The Study On The Therapeutic Effects Of The Pueraria Anti-inebriation Solution And Puerarin On The Alcoholic Liver Disease In Rats

Objective: The study aims to establish alcoholic liver disease model rat, to discuss new insight on the pathogenesis of alcoholic liver diseases and to probe into the therapeutic effects of the Pueraria & puerarin on the alcoholic liver injury in rat.Methods: 108 SD rats, that the numbers of male and female rats were equal, 180～220gweight, were randomly divided into 5 groups. Pueraria anti-inebriation solution (self-made) and puerarin injection (shandong ruiyang biopharmaceutical limited company) were used in the treatment groups.Group A (the model matched control group):36 rats were infused and taken sterile survival saline infusion 10ml/kg.day in gastric that continuedl2w. Every 12 rats were sacrificed at the end of the 4th, 8th and 12th week. The parameters were also determined.Group B (model group) 36 rats, in gastric, were infused and taken 56% ethanol infusion 10ml/kg.day. Every 12 rats were sacrificed at the end of the 4th, 8th and 12th week. The parameters were also determined.Group C (Pueraria anti-inebriation solution treated group) 12 rats, in gastric, were infused and taken 56% ethanol infusion 10ml/kg.day till 12w. From 9thw, rats were treated or injected with Pueraria anti-inebriation solution in peritoneal cavity; at the end of 12th week, all the rats were sacrificed and the parameters were also determinedGroup D (puerarin treated group) 12 rats, in gastric, were infused and taken 56% ethanol infusion 10ml/kg.day till 12w. From 9thw, rats were treated or injected with the puerarin in peritoneal cavity; at the end of 12th week, all the rats were sacrificed and the parameters were also determined.Group E (the treatment control group) 12 rats, in gastric, were infused and taken 56% ethanol infusion 10ml/kg.day till 12w. From 9thw, rats were injected with sterile survival saline in peritoneal cavity; at the end of 12th week, all the rats were sacrificed and the parameters were also determined.ALT, AST, superoxide dismutase (SOD) activity, trace amount of iron & malonaldehyde (MDA) content were determined in serum of rats. SOD activity and MDA content were determined in hepatic tissue. RT-PCR was employed to check the expression of hepcidin in liver. The same part of each liver was fixed respectively in 4% paraformaldehyde for 24 hours, then processed by standard histological procedures, embedded in paraffin, cut into 5 urn pieces and mounted on the slide. The samples were stained with H.E for histopathological examination and immunohistochemical staining, and another same part of each liver was operated by frozen section and cut into 20μm pieces and mounted on the slide. The samples were stained with SudanⅣfor histopathological examination, and then slides were observed with optical microscope.Results:(1) General information of rats103 rats of the total 108 rats were survivors during the experimental period. There were 2 rats died during the gastric perfusion, and the reason was that the tube went through the wrong way. There were 3 rats died because of the deficiency of alcohol resistant capacity. All the rats from the group A finished the test completely. During the test, compared with the group A rats, the rats from group B and E had worse mental state, clumsy reaction, the times of movement reduced. With the increase of in gastric, 56% ethanol infusion done, food intake of the group B and E gradually decreased with the diarrhea, meanwhile their coat lost the gloss. The body weight gains of the group B and E rats were less than those of the group A (P＜0.01), and the liver index number were more than those of the group A. During the treatment test(from 9W to 12W), the rats from group C and D had better the mental state, the times of movement were more than group B and E. The body weight gains of the group C and D rats were more than those of the group B and E (P＜0.01), and the liver index number were more than those of the group A(P＜0.01). Group C compared with the Group D, The body weight gains and the liver index number of two groups rats had no significant difference(P＞0.05).(2) Determination of ALT and AST level in serumALT, AST and iron in rats serum in group B were higher than that in group A(P＜0.01);compared with group B and E, ALT, AST and iron in rats serum were lower than both in group C and group D; group C and group D had no significant difference of ALT,AST and iron in rats serum (P＞0.05).(3) Determination of SOD activity, MDA level in serumCompared with group A, SOD activity in group B was significant decreased (P＜0.01); MDA level was significant increased in group B (P＜0.01). Compared with group B, SOD activity was significant increased (P＜0.05) and MDA level was significant decreased (P＜0.01) in both group C and group D. No significant differences in SOD activity and MDA Level were observed between group C and group D(P＞0.05).(4) Determination of SOD activity and MDA Level in hepatic tissue Compared with group A, SOD activity was significant decreased in group B (P＜0.01),MDA level was significant increased in group B (P＜0.01). Compared with group B, SOD activity was significant increased (P＜0.05) and MDA Level was significant decreased (P＜0.01) in both Group C and Group D. No significant differences in SOD activity and MDA level were observed between group C and group D(P＞0.05).(5) Observed result of naked eye viewing liver and the hematoxylin and eosin (H.E) staining of the liver tissue:Group A: The surface of the liver of the model matched control group rats were dark red and had the glossy appearance. The complete structures of hepatic lobule were found under low power field. Hepatocytes ranged in plates that anastomose with one another and arrayed radically around the central vein. The cells were polygonal in shapes which were quite uniform.Group B: The livers of the model group rats enlarged and had rough surface, and the color were grey-yellow. At the end of 4th weeks, H.E staining revealed light steatosis and focal cytoplasmic degeneration. One to several large fat droplets could be found in the cytoplasm. At the end of 8th weeks, H.E staining showed that there were moderate and severe steatosis and a lump of inflammatory cells. The lobular structure destroyed, and the hepatic plates were disordered and the large fat droplets could be found in the cytoplasm. At the end of 12th weeks, local hepatocellular necrosis and increasing inflammatory cells were shown by H.E staining and the large fat droplets that were almost as big as the hepatocytes could be found in the cytoplasm. The proportion of fatty degeneration was 85%, the proportion was higher than those in group A,C and D and the different was significant (P＜0.05).Group C: At the end of 12th week, the hepatomegaly degree was lower in this group, compared with group B, the livers were light-yellow. Hepatocytes were ranged in plates, several fat droplets could be found in the cytoplasm and the proportion of fatty degeneration was lower than those in group B and group E. The different was significant (P＜0.05). Group D: At the end of 12th week, the hepatomegaly degree was lower in this group. Compared with group B, the livers were light-yellow. Hepatocytes were ranged in plates. Several fat droplets could be found in the cytoplasm and the proportion of fatty degeneration was lower than those in group B and group E. The different was significant (P＜0.05).Group E: The results were similar as group B. At the end of 12th weeks, local hepatocellular necrosis and increasing inflammatory cells were shown by H.E. staining and large fat droplets that were almost as big as the hepatocytes could be found in the cytoplasm. The proportion of fatty degeneration was 90%. The proportion was higher than those in group A, C and D and the different was significant (P＜0.05).(6) The SudanⅣstaining of the liver tissue at the end of 12th weeksGroup A: The result revealed little red tangerine staining sectors could be found in the cytoplasm.Group B: The result revealed a great number of red tangerine staining sectors could be found in the cytoplasm.Group C: The result revealed many red tangerine staining sectors could be found in the cytoplasm.Group D: The result revealed many red tangerine staining sectors could be found in the cytoplasm.Group E: The result revealed a great number of red tangerine staining sectors could be found in the cytoplasm.(7) The immunohistochemistry results of TNF-a and IL-6 in liver tissueGroup A: A lot of memberane and plasmas of the cells of liver were negatively stained with target antibody--Tumor Necrosis Factor-alpha (TNF-a) and interleukin-6 (IL-6).The light yellow particles were not observed in the hepatocytes except only two rats' liver tissue in this group rats. Group B: The number of positive cells that were stained positively with the antibody and identified by the yellow-brown particles could be found in the memberane and cytoplasm and were increased at the end of 8th week than those in the 4th week. The rates of positive expression were increased at the end of the 12th week than those in the 8 week.Group C: The positive cells that were stained positively with the antibody and identified by a few of yellow-brown particles could be found in the memberane and cytoplasm.Group D: The positive cells that were stained positively with the antibody and identified by a few of yellow-brown particles could be found in the memberane and cytoplasm.Group E: The number of positive cells that were stained positive with the antibody identified by a great number of yellow-brown particles could be found in the memberane and cytoplasm.Intergroup comparison: About positive expression of TNF-α, in every group, at the end of 12th week, group B was higher than group A(P＜0.01), group C and group D (P＜0.05); group E was higher than group A (P＜0.01),group C and group D (P＜0.05). No significant differences between group C and group D(P＞0.05). About positive expression of IL-6, in every group, at the end of 12th week, group B was higher than group A, group C and group D(P＜0.01); group E was higher than group A,group C and group D (P＜0.01). No significant differences was between group C and group D(P＞0.05).(8) The results of the RT-PCRIt can be seen that theβ-actin expression of all five groups were similar (P＞0.05); mRNA levels of hepcidin in group B were significantly lower than those in the group A (P＜0.01). Compared with group B and group E, the expression of hepcidin in group C and group D were higher (P＜0.01). There is no significantly different between group C and group D (P＞0.05). Conclusions:(1) Gastric perfusion is a good method of induction of alcoholic liver injury model in rat.(2) In the liver tissue of the rats of alcoholic liver injury, the increase in expression of TNF-α, IL-6 and the decrease in the expression of hepcidin may play an important role in the course of the occurrence and development of alcoholic liver injury.(3) Pueraria anti-inebriation solution and puerarin had therapeutic effects of the on the alcoholic liver injury in rats, and no difference was between those two medicine groups in therapy.