The Effect Of Alcoholic Liver Injury In Rats And Mechanism Of Aplysia Prime Improved

Objective Aplysin, belonging to one of the bromine-sesquiterpene compounds and mainly extracted from red alga Laurencia tristicha, had attracted much attention due to its potent biological activities, including anti-bacteria, anti-inflammatory, anti-tumor, immunopotentiation and antixdiant. This study intended to explore the hepatic protective effects of Aplysin against alcohol-induced liver injury in rats through regulating ethanol metabolic enzyme system, antioxidant capacity, DNA damage and repair, hepatocyte apoptosis, oxidative/nitrative stress, mitochondrial function and endogenous apoptotic signaling pathways. Thus, the potential of liver injury preventive activity and possibly related mechanism of Aplysin will be illustrated in this paper.Methods1. Animal grouping and Model building:there were100healthy male Wistar rats aged two months, weighing180-220g, which were randomly divided into five equal groups with20in each by weight:control group, normal diet with normal saline; alcohol-model group, normal diet with alcohol administration; and three low-, medium-, and high-dose Aplysin plus alcohol treatment groups. Except the control group, all the alcohol-treated group were initially given with50%(v/v) alcohol8ml·kg-1·day-1for two weeks by intragastric administration. Then the dose of alcohol would be increased to12ml·kg-1·day-1for the remaining four weeks. In addition, three low-, medium-, and high-dose Aplysin groups were respectively given Aplysin50,100,150mg·kg-1·day-1for entire six weeks by intragastric administration. Control group would be given normal saline with the same volume for whole six weeks by intragastric administration. The rats were weighed and anesthetized with7%chloral hydrate at the twelve hours after the last treatment. Then blood samples were collected by aorta ventralis puncture to determine biochemical parameters. Liver tissue was rapidly dissected, then cut and fixed in formaldehyde saline (10%) solution for histopathological analysis and liver index. Mitochondria and microsome were extracted from isolated erythrocyte membranes.2. Pathological evaluation of liver:Pathological changes of liver tissue were evaluated by hematoxylin-eosin (H-E) staining; the changes of hepatocellular ultrastructure were observed by using transmission electron microscopy.3. Evaluation of hepatic function and level of lipid metabolism:serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activities were measured by using Yoriuji method; serum alkaline phosphatase (ALP) activity was tested by using enzyme linked immunosorbent assay (ELISA); serum level of total cholesterol (TC) was measured by using COD-PAP method; serum and liver tissue level of triglyceride (TG) was tested by using GPO-PAP method. 4. Evaluation of the extent of nitrative stress:serum nitric oxide synthase (NOS) activity was tested by using chemical colorimetry; protein expression of iNOS was measured by using western blotting.5. Measurement of hepatic ethanol metabolic enzymes activity:prepared10%liver homogenate was used to test the activity of alcohol dehydrogenase (ADH); microsome was prepared by using differential centrifugation Ca2+precipitation method; the activity of CYP2E1in liver microsome was measured by using p-nitrophenol hydroxylase (PNPH) method.6. Evaluation of the extent of DNA oxidative damage:an in situ Ⅳ collagenase two-step perfusion method were applied to isolate hepatocytes which were used to prepare liver cell suspension; the extent of DNA oxidative damage was measured by using the alkaline comet assay (single cell gel electrophoresis, SCGE); plasma level of8-OHdG was determined by using an8-hydroxy-desoxyguanosine (8-OHdG) ELISA kit.7. Analysis of comprehensive antioxidant abilities:cytoplasmic redox state ([NAD+]/[NADH] ratio) was estimated from the corresponding [lactate]/[pyruvate](L/P) ratio and the equation of the chemical equilibrium; the fluidity of erythrocyte membrane was performed by the fluorescence polarization technique with1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescent probe; the concentration of lipid peroxide (LPO) was determined by using lipid peroxidation assay kit; the concentration of malondialdehyde (MDA) in plasma and liver tissue was measured by using thiobarbituric acid (TBA); the activities of serous superoxide dismutase (SOD) was tested by using xanthine oxidase method; the activities of plasmic glutathione peroxidase (GSH-Px) was determined by using DTNB method; the activity of hepatic catalase (CAT) in liver tissue was tested by using visible spectrophotometry method.8. Evaluation of mitochondrial function:mitochondria were prepared by using differential centrifugation method and were used to determine the activity of Mn-SOD and the concentration of GSH in mitochondrial suspension; colorimetric method was utilized to measure the activity of mitochondrial respiratory chain complex (MRC).9. Evaluation of hepatocyte apoptosis:the extent of hepatocyte apoptosis was determined by using Annexin V-FITC/PI Apoptosis Detection Kit.10. Western blotting technology was utilized to determine the changes of protein expression of iNOS, CYP2E1and critical protein of mitochondria-induced endogenous apoptosis pathway in liver tissue, such as Bcl-2, Bax, cytochrome c and caspase-3.11. Total RNA was isolated from liver tissues using TRIzol reagent and cDNA was prepared via reverse transcription. Then the real-time PCR (qPCR) method was utilized to determine the changes of mRNA expression of CYP2E1and endogenous apoptosis related genes in liver tissue, such as Bcl-2, Bax, cytochrome c, caspase-9and caspase-3.Results 1. Evaluation of protective effect of Aplysin improving alcohol-induced liver damageCompared with normal group, weekly-weight in alcohol-model group was slightly decreased and liver index was siginificantly increased (P<0.05), compared with alcohol-model group, weekly-weight in three low-, medium-, and high-dose Aplysin groups all went up, but liver index among medium-, and high-dose Aplysin groups was significantly decreased (P<0.05). The results from HE staining pathological observation revealed that hepatic steatosis in three low-, medium-, and high-dose Aplysin groups was obviously improved, such as reducing of inflammatory cell infiltration. In addition, compared with alcohol-model group, hepatic cords arranged orderly and hepatocyte morphology was normal in medium-and high-dose Aplysin groups. Through the observation of transmission electron microscope, among medium-and high-dose Aplysin groups, the number of lipid droplets within cytoplasm was decreased; mitochondrial pathological changes was significantly alleviated in the liver and the number of mitochondria was obviously increased; degeneration of rough endoplasmic reticulum and disorder arrangement were both improved. In this study, serum activities of ALT, AST and ALP in alcohol-model group were all prominently increased (P<0.05), but Aplysin could effectively inhibit the increasing of serum activities of ALT, AST and ALP. In addition, long-term massive alcohol-treatment could disorder lipid metabolism via intragastric administration and make the level of TC, TG within serum and liver tissue increase. Aplysin-treatment successfully inhibited these changes caused by alcohol and seemed to have a positive moderating effect of blood lipid.2. The effect of Aplysin against alcohol-exposed nitrative stress and ethanol metabolic enzymes in ratsCompared with the normal group, serum activities of TNOS and iNOS in alcohol-model group was significantly increased, the concentration of NO went up, and liver activity of ADH and liver microsome activity of CYP2E1was increased (P<0.05); compared with alcohol-model group, serum activities of TNOS, iNOS and NO within three low-, medium-, and high-dose Aplysin groups were all decreased and had dose-dependent effect. However, activity of ADH and CYP2E1was only inhibited in high-dose Aplysin group. In addition, medium-, and high-dose Aplysin group could obviously inhibit protein expression of iNOS in liver tissue (P<0.05); intaking of Aplysin significantly reduce the protein or mRNA expression of CYP2E1.3. The effect of Aplysin against alcohol-exposed liver oxidative damage and oxidative stressCompared with the normal group, cytoplasm NAD+/NADH ratio and the fluidity of erythrocyte membrane in alcohol-model group were significantly decreased (P<0.05); Aplysin-treatment groups could inhibit the changes of NAD"/NADH and the fluidity of erythrocyte membrane caused by alcohol, especially in medium-, and high-dose Aplysin groups. Aplysin-treatment could significantly relieve the increasing of plasma8-OHdG induced by alcohol. On the other hand, the results of comet assay test indicated that the extent of DNA damage isolated from liver was significantly attenuated, for example, tail DNA percent, tail length, tail moment and olive tail moment were all significantly decreased compared with alcohol-model group(P<0.05) Concentration of LPO and MDA in serum or liver was both obviously increased. In contrast, concentration of LPO and MDA in all Aplysin-treatment groups significantly went down. In addition, Aplysin-treatment groups could obviously inhibit the decreasing of GSH and recover the activity of SOD, GSH-Px and CAT.4. The role of mitochondria-mediated endogenous apoptosis pathway for alcohol-induced liver damage interfered by AplysinAccording to the measurement of Annexin V-FITC/PI method by using fluorescence microscope, the number of liver apoptosis was significantly increased, and most of them were the late stage apoptotic cells. In contrast, the number of liver apoptosis in Aplysin-treatment groups was obviously decreased, and most of them were the early stage apoptotic cells. During the test of activity of mitochondrial respiratory chain complex (MRC), it could be seen that Aplysin-treatment reverse the decreasing of activities of MRC I, III and IV resulted from alcohol. Compare with the alcohol-model group, the differences had significance (P<0.05). In addition, the results of Western blotting demonstrated that Aplysin could up-regulate protein expression of Bcl-2, down-regulate expression of Bax, reduce the release of cytochrome C, and inhibit the activation of caspase-3and caspase-9. The results of real-time PCR indicated that mRNA expression of Bax, cytochrome C, caspase-3, caspase-9in the liver was gradually decreased with the development of dose of Aplysin, compared with alcohol-model group. But mRNA expression of Bcl-2was gradually increased (P<0.05).Conclusion1.Based on the results of liver index, observation of hepatic histopathology, the evaluation of related enzymes of liver function, and measurement of lipid metabolism, it was initially proved that Aplysin had significantly protective effect on the improvement of alcohol-induced liver damage in rats.2.The mechanism of hepatic protective effect of Aplysin against alcohol-induced liver damage in rats could be relevant with some factors, such as:1. Inhibit the activity of iNOS, down-regulate protein expression of iNOS, reduce the formation of NO;2. Inhibit the activity of ADH in liver and the activity of CYP2E1in microsome, down-regulate protein or mRNA expression of CYP2E1. But whether Aplysin would effectively prevent alcohol-induced liver damage, as one of drugs which had inhibiting effect against iNOS and regulate ethanol metabolic enzymes in liver, still needed more and further experiments to confirm. 3.Supplement of Aplysin could enhance the ability of antioxidant to resist alcohol-induced oxidative stress, and alleviate lipid peroxidation and DNA oxidative damage. This could be one of the mechanisms of Aplysin against alcohol-induced liver damage.4.Overdose of intaking alcohol could lead to the liver apoptosis, but Aplysin would protect alcohol-induced liver damage, through regulating protein or mRNA expression of Bcl-2family, inhibiting the release of cytochrome C, inhibiting the activation of caspase-3, reducing the mRNA expression of cytochrome C, caspase-9and caspase-3, inhibiting the activation of mitochondria-mediated apopsis pathway.