Objective

2

To investigate the therapeutic effect of

Modified Dachaihu Decoction

（加减大柴胡汤， MDD） on cholesterol gallstones （CS） mice with damp-heat syndrome and the possible mechanism.

Methods

2

Forty-eight mice were randomly divided into blank group， model group， MDD group and ursodeoxycholic acid group， with 12 mice in each group. Except for the blank group， the other three groups were administered with “internal damp + external damp+ high cholesterol stone-inducing diet” to establish the CS damp-heat syndrome mouse model. After successful modeling， MDD group and ursodeoxycholic acid group were given 0.02 ml/g of MDD at a concentration of 1.17 g/ml and 0.02 ml/g of ursodeoxycholic acid solution at a concentration of 2.5 mg/ml by gavage， respectively， while the model group and blank group were given 0.02 ml/g of normal saline by gavage， once daily， for a total of 4 weeks. The general changes of body mass， diet， fur， stool， urine and otherconditions of mice in each group were observed and recorded every week since the establishment of the model. Open field test including the number of crossings， the rearing times and resting time was performed to measure the activity of mice in each group before and after modeling and after treatment. After treatment， the contents of total cholesterol （TC） and total bile acid （TBA） in bile were detected； hematoxylin and eosin （HE） staining was used to observe the histopathological changes of the ileum；and 16SrDNA amplification sequencing was used to detect intestinal flora biodiversity in each group.

Results

2

Compared to the blank group， the mice in the model group had decreased number of crossings and rearing times， prolonged resting time， increased TC content in bile and decreased TBA content （

P

＜0.01）. Compared to the model group and ursodeoxycholic acid group， the MDD group had increased number of crossings and rearing times， shortened resting time， decreased TC content in bile and increased TBA content （

P

＜0.05 or

P

＜0.01）. HE staining showed that the structural integrity of the ileum was destroyed in the model group， while the intestinal villi were loose， messy and broken， and the lamina propria was significantly thickened. Compared to those in the model group， the density of intestinal villi of mice was increased， while the conditions of intestinal villi rupture were improved in the MDD group and ursodeoxycholic acid group， and the pathological morphology of ileum was improved more obviously in the MDD group. Alpha diversity analysis showed that mice in the model group had decreased intestinal Chao1， Observed species， Pielou_e， Shannon， and Simpson indexes than the blank group （

P

＜0.01）， and the MDD group had increased intestinal Shannon and Pielou_e indexes than the model group and ursodeoxycholic acid group （

P

＜0.05 or

P

＜0.01）. Beta diversity analysis showed that there were no significant differences between model group and ursodeoxycholic acid group which was no larger than the within-group difference （

P

＞0.05）， and significant differences between other groups that larger than within-group difference （

P

＜0.01）. There were 66 significantly different bacteria among the four groups of mice at the taxonomic level of kingdom， phylum， class， order， family and genus， of which 25 were at the genus level. Compared to the model group and ursodeoxycholic acid group， Heterobacterium， Lactobacillus and Zurichella were significantly different genera in the MDD group.

Conclusion

2

MDD can improve the activity level and damp-heat syndrome and regulate lipid metabolism in CS mice with damp-heat syndrome， so as to promote gallbladder function and remove stones. The mechanism may be related to improving intestinal flora disorder and ileal tissue pathological damage.