逍遥散对雷公藤致大鼠肝毒性的保护作用及其机制研究
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摘要
雷公藤系卫矛科雷公藤属一年生藤本植物,性辛、凉,味苦,归肝、肾经,具有祛风除湿,活血通络,消肿止痛,杀虫解毒等功效。该药对类风湿性关节炎、慢性肾炎、系统性红斑狼疮、强直性脊柱炎等自身免疫性疾病具有很好的治疗效果。但雷公藤有大毒,其所致肝毒性在相关文献报道中居单味肝毒性中药的首位,很大程度上限制了其在临床上推广应用。但有关雷公藤致肝毒性的病因和发病机制尚不完全清楚。所以,本课题在用雷公藤建立大鼠急性肝毒性模型的基础上,运用现代分子生物学技术,探索雷公藤导致肝毒性的分子机制,并观察中医方剂逍遥散对雷公藤致大鼠肝毒性的保护作用及其作用机制,以此为逍遥散防治雷公藤所致肝毒性提供理论和实验依据。
实验一雷公藤致大鼠急性肝毒性模型的制备
目的：建立雷公藤水煎剂致大鼠急性肝毒性模型。
方法：分别设低、中、高给药剂量(2.5、3.75、5.0g.kg-1)和不同给药时间(1、2、3、4、5、6d),大鼠经口灌服雷公藤水煎剂,同时设正常对照组。采用称重法测定大鼠肝脏指数,干化学法检测血清ALT、AST活力,HE染色观察肝组织病理改变并进行半定量分析。
结果：与正常组比较,雷公藤水煎剂中、高剂量组大鼠的肝脏指数及血清ALT、AST的活力显著升高,且肝组织均有明显病理改变,肝脏严重受损,差异均有统计学意义；随着给药时间的延长,雷公藤水煎剂的毒性呈加重趋势,干预4d后达到峰值。
结论：雷公藤中剂量(3.75g.kg-1)干预4d可作为制备大鼠急性肝毒性模型的相对合适剂量和时间。实验二逍遥散对雷公藤致大鼠急性肝毒性的保护作用
目的：观察不同剂量和不同给药方案的逍遥散对雷公藤水煎剂致大鼠急性肝毒性的保护作用。
方法：不同剂量实验分组：大鼠经口灌服低、中、高剂量(3.375、6.75、13.5g.kg-1)的逍遥散进行预防性干预,每天1次,连续4d,从第5d开始经口灌服造模剂量的雷公藤水煎剂,每天1次,连续4d,同时设正常对照组、模型组和甘利欣阳性对照组。不同给药方案实验分组：分别在造模剂量的雷公藤水煎剂给药4d前、同时、4d后(预防、对抗和治疗)经口灌服大鼠逍遥散(6.75g.kg-1)进行干预,每天1次,连续4d,同时设正常对照组和模型组。采用称重法测定大鼠肝脏指数,干化学法检测血清ALT、AST活力,HE染色观察肝组织病理改变并进行半定量分析。
结果：与模型组比较,逍遥散中、高剂量组大鼠的肝脏指数及血清ALT、AST活力明显降低,肝脏病理损伤明显减轻,差异均有统计学意义与正常组和甘利欣组比较,差异均无统计学意义。与模型组比较,逍遥散预防组大鼠肝脏指数及血清ALT、AST的活力明显降低,大鼠肝脏病理损伤明显减轻,差异均有统计学意义,与正常组比较,差异均无统计学意义
结论：逍遥散中剂量(6.75g.kg-1)可作为其保护雷公藤致大鼠急性肝毒性的相对合适剂量,且预防作用效果最好。
实验三雷公藤致大鼠急性肝毒性及逍遥散对其保护作用的机制研究
目的：探讨雷公藤水煎剂致大鼠急性肝毒性及逍遥散对其保护作用的分子机制。
方法：分别在造模剂量的雷公藤水煎剂给药4d前经口灌服大鼠逍遥散(6.75g.kg-1),腹腔注射CYP450诱导剂苯巴比妥(100mg·kg-1)和CYP450抑制剂酮康唑(80mg·kg-1),每天1次,连续4d,同时设正常对照组和模型组。采用称重法测定大鼠肝脏指数,干化学法检测血清ALT、AST活力,HE染色观察肝组织病理改变并进行半定量分析；CO还原分光光度法测定肝微粒体CYP450含量,化学比色法测定b5含量；Western-immunobloting检测肝微粒体CYP3A4、CYP2C19、肝线粒体、胞质Cyt-C蛋白水平；HPLC检测肝微粒体CYP3A4、CYP2C19的活性；Real-Time PCR检测肝组织中Bcl-2、Bax、Caspase-3的mRNA水平；免疫组织化学法检测肝组织中Bcl-2、Bax、Caspase-3的蛋白水平；流式细胞术检测肝细胞线粒体膜电位水平；TUNEL法检测肝细胞的凋亡率；化学比色法检测血清SOD、GSH-Px活力及MDA含量；ELISA法检测大鼠血清CD68、TNF-α的含量。
结果：
(1)雷公藤致大鼠急性肝毒性及逍遥散对其行使保护作用时肝脏CYP450、b5、CYP3A4及CYP2C19的变化
与正常组比较,模型组大鼠肝脏CYP450、b5含量明显降低(P<0.05或P<0.01),CYP3A4、CYP2C19蛋白水平和活性均明显降低(P<0.05或P<0.01)；
与模型组比较,逍遥散预防组、CYP450诱导组大鼠肝脏CYP450含量明显升高(P<0.01),b5含量有升高趋势(P>0.05),CYP3A4、CYP2C19蛋白水平和活性均明显升高(P<0.05或P<0.01),CYP450抑制组大鼠CYP450含量明显降低(<0.05),b5含量有降低趋势(P>0.05),CYP3A4、 CYP2C19蛋白水平和活性均明显降低(P<0.05)；
与正常组比较,逍遥散预防组、CYP450诱导组大鼠肝脏CYP450含量明显升高(P<0.01),b5含量有降低趋势(P>0.05)；
逍遥散预防组与CYP450诱导组比较,大鼠肝脏CYP450、b5含量差异不明显(P>0.05)。
(2)逍遥散通过诱导大鼠肝脏CYP450及其亚酶CYP3A4、CYP2C19而对雷公藤致大鼠急性肝毒性的影响
与正常组比较,模型组大鼠的肝脏指数及血清ALT、AST的活力明显升高(P<0.01),肝组织有明显病理改变(P<0.05)；
与模型组比较,逍遥散预防组、CYP450诱导组大鼠肝脏指数及血清ALT、AST的活力明显降低(P<0.05或P<0.01),肝组织病理改变明显减轻(P<0.05),CYP450抑制组大鼠的肝脏指数及血清ALT、AST的活力明显升高(P<0.05),肝组织病理改变明显加重(P<0.05)；
与正常组比较,逍遥散预防组、CYP450诱导组大鼠的肝脏指数及血清ALT、AST的活力差异不明显(P>0.05),肝组织无明显病理改变；
与CYP450诱导组比较,逍遥散预防组大鼠的肝脏指数及血清ALT、AST的活力有降低趋势(P>0.05),肝组织病变程度更轻(P＜0.05)。
(3)逍遥散通过诱导大鼠肝脏CYP450及其亚酶CYP3A4、CYP2C19而对雷公藤致大鼠肝细胞过度凋亡的影响
与正常组比较,模型组大鼠肝脏Bcl-2mRNA水平明显降低,Bax、Caspase-3mRNA水平明显升高(P<0.05或P<0.01),Bcl-2蛋白水平明显降低,Bax、Caspase-3蛋白水平明显升高(P<0.01),肝细胞线粒体膜电位水平明显降低(P<0.01),肝线粒体内Cyt-C水平明显降低,胞质内Cyt-C水平明显升高(P<0.01),肝细胞凋亡率明显升高(P<0.01)；
与模型组比较,逍遥散预防组、CYP450诱导组大鼠肝脏Bcl-2mRNA水平明显升高,Bax、Caspase-3mRNA水平明显降低(P<0.05或P<0.01),Bcl-2蛋白水平明显升高,Bax、Caspase-3蛋白水平明显降低(P<0.01),肝细胞线粒体膜电位水平明显升高(P<0.01),肝线粒体内Cyt-C水平明显升高,胞质内Cyt-C水平明显降低(P<0.01),肝细胞凋亡率明显降低(P<0.01),CYP450抑制组大鼠肝脏Bcl-2mRNA水平明显降低,Bax、Caspase-3mRNA水平明显升高(P<0.05),Bcl-2蛋白水平明显降低,Bax、Caspase-3蛋白水平明显升高(P<0.05或P<0.01),肝细胞线粒体膜电位水平明显降低(P<0.05),肝线粒体内Cyt-C水平明显降低,胞质内Cyt-C水平明显升高(P<0.05),肝细胞凋亡率明显升高(P<0.05)；
与正常组比较,逍遥散预防组、CYP450诱导组大鼠肝脏Bcl-2、Bax、Caspase-3的mRNA水平差异不明显(P>0.05),Bcl-2、Bax蛋白水平差异不明显(P>0.05),肝细胞线粒体膜电位水平差异不明显(P>0.05),肝线粒体内、胞质内Cyt-C水平差异不明显(P>0.05),逍遥散预防组大鼠肝脏Caspase-3蛋白水平及肝细胞凋亡率差异不明显(P>0.05),CYP450诱导组大鼠肝脏Caspase-3蛋白水平及肝细胞凋亡率明显升高(P<0.05)；
与CYP450诱导组比较,逍遥散预防组大鼠肝脏Bcl-2mRNA水平有升高趋势,Bax、Caspase-3mRNA水平有降低趋势(P>0.05),Bcl-2蛋白水平明显升高(P<0.05),Bax、Caspase-3蛋白水平有降低趋势(P>0.05),肝细胞线粒体膜电位水平明显升高(P<0.05),肝线粒体内Cyt-C水平明显升高,胞质内Cyt-C水平明显降低(P<0.05),肝细胞凋亡率有降低趋势(P>0.05)。
(4)逍遥散通过诱导大鼠肝脏CYP450及其亚酶CYP3A4、CYP2C19而对雷公藤致急性肝毒性大鼠血清SOD、GSH-Px活力及MDA含量的影响
与正常组比较,模型组大鼠血清SOD、GSH-Px活力明显降低,MDA含量明显升高(P<0.01)；
与模型组比较,逍遥散预防组、CYP450诱导组大鼠血清SOD、GSH-Px活力明显升高,MDA含量明显降低(P<0.01),CYP450抑制组大鼠血清SOD、GSH-Px活力明显降低,MDA含量明显升高(P<0.05)；
与正常组比较,逍遥散预防组、CYP450诱导组大鼠血清SOD、GSH-Px活力及MDA含量差异不明显(P>0.05)；
与CYP450诱导组比较,逍遥散预防组大鼠血清SOD、GSH-Px活力有升高趋势,MDA含量有降低趋势(P>0.05)。
(5)逍遥散通过诱导大鼠肝脏CYP450及其亚酶CYP3A4、CYP2C19而对雷公藤致急性肝毒性大鼠血清CD68、TNF-α的含量的影响
与正常组比较,模型组大鼠血清CD68、TNF-α的含量明显升高(P<0.05);
与模型组比较,逍遥散预防组、CYP450诱导组大鼠血清CD68、TNF-α的含量明显降低(P<0.05或P<0.01),CYP450抑制组大鼠血清CD68、TNF-α的含量明显升高(P<0.05)；
与正常组比较,逍遥散预防组、CYP450诱导组大鼠血清CD68、TNF-α的含量差异不明显(P>0.05)；
与CYP450诱导组比较,逍遥散预防组大鼠血清CD68、TNF-α的含量有降低趋势(P>0.05)。
结论：
(1)雷公藤致肝毒性的机制：雷公藤在代谢的同时抑制了CYP450酶含量及其亚酶CYP3A4和CYP2C19的含量和活性,导致自身代谢减慢,毒性增强,进而引起下游损伤途径,最终导致肝毒性的发生。具体机制表现在以下三个方面：
1)Bcl-2的基因和蛋白表达水平下调, Bax、Caspase-3的基因和蛋白表达水平上调,引起线粒体膜电位水平下降,Cyt-C释放增加,导致肝细胞过度凋亡。
2)SOD、GSH-Px活力下降, MDA含量增加,导致脂质过氧化反应性肝损伤。
3)CD68、TNF-α的含量增加,导致免疫性肝损伤。
(2)逍遥散保护雷公藤致肝毒性的机制：通过诱导CYP450酶含量及其亚酶CYP3A4和CYP2C19的含量和活性,提高CYP450酶对雷公藤的代谢解毒能力,进而抑制了下游损伤途径,保护肝脏。具体机制表现在以下三个方面：
1)Bcl-2的基因和蛋白表达水平上调, Bax、Caspase-3的基因和蛋白表达水平下调,引起线粒体膜电位水平升高,Cyt-C释放减少,抑制了肝细胞过度凋亡
2)SOD、GSH-Px活力升高, MDA含量减少,抑制了脂质过氧化反应性肝损伤。
3)CD68、TNF-α的含量减少,抑制了免疫性肝损伤。
Tripterygium wilfordii Hook. f.(TWHF) belongs to Celastraceae. Its taste is bitter and belongs to liver and kidney channel. It has a relatively good therapeutic effect for many diseases, such as nephrotic syndrome, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, skin diseases and so on. But, the toxicity of TWHF is fierce. Its liver toxicity is in the first place in documents reported about single traditional Chinese medicine, which limits its clinical application to a large extent. But the etiology and pathogenesis of TWHF-induced liver toxicity is not entirely clear. So, this object aims to explore the molecular mechanism of liver toxicity caused by TWHF and the protective effects and mechanism of Xiaoyao San on it by the modern molecular biology techniques, thus to provide theoretical and experimental basis for the prevention and cure of Xiaoyao San on liver toxicity caused by TWHF.
Part One Preparation for the acute liver toxicity Model in Rats Induced by TWHF Water Decoction
Objective:To establish acute liver toxicity model in rats by TWHF water decoction. Methods:Rats were given TWHF water decoction by gavage and divided into different groups by doses of2.5,3.75and5.0g·kg-1and time of1,2,3,4,5and6d. The liver index of rats was detected by weighing method. The activities of serum ALT and AST were detected by dry chemical method. Liver tissue pathological changes were analyzed by HE staining and Semi-quantitative analysis was combined. Results:Compared with the control group, the liver index and the activities of serum ALT and AST were increased significantly at the middle and high doses of TWHF water decoction. At the same time, the pathological changes and serious liver damage were found in liver tissue too. With the extension of the intervention time, the toxic effects of TWHF have a increasing trend and arrived the peak after4days. Conclusion: Intervening rats for4days at the middle dose of3.75g.kg-1of TWHF could be considered as relative right methods to induce acute liver toxicity model.
Part Two Protective Effect of Xiaoyao San on the TWHF Water Decoction Induced Acute Liver Toxicity
Objective:To observe the protective effect of different doses and dosing regimens of Xiaoyao San on the acute liver toxicity induced by the TWHF water decoction. Methods:Rats were given TWHF water decoction by gavage to induce the acute liver toxicity model and were intervented by different doses of3.375,6.75and13.5g. kg-1and dosing regimens of prevention, combat and treatment of Xiaoyao San for4days at the same time. The liver index of rats was detected by weighing method. The activities of serum ALT and AST were detected by dry chemical method. Liver tissue pathological changes were analyzed by HE staining and Semi-quantitative analysis was combined. Results:Compared with the model group, the Liver index, the activities of serum ALT, AST and liver pathological changes in rats of middle and high doses of Xiaoyao San group were reduced significantly and had no significant difference compared with the control group. Compared with the model group, the Liver index, the activity of serum ALT, AST and liver pathological changes in rats of Xiaoyao San prevention group were reduced significantly and had no significant difference compared with the control group. Conclusion:It was showed that interventing rats at the middle dose of Xiaoyao San ahead of TWHF could be considered as relative right methods to protect the acute liver toxicity caused by TWHF.
Part Three Study on Mechanism of the TWHF Induced Acute Liver Toxicity and Protective Effect of Xiaoyao San
Objective:To observe the mechanism of the TWHF induced acute liver toxicity and the protective effect of Xiaoyao San. Methods:Rats were respectively given Xiaoyao San by gavage of6.75g.kg-1and Phenobarbital of1010mg·kg-1and ketoconazole of80mg·kg-1by abdomen injection respectively for4days before being given TWHF at the dose of model. The control group and model group were set. The content of CYP450was detected by CO reduction Spectrophotometric method and the content of b5was detected by the same method except without adding CO. The content of protein of CYP3A4, CYP2C19and Cyt-C of liver were detected by Western-immunoblotting. The activity of CYP3A4and CYP2C19of liver were detected by HPLC. The liver index of rats was detected by weighing method. The activities of serum ALT and AST were detected by dry chemical method. Liver tissue pathological changes were analyzed by HE staining and Semi-quantitative analysis was combined. The mRNA levels of Bcl-2, Bax and Caspase-3genes in the liver tissue were detected by Real-Time PCR. The protein expression levels of Bcl-2, Bax and Caspase-3in the liver tissue were detected by immunohistochemistry. The membrane potential of mitochondrial in liver cells was detected by FCM. The apoptosis of liver cells was detected by TUNEL assay. The activities of SOD and GSH-Px and the content of MDA in serum were detected by Chemical colorimetric. The content of CD68and TNF-a in serum were detected by ELISA assay. Results:Compared with the control group, the content of liver CYP450and b5of model group were reduced significantly, both the activities and protein expression levels of CYP3A4and CYP2C19in rats liver of the model group were increased significantly, the expression of Bcl-2mRNA and protein in rats liver of the model group were reduced significantly and that of Bax, Caspase-3mRNA and protein were increased significantly, the ΔΨm level of liver cells of the model group was reduced significantly, the release of Cyt-C level of the model group was increased significantly, the apoptosis rate of liver cells of the model group was increased significantly, the activities of SOD, GSH-Px in serum of rats of the model group were reduced significantly, however, the content of MDA was increased significantly, the content of CD68, TNF-a in serum of rats of the model group were increased significantly. Compared with the model group, the content of liver CYP450of Xiaoyao San prevention group and CYP450induction group were increased significantly, the Liver index and the activities of serum ALT, AST of these groups were reduced too.There were no obvious pathological changes in liver tissue of Xiaoyao San prevention group and CYP450induction group. Compared with the model group, both the activities and protein expression levels of CYP3A4and CYP2C19in rats liver of Xiaoyao San prevention group and CYP450induction group were increased significantly, the expression of Bcl-2mRNA and protein in rats liver were increased significantly and that of Bax, Caspase-3mRNA and protein were reduced significantly of the Xiaoyao San prevention group and CYP450induction group, the ΔΨm level of liver cells was increased significantly, the release of Cyt-C level was reduced significantly, the apoptosis rate of liver cells was reduced significantly, the activities of SOD, GSH-Px in serum of rats of Xiaoyao San prevention group and CYP450induction group were increased significantly, however, the content of MDA was reduced significantly, the content of CD68, TNF-α in serum of rats of Xiaoyao San prevention group and CYP450induction group were reduced significantly. Conclusion:There is some association between CYP450enzymes metabolic disorders and liver toxicity induced by TWHF. The possible protection mechanism of Xiaoyao San against TWHF induced liver toxicity may be related with inducting of CYP450enzymes and further to inhibit excessive apoptosis of liver cells, lipid peroxidation induced liver toxicity and cytokine induced immune liver toxicity.
实验一雷公藤致大鼠急性肝毒性模型的制备
目的：建立雷公藤水煎剂致大鼠急性肝毒性模型。
方法：分别设低、中、高给药剂量(2.5、3.75、5.0g.kg-1)和不同给药时间(1、2、3、4、5、6d),大鼠经口灌服雷公藤水煎剂,同时设正常对照组。采用称重法测定大鼠肝脏指数,干化学法检测血清ALT、AST活力,HE染色观察肝组织病理改变并进行半定量分析。
结果：与正常组比较,雷公藤水煎剂中、高剂量组大鼠的肝脏指数及血清ALT、AST的活力显著升高,且肝组织均有明显病理改变,肝脏严重受损,差异均有统计学意义；随着给药时间的延长,雷公藤水煎剂的毒性呈加重趋势,干预4d后达到峰值。
结论：雷公藤中剂量(3.75g.kg-1)干预4d可作为制备大鼠急性肝毒性模型的相对合适剂量和时间。实验二逍遥散对雷公藤致大鼠急性肝毒性的保护作用
目的：观察不同剂量和不同给药方案的逍遥散对雷公藤水煎剂致大鼠急性肝毒性的保护作用。
方法：不同剂量实验分组：大鼠经口灌服低、中、高剂量(3.375、6.75、13.5g.kg-1)的逍遥散进行预防性干预,每天1次,连续4d,从第5d开始经口灌服造模剂量的雷公藤水煎剂,每天1次,连续4d,同时设正常对照组、模型组和甘利欣阳性对照组。不同给药方案实验分组：分别在造模剂量的雷公藤水煎剂给药4d前、同时、4d后(预防、对抗和治疗)经口灌服大鼠逍遥散(6.75g.kg-1)进行干预,每天1次,连续4d,同时设正常对照组和模型组。采用称重法测定大鼠肝脏指数,干化学法检测血清ALT、AST活力,HE染色观察肝组织病理改变并进行半定量分析。
结果：与模型组比较,逍遥散中、高剂量组大鼠的肝脏指数及血清ALT、AST活力明显降低,肝脏病理损伤明显减轻,差异均有统计学意义与正常组和甘利欣组比较,差异均无统计学意义。与模型组比较,逍遥散预防组大鼠肝脏指数及血清ALT、AST的活力明显降低,大鼠肝脏病理损伤明显减轻,差异均有统计学意义,与正常组比较,差异均无统计学意义
结论：逍遥散中剂量(6.75g.kg-1)可作为其保护雷公藤致大鼠急性肝毒性的相对合适剂量,且预防作用效果最好。
实验三雷公藤致大鼠急性肝毒性及逍遥散对其保护作用的机制研究
目的：探讨雷公藤水煎剂致大鼠急性肝毒性及逍遥散对其保护作用的分子机制。
方法：分别在造模剂量的雷公藤水煎剂给药4d前经口灌服大鼠逍遥散(6.75g.kg-1),腹腔注射CYP450诱导剂苯巴比妥(100mg·kg-1)和CYP450抑制剂酮康唑(80mg·kg-1),每天1次,连续4d,同时设正常对照组和模型组。采用称重法测定大鼠肝脏指数,干化学法检测血清ALT、AST活力,HE染色观察肝组织病理改变并进行半定量分析；CO还原分光光度法测定肝微粒体CYP450含量,化学比色法测定b5含量；Western-immunobloting检测肝微粒体CYP3A4、CYP2C19、肝线粒体、胞质Cyt-C蛋白水平；HPLC检测肝微粒体CYP3A4、CYP2C19的活性；Real-Time PCR检测肝组织中Bcl-2、Bax、Caspase-3的mRNA水平；免疫组织化学法检测肝组织中Bcl-2、Bax、Caspase-3的蛋白水平；流式细胞术检测肝细胞线粒体膜电位水平；TUNEL法检测肝细胞的凋亡率；化学比色法检测血清SOD、GSH-Px活力及MDA含量；ELISA法检测大鼠血清CD68、TNF-α的含量。
结果：
(1)雷公藤致大鼠急性肝毒性及逍遥散对其行使保护作用时肝脏CYP450、b5、CYP3A4及CYP2C19的变化
与正常组比较,模型组大鼠肝脏CYP450、b5含量明显降低(P<0.05或P<0.01),CYP3A4、CYP2C19蛋白水平和活性均明显降低(P<0.05或P<0.01)；
与模型组比较,逍遥散预防组、CYP450诱导组大鼠肝脏CYP450含量明显升高(P<0.01),b5含量有升高趋势(P>0.05),CYP3A4、CYP2C19蛋白水平和活性均明显升高(P<0.05或P<0.01),CYP450抑制组大鼠CYP450含量明显降低(<0.05),b5含量有降低趋势(P>0.05),CYP3A4、 CYP2C19蛋白水平和活性均明显降低(P<0.05)；
与正常组比较,逍遥散预防组、CYP450诱导组大鼠肝脏CYP450含量明显升高(P<0.01),b5含量有降低趋势(P>0.05)；
逍遥散预防组与CYP450诱导组比较,大鼠肝脏CYP450、b5含量差异不明显(P>0.05)。
(2)逍遥散通过诱导大鼠肝脏CYP450及其亚酶CYP3A4、CYP2C19而对雷公藤致大鼠急性肝毒性的影响
与正常组比较,模型组大鼠的肝脏指数及血清ALT、AST的活力明显升高(P<0.01),肝组织有明显病理改变(P<0.05)；
与模型组比较,逍遥散预防组、CYP450诱导组大鼠肝脏指数及血清ALT、AST的活力明显降低(P<0.05或P<0.01),肝组织病理改变明显减轻(P<0.05),CYP450抑制组大鼠的肝脏指数及血清ALT、AST的活力明显升高(P<0.05),肝组织病理改变明显加重(P<0.05)；
与正常组比较,逍遥散预防组、CYP450诱导组大鼠的肝脏指数及血清ALT、AST的活力差异不明显(P>0.05),肝组织无明显病理改变；
与CYP450诱导组比较,逍遥散预防组大鼠的肝脏指数及血清ALT、AST的活力有降低趋势(P>0.05),肝组织病变程度更轻(P＜0.05)。
(3)逍遥散通过诱导大鼠肝脏CYP450及其亚酶CYP3A4、CYP2C19而对雷公藤致大鼠肝细胞过度凋亡的影响
与正常组比较,模型组大鼠肝脏Bcl-2mRNA水平明显降低,Bax、Caspase-3mRNA水平明显升高(P<0.05或P<0.01),Bcl-2蛋白水平明显降低,Bax、Caspase-3蛋白水平明显升高(P<0.01),肝细胞线粒体膜电位水平明显降低(P<0.01),肝线粒体内Cyt-C水平明显降低,胞质内Cyt-C水平明显升高(P<0.01),肝细胞凋亡率明显升高(P<0.01)；
与模型组比较,逍遥散预防组、CYP450诱导组大鼠肝脏Bcl-2mRNA水平明显升高,Bax、Caspase-3mRNA水平明显降低(P<0.05或P<0.01),Bcl-2蛋白水平明显升高,Bax、Caspase-3蛋白水平明显降低(P<0.01),肝细胞线粒体膜电位水平明显升高(P<0.01),肝线粒体内Cyt-C水平明显升高,胞质内Cyt-C水平明显降低(P<0.01),肝细胞凋亡率明显降低(P<0.01),CYP450抑制组大鼠肝脏Bcl-2mRNA水平明显降低,Bax、Caspase-3mRNA水平明显升高(P<0.05),Bcl-2蛋白水平明显降低,Bax、Caspase-3蛋白水平明显升高(P<0.05或P<0.01),肝细胞线粒体膜电位水平明显降低(P<0.05),肝线粒体内Cyt-C水平明显降低,胞质内Cyt-C水平明显升高(P<0.05),肝细胞凋亡率明显升高(P<0.05)；
与正常组比较,逍遥散预防组、CYP450诱导组大鼠肝脏Bcl-2、Bax、Caspase-3的mRNA水平差异不明显(P>0.05),Bcl-2、Bax蛋白水平差异不明显(P>0.05),肝细胞线粒体膜电位水平差异不明显(P>0.05),肝线粒体内、胞质内Cyt-C水平差异不明显(P>0.05),逍遥散预防组大鼠肝脏Caspase-3蛋白水平及肝细胞凋亡率差异不明显(P>0.05),CYP450诱导组大鼠肝脏Caspase-3蛋白水平及肝细胞凋亡率明显升高(P<0.05)；
与CYP450诱导组比较,逍遥散预防组大鼠肝脏Bcl-2mRNA水平有升高趋势,Bax、Caspase-3mRNA水平有降低趋势(P>0.05),Bcl-2蛋白水平明显升高(P<0.05),Bax、Caspase-3蛋白水平有降低趋势(P>0.05),肝细胞线粒体膜电位水平明显升高(P<0.05),肝线粒体内Cyt-C水平明显升高,胞质内Cyt-C水平明显降低(P<0.05),肝细胞凋亡率有降低趋势(P>0.05)。
(4)逍遥散通过诱导大鼠肝脏CYP450及其亚酶CYP3A4、CYP2C19而对雷公藤致急性肝毒性大鼠血清SOD、GSH-Px活力及MDA含量的影响
与正常组比较,模型组大鼠血清SOD、GSH-Px活力明显降低,MDA含量明显升高(P<0.01)；
与模型组比较,逍遥散预防组、CYP450诱导组大鼠血清SOD、GSH-Px活力明显升高,MDA含量明显降低(P<0.01),CYP450抑制组大鼠血清SOD、GSH-Px活力明显降低,MDA含量明显升高(P<0.05)；
与正常组比较,逍遥散预防组、CYP450诱导组大鼠血清SOD、GSH-Px活力及MDA含量差异不明显(P>0.05)；
与CYP450诱导组比较,逍遥散预防组大鼠血清SOD、GSH-Px活力有升高趋势,MDA含量有降低趋势(P>0.05)。
(5)逍遥散通过诱导大鼠肝脏CYP450及其亚酶CYP3A4、CYP2C19而对雷公藤致急性肝毒性大鼠血清CD68、TNF-α的含量的影响
与正常组比较,模型组大鼠血清CD68、TNF-α的含量明显升高(P<0.05);
与模型组比较,逍遥散预防组、CYP450诱导组大鼠血清CD68、TNF-α的含量明显降低(P<0.05或P<0.01),CYP450抑制组大鼠血清CD68、TNF-α的含量明显升高(P<0.05)；
与正常组比较,逍遥散预防组、CYP450诱导组大鼠血清CD68、TNF-α的含量差异不明显(P>0.05)；
与CYP450诱导组比较,逍遥散预防组大鼠血清CD68、TNF-α的含量有降低趋势(P>0.05)。
结论：
(1)雷公藤致肝毒性的机制：雷公藤在代谢的同时抑制了CYP450酶含量及其亚酶CYP3A4和CYP2C19的含量和活性,导致自身代谢减慢,毒性增强,进而引起下游损伤途径,最终导致肝毒性的发生。具体机制表现在以下三个方面：
1)Bcl-2的基因和蛋白表达水平下调, Bax、Caspase-3的基因和蛋白表达水平上调,引起线粒体膜电位水平下降,Cyt-C释放增加,导致肝细胞过度凋亡。
2)SOD、GSH-Px活力下降, MDA含量增加,导致脂质过氧化反应性肝损伤。
3)CD68、TNF-α的含量增加,导致免疫性肝损伤。
(2)逍遥散保护雷公藤致肝毒性的机制：通过诱导CYP450酶含量及其亚酶CYP3A4和CYP2C19的含量和活性,提高CYP450酶对雷公藤的代谢解毒能力,进而抑制了下游损伤途径,保护肝脏。具体机制表现在以下三个方面：
1)Bcl-2的基因和蛋白表达水平上调, Bax、Caspase-3的基因和蛋白表达水平下调,引起线粒体膜电位水平升高,Cyt-C释放减少,抑制了肝细胞过度凋亡
2)SOD、GSH-Px活力升高, MDA含量减少,抑制了脂质过氧化反应性肝损伤。
3)CD68、TNF-α的含量减少,抑制了免疫性肝损伤。
Tripterygium wilfordii Hook. f.(TWHF) belongs to Celastraceae. Its taste is bitter and belongs to liver and kidney channel. It has a relatively good therapeutic effect for many diseases, such as nephrotic syndrome, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, skin diseases and so on. But, the toxicity of TWHF is fierce. Its liver toxicity is in the first place in documents reported about single traditional Chinese medicine, which limits its clinical application to a large extent. But the etiology and pathogenesis of TWHF-induced liver toxicity is not entirely clear. So, this object aims to explore the molecular mechanism of liver toxicity caused by TWHF and the protective effects and mechanism of Xiaoyao San on it by the modern molecular biology techniques, thus to provide theoretical and experimental basis for the prevention and cure of Xiaoyao San on liver toxicity caused by TWHF.
Part One Preparation for the acute liver toxicity Model in Rats Induced by TWHF Water Decoction
Objective:To establish acute liver toxicity model in rats by TWHF water decoction. Methods:Rats were given TWHF water decoction by gavage and divided into different groups by doses of2.5,3.75and5.0g·kg-1and time of1,2,3,4,5and6d. The liver index of rats was detected by weighing method. The activities of serum ALT and AST were detected by dry chemical method. Liver tissue pathological changes were analyzed by HE staining and Semi-quantitative analysis was combined. Results:Compared with the control group, the liver index and the activities of serum ALT and AST were increased significantly at the middle and high doses of TWHF water decoction. At the same time, the pathological changes and serious liver damage were found in liver tissue too. With the extension of the intervention time, the toxic effects of TWHF have a increasing trend and arrived the peak after4days. Conclusion: Intervening rats for4days at the middle dose of3.75g.kg-1of TWHF could be considered as relative right methods to induce acute liver toxicity model.
Part Two Protective Effect of Xiaoyao San on the TWHF Water Decoction Induced Acute Liver Toxicity
Objective:To observe the protective effect of different doses and dosing regimens of Xiaoyao San on the acute liver toxicity induced by the TWHF water decoction. Methods:Rats were given TWHF water decoction by gavage to induce the acute liver toxicity model and were intervented by different doses of3.375,6.75and13.5g. kg-1and dosing regimens of prevention, combat and treatment of Xiaoyao San for4days at the same time. The liver index of rats was detected by weighing method. The activities of serum ALT and AST were detected by dry chemical method. Liver tissue pathological changes were analyzed by HE staining and Semi-quantitative analysis was combined. Results:Compared with the model group, the Liver index, the activities of serum ALT, AST and liver pathological changes in rats of middle and high doses of Xiaoyao San group were reduced significantly and had no significant difference compared with the control group. Compared with the model group, the Liver index, the activity of serum ALT, AST and liver pathological changes in rats of Xiaoyao San prevention group were reduced significantly and had no significant difference compared with the control group. Conclusion:It was showed that interventing rats at the middle dose of Xiaoyao San ahead of TWHF could be considered as relative right methods to protect the acute liver toxicity caused by TWHF.
Part Three Study on Mechanism of the TWHF Induced Acute Liver Toxicity and Protective Effect of Xiaoyao San
Objective:To observe the mechanism of the TWHF induced acute liver toxicity and the protective effect of Xiaoyao San. Methods:Rats were respectively given Xiaoyao San by gavage of6.75g.kg-1and Phenobarbital of1010mg·kg-1and ketoconazole of80mg·kg-1by abdomen injection respectively for4days before being given TWHF at the dose of model. The control group and model group were set. The content of CYP450was detected by CO reduction Spectrophotometric method and the content of b5was detected by the same method except without adding CO. The content of protein of CYP3A4, CYP2C19and Cyt-C of liver were detected by Western-immunoblotting. The activity of CYP3A4and CYP2C19of liver were detected by HPLC. The liver index of rats was detected by weighing method. The activities of serum ALT and AST were detected by dry chemical method. Liver tissue pathological changes were analyzed by HE staining and Semi-quantitative analysis was combined. The mRNA levels of Bcl-2, Bax and Caspase-3genes in the liver tissue were detected by Real-Time PCR. The protein expression levels of Bcl-2, Bax and Caspase-3in the liver tissue were detected by immunohistochemistry. The membrane potential of mitochondrial in liver cells was detected by FCM. The apoptosis of liver cells was detected by TUNEL assay. The activities of SOD and GSH-Px and the content of MDA in serum were detected by Chemical colorimetric. The content of CD68and TNF-a in serum were detected by ELISA assay. Results:Compared with the control group, the content of liver CYP450and b5of model group were reduced significantly, both the activities and protein expression levels of CYP3A4and CYP2C19in rats liver of the model group were increased significantly, the expression of Bcl-2mRNA and protein in rats liver of the model group were reduced significantly and that of Bax, Caspase-3mRNA and protein were increased significantly, the ΔΨm level of liver cells of the model group was reduced significantly, the release of Cyt-C level of the model group was increased significantly, the apoptosis rate of liver cells of the model group was increased significantly, the activities of SOD, GSH-Px in serum of rats of the model group were reduced significantly, however, the content of MDA was increased significantly, the content of CD68, TNF-a in serum of rats of the model group were increased significantly. Compared with the model group, the content of liver CYP450of Xiaoyao San prevention group and CYP450induction group were increased significantly, the Liver index and the activities of serum ALT, AST of these groups were reduced too.There were no obvious pathological changes in liver tissue of Xiaoyao San prevention group and CYP450induction group. Compared with the model group, both the activities and protein expression levels of CYP3A4and CYP2C19in rats liver of Xiaoyao San prevention group and CYP450induction group were increased significantly, the expression of Bcl-2mRNA and protein in rats liver were increased significantly and that of Bax, Caspase-3mRNA and protein were reduced significantly of the Xiaoyao San prevention group and CYP450induction group, the ΔΨm level of liver cells was increased significantly, the release of Cyt-C level was reduced significantly, the apoptosis rate of liver cells was reduced significantly, the activities of SOD, GSH-Px in serum of rats of Xiaoyao San prevention group and CYP450induction group were increased significantly, however, the content of MDA was reduced significantly, the content of CD68, TNF-α in serum of rats of Xiaoyao San prevention group and CYP450induction group were reduced significantly. Conclusion:There is some association between CYP450enzymes metabolic disorders and liver toxicity induced by TWHF. The possible protection mechanism of Xiaoyao San against TWHF induced liver toxicity may be related with inducting of CYP450enzymes and further to inhibit excessive apoptosis of liver cells, lipid peroxidation induced liver toxicity and cytokine induced immune liver toxicity.
引文
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