不同频率摩腹对脾虚型功能性消化不良家兔胃肠动力的调控规律及其与CaM-MLCK信号通路相关性研究

Research on the regulation of gastrointestinal motility in rabbits with functional dyspepsia due to spleen deficiency by different frequency Abdominal Rubbing and its correlation with CaM-MLCK signaling pathway

目的观察不同频率摩腹对脾虚型功能性消化不良家兔胃肠动力的调控规律,以钙调蛋白—肌球蛋白轻链激酶(caImodulin-myosin light chain kinase,CaM-MLCK)信号通路为切入点探讨摩腹调控胃肠动力的作用机制。方法随机将60只健康成年新西兰家兔均分为空白组、模型组、低频摩腹治疗组、高频摩腹治疗组、低频摩腹+抑制剂组及高频摩腹+抑制剂组。除空白组外,其余各组家兔均造脾虚型功能性消化不良模型,空白组及模型组均不施加干预。治疗组采用人工智能机械臂以相应频率摩腹作用于各组家兔中脘、天枢穴(双),保持摩腹压力与方向一致,低频摩腹治疗组及低频摩腹+抑制剂组摩腹频率为101~150次/分钟,高频摩腹治疗组及高频摩腹+抑制剂组频率为201~250次/分钟,每只家兔每穴推5分钟,共计15分钟,每日1次。低频摩腹+抑制剂组及高频摩腹+抑制剂组均在每日摩腹治疗给家兔腹腔注射肌球蛋白轻链激酶抑制剂(ML 9 hydrochloride,ML-9),连续干预10天。干预结束后采用炭末灌胃法测定各组家兔小肠推进率,并测定各组家兔胃肠组织一氧化氮(nitric oxide,NO)、P物质(Substance P,SP)、腺嘌呤核苷三磷酸(adenosine triphosphate,ATP)、三磷酸肌醇(Inositol Triphosphate,IP3)及环磷酸腺苷(cyclic adenosine monophosphate,cAMP)的含量。结果与空白组相比,模型组小肠推进率显著降低(P<0.01),胃NO含量显著升高(P<0.01),胃SP含量显著下降(P<0.01),胃、肠IP3无统计学差异(P>0.05),胃、肠ATP含量下降(P<0.05),胃、肠cAMP含量升高(P<0.05)。干预结束后,与模型组相比,低频摩腹治疗组、低频摩腹+抑制剂组及高频摩腹+抑制剂组小肠推进率均显著提高(P<0.01),高频摩腹治疗组无统计学差异(P>0.05);与模型组相比,低频摩腹治疗组胃NO含量显著下降(P<0.01),胃SP含量显著升高(P<0.01),肠IP3下降(P<0.05),胃、肠ATP含量升高(P<0.05),胃、肠cAMP含量下降(P<0.05);高频率摩腹治疗组胃SP含量升高(P<0.01),肠IP3含量下降(P<0.05),肠ATP含量升高(P<0.05),其余指标无统计学差异(P>0.05)。与低频摩腹治疗组比较,低频摩腹+抑制剂组胃NO含量升高(P<0.05),胃SP含量无统计学差异(P>0.05),肠IP3显著下降(P<0.01),胃、肠ATP含量显著下降(P<0.01),胃、肠cAMP含量升高(P>0.05);与高频摩腹治疗组比较,高频摩腹+抑制剂组胃NO含量升高(P<0.05),胃SP含量无统计学差异(P>0.05),肠IP3显著下降(P<0.01),胃、肠ATP含量下降(P<0.05),胃、肠cAMP含量升高(P<0.05)。结论摩腹对胃肠动力的影响存在频率—效应规律,摩腹在101~150次/分钟频率段时可显著促进胃肠动力,当频率超过200次/分时,摩腹对胃肠动力无改善作用,甚至有损害趋势。其作用机制可能是:(1)通过降低胃肠组织中NO及cAMP的含量,抑制cAMP/PKA途径及cGMP/PKG途径,减少对Ca 2+-CaM的竞争性消耗,从而促进上游CaM-MLCK信号通路中Ca 2+、Ca 2+-CaM与MLCK的结合。(2)当CaM-MLCK信号通路被阻断后,一方面,摩腹可通过提高家兔胃SP含量,发挥其促进胃肠动力的直接效应;另一方面,IP3的显著下降提示,阻断CaM-MLCK信号通路,可能增加IP3的消耗促使SOCE发生,从而改变胞内Ca 2+水平,激发Ca 2+介导的其它信号通路来调节胃肠动力。

Objective To observe the regulation of gastrointestinal motility in rabbits with functional dyspepsia due to spleen deficiency by different frequencies of Abdominal Rubbing,and to explore the mechanism of this regulation with CaM-MLCK signaling pathway.Methods Sixty healthy adult New Zealand rabbits were randomly divided into blank group,model group,low-frequency abdominal rubbing treatment group,high-frequency abdominal rubbing treatment group,low-frequency abdominal rubbing+inhibitor group and high-frequency abdominal rubbing+inhibitor group.Except for the blank group,all other groups of rabbits were all created as spleen deficiency FD model,and neither the blank group nor the model group had any intervention.Artificial intelligence robotic arm is used to massage the rabbits at the corresponding frequency on Zhongwan and Tianshu points(both sides),keep the pressure and the direction Unchanged,the low-frequency was 101~150 times/minute,the high-frequency was 201~250 times/minute.Push each point for 5 minutes,15 minutes in total each time,once a day.Both rabbits in the low-frequency abdominal+inhibitor group and the high-frequency abdominal+inhibitor group were injected intraperitoneally with ML-9 after daily abdominal rubbing treatment.After the intervention,the small intestine propulsion rate of the rabbits in each group was measured by gavage with charcoal powder,and the contents of NO,SP,ATP,IP3 and cAMP were measured in the gastrointestinal tissues of the rabbits in each group.Results Compared with the blank group,the small intestinal advancement rate was significantly decreased(P<0.01),the gastric NO content was significantly increased(P<0.01),the gastric SP content was significantly decreased(P<0.01),there was no statistical difference in gastric and intestinal IP3(P>0.05),the stomach and intestinal ATP content was decreased(P<0.05),and the gastric and intestinal cAMP content was increased(P<0.05).After the end of the intervention,compared with the model group,the small intestinal advancement rate was significantly increased in the low-frequency rubbing group,the low-frequency rubbing+inhibitor group and the high-frequency rubbing+inhibitor group(P<0.01),and there was no significant difference in the high-frequency rubbing group(P>0.05);compared with the model group,the gastric NO content was significantly decreased(P<0.01),gastric SP content was significantly increased(P<0.01),intestinal IP3 was decreased(P<0.05),stomach and intestinal ATP content was increased(P<0.05),stomach and intestinal ATP content was increased(P<0.05),stomach and the content of intestinal cAMP was decreased(P<0.05),and the gastric SP content was increased(P<0.01),the intestinal IP3 content wasdecreased(P<0.05),and the intestinal ATP content was increased(P<0.05)in the high-frequency rubbing treatment group,and there was no statistical difference in the rest of the indicators(P>0.05).Compared with the low-frequency rubbing treatment group,the gastric NO content in the low-frequency rubbing+inhibitor group was increased(P<0.05),the gastric SP content was not statistically different(P>0.05),the intestinal IP3 significantly was decreased(P<0.01),the gastric and intestinal ATP content was decreased significantly(P<0.01),and the gastric and intestinal cAMP content was increased(P>0.05).Compared with the high-frequency rubbing massage treatment group,the gastric NO content in the high-frequency rubbing+inhibitor group was increased(P<0.05),gastric SP content was not statistically different(P>0.05),intestinal IP3 significantly decreased(P<0.01),gastric and intestinal ATP content decreased(P<0.05),and gastric and intestinal cAMP content was increased(P<0.05).Conclusion The influence of different frequencies of Abdominal Rubbing on gastrointestinal motility is different and there is a certain rule.When the frequency of manipulation is 101~150 times/min,rubbing has the benign effect of significantly improving the symptoms of spleen deficiency and promoting gastrointestinal motility,when the frequency exceeds 200 times/min,it could not restore gastrointestinal motility to normal levels,and there was a potential risk of damaging gastrointestinal function.The mechanism of it may be:(1)By reducing the content of NO and cAMP in gastrointestinal tissue,inhibiting the cAMP/PKA pathway and cGMP/PKG pathway,the competitive consumption of Ca 2+-CaM is reduced,thereby promoting the binding of Ca 2+,Ca 2+-CaM and MLCK in the upstream CaM-MLCK signaling pathway.(2)When the CaM-MLCK signaling pathway is blocked,abdomen rubbing can up-regulate the gastric SP content in rabbits and exert its direct effect of promoting gastrointestinal motility;on the other hand,a significant decrease in IP3 suggests that blocking the CaM-MLCK signaling pathway may up-regulate the consumption of IP3 and promote SOCE,thereby changing intracellular Ca 2+levels and stimulating other Ca 2+-mediated signaling pathways to regulate gastrointestinal motility.