| Objectives:1.Clinical ApplicationTo explain the source of "Jianpiliqi Granule",to discuss its theoretical basis in treating functional dyspepsia(FD),and to briefly expound the processing technique of "Jianpiliqi Granule".2.Animal ExperimentsTo investigate the therapeutic effect of "Jianpiliqi Granule" on FD rat model,and reveal some of the mechanisms.Methods:1.Clinical ApplicationThe clinical experience of Professor Zhang in the treatment of FD was summarized,the theoretical source and processing technique of "Jianpiliqi Granule" was expounded.2.Animal ExperimentsExperiment 1:90 seven-day-old male SD rats were randomly divided into normal group,model group,"Jianpiliqi Granule" high-dose group(high-dose group),"Jianpiliqi Granule" medium-dose group(medium-dose group),"Jianpiliqi Granule" low-dose group(low-dose group),and"Qizhiweitong Granule group"(positive drug group).There were 15 rats in each group.FD rat model was established with iodoacetamide(IA)treatment and tail-squeezed.At 8 weeks of age,rats in high-dose group,medium-dose group and low-dose group received 8.4g/kg,4.2g/kg,and 2.1g/kg of "Jianpiliqi Granule" daily by oral gavages respectively for 7 days;rats in positive drug group received 1.56g/kg.d of "Qizhiweitong Granule" by oral gavages for 7 days;and rats in the normal group and the model group received normal saline(NS).At 9 weeks of age,each rat was anesthetized,one balloon was surgically implanted in the stomach,and a pair of stainless steel wires was implanted into the acromiotrapezius(a superficial neck muscle)and externalized at the back of the neck.At 10 weeks of age,all rats received gastric balloon distentions,graded intragastric pressure(20,40,60,and 80 mmHg)was achieved by inflating the balloon for 30 seconds with 3 minutes rest.Behavioral responses(based on the abdominal withdrawal reflex[AWR])were graded.Electromyogram(EMG)was recorded simultaneously with grading behavior.Gastric accommodation was calculated by monitoring the change of gastric balloon volume.Experiment 2:30 seven-day-old male SD rats were randomly divided into normal group,model group,and "Jianpiliqi Granule" medium-dose group.Model establishment was same with experiment 1.At 8 weeks of age,rats in "Jianpiliqi Granule" medium-dose group received 4.2g/kg of "Jianpiliqi Granule" daily by oral gavages respectively for 7 days,and rats in the normal group and the model group received NS.At 9 weeks of age,gastric balloon was surgically implanted in the stomach of each rat.At 10 weeks of age,all rats received gastric balloon distentions,graded intragastric pressure(40 mmHg)was achieved by inflating the balloon for 60 seconds.Then the T8-T10 of spinal cord and the stomach were rapidly collected.HE staining was used to observe the pathological changes of gastric tissue in rats.The distribution of FOS positive neurons in the dorsal horn of the spinal cord was determined by immunohistochemistry technique.Real Time PCR was used to detect the relative mRNA expression of c-fos,iNOS and GABAb.Western Blot was used to detect the relative protein expression of FOS,iNOS and GABAb.;Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of cGMP and PKG.Experiment 3:12 male SD rats were randomly divided into NS group and L-NAME group.Each gastric circular smooth muscle strip(2mm × 8mm)was collected and put into a chamber containing 15ul Krebs solution and babbled with a mix gas of 95%02 and 5%C02.Then 5ul of NS or L-NAME(10-4mol/L)were added to the bath of the two groups respectively,and each muscle strip was incubated for 30min.The effects of "Jianpiliqi Granule" at concentrations of 3μg/ml,30μg/ml,300μg/ml,3000μg/ml and 6000μg/ml on the decline ratio of average tension of muscle strips were observed.Results:1.Clinical Application"Jianpiliqi Granule" was derived from the clinical experience of my tutor,Professor Zhang.Its clinical application has a solid basis and a reliable effect."Jianpiliqi Granule" has a normative preparation process,and provides the support for animal experiments.2.Animal ExperimentsExperiment 1:When the pressure of gastric balloon was 40mmHg,60mmHg or 80mmHg,the AWR score and cervical EMG change of rats in the model group were significantly higher than those in the normal group(P<0.05,P<0.01).When the pressure was 40mmHg or 60mmHg,the AWR score and cervical EMG change of rats in the high-dose group,medium-dose group,and low-dose group were significantly decreased compared with those in the model group(P<0.05,P<0.01).When the pressure was 80mmHg,the cervical EMG change of rats in the high-dose group and medium-dose group were significantly decreased compared with those in the model group.The gastric compliance of the rats in the model group was significantly lower than that in the normal group,and the compliance of the rats in the treatment groups was significantly higher than that in the model group(P<0.05,P<0.01).Experiment 2:No ulcer,erosion,bleeding,mucosal atrophy or other organic diseases was found in the stomach.FOS positive neurons in the spinal cord were mainly distributed in the superficial dorsal horn.The relative expression of c-fos mRNA in the spinal dorsal horn of the model group was significantly higher than that of the normal group(P<0.01),and the c-fos mRNA relative expression of the treatment group was significantly lower than that of the model group(P<0.01).The relative expression levels of iNOS mRNA and iNOS protein in the spinal dorsal horn of the model group were higher than those in the normal group(P<0.01,P<0.05),and the relative expression of iNOS mRNA and iNOS protein of the treatment group were significantly lower than those of the model group(P<0.01,P<0.05).The relative expression of GABAb mRNA and protein in the spinal dorsal horn of the model group were lower than those in the normal group(P<0.01,P<0.05),and the relative expression of GABAb mRNA and protein of the treatment group were significantly higher than those of the model group(P<0.01,P<0.05).The levels of cGMP and PKG in the spinal dorsal horn of the model group were higher than those in the normal group(P<0.01),and the levels of cGMP and PKG of the treatment group were significantly lower than those of the model group(P<0.01).Experiment 3:NS incubation did not affect the average tension of gastric smooth muscle strips,and 300μg/ml,3000μg/ml,and 6000μg/ml of "Jianpiliqi Granule" reduced the average tension significantly(P<0.01).L-NAME incubation for 30min inhibited the effect of "Jianpiliqi Granule" significantly(P<0.01).Conclusions:There are adequate theoretical basis and clinical foundation for the application of "Jianpiliqi Granule" in FD patients."Jianpiliqi Granule" combines the clinical experience of Professor Zhang,and has a normative preparation process.It can effectively regulate the gastric sensitivity and compliance in FD rats.The mechanism of its action is closely related to the regulation of NO,cGMP,PKG and GABAb levels in spinal cord and the tension of gastric smooth muscle. |
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