| Objective:（1）The aim of this study was to establish an optimized an ultrasonic assisted enzyme pretreatment process to improve the ultrasonic extraction efficiency of baicalin.（2）Using immunohistochemistry,Western Blot and ELISA techniques to investigate the therapeutic effect of baicalin on DSS induced UC mice and the protective mechanism of colon mucosal immune barrier and mechanical barrier.Methods:（1）Taking 1 g of Scutellaria baicalensis Georgi powder,adding 10 m L of60%ethanol solvent,adding 1 mg of cellulase,and putting it into an ultrasonic cleaner to set a specific temperature for ultrasonic radiation,according to the single-factor experimental design,three main factors（p H value,temperature and ultrasonic time）of ultrasonic-assisted enzyme pretreatment were controlled to evaluate their effects on the extraction rate of baicalein,response Surface Methodology（RSM）was used to optimize the pretreatment process;（2）42 male BALB/c mice were randomly divided into 6 groups（N=7 in each group）,the rats were divided into blank group,model group,Baicalein Low,middle,high dose groups and mesalazine positive drug control group.The UC mouse model was established by 4%DSS.After successful modeling,the mice in the blank group and the model group drank distilled water freely,baicalein suspension(10 mg·kg-1、20 mg·kg-1、40 mg·kg-1)was given to the low,middle and high dose groups respectively,and mesalazine(600 mg·kg-1)was given to the positive drug group for 14 days,the changes of colonic histopathology were observed by HE staining,the levels of WBC and MONO in blood were analyzed by hematology analyzer,and the levels of LPS and SIGA in serum were measured by Elisa kit,the expression of IL-6,TNF-αand NF-κB was detected by Western blotting,and the distribution and expression of Claudin-1,Occludin and ZO-1 were detected by IHC.Results:（1）The optimal extraction time of baicalin was 40 min,hydrolysis temperature was 50℃,p H was 5,and the extraction rate was 16.16 mg/g through single factor investigation and response surface design experiment.（2）After successful modeling,the mice treated with baicalin showed good mental state,increased body weight,increased colon length,decreased DAI score and decreased histopathological score.The results of blood analysis showed that the levels of WBC and MONO in the model group were significantly higher than those in the control group（p<0.01）,the level of WBC in blood of mice with positive drug,low dose and middle dose decreased significantly（p<0.01）,while that of mice with high dose had no significant difference（p>0.05）,the proportion of MONO in blood of mice with positive drug,low dose and middle dose decreased significantly（p<0.01）.ELISA results showed that compared with blank group,serum LPS concentration in model group was significantly increased（P<0.01）,while serum LPS concentration in treatment group was significantly decreased（P<0.01）.Compared with blank group,s Ig A concentration was significantly decreased（P<0.01）.Compared with model group,s Ig A concentration of positive drug and baicalin low-dose and medium-dose groups was increased（P<0.05）,while there was no significant difference in s Ig A concentration of baicalin high-dose groups（P>0.05）.Western blot results showed that compared with blank group,the expressions of IL-6,TNF-αand NF-κB in colon tissue of mice in model group were significantly up-regulated（P<0.01）.Compared with model group,the expressions of IL-6,TNF-αand NF-κB in colon tissue of mice in treatment groups were significantly down-regulated（P<0.01）.Immunohistochemical results showed that compared with blank group,the protein expression of Claudin-1,Occludin and ZO-1 in model group significantly decreased（P<0.01）.Compared with model group,the protein expression of Claudin-1,Occludin and ZO-1 in positive and medium dose groups was significantly increased（P<0.01）.In low-dose group,the protein expression of Claudin-1 and Occludin increased significantly（P<0.01）,and the protein expression of ZO-1 increased significantly（P<0.05）.The protein expression of Claudin-1 and Occludin in high-dose group increased significantly（P<0.01）,while the protein expression of ZO-1 had no statistical significance（P>0.05）.Conclusion:（1）The extraction rate of Baicalein could be increased by ultrasound-assisted enzymatic pretreatment and the yield of Baicalein was increased about one-fold by ultrasound-assisted enzymatic pretreatment;（2）Baicalin could reduce the total number of WBC and MONO ratio in blood,increase the serum s Ig A level and reduce the serum LPS level,thus reducing the expression of TNF-αin the colonic tissues of UC mice,alleviating the expression of inflammatory factor IL-6,further regulating the NF-κB signaling pathway and exerting the protective effect on the immune barrier;meanwhile,it could increase the expression levels of Claudin-1,Occludin and ZO-1 in the colonic tissues of UC mice,which effectively improved the damage of intestinal mucosal mechanical barrier. |
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