Research The Effect Of Modified Minor Radix Bupleuri Decoction On Hepatic Injury And Apoptosis

1. Objective: Hepatic injury was a kind of common pathologic change of more liver diseases. Its long-term existence was the importantly initiating agent of hepatic fibrosis , cirrhosis and hepatocarcinogenesis . So , treating hepatic injury was the mail point of curing clinical liver ailment. Although mechanism of Hepatic injury was complex, by the developing of modern medicine, recognition of hepatic injury had been reached the molecular level and gnosia of mechanism of action of traditional Chinese medicine effecting on Hepatic injury was surpassed to protecting membrane of hepatic cell and , depressing aminopherase. Research in recent years manifest that apoptosis was more important in hepatic cell injury than other factors. Regulative iter of apoptosis mediated by Caspase was divided into endo-cell channel (through cellular membrane) and ecto-cell channel (through chondriosome) which was an importantly researching content in apoptosis mechanism of Hepatic injury. Because the research of Chinese medical science on was dispersed,the theory did not emerge a system yet.Minor Radix Bupleuri Decoction was from Treatise on Febrile Diseases which was used in long-term medical practice in Systemic disease such as liver and gall, digestion, immunization, and so on. Base on clinical experience of my advisor, Splenic asthenia, yin asthenia and stagnant blood was frequent patho-change in hepatic injury, but potency of a drug of invigorating the spleen, raising yin and promoting blood flow was insufficient in prescription.So, I added Chinese medicinal materialssuch as hoelen, atractylodes macrocephala, paeoniae lactiflorae .angelicae sinensis, danshen root to composite modified Minor Radix Bupleuri Decoction. According to tendency of researching mechanism of action of Chinese medical science treating hepatic injury, I evaluated the effect of modified Minor Radix Bupleuri Decoction on hepatic injury caused by conA, paracetamol, D—galactosamine, alcohol. My research would study the regulative mechanism of action of Modified Minor Radix Bupleuri Decoction effect on hepatocyte damage and apoptosis signal iter by detecting expression of related gene of apoptosis signal iter. 2. Methodology:2.1 Acute toxicity testing of two kinds of Minor Radix Bupleuri Decoction (modified Karber' s method): Before experiment Randomly divided mice into groups of Minor Radix Bupleuri Decoction 80g/kg/d, 40g/kg/d, 20g/kg/d, lOg/kg/d and modified Minor Radix Bupleuri Decoction 80g/kg/d, 40g/kg/d, 20g/kg/d, lOg/kg/d and normal control. Modified Minor Radix Bupleuri Decoction and Minor Radix Bupleuri Decoction group were both intragastric administrated according to 80g/kg/d ^ 40g/kg/d, 20g/kg/d, lOg/kg/d and normal control was administered with tales dose of isotonic Na chloride. Record toxic symptom, death information everyday after intragastric administrated for 5 days. The dead mice must be autopsied in time and noted pathological changes. The tissue must be checked pathological changes if it' s changes were observed by naked eye. Observed number of death, piloerection, tired to lie down, auricle pale or congest exorbitism, walk haltingly, information of urination and defecation, plegia, cataphora, convulsion, convulsion ,anhelation and carriage at the same time. Determined toxicity of different density of modified Minor Radix Bupleuri Decoction and Minor Radix Bupleuri Decoction on mice by observation of the experiment to decide adoptive dosage range in experiments.2.2 Protection of hepatic injury caused by concanavalin A treated with two kinds of Minor Radix Bupleuri Decoction : Randomly divided mice into groups of modified Minor Radix Bupleuri Decoction 40g/kg/d, 20g/kg/d, lOg/kg/d and normal control, bifendate, model, Minor Radix Bupleuri Decoction 40g/kg/d. Intragastric administrated according to modified Minor Radix Bupleuri Decoction 40g/kg/d, 20g/kg/d, lOg/kg/dand Minor Radix Bupleuri Decoction 40g/kg/d, bifendate 150mg/kg/d in the morning and afternoon in the first day and afternoon in the second day, 0.2ml every time, but the mice in normal control and model group were intragastric administrated with distilled water. Four hours after administrated last time, all mice were intravenously injected ConA 20mg/kg through tail except normal control group, abrosia, cannot refrain from water, to establish the animal model of hepatic injury caused by ConA. Obtained blood from eyeball of mice after 8 hours and got serum in coagulation which was divided into two parts conservated in -20°C and -70 °C refrigerator. Then got tissues from left hepatic lobe which were retained by 10% formaldehyde solution. By detecting AST, ALT, IFN- Y, TNF-a of Hepatic injury mice and pathologic check of hepatic tissue to approach and compare with protecting liver, depressing aminopherase ,changing content of IFN-Y , TNF-a of two kinds of Minor Radix Bupleuri Decoction and researching the effect and mechanism of preventing hepatic injury.2.3 Protection of hepatic injury caused by paracetamol treated with modified Minor Radix Bupleuri Decoction : Randomly divided mice into groups of modified Minor Radix Bupleuri Decoction 40g/kg/d, 20g/kg/d, lOg/kg/d and normal control , bifendate , model. Intragastric administrated according to modified Minor Radix Bupleuri Decoction 40g/kg/d, 20g/kg/d, lOg/kg/d and bifendate 150mg/kg/d for 10 days, but the mice in normal control and model group were intragastric administrated with distilled water 0. 2ml per mouse at same time. Six hours after intragastric administrated last time, the mice in normal control were intraperitoneal injected with isotonic Na chloride and the rest were intraperitoneal injected with paracetamol 300m g/Kg to establish the animal model of hepatic injury caused by paracetamol.All mice were got serum from eyeball for biochemistry test 16 hours after intraperitoneal injected with isotonic Na chloride or paracetamol and obtained hepatic tissue for pathologic check. By detecting LPO, GSH, IL-2, IL-6, DNA ladder pathologic check of hepatic tissue to approach the effect and mechanism of modified Minor Radix Bupleuri Decoction antioxidation and preventing hepatic injury. 2. 4 Protection of hepatic injury caused by D-galactosamine treated withModified Minor Radix Bupleuri Decoction : Randomly divided mice into groups of modified Minor Radix Bupleuri Decoction 40g/kg/d,20g/kg/d, 10g/kg/d and normal control , bifendate , model. Intragastric administrated according to modified Minor Radix Bupleuri Decoction 40g/kg/d, 20g/kg/d, 10g/kg/d and bifendate 150mg/kg/d, but the mice in normal control and model group were intragastric administrated with distilled water 0.2ml per mouse at same time for 7 days. In the 6th day, the mice in normal control were intraperitoneal injected with isotonic Na chloride and the rest were intraperitoneal injected intragastric administrated with D-galactosamine 0. 5g/kg to establish the animal model of hepatic injury caused by D-galactosamine. All mice were got serum from eyeball 24 hours after intraperitoneal injected with isotonic Na chloride or D—galactosamine and obtained hepatic tissue for pathologic check. By detecting AST, ALT, SOD, MDA, NO, mRNA expression of gene of Fas and FasL .pathologic check of hepatic tissue to approach the effect and mechanism of modified Minor Radix Bupleuri Decoction potentially protecting liver, depressing aminopherase, antioxidation, preventing hepatic injury and apoptosis.2. 5 Protection of hepatic injury caused by alcohol treated with Modified Minor Radix Bupleuri Decoction : Randomly divided mice into groups of modified Minor Radix Bupleuri Decoction 40g/kg/d, 20g/kg/d, lOg/kg/d and normal control, model. Intragastric administrated according to modified Minor Radix Bupleuri Decoction 40g/kg/d, 20g/kg/d, lOg/kg/d, but the mice in normal control and model group were intragastric administrated with distilled water 0.2ml per mouse at same time for 30 days. 20 minutes after last intragastric administrated, the mice in normal control were intragastric administrated with isotonic Na chloride and the rest were intragastric administrated with alcohol 6. 5g/kg to establish the animal model of hepatic injury caused by alcohol. All mice were got serum from eyeball 16 hours after intragastric administrated with isotonic Na chloride or alcohol and obtained hepatic tissue for pathologic check. By detecting MDA, GSH, mRNA expression of gene of Bax and Bcl-2, pathologic check of hepatic tissue to approach the effect and mechanism of modified Minor Radix Bupleuri Decoction potentially preventing hepatic injury and apoptosis and provided scientificly evidence for modified Minor RadixBupleuri Decoction used in clinical work. 3. Results:3.1 Acute toxicity testing of two kinds of Minor Radix Bupleuri Decoction (modified Karber's method): The mice in groups of Minor Radix BupleuriDecoction and modified Minor Radix Bupleuri Decoction 8Og/kg/d showed invariabe adverse effect. From the second day, the mice showed tired to lie down, debilitation, seldom eat, moved slowly, seldom drink water, moist pelage, loose stool. Two mice died in 4th day and one mouse died in the 5th day in group of Minor Radix Bupleuri Decoction 80g/kg/d. One mouse died in the 4th day and one mouse died in the 5th day in group of modified Minor Radix Bupleuri Decoction 80g/kg/d which had not obviously pathological changes by dissected. Maybe it' s reason was that medicamentous density was justo major and thick which effected drink and eat of mice and resulted above symptom. Minor Radix Bupleuri Decoction and modified Minor Radix Bupleuri Decoction AOg/kg/6^ 20g/kg/^ lOg/kg/d had not obviously effected on mice. They moved freedom, kept trichoxerosis, ate justoly, drinked more water, urinated and defecated mormally. It is thus evident that Minor Radix Bupleuri Decoction and modified Minor Radix Bupleuri Decoction 80g/kg/d maybe had more effection on mice, but the dose of 40g/kg/d maybe had less effection on mice. So the dose used in follow experiment was all below 40g/kg/d.3.2 Protection of hepatic injury caused by concanavalin A treated with two kinds of Minor Radix Bupleuri Decoction : Intravenously injecting ConA from mice tail to build model, the level of TNF- a and IFN- Y in serum of mice in model group were significantly heightened which synchronizaed with activity of ALT% AST and patho-change of Hepatic injury. Hepatic histology check confirmed Hepatic cellular damage was Hepatic cell degeneration, necrosis. So it manifested experimental mice injected ConA were conspicuous Hepatic injury caused by ConA and made aminopherase, TNF-a , IFN-y in serum obviously heightened. But high and middle dosage of modified Minor Radix Bupleuri Decoction could lessen degree of Hepatic injury induced by ConA and it' s effectiveness was surpassed Minor Radix Bupleuri Decoction . The low dosage of modified Minor Radix Bupleuri Decoction had no effect which may be concerned with justo minor and unable to treat immunogenic Hepatic injury obviously inducedby ConA. Modified Minor Radix Bupleuri Decoction had no. reinforce or cooperation with the high level of IFN- Y caused by ConA, on the contrary it depressed the level of IFN- y caused by ConA and this phenomenon may be concerned with coordination and Hepatic injury of inner cellular network cytokine.Decreased secreting IFN-y could depress enhancement bioactive property of IFN-y on TNF—a to achieve decreasing hepatic cellimpaired effect caused by ConA. The experimental result provided new evidence to explain mechanism of action of Hepatic injury of modified Minor Radix Bupleuri Decoction .The next stage,we would make penetrating research surround;mechanism of protecting liver effect of modified Minor Radix Bupleuri Decoction .3.3 Protection of hepatic injury caused by paracetamol treated with modified Minor Radix Bupleuri Decoction : The high and middle dosage of modified Minor Radix Bupleuri Decoction had obviously protection on chemically Hepatic injury induced by high dosage of paracetamol. It could significantly depress the level of LPO, heighten the level of GSH which possible concerned with anti oxygen free radical,restraining lipid peroxidation, inducing activity of liver drug enzyme, promoting metabolism neutralize poison capability of liver drug enzyme effecting on toxic. In addition, it still heighten the level of IL-2, depress the level of IL-6 , mitigate Hepatic injury, which may be concerned with function of regulating endosomatic immune response. From detection of pathological section and apoptotic DNA ladder, we knew that it had Hepatic injury, hepatonecrosis, still hepatocyte apoptosisin hepatic injury induced by paracetamol. But the high and middle dosage of modified Minor Radix Bupleuri Decoction could obviously lessen degree of Hepatic injury and lessen gragmentation strap of Hepatic injury cell caused by paracetamol which illustrated modified Minor Radix Bupleuri Decoction maybe had protection liver by lessen apoptotis.3. 4 Protection of hepatic injury caused by D—galactosamine treated with Modified Minor Radix Bupleuri Decoction .- Modified Minor Radix Bupleuri Decoction maybe had obvious protection on acute Hepatic injury caused by D-galactosamine. It could obviously highten activity of SOD and depress the level of MDA and lessen pathological change of hepatic tissue which showed protection of hepatic injury of modified Minor Radix BupleuriDecoction maybe concerned with opposing free radical and lipid. peroxidation. In addition, modified Minor Radix Bupleuri Decoction could depress the level of NO and lessen the gene expression of Fas and FasL which showed that modified Minor Radix Bupleuri Decoction educed refrain apoptosis by lessening the level of NO .refraining ento-cyte iter (bioblast channel), reducing mRNA expression of gene of Fas and FasL, refraining ecto- cyte iter(cellular membrane channel)apoptosis signal, decreasing apoptosis so that it could educe protection of Hepatic injury.3. 5 Protection of hepatic injury caused by alcohol treated with Modified Minor Radix Bupleuri Decoction .- The result showed that, after alcohol was drunk by mice, MDA in serum was obviously hightened, GSH was obviously lessened. But modified Minor Radix Bupleuri Decoction which maybe had capability of resisting lipid peroxidation could lessen the level of MDA and highten the level of GSH. These manifested protection of hepatic injury treated with modified Minor Radix Bupleuri Decoction maybe concerned with it' s antioxidize.It was not only a kind of mundificant but also catastaltic of oxygen free radical and could efficiently change the pathological change of hepatic injury caused by alcohol. We also discovered that modified Minor Radix Bupleuri Decoction could up-regulation the gene expression of Bax and Bcl-2 and made the specific value of Bcl-2/Bax get higher which confirmed modified that, when Minor Radix Bupleuri Decoction used to treat hepatic injury caused by alcohol, because of specific value of Bcl-2/Bax get higher , it elevated capacity of hepatic tissue of hepatic injury mice resisting Apoptosis. So, modified Minor Radix Bupleuri Decoction could reduce hepatic injury by up-regulation the specific value of Bcl-2/Bax. It maybe illustrate modified Minor Radix Bupleuri Decoction could educe protection of Hepatic injury through refraining ento-cyte iter (bioblast channel) to reduce hpoptosis by up-regulation the specific value of Bcl-2/Bax. 4. Conclusion:4.1 Modified Minor Radix Bupleuri Decoction might has protection on hepatic injury caused by many reasons and it' s effect was better than Minor Radix Bupleuri Decoction. Modified Minor Radix Bupleuri Decoctioncoujd educe protection of Hepatic injury through obviously depress aminopherase, resist oxygen free radical, refrain lipid peroxidation and regulate immune function.4.2 Modified Minor Radix Bupleuri Decoction could educe protection of hepatic injury through depressing the level of NO, refraining ento-cyte iter (bioblast channel) by up-regulation the specific value of Bcl-2/Bax. , refraining ento-cyte iter (bioblast channel) by reducing mRNA expression of gene of Fas and FasL to reduce apoptosis. It will provide an original research route for Chinese medical science treating hepatic injury in the future and substantial foundation for developing the study of tradition prescription by researching the regulative mechanism of action of Modified Minor Radix Bupleuri Decoction effect on hepatocyte damage and apoptosis signal iter. It was worth continue researching the pharmaco-value of modified Minor Radix Bupleuri Decoction resisting hepatic injury.