Huangqin Decoction Ameliorates Ulcerative Colitis In Mice By Remodeling Intestinal Microecology

ObjectiveHuangqin Decoction(HQD)is a classical TCM formula chronicled in the Shang Han Lun.It is mainly aimed at symptoms such as hot dysentery and abdominal pain,dysentery and abdominal pain,red tongue,yellow fur and pulse string number.HQD is used for the treatment of dysentery and abdominal pain caused by shaoyang evil heat.It is intended to be a classic prescription for early dysentery,and possesses a various pharceutical effects such as clearing heat,removing dampness,and stopping dysentery.HQD is widely used in clinical and experimental studies to treat digestive diseases.Ulcerative colitis(UC)is a chronic nonspecific inflammatory bowel disease(IBD)with unknown etiology.It is a difficult disease in the Department of Gastroenterology,and belongs to the category of“Li ji”(dysentery)in Traditional Chinese medicine(TCM).The main manifestations of UC are abdominal pain,mucous pus and blood stool with tenesmus,and the lesions are mainly involved in colorectal mucosa and submucosa.The incidence of UC is increasing year by year in Asia,but there is still a lack of effective treatment.Recent studies believe that the incidence of ulcerative colitis is mainly related to genetic,environmental and intestinal mucosal barrier damage and other factors,and it indicated that intestinal microecology disorder is a key factor in the occurrence and development of UC.The intestinal microecology disorder causes the intestinal flora disequilibrium and then results in flora metabolism dysfunction.The endotoxin secreted by bacteria would destory the intestinal barrier and disturb intestinal permeability.The increasing permeability translocates the antigen,endotoxin,and other proinflammatory cytokines from the bowel lumen into the lamina propria of intestinal mucosa,resulting in intestinal injury and exacerbating UC deterioration.HQD is mostly used to treat gastrointestinal disorders in clinical trails and experimental studies.At present,the effect of HQD on UC has been reported,and most of them were focus on intestinal immune system improvement.However,it is not clear whether HQD can alleviate UC by improving the intestinal microecology,another key factor inducing UC.Therefore,this paper is to explore the the intestinal microecology regulation of HQD in UC mice and FHC cell injury model,to clarify the mechanism of HQD in alleviating colonic injury in UC mice.Methods1.Identification and analysis of the main compounds of HQDThe compounds of HQD were identified by high performance liquid chromatographyquadrupole time of flight mass spectrometry(HPLC-QTOF/MS).The main components concentration of HQD were analyzed by high performance liquid chromatography(HPLC).The composition of HQD was analyzed by high-performance liquid chromatographyquadrupole-time of flight mass spectrometry(HPLC-QTOF/MS).For HPLC separation,a C18 analytical column(250 mm × 4.6 mm,5 mm)was used.The mobile phase was acetonitrile(solvent A)and 0.1%(v/v)formic acid(solvent B).The gradient elution program was as follows:0-15 min,5-5%A,15-30 min;5-15%A,30-60 min;15-23%A,60-90 min;23-45%A,90-100 min;45-60%A.The flow rate was 1.0 mL/min.The column oven was set at 30℃.The injection volume was 10μL.The wavelength was 280 nm.The ESI source parameters were as follows:drying gas temperature-350℃,capillary voltage=3.2 kV,drying gas flow=700 L/h of nitrogen,extraction cone voltage=3 V,sampling cone voltage=50 V,high-pressure RF voltage=30 V,and low-pressure RF voltage=4 V.QTOF/MS operates in full scan mode,collecting data in the range of 50-1000 mass-to-charge ratio(m/z)at a scanning rate of 2 spectra/s.2.The effect of HQD on colon injury and inflammatory lesions in UC miceThe experimental mice were free access to 3%(weight/volume)DSS solution for a week to estabolish UC model.Simultaneously,the mice in treatment groups were administrated with 5-amino salicylic acid(5-ASA,200 mg/kg)and HQD(2.275,4.55 g/kg),respectively.During the experiment,the health status,body weight,food intake and water consumption of mice was monitored and recorded daily,and these were used to score according to the specific grading criterion.In the end of experiment,to measure the colon length of mice,and evaluate the colon macroscopic and histopathological difference.Furthermore,to detecte intestinal barrier junction proteins by Western Blotting(WB),including ZO-1,E-cadherin,Occludin and Claudin-1,and detecte the levels of inflammatory factors by enzyme-linked immunosorbent assay(ELISA),including LPS,IL-2,IL-4,IL-17A,IFN-γ and TNF-α.3.The regulatory effect of HQD on gut microbiota and microbial metabolites in UC miceThe Illumina HiSeq high-throughput sequencing platform was used to sequence the intestinal contents of experimental mice.According to the sequencing results,the composition of dominant species,the function of the microflora and their related metabolic pathways in experimental mice were analyzed.These results were used to evaluated the regulatory effect of HQD on the intestinal microflora in UC mice.The Q300TM platform was used to detect the intestinal content metabolites in mice.The metabolites were quantified by ultra-performance liquid chromatography coupled to tandem mass spectrometry(UPLC-MS/MS)system,and the chemical composition of them were analyzed by positive and negative ion scanning.The effects of HQD on metabolites and related molecular signaling pathways of UC mice were analyzed according to the detection results.The mTOR signaling pathway proteins were detected by WB,including mTOR,PI3K,Akt,S6 and 4E-BP1 proteins,as well as their corresponding phosphorylated proteins.4.The effect of differential metabolites-amino acid in cell model in vitroFHC cells injury model was induced by 0.5%DSS incubation for 4h.The differential metabolites(including L-lysine,L-alanine,L-proline,L-leucine,glycine,L-valine,Ltyrosine,keto-leucine,L-glutamate and L-isoleucine)after HQD treatment in UC mice were screen by metabolome,and these differential metabolites-amino acid compounds were added into the injuried FHC cells for 48h.The effect of differential amino acids on DSSdamaged FHC cells viability was detected by CCK-8 assay.The effect of differential amino acids on the morphology of damaged FHC cells was observed by gentian violet staining.The effect of differential amino acids on apoptosis of damaged FHC cells was detected by flow cytometry(FCM).The effect of differential amino acids on ZO-1,E-cadherin,Occludin,Claudin-1 and mTOR signaling pathway proteins mTOR,PI3K,Akt,S6,4E-BP1 and their phosphorylated proteins in FHC cells was detected by WB.The effects of differential amino acids on the expression of IFN-γ,TNF-α,IL-2 and IL-6 were determined by ELISA.The helper T cells(Th cells)from mouse spleen were purified by immunomagnetic bead sorting,and then activated by anti-mouse CD3ε and anti-mouse CD28 functional antibodies to simulate the immune hyperactivation model of UC mice.The differential metabolitesamino acids(including L-lysine,L-leucine,L-glutamate and L-isoleucine)were added into Th cells for 72 hours.The effect of differential amino acids on Th cells viability was detected by cck-8 assay Cck-8 assay was used to detect the effect of different amino acids on Th cell viability.The effect of differential amino acids on apoptosis of damaged Th cells was detected by FCM.The effects of differential amino acids on the expression of IFN-y,TNF-α,IL-2 and IL-4 in Th cells were determined by ELISA.Results1.The composition analysis of HQDBy HPLC-QTOF/MS indefication and HPLC analysis,the main compounds of HQD were Baicalein,Wogonoside,Glycyrrhizic acid,Oroxylin A 7-O-glucuronide,Wogonin,Liquiritigenin,Oroxylin A,Albiflorin,Baicalin and Paeoniflorin.2.The effect of HQD on colon injury and inflammatory lesions in UC miceHQD prevented weight loss,reduced DAI score and reverse colon length shortening in UC mice without interfering with their diet.HQD could improve the macroscopic pathological characteristics of colon tissue in UC mice,relieve colonic edema,congestion,adhesion,ulcer spots.HQD also improved colonic histopathological characteristics,making the structure complete,villi orderly arranged,intestinal crypt branches obvious,and no obvious inflammatory infiltration.Further studies showed that HQD could restore intestinal barrier integrity in UC mice by upregulating the expression of intestinal barrier junctions ZO1,Occludin,e-cadherin and Claudin-1,and alleviate the intestinal inflammatory injury by downregulating the expression of LPS,IL-2,IL-4,IL-17A,IFN-y and TNF-α.3.HQD regulated the gut microbiota and mictobial metabolites in UC miceHQD could significantly increase the relative abundance of Firmicutes and lower that of Bacteroidetes,and alter the composition of dominant microbiota.At the same time,HQD regulated the microbial metabolism of intestinal flora in UC mice,especially carbohydrate metabolism and amino acid metabolism.HQD could significantly upregulate the levels of various amino acids,including Llysine,L-alanine,L-proline,L-leucine,glycine,L-valine,L-tyrosine,keto-leucine,Lglutamate,L-isoleucine,and regulate the biosynthesis and metabolic pathways related to amino acids in UC mice.Correlation analysis showed that HQD-regulated amino acid metabolites were positively correlated with the relative abundance of Firmicutes,while positive correlated with that of Bacteroides.In addition,protein detection results showed that HQD activated mTOR signaling pathway and promoted the expression of phosphorylated proteins of mTOR,PI3K,Akt,S6 and 4E-BP1.4.HQD-regulated differential metabolites-amino acids functioned on cell in vitroAmong the differential amino acids regulated by HQD,L-lysine,L-leucine,Lisoleucine and L-glutamate could significantly upregulate the viability of FHC cells damaged by DSS at 200 μmol/L,while the other differential amino acid compounds showed no effect.In addition,L-lysine,L-leucine,L-isoleucine and L-glutamine promoted the damage of intercellular junctions,inhibited apoptosis and restored cell morphology in FHC cells.Further studies showed that these four amino acids could activate the mTOR signaling pathway in FHC cells,upregulate the expression of mTOR,PI3K,Akt,S6 and 4E-BP1 phosphorylated proteins,and also promote the expression of intestinal barrier proteins ZO-1,Occludin,E-cadherin and Claudin-1.Conclusion1.HQD could increase the expression of intestinal barrier protein,restore the integrity of colon tissue structure to improved colon injury in UC mice.Simultaneously,HQD ccould downregulate the levels of inflammatory factors to improve the colitis in UC mice.2.HQD could restore the homeostasis of intestinal flora and regulate the miecrobiamediated amino acids metabolism.By improving microbiota and their metabolites,HQD thus activated the mTO signaling pathway to upregulate the expression of intestinal barrier protein,and improved colon injury in UC mice.3.HQD-regulating differential amino acids(including L-lysine,L-alanine,L-proline,L-leucine,glycine,L-valine,L-tyrosine,keto-leucine,L-glutamate,and L-isoleucine)could inhibit the cell apoptosis,activate mTOR signaling pathway to restore intercellular junction.4.HQD-regulating differential amino acids(including L-lysine,L-leucine,L-glutamate,and L-isoleucine)had no pharmacodynamic effect on Th cells,suggesting these differential amino acids could not function on improving the intestinal immune system in UC mice.