| ObjectivesDelayed gastric emptying is considered to be one of the major pathophysiological mechanisms of functional dyspepsia(FD),accounting for 40%of all FD patients.At the same time,delayed gastric emptying is associated with dyspepsia such as bloating and early satiety.The method of promoting gastric emptying treatment is limited,the effect is poor,and there has been no clinical attempt to stop looking for an effective treatment method.Brain-derived neurotrophic factor(BDNF),a peptide for neural nutrition,is a natural agonist specific for tyrosine kinase receptor B(TrkB).Following the activation of TrkB by BDNF,it is well-known that in the nervous system,one or more of three intracellular signaling pathways are turned on:phospholipase C(PLC),phosphatidylinositol-3-kinase(PI3K)/Akt(protein kinase B)and the Ras/extracellular signal-regulated kinase(ERK).Activation of the PLC pathway generally leads to elevation of intracellular Ca2~+ concentration and strengthens excitatory synapse transduction at the neuromuscular junction.Studies in recent years indicate that BDNF and TrkB receptors were highly expressed in the intestinal tract as well,promoting the peristalsis of adult intestinal tract and enhancing the electrophysiological activities of rat gastrointestinal smooth muscles when both bind together.Therefore,in the digestive system,BDNF/TrkB signaling pathway may play an important role in regulating intracellular Ca2+concentration and promoting gastrointestinal motility.7,8-dihydroxyflavone(7,8-DHF)is a member in the flavone family.The synthetic flavonoid compound is widely distributed in various plants and possesses multiple biological functions,such as antioxidation,anti-inflammation,anti-carcinoma,neural protection,anti-hypertension,anti-diabetes and so on.The molecular mechanism of7,8-DHF has not been understood until recently.7,8-DHF has been revealed to be an agonist specific for TrkB and the first drug which can simulate the biological effect of BDNF.Its interaction with TrkB activates PLC-γ,AKT and ERK1/2 signaling pathways,plays roles in neural protection and leads to behavioral changes,for instance,improving memory and counteracting depression.Besides the nervous system,since TrkB receptors are also widely distributed in the gastrointestinal tract,it is presumed that 7,8-DHF is probably capable of activating TrkB and exerts biological effect on gastrointestinal dynamics.One report has indicated that like BDNF,7,8-DHF was able to promote intestinal dynamics.However,in fact,the roles of BDNF,7,8-DHF and TrkB in the intestine are controversial at present.Al-Qudah et al found that both 7,8-DHF and BDNF might increase the carbachol(CCh)-induced intestinal contraction of rabbits while the molecular mechanism for the effect remained unclear.Therefore,7,8-DHF may promote contraction of gastric smooth muscle by activating TrkB receptor and promote gastric emptying.At the same time,there is no research on the molecular mechanism of 7,8-DHF acting on gastric smooth muscle.Therefore,this experiment will study the following aspects:1.To study on the effect of 7,8-DHF on the tension of isolated gastric ring muscle strips2.To study the molecular mechanism of 7,8-DHF-induced changes in the tension of the gastric ring muscle.3.Effect of 7,8-DHF on gastric emptying in rats.Methods1.Preparation of gastric circular smooth muscle stripsBefore experiments,rats were fasted for 12 hours but allowed to drink water ad libitum and were then anesthetized with thiobutabarbitol(100 mg/kg)intraperitoneally injected.The entire stomach,with incisal edges being 1 cm above the cardia at near-end and 1 cm below the pylorus at far-end,was taken out rapidly through an abdominal incision and placed in 37°C Krebs solution bubbled continuously with a mixed gas of 95%O2 and 5%CO2.The gastric lumen was cut open along the greater curvature,and the gastric mucosa and serosa were gently removed with tweezers.Gastric circular smooth muscle strips were prepared by cutting the antrum perpendicular to the long axis of the gastric lumen.A circular muscle strip(15 mm long×5 mm wide)was then taken from~5mm above the pylorus,hung lengthwise after being tied with surgical silk thread at ends and placed in a chamber perfused with 6 mL of 95%O2/5%CO2-being-equilibrated Krebs solution at 37°C.Initial tension of 0.5 g was applied to the strips for 60 minutes,which was rinsed with fresh 37°C Krebs solution every 15 minutes until the spontaneous contraction of muscle strips stabilized.The strips were rinsed with 37°C Krebs solution three times with interval time of 10 minutes beforea drug was applied or substituted.2.Effects of CCh and 7,8-DHF on gastric circular muscle strips2.1 The muscle strips were incubated with 7,8-DHF(1,10,30,100μmol/L)for 30 minutes,and the changes in the tension of the gastric ring muscles were observed.The muscle strips were incubated for 3 minutes with different concentrations of CCh(1,10,30,100μmol/L),and the changes of CCh on the tension of the gastric ring muscle were observed to determine the concentration of CCh in the subsequent experiments.2.2 Different concentrations of 7,8-DHF(1,10,30,100μmol/L)were incubated for 30 minutes,then CCh was incubated for 3 minutes to observe changes in gastric ring muscle tension,and the concentration of 7,8-DHF in subsequent experiments was determined..2.3 After incubation with 7,8-DHF for 30 minutes,the muscle strips were incubated with 1μmol/L SP and 10 mmol/L k+for 8 minutes to detect changes in gastric muscle strip tension.It is clear whether 7,8-DHF has an effect on SP and high concentration of k+induced gastric muscle strip tension.3.Molecular mechanism of 7,8-DHF enhancing CCh-induced gastric muscle strip tension3.1 Molecular pathway protein inhibition experiments:Studies have shown that7,8-DHF may activate Akt,ERK1/2 and PLC-γsignaling pathways by activating TrkB to produce biological effects.To further investigate the molecular mechanism of 7,8-DHF action,an antagonist of the pathway protein was used to see if the antagonist could inhibit the CCh-induced gastric muscle strip contraction effect by 7,8-DHF.3.1.1 Different concentrations of the TrkB receptor antagonist ANA-12(1μmol/ L or 10μmol/L)incubated muscle strips 15 minutes,and 10μmol/L 7,8-DHF incubated for 30 minutes,and then 10μmol/L CCh incubated for 3 minutes,the changes in the tension of the gastric circular muscles were observed.3.1.2 The muscle strips were incubated with the Erk1/2 antagonist PD98059(10μmol/L)for 15 minutes,then incubated with 10μmol/L 7,8-DHF for 30 minutes,and then the muscle strips were incubated with 10μmol/L CCh for 3 minutes to observe the change in tension of the gastric ring muscle strips.3.1.3 The muscle strips were incubated with the Akt antagonist LY294002(10μmol/L)for 15 minutes,then incubated with 10μmol/L 7,8-DHF for 30 minutes,and then the muscle strips were incubated with 10μmol/L CCh for 3 minutes to observe the change in tension of the gastric ring muscle strips.3.1.4 The muscle strips were incubated with the PLC-γantagonist U73122(10μmol/L)for 15 minutes,then incubated with 10μmol/L 7,8-DHF for 30 minutes,and then the muscle strips were incubated with 10μmol/L CCh for 3 minutes to observe the change in tension of the gastric ring muscle strips.3.2 For western blots 1:the strip tissues were collected after the following four experiments.(1).Control:muscle strips before any treatment;(2).CCh:After incubating the strips with Krebs solution for 60 minutes,a final concentration of 10μmol CCh was added the incubation chamber.The strip tissue was collected after CCh stimulated muscle strips for 3 minutes;(3)DHF:After stabilization,10μmol/L 7,8-DHF was added and remained in the solution for 30 minutes before the tissue was collected;(4)CCh+DHF:muscle strips incubated for 30 min with7,8-DHF above in step 3 was exposed to 10μmol/L CCh for 3 min and then was harvested for western blots.The tissues collected were used for detection by western blots of TrkB,Erk1/2,Akt andPLCγreceptor expression changes and protein phosphorylation and M3、M2 receptor expression changes.3.3 Western blotting experiment 2:To test the effect of AKT antagonist on the expression level of muscle strip M3 receptor:group:(1)Control:after treatment of muscle strip,apply 0.5 g pre-initial tension,balance for 60 minutes(fresh 37°C every 15 minutes)Kerbs solution to wash the muscle strip);(2)LY294002+CCh group:antagonist LY294002(10μmol/L)incubate the muscle strip for 15 minutes,add the final concentration of 10μmol/L 7,8-DHF to incubate the muscle strip for 30 minutes;(3)DHF:give muscle After a 0.5 g tension and stable equilibrium,incubate for 10 minutes by adding 10μmol/L 7,8-DHF.4.Rats were fed by gavage with 7,8-DHF solution(0.2,0.8 or 3.2 mmol/L)or equal volume(1 mL)of normal saline(Control group)once a day for 7 days.To measure the rate of gastric emptying,phenol red was administered.After being fasted but with water available for drinking ad libitum for 16 hours,rats were fed by gavage with 2 mL of phenol red solution(1.5 mmol/L)and then sacrificed 15 minutes later.The gastric contents were collected for measuring residual amounts of phenol red by spectrophotometry.The percentage of discharged amount of phenol red over the administered amount in 15 minutes indicates the gastric emptying rate.Results1.The study found that 7,8-DHF did not affect the contraction of gastric strips.2.We treated the gastric strips with CCh from 1,10,30 to 100μmol/L shows that CCh induced dose-dependent contraction of the strips.We determined the optimal concentration of CCh to produce the contractile effect on the muscle strips.when muscle strips was incubated in 30 and 100μmol/L CCh,did not readily and quickly recover to the baseline by washing,the optimal concentration was 10μmol/L CCh that was selected for the subsequent experiments.3.The muscular strips were incubated with 7,8-DHF(1,10,30 and 100μmol/L)in Krebs solution for 30 minutes and then CCh(10μmol/L)was added to the incubating solution to contract the strips,we found that 7,8-DHF enhanced CCh-induced contraction of rat gastric muscle.The effect of 7,8-DHF was apparently dose-dependent.But 7,8-DHF did not significantly alter the strip contraction induced by either SP or high[K+].4.The TrkB receptor antagonist ANA-12 was applied to the gastric muscle strips,and it was found that 10μmol/L could inhibit the gastric muscle strip tension induced by7,8-DHF,but the inhibitory effect of 1μmol/L ANA-12 was not obvious.This indicates that 7,8-DHF acts to promote muscle contraction through the TrkB receptor,but ANA-12can inhibit TrkB receptor after reaching a certain concentration.5.LY294002 is an antagonist specific for AKT was applied to the muscular strips.We found that LY294002 almost completely blocked the 7,8-DHF-Enhanced contraction although it did not affect the CCh-induced contraction.PLC-γantagonist U73122,significantly weakened the strip contraction induced by CCh or CCh+7,8-DHF treatment.PD98059 is an antagonist specific for ERK1/2,The antagonist did not show any significant influence on the CCh-induced or 7,8-DHF+CCh-induced contraction of gastric strips This indicated that 7,8-DHF affects carbachol(CCh)-induced rat gastric muscle contraction via AKT and PLC-γpathways.6.By way of western blots,we found that the expression levels of TrkB protein were not affected by 7,8-DHF,CCh and 7,8-DHF+CCh treatment.whereas the signals of p-TrkB were significantly intensified by either 7,8-DHF or 7,8-DHF+CCh treatment.The experiments provided evidence that 7,8-DHF did activate TrkB.7.PLC-γ,akt and ERK1/2 protein levels were not significantly changed in CCh,7,8-DHF and 7,8-DHF+CCh treatments.p-EKR1/2 protein levels in gastric strips were not altered by single or combined treatment of 7,8-DHF and CCh.We did not see any alteration of PLC-γphosphorylation in gastric muscle strips treated with 7,8-DHF alone,indicating that PLC-γmight not be turned on by TrkB activation in gastric muscles.CCh treatment induced phosphorylation of PLC-γ,a combined treatment with CCh+7,8-DHF evidently enhanced the CCh-induced PLC-γphosphorylation.This indicatesd that 7,8-DHF could not directly activate PLC-γprotein,but has other ways to affect carbachol(CCh)-induced rat gastric muscle contraction.8.The study found that 7,8-DHF treatment evidently made Akt phosphorylated,CCh alone did not activate Akt and the combination of 7,8-DHF+CCh did not further increase p-Akt levels.7,8-DHF significantly increased the M3 receptor expression in the gastric muscular strips CCh+7,8-DHF treatment produced a similar result,but CCh could not increased the M3 receptor expression.Akt antagonist LY294002 blocked the7,8-DHF-augmented M3 receptor expression.This indicatesd that 7,8-DHF increases the expression level of M3 receptor through the TrkB/AKT pathway.7,8-DHF did not significantly change the expression of M2 receptor expression.9.We tested three concentrations of 7,8-DHF(0.2,0.8 and 3.2 mmol/L)fed orally to observe its effect on the gastric emptying rate.After feeding once a day for 7 days,we found that 7,8-DHF fed at 0.8 and 3.2 mmol/L significantly increased gastric emptying rate.Conclusions1.7,8-DHF alone does not promote contraction of gastric muscle strips,but 7,8-DHF can enhance the tension of CCh-induced gastric strips.2.7,8-DHF does not directly act on the CCh/M3/PLC-γ/Ca+pathway to increase gastric muscle strip tension,while the 7,8-DHF/TrkB/Akt pathway increases M3 receptor expression levels,thereby increasing The effect of CCh on M3 increases the tension of the gastric ring muscle strip,but the specific mechanism of increased M3 receptor expression needs further study.3.7,8-DHF can increase gastric emptying in rats,suggesting that 7,8-DHF promotes gastric motility in whole animals,indicating that 7,8-DHF has the potential to develop a therapeutic drug that enhances gastric motility. |
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