| Objective At present,due to the deepening research of Functional dyspepsia(FD),the research direction of its mechanism has gradually shifted from gastric dysfunction to duodenal dysfunction,and its treatment mode has also gradually shifted from drug therapy to green therapy.Low-grade inflammation is closely related to the onset of FD,this research is focused on the duodenum low-grade inflammation,TLR4/NF-κB p65 signal pathway effect of duodenal mucosa barrier as the breakthrough point,to explore the curative intervention ST 36 holes on the mechanism of action of FD model rats with low-grade inflammation,including preliminary clarify foot three holes by inhibiting TLR4/NF-κB p65 signal pathway,repair duodenal mucosal barriers,reduce the inflammation,and the mechanism of action of FD treatment,providing theoretical basis for the curative treatment of FD.Methods 63 male Sprague Dawley rats aged 40 days and weighing 200±20g were randomly divided into a blank group(12)and a model group(51).The rats in the modeling group were randomly divided into model group,electroacupuncture group,electroacupuncture + inhibitor group,and inhibitor group,with 12 rats in each group,after 20 days of modeling with modified multi-factor intervention method(clip-tail stimulation +0° normal saline + fasting).The rats in the electroacupuncture group were acupunctured to bilateral "Zusanli" point followed by Han’s acupoint nerve stimulator,with continuous wave,frequency of 2Hz,intensity of 1m A.Each intervention lasted for 30 min,once a day,and lasted for 14 days.The rats in the inhibitor group were given the TLR4 inhibitor TAK242(0.5mg/kg)once a day for 14 consecutive days through tail vein injection.The rats in the electroacupuncture+inhibitor group were given electroacupuncture and inhibitor intervention successively,and the intervention method was the same as that in the electroacupuncture and inhibitor group,and the intervention method was 14 days.During the intervention,the rats in each group were raised in the same environment and the experimental conditions were kept consistent except for the different intervention methods.Before and after modeling as well as after intervention,the changes of general conditions,food intake and body mass of rats in each group were recorded.After the intervention,the rats in each group were tested by cotton nest experiment,and then were treated with fasting for 24 h to avoid water.After 24 h,6 rats in each group were randomly selected to receive nutritive semi-solid paste gavage(3ml/100g)according to their body mass.After 30 min,the rats were anesthetized with 10% chloral hydrate(0.35 ml /100g)in the sequence of gavage,and then the gastric emptying and small intestine propulsion rate were detected rapidly.After the conclusion of gastric emptying and small intestine propulsion rate detection,the duodenum tissues were taken immediately and treated according to different detection methods(HE staining,Western Blot,RT-PCR).Then,3 rats were randomly selected to receive blood samples from abdominal aorta after anesthesia for ELISA.Finally,the duodenal tissues of the 3 rats were collected after anesthesia for electron microscopy and immunofluorescence detection.The histopathological changes of duodenum were observed by HE staining.Serum IL-6,TNF-α and SIg A were detected by ELISA.Western Blot was used to detect TLR4,Myd88,TRAF6,NF-κB p65,NF-κB p-p65,ZO1,Occludin,Claudin-1 protein expressions in the duodenum.TLR4,NF-κB p65,ZO-1 and Claudin-1 expression levels in duodenum were detected by RT-PCR.Immunofluorescence was used to detect the location of NF-κB p65 in duodenum.The ultrastructural changes of duodenal mucosa were observed by electron microscopy.Results 1.Electroacupuncture could increase food intake and body mass of FD model rats for 3h,improve depression and promote gastrointestinal motility.(1)General conditions changed after modeling,the rats were in poor spirit,with withered and yellow fur,messy and stretched,and sparse fur,spontaneous fighting developed to piles of crouching,slow response to capture,yellow-green feces,shapeless,containing undigested feed particles,and smelly.After electroacupuncture intervention,the rats were in better mental state,with apricot yellow,smooth and dense fur.Increased activity,sensitive capture response;Manure is brownish yellow,dry,soft and odorless.(2)Body mass of rats in each group was recorded after 7 days of adaptive feeding(before modeling),20 days after modeling(after modeling),and 14 days after intervention(after intervention).Before modeling,there was no significant difference in body mass between the groups(P > 0.05).After modeling,the body mass of rats in the modeling group decreased significantly(P < 0.01),but there was no significant difference in the body mass between the modeling groups(P>0.05).After the electroacupuncture intervention,the body mass of the rats in the electroacupuncture group was significantly increased compared with that in the model group(P < 0.01),indicating that the electroacupuncture could increase the reduced body mass of the FD rats and restore the weight of the rats.In addition,the body mass of the electroacupuncture + inhibitor rats increased more(P<0.01),but was still lower than that of the blank group(P<0.01).(3)Food intake for 3h Before modeling,there was no significant difference in food intake between the groups(P> 0.05).Compared with the blank group,the food intake of rats in the model group decreased significantly(P<0.01)at 3h,indicating that the rats ate less after modeling.Compared with the model group,the feeding amount of the rats in the electroacupuncture group,the electroacupuncture + inhibitor group and the inhibitor group was increased for 3h(P<0.01),but all of them were lower than that in the blank group(P<0.01),indicating that both the electroacupuncture and the inhibitor could increase the feeding amount of the rats in FD.Compared with the electroacupuncture group,the feeding amount of rats in the electroacupuncture + inhibitor group increased more at 3h(P<0.01),but there was no statistical significance(P>0.05)compared with that in the inhibitor group at 3h.4)Cotton nestle shredding test Compared with the blank group,the amount of crushed cotton increased significantly after the intervention of compound factors(P < 0.01),indicating that FD rats showed signs of depression,which was consistent with the observed results.Compared with the model group,the cotton crushed in the electroacupuncture group,electroacupuncture + inhibitor group and inhibitor group decreased(P<0.01),but was still higher than that in the blank group(P<0.01).Compared with the electroacupuncture group,the amount of cotton crushed in the electroacupuncture + inhibitor group decreased more(P <0.05).The results showed that electroacupuncture could improve the depression of FD rats.5)Gastric emptying rate Compared with the blank group,the gastric emptying rate of the model group was reduced(P=0.048).Compared with the model group,the gastric emptying rate of rats in the electroacupuncture group was significantly increased(P=0.043),but there was no statistically significant difference in the gastric emptying rate between the electroacupuncture + inhibitor group and the inhibitor group(P>0.05).There was no significant difference between the electroacupuncture group and the electroacupuncture+inhibitor group and the inhibitor group(P>0.05).(6)After the modeling of small intestinal propulsion rate FD,the small intestinal propulsion rate in the model group was significantly reduced(P < 0.01),indicating that the small intestinal motility of FD rats was insufficient and intestinal peristalsis slowed down.After intervention,the small intestine propulsion rate of rats in electroacupuncture group,electroacupuncture+inhibitor group and inhibitor group was significantly higher than that in model group(P < 0.01),indicating that both electroacupuncture and inhibitor intervention could accelerate small intestine peristalsis.Compared with the electroacupuncture group,the electroacupuncture + inhibitor group improved the small intestine propulsion rate more significantly(P<0.05).There was no significant difference between the inhibitor group and the electroacupuncture group(P > 0.05).(7)Morphological changes of the duodenum.HE staining of duodenal tissue.No significant organic changes were observed in duodenum of each group.In the blank group,the duodenal mucosa was intact,the intestinal villi were orderly,and no inflammatory cell infiltration was observed.The structure of duodenal mucosa in the model group was relatively complete,but the villi were disordered,some of them were lodging,fusion and shedding,and the mucosa was infiltrated by inflammatory cells.The duodenal mucosa structure of the electroacupuncture group was more complete,the intestinal villi were more orderly arranged,the tips of some villi were detached,and the infiltration of inflammatory cells in the mucosa was reduced.The duodenal mucosa structure of the electroacupuncture + inhibitor group was more complete,the intestinal villi were arranged more orderly,and the infiltration of inflammatory cells in the mucosa was reduced.In the inhibitor group,the duodenal mucosal structure was more complete,the intestinal villi were more orderly,and the infiltration of inflammatory cells in the mucosal layer was reduced.2.Electroacupuncture can reduce the inflammation of duodenal mucosa in FD rats.(1)Changes of serum IL-6 and TNF-α Compared with the blank group,the serum of IL-6 and TNF-α in the model group were significantly increased(P < 0.01),suggesting that inflammatory factors and inflammatory response were increased in the serum of FD model rats after modeling.After the end of the intervention,serum IL-6 of the electroacupuncture group,the inhibitor group and the electroacupuncture+inhibitor group were significantly decreased(P<0.01).Compared with the electroacupuncture group,the effect of electroacupuncture + inhibitors on the reduction of serum IL-6 was more significant(P<0.01),but the reduction of serum IL-6 by inhibitors was lower than that by electroacupuncture group(P < 0.01),suggesting that both electroacupuncture and inhibitors can reduce the content of serum IL-6 and have the effect of reducing inflammation. Compared with the blank group,the serum TNF-α of the model group was significantly increased(P<0.01),suggesting that the serum inflammatory factors and inflammatory response were increased in the FD model rats after modeling.After the end of the intervention,serum IL-6 of the electroacupuncture group,the inhibitor group and the electroacupuncture+inhibitor group were significantly decreased(P < 0.01).Compared with the electroacupuncture group,the electroacupuncture + inhibitor group had a more significant effect in reducing serum IL-6(P<0.01),but the inhibitor reduced the serum TNF-α content less than that of the electroacupuncture group(P < 0.05),suggesting that both the electroacupuncture and the inhibitor could reduce the serum TNF-α content and reduce the inflammatory reaction.(2)The expression of IL-6 and TNF-α in duodenum Compared with the blank group,the relative expression levels of IL-6 in the duodenum of the model group were significantly increased(P<0.01),indicating the presence of inflammation in the duodenum of FD rats.After intervention,the relative expression levels of IL-6 and TNF-α in the duodenum of the electroacupuncture group,the electroacupuncture + inhibitor group and the inhibitor group were significantly decreased(P < 0.05),indicating that the electroacupuncture and the inhibitor intervention can reduce the inflammation of the duodenum of the FD rats.Compared with the electroacupuncture group,the relative expression level of TNF-α in the duodenum of the electroacupuncture + inhibitor group and the inhibitor group was not statistically significant(P>0.05).Compared with the blank group,the relative expression of TNF-α in the model group was significantly increased(P < 0.01),indicating the presence of inflammation in the duodenum of FD rats.After the intervention,the relative expression of TNF-α in the duodenum of the electroacupuncture group,the electroacupuncture+inhibitor group,and the inhibitor group was significantly decreased(P < 0.01),indicating that the electroacupuncture and the inhibitor intervention can reduce the inflammation of the duodenum of the FD rats.Compared with the electroacupuncture group,the relative expression levels of TNF-α in the duodenum of the electroacupuncture + inhibitor group and the inhibitor group were not statistically significant(P>0.05).3.Electroacupuncture can improve the damage of duodenal mucosal barrier in FD rats. (1)Changes of serum DAO and D-lactic acid Compared with the blank group,the serum DAO and D-lactic acid levels in each group were significantly increased after modeling(P<0.01),indicating that the mucosal permeability of FD rats increased.After intervention,the serum DAO level of rats in electroacupuncture group,electroacupuncture + inhibitor group and inhibitor group decreased to different degrees(P<0.01),suggesting that both electroacupuncture and inhibitor can improve mucosal permeability.Compared with the electroacupuncture group,the serum DAO level in the electroacupuncture + inhibitor group decreased more significantly(P < 0.05).Compared with the blank group,the serum D-lactic acid level of the FD model rats increased significantly(P < 0.05),indicating that the mucosal permeability of FD model rats increased.After intervention,the serum D-lactic acid level of rats in electroacupuncture group,electroacupuncture+inhibitor group and inhibitor group decreased to different degrees(P<0.01),suggesting that both electroacupuncture and inhibitor can improve mucosal permeability.Compared with the electroacupuncture group,the decrease of serum d-lactic acid level in the inhibitor group was lower(P<0.01),and there was no significant difference in the serum d-lactic acid level in the electroacupuncture + inhibitor group(P>0.05).(2)Changes of serum SIg A Compared with the blank group,the serum SIg A of the model group was significantly reduced(P<0.01),indicating that the mucosal immune barrier of FD duodenum was impaired.After intervention,the serum SIg A of the electroacupuncture group,electroacupuncture + inhibitor group and inhibitor group were increased(P < 0.01),suggesting that both electroacupuncture and inhibitor can improve the duodenal immune barrier.Compared with the electroacupuncture group,there was no significant difference in serum SIg A in the electroacupuncture + inhibitor group(P>0.05),and the increase of serum SIg A in the inhibitor group was less than that in the electroacupuncture group(P<0.05).(3)The expression of ZO-1,Occludin and Claudin-1 protein in duodenum Compared with the blank group,the relative expressions of ZO-1,Occludin and Claudin1 in the duodenum of the model group were significantly decreased(P < 0.01),indicating that the expression of Occludin and Claudin1 in the duodenum of the rats after FD modeling was decreased.Compared with the model group, the relative expression levels of ZO-1,Occludin and Claudin-1 in the duodenum of rats in the electroacupuncture group,electroacupuncture+inhibitor group and inhibitor group were increased(P < 0.01,P < 0.05),suggesting that electroacupuncture could increase the expression of interconnective protein in the duodenal mucosa and improve the mucosal permeability.Electroacupuncture+inhibitor increased the relative expression levels of ZO-1,Occludin,Claudin-1 in the duodenum(P < 0.01,P < 0.05),while electroacupuncture increased the relative expression levels of ZO-1,Occludin,Claudin-1 in the duodenum without significant difference from the inhibitor(P>0.05).(4)The relative expression of ZO-1 and Claudin-1 m RNA in duodenum Compared with the blank group,the relative expression level of ZO-1 in the duodenal mucosa of the model group was significantly decreased(P < 0.01),indicating that the transcription level of ZO-1 in the duodenum of the FD rats was decreased after multi-factor intervention.Compared with the model group,the relative expression level of ZO-1 in duodenal mucosa of electroacupuncture group,electroacupuncture + inhibitor group and inhibitor group was significantly increased(P<0.01,P<0.05),but still lower than that of the blank group(P<0.01,P<0.05).These results indicated that electroacupuncture intervention could increase the transcription level of ZO-1 in the duodenum of FD rats.Compared with the blank group,the relative expression level of Claudin-1 in the duodenal mucosa of the model group was significantly decreased(P < 0.01),indicating that the transcription level of Claudin-1 in the duodenum of the FD rats was decreased after multi-factor intervention.Compared with the model group,the relative expression level of Claudin-1 in duodenal mucosa of the electroacupuncture group,the electroacupuncture+inhibitor group and the inhibitor group was significantly increased(P<0.01),but still lower than that of the blank group(P<0.01,P<0.05).The above indicated that electroacupuncture intervention could increase the transcription level of Claudin-1 in the duodenum of FD rats.(5)Changes in the ultrastructure of duodenal mucosa.In the blank group,the microvilli of duodenal mucosa were arranged neatly.The gap of tight junction between cells is clear without broadening.Organelle structure did not show obvious abnormality.After modeling,the microvilli of duodenal mucosa were not arranged properly,and some of them were broken and lodging.The gap of tight junctions between cells was widened and blurred.Mitochondria swell and endoplasmic reticulum expands.After electroacupuncture intervention,the microvilli were arranged more neatly,the tight connections between cells were more clear,and the swelling of mitochondria and endoplasmic reticulum was less.In the electroacupuncture + inhibitor group,the duodenal mucosa villi of the rats were orderly arranged,the cell connections were tight and clear,and the gaps were not widened.The swelling of mitochondria and endoplasmic reticulum was not obvious.In the inhibitor group,the microvilli of duodenal mucosa were arranged neatly and some of them were broken.The tight connection between cells was clear and the gap was slightly wider.Mitochondria and endoplasmic reticulum are slightly swollen.It is suggested that electroacupuncture can repair the duodenal mucosal barrier.4.Electroacupuncture inhibits TLR4/NF-κB p65 pathway in duodenum of FD rats(1)Changes of TLR4/ NF-κB p65 pathway related protein expression in duodenum The duodenum tissue TLR4/NF-κB p65 pathway related protein expression changes compared with the blank group,model group rats duodenal TLR4,Myd88,TFAF6,NF-κB p65 and NF-κB p-p65 relative expression increased significantly(P<0.01,P<0.05),suggesting that the FD rats duodenal TLR4/NF-κB p65 pathway related protein increased,mediating inflammation.The relative expression levels of TLR4,Myd88,TFAF6,NF-Bp65 and NF-BP-P65 in the duodenum of the electroacupuncture group,the electroacupuncture + inhibitor group,and the inhibitor group were significantly decreased(P < 0.01,P < 0.05),indicating that the electroacupuncture intervention had similar effects with the inhibitor,and could inhibit the TLR4/NF-Bp65 pathway and reduce inflammation by inhibiting the expression of TLR4 protein.Compared with the electroacupuncture group,the TLR4/ NF-κB p65 pathway related protein expression in duodenum of the electroacupuncture + inhibitor group was lower,but still higher than that of the normal group(P < 0.01,P < 0.05).The relative expression levels of TLR4,Myd88,NF-κB p65 and NF-κB p-p65 in the duodenum of the inhibitor group were slightly higher than those of the electroacupuncture group(P<0.01,P<0.05),and there was no significant difference in TFAF6 expression between the inhibitor group and the electroacupuncture group(P>0.05).These results suggest that electroacupuncture reduces inflammation by inhibiting activation of the TLR4/ NF-κB p65 pathway,which is associated with blocking effects of TLR4 inhibitors.(2)Transcriptional expression of TLR4,NF-κB p65 gene in duodenum The expression levels of TLR4,NF-κB p65 in the duodenum were significantly higher in the model group than those in the blank group(P<0.01),indicating that the transcription levels of TLR4,NF-κB p65 in the model group were significantly increased after modeling.Compared with the model group,the relative expression levels of TLR4,NF-κB p65 in the duodenum of the electroacupuncture group,the electroacupuncture + inhibitor group and the inhibitor group were significantly lower(P<0.01),but still higher than that of the blank group(P<0.01),indicating that the electroacupuncture has the effect of reducing the transcription level of TLR4,NF-κB p65 gene in the duodenum of the FD rats.TLR4,NF-κB p65 m RNA levels were significantly decreased in the electroacupuncture + inhibitor group(P<0.01),but the effect of the inhibitor group was not as significant as that of the electroacupuncture group(P <0.01).(3)Transposition of the NF-κB nucleus of the duodenum NF-κB p65(red fluorescence)was mainly distributed in the cytoplasm of the rats in the blank group.After modeling,NF-κB p65 was concentrated in the nucleus(red fluorescence and blue),indicating activation of NF-κB in FD rat model.NF-κB p65 was transported from the cytoplasm to the nucleus to increase transcription expression of IL-6 and TNF-α.NF-κB p65 was significantly expressed in the cytoplasm after intervention with electroacupuncture or TLR4 inhibitor,indicating that the transport of activated NF-κB nuclear was inhibited in FD rats.Electroacupuncture inhibited the TLR4/NF-Bp65 signaling pathway in FD rats.Conclusion 1.FD rats showed low mood,reduced food intake,decreased body mass,weakened intestinal motility,impaired duodenal mucosal barrier,and systemic inflammation and duodenal inflammatory cell infiltration.The difference of gastric emptying delay was not observed.2.Electroacupuncture intervention can improve the depression of FD rats,increase the food intake and body mass for 3h,promote gastrointestinal motility,improve the damage of duodenal mucosal barrier,and reduce inflammation.3.Electroacupuncture intervention can reduce the levels of downstream inflammatory cytokines IL-6 and TNF-α in serum NF-κB of FD rats,and down-regulate the expression levels of IL-6 and TNF-α in duodenal mucosa.TLR4 inhibitors are synergistic with electroacupuncture.4.Electroacupuncture intervention can reduce serum DAO and D-lactic acid and improve duodenal mucosal permeability;Increased serum SIg A and improved duodenal mucosal immune barrier injury.Electroacupuncture intervention in FD model rats could also increase the expressions of tight junction proteins ZO-1,Occludin,claudin-1,and increase the expressions of ZO-1 and Claudin-1.The gap between the duodenal mucosal cells was narrowed,the villi were restored,and the mechanical barrier of the duodenal mucosa was improved.TLR4 inhibitor intervention can also achieve the above effects,suggesting that TLR4 is involved in improving duodenal barrier injury.5.Electroacupuncture reduce the FD model rats duodenal TLR4,Myd88,TFAF6,NF-κB p65,NF-κB p-p65 protein expression and the TLR4,NF-κB p65 gene transcription,inhibiting activation of NF-κB,prevent the NF-κB p65 to the displacement of the nuclei,thus inhibiting TLR4/NF-κB p65 signal pathway,reduce downstream inflammatory cytokines IL-6,the expression of TNF-α,improve the duodenal mucosa barrier damage,reduce inflammation reactions. |
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