| ObjectiveIn this study,the method of "stone-causing feed + internal dampness + external dampness" was used to establish a mouse model of Cholesterol gallstones(CS)with damp-heat syndrome,and the intervention of Modified Dachaihu Decoction from the perspective of prescription and syndrome was performed.Cholesterol-bile acid metabolism abnormality caused by bacterial flora disorder to explore the molecular biological mechanism of CS damp-heat syndrome,and provide a new breakthrough point and scientific basis for the objective research and clinical diagnosis and treatment of CS damp-heat syndrome.Methods1.The CS animal model was established by high cholesterol lithogenic diet induction method.Fifty-four 6-week-old SPF C57BL/6J male mice were randomly divided into blank group of 15 and modeling group of 39 according to body weight.The mice in the blank group were given ordinary feed,and the mice in the model group were given high cholesterol stone-causing feed;after 9 weeks of modeling,3 mice in each group were randomly selected for sampling to check their gallstones to determine whether the CS model was successful.After successful modeling,the mice in the modeling group were randomly and equally divided into the model group,the modified Dachaihu decoction group,and the ursodeoxycholic acid group according to body weight,with 12 mice in each group.2.To establish the animal model of damp-heat syndrome by the method of "internal dampness+external dampness",namely,the method of "stone-causing feed+internal dampness+external dampness" to establish the model of damp-heat syndrome in CS mice.From the 10 th to the 13 th week of modeling,the mice in the blank group continued the previous feeding mode.The mice in the model group,the Modified Dachaihu Decoction group and the ursodeoxycholic acid group were fed with high cholesterol lithogenic diets around the clock.On the basis of this,they were placed in a humid and hot environment simulated by an artificial climate box(temperature 32±1℃,humidity 95%)for 10 h/d,given20% sucrose water for drinking while in the humid and hot environment,and changed to normal drinking water for the rest of the time.Before and after modeling,the general conditions of the mice were observed and recorded,and open field behavioral experiments were performed.From the 14 th to the 17 th week of modeling,under the premise of unchanged modeling conditions,mice in the modified Dachaihu decoction group and ursodeoxycholic acid group were given corresponding drug interventions,while the blank group and model group were given the same dose of gavage.saline.After the intervention,the mice in each group were collected for subsequent detection of relevant indicators.3.HE staining was used to observe the morphological differences of liver,ileum and tongue tissues of mice in each group.4.Serum ALP,GGT and TBIL levels were detected by NPP substrate-AMP buffer method,GCNA substrate method and vanadium hydrochloric acid oxidation method,respectively.5.ELISA was used to detect the content of TC and TBA in the bile of mice in each group.6.16 Sr DNA amplification and sequencing method was used to detect the biological diversity of intestinal flora of mice in each group.7.The m RNA and protein expression levels of FXR,FGF15 in mouse ileum tissue and FGFR4,ERK1/2,JNK in liver tissue were detected by q-PCR and Western Blot,respectively.Results1.General conditions: The mice in the blank group were in good mental state,climbed well,had a high degree of activity,had dense,supple and bright fur,showed an overall steady increase in body weight,had a good diet,and had normal bowel movements.In the model group,the modified Dachaihu decoction group and the ursodeoxycholic acid group,the weight gain of the mice in the model group was significantly higher than that in the blank group,and the amount of food intake was higher than that in the blank group.,the weight gain was lower than that of the blank group,the food intake was reduced,the hair was shiny and sparse,the patients were prone to lying and sluggish,excessive squatting,lack of energy,yellow urine,loose stools,and obvious symptoms of damp-heat syndrome.After the 17 th week,the state of the mice in the model group and the ursodeoxycholic acid group had little difference compared with the previous ones;the mice in the modified Dachaihu decoction group had a better mental state,often scattered in the cage,and had rough but dense fur.Fewer urination,normal urination,loose stool when dry,body weight increased significantly compared with the model group and ursodeoxycholic acid group(P<0.01),the amount of food intake was increased,and the damp-heat syndrome was improved.2.Open-field behavioral experiment: Before modeling,there were no significant differences in the number of horizontal crossing grids,the number of uprights and the resting time in the open-field behavioral experiment of mice in each group(P>0.05).After 13 weeks of modeling,compared with the blank group,the number of horizontal crossing grids and the number of erection times of the mice in the model group were significantly reduced(P<0.01),and the resting time was significantly prolonged(P<0.05).After the intervention treatment,compared with the blank group,the number of horizontal crossing grids and the number of standing uprights of the mice in the model group were still significantly reduced(P<0.01),and the stationary time was still significantly prolonged(P<0.01).Compared with the model group,after the intervention of the modified Dachaihu decoction group,the number of horizontal crossing grids was significantly increased(P<0.01),the number of erections was significantly increased(P<0.05),and the stationary time was significantly shortened(P<0.01).Compared with the ursodeoxycholic acid group,after the intervention of the modified Dachaihu decoction group,the number of horizontal crossing grids and the number of erections were significantly increased(P<0.01),and the resting time was significantly shortened(P<0.05).3.Cholecystolithiasis: Compared with the blank group,the gallbladder of the mice in the model group was significantly enlarged,the bile was yellow-brown turbid,and there was a small amount of sediment-like substances precipitated;the mice in the modified Dachaihu decoction group and ursodeoxycholic acid Compared with the model group,the bile turbidity of the mice in the group was relieved to different degrees.4.Serum ALP,GGT and TBIL contents: Compared with the blank group,the serum ALP,GGT and TBIL contents of mice in the model group were significantly increased(P<0.01).Compared with the model group,the serum ALP,GGT and TBIL contents of the mice in the modified Dachaihu decoction group and the ursodeoxycholic acid group were significantly decreased(P<0.01).Compared with the ursodeoxycholic acid group,the serum ALP and TBIL contents of the mice in the modified Dachaihu decoction group were significantly decreased(P<0.01).5.Contents of TC and TBA in bile: Compared with the blank group,the content of TC in the bile of the mice in the model group was significantly increased(P<0.01),and the content of TBA was significantly decreased(P<0.01);The content of TC in the bile of the decoction group and the ursodeoxycholic acid group was significantly decreased(P<0.01),and the content of TBA was significantly increased(P<0.01);The content of TC in the bile of mice in the Hutang group was significantly decreased(P<0.01),and the content of TBA was significantly increased(P<0.01).6.Intestinal flora biodiversity:(1)Compared with the blank group,the intestinal flora richness(Chao1,Observed species),diversity(Shannon,Simpson)and uniformity(Pielou＿e)of mice in the model group were significantly reduced(P<0.01);compared with the model group,the richness(Chao1,Observed species),diversity(Shannon,Simpson)and evenness(Pielou＿e)of the mice in the modified Dachaihu decoction group had an upward trend(P>0.05).(2)The results of Beta diversity and phylum and genus level flora species composition analysis showed that,except for the model group and the ursodeoxycholic acid group,there was no significant difference in the flora(P>0.05),and the other component groups had significant differences(P<0.01).(3)There were 66 significantly different bacteria(LDA threshold>3)in 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.The blank group included9 different genera including Bacteroides,Gelidibacter,Thalassospira,and Blautia;Melissococcus was the significantly different genera in the model group;11 genera were significantly different in the modified Dachaihu decoction group,including Aerococcus,Candidatus＿Arthromitus,Lactobacillus,etc.;In the oxycholic acid group,there were four distinct genera of Chitinophaga,Kurthia,Vagococcus and Weissella.7.The m RNA and protein expressions of ileal FXR,FGF15 and liver FGFR4,ERK1/2,JNK:(1)FXR: Compared with the blank group,the m RNA and protein expressions of ileal FXR in the model group showed a significant upward trend(P< 0.01);Compared with the model group,the m RNA and protein expressions of FXR in the ileum of the mice in the modified Dachaihu decoction group were significantly decreased(P<0.01),and the m RNA expression of FXR in the ileum of the mice in the ursodeoxycholic acid group was significantly decreased(P<0.05).Compared with the ursodeoxycholic acid group,the protein expression of FXR in the ileum of the mice in the modified Dachaihu decoction group was significantly decreased(P<0.05).(2)FGF15: Compared with the blank group,the m RNA expression of FGF15 in the ileum of the mice in the model group was significantly increased(P<0.01),and the protein expression was significantly increased(P<0.05).The m RNA expression of FGF15 was significantly decreased(P<0.05).(3)FGFR4: Compared with the blank group,the m RNA and protein expressions of FGFR4 in the liver of the mice in the model group were significantly increased(P<0.05).The m RNA expression of FGFR4 was significantly decreased(P<0.05).(4)ERK1/2: Compared with the blank group,the m RNA(P<0.01)and protein expression of ERK1/2 in the liver of mice in the model group were significantly increased(P<0.05);The m RNA expression of ERK1/2 in the liver of mice in the deoxycholic acid group decreased significantly(P<0.01),and the protein expression showed a downward trend(P>0.05).(5)JNK: Compared with the blank group,the m RNA and protein expressions of JNK in the liver of the mice in the model group were significantly increased(P<0.01);The protein expression was significantly decreased(P<0.05).8.Correlation between different bacterial genera and indicators related to cholesterol-bile acid metabolism: Kurthia,Aerococcus,Weissella and Melissococcus There was a significant positive correlation with the expression of FXR,FGF15,FGFR4,ERK1/2 and JNK(P<0.01);the expression of FXR,FGF15,FGFR4,ERK1/2 and JNK was significantly correlated with Alistipes and Prevotella,Coprococcus and Barnesiella were significantly negatively correlated(P<0.01,P<0.05).Conclusions1.The abnormal expression of FXR/FGF15/FGFR4 pathway caused by intestinal flora disturbance is one of the molecular biological mechanisms of CS damp-heat syndrome.2.Modified Dachaihu Decoction can regulate intestinal flora disturbance,regulate the expression of FXR/FGF15/FGFR4 pathway related factors,regulate cholesterol-bile acid metabolism,improve liver function and abnormal bile composition,and play a role in clearing heat and dampness,eliminating stone and promoting gallbladder.effect. |
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