| ObjectiveUlcerative colitis is a lifelong intestinal inflammatory disease that is difficult to treat,its clinical symptoms are persistent and recurrent abdominal pain,diarrhea,and even mucous purulent stool,severe cases accompanied by fever,weight loss,constipation and the development of colorectal cancer,and some patients may have parenteral diseases,such as primary sclerosing cholangitis,gangrenous pyoderma and so on.In recent years,the incidence and prevalence of ulcerative colitis continue to rise.However,the current drugs and treatments can only limited relieve symptoms and are difficult to cure,because the complexity and diversity of its pathogenic factors and pathogenesis.It was found that the expression levels of peroxisome proliferation-activated receptor γ（PPAR γ）gene and protein in colon of patients with UC were significantly decreased.PPARγ deficiency in colonic epithelium can lead to a significant increase in the expression of nitric oxide synthase 2（Nos2）.After that,the increase of inducible nitric oxide synthase encoded by Nos2 led to the increase of NO production in colon.NO was easily converted to NO2 and NO3,resulting in a significant increase in nitrate synthesis in colonic mucosa.Nitrate as an energy source of Enterobacteriaceae made it proliferate rapidly and restricts the growth of probiotics,resulting in intestinal flora disorder.In addition,PPAR γ ligand inhibited the activation of NF-κB signal in many ways,that is,inhibited the production of inflammation.Therefore,PPAR γ ligand can inhibit the synthesis of intestinal iNOS,reduce the production of intestinal NO,and then inhibit the production of intestinal nitrate,thus limit the growth of pathogenic Enterobacteriaceae bacteria,regulate the balance of intestinal flora,and inhibit the activation of NF-κB signal and the production of inflammation.Emodin is the main chemical component of traditional Chinese medicine rhubarb and Polygonum cuspidatum,which has many pharmacological activities,such as antiinflammation,anti-fibrosis,anti-tumor,protection of gastrointestinal mucosa and so on.It was used in the treatment of heat constipation,gastrointestinal failure,digestive system cancer and so on.In addition,emodin was well absorbed in different intestinal segments and had a protective effect on intestinal injury caused by many factors.It was found that emodin was the ligand of PPARγ,and the previous study of our group found that emodin significantly alleviated ulcerative colitis induced by sodium dextran sulfate（DSS）,but its mechanism was still unknown.Therefore,in this study,the mouse model induced by DSS and the Caco-2 cell model induced by IFNγ and IL-22 were used to explore the effects of emodin on intestinal flora,PPAR γ signal pathway and inflammation,in order to clarify its mechanism.Methods1.Establishment of a mouse model of ulcerative colitis and its administration regimenThe random number table was generated by SPSS software,and the male C57BL/6 mice were randomly divided into control group（Control）,model group（Model）,emodin group（5mg/kg）,emodin group（10mg/kg）,emodin group（20mg/kg）,emodin+GW992 group,mesalazine group（Mesalazine）and rosiglitazone group（Rosigltazone）.The control group was given distilled water,and the other groups were given 3%DSS by free drinking,and then changed to distilled water for 4 days to induce UC model.At the same time,the mice in the normal group and model group were given distilled water by intragastric administration,and the other groups were given corresponding compounds or drugs by intragastric administration for 10 days.2.Therapeutic effect of emodin on ulcerative colitis in miceThe activity status,body weight,diarrhea and hematochezia of mice were recorded every day,and the disease activity index was calculated according to the corresponding scoring criteria.In the meantime,the therapeutic effect of emodin on UC mice was investigated by colonic morphology,colonic length,colonic tissue Haematoxylin-Eosin staining and pathological score,thymus and spleen index,number and proportion of peripheral blood cells and so on.3.Regulatory effect of emodin on PPARγ signal pathwayThis part of the study included detected the expression of PPARγ,iNOS and NF-kB p65 protein in mouse colon with Western Blot.The expression levels of Angptl4 and NOS2mRNA in colonic tissue of mice were detected by RT-PCR.The neutrophil infiltration in mouse colon was detected by immunohistochemistry.Detection of the levels of cytokines IFNγ,IL-22,TNF-α,CXCL8 and TGF-in serum of mice by ELISA.Intestinal nitrate was detected with Griess reagent.MTT was used to detect the effect of emodin on the Caco-2 cell viability in order to screen the appropriate concentration of emodin.The expression of PPARγ and iNOS protein in Caco-2 cells were measured by Western Blot.Detection of PPAR γand NOS2mRNA expression in Caco-2 cells by RT-PCR.The level of IL-8 in the supernatant of cells was detected by ELISA.Identification of PPAR γ protein and gene expression in Caco-2 transfected cells by Western Blot and RT-PCR.Molecular docking of emodin with PPAR γ by Autodock Vina software.4.Regulation of emodin on intestinal microbiotaDetection of the number and abundance of intestinal microflora in mice by 16s rDNA.The plate counting method was used to detect the number of Escherichia coli,Enterobacter cloacae subspecies and Proteusbacillus vulgaris.5.Data analysis and statisticsData was collected by Excel,and analyzed using SPSS 21 software.If the measurement data satisfied the normal distribution,it was described by Mean±standard deviation.If it does not satisfy the normal distribution,it was described by M（P25～P75）.The data satisfied both normal distribution and homogeneity of variance were compared by t-test and multiple groups by single factor analysis of variance.Grade data,described by median and rank sum test.The difference was statistically significant（P<0.05）.Drawing with GraphPad Prism 5 software.Results1.Therapeutic effect of emodin on ulcerative colitis in miceCompared with the control group,the body weight of mice in the model group decreased significantly after the fifth day,and emodin alleviated the body weight in a dose-dependent manner.However,Emodin 20mg/kg combined with GW9662（PPAR γ inhibitor）had no effect on weight loss.The disease activity index of the model group was significantly higher than that of the control group,and after administration of emodin 20mg/kg and rosiglitazone,the disease activity index decreased,and the difference was statistically significant.After administration of PPAR γ inhibitor,emodin had no significant effect on disease activity index in mice.Observing the morphology of the colon of mice,it was found that the colon of the model group was obviously shortened,and the phenomenon of hyperemia and edema appeared.Administration of mesalazine,rosiglitazone and emodin significantly improved the morphology and length of colon in UC mice,especially emodin 20mg/kg.The colon morphology and length of mice treated with emodin 20mg/kg and GW9662 were not ameliorated.The results showed that in the control group,showing that the structure of colon was intact,the intestinal glands were arranged neatly,and there was no hyperemia and edema.In the model group,there were obvious ulcers in the colon,loose and disordered arrangement of intestinal glands,infiltration of a large number of inflammatory cells,local hyperemia and edema.The structural integrity of colon in mice treated with mesalazine,rosiglitazone and emodin was significantly recovered,ulcer and inflammatory cell infiltration were reduced.The pathological state of colon in emodin combined with GW9662 group was similar to that in model group.2.Regulation of emodin on PPAR γ signal pathway in mice with ulcerative colitisCompared with control mice,the expression of PPAR γ protein in colonic tissue of UC mice was significantly decreased（P<0.05）,iNOS and NF-kB p65 protein expression was significantly increased（P<0.05,P<0.01）,and emodin significantly restored the expression of PPAR γ protein and inhibited the expression of iNOS and NF-kB p65 protein（P<0.05,P<0.01,P<0.001）.However,emodin 20mg/kg combined with GW9662 had no such effect.The expression of Angptl4 and NOS2 mRNA in colonic tissue of UC mice was detected,and the results showed that compared with control mice,the expression of Angptl4 mRNA in colonic tissue of UC mice decreased and the expression of NOS2 mRNA increased.After administration of emodin,mesalazine and rosiglitazone,the expression of Angptl4 mRNA increased and NOS2 mRNA decreased in colonic tissue of UC mice,but the difference was not statistically significant.Compared with emodin 20mg/kg,the expression of Angprl4 mRNA decreased and NOS2 mRNA increased in colonic tissue of mice treated with emodin 20mg/kg and GW9662.The neutrophil infiltration in the colon of mice was analyzed,and the results showed that the neutrophil infiltration in the colon of the model group induced by DSS was significantly higher than that in the normal group.Neutrophil infiltration in colon decreased after emodin administration in UC mice.However,compared with emodin 20mg/kg,emodin 20mg/kg combined with GW9662 had no significant effect on neutrophil infiltration in colon of UC mice.In addition,compared with control mice,the levels of cytokines IFN γ,IL-22,TNF-α and CXCL8 in serum of UC mice were significantly increased（P<0.001）,while the level of TGF-cytokines decreased significantly（P<0.001）.After UC mice were treated with emodin,the levels of IFN γ,IL-22,TNF-α and CXCL8 in serum decreased significantly（P<0.05,P<0.01,P<0.001）,and the level of TGF-receptor increased significantly（P<0.05,P<0.01,P<0.001）.There was significant difference between emodin 20mg/kg group and emodin 20mg/kg+GW9662 group in the effects of serum cytokines IFN γ and IL-22,but there was no significant difference in serum TNF-α,CXCL8 and TGF-levels in mice.That is,the effect of emodin on serum cytokines IFN γ and IL-22 in UC mice is related to PPARγ.Compared with the control,the intestinal mucosal nitrate level in the model group increased significantly（P<0.05）and decreased after emodin intervention（P<0.05）,and the difference of intestinal mucosal nitrate between emodin 20mg/kg and emodin 20mg/kg+GW9662 was statistically significant（P<0.05）.3.Emodin regulated PPAR γ signal pathway induced by IFN γ and IL-22 in Caco-2 cellsFirst of all,the effect of emodin on the activity of Caco-2 cells was investigated by MTT method.The results showed that emodin had no significant effect on the activity of Caco-2 cells when the concentration of emodin was 0.15,0.313,0.625,1.25,5,10 and 20μM.After comprehensive consideration,emodin 5,10 and 20 M would be used in the experiment.Then,the effects of emodin or Rhein on PPAR γ protein and gene expression in Caco-2 cells were investigated.The results showed that emodin 5,10,20μM directly increased the expression of PPAR γ protein and gene in Caco-2 cells,while Rhein could not increase the expression of PPAR γ gene in Caco-2 cells.That is to say,emodin played a direct role in the regulation of PPAR γ signal pathway,rather than metabolism into Rhein.Furthermore,Compared with the control,the expression of PPAR γ mRNA decreased and the expression of NOS2 mRNA increased in Caco-2 cells induced by IFN γ and IL-22（P<0.01）.The expression of PPAR γmRNA increased and the expression of NOS2 mRNA decreased after emodin 10 μM or 20μM,and the difference was statistically significant（P<0.01）.In addition,the secretion of IL8 in Caco-2 cells stimulated by IFN γ and IL-22 increased compared with the control group.Compared with the model group,emodin or rosiglitazone significantly decreased the secretion of IL-8 by Caco-2 cells,and the difference was statistically significant（P<0.05,P<0.001）.4.Regulation of emodin on PPAR γ signal pathway induced by IFN γ and IL-22 in shPPARγ-Caco-2 cellsFour plasmids with different PPARy shRNA sequences were designed,and four transfected cells were obtained.The optimal transfected cells were screened by RT-PCR and Weatern Blot methods.The results showed that the expression of PPAR γ protein in shPPARγ-Caco-2-1 and shPPAR γ-Caco-2-2 cells was significantly lower than that in control cell.Compared with control,the expression of PPAR γ mRNA in shPPAR γ-Caco-2-1,shPPAR γCaco-2-2 and shPPAR γ-Caco-2-3 cells decreased significantly（P<0.01,P<0.001）,and the expression of PPAR γ mRNA in shPPAR γ-Caco-2-2 cells was the lowest.Therefore,the cell line was used for the next experiment,and it was directly expressed by shPPAR γ-Caco-2 cells.Compared with the control,the expression of iNOS protein in Caco-2-NC,Caco-2-SC and shPPAR γ-Caco-2 cells stimulated by IFN γ and IL-22 was significantly increased（P<0.05,P<0.01,P<0.001）.Moreover,Compared with the model group,the expression of iNOS protein in Caco-2-NC cells and Caco-2-SC cells decreased significantly after emodin administration,especially emodin 10 μM and 20 μM.However,compared with the model group,emodin 10 μM and 20 μM had no inhibitory effect on the expression of iNOS protein in shPPAR γ-Caco-2 cells,which further confirmed that the target of emodin was PPAR γ.5.Molecular docking of emodin with PPAR γMolecular docking of emodin with PPAR γ by Autodock Vina software.The results showed that emodin bound to the active site of PPAR γ protein and formed hydrogen bond interaction with ARG 288 and CYS 285 amino acids.These interactions promoted the stable binding of peptides to protein active sites and exerted their biological activity.6.Regulation of emodin on intestinal microbiotaCompared with the control group,the number of OUT and the diversity of a in the model group decreased,and Emodin obviously regulated OUT and the diversity of α.The results ofβ diversity detection showed that the model group was completely separated from the other groups,while the control group,emodin group,mesalazine group and rosiglitazone group were crossed,indicating that there were great differences in diversity between the model group and the other groups.the samples of control group,emodin group,mesalazine group and rosiglitazone group were similar.As for phylum level,compared with the control group,the relative abundance of Firmicutes,Actinobacteria and Cyanobacteria decreased in the model group,while the relative abundance of Proteobacteria and Deferribacteres increased significantly.Compared with the model group,emodin increased the relative abundance of Cyanobacteria and decreased the abundance of Proteobacteria.At the class level,Compared with the control group,the relative abundance of Alphaproteobacteria,Bacilli,Betaproteobacteria,Deferribacteres,Epsilonproteobacteria,Gammaproteobacteria in the model group increased,while the relative abundance of Actinobacteria,Clostridia,4C0d2 decreased.Compared with the model group,emodin significantly reduced the number of Gammaproteobacteria and increased the number of 4C0d2.At the Order level,compared with the control group,the relative abundance of Burkholderiales,Campylobacterales,Deferribacterales,Enterobacteriales,Lactobacillales,Pseudomonadales,and RF32 bacteria in UC mice increased,while the relative abundance of Bifidobacteriales,Clostridiales,coriobacteriales,RF39 and YS2 decreased.Emodin decreased the abundance of Enterobacteriales and Pseudomonadales,and increased the abundance of YS2 in the intestinal tract of UC mice.At the family level,compared with the normal group,in the model group,the abundance of Alcaligenaceae,Bacteroidaceae,Clostridiaceae,Deferribacteraceae,Enterobacteriaceae,Mycoplasmataceae,Pasteurellaceae,Propionibacteriaceae,Pseudomonadac eae,Sphingomonadaceae,Streptococcaceae increased,while the abundance of Aerococcaceae,Bifidobacteriaceae,Chthoniobacteraceae,F16,Lachnospiraceae,Odoribacteraceae,and Rikenellaceae decreased.In addition,Compared with the model group,emodin reduced the abundance of Enterobacteriaceae,Enterococcaceae,Lactobacillaceae,Aerococcaceae,and increased the abundance of Peptococcaceae and Rikenellaceae.The plate counting method was used to investigate the effects of emodin on the growth of Escherichia coli,Enterobacter cloacae subspecies and Proteusbacillus vulgaris.The results showed that different doses of emodin had no obvious inhibitory effect on Escherichia coli,Enterobacter cloacae subspecies and Proteusbacillus vulgaris.Conclusion1.Emodin has obvious therapeutic effect on DSS-induced ulcerative colitis in mice.2.Emodin inhibits intestinal inflammation by activating PPAR γ signal,on the one hand,inhibiting the expression of NOS2 and iNOS,reducing the level of intestinal nitrate,and then selectively inhibiting the growth of Enterobacteriaceae,hindering the deterioration of ulcerative colitis.on the other hand,inhibiting the activation of NF-κB signal pathway to inhibit intestinal inflammation. |
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