| ObjectiveClinical Research:Based on the previous clinical research,we evaluate the clinical efficacy of Anchang Yuyang Decoction combined with Mesalazine in the treatment of ulcerative colitis of spleen deficiency and toxin-accumulation syndrome and the endoscopic and histopathological effects of different intestinal segments.The effect of Anchang Yuyang Decoction combined with mesalazine on the regulation of serum inflammatory factors TNF-? and IL-10 to explain its mechanism of action.Experimental research:Based on the pre-clinical clinical and experimental research,we study the protective effect of Anchang Yuyang Decoction on IL-13/JAK1/STAT6 pathway in LPS-induced inflammatory injury model of HT-29 cells with the theory of"virulent toxicity",in order to explore its role in the treatment of ulcerative colitis to reveal its deeper mechanism of action in the further step.Method1.Clinical Research:All patients come from the department and ward of the Spleen and Gastroenterology Department of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine.44 patients with ulcerative colitis of spleen deficiency and dampness syndrome were included in the period from April 2017 to January 2019.The self-controlled clinical study before and after treatment was used.There are 24 cases of E1 type(rectal type)and 20 cases of E2 type(left semi-colon type)according to Montreal classification.E1 type was given Anchang Yuyang Decoction combined with Mesalazine suppository.and E2 type was given Anchang Yuyang Decoction combined with Mesalazine enteric-coated tablets for 12 weeks.To evaluate the total clinical efficacy after treatment,compare the TCM symptom scores before treatment,after 4 weeks,8 weeks,and 12 weeks,the Mayo scores?the expression of serum TNF-? and IL-10 mRNA?endoscopic Baron scores and pathological Geboes score before and after treatment,safety was observe.Statistical analysis was performed by SPSS22.0 software.2.Experimental research:(1)Establishment and evaluation of cell model in vitro of ulcerative colitis:exploring the culture conditions of HT-29 cells,cryopreservation and resuscitation conditions,and establishing inflammatory inflammatory cells in vitro with ulcerative colitis induced by TNF-?,LPS,TNF-? and LPS,respectively.The model was used to evaluate the establishment of an in vitro cell model of ulcerative colitis using MTT assay and q-RT-PCR assay.(2)The protective effect of Anchang Yuyang Decoction on LPS-induced inflammatory injury of HT-29 cells:HT-29 cells were cultured in vitro,and HT-29 cells were induced by LPS 20ug/ml for 24h to induce inflammatory cell model.Anchang Yuyang Decoction was divided into 7 concentration gradients of 0.01mg/ml,0.1mg/ml,1mg/ml,5mg/ml,10mg/ml,25mg/ml and 50mg/ml.Mesalazine was divided into 8 concentration gradients of 0.001mg/L,0.01mg/L,0.1mg/L,1mg/L,10mg/L,25mg/L,50mg/L,100mg/L.MTT assay was used to detect the activity of LPS-induced HT-29 cells by Anchang Yuyang Decoction and Mesalazine.The effect of q-RT-PCR was used to detect the expression of IL-8 mRNA,and the optimal concentration of Anchang Yuyang Decoction and Mesalazine was screened.The apoptosis experiment and reactive oxygen species test were used to observe the induction of LPS by Anchang Yuyang Decoction.The effects of HT-29 cells on apoptosis and reactive oxygen species were observed.The effects of Anchang Yuyang Decoction on the expression of IL-13 and TFF3 in LPS-induced HT-29 cells were observed by ELISA and immunofluorescence experiments..(3)To explore the molecular mechanism of Anchang Yuyang Decoction in protecting inflammatory injury of HT-29 cells based on JAK1/STAT6 signaling pathway:using q-RT-PCR and Western Blot assay to detect IL-13,TFF3,JAK1,STAT6 after intervention of Anchang Yuyang Decoction.ResultClinical Research:1.The total effective rate of Anchang Yuyang Decoction combined with Mesalazine was 93.2%.2.The different evaluation time nodes were selected.and the scores of TCM symptoms at 4 week.8 week,and 12 week after treatment were significantly lower than those before treatment.The difference was statistically significant(P<0.01),and the symptom score at 4 week after treatment was lower than 8 week after treatment(P<0.01),the symptom score at 8 week after treatment were lower than 12 week after treatment(P<0.01).3.The Mayo score after treatment was significantly lower than that before treatment(P<0.01).4.The expression of TNF-? mRNA was significantly down-regulated after treatment(P<0.01),and the expression of IL-10 mRNA was significantly up-regulated before treatment(P<0.01).5.The Baron score of rectum colonoscopy after treatment was significantly lower than that before treatment of E1 type(P<0.01).The Baron score of rectum,sigmoid colon and descending colon after treatment were significantly lower than that before treatment of E2 type(P<0.01).6.Geboes scores of different intestinal segments before treatment:the rectum was higher than the sigmoid colon,descending colon,transverse colon,and ascending colon(P<0.01).The sigmoid colon was higher than the descending colon,transverse colon,and ascending colon(P<0.05),and no significant difference was found between descending colon,transverse colon,and ascending colon(P>0.05).It can be seen that the pathological scores of the intestinal segments before treatment are ranked from high to low:rectum>sigmoid colon>descending colon>transverse colon,ascending colon.The pathological Geboes scores of rectum,sigmoid colon were significantly lower than befo re(P<0.01).The descending colon,transverse colon,and ascending colon were all lower than before treatment(P<0.05).Geboes scores of different intestinal segments before treatment:rectum was higher than sigmoid colon(P<0.05).Rectum was significantly higher than descending colon,transverse colon,and ascending colon(P<0.01).Sigmoid colon was higher than descending colon,transverse colon,ascending colon(P<0.05).The descending colon was higher than the transverse colon and ascending colon(P<0.05).There was no significant difference between the transverse colon and ascending colon(P>0.05).It can be seen that the pathological scores of each intestinal segments before treatment are ranked from high to low:rectum>sigmoid colon>descending colon>transverse colon,ascending colon.Geboes score of rectum,sigmoid colon,descending colon,transverse colon and ascending,colon after treatment were significantly lower than those before treatment(P<0.01).7.Reduction of pathological Geboes scores:intestinal segment comparison of E1 type:The reduction of rectum was the highest,the rectum was higher than the igmoid colon,descending colon,transverse colon,ascending colon(P<0.01,P<0.05).the sigmoid colon was higher than transverse colon and ascending colon(P<0.05).The sigmoid colon was higher than the descending colon but the difference was not statistically significant(P>0.05).There was no significant difference between the descending colon,transverse colon and ascending colon(P>0.05).The score reduction of each intestinal segment was ranked from high to low:rectum>sigmoid colon>descending colon,transverse colon,ascending colon.Intestinal segment comparison of E2 type:The reduction of rectum was the highest,it was higher than the descending colon,transverse colon,ascending colon(P<0.01,P<0.05).The sigmoid colon was higher than the descending colon,transverse colon,ascending colon(P<0.05).The sigmoid colon was higher than the descending colon but the difference was not statistically significant(P>0.05).The descending colon was higher than transverse colon and ascending colon(P<0.05).There was no significant difference between the rectum and sigmoid colon(P>0.05).The score reduction of each intestinal segment was ranked from high to low:rectum,sigmoid colon>descending colon>transverse colon,ascending colon.8.There were no adverse reactions such as stomach discomfort,nausea,vomiting,headache,etc.during the treatment.No obvious blood routine,liver and kidney function,or abnormal electrocardiogram.Experimental research:1.Establishment and evaluation in vitro cell model of ulcerative colitis:20ug/ml LPS-induced for HT-29 cells after 24h can significantly up-regulated IL-8 mRNA expression and down-regulated IL-13 mRNA expression.This successfully establish the inflammation injury model of HT-29 cells.2.The protective effect of Anchang Yuyang Decoction on LPS-induced inflammatory injury of HT-29 cells:Anchang Yuyang Decoction has a proliferative effect on HT-29 at a concentration of 0.01?10mg/ml,at concentration of 0.1 mg/ml,1mg/ml,and 10 mg/ml inhibited IL-8 mRNA expression in a dose-dependent manner.Therefore,0.1mg/ml,1 mg/ml,and 10 mg/ml were selected as low.medium,and high intervention concentrations for subsequent experiments.Mesalazine has a significant proliferative effect on HT-29 at concentrations of 0.1 mg/L,1mg/L,and 10 mg/L,and inhibits IL-8 mRNA expression at a concentration of 1 mg/L.Therefore,The optimal drug concentration of 1mg/L as the positive drug control mesalazine was chosen.LPS induces inflammatory injury of HT-29 cells,causing an increase in intracellular reactive oxygen species.Anchang Yuyang Decoction could reduce the level of intracellular reactive oxygen species.The high concentration group was better than the low and middle concentration groups.There was no significant difference between high concentration group and the mesalazine group.HT-29 cells induced with LPS decrease the expression of IL-13 and TFF3.Anchang Yuyang Decoction could increase the expression of IL-13 and TFF3.The high concentration of Anchang Yuyang Decoction group was better than the low and middle concentration group.There was no significant difference between high concentration group and the mesalazine group.3.Based on JAK1/STAT6 signaling pathway,explore the molecular mechanism of Anchang Yuyang Decoction in protecting inflammatory injury of HT-29 cells:the expression of IL-13,JAK1,STAT6 and TFF3 mRNA and protein decreased after LPS intervention in HT-29 cells.Both Anchang Yuyang Decoction and Mesalazine can up-regulate the expression of IL-13,JAK1,STAT6 and TFF3 mRNA and protein.The high concentration group is superior to the low and medium concentration group.There was no significant difference between high concentration group and the mesalazine group.ConclusionAnchang Yuyang Decoction combined with Mesalazine has achieved satisfactory clinical efficacy in the treatment of ulcerative colitis,which can significantly relieve the symptoms,down-regulate pro-inflammatory factor TNF-?,up-regulate anti-inlammatory factor IL-10.The diseased and non-diseased intestinal sections of endoscopy showed different deg,rees of inflammatory injury in histopathology.Multi-segment and multi-point sampling on the biopsy for pathological scoring can accurately assess the intestinal mucosa deep healing.but also achieves histopathological inflammation relief,and is safe and reliable.Anchang Yuyang Decoction combined with mesalazine not only achieved endoscopic relief,but also achieved inflammation relief of histopathology of the whole intestinal segments especially of rectal and sigmoid colon,and it's safe and reliable.The combination of Anchang Yuyang Decoction and Mesalazine utilizes the advantages of integration of traditional Chinese and western medicine.The characteristics of multi-path and multi-target of traditional Chinese medicine make up for the shortcomings of single target of western medicine.Western medicine has fast onset and can induce rapid remission,which makes up for the shortcoming of traditional Chinese medicine.Traditional Chinese medicine and western medicine complement each other,improving efficacy and reducing side effects,accelerating and maintaining clinical remission,promoting the deep healing of the mucosa.Anchang Yuyang Decoction has a significant protective effect on LPS-induced HT-29 cells and promoted the proliferation of HT-29 cells.It can alleviate oxidative stress damage,promote the release of anti-inflammatory factors,and reduce the release of pro-inflaminatory factors.The mechanism may probably protect the intestinal mucosal barrier by up-regulating IL-13 secretion,activating JAK1/STAT6 pathway,promoting the secretion of TFF3. |
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