| Objective:To observe the therapeutic effect of acupoint catgut embedding on ulcerative colitis(UC)in rats with "deficiency and stasis" and the effect on intestinal mucosal epithelial barrier;To explore the protective mechanism of acupoint catgut embedding on intestinal mucosal epithelial barrier injury based on PLC dependent signal pathway.Methods:1.In vivo experimentation: Effect of Catgut Embedding at acupoints on intestinal mucosal epithelial barrier in UC rats with “deficiency and stasis”.60 male SD rats were randomly divided into the normal group(n=12)and the model group(n=48).In the model group,adenine and senna were used to instill the "deficiency and stasis" state of the rats by stage instillation,and then 5%2,4,6-trinitrobenzenesulfonic acid(TNBS,100mg/kg)dissolved in 0.25 ml of 50% ethanol enema were used to establish the rat UC model.The model group was randomly divided into model group,salazopyridinium(SASP)group and acupoint embedding group each with 10 rats after successful modeling.The catgut embedding was applied to bilateral "Tianshu","Zusanli","Geshu","Pishu","Shenshu" and "Dachang shu" points in the line embedding group,once every 14 days,3 times altogether.Rats of the salazopyridine group were treated 42 days by intragastric perfusion of salazopyridine once a day.The general condition,body mass,anal temperature and hematochezia were observed,and the disease activity index(DAI)was calculated.The colon tissues were collected for observing appearance and hispathological changes with the naked eye and HE staining at the end of experiments.The contents of serum D-lactic acid(D-LA),diamine oxidase(DAO),protein kinase C(PKC),calcium-calmodulin dependent protein kinases II(CAMK2)and myosin light chain kinase(MLCK)were detected by ELISA.The mRNA expression levels of occludin,claudin1,claudin2,claudin8,zo-1,cingulin and zonulin in the colon tissues of rats were detected using quantitative RT-PCR and the protein expression levels of occludin,claudin1,claudin2,claudin8,zo-1,cingulin,zonulin,PKC,CAMK2 and MLCK in the colon tissues of rats were detected by Western blot.2.In vitro experiment: The regulatory effect of serum on Caco-2 cell barrier injury based on PLC dependent signal pathway after Catgut Embedding at acupoints.Caco-2cells were cultured for 23 days to establish intestinal epithelial barrier model in vitro,then the cells were divided into 7 groups: control group,model group,control rats serum group,acupoint catgut embedding rats serum group,acupoint catgut embedding rats serum plus PLC inhibitor group,acupoint catgut embedding rats serum plus negative inhibitor group,and PLC inhibitor group.No treatment was given to the control group and the other 6 groups were incubated with 50mmol/L ethanol for6 hours,resulting in impaired intestinal mucosal epithelial barrier function.In addition to the model group,the other 5 groups adopted the corresponding treatment intervention program,and at least 3 different generations of cells were selected for each experiment.The structure of Caco-2 cells in each group was observed by transmission electron microscope.The intestinal epithelial barrier function of each group was evaluated by the measurement of transepithelial electrical resistance(TEER)and sodium fluorescein transmittance,the protein expressions of protein kinase C(PKC),calcium-calmodulin dependent protein kinases(CAMK2)and myosin light chain kinase(MLCK)were detected by Western Blot.Results1.Compared with normal group,body quality of rats in model group significantly reduced(P<0.01),colon tissues of rats in model group showed markedly swollen,disordered arrangement of intestinal mucosal cells,hemorrhage with infiltration of inflammatory cells,the DAI score,the colonic histological injury index score and the colonic mucosa damage index score were remarkably higher(P<0.01),the content of serum D-LA,DAO,PKC,CAMK2 and MLCK were significantly increased in the model group(P<0.01),the mRNA and protein expression levels in colonic tissues of occludin,claudin1,claudin8 zo-1 and cingulin were significantly decreased(P<0.01),the claudin2 and zonulin mRNA and protein expression level were significantly increased(P<0.01),and the protein expression level of PKC,CAMK2 and MLCK were significantly increased(P<0.01).Compared with model group,body mass of rats in SASP group and acupoint embedding group were significantly increased(P<0.01),colon tissues lesion of rats in SASP group and acupoint embedding group was relatively milder,the DAI score,the colonic histological injury index score and the colonic mucosa damage index score were remarkably lower(P<0.01,P<0.05),the content of serum D-LA,DAO,PKC,CAMK2 and MLCK were significantly decreased(P<0.01),the mRNA and protein expression levels in colonic tissues of occludin,claudin1,claudin8 zo-1 and cingulin were significantly increased(P<0.05,P<0.01),the claudin2 and zonulin mRNA and protein expression level was significantly decreased(P<0.01),and the protein expression level of PKC,CAMK2 and MLCK were significantly decreased(P<0.01,P<0.05).No significant differences were found in all indexes between the acupoint line embedding group and the salazosulfopyridine group(P>0.05).Conclusion Acupoint catgut embedding can regulate the tight junction of colonic epithelial,repair the intestinal mucosal epithelial barrier,and promote intestinal mucosal healing,which may be related to inhibiting the activation of PKC,CAMK2 and MLCK.2.Compared with control group,the cell gap in the model group and the control rats serum group was widened,the arrangement of cells was loose,the mitochondria was mostly denatured,and the cell nucleus and cytoplasm were also interstitial.TEER was significantly reduced(P<0.01),the transmittance of luciferin sodium was significantly increased(P<0.01),and the protein expressions of PKC,CAMK2 and MLCK were significantly increased(P<0.01).Compared with model group,tight junction of cells in acupoint catgut embedding rats serum group,acupoint catgut embedding rats serum plus PLC inhibitor group,acupoint catgut embedding rats serum plus negative inhibitor group,and PLC inhibitor group were increased and closed,with some expansion of rough endoplasmic reticulum and increased mitochondria,TEER are significantly increased(P<0.01),sodium fluorescein transmittance decreased significantly(P<0.01),PKC,CAMK2 and MLCK protein expression level decreased significantly(P < 0.01,0.05);Compared with the acupoint catgut embedding rats serum group and PLC inhibitor group,cells in the acupoint catgut embedding rats serum plus PLC inhibitor group showed more tight junction under endoscopy,significantly increased TEER(P<0.01),significantly reduced the transmittance of luciferin sodium(P<0.01,0.05),and significantly decreased the protein expressions of PKC,CAMK2 and MLCK(P<0.01,0.05).There was no significant difference between the model group and the control rats serum group,and between the acupoint catgut embedding rats serum group and the acupoint catgut embedding rats serum plus negative inhibitor group(P>0.05).Conclusion1.Acupoint catgut embedding can improve the blood and body mass of rats with ulcerative colitis under the state of "deficiency and stasis",reduce DAI,mucosal damage index and tissue morphological damage index score;2.Acupoint catgut embedding can up regulate the expression of occludin,claudin1,claudin8,ZO-1,cingulin,down regulate the expression of claudin2 and zonulin of ulcerative colitis rats.It can reduce the permeability of mucosal epithelium and promote the healing of intestinal mucosa.3.Acupoint catgut embedding can reduce the expression of PKC,CAMK2 and MLCK in serum and colon tissue of rats with ulcerative colitis;4.The serum of rats after catgut embedding at acupoint can increase the TEER of Caco-2cells damaged by ethanol,reduce the transmission rate of fluorescein sodium,and reduce the expression of PKC,CAMK2 and MLCK;5.Catgut embedding at acupoint may inhibit protein expression of PKC,CAMK2 and MLCK by inhibiting PLC signal pathway,thereby regulating tight junction protein to repair the intestinal mucosal barrier in the treatment of ulcerative colitis. |
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