| Objective:To study the enzyme kinetics of Aplysin metabolism in rat liver microsome, and the effect of the CYP3A inhibitor Ketoconazole on the metabolism of Aplysin in rat liver microsome. To observe the effect of Aplysin on the Fas/FasL expression in liver and serum TNF-a concentration in rats of alcohol-induced hepatic injury.Methods:1. Fifteen male Wistar rats were killed by cervical dislocation, calcium precipition method was used to make rats liver microsomes, and the protein concentrations were measured by BCA method. The effects of different incubation time(10,20,30,45,60,90min), liver microsomal protein concentration (0.1,0.2,0.5,1.0,1.5,2.0mg/mL) and Aplysin concentration (1,2,5,10,20,40,80mg/L) to the metabolism in rat liver microsome were observed by using a HPLC method for the determination of Aplysin in rat liver microsome. Lineweave-Burk graphic method was used to calculate the enzyme kinetics parameters. Ketoconazole as CYP3A inhibitors was used (0.5,2,5,10,20,40mg/L) to investigate inhibitory effects on the metabolism of Aplysin in vitro.2. Fifty male Wistar rats were randomly divided into the following five groups:control group, normal diet with normal saline; ethanol-treated group, normal diet with ethanol administration; and three low-, medium-, and high-dose Aplysin[50,100, and150mg/(kg·bw·d)] plus ethanol treatment groups. Excluding the rats in the normal control group, the other animals were initially administered orally with50%(v/v) ethanol8mL kg-1day-1for2weeks following by an increasing intake of ethanol up to12mL kg-1day-1for the remaining4weeks. Twelve hours after the last ethanol treatment, the rats were anesthetized, and blood was collected. The liver was used to calculate liver index and obtained for histopathology by HE staining; The expressions of Fas、FasL in liver were detected by immunohistochemistry; Serum TNF-a concentration was measured by enzyme linked immunosorbent assay.Results:1. Aplysia linear range was0.5-100mg/L with good linear relationship. The intra-and inter-day accuracy was92.42%-100.58%, and RSDs were less than3.18%.The recovery was96.19%-97.83%, and the stability of room temperature for24h and a week at-20℃were good with RSDs less than1.941%. The best incubation conditions of Aplysin metabolism in rat liver microsome were incubation time for30min and protein concentration of1mg/mL. The enzyme kinetic parameters were as follows:Km of19.72mg/L, Vmax of0.35mg/min/g Protein, CLint of17.75mL/min/g Protein. Ketoconazole as CYP3A inhibitors obviously inhibited on the metabolism of Aplysin in vitro, and the maximum inhibition was68%.2. Compared with control group, liver index was increased in the ethanol-treated group(P<0.05); Compared with ethanol-treated group, liver index in medium and high-dose Aplysin were decreased(.P<0.05); Pathological observation of ethanol-treated group liver tissue by HE staining showed the epatic lobule structure was Fuzzy, and there were different sizes, quantity of round fat vacuoles in cytoplasmic and iflammatory cells infiltration; Epatic lobule structure had different degree of damage in each dose Aplysin, and hepatocyte necrosis was significantly reduced compared with ethanol-treated group; The expressions of Fas and FasL in ethanol-treated group were increased compared with control group, and serum TNF-a concentration was higher (P＜0.05); The expression of Fas/FasL and TNF-a concentration were more significantly reduced than ethanol-treated group(P<0.05); With Aplysin ingested dose increasing, the expression of Fas was correspondingly reduced and a dose dependent(P<0.05).Conclusion:The established HPLC method is fast, simple, specific and sensitive, it is applicable to investigate the enzyme kinetics of Aplysin metabolism in rat liver microsome, and CYP3A may be involved in the metabolism of Aplysin. Aplysin can improve liver damage caused by alcohol exposure, and its mechanism may be related to by reducing expression of Fas and FasL in liver tissue and decreased serum levels of TNF-a to inhibit the apoptosis of liver cell. |
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