天津医药

天津医药 ›› 2021, Vol. 49 ›› Issue (9): 938-943.doi: 10.11958/20203142

• 实验研究 • 上一篇    下一篇

17β-HSD3抑制剂抗前列腺癌活性及急性毒性的研究

韩杨1，包海花2，柏合2，刘洁婷2，孙文慧2，袁晓环2△   

  1. 1牡丹江医学院第一临床医学院（邮编157011）；2牡丹江医学院医药研究中心
  • 收稿日期:2020-11-12 修回日期:2020-12-15 出版日期:2021-09-15 发布日期:2021-09-18
  • 通讯作者: 袁晓环 E-mail:yuanxiaohuan1969@163.com
  • 基金资助:
    2018年度黑龙江省省属高等学校基本科研业务费科研项目

Study on the anti-prostate cancer activity and acute toxicity of 17β-HSD3 inhibitor

HAN Yang1, BAO Hai-hua2, BAI He2, LIU Jie-ting2, SUN Wen-hui2, YUAN Xiao-huan2△   

  1. 1 The First Clinical Medical College, Mudanjiang Medical University, Mudanjiang 157011, China; 
    2 Medical Research Center of Mudanjiang Medical University
  • Received:2020-11-12 Revised:2020-12-15 Published:2021-09-15 Online:2021-09-18

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摘要： 摘要：目的　探讨17β-羟基类固醇脱氢酶3（17β-HSD3）抑制剂的抗前列腺癌活性、药代动力学及其急性毒性。方法　取对数生长期人雄激素依赖性前列腺癌LNCaP细胞，设置空白对照组（Con组）、姜黄素组（Cur组，Cur 150 µmol/L）、雄激素受体（AR）抑制剂组（AR inhibitor组，AR抑制剂5 µmol/L）和姜黄素类似物H7组（H7组，H7 150 µmol/L），分组处理后采用xCELLience RTCA DP细胞分析仪检测各组细胞活性、流式细胞术检测细胞凋亡率、Western blot法检测细胞中Caspase-3、AR蛋白表达。C57小鼠32只，采用随机数字表法分成4组（n=8），分别为空白对照组和姜黄素类似物H7高、中、低剂量组。用1%羧甲基纤维素钠（CMC-Na）溶解H7（100、50、25 mg/kg），每天灌胃1次，空白对照组小鼠灌胃1% CMC-Na，观察小鼠有无死亡及异常现象，14 d后取材，苏木素-伊红（HE）染色，观察肝脏和肾脏的病理形态学变化。SD大鼠10只，用1% CMC-Na溶解H7（5 mg/kg）灌胃后，高效液相色谱仪（HPLC）检测血药浓度，分析药代动力学参数。另取SD雄性大鼠10只，H7（5 mg/kg）灌胃30 min后，HPLC检测大鼠脑、肝、脾、肺、小肠、胃、肾、心和睾丸中药物浓度，考察药物组织分布。结果　与Con组比较，Cur组、AR inhibitor组和H7组LNCaP细胞的细胞指数（CI值）显著下降，凋亡率明显升高，Caspase-3蛋白表达水平明显升高，而AR蛋白表达水平降低（P＜0.05）；H7组凋亡率较Cur组和AR inhibitor组明显升高（P＜0.05）。连续灌胃H7 14 d，小鼠未出现活动增加、流涎、抽搐、昏迷等异常现象。与Con组相比，小鼠肝脏和肾脏未见明显的病理形态学变化。药代动力学参数结果显示，测出血药浓度-时间曲线下面积（AUC）为（557.31±36.12）mg/（L·h）；峰浓度（Cmax）为（36.92±1.29）mg/L；最高峰时间（tmax）为2 h；消除半衰期（t1/2）为（22.13±1.74）h。H7在小肠中的浓度约是脑组织中的5 000倍。结论　H7作为17β-HSD3抑制剂，具有明显抗激素依赖性前列腺癌的活性和良好的药代动力学参数，无明显的急性毒性作用，以胃肠吸收为主，具有进一步研究开发前景。

关键词: 前列腺肿瘤；受体, 雄激素；17-羟甾类脱氢酶类；毒性试验, 急性；药代动力学；17β-羟基类固醇脱氢酶3；姜黄素类似物

Abstract: Abstract: Objective　To investigate the anti-prostate cancer activity, pharmacokinetic analysis and acute toxicity of 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) inhibitor. Methods　The logarithmic growth phase androgen-dependent prostate cancer LNCaP cells were divided into the blank control group (Con group), curcumin group (Cur group, Cur 150 µmol/L), androgen receptor (AR) inhibitor group (AR inhibitor group, 5 µmol/L) and curcumin analog H7 group (H7 group, H7 150 µmol/L). After grouping treatment, the xCELLience RTCA DP cell analyzer was used to detect cell viability, and flow cytometry was used to detect cell apoptosis. The changes of Caspase-3 and AR protein expression levels in cells were detected by Western blot assay. Thirty-two C57 mice were divided into 4 groups (n=8 for each group), namely the blank control group and the high, medium and low-dose curcumin analog H7 groups. The high, medium and low-dose groups of mice were treated with 1% carboxymethyl sodium cellulose (CMC-Na) dissolved H7 (100, 50 and 25 mg/kg), and gavage once a day. At the same time, mice in the blank control group were gavage 1% CMC-Na to observe whether the mice were dead or abnormal. After 14 days, samples were taken and stained with hematoxylin-eosin (HE) to observe the pathological changes of liver and kidney. Ten SD rats were used to gavage with 1% CMC-Na dissolved H7 (5 mg/kg), and then the plasma concentration was detected by high performance liquid chromatography (HPLC) to analyze the pharmacokinetic parameters. In addition, another 10 SD rats were taken and H7 (5 mg/kg) was gavage for 30 min. The drug concentrations in rat brain, liver, spleen, lung, small intestine, stomach, kidney, heart and testis were detected by HPLC, and the distribution of the drug tissue was investigated. Results　Compared with the Con group, the cell index (CI value) of LNCaP cells significantly decreased in the Cur group, AR inhibitor group and H7 group. The flow cytometry results showed that the apoptosis rate was significantly increased (P＜0.05). The Western blot results showed that Caspase-3 protein expression levels were significantly increased (P＜0.05), while AR protein expression levels decreased (P＜0.05). Compared with Cur group and AR inhibitor group, flow cytometry results showed that the apoptosis rate was significantly increased in H7 group (P＜0.05). After treatment with H7 for 14 days, no abnormalities such as increased activity, salivation, convulsions and coma were found in the mice. Compared with the control group, HE staining showed no obvious pathological changes in mouse liver and kidney. The results of pharmacokinetic parameters showed that the area under the curve (AUC) was (557.31±36.12) mg/(L·h), the peak concentration (Cmax) was (36.92±1.29) mg/L, the highest peak time (tmax) was 2 h and the elimination half-life (t1/2) was (22.13±1.74) h. The H7 concentrations in the small intestine were 5 000 times higher than that in brain tissue. Conclusion　As a 17β-HSD3 inhibitor, H7 has obvious anti-hormone-dependent prostate cancer activity and good pharmacokinetic parameters, no obvious acute toxicity, mainly gastrointestinal absorption, and has further research and development prospects.

Key words: prostatic neoplasms, receptors, androgen, 17-hydroxysteroid dehydrogenases, toxicity tests, acute, pharmacokinetics, 17β-hydroxysteroid dehydrogenase 3, curcumin analogue