Establishment Of The Model Of Wetness-Heat Of Liver And Gallbladder Syndrome And The Mechanism Of Longdanxiegan Pills In The Treatment Of Wetness-Heat Of Liver And Gallbladder Syndrome

Wetness-Heat of Liver and Gallbladder Syndrome is one of the common syndromes in TCM. It's usually caused by damp-heat accumulated of liver. The main pathogenesis of this syndrome is disorder of liver discharge, and it invoves in vary kinds of diseases, the hepatobiliary diseases occupied the first place. The major clinical manifestations were eye jaundice, body jaundice and hypochondriac pain. On modern medicine, body jaundice and eye jaundice was caused by cholistasis. However, intrahaptic cholistasis(IC) are basic lesion on many kinds of liver diseases. Although traditional Chinese medicine has no relative names of diseases corresponding "Intrahepatic Cholestasis", this syndrome belongs to "Jaundice", "Abdominal mass", "Abdominal Tympanites" on the Category of diseases of TCM. Wetness, heat, blocking stasis and liver failing to discharge is the basic pathogenesis of IC.Animal models are the important tools in study of pathogenesis, drug screening, evaluation of efficacy and mechanism of action. There , however, hasn't an ideal animal model of Wetness-Heat of Liver and Gallbladder Syndrome. These bring difficulty to investigation on the pathogeny and pathogenesis, drug screening, evaluation of efficacy and mechanism of action. Clinical manifestations of Wetness-Heat of Liver and Gallbladder Syndrome usually indicate body jaundice and eye jaundice. There is similarity in symptoms of Wetness-Heat of Liver and Gallbladder Syndrome and cholestasis. Therefore , it is practicable to establish model of Wetness-Heat of Liver and Gallbladder Syndrome by means of induced cholestasis. There are many transporters conveying bile ingredients in hepatocyte membrane and biliary canaliculi membrane. On various kinds cholistasis heptic lesion, It has been proved that abnormality or deficiency of transporters expression and functions can lead to a variety of disorders. Multidrug resistance protein (MDR) and multidrug resistance-associated protein (MRP) are two classes of proteins associated with bile transporting. Present studies have already proved that the formation of cholistasis might be related to MDR or MRP gene defect.Alpha-naphthylisothiocyanate (ANIT) may converse liver toxicity metabolites in animals . ANIT-induced murine liver injury is similar of human intrahaptic cholistasis in biochemical and pathomorphological changes.Longdanxiegan pills (LD) consists of Radix Gentianae, Radix Bupleuri, Radix Scutellariae, Gardenia, Alisma Orientale, Lardizabalaceae, Semen Plantaginis, Angelica sinensis, Rehmanniae Fusarium Wilt and Radix Glycyrrhizae Preparate. Longdanxiegan pills , which is one of the prescription formulas on treatment of the pattern of Wetness-Heat of Liver and Gallbladder Syndrome, can clear Liver, normalize and Severe damp-heat syndrome . Moreover there are not reports about its mechanism now.We would established the animal model accoding to the major symptoms of Wetness-Heat of Liver and Gallbladder Syndrome and the mechanism of cholistasis, explore pathogenesis of the model and study the effective and regulating mechanism of LD on Wetness-Heat of Liver and Gall Bladder Syndrome.ObjectiveWe attempt to set up an animal model with a Syndrome of Wetness-Heat of Liver and Gall Bladder induced by ANIT and observe the effect of Longdanxiegan Pills (LD) To explore the effective and regulating mechanism of LD on Wetness-Heat of Liver and Gall Bladder Syndrome. It can supply scientific basis for the treatment of Wetness-Heat of Liver and Gallbladder Syndrome. MethodsExperiment 1, Establishment of the animal model of Wetness-Heat of Liver and Gallbladder Syndrome10 healthy Wistar rats were divided into 2 groups, namely normal control group, ANIT-treated group. There were 5 aniamls in each group. Rats of normal control group were intragastrically administered with peanut oil, and other groups were treated with 10mg/ml ANIT peanut oil(100mg·kg-1 weight) once. Bile from each rat was collected for 4 hour at 72 hours after oral administration, and serum AST ,ALT, ALP, TBIL, DBIL, and CHO were measured respectively. At the same time, we observed the pathological changes of lung tissue. In addition, 5 healthy Wistar rats were treated 10mg/ml ANIT Peanut oil(100mg·kg-1 weight) once. Then rats were killed at 12h, 24 h , 48 h ,72h,and 96h respectivelly after oral administration ANIT. Another rat which was treated with peanut oil served as normal control. Each rat's livers were taken for total RNA extraction. The levels of MDR1b and MRP2 mRNA in liver tissue were investigated by reverse transcription polymerase chain reaction (RT-PCR).Experiment 2, Effect of Longdanxiegan Pills on Wetness-Heat of Liver and Syndrome Model72 healthy Wistar rats were randomly divided into 6 groups including control group, model group, DanLe caps (DL) group, high dosage of LD group, middle dosage of LD group and low dosage of LD group, 12 rats for each group. DL group and LD groups of three doses were treated for 8 successive days, while control group were administered with peanut oil. At day 5, model group, DL group and three LD groups were treated with 10mg/ml ANIT peanut oil(100mg·kg-1 weight) once. Bile of all rats from each group was collected continiously for 4 hour at 72 hours after oral administration of ANIT. Serum AST ,ALT, ALP, TBIL, DBIL, and CHO were measured respectively. At the same time , we observed the pathological changes of liver tissue. In addition, 72 healthy ICR mouse, were treated with the same method as did in rats. Serum TBIL and DBIL were measured too.Experiment 3, Study on the machanism of Longdanxiegan Pills in expression of MDR1a, MDR1b, MDR2, MRP1,MRP2 mRNA of Wetness-Heat of Liver and Syndrome Model20 healthy Wistar rats were randomly divided into 4 groups, 5 rats for each group, including control group, ANIT-treated group, high dosage of LD group , and low dosage of LD group . Three LD groups were treated for 8 successive days, while control group were administered with peanut oil. At day 5, model group, three LD groups were treated with 10mg/ml ANIT peanut oil(100mg·kg-1 weight) once. At 72 hours after oral administration of ANIT, livers of all rats in each groups were taken for total RNA extraction. The levels of MDR1a, MDR1b, MDR2, MRP1 and MRP2 mRNA in liver tissue were investigated by reverse transcription polymerase chain reaction (RT-PCR).Experiment 4, Effect of Longdanxiegan Pills on expression of CD18 of neutrophilic granulocyte in Wetness-Heat of Liver and Syndrome model20 healthy Wistar rats were divided into 4 groups, namely normal control group, ANIT-treated group, high dosage of LDT group , and low dosage of LDT group, 5 aniamls for each group. Each group was treated with the same method as mention in experiment 3 . At 72 hours after oral administration of ANIT, 100μl anticoagulated blood was collected from each rat's orbit, and the blood was incubated with phycoerythrin (PE)-labelled CD18 antibody for 20 min. Expression of CD18 was measured by using flow cytometer. Moreover, Total leukocytes and granular leukocytes were counted by using microcomputer controlled hematology analyzer.ResultsExperiment 1: Establishment of animal model of Wetness-Heat of Liver and Gallbladder Syndrome The results showed that bile secretion of model group was obviously lower than that of control group after oral administration of ANIT for 72h. The levels of serum TBIL, DBIL, ALT, AST, and ALP in model group increased obviously as compared with control group. Hepatocytes necrosis and bile duct injury could be observed in model group. Few MDR1b mRNA and MRP2 mRNA was expressed in control group and ANIT-treated 24h group. The MDR1b expression was increased at 48-96 h after ANIT administration. MRP2 expressions were increased at 48 h, 72h, and 96h after ANIT administration.Experiment 2: Effect of Longdanxiegan Pills on Wetness-Heat of Liver and Syndrome Model1. Bile secretionThe results showed that bile secretion of LD 5.0 and LD 2.5g crude herbs/kg was obviously higher than that of model group.2. The level of serum TBIL, DBIL and CHOThe serum concentration of TBIL, DBIL and CHO of model group significantly increased as compared with control group (P < 0.01) . Serum TBIL, DBIL, CHO of LD groups had the tendency to reduce (P>0.05) .3. The level of serum ALT, AST, and ALPThe level of serum ALT, AST, and ALP of model group significantly increased as compared with control group(P < 0.01). The level of serum TBIL, DBIL, CHO of LD groups were lower than that of model group (P>0.05) .4. The level of serum MDA, SOD, GSHThe level of serum MDA of model group significantly increased as compared with control group (P < 0.01. The level of serum MDA of LD groups were lower than model group (P>0.05) .5. HistomorphologyHepatic necrosis and bile duct injury could be observed in ANIT-treated group. Injury extent of the LD groups was reduced compared with model group.Experiment 3: Study on the machanism of Longdanxiegan Pills in expression of MDR1a, MDR1b, MDR2, MRP1,MRP2 mRNA of Wetness-Heat of Liver and Syndrome Model1. The expresion of MDR1a, MDR1b mRNA in model group Experimental results showed that expresion of MDR1a,MDR1b mRNA inmodel group were obviously higher than that of normal control group with different denervation degree (P<0.05, P<0.01) .So do the expresion of MDR2 mRNA (P>0.05). The expresion of MDR1a, MDR1b and MDR2 mRNA of LD 5.0g pharmacognosy /kg group and LD 2.5g pharmacognosy /kg were similar than those of model group (P>0.05).2. The expresion of MRP1,MRP2 mRNA in model group ratsThe expresion of MRP2 mRNA of model group was lower than the control group(P>0.05 ) . The expresion of MRP2 mRNA of model group was unchaged(P>0.05) .The level of serum TBIL, DBIL, CHO of model group was lower than thatof model group (P>0.05) . The expresion of MRP1 and MRP2 mRNA of LD groupwas not obviously changed than that of model group (P>0.05) .Experiment 4: Effect of Longdanxiegan Pills on expression of CD18 of neutrophilic granulocyte in Wetness-Heat of Liver and Syndrome modelExperimental results showed that there was a downtrend of total leukocytes and granular leukocytes in LD groups. CD18 fluorescence intensity of LD groups was obviously lower than that of model group.ConclusionFrom this study, we concluded that: the animal model established by oral ANIT administration has basic pathophysiologic characteristics of intrahepatic cholestasis, increasing of the activity of liver enzyme and hepatocyte injury. It was similar to the Wetness-Heat of Liver and Gallbladder Syndrome. So, this model can be used as the Wetness-Heat of Liver and Gallbladder Syndrome model. The expression of liver MDR and MRP mRNA of this model had changed in a time-dependent manner. The results indicated that MDR and MRP involed in the pathogenesis of the Wetness-Heat of Liver and Gallbladder Syndrome model. LD may improve Wetness-Heat of Liver and Gallbladder Syndrome by the following mechanisms: Increasing bile flow of intrahepatic biliary canaliculi; alleviating lipid peroxidation and stabilizing the cell membrane; Inhibiting endothelial-leukocyte adhesion- mediated CD11/CD18 expression.