Protective Effect Of Traditional Chinese Medicine Yigan Fuzheng Paidu Capsules Combined With Medical Ozone Treatment On CCl₄-induced Acute Hepatic Injury Dogs

Supported bySuperintendent foundation of Nanfang Hospital in Guangzhou city and foundation for studies of the Technology Bureau in Shenzhen City(200204176).Objects and significancesYigan Fuzheng Paidu Capsule is a compound preparation of TCM for treating chronic viral hepatitis type B.Medical ozone(O₃),in which the strong effects of immunologic induction and immunologic assault had been found,is the allotropic form of oxygen with three atoms and two unpaired electrons,which has a higher oxidizing capacity than oxygen.Using large animal canine models,this study was aiming at how did TCM Yigan Fuzheng Paidu combined with medical ozone affect on CCl₄-induced acute hepatic injury dogs.In order to supply a reliable basement on their clinical application,we have tried to found out whether this united medication exerted a protective effect on livers according to experimental way of pharmacodynamics. MethodsDrugs and equipmentYigan Fuzheng Paidu Capsules,Chinese formulated products prepared based on one of the empirical formulae of Professor Yang Yun-gao.Oleanolic Acid Tablets,produced by Guangdong Nanguo Biological Pharmacy Co.,Ltd.The medical generator of ozone,made in Germany,was registered and licensed for medical therapy by FDA of PRC.ModelThe acute hepatic injury canine models were made by being injected carbon tetrachloride(CCl₄).CCl₄ mixed with peanut oil(1:1,W/W)in the dosage of 0.9ml per kilogram of body weight was injected intraperitoneally to dogs.Groups and AdministrationsTwenty-four healthy dogs were divided randomly into four groups(n=6 in each group).Dogs in Group Model received nothing before CCl₄ intraperitoneally injection. Dogs in Group OAT received Oleanolic Acid Tablets(10mg/d·kg body wt,30 days). Group YC,dogs that received Yigan Fuzheng Paidu Capsules(0.2g/d·kg body wt, 30days).Dogs in Group YC+O₃ received both Yigan Fuzheng Paidu Capsules(applied as aforementioned,30 days)and medical ozone(20μg/ml,150ml per dog,transrectum insufflation every other day,15 times).The second day after the last time's preconditioning administrations,dogs of all groups were injected CCl₄ as the methods aforementioned to be made into acute hepatic injured canine models.These dogs were given full diet.If they got poor appetite,they would be given,fluid infusion.Observation and Determination IndexesObservation of dogs' presentation:Record the presentation of each dog before and after the injection,I.e.motoricity,mental state,eating,urination and defecation etc.Survival time:Record the live time of each dog,accurate to hours.The biochemical and hematological indexes determination:intravenous blood was sampled pre-injection and post-injection 24h,2d,3d,4d,7d,14d.All the blood samples were determined immediately by clinical laboratory of NanFang Hospital in the values of liver function test(ALT,AST/ALT,TBIL),blood ammonia(AMMO), and renal function test(BUN).Histopathology:Multiple liver samples were obtained through ultrasound-guided percutaneous needle biopsy from each dogs for the evidence of no liver pathological changes before injection.After the injection,each dog died or living for 15 days was submitted to livers.The liver tissues were fixed by 10%formaldehyde solution, wrapped by the paraffin,then sliced and received regular HE and Gomori silver staining.Statistical analysisSPSS 12.0 was used,by which Kaplan Meier was used for survival analysis.Data in a group of certain time was expressed by Mean±SD((?)±s).Independent-Samples Tests were used for comparisons within a group,and of different groups.The docimastic significance level is 'a=0.05'.ResultsDogs' presentation ObservationPre-injection,all twenty-four dogs were in good mental and physical status,and had sensitive reactions and yellow sheen hairs.Their diet,stool and urine were normal. Post-injection,all dogs began to vomite and lost balance immediately.Except for Group YC+O₃,all dogs of other three groups appeared to prefer either staying still alone,or being restless and pacing up and down,some of which showed bad mental status and appetites,and yellow urination.It showed nothing different in dogs of group YC+O₃ before and after injection,except that two of them showed yellow urination.Survival analysisLog Rank,Breslow and Tarone-Ware tests were used for comparisons of four groups surviving times.All three tests showed that Group YC+O₃ was significantly(P＜0.05)different from Group Model,while there was no significance (P＞0.05)on pairwise comparison of four groups.Adding survival curves of four groups,results of aforementioned showed that Yigan Fuzheng Paidu Capsule combined with medical ozone could significantly raise the survival rates of acute hepatic injury canines.The biochemical and hematological indexesAll the indexes except for AST/ALT of four groups had the tendency of rising at first and then falling.(1)Pre-injection,there were no significances(P＞0.05)between Group Model and other three groups respectively in each indexes(AST/ALT was out of analysis at the time).(2)Post-injection,Group Model was significantly(P＜0.05) higher than any other three groups in the indexes of ALT,TBIL,PT and AMMO,while Group YC and Group YC+O₃ was significantly(P＜0.05)higher than Group Model in the indexes of 4^thday's BUN.Post-injection,Group YC was significantly(P＜0.05) higher than Group OAT in the index of ALT,while Group OAT was significantly(P＜0.05)higher than Group YC+O₃ in the indexes of ALT,TBIL,PT and significantly(P＜0.05)lower in the index of AMMO.Post-injection,the values of AST/ALT in Group Model and Group YC were a little 1.0 below at 24^thh,then rising(AST/ALT＞1.5,Group Model was rising more significantly),then fall to 0.5 below in 3-4 days.In 5-14 days,the values of AST/ALT in group Model continued falling(to about 0.1 in 14^thday),nevertheless group YC started to rise after 4^thd(to the level of 14^thday,I.e.a little 1.0 above).Values of AST/ALT in Group OAT were between 1.5-2.0 in 24^thday,and then started to fall(to about 0.5),and began to rise from 7^thday(to 1.0 above in 14^thd).Group YC+O₃ was always 1.0 above.Histopathology of LiversIn light microscope normal liver tissues showed that the structures of hepatic cords,hepatic sinusoid and hepatic lobule were clear.There was not any hepatocellular degeneration,necrosis or other pathological change in the liver tissues.The structure of reticular fibers existed normally.In light microscope,liver tissues of Group Model showed:(a)Hepatocellular necrosis and hemorrhage occurred in centrilobular and midlobular zones with many different fat vacuoles in and around them appearing medium or serious steatosis.There were infiltration of inflammatory cells in necrosis zones.Retained hepatocytes in peripheral zone formed pseudolobule similarities.The structures of reticular fibers in necrosis zones had collapsed.(b)There were hepatocellular bridged necrosis and hemorrhage.In centrilobular zone there were some proliferated bile canaliculi,bile embolism and bile pigmentation.In portal area there was cholestasis.The structures of reticular fibers in necrosis zones had collapsed.(c)There were hepatocellular vacuolar degeneration and hepatocellular punctiform necrosis in liver.(d)There were hepatocellular piecemeal necrosis in portal area where inflammatory cells splashed out, and some councilman bodies around the perimeter of hepatic lobules.In light microscope,liver tissues of Group OAT and Group YC showed:(a)There were hepatocellular focal necrosis,where structures of reticular fibers had collapsed.(b) There were hepatocellular piecemeal necrosis in portal area where inflammatory cells splashed out.(c)There were fatty degenerations occurred in most areas of liver.In light microscope,liver tissues of Group YC+O₃ showed that there were a few hepatocellular punctiform necrosis and fatty degenerations in livers.DiscussionAccording to the results of this study,effects of Yigan Fuzheng Paidu Capsule combined with medical ozone on acute hepatic injury dogs were manifested as followings:(a)They can decrease the incidence rates of jaundice and hepatic encephalopathy,promote the recovery of mental state and diet,and improve the quality of life.(b)They can prolong survival time of canine models.(c)They can decrease ALT,TBIL,PT,AMMO values of canine models which reflected its effects of decreasing transaminase,relieving jaundice and promoting the recovery of hepatic functions.Compared with OAT,the effects of YC's decreasing transaminase, relieving jaundice and promoting blood clotting functional recovery is better.Although Group YC was significantly(P＜0.05)higher than Group OAT in ALT value,there were no significant(P＞0.05)differences between them in TBIL,PT and AMMO values. Therefore,it is proper to get the conclusion that OAT is better in decreasing transaminase than YC.But their total curative effects have no significance. Comparing AST/ALT values of four groups,it showed the single administration of YC or OAT could alleviate the hepatic injuries and promote the recovery of acute hepatitis, while combined administration could protect livers and significantly reduce the degrees of liver injuries.(d)Hepatic histopathology results showed the liver necrosis were much serious in dogs of group Model with the administration of nothing,even though in those living for 15 days or more.But livers of dogs with single administration of YC or OAT have fewer scales of necrosis than that of Group Model.However,there were only a few hepatic punctiform necrosis,mild degeneration and a few infiltrations of inflammatory cells in dogs with combined administration.In conclusion,Yigan Fuzheng Paidu Capsules combined with medical ozone was able to decrease transaminase and blood ammonia levels,relieve jaundice,prolong the live time of CCl₄-induced hepatic injury dogs,and exert a protective effect on the liver injury.