| Objective: This study mainly investigated the efficacy of Fufangxiaopi Formula in the clinical treatment of ulcerative colitis and explored its mechanism of action through in vitro and in vivo experiments.Methods: In the clinical experiment,we collected 70 patients with ulcerative colitis of spleen and kidney yang deficiency type and randomly divided them into the control group and the treatment group.The patients in the control group were given mesalazine enteric-coated tablets,and the patients in the treatment group were given Compound Elimination Duodian Formula orally on the basis of the control group,and compared and observed the overall effective rate of the two groups,as well as the changes of Chinese medicine index score,Modified Mayo Score,colonoscopic mucosal score,blood sedimentation and C-reactive protein before and after treatment to evaluate the clinical efficacy of the patients.In order to further investigate the mechanism of action of Fufangxiaopi Formula,we utilized network pharmacology to predict the key targets and major signaling pathways of Fufangxiaopi Formula in ulcerative colitis.After that,we verified the mechanism of action of Fufangxiaopi Formula through both in vivo and in vitro experiments.In vivo,we established a DSS-induced colitis model in mice,and clarified the efficacy of Fufangxiaopi Formula on the symptoms associated with DSS-induced colitis by observing the body weight change,colon length,DAI score and H&E staining in mice.Afterwards,the release of inflammatory mediators in mouse colon tissues was detected by ELISA and MPO,and the expression of TLR4/NF-κB signaling pathway and MAPK signaling pathway-related proteins as well as intestinal barrier-related proteins were detected in mouse colon tissues by western blotting.Changes in the content of barrier proteins in mouse colon tissues were detected by immunofluorescence.In order to clarify the effects of Fufangxiaopi Formula on the intestinal flora,we performed 16 S r RNA high-throughput sequencing and fecal microbial transplantation experiments.In vitro,in order to further clarify the mechanism of action of Fufangxiaopi Formula,we established an LPS-induced cellular inflammation model.The effect of Fufangxiaopi Formula-containing serum on the release of inflammatory mediators from LPS-induced RAW264.7 cells was detected by q RT-PCR.The effects of Fufangxiaopi Formula-containing serum on the expression of proteins related to TLR4/NF-κB signaling pathway and MAPK signaling pathway in RAW264.7 cells as well as intestinal barrier-associated proteins in Caco-2 cells were detected by western blotting.The effects of Fufangxiaopi Formula-containing serum on LPS-induced nuclear translocation of NF-κB-p65 proteins in RAW264.7 cells and barrier-associated proteins in Caco-2 cells were examined by immunofluorescence.Results: The clinical study showed that both the treatment group and the control group improved the symptoms of the patients,and the total effective rate of the treatment group was 90.91% and the total effective rate of the control group was 74.19%(P < 0.05),the difference was statistically significant.After treatment,the TCM evidence points,modified Mayo score,colonoscopic mucosal score,blood sedimentation and C-reactive protein of the two groups improved compared with those before treatment,and the treatment group was better than the control group in terms of TCM evidence points,colonoscopic mucosal score,and blood sedimentation(P < 0.05),with a statistically significant difference.In terms of the reduction of modified Mayo score and C-reactive protein the two groups had comparable effects after treatment(P > 0.05),and the difference was not statistically significant.Through the study of network pharmacology we found that there are 198 key components of Fufangxiaopi Formula intervention in ulcerative colitis and 313 targets of action,including149 key targets related to ulcerative colitis disease.The enrichment analysis of KEGG signaling pathway showed that Fufangxiaopi Formula may intervene in ulcerative colitis through two classical signaling pathways,MAPK signaling pathway and NF-kappa B signaling pathway.In vivo studies showed that Fufangxiaopi Formula significantly reduced the symptoms of colitis in mice,including shortened colon length,weight loss,increased clinical scores,and colonic tissue damage.In addition,Fufangxiaopi Formula inhibited the activation of TLR4/NF-κB and MAPK signaling pathways and reduced the release of pro-inflammatory mediators.In addition,Fufangxiaopi Formula increased the expression levels of claudin-3,occludin,and ZO-1,thereby protecting the integrity of the intestinal barrier.Moreover,Fufangxiaopi Formula can alleviate the symptoms of colitis in mice by regulating the balance of intestinal flora.In vitro studies have shown that Fufangxiaopi Formula-containing serum inhibited the activation of TLR4/NF-κB and MAPK signaling pathways and reduced the secretion of pro-inflammatory mediators,such as i NOS,IL-1β,TNF-α,IL-6 and COX-2 in RAW264.7 cells.In addition,Fufangxiaopi Formula-containing serum increased the expression of epithelial barrier proteins in Caco-2 cells.Conclusion: Fufangxiaopi Formula is clinically effective in the treatment of ulcerative colitis.Its mechanism of action may be to protect the intestinal barrier function by regulating the balance of intestinal flora,and to alleviate the symptoms of ulcerative colitis by inhibiting the activation of the TLR4/NF-κB and MAPK signaling pathways and thereby reducing the release of pro-inflammatory mediators. |
