| Objective1.To study the effect of electroacupuncture at "Zusanli","Liangmen" and"Sanyinjiao" on gastrointestinal motility disorder in Diabetic Gastroparesis(DGP)rats.2.To observe the effect of eleetroacupuncture on the ultrastructure of interstitial cells of Cajal(ICC)in gastric antrum of DGP rats and the expression of SCF,c-kit protein in the gastric antrum of DGP rats.3.To investigate whether the effects of electroacupuncture(EA)performed on gastrointestinal motility in DGP rats based on the concentration of intracellular Ca2+and the currents of Calcium-activated-chloride channels(CaCCs)as well as the expression of its Channel protein TMEM16A of interstitial cells of Cajal(ICC)or not.MethodsAccording to the literature modeling method,DGP model was established by intraperitoneal injection of streptozotocin(STZ,55mg/kg,2%),and raised with high-sugar high-fat diet irregularly for 8 weeks;EA was applied to Zusanli(ST36),Liangmen(ST21)and Sanyinjiao(SP6),sparse-dense wave were used in the EA group(sparse wave:10HZ,dense wave:50HZ).The rats in metoclopramide group,received an oral gavage of metoclopramide(1mL/100g).Blood sugar was determined using a OneTouch blood glucose meter.The gastric emptying rate(GER)and the intestinal propulsion rate(IPR)were measured by an oral gavage of phenol red.The ultrastructure of ICC was detected by electron microscopy.The expression levels of SCF,c-kit protein of gastric antrum tissue were examined respectively by Western blot.Interstitial cells of Cajal were isolated and cultured from gastric antral tissue.The changes of Ca2+ concentration in Cajal interstitial cells were detected by immunofluorescence.Whole-cell patch clamp was used to detect the calcium-activated chloride channel(CaCCs)current in Cajal interstitial cells.Inmunohistochemistry was used to detect the expression level of TMEM16A which is the specific protein of calcium-activated chloride channel.Results1.DGP models were made successfully,Compared with the normal control group,symptoms integral and blood sugar level in the model group were significantly higher(P<0.01),the weight decreased significantly(P<0.01),the GER and IPR as well as the percentage of normal slow wave and the main power were significantly decreased(P<0.01);Compared with the model group,symptoms integral and blood sugar level in the EA-acupoint group were significantly decreased(P<0.01),The GER and IPR in the EA-acupoint group were significantly increased(P<0.05,P<0.01),as well as the percentage of normal slow wave and the main power were significantly increased(P<0.05);Compared with the model group,The GER and IPR in the metoclopramide group were significantly increased(P<0.05,P<0.01),as well as the percentage of normal slow wave and the main power were significantly increased(P<0.05);Compared with the metoclopramide group,symptoms integral in the EA-acupoint group were significantly decreased(P<0.05).2.Comparison of the ultrastructural of ICC of gastric antral among the groups:Compared with the normal control group,ICC ultrastructure of the model group showed apoptotic state,ICC ultrastructure of the EA acupoint group has a complete basement membrane,there was a great number of mitochondria,Golgi apparatus and rough endoplasmic reticulum in cytoplasm,and the structure is still clear,occasionally swelling of mitochondria,and gap junctions between adjacent smooth muscle cells still exist;there were no significant differences between the metoclopramide group and the model group.Comparison of the expression level of SCF,c-kit protein in gastric antrum of rats:Compared with the normal control group,the expression level of SCF,c-kit protein in the gastric antrum were significantly increased in the model and metoclopramide group(P<0.01,P<0.05),while there is no significant difference on the expression level of SCF,c-kit protein between the EA-acupoint group and the control group(P>0.05).3.The primary cultured cells of ICC was irregular triangular,star-shaped,or spindle-shaped,with multiple protrusions.With the prolongation of culture period,The protrusions are connected to each other to form a network.The cells were positive for immunofluorescence staining of c-kit antibody,indicated thatin vitro culture of primary ICC was successful.Compared with the normal control group,the intracellular Ca2+ concentration of the ICC was lower in the model group(P<0.01),and the calcium chloride channel current(CaCCs)decreased in the model group.Compared with the model group,the intracellular Ca2+ concentration of the ICC was increasedin the EA-acupoint group and themetoclopramide group(P<0.05),and the CaCCs increased in the two groups.Compared with the EA-acupoint group,the CaCCs decreased in the metoclopramidegroup.Compared with the normal control group,the expression level of TMEM16Ain gastric antrum was significantly decreasedin the model group(P<0.01),and there was no significant differences among other groups(P>0.05).Conclusions1.Electroacupuncture at Zusanli,Liangmen,Sanyinjiao on DGP rats can regulate the gastrointestinal motility.2.The mechanism of DGP gastrointestinal motility disorder may be related to the inhibition of abnormal expression of SCF-kit protein so as to restore the ultrastructure of gastric antrum Cajal interstitial cells.3.The theraputic effect of gastrointestinal motility disorder in DGP rats by electroacupuncture may be related to the concentration of intracellular Ca2+ and calcium-activated chloride channel current(CaCCs)in ICC of gastric antrum in DGP rats. |
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