| Objective:Functional dyspepsia(FD)is a clinical syndrome characterized by upper abdominal,distension,belching,early satiety,nausea vomiting and some relative symptoms,but there are not organic disease examined by gastrointestinal endoscope.The morbidity of FD is high,but the clinical treatment methods is limited.FD seriously affects the quality of life and brings a great financial burden to the medical system.The underling pathogenesis of FD is not well understood yet,and many factors may be involved in the pathogenesis,including abnormal of the stomach and duodenum,decreased compliance of the proximal stomach and the increased sensitivity of the stomach and duodenum.Recently,a great number of studies have found that FD is closely related to the duodenum,especially the duodenal inflammation,including the infiltration of mast cells,eosinophils,intraepithelial lymphocyte and some inflammatory factors like TNF-alpha.Recent research have demonstrated that the impaired duodenal mechanical barrier function is associated with the onset of FD and the duodenal microinflammatory.The increase of some inflammation factor in the duodenum,such as TNF-alpha,IFN-gamma,iNOS,can activate the NF-kappa B signaling pathway,which can enhance the level of MLCK in the epithelium cells.MLCK could promote the contraction of the cytoskeleton and lead to the change of tight junction proteins claudin-1,occluidn,resulting in the opening up of the paracellular pathway of duodenal epithelial cells and the impaired mechanical barrier function.PGE2 plays an important role in the modulation of the gastrointestinal tract.PGE2 can protect the gastric mucosa,promote the secretion of HCO3 in the duodenum.Besides,PGE2 can promote the expression of duodenal tight junction proteins and protect the duodenal tight junction barrier by EP1 or EP4 and cAMP-PKA related signaling pathways.The modern medicine has few methods in the modulation of intestinal mechanical barrier and tight junction proteins.But some studies have found that some traditional Chinese medicine(TCM),mainly the spleen-strengthening,liver qi regulating and heat-clearing play a key role in the regulation of intestinal tight junction proteins.The Jianpiliqi(JPLQ)formula is a traditional Chinese medicine compound,which is based on the TCM theory and my tutor's clinical practice.The previous clinical studies and animal experiments have showed that the JPLQ was effective in the treatment of FD,but its effect on FD duodenal tight junction proteins and the underling molecular mechanism is not well understood.Therefore,this study investigated the effect of the JPLQ in the treatment of FD and its impact on the duodenal mechanical barrier and tight junction proteins,and explored the underling molecular mechanisms and the possible signaling pathways.Methods:Research one:The effect of JPLQ on gastric compliance and sensitivity of FD ratsEighteen healthy male Sprague-Dawley rats(SPF grade)were bought from Beijing Vital River Laboratory Animal Technology Company.Twelve rats were used to build the FD model by tail clamping.Then the FD rats were randomly divided into model group and JPLQ treatment group.The JPLQ group rats received JPLQ oral administration while the control group and model group received water as a vehicle control for total 7days.At the end of treatment,A polyethylene balloon was fixed in the stomach by introducing from the mouth,and two electrodes were fixed into the trapezius of these rats.The balloon and the electrodes were connected to the barostat and electrophysiological recorder respectively.The pressure of the balloon was increased stepwise from 20 to 80 mmHg.The gastric compliance was detected based on the volume changes per mmHg during the distension experiment,while the gastric sensitivity is revealed as the rate of change root mean square of the myoelectricity.Research two:The effect of JPLQ on the duodenal mucosa permeability and the expression of tight junction proteins in FD ratsEighteen healthy male Sprague-Dawley rats(SPF grade)were bought from Beijing Vital River Laboratory Animal Technology Company.Twelve rats were used to build the FD model by tail clamping.Then the FD rats were randomly divided into model group and JPLQ treatment group.The JPLQ group rats received JPLQ oral administration while the control group and model group received water as a vehicle control for total 7days.At the end of treatment,all of the rats in each group were anesthetized using 2%pentobarbital sodium and the 3 cm duodenum was excised for the following experiments.The expression of TJ proteins,claudin-1 and occludin was detected by Western blot.The duodenal tight junction barrier was revealed by transepithelial electrical resistance(TEER)testing by Ussing chamber.The expression of cytoskeleton F-actin was measured by phalloidine staining.Research three:Effect of JPLQ on the cAMP and NF-?B related signaling pathwaysEighteen healthy male Sprague-Dawley rats(SPF grade)were bought from Beijing Vital River Laboratory Animal Technology Company.Twelve rats were used to build the FD model by tail clamping.Then the FD rats were randomly divided into model group and JPLQ treatment group.The JPLQ group rats received JPLQ oral administration while the control group and model group received water as a vehicle control for total 7days.At the end of treatment,all of the rats in each group were anesthetized using 2%pentobarbital sodium and the 3 cm duodenum was excised for the following experiments.The morphology of duodenum was measured by HE staining.The concentrations of TNF-?,IFN-y,PGE2 and cAMP were detected by Elisa.The expression of iNOS was revealed by Western blot.The mRNA of NF-?B,MLCK,EP1,EP4 was tested by qPCR.Results:Research one:The effect of JPLQ on gastric compliance and sensitivity of FD ratsCompared with the control animals FD rats showed a significant decrease in gastric compliance at 40 mmHg,60 mmHg and 80 mmHg(40mmHg:0.18±0.01 vs.0.29±0.01,P<0.01,60mmHg:0.21±0.01 vs.0.31±0.01,P<0.01,80mmHg:0.23±0.01 vs.0.29±0.01,P<0.01).Treating FD rats with SNS significantly improved gastric compliance(40mmHg:0.28±0.01 vs.0.18±0.01,P<0.011,60mmHg:0.31±0.01 vs.0.21±0.01,P<0.01,80mmHg0.29±0.01 vs.0.23±0.01,P<0.01).On the other hand,FD rats elicited VH under all distention pressures of 20 mmHg,40 mmHg,60 mmHg and 80 mmHg compared with the control group(20mmHg:28.05±3.26 vs.12.07±1.58,P<0.01,40mmHg:73.97±7.94 vs.31.77±5.15,P<0.01,60mmHg:196.72±23.55 vs.117.08±14.25,P<0.01,80mmHg:239.65±35.01 vs.114.73±16.91,P<0.01).JPLQ treatment significantly decreased visceral sensitivity in FD rats(20mmHg:15.47±2.20 vs.28.05±3.26,P<0.01,40mmHg:31.67±4.37 vs.73.97±7.94,P<0.01,60mmHg:111.54±8.90 vs.196.72±23.55,P<0.01,80mmHg:135.83±19.27 vs.239.65±35.01,P<0.01).Research two:The effect of JPLQ on the duodenal mucosa permeability and the expression of tight junction proteins in FD ratsThe FD model group showed a significantly decreased TEER(20.21±0.88 vs.39.72±0.60,P<0.01)and the expression of claudin-1,occludin was remarkably lower compared with the control group(claudin-1:0.02±0.01 vs.0.07±0.01,P<0.01,occludin:0.05±0.01 vs.0.14±0.01,P<0.01).The JPLQ treatment could prominently improve the TEER(37.03±1.61 vs.20.21±0.88,P<0.01)and enhance the expression of claudin-1 and occludin(claudin-1:0.07±0.01 vs.0.02±0.01,P<0.01,occludin:0.16±0.01 vs.0.05±0.01,P<0.01).But the expression of F-actin was not different significantly.Research three:Effect of JPLQ on the cAMP and NF-?B related signaling pathwaysCompared with the control rats,the concentration of TNF-?,IFN-y in duodenum was notably higher in FD model group(TNF-?:1.54±0.11 vs.0.79±0.05,P<0.01,INF-?:0.37±0.02 vs.0.15±0.02,P<0.01).The mRNA of NF-?B,MLCK and iNOS protein level were remarkably increased in model group(NF-?B:1.28±0.10 vs.1.01±0.05,P<0.05;,MLCK mRNA:1.59±0.12 vs.1.01±0.07,P<0.01;iNOS:0.1210.01 vs.0.07±0.01,P<0.01).The concentration of PGE2,cAMP and mRNA of EP4 were significantly decreased in model group(PGE2:0.74±0.08 vs.1.35±0.06,P<0.01,cAMP:10.861±0.20 vs.14.38±0.35,P<0.05;EP4 mRNA:0.43±0.11 vs.1.09±0.18,P<0.05,).The JPLQ treatment could notably decrease the expression of TNF-?,IFN-y(TNF-?:0.83±0.06 vs.1.54±0.11,P<0.01,IFN-?:0.14±0.02 vs.0.37±0.02,P<0.01),mRNA of NF-?B,MLCK,iNOS protein level(NF-?B:0.98±0.07 vs.1.28±0.10,P<0.05;MLCK mRNA:0.84±0.09 vs.1.59±0.12,P<0.05;iNOS:0.07±0.01 vs.0.12±0.01,P<0.01)and enhance the expression of PGE2,EP4 mRNA,cAMP(PGE2:1.26±0.04 vs.0.74±0.08,P<0.01;EP4 mRNA:1.11±0.19 vs.0.43±0.11,P<0.05,cAMP:14.01±0.30 vs.10.86±0.20,P<0.01).But the mRNA of EP1 was not different among three group.Conclusion:JPLQ was effective for the treatment of FD rats and could improve the gastric compliance and visceral sensitivity.JPLQ was able to restore the duodenal TJ barrier and up-regulate the expression of TJ proteins.TheTNF-?,IFN-?-NF-?B and the PGE2-cAMP were the underlying signal pathways. |
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