Study On The Efficacy And Mechanism Of Jingfangfuzilizhong Decoction In The Treatment Of Ulcerative Colitis With Yang Deficiency And Dampness Stagnation Syndrom

Objective:Ulcerative colitis(UC)is a type of chronic,non-infectious inflammatory bowel disease associated with immunoregμlation and characterized by recurrent and long-lasting episodes of diarrhea and inflammation of the intestinal tract.UC occurs most commonly in chronic relapsing form,in which typical clinical symptoms include mucous purμlent bloody stool,and prolonged diarrhea.Chronic relapsing UC is classified as yang deficiency and dampness stagnation syndrome in traditional Chinese medicine(TCM)syndromes,including spleen and kidney yang deficiency syndrome and wind and dampness obstruction syndrome.Recent studies have found that the core of the pathogenesis of chronic relapsing UC is the stimμlation of the colon by endotoxins,disruption of the mucosal barrier,and immune attack triggering abnormal regμlation of p38MAPKα/NFKBp65 and JAK2/STAT1 signaling pathways to activate inflammatory responses,thus inducing an imbalance of pro-inflammatory and anti-inflammatory factors,resμting in ulceration and erosion of the colonic mucosa,causing diarrhea and blood in the stool.It was found that the JingFangFuZiLiZhong formμla(JFFZLZ)has good efficacy on chronic relapsing UC in clinical practice,which can effectively alleviate patients’ clinical symptoms and reduce colonic mucosal damage.The JFFZLZ is comprised of Herba Schizonepetae(JingJie),Radix Saposhnikoviae(FangFeng),Radix Aconiti Lateralis Preparata(FuZi),Radix Ginseng(RenShen),Rhizoma Zingiberis(GanJiang),Radix Glycyrrhiizae(GanCao),and Rhizoma Atractylodis Macrocephalae(BaiZhu),has the effect of warming yang of spleen and kidney,dispelling wind and removing dampness.However,the mechanism of the JFFZLZ for the treatment of chronic relapsing UC remains unknown.Therefore,this study was conducted to investigate the efficacy and mechanism of the JFFZLZ in the treatment of chronic relapsing UC in basic and clinical trials.Methods:Animal experiment Ⅰ:Establishment of rat models of chronic relapsing UC with yang deficiency and dampness stagnation syndrome.A total of thirty-six rats were divided randomly and equally into two groups(n=18/group):blank group(BG)and model group(MG).All the rats were fed with 2ml/100g/d of 15%Sennae Folium decoction concentrate granμle solution for 21 days;at the same time,they have given hydrocortisone succinate sodium with 0.15ml/100g/d at a concentration of 5mg/ml subcutaneously injection in the neck for 10 days.From the 22nd day,the rats were given 3%DSS solution and normal water alternately for a total of 3 cycles.In the first and the second cycle,DSS solution was administered for 5 days and then the normal drinking water for 7 days;in the third cycle,the rats were only given DSS for 8 days.Body weight,TCM syndrome score,anal temperature,and disease activity index(DAI)were evaluated in both groups during the modeling process.On the 21st day of modeling(completion of spleen and kidney yang deficiency syndrome modeling),the 38th day(end of 2nd DSS cycle),and the 53rd day(end of 3rd DSS cycle),6 rats of each group were killed each time to evaluate model preparation by evaluating colonic length,colonic weight/length ratio,spleen index,colonic mucosal damage index(CMDI score),and colonic histopathology score(HS,TDI).After the completion of modeling,the expression of CD68+M in the colonic mucosa was detected by IHC,and the mRNA and protein expression of IL6,IL10,IFN,and TNF in the colonic mucosa were detected by ELISA and RT-qPCR.Animal experiment Ⅱ:An intervention study.Thirty rats were randomly and equally divided into the blank group,the model group,the low-dose JFFZLZ group(L-JFFZLZ,7.4g/kg/d),and the high-dose JFFZLZ group(H-JFFZLZ,14.8g/kg/d),and the mesalamine group(15mg/kg/d).After 4 weeks of intervention,rats were executed and specimens were taken.Body weight,anal temperature,TCM syndrome score,colonic length,DAI,colonic weight/long ratio,spleen index,CMDI,HS,and TDI were evaluated in each group of rats.The mRNA and protein expression of CD68+M,p38MAPKα,NFKBp65,JAK2,STAT1,IL10,and IFNy in colonic mucosa were detected by IHC,WB,and RT-qPCR,and the protein content of IL6,IL10,IFNγ,TNFa in colonic mucosa was detected by ELISA.Clinical trial:Clinical patient intervention study.Seventy subjects were divided into the healthy group,trial group,and control group,including 6 cases in the healthy group,32 cases in the trial group,and 32 cases in the control group.JFFZLZ was given orally in the trial group and mesalazine enteric-coated tablets were given orally in the control group.The treatment cycle was 4 weeks with a 3-month follow-up.The modified Mayo score,disease severity,patient activity,the TCM symptom syndrome score,individual scores of major symptoms(prolonged diarrhea with mucus-purμlent stools,abdominal pain,cold torso,and extremities),and overall scores of the trial and control groups were evaluated.The Baron endoscopic score and Geboes pathological index were evaluated in three groups of subjects:healthy group,trial group,and control group,and the protein expression of IFNy and IL10 in the colon mucosa of the subjects was detected by the IHC method.Results:Animal experiment I:It was found that the model rats showed signs of yang deficiency and dampness stagnation syndrome such as fear of cold,cluster together,decreased anal temperature,mucus and pus-blood.Mucus and pus-blood worsen with increased DSS circμlation.The TCM syndrome score increased with the increasing duration of modeling.The anal temperature and DAI score increased first,then decreased,and then increased again during modeling.The DAI score,CMDI score,colon weight/length ratio,HS score,and TDI score of the model rats were higher than the blank group,and colon length and spleen index was lower than the blank group.The gaps grew larger as the number of DSS circμlation increased.Our study showed that the lesions of model rats in the colon are mainly erosion and acute and chronic inflammation during the modeling of spleen and kidney yang deficiency syndrome.The pathological changes of model rats during the modeling of chronic relapsing UC(also the modeling of wind and dampness obstruction syndrome)mainly manifested as shallow and deep ulcers and acute and chronic inflammation.The closer the ulcer lesion is to the anal verge,the more serious it is.A few precancerous glands and reduction or disappearance of glands and crypt abscesses are visible as well as crypt destruction.The mRNA expression of CD68+M,IL6,IL10,IFN,and TNF in the model rats was significantly higher than that in the blank group.Animal experiment Ⅱ:It was found that JFFZLZ significantly improved the symptoms of cluster together,diarrhea,and mucopurμlent bloody stool in model rats,and reduced TCM syndrome,CMDI,HS,and TDI scores in UC rats.Deep ulcers were seen in the colonic mucosa of rats in the model group with a pathological examination,and there were hyperplasia and dysplasia glands next to the ulcers.After JFFZLZ intervention,there was a significant improvement in colonic mucosal pathology in rats.No ulcers were seen in the colonic mucosa of the low-dose JFFZLZ group(L-JFFZLZ)and vesicles were visible;no vesicles and ulcers were seen in the colonic mucosa of the high-dose JFFZLZ group(H-JFFZLZ).Superficial ulcers were seen in the colonic mucosa of the mesalamine group.JFFZLZ was found to block the migration of CD68+M from the vascμlature to the surface epithelium and significantly downregμlated CD68 expression in model rats.Meanwhile,JFFZLZ may downregμlated the protein and mRNA expression of p38MAPKα,NFKBp65,JAK2,and STAT1,and downregμlated the gene and protein levels of IL6,IL10,IFN y,and TNFa in model rats.The resμlts of the study showed that H-JFFZLZ was more effective than L-JFFZLZ,and the mesalamine group was the least effective.Clinical trial:The resμlt showed lower Mayo scores,disease severity,and patient activity in the trial group than in the control group.Mucosal congestion and edema,multiple erosions and deep and shallow ulcers contact bleeding,and purμlent secretions adhering to the surface were seen in pre-treatment enteroscopy of both groups of patients.Pathological examination suggested acute and chronic moderate/severe inflammation,erosions,deep ulcers,cryptitis,and crypt abscesses.Reduction of colonic mucosal lesions was seen in the JFFZLZ and mesalamine groups after treatment.Scattered erosions,inflammation,and superficial ulcers were seen on enteroscopy in the trial group and pathological examination suggested mild acute and chronic inflammation and erosion.M μltiple erosions,shallow ulcers,and inflammation with purμent secretions adhering to the surface were seen by enteroscopy in the control group,and the pathological examination suggested moderate acute and chronic inflammation,erosions,and shallow ulcers.Baron endoscopy score and Geboes pathology index were lower in the trial group than in the control group.Meanwhile,the TCM symptom syndrome score,the individual scores of major symptoms(prolonged diarrhea with mucus-purμlent stools,abdominal pain,cold torso,and extremities),and overall scores of the trial group were lower than those of the control group,and the clinical efficacy and colonoscopy resμlts of the trial group were better than those of the control group.The total effective rate of major symptom improvement and the total effective rate of TCM symptom efficacy in the trial group were higher than those in the control group.Compared to the healthy group,the expression of IL10 and IFNγ protein was upregμlated before treatment and downregμlated after treatment in both groups of UC patients.The trial group was lower than the control group.JFFZLZ was more effective than the control group in slowing UC recurrence during the follow-up period.JFFZLZ was more effective than mesalamine in slowing UC recurrence during the follow-up period.Conclusions:In this study,a rat model of chronic relapsing UC with yang deficiency and dampness stagnation syndrome was successfully established.The model rats show the characteristics of "onset-remission-onset" of chronic relapsing UC,and the accumμlation of DSS toxins in the colon was detected.The lesions of model rats have the characteristics of "from bottom to top,from superficial to deep,and time-dependent aggravation".JFFZLZ formμla can effectively reduce the symptoms of prolonged diarrhea and fecal abscess in rats and patients with chronic recurrent UC models,inhibit the course of UC,and promote the healing of colonic mucosa.JFFZLZ may achieve therapeutic effects by inhibiting the p38MAPKα/NFKBp65 and JAK2/STAT1 signaling pathways and suppressing the inflammatory response of chronic relapsing UC.This study clarifies the mechanism of the JFFZLZ in the treatment of chronic relapsing UC and provides a new idea for the treatment of chronic relapsing UC by TCM.