| Objective(1)To observe whether the flora after electroacupuncture could promote the recovery of intestinal function in rats with spinal cord injury after transplanting the flora after electroacupuncture to rats.(2)To detect whether the flora after electroacupuncture can promote the expression of 5-HT and 5-HT receptor in colon of rats with spinal cord injury after transplanting the flora after electroacupuncture into rats.(3)To determine whether the improvement of intestinal dysfunction by electroacupuncture regulation of colon 5-HT system is mediated by intestinal flora.MethodEighty female SD rats aged 2-3 months were fed adaptively for one week.Twenty rats were randomly selected as control group(sham operation group).In the sham operation group,only the spinous process and lamina from T10 to T13 were exposed for 5 minutes,and no other injury treatment was done,and then the wound was infiltrated with gentamicin and sutured directly.The remaining 60 rats were used to establish a severe spinal cord injury model by heavy object falling method.After 24 hours,the BBB score was 0,and the model was successfully established.Then the successful rats were randomly divided into 3 groups: model group(n=20),electroacupuncture group(n=20)and flora transplantation group(n=20).The rats in the sham operation group were fixed for 15 minutes a day,the electroacupuncture group was electroacupuncture at Zusanli points on both sides for 15 minutes a day,and the flora transplantation group was treated with 10% flora suspension prepared from the collected fresh feces of the rats in the electroacupuncture group by intragastric administration.The daily dose is calculated according to the weight of the rats in the flora transplantation group(1ml/100g).All rats were given routine postoperative nursing,and the intervention time lasted for 2 weeks.After the intervention,rats in each group underwent behavioral test.Finally,the colon tissues were taken uniformly,and the pathological changes of the colon tissues were observed by HE staining.Immunohistochemical staining was used to observe the expression of5-HT3 AR and 5-HT4 R in the myenteric plexus and mucosa of the colon.The content of 5-HT in the colon tissue was detected by ELISA,and the relative expression of5-HT3 ARm RNA and 5-HT4 Rm RNA in the colon tissue was detected by real-time PCR.Results(1)Changes of intestinal transport function1)Duration of the first black stool: Compared with the model group,the first black stool excretion time of the electroacupuncture group and the flora transplantation group was significantly shortened(P<0.001),and compared with the sham operation group,the first black stool excretion time of the two groups was also significantly shortened(P<0.001).2)24-hour fecal wet weight,dry weight and water content: Compared with the sham operation group,the 24-hour fecal wet weight of the model group was significantly reduced(P<0.001),and the fecal wet weight of the electroacupuncture group and the flora transplantation group was also reduced(P<0.01),but compared with the model group,the electroacupuncture group The wet weight of feces increased(P<0.05);Compared with the sham operation group,the dry weight of feces in the model group decreased significantly(P<0.001),the electroacupuncture group also decreased(P<0.05),and the flora transplantation group also decreased significantly(P<0.01).Compared with the model group,the dry weight of feces in the electroacupuncture group increased significantly(P<0.01),and the dry weight of feces in the flora transplantation group also increased(P<0.05).In terms of water content,there was no significant difference between the four groups.(2)Changes of intestinal sensory functionThrough AWR score statistics,it was found that when the injection volume was1 ml and 1.5ml,there was a statistical difference between the sham operation group and the model group(P<0.05).The AWR score of the model group was higher,and there was no statistical difference between other groups.When the injection volume was 1.25 ml,there was no statistical difference in the AWR score of the four groups.(3)Morphological changes of colon tissueAfter HE staining,it was observed under the optical microscope that the mucosal layer and muscular layer of the colon in the sham operation group were intact,the myenteric plexus shedded less,the glands were arranged regularly,and there was no inflammatory cell infiltration.In the model group,the intestinal wall was obviously atrophic,mucosal erosion was obvious,inflammatory cells infiltrated,glands were reduced and arranged irregularly,and interstitial edema was found.Spinal cord injury leads to significant changes in colon histology.The colon tissue morphology of the electroacupuncture group was similar to that of the flora group.Compared with the model group,the morphology of the two groups was significantly improved,only slight muscular atrophy and a small amount of inflammatory cell infiltration occurred,and the number of glands increased significantly.(4)Changes of 5-HT content in colon tissue There was significant difference between sham operation group and model group(P<0.05).The content of 5-HT in the model group decreased,and there was a significant difference between the electroacupuncture group and the model group(P<0.01).The content of 5-HT in the electroacupuncture group increased.There was a statistical difference between the flora transplantation group and the model group(P<0.05),the content of 5-HT in the flora transplantation group increased.Immunohistochemical analysis1)Immunohistochemical results of 5-HT3 AR and 5-HT4 R in the myenteric plexus of the colon By immunohistochemical staining,it was found that in the myenteric plexus of the colon,the expression of 5-HT3 AR and 5-HT4 R in the sham operation group was strongly positive,showing brown coarse particles.The expression of the model group was weakly positive,showing light yellow fine particles.In contrast,the electroacupuncture group and the flora transplantation group showed positive expression,showing brown granules.Statistical analysis of the average optical density(AOD value)of the positive staining area detected by the image analysis system found that there was a significant difference between the sham operation group and the model group(P<0.001).The AOD values of 5-HT3 AR and 5-HT4 R staining in the model group were significantly reduced,that is,the expression of 5-HT3 AR and5-HT4 R in the model group was significantly reduced.Compared with the model group,the electroacupuncture group and the flora transplantation group have significant differences(P<0.001).The contents of 5-HT3 AR and 5-HT4 R in the electroacupuncture group and the flora transplantation group are significantly increased.The electroacupuncture group is significantly different from the sham operation group(P<0.001).The content of 5-HT3 AR in the electroacupuncture group is increased,and the difference between the electroacupuncture group and the flora transplantation group is statistically significant(P<0.05),the content of 5-HT4 R in the electroacupuncture group is higher.2)Immunohistochemical results of 5-HT3 AR and 5-HT4 R in colonic mucosa By immunohistochemical staining,it was found that 5-HT3 AR and 5-HT4 R were strongly positive in the colonic mucosa of the sham operation group,and brown coarse particles could be seen under the microscope.The model group showed weak positive expression,and only light yellow fine particles could be seen under the microscope.In contrast,the electroacupuncture group and the flora transplantation group showed positive expression,and obvious brown granules could be seen under the microscope.The statistical analysis of the AOD value of the positive staining area detected by the image analysis system found that there was a significant difference between the sham operation group and the model group(P<0.001).The AOD values of 5-HT3 AR and 5-HT4 R staining in the model group were significantly reduced.Compared with the model group,the electroacupuncture group and the flora transplantation group had significant differences(P<0.001).The AOD values of5-HT3 AR and 5-HT4 R staining in the electroacupuncture group and the flora transplantation group were significantly increased.When the AOD value of 5-HT4 R was compared between sham operation group,electroacupuncture group and flora transplantation,there were significant differences(P<0.001).The AOD value of5-HT4 R staining in the sham operation group was significantly higher than those in the two groups.When the AOD value of 5-HT3 AR was compared between the sham operation group and the electroacupuncture group and the flora transplantation group,the difference between the sham operation group and the electroacupuncture group was not statistically significant,but there was a significant difference between the sham operation group and the flora transplantation group(P<0.01).The AOD value of5-HT3 AR in the flora transplantation group decreased.(5)Analysis of PCR ResultsThe relative expression of 5-HT3 ARm RNA and 5-HT4 Rm RNA in colon was detected by PCR.It was found that there was a significant difference between the sham operation group and the model group(P<0.001).The expression levels of5-HT3 ARm RNA and 5-HT4 Rm RNA in the model group decreased significantly,and the difference between the electroacupuncture group and the model group was statistically significant(P<0.05).The expression levels of 5-HT3 ARm RNA and5-HT4 Rm RNA in the electroacupuncture group increased,and the difference between the flora transplantation group and the model group was statistically significant(P<0.05).The expression levels of 5-HT3 ARm RNA and 5-HT4 Rm RNA in the flora transplantation group increased.ConclusionBy regulating intestinal flora and its metabolites,electroacupuncture can further benign regulation of colon 5-HT system,promote intestinal motility,and improve defecation dysfunction after spinal cord injury. |
