| 1. Objective1.1 Animal models were used whose livers were chemically and immunologically damaged to study the hepatoprotective effect of Trans-Resveratrol as well as its hepatic transaminase lowering capability.1.2 To study the protective effect of Trans-Resveratrol on H2O2 induced hepatic injury in order to develop and provide new ideas and methods by which to screen Chinese medicinal herbs for HBV.1.3 To observe the immunomodulating function of Trans-Resveratrol and analyze its mechanism.1.4 To study resveratrol inside DHBV function.2. Method2.1 Studying the effects of Trans-Resveratrol on chemically induced hepatic injury2.1.1 60 mice were randomly divided into five groups: A) normal or healthy mice, B) mice model, C) large herbal dose, D) small herbal dose and E) the positive group.The groups of mice that were administered herbs was given doses at 30mg/kg, 15 mg/kg, and 7.5 mg/kg. The positive control group was fed 150mg/kg of bifendate. The healthy and model groups were fed equal amounts of saline water. The above doses were administered for 7 days. On the 6th day, the model group and the herbal groups were given 0.5 g/kg D-Gal by abdominal injection. The healthy group was given an identical dose of salinesolution by abdominal injection. 24 hours after the dose of D-Gal the eye was excised andblood removed. Colorimetric method was used to measure the activity of AST, ALT, blood serum, SOD activity as well as MDA water content.2.1.2 The mouse model with Bacille Calmette-Guerin (BCG) and lipopolysaccaride (LPS)induced hepatic injury was divided into a blank control group (A), a model group (B), a bifendate group (C), a Trans-Resveratrol high dosage group (D), medium dosage group (E) and low dosage group (F). The blank control group was injected with saline solution through the tail vein. Injections were given at identical times as the model mice. Beginning on the day that the experimental model was created, the blank control group and the model control group were each fed 0.2ml/mouse/time of saline solution. The C group were fed 150mg/kg/time of bifendate, and the D, E, and F groups were fed large, medium and low doses of Trans-Resveratrol, respectively. All groups were fed once a day for 10 days after which Lai method was used to analyze ALT and AST levels. After the end of the experiment, tissue biopsy slices from identical regions of the liver were obtained, observed under light microscope and photographed.2.1.3 The mice model with BCG and LPS induced hepatic injury were divided into the (A) blank control group, the (B) model control group, the (C) bifendate group and the (D) high, (E) medium and (F) low dose of Trans-Resveratrol groups. The blank group was not subjected to the model and was injected with saline solution via tail vein at the identical times as the injections given to the other mice groups. Each group began feeding on the day of the model creation. The blank control group and the model control group were each fed saline solution (0.2ml/mouse/time). The C group were fed bifendate at 150mg/kg/time. The D, E, and F. groups were fed large, medium and low doses of Trans-Resveratrol equivalent to 20g/kg/d, 10g/kg/d, and 5g/kg/d of raw herb. Each group was fed once a day for a total of 10 days. ALT and AST was measured by the Lai's method. Preserved liver tissue was pureed after which the levels of SOD, MDA and GSH were measured using NO levels were measured by liver tissue.2.1.4 The animals were randomly divided into 6 groups of 10 animals each. The groups were separated into the healthy control group, model group, bifendate group and the Trans-Resveratrol high, medium and low dose groups. Except for the healthy group, the other mice were injected with ConA20mg/kg via tail vein. Subsequently, mice were fed medicine orally twice on the first day (morning and afternoon) and in the afternoon of the second day. 4-6 hours after the last dose, except for the healthy group, each of the groups was injected once with ConA20mg/kg via tail vein. After 12 hours, blood was obtained from the eye and the animals were sacrificed by decapitation. Blood centrifuged to get serum and stored for later analysis and the organs were preserved in formaldehyde solution.2.1.5 NIH rats were selected and sacrificed after obtaining blood from the eye. Livers were quickly removed in a sterile environment and washed clean of blood. They were then placed on a 200 stainless steel mesh and lightly ground using a glass injection bottle afterwhich the hepatic cell fluid was collected and centrifuged at 1000rpm X 5min and 4℃. They were washed twice with cold Hanks water and then suspended in the Hanks fluid adjusting the cell concentration to approximately 4.5 × 108~5.5 × 108 L-1. This research project counted the viable cells of liver cell suspension after dyeing with trypanblue. With viable cells measuring >90%, 1.5ml of hepatic cell suspension was placed in sterile glass test tubes. A series of Trans-Resveratrol concentrations (0.05, 0.1, 0.5, 1, 2, 4 mmol· L-1) were added and incubated for 1 hour at 37℃ in a warm water container . Then 2 mmol·L-1 H2O2 was added. An equal volume of saline solution was added to the blank control group. Incubation resumed for 30 minutes after which the following markers from each cell suspension tube were measured: ALT activity, NO amounts, NOS activity (measurements were performed according to the kit's instructions), SOD activity (pyrogallol uuto-oxidion method), MDA levels (TBA colorimetric method), and GSH levels (modified Hafeman method in which an enzyme activity unit was measured by the amout every mg of protein reduced GSH concentration lumol each minute). Protein content in the hepatic cell suspension was measured using the Lowry method.3.Results3.1 Research on Trans-Resveratrol's immunomodulatory effect on mice3.1. Trans-Resveratrol's effect on the function of the macrophage cells in the abdominalcavity of mice20-25g NIH white mice were randomly divided into 4 groups: the experimental controlgroup was fed 0.2ml/mouse/day of distilled water. There were three Trans-Resveratrolgroups distinguished by doses of 20mg/kg, 15mg/kg and 7.5mg/kg. Each group was givenan herbal dose of 0.2ml/mouse/day. All groups were fed a total of 7 days. Except for thenormal group, 1 hour after the last dose was administered, the other three groups wereinjected with 0.1 ml/10g of India ink diluted with saline solution (at a ratio of 1:5) into thetail vein. 2 and 10 minutes later, 20μl blood was acquired from the vein behind the eyesocket. The blood sample was added to 2 ml of 0.1%Na2CO3 and mixed thoroughly. Assayopitical density(OD) in 600nm by 752 type ultraviolet spectrophotometer. The mice weredissected and their livers and spleens were weighed. K values of clearance index and the avalue of the liver and spleen phagocytic system calculated.3..2 The effect of Trans-Resveratrol on circulatory antibodies caused by chicken RBC20-25g NIH small mice were randomly divided into 4 groups. The experimental control group was fed 0.2ml/mice/day of saline solution. Three Trans-Resveratrol groups were fed 0.2ml/mice 30mg/kg, 15mg/kg and 7.5mg/kg, respectively. All groups were fedfor a total of 7 days. On days 2, 4, and 6 each mouse was abdominally injected once with 50mg/10ml/kg of cyclophosphamide. The blank control group was injected with equal volumes on saline solution. On day 3, every mouse was injected with a 0.2ml solution of 5% suspension of chicken RBC in saline solution to initiate immunity. 5 days after immunity, blood was obtained from the eye and centrifuged. The serum was diluted with 100x the amount of saline solution. 1ml of the diluted serum was mixed with 5% chicken RBC solution (0.5ml) and 10% complement (0.5ml). After 30 minutes incubation at 37℃ the reaction was stopped by placing in a 0℃ refrigerator. Supernatant was obtained after centrifuge and subjected to 540ηm of 721 type ultraviolet spectrophotometer.The green light density was measured(OD). A blank control group without blood serum was set for which the colorimetry of the supernatant was calibrated to 0. The Light Density Meter(OD) reading acted as the marker for the blood serum hemolysin. Differences between the groups were compared.3.3varying degree if resistance to the mice with cyclophosphamide inhibited immune systems and were able to restore the number of phagocytes and the phagocytic system function in immunocompromised mice.3.4 Cyclophosphamide is able to significantly reduce the serum hemolysin antibody generation (P<0.01) in mice indicating that the model was successful. High doses of Trans-Resveratrol showed a positive trend in these immunocompromised mice. The other two herbal groups had weaker reactions with lesser statistical difference. Levamisole showed a more significant effect (P<0.01).3.5The hepatoprotective effect of Trans-Resveratrol on hydrogen peroxide injured mice liver cells in vitro.Trans-Resveratrol has an obvious antioxidating effect on free radicals on H2O2 injured hepatic cells. It is able to enhance hepatic cellular SOD and NO activity, reduce GSH consumption, lower MDA levels, inhibit NOS activity, and reduce NO production and oxidizing effect. As such it reduced the hepatic damage caused by oxidization and significantly lowered ALT levels in the hepatic cell suspension. This indicates that Trans-Resveratrol has a definite hepatoprotective effect on H2O2 induced liver injury. Our results also show that Trans-Resveratrol's anti-oxidant effect is dose dependant with concentrations ranging from 0.05-2 mmol ·L-1. At concentrations of 4 mmol·L-1, Trans-Resveratrol exhibited a certain degree of inhibitory effect on the hepatic intracellular transaminase system. Simultaneously it showed a trend to increase the ALT levels in the hepatic cell suspension. At the dosages used in this research project, Trans-Resveratrol showed no apparent damage to hepatic cells. However, at dosages of 4 mmol·L-1, showed...
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