Study On The Anti-inflammatory And Anti-ulcerative Colitis Effects And Mechanisms Of The Intestinal Oxidative Metabolite Of Berberine

Objective:Berberine(BBR),an isoquinoline alkaloid isolated from important traditional Chinese medicinal herbs like Coptidis chinensis Franch.,Phellodendron chinense Schneid.,which are commonly used to treat colitis and dysentery.BBR has a wide range of applications,especially in the treatment of gastroenteritis,diarrhea,dysentery and other intestinal infectious diseases.Studies have found that BBR has many bioactivities including anti-inflamrnatory,anti-colitis,anti-bacterial effect,anti-tumor effects,etc.Therefore,in recent years,it has been widely concerned as a "panacea" with great potential for application and pushed to the forefront of the monomer development of traditional Chinese medicine.However,its pharmacological mechanism is still not clear,and there is enigma since its absolute bioavailability is less than 1%.Studies have reported that metabolites,especially its intestinal microflora-mediated metabolites are important material basis for the pharmacological activity of BBR.It has been found that after oral administration of BBR,intestinal bacteria can convert BBR into metabolites like dihydroberberine,berberubine,demethyleneberberine,Jatrorrhizine by reduction,methylation,demethylation,dehydroxylation and other reactions.However,the pharmacological activities of these metabolltes are not significant as compared with those of BBR,which is difficult to explain the broad and significant pharmacological activities of BBR.Hence,other metabolic pathways may produce more active metabolites.Nowadays,the oxidative metabolites and pharmacological activities have not been reported.In the present study,we found for the first time that the intestinal microflora could transform BBR into oxyberberberine(OBB)by oxidation reaction.It has been found that OBB has superior activities to BBR,including anti-inflammatory,anti-fungal,anti-tumor and anti-arrhythmic effects.Based on the anti-Inflammatory and anti-ulcerative colitis activity of BBR,which is widely used in the prevention and treatment of intestinal infectious diseases such as gastroenter-tis and dysentery,this study aimed to investigate the anti-inflammatory and anti-ulcerative colitis effects and mechanisms of BBR intestinal oxidative metabolite-OBB.This study is envisaged to provide scientific basis for the traditional application of Coptidis chinensis and BBR in the treatment of abdominal pain,diarrhea,dysentery and so on.Furthermore,it may provide support for the research and dex,elopment of potential novel anti-UC lead compound.Methods 1.The biotransformation of BBR by intestinal microflora Firstly,the fresh feces from normal SD rats and KM mice,and six single intestinal strains,were incubated with BBR under 37? anaerobic conditions for 24 h respectively.After incubation,the main metabolites were separated by column chromatography.The chemical structure and content of metabolites of BBR were identified and analyzed by LC-MS-MS and NMR methods.Secondly,the metabolites of BBR were detected and Identified in the feces of normal SD rats and KM mice,respectively,within 24 hours after oral administration.Finally,pseudo-germfree SD rat or KM mice model was established by oral antibiotics following established regime.The metabolic transformation of BBR in vivo was studied in pseudo-germfree mice to verify the effect of intestinal microflora on its metabolite content.2.Anti-inflammatory effect and mechanism of OBB in vitro and in vivoThe potential anti-inflammatory properties of BBR and its gut microbiota metabolites,OBB and dihydroberberine(DHBB)were comparatively evaluated in vitro by lipopolysaccharide(LPS)-induced RAW264.7 macrophages cells,and in vivo via three typical acute inflammation murine models,xylene-induced mice ear edema,acetic acid-induced vascular permeability and carrageenan-induced paw edema in mice.The levels and expression of some inflammation-related molecules such as TNF-?,IL-6,IL-1?,and so on,were analyzed by Elisa,qRT-PCR and Western blotting.3.Anti-ulcerative colitis effect and mechanism of OBB on dextran sulfate sodium-induced ulcerative colitis Ulcerative colitis(UC)model was induced in SPF male BALB/c mice by giving 3%DSS drinking water for 8 days.At the same time,the mice in each group were given corresponding drugs:OBB(12.5,25,50 mg/kg),BBR(50 ng/kg),AZA(13 mg/kg),and the corresponding solvents(2%Tween-80 aqueous solution).The effect of OBB on general symptoms(body weight change,rectal bleeding,stool consistency,disease activity index(DAI)scores and colon length)and colon histopathological changes of colon were observed to evaluate the therapeutic effeect of OBB on UC induced by DSS in mice.Then,several techniques and methods have been applied to investigate the possible protective mechanism of OBB against DSS-induced UC. Firstly,the content of MPO was measured by assay kit.Secondly,the colonic levels of inflammatory factors(TNF-?,IL-1?,IL-6,IL-17,IFN-? and IL-10)and serum immunoglobulin(IgA,IgG and IgM)were detected by Elisa.Thirdly,the mRNA expressions of mucin-1 and mucin-2 in colon were detected by qRT-PCR.Fourthly,Western blot was used to detect the expressions of TLR4-MyD88-NF-KB signaling pathway protein(TLR4,MyD88,NF-?B p65,p-IKB? and I?B?)and intestinal barrier protein(ZO-1,ZO-2,JAM-A,Claudin-1 and Occludin)in intestinal tissue.Lastly,the total feces DNA of samples was extracted and the 16s rDNA high-pass sequencing analysis was used for sequencing.The regulation of OBB on intestinal microflora diversity and population structure in UC mice induced by DSS was investigated by OTU Wenn diagram,species abundance analysis and cluster analysis,respectively.Results 1.The metabolism of BBR by intestinal microflora LC-MS-MS and NMR showed that BBR could be converted to OBB by gut microflora in normal and pseudo-germfree mice or rat.The conversion rates of OBB by intestinal microflora in normal SD rats and KM mice were 12.42%and 17.03%.respectively.After oral administration of berberine,the contents of OBB were 1.49%and 1.75%in fresh fecal of normal SD rats,normal KM mice within 24 hours,respectively.And the content of OBB was significantly decreased in pseudo-germfree mice,which was 0.05%and 0.21%in pseudo-germfree SD rats and KM mice within 24 hours,respectively.Mover,6 kinds of single intestinal bacteria(beneficial bacteria:Bifidobacterium loogum,Lactobacillus acidophilus;intermediate bacteria:Streptococcus Faecalis,Escherichia coli;harmful bacteria:Pseudomonas aeruginosa,Staphlococcus aureus)all could convert BBR to OBB,among which Streptococcus Faecalis from intermediate bacteria provided the most productivity.2.Anti-inflammatory effect and mechanism of OBB in vitro and in vivo The cell viability of BBR,OBB and DHBB on RAW264.7 macrophages were evaluated by MTT assay which indicated that the safety margin was OBB>BBR>DHBB.The in vitro assay indicated that BBR,OBB and DHBB(1.25,2.5 and 5 ?M)pretreatment significantly decreased the levels of pro-inflammatory cytokines TNF-?,IL-1? and IL-6 in a dose-dependent manner.Besides,real-time PCR also revealed that BBR,OBB and DHBB could significantly inhibit the mRNA expressions of COX-2 and iNOS and thus,decreased the levels of PGE2 and NO in a dose-dependent manner in LPS-stimulated RAW264.7 macrophages,with relative efficiency of OBB>BBR>DHBB.Furthermore,OBB,BBR and DHBB remarkably inhibited the phosphorylation of NF-?B p65 and I?B?. In vivo,acute toxicity study revealed that the LD50 value of OBB in mice was estimated to be above 5243.6 mg/kg,which was far greater than that of DHBB(503.80 mg/kg),indicating OBB presumably possessed more favorable safety profile as compared with DHBB.Besides,OBB(5,10,and 20 mg/kg),BBR(20 mg/kg)and DHBB(20 mg/kg)pretreatment significantly ameliorated the xylene-induced ear edema,carrageenan-stimulated paw edema,and acetic acid-elicited vascular permeability in mice in a dose-dependent manner with relative efficiency of OBB>BBR>DHBB.Histopathological analysis indicated that OBB,BBR and DHBB could markedly attenuate the inflammatory deterioration and decrease the cellular infiltration in paw tissues.Further mechanism study indicated OBB,BBR and DHBB significantly decreased the protein level and mRNA expressions of TNF-a,IL-1? and IL-6,but increased IL-10 in carrageenan-induced paw tissue.Additionally,the COX-2 and INOS mRNA expressions were effectually and concentration-dependently suppressed by OBB,BBR and DHBB pretreatment,thereby reducing the contents of PGE2 and NO. 3.Anti-ulcerative colitis effect and mechanism of OBB on dextran sulfate sodium-induced ulcerative colitis In the model of ulcerative colitis induced by DSS in mice,different doses of OBB(12.5,25,50 ng/kg)could slgnificantly improve the body weight loss,rectal bleeding,stool consistency,colon length,colon tissue epitlielial barrier damage,inflammatory infiltration,and reduce DAI index.And the anti-colitis effect of OBB-M(25 mg/kg)was similar to AZA(13 mg/kg)and superior to BBR(50 mg/kg). The results of protective mechanism study showed that OBB pretreatment significantly up-regulated the mRNA expression of mucin-1 and mucin-2 and increased the protein expression of ZO-1,ZO-2,JAM-A,Claudin-1 and Occludin to protect intestinal mucosal barrier functlon.Meanwhile,OBB significantly down-regulated inflammatory cytokines(TNF-a,IL-1?,IL-6,IL-17,IFN-? and IL-10),and reduced the contents of serum immunoglobulins IgA,IgG and IgM in DSS-induced colitis mice.Moreover,OBB treatment down-regulated the protein expression of TLR4 and MyD88,inhibited the phosphorylation of I?B?,and the migration of NF-?B p65 from cytoplasm to nucleus,thereby inhibiting the TLR4-MyD88-NF-?B signaling pathway and regulating inflammation and immune function to reduce intestinal mucosal inflammation and colonic mucosal injury. Besides,gut microbiota analysis of OBB on DSS-induced mice colitis showed that OBB significantly increased the relative abundance of Bacteroidetes,Bacteroides and Prevotella,but inhibited the relative abundance of Firmicutes,Clostridium,Coprobacillus,Parabacteroides,Paraprevotella,Ruminococcus and Staphylococcus in colitis mice induced by DSS.Principal component analysis(PCA),principal coordinate analysis(PCoA)and cluster analysis also showed that OBB could significantly reverse the changes of intestlnal microflora composition in UC mice Induced by DSS.The results indicated that OBB could significantly regulate the dysbacteriosis induced by DSS and restore the dysbacterium to normal level.Conclusion 1.BBR can be transformed Into its oxidized derivative OBB through in vivo and in vivo intestinal microflora metabolic experiments.Its conversion rate may be related to different intestinal microflora structure,strains,etc. 2.By comparing the anti-inflammatory effects of BBR and its oxidized and reduced derivatives from the metabolites of BBR by intestinal microflora,it was found that they have obvious anti-inflammatory effects in vitro and in vivo.And the anti-inflammatory activity of BBR and its natural derivatives was in the order of OBB>BBR>DHBB.Further mechanism study indicated that they were probably associated with the inhibition of NF-?B signaling pathway,and subsequent down-regulation the productions of pro-inflammatory mediators,and up-regulation anti-inflammatory factors. 3.It was found that OBB had a significant protective effect against ulcerative colitis,and its efficacy was similar to AZA,and superior to BBR.The potential mechanism was related to its role in protecting intestinal mucosal barrier function,inhibiting inflammation and immunoglobulin levels,and regulating intestinal microflora structure.In conclusion,the interaction between intestinal microorganisms and drugs can change the structure and efficacy of drugs.OBB,a metabolite of intestinal microflora of BBR,may be one of the main pharmacodynamic active substances of Coptis chinensis and its main component berberine in the treatment of intestinal inflammatory diseases.This study may serve to furthermore illuminate the relationship between intestinal microflora and drug action mechanism,clarify the pharmacodynanic active substances of BBR,and provide support for the development of OBB into a safe and effective natural lead compound for the treatment of UC.In addition,this study reveals that BBR has the properties of natural precursor effectors,and studies on its intestinal mechanism may help to provide a new perspective and example for elucidating the action mechanism of active components originated from traditional Chinese medicine with low bioavailability but significant efficacy.