Effct Of Shenling Baizhu Powder On Expression Of Gene And Protein In Tissue Of Ulcerative Colitis Disease In Syndrome Of Dampness Stagnancy Due To Spleen Deficiency

Objective: To study the intervention effects of Shenling Baizhu powder on p38 MAPK signal pathway in rats tissue of ulcerative colitis disease in syndrome of dampness stagnancy due to spleen deficiency. In order to detect the expression of p38 MAPK gene, protein and associated inflammatory IL-1β and IL-4.With the study, With these methods,to detect the treatment mechanism of Shenling Baizhu powder on UC in syndrome of dampness stagnancy due to spleen deficiency.Methods: According to random number table, 60 SPF Witar rats(weight:180±20g) are divided into blank group and model maded group. and then blank group and model group(10 per group)blank group rats are feeding in normal conditions, and do not give to any interventional effects. Model maded group rats were made using comprehensive factors both inside and outside to copy ulcerative colitis model: 1 Model group rats were fed refined lard 2ml every other day;2Make model group rats standing in water about 2cm depth for eight hours every day;3The rats in the control group standing using irregular diet feeding method, every other day foods are forbidden. These methods are continued for 21 days in the progress;4 And then the model group rats were numbered with randomly divided into model group, Shenling Baizhu powder high dose, medium and low dose group and positive control group(SASP: SASP group) 10 each group, male and female is divided into different cases in each group. Model group is determined to establisheb ulcerative colitis disease in syndrome of dampness stagnancy due to spleen deficiency. Model group was maded by means of acomprehensive methods. The comprehensive factors both inside and outside in order to replicate model of Ulcerative Colitis of dampness stagnancy due to spleen deficiency. Make the group outside of blank group rats in water about 2cm depth eight hours each day;5Treatment: After the model of disease is successfully replicated in animals, blank group and model group were given distilled water treatment each day 2ml·d; 6Shenling Baizhu powder treatment groups were given a high, medium and low dose oral therapy each day(dose 24 、 12 、 6g·kg-1·d-1); 7The SASP group was equally treated by weight with SASP(0.2g·Kg-1·d-1). The rats in positive group were treated with SASP gavage, and the rats of blank group and model group were treated distilled water gavage in the same volume. All rats were treated by oral gavage per day for 21 days. After completing these treatment steps, All rats in every group are water-deprived for 24 h after last gavage.Materials and testing: after the last treatment every rats in each group are deprived food for 24 hours, To anesthesia animals with 5% chloral hydrate(0.3m L/100g).1 Taking blood from Femoral artery,and the blood is placed into the centrifuge tube centrifuge(3000rm/min·5min), serum was extracted from the upper level of blood and frozen and stored in a clean tube-80℃ degrees below zero for ELISA detection.2 To cut off colonic tissue, cleaning colonic tissue with Saline solution carefully. Preferred to the naked eye and recorded colonic mucosa of rats in each group model congestion, edema, necrosis, ulcers and other lesions;3 And then colonic tissue about 2~3cm was taken and fixed with paraformaldehyde, paraffin-embedded tissue sections, HE staining, Light microscope observations, the pathological changes of the rats colon tissue, colon tissue placed stored at condition of-80℃ degrees, these tissues are used for the subsequent research of RT-PCR and Western-blot or other detection methods.Results:1 Compared with the blank group, Colonic mucosa in model rats are different degrees of congestion, edema, erosion and ulcers and other diseases. Differences between groups were significant(P<0.01), There are statistically significant; Microscope: colonic mucosa of rats in the model group were accompanied by a large number of inflammatory cell infiltration, granulation, fibrous hyperplasia. Partial inflammation, necrosis, granulation and fibrous hyperplasia and other pathological changes of Shenling Baizhu powder group and SASP group rat colon tissue were reduced than the model group. The colon pathology and injury score that was observed lower than the model group, Significant differences between groups(P<0.01), The difference was significant.2 Compare with the control group, levels of p38 MAPK and IL-1β in serum and colon tissue of model group were significantly increased,Content of IL-4 was significantly decrease, significantly differences between groups(P<0.01).After treatment,, the content of p38 MAPK and IL-1β in Shenling Baizhu powder high dose group were significantly decreased, the content of IL-4 was significantly increased(P<0.01),there were significantly different between two groups. Compared within the Sulfasalazine group and Shenling Baizhu powder high-dose group, The expression content of p38 MAPK, IL-1β and IL-4 was no significant difference between groups of high dose group and Sulfasalazine group(P>0.05), results have no statistical significance.3 The comparison of gene expression levels of p38MAPK、IL-1β and IL-4: Compared with the control group, the content of p38 MAPK and IL-1β in model group increased significantly.The content of IL-4 was decreased significantly(P<0.01). There was significantly different between groups. Gene expression levels of p38 MAPK, IL-1β and IL-4 in colon tissue in each Shenling Baizhu powder group are different declined, the change in middle and high dose was decreased significantly,(P<0.01 or P<0.05); In the high dose group of Shenling Baizhu powder these expression of gene was decreased obvious, there are significantly differences between groups. Gene expression of IL-4 was significantly increased(P<0.01 or P<0.05), statistically significant. Effcts in group Shenling Baizhu powder high-dose treatment compared with SASP therapy group, no significant difference, and no statistically significant between groups.4 Comparison of p38 MAPK protein expression level: Compared with the control group of rats in model group, expression levels of p38 MAPK protein were significantly increased.(P<0.01), there are significantly differences,there are statistically significant between two group(P<0.01). Protein content of p38 MAPK in colon tissue of each treatment group was decreased on different levels.The difference between groups of model group and the high dose was significant(P<0.01),the difference was significant, statistically significant. The differences between high-dose group and SASP group showed no statistical significance(P>0.05). The level of protein expression in colon tissue of each Shenling Baizhu powder group decreased in varying degrees,the effcct was significant in middle group and high group(P<0.01 or P<0.05), differences between groups were significant.Compared with group of SASP, no significant differences was observed between Shenling Baizhu powder group and SASP group, differences between groups were not significant(P>0.05).Conclusion: 1 Shenling Baizhu powder can interven the expresssion level of IL-4 and IL-1β in rat serum, or p38 MAPK expression level in colon tissue. Shenling Baizhu powder can reduce inflammatory cell infiltration, reducing local inflammation and promote tissue repair and so on. Observation optical by microscope showed that colon tissues in Shenling Baizhu powder high group and middle group was decreased significantly, and cell morphology of intestinal mucosa was close to a normal cell morphology. Shenling Baizhu powder can contribute to the function of restoration and secreting in mucous of colon which can contribute to the self-protection of mucous of colon.2 The expression levels of protein and m RNA of p38 MAPK, IL-1β and IL-4 in Serum and colon tissue in rats with ulcerativecolitis disease in syndrome of dampness stagnancy due to spleen deficiency may reflect the degree of severity of disease rats in model group which maybe has a certain significance of detection.3 The therapeutic effect of Shenling Baizhu powder may decrease the expression levels of IL-1β protein and gene in the organization colonic mucosa of rats which be related to UC in model group, increased IL-4 protein and gene expression levels, promote the recovery of colonic mucosa tissue.4 Shenling Baizhu powder can reduce p38 MAPK protein content in colon tissues of rats suffered from UC. Shenling Baizhu powder can inhibit inflammatory injury and Cell Apoptosis, meanwhile itcan promote mucosal repair injury site of the colon, It means that therapeutic effect of Shenlingbaizhu powder may be related to decrease the protein expression of p38 MAPK, it can decrease the cytokine expression of IL-1β and IL-4 in colon tissue at the same time.