Study On The Mechanism Of Wumei Pills With Four Legal Ingredients In Treating Ulcerative Coliti

Object To explore the potential mechanism of Fructus Mume pills(FMP)four flavors composition via network pharmacology;to explore flavors-biological effects through observing the effects of FMP and its four kinds of compositions on colonic permeability,inflammatory factors,oxidative stress,neuropeptides and taste receptors on ulcerative colitis rats.Methods ①Collect FMP active components by TCMSP database,potential targets were obtained from PubChem and Swiss target prediction.FMP four flavors compounds-targets graphs were constructed by Cytoscape 3.7.2,target protein interaction(PPI)network was constructed by STRING database.GO and KEGG pathway enrichment was obtained and potential biological pathways and signal pathways analyzed by Omicshare platform.②According to four flavors composition,FMP was disassembled into four compositions.83 SD male rats were divided into control group,model group,whole recipe group,sour flavor group,bitter flavor group,pungent flavor group,sweet flavor group,and SASP group.4%acetic acid enema was used to replicate the rat model of ulcerative colitis.24 h after modeling,10 ml/kg(B.W.)drug was administrated twice a day for consecutive 7 days.Model group and control group were given the same amount of distilled water every day.After the last administration,rats in each group were fasted for 12h before sampling.After Sodium pentobarbital anesthesia,blood plasma were collected by abdominal aortic method,3 ml blood was taken by sodium citrate(3.8%)1:9 anticoagulation tube,1.2 ml blood was taken by sodium citrate(3.2%)1:4 anticoagulation tube,heparin anticoagulation tube was used to extract 2ml for hematocrit detection.Blood samples are processed according to detection methods.The rest of the blood was extracted with ordinary serum tubes,and the serum was separated and stored at-20℃ for testing.The distal colon was taken 6-8cm,ulcers were observed and photographed.A section of the distal colon was taken and fixed in 10%paraformaldehyde.The remaining colon was washed with pre-cooled PBS,dried with filter paper,and frozen at-80℃ for detection.H&E,AB-PAS,masson staining were used to observe the pathological injury of colon tissue.Immunohistochemical method was used to observe the expression of occludin-1 in colon tissue,ELISA was used to detect the levels of inflammatory factors and neuropeptide factors,ELISA and WB were used to detect the expression of oxidative stress factors,WB was used to detect the changes of VEGF signaling pathway.MRNA expressions of taste receptors were detected by PCR.The other indicators were operated according to the instructions of the corresponding kit,and the coagulation indicators were detected by the coagulation analyzer.Results ①Totally 909 compounds and 381 active compounds were obtained from FMP.Among them,21 active compounds and 140 potential targets were obtained from sour flavor group.In bitter flavor group,82 active compounds and 161 potential targets were obtained from Rhizoma Coptidis and Cortex Phellodendri Chinensis.A total of 225 active compounds and 138 potential targets were obtained from rhizoma zingiberis,Ramulus Cinnamomi,Aconitum carmichaeli Debx,Zanthoxylum bungeanum Maxim and Asarum sieboldii in pungent flavor group.A total of 93 active compounds and 76 potential targets were obtained from Angelicae Sinensis Radix and Codonopsis Radix in the sweet flavor group.The sour flavor group was enriched with 3264 GO and 104 signal pathways,the bitter flavor group was enriched with 3806 GO and 128 signal pathways,the pungent flavor group was enriched with 3261 GO and 109 signal pathways,and the sweet flavor drug was enriched with 3116 GO and 129 signal pathways.②DAI score in model group was significantly higher than in control group.Intestinal wall thickening,hyperemia and adhesion were observed in the colon tissue of rats.H&E,ABPAS and Masson staining results showed that part of colon epithelial cells were lost,crypt structure changed,mucus decreased,fibroblast increased and inflammatory infiltration in the colon tissue of model group.Compared with the control group,the expression of occludin-1 protein in colon tissue was decreased,and the expression of VEGFA,VEGFR2,Src and eNOS proteins were increased.Among the four coagulation indexes,PT,APTT and TT time showed shortening tendency,FIB showed a trend of increasing,erythrocyte sedimentation rate was accelerated and hematocrit decreased.24h urine volume decreased,d-xylose excretion decreased.The levels of IL-6,IL-8,TNF-α,CXCR-1 and MMP9 in model group increased,while the serum IL-10 level decreased.The levels of ROS,MDA and LPO in model group increased,while the levels of SOD,PRDX2 and SIRT3 decreased.The levels of VIP,SP,ghrelin,5-HT and NPY in colon tissue were significantly increased,while the level of CGRY was significantly decreased.PKD2L1,T1R3 and TRPV6 mRNA increased in model group.DAI scores of each administration group were lower than those of model group.Hyperemia and adhesion of part colon were observed in administration groups.The destruction of colonic epithelial structure was reduced,crypts were arranged closely,a large amount of mucus was retained,morphology changed,and capillary fibroblasts were reduced.IL-6,TNF-α,IL-8,CXCR-1 and MMP9 were all decreased,while IL-10 was increased in each administration group.ROS,MDA,LPO decreased,SOD,SIRT3 increased.Compared with model group,the levels of VEGFA,VEGFR2 and eNOS were decreased,ESR decreased,hematocrit increased,d-xylose excretion increased,VIP,SP,ghrelin,5-HT,NPY decreased,PRDX2,CGRY increased in the whole recipe group.In the whole group,T2R1 mRNA expression were increased and TRPV6 mRNA in colon tissues were decreased.Compared with model group,levels of eNOS,VIP,SP,ghrelin,5-HT,NPY were decreased,PRDX2 were increased in sour flavor taste group.Besides,PKD2L1 mRNA expression increased,TRPV6 mRNA expression decreased in this group.In bitter group,VEGFR2,Src,eNOS decreased,TT time prolonged,hematocrit increased,PRDX2 level increased,the levels of VIP,SP,ghrelin,5-HT,NPY in colon tissue decreased,and CGRY increased.Besides,colonic levels of T2R1 mRNA,T1R3 mRNA and TRPV6 mRNA were increased in bitter group.In pungent flavor taste group,VEGFR2 level decreased,hematocrit increased,PRDX2 increased,colonic levels of VIP,ghrelin,5-HT decreased,SP,CGRY increased,the expression of PKD2L1,T1R3,TRPV6 mRNA decreased,T2R1 mRNA increased.In sweet flavor taste group,colonic levels of VEGFR2,Src decreased,24h urine volume increased,PT time prolonged.VIP,SP,ghrelin,NPY in colon tissue were decreased,and CGRY increased.The taste receptors PKD2L1,T1R3 mRNA expression were decreased.In SASP group,colonic levels of VEGFR2,eNOS decreased,PRDX2 increased,VIP,SP,ghrelin,5-HT,NPY decreased and CGPY increased.Colonic level of PKD2L1 mRNA decreased in SASP group.Conclusion The four flavors in FMP own their unique biological modules and signaling pathways.FMP and its decomposed recipes can alleviate the damage of ulcerative colitis model through different aspects.Sour flavor excels in improving defecation,bitter and pungent flavor are skilled in regulating ESR,inflammatory factors,sweet flavor is adept at recover body weight.There are differences in neuropeptide regulation among the four groups.In addition,FMP and four taste groups take effect on different taste receptors.