| Objective: Functional constipation(FC)is a common refractory disease in clinical practice.Acupuncture has long-lasting,safer effects and outstanding advantages on the treatment of FC.However,the concrete mechanism about acupuncture treatment for FC remains unclear.Basic researches have shown that gastrointestinal transmission dysfunction caused by imbalance of intestinal flora is the main pathological reason of protracted FC.Therefore,in this study,intestinal microflora was taken as the starting point,from the short chain fatty acids(SCFAs)and 5-HT,which are closely related to gastrointestinal transport function,to compare and observe the effect of acupuncture on the different between a mice FC model and a mice pseudo-germfree(PGF)model,to deeply explore and reveal molecular mechanism of He-Mu points acupuncture treatment for FC with regulating intestinal flora and then improving gastrointestinal transport function on both sides.Which provides a scientific evidence for the clinical application of acupuncture in FC.Methods: Forty female Kunming mice were randomly divided into Normal Control(NC)group,FC model group,FC model + Electroacupuncture(FC + EA)group,PGF model group,PGF model + EA(PGF+EA)group.FC model group and FC+EA group were given compound diphenoxylate by gavage to establish a mice FC model.Antibiotic cocktail method(ampicillin,neomycin sulfate,metronidazole and vancomycin hydrochloride were mixed into drinking water)was used to eliminate the intestinal flora of mice to establish a mice PGF model in PGF model group and PGF +EA group.After 14 days of continuous modeling,mice in each group were given 0.1 ml of activated carbon suspension by gavage after fasting but not water for 12 h,and the first black stool discharge time of mice was recorded.Comparing with the data of mice in the control group has a statistical significance difference(P<0.01)was used as the criterion to determine the success of the model.After successful modeling,the mice in each group continued to model according to the above methods to maintain the stability of the model.At 1h after modeling,FC+EA group and PGF+EA group were treated with electroacupuncture.The ipsilateral "Tianshu" and "Shangjuxu" points were selected to sequentially insert needles into the left and right sides.After inserting needles,the EA apparatus was connected with a dilatational wave.The current intensity was appropriate for slight shaking of the end of the limbs of the mice,and the needles were retained for 30 min once a day.5 times a course of treatment,2 days rest between courses,a total of 2 courses of treatment.After the last treatment,to observe and detect the following four closely related research contents.1.The time of excretion of the first black feces,the number and weight of fecal grains in 8h,and calculate the fecal water content based on the fecal quality were gathered and recorded in combination with gastric emptying rate,small intestinal propulsion rate to evaluate the gastrointestinal transit function of mice.And the pathological morphological changes of distal colon were detected by HE staining.To observe the difference of He-Mu points acupuncture effects directly on the gastrointestinal transit function of mice between FC model group and PGF model group.2.DNA was extracted from colon contents of mice,and 16 S r RNA high-throughput sequencing was used to detect and analyze the diversity and structural composition of intestinal flora of mice in each group.To analyze and evaluate the difference of He-Mu points acupuncture regulation on the intestinal flora of mice between FC model group and PGF model group.3.Changes of SCFAs content in colon contents of mice in each group,were detected by GC-MS(gas chromatography/mass spectrometry).To analyze and evaluate the difference of He-Mu points acupuncture effects on the main metabolites of intestinal flora SCFAs of mice between FC model group and PGF model group.4.The content of 5-HT in colon of mice in each group were detected by ELISA,the protein expression of 5-HT4 R and SERT in colon of mice were detected by Western blot,and the gene expression of 5-HT4 R,SERT in colon of mice were detected by RT-q PCR.To analyze and evaluate the difference of He-Mu points acupuncture effects on the 5-HT and its receptor and transporter in colon of mice between FC model group and PGF model group.Result: 1.Changes of gastrointestinal transport function:(1)Compared with NC group,the time of excretion of the first black feces in FC model group was significantly prolonged(P<0.01),the number and weight of fecal particles and fecal water content in FC model group decreased significantly in 8 hours(all P<0.01),the small intestine pushing rate and gastric emptying rate in FC model group decreased significantly(all P<0.01),meanwhile,the colon tissue of FC model group had no serious pathological damage.Compared with FC model group,the time of excretion of the first black feces in FC+EA group was significantly shortened(P<0.05),the number and weight of fecal particles and fecal water content in FC+EA group increased significantly in 8 hours(all P<0.01),the small intestine pushing rate and gastric emptying rate in FC model group increased significantly(all P<0.01).The results indicated that He-Mu points acupuncture could enhance the gastrointestinal transport function,and then improve the constipation symptoms in a mice FC model.(2)Compared with NC group,the number of fecal particles in 8 hours,the small intestine pushing rate and gastric emptying rate in PGF model group decreased significantly(all P<0.01),the time of excretion of the first black feces in PGF model group group was significantly prolonged(P<0.01).The results indicated that a mice PGF model have gastrointestinal transport dysfunction.Compared with PGF model group,the small intestine pushing rate in PGF+EA model group was significantly increased(P<0.01),there were no significant difference in the first black stool excretion time and gastric emptying rate(all P>0.05)of PGF+EA model group.The results indicated that He-Mu points acupuncture enhancement on gastrointestinal transmission function is inhibited in the absence of intestinal flora.The regulation of intestinal flora is the core point for acupuncture to enhance gastrointestinal transmission function.2.Changes in flora diversity:(1)Compared with NC group,the observed species and Chao1 of intestinal flora in FC model group were significantly downregulated(all P<0.05),there was no significant difference in the diversity index(Shannon and Simpson)of FC model group(all P>0.05),but it showed a downregulated tendency,the relative abundance of Firmicutes,Lactobacillaceae and Staphylococcaceae decreased,while the relative abundance of Bacteroidetes,Proteobacteria,Murobacteriaceae and Enterobacteriaceae increased in FC model mice.The changes in the species structure and composition of intestinal flora were characterized by reducing the relative abundance of bacteria with a higher proportion in the intestine of normal mice and increasing the relative abundance of bacteria with a lower proportion in the intestine of normal mice.Compared with FC model group,there were no significant difference in the observed species and Chao1 of intestinal flora,the diversity index(Shannon and Simpson)of FC+EA group(all P>0.05), but they showed an upregulated tendency,microorganisms in Bacteroidetes and its classified Murine and Escherichia coli families in FC+EA group decreased,while microorganisms in Firmicutes and its classified Staphylococcic in FC+EA group increased,which restored the trend of Firmicutes/Bacteroidetes ratio to NC group mice.(2)Compared with NC group,the observed species and Shannon in PGF model group were significantly downregulated(all P<0.05),and the most of intestinal flora such as Firmicutes,Bacteroidetes were eliminated in PGF model group.Compared with PGF model group,there were no significant difference in the Alpha diversity index and composition of species structure of FC model group(all P>0.05).The results indicated that He-Mu points acupuncture regulation on intestinal flora of mice is inhibited successfully by antibiotic.3.Changes in short chain fatty acids:(1)Compared with NC group,there were no significant difference in the SCFAs of colon contents of FC model mice(all P>0.05).Compared with FC model group,butyric acid was significantly upregulated(P<0.05)in FC+EA group,while there were no significant difference in remaining SCFAs.According to correlation analysis,there was a significant positive correlation between Staphylococcaceae and butyric acid.The results indicated that He-Mu points acupuncture may promote the production of butyric acid by upregulating the microorganisms in Staphylococcaceae.(2)Compared with NC group,the SCFAs of colon contents in PGF model group decreased significantly(all P<0.01),there were no significant difference in the SCFAs of colon contents of PGF+EA group(all P>0.05).The results indicated that intestinal flora is the basic condition of He-Mu points acupuncture mediated SCFAs production.4.Changes of 5-HT and its receptor and transporter:(1)Compared with NC group,the expression levels of 5-HT,5-HT4 R m RNA and protein in the colon of FC model group were significantly decreased(all P<0.01),the expression levels of SERT m RNA and protein increased significantly(all P<0.01).Compared with FC model group,the expression levels of 5-HT,5-HT4 R m RNA and protein in the colon of FC+EA group were significantly increased(all P<0.01),there were no significant difference in expression levels of SERT m RNA and protein(all P>0.05),but they showed a decreasing tendency.The results indicated that He-Mu points acupuncture can promote 5-HT synthesis and secretion,inhibit its transport and inactivation,and then activate the gene and protein expression of 5-HT4 receptor closely related to promoting intestinal motility.(2)Compared with NC group,the expression levels of 5-HT,5-HT4 R m RNA and protein in the colon of PGF model group were significantly decreased(all P<0.01).Compared with PGF model group,there were no significant difference in the expression levels of 5-HT,5-HT4 R m RNA and protein in the colon of PGF+EA group(all P>0.05).The results indicated that effects of He-Mu points acupuncture on 5-HT and 5-HT4 receptor are inhibited in the absence of intestinal flora.Intestinal microflora is the key factor for He-Mu points acupuncture to affect the expression of 5-HT and 5-HT4 receptors in colon of mice.Conclusion: 1.He-Mu points acupuncture could improve the gastrointestinal transport function in a mice FC model,which is closely related to promoting the disorder recovery of the overall structure of intestinal flora.2.He-Mu points acupuncture may mediate the production of butyric acid mainly by upregulating the microorganisms in Staphylococcaceae,and then stimulate the expression of 5-HT and 5-HT4 receptor.Which improves gastrointestinal transport function,and thus playing a role in the treatment of FC.3.The regulation of He-Mu points acupuncture on gastrointestinal transport function,SCFAs,5-HT and 5-HT4 receptor is inhibited in the absence of intestinal flora.This is confirmed the regulation of intestinal flora is the core point in acupuncture treatment of FC from the opposite angle. |
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