Exploration Of The Relationship Between The Nonalcoholic Fatty Liver Disease In Rats And The Correlation Of Dampness And Blood Stasis Based On The OATP2a1 And OATP2b1

Objectives:The objectives of this Master project include: a)to establish a nonalcoholic fatty liver rat model and characterize the changes of the mRNA and protein expression of OATP2a1 and OATP2b1 in nonalcoholic-fatty liver in rats,in order to explore the relationship between the nonalcoholic fatty liver and dampness;b)to investigate whether there is dampness together with blood stasis in the rats with nonalcoholic fatty livers by observing the hemorheological parameters;c)to experimentally validate whether Jianpi Huoxue being helpful to strengthen spleen,ooze and promote blood circulation will affect OATP2a1,OATP2b1,and the hemorheological parameters of the nonalcoholic-fatty model rats in order to further verify the relationship between the nonalcoholic fatty liver and dampness as well as the blood stasis.It is expected to provide a theoretical and experimental foundation to the clinical formation of more reasonable and effective Chinese medicine treatment to some extent.Methodologies:The study has been carried out through the following methodologies:1.Selection and Grouping : Eighty male sprague-dawley(SD)rats were selected in the study,and they were randomly divided into two groups: 20 in the normal group and 60 in the model group.After eight weeks,20 rats from the two groups(10 each)were killed.The rest of the model group were further grouped into: 10 in the positive comparison group,10 in the Jianpi Huoxue low-dose group,10 in the Jianpi Huoxue medium-dose group,and 10 in the Jianpi Huoxue high-dose group.2.Model Preparation : The normal group was fed with ordinary feed,and the other groups were fed with high-fat feed(ordinary feed with 10% lard and 2% cholesterol.The feeding time was 12 weeks with normal drinking water for all.3.Group Drug-Feeding : At the end of the eighth week,the normal group and the model group were fed by normal saline,while the positive comparison group,and the low-,medium-,and high-dose groups were fed by simvastatin and Jianpi Huoxue with different levels of concentration.Once a day for four weeks.4.Indicator Detection : Using the hepatic tissue paraffin section hematoxylin-eosin(HE)staining(light microscopy),frozen section Sudan III staining(light microscopy)to observe pathological changes.Basic detection: cholesterol(TC),triglyceride(TG),ALT,and AST.Full hemorheological detection: full-blood viscosity(low cut,high cut),plasma viscosity,aggregation index of the red blood cells,full-blood viscosity(1s,60 s,200 s),and fibrinogen.The relative protein expression levels of mRNA and protein in OATP2a1 and OATP2b1 in liver tissues.Results:1.Modeling of the nonalcoholic fatty liver rats induced by high-fat feed At the end of the eighth week,compared with the normal group,the blood lipid(serum cholesterol,serum triglyceride)and liver function(ALT,AST)of the model group rats increased,and the results were statistically significant(P < 0.05).Observations from the hepatic tissue paraffin section HE staining showed that fat vacuoles appeared in the cytoplasm of hepatic parenchyma cells and certain vacuoles merged,which pushed the nucleus to the edge of the cell.It was found in the observations from the frozen section Sudan III staining that the rats had orange-red fat-drops in the cytoplasm of hepatocytes.2.High-fat feed-induced nonalcoholic fatty liver rats and dampness indicators2.1General observation:Compared with the normal group,the model group were of significant obesity,becoming obsessing in huddling and lying down with less movements.Moreover,they had thinning stool(anal was not clean).2.2Protein expression changes of the OATP2a1 and OATP2b1 mRNA:Compared with the normal group,the relative expression levels of the OATP2a1 and OATP2b1 mRNA in the model group increased at the 8th and 12 th week.The results were statistically significant(P < 0.05).2.3Protein expression changes of the OATP2a1 and OATP2b1:Compared with the normal group,the relative expression levels of the OATP2b1 and OATP2a1 in the model group also increased at the 8th and 12 th week.The results were also of statistical significance(P < 0.05).3.Changes in the hemorheological parameters By contrast to the normal group,at the end of the 8th and 12 th week,in the model group,the full-blood viscosity(low-,high-cut),full-blood viscosity(1 s,60 s),plasma viscosity,red blood cell aggregation index,and fibrinogen increased significantly.The results were also meaningful.4.Evidence of experimental results After the intervention of drug-feeding,compared with the model group,the Jianpi Huoxue groups(medium-and high-dose)and the positive comparison group showed a lower expression level of the OATP2a1 and OATP2b1 mRNA at the end of the eighth week.The relative expression level of the OATP2a1 and OATP2b1 among all the drug-feeding groups decreased.In the Jianpi Huoxue groups(medium-and high-dose)and the positive comparison group,the full-blood viscosity(low-and high-cut)and the fibrinogen were lower.The full-blood viscosity(1 s,60 s,200 s)and the plasma viscosity also decreased in all the drug-feeding groups.Moreover,the Jianpian Huoxue high-dose group and the positive comparison group showed a lower red blood cell aggregation index.All the results had a significant statistical meaning(P < 0.05).Conclusion:The project was successfully completed.The following conclusions can be drawn from the Master project:1.The nonalcoholic fatty liver disease was successfully induced by feeding high-fat feed to the model rats for 8 weeks.The model rats showed an upward trend in the cholesterol(TC),triglyceride(TG),ALT and AST,compared with normal rats.Moreover,the hepatic tissue paraffin section hematoxylin-eosin(HE) staining and the frozen section Sudan III staining showed that the livers of rats had steatosis;2.The mRNA and protein expression level of the OATP2a1 and OATP2b1 in nonalcoholic fatty liver rats increased as well compared with the normal rats.This has indicated that the OATP2a1 and OATP2b1 may be involved in the process of dampness,which implies that the nonalcoholic fatty liver of model rats may be related to the dampness.In addition,the increase of the hemorheological index of the nonalcoholic fatty liver rats was also observed,showing that there may be blood stasis in the nonalcoholic fatty liver rats;3.Jianpi Huoxue helps to strengthen spleen,ooze and promote blood circulation.Thus,it can be used to improve the mRNA and protein expression of the OATP2b1 and OATP2a1 as well as the hemorheological parameters.This further demonstrates that the dampness and blood stasis may be closely related to the nonalcoholic fatty livers.The above investigation may provide a theoretical and experimental foundation to the clinical formation of more reasonable and effective Chinese medicine treatment.