| Purpose:1.TNBS-induced UC rats and TNF-induced HIMEC were intervened by FA to clarify the effect of FA on inhibiting inflammatory damage of UC.2.To explore the curative effect and mechanism of Tou Nong San in the treatment of ulcerative colitis of Da Chang Shi Re.Methods:1.In the in vivo experiment section,the SD rats were divided into control group,model group(100 mg/kg TNBS),solvent group,low-dose group(10mg/kg FA),medium-dose group(20mg/kg FA),and high-dose group(50mg/kg FA).After 14 days,samples were taken to observe colon length,colon tissue morphology was visualized by HE staining microscope,concentration of cytokines such as IL-1,IL-6 and IL-12 were determined by ELISA,and protein expression of caspase-1,caspase-3,bcl-2,TXNIP and NLRP3 by western blot,immunohistochemistry and immunofluorescence.In the in vitro experiment section,HIMEC was divided into control group,model group(10 ng/ml TNF-α),solvent group,low-dose group(125 μM FA),medium-dose group(250 μM FA),and high-dose group(500 μM FA).Cells were cultured and passaged to reach certain density,HIMEC morphology by microscope and HE staining,HIMEC activity by MTT,HIMEC apoptosis by flow cytometry,concentration of cytokines including IL-1,IL-6 and IL-12 by ELISA,and caspase-1,caspase-3,bcl-2,TXNIP and NLRP3 by western blot,immunohistochemistry and immunofluorescence.2.60 patients were selected for the clinical trial,and 52 patients completed the trial eventually.Among them,25 control group,27 experimental group,control group were treated with mesalazine,experimental group was treated with Tou Nong San,and the methods of administration were different according to the location of the disease.All of them were given for 2 months.The indexes were observed and the contents were as follows:(1)The patients were given symptom scores and efficient evaluation,and the selected symptoms included four major factors,including diarrhea,abdominal pain,purulent stool and stool frequency.(2)Colonoscopy was performed on patients,including the Montreal typing study,the mucosal status score and mucosal appearance score,and the pathological Geboes score.(3)The patient was given a Mayo score based on the patient’s clinical symptoms,endoscopic findings and physician assessment.(4)The contents of hemoglobin(Hb),C-reactive protein(CRP),erythrocyte sedimentation rate(ESR),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and interleukin-10(IL-10)were detected.Results:1.In terms of in vivo experiments,the colon length was normal in the control and solvent group,and the model group was significantly smaller than the control and solvent group(P<0.01),while FA reversed the shortening,with the high dose group(P<0.01),followed by the middle dose group(P<0.01)and the low dose group(P<0.05).Microscopic colon tissue morphology after HE staining,found that the control and solvent group had intestinal mucosal barrier intact,normal gland structure,no inflammatory cell infiltration,and model group,colon barrier destruction,large inflammatory cell infiltration,FA intervention,the high dose group is the closest to the colonic morphology of the control group.Concentrations of IL-1,IL-6,and IL-12 in colonic tissues were determined by ELISA,Low concentrations of IL-1,IL-6,and IL-12 were found in the control and solvent groups,The concentration of these cytokines was significantly increased in the model group(P<0.01),FA was able to reduce the concentration of cytokines in the tissue,The largest reduction in the high-dose group(P<0.01),Second in the middle-dose group(P<0.01),there was no significant statistical difference in IL-1 in the low-dose group(P>0.05),The IL-6 was statistically different(P<0.05),The IL-12 difference was even more distinct(P<0.01).western blot,immunohistochemistry and immunofluorescence detection of caspase-1,caspase-3,bcl-2,TXNIP and NLRP3,the control and solvent groups,while FA could inhibit the expression of each protein.Among them,the high-dose group had the most inhibition,the least in the low-dose group.In vitro experiments,through the microscope and HE cell morphology,found that control and solvent group cell viability,normal morphology,cells cover the whole field,and model group cell vitality,cell circle,low density,visual field in obvious area,FA can reverse this change,the high dose group reversal trend is the most obvious,,the smallest degree of reversal is the low dose group.HIMEC activity was detected by MTT,with strong cell viability in the control and solvent groups,and the worst cell viability in the model group,while FA could improve cell activity,the most obvious in the large dose group(P<0.01),followed in the medium dose group(P<0.01),and not in the low dose group(P>0.05).The degree of apoptosis in HIMEC was detected by flow cytometry,there was no significant apoptosis in the control and solvent groups,and the model group showed great apoptosis,while FA inhibited this apoptosis in the high-dose group(P<0.01),followed in the medium-dose group(P<0.05),and not in the low-dose group(P>0.05).2.The clinical trial results are as follows:(1)In terms of effective rate,the treatment group was statistically significant compared to the control group(P<0.01),the control group and the experimental group after treatment were lower than those before treatment in terms of diarrhea,abdominal pain,purulent stool and stool frequency scores(P<0.01),and the experimental group after treatment was lower than the control group in terms of diarrhea score(P<0.05).The other scores,there was no significant difference between the experimental group and the control group(P>0.05).(2)Montreal typing study,the experimental group after treatment compared with the experimental group before treatment,had statistical differences(P<0.05),the control group before treatment compared with the experimental group before treatment,the control group after treatment compared with the control group before treatment,the experimental group after treatment compared with the control group after treatment,there was no significant difference(P>0.05);The mucosal state scores and mucosal appearance scores of colonoscopy,the control group and the experimental group after treatment compared with theirs before treatment decreased,there was significant statistical difference(P<0.01),there was no significant difference between the experimental group and the control group after treatment(P>0.05);The pathological Geboes score,the control group and the experimental group after treatment compared with theirs before treatment scores decreased,there was significant statistical difference(P<0.01),the experimental group after treatment compared with the control group after treatment,the score decreased,there was statistical difference(P<0.05).(3)There was significant difference in Mayo score(P<0.01)between the control group and the experimental group after treatment and theirs before treatment,the experimental group after treatment compared with the control group after treatment had statistical difference(P<0.05).(4)Compared with the control group and experimental group before treatment,the Hb of theirs after treatment increased significantly(P<0.01),compared with the control group after treatment and the experimental group after treatment,there was no significant difference(P>0.05);compared with the control group and experimental group before treatment,the CRP of theirs after treatment decreased significantly(P<0.01),compared with the control group after treatment and the experimental group after treatment,there had statistical difference(P<0.05);compared with the control group and experimental group before treatment,the ESR of theirs after treatment decreased significantly(P<0.01),compared with the control group after treatment and the experimental group after treatment,there was no significant difference(P>0.05);IL-1β,compared with the control group and experimental group before treatment,the IL-1β of theirs after treatment decreased significantly(P<0.01),compared with the control group after treatment and the experimental group after treatment,there had significant statistical difference(P<0.01);TNF-α,compared with the control group and experimental group before treatment,the TNF-α of theirs after treatment decreased significantly(P<0.01),compared with the control group after treatment and the experimental group after treatment,there had significant statistical difference(P<0.01);IL-10,compared with the control group and experimental group before treatment,the IL-10 of theirs after treatment decreased significantly(P<0.01),compared with the control group after treatment and the experimental group after treatment,there had significant statistical difference(P<0.01).Conclusions:1.In vivo,FA can improve colon mucosal tissue damage in SD rats,mainly by reducing colon shortening,improving its morphological structure,and reducing the concentration of cytokines such as IL-1,IL-6 and IL-12,it can also delayed elevation of bcl-2 proteins,and reduced proteins such as caspase-1,caspase-3,TXNIP,and NLRP3.In vitro,FA can improve the inflammatory damage of HIMEC,mainly by improving cell morphology,viability,by reducing apoptosis rate,and by reducing cytokines such as IL-1,IL-6,and IL-12,and by reducing proteins such as caspase-1,caspase-3,TXNIP,and NLRP3,and by regulating bcl-2 protein.2.Tou Nong San has significant efficacy in the treatment of UC,improving clinical efficiency,reducing clinical score,reducing endoscopic score,reducing lesion site,reducing Mayo score and Geobes score,increasing Hb,reducing CPR and ESR,reducing IL-1β,and TNF-α,and raising IL-10,which is better than mesalazine,so it is an ideal alternative therapy. |
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