目的 基于脂多糖(LPS)誘導(dǎo)大鼠肝臟損傷建立何首烏肝毒性模型,探究何首烏乙醇提取液(AEP)對(duì)大鼠細(xì)胞色素P450(CYP450)酶主要亞型活性及蛋白表達(dá)的影響。方法 雄性SD大鼠100只,隨機(jī)分為6組:對(duì)照組、LPS組、對(duì)乙酰氨基酚(APAP)組、(LPS+APAP)組、AEP組和(LPS+AEP)組。尾iv 4 mg/kg LPS,2 h后,按組別分別ig 625 mg/kg APAP和6 g/kg AEP,每天1次,連續(xù)給藥7 d,每天觀察大鼠體質(zhì)量變化。在建模過程中,取第2、14小時(shí)和5、8天4個(gè)不同時(shí)間點(diǎn),分別對(duì)各組別動(dòng)物麻醉后取血,檢測(cè)肝功能生化指標(biāo)丙氨酸氨基轉(zhuǎn)移酶(ALT)、天門冬氨酸氨基轉(zhuǎn)移酶(AST)和堿性磷酸酶(ALP)活性;解剖,記錄肝臟質(zhì)量;蘇木精-伊紅(HE)染色進(jìn)行組織病理學(xué)檢查;試劑盒法測(cè)定肝細(xì)胞中細(xì)胞色素CYP1A2、CYP2E1、CYP3A1活性的變化;提取肝臟蛋白,應(yīng)用Western blotting法檢測(cè)CYP1A2、CYP2E1、CYP3A1蛋白表達(dá)情況。結(jié)果 與對(duì)照組比較,第2和14小時(shí),LPS組、(LPS+APAP)組和(LPS+AEP)組ALT、AST和ALP活性顯著升高;組織病理學(xué)檢查發(fā)現(xiàn),肝細(xì)胞灶狀壞死,伴炎細(xì)胞浸潤(rùn);給藥后第8天,LPS組組織病理學(xué)檢查正常,但(LPS+AEP)組可見顯著肝細(xì)胞變性,局部慢性炎性灶。第8天,AEP組大鼠肝臟CYP1A2、CYP2E1、CYP3A1的活性明顯降低;Western blotting法檢測(cè)發(fā)現(xiàn),AEP能顯著降低大鼠肝臟CYP1A2蛋白的表達(dá),而對(duì)CYP2E1和CYP3A1蛋白表達(dá)沒有顯著影響。結(jié)論 經(jīng)LPS誘導(dǎo),AEP對(duì)SD大鼠產(chǎn)生明顯的肝臟毒性,毒性的發(fā)生及LPS誘發(fā)的免疫作用與抑制CYP1A2、CYP2E1、CYP3A1的活性和抑制CYP1A2蛋白表達(dá)有關(guān)。

Objective Based on LPS-induced animal model of the hepatotoicity of Polygonum multiflorum in rat liver, we aimed to study the influence of alcohol extract of P. multiflorum (AEP) on the activity of main subtypes of CYP450s and the expression of those protein during activated in the rats. Methods Male SD rats (120 rats) were randomly divided into 6 groups: control, LPS, acetaminophen (APAP), LPS + APAP, AEP, and LPS + AEP group, iv injected LPS (4 mg/kg). After 2 h, the rats in different groups were orally administered with APAP (625 mg/kg) and AEP (6 g/kg) respectively, once daily, continuous administration for 7 d. We observed the change in their weight every day and collected their abdominal aortic blood respectively at 2 h, 14 h, 5 d, and 8 d for each group. Then the biochemical indicators were detected and the body weight ratio of liver was recorded using histopathological HE staining. Furthermore, we detected the activities of CYP1A2, CYP2E1, and CYP3A1 cytochrome in liver cells; Western blotting method was used to analyze the expression of CYP1A2, CYP2E1, and CYP3A protein. Results Compared with the control group, the level of ALT, AST, and ALP in LPS group, LPS + APAP group, and LPS + AEP group increased significantly at 2 h and 14 h after injection of LPS; Histopathological examination revealed that LPS group, LPS + APAP group, and LPS + AEP group showed focal necrosis of liver cells, with inflammatory cell infiltration 2 h and 14 h after injection of LPS, 8 d after the administration, In LPS group, histopathological examination was normal, but LPS + AEP group showed significant liver cell degeneration and local chronic inflammatory lesions. Fluorescence assay found in rats induced by LPS in 8 d, AEP could significantly reduce the activities of CYP1A2, CYP2E1, and CYP3A1 in rat liver; Western blotting method was used to detect the protein expression of CYP1A2, CYP2E1, and CYP3A1, AEP can significantly reduce the protein expression of CYP1A2 in rat liver, whereas without significant effect on the expression of CYP2E1 and CYP3A1. Conclusion After induction by LPS, AEP produces significant liver toxicity to SD rats, the occurrence of toxicity and LPS-induced immune function were related to inhibition of CYP1A2, CYP2E1, and CYP3A1 activity and inhibition of CYP1A2 protein expression.

重大新藥創(chuàng)制科技重大專項(xiàng)(2013ZX09302303);重大新藥創(chuàng)制科技重大專項(xiàng)(2012ZX09301-001-008);北京市科委基金項(xiàng)目(Z131100006513010)