W3D固体分散体的制备及其抗对乙酰氨基酚诱导的急性肝损伤的保护作用

Preparation of W3D Solid Dispersion and Its Protective Effect Against Activity on Acute Liver Injury Induced by N-acetyl-para-aminophenol

关键词:  4-(5'-二甲氨基)-萘磺酰氧基苯并噁唑酮  对乙酰氨基酚  急性肝损伤  炎症因子  MD2

Key words:  4-(5'-dimethylamino)-naphthalenesulfonyl-2(3H)-benzoxazolone(W3D)  N-acetyl-para-aminophenol  acute liver injury  inflammatory factors  MD2

摘要:

目的 将4-(5'-二甲氨基)-萘磺酰氧基苯并噁唑酮(W3D)制成固体分散体,并研究其对对乙酰氨基酚(N-acetyl-para-aminophenol,APAP)诱导的急性肝损伤(acute liver injury,ALI)的保护作用。方法 采用溶剂法将原料药W3D及辅料PVP k30按1:7反应得W3D固体分散体。采用差示扫描量热法(differential scanning calorimetry,DSC)和X-粉末射线衍射(X-ray diffraction,XRD)手段表征固体分散体的物相变化,同时对其饱和溶解度、溶出重现性进行考察。建立对乙酰氨基酚诱导的ALI模型,分别灌胃给予阳性药N-乙酰半胱氨酸(n-acetyl-L-cysteine,NAC,12.5 mg·kg^-1)、W3D原料药(12.5 mg·kg^-1)、W3D固体分散体(12.5,6.25和3.125 mg·kg^-1),24 h后处死小鼠,取出肝脏,计算肝脏指数;HE染色观察肝组织病理变化,微孔板法检测血清中ALT、AST的含量及肝组织中SOD活力、微量GSH含量,TBA法检测MDA的含量,ELISA法检测血清中TNF-α、IL-6的含量,免疫组化检测肝组织MD2蛋白的表达。结果 W3D固体分散体为无定形形态,10 min的溶出度即可达70%;W3D可降低APAP诱导的ALI小鼠肝脏指数,减少肝组织血管周围细胞胞核固缩、碎裂或溶解等现象,剂量依赖性地减少血清中ALT、AST、TNF-α、IL-6的含量,升高肝组织中GSH含量及SOD活力,降低MDA含量而呈现出优于临床用药NAC的肝保护活性;W3D亦可降低ALI小鼠肝组织中MD2蛋白的含量。结论 W3D固体分散体可通过抑制氧化应激和炎症而发挥抗APAP诱导的ALI作用,其作用机制可能与降低MD2蛋白表达有关。

Abstract:

OBJECTIVE To prepare 4-(5'-dimethylamino)-naphthalenesulfonyl-2(3H)-benzoxazolone(W3D) as solid dispersion to investigate its protective effect on N-acetyl-para-aminophenol(APAP) induced acute liver injury(ALI). METHODS The solid dispersion of W3D was prepared by the reaction of W3D and PVP k30 at a ratio of 1:7 using solvent method. Differential scanning calorimetry(DSC) and X-ray diffraction(XRD) analysis were used to characterize the phase change of the solid dispersion. The saturation solubility and dissolution reproducibility were also determined. After APAP induced ALI model mice established, the mice were administered intragastrically with n-acetyl-L-cysteine(NAC), W3D(12.5 mg·kg^-1), W3D solid dispersion (12.5, 6.25 and 3.125 mg·kg^-1). Twenty-four hours later, the liver tissues were collected to calculate the liver index, and the pathological changes were evaluated by HE staining. The expression of ALT and AST in serum, SOD activity and GSH in liver tissue were detected by microporosity plate, MDA content was detected by TBA method, TNF-α and IL-6 contents in serum were detected by ELISA, and the expression of MD2 protein in liver tissue was detected by immunohistochemistry. RESULTS The W3D solid dispersion was amorphous and the dissolution rate was ＞70% in 10 min. W3D could decrease the liver index, reduce the phenomenon of nucleus pycnosis, fragmentation and dissolution compared with APAP treated group. The levels of ALT, AST, TNF-α and IL-6 in serum were decreased dose-dependently, and the activities of GSH and SOD in liver tissue were increased in W3D treat groups. On the contrary, W3D could reduce the production of MDA significantly, which was better than NAC. Meanwhile, the expressions of MD2 protein in liver tissues were also decreased after the treatment of W3D. CONCLUSION W3D solid dispersion can protect APAP-induced ALI by regulate the oxidative stress and inflammation reaction, and the mechanism might be related to the inhibiting effect on MD2 protein.