK in the colon of colitis mice and in LPS-stimulated RAW264.7 cells.At the same time,luteolin,apocynin,caffeic acid,caffeic acid methyl ester,luteoloside,picroside Ⅱ,aucubin,cinnamic acid,vanillic acid,and sweroside were predicted based on network pharmacology analysis.Glycosides may be the main active ingredient of GPS to exert anti-inflammatory effect.The above compounds were verified by in vitro experiments to significantly inhibit the release of NO in RAW264.7 cells induced by LPS;the molecular docking results showed that these compounds could bind well to the target proteins Akt,p38,ERK and JNK,especially picroside Ⅱ,luteolin and luteoloside.Conclusions:GPS has a significant anti-inflammatory effect on LPS-induced RAW264.7macrophage inflammation model and can obviously improve the clinical symptoms of experimental colitis in mice.The anti-inflammatory mechanism is related to the inhibition of PI3K/Akt and MAPK signaling pathways.Luteolin,apocynin,caffeic acid,caffeic acid methyl ester,luteoloside,picroside Ⅱ,aucubin,cinnamic acid,vanillic acid,and sweroside are responsible for GPS anti-inflammatory effects of the main components.