Effect Of Jianpihuoxiequshi Prescription On The AQP8、AQP9and Hepatic Mitochondrial Of Cirrhotic Rats Complicated With Ascites

ObjectiveThe cirrhosis with aseites is a common complication of several liver diseases at decompensation stage. It will cause serious damage to human he alth while its formation. There is no "cirrhosis with aseites" record in TC M, it belongs to "drum belly"、"flank pain" in TCM. The symptomatic treatm ents is the main therapeutic method for cirrhosis with aseites, but the ef feet is not satisfying. My tutor has been treated cirrhosis with aseites with formula based on invigorating spleen and tonifying liver and spleen-mitochondria correlation theory (Jianpihuoxiequshi prescription).The pre scription has good clinical efficacy to the cirrhosis with aseites. And th e most medicines of the prescription were characteristic Lingnan Chinese herbal medicine. for years, and has achieved significant curative effect. The study aims to further prove the therapeutic effect to Jianpihuoxieq ushi prescription by experiment of rats and catch the mechanism of Ji anpihuoxiequshi prescri-pt ion.MethodsThere is two parts of the is research. In the first literature researeh part, we summarize the studies of hepatocirrhosis aseites(drum belly) and liver in TCM. Then analyze the mechanisms of how hepatocirrhosis aseites occurs and therapeutic method, and animal modeling.In the second animal experiment part we choosed90healthy specific pathogen free(SPF) Wistar rats. Selected ten rats randomly as blank group(groupA). Eighty other rats were used to preparate cirrhosis ascites model with an improved CC14combined with alcohol method. On the first day of experiment, the rats were injected with CCL4hypodermically,5mini-liter per kilogram(mL/kg) of the rats’ weight. Next we injected the rats subcutaneous with40%CCl4mixed with olive Oil. We give the rats with alcohol only for drinking. The molding for cirrhosis with ascites goes on8weeks. After it, there were65rats left. Used diagnostic peritoneocentesises for the rats,50rats were comformed with ascites. Then divided the rats into5groups(each group had10rats) and began a4week intragastric administration. The blank and model group were given normal saline with the same capacity of the herbal medicine.The Jianpihuoxiequshi prescription large dose group(group C) was given the dose of52g/kg (BW). The Jianpihuoxiequshi prescription medium dose group(group D) was given the dose of26g/kg (BW).The Jianpihuoxiequshi prescription small dose group (group E) was given the dose of16.25g/kg (BW) Given colchicine to the colchicine group with the dosage as100ug/kg (Body Weight). The intragastrical amount was10ml/kg (Body Weight). Observe the rats’ body weight, physical and mental condition, and the amount of ascites at the end of the study. The indexes(ALT、AST、ALB) of hepatic function were detected from portal vein blood of these rats. The rats’ livers’ AQP8、AQP9and mitochondria cytochrome c oxidase activity, ATP content, ATPase activity were observed. Then use the result to analyze the data.The results show that colchicine can improve the cytochrome c oxidase activity (P<0.05), but can not improve the expression of ATP content and ATPase (P>0.05). It indicate that the effectiveness of liver protection by colchicine is not based on the mechanism of regulating the energy metabolism of mito-chondria, but others.Results1. General conditions of the rats:the modeling rats’ mortality was18.75%. And the formation rate of the Ascites was76.92%.The rats of the model g roup were irritable and apathetic, loss of body weight, decrease of food, d ecrease of activity. Through4weeks’treatment with the Jianpihuoxiequshi prescription, the symptoms above were all improved.2. The condition of the ascites:At the end of the experiment, the blank group (A) has no ascites, and the model group(B) and the colchicine group(F) has much ascites which colour was light yellow. After the treatment by the Jianpihuoxiequshi prescription, the amount of ascites of each therapeutic group decreased remarkablely. The differences were significantly compared with model group (P<0.01). But compared groupB with group F, there was no significant differences(P>0.05).3. The contents of indexes of hepatic function including ALT、AST heightened obviously in model group rats (compared to groupA, P<0.01). After the treatment in groupC, D, E, F the contents of ALT、AST decreased unequally, and the contents of ALB heightened. The effectiveness in groupC was the best. Compared with groupA, there was no significant differences after the treatment in those indexes(P>0.05).But there was significant differences between each group(D, E, F)and group A after the treatment (P<0.01).4. Effect of Jian Pi Huo Xue Qu Shi prescription on the AQP8RT-PCR results show that the AQP8mRNA expression has remarkable diffe rence with the blank group(P<0.01). The marks of AQP8mRNA rise tremendousl y after treating by JianPiHuoXieQusShi decoction. There are remarkable di fference between high dosage JianPiHuoXieQusShi decoction group and the m odel group (P<0.01), and significant difference between medium dosage grou p, colchicines group and model group(P<0.05). ICH results show that the AQ P8quality of blank group has remarkable differences with the model group (P<0.01). Western Blotting results show the quality of AQP8are remarkabl e difference between high dosage JianPiHuoXieQusShi decoction group and t he model group (P<0.01). In the medium dosage group, the quality of AQP8i s little higher than the model group(P>0.05). In the low dosage group, the quality of AQP8is little lower than the model group. Significant differ ence exists between colchicines group and the model group(P<0.05). The eff ects is related to the dosage use of the TCM.5. Effect of Jian Pi Huo Xue Qu Shi prescription on the AQP9RT-PCR results show that The AQP9mRNA expression has remarkable difference with the blank group(P<0.01). The marks of AQP9mRNA rise tremendously after treating by JianPiHuoXieQusShi decoction, the effects among them in positive correlation with the dosage use of the TCM. There are remarkable difference between high dosage JianPiHuoXueQusShi decoction group and the model group (P<0.01), and significant difference between medium dosage group, colchicines group and model group(P<0.05). ICH results show that The AQP9quality of blank group has remarkable differences with the model group(P<0.01). Western Blotting results show The quality of AQP9are remarkable difference between high, medium dosage group and the model group (P<0.01). But no significant difference between low dosage group and model group (P>0.05). Significant difference exists between colchicines group and the model group(P<0.01). The effects is related to the dosage use of the TCM.6. Experiment results of Jian Pi Huo Xue Qu Shi prescription on the hepatic mitochondrial(1)The expression of cytochrome c oxidase activity of blank group has sig nificant difference with the model group(P<0.01). The marks of cytochrome c oxidase activity expression rise tremendously after treated by JianPiH uoXieQusShi decoction, the effects among them is related to the dosage use of the TCM. There are significant difference between high, medium dosage g roup and model group(P<0.01). And significant difference between low dosag e group, colchicines group and model group(P<0.01).(2)The expression of ATP content of blank group has significant difference with the model group(P<0.01). The marks of ATP content expression rise tremendously after treated by JianPiHuoXieQusShi decoction, the effects among them is related to the dosage use of the TCM. There are significant difference between high, medium dosage group and model group(P<0.01). And significant difference between low dosage group and model group (P<0.01). But no significant difference between colchicines group and model group(P>0.05).(3) The expression of ATPase activity of blank group has significant difference with the model group(P<0.01). The marks of ATPase activity expression rise tremendously after treated by JianPiHuoXieQusShi decoction, the effects among them is related to the dosage use of the TCM. There are significant difference between high, medium dosage group and model group(P<0.01). And significant difference between low dosage group and model group (P<0.01). But no significant difference between colchicines group and model group (P>0.05).Conelusions:1. From the results of the study, we found the improved CC14combined with alcohol method is a ideal method of preparation of animal hepatocirrhosis aseites models.2. Jianpihuoxiequshi prescription could improved the rats’mental status, desire for foods, body weight and so on. It also could decreased the rats’ aseites’amount.3. Jianpihuoxiequshi prescription could obviously decrease the content of the ALT, AST and increase the content of ALB which showed better effectiveness on liver protection.4. Jianpihuoxiequshi prescription can increase the expression of AQP8.5. Jianpihuoxiequshi prescription can increase the expression of AQP9.6. Jianpihuoxiequshi prescription can increase the expression of ATP conten、 ATPase activity and cytochrome c oxidase activity.7. The Jianpihuoxiequshi prescription could tonify spleen and replenish qi,activate blood and dissolve stasis, clear damp and promote diuresis, an d tonify stomach and regulate qi.With the appliance of Jianpihuoxiequshi prescription, symptoms and ascites of the cirrhotic rats complicated wit h ascites could be improved significantly. It also can improve the express ion of AQP8, AQP9and the disorder of energy metabolism of mitochondria of the rats with ascites in cirrhosis. And relieve the liver cells and mitoc hondria edema,maintain the normal function of liver cells, so as to facil itate the ascites retrogression. There maybe a certain correlation between the liver AQP8, AQP9and the liver mitochondrial energy metabolism.