| Objective: To explore the effect of Danshi Ⅵ granules on cholesterol gallstones by high cholesterol-induced guinea pigs cholesterol stone;and further to explore the key proteins of cholesterol metabolism as well as the therapeutic effect of DanshiⅥ granules on nonalcoholic fatty liver.It will provide a basis for new indications of DanshiⅥ granules.Methods:(1)Effect of Danshi Ⅵ granules on cholesterol-induced calculus in guinea pig.Sixty guinea pigs(half female and male)were randomly divided into 6 groups(10/group): control group(equal volume purified water),model group(equal volume purified water),and ursodeoxycholic acid group(50 mg/kg)and DanshiⅥ granules low,medium and high dose groups(2.9,5.8,11.6 g/kg),10 per group.Except for the control group,the others were fed with high cholesterol food for 8 weeks.Thereafter,the animals were treated with DanshiⅥ granules or the positive drug for 4 weeks.The general growth of the guinea pigs was observed weekly and weighed,as well as the stone formation rate was calculated.The morphology of liver and gallbladder of each group were observed by naked eyes;the stones were detected by infrared spectroscopy;the blood was collected to test low-density lipoprotein cholesterol(LDL),total cholesterol(TC)and glycerol,as well as triglyceride(TG),high density lipoprotein(HDL)content and alanine aminotransferase(ALT),aspartate aminotransferase(AST)activity;the pathological changes of gallbladder and liver were stained with Hematoxylin-eosin(HE),and observed using light microscope.Whether low-,medium-,and high-dose DanshiⅥ granules have therapeutic effects on cholesterol gallstones induced by high cholesterol diet were investigated as decribed above experiments.(2)Effect of DanshiⅥ granules on key proteins of cholesterol metabolism in guinea pigs.Western-blot(WB)was used to detect the key proteins in cholesterol synthesis,transport and metabolism process in liver,including trishydroxymethyl glutaryl coenzyme reductase(HMGCR)and cholesterol carrier protein 2(SCP2),cholesterol 7α-hydroxylase (CYP7A1),and the Niemann-Pick C1 type protein 1(NPC1L1)in upper jejunal mucosal epithelial cells.The results from the above assays were used to explore the possible mechanism of DanshiⅥ granules in treating guinea pigs cholesterol gallstones.Further to provide a theoretical basis for clinical applications of Danshi Ⅵ granules.(3)Effect of DanshiⅥ granules on the nonalcoholic fatty liver in mice.Sixty female Kunming mice were randomly divided into normal control group(equal volume 0.9% sodium chloride solution),model group(equal volume 0.9% sodium chloride solution),simvastatin group(3 mg/kg)and Model + DanshiⅥ granules Low,medium and high dose groups(5.5,11,22 g/kg),10 mice in each group.Except for the control group,the others were fed with high-fat diet for 8 weeks.After 8 weeks,detection of mice nonalcoholic fatty liver model by B-ultrasound.After successful,the drugs were administered once a day for 4 weeks.The body weight and liver wet weight of the mice were weighed to calculate the liver index;the pathological morphology of liver was observed and the pathological score was obtained;TC,TG,HDL,LDL contents and AST and ALT activities in serum were determined by kits.Superoxide dismutase(SOD)activity and malondialdehyde(MDA)content in mice liver tissue were tested by kits.Results:(1)Effect of DanshiⅥ granules on cholesterol-induced calculus in guinea pigs.The stone formation rate was 88.9% in model group.No stones were found in control group.The stone formation rate was 55.6%,37.5% and 33.3% in low-dose,medium-dose and the high-dose,respectively.UDCA group composition stone rate was 22.0%;the stones were cholesterol stones identified by infrared spectrum assay;the serum TC,TG,LDL of the model animals increased significantly compared with the normal group(P<0.01).TC,TG,LDL showed no difference in the low-dose group,but showed significant difference in the middle-dose and high-dose groups(P<0.05,0.01).HDL was not obvious in the low,medium and high doses treatment groups.The changes of serum ALT and AST in the model group were significantly higher than those in the normal control group(P<0.01).After drug treatment,serum ALT and AST were not found in the low-dose group compared with the model group.The medium-dose and high-dose groups showed significant differences(P<0.05,0.01).HE staining showed that the model group had severe liver damage,disordered cell arrangement,and cytoplasmic fat.After filling the field of view,the nucleus was squeezed to one side,and the nucleus was loose.The results showed a dose-dependent improvement followed treatment.The gallbladder pathological observation showed that the connective tissue of the gallbladder tissue in model group was proliferated obviously,and the pathological state after drug treatment was given.There is also a dose dependent improvement.(2)Effect of DanshiⅥ granules on key proteins of cholesterol metabolism in guinea pigs.In model group,HMGCR,SCP2 and NPC1L1 were significantly increased compared with the normal group(P<0.01).After drug treatment,the expression level was significantly lower than that of the model group(P<0.05).At the same time,the model group had a better effect on CYP7A1.The group comparison was lower(P<0.05),and the expression of CYP7A1 was significantly up-regulated compared with the model group after drug treatment(P<0.01).(3)The beneficial effect of DanshiⅥ granules on the nonalcoholic fatty liver in mice.Compared with the control group,the liver index of model group was significantly increased(P<0.01);the appearance showed that the liver enlargement was grayish white,the texture was hard,the pinch had granules and greasy feeling,and the edge was round.Pathological morphology can be seen that the boundaries of hepatic lobule were not clear,hepatocytes were swollen,a large number of spherical lipid droplets appeared in the cytoplasm,and non-alcoholic steatohepatitis had progressed;pathological morphology scores were significantly increased;serum TC,TG,LDL levels were significantly increased,HDL content was significantly reduced,AST and ALT activities were significantly enhanced;SOD was significantly attenuated and MDA content was significantly increased;(P<0.01).Compared with model group,the liver index of simvastatin group and the middle and high doses of DanshiⅥ granules groups decreased significantly(P<0.05);the liver color appeared to be normal;the pathological morphology improved significantly.The pathological morphology score was significantly decreased;the serum TG and LDL levels were significantly decreased,the HDL content was significantly increased;SOD activity in liver tissue was significantly increased,and MDA content was significantly decreased;the difference was statistically significant(P<0.01 or P<0.05).Serum TC levels in the low,middle and high dose groups of simvastatin group and Danshi Ⅵ granules were significantly decreased,and AST and ALT activities were significantly decreased(P<0.01 or P<0.05).Conclusion:(1)DanshiⅥ granules reduced the occurrence of cholesterol gallstones in guinea pigs and have a therapeutic effect on cholesterol stones.(2)DanshiⅥ granules reduced the formation mechanism of guinea pig cholesterol stones.It may be related to decrease the protein expression of HCGCR,SCP2,NPC1L1 and increase the protein expression of CYP7A1.(3)DanshiⅥ granules reduced the non-alcoholic fatty liver disease induced by high-fat in mice,and its mechanism was related to the improvement of lipid metabolism disorder,anti-oxidative stress and lipid peroxidation,and provides a basis for clinical applications in the treatment of NAFLD. |
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