The Effect And Mechanism Of Dihydromyricin On Intestinal Mucosal Barrier In Dextran Sulfate Sodium-induced Colitis In Mice

Objective: To explore the effect and possible mechanisms of DHM on the experimental mouse model of colitis.Methods: Fifty C57bl/6 mice were randomly divided into five groups: control group,dextran sulfate sodium group(DSS),DSS+20mg/kg DHM group,DSS+40mg/kg DHM group,and Salazosulfapyridine(SASP)group.The control group mice were free to drink the PBS for 7 days,while the other mice freely drank2.5% DSS for 7 days to establish an experimental colitis model.And then,the control group and the DSS group mice were given PBS for 7 days by gavage,and the other groups were given different concentrations of DHM or SASP at a certain concentration for 7 days.During the period,the weight and fecal character of every mouse were recorded to calculate the disease activity index(DAI).On the 15 th day after chloral hydrate anesthesia,portal venous blood was collected,mice were sacrificed,and colon tissues of mice in each group were collected.Hematoxylin and eosin staining was used to detect intestinal tissue damage in mice.The expression level of the Tight junction proteins ZO-1,Occludin,Claudin-2 was detected by immunohistochemistry.The expression of apoptosis related proteins BAX,Bcl-2,caspase3 and the number of apoptotic intestinal cells were detected by TUNEL assay kit and western blotting.The effect of DHM on ectopic of intestinal flora in mice with colitis induced by DSS was quantifying by the number of bacteria in portal venous blood of mice.The concentration of FITC-Dextran in peripheral blood of mice was measured to further reflect the protective effect of DHM on intestinal mucosal barrier function in colitis mice.Rat intestinal epithelial cell line IEC6 were divided into four groups: control group,lipopolysaccharide(LPS)group(350ug/mL),LPS+DHM group(0.5ug/mL),and DHM group(0.5ug/mL).The cells in the control group were cultured with complete medium for 72 hours,the cells in the DHM group and the LPS group were treated with 0.5ug/ml DHM or 350ug/ml LPS for 72 hours,while the cells of the LPS+DHM group were treated with 0.5ug/ml DHM+350ug/mL LPS for 72 hours.Reverse transcriptase-polymerase chain reaction(RT-PCR)was used to detect the expression of inflammatory factors IL-6 and IL-1?.Results: DHM can improve the symptoms of colitis mice and reduce the Disease activity index(DAI)in mice.HE staining results suggested that DHM could protect the intestinal tissue structure destruction caused by DSS in mice.Histological score showed that: compared with the control group,the histological score of DSS group is significantly higher(p<0.001),and 20mg/kg,40mg/kg DHM and SASP can reduce the histological score(p<0.001).Compared with the SASP group,there was no statistical significance in the 20mg/kg DHM group or the 40mg/kg DHM group(p>0.05).The number of apoptotic cells in intestinal tissue was detected by TUNEL staining and the results were analyzed quantitatively.Small apoptosis cells were seen in the control group.Compared with the control group,more apoptotic cells were seen in DSS group and the difference was not statistically(p < 0.001).2 DHM at the dose of 20 mg/kg and 40 mg/kg and SASP can reduce the number of apoptosis cells(p <0.001).The expression level of apoptosis related proteins BAX,Bcl-2,Caspase3 were detected by western blot,and the expression level of BAX was not statistically significant among the groups.DSS can decreased expression level of Bcl-2(p<0.001)but increased caspase-3(p<0.001)in colitis mice.Furthermore,the expression level of caspase-3 was observed increased in DHM groups(20mg/kg and 40mg/kg)but decreased in SASP group.The expression of tight junction protein in intestinal tissues of mice with DSS-colitis was detected by immunohistochemical.DSS could lead to the decrease of expression of the tight junction protein ZO-1 and Occludin in mice,but the increase of claudin-2 expression(p<0.05).40mg/kg DHM could increase the expression of ZO-1 and Occludin(p<0.001).20mg/kg DHM could increase the expression of ZO-1and decrease the expression of Claudin-2(p<0.05).Moreover,SASP can only reduce the expression of Claudin-2(p<0.05).There are two methods:bacterial activity detection and bacterial culture were used to detected the bacterial activity in the portal venous blood in mice.Compared with the control group,the bacterial activity in the portal venous blood of the DSS group was higher than the control group(p < 0.001).DHM can reduce the bacterial number in the portal venous blood and the results are statistically significant(p < 0.05).Although SASP can also reduce bacterial number,there is no statistical significance as compared to the control group(p>0.05).Compared with the control group,the concentration of FITC-Dextran in the venous blood of the DSS group increased(P <0.01),and 20 mg/kg DHM could reduce the content of FITC-Dextran in the venous blood of the mice with DSS colitis(P <0.001).The effect of DHM on intestinal epithelial cell proliferation was detected by MTT assay,DHM at high doses(10 ug/mL,5 ug/mL,2 ug/mL,and 1 ug/mL)of DHM significantly inhibited cell proliferation,while low doses(0.5 ug/mL,0.25 ug/mL,0.125 ug/mL,0.0625 ug/mL),the same effect cannot be observed.The effect of LPS on the proliferation of IEC6 was further detected by the MTT experiment.The results showed that the LPS concentration had a non-linear relationship with the proliferation of IEC6.The inhibitory effect of LPS of 300 ug/mL,350 ug/mL and 500 ug/mL on the proliferation rate of IEC6 was significantly stronger than that of LPS of 400ug/mL and 450 ug/mL,and the IC50 was 357.7ug/mL.LPS significantly increased the secretion of inflammatory factors IL-1? and IL-6 for IEC-6(p<0.001),and DHM reduced the secretion of IL-1?(p<0.01).Conclusions: It has been proved that the DHM plays a critical role in the treatment of DSS colitis by reducing the secretion of inflammatory factors and promoting the integrity of the intestinal mucosal barrier.Thus,DHM may become a candidate drug for the treatment of colitis.