| ObjectiveTo investigate the protective effect ofα-Lipoic Acid (α-LA) against acetaminophen (AAP)-induced hepatic injury and the effect of cytochrome P450 (CYP450) content and cytochrome P450 2E1(CYP2E1) activity in mice.Methods1. Establishment of in vitro free radical models: Hydroxyl free radical (?OH) was generated using H2O2/Fe2+ system via Fenton reaction. The inhibitory effect ofα-LA on lipid peroxidation was examined by use of ?OH-caused hepatic homogenate lipid peroxidation. The protective effect ofα-LA on RBC membrane was investigated utilizing ?OH-caused rupture of RBC membrane. Absorbence was measured with UV-754 spectrophotometer, and free radical scavenging percenttage, SC50 and IC50 ofα-LA was calculated.2. Establishment of in vivo mouse liver damage models: The model was established by ig administration of AAP to mice at a single dose of 500 mg/kg. In hepato-protective experiment, mice were given AAP subsequent to ig administration ofα-LA as a single dose or as a multi-dose (bid) for consecutive three days; and 12 hours later the blood and liver were removed immediately. GPT activity in serum, MDA and GSH content of liver tissue were measured. Gross pathologic changes of liver in the aforementioned model were observed with light microscope.3. Liver microsome preparation: Liver microsome suspensions were prepared by means of calcium precipitation method; Microsomal protein levels were measured by Lowry method.4. Determination of CYP450 content and CYP2E1 activity: CYP450 contents were examined using carbon monoxide reduction differential spectrophotometry; Aniline hydroxylase (ANH) activity as a specific marker of CYP2E1 enzymatic activity was spectrophotometrically measured with phenol as a coloring reagent.5. Determination of Pentobarbital sodium sleep time in mice: Mice were ip pentobarbital sodium (35 mg/kg) at 1 h after igα-LA. The time lasted from administration to the disappearance of righting reflex was recorded and regarded as latent period of sleep; and the time between the disappearance to recovery of righting reflex as sleep time.Results1. Free radical scavenging activity ofα-LA: (1)α-LA showed obvious dose-dependent ?OH scavenging activity. It was demonstrated thatα-LA has much more potency than VitC, with SC50 being 0.028mol/L and 0.049mol/L, respectively. (2) Obvious and dose-dependent inhibition of hepatocytic lipid peroxidation byα-LA was observed. IC50 ofα-LA on hepatocytic lipid peroxidation was 0.269mol/L as compared with IC50 of 0.422mol/L for VitC, indicating thatα-LA has much greater ?OH scavenging activity than VitC. (3) ?OH-caused RBC hemolysis was profoundly prevented byα-LA. IC50 ofα-LA on ?OH-caused RBC hemolysis was 0.216mmol/L, a value less than IC50 (0.358mmol/L) of VitC.α-LA should be considered to have much greater potency than VitC.2. Hepato-protective effects ofα-LA in mice: In the single dose experiment, mice pretreated withα-LA at a single ig dose of 50, 100, 200 and 400mg/kg showed a dose-dependently decreased MDA content and increased GSH of liver tissue in comparison with model group mice, with ID50 ofα-LA on increased MDA being 174.52mg/kg and ID50 ofα-LA on decreased GSH content being 190.29mg/kg; butα-LA had no effect on GPT activity in serum (P>0.05). In the multi-dose experiment,α-LA pretreatment for bid three days not only dose-dependently increased GSH of liver tissue,decreased MDA conten but also diminished GPT activity in serum with ID50 being 159.84mg/kg,151.77 mg/kg and 167.31 mg/kg, respectively.3. Effect ofα-LA on CYP450 and CYP2E1 in hepatic injury mice: In the single dose experiment,α-LA pretreatment at a single dose of 50, 100, 200 and 400mg/kg had no effect on elevated hepatic microsomes CYP450 content and CYP2E1 isozyme activity (P>0.05). By contrast, in the multi-dose experiment, pretreatment withα-LA for consecutive three days resulted in a obvious and dose-dependent decrease in P450 content and CYP2E1 activity (P<0.01), with the inhibitory rate of 48.4% and 39.3% relative to injured group, respectively.4. Effect ofα-LA on CYP450 and CYP2E1 in normal mice: In the single dose experiment,α-LA pretreatment had no effect on hepatic microsomes CYP450 content and CYP2E1 isozyme activity (P>0.05). While in the multi-dose experiment,α-LA obviously decreased CYP450 content and inhibited CYP2E1 activity in a dose-dependent mannar (P<0.01).Data showed that the inhibitory rate were 41.1% and 32.3% compared with normal control group, respectively.5. Pentobarbital sodium sleep time as supportive evidence: No significant effect on pentobarbital sodium sleep latent period and sleep time was observed in mice receiving single dose ofα-LA (P>0.05). While obviously prolonged sleep time was found in mice receiving the multi-dose ofα-LA, the sleep time being from 35.84 min in control group untreated withα-LA to 81.20 min and 158.42 min in 100 mg/kg and 400mg/kgα-LA pretreatment groups (P<0.01).Conclusions1.α-LA has greatly potent and dose-dependent free radical-scavenging activity. As compared with VitC,α-LA has much powerful anti-free radical effects, as evidenced by SC50 and IC50.2.α-LA administrated for consecutive three days to mice with liver injury induced by AAP demonstrated obvious and dose-dependent hepato-protective effects reflected by substantially elevated GSH content, lowered GPT activity and MDA content and deminished hepatic pathologic lesions.3.α-LA administrated for consecutive three days to the above mentioned liver injury mice dose-dependently decreased P450 content and inhibited CYP2E1 activity, suggesting thatα-LA could reduce AAP metabolism and decrease the production of the hepatotoxic N-acetyl- p-benzoquinoneimine (NAPQI). This might be one of the important mechanisms by whichα-LA exerts its hepato-protective effects.4.α-LA administrated to normal mice for three days could obviously decrease P450 content and inhibit CYP2E1 activity and prolong pentobarbital sodium sleep time, showing thatα-LA continuously administrated at multi-dosage could inhibit liver microsomal cytochrome oxidase.5.α-LA pretreatment at a single dose had no obvious hepato-protective effects in liver injury mice induced by AAP, and had no inhibitory effects on CYP450 and CYP2E1 in liver injury mice and in normal mice.Overall, it can be concluded thatα-LA has obvious hepato-protective effects by anti-free radical, GSH regeneration and reduction in CYP450 content and inhibition in CYP2E1 activity.
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