Effects Of Wu Mei Wan And Its Recipe On TLR9 / MyD88 / NF-κBp65 And IL-6 / JAK / STAT3 Signaling Pathway In Rats With Ulcerative Colitis

Purpose:By researching the characteristics of compatibility and treating syndrome of Wu Mei pill, to observe the effects on pathological changes and inflammation cytokines and TLR9/My D88/NF-κBp65 and IL-6/JAK/STAT3 signaling pathway in ulcer colitis rat colon mucosa treated by Wu Mei Pill and its disassembled parties, and then analyze immunological mechanisms of ulcer colitis, and discuss the targets of Wu Mei Pill in the treatment of ulcerative colitis, and reveal the meaning of compatibility of sour herbal medicines with pungent and bitter herbal medicines to regulate the liver and spleen.Material and method:Collated the relevant literatures and analyzed rules and characteristics of the prescriptions of Wu Mei Pill. Model of ulcerative colitis were made with SPF healthy adult wistar rats by 2, 4, 6- three nitrobenzene sulfonic acid / ethanol method.The rats were randomly divided into normal group, model group, SASP group, Wu Mei Pill group, Xin Kai Ku Jiang with Wu Mei group, Xin Kai with Wu Mei group, Ku Jiang with Wu Mei group and Bu Yi with Wu Mei group(the abbreviated names of each disassembled recipe party group were used with Xin Kai Ku Jiang group, Xin Kai group, Ku Jiang group and Bu Yi group in the following conten). Rats in each treatment group were given by the corresponding drugs, and the treatment period was 28 days. The anesthetized rats by the method of injection of 10% chloral hydrate in abdominal cavity were killed and their colonic tissue and blood were drawn. Observed disease activity index(DAI) and colon mucosa injury of the rats; observed pathological sections of colon mucosa under the microscope; detected SOD and MDA levels in colonic mucosa; detected interleukin-10(IL-10) and tumor necrosis factor-alpha(TNF-α) level in serum and interleukin- 4(IL-4) and interference factor gamma(IFN-γ) and interleukin 6(IL-6) level in colon mucosa tissue by the method of ELISA;investigated the expression of Toll like receptor 9(TLR9) and myeloid differentiation factor88(My D88) and nuclear factor kappa Bp65(NF-κBp65) expression in colonic mucosa tissue by immunohistochemical method; detected IL-6 m RNA and JAK2 m RNA and STAT3 m RNA expression in colonic mucosa tissue by RT-PCR method. All the experimental datas were analyzed by using SPSS18.0 statistical software analysis. The comparison between the groups used the method of one factor analysis of variance(ANOVA), and the difference was statistically significant with P < 0.05.Results:1 General status and DAI and intestinal mucosal damage degree scoreStool of rats became loose and bloody, and the crissum was dirty in the second day after making model. And then diet amount of the model rats was reduced. Hair of the model rats became dirty and dull. The model rats chilled and curled up and became lazy, whose weight was loss. By intragastric administration with the drugs of different groups, rats of SASP group,Wu Mei pill group and Xin Kai Ku Jiang group had improvement in symptoms with different degrees. DAI and colonic mucosal injury score in these three groups were significantly lower than those of model group(P < 0.01). DAI and colonic mucosal injury score in Xin Kai group,Ku Jiang group and Bu Yi group were not significant different with those in model group(P >0.05).2 Pathological changes of colonic mucosaThe colonic mucosa of rats in model group was missing. Glands in the lamina propria of colonic mucosa were damaged or disappeared, and goblet cells decreased. There were a large number of inflammatory cells infiltrate in colonic mucosa. Around epithelial hyperplasia of colon mucosa in SASP group and Wu Mei pill group repaired, whose epithelial defect was not obvious, and goblet cells were rich. Their basic form was similar to normal group. Colon mucosa defect in Xin Kai Ku Jiang group rats was superficial, and gland hyperplasia was active, and there was a small amount of inflammatory cell infiltration in lamina propria. The microsopic colon mucosa structures were similar in Xin Kai group, Ku Jiang group, Bu Yi group rats, and there were still some defects in the colonic mucosa, and gland structures were not complete, and goblet cells decreased obviously, and there were still a large number of inflammatory cells infiltrated in lamina propria.3 The level of SOD and MDA in Colonic mucosalSOD level significantly decreased and MDA level significantly increased in colonic mucosa tissue of the model group rats compared with the normal group rats(P < 0.01). SOD level significantly increased and MDA level significantly reduced in colonic mucosa tissue of each treatment group rat compared with the model group rats(P < 0.01); and the level of SOD had no significant difference among SASP group and Wu Mei Pill group and Xin Kai Ku Jiang group(P > 0.05),which was significantly higher than that of the Xin Kai group, Ku Jiang group and Bu Yi group(P < 0.01); and the level of MDA had no significant difference among SASP group and Wu Mei Pill group and Xin Kai Ku Jiang group(P > 0.05),which was significantly lower than that of the Xin Kai group, Ku Jiang group and Bu Yi group(P <0.01).4 The level of IL-10 and TNF-α in Serum and the level of IL-4 and IFN-γ and IL-6 in colonic mucosa4.1 The level of IL-10 and TNF-α in SerumCompared with the normal group rats, the level of IL-10 in serum was significantly lower in model group rats(P < 0.01). Compared with the model group rats, the level of IL-10 in serum significantly increased in the SASP group and Wu Mei Pill group and Xin Kai Ku Jiang group and Bu Yi group rats(P < 0.01), but Xin Kai group and Ku Jiang group had no statistical difference with model group(P > 0.05). The level of IL-10 in serum in SASP group rats was significantly higher than that of other treatment groups(P < 0.01). The level of IL-10 in serum of Wu Mei Pill group rats was significantly higher than that of(P < 0.01). The level of IL-10 in serum of Xin Kai Ku Jiang group rats showed no significant difference with Wu Mei pill group, Xin Kai group, Ku Jiang group and Bu Yi group rats(P > 0.05).Compared with the normal group rats, the level of TNF-α in serum was significantly higher in model group rats(P < 0.01). Compared with the model group rats, the level of TNF-α in serum significantly decreased in each treatment group rats(P < 0.01), there was not statistical difference between the level of TNF-α in serum of Wu Mei Group rats and that of SASP group rats(P > 0.05). The level of TNF-α in serum of Wu Mei group rats was significantly lower than that of Xin Kai group and Ku Jiang group and Bu Yi group rats(P <0.01). The level of TNF-α in serum of Xin Kai Ku Jiang group rats showed no significant difference with Wu Mei pill group, Xin Kai group, Ku Jiang group and Bu Yi group rats(P >0.05).4.2 The level of IFN-γ, IL-4 and IL-6 in colonic mucosaCompared with the normal group rats, the level of IFN-γ in the colonic mucosa of the model group was significantly increased(P<0.01). Compared with the model group rats, the level of IFN-γ in the colonic mucosa of other treatment group rats except the Bu Yi group all decreased significantly(P < 0.01). There was no statistical difference between the level of IFN-γ in the colonic mucosa of the SASP group rats and that of the Wu Mei Pill group(P >0.05), but that of other disassembled parties group rats of Wu Mei Pill was significantly increased compared with SASP group(P < 0.01). Compared with Wu Mei Pill group rats, the level of IFN-γ in the colonic mucosa of Xin Kai Ku Jiang group rats, Xin Kai group rats and Ku Jiang group rats all had no significant difference(P > 0.05), but that of Bu Yi group rats was significantly increased. Compared with Xin Kai Ku Jiang group rats, the level of IFN-γ in the colonic mucosa of Xin Kai group rats and Ku Jiang group rats all showed no significant difference(P > 0.05), but that of Bu Yi was significantly increased(P < 0.01).Compared with the normal group rats, the level of IL-4 in the colonic mucosa of the model group was significantly decreased(P<0.01). Compared with the model group rats, the level of IL-4 in the colonic mucosa of each treatment group rats increased significantly(P <0.01). Compared with the SASP group rats, the level of IL-4 in the colonic mucosa of Wu Mei pill and its disassembled party group rats all decreased significantly(P < 0.01). There was no statistical difference between the level of IL-4 in the colonic mucosa of the Wu Mei pill group rats and that of Xin Kai Ku Jiang group rats(P > 0.05), but the level of IL-4 in the colonic mucosa of Xin Kai group rats and Ku Jiang group rats and Bu Yi group rats decreased significantly compared with Wu Mei Pill group rats(P < 0.01). The level of IL-4 in the colonic mucosa of Xin Kai group rats and Ku Jiang group rats and Bu Yi group rats all decreased significantly compared with Xin Kai Ku Jiang group rats(P < 0.01).Compared with the normal group rats, the level of IL-6 in the colonic mucosa of the model group was significantly increased(P<0.01). Compared with the model group rats, the level of IL-6 in the colonic mucosa of each treatment group rats decreased significantly(P <0.01). The level of IL-6 in the colonic mucosa of Wu Mei pill group rats and its disassembled party group rats all increased significantly compared with SASP group rats(P < 0.01).Compared with Wu Mei Pill group rats, the level of IL-6 in the colonic mucosa of Xin Kai Ku Jiang group rats, Xin Kai group rats, Ku Jiang group rats(P < 0.01) and Bu Yi group rats(P <0.05) all significantly increased. Compared with Xin Kai Ku Jiang group rats, the level of IL-6 in the colonic mucosa of Xin Kai group rats and Bu Yi group rats all showed no significant difference(P > 0.05), but that of Ku Jiang was significantly increased(P < 0.01).5 The level of TLR9, My D88, NF-κBp65 in colonic mucosa5.1 The level of TLR9 in colonic mucosaCompared with the normal group rats, the level of TLR9 in the colonic mucosa of the model group was significantly increased(P<0.01). Compared with the model group rats, the level of TLR9 in the colonic mucosa of each treatment group rats all decreased significantly(P < 0.01). There was no difference between the level of TLR9 in the colonic mucosa of Wu Mei pill group rats and that of SASP group, but the level of TLR9 in the colonic mucosa of each disassembled party group rats all increased significantly compared with SASP group rats(P < 0.01). Compared with rats, the level of TLR9 in the colonic mucosa of Xin Kai Ku Jiang group rats showed no significant difference(P > 0.05). Compared with Wu Mei Pill group rats or Xin Kai Ku Jiang group rats, the level of TLR9 in the colonic mucosa of other disassembled party group rats significantly increased(P < 0.01).5.2 The level of My D88 in colonic mucosaCompared with the normal group rats, the level of My D88 in the colonic mucosa of the model group was significantly increased(P<0.01). Compared with the model group rats, the level of My D88 in the colonic mucosa of each treatment group rats all decreased significantly(P < 0.01). There was no difference between the level of My D88 in the colonic mucosa of Wu Mei pill group rats and that of SASP group, but the level of My D88 in the colonic mucosa of each disassembled party group rats all increased significantly compared with SASP group rats(P < 0.01). Compared with Wu Mei Pill group rats, the level of My D88 in the colonic mucosa of Xin Kai Ku Jiang group rats showed no significant difference(P > 0.05). Compared with Wu Mei Pill group rats or Xin Kai Ku Jiang group rats, the level of My D88 in the colonic mucosa of other disassembled party group rats significantly increased(P < 0.01).5.3 The level of NF-κBp65 in colonic mucosaCompared with the normal group rats, the level of NF-κBp65 in the colonic mucosa of the model group was significantly increased(P<0.01). Compared with the model group rats,the level of NF-κBp65 in the colonic mucosa of SASP group rats, Wu Mei pill group rats, Xin Kai Ku Jiang group rats and Bu Yi group rats all decreased significantly(P < 0.01), but the level of NF-κBp65 in the colonic mucosa of Xin Kai group and Ku Jiang group rats showed no significant difference(P > 0.05). There was no difference between the level of NF-κBp65in the colonic mucosa of Wu Mei pill group rats and that of SASP group, but the level of NF-κBp65 in the colonic mucosa of each disassembled party group rats all increased significantly compared with SASP group rats(P < 0.01). Compared with Wu Mei Pill group rats, the level of NF-κBp65 in the colonic mucosa of Xin Kai Ku Jiang group rats showed no significant difference(P > 0.05). Compared with Wu Mei Pill group rats or Xin Kai Ku Jiang group rats, the level of NF-κBp65 in the colonic mucosa of other disassembled party group rats significantly increased(P < 0.01).6 The level of IL-6 mRNA, JAK mRNA, STAT3 mRNA in colonic mucosa6.1 The level of IL-6 m RNA in colonic mucosaCompared with the normal group rats, the level of IL-6 m RNA in the colonic mucosa of the model group was significantly increased(P<0.01). Compared with the model group rats,the level of IL-6 m RNA in the colonic mucosa of SASP group, Wu Mei pill group and Xin Kai Ku Jiang group rats all decreased significantly(P < 0.01), but the level of IL-6 m RNA in the colonic mucosa of Xin Kai group, Ku Jiang group and Bu Yi group rats showed no significant difference(P > 0.05). There was no difference between the levels of IL-6 m RNA in the colonic mucosa of Wu Mei pill group rats and that of SASP group, but the level of IL-6m RNA in the colonic mucosa of Wu Mei pill group rats and SASP group rats was lower than that of Xin Kai Ku Jiang group rats. And the level of IL-6 m RNA in the colonic mucosa of these three group rats was significantly lower than that of Xin Kai group, Ku Jiang group and Bu Yi group rats(P < 0.01).6.2 The level of JAK m RNA in colonic mucosaCompared with the normal group rats, the level of JAK m RNA in the colonic mucosa of the model group was significantly increased(P<0.01). Compared with the model group rats,the level of JAK m RNA in the colonic mucosa of SASP group, Wu Mei pill group and Xin Kai Ku Jiang group rats all decreased significantly(P < 0.01), but the level of JAK m RNA in the colonic mucosa of Xin Kai group, Ku Jiang group and Bu Yi group rats showed no significant difference(P > 0.05). There was no difference among of the levels of JAK m RNA in the colonic mucosa of Wu Mei pill group rats, Xin Kai Ku Jiang group rats and SASP group rats, but the level of JAK m RNA in the colonic mucosa of these three group rats was significantly lower than that of Xin Kai group, Ku Jiang group and Bu Yi group rats(P <0.01).6.3 The level of STAT3 m RNA in colonic mucosaCompared with the normal group rats, the level of STAT3 m RNA in the colonic mucosa of the model group was significantly increased(P<0.01). Compared with the model group rats,the level of STAT3 m RNA in the colonic mucosa of each treatment group rats decreased significantly(P < 0.01). There was no difference among of the levels of STAT3 m RNA in the colonic mucosa of Wu Mei pill group rats, Xin Kai Ku Jiang group rats and SASP group rats,but the level of STAT3 m RNA in the colonic mucosa of these three group rats was significantly lower than that of Xin Kai group, Ku Jiang group and Bu Yi group rats(P <0.01).Conclusion:1 By the theoretical research on Wu Mei Pill, the key of drug compatibility mechanism of treating ascariasis and dysentery and diarrhea and Jue Yin disease perhaps lies in regulating liver and spleen and stomach. Combination of sour and pungent and bitter drugs may be the fundamental form of legal prescription of Wu Mei pill.2 The rat model of ulcerative colitis induced by 2, 4, 6- three nitrobenzene sulfonic acid /ethanol method was successfully constructed.3 There is a close relationship between the occurrence of ulcerative colitis and lipid per-oxidation, imbalance of pro-inflammatory factors and anti-inflammatory factors, and the abnormal activation of TLR9/My D88/NF- κBp65 and IL-6/JAK/STAT3 two signaling pathway.4 Xin Kai Ku Jiang group could inhibit abnormal activation in TLR9/My D88/NF- κbp65 and IL-6/JAK/STAT3 signaling pathway and regulate immune dysfunction, and their effects were better than that of the other disassembled party group rats.5 Immune regulation mechanism of Wu Mei pill on ulcerative colitis is perhaps combination of Suan Ku and Xin Ku drugs to regulate liver and spleen and stomach.