Effects Of Jiuxieling Granules On The Express-ion Of TLR4 And NF-κB P65 And Inflammatory Cytokines On Ulcerative Colitis Model Ra-ts Of The Spleen And Kidney Yang Deficiency

ObjectiveTo clarify the histopathological changing effects and the expressions of IL-1, IL-6,TNF-α, IFN-γ inflammatory cytokines and TLR4, NF-κB p65 genes and proteins in UC rats of the spleen and kidney yang deficiency by Jiuxieling granules and preliminary interpret the action mechanisms and scientific connotation of Jiuxieling granules that was guided by warming and tonifying the spleen and kidney to treat UC and it will provide effective experimental data for exploring the treatment of UC new clinical medication.Methods1. All rats(n=60) SPF Wistar rats were divided into blank group and model group. The perfusion of Rhubarb Decoction plus intramuscular injection of hydrocortisone and combined with TNBS(2, 4, 6- three trinitrobenzene sulfonic acid) and ethanol enema were employed to establish UC animal model. After successful modeling model group was divided into UC model of spleen and kidney yang deficiency groups for 7 days, 14 days and 21 days. After materials were taked, we observed each group rats colon tissue pathological morphological changes by adopting pathology method, detected the expression of FT3, FT4, T in the rat serum by ELISA method.2. All rats(n=90) SPF Wistar rats were divided into blank group and model group.The method was same as the first lab. After successful modeling model group was divided into UC model of spleen and kidney yang deficiency group, treatment groups by Jiuxieling granules for 7 days, 14 days and 21 days respectively and sulfasalazine(SASP) group. According to the requirements of each group respectively for7 days,14 days, 21 days and the rats in positive group were treated with SASP. After materials were taked, we observed each group rats colon tissue pathological morphological changes by adopting pathology method(macroscopic observation and HE staining microscopic observation), detected the expression of IL-1, IL-6,TNF-α, IFN-γ in the rat serum and tissue by ELISA method, and also detected the expression of TLR4, NF-κB p65 protein and gene by SP and RT-q PCR.Results1. ELISA method detected rats serum FT3, FT4, T expressions: compared with the blankgroup, the expressions of FT3, FT4, T level decreased in the model group rats serum for 7days, 14 days, 21 days, and model group for 21 days decreased obviously(P<0.01).2. ELISA method detected rats serum and tissue IL-1, IL-6, TNF-α, IFN-γ expressions:compared with the blank group, the expressions of IL-1, IL-6, TNF-α, IFN-γ level increased averagely in the model group rats serum and tissue(P<0.01); After treatment, compared with model group, the expressions of IL-1, IL-6, TNF-α, IFN-γ level in the treatment of 7 days, 14 days, 21 days groups and positive group decreased averagely(P<0.01 or P<0.05), and the treatment of 21 days groups decreased obviously(P<0.01).3. RT-q PCR method detected the expression of rats colonic tissue TLR4, NF-κB p65 m RNA, compared with model group, the expression of TLR4, NF-κB p65 in blank group were significantly lower than model group(P<0.01), After treatment, compared with model group, the expression of TLR4, NF-κB p65 in the rest of the group reduced.4. Immunohistochemical method detected the proteins expression of TLR4, NF-κB p65 in rats colonic tissue,TLR4 expression,(1)Microscopically: the positive TLR4 expression was found in model group intestinal mucosal epithelium and lamina propria mononuclear cells, which expressed higher than that of blank group, and the closer to the lumen, the stronger to the expression.The expression of TLR4 in the rest of treatment group rats were weak or medium strength.(2)The TLR4 positive cells average grey value results showed: compared with the blank group the expression of TLR4 in model group significantly enhanced(P<0.01), after treatment, compared with model group, the expression of TLR4 in the rest of the group decreased significantly(P<0.01 or P<0.05).NF-κB p65 expression,(1)Microscopically: In blank group, NF-κB p65 positive cells expression were negative, in the model group NF-κB p65 positive cells were mononuclear macrophages, they were expressed in cytoplasm, intestinal mucosa epithelium and lamina propria mononuclear cells, and the level of expression was higher than that of blank group.The expression of NF-κB p65 in the rest of treatment group rats were weak or moderate intensity expression.(2)The NF-κB p65 positive cells average grey value results showed:compared with the blank group, the expression of NF-κB p65 in the model group significantlyenhanced(P<0.01), compared with model group, after treatment,the expression of other groups significantly reduced(P < 0.01 or P < 0.05).Conclusion1. Spleen and kidney Yang deficiency is the key pathogenesis of UC which is repeatedly attacking and refractoring. Guided by the method of removing the spleen kidney of Jiuxieling granule has a good effect in the treatment of UC, which can provide effective basis for clinical application.2. Jiuxieling granules can reduce the expression of IL-1, IL-6, TNF-α, IFN-γ in the spleen and kidney yang deficiency type of UC model rats serum and tissue. it inhibits the release of inflammatory cells and Inflammatory activity products, and reduces the body’s inflammation of the intestinal mucosa reaction and issue damage.It plays a role to promote Intestinal mucosa repairing.3. Jiuxieling granules reduces the gene transcription and the protein expression of TLR4,NF-κB p65 in UC model rats of spleen and kidney yang deficiency, relieves the mucosal inflammation, play an important role in repairing mucous membrane.