The Preparation Of Tripterygium Glycosides Loaded Chitosan Microspheres And Its Effects In Mice Model With Experimental Colitis

Background and objecyive:Tripterygium glycosides(TG), as an immunosuppressant, has a strong antiinflammatory and anti-immune function. Although the etiology and pathogenesis of Inflammatory bowel disease(IBD) is still unclear, the researches found that over-activation of the Toll-like receptor(TLRs)/NF-κB signaling pathway has play an important role in this disease. First preparation of chitosan microspheres containing tripterygium glycosides and coating eudragit, and then use the microspheres to treat DSS-induced colitis in mice, detect the expression of TLR4, My D88, NF-κB p65, in order to determine the effect of Tripterygium glycosides on DSS-induced colitis in mice and its possible mechanism. Methods:1. Prepare chitosan microspheres containing tripterygium glycosides and detect its release in vitroThe chitosan microspheres were prepared by the O/W/O multiple emulsion method, Based on the concentration of the chitosan, cross-linking agent usage, the ratio of oil and colostrum, vortexed velocity to design orthogonal experiment to optimize preparation condition, and then coat microspheres with eudragit. Use the method of scanning electron microscopy and light microscope to observe the morphology of microspheres and determine the size of the microspheres. Use the HPLC to analyze encapsulating efficiency and drug-loading rate, the release rate of microspheres in vitro was determined in the different medium solutions.2. The therapeutic effects on DSS-induced colitis in mice of chitosan microspheres containing tripterygium glycosides2.1 The DSS induced-acute ulcerative colitis model was established in mice by drinking 5%DSS freely for 7 days, the control group was drunk the pure water.2.2 Experiment group: normal control group(N); model group(M); Salicylazosulfapyridine group(SASP); Chitosan microsphere group(C); TG high,medium and low dose group(HTG, MTG, LTG); TGM high, medium and low dose group,(HTGM, MTGM, LTGM). N group: normal mice were given 0.5%CMC solution. M group: model mice were given 0.5%CMC solution. SASP group: model mice were given 100mg/kg SASP 0.5% CMC solution. C group: model mice were given 120mg/kg chitosan microspheres 0.5% CMC solution. HTG, MTG, LTG group: model mice were given 40mg/kg, 20mg/kg, 10mg/kg TG 0.5% CMC solution. HTGM, MTGM, LTGM group: model mice were given 240mg/kg, 120mg/kg, 60mg/kg TGM 0.5% CMC solution.2.3 Indicators of detection:①Observed disease activity index(DAI) and the colon length everyday; ②Observed the colon change of histopathology by HE stain;③Detected the levels of My D88, TLR4 by Western blotting. ④The levels of My D88, TLR4 and NF-κB p65 were measured by Immunohistochemistry; ⑤The level of IL-6 in serum was detected by ELISA. Results:1. Prepare chitosan microspheres containing tripterygium glycosides and detect its release in vitroThe optimized formulation was as follows: The concentration of chitosan(15mg/ml), Colostrum-oil phase(1:15), Crosslinking agent(4m L)and the stirring speed(600rpm/min). Microspheres prepared according to the above condition were well-shaped, the mean drug-loading rate was17.12%±1.74,the average encapsulation efficiency was 54.97%±4.10, and the mean diameter was 7.16±2.10 μm. The uncoated microspheres were released all of the tripterygium glycosides in the first 5h. But the coated microspheres were almost not released in the artificial gastric juice(p H1.2), either in the artificial intestinal juice(p H4.8), however, the release of tripterygium glycosides was increased in artificial intestinal juice(p H6.8), and in the artificial large intestinal juice(p H7.4) and containing rat colon contents the release ratio nearly reached to 70%. The release in vitro of microspheres meet the requirements of colon-spcific drug delivery.2. The therapeutic effects on DSS-induced colitis in mice of chitosan microspheres containing tripterygium glycosides2.1 Colon length and DAI score of mice : Compared with the N group, the Colon length of M group and C group shorten significantly(P<0.05); were no significant difference when the colon length of the LTG group and LTGM group compared with the M group(P>0.05);compared with the M group,the rest of the treatment groups were significant difference(P<0.05). The DAI scores of M group and C group were gradually increased with the increase of experimental time;The DAI scores of SASP group, TGM groups and TG groups compared with the M group were significantly decreased, but higher than N group.2.2 The histopathology score by HE stain of colon: The score of N group was significantly lower than other groups(P<0.01); Compared with the M group, the score of TGM groups and TG groups were significant difference(P<0.05,0.01); The score of TGM groups and TG groups were dose-dependent, TGM groups were significantly lower than that of TG groups corresponding with the same dosage(P<0.05).2.3 Expression of My D88, TLR4 and NF-κBp65: Compared with the N group, the expressions of My D88, TLR4 and NF-κB protein M group and C group were raised significantly(P<0.01); Compared with the M group, the expression of each drug groups were decreased, and SASP, HTG, MTG and TGM groups most obvious decline(P<0.01); with the different dosage, the expressions of My D88, TLR4 and NF-κB protein reduced as the dose increased; the TGM groups was significantly lower than the same dose of TG groups(P<0.05).2.4 IL-6 level: In the M group, IL-6 content in serum increased significantly(P < 0.01); In groups of TG and TGM, IL-6 were lower, secretion reduced with the increase of dose to medicine. Conclusion:1. In this experiment, Preparation of chitosan microsphere containing TG was well-shaped, complied with the requirements of the colon targeted release formulations.2. The expression of My D88, TLR4 and NF-κB p65 were increased on DSS-induced colitis in mice, therefore the over-activation of TLRs/ NF-κB signaling pathway partake in the pathogenesis of IBD.3. TG could relieve the symptoms of inflammatory on DSS-induced colitis in mice. TG may be through the inhibition of TLRs / NF-κB activation to reduce the inflammation of the colon tissue.4. The same dose of TGM on DSS-induced colitis in mice and effect of TLR signaling pathway is better than TG, suggesting that the dose can be reduced by the drug-loaded microspheres and improved drug treatment in the colon concentration. The role of the sustained release and targeted can be expected to become the new ways of treatment for IBD.