目的 探讨鹿血晶保护肾脏以及抑制顺铂诱导肾损伤的作用机制。方法 人肾小管上皮HK-2细胞设对照组、模型组及鹿血晶（160、400、1000 μg/mL）组和单胺氧化酶A（monoamine oxidase A，MAO-A）抑制剂氯吉兰（15 μmol/L）组，加入药物预处理1 h后加入顺铂（20 μmol/L）刺激24 h诱导细胞损伤。雄性ICR小鼠随机分为对照组、模型组及鹿血晶（0.78 g/kg）组，鹿血晶组预给药7 d，然后模型组及鹿血晶组ip顺铂（25 mg/kg）诱导肾损伤，继续治疗3 d后处死动物。检测细胞内丙二醛（malondialdehyde，MDA）、还原型谷胱甘肽（glutathione，GSH）、三磷酸腺苷（adenosine triphosphate，ATP）水平、超氧化物歧化酶（superoxide dismutase，SOD）活性、上清液中肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）水平，以及细胞及小鼠肾组织活性氧（reactive oxygen species，ROS）、5-羟色胺（5-hydroxytryptamine，5-HT）水平；采用荧光探针DCFH-DA、Mito-Tracker Red CMXRos及荧光染料Hoechst 33342分别检测细胞内ROS含量、定位及细胞凋亡情况；Western blotting法检测细胞B淋巴细胞瘤-2（B cell lymphoma-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X，Bax）、半胱氨酸蛋白酶-3（Caspase-3）、Caspase-9、NADH脱氢酶1（NADH dehydrogenase 1，ND1）、细胞色素B（cytochrome b，CYTB）、ATP合成酶亚单位6（ATP synthase 6，ATP6）、核因子-κB p65（nuclear factor kappa-B，NF-κB p65）、磷酸化NF-κB p65（phosphorylated NF-κB p65，p-NF-κB p65）、抑制子κBα（inhibitor of NF-κB，IκBα）、p-IκBα蛋白表达，细胞及小鼠肾组织色氨酸羟化酶1（tryptophan hydroxylase，Tph1）、芳香簇氨基酸脱羧酶（aromatic amino acid decarboxylase，AADC）、5-HT_2A受体（5-HT_2Areceptors，5-HT_2AR）、MAO-A蛋白表达；采用苏木素-伊红（HE）染色检测小鼠肾组织病理损伤；采用免疫组化法检测小鼠肾组织Tph1、AADC、5-HT_2AR、MAO-A表达。结果 与模型组比较，鹿血晶可抑制HK-2细胞内ROS、MDA及上清液中TNF-α、IL-1β水平（P<0.05、0.01），降低细胞内GSH、SOD水平（P<0.05、0.01），抑制细胞凋亡。鹿血晶（1000 μg/mL）组与氯吉兰组的作用效果接近，两组对顺铂诱导NF-κB p65磷酸化、细胞凋亡激活（Bax、Caspase-3、Caspase-9表达上调及Bcl-2表达下调）、呼吸链功能下降（ND1、CYTB、ATP6表达下调及ATP水平降低）的逆转作用也接近。并且，鹿血晶（1000 μg/mL）组可明显抑制顺铂诱导的HK-2细胞Tph1、AADC、5-HT_2AR、MAO-A表达上调以及5-HT含量升高（P<0.05、0.01），而氯吉兰组使5-HT水平进一步升高（P<0.01）。荧光探针检测表明，线粒体是顺铂刺激时HK-2细胞产生ROS的部位，且鹿血晶（1000 μg/mL）组和氯吉兰组对其有相似的抑制效果。HE染色显示，顺铂致小鼠肾组织损伤的部位主要在近端肾小管上皮细胞，免疫组化显示该部位也是Tph1、AADC、5-HT_2AR、MAO-A表达上调最明显的部位。鹿血晶可抑制顺铂诱导的Tph1、AADC、5-HT_2AR、MAO-A蛋白表达的上调及肾组织5-HT、ROS含量升高（P<0.01）。结论 鹿血晶活性成分抑制顺铂诱导肾损伤的机制可能是直接作用于肾脏近端肾小管上皮细胞，抑制顺铂对细胞Tph1、AADC、5-HT_2AR、MAO-A表达的上调，从而抑制细胞的5-HT合成及线粒体5-HT降解，达到抑制线粒体ROS生成、保护呼吸链，最终抑制氧化应激、炎症、细胞凋亡的效果。

Objective To explore the mechanism of deer blood crystal (DBC) on protecting the kidney and inhibiting cisplatin-induced kidney injury. Methods HK-2 cells were divided into control group, model group, DBC (160, 400, 1000 μg/mL) groups and monoamine oxidase A (MAO-A) inhibitor clorgyline (15 μmol/L) group, drug were added to pretreat for 1 h, and then the cells were exposed to cisplatin (20 μmol/L) for 24 h to induce cell damage. Male ICR mice were randomly divided into control group, model group and DBC (0.78 g/kg) group, after pretreatment with DBC for 7 d, kidney injury was induced by ip cisplatin (25 mg/kg) in model and DBC group; Furthermore, mice were still treated for 3 d and then killed. Malondialdehyde (MDA), glutathione (GSH), adenosine triphosphate (ATP) levels, superoxide dismutase (SOD) activity in cells and tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) levels in supernatant were measured, reactive oxygen species (ROS) and 5-hydroxytryptamine (5-HT) levels in cells and kidney tissue of mice were measured. ROS content, location and apoptosis of HK-2 cells were respectively distinguished by fluorescent probe DCFH-DA, Mito-Tracker Red CMXRos and fluorescent dye Hoechst 33342.Western blotting was used to analyze the protein expressions of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Caspase-9, NADH dehydrogenase 1 (ND1), cytochrome B (CYTB), ATPase 6 (ATP6), nuclear factor-κB p65 (NF-κB p65), phosphorylated NF-κB p65 (p-NF-κB p65), inhibitor κBα (IκBα) and p-IκBα in cells, and tryptophan hydroxylase 1 (Tph1), aromatic cluster amino acid decarboxylase (AADC), 5-HT_2A receptor (5-HT_2AR), MAO-A protein expressions in cells and kidney tissue of mice. Hematoxylin-eosin (HE) staining was used to detect the pathological damage of renal tissues; Immunohistochemistry was used to detect expressions of Tph1, AADC, 5-HT_2AR, and MAO-A in renal tissues. Results Compared with model group, elevation of intracellular ROS, MDA levels and supernatant TNF-α, IL-1β levels were inhibited by DBC (P < 0.05, 0.01), intracellular GSH, SOD levels were reduced (P < 0.05, 0.01), and apoptosis in HK-2 cells were reduced. Effect of DBC (1000 μg/mL) group was similar to that of clorgyline group, and reversal effects of both on cisplatin-induced phosphorylation of NF-κB p65, activation of apoptosis (upregulation of Bax, Caspase-3, Caspase-9 expressions, and downregulation of Bcl-2 expression), and decline of respiratory chain function (downregulation of ND1, CYTB and ATP6 expressions, and reduction of ATP level) were also similar in HK-2 cells. Additionally, the upregulation of Tph1, AADC, 5-HT_2AR, MAO-A expression, and increased 5-HT levels in HK-2 cells induced by cisplatin were significantly inhibited in DBC (1000 μg/mL) group (P < 0.05, 0.01), while the level of 5-HT was even higher in clorgyline group (P < 0.01). Fluorescence probe detection showed that mitochondria was the site of ROS production in HK-2 cells induced by cisplatin, and the inhibitory effect of DBC (1000 μg/mL) group and clorgyline group on ROS production was similar. HE staining showed that the damage site of renal tissue induced by cisplatin was mainly in the proximal renal tubular epithelial cells, and it was the site of the highest upregulation of Tph1, AADC, 5-HT_2AR and MAO-A expressions induced by cisplatin as shown by immunohistochemistry. DBC could suppressed the upregulation of Tph1, AADC, 5-HT_2AR and MAO-A protein expressions and the increase of 5-HT and ROS levels induced by cisplatin in renal tissue (P < 0.01). Conclusion The mechanism of inhibiting cisplatin-induced kidney injury by active components of DBC is probably to directly act on the renal proximal tubular epithelial cells, suppressing the upregulation of Tph1, AADC, 5-HT_2AR and MAO-A expressions induced by cisplatin, thus inhibiting the synthesis of 5-HT and mitochondrial 5-HT degradation, inhibiting ROS production and protecting mitochondrial respiratory chain, so as to inhibit oxidative stress, inflammation and apoptosis.

R285.5

国家自然科学基金资助项目（81570720）