| BackgroundCholestasis is one kind of synthetical disease with a complex pathogenesis and varied etiology,which is caused by the formation and excretion of bile disorder.At the same time,we can see some related diseases such as jaundice,skin itching,abnormal glucose metabolism and abnormal lipid metabolism,etc.Cholelith disease will cause abdominal pain and biliary infection clinical signs and symptoms,because of the bile duct obstruction by the gallbladder tissue appeared similar stone or sediment sample material.Both of these two diseases are caused by liver and gallbladder as the biliary disorderly drainage into duodenal.The middle aged and elderly people(cholecystitis or cholelith disease),pregnant women(intrahepatic cholestasis during pregnancy)and people who live in LingNan area are easy to get this disease.Now as the diet structure change,the number of disease is growing.However,as the single clinical medication application and the lack of comprehensive treatment system,it is urgent to development new drugs.Snowbell-leaf Tickclover Herb[Desmodium styracifolium(Osheck)Merr.]as the leguminous plants,has the abi 1 ity of clear away heat,remove dampness and induce diuresis f'or treating stranguria.So it can treat with the difficulty and pain in micturition,lithangiuria,etc.Modern pharmacology study find that Snowbell-leaf Tickclover Herb has the pharmacologic action to treatment of urinary tract stones,anti ?inflammatory and cholagogic,diuretic,etc.Snowbell-leaf Tickclover Herb as the main raw material of proprietary Chinese medicine,such as"XiaoShiYin","ShiLinTong","XiaoYanLiDanPian","XiaoShiPian","JinQianCaoChongJi",etc.YongQi Miao,a famous western medicine doctor,said that the Snowbell-leaf Tickclover Herb was wonderful to treatment of bladder stones and gallbladder stones.So it has important practical significance to study the Snowbell-leaf Tickclover Herb treat with cholelith disease,cholestasis disease,and so on.ObjectiveBased on the expression of the gene and protein,We get the mechanism of Snowbell-leaf Tickclover Herb Extract how to regulate the bile acid and the cholesterol metabolism effect and mechanism.And it can expound the mechanism and scientific connotation of the Snowbell-leaf Tickclover Herb for protecting the liver and gallbladder.Methods1.Screening the active extracts of the Snowbell-leaf Tickclover Herb5 kg powder of Snowbell-leaf Tickclover Herb(20 mesh),added 20 times volume of 60%ethanol.Ultrasonic extraction 2 times,90 min each time.To concentrate without alcohol flavor,then respectively with three times the amount of ethyl acetate and n-butanol to extract 5 times.After dried the organic solvent,we got the ethyl acetate extract and n-butanol extract.The extract would suspend in 0.5%CMC-Na solution.Designed 6 groups of 40 experimental rats as follows:the normal control group,the model group,the ethyl acetate extract group(1.02g · kg-1),the n-butyl alcohol extract group(2.06g.kg-1),the Snowbell-leaf Tickclover Herb aqueous extract(crude drugs 3.6 g · kg-1)and the positive control group(ursodesoxycholic acid,UDCA,100mg · kg-1).Intragastric administration of medication was given for seven days respectively,and built the model by intragastric administration of a-nai isothiocyanates(ANIT,80mg · kg-1)at the fifth day.After 30 min of the last treatment,observed the bile flow,detected the changes of serum biochemical indexes,such as cereal third transaminase(ALT),aspertate aminotransferase(AST),total bilirubin(TBIL),alkaline phosphatase(ALP)and total bile acid(TBA).2.The acute toxicity test of the active extracts of the Snowbell-leaf Tickclover HerbWe treated 40 mice(half male and female,and body weight is 18?22g)with the Snowbell-leaf Tickclover Herb n-butyl alcohol extract 2 times a day and each time interval 8 h and continue observation for 14 d.The maximum concentration was 31.25 g · ml-1(equivalent of crude drugs)with the animal largest tolerated and the maximum volume(0.04mL · g-1,).3.Different doses of Snowbell-leaf Tickclover Herb n-butyl alcohol extract effect on the expression of gene and protein,the bile flow and the change of histomorphology in rats.Designed 7 groups of 56 experimental rats as follows:Snowbell-leaf Tickclover Herb N-butyl alcohol extract group(high,medium and low dose groups),the positive control group(Ursodeoxycholic Acid,UDCA,100mg · kg-1),Snowbell-leaf Tickclover Herb aqueous extract(crude drugs 3.6 g · kg-1,equivalent to 6 times of clinical dosage for adults),the normal control group(0.5%CMC-Na)and the model group(0.5%CMC-Na).Intragastric administration of medication was given for seven days respectively.After 30 min of the last time treatment,we narcotized the rats by intraperitoneal injection of 25%wulai sugar(0.4mL·100g-1),then did the surgery to collect the bile,the whole body blood,ileum,kidney and liver.Measured the volume of bile,detected the biochemical indicators of liver,such as cereal third transaminase(ALT),aspertate aminotransferase(AST),alkaline phosphatase(ALP),total bilirubin(TBIL),direct bil irubin(DBIL),total bile acid(associates),gamma glutamyl transpeptidase(?-GT),nitric oxide(NO)and ring adenosine phosphate(cAMP),and the level of TBA and GSH in bile.Detected the amount of the gene and protein expression of FXR,Bsep and MRP2 by RT-PCR and Western Blot method.Then observed the morphology changes of liver,ileum and kidney.4.The molecular mechanism of the Snowbell-leaf Tickclover Herb n-butanol extract effect on anti-formed of cholesterol stone105 C57BL/6 mice were randomly divided into 7 groups:Blank contrcol group,stone group,the bear deoxycholic acid(ursodoxychol ic acid,UCDA)in the control group,the Snowbell-leaf Tickclover Herb water decoction group,the Snowbell-leaf Tickclover Herb n-butyl alcohol extract(high,medium and low dose group).In addition to the blank control group,the rest of the group were given high cholesterol diet(add 15%fat,2%cholesterol and 0.5%cholic acid in normal feed).After treated 8 weeks,observed the change of the formed of stone,TC in mice serum cholesterol,bile acid(TBA),triglycerides(TG),high-density lipoprotein cholesterol(HDL-C)and the low density lipoprotein cholesterol(LDL-C).And detected the change of liver tissue liver X receptor(liver X receptor,LXR),cholesterol carrier protein(Scp2)and ileum protein cholesterol Niemann-Pick Cl Like 1(NPC1L1)by chemical,RT-PCR and Western Blot methods.Results1.Compared with the model group,the bile flow of the n-butanol extract groups at each detected time were significantly increased(P<0.05 or P<0.01).And the liver function index of glutamic-pyruvic transaminase(ALT),glutamic oxalacetic transaminase(GOP),total bile acid(TBA)and alkaline phosphatase(ALP)were significantly decreased(P<0.05 or P<0.01).However,the ethyl acetate extract groups have no difference change(P 0.05).2.Observed for 7 days,there was no death after administrated the maxifmum concentration of the Snowbell—leaf Tickclover Herb n-butyl alcohol extract 31.25 g· ml-1(equivalent of crude drugs)one time in a day.And for 14 days,there was also no death for administration 2 times in a day.We could get the maximum tolerated dose equivalent to 1400 times of the adult dosage.It means that the Snowbell-leaf Tickclover Herb n-butyl alcohol extract has the lower toxicity and is relatively safe in the scope of the dosage.3.Compared with the model group,the Snowbell-leaf Tickclover Herb n-butyl alcohol extract of high,medium and low dose groups could significantly increase the bile secretion,the levels of NO,cAMP and GSH in serum,the TBA in bile and the expression of mRNA and protein of FXR,Bsep and MRP2.However,the levels of ALT,AST,ALP and TBA in serum and the GSH in bile were significantly reduced.4.Compared with the stone group,the Snowbell-leaf Tickclover Herb water decoction group and the Snowbell-leaf Tickclover Herb n-butyl alcohol extract group could reduce the levels of TC,TG,LDL-C in serum,the CSI in gallbladder bile and the expression of mRNA and protein of Scp2 and NPCIL1.However,increased the levels of TBA and HDL-C in serum and the expression of mRNA and protein of LXR.conclusion1.The Snowbell-leaf Tickclover Herb n-butanol extract can act the ability as the Snowbell-leaf Tickclover Herb to anti-cholestasis.2.In order to anti-cholestasis,The Snowbell-leaf Tickclover Herb n-butanol extract can protect liver cells from damage,promote the secretion of bile,but reduce the accumulation of TBA and other metabolites.3.The Snowbell-leaf Tickclover Herb n-butanol extract can promote the transformation of cholesterol and excretion through up-regulation the expression of LXR and NPC1L1,and reduce SCP2.Which can resi.stance to the role of cholesterol calculus formation. |
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