| Objective The aim of this study is to observe the effects of Sparganii Pill Decoction treating intestinal fibrosis in rats with rats model of intestinal fibrosis of Crohn's disease,to investigate the effects of Sparganii Pill Decoction on the expressions of E-cadherin,a-SMA and PPAR-y in the epithelial mesenchymal transition(EMT)process of transforming growth factor betal(TGF?1)-induced intestinal epithelial cells(IEC-6).and to explore the mechanism of reversing EMT of intestinal fibrosis through TGF-?1/Smads signal pathway by Sparganii Pill Decoction.Methods Vivo experimental part:48 SD rats were randomly divided into six groups in this study:blank control group(n=8);TNBS model group:TNBS rats without any treatment except oral saline(n=8);Rosiglitazone control treatment group:TNBS rats orally treated with Rosiglitazone(8mg kg-1day-1)(n=8);low-dose treatment group:TNBS rats orally treated with Sparganii Pill(2.5g kg-1day-1)(n=8);middle-dose treatment group:TNBS rats orally treated with Sparganii Pill(5g kg-1day-1)(n=8);high-dose treatment group:TNBS rats orally treated with Sparganii Pill(10g kg-1day-1)(n=8).All the treatment groups of rats were anesthetized with ketamine hydrochloride and received a weekly intracolonic instillation of 5%TNBS with a dose of 10 mg,15 mg,20 mg,25 mg and 30mg on the 1st,8th,15th,22th and 29th day respectively,with 50%ethanol(0.8mL)via a 2 mm diameter catheter inserted approximately 8 cm proximal to the anus.The blank control rats were given the enema with an equal volume of 50%ethanol.The rats were held in a vertical position for 1 minitue after the intrarectal injection to ensure the distribution of TNBS within the entire colon and cecum.Daily administration started on 1st day and continued for 5 weeks.On 35th day after TNBS administration and Sparganii Pill treatment,all the rats were killed.The general condition of the rats in each group was observed,and the colon specimens were collected for the pathological evaluation.The levels of TGF-?1,E-cadherin,?-SMA,FN,CTGF and PPAR-y were detected.Vitro experimental part:In the EMT model of TGF-?1-induced IEC-6 cells,Western blot method was used for the detecting PPAR-y,Smad2/3 and psmad3 protein levels of high,middle and low dose groups of Sparganii Pill-medicated serum and rosiglitazone,the mRNA expression levels of E-cadherin and a-SMA were tested by RT-PCR,and the effects of on the nuclear translocation level of PPAR-?,pSmad3 by Sparganii Pill-medicated serum and rosiglitazone groups were observed.After adding PPAR-y antagonist GW9662,the changes of the same related indicators were detected.Results Vivo experimental part:Compared with model group,disease activity index(DAI),fibrosis and the general colon tissue scores of Sparganii Pill high dose group were significantly lower than in the model group(P<0.05),and the Masson staining showed that collagen fiber content of the Sparganii Pill high dose group was significantly lower than in the model group;E-cadherin,PPAR-y protein levels in Sparganii Pill high dose group were significantly increased(P<0.05),while TGF-?1,FN,a-SMA levels were significantly decreased(P<0.05).Vitro experimental part:Compared with 10%normal serum of rats group,Sparganii Pill-medicated serum group inhibited the level of a-SMA mRNA expression,improved the protein expression levels of E-cadherin and PPAR-y,suppressed the protein expressions of pSmad3,?-SMA,promoted the mRNA expressions of E-cadherin and PPAR-y;Immunofluorescence results showed that the 10%Sparganii Pill-medicated serum group promoted the nuclear import of PPAR-y,and inhibited Nuclear entry of the pSmad3,while adding PPAR-y antagonist GW9662 reversed the trend of change for the above indicators.Conclusion Sparganii Pill Decoction can show an effect of anti-intestinal fibrosis in rats intestinal fibrosis model with CD,and also show an inhibited effect of the EMT for IEC-6 cells.The mechanism of Sparganii Pill Decoction treating intestinal fibrosis of Crohn's disease may be related to inhibition of TGF-(31/Smads signaling pathway by activated PPAR-?. |
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