Study On Alleviating Effects Of Purslane On Ulcerative Colitis Induced By DSS And Its Mechanism

Ulcerative colitis（UC）,a type of inflammatory bowel disease（IBD）,occurs mostly in the sigmoid colon and rectum,and in severe cases can extend to the entire colorectal region.With the change of people’s living environment,diet structure and other factors,the number of UC patients is increasing rapidly in the world.At present,there are still defects in the clinical treatment drugs for UC（drug side effects and limited treatment effects,etc.）,which makes some patients have to accept surgical treatment finally.Therefore,it is of great significance for the development of natural and safe drugs to treat UC.Purslane（Portulaca oleracea L.）is an annual herb in the order Caryophyllaceae and the family Portulaca,which has been used as a food or medicine for thousands of years in China.It has many pharmacological effects such as anti-inflammatory,antioxidant,hypoglycemic,lipid-lowering,and dysentery relieving.However,there are few reports on the effects of aqueous extracts of purslane on UC,and the composition of effective components in aqueous extracts and their mechanisms of alleviating UC have not been clearly analyzed.In this study,the membrane separation system was first used to obtain purslane aqueous extracts with different molecular weight ranges,and chemical reagent colorimetry and liquid chromatography mass spectrometry（LC/MS）were used to analyze the differences in substance composition.Combined with dextran sulfate sodium（DSS）induced ulcerative colitis mouse model,the alleviating effects and differences in action of purslane aqueous extracts with different molecular weight ranges were explored.Based on the experimental results of the aqueous extracts,it was speculated that polysaccharide had strong advantages and potential in the treatment of UC.Therefore,Portulaca oleracea L.polysaccharides（POP）was isolated from the aqueous extract of purslane.The polysaccharide was characterized by gel permeation chromatography,high performance liquid chromatography,scanning electron microscopy,fourier transform infrared spectroscopy and antioxidant capacity in vitro.The alleviating effects of POP on UC was explored using UC mouse model.Finally,combining transcriptome and microbiome analysis,the mechanism of POP alleviating UC was further explored.The main findings are as follows:1.Study on the composition characteristics of different components of aqueous extract of purslane.According to the molecular weight,four components were separated using a membrane separation system,namely the Portulaca oleracea L.extract macromolecule（POEM,molecular weight>10 k Da）,the 3–10 k Da component,the 1–3 k Da component,and the Portulaca oleracea L.extract small molecule（POES,molecular weight<1 k Da）.Through component analysis,it was found that POEM contains a higher proportion of polysaccharides and proteins,while POES contains more kinds of organic acids and alkaloids.By comparison,it was found that the proportion of substances in the other two components was less than 1%,and the types of substances detected by LC/MS were mostly covered by POES and POEM.In vitro antioxidant experiments,POEM and POES also showed more prominent effects.By using scanning electron microscopy to observe the microstructure of POEM and POES,it was found that the surface of POEM is more compact,while POES contains more pores.2.Study on the improving effects of POEM and POES on UC mice.The animal experiments were divided into 5 groups,which were control group（CON）,model group（DSS）,Portulaca oleracea L.extract macromolecule group（POEM,400 mg/kg/day）,Portulaca oleracea L.extract small molecule group（POES,400 mg/kg/day）and positive drug group（SASP,200 mg/kg/day）.Except CON group,the other groups added 4%DSS to drinking water to build UC model,to study the relieving effects of aqueous extracts on UC.The results showed that both POEM and POES could reduce the colon shortening and weight loss caused by DSS;increased the activities of antioxidant enzymes(superoxide dismutase（SOD）,catalase（CAT）and glutathione peroxidase（GSH-px）in vivo;decreased the content of tumor necrosis factor-α（TNF-α）and interleukins（IL-6）;increased the content of IL-10;maintained intestinal integrity,reduced mucin loss and restored tight junction protein expression;regulated gut microbiota and increased the content of short-chain fatty acids（SCFAs）in colon contents.However,it was found that POEM and POES alleviate UC in different ways.In addition to anti-inflammatory and antioxidant capacity regulation,POEM had a closer interaction with gut microbiota and relieved UC by reshaping the gut microbiota.POES mainly acted on anti-inflammatory and antioxidant abilities.3.Extraction and characterization of Portulaca oleracea L.polysaccharide.Due to the increase of SCFAs after POEM intervention and the analysis of microbiome results,and combined with the composition characteristics of POEM,the study focused on purslane polysaccharides.After using hot water extraction,ethanol precipitation,and Sevag method to remove protein,a polysaccharide with a molecular weight of 2.65×10⁴ Da was extracted from the aqueous extract of purslane separated by membrane system,mainly containing galactose,arabinose,galacturonic acid,and glucose.By fourier transform infrared spectroscopy,proving that the POP contained 3279 cm^-1,2936 cm^-1,1738 cm^-1,and 1607 cm^-1 of specific absorption peak.In addition,through the analysis of scanning electron microscopy results,it was found that the microstructure surface of POP was relatively complete and smooth.The results of antioxidant experiments showed that each mg POP was equivalent to 20.92±1.92μg VE according to the IC₅₀ calculated by 2,2′-Azino-bis（3-ethylbenzothiazoline-6-sulfonic acid）,diammonium salt（ABTS）scavenging ability experiment.Ferric reducing antioxidant power（FRAP）results showed that each mg POP was equivalent to 5.15±0.89μg VE.4.Study on the ability and mechanism of POP to alleviate UC.The animal experiments were divided into 5 groups,which were normal control group（CON）,model group（DSS）,POP low-dose group（POPL,400 mg/kg/day）and POP high-dose group（POPH,800 mg/kg/day）and positive drug group（SASP,200 mg/kg/day）.Except CON group,the other groups added 4%DSS to drinking water.The results showed that POP could also reduce the typical pathological symptoms of UC models such as colon shortening and weight lossing caused by DSS;regulated oxidative stress in vivo,increased the activities of antioxidant enzymes（SOD,CAT and GSH-px）;reduced the pro-inflammatory factors TNF-αand IL-6,increased the anti-inflammatory factor IL-10;maintained intestinal integrity,reduced the loss of mucin and restored the expression of tight junction protein.POP could regulate gut microbiota,especially with high dose intervention,its phylum level clustering is closer to that of CON group.POPH intervention significantly enriched intestinal beneficial bacteria such as:Clostridia＿UCG＿014,Paraprevotella,Lachnospiraceae＿NK4A136＿group,Prevotellaceae＿UCG＿001,Rikenellaceae＿RC9＿gut＿group,Oscillibacter and Romboutsia.In addition,POP increased the content of short-chain fatty acids in colon contents.The transcriptome was used to analyze the transcription status of colon samples of mice.The results showed that POP down-regulated the transcription related to retinol metabolism and increased the transcription of B cell proliferation and differentiation.The content of retinol in feces was detected by high performance liquid chromatography,and it was found that POP could increase the content of retinol.The results of reverse transcription quantitative real-time polymerase chain reaction（RT-q PCR）and immunofluorescence also confirmed that POP affected the maturation and differentiation of B cells,such as increasing the m RNA and protein expression of CD19,and increasing m RNA expression of Ms4a1,Cr2,Cd22,Fcer2a,and Cd37（period-specific surface markers of B cells）.Meanwhile,the intervention of POP reduced the expression of inflammatory plasma cell marker CD138 and increased the expression of immunoglobulin A（Ig A）.The results showed that POP regulated host intestinal homeostasis to alleviate UC.In summary,in this study,purslane was taken as the research object,and different components were obtained from purslane aqueous extract.Based on the characteristics of substance composition,the differences in their ability to alleviate UC were analyzed.In this study,a purslane polysaccharide was also extracted from purslane,confirming the improvement effects of POP on UC.Analysis of the results of intestinal microbiome and transcriptomics confirmed the effects of POP on the proliferation and differentiation of B cells and its ability to reshape intestinal homeostasis.This study supplements the theoretical basis for the development of purslane as a nutritional/functional food,providing ideas for the development of effective ingredients and functional exploration of more similar medicinal and edible plants in the future.