| BackgroundUlcerative colitis(UC)is a kind of chronic non-specific inflammatory colorectal disease that occurs in people among 18-50 age group.Abdominal pain,diarrhea,and mucopurulent bloody stools affects their lives seriously.Because it is difficult to be cured,repeated,and the high risk of cancer,UC has been classified as a refractory disease by the WHO.The pathogenesis of UC involves a variety of complex mechanisms,of which the destruction of integrity of the intestinal barrier is currently recognized as an important link.Oxidative stress,inflammation,and apoptosis are important mechanisms in the development of many diseases.Studies on the excessive activation of these mechanisms jointly destroying the epithelium of lungs,renal tubules,and cardiovascular have been reported.But the effects of over-activation of them,especially oxidative stress and inflammation,on the intestinal physical barrier are rarely reported in UC.Qingchang Wenzhong formula was modified and created,from thought of academician Dong Jianhua,LianLi Decoction,and his clinical experience,by Professor Li Junxiang,Dongfang Hospital of Beijing University of Chinese Medicine.In the previous study of single-center double-blind double-simulation RCT clinical observation,it was found that Qingchang Wenzhong had the clearly effect of improving clinical symptoms,reducing disease activity and improving the quality of lives in patients in mild to moderate active UC.Basic experiments improved evidences of repairing the colonic mucosa and reducing the inflammation of UC.However,these experiments mainly relies on the Qingchang Wenzhong formula how to regulate the inflammatory response,but lacks further verification of other potential mechanisms.Objective1.Exploring the potential effects of oxidative stress,inflammation and apoptosis,especially from oxidative stress and inflammation,based on the physical barrier in UC.To reveal the pathogenesis of UC based on the above mechanisms,and to clarify the regulatory effects of Qingchang Wenzhong on the above mechanism from multiple dimensions in UC.2.Decomposed study of Qingchang Wenzhong formula from the perspective of hypoxic environment,skeleton destruction and inflammatory reaction,to clarify the more effective composition unit for different pathogenesis in the treatment of UC,and to provide scientific evidence on compound compatibility and clinical applications.MethodsExperiment 1:A rat model of acute UC was established by drinking DSS solution.The rats were treated with different doses of Qingchang Wenzhong extract powder solution and mesalazine suspension.Disease activity index(DAI),colon wet weight,colonic mucosal disease index(CMDI),histological lesion score,immunohistochemical staining of Occlundin and NF-κB p50 proteins in colon,oxidative stress substances in serum and colon tissue(SOD,MDA,GSH-px),serum inflammatory mediator(IL-1α,IL-6,TNF-α)and colon(MPO)by Elisa,apoptosis-related proteins(Bax,Bcl-2,Akt,p-Akt,Caspase-3)in colon by Western blot,Alcian blue staining of goblet cells and TUNEL apoptosis assay methods were used to study the regulation of oxidative stress,inflammation,and apoptosis in acute UC rats based on the physical barrier.Experiment 2:A rat model of acute UC was established by enema with TNBS/anhydrous ethanol.The rats were treated with different doses of Qingchang Wenzhong extract powder solution and mesalazine suspension.DAI,colon wet weight and length,CMDI,histological lesion score,immunohistochemical staining of Occlundin and NF-κB p50 proteins in colon,oxidative stress substances in serum and colon tissue(SOD,MDA,GSH-px),serum inflammatory mediator(IL-6、IL-13、TNF-α、IFN-γ)by Elisa were used to study the regulation of oxidative stress,inflammation,and apoptosis in acute UC rats based on the physical barrier.Experiment 3:A mouse model of chronic UC was established by drinking DSS solution.The mice were treated with different doses of Qingchang Wenzhong extract powder solution and mesalazine suspension.DAI,colon wet weight,CMDI,histological lesion score,immunohistochemical staining of Occlundin and NF-κB p50 proteins in colon,oxidative stress substances in serum and colon tissue(SOD,MDA,GSH-px),serum inflammatory mediator(IL-1β、IL-4、IL-5、IL-6、TNF-α)by Elisa were used to study the regulation of oxidative stress,inflammation,and apoptosis in chronic UC mice based on the physical barrier.Experiment 4:A rat model of acute UC was established by drinking DSS solution.The rats were treated with decomposed formula of Qingchang Wenzhong extract powder solution and mesalazine suspension.DAI,CMDI,histological lesion score,immunohistochemical staining of VIM and K8 proteins in colon,serum inflammatory mediator(IP-10,CXCR3)by Elisa,HIF-1α in colon by Western blot were used to carry out the regulation of hypothermia environment,skeleton destruction and inflammation in acute UC rats by Qingchang Wenzhong and its decomposed formula.ResultsExperiment 1:Compared with the model group(p<0.05),Qingchang Wenzhong formula could reduce DAI,CMDI,histological lesion scores significantly in acute UC rats.It could lighten wet weight of colons,promote Occludin but inhibit NF-κB p50 expression in IHC.MDA was cut but SOD and GSH-PX activity was boosted in serum and colons.It reduced level of IL-1α,IL-6,TNF-α in serum but decrease MPO activity in colons,decreased expression of apoptosis-related proteins Bax,Caspase-3 and ratio of phosphorylated Akt/Akt in colons and stimulated the expression of anti-apoptosis protein Bcl-2.Expression of goblet cells was encouraged the apoptosis of intestinal epithelium was inhibited.Mild-dose of Qingchang Wenzhong formula had more obvious effects.Experiment 2:Compared with the model group(p<0.05),Qingchang Wenzhong formula could reduce DAI,CMDI,histological lesion scores significantly in acute UC rats.It could lighten wet weight and grow the length of colons,promote Occludin but inhibit NF-κB p50 expression in IHC.MDA was cut but SOD and GSH-PX activity was boosted in serum and colons.It reduced IL-6,TNF-α but promoted IL-13 levels in serum.Mild-dose of Qingchang Wenzhong formula played a more effective role among all the groups.Experiment 3:Compared with the model group(p<0.05),Qingchang Wenzhong formula could reduce CMDI,histological lesion scores significantly in chronic UC mice,but DAI was ineffective.It could add wet weight of colons,promote Occludin but inhibit NF-κB p50 expression in IHC.MDA was cut but SOD and GSH-PX activity was boosted in serum and colons.It reduced IL-1β,IL-6,TNF-α whereas boosted IL-4,IL-5 evels in serum.Mild-dose of Qingchang Wenzhong formula had better effects.Experiment 4:Compared with the acute UC model group(p<0.05),Qingchang Wenzhong formula and decomposed groups could all reduce DAI and CMDI score,that H,PH,PS and the entire groups cut down histological lesion scores significantly.It could promote K8 but decrease VIM expression and ratio of VIM/K8 by IHC,which PS group played better.Each group decreased IP-10 inserum,only P,H,PH,PS,HS and entire groups could reduce CXCR3,HS and PS had more effects.HIF-1 a could be inhibit by each group,especially the PH and PS,whereas there had no statistically dintinguish among each groups.Conclusions1.Qingchang Wenzhong formula can reduce lipid peroxidealle,promote activity of anti-oxidative enzymes,and to restore the oxidative-antioxidant balance.It can also reduce mucosal inflammation via regulating different cytokines.Meanwhile,it can inhibit apoptosis of intestinal epithelium by decrease apoptosis-related proteins and increase the opposite ones.It could reduce the impact of physical barriers from multi-dimensionality.In addition,it can improve expression of tight junctions and goblet cells,repaire mucosal structures,and restore the damaged physical barriers to treat UC.2.Combined with effects of cytoskeleton,inflammation and the mucosal hypoxia,entire of Qingchang Wenzhong formula had a better function than PH and PS,the latter two groups made the more apparent effect than P,H,and S.Only combined with Zhong Jiao-warming and spleen-strengthening therapy,heat-reducing and dampness-relieving or blood-cooling and stasis-eliminating thepapies would play the better efficacy,improving,repairing the cytoskeleton and alleviating inflammation,like the entire formula.It also suggested that in the clinical treatment of UC,we should pay more attention to taking care of Zheng Qi and strengthening the spleen and warming the Middle Jiao. |
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