| ObjectivePrevious studies in our research group show that acupuncture has a definite therapeutic effect on the ulcerative colitis(UC)model induced by TNBS(2,4,6-Trinitrobenzene sulfonic acid,TNBS)in SD rats.Based on this,in this study,we focused on investigating the effect mechanism of acupuncture on ultrastructural alterations of colon with UC,oxidative stress factors,the expressions of key proteins in inflammatory signal pathway and endoplasma reticulum stress in rat UC models,hoping to provide a new experimental data for revealing the mechanism of acupuncture treatment on UC model.MethodWe established the UC model by intrarectally administrating TNBS.After four days,both"Zusanli" and "Quchi" were manual-acupunctured(MA)or electro-acupunctured(EA)with stainless steel needle for 20 min for one time every other day,and Methanazine(500mg/kg)by intragastric administration as western medicine group.General clinical symptoms of rats in each group were carely observed and recorded during the treatment period.Rats after anesthesia were executed to shear the colon tissue and observe the appearance and pathological changes by HE staining.The ultrastructural changes of the colon were observed by scanning electron microscope(SEM)and transmission electron microscope(TEM),the content of inflammatory cytokines and factors related to oxidative-stress and analgesia in rat colon tissues were detected by ELISA,Bcl-2,Bax,cleaved-caspase3 and phosphorylated(p)-inhibitor of nuclear factor kappa B kinase a(I?B?),p-p65 and the key proteins of the endoplasmic reticulum stress(ERS)of colonic tissues were detected by Western blot separately.Results1.Acupunture treatment alleviates the TNBS induced pathologic changes of UC(1)The general condition of UC model rat was worse than the control,with more severe diarrhea and a lot of feces in the perianal area,and the body weight increased more slowly.At the end of the experiment,the stool was still soft.Both MA and EA treatment significantly improved the clinical symptoms of the rats,and the effect of EA was better than that in MA and the Mesalazine group.(2)After necropsy,rats in the model group had large colon and severe cecal swelling with a small amount of hemorrhage.The morphological score of the colon was significantly higher than that of the control(P<0.01).The colon histomorphological score of the EA group was the lowest,followed by MA group and Mesalazine group.(3)Light microscopy results:compared with the control,the colonic mucosal cells were arranged irregularly with a large number of inflammatory cells infiltrated,congestion and edema.The colonic mucosa of other three treatment groups was almostly intact with infiltration of a small amount of inflammatory cell and hyperemia.2.Acupuncture treatment abates the TNBS induced ultrastructural damage of colon tissue in UC models(1)Scanning electron microscopy results showed that compared with the control,the rats in the model group had severe damage to the colon mucosa,damaged villi,severe microvilli defects and even disappeared,and a large number of bacterial attachments were observed at the damaged sites.The MA group showed a significant reduction in intestinal mucosal lesions,but the mucosal surface still had various degrees of damage,and some microvilli were in lodging.The Mesalazine group was similar to MA group.The status of the intestinal mucosa in the EA group was close to the control group.(2)Transmission electron microscopy results showed that the microvilli were more sparse and shorter in the model group relative to those of the control.Meanwhile,there were unevenly distributed nuclear chromatin,swollen mitochondria,breaked or disappeared mitochondrial ridges and expanding endoplasmic reticulum in the colon tissue of model group.After treatment with EA or MA,microvilli appearred neatly and mitochondria and endoplasmic reticulum were slightly swollen,which was closer to the control.The mesalazine group also has similar changes.3.Effects of acupuncture on oxidative stress factors,proinflammatory cytokines and pain related factors in UC models(1)In terms of antioxidant level,EA treatment can significantly increased the expression of SOD,CAT,and GSH-Px in the colon tissue and reduce the content of MDA(P<0.01,P<0.05),and the effect is better than the other treatment group.(2)Compared with the model group,EA treatment significantly decreased the levels of TNF-a,IL-1?,myeloperoxidase(MPO)and homocysteine(Hcy)in the colon of rats(P<0.01,P<0.05),while increasing the expression of IL-10.The MA and the Mesalazine group have similar trends.(3)Compared with the control,the level of ?-EP in the rats of the model group was significantly reduced,content of 5-HT were significantly higher.The treatment of acupunture can reverse the level of the above factors.4.Effect of acupuncture on the expressions of NF-?B and ERS signals in UC model rats(1)Compared with the control,the ratio of Bcl-2/Bax in the model group was significantly decreased(P<0.01),and the expression level of cleaved-caspase3 was significantly increased(P<0.01).Acupuncture treatment could reverse this change,and the EA group was closer to the control.(2)The level of p-lKBa and p-p65 in the model group was significantly increased(P<0.01).EA or MA treatment significantly inhibited their expression(P<0.01,P<0.05).(3)The expression of GRP78,p-PERK and p-eIF2a were significantly increased in the model group compared with the control(P<0.01,P<0.05),EA treatment significantly inhibited the expression of those(P<0.01).There is a similar trend in the MA group and Mesalazine group.However,levels of p-IRE1,XBP-1s and ATF6 were not significantly different among the groups(P>0.05).Conclusions1.Acupuncture can protect UC model induced by TNBS in rats,and EA has the best effect and it is better than the western medicine(Mesalazine).2.Acupuncture can downregulate the phosphorylation of key proteins in the NF-?B signaling pathway and descrese the levels of pro-inflammatory cytokines,increasing the secretion of anti-inflammatory cytokines,and at the same time,regulating the levels of oxidative stress,analgesic and pain-causing factors to achieve the role of protecting UC model.3.TNBS can activate the unfolded protein response(UPR)signal pathway in ERS.Acupuncture maybe reduce the endoplasmic reticulum stress via regulating the UPR-PERK-eIF2a pathway and protect the colonic mucosa. |
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