Evaluation And Mechanism Of Qianjin Xiaopi Enema In The Treatment Of Ulcerative Colitis

Objective:The purpose of this study is to evaluate its therapeutic effect in clinical application,And in vivo and in vitro experimental study to explore the intervention mechanism of Qianjin xiaopi enema on ulcerative colitis(UC).Methods:Patients diagnosed with UC from June 2019 to September 2021 and the dialectical type belonged to the syndrome of heat stagnation and blood stasis were collected from the Anorectal Outpatient Clinic and Treatment Area of the Affiliated Hospital of Changchun University of Chinese Medicine,and treated with QJXP retention enema,treatment duration is 12 weeks.The research method is self-control before and after treatment.Quantitative criteria for intestinal symptoms were collected before treatment,at the 6th week and at the 12th week.The modified mayo score,platelet count,erythrocyte sedimentation rate,c-reactive protein,ANCA spectroproteinase 3 antibody,and serum TNF-α and IL-6 levels were compared before and after treatment.To further explore the intervention mechanism of QJXP on UC,a network pharmacology analysis was performed.With the help of TCMSP database,the screening conditions are bioavailability OB≥ 30,drug-like DL≥0.18,and intestinal epithelial permeability CACO≥-0.4 to collect all active ingredients and related gene target information,and combine them with UC disease-related gene targets,then construct a network diagram of drug-ingredient-target".The GO and KEGG enrichment analysis was performed on the Metascape platform to analyze the signaling pathways and biological processes of QJXP in the treatment of UC.In order to verify the accuracy of the prediction results of network pharmacology,an in vitro inflammatory cell model was established by stimulating mouse peritoneal macrophage RAW 264.7 with LSP1 μg/ml.The CCK-8 cell activity detection method clarifies the appropriate concentration of the experimental drug,and the experiment is carried out in the way of pre-protection of the therapeutic drug for 1 h and stimulation with LPS for 4 h.The total protein and mRNA of the cells after intervention were extracted,and the contents of IKK-α,IκB-α,P-p65,JAK2,P-JAK2,STAT3,and P-STAT3 proteins were detected by Western-bolt method.TNF-α,IL-6,IL-1β were amplified by PCR.Their relative contents were detected by agarose gel electrophoresis.In addition,an in vitro model of intestinal epithelial injury was established by stimulating human colonic epithelial cells caco-2 with 10 μg/ml LSP.The same pre-protection experiment method was adopted as before(LPS stimulation time was 24h),and the relative contents of zo-1,occludin and claudinl were tightly connected by Western-bolt method.In addition,2%(w/v)DSS was induced for 7 days to create a UC mouse model.After 3 days of DSS intervention,different doses of QJXP enema protection were treated for 7 days.The changes in body weight and disease activity index(DAI)of mice before and after treatment were compared.In addition,2%(w/v)DSS was induced for 7 days to create a UC mouse model.After DSS intervention for 3 days,different doses of QJXP enema were used to protect the mice for 7 days.The changes in body weight and disease activity index(DAI)of mice before and after treatment were compared.The colon length,mass,colon index and mucosal mucosal injury score(CMDI,TDI)of mice were used to analyze the protective effect of QJXP on the mucosa of UC mouse model.Results:1.Prospective clinical study:① The total clinical effective rate of UC patients before and after treatment was 92%;②The patient’s intestinal symptoms were significantly improved in the 6th week after treatment compared with before treatment(p<0.05);There is also a very significant difference between the 12th week and the 6th week(p<0.05);③The modified mayo score was significantly improved after treatment,(P<0.05);④There was a very significant difference in platelet count before and after treatment;there was a very significant difference in erythrocyte sedimentation rate before and after(p<0.05);there was a significant difference in c-reactive protein before and after(p<0.05);ANCA spectrum 3 antibody was compared before and after(p<0.05);⑤After 12 weeks of treatment,the patient The levels of TNF-α and IL-6 in serum were lower than those before treatment,with statistical significance(p<0.05).2.pharmacology research:①A total of 45 active components could play a role in QJXP enema administration,and with 195 therapeutic targets;②There were 33 active components and 76 related targets in the treatment of UC by QJXP;③GO Biological Processes analysis showed that QJXP had a therapeutic effect on UC through trauma response and angiogenesis;④KEGG PATHWAY enrichment analysis showed that QJXP could treat UC by intervening NF-κB signaling pathway and JAK-Stat signaling pathway.3.Effects of Qianjin xiaopi enema on IKK-IκB-NF-κB and JAK2-STAT3 signaling pathway:①QJXP 5,2.5,1μg/μ L and SASP1mmol/L had no significant effect on RAW264.7 cell viability;②The levels of IκB-α in each group decreased after stimulation with LPS,which,combined with the results of P-p65,confirmed that RAW264.7 cells activated the NF-κB signaling pathway under LPS stimulation,and it was positively activated.Compared with the model group,QJXP(2.5 μg/μl)down-regulated the content of P-p65,blocked the phosphorylation of NF-κB into the nucleus,and relieved the symptoms of UC,but QJXP(5 μg/μl)promoted NF-κB Phosphorylation showed that different concentrations of QJXP produced different effects,which deduced that QJXP may have a bidirectional regulatory effect on the NF-κB signaling pathway.In the subsequent PCR detection of TNF-α and IL-1βmRNA content,TNF-α and IL-1β mRNA were P-p65 nuclear transcription products.1 μg/μl)group decreased in content,while QJXP(5 μg/μl),tended to promote the production of TNF-α mRNA.In the analysis of IL-1β mRNA,although QJXP(5 μg/μl)also significantly decreased the production of IL-1βunder LPS stimulation,the degree was significantly different from that of SASP,QJXP(2.5,1 μg/μl),and QJXP(1 μg/μl)Suppression trend is also significantly better than SASP.③ Compared with the NT group,the content of P-STAT3 in the model group was significantly increased,which was consistent with the previous results of P-JAK2,which also confirmed that the JAK/STAT signaling pathway in RAW 264.7 cells was activated under the influence of LPS.There was a significant difference between QJXP(1 μg/μl)and the model group,which indicated that low-dose QJXP could inhibit the JAK/STAT signaling pathway.However,QJXP(5 μg/μl)suggested that the content of P-STAT3 was increased,which promoted the phosphorylation of STAT3.Combined with the results of P-STAT3/STAT3 and the analysis of the relative expression of P-JAK2,it was inferred that high doses of QJXP could increase the effect of P-JAK2 on the The phosphorylation efficiency of STAT3,thereby promoting the JAK/STAT signaling pathway.4.Effect of Qianjin xiaopi enema on colonic mucosal barrier ①QJXP 5,2.5,1 μg/μl,SASP 1mmol/L and LPS 10 μg/mL had no significant effect on caco-2 cell viability.②Appropriate concentration of QJXP(2.5 μg/μl)has protective effect on tight junction proteins zo-1,occludin and claudin-1 in caco-2 intestinal mucosal injury model in vitro,and the effect is not significantly different from that of SASP;③QJXP can effectively improve the general state of 2%DSS-induced UC model mice,and improve their digestion and absorption function;④ QJXP can effectively improve the disease activity of 2%DSS-induced UC model mice,mainly by maintaining mouse body weight and improving defecation.,reduce the amount of mucus and blood in the feces;⑤QJXP can effectively inhibit the intestinal inflammatory infiltration in the mouse ulcerative colitis model,promote the healing of mucosal ulcers,and improve intestinal edema.The effect of the middle dose group was similar to that of SASP.Conclusion:QJXP retention enema in the treatment of ulcerative colitis has a significant clinical effect,can significantly improve intestinal symptoms,down-regulate pro-inflammatory factors TNF-α,IL-6,after 3 months of clinical observation,no definite adverse reactions were found,which is worthy of clinical promotion application.Appropriate concentration of QJXP can effectively inhibit IKK/IκB/NF-κB and Jak2-STAT3 signaling pathways,reduce the release of pro-inflammatory factors,and restore the balance of immune regulation.QJXP has a protective effect on intestinal epithelial tight junction proteins,thereby strengthening the intestinal mucosal mechanical barrier and reducing further damage to the intestinal mucosa by inflammation.