Effect Of Xiao-Yu-Hua-Tan-Yin On Hepatic Tissue PPARαmRAN Of Nonalchoholic-Fatty Liver Rat

Objective: Xiao-Yu-Hua-Tan-Yin (It was composed of danshen, yujin, zexie, zhibanxia, chaihu, juemingzi, shenghuangqi, chaobaizhu medicine etc.) has the function of regulating liver and removing stasis and phlegm, strengthening spleen. And it has been proved significantly effective in treating nonalcoholic fatty liver disease (NAFLD) through years of clinical observation. In order to futher probe its action mechanism, we duplicated the NAFLD animal model with high-fat forage. At the same time, the animal was interfered with medicine. And to explore the action mechanism of this formula in regulating lipid metabolism, protecting liver function and enhancing the expression of PPARαmRNA in liver, we observed it's effect on lipid metabolism and expression of PPARαmRNA in liver. Futhermore, we made observation with light microscope and electron microscope on effect of this formula on pathomorphology of liver tissue.MethodsPartⅠEffect of Xiao-Yu-Hua-Tan-Yin on Lipid metabolism & Pathomorphology of Nonalchoholic-Fatty Liver RatSelect male SD rats 50, weight from 160g to 180g. Rats drunk water and took food freely, and were raised in sanitary experiment lab that temperature was 18℃～22℃, bright and dim alternate every 12 hours. After 1 week, they were divided into 5 groups at random: Normal control group, Model control group, High-dose of Xiao-Yu-Hua-Tan-Yin group, Low-dose of Xiao-Yu-Hua-Tan-Yin group, Dong-Bao-Gan-Tai control group. Rats except for normal control group are treated as well as set up models. Except for the ordinary forage in normal group, the rest of groups were all fed with high-fat forage. Meanwhile control drug or treatment drug was given in each treatment group and Saline was given to normal and model group, which lasted for 8 weeks of stomach-perfusion, every time the drug volume was calculated by 1ml/100g. Confirmed the formation of fatty liver, then 12 hours after fasting of the last time, the rats were anaesthetized and their blood were drawn out, the blood sample was made centrifugation with low temperature and the blood serum was isolated. Then unfold abdominal cavity quickly to get liver, finally the contents of TC, TG and FFA were measured with enzyme method; the contents of AST and ALT were measured with Lai's method. In addition, to obtain 0.1×0.1×0.1cm~3 hepatic tissue at the 0.5cm borderline of the max leaf of the liver, soak in 4% Glutaric Dialdehyde, affix to provide for the observation of the light microscope. To obtain 0.5×0.5×0.5cm~3 hepatic tissue at the same genetic locus, soak in 4% paraform, affix, paraffin imbedding, microtome section, HE dyeing to provide for the observation of the electron microscope. In the end, the rest of the max leaf of the liver was made into 10% liver tissue homogenate and then the supernatant fluid was drawn out for measurement of contents of TC, TG with color compared method.PartⅡEffect of Xiao-Yu-Hua-Tan-Yin on Hepatic Tissue PPARαmRAN of Nonalchoholic-Fatty Liver RatThe group distribution and drug-given were the same as above. At the 9th week since the experiment formally started, when confirmed the formation of fatty liver, the rats were killed 12 hours after fasting of the last time. The rats were anaesthetized, then unfold abdominal cavity quickly to get liver to obtain a little hepatic tissue at the 0.5cm borderline of the max leaf of the liver, liquid nitrogen frozen, to detect the expression of PPARαmRAN in liver through ways of RT-PC.ResultPartⅠEffect of Xiao-Yu-Hua-Tan-Yin on Lipid metabolism & Pathomorphology of Nonalchoholic-Fatty Liver Rat1 Effect of Xiao-Yu-Hua-Tan-Yin on contents of TC, TG,FFA of Nonalchoholic-fatty liver disease ratThe contents of TG, TC, FFA (1.17±0.096,3.27±0.18, 480.475±18.94) in serum of model rats were significantly increased than normal group (0.56±0.084,1.45±0.20,225.71±15.98) (P < 0.01), which showed that NAFLD rats had significant blood-fat metabolic disorder. After the intervention of medicine, the contents of TG, TC, FFA in serum of each treatment group were significantly reduced. There was significant difference between treatment group and model group (P<0.01). And the contents of TG, TC, FFA in high-dose group (0.52±0.065,1.44±0.11,221.43±15.32) and low-dose group (0.57±0.059,1.56±0.13,241.42±16.96) were obviously reduced than the control group (0.76±0.070,2.06±0.28,331.90±16.32) (P<0.01). There was no marked difference between the high-dose and low-dose group(P>0.05).2 Effect of Xiao-Yu-Hua-Tan-Yin on contents of ALT and AST in serum of Nonalchoholic-fatty liver disease ratThe contents of AST and ALT in the rats'serum of model group (32.37±2.45,15.84±1.01) were markedly increased than the normal group (26.59±2.33,10.89±0.86) (P<0.01). After the intervention of medicine, the contents of AST and ALT in the rats'serum of each treatment group were all obviously reduced, and had significant difference with model group (P<0.01). The AST contents in the rats'serum of low-dose group (19.45±1.90) was obviously reduced than the control group (23.19±1.78) (P<0.05 ); The ALT contents in the rats'serum of High-dose group (4.16±0.89)was obviously reduced than the control group (7.32±0.45) (P<0.05); There was no marked difference betwe- en the high-dose and low-dose group(P>0.05).3 Effect of Xiao-Yu-Hua-Tan-Yin on contents of TC, TG in liver tissue of Nonalchoholic-fatty liver disease ratThe contents of TG and TC in liver tissue of model rats (0.82±0.08,0.44±0.07) were obviously increased than normal group (0.45±0.05,0.27±0.04) (P<0.01), which showed that NAFLD rats had significant blood-fat metabolic disorder in liver tissue. After the intervention of medicine, low-dose group, high-dose group and positive drug control group could all significantly reduced the contents of TC, TG in liver tissue and all had significant difference with model group (P<0.01). And the contents of TC in liver tissue of high-dose group (0.27±0.04) were obviously reduced than control group (0.34±0.05) (P<0.05);There was no marked difference between the high-dose and low-dose group at the contents of TC, TG (P>0.05).4 Effect of Xiao-Yu-Hua-Tan-Yin on liver tissue morphology of Nonalchoholic-fatty liver disease ratIt was clear that by photo scope: In the normal group, constitution of hepatic tissue was clear and Integrity, constitution of hepatic lobules was normal, central veins was big and had thin paries. Hepatic cells were polygon which were arranged hepatic cord,which were distributed as radial around central veins.As for the rats in model group, they have unclear limit of hepatic lobules and disordered arrangement of hepatic cord; major part of the hepatic sinus disappeared; majority of the hepatic cells appeared severe fatty degeneration, especially in the area around the lobules; hepatic cells became extremely swollen and formed round shape; lipid droplet vacuoles (fatty degeneration) in different sizes and numbers was visible inside the cytoplasm, and sometimes the lipid droplets merged together and squeezed the nucleus to the cell side, which made it look very similar with fatty cells; the rest of the hepatic cells had focal necrosis and hepatic interstitials had no fibrosis changes; While as for the rats in high-dose and low-dose group, they had very clear structure of hepatic lobules and sinuses; the cells arranged orderly; lipid droplet vacuoles basically disappeared; the morphology and structure of nucleus became normal and regenerated hepatic cells were visible; and as for the rats in the control group, they had clear structure of hepatic lobules; hepatic sinus became mildly expanded; fatty degeneration of hepatic cells became mild; scanty lipid droplet vacuoles were still visible; the structure of nucleus was comparatively clear; Compared with the model group, the degree of fatty degeneration in each drug group got significant improvement, but the high and low-dose group were superior to the control group.It was clear that electron microscope: As for the rats in normal group, they had basically normal chondrosom and rough endoplasmic reticulum. In the model group, lipid droplet vacuoles (fatty degeneration) in different sizes and numbers was visible inside the cytoplasm, all lophos of chondrosome confluence or disappeared. Rough endoplasmic reticulum had degranulation. Intracytoplasm were full of big or small lipids droplet, Chondrosome tumescenced, part of rough endoplasmic reticulum were disrupted. As is mentioned above between the high dose and the low dose obviously improved, endochylema had small amounts lipids droplet, Chondrosome was normal, the number of lophos were mult, only portion of which lightly tumescenced or deformed, rough endoplasmic reticulum were basically normal.PartⅡEffect of Xiao-Yu-Hua-Tan-Yin on Hepatic Tissue PPARαmRAN of Nonalchoholic-Fatty Liver RatWith RT-PCR, electrophoresis band of PPARαmRNA andβ-actin were showed respectively at 239bp and 587bp,which were coincident with the respected result. The expression of PPARαmRNA of model rats (0.220±0.020) was significantly reduced than normal group (0.419±0.023) (P<0.01). After the intervention of medicine, the expression of PPARαmRNA of model group was significantly increased than treatment group. There was significant difference between treatment group and model group (P<0.01). And the expression of PPARαmRNA of high-dose group(0.341±0.020)and low-dose group(0.351±0.017) were obviously increased than the control group (0.276±0.017) (P < 0.01). There was no marked difference between the high-dose and low-dose group(P>0.05).Conclusions1 Xiao-Yu-Hua-Tan-Yin could sigmificangtly reduce the contents of TC, TG, FFA in model group, restrain the activity of ALT, AST in serum. It had marked function in regulating blood fat and protecting liver.2 Xiao-Yu-Hua-Tan-Yin could obviously improve the liver tissue lesion degree of Nonalchoholic-fatty liver disease, it manifested like this: after the drug was given, the hepatic lobule and sinus got clear structure; the cells arranged orderly, no obvious lipid droplet vacuoles inside the hepatic cells; only scanty hepatic cells appeared mild swollen, chondrosom were basically normal, and the amounts of lophos were increased.3 Xiao-Yu-Hua-Tan-Yin which could promote conveying of lipid and oxygenolysis of fat, could enhance the expression of PPARαmRNA in hepatic tissue. At the same time it could reduce the deposite in liver of lipid. In a word, it has the function of treating NAFLD.