| ObjectiveHuanglian Wendan Decoction is made of the classic recipe Wendan Decoction plus Coptis,which has the effect of heat-clearing,regulating qi,eliminating dampness and phlegm.There are clinical reports that it can treat gastritis and liver disease,and its addition and reduction formula can treat viral enteritis,but there is no research about colitis and colitis-associated colon cancer.Based on this,this paper intends to verify through experiments as follws:1.Whether Huanglian Wendan Decoction can anti-colitis;2.What are the main anti-colitis mechanisms of Huanglian Wendan Decoction and whether it depends on the regulation of flora?3.Whether Huanglian Wendan Decoction can anti-colitis-associated colon cancer,and what inflammation and immune cell group changes;4.The main target of Huanglian Wendan Decoction in regulating the development of colitis and colitis-associated colon cancer by proteomics.Methods1.Effect of Huanglian Wendan Decoction on colitis in mice(1)Efficacy evaluationAfter administration of HLWDD 5days,2.25%DSS was given for 7 days to construct an inflammatory enteritis model.Body weight and stool were observed,calculate DAI score,the length of colon was recorded.The contents of DAO in serum and MPO and NAG in colon tissue were determined by Elisa.The structure and pathological changes of colon tissue were observed by HE staining.(2)Mechanism researchThe contents of IL-1β,IL-10,TNFα,LPS and IL-6 in colon tissue were determined by Elisa.The intestinal barrier proteins ZO1,Occludin and Claudinl;inflammatory pathways TLR4/NF-κB and JAK2/STAT3;endoplasmic reticulum stress-related proteins PDI,(p)eIF2α,CHOP and apoptosis-related proteins,cleaved PARP,cleaved caspase3,Bcl2,and BAX in colon tissue were detected by Western blot.The changes of MUC2,NLRP3,CHOP.and cleaved caspase3 in colon tissue were detected by IHC.2.Effect of Huanglian Wendan Decoction on intestinal flora of colitis mice(1)Detection of intestinal flora and short-chain fatty acids in miceIntestinal flora richness of colitis mice was detected by 16S rDNA,and a and βdiversity and species difference were analyzed.Then,seven short-chain fatty acids in intestinal contents were detected by HPLC-MS.(2)Antibiotic bacterial clearing experimentAfter five days of complex antibiotics clearing intestinal flora,colitis model was constructed to evaluate whether Huanglian Wendan Decoction was dependent on intestinal flora in anti-colitis.Body weight and stool were observed,calculate DAI score,the length of colon was recorded.The contents of DAO,MPO,IL-1β and LPS in colon tissues were determined by Elisa.The proteins expression of ZOl,cleaved PARP,cleaved caspase3,PDI,eIF2a and CHOP were detected by Western blot.The structure and pathological changes of colon tissues were observed by HE staining,and the changes of MUC2,NLRP3,CHOP,and cleaved caspase3 in colon tissue were detected by IHC.3.Effect of Huanglian Wendan Decoction on colitis-related colon cancer in miceAOM(10mg/kg)was injected intraperitoneally for the first time,then recovered for 7 days.1.5%DSS were given for 7 days by free drinking and recovered for 14 days,this cycle was repeated 4 times.The weight changes of mice and DAI score were recorded.Starting from the end of the second cycle,mice in each group were dissected,colon length was measured,the number of tumors>2mm,1-2mm and<1mm in diameter were recorded,and organ index and tumor load was calculated.The pathological changes of colon tumor tissue,paracancerous and spleen were observed by HE.The contents of LPS,IL-23,IL-8,IL-17 and COX2,and the contents of CCL20,MCP1,G-CSF,M-CSF and GM-CSF in colon tissue was detected by Elisa.The changes of cytotoxic T cells,myeloid-derived suppressor cells,Treg cells and macrophage cells were detected by flow cytometry.4.The effect of Huanglian Wendan Decoction on colitis-related colon cancer by ProteomicDIA chemical labeling method was used to conduct proteomic analysis of tumor tissues and paracancerous tissues in each group.Cluster,correlation,principal component and protein molecular weight analysis were carried out to determine the differential proteins,and then GO,KEGG and Reactome Pathway enrichment molecules were carried out.Potential target protein was identified by interaction network analysis.Western blot was used to confirm whether the target protein MLCK mediates the inflammatory-cancer process in colitis mice.The changes of MLCK in colitis mice,colitis-related colon cancer mice,clinical samples and paracancerous tissues was detected by IHC.Results1.Effect of Huanglian Wendan Decoction on colitis in mice(1)Efficacy evaluationHLWDD could significantly delay the weight loss of mice induced by DSS(P<0.01),alleviated diarrhea and rectal bleeding,and reduce DAI score(P<0.01).Compared with DSS group,HLWDD could effectively restore colon length(P<0.01).HLWDD significantly increased the level of DAO(P<0.01),and inhibited the levels of MPO and NAG in colon tissues(P<0.01 in HLWDD-H,0.01<P<0.05 in HLWDD-L).After HLWDD treatment,intestinal epithelial cells recovered,with neat arrangement and clear boundary,goblet cells were visible in them,and mucosal layer also recovered significantly.(2)Mechanism researchIn DSS group,the expressions of intestinal tight junction proteins Claudinl(0.01<P<0.05),Occludin and ZO1(P<0.01)were significantly decreased.HLWDD significantly increased the expressions of tight-junction proteins(0.01<P<0.05,P<0.01).At the same time,IHC results showed that the expression of MUC2 in DSS group was significantly decreased,but it was reversed after HLWDD administration.In conclusion,HLWDD can effectively protect tight junction protein and restore intestinal barrier function.Compared with Control group,DSS significantly increased the content of IL-1β(P<0.01),TNFα(0.01<P<0.05),and decreased the content of IL-10(P<0.01)in colon tissues.However,after HLWDD treatment,the contents of IL-1β(P<0.01)and TNF-α(P<0.01)in colon tissues were significantly decreased,while the content of IL-10 were increased(P<0.01).The results of IHC showed that the expression of NLRP3 was significantly increased in the colon tissues,and HLWDD could effectively inhibit it.Meanwhile,the contents of LPS and IL-6 in DSS group were significantly increased(P<0.01),while HLWDD could significantly reduce them(0.01<P<0.05,P<0.01).Western blot results showed that the expressions of TLR4/MyD88/p-NF-κB and(p)-JAK2/(p)STAT3 in DSS group were significantly increased,while HLWDD could significantly inhibit the expressions of them(0.01<P<0.05).These results indicated that HLWDD could effectively inhibit intestinal inflammation.The expressions of CHOP(0.01<P<0.05),p-eIF2α and eIF2α in DSS group was significantly,while the expression of PDI was decreased(P>0.05).Compared with DSS group,HLWDD could significantly reduce the expressions of CHOP and eIF2α in colon tissues(0.01<P<0.05),decrease the expression of p-eIF2α and increase the expression of PDI,but had no significant difference.Combined with the IHC results,it was found that HLWDD could effectively inhibit the expression of CHOP in colon tissues.In DSS group,the expressions of cleaved PARP,cleaved caspase3 and Bax were significantly increased,while the expression of Bcl2 was significantly decreased(0.01<P<0.05).HLWDD significantly inhibited the expressions of cleaved PARP,cleaved caspase3 and Bax,while promoted the expression of Bcl2(0.01<P<0.05).IHC results also showed that,the expression of cleaved caspase3 was increased in the DSS group,while HLWDD inhibited it.It can be inferred that HLWDD can effectively relieve endoplasmic reticulum stress,inhibit the apoptosis of intestinal epithelial cells,and protect the intestinal tract.2.Effect of Huanglian Wendan Decoction on intestinal flora of colitis mice(1)Detection of intestinal flora and short-chain fatty acids in mice16S rDNA assay showed that 354 common OTUs sequences were obtained,including 139 in the Control group,25 in the DSS group,5 in the HLWDD-L group,and 11 in the HLWDD-H group.Non-metric multidimensional scaling analysis(NMDS),principal component analysis(PCA)and microflora typing analysis showed differences between HLWDD group and DSS group.HLWDD decreased the Chao index and increased the Shannon index by a diversity analysis,(0.01<P<0.05).The results of species difference analysis showed that the abundance of Bacteroidaceae,Enterobacteriaceae,Alistipes_finegoldii and Paraprevotella_clara was significantly increased in DSS group,while the abundance of Akkermansia was significantly decreased.Compared wit DSS group,after HLWDD intervention,the abundance of Escherichia,Paraprevotella__clara and Alistipes_finegoldii was significantly decreased,while the abundance of Ruminococcaceae,Verrucomicrobiaceae and Akkermansia was significantly increased.Compared with Control group,the contents of acetate,isobutyrate,valerate and total short-chain fatty acids in DSS group were significantly decreased(0.01<P<0.05),while the contents of butyrate,caproate and isovalerate were decreased but had no significant differences(P>0.05).Compared with DSS group,HLWDD-L significantly increased the content of valerate(0.01<P<0.05),HLWDD-H significantly increased the contents of acetate,butyrate,isobutyrate,valerate and total short-chain fatty acids(0.01<P<0.05),and the contents of other short-chain fatty acids had an increasing trend but with no significant difference(P>0.05).(2)Antibiotic bacterial clearing experimentAntibiotic administration could significantly reduce the intestinal flora of mice.After bacteria removal,the effect of HLWDD on alleviating weight loss was reversed,and the degree of weight loss was not significantly different from DSS group(P>0.05),but it was significantly decreased compared with group(0.01<P<0.05).DAI score results showed that it was significantly higher in HLWDD-H(ABX)group than that in HLWDD-H group(P<0.01).Compared with HLWDD-H group,the colon length was significantly shortened(P<0.01)in HLWDD-H(ABX)group.HE results showed that the colon structure of mice in HLWDD(ABX)group showed the same result as that in DSS group,and the mucosa was seriously damaged,goblet cells were almost invisible,and the permeability of colon was increased.In conclusion,the protective effect of HLWDD on colon was reversed after removal of bacterial flora.Compared with Control(ABX)group,the content of DAO in DSS(ABX)group was decreased with no significant difference(P>0.05).Compared with DSS(ABX)group,the content of DAO in HLWDD-H(ABX)group was significantly increased(0.01<P<0.05).,There was no significantly difference in the content of DAO between HLWDD-H(ABX)group and HLWDD-H group(P>0.05).Compared with Control group(ABX),the contents of MPO,LPS and IL-1β in DSS(ABX)group were significantly increased(P<0.01 in MPO and LPS;0.01<P<0.05 in IL-1β).Compared with DSS(ABX)group,the contents of MPO,LPS and IL-1β in HLWDD,H(ABX)group were significantly decreased(0.01<P<0.05 in MPO;P<0.01 in LPS and IL-1β).Compared with HLWDD-H group,the contents of MPO and IL-1β in HLWDD-H(ABX)group were increased with no significant difference(P>0.05),and the content of LPS was significantly increased(P<0.01).In conclusion,it can be found that the inhibitory effect of HLWDD on inflammation is significantly weakened after antibiotics clear intestinal flora.Western blot results showed that the expressions of ZOl and PDI in HLWDD-H(ABX)group were significantly decreased,and the expressions of ZO1 and PDI in HLWDDH(ABX)group were significantly increased(0.01<P<0.05),the expressions of cleaved caspase3,cleaved PARP,eIF2α and CHOP were significantly increased(0.01<P<0.05).The IHC results showed similar results.Thus,it can be inferred that antibiotics reverse the anticolitis effect of HLWDD by inhibiting ERS and intestinal epithelial cell apoptosis and protecting the intestinal barrier3.Effect of Huanglian Wendan Decoction on colitis-related colon cancer in miceDuring the whole experiment period,there was no significant change in body weight and colon length(P>0.05).DAI score peaked at 7days after DSS administration,and DAI threshold gradually increased in each subsequent cycle.During the whole process.DAI score could be decreased in HLWDD group,which was consistent with the results of colitis experiment.In cycle2,there was no significant difference in organ index among all groups(P>0.05).The number of>2mm tumors in Model group was higher than that in HLWDD group,and there was no significant difference between 1-2mm and<1mm tumors(P>0.05).After HLWDD administration,the tumor load was decreased with no significant difference(P>0.05).Compared with Model group,HLWDD could significantly reduce the content of LPS(P<0.01)and IL-23(0.01<P<0.05 in HLWDD-L,P<0.01 in HLWDD-H)in colon tissues.But there were no significant effect on the contents of IL-8,IL-17 and COX2(P>0.05).Compared with Model group,HLWDD had no effect on the contents of CCL20,MCP1,G-CSF,M-CSF and GM-CSF in colon tissues(P>0.05).In cycle3,there were no significant differences in the indexes of heart,lung and thymus among all groups(P>0.05).Compared with Control group,the liver index,spleen and colon index were significantly increased in Model group(0.01<P<0.05),while kidney index was significantly decreased(P<0.01).Compared with Model group,there were no significant differences in liver,spleen,kidney and colon indexes among all administration groups(P>0.05).Compared with Model group,the number of>2mm,1-2mm and<1mm tumors and tumor load were significantly decreased in HLWDD-L group(0.01<P<0.05;P<0.01);The number of>2mm and<1mm tumors and tumor load were significantly decreased in HLWDD-H group(0.01<P<0.05;P<0.01),there was no significant difference in the number of tumors at 1-2mm(P>0.05).Compared with Model group,HLWDD significantly decreased the content of LPS(0.01<P<0.05),IL-23(0.01<P<0.05 in HLWDD-H,P>0.05 in HLWDD-L)and IL-8(P<0.01 in HLWDD-L,0.01<P<0.05 in HLWDD-H),but there was no significant effect on the contents of IL-17 and COX2(P>0.05).Compared with Model group,HLWDD had no effect on the contents of CCL20,MCP1,G-CSF and GM-CSF in colon tissues(P>0.05),but the contents of M-CSF was significantly decreased(0.01<P<0.05).In cycle4,compared with Control group,the indexes of heart,liver,spleen,lung,kidney and colon were significantly increased in Model group(0.01<P<0.05;P<0.01),and the index of thymus was significantly decreased(0.01<P<0.05).Compared with Model group,HLWDD-L significantly decreased the indexes of heart,liver,spleen,thymus and colon(0.01<P<0.05;P<0.01),HLWDD-H significantly decreased the indexes of liver,spleen,lung,kidney,thymus and colon(0.01<P<0.05;P<0.01),there was no significant difference in heart index(P>0.05).Compared with Model group,the number of>2mm tumors was significantly decreased(P<0.01),the number of 1-2mm and<1mm tumors was decreased with no significant difference(P>0.05),and the tumor load was also significantly decreased(P<0.01)in HLWDD-L group.The number of tumors>2mm and 12mm was significantly decreased(P<0.01),the number of tumors<1mm was decreased with no significant difference(P>0.05),and the tumor load was also significantly decreased(P<0.01)in HLWDD-H group.Compared with Model group,HLWDD could significantly reduce the contents of LPS,IL-8,IL-23,IL-17 and COX2 in colon tissues(P<0.01),but had no significant effect on the content of IL-17(P>0.05).Compared with Model group,HLWDD could significantly reduce the contents of CCL20,MCP1,G-CSF,M-CSF and GM-CSF in colon tissue(P<0.01).The percentage of Tc cells,M1 macrophage cells in blood,spleen and tumor tissue was significantly decreased(0.01<P<0.05;P<0.01),and the percentage of MDSCs cells,M2 macrophage cells and Treg cells was significantly increased(0.01<P<0.05;P<0.01)in Model group.HLWDD significantly increased the percentage of Tc cells and M1 macrophage cells in blood,spleen and tumor tissues(0.01<P<0.05;P<0.01),decreased the percentage of MDSCs cells,M2 macrophage cells and Treg cells(0.01<P<0.05;P<0.01).These results indicated that Huanglian Wendan Decoction has obvious anti-colitisassociated colon cancer effect.4.The effect of Huanglian Wendan Decoction on colitis-related colon cancer by ProteomicProteomic analysis showed that 85 different proteins were obtained.GO enrichment analysis showed that the differential proteins were mainly located in supramolecular fibers,muscle segments,myosin filaments,actin myosin complex,myofibrillar fibers,actin cytoskeleton and contractile fibers.It mainly has binding functions,including protein binding,metal ion binding,cytoskeletal protein binding,calmodulin binding,calcium ion binding,actin filament binding and other molecular functions.It is involved in biological processes such as free skeletal muscle contraction,twitch skeletal muscle contraction,striated muscle contraction,skeletal muscle contraction,muscle contraction regulation,musculoskeletal movement,muscular system processes,muscle structure development and multicellular biological movement.KEGG results showed that differential proteins were mainly involved in tight junction,cellular endocytosis,calcium signaling pathway,myocardial contraction,hypertrophic cardiomyopathy,glucagon signaling pathway,glutaminergic synapses and phosphatidylinositol signaling system.Reactome enrichment analysis showed that the enriched protein was mainly involved in muscle contraction,but also involved in cancer gene-induced aging,histocytosis-lymphadenopathy and syndrome caused by SLC29A3 defect,and intracellular oxygen transport.The differential proteins were closely related,forming a complex interaction network,in which 19 proteins were key nodes,including Actn3,Ldb3,Tnnt3,Myll,Myh1,Myl3 and Myh7(String network degree≥10).Myosin family proteins were detected by Western blot.Compared with Control group,The expressions of MYL1,MYL3 and MYL4 were significantly decreased(P<0.01),the expressions of MYLPF,MYH1,MYH4 and MYH7 were significantly decreased(0.01<P<0.05),the expressions of MYBPC2 and MLCK were significantly increased(P<0.01)in Model group.Compared with Model group,the expressions of MYL1,MYL4 and MYH4 were significantly increased(P<0.01),the expressions of MYL3,MYLPF,MYH1 and MYH7 were significantly increased(0.01<P<0.05),the expressions of MYBPC2 and MLCK were significantly decreased(0.01<P<0.05)in colon tumor tissues of HLWDD-L group.The expressions of MYL3,MYL4,MYLPF and MYH7 were significantly increased(P<0.01),the expressions of MYL1,MYH1 and MYH4 were significantly increased(0.01<P<0.05),the expressions of MYBPC2 and MLCK were significantly decreased(0.01<P<0.05;P<0.01)in colon tumor tissues of HLWDD-H group.In colitis mice,HLWDD also decreased the expression of MLCK in colon tissues(0.01<P<0.05).The results of IHC detection also showed that the expression of MLCK was up-regulated in the colon tissues of CAC and IBD mice,while HLWDD could reverse it.In clinical samples,the expression MLCK in paracancerous tissue was significantly decreased compared with that in tumor tissue.Conclusion1.HLWDD can effectively protect intestinal barrier function and has a good effect on IBD2.The mechanism of HLWDD on anti-IBD may be related to relieving endoplasmic reticulum stress,inhibiting TLR4/MyD88/NF-B and IL-6/JAK2/STAT3 and inhibiting intestinal epithelial cell apoptosis3.HLWDD can significantly inhibit CAC and activate Tc cells,M1 macrophage cells and inhibit MDSCs cells,M2 macrophage cells and Treg cells4.The inhibition of HLWDD on IBD-CAC development may be related to MLCK... |
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