UPLC-QTOF-MS Analysis Of Huanglian Jiedu Decoction And Its Effect On JAK2/STAT3 Pathway In Mice With Acute Ulcerative Colitis

Objective:To investigate the effect of Huang Lian Jie Du decoction（HLJD）in models of Ulcerative Colitis（UC）by Dextran sulphate sodium（DSS）,and study it the mechanism of JAK2/STAT3 pathway.Methods:1.Establishment and evaluation of acute UC model in miceThe experimental UC model was established by Dextran sulphate sodium（DSS）.Balb/c mice aged 4 weeks were randomly divided into control group and DSS solution groups（2%,2.5%,3%）,with 10 rats in each group.The rats drank the DSS solution freely for 8 days,Then Observed condition of rats including weight、mortality、fecal character、detection of fecal occult blood in order to measure disease activity index（DAI）score.After fetched colon from the rats at the end of experiment,colon length was measured,and the staining with HE conducted Colon Macroscopic Damage Index（CMDI）score to screen the modeling conditions of acute UC in mice.2 Effect of HLJD on acute UC colonic mucosal injury in mice.Balb/c mice were randomly divided into control group,model group（2.5%DSS）,high-dose HLJD group（24.66 g/kg）,medium-dose HLJD group（12.33 g/kg）,low-dose HLJD group（6.17 g/kg）,and mesalazine group（USAN,0.3 g/kg）,with 10 mice in each group.Except control,UC model was induced by DSS in other groups.At the same time,the corresponding drugs were given by gavage.During the experiment,the weight change,fecal characteristics and occult blood test of mice were measured and recorded,and the DAI score was counted.After the experiment,the mice colon was dissected and graded by HE staining section to investigate the effect of different concentrations of HLJD on acute UC,and finally to evaluate the alleviating effect of HLJD on acute ulcerative colitis.3.Effect of HLJD on JAK2/STAT3 pathway in acute UC mice.Balb/c mice were randomly divided into control,model,AG490 group,AG490+HLJD group and HLJD group with 10 mice in each group.Subsequently,except control,all the other groups were given 2.5%DSS solution continuously and freely until the model of acute ulcerative colitis was successfully established.The JAK2/STAT3 pathway in mice was blocked by intraperitoneal injection of AG490 two days before the start of the experiment until the end of the experiment.During this period,HLJD was administered intragastrically to mice in HLJD group and AG490+HLJD group.The changes of body weight,fecal characteristics and occult blood were measured and recorded during the experiment.The health status of mice was judged and the DAI score was counted.At the end of the experiment,HE staining and Masson staining were used to evaluate the symptoms and colon injury of mice with acute UC.Then JAK2,STAT3 and p-STAT3 were detected by Western Blot.Finally,the expression of Ki67 and TUNEL in colon tissue was evaluated by immunohistochemistry.To determine the impact of HLJD on the occurrence and development of acute ulcerative colitis by regulating JAK2/STAT3 pathway.4.Analysis of Intake Components of HLJD in Mice with Acute UC Colon Injury.The serum containing HLJD was detected by UPLC-QTOF-MS,and the compounds in the serum of HLJD-H group were screened and compared,in order to find the blood-entry components of HLJD and identify its main active components.Results:1.Screening and Investigation of Acute UC Model:On the 8th day of the experiment,except control,all the mice in the administration group showed acute ulcerative colitis symptoms of varying degrees,such as hematochezia,diarrhea,weight loss,etc.The most obvious symptoms were in the 3%DSS concentration group.The DAI score showed that the final score was positively correlated with the concentration of DSS.The score of 3%DSS concentration group was significantly different from that of control group（P<0.05）,but the pathological section showed that the inflammatory infiltration and injury had reached serosa layer,and the animals were dying in the experiment.Therefore,3%DSS concentration was not adopted as the optimal concentration for modeling;the HE score of 2%DSS concentration group was significantly different from that of control group（P<0.05）.However,in pathological section,it was found that the inflammation injury was limited to the mucosa,and the area of the injury was small,which did not meet the criteria of acute UC model.Therefore,2%DSS concentration was not adopted as the optimal concentration for modeling;HE score of 2.5%DSS concentration group was significantly different from that of control group（P<0.05）,and pathological section showed that the inflammation invasion and injury were concentrated near the submucosa and basal layer,and adjacent to the acute UC model.The cases of acute ulcerative colitis in bed were similar,which met the requirements of acute UC model.Therefore,free drinking of 2.5%DSS solution for 8 days is the best condition for acute UC.2.Observation of the effect of HLJD:On the 8^th day of the experiment,except for control,all the mice in the administration group had acute ulcerative colitis symptoms of varying degrees,the most typical of which was model.Most of the model mice had hematochezia,diarrhea,weight loss and other symptoms.However,the symptoms of colitis in HLJD group were alleviated effectively compared with model,and the DAI scores were decreased compared with model.Compared with HE pathological sections,the damage of colonic tissue in HLJD group was improved,especially in mucosal tissue.The final score of HE slices in all HLJD groups was significantly different from that in model group（P<0.05）,and the HLJD-H concentration group was the best alleviating effect.3.The effect of HLJD on JAK2/STAT3 signaling pathway:The DAI score of AG490 group was lower than that of model group at the end of 8^thday.HE score showed that the degree of colonic inflammatory infiltration of mice treated with AG490 was significantly improved compared with that of model（P<0.05）.The results of Mason staining showed that collagen deposition in model mucosa was significantly higher than that in HLJD group and control group（P<0.01）.WB assay of colonic homogenate showed that the expression of JAK2,STAT3 and p-STAT3 in model group was significantly higher than that in control group and all other groups（P<0.05）.The expression of JAK2 and STAT3 in the HLJD group was lower than that in the control group,but the expression of JAK2 and STAT3 in the HLJD group was similar to that in the control group,and there was no significant difference（P>0.05）.It was found in the results of immunohistochemistry.The expression of Ki67 in HLJD group was higher than that in control group（P<0.05）.The expression of Ki67 in HLJD group was similar to that in HLJD group（P>0.05）.The expression of Tunel in HLJD group was significantly higher than that in HLJD group and control group（P<0.05）.4.By extracting theoretical excimer ion peaks and fragment information of chemical constituents and comparing with related literatures and reference substances,it was found that 10 kinds of genipin-1-O-beta-D gentian disaccharides,phellodendrine,magnoliine,dihydroberberberine,palmatine,berberine,phellodendron,baicalin,baicalin and baicalin were found in the positive ion mode.Scores;display of baicalin and Baicalin in negative ion modeConclusion:1.Male Blab/c mice drank 2.5%DSS solution freely for 8days,which could induce a reliable and stable animal model of acute ulcerative colitis.2.HLJD can effectively alleviate DSS-induced acute UC colonic mucosal injury,which is related to down-regulation of JAK2,STAT3,p-STAT3 protein expression,inhibition of intestinal recess cell apoptosis,and promotion of mucocolic mucosal healing.3.There are 10 components of HLJD,including berberine and baicalin.The pharmacodynamic effects of HLJD may be related to these components.