The clinical symptoms of Functional Dyspepsia(FD)are complex and diverse,and the prevalence of FD is high.Although the current conventional drugs are effective,the long-term efficacy is not good enough,which requires long-term medication with many side effects.Thus,it is necessary to seek an effective new treatment for FD.Many external treatments of traditional Chinese medicine,especially massage therapy,can effectively relieve a variety of symptoms of FD on the basis of no medication and few side effects.It is a commonly used clinical treatment in China,and it is worth exploring in depth.Gastric motility deficiency is one of the basic pathogenesis of FD,and IP3-CaM-MLCK signaling pathway,which regulates the contraction of gastrointestinal smooth muscle,is an important part of it.Previous studies have shown that multiple traditional Chinese medicine therapies can regulate gastric motility through this pathway to varying degrees.ObjectiveOn the one hand,established in the basic theory of "reinforcing or reducing with slow or rapid frequency" of Tuina manipulation,this research studied the effect of pushing manipulation with one-finger meditation with different fast or rapid frequency on the gastrointestinal function of FD rabbits with spleen deficiency syndrome.On the other hand,based on IP3-CaM-MLCK signaling pathway,the mechanism of pushing manipulation with one-finger meditation in improving the gastric motility of FD rabbits with spleen deficiency syndrome was explored.MethodsExperiment 1:The normal rabbits were fed with low,medium or high concentration of bitter and cold herbal liquid,which contained rhubarb,mirabilite and senna leaf.At the same time,the rabbits were given with the method of irregular starvation for continuous 28 days to set up FD models with spleen deficiency syndrome.We recorded the appearance,body weight,food intake,water intake and rectal temperature of the rabbits in each group every 7 days.The gastric emptying rate and small intestinal propulsion rate after modeling were also recorded.Through observing the changes of gastrointestinal function in rabbits with different concentrations of herbal liquid gavage and different modeling cycles,we explore the appropriate gavage dose and modeling time for successful establishment of FD rabbit models with spleen deficiency syndrome,so as to guide this project to formulate a modeling program with optimized drug dose and modeling cycle.Experiment 2:Three massage physicians were invited to perform pushing manipulation with one-finger meditation of light,medium or severe pressure at Zhongwan(RN12)of 8 FD rabbits with spleen deficiency syndrome according to the observation of rabbit behavior.And MFF multi-point membrane pressure test system was used to measure and collect the pressure value of each manipulation.At the same time,the number of positive behavioral response items of each rabbit was observed and recorded.Then,from the horizontal and vertical perspectives,the difference degree of different levels of manipulation pressure of the three physicians as well as the correspondence between different levels of manipulation pressure and positive behavioral response of rabbits were observed.The mild,moderate and severe pressure ranges of manipulation by three Tuina physicians were set as the pressure parameters of the mechanical arm simulation.And the mechanical arm was used to simulate manipulation at Zhongwan(CV 12)in 8 FD rabbits with spleen deficiency syndrome.Later,the number of positive items in the rabbits was observed and recorded to be compared with the number of positive items of manual manipulation,to observe the difference of manual pressure under artificial/mechanical operation on FD rabbits with spleen deficiency syndrome.From this,the moderate pressure parameter range of the mechanical arm to simulate the pushing manipulation with one-finger meditation was sought,so as to provide stable and quantifiable research conditions for the frequence-reinforcing and reducing effect experiment of pushing manipulation with one-finger meditation.Experiment 3:Before modeling,12 rabbits were randomly selected as the blank group(group K).After modeling,50 FD rabbits with spleen deficiency syndrome were randomly divided into 5 groups:model group(group P),low-frequency manipulation group(group A),high-frequency manipulation group(group B),low-frequency manipulation+inhibitor group(group Ⅰ-1),and high-frequency manipulation+inhibitor group(group Ⅰ-2).The mechanical arm was used to operate the pushing manipulation with one-finger meditation with different frequencies at Zhongwan(CV 12)and Tianshu(ST 25)(bilateral)in rabbits,and the MLCK inhibitor(ML-9)was selectively injected intraperitoneally.The appearance,body weight,food intake,water intake and rectal temperature of rabbits in each group at different time points,as well as gastric emptying rate and small intestine propulsion rate after intervention,were observed.From the above,we explored the effects of different frequencies of pushing manipulation with one-finger meditation on the macroscopic manifestations of gastrointestinal motility in rabbits with FD and spleen deficiency syndrome.Experiment 4:The grouping and intervention methods of rabbits were the same as"Experiment 3".And ELISA was used to detect the contents of MTL and GAS in serum as well as the contents of IP3,cAMP and ATP in gastric antrum tissue of rabbits in each group.Real-Time PCR was utilized to detect the gene expression levels of CaM,MLCK and MLCP in gastric smooth muscle tissue.The relative protein expression levels of CaM,MLCK and MLCP in gastric smooth muscle tissue were detected by Western Blot.Then the comparison of the above indexes in each group was obtained.Based on these experimental results,we discussed the molecular biological mechanism of improving gastrointestinal motility in FD rabbits with spleen deficiency syndrome by pushing manipulation with one-finger meditation with different frequencies.ResultsExperiment 1:By comparing the model effects formed by different drug doses and modeling periods,we established an optimized modeling scheme of FD rabbit with spleen deficiency syndrome.The results showed that the rabbits in the medium-dose and high-dose groups had distinct changes in appearance on the 21st and 28th days of modeling,while the rabbits in the low dose group had no obvious spleen deficiency.In terms of body weight,food intake and rectal temperature,the high-dose group showed significant changes on the 14th day of modeling with a downward trend as modeling time extension.The body weight and food intake of rabbits in the medium-dose group also decreased significantly on the 14th day of modeling and showed a downward trend.The water intake of the medium-dose and high-dose groups began to decrease significantly from the 21st day of modeling.Compared with the control group,the body weight of the low-dose group decreased significantly on the 28th day of modeling,and the food intake reduced on the 21st day of modeling,but then rebounded.There was few changes in water intake and rectal temperature of low-dose group.At the end of modeling,the gastric emptying rate of rabbits in the medium-dose and high-dose groups was significantly lower than that in the blank group,and the small intestinal propulsion rate of rabbits in the three gavage groups was lower than that in the blank group.In addition,two rabbits died in the high-dose group at the final stage of modeling.Therefore,we finally determined that the protocol with 40%herbal liquid for intragastric administration and 21 days of modeling period could successfully construct FD rabbits with spleen deficiency syndrome that meet the model evaluation criteria,which was an optimized protocol for establishing this animal model.According to the macroscopic performance of rabbits in the medium-dose group,and referring to several relevant guidelines for diagnosis and treatment and literature containing model evaluation criteria,we initially refined the scoring criteria for the animal model of FD with spleen deficiency syndrome.The total score was 13 points,and the score≥7 points was considered to be successful.And this would be used as the scoring standard to judge whether the model was successfully established in subsequent experiments.Experiment 2:By comparing the pressure values and the corresponding behavioral responses of rabbits between different operators as well as between artificial and mechanical operation,the moderate pressure parameter of the mechanical arm for simulating the pushing manipulation with one-finger meditation was explored and determined.The results showed that there were significant differences in the pressure values of the manipulation performed by the same physician at different pressure levels,while there was no differences in the pressure values of the technique performed by the three physicians at the same pressure level in rabbits.These indicated that there were clear distinctions in the pressure values produced by different manipulation pressure levels,but the maneuvers of the three physicians had certain stability and convergence in the pressure of manipulation.In addition,the similarities and differences in the number of positive items generated by each doctor’s manipulations with different pressure levels were basically consistent with the pressure level,which further supported the above results.Then,the manipulation pressure values of the three doctors at each pressure level were integrated(mild:1.23±0.14N,moderate:2.66±0.42N,severe:4.71±0.31N)and set in the robotic arm control system to simulate pushing manipulation with one-finger meditation at Zhongwan(CV 12)in rabbits.Finally,91.67%of the numbers of positive behavior items were in accordance with the classification standard of different levels of manipulation pressure.And there was no difference between the numbers of positive behavior items from mechanical operation and those produced by artificial manipulation at the same pressure level.It manifested that under the same pressure level,the effects of manual operation and robotic arm operation on the behavioral response of FD rabbits with spleen deficiency syndrome were very close,which implied that there was homogeneity between the pressure values of man-machine manipulation.Therefore,in the subsequent experiments,the moderate pressure range of human manipulation(2.66±0.42N)would be set as the standard pressure parameter for the mechanical arm to simulate the pushing manipulation with one-finger meditation.Experiment 3:The results showed that the appearances of rabbits in groups A and group B gradually improved with the extension of intervention time.And they were significantly improved on the 10th day of intervention,but the final effect of group B was not as good as that of group A.In addition,there was little change in rabbits in groups Ⅰ-1 and group Ⅰ-2.After intervention,the body weight of rabbits in group A was distinctly higher than that in group P,while group B,Ⅰ-1 and Ⅰ-2 showed no change,which were obviously lower than group A on the 10th day of intervention.The food intake of rabbits in groups A and B were more than those in group P after manual intervention,while there was no change in Ⅰ-1 and Ⅰ-2 groups.In terms of total water intake,group A and Ⅰ-1 were significantly higher than group P after the intervention,while group B had less total water intake than group A.These results indicated that the pushing manipulation with one-finger meditation could improve the macroscopic performance of rabbits,such as appearance representation,body weight,food intake and water intake,and the effect of the low-frequency pushing manipulation with one-finger meditation was better than that of the high-frequency technique.Except for water intake,ML-9 may block the improvement of all other macroscopic manifestations in FD rabbits with spleen deficiency syndrome.After the intervention,the gastric emptying rate of rabbits in groups A and B was higher than that in group P,while the small intestinal propulsion rate of rabbits in group A was much higher than that in other four groups.It manifested that the pushing manipulation with one-finger meditation in low frequency can effectively improve the gastrointestinal motility of rabbits,and its promoting effect was distinctly better than that of the high-frequency pushing manipulation with one-finger meditation.Furthermore,ML-9 may have a blocking effect on the gastrointestinal motility of FD rabbits with spleen deficiency syndrome.Experiment 4:After manual intervention,the results of ELISA showed that the serum MTL of the four intervention groups was significantly higher than that of the P group,while the serum GAS was obviously decreased.And the serum MTL of group B was lower than that of group A,while the serum GAS was higher than that of group A.In addition,differences similar to those between groups A and B were also observed between groups Ⅰ-1 and Ⅰ-2.The levels of IP3,cAMP and ATP in gastric antrum tissue of group A and group B were distinctly higher than those of group P;The index levels above of group B and Ⅰ-1 were lower than those of group A;However,they were also lower in group Ⅰ-2 than in group B.These results indicated that the pushing manipulation with one-finger meditation could effectively regulate the serum MTL,GAS and the levels of IP3,cAMP and ATP in gastric antrum tissue of rabbits,and the improvement effect of the low-frequency pushing manipulation with one-finger meditation was much better than that of the high-frequency technique.They also showed that ML-9 did not affect the levels of MTL and GAS in serum,but could inhibit the levels of IP3,cAMP and ATP in gastric antrum tissue.The results from RT-PCR and Western-blot showed that compared with group P,the mRNA expressions of CaM in the four intervention groups were sharply increased,and the changes between groups A and B,Ⅰ-1 and Ⅰ-2 showed basically the same trend.The expression of CaM protein in groups A and Ⅰ-1 was higher than that in group P,and there were also wide differences between groups A and B,Ⅰ-1 and Ⅰ-2.These indicated that low-frequency pushing manipulation with one-finger meditation could up-regulate CaM expression in gastric body smooth muscle of rabbits better than high-frequency one,and this expression is not affected by ML-9.The expression of MLCK and MLCP mRNA and protein in gastric body tissues of rabbits in group A and group B were clearly higher than those in group P,and group A was better than group B,which indicated that the low-frequency pushing manipulation with one-finger meditation had better effects on up-regulating the expression of MLCK and MLCP in gastric body tissues of rabbits.However,group A was much higher than group Ⅰ-1,and group B was also higher than group Ⅰ-2,indicating that ML-9 may have a blocking effect on the expression of MLCK and MLCP in rabbit gastric body smooth muscle.ConclusionThe pushing manipulation with one-finger meditation could effectively improve the gastrointestinal motility status of FD rabbits with spleen deficiency syndrome,including the body weight,food intake,water intake,and gastrointestinal transit capacity.However,the low-frequency manipulation had a better effect on the improvement of gastrointestinal function in FD rabbits with spleen deficiency syndrome than the high-frequency one,and the mechanism may be related to the regulation of IP3-CaM-MLCK signaling pathway.And pushing manipulation with one-finger meditation could increase the release of related brain-gut peptides,up-regulate the expression level of IP3-CaM-MLCK signaling pathway related factors,and then promote the contraction of gastric body smooth muscle.This study revealed the mechanism of pushing manipulation with one-finger meditation in regulating gastric motility of FD rabbits with spleen deficiency syndrome. |