| Objective:To explore the mechanism of electroacupuncture (EA) Back-shu and Front-mu points on EGC and GDNF-PI3K-Akt signal pathway in colon in functional constipation(FC), based on the effect of acupuncture, so as to provide a strong theoretical basis for acupuncture Back-shu and Front-mu points treating the disease.Methods:1-. In experiment 1, A total of 80 KM mice were randomly assigned to five groups:blank control (n=16),model (n=16),drug (n=16),electroacupuncture (EA; n= 16), blank acupuncture (n=16) groups. We established the model by compound diphenoxylate orally. The animals underwent electroacupuncture (EA) treatment at Tianshu (ST 25), Dachangshu (BL 25) on each side about alternate acupuncture for 30 minutes, once a day, a course of five treatments, and rest for two days between two courses. The Gastric emptying, The small intestine transmitting rate, the amplitude of the colon contraction were examined after each course.2、In experiment 2 and 3, A total of 80 KM mice were randomly assigned to five groups:control (n= 16), model (n= 16), electroacupuncture (EA; n=16), EGC control (n=16), EGC acupuncture (n=16). The method of establishing the model and make EA treatment were the same as experiment 1. We examined the morphology of the distal colon, the ultrastructure and apoptosis of SMC, the GDNF-PI3K-Akt and mTOR after each course by Digital scanning microscope, Electron microscope, TUNEL, Immunohistochemistry, in situ hybridization, PCR and Western Blot in the distal colon.Results:1、Gastrointestinal Transmitting Function:(1) Gastric emptying rateThe Gastric emptying rate in model group was significantly delayed (P<0.01), EA back-Shu and front-Mu points and drug can improve Gastric emptying rate (P<0.01, P<0.01). In the condition of health, the effect of acupuncture on gastric emptying rate was not obvious (P>0.05). Compared between 1,2 period of treatment, there was no statistically significant difference within each group (P>0.05).(2) Small intestine transmitting rateThe small intestine transmitting rate in model group was significantly decelerated (P<0.01). EA back-Shu and front-Mu points and drug can improve Small intestine transmitting rate (P<0.01, P< 0.01). In the condition of health, the effect of acupuncture on small intestine transmitting rate was not obvious (P>0.05). Compared between 1,2 period of treatment, there was no statistically significant difference within each group (P>0.05).(3) The percentage of average muscle tension change in colonic smooth muscleIn model group, the percentage of tension change in colonic smooth muscle was reduced (P>0.05), EA back-Shu and front-Mu points can improve the tension change percentage (P<0.05). In the condition of health, the effect of acupuncture on the percentage of average muscle tension change was not obvious (P>0.05). Compared between 1,2 period of treatment, there was no statistically significant difference within each group (P>0.05).2、The morphological changes of the distal colonIn control group, ganglion cells in the colonic myenteric plexus were visible. In model group, the number of nerve cells was reduced with vacuoles degeneration in nerve fibers. In acupuncture group, colonic morphologic change was not obvious compared with the control group. The change was not obvious between EGC control group and EGC acupuncture group. Compared between 1,2 period of treatment, there was no significantly difference within each group.3、The morphological changes of SMCIn control group, smooth muscle cells with evenly distributed chromatin, mitochondria, rough endoplasmic reticulum, ribosome were clearly observed. In model group, the nuclei showed chromatin pycnosis, with mitochondrial swelling, dilation of rough endoplasmic reticulum. In acupuncture group, the chromatin was distributed evenly, with clear mitochondria, rough endoplasmic reticulum, ribosome in the cytoplasm. After the inhibition of EGC cell, the morphological changes of smooth muscle cells were not obvious in EGC control and EGC acupuncture.4, SMC apoptosisThe positive expression rate of apoptosis cells was significantly increased in model group (P<0.01); EA back-Shu and front-Mu points can reduce the positive expression rate of apoptosis cells (P<0.01); after the inhibition of EGC cells, acupuncture can not reduce the positive expression rate of SMC apoptosis cells (P>0.05). Compared between 1,2 period of treatment, there was no statistically significant difference within each group (P>0.05).5, GDNF-PI3K-Akt(1) The expression of GDNF proteinIn model group, the content of GDNF protein was significantly reduced than that in control group (P<0.01). In acupuncture group, the levels of GDNF was increased (P<0.01). After the inhibition of EGC cell, acupuncture can not increase the expression of GDNF protein (P>0.05). Compared between 1,2 period of treatment, the content of GDNF protein was significantly increased (P<0.05), withnot significantly difference in other groups (P>0.05).(2) The expression of Rai protein and mRNAIn model group, the content of Rai protein and mRNA was significantly reduced than those in control group (P<0.01, P<0.05). After the inhibition of EGC cell, acupuncture can increase the expression of Rai protein and mRNA (P<0.05). Compared between 1,2 period of treatment, the content of Rai protein was significantly increased (P<0.01).(3) The expression of RET protein and mRNAIn model group, the content of RET protein and mRNA was significantly reduced than those in control group (P<0.01, P<0.01). In acupuncture group, the levels of them were increased (P<0.01, P<0.01). After the inhibition of EGC cell, the change was not obvious between EGC control group and EGC acupuncture group (P>0.05, P>0.05). Compared between 1,2 period of treatment, there was no statistically significant difference within each group (P> 0.05, P>0.05).(4) The expression of PI3K and AktIn model group, the content of PI3K and Akt was significantlyincreased than that of control group (P<0.01, P<0.01). In acupuncture group, the level of Akt was decreased (P<0.05). After the inhibition of EGC cell, the change was not obvious between EGC control group and EGC acupuncture group (P>0.05). Compared between 1,2 period of treatment, there was no statistically significant difference within each group (P>0.05, P>0.05).(5) The expression of mTORIn model group, the content of mTOR was significantly increased (P<0.01). Acupuncture can inhibit the expression of mTOR in colon, but there was no statistically significant difference compared with the model group (P>0.05). After the inhibition of EGC cell, the change was not obvious between EGC control group and EGC acupuncture group (P> 0.05). Compared between 1,2 period of treatment, there was no statistically significant difference within each group (P>0.05).Conclusions:1、In the condition of health, the effect of EA back-Shu and front-Mu points was not obvious on gastrointestinal transmitting function.2、In the condition of functional constipation, EA back-Shu and front-Mu points can accelerate the GDNF secretion in EGC cells, regulate PI3K-Akt signaling pathway, inhibit the expression of mTOR, and reduce the cell apoptosis, improve the colon morphology and gastrointestinal transmission function.3、After the inhibition of EGC cell, the effect of EA back-Shu and front-Mu points was not obvious on the morphological changes of the colon and SMC, the expression of GDNF-PI3K-Akt. The study confirmed the effect of EGC from the opposite side. |
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