The Clinical And Experimental Study On Soothing Liver And Trengthening Spleen Intervention Of Diarrhea-predominant Irritable Bowel Syndrome

ObjectiveBase on the colon and hippocampus of brain-gut axis, the regulatory pathway of visceral hypersensitivity in IBS-D was explored through the clinical and experimental study, and the effect mechanism of soothing liver and trengthening spleen intervention of diarrhea-predominant irritable bowel syndrome was also researched from this. At a certain extent,the biological basis of liver controlling dispersion could assist spleen in controlling digestion was illuminated by means of the research thought that"prescription of TCM(combined prescription of sijunzi decoction and sini powder)�'therapy of TCM(soothing liver and strengthening spleen)�'etiology and pathogenesis(liver dysfunction result in dys-splenism)�'physiological function (liver controlling dispersion could assist spleen in controlling digestion)".Accordingly, this study not only richen the pathogenesis of IBS-D, but also the theory of visceral outward manifestation.Methods1. The clinical researchIn this section, a double-blind randomized controlled trial was designed. Patients who met the diagnostic criteria were randomly divided into soothing the liver group, tonifying spleen group, soothing liver and strengthening spleen group, placebo group, and the sijunzi decoction、sini powder、combined prescription of sijunzi decoction and sini powder、placebo were given respectively for treating 28±3 days. Before and after treatment, the patient information was collect according to case report form, also the brain-gut peptides were tested by means of the ELISA in serum of IBS-D patient. Then the TCM syndrome integrals、bellyache scores、Bristol faeces character scores、abdominal distension scores、5-HT、SP、NPY were counted by statistical analysis.2. The experimental researchThe IBS-D liver-stagnation and spleen-deficiency syndrome rat model was made by using 4% acetic acid enema and combining chronically psychological stress, then the modeled rat were given soothing liver and strengthening spleen intervention. By means of weighing rat weight、open field test、sucrose preference test、Bristol faeces character scores、abdominal withdrawal reflex scores, the manifestation of rat behavior was gathered. At the end of the experiment,5-HT、SP and NPY which in rat serum、colon and hippocampus were tested by ELISA. The ERK1、ERK2、c-fos of MAPK-ERK signal transduction pathways which in colon and hippocampus were tested by RT-PCR and western blotting. Then counted all these indexes by statistical analysis.Results1. The clinical research1.1 The effect of soothing liver and strengthening spleen intervention of IBS-D liver-stagnation and spleen-deficiency syndrome1.1.1 Age、course of disease and gender There is no statistic differencein age、course of disease、gender among the tonifying spleen group, soothing the liver group, soothing liver and strengthening spleen group and placebo group (P>0.05).1.1.2 The TCM syndrome integrals In soothing liver and strengthening spleen group, also in the placebo group, the difference in the TCM syndrome integrals was statistically significant before and after treatment (P<0.05). There is a significant differencein the D-value of TCM syndrome integrals between the soothing liver and strengthening spleen group and the placebo group (P<0.05).1.1.3 Bellyache scores in soothing liver and strengthening spleen group, also in the placebo group, the difference in the bellyache scores was statistically significantbefore and after treatment (P<0.05).There is a significant differencein the D-value of bellyache scores between the soothing liver and strengthening spleen group and the placebo group (P<0.05)1.1.4 Bristol faeces character scores In soothing liver and strengthening spleen group, also in the placebo group, the difference in the Bristol faeces character scores was statistically significant before and after treatment (P<0.05). There is a significant differencein the D-value of Bristol faeces character scores between the soothing liver and strengthening spleen group and the placebo group (P<0.05)1.1.5 Abdominal distension scores In soothing liver and strengthening spleen group, also in the placebo group, the difference in the abdominal distension scores was statistically significant before and after treatment (P<0.05).There is a significant difference in the D-value of abdominal distension scores between the soothing liver and strengthening spleen group and the placebo group (P<0.05)1.2 The effect of soothing liver and strengthening spleen Intervention of brain-gut peptides in IBS-D liver-stagnation and spleen-deficiency syndrome patient serum1.2.1 5-HT In soothing liver and strengthening spleen group, also in the placebo group, the difference in the 5-HT was statistically significant before and after treatment (P<0.05). There is a significant difference in the D-value of 5-HT between the soothing liver and strengthening spleen group and the placebo group (P>0.05)1.2.2 SP In soothing liver and strengthening spleen group, the difference in the SP was statistically significant before and after treatment (P<0.05) In placebo group, the difference in the SP was not statistically significant before and after treatment (P>0.05).There is a significant difference in the D-value of SP between the soothing liver and strengthening spleen group and the placebo group (P<0.05)1.2.3 NPY In soothing liver and strengthening spleen group, the difference in the NPY was statistically significant before and after treatment (P<0.05). In placebo group, the difference in the NPY was not statistically significant before and after treatment (P>0.05).There is a significant difference in the D-value of NPY between the soothing liver and strengthening spleen group and the placebo group (P>0.05)1.3 Comparison of total effective rate in four groupsThe total effective rate in the tonifying spleen group, soothing the liver group, soothing liver and strengthening spleen group, placebo group is respectively 90%、83.33%、76.67% and 70%. After treatment, the difference in the total effective rate was not statistically significant among the tonifying spleen group, soothing the liver group, soothing liver and strengthening spleen group, placebo group (P>0.05)2. The experimental study2.1 The effect of soothing liver and strengthening spleen intervention of the manifestation of rat behavior of IBS-D liver-stagnation and spleen-deficiency syndrome2.1.1 General condition The modeled rats showed inactivity-mousefur matt and sticking feces、licking abdomen、back of a bow while belly in、crissum with stain、loose stools、mucous stool、smelly sawdust、bad cleanliness of rat’s cage, but the rats in normal control group rats and soothing liver and strengthening spleen group were not.2.1.2 Weight There is a significant difference in the D-value of weight between the normal control group and the model control group (P<0.05),also between the soothing liver and strengthening spleen group and the model control group (P<0.05), between the tonifying spleen group and the model control group (P<0.05).between soothing the liver group and the model control group (P <0.05).between the tonifying spleen group and the model control group (P <0.05)2.1.3 Sucrose preference test There is a significant difference in the sucrose consumption between the normal control group and the model control group (P<0.05), also between the soothing liver and strengthening spleen group and the model control group (P<0.05). But there is not significant difference in the sucrose consumption between the tonifying spleen group and the model control group (P>0.05), between soothing the liver group and the model control group (P>0.05)2.1.4 Open field test There is a significant difference in the total distance between the normal control group and the model control group (P< 0.05), also between the soothing liver and strengthening spleen group and the model control group (P<0.05). But there is not significant difference in the total distance between the tonifying spleen group and the model control group (P>0.05).between soothing the liver group and the model control group (P >0.05).2.1.5 Water content in feces There is a significant difference in the water content in feces between the normal control group and the model control group(P<0.05), also between the soothing liver and strengthening spleen group and the model control group (P<0.05), between the tonifying spleen group and the model control group (P<0.05).between soothing the liver group and the model control group (P<0.05), between the tonifying spleen group and the model control group (P<0.05)2.1.6 Bristol faeces character scores There is a significant difference in the Bristol faeces character scores between the normal control group and the model control group (P<0.05),also between the soothing liver and strengthening spleen group and the model control group (P<0.05), between the tonifying spleen group and the model control group(P<0.05), between soothing the liver group and the model control group (P<0.05), between the tonifying spleen group and the model control group (P<0.05)2.1.7 Abdominal withdrawal reflex scores AWR under the 20mmHg pressure of balloon inflation:After the treatment, there is not significant difference in the AWR under the 20mmHg pressure of balloon inflation between the five groups (P>0.05).AWR under the 40mmHg pressure of balloon inflation:After the treatment, there is a significant difference in the AWR under the 40mmHg pressure of balloon inflation between the normal control group and the model control group (P<0.05),also between the soothing liver and strengthening spleen group and the model control group (P<0.05). But there is not significant difference in the total distance between the tonifying spleen group and the model control group (P>0.05), between soothing the liver group and the model control group (P>0.05).AWR under the 60mmHg pressure of balloon inflation: After the treatment, there is a significant difference in the AWR under the 60mmHg pressure of balloon inflation between the normal control group and the model control group (P<0.05), between soothing the liver group and the model control group (P<0.05), also between the soothing liver and strengthening spleen group and the model control group (P<0.05). But there is not significant difference in the AWR under the 60mmHg pressure of balloon inflation between the tonifying spleen group and the model control group (P>0.05).AWR under the 80mmHg pressure of balloon inflation:After the treatment, there is a significant difference in the AWR under the 80mmHg pressure of balloon inflation between the normal control group and the model control group (.P <0.05).between soothing the liver group and the model control group (P< 0.05), also between the soothing liver and strengthening spleen group and the model control group (P<0.05).But there is not significant difference in the AWR under the 80mmHg pressure of balloon inflation between the tonifying spleen group and the model control group (P>0.05)2.2 The effect of soothing liver and strengthening spleen intervention of the peripheral sensitization of IBS-D liver-stagnation and spleen-deficiency syndromeBrain-gut peptides in rat serum2.2.1 5-HT in rat serum There is a significant difference in the 5-HT between the normal control group and the model control group (P<0.05), between soothing the liver group and the model control group (P<0.05),also between the soothing liver and strengthening spleen group and the model control group (P<0.05).But there is not significant difference in the 5-HT between the tonifying spleen group and the model control group (P>0.05)2.2.2 SP in rat serum There is a significant difference in the SP between the normal control group and the model control group (P<0.05),between the soothing liver and strengthening spleen group and the model control group (P<0.05),between the tonifying spleen group and the model control group (P<0.05),between soothing the liver group and the model control group (P<0.05), also between the tonifying spleen group and the model control group (P<0.05).2.2.3 NPY in rat serum There is a significant difference in the NPY between the normal control group and the model control group (P<0.05),also between the soothing liver and strengthening spleen group and the model control group (P<0.05).But there is not significant difference in the NPY between the tonifying spleen group and the model control group (P>0.05),between soothing the liver group and the model control group (P>0.05)The MAPK-ERK signal transduction pathway in rat colon2.2.4 ERK1 in rat colon ERK1 mRNA:There is a significant difference in the ERK1 mRNA between the normal control group and the model control group (P<0.05),between soothing the liver group and the model control group (P <0.05),also between the soothing liver and strengthening spleen group and the model control group (P<0.05).But there is not significant difference in the ERKl mRNA between the tonifying spleen group and the model control group (P>0.05). ERK1 protein:There is a significant difference in the ERK1 protein between the normal control group and the model control group (P<0.05), between soothing the liver group and the model control group (P<0.05).also between the soothing liver and strengthening spleen group and the model control group (P<0.05). But there is not significant difference in the ERK1 protein between the tonifying spleen group and the model control group (P>0.05)2.2.5 ERK2 in rat colon ERK2 mRNA:There is a significant difference in the ERK2 mRNA between the normal control group and the model control group (P<0.05).between soothing the liver group and the model control group (P <0.05).between the soothing liver and strengthening spleen group and the model control group (P<0.05).also between the tonifying spleen group and the model control group (P<0.05). ERK2 protein:There is a significant difference in the ERK2 protein between the soothing liver and strengthening spleen group and the model control group(P<0.05). But there is not significant difference in the ERK2 protein between the tonifying spleen group and the model control group (P>0.05), between soothing the liver group and the model control group (P>0.05).also between the normal control group and the model control group (P>0.05)2.2.6 c-fos in rat colon c-fos mRNA:There is a significant difference in the c-fos mRNA between the soothing liver and strengthening spleen group and the model control group (P<0.05).between soothing the liver group and the model control group (P<0.05).But there is not significant difference in the c-fos mRNA between the tonifying spleen group and the model control group (P>0.05).also between the normal control group and the model control group (P>0.05). c-fos protein:There is a significant difference in the c-fos protein between the normal control group and the model control group (P< 0.05), between soothing the liver group and the model control group (P<0.05), between the soothing liver and strengthening spleen group and the model control group (P<0.05), also between the tonifying spleen group and the model control group (P<0.05)2.3 The effect of soothing liver and strengthening spleen intervention of the central sensitization of IBS-D liver-stagnation and spleen-deficiency syndromeBrain-gut peptides in hippocampus2.3.15-HT in rat hippocampus:There is a significant difference in the 5-HT between the normal control group and the model control group (P<0.05), between the tonifying spleen group and the model control group (P<0.05), also between the soothing liver and strengthening spleen group and the model control group (P<0.05).But there is not significant difference in the 5-HT between soothing the liver group and the model control group (P>0.05)2.3.2 SP in rat hippocampus:There is a significant difference in the SP between the normal control group and the model control group (P<0.05), between soothing the liver group and the model control group (P<0.05).also between the soothing liver and strengthening spleen group and the model control group (P<0.05).But there is not significant difference in the SP between the tonifying spleen group and the model control group (P>0.05)2.3.3 NPY in rat hippocampus:There is a significant difference in the NPY between the normal control group and the model control group (P<0.05), between soothing the liver group and the model control group (P<0.05), between the soothing liver and strengthening spleen group and the model control group (P<0.05), also between the tonifying spleen group and the model control group (P>0.05).The MAPK-ERK signal transduction pathway in rat hippocampus2.3.4 ERKl in rat hippocampus ERK1 mRNA:There is a significant difference in the ERKl mRNA between the normal control group and the model control group (P<0.05), between soothing the liver group and the model control group (P<0.05).between the soothing liver and strengthening spleen group and the model control group (P<0.05), also between the tonifying spleen group and the model control group (P<0.05).ERK1 protein:There is a significant difference in the ERKl protein between the normal control group and the model control group (P<0.05), also between the soothing liver and strengthening spleen group and the model control group(P<0.05). But there is not significant difference in the ERKl protein between the tonifying spleen group and the model control group (P>0.05), also between soothing the liver group and the model control group (P>0.05)2.3.5 ERK2 in rat hippocampus ERK2 mRNA:There is a significant difference in the ERK2 mRNA between the normal control group and the model control group (P<0.05), between soothing the liver group and the model control group (P<0.05).between the soothing liver and strengthening spleen group and the model control group (P<0.05), also between the tonifying spleen group and the model control group (P<0.05). ERK2 protein:There is a significant difference in the ERK2 protein between the normal control group and the model control group (P<0.05).between the soothing liver and strengthening spleen group and the model control group (P<0.05), between the tonifying spleen group and the model control group (P<0.05).also between soothing the liver group and the model control group (P<0.05)2.3.6 c-fos in rat hippocampus c-fos mRNA:There is not significant difference in the c-fos mRNA between the normal control group and the model control group (P>0.05), between soothing the liver group and the model control group (P>0.05),between the soothing liver and strengthening spleen group and the model control group (P>0.05), also between the tonifying spleen group and the model control group (P>0.05).c-fos protein:There is a significant difference in the c-fos protein between the normal control group and the model control group (P<0.05), between the soothing liver and strengthening spleen group and the model control group (P<0.05), between the tonifying spleen group and the model control group (P<0.05),also between soothing the liver group and the model control group (P<0.05) Conclusion1.4% acetic acid enema and combining chronically psychological stress could lead to the visceral hypersensitivity, through the up-regulation of c-fos that raised by MAPK-ERK signal transduction pathway which anomaly activated by 5-HT、SP and NPY in colon and hippocampus of brain-gut axis.2. The effect mechanism of soothing liver and strengthening spleen intervention of IBS-D liver-stagnation and spleen-deficiency syndrome correlate with improvement of peripheral sensitization and central sensitization, through the down-regulation of c-fos that caused by modulation of MAPK-ERK signal transduction pathway which induced by 5-HT、SP and NPY in colon and hippocampus of brain-gut axis.3. The TCM pathogenesis of IBS-D stagnation of liver qi and spleen deficiency in molecular level is related to MAPK-ERK signal transduction pathway which excessively activated by abnormal 5-HT、SP and NPY in colon and hippocampus of brain-gut axis.4. The biological mechanism of liver controlling dispersion could assist spleen in controlling digestion correlate with modulation of signal transduction pathway which induced by abnormal brain-gut peptides in central nervous system and peripheral tissues of brain-gut axis.innovation point:1. This study enriches the pathogenesis of IBS-D, and confirms that the abnormal changes of MAPK-ERK signal transduction pathway is a important way which arouse the visceral hypersensitivity, base on this, the effect mechanism of soothing liver and strengthening spleen intervention of IBS-D correlate with improvement of visceral hypersensitivity, through the down-regulation of c-fos that caused by modulation of MAPK-ERK signal transduction pathway.2. The TCM pathogenesis of IBS-D stagnation of liver qi and spleen deficiency in molecular level is related to activation of MAPK-ERK signal transduction pathway which in central nervous system and peripheral tissues of brain-gut axis. base on this, the biological mechanism of liver controlling dispersion could assist spleen in controlling digestion was probed into by means of the idea which "from TCM pathogenesis to infer the TCM physiological function"3. In this study, the similarities and differences of molecular mechanism which the soothing liver therapy、tonifying spleen therapy、soothing liver and strengthening spleen therapy intervention of IBS-D was Compared under the brain-gut axis frame, the result could help understanding the TCM treating the same disease with different methods.