| As a representative formula of the middle energizer damp-heat symptom,Gegen Qinlian Decoction(GQD)had the effect of releasing the exterior and clearing heat,which was mainly used in the treatment of interior heat.It was the essential medicine for the treatment of intestinal heat syndrome caused by the invagination of the Tai-yang.Recently,it has been clinically proven to be effective in treating inflammatory bowel disease(IBD).The preliminary study of our research group found that GQD had a good therapeutic effect on IBD.Besides,we found that the TNBS/ethanol enema method could replicate the IBD model.Therefore,this article applied TNBS/ethanol enema to replicate the IBD model.After the model was created,GQD was given by gavage.UPLC-QTOF-MS technology was used to study the metabolome of GQD on the intervention of IBD in order to determine the endogenous biomarkers and related endogenous metabolic pathways of the intervention on IBD.At the same time,the 16S rRNA sequencing method was used to study the effect of GQD on the gut microbiota of IBD rats,so that determined the iconic bacteria and related pathways that exerted its effects.Gas chromatography(GC)was used to study the effect of GQD on gut microbiota metabolites short chain fatty acids(SCFAs)in feces of IBD rats.The main research methods and results were as follows:(1)The TNBS/ethanol enema method was used to make the IBD rat model.After gavage of GQD,the general state and Disease activity index score of the rats were used to evaluate the model and the effect after administration.The results showed that the IBD rat model was successfully made,and GQD had a good therapeutic effect on IBD.UPLC-QTOF-MS technology was used to analyze themetabolome of the intervention of GQD in rats with IBD.By comparing the metabolic profile between model group and control group,81 differential metabolites related to IBD were screened out,including amino acids,sphingosines,sterols,etc.,and 8 related metabolic pathways were enriched.After that,the metabolic profile of the intervention of GQD on IBD was analyzed,and 27 differential metabolites(such as arachidonic acid,sphingosine,etc.)with a callback trend were screened,which might interfere with IBD,and 2 related metabolic pathways including sphingolipid metabolism,and porphyrin and chlorophyll metabolism were enriched.8 medicinal components of GQD’s intervention on IBD,which have been screened in the previous stage and the two proteins,including sphingosine kinase-1(Sphk1)and sphingosine-1-phosphate receptor-1(S1PR1),related to the sphingolipid metabolism pathway,were applied by molecular docking.The results showed that the 7 effective components of GQD had strong binding ability with Sphk1 and S1PR1,and the binding energy was less than 0,that was,they could be combined spontaneously.Furthermore,Western Blot was used to detect the expression of Sphk1 and S1PR1 protein in the colon tissue of IBD model before and after the administration of GQD.The results showed that compared with the control group,the protein expression of Sphk1 and S1PR1 in IBD model could be significantly increased(p<0.01).After the administration of GQD,the protein expression of Sphk1 and S1PR1 in IBD model was decreased,and the trend of S1PR1 was more obvious(p<0.05).(2)16S rRNA sequencing was applied to study the effect of GQD on the gut microbiota of IBD model.The results showed that GQD could improve the imbalance of gut microbiota in the stool of rats with IBD,which could reduce the abundance of harmful bacteria such as Proteobacteria and increase the abundance of dominant bacteria such as Firmicutes and Clostridium,and it could also increase the abundance of SCFAs-producing bacteria including Dorea,Anaerostipes and Lachnospiraceae.The correlation analysis between the 6 different gut microbiota at the genus level and the 27 different metabolites of fecal metabolome showed that Anaerostipes had a extremely strong positive correlation with 3-Hydroxydodecanedioic acid,Mesoporphyrin Ⅸ,Ricinoleic acid and Cholic acid.And it also had a extremely strong negative correlation with Cer(d18:0/18:0),Cer(d18:0/20:0)and Protoporphyrin Ⅸ.Allobaculum had a extremely strong positive correlation with 25-Hydroxytachysterol 3,and it also had a extremely strong negative correlation with 4-(2-Aminophenyl)-2,4-dioxobutanoic acid.Anaerostipes had a strong correlation with 10 differential metabolites such as Indole-3carbinol,and it also had a negative correlation with Tetracosapentaenoic acid(24:5n-3),Sphinganine,N-Palmitoylsphingosine and Heptadecanoic acid;Allobaculum had a positive correlation with 5 different metabolites such as Kynurenic acid,and it also had a negative correlation with 11 different metabolites such as 3-(3-Hydroxyphenyl)propanoic acid and 8(S)-HPETE;Dorea had positive correlation with 3-Hydroxydodecanedioic acid and Betatocopherol,and it also had negative correlation with Heptadecanoic acid;Sutterella had a positive correlation with 4-(2-Aminophenyl)-2,4-dioxobutanoic acid,Nabilone and 25Hydroxytachysterol 3;Clostridium and 9-HODE,Desulfovibrio and 25-Hydroxytachysterol 3 all had positive correlation.(3)GC was used to study the effect of GQD on the contents of histone deacetylase-1(HDAC1,SCFAs were the inhabitors of HDAC1),western blot was used to detect the expression of HDAC1.The results showed that compared with the control group,the contents of acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid and valeric acid in the feces of the model group were decreased,and the content of acetic acid,propionic acid and butyric acid decreased significantly(p<0.05).Compared with the model group,the contents of acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid and valeric acid increased significantly after gavage of GQD(p<0.05),and they were significantly higher than those in the feces of rats in the control group,and the contents of acetic acid,propionic acid,isobutyric acid and isovaleric acid increased significantly(p<0.01).The results of western blot showed that compared with the control group,the expression of HD AC1 was significantly upregulated in the IBD model(p<0.01).After the administration of GQD,the expression of HDAC1 was significantly reduced compared with the model group(p<0.01).(4)The rats were treated with broad-spectrum antibiotics(Antibiotic cocktail,ABX),to make germ-free rats,and the content of SCFAs in feces was determined by GC.Moreover,Western Blot was used to detect the expression of HD AC1.The results showed that compared with the GQD group,the content of acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid and valeric acid in the feces of rats in the G-ABX group was reduced significantly(p<0.05),and the changes of acetic acid,propionic acid,isobutyric acid,butyric acid and isovaleric acid were extremely significant different(p<0.01).Compared with rats in the G-ABX group,the content of above 6 SCFAs in the feces of the complementary SCFAs group were increased,and the content of acetic acid,butyric acid and valeric acid changed significantly(p<0.05).The results of western blot showed that compared with the GQD group,the expression of HDAC1 in the colon of rats treated with antibiotics was significantly upregulated(p<0.05),and after the complementary of SCFAs,the expression of HDAC1 was reduced compared with the antibiotic group. |
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