Study On The Mechanism Of Anti-UC Intestinal Fibrosis By Total Xanthones From Gentianopsis Paludosa And Preparation Of Colon-specific Pellets

Colonic fibrosis is one of the most common complications of ulcerative colitis（UC）with unknown etiology and incurable treatment.At present,medicines such as aminosalicylic acid,steroids,and immunosuppressants are mainly used in clinical treatment of UC.However,the purpose of these medicines is to relieve the symptoms of UC,not to cure UC completely,and they are prone to develop medicine resistance,and side effects are large.Therefore,clarifying the pathogenesis and developing new medicines are the current research priorities to deal with UC and its intestinal fibrosis.It was found that the xanthones compounds from Tibetan medicine Gentianopsis paludosa（Hook.f.）Ma have anti-UC intestinal fibrosis activity.Therefore,the in-depth development and research of these compounds are of great significance for the prevention and treatment of UC intestinal fibrosis.The purpose of this study was to isolate and purify the effective fractions of total xanthones from Gentianopsis paludosa（TXG）,to study the pharmacological mechanism of its anti-UC intestinal fibrosis,to explore the regulating effect between autophagy and epithelial-mesenchymal transition（EMT）of colon cells in the process of UC intestinal fibrosis,to design and prepare a p H-dependent colon-specific pellets of TXG,and to provide a research reference for further clarifying the pathogenesis of UC intestinal fibrosis and developing new medicines derived from national medicines sources.Methods1.Isolation and purification of TXGThe Zr OCl₂colorimetric method was used to determine the content of TXG,and the method was validated.The most suitable macroporous adsorption resin was selected by static adsorption and desorption tests.Using the optimal macroporous adsorption resin as the stationary phase,the adsorption mechanism was discussed through the adsorption kinetics and thermodynamics,and the isolation and purification technology was optimized.2.Pharmacological mechanism research60 Wistar rats were randomly divided into normal control group,model control group,pirfenidone positive control group,and high,medium and low dose groups of TXG(0.1386,0.0693,0.0347 mg.g^-1.d^-1).Except for the normal control group,rats in other groups were induced UC intestinal fibrosis model by dinitrobenzene sulfonic acid（DNBS）twice at an interval of 10 days.The medicine was administered for 20 days from the second day of the first modeling,and the general state of the rats was observed and the disease activity index（DAI）score was evaluated.The rats were sacrificed after the last medicine administration,and the general morphological changes of colonic mucosa were observed after taking the materials.The colonic mucosal damage index（CMDI）was scored,and the colonic wet mass index was calculated.Hematoxylin and eosin（HE）staining and Masson staining were performed to observe the histopathological changes and fibrosis degree of the colon of rats,respectively.Western blot（WB）was used to detect the relative expressions of E-cadherin,α-SMA,N-cadherin,Col I,Col III,LC3-II/LC3-I,Beclin-1 and p62 proteins.The m RNA relative expressions of TGF-β1,Smad2,Smad3 and Snail 1 were detected by real time fluorescence quantitative polymerase chain reaction（q PCR）.3.Preparation of p H-dependent colon-specific pellets of TXGThe formulation of pellets was screened.The drug-loaded pellet cores were prepared by extrusion-spheronization method,and the extrusion-spheronization process parameters were optimized by Box-Behnken response surface method.Fluidization bed bottom spraying method was used for coating.Using the TXG as index,the basket method was used to determine the release of p H-dependent TXG pellets in vitro.The optimal weight gain percentage of pellet cores coating was investigated by in vitro release rate.The quality of coated pellets was evaluated,and the similarity of in vitro release process of coated pellets between batches was evaluated by f₂similarity factor method.Results1.The established method for the determination of TXG was precise and accurate.The optimal macroporous adsorption resin was DM130 resin.The adsorption process could be well explained by pseudo-second-order kinetics,particle diffusion kinetics,Langmuir and Freundlich models,and the process was a spontaneous,endothermic,entropy-increasing adsorption process.The optimized technology for the isolation and purification of TXG were as follow:the mass concentration of loading liquid was 1.5 mg·m L^-1,the eluent was 80%ethanol,the p H of the sample liquid was 5,the volumes of eluent were 10 BV,and the flow rate of eluent was 2 BV/h.After isolation and purification according to the optimized technology,the total xanthones content from Gentianopsis paludosa increased from 13.39%to51.26%（n=3）.2.（1）General state and DAI score of rats:compared with model control group,the general state of rats in each dose groups of TXG were improved to varying degrees,and DAI scores were significantly decreased（P<0.01）.（2）CMDI score and colonic wet mass index of rats:compared with the model control group,the CMDI scores of rats in each dose groups of TXG were significantly decreased（P<0.01）,and the colon wet mass indexes were decreased to varying degrees（P<0.01）.（3）Histopathological change and fibrosis degree of rat colon:HE staining results showed that,compared with model control group,with the increase of drug dose,the colonic villi gradually tended to be longer and connected with the basement membrane more compact,the villi tended to be arranged neatly,the goblet cells increased,the inflammatory infiltration and hemorrhage in the whole tissue were significantly reduced,and colon histopathological score were significantly decreased（P<0.01）in the medium and high dose groups.Masson staining showed that,compared with model control group,the arrangement of colonic muscle fibers tended to be neat in different degrees and the area ratio of collagen fibers decreased significantly in high and medium dose groups（P<0.01 or P<0.05）.（4）The effect of TXG on the EMT of colon tissue in rats:WB test results showed that,compared with the model control group,the relative expression of E-cadherin protein of rats in each dose groups of TXG,a marker of normal colon epithelial cells,were significantly increased（P<0.01）,and the relative expression ofα-SMA protein of rats in each dose groups of TXG,a marker of mesenchymal cells,were significantly decreased（P<0.01 or P<0.05）,and the relative expression of N-cadherin protein of rats in high and medium dose groups of TXG,a marker of colonic mesenchymal cells,were significantly decreased（P<0.01）,which slowed down the EMT process to varying degrees.Compared with the model control group,the relative expression of Col I and Col III of rats in each dose groups of TXG were significantly decreased（P<0.01 or P<0.05）,and the accumulation of col I and col III in the process of EMT of UC intestinal fibrosis were reduced.（5）The effect of TXG on autophagy level in rats colon tissue:WB test results showed that,compared with the model control group,the relative expression of LC3-II/LC3-I and beclin-1 protein of rats in high and medium dose groups were significantly increased（P<0.01 or P<0.05）,and the relative expression of p62protein of rats in high dose group was significantly decreased（P<0.01）.The ratio of LC3-II/LC3-I can indicate the degree of autophagy.Beclin 1 is a mammalian autophagy effector protein.P62 is a selective substrate of autophagy,which is degraded by autophagy,and its expression level reflects the strength of autophagy.Therefore,the WB results indicated that TXG could up-regulate the autophagy level of rats colon cells.（6）The effect of TXG on EMT signal pathway of rats colon tissue:Compared with the model control group,the m RNA relative expressions of TGF-β1,Smad2,Smad3 and Snail 1 m RNA in the colon tissue of rats in each dose groups of TXG were significantly decreased（P<0.01 or P<0.05）,indicating that TXG could inhibit the TGF-β/Smad signal pathway and the relative expression of Snail 1m RNA,a key EMT transcription factor regulated by TGF-β/Smad signal pathway.3.（1）Prescription of pellets:the mass ratio of medicine components（TXG）,MCC and micro powder silica gel was 5:3:2,and 50%ethanol solution of 3%PVP was used as wetting agent and adhesive.Coating material Eudragit?S100,plasticizer TEC and coating anti-sticking agent talc were dissolved in 80%ethanol solution as colon-specific coating solution.Each 100 m L of coating solution contained Eudragit?S100 5 g,TEC 1 g,and talc 2g.（2）The optimal process for preparing drug-loaded cores of pellets by extrusion-spheronization was as follows:extrusion speed of 40 r.min^-1,spheronization speed of 1100 r.min^-1,and spheronization time of 3 min.Under these conditions,the comprehensive score of drug-loaded cores of pellets could reach 70.4.（3）The final coated pellets were prepared by coating weight gain of 20%.（4）The results of quality evaluation and in vitro release evaluation showed that the obtained coated pellets met the quality requirements.The TXG in the pellets was rarely released in simulated gastric fluid（p H 1.2 hydrochloric acid solution）and simulated small intestinal fluid（p H 6.8 phosphate buffer）（the cumulative release in simulated gastric fluid and simulated small intestinal fluid were less than 5%）,and the cumulative release in simulated colonic fluid（p H 7.8 phosphate buffer）was more than90%after 2 hours.The evaluation results of the f₂similarity factor method showed that in vitro release process of different batches of coated pellets were similar.ConclusionIn conclusion,in this paper,the effective fractions of total xanthones were isolated and purified by macroporous adsorption resin technology for the first time,and its anti-UC intestinal fibrosis activity and pharmacological mechanism were studied.The results showed that TXG have anti-UC intestinal fibrosis activity,and its mechanism of action may be through inhibiting TGF-β/Smad signaling pathway and enhancing autophagy,so as to inhibiting EMT in rats colon tissue,and finally effectively inhibiting UC intestinal fibrosis.For the first time,it is clear that in the process of UC intestinal fibrosis,induction of autophagy in colon cells can reduce the expression of EMT,and new progress was made in the pathological research.In order to overcome the shortcomings of poor water solubility and low oral bioavailability of TXG,a p H-dependent colon-specific pellets of TXG were prepared.The preparation process was reasonable and feasible,and the obtained pellets had good in vitro colon localization release characteristics.The above research results can provide a reference basis for further understanding the pathogenesis of UC intestinal fibrosis and developing new anti-UC intestinal fibrosis drugs from national medicines sources.