| BackgroundsThe mucosal epithelium of the alimentary tract represents a crucial barrier to a broad spectrum of noxious and immunogenic substances within the intestinal lumen.An impairment of the integrity of the mucosal epithelial barrier is observed in the course of various intestinal disorders including inflammatory bowel diseases(IBD),celiac disease,intestinal infections,and various other diseases.The integrity of the intestinal mucosal surface barrier is rapidly reestablished even after extensive destruction because of an enormous regenerative capability of the mucosal surface epithelium.The process of mucosal restitution refers to resealing of superficial wounds to this barrier,which occurs as a consequence of epithelial cell migration into the defect,a process that does not require epithelial cell proliferation.Epithelial restitution absolutely need intracellular polyamines.In an in vitro IEC-6 cell model which simulated mucosal restitution,polyamine was also needed to stimulate cell migration.Studies have shown that polyamine-mediated intracellular calcium signaling pathway is the core factor to promote IEC-6 cell migration.As the IEC-6 cell is a non-excitable cell,the cell membrane will not express voltage-gated calcium channels,the increase of the intracellular calcium ion concentration relies on potassiu-m ion channel expression and activation.This study will focus on the upstream potassium ion channel expression and downstream Rho GTPases changes of intracellular calcium,so as to investigate the mechanism of Atractylodes Macrocephala Koidz(AMK)promoting IEC-6 cell migration.Chinese medicine believes that the spleen is the source of Qi and Blood,spleen is the root of later heaven.Strong function of the spleen is an important factor in health and guaranteeing against the evils of the body.In recent years,the protection function of the gastrointestinal mucosa has been received more attention.The closely spaced epithelial cells of gastrointestinal mucosa and the integrity and continuity of the mucous layer of the epithelial surface constitute two important anatomical basis of gastrointestinal mucosal barrier.Gastrointestinal mucosal injury is an important pathological basis of certain syndro-mes,such as spleen deficiency syndrome,gastrointestinal mucosa protection is one of the major mechanisms of action of Traditional Chinese medicine replenishing qi to invigorate the spleen.Protection of the gastrointestinal mucosa is important content of the function of"Spleen",it is also an important target of Traditional Chinese medicine replenishing qi to invigorate the spleen.ObjectiveThe aim of this study was to observe the effect of AMK(one of the traditional Chinese medicines replenishing qi to invigorate the spleen)on IEC-6 cell migration and different sites of polyamine-mediated calcimu channel signaling pathway,so as to investigate the mechanism of AMK on repairing gastrointestinal mucosal injury.Methods1.AMK was ultrasonic extracted with 100%methanol,High performance liquid chro-matography(HPLC)was used for qualitative analysis of the main components of AMK extracts.2.The migration model of IEC-6 cells was established with a operating knife blade,fluorescence inverted microscope was used to observe and photograph the cell migration images.3.MTT method and RTCA(Real-Time Cell Analyzer)were used to investigate the effects of AMK on the proliferation of IEC-6 cells.4.Pre-column derivatization HPLC was applied to detect the polyamines(putrescine,spermidine and spermine)content during IEC-6 cell migration.5.Fluorescent dyes DiBAC4(3)and Fluo-3/AM were used to detect membrane pote-ntial(Em)and cytosolic free Ca2+ concentration([Ca2+]cyt)of IEC-6 cells by flow cytom-etry,respectively.6.The expression level of Kv1.1,TRPC1,PLC-β1 RhoA,Rac1,and Cdc42 mRNAs of IEC-6 cells was detected by RT-qPCR.7.The expression level of Kv1.1,TRPC1,PLC-γ1,RhoA,Rac1,and Cdc42 proteins of IEC-6 cells was detected by Western Blot analysis.8.The expression and distribution of Myosin II proteins were detected by Laser scann-ing confocal microscope.Results1.The yield of AMK after 100%methanol extraction by ultrasound was 10.5%,10.05%and 11.05%,respectively.Three main components of AMK,atractylenolide Ⅰ,Ⅱ,Ⅲ,were identified by HPLC.The fingerprint similarity of three batches of AMK was 0.995,0.989,and 0.995,respectively.These results indicated that the quality of AMK used in this study was stable and controllable.2.After establishment of the IEC-6 cell migration model,we screened three doses of 50,100 and 200 mg/L for subsequent migration experiments.Proliferation experiments showed that the three doses had no obvious effects on promoting IEC-6 proliferation within 24 or 36 h,which indicated that the epithelial restitution was mainly dependent on cell migration,these experiments also provided a good foundation for follow-up study.3.AMK 100 and 200 mg/L dosage significantly increased EEC-6 cell migration and improved polyamines(putrescine,spermidine,and spermine)content to varying degrees both in normal and polyamine dificient(plus DFMO)cases,which preliminary showed that AMK promoted IEC-6 cell migration may be related to its role of increasing intracellular polyamines content.4.AMK significantly increased Em and[Ca2+]cyt in normal and polyamine-deleted IEC-6 cells.5.IEC-6 cell migrationn was dramatically inhibited when 4-AP(a potassium channel inhibitor)was added to the culture,AMK 100 and 200 mg/L dosages reversed the inhibitory effects on cell migration induced by 4-AP;IEC-6 cell migration dramatically decreased in Ca2+-free media,100 and 200 mg/L AMK showed a weak effect to promote IEC-6 cell migration in Ca2+-free media.6.The mRNA and protein expression of Kv1.1,TRPC1,PLC-γ1,RhoA,Rac1,and Cdc42 were significantly increased by AMK(100 and 200 mg/L)treatment both in nomal and polyamine-dificient IEC-6 cells.In addition,AMK(100 and 200 mg/L)enhanced myosin Ⅱ protein expression,thus increased the formation of myosin Ⅱ stress fibers.ConclusionThe effect of AMK(one of the traditional Chinese medicines replenishing qi to invigorate the spleen)on repairing gastrointestinal mucosal injury was related to its role of promoting intestinal epithelial cell migration after wounding,the effective materia basis may be Atractylenolide Ⅰ,Ⅱ,Ⅲ present in AMK extract,the mechanism may be related to its role of affecting indexes of the polyamine-mediated calcium channel signaling pathway. |
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