| ObjectiveBased on the relationship between the heart and the small intestine in Traditional Chinese Medicine and the idea of "treating the disease at its source",this experiment used a combination of acute and chronic stress and senna gavage to prepare a rat model of Irritable Bowel Syndrome with predominant Diarrhea(IBS-D)and anxiety.By acupuncture and moxibustion of "Biaoben Acupoints Combination",this method was used to observe anxiety,intestinal pathology,expression of Atrial Natriuretic Peptide(ANP)and ileal ANP receptors,and expression of ileal Peptide tyrosine tyrosine(PYY)and myocardial PYY receptors in rats,and to explore the effects and mechanisms of "Biaoben Acupoints Combination" acupuncture and moxibustion for the overall prevention and treatment of cardiac and physical diseases by harmonizing the heart and intestines and treating both the symptoms and the root cause.This study will provide a basis for the clinical treatment of IBS-D from the "The heart and the small intestine are internally and externally related to each other".MethodsOf the 140 SPF-rated SD female rats,8 were randomly selected as the blank group,and the remaining 132 rats were subjected to the IBS-D with anxiety model.After excluding the rats that died unexpectedly during the modeling process,those with insignificant diarrhea,and those without anxiety-like behavioral changes,the rats were randomly divided into 13 groups by random number table method:model group,convention acupuncture and moxibustion group,Biaoben acupuncture and moxibustion group,ANP-negative virus control group,model+ANP inhibitor group,model+ANP overexpression group,PYY-negative virus control group,model+PYY agonist group,model+PYY agonist group,model+ANP inhibitor group,and Biaoben+ANP inhibitor group,model+PYY knockdown group,Biaoben+ANP inhibitor group,Biaoben+ANP overexpression group,Biaoben+PYY agonist group,and Biaoben+PYY knockdown group,8 rats in each group.Different interventions were given to each group.(1)Blank group:normal rats.(2)Model group:IBS-D with anxiety model rats.(3)Convention acupuncture and moxibustion group(referred to as convention group):IBS-D with anxiety model rats+acupuncture and moxibustion of bilateral ST25,ST36,and ST37,1 time/day for 21 days.(4)Biaoben Acupoints Combination acupuncture and moxibustion group(referred to as Biaoben group):IBS-D with anxiety model rats+acupuncture and moxibustion of bilateral PC6,ST36,and CV4,1 time/day for 21 days.(5)ANP-negative virus control group:IBS-D with anxiety model+rats microfed with 100μL of ANP-negative control virus for tail vein injection,only one injection,and regular feeding for 21 days.(6)Model+ANP overexpression group:IBS-D with anxiety model rats+100μL of ANP overexpression virus taken by microsampler for tail vein injection,only one injection,regular feeding for 21 days.(7)Model+ANP inhibitor group:IBS-D model rats+tail vein injection of ANP inhibitor at a concentration of 0.5μg/g each,1 time/day,for 21 days.(8)Biaoben+ANP overexpression group:IBS-D with anxiety model rats+100μL of ANP overexpression virus extracted by microsampler for tail vein injection only once;followed by acupuncture and moxibustion of bilateral PC6,ST36 and CV4,1 time/day for 21 days.(9)Biaoben+ANP inhibitor group:IBS-D with anxiety model rats+tail vein injection of ANP inhibitor at a concentration of 0.5μg/g each,1 time/day,followed by acupuncture and moxibustion of bilateral PC6,ST36,and CV4,1 time/day,for 21 days.(10)PYY-negative virus control group:IBS-D with anxiety model rats+100μL of PYY-negative control virus extracted by microsampler for tail vein injection,only one injection,and regular feeding for 21 days.(11)Model+PYY knockdown group:IBS-D with anxiety model rats+100μL of PYY knockdown virus extracted by microsampler for tail vein injection,one injection only,and regular feeding for 21 days.(12)Model+PYY agonist group:IBS-D model rats+tail vein injection of PYY agonist at a concentration of 0.5μg/g each,1 time/day for 21 days.(13)Biaoben+PYY knockdown group:IBS-D with anxiety model rats+microsampler to extract 100μL of PYY knockdown virus for tail vein injection,only once;then acupuncture and moxibustion with bilateral PC6,ST36,and CV4,1 time/day for 21 days.(14)Biaoben+PYY agonist group:IBS-D model rats+tail vein injection of PYY agonist at a concentration of 0.5μg/g each,1 time/day,followed by acupuncture and moxibustion of bilateral PC6,ST36,and CV4,1 time/day,for 21 days.After the intervention,body weight,Bristol fecal trait score,and fecal water content were recorded;abdominal withdrawal reflex was used to detect visceral hypersensitivity;behaviorally related experiments were performed to detect anxiety and depression in rats;heart rate variability was measured by isoflurane anesthesia;blood was collected from the abdominal aorta,fresh heart and ileum tissues were taken,and the pathological structure of ileum was observed by HE and transmission electron microscopy,and ELISA was used to detect serum,heart and ileal ANP and serum and ileal PYY.ELISA was performed to detect the levels of ANP and serum and ileal PYY.Real time qPCR,Western Blot,and immunohistochemistry were performed to detect the expression of myocardial ANP and ileal NPR-A receptors,and ileal PYY and myocardial NPY5R receptors,and all data were statistically analyzed.Results1.Mechanism of anxiety-like behaviors in rats with IBS-D by "Biaoben Acupoints Combination" acupuncture and moxibustion1.1 Effect of "Biaoben Acupoints Combination" acupuncture and moxibustion on anxiety and depression in IBS-D rats with anxiety model(1)Results of the open field test:Compared with the blank group,the distance of movement,the number of uprightness and the dwell time in the central area of the rats in the remaining groups were reduced(P<0.05).Compared with the model group,the exercise distance,the number of upright and central area residence times of rats in the convention and Biaoben groups increased(P<0.05).Compared with the convention group,the distance of movement,the number of uprightness and the dwell time in the central area increased in the Biaoben group(P<0.05),indicating that "Biaoben Acupoints Combination" acupuncture and moxibustion could better improve the negative emotions such as anxiety in IBS-D rats.(2)Results of the elevated plus-maze:Compared with the blank group,the open arm dwell time,the number of times into the open arm,and the total exercise distance were significantly reduced in the model group(P<0.05);compared with the model group,the open arm dwell time,the number of times into the open arm,and the total exercise distance were significantly increased in the convention group and the Biaoben group(P<0.05);compared with the convention group,the open arm dwell time,the number of times into the open arm,and the total exercise distance were increased in the Biaoben group(P<0.05),indicating less anxiety and depression in the Biaoben group.This suggests that the "Biaoben Acupoints Combination"acupuncture and moxibustion can better alleviate the anxiety and depression in the IBS-D with anxiety model rats.(3)Results of the light-dark box:Compared with the blank group,the duration of stay in the open box,the number of times of going through the box and the total distance of movement of the rats in the model group decreased(P<0.05),indicating that the rats in the model group were at anxiety level and lost interest in exploration;compared with the model group,the duration of stay in the open box,the number of times of going through the box and the total distance of movement of the rats in the convention and Biaoben groups increased(P<0.05).Compared with the convention group,the duration of stay in the open box,the number of times of going through the open and dark boxes,and the total distance of exercise were all increased in the Biaoben group(P<0.05),indicating that "Biaoben Acupoints Combination"acupuncture and moxibustion has a better anxiolytic effect.1.2Effect of "Biaoben Acupoints Combination" acupuncture and moxibustion on heart rate variability in IBS-D and anxiety model ratsCompared with the blank group,SDNN and RMSSD decreased(P<0.05)and LF/HF increased(P<0.05)in the model group;compared with the model group,SDNN and RMSSD increased(P<0.05)and LF/HF decreased(P<0.05)in both the convention and Biaoben groups;compared with the convention group,SDNN and RMSSD increased(P<0.05)and LF/HF decreased(P<0.05)in the Biaoben group(P<0.05).It is suggested that the"Biaoben Acupoints Combination" acupuncture and moxibustion can better regulate the alteration of heart rate variability in rats with IBS-D and anxiety model.1.3Effects of "Biaoben Acupoints Combination" acupuncture and moxibustion on serum and myocardial ANP in IBS-D and anxiety model ratsCompared with the blank group,the serum and myocardial ANP concentrations in the model group increased(P<0.05);compared with the model group,the serum and myocardial ANP concentrations in both the convention and Biaoben groups decreased(P<0.05);compared with the convention group,the serum and myocardial ANP concentrations in the Biaoben group decreased(P<0.05).It was suggested that "Biaoben Acupoints Combination" acupuncture and moxibustion could reduce the serum and myocardial tissue ANP concentrations more effectively in IBS-D and anxiety model rats.2.Mechanism of "Biaoben Acupoints Combination" acupuncture and moxibustion to regulate the function of intestinal organs of rats in IBS-D with anxiety model2.1 Effects of "Biaoben Acupoints Combination" acupuncture and moxibustion on body weight,fecal trait score and fecal water content of rats in IBS-D with anxiety modelAfter the intervention,the body weight of rats in the model group was still significantly lower than that of the blank group(P<0.05);compared with the model group,the body weight of rats in both the convention and Biaoben groups increased,the Bristol fecal trait score decreased,and the fecal water content decreased significantly(P<0.05);compared with the convention group,the body weight of rats in the Biaoben group increased,the fecal water content decreased(P<0.05),and the Bristol fecal trait score was not statistically significant(P>0.05).It was suggested that both "Convention Acupoints Selection" and "Biaoben Acupoints Combination" could alleviate the diarrhea symptoms in the model rats.2.2Effect of "Biaoben Acupoints Combination" acupuncture and moxibustion on visceral hypersensitivity in IBS-D with anxiety model ratsThe difference in AWR scores between the rats in each group was not statistically significant at a dilatation pressure of 20 mmHg(P>0.05).Compared with the blank group,rats in the model group had higher AWR scores and lower visceral pain thresholds at dilating pressures of 40,60,and 80 mmHg(P<0.05).Compared with the model group,the AWR scores were lower and the visceral pain thresholds were higher in both the convention and Biaoben groups at 40,60,and 80 mmHg of dilatation pressure(P<0.05).Compared with the convention group,the difference of AWR score in the Biaoben group was not statistically significant(P>0.05)at 40 and 60 mmHg dilatation pressure,and the AWR score was significantly lower and the visceral pain threshold was higher(P<0.05)at 80 mmHg dilatation pressure.It was suggested that both "Convention Acupoints Selection" and "Biaoben Acupoints Combination" could relieve the abdominal pain symptoms in the model rats.However,"Biaoben Acupoints Combination" acupuncture and moxibustion were more effective in reducing visceral hypersensitivity at 80 mmHg dilatation pressure in the model rats.2.3Effect of "Biaoben Acupoints Combination" acupuncture and moxibustion on the intestinal pathological structure of IBS-D and anxiety model ratsHE staining:the ileal villi of rats in the blank group were intact and dense,with the regular and uniform arrangement,and the submucosal layer and muscle layer were clearly demarcated.In the model group,the ileal villi were slightly defective,with interstitial breaks and low inflammatory reaction in the epithelial tissue,with a small amount of neutrophils infiltrating the mucosal layer and submucosal layer and a decrease in cup cells;in the convention group,the morphology was basically similar to that of the Biaoben group,with the ileal villi relatively intact,the crypt intact,and no obvious neutrophil infiltration.Transmission electron microscopy:The intestinal mucosal epithelial cells in the blank group were normal in morphology,with normal gap connection,and the intestinal villi were neatly arranged,uniform in density,consistent in length,tightly connected and intact,and clearly visible.In the model group,there were degranulation and vacuolation in the cytoplasm of intestinal epithelial cells,destruction of tight junctions,widening of gap junctions,fracture and atrophy of intestinal villi,disorganization and uneven distribution,and mitochondrial lysis and swelling.In the convention group,there were some vacuoles in the cytoplasm of the cells,and a few intestinal villi were broken,incomplete tight junctions,and slightly damaged bridge granules.In the Biaoben group,the epithelial cell gap was clearer,no widening was seen and the microvilli were neatly arranged,tight junctions were intact and the bridging granules were normal.2.4Effects of "Biaoben Acupoints Combination" acupuncture and moxibustion on serum and ileal PYY in rats with IBS-D and anxiety modelCompared with the blank group,the serum and ileal PYY concentrations in the model group decreased(P<0.05);compared with the model group,the serum and ileal PYY concentrations in the convention and Biaoben groups increased(P<0.05);compared with the convention group,the serum and ileal PYY concentrations in the Biaoben group increased(P<0.05).It was suggested that "Biaoben Acupoints Combination" acupuncture and moxibustion could increase the serum and ileal tissue PYY concentrations in the IBS-D and anxiety model rats.3.Mechanism and effect of "Biaoben Acupoints Combination" acupuncture and moxibustion in regulating the expression of myocardial ANP and ileal ANP receptors to improve IBS-D3.1Effect of "Biaoben Acupoints Combination" acupuncture and moxibustion on ANP overexpression virus in the heart of model ratsCompared with the model+ANP overexpression group,the myocardial ANP mRNA and protein expression were both reduced in the Biaoben+ANP overexpression group(P<0.05),indicating that "Biaoben Acupoints Combination" acupuncture and moxibustion could down-regulate the cardiac ANP mRNA and protein expression in the IBS-D with anxiety model rats.3.2Effect of ANP in the IBS-D with anxiety model linking the heart and small intestine(1)Effect of ANP on diarrhea and abdominal pain symptoms as well as anxiety-like mood in model ratsCompared with the model group,the rats in the model+ANP overexpression group had lower body weight(P<0.05),significantly higher fecal water content(P<0.05),lower visceral pain threshold(P<0.05),and significantly lower open arm residence time,number of times into the open arm,and total exercise distance(P<0.05),indicating that the diarrhea and abdominal pain,anxiety,and depression in the model+ANP overexpression group became more.The rats in the model+ANP inhibitor group had increased body weight(P<0.05),decreased fecal water content(P<0.05),increased visceral pain threshold(P<0.05),and significantly increased open arm dwell time,number of times into the open arm,and total exercise distance(P<0.05),suggesting that the rats in the model+ANP inhibitor group had less diarrhea and abdominal pain and less anxiety and depression.(2)Changes in myocardial ANP and ileal NPR-A gene and protein expression in model ratsCompared with the model group,myocardial ANP gene and protein expression decreased(P<0.05)and ileal NPR-A gene and protein expression decreased(P<0.05)in the model+ANP inhibitor group,while myocardial ANP gene and protein expression increased(P<0.05)and ileal NPR-A gene and protein expression increased(P<0.05)in the model+ANP overexpression group.It indicates that ANP is involved in the development of IBS-D,closely linking the heart and small intestine.3.3Effects of "Biaoben Acupoints Combination" acupuncture and moxibustion on myocardial ANP and ileal NPR-A expression in IBS-D and anxiety model rats(1)The relief of symptoms in model rats may be related to the regulation of ANP with "Biaoben Acupoints Combination" acupuncture and moxibustion.Both acupuncture and ANP inhibitors increased body weight,decreased Bristol stool trait score,decreased stool water content(P<0.05),decreased AWR score,increased visceral pain threshold(P<0.05),and improved diarrhea and abdominal pain in IBS-D and anxiety model rats.The symptoms of diarrhea and abdominal pain in rats were improved by increasing the open arm dwell time,the number of times of entering the open arm,and the total distance of exercise(P<0.05),and relieving the anxiety and depression in rats with IBS-D and anxiety model.The rats in the Biaoben+ANP overexpression group had higher body weight,lower fecal water content(P<0.05),higher visceral pain threshold(P<0.05),and higher open arm dwell time,number of times into the open arm,and total movement distance(P<0.05)than those in the model group,indicating that the Biaoben+ANP overexpression group reduced diarrhea and abdominal pain symptoms and anxiety than the model group.It is suggested that the significant improvement of diarrhea,abdominal pain symptoms and anxiety-like emotion in IBS-D with anxiety model rats by "Biaoben Acupoints Combination",which is probably related to ANP expression.(2)Acupuncture and moxibustion with "Biaoben Acupoints Combination"down-regulated myocardial ANP,ileal NPR-A gene and protein expression in model ratsCompared with the model group,myocardial ANP and ileal NPR-A gene and protein expression were reduced in the Biaoben group and the Biaoben+ANP inhibitor group(P<0.05),suggesting that "Biaoben Acupoints Combination" and ANP inhibitor can both benignly regulate the secretion of cardiac ANP,and both have the same effect;compared with the model group,myocardial ANP and ileal NPR-A gene and protein expression were reduced in the Biaoben+ANP overexpression group.Compared with the model group,myocardial ANP and ileal NPR-A gene and protein expression were reduced(P<0.05),further suggesting that "Biaoben Acupoints Combination"acupuncture and moxibustion may improve diarrhea,abdominal pain symptoms and anxiety-like mood of rats with IBS-D and anxiety model by down-regulating myocardial ANP secretion and inducing a reduction in ileal NPR-A expression.4.The effect mechanism of ileal PYY and myocardial PYY receptor expression modulation by "Biaoben Acupoints Combination" acupuncture and moxibustion to improve IBS-D symptoms4.1 Effect of "Biaoben Acupoints Combination" acupuncture and moxibustion on ileal PYY knockdown virus in model ratsCompared with the model+PYY knockdown group,the ileal PYY mRNA and protein expression in the Biaoben+PYY knockdown group were both increased(P<0.05),indicating that "Biaoben Acupoints Combination"acupuncture and moxibustion could up-regulate the ileal PYY mRNA and protein expression in IBS-D with anxiety model rats.4.2Effect of PYY linking small intestine and heart in the IBS-D with anxiety model(1)Effect of PYY on diarrhea and abdominal pain symptoms as well as anxiety-like mood in model ratsCompared with the model group,rats in the model+PYY knockdown group had lower body weight(P<0.05),significantly higher fecal water content(P<0.05),lower visceral pain threshold(P<0.05),and significantly lower open arm dwell time,number of times into the open arm,and total exercise distance(P<0.05),indicating that diarrhea and abdominal pain as well anxiety depression became more severe in the model+PYY knockdown group,while rats in the model+PYY agonist group had more severe abdominal pain and anxiety depression.The rats in the model+PYY agonist group had more body weight(P<0.05),less fecal water content(P<0.05),higher visceral pain threshold(P<0.05),and significantly more open-arm dwell time,the number of open-arm entries,and total exercise distance(P<0.05),indicating that the model+PYY agonist group had less diarrhea,less abdominal pain,less anxiety and less depression.(2)Changes in gene and protein expression of ileal PYY and myocardial NPY5R in model ratsCompared with the model group,rats in the model+PYY agonist group had increased ileal PYY gene and protein expression(P<0.05)as well as increasing myocardial NPY5R gene and protein expression(P<0.05);rats in the model+PYY knockdown group had decreased ileal PYY gene and protein expression(P<0.05)as well as decreasing myocardial NPY5R gene and protein expression(P<0.05).It indicates that PYY is involved in the development of IBS-D,closely linking the small intestine with the heart.4.3Effects of "Biaoben Acupoints Combination" acupuncture and moxibustion on ileal PYY and myocardial NPY5R expression in rats with anxiety model of IBS-D(1)The relief of symptoms in model rats may be related to the regulation of PYY with "Biaoben Acupoints Combination" acupuncture and moxibustionBoth "Biaoben Acupoints Combination" and PYY agonist increased body weight,decreased Bristol stool typing score and fecal water content(P<0.05),decreased AWR score,increased visceral pain threshold(P<0.05),and improved diarrhea and abdominal pain symptoms in the model rats;increased open arm dwell time,the number of entries into the open arm,and total distance traveled for exercise(P<0.05),and relieved the anxiety and depression of the model rats;however,the difference between the Biaoben+PYY knockdown group and the model group was not statistically significant(P>0.05),indicating that the effect of "Biaoben Acupoints Combination" acupuncture and moxibustion to prevent and treat IBS-D was partially suppressed by the PYY knockdown virus.It is further suggested that"Biaoben Acupoints Combination" acupuncture and moxibustion may induce an increase in myocardial NPY5R expression through upregulating the expression of ileal PYY,and improve diarrhea,abdominal pain and anxiety-like mood in the IBS-D with anxiety model rats.Conclusion1.Acupuncture and moxibustion with "Biaoben Acupoints Combination" can better regulate the heart and mind of IBS-D with anxiety model rats.The reason for the superior effect of Biaoben Acupoints Combination on the prevention and treatment of IBS-D is that it can solidly protect the congenital qi and acquired qi,also can regulate the heart and mind and protect the intestines,and can better alleviate the negative emotions such as anxiety and depression as well as the symptoms of abdominal pain and diarrhea in IBS-D rats,protect the body’s positive energy and improve the resistance to disease.2.The heart and the small intestine form an internal and external relationship with each other through the meridians.The gene and protein expression of myocardial ANP and ileal NPR-A were increased,and the gene and protein expression of ileal PYY and myocardial NPY5R were decreased in the IBS-D with anxiety model rats,suggesting that irritable bowel syndrome can lead to dysfunction of the heart and intestine and disorder of ANP and PYY metabolism in vivo.3.Acupuncture and moxibustion with "Biaoben Acupoints Combination" can down-regulate the gene and protein expression of ANP in myocardial tissue and reduce the gene and protein expression of NPR-A receptor in ileum of IBS-D with anxiety model rats,suggesting that acupuncture and moxibustion with "Biaoben Acupoints Combination" can effectively relieve anxiety,improve diarrhea and abdominal pain.The effect mechanism may be related to the regulation of cardiac ANP expression.4.Acupuncture and moxibustion with "Biaoben Acupoints Combination" can up-regulate the gene and protein expression of PYY in ileum tissue and increase the gene and protein expression of NPY5R receptor in heart of IBS-D with anxiety model rats,suggesting that acupuncture and moxibustion with "Biaoben Acupoints Combination" can effectively improve diarrhea,abdominal pain and relieve anxiety.The effect mechanism may be related to the regulation of ileal PYY expression. |
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