| Huang-lian-Jie-Du decoction(HLJDD)is a representative prescription of clearing away heat and detoxification,which can be used to treat gastrointestinal diseases.Ulcerative colitis(UC)is a kind of inflammatory bowel disease,which is characterized by inflammation and ulcerative lesions in colorectal mucosa and submucosa layer.In order to explore the therapeutic effect and mechanism of HLJDD on acute UC,this study comprehensively evaluated the therapeutic effect of HLJDD on acute UC in mice,and explored some of its mechanisms at the molecular level.Furthermore,the antiulcer effective fraction of HLJDD was screened by pharmacodynamics experiment in vivo,and the effect of HLJDD and its effective fraction on intestinal flora of UC mice were investigated.On the basis of these,we combined metabolomics and molecular simulation docking technology to further explore the antiulcer mechanism of HLJDD and its effective fraction.Methods and results:(1)The acute UC model of BABL/c mice were established by feeding 3.5%(w/v)dextran sodium sulfate drinking water for seven consecutive days.At the same time,high(9.2 g/kg),medium(4.6 g/kg)and low(2.3 g/kg)doses of HLJDD were administered orally for treatment,and sulfasalazine(0.45 g/kg)was used as a positive control drug.During the experimental period,the clinical symptoms of each group of mice were evaluated by disease activity index(DAI)score.After the experiment,the plasma of mice in each group were collected for cytokines(IL-1?,TNF-a and IL-10)detection,and colon tissue was collected for histopathology and oxidative stress detection.Finally,the key proteins of NF-?B,Nrf2 signaling pathway and intestinal mucosa tight junction protein in the colon tissue of each group of mice were detected by WB and immunofluorescence.The results showed that HLJDD could significantly alleviate the weight loss and DAI increase of UC mice(p < 0.05);significantly inhibit the colon shortening and reduce the pathological damage of colon(p < 0.05);significantly reduce the plasma and colon MPO level(p < 0.05).Besides,HLJDD treatment could significantly increase the plasma IL-10 content in UC mice,reduce IL-1? and TNF-a content,and significantly inhibit colonic NF-?B p65,p-I?K?/? and p-I?B? protein expression(p < 0.05).Additionally,after HLJDD treatment,the content of NO and MDA in the colon of UC mice decreased significantly,while the content of GSH,SOD and Nrf2,Keap1 protein expression increased significantly(p < 0.05).Colonic mucosal barrier test showed that HLJDD treatment also increased the secretion of mucin protein in UC mice and significantly restored the expression of ZO-1 and Occludin-1 in colonic mucosa(p < 0.05).Among the above results,the HLJDD middle dose group has the best effect.(2)Soxhlet extractor combined with system solvent extraction method was used to extract the freeze-dried powder of HLJDD with petroleum ether,ethyl acetate,n-butanol and purified water in order to obtain different polar fractions of HLJDD.At the same time of establishing acute UC model in mice,HLJDD and its different polar fractions(4.6 g/kg,calculated by raw herbs)were administrated by gavage for 7 consecutive days to screen the effective anti-ulcer fractions of HLJDD.The cecum contents of mice after treatment of HLJDD and its effective fraction were collected.The V3-V4 hypervariable region gene of 16 S rRNA of intestinal flora were sequenced and analyzed.The results showed that the yield of HLJDD-n-butanol(HLJDD-NBA)fraction was the highest(yield: 64.45%).The results of in vivo pharmacodynamic experiments showed that HLJDD-NBA treatment could not only significantly reduce DAI and improve histopathological damage of colon in UC mice,but also had significant anti-inflammatory and antioxidant effects(p < 0.05),and there was no significant difference between these effects and HLJDD(p > 0.05).Furthermore,intestinal flora detection results showed that both HLDD and HLJDD-NBA treatments can significantly reduce the alpha diversity of the intestinal flora of UC mice,and both of them can inhibit the decrease of the relative abundance of beneficial flora(e.g.Lactobacillus,Parabacteroides,Prevotella,etc.)and the growth of pathogenic bacteria(e.g.Escherichia,Odoribacter,Alloprevotella,etc.).In addition,HLJDD and HLJDD-NBA can also significantly correct abnormal disorder of intestinal flora in UC mice.(3)Metabolomics techniques and multivariate statistical analysis were used to detect and analyze the plasma metabolites of UC mice after HLJDD and HLJDD-NBA treatment.Subsequently,the differential metabolites between the model group and the NC,HLJDD and HLJDD-NBA treatment groups were screened respectively(screening criteria: FC ? 1.2 or ? 0.833,p-value < 0.05 and VIP ? 1).Further,enrichment analysis of metabolic pathways was performed on the common metabolites between groups,and potential metabolic pathways were screened [selection criteria: Impact ? 0.1 and-log(p)> 2].Finally,the molecular regulatory mechanism of the main active components in HLJDD or HLJDD-NBA on the potential target metabolic pathway was investigated by using molecular simulation docking technology and literature review.The results showed that HLJDD and HLJDD-NBA could significantly improve the clinical symptoms and colonic pathological damage of UC mice,and significantly inhibit the anemia and inflammatory response in UC mice(p < 0.05).In addition,HLJDD and HLJDD-NBA can significantly improve the metabolic dysfunction of UC mice by reversing the abnormal changes of 24 different metabolites in UC mice,and arachidonic acid metabolic pathway and glycerophospholipid metabolic pathway are their target metabolic pathways.Further literature review and molecular simulation docking analysis showed that HLJDD could inhibit the expression of COX-2 protein,and 13 active components in HLJDD or HLJDD-NBA had the potential to inhibit the activities of PLA2 and 5-LOX,the key protease in the above target metabolic pathway.Conclusion:(1)HLJDD can effectively therapy acute UC in mice by inhibiting the NF-?B signaling pathway,activating the Nrf2 signaling pathway,and enhancing the intestinal mucosal barrier function;(2)HLJDD-NBA is the effective fraction of HLJDD in treating acute UC in mice;(3)HLJDD and HLJDD-NBA can maintain the intestinal microflora homeostasis of UC mice by inhibiting the disorder of intestinal microflora structure and restoring the normal function of intestinal microflora;(4)Arachidonic acid metabolic pathway and glycerophospholipid metabolic pathway are the target metabolic pathways of HLJDD and HLJDD-NBA in the treatment of acute UC in mice;(5)HLJDD and HLJDD-NBA may inhibit the disorder of arachidonic acid metabolic pathway and glycerophospholipid metabolic pathway by inhibiting COX-2 protein expression and inhibiting 5-LOX,PLA2 enzyme activity.In conclusion,this study confirmed the therapeutic effect and effective fraction of HLJDD on acute UC in mice,and also revealed part of its mechanism,which provided a scientific basis for expanding the application of HLJDD,and laid a foundation for the refining of traditional Chinese medicine prescription and development of new anti-ulcer drugs. |
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