Comparative Study Of CAMP / PKA Signaling Pathway In Brain - Gut Peptide And Small Intestine Of Rats With Spleen - Qi Deficiency Syndrome And Spleen - Yang Deficiency Syndrome

Purpose:In this research, PCR-array method is used to test energy metabolism related genes in small intestine of Spleen Qi deficiency and Spleen Yang deficiency syndrome model rats, which can we learn the genes change of both Spleen Qi deficiency and Spleen Yang deficiency syndrome model rats and their differences of expression. We choose three energy metabolism related brain-gut peptide, CCK、Ghrelin and VIP, to explore the differences among their changes of genes and protein in Spleen Qi deficiency and Spleen Yang deficiency syndrome model rats. And to further explore the functional differences between Spleen Qi deficiency and Spleen Yang deficiency syndrome model rats via c AMP/PKA signaling pathway in small intestine. To investigate the biological basis of Spleen Qi deficiency and Spleen Yang deficiency syndrome model rats and comparative analysis of the differences between the candidates, in order to try to explain the scientific connotation of Spleen Qi deficiency and Spleen Yang deficiency syndrome model. In order to compare the differences between the two syndromes, we set up sijunzi decoction group and fuzilizhong pill group, through which we can further explore the curative effect and mechanism of sijunzi decoction and fuzilizhong pill to treat spleen qi deficiency and spleen yang deficiency syndrome.Materials and methods:60 male SD rats were randomly devided into 5 groups:normal group, Spleen Qi deficiency group(referred to as Qi deficiency group), Spleen Yang deficiency group(referred to as yang deficiency group),sijunzi decoction group and fuzilizhong pill group(n=10 in each group). All rats were fed adaptive for 1 week in the Experimental Animal Center.Rats in normal group were fed with noamal diet. The other rats were treated with complex factors of improper diet and overstrain to establish Spleen Qi deficiency syndrome.Rats satiation 1d, and then fasting 2d, but water is free, 3 days for a period, recording food intake after satiation. And daily swim in water temperature of 35℃-37℃ to be exhaustive.After five periods to evaluate Spleen Qi deficiency syndrome model. Spleen Qi deficiency evaluation criteria: Shenpi, fatigue, dull fur or bent, and weight loss. Rats in spleen yang deficiency group and fuzilizhong pill group are lavaged by Senna decoction 2 times a dayafter five periods. After 7 days the Spleen Yang deficiency model are completed. Spleen Yang deficiency model evaluation criteria was added in the Spleen Qi deficiency model evaluation criteria: loose stools, lay curled up and flock together and crissum filth,temperature drop or slightly decreased. The rats outward manifestation be quantified using behavioral experiments to observe the movement from the system within 5min rats, exercise time, average speed of movement(movement distance / movement time), activity area, the number of quantitative indicators and other vertical activity of rats Shenpi symptoms. Using a large mouse grip tester rat forelimb grip strength of fatigue symptoms in rats. Rat stool water content in rats to quantify it soft or loose stools. based on the recorded temperature data to quantify lying curled happy to get together, the temperature drops and other performance.Rats were observed the changes of feed intake and body weight; Rats were observed to determine its withered state or degree of matte. After the model were established, rats in sijunzi decoction group are treated with sijunzi decoction, rats in fuzilizhong pill group are treated with fuzilizhong pill, while other rats are fed normally, 1time a day, lasting 7days.Until the end of the treatment, all rats were fasting and free drinking water for 24 hours.The rats were weighed and 10% hydrated with chlorine aldehyde and intraperitoneal anesthesia(3ml/kg body weight). After the rats were anesthetized fixed in rats, using 0.5%Iodophor disinfection rat abdomen and neck skin, cut open the skin and peritoneum, take small intestine washing with PBS solution, decapitated, hypothalamus specimens were frozen in liquid nitrogen under 80 degrees refrigerator preservation reserve. PCR-array to test energy metabolism related genes in small intestine of rats. We use Elisa method to test ATP、c AMP、CCK 、 Ghrelin and VIP contentin rat’s small intestine. Q-PCR method for detecting PKA,Ghrelin and the Ghrelin receptor, CCK and CCK receptor, VIP and VIP receptor gene expression of small intestine and hypothalamus. Western blot was used to detect the small intestine PKA protein expression.PCR array Statistics Statistical analysis was performed on the PCR array QIAGEN company website, calculated differentially expressed genes between the two groups through the 2-△△Ct. Multiples≥2 or ≤0.5 multiples represent a statistically significant difference.SPSS19.0 experimental data using statistical software for experimental data analysis,data through the test of normality and homogeneity of variance test, and then the one-way ANOVA. Results are expressed as mean ± standard deviation( x ±s) represented, P <0.05 was considered statistically significant.Results:1. Differences between expression of genes related to energy metabolism in small intestine of Spleen Qi deficiency and Spleen Yang deficiency syndrome model ratsCompared with the normal control group, there are 49 metabolism related genes’ expression of qi deficiency group have significantly changed. Among them, 10 genes were two times up-regulated, 39 genes were two times down-regulated. There are 38 metabolism related genes expression of Spleen Yang deficiency group have significantly changed. Among them, 13 genes were two times up-regulated, 25 genes were two times down- regulated.Compared with Spleen Qi deficiency group, there are 45 metabolism related genes expression of sijunzi decoction group have significantly changed. Among them, 27 genes were two times up-regulated, 18 genes were two times down-regulated. There are 48 metabolism related genes expression of Spleen Yang deficiency group have significantly changed. Among them,33 genes were two times up-regulated, 15 genes two times down-regulated.2. Study on the content of brain-gut peptide and its receptor’s m RNA in rats with Spleen Qi deficiency and Spleen Yang deficiency syndrome2.1 CCK protein content and gene expression and its receptor gene expression in rats of each groupCompared with the normal control group, the contents of CCK in small intestine of rats with Spleen Qi deficiency group and Spleen Yang deficiency group are increased(P<0.05);m RNA expression of CCK and its receptor in the small intestine and hypothalamus are up-regulate(P<0.05). Compared with the Spleen Qi deficiency group, the contents of CCK in small intestine of rats with Spleen Yang deficiency group are increased(P<0.05); m RNA expression of CCK and its receptor in the small intestine and hypothalamus are up-regulated(P<0.05); the contents of CCK in small intestine of sijunzi decoction group are decreased(P<0.05); m RNA expression of CCK and its receptor in the small intestine and hypothalamus are down-regulated(P<0.05). Compared with the Spleen Yang deficiency group,the contents of CCK in small intestine of fuzilizhong pill group are decreased(P<0.05);m RNA expression of CCK and its receptor in the small intestine and hypothalamus are down-regulated(P<0.05).2.2 Ghrelin protein content and gene expression and its receptor gene expression in rats of each groupCompared with the normal control group, the contents of Ghrelin in small intestine of rats in Spleen Qi deficiency group and Spleen Yang deficiency group are decreased(P<0.05);m RNA expression of Ghrelin and its receptor in the small intestine and hypothalamus aredown-regulated(P<0.05). Compared with the Spleen Qi deficiency group, the contents of Ghrelin in small intestine of rats with Spleen Yang deficiency group are decreased(P<0.05);m RNA expression of Ghrelin and its receptor in the small intestine and hypothalamus are down-regulated(P<0.05); the contents of Ghrelin in small intestine of sijunzi decoction group are increased(P<0.05); m RNA expression of Ghrelin and its receptor in the small intestine and hypothalamus are up-regulated(P<0.05). Compared with the Spleen Yang deficiency group,the contents of Ghrelin in small intestine of fuzilizhong pill group are increased(P<0.05);m RNA expression of Ghrelin and its receptor in the small intestine and hypothalamus are up-regulated(P<0.05).2.3 VIP protein content and gene expression and its receptor gene expression in rats of each groupCompared with the normal control group, the contents of VIP in small intestine of rats in Spleen Qi deficiency group and Spleen Yang deficiency group are decreased(P<0.05); m RNA expression of VIP and its receptor in the small intestine and hypothalamus are down-regulated(P<0.05). Compared with the Spleen Qi deficiency group, the contents of Ghrelin in small intestine of rats with Spleen Yang deficiency group are decreased(P<0.05);m RNA expression of VIP and its receptor in the small intestine and hypothalamus are down-regulated(P<0.05); the contents of VIP in small intestine of sijunzi decoction group are increased(P<0.05); m RNA expression of VIP and its receptor in the small intestine and hypothalamus are up-regulated(P<0.05). Compared with the Spleen Yang deficiency group,the contents of VIP in small intestine of fuzilizhong pill group are increased(P<0.05); m RNA expression of VIP and its receptor in the small intestine and hypothalamus are up-regulated(P<0.05).3. Study on the Ghrelin/c AMP/PKA signal pathway in the small intestine of Spleen Qi deficiency and Spleen Yang deficiency syndrome model rat3.1 Changes of ATP and c AMP protein in the small intestine of rats in each groupCompared with the normal control group, the contents of ATP and c AMP in small intestine of rats in Spleen Qi deficiency group and Spleen Yang deficiency group are decreased, the difference was statistically significant(P<0.05). Compared with the Spleen Qi deficiency group, the contents of ATP and c AMP in small intestine of rats with Spleen Yang deficiency group are decreased(P<0.05); the contents of ATP and c AMP in small intestine of sijunzi decoction group are increased, the difference was statistically significant(both P<0.05).Compared with the Spleen Yang deficiency group, the contents of ATP and c AMP in small intestine of fuzilizhong pill group are increased, the difference was statisticallysignificant(P<0.05).3.2 Changes of PKA protein and mRNA expression in the small intestine of rats in each groupCompared with the normal control group, the contents of PKA protein and m RNA expression in small intestine of rats in Spleen Qi deficiency group and Spleen Yang deficiency group are decreased, the difference was statistically significant(P<0.05). Compared with Spleen Qi deficiency group, the PKA protein and m RNA expression in small intestine of rats with Spleen Yang deficiency group are decreased(P<0.05); the PKA protein and m RNA expression in small intestine of sijunzi decoction group are increased, the difference was statistically significant(both P<0.05). Compared with Spleen Yang deficiency group, the PKA protein and m RNA expression in small intestine of fuzilizhong pill group are increased, the difference was significant(P<0.05).Conclusion:1. The difference of Spleen Qi deficiency syndrome and Spleen Yang deficiency syndrome is closely related to the expression of energy metabolism genes.2. The expression of CCK, VIP and Ghrelin of hypothalamus and intestinal tissue in Spleen Yang deficiency model rat changed more significant than in Spleen Qi deficiency model rat,which means the difference between Spleen Qi deficiency syndrome and Spleen Yang deficiency syndrome in regulating energy metabolism with c AMP/PKA signaling pathway.3. Sijunzi decoction and fuzilizhong pill can adjust the CCK, VIP and Ghrelin of Spleen Qi deficiency and Spleen Yang deficiency syndrome rats via participate in c AMP/PKA signaling pathway.4. The biological basis of the two syndromes is the difference expression of CCK, VIP,Ghrelin and c AMP/PKA signaling pathway in hypothalamus and intestinal tissue.