Study On Rectal Administration Of Thermosensitive Gel Containing Berberine Hydrochloride-Calcium Alginate Microspheres In The Treatment Of Ulcerative Colitis

Inflammatory bowel disease(IBD)is a chronic idiopathic disease with two main forms:ulcerative colitis(UC)and Crohn’s disease(CD).In this thesis,berberine hydrochloridecalcium alginate microspheres were designed and prepared by using berberine hydrochloride as therapeutic drug and glutaraldehyde as cross-linking agent,and loaded into a temperaturesensitive gel composed of guanidine chitosan and pentahydrate-β-glycerophosphate sodium.The colitis model mice were treated by rectal administration,and its therapeutic effect was investigated.Berberine hydrochloride-calcium alginate microspheres were prepared by inverse emulsification / internal gelation method.taking the particle size of the microspheres as the index,the formulation and preparation process of the preparation were investigated by single factor-response surface method.The microstructure and material characteristics of the microspheres were characterized by scanning electron microscope(SEM),Fourier transform infrared spectroscopy(FT-IR)and differential scanning calorimetry(DSC).The drug loading rate,drug release and swelling characteristics of the microspheres were studied by high performance liquid chromatography(HPLC),in vitro release and swelling experiments,so as to provide reference for in vivo research.The natural polymer material chitosan was guanidine,the guanidine chitosan was characterized,and then combined with sodium pentahydrate-β-glycerophosphate,the best ratio of temperature-sensitive gel was selected by single factororthogonal experiment.the gelling properties of the best combination of temperature-sensitive gels were investigated in order to lay a foundation for follow-up experiments.Berberine hydrochloride-calcium alginate microspheres were loaded into temperature-sensitive gel to prepare berberine hydrochloride-calcium alginate microsphere temperature-sensitive gel.The mouse colitis model was established by simple and reliable sodium glucose sulfate(DSS)induction method.The therapeutic effect of berberine hydrochloride-calcium alginate microsphere thermosensitive gel on ulcerative colitis was investigated by evaluating the body weight,clinical signs,colon length and histopathological damage of DSS mice.Real-time RTPCR and ELISA techniques were used to study the molecular pharmacological mechanism of rectal administration of berberine hydrochloride-calcium alginate microspheres in the treatment of ulcerative colitis.The results show that the berberine hydrochloride-calcium alginate microspheres prepared by inverse emulsification / internal gelation method have smooth surface,good sphericity and narrow particle size distribution,and the average particle size is 65.39 ±10.34 μ m.The results of scanning electron microscope showed that the surface of the freeze-dried berberine hydrochloride-calcium alginate microspheres became rough and had obvious folds and depressions.The results of DSC and FT-IR spectra showed that the characteristic endothermic peak of berberine hydrochloride in the microspheres disappeared completely,indicating that berberine hydrochloride had been encapsulated in the microspheres.The results of drug loading rate showed that the drug loading rate of microspheres was 7.9%.The results of swelling experiment in vitro showed that the cross-linking of glutaraldehyde could significantly reduce the swelling rate of microspheres and improve the mechanical stability of microspheres.The results of in vitro release showed that p H-sensitive berberine hydrochloride-calcium alginate microspheres could rapidly release a large amount of drugs from the colon.Entrapment in the temperature-sensitive gel can slow down the release of the drug and prevent sudden release.The results of pharmacodynamic experiments in vivo showed that berberine hydrochloridecalcium alginate microsphere temperature-sensitive gel could effectively relieve the clinical signs such as weight loss,hematochezia and diarrhea in mice,and improve the shortening of colon length.The results of histopathological analysis of mouse colon showed that compared with the model group,berberine microsphere thermosensitive gel group had low level of inflammatory cell infiltration,regular crypt shape and good mucosal structure.the results showed that berberine microsphere thermosensitive gel could effectively reduce colonic inflammation and mucosal injury in colitis mice.The Real-time RT-PCR results showed that berberine hydrochloride microsphere thermosensitive gel could significantly reduce the inflammatory reaction by down-regulating the m RNA expression of pro-inflammatory cytokines tumor necrosis factor-α(TNF-α),interleukin-1 β(IL-1 β),interleukin-6(IL-6),nitric oxide synthase(i-NOS)and cyclooxygenase(COX-Ⅱ)in the colon of colitis mice.Immunohistochemical experiments showed that the thermosensitive gel containing microspheres protected the intestinal mucosa by protecting the intestinal mechanical barrier.The overall results show that berberine hydrochloride-calcium alginate microspheres temperature-sensitive gel can effectively prevent the occurrence and development of ulcerative colitis and is expected to be a new effective and safe strategy for the treatment of colitis.