Based On The TNF-?/NF-?B/Snail1 And TGF-?1/Smad3/Snail1 Pathways, The Regulatory Mechanism Of Acupuncture On Crohn's Disease Intestinal Epithelial-mesenchymal Transition Was Studied

ObjectTo elucidate whether acupuncture and moxibustion can inhibit the overexpression of TGF-?and TGF-?1 in intestinal epithelial cells of patients with Crohn's disease,and then inhibit intestinal epithelial-mesenchymal transition mediated by TNF-?/NF-?B/Snail1 and TGF-?1/Smad3/Snail1 pathways to provide scientific basis for acupuncture and moxibustion to regulate the intestinal epithelial-mesenchymal transition in CD.Methods1.63 patients with CD were randomly divided into observation group and control group.Patients in the observation group were treated with 43?mild moxibustion combined with acupuncture,while the control group was treated with 37?mild moxibustion combined with sham acupuncture.The acupoints in both groups were same.Moxibustion acupoints were bilateral Tianshu?ST25?,Zusanli?ST36?and the acupuncture points were bilateral Shangjuxu?ST37?,Sanyinjiao?SP6?,Gongsun?SP4?,Taichong?LR3?,Taixi?KI3?,Hegu?LI4?,Quchi?LI11?.The two groups were treated once every other day for 30 minutes,3 times a week for 12 weeks.CDAI score,abdominal pain,diarrhea,abdominal distension,fatigue,poor appetite,soft waist and knee,cold limbs,intestinal sound,sigh and tenesmus were used to evaluate the clinical effect.2.10 volunteers in each group were taken a little intestinal mucosa tissue before and after treatment.H&E stain and electron microscope were used to evaluate intestinal morphology and microstructure;immunohistochemistry was used to observe TNFR1?TNFR2?NF-?B?T?R1?T?R2?smad3?Snail1?E-cadherin?Fibronectin levels in TNF-?/NF-?B/Snail1 and TGF-?1/Smad3/Snail1 pathway accordingly;In addition,RT-q PCR was used to observe Snail1?E-cadherin?Fibronectin m RNA,and double immunofluorescent staining was used to observe coexpression of Snail1/E-cadherin and Snail1/Fibronectin.Results1.CDAI score showed that:compared with pre-treatment,the CDAI score of patients in both of observation group and control group decreased significantly after treatment and 3-month follow-up period?both P<0.01?,and improvement of CDAI score in observation group was significantly better than that in control group after treatment and 3-month follow-up period?P<0.01?.The improvement of CDAI score in the active stage and remission stage was better than that in the control group after treatment and3-month follow-up period(P_active<0.05,P_remission<0.01).2.Tradtional Chinese medicinal symptom score evaluation showed that the abdominal pain?degree,frequency,time?,diarrhea?number of diarrhea,character,frequency?,abdominal distension score,fatigue,poor appetite,soft waist and knee,cold limbs,intestinal sound,sigh and tenesmus were improved in observation group after treatment?P<0.01-0.05?;abdominal pain?degree,frequency,time?,diarrhea?number of diarrhea,character,frequency?,abdominal distension score,fatigue,poor appetite,intestinal sound,sigh and tenesmus were also improved in control group?P<0.01-0.05?.After treatment,the improvement values of abdominal pain?degree,frequency?,diarrhea?number of diarrhea,character,frequency?,fatigue and cold limbs in observation group were better than those in control group?all P<0.05?.3.Histological observation of intestinal mucosa in observation group showed that the intestinal mucosal epithelial cell proliferation and repair,mild hyperemia in the lamina propria,significantly decreased inflammatory cell infiltration and a small amount of fibrous tissue hyperplasia could be seen after treatment;ultrastructural observation showed that the tight junctions between intestinal epithelial cells were arranged in order,swelling of mitochondria and endoplasmic reticulum were reduced,microvilli were arranged neatly,and mucus vacuoles were seen in the cells under electron microscope.After treatment in the control group,the lamina propria of intestinal mucosa was hyperemia and edema and a small amount of inflammatory cells infiltrated under light microscope,and the tight junctions between intestinal epithelial cells were arranged in an orderly manner,and mitochondria and endoplasmic reticulum swelled slightly under electron microscope.4.Immunohistochemical results showed that compared with pre-treatment,expressions of TNF-?,TNFR1,NF-?B in the observation group was decreased(P_TNF-?<0.01,P_TNFR1<0.05,P_NF-?B<0.05).Expressions of TNF-?,TNFR1,decreased in control group?all P<0.05?.After treatment,the expressions of TNF-?and TNFR1 in observation group were lower than those in control group?all P<0.05?.5.Immunohistochemical results showed that compared with pre-treatment,expressions of TGF-?1,T?R2,Smad3 in observation group was significantly decreased(P_TGF-?₁<0.05,PT?_R2<0.01,P_Smad3<0.05);in control group,expressions of T?R2 and Smad3 were decreased?all P<0.05?.After treatment,compared with control group,expressions of TGF-?1 and T?R2 in observation group were decreased?all P<0.05?.6.Immunohistochemical results showed that compared with pre-treatment,expressions of Snail1?E-cadherin?Fibronectin in observation group was significantly decreased(P_Snail1<0.01,P_Fibronectin<0.01),expression of E-cadherin was significantly increased?P<0.01?;in control group,expressions of Snail1 and Fibronectin were decreased?all P<0.05?,and the expression of E-cadherin was increased?P<0.05?.After treatment,compared with control group,expressions of Snail1 and Fibronectin in observation group were decreased?all P<0.05?,and expression of E-cadherin was increased?P<0.05?.7.RT-q PCR results showed that,compared with pre-treatment,Snail1 and Fibronectin m RNA in observation group were decreased(P_Snail1<0.01,P_Fibronectin<0.05),and E-cadherin m RNA was increased?P<0.05?.In control group,compared with pre-treatment,expressions of Snail1 and Fibronectin m RNA in intestinal mucosa epithelium were decreased?all P<0.05?,and the E-cadherin m RNA was increased?P<0.05?.After treatment,compared with control group,Snail1 and Fibronectin m RNA in observation group were decreased?all P<0.05?,and E-cadherin m RNA was increased?P<0.05?.8.The co-expressions of Snail1/E-cadherin and Snail1/Fibronectin in intestinal epithelium of all groups showed that compared with pre-treatment,expressions of Snail1and Fibronectin were significantly decreased in observation group(P_Snail1<0.01,P_Fibronectin<0.01),expression of E-cadherin was increased?P<0.05?;In control group,expressions of Snail1 and Fibronectin were decreased?P<0.05?,while the expression of E-cadherin was increased?P<0.05?.After treatment,compared with control group,expressions of Snail1and Fibronectin in observation group were decreased?all P<0.05?,and expression of E-cadherin was increased?P<0.05?.Conclusions:1.Acupuncuture and moxibusion can decrease CDAI score in both active and remission stage patients and improve symptoms of abdominal pain,diarriah,fatigue and limbs cold.2.Acupuncture and moxibustion?mild moxibustion combined with acupuncture?can inhibit the overexpression of TGF-?and TGF-?1 in intestinal epithelial cells of patients with Crohn's disease,and then inhibit intestinal epithelial-mesenchymal transition mediated by TNF-?/NF-?B/Snail1 and TGF-?1/Smad3/Snail1 pathways...