肝靶向一氧化氮释放药物对肝损伤的抑制作用及其细胞毒性的评价

Efficacy and cytotoxicity of a liver-targeted nitric oxidereleasing drug in the treatment of liver injury in mice

目的:评价新型肝靶向一氧化氮释放药物NO-040527抗小鼠肝损伤的药效学及细胞毒性,为进一步的临床前研究开发奠定基础.方法:通过腹腔注射四氯化碳、D-氨基半乳糖或对乙酰氨基酚,制备肝损伤动物模型;分别于造模前1h和造模后12h灌胃给予不同剂量的待测药物,于第2次给药后24h,眼眶取血,留取血清标本,测定血清中的ALT,AST水平.取对数生长期的HepG2细胞,接种于96孔培养板,长成单层,加入不同浓度的待测药物.继续培养24h,用MTT法,测定细胞的存活率.结果:肝靶向药物NO-040527能够显著降低CCl4所致小鼠肝损伤的转氨酶升高(P<0.01),降酶作用具有良好的剂量依赖性;在相同剂量(55mg/kg)下,NO-040527的降酶作用强于阳性对照药NCX-1000(P<0.05).同样,肝靶向药物NO-040527能够显著降低对乙酰氨基酚所引起的小鼠转氨酶升高(P<0.01),各剂量组之间降酶作用无显著性差异,在相同剂量(55mg/kg)下,NO-040527的降酶作用与NCX-1000相当.NO-040527对D-氨基半乳糖所致小鼠肝损伤没有保护作用,中低剂量组的小鼠血清ALT、AST值较模型组相比没有显著差异,高剂量组转氨酶甚至略高于模型组.NO-040527在500μmol/L浓度显示出细胞毒性,细胞存活率为45.96%±29.46%(P=0.058);在1000μmol/L浓度具有显著的细胞毒性(P<0.005);在50μmol/L和100μmol/L浓度时,NO-040527显示出促细胞生长的作用,细胞存活率分别为137.67%±8.47%和152.65%±10.084%,显著高于溶剂组(P<0.05).结论:NO-040527能够显著降低CCl4和对乙酰氨基酚所引起的小鼠转氨酶升高,但NO-040527和NCX-1000对D-氨基半乳糖所致小鼠肝损伤没有保护作用,高剂量甚至加重转氨酶升高.NO-040527在高浓度显示出细胞毒性,低浓度显示出促细胞生长的作用.

AIM：To evaluate the efficacy and cytotoxicity of NO-040527,a new liver-targeted nitric oxide-releasing drug,in the treatment of liver injury induced with carbon tetrachloride,acetaminophen or D-galactosamine in mice. METHODS：Liver injury was induced in mice by intraperitoneal injection of carbon tetrachloride,acetaminophen or D-galactosamine. NO-040527 was orally administered to animals 1 h before and 12 h after the induction of liver injury. The animals were killed 24 h after drug delivery,and blood samples were taken to determine serum ALT and AST levels using an automatic biochemical analyzer. The cytotoxicity of the targeted conjugate was determined by methyl thiazol tetrazolium （MTT） assay. RESULTS：NO-040527 could significantly inhibit carbon tetrachloride-induced elevation of ALT and AST in mice （both P 0.01） in a dosedependent manner. At the same dose （55 mg/kg）,NO-040527 exhibited better efficacy than control drug NCX-1000 （P 0.05） in lowering carbon tetrachloride-induced elevation of serum ALT and AST levels. Similarly,NO-040527 could also significantly inhibit acetaminophen-induced elevation of ALT and AST （both P 0.01） though no dose-dependent effect was noted. At the same dose （55 mg/kg）,NO-040527 exhibited similar efficacy to NCX-1000 in lowering acetaminophen-induced elevation of serum ALT and AST levels. NO-040527 showed no protective effects against liver injury induced with D-galactosamine. No significant differences were noted in serum ALT and AST levels between untreated mice with D-galactosamine-induced liver injury and those treated with medium-or low-dose NO-040527. However,the levels of serum ALT and AST in mice with D-galactosamine-induced liver injury treated with high-dose NO-040527 was higher than those in untreated ones. NO-040527 at a dose of 500 μmol/ L began to show cytotoxicity （cell viability rate：45.96% ± 29.46%; P = 0.058）. Signif icant cytotoxicity was noted when the dose of NO-040527 rose to 1000 μmol/L （P 0.005）. NO-040527 at 50 and 100 μmol/L could accelerate cell growth （cell viability rate：137.67% ± 8.47% and 152.65% ± 10.084%,respectively; both P 0.05）. CONCLUSION：NO-040527 has good protective effects against carbon tetrachloride-or acetamin-ophen-induced liver injury in mice. NO-040527 is cytotoxic when used at high dose.