Unveiling the Dark Side of Flavonoid：Rutin Provokes Hepatotoxicity in Low－Dose 2－Amino－3－methylimidazo［4，5－f］Quinoline－Exposed Mice via Regulating Gut Microbiota and Liver Metabolism
揭示类黄酮的黑暗面：槲皮素通过调节肠道菌群和肝脏代谢在低剂量 2-氨基-3-甲基咪唑[4,5-f]喹啉暴露的小鼠中诱发肝毒性

Cite This：https：／／doi．org／10．1021／acs．jafc．4c07330
引用此文献：https：／／doi．org／10．1021／acs．jafc．4c07330

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KEYWORDS：2－amino－3－methylimidazo［4，5－f］quinoline，heterocyclic amines，liver injury，liver metabolism，gut microbiota
关键词：2-氨基-3-甲基咪唑[4,5-f]喹啉，杂环胺，肝损伤，肝脏代谢，肠道菌群

Heterocyclic amines（HCAs）are harmful compounds asso－ ciated with cancer and genetic mutations，which are always produced when protein－rich，animal－based foods are cooked at high temperatures，particularly in fried or grilled meat dishes．At present，more than 30 types of HCAs have been identified in daily food．${}^1$${}^1$^(1){ }^{1} A case study at the hospital revealed that consuming red meat with high levels of HCAs can increase the risk of colon tumors by approximately $7-9\mathrm{\%}{.}^2$$7-9\mathrm{\%}{.}^2$7-9%.^(2)7-9 \% .^{2} 2－Amino－3－methylimidazo ［4，5－f］quinoline（IQ），a compound formed through a condensation process involving creatine and amino acids while meat or fish is being cooked，is one of the most extensively researched HCAs due to its highly carcinogenic and genotoxic properties．${}^3$${}^3$^(3){ }^{3} Zebrafish exposed to IQ for 35 days showed apoptosis of hepatocytes，along with a notable decrease in Bcl－2 protein expression．${}^4$${}^4$^(4){ }^{4} Studies have demonstrated that a single dose of $50\mathrm{mg}/\mathrm{kg}$$50\mathrm{mg}/\mathrm{kg}$50mg//kg50 \mathrm{mg} / \mathrm{kg} IQ can induce colon damage and cancer precursors in mice．${}^{5,6}$${}^{5,6}$^(5,6){ }^{5,6} As the main component of HCAs commonly found in the diet，the impact of IQ on human health presents a challenge that is unable to be disregarded．
杂环胺（HCAs）是与癌症和基因突变相关的有害化合物，通常在富含蛋白质的动物性食物高温烹饪时产生，尤其是在炸或烤的肉类菜肴中。目前，已鉴定出日常食物中超过 30 种 HCAs。 ${}^1$${}^1$^(1){ }^{1} 医院的案例研究表明，摄入 HCAs 含量高的红肉会增加结肠肿瘤的风险，大约为 $7-9\mathrm{\%}{.}^2$$7-9\mathrm{\%}{.}^2$7-9%.^(2)7-9 \% .^{2} 2-氨基-3-甲基咪唑[4,5-f]喹啉（IQ），这种化合物是在肉类或鱼类烹饪过程中通过肌酸和氨基酸的缩合过程形成的，由于其高度致癌和遗传毒性的特性，IQ 是研究最广泛的 HCAs 之一。 ${}^3$${}^3$^(3){ }^{3} 暴露于 IQ 35 天的斑马鱼显示出肝细胞凋亡，以及 Bcl-2 蛋白表达的显著下降。 ${}^4$${}^4$^(4){ }^{4} 研究表明， $50\mathrm{mg}/\mathrm{kg}$$50\mathrm{mg}/\mathrm{kg}$50mg//kg50 \mathrm{mg} / \mathrm{kg} IQ 的单次剂量可以诱导小鼠的结肠损伤和癌症前体。 ${}^{5,6}$${}^{5,6}$^(5,6){ }^{5,6} 作为日常食物中常见的 HCAs 主要成分，IQ 对人类健康的影响是一个不能忽视的挑战

There is rising interest in the potential benefits of dietary flavonoids on human health．Rutin（Ru），a flavanol derivative of quercetin（Que），has been shown to possess strong anti－ oxidation and anti－inflammatory properties，which mainly exist in hops，tartary buckwheat，citrus，tomatoes，and other fruits and vegetables with a rough content of $80.37\mathrm{mg}/100\mathrm{g}$$80.37\mathrm{mg}/100\mathrm{g}$80.37mg//100g80.37 \mathrm{mg} / 100 \mathrm{~g} to 1529.51 $\mathrm{mg}/100{\mathrm{g}}^7$$\mathrm{mg}/100{\mathrm{g}}^7$mg//100g^(7)\mathrm{mg} / 100 \mathrm{~g}^{7} Our previous studies found that Ru has no significant effect on IQ cytotoxicity in vitro．${}^8$${}^8$^(8){ }^{8} Actually，Ru cannot be directly absorbed in the enterocytes and requires the
人们对膳食类黄酮对人类健康的潜在益处日益关注。槲皮素（Ru）是槲皮素（Que）的类黄酮衍生物，已被证明具有强大的抗氧化和抗炎特性，主要存在于啤酒花、苦荞、柑橘、番茄等粗含量为 0 到 1529.51 的水果和蔬菜中。我们之前的研究发现，槲皮素在体外对 IQ 细胞毒性没有显著影响。实际上，槲皮素不能直接被肠细胞吸收，需要
action of intestinal microbes to produce absorbable products， such as Que and a series of flavonoids，${}^9$${}^9$^(9){ }^{9} thus resulting in varying effects in vitro and in vivo．Without any doubt，it is not harmful to ingest Ru below the standard level under healthy conditions． However，whether ingestion of Ru is scatheless under IQ－ addressed potential unhealthy conditions，especially with intestinal and liver injury in vivo，is unknown．
肠道微生物的作用可产生可吸收的产物，如槲皮素和一系列类黄酮， ${}^9$${}^9$^(9){ }^{9} 从而导致体外和体内产生不同的效果。毫无疑问，在健康条件下，低于标准水平摄入 R 乌头碱是无害的。然而，在 IQ 所关注的潜在不健康条件下，尤其是体内存在肠道和肝脏损伤时，摄入 R 乌头碱是否无损害尚不清楚

Growing evidence has pointed out the importance of gut microbiota and its metabolites on maintaining the intestinal barrier function and immune homeostasis，${}^{10,11}$${}^{10,11}$^(10,11){ }^{10,11} and disrupted gut microbiota always led to intestinal mucosa damage．${}^{12}$${}^{12}$^(12){ }^{12} The communication between the gut and liver is through the portal vein，known as the gut－liver axis．Increased intestinal barrier permeability promotes the movement of bacterial components， such as lipopolysaccharide（LPS）and metabolites，to the liver． When LPS reached the liver，the Kupffer cells could be triggered by binding to CD14 along with TLR4．This stimulation leads to inflammatory cytokine secretion such as TNF－$\alpha$$\alpha$alpha\alpha ，IL－6，and IL－1 $\beta$$\beta$beta\beta and finally stimulates liver inflammation．${}^{13,14}$${}^{13,14}$^(13,14){ }^{13,14} The gut－liver axis highlights the bidirectional communication between the intestine and liver，which is influenced by the liver＇s secretory
越来越多的证据表明肠道菌群及其代谢产物在维持肠道屏障功能和免疫稳态方面的重要性， ${}^{10,11}$${}^{10,11}$^(10,11){ }^{10,11} 而肠道菌群失调总是会导致肠黏膜损伤。 ${}^{12}$${}^{12}$^(12){ }^{12} 肠道与肝脏之间的通讯通过门静脉进行，称为肠-肝轴。肠道屏障通透性增加会促进细菌成分（如脂多糖（LPS）和代谢产物）进入肝脏。当 LPS 到达肝脏时，库普弗细胞可以通过与 CD14 和 TLR4 结合而被激活。这种刺激会导致炎症细胞因子的分泌，如 TNF- $\alpha$$\alpha$alpha\alpha 、IL-6 和 IL-1 $\beta$$\beta$beta\beta ，最终刺激肝脏炎症。 ${}^{13,14}$${}^{13,14}$^(13,14){ }^{13,14} 肠-肝轴突出了肠道和肝脏之间的双向通讯，这种通讯受肝脏分泌的影响

Figure 1. Effect of Ru on IQ uptake and cytotoxicity in Caco-2 cells (A) Effect of Ru on IQ uptake by Caco2 cells. (B) Effect of cotreatment with Ru and IQ on the viability of Caco2 cells. © Effect of Que on IQ uptake in Caco2 cells. (D) Effect of cotreatment with Que and IQ on the viability of Caco2 cells. ${}^{\ast }P<0.05,{}^{\ast \ast }P<0.01$${}^{\ast }P<0.05,{}^{\ast \ast }P<0.01$^(**)P < 0.05,^(****)P < 0.01{ }^{*} P<0.05,{ }^{* *} P<0.01, and ${}^{\ast \ast \ast }P<0.001$${}^{\ast \ast \ast }P<0.001$^(******)P < 0.001{ }^{* * *} P<0.001.
图 1. Ru 对 Caco-2 细胞中 IQ 摄取和细胞毒性的影响 (A) Ru 对 Caco2 细胞 IQ 摄取的影响。(B) Ru 和 IQ 联合处理对 Caco2 细胞活力的影响。© Que 对 Caco2 细胞 IQ 摄取的影响。(D) Que 和 IQ 联合处理对 Caco2 细胞活力的影响。 ${}^{\ast }P<0.05,{}^{\ast \ast }P<0.01$${}^{\ast }P<0.05,{}^{\ast \ast }P<0.01$^(**)P < 0.05,^(****)P < 0.01{ }^{*} P<0.05,{ }^{* *} P<0.01 ，和 ${}^{\ast \ast \ast }P<0.001$${}^{\ast \ast \ast }P<0.001$^(******)P < 0.001{ }^{* * *} P<0.001 。
function and the microbial activity in the small intestine, impacted by factors such as diet, environment, and pathogens. ${}^{15,16}$${}^{15,16}$^(15,16){ }^{15,16} A large number of studies have demonstrated that flavonoids could improve liver function by regulating the gutliver axis. Kaempferol has been shown to improve alcoholinduced liver injury by regulating short-chain fatty acids (SCFAs) and gut microbiota in mice. ${}^{17}$${}^{17}$^(17){ }^{17} Similarly, luteolin has been demonstrated to alleviate liver damage by restoring and repairing the damaged intestinal mucosal barrier and rectifying microbiota imbalances in rats. ${}^{18}$${}^{18}$^(18){ }^{18} However, the effect of the coexposure of Ru and IQ on gut microbiota changes and gutliver regulation remains unclear.
功能及小肠微生物活性，受饮食、环境、病原体等因素影响。 ${}^{15,16}$${}^{15,16}$^(15,16){ }^{15,16} 大量研究表明，类黄酮可以通过调节肠-肝轴来改善肝功能。山奈酚已被证明可以通过调节小鼠的短链脂肪酸（SCFA）和肠道菌群来改善酒精性肝损伤。 ${}^{17}$${}^{17}$^(17){ }^{17} 类似地，木犀草素已被证明可以通过恢复和修复受损的肠道黏膜屏障以及纠正大鼠肠道菌群失衡来减轻肝损伤。 ${}^{18}$${}^{18}$^(18){ }^{18} 然而，铑和 IQ 共同暴露对肠道菌群变化和肠-肝调节的影响尚不清楚。

This study aimed to explore the potential role and mechanism of Ru on low-dose IQ-regulated liver function by evaluating the liver nontargeted metabolomics, SCFAs, intestinal barrier, and gut microbiota in mice. Overall, this research provides novel insight and a vital theoretical basis for the application of Ru- and IQ-related food safety issues and could preliminarily provide a daily diet reference for human health.
本研究旨在通过评估小鼠的肝脏非靶向代谢组学、SCFA、肠道屏障和肠道菌群，探索铑在低剂量 IQ 调节肝功能中的潜在作用和机制。总体而言，这项研究为铑和 IQ 相关食品安全问题的应用提供了新的见解和重要的理论依据，并初步为人类健康提供了日常饮食参考。

2.1. Animal Experiments. 4-6 week-old male C57BL/6 mice ( $18-20\mathrm{g}$$18-20\mathrm{g}$18-20g18-20 \mathrm{~g} ) were bought from Zhuhai Best Test Biotechnology Co., Ltd. (Zhuhai, China). The animal experiments were approved by the Animal Experiment Committee of Guangdong Ocean University (no. GDOU-LAE-2022-012). Rutin (CAS: 153-18-4) was acquired from Solarbio (Beijing, China). IQ (CAS no. 5346-56-5) was acquired from Yuanye Biotechnology Co., LTD (Shanghai, China). All mice underwent a 7 day acclimatization period in the animal facility before being randomly assigned to one of four groups ( $n=7$$n=7$n=7n=7 ): control group (normal saline, 10
2.1. 动物实验。4-6 周龄雄性 C57BL/6 小鼠（ $18-20\mathrm{g}$$18-20\mathrm{g}$18-20g18-20 \mathrm{~g} ）购自珠海百思特生物科技有限公司（珠海，中国）。动物实验获得广东海洋大学动物实验委员会批准（编号 GDOU-LAE-2022-012）。槲皮素（CAS：153-18-4）购自 Solarbio（北京，中国）。IQ（CAS 编号 5346-56-5）购自友诺生物科技有限公司（上海，中国）。所有小鼠在动物设施中进行 7 天的适应期后，随机分配到四个组（ $n=7$$n=7$n=7n=7 ）：对照组（生理盐水，10
$\mathrm{mL}/\mathrm{kg}$$\mathrm{mL}/\mathrm{kg}$mL//kg\mathrm{mL} / \mathrm{kg} bw), IQ group (IQ, $7\mathrm{mg}/\mathrm{kg}$$7\mathrm{mg}/\mathrm{kg}$7mg//kg7 \mathrm{mg} / \mathrm{kg} bw), Ru (Ru, $70\mathrm{mg}/\mathrm{kg}$$70\mathrm{mg}/\mathrm{kg}$70mg//kg70 \mathrm{mg} / \mathrm{kg} bw) group, and IQ + Ru (Ru, $70\mathrm{mg}/\mathrm{kg}$$70\mathrm{mg}/\mathrm{kg}$70mg//kg70 \mathrm{mg} / \mathrm{kg} bw + IQ, $7\mathrm{mg}/\mathrm{kg}$$7\mathrm{mg}/\mathrm{kg}$7mg//kg7 \mathrm{mg} / \mathrm{kg} bw) group. Mice in the IQ group and Ru group were administered $7\mathrm{mg}/\mathrm{kg}$$7\mathrm{mg}/\mathrm{kg}$7mg//kg7 \mathrm{mg} / \mathrm{kg} of IQ and $70\mathrm{mg}/\mathrm{kg}$$70\mathrm{mg}/\mathrm{kg}$70mg//kg70 \mathrm{mg} / \mathrm{kg} of Ru per day, respectively, and the IQ + Ru group was administered a mixed solution of Ru and IQ. The concentrations of IQ and Ru were determined according to previous publications. ${}^{19,20}$${}^{19,20}$^(19,20){ }^{19,20} The SPF experimental animal room housed the mice with a 12 h light/dark cycle, maintaining a controlled temperature of $20\pm 2{}^{\circ }\mathrm{C}$$20\pm 2{}^{\circ }\mathrm{C}$20+-2^(@)C20 \pm 2{ }^{\circ} \mathrm{C}, and relative humidity between $60\mathrm{\%}$$60\mathrm{\%}$60%60 \% and $70\mathrm{\%}$$70\mathrm{\%}$70%70 \%. After 6 weeks of feeding, all mice were euthanized. The samples of serum, liver, colon, and coccal contents were collected for further investigation.
$\mathrm{mL}/\mathrm{kg}$$\mathrm{mL}/\mathrm{kg}$mL//kg\mathrm{mL} / \mathrm{kg} bw), IQ 组（IQ, $7\mathrm{mg}/\mathrm{kg}$$7\mathrm{mg}/\mathrm{kg}$7mg//kg7 \mathrm{mg} / \mathrm{kg} bw), Ru 组（Ru, $70\mathrm{mg}/\mathrm{kg}$$70\mathrm{mg}/\mathrm{kg}$70mg//kg70 \mathrm{mg} / \mathrm{kg} bw)和 IQ + Ru 组（Ru, $70\mathrm{mg}/\mathrm{kg}$$70\mathrm{mg}/\mathrm{kg}$70mg//kg70 \mathrm{mg} / \mathrm{kg} bw + IQ, $7\mathrm{mg}/\mathrm{kg}$$7\mathrm{mg}/\mathrm{kg}$7mg//kg7 \mathrm{mg} / \mathrm{kg} bw)。IQ 组和 Ru 组的每只小鼠分别每天给予 $7\mathrm{mg}/\mathrm{kg}$$7\mathrm{mg}/\mathrm{kg}$7mg//kg7 \mathrm{mg} / \mathrm{kg} 的 IQ 和 $70\mathrm{mg}/\mathrm{kg}$$70\mathrm{mg}/\mathrm{kg}$70mg//kg70 \mathrm{mg} / \mathrm{kg} 的 Ru，而 IQ + Ru 组则给予 Ru 和 IQ 的混合溶液。IQ 和 Ru 的浓度根据先前文献确定。 ${}^{19,20}$${}^{19,20}$^(19,20){ }^{19,20} SPF 实验动物房内饲养小鼠，保持 12 小时光照/黑暗周期，温度控制在 $20\pm 2{}^{\circ }\mathrm{C}$$20\pm 2{}^{\circ }\mathrm{C}$20+-2^(@)C20 \pm 2{ }^{\circ} \mathrm{C} ，相对湿度在 $60\mathrm{\%}$$60\mathrm{\%}$60%60 \% 至 $70\mathrm{\%}$$70\mathrm{\%}$70%70 \% 之间。喂养 6 周后，所有小鼠被安乐死。采集血清、肝脏、结肠和盲肠内容物样本进行进一步研究。
2.2. Biochemical Analysis and Histological Analysis. Thirty mg of fresh liver or colon tissue was fragmented and centrifuged at 3000 rpm at $4{}^{\circ }\mathrm{C}$$4{}^{\circ }\mathrm{C}$4^(@)C4{ }^{\circ} \mathrm{C} for 10 min . ALT, AST, GSH, CAT, SOD, and MDA were determined in accordance with the kit instructions of Suzhou Gris Biotechnology Co., Ltd. (Suzhou, China). The liver and colon were immobilized in 4% paraformaldehyde, and after dehydration, samples were embedded in paraffin. Liver samples were sliced into $5\mu \mathrm{m}$$5\mu \mathrm{m}$5mum5 \mu \mathrm{~m} thick sections, and colon samples were sliced into $4\mu \mathrm{m}$$4\mu \mathrm{m}$4mum4 \mu \mathrm{~m} thick sections. Sections were stained with hematoxylin and eosin (H&E) after being dewaxed and rehydrated.
2.2. 生化分析和组织学分析。取 30 mg 新鲜肝脏或结肠组织进行粉碎，并在 $4{}^{\circ }\mathrm{C}$$4{}^{\circ }\mathrm{C}$4^(@)C4{ }^{\circ} \mathrm{C} 转速下离心 10 分钟。ALT、AST、GSH、CAT、SOD 和 MDA 的测定按照苏州格瑞斯生物科技有限公司（苏州，中国）的试剂盒说明书进行。肝脏和结肠组织固定于 4%多聚甲醛中，经脱水后进行石蜡包埋。肝脏样本切片厚度为 $5\mu \mathrm{m}$$5\mu \mathrm{m}$5mum5 \mu \mathrm{~m} ，结肠样本切片厚度为 $4\mu \mathrm{m}$$4\mu \mathrm{m}$4mum4 \mu \mathrm{~m} 。切片经脱蜡和再水化后，用苏木精和伊红（H&E）染色。
2.3. UHPLC-QE-MS Nontarget Metabolomics Detection of Liver. The $2\mu \mathrm{L}$$2\mu \mathrm{L}$2muL2 \mu \mathrm{~L} sample injection volume was used in this study. Electrospray ionization served as the ion source for mass spectral signal collection, with scanning conducted separately for positive and negative ions. The LC-MS raw data underwent filtering, identification, integration, retention time correction, and alignment using Progenesis IQ software from Waters Corporation (Milford, USA). The output includes a data matrix containing retention time, mass-to-charge ratio, and peak intensity, as well as MS and MSMS information. The metabolite information was obtained from public databases such as HMDB and Metlin as well as a custom-built database for metabolite matching.
2.3. UHPLC-QE-MS 非靶向代谢组学检测肝脏。本研究使用了 $2\mu \mathrm{L}$$2\mu \mathrm{L}$2muL2 \mu \mathrm{~L} 样品的进样体积。电喷雾电离作为质谱信号采集的离子源，分别对正离子和负离子进行扫描。LC-MS 原始数据通过 Waters 公司（美国米福德）的 Progenesis IQ 软件进行过滤、鉴定、积分、保留时间校正和校准。输出结果包括一个数据矩阵，其中包含保留时间、质荷比和峰强度，以及 MS 和 MSMS 信息。代谢物信息从 HMDB 和 Metlin 等公共数据库以及一个定制的代谢物数据库中获取。

Figure 2. Effect of combination of IQ and Ru on serum biochemical indexes. (A) Animal experiment protocol. (B) Level of serum CAT. © Level of serum GSH. (D) Level of serum SOD. * indicated $P<0.05$$P<0.05$P < 0.05P<0.05 and ** indicated $P<0.01$$P<0.01$P < 0.01P<0.01 compared with the control group. # indicated $P<0.05$$P<0.05$P < 0.05P<0.05 and ## indicated $P<0.01$$P<0.01$P < 0.01P<0.01 compared with the IQ group.
图 2. IQ 和 Ru 联合对血清生化指标的影响。(A)动物实验方案。(B)血清 CAT 水平。(C)血清 GSH 水平。(D)血清 SOD 水平。*表示与对照组比较的 $P<0.05$$P<0.05$P < 0.05P<0.05 ，**表示与对照组比较的 $P<0.01$$P<0.01$P < 0.01P<0.01 。#表示与 IQ 组比较的 $P<0.05$$P<0.05$P < 0.05P<0.05 ，##表示与 IQ 组比较的 $P<0.01$$P<0.01$P < 0.01P<0.01 。
2.4. Gut Microbiome Analysis. Genomic DNA was extracted from the cecum contents and detected by agarose gel electrophoresis. Primers 338F (ACTCCTACGGGAGGCAGCAG) and 806R (GGACTACHVGGGTWTCTAAT) were selected for PCR amplification. The amplified product was quantified. The samples were sequenced using an Illumina mise sequencer with $2\times 300\mathrm{bp}$$2\times 300\mathrm{bp}$2xx300bp2 \times 300 \mathrm{bp} paired ends and bioinformatics (STAM, Ver.2.1.3; R software, version 3.3.3; Cytoscape, Ver. 3.6.0).
2.4. 肠道菌群分析。从盲肠内容物中提取基因组 DNA，并通过琼脂糖凝胶电泳检测。选择引物 338F（ACTCCTACGGGAGGCAGCAG）和 806R（GGACTACHVGGGTWTCTAAT）进行 PCR 扩增。扩增产物进行定量。使用 Illumina mise 测序仪进行测序，采用 $2\times 300\mathrm{bp}$$2\times 300\mathrm{bp}$2xx300bp2 \times 300 \mathrm{bp} 双端测序，并结合生物信息学（STAM, Ver.2.1.3; R 软件, version 3.3.3; Cytoscape, Ver. 3.6.0）。
2.5. Determination of SCFAs in Feces. Feces were collected weekly starting from the third week and stored at $-{80}^{\circ }\mathrm{C}$$-{80}^{\circ }\mathrm{C}$-80^(@)C-80^{\circ} \mathrm{C}. SCFAs were quantitatively analyzed by using gas chromatography. A 50 mg sample of feces was combined with 1 mL of ethyl acetate containing $0.0075\mathrm{\%}$$0.0075\mathrm{\%}$0.0075%0.0075 \% phosphoric acid in a 2 mL grinding tube with grinding beads. The mixture was then ground in a cryo grinder for 1 min , followed by centrifugation for 10 min . The supernatant was transferred to a vial for further analysis.
2.5. 粪便中 SCFAs 的测定。从第三周开始每周收集粪便并储存在 $-{80}^{\circ }\mathrm{C}$$-{80}^{\circ }\mathrm{C}$-80^(@)C-80^{\circ} \mathrm{C} 。通过气相色谱法定量分析 SCFAs。取 50 mg 粪便样品与含 $0.0075\mathrm{\%}$$0.0075\mathrm{\%}$0.0075%0.0075 \% 磷酸的 1 mL 乙酸乙酯混合于 2 mL 研磨管中，加入研磨珠。将混合物在冷冻研磨机中研磨 1 分钟，然后离心 10 分钟。将上清液转移至试管中进行进一步分析。
2.6. Cell Culture and Transfection. Caco-2 cells were sourced from the ATCC Corporation of America. The cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with $10\mathrm{\%}$$10\mathrm{\%}$10%10 \% fetal bovine serum (FBS, Gibco), $1\mathrm{\%}$$1\mathrm{\%}$1%1 \% antibiotics (penicillin and streptomycin), and $1\mathrm{\%}$$1\mathrm{\%}$1%1 \% MEM minimal essential medium. Culturing took place at ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} in a humidifier with $5\mathrm{\%}{\mathrm{CO}}_2$$5\mathrm{\%}{\mathrm{CO}}_2$5%CO_(2)5 \% \mathrm{CO}_{2}.
2.6. 细胞培养和转染。Caco-2 细胞来源于美国 ATCC 公司。细胞在含 $10\mathrm{\%}$$10\mathrm{\%}$10%10 \% 胎牛血清（FBS，Gibco）、 $1\mathrm{\%}$$1\mathrm{\%}$1%1 \% 抗生素（青霉素和链霉素）和 $1\mathrm{\%}$$1\mathrm{\%}$1%1 \% MEM 最小必需培养基的改良 Eagle 培养基中培养。培养在 ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} 的加湿器中进行， $5\mathrm{\%}{\mathrm{CO}}_2$$5\mathrm{\%}{\mathrm{CO}}_2$5%CO_(2)5 \% \mathrm{CO}_{2} 。
2.7. Determination of Cell Viability. The cells were seeded into 96-well plates at a density of 10,000 cells per well. After 24 h , the cells were treated with 50 and $100\mu \mathrm{M}$$100\mu \mathrm{M}$100 muM100 \mu \mathrm{M} of Ru or 50 and $100\mu \mathrm{M}$$100\mu \mathrm{M}$100 muM100 \mu \mathrm{M} of IQ. After another $24\mathrm{h},5\mathrm{mg}/\mathrm{mL}$$24\mathrm{h},5\mathrm{mg}/\mathrm{mL}$24h,5mg//mL24 \mathrm{~h}, 5 \mathrm{mg} / \mathrm{mL} of MTT (Shanghai Scigrace Biotech. Co., Ltd.) was added, and the plates were incubated in the dark for 4 h . Following incubation, the old medium was discarded, and DMSO was added and mixed to dissolve the formazan crystals. The optical density was then measured at a wavelength of 490 nm . The method for assessing the effect of Que on cell viability was the same as that described above.
2.7. 细胞活力测定。细胞以每孔 10,000 个细胞的密度接种到 96 孔板中。24 小时后，用 50 和 $100\mu \mathrm{M}$$100\mu \mathrm{M}$100 muM100 \mu \mathrm{M} 的 Ru 或 50 和 $100\mu \mathrm{M}$$100\mu \mathrm{M}$100 muM100 \mu \mathrm{M} 的 IQ 处理细胞。然后加入 $24\mathrm{h},5\mathrm{mg}/\mathrm{mL}$$24\mathrm{h},5\mathrm{mg}/\mathrm{mL}$24h,5mg//mL24 \mathrm{~h}, 5 \mathrm{mg} / \mathrm{mL} 的 MTT（上海斯可佳生物科技有限公司），并将 plates 在黑暗中孵育 4 小时。孵育后，弃去旧培养基，加入 DMSO 并混合溶解甲臜晶体。然后在 490 nm 波长下测量吸光度。评估 Que 对细胞活力影响的测定方法与上述方法相同。
2.8. Determination of IQ Absorption in Caco-2 Cells by HPLC. Cells were plated at a density of $2\times {10}^5$$2\times {10}^5$2xx10^(5)2 \times 10^{5} cells/well in a 6-well plate and treated with $100\mu \mathrm{M}$$100\mu \mathrm{M}$100 muM100 \mu \mathrm{M} IQ or a combination of $50\mu \mathrm{M}\mathrm{Ru}$$50\mu \mathrm{M}\mathrm{Ru}$50 muMRu50 \mu \mathrm{M} \mathrm{Ru} and $100\mu \mathrm{M}$$100\mu \mathrm{M}$100 muM100 \mu \mathrm{M} IQ
2.8. 通过 HPLC 测定 Caco-2 细胞中 IQ 的吸收。细胞以 $2\times {10}^5$$2\times {10}^5$2xx10^(5)2 \times 10^{5} 个细胞/孔的密度接种到 6 孔板中，并用 $100\mu \mathrm{M}$$100\mu \mathrm{M}$100 muM100 \mu \mathrm{M} 的 IQ 或 $50\mu \mathrm{M}\mathrm{Ru}$$50\mu \mathrm{M}\mathrm{Ru}$50 muMRu50 \mu \mathrm{M} \mathrm{Ru} 和 $100\mu \mathrm{M}$$100\mu \mathrm{M}$100 muM100 \mu \mathrm{M} 的 IQ 组合处理。
for varying time intervals. Following treatment, cells were washed with phosphate-buffered saline (PBS, pH 7.4), lysed with $60\mu \mathrm{L}$$60\mu \mathrm{L}$60 muL60 \mu \mathrm{~L} of ultrapure water, and harvested by scraping. The cellular lysate was then subjected to ultrasound treatment in an ice bath for 20 min and subsequently centrifuged at $10,000\mathrm{rpm}$$10,000\mathrm{rpm}$10,000rpm10,000 \mathrm{rpm} for 10 min at $4{}^{\circ }\mathrm{C}$$4{}^{\circ }\mathrm{C}$4^(@)C4{ }^{\circ} \mathrm{C}. HPLC analysis was performed under consistent conditions for all samples, with acetonitrile as phase $A$$A$AA and a mixture of $0.1\mathrm{\%}(\mathrm{v}/\mathrm{v})$$0.1\mathrm{\%}(\mathrm{v}/\mathrm{v})$0.1%(v//v)0.1 \%(\mathrm{v} / \mathrm{v}) formic acid and water as phase $B$$B$BB. The gradient elution profile was as follows: $0-3\min ,20-35\mathrm{\%}\mathrm{A}$$0-3\min ,20-35\mathrm{\%}\mathrm{A}$0-3min,20-35%A0-3 \mathrm{~min}, 20-35 \% \mathrm{~A} in B ; $3-7\min ,35\mathrm{\%}\mathrm{A}$$3-7\min ,35\mathrm{\%}\mathrm{A}$3-7min,35%A3-7 \mathrm{~min}, 35 \% \mathrm{~A} in $\mathrm{B};7-12\min ,35-60\mathrm{\%}\mathrm{A}$$\mathrm{B};7-12\min ,35-60\mathrm{\%}\mathrm{A}$B;7-12min,35-60%A\mathrm{B} ; 7-12 \mathrm{~min}, 35-60 \% \mathrm{~A} in $\mathrm{B};12-15\min ,60-35\mathrm{\%}$$\mathrm{B};12-15\min ,60-35\mathrm{\%}$B;12-15min,60-35%\mathrm{B} ; 12-15 \mathrm{~min}, 60-35 \% A in B; $15-18\min ,35-20\mathrm{\%}\mathrm{A}$$15-18\min ,35-20\mathrm{\%}\mathrm{A}$15-18min,35-20%A15-18 \mathrm{~min}, 35-20 \% \mathrm{~A} in B; and $15-18\min ,70-55\mathrm{\%}\mathrm{A}$$15-18\min ,70-55\mathrm{\%}\mathrm{A}$15-18min,70-55%A15-18 \mathrm{~min}, 70-55 \% \mathrm{~A} in B. The column temperature was maintained at ${30}^{\circ }\mathrm{C}$${30}^{\circ }\mathrm{C}$30^(@)C30^{\circ} \mathrm{C} with a flow rate of 0.2 $\mathrm{mL}/\min$$\mathrm{mL}/\min$mL//min\mathrm{mL} / \mathrm{min} and detection at 262 nm using a diode array detector (Agilent, Tianjin, China).
对于不同的时间间隔。治疗后，细胞用磷酸盐缓冲液（PBS，pH 7.4）清洗，用超纯水裂解，并通过刮取收获。细胞裂解物然后在冰浴中进行超声处理 20 分钟，随后在 $10,000\mathrm{rpm}$$10,000\mathrm{rpm}$10,000rpm10,000 \mathrm{rpm} 下以 $4{}^{\circ }\mathrm{C}$$4{}^{\circ }\mathrm{C}$4^(@)C4{ }^{\circ} \mathrm{C} 的速度离心 10 分钟。所有样品均在相同条件下进行高效液相色谱（HPLC）分析，以乙腈为相 $A$$A$AA ，以甲酸和水混合物为相 $B$$B$BB 。梯度洗脱曲线如下： $0-3\min ,20-35\mathrm{\%}\mathrm{A}$$0-3\min ,20-35\mathrm{\%}\mathrm{A}$0-3min,20-35%A0-3 \mathrm{~min}, 20-35 \% \mathrm{~A} 在 B 中； $3-7\min ,35\mathrm{\%}\mathrm{A}$$3-7\min ,35\mathrm{\%}\mathrm{A}$3-7min,35%A3-7 \mathrm{~min}, 35 \% \mathrm{~A} 在 $\mathrm{B};7-12\min ,35-60\mathrm{\%}\mathrm{A}$$\mathrm{B};7-12\min ,35-60\mathrm{\%}\mathrm{A}$B;7-12min,35-60%A\mathrm{B} ; 7-12 \mathrm{~min}, 35-60 \% \mathrm{~A} 在 $\mathrm{B};12-15\min ,60-35\mathrm{\%}$$\mathrm{B};12-15\min ,60-35\mathrm{\%}$B;12-15min,60-35%\mathrm{B} ; 12-15 \mathrm{~min}, 60-35 \% A 在 B 中； $15-18\min ,35-20\mathrm{\%}\mathrm{A}$$15-18\min ,35-20\mathrm{\%}\mathrm{A}$15-18min,35-20%A15-18 \mathrm{~min}, 35-20 \% \mathrm{~A} 在 B 中；以及 $15-18\min ,70-55\mathrm{\%}\mathrm{A}$$15-18\min ,70-55\mathrm{\%}\mathrm{A}$15-18min,70-55%A15-18 \mathrm{~min}, 70-55 \% \mathrm{~A} 在 B 中。柱温维持在 ${30}^{\circ }\mathrm{C}$${30}^{\circ }\mathrm{C}$30^(@)C30^{\circ} \mathrm{C} ，流速为 0.2 $\mathrm{mL}/\min$$\mathrm{mL}/\min$mL//min\mathrm{mL} / \mathrm{min} ，使用二极管阵列检测器（Agilent，天津，中国）在 262 nm 处进行检测。
2.9. Statistical Analysis. The data obtained from the experiment underwent statistical analysis through a one-way ANOVA, followed by Tukey’s post hoc test utilizing SPSS 19 software (IBM Corporation, Armonk, NY, USA). Mean values are presented as mean $\pm$$\pm$+-\pm SEM or* denotes $p<0.05$$p<0.05$p < 0.05p<0.05 or $p<0.01$$p<0.01$p < 0.01p<0.01 compared to the control group; "or ## denotes $p<0.05$$p<0.05$p < 0.05p<0.05 or $p<0.01$$p<0.01$p < 0.01p<0.01 compared to the IQ group.
2.9. 统计分析。实验获得的数据通过单因素方差分析（ANOVA）进行统计分析，随后使用 SPSS 19 软件（IBM 公司，纽约州阿蒙克，美国）进行 Tukey 事后检验。平均值以平均值 $\pm$$\pm$+-\pm 标准误（SEM）表示或*表示与对照组相比的 $p<0.05$$p<0.05$p < 0.05p<0.05 或 $p<0.01$$p<0.01$p < 0.01p<0.01 ；"或##表示与 IQ 组相比的 $p<0.05$$p<0.05$p < 0.05p<0.05 或 $p<0.01$$p<0.01$p < 0.01p<0.01 。