Elsevier

Phytomedicine  植物药

Volume 109, January 2023, 154563
第 109 卷 ，2023 年 1 月，154563
Phytomedicine

Original Article  原创文章
Curcumin treatment suppresses cachexia-associated adipose wasting in mice by blocking the cAMP/PKA/CREB signaling pathway
姜黄素治疗通过阻断 cAMP/PKA/CREB 信号通路抑制小鼠恶病质相关的脂肪消耗

https://doi.org/10.1016/j.phymed.2022.154563Get rights and content  获取权利和内容
Full text access  全文访问

Highlights  突出

  • •
    Curcumin protected against cachectic weight loss in C26 tumor-bearing mice.
    姜黄素可防止 C26 荷瘤小鼠的恶病质体重减轻。
  • •
    Curcumin alleviated cancer cachexia-associated adipocyte atrophy and lipid degradation.
    姜黄素减轻了癌症恶病质相关的脂肪细胞萎缩和脂质降解 。
  • •
    The protective effects of curcumin on cachectic fat loss are mediated by the cAMP/PKA/CREB signaling pathway.
    姜黄素对恶病质脂肪减少的保护作用由 cAMP/PKA/CREB 信号通路介导。

Abstract  抽象

Background  背景

Cachexia is a multifactorial debilitating syndrome that is responsible for 22% of mortality among cancer patients, and there are no effective therapeutic agents available. Curcumin, a polyphenolic compound derived from the plant turmeric, has been shown to have anti-inflammatory, antioxidant, anti-autophagic, and antitumor activities. However, its function in cancer cachexia remains largely unexplored.
恶病质是一种多因素使人衰弱的综合征，占癌症患者死亡率的 22%，并且没有有效的治疗药物可用。姜黄素是一种源自植物姜黄的多酚化合物，已被证明具有抗炎、抗氧化、抗自噬和抗肿瘤活性。然而，它在癌症恶病质中的功能在很大程度上仍未得到探索。

Purpose  目的

This study aimed to elucidate the mechanisms by which curcumin improves adipose atrophy in cancer cachexia.
本研究旨在阐明姜黄素改善癌症恶病质脂肪萎缩的机制。

Methods  方法

C26 tumor-bearing BALB/c mice and β3-adrenoceptor agonist CL316243 stimulated BALB/c mice were used to observe the therapeutic effects of curcumin on the lipid degradation of cancer cachexia in vivo. The effects of curcumin in vitro were examined using mature 3T3-L1 adipocytes treated with a conditioned medium of C26 tumor cells or CL316243.
采用 C26 荷瘤 BALB/c 小鼠和 CL316243 刺激的 BALB/c 小鼠的 β3-肾上腺素能受体激动剂观察姜黄素对体内癌恶病质脂质降解的治疗效果。使用用 C26 肿瘤细胞或 CL316243 的条件培养基处理的成熟 3T3-L1 脂肪细胞检查姜黄素在体外的影响。

Results  结果

Mice with C26 tumors and cachexia were protected from weight loss and adipose atrophy by curcumin (50 mg/kg, i.g.). Curcumin significantly reduced serum levels of free fatty acids and increased triglyceride levels. In addition, curcumin significantly inhibited PKA and CREB activation in the adipose tissue of cancer cachectic mice. Curcumin also ameliorated CL316243-induced adipose atrophy and inhibited hormone-mediated PKA and CREB activation in mice. Moreover, the lipid droplet degradation induced by C26 tumor cell conditioned medium in mature 3T3-L1 adipocytes was ameliorated by curcumin (20 µM) treatment. Curcumin also improved the lipid droplet degradation of mature 3T3-L1 adipocytes induced by CL316243.
姜黄素 （50 mg/kg，即 50 mg/kg） 保护患有 C26 肿瘤和恶病质的小鼠免受体重减轻和脂肪萎缩。姜黄素显着降低血清游离脂肪酸水平并增加甘油三酯水平。此外，姜黄素显著抑制癌症恶病质小鼠脂肪组织中的 PKA 和 CREB 活化。姜黄素还改善了小鼠 CL316243 诱导的脂肪萎缩，并抑制了激素介导的 PKA 和 CREB 激活。此外，姜黄素 （20 μM） 处理改善了成熟 3T3-L1 脂肪细胞中 C26 肿瘤细胞条件培养基诱导的脂滴降解。姜黄素还改善了 CL316243 诱导的成熟 3T3-L1 脂肪细胞的脂滴降解。

Conclusion  结论

Curcumin might be expected to be a therapeutic supplement for cancer cachexia patients, primarily through inhibiting adipose tissue loss via the cAMP/PKA/CREB signaling pathway.
姜黄素有望成为癌症恶病质患者的治疗补充剂，主要是通过 cAMP/PKA/CREB 信号通路抑制脂肪组织损失。

Keywords  关键字

Curcumin
Cancer cachexia
Lipolysis
CL316243
cAMP/PKA/CREB

姜黄素
癌恶病质
溶脂
CL316243
cAMP/PKA/CREB

Abbreviations  缩写

AC
adenylate cyclase
ATGL
adipose triglyceride lipase
CCK-8
Cell Counting Kit-8
cAMP
cyclic adenosine monophosphate
CL
CL316243
CREB
cAMP-response element binding protein
Cur
curcumin
DG
diacylglycerol
DEX
dexamethasone
DMEM
Dulbecco's modified Eagle's medium
eWAT
epididymal adipose tissue
EPI
epinephrine
FFA
free fatty acids
FBS
fetal bovine serum
FASN
fatty acid synthase
HSL
hormone-sensitive lipase
IBMX
3-isobutyl-1-methylxanthine
LDL
low-density lipoprotein
Lu
luteolin
MG
monoacylglycerol
MGL
monoacylglycerol lipase
P/S
penicillin-streptomycin solution
PTHrP
parathyroid hormone-related protein
PKA
protein kinase A
RPMI-1640
Roswell Park Memorial Institute-1640
TG
triglyceride
UCP1
uncoupling protein 1
WAT
white adipose tissue
β3-AR
β3-adrenoceptor
5Fu
5-fluorouracil

AC
腺苷酸环化酶
ATGL
脂肪甘油三酯脂肪酶
CCK-8
细胞计数试剂盒-8
cAMP
环磷酸腺苷
CL
CL316243
CREB
cAMP 反应元件结合蛋白
Cur
姜黄素
DG
甘油二酯
DEX
地塞米松
DMEM
、Dulbecco 改良 Eagle 培养基
eWAT
附睾脂肪组织
EPI
肾上腺素
FFA
游离脂肪酸
FBS
胎牛血清
FASN
脂肪酸合酶
HSL
激素敏感脂肪酶
IBMX
3-异丁基-1-甲基黄嘌呤
LDL
低密度脂蛋白
Lu
木犀草素
MG
单酰基甘油
MGL
单酰基甘油脂肪酶
P/S
青霉素-链霉素溶液
PTHrP
甲状旁腺激素相关蛋白
PKA
蛋白激酶 A
RPMI-1640
罗斯威尔公园纪念馆 研究所-1640
TG
甘油三酯
UCP1
解偶联蛋白 1
WAT
白色脂肪组织
β3-AR
β3-肾上腺素能受体
5Fu
5-氟尿嘧啶

Introduction  介绍

Cancer cachexia is a common complication of malignancy and is characterized by progressive depletion of fat and muscle tissue, weight loss, anorexia, anemia, and physical weakness due to metabolic changes (Argilés et al., 2018). Cancer cachexia negatively impacts patients' quality of life, decreases the effectiveness of tumor treatment, and shortens their survival (Vaughan et al., 2013). Malignant tumor patients are more likely to experience cancer cachexia, with an incidence rate of between 50% and 80%, and 22% of all cancer deaths are caused by cancer cachexia (Argilés et al., 2014). Cancer cachexia remains untreatable in spite of great progress in understanding its pathogenesis in recent decades.
癌症恶病质是恶性肿瘤的常见并发症，其特征是脂肪和肌肉组织进行性消耗、体重减轻、厌食、 贫血和代谢变化导致的身体虚弱 （Argilés 等 人，2018 年）。癌症恶病质会对患者的生活质量产生负面影响，降低肿瘤治疗的有效性，并缩短他们的生存期（Vaughan 等 人，2013 年）。恶性肿瘤患者更容易出现癌症恶病质，发病率在 50% 到 80% 之间，所有癌症死亡的 22% 是由癌症恶病质引起的（Argilés 等 人，2014 年）。尽管近几十年来在了解癌症恶病质的发病机制方面取得了巨大进展，但癌症恶病质仍然无法治疗。
Adipose tissue atrophy is a common symptom of cancer cachexia, which occurs earlier than muscle atrophy. In patients with advanced cachexia, the loss of adipose tissue has been associated with a significant increase in mortality (Rohm et al., 2016). Previous research has shown that cancer patients with normal and low body mass indexes have higher mortality rates than obese patients (Antoun et al., 2015). Besides increasing free fatty acids (FFAs) in the blood, lipolysis may also enhance the effects of cancer cachexia by activating ubiquitin lipase in muscle cells (Vaitkus and Celi, 2017).
脂肪组织萎缩是癌症恶病质的常见症状，比肌肉萎缩更早发生。在晚期恶病质患者中，脂肪组织的损失与死亡率的显着增加有关（Rohm et al.， 2016）。 先前的研究表明， 体重指数正常和低的癌症患者的死亡率高于肥胖患者（Antoun et al.， 2015）。 除了增加血液中的游离脂肪酸 （FFA） 外， 脂肪分解还可以通过激活肌肉细胞中的泛素脂肪酶来增强癌症恶病质的作用 （Vaitkus 和 Celi，2017  年）。
Adipocyte lipolysis, rather than reduced fat synthesis or lipogenesis, mainly accounts for fat loss in cancer cachexia (Rydén et al., 2008). Lipolysis is the breakdown of triacylglycerols stored in cellular lipid droplets, which convert triglycerides (TGs) into fatty acids and glycerol. At least three enzymes are required to complete lipolysis in three consecutive steps, including adipose triglyceride lipase (ATGL), which catalyzes TGs to diacylglycerols (DGs); hormone-sensitive lipase (HSL), which converts DGs to monoacylglycerols (MGs); and MG lipase (MGL), which hydrolyzes MGs. Over 90% of TG hydrolysis occurs in adipose tissue by HSL and ATGL (Schweiger et al., 2006), which are regulated by multiple pathways, including the cAMP/PKA pathway (Djouder et al., 2010). Apart from lipolysis, the browning of white adipose tissue (WAT) also causes fat loss associated with cancer cachexia. Uncoupling protein 1 (UCP1) expressed by brown adipocytes decreases fat mass by increasing energy expenditure (Han et al., 2018).
脂肪细胞脂肪分解，而不是减少脂肪合成或脂肪生成，主要是癌症恶病质中脂肪流失的原因（Rydén et al.， 2008）。脂肪分解是储存在细胞脂质液滴中的三酰基甘油的分解，它将甘油三酯 （TG） 转化为脂肪酸和甘油。至少需要三种酶才能连续三个步骤完成脂肪分解，包括脂肪甘油三酯脂肪酶 （ATGL），它催化 TG 转化为甘油二酯 （DG）;激素敏感脂肪酶 （HSL），将 DG 转化为单酰基甘油 （MG）;和 MG 脂肪酶 （MGL），可水解 MG。超过 90% 的 TG 水解发生在脂肪组织中，由 HSL 和 ATGL （Schweiger et al.， 2006） 进行，它们受多种途径调节，包括 cAMP/PKA 途径 （Djouder et al.， 2010）。除了脂肪分解外，白色脂肪组织 （WAT） 的褐变也会导致与癌症恶病质相关的脂肪流失。棕色脂肪细胞表达的解偶联蛋白 1 （UCP1） 通过增加能量消耗来减少脂肪量 （Han et al.， 2018）。
Curcumin is a polyphenol extracted from the root of Curcuma longa Linn. Curcumin possesses antioxidant, anti-autophagic, anti-inflammatory, and antitumor properties that can help in preventing and treating a number of diseases (Hewlings and Kalman, 2017). Curcumin also suppresses blood glucose levels and improves insulin sensitivity in diabetic and obese rodents. Morever, curcumin inhibits lipolysis in adipocytes by reducing circulating free fatty acid levels (Xie et al., 2012a). It has been shown that curcumin limits FFA flow to the liver by inhibiting FFA transport and reduces lipolysis by attenuating ER stress via the cAMP/PKA pathway, thereby enhancing insulin sensitivity to suppress hepatic glucose production (Wang et al., 2016). Curcumin also improved total body weight and muscle phenotype in cachectic mice by inhibiting multiple signaling mechanisms and protein hydrolysis in muscle (Penedo-Vázquez et al., 2021).
姜黄素是从姜黄根中提取的多酚。姜黄素具有抗氧化、抗自噬、抗炎和抗肿瘤特性，有助于预防和治疗多种疾病（Hewlings 和 Kalman，2017  年 ）。姜黄素还可以抑制血糖水平并提高 糖尿病和肥胖啮齿动物的胰岛素敏感性。此外，姜黄素通过降低循环游离脂肪酸水平来抑制脂肪细胞中的脂肪分解（Xie 等 人，2012a）。已经表明，姜黄素通过抑制 FFA 转运来限制 FFA 流向肝脏 ，并通过 cAMP/PKA 通路减轻 ER 应激来减少脂肪分解 ，从而增强胰岛素敏感性以抑制肝葡萄糖产生 （Wang et al.， 2016）。 姜黄素还通过抑制肌肉中的多种信号传导机制和蛋白质水解来改善恶病质小鼠的总体重量和肌肉表型 （Penedo-Vázquez 等 人，2021 年）。
In this study, curcumin effectively alleviated the symptoms of cancer cachexia, particularly adipose atrophy, in mice inoculated with C26 colon tumor cells. cAMP/PKA/CREB pathway activation in the epididymis of mice bearing C26 tumors was significantly inhibited by curcumin. The potential of curcumin in ameliorating adipose atrophy was further confirmed using the CL316243-induced adipose atrophy animal model. Curcumin significantly ameliorated C26 tumor cell conditioned medium and CL316243-induced lipid droplet degradation in mature 3T3-L1 adipocytes. Our study showed that curcumin effectively ameliorated adipose atrophy in a model of cancer cachexia primarily by hindering cAMP/PKA/CREB pathway activation in adipose tissue. Our study suggests that curcumin could be a promising drug for patients with cachexia who suffer from lipid degradation due to its ability to inhibit cAMP/PKA/CREB signaling pathways.
在这项研究中，姜黄素有效缓解了接种 C26 结肠肿瘤细胞的小鼠的癌症恶病质症状，尤其是脂肪萎缩。姜黄素显著抑制了携带 C26 肿瘤的小鼠附睾中的 cAMP/PKA/CREB 通路激活。使用 CL316243 诱导的脂肪萎缩动物模型进一步证实了姜黄素在改善脂肪萎缩方面的潜力 。姜黄素显着改善 C26 肿瘤细胞条件培养基和 CL316243 诱导的成熟 3T3-L1 脂肪细胞中的脂滴降解。我们的研究表明，姜黄素主要通过阻碍脂肪组织中的 cAMP/PKA/CREB 通路激活，有效改善癌症恶病质模型中的脂肪萎缩。我们的研究表明，姜黄素可能是一种很有前途的药物，适用于患有脂质降解的恶病质患者，因为它能够抑制 cAMP/PKA/CREB 信号通路。

Materials and methods  材料和方法

Reagents and antibodies  试剂和抗体

Curcumin (Cur, CAS No. 458–37–7, with a purity ≥ 98%), CL316243 (CL, CAS No. 138908-40-4, with a purity of 98.31%), 5-fluorouracil (5Fu, CAS No. 51-21-8, with a purity of 99.67%), insulin, Cell Counting Kit-8 (CCK-8), phosphatase inhibitor cocktail I, phosphatase inhibitor cocktail, and protease inhibitor cocktail were purchased from Med Chem Express (Shanghai, P.R. China). The chemical structure of Cur is depicted in Fig. 1A. Sigma–Aldrich (St. Louis, MO, USA) provided dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), and antibodies against phospho-cAMP-response element binding protein (p-CREB) and phospho-protein kinase A (p-PKA). Solarbio (Beijing, P.R. China) provided the TG and FFA quantification kits. Cyagen Biosciences (Guangzhou, P.R. China) provided Oil Red O solution. Antibodies against CREB were obtained from Beyotime Biotechnology (Shanghai, P.R. China). Antibodies against PKA and UCP1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell Signaling Technology (Danvers, MA, USA) provided antibodies against ATGL. ABclonal Technology (Wuhan, P.R. China) provided antibodies against phospho-HSL and fatty acid synthase (FASN).
姜黄素 （Cur，CAS 号 458–37–7，纯度≥ 98%）、CL316243（CL，CAS 号 138908-40-4，纯度为 98.31%）、5-氟尿嘧啶（5Fu，CAS 号 51-21-8，纯度为 99.67%）、胰岛素、细胞计数试剂盒-8 （CCK-8）、 磷酸酶抑制剂混合物 I、磷酸酶抑制剂混合物和蛋白酶抑制剂混合物购自 Med Chem Express（上海， 中国）。Cur 的化学结构如图 1 所示 A. Sigma-Aldrich（美国密苏里州圣路易斯）提供地塞米松 （DEX）、3-异丁基-1-甲基黄嘌呤 （IBMX） 以及针对磷酸化 cAMP 反应元件结合蛋白 （p-CREB） 和磷酸化蛋白激酶 A （p-PKA） 的抗体。Solarbio（中国北京）提供了 TG 和 FFA 定量试剂盒。Cyagen Biosciences（中国广州）提供 Oil Red O 溶液。CREB 抗体购自 Beyotime Biotechnology（中国上海）。抗 PKA 和 UCP1 的抗体购自圣克鲁斯生物技术公司（美国加利福尼亚州圣克鲁斯）。Cell Signaling Technology （Danvers， MA， USA） 提供了抗 ATGL 的抗体。ABclonal Technology（中国武汉）提供针对磷酸化 HSL 和脂肪酸合酶 （FASN） 的抗体。
Fig 1
  1. Download: Download high-res image (656KB)
    下载： 下载高分辨率图片 （656KB）
  2. Download: Download full-size image
    下载： 下载全尺寸图像

Fig. 1. Treatment with curcumin prevented cachectic body weight loss in mice bearing the C26 tumor. (A). The structure of curcumin. (B). Schematic diagram of the experimental protocol. (C). The average body weight was calculated every two days in each mouse of the four groups (control, C26, C26+Cur, and control+Cur). (E). Over the course of the experiment, changes in the cumulative intake of food were observed in each group. Following the sacrifice of the mice, the tumor-free body weight (D) and tumor weight (n  =  5) (F) were measured. Data are expressed as the mean ± SD (*p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001).
图 1.姜黄素治疗可防止携带 C26 肿瘤的小鼠出现恶病质体重减轻。姜黄素的结构。实验方案示意图。每两天计算四组 （对照组、C26、C26+Cur 和对照组 + Cur） 每只小鼠的平均体重。在实验过程中，观察到每组食物累积摄入量的变化。在小鼠处死后，测量无肿瘤体重 （D） 和肿瘤重量 （n = 5） （F）。数据表示为均值± SD （*p < 0.05， ** p < 0.01， ***p < 0.001， **** p < 0.0001）。

Cell culture  细胞培养

The colon-26 (C26) murine adenocarcinoma cell line ATCC was cultured as a monolayer with RPMI-1640 medium supplemented with 10% FBS, L-glutamine, and 1% P/S in a humidified atmosphere containing 5% CO2 at 37 °C. In adipocyte medium (AM, high-glucose DMEM with 10% FBS and 1% P/S), 3T3-L1 preadipocytes were cultured with 5% CO2 at 37 °C. On culture plates coated with 0.1% gelatin, 3T3-L1 preadipocytes (ATCC) were plated and grown for in AM 48 h until they reached approximately 100% confluency. The 3T3-L1 preadipocytes were then treated for 48 h with differentiation media (DM I and DM II) to induce differentiation. The DM I medium was AM containing 10 μg/ml insulin (dissolved in ddH2O, ultrasonicated and adjusted pH to 5 with HCl), 1 μM DEX (dissolved in ethanol), and 0.5 mM IBMX (dissolved in ethanol), while the DM II medium was DEX-free and IBMX-free DM I. A subsequent change of AM every 2 days was performed on the differentiated cells until they were used for inducing lipolysis. There was no contamination of any cells with mycoplasma before use.
在 37 °C 下，用补充有 10% FBS、L-谷氨酰胺和 1% P/S 的 RPMI-1640 培养基，将结肠-26 （C26） 小鼠腺癌细胞系 ATCC 与补充有 10% FBS、L-谷氨酰胺和 1% P/S 的 RPMI-1640 培养基作为单层培养 。  在脂肪细胞培养基 （AM，含 10% FBS 和 1% P/S 的高葡萄糖 DMEM） 中，用 5% CO 2 在 37 °C 下培养 3T3-L1 前脂肪细胞。 在涂有 0.1% 明胶的培养板上，将 3T3-L1 前脂肪细胞 （ATCC） 接种并在 AM 48 小时内生长，直至它们达到大约 100% 汇合。然后用分化培养基 （DM I 和 DM II） 处理 3T3-L1 前脂肪细胞 48 h 以诱导分化。DM I 培养基是含有 10 μg/ml 胰岛素（溶于 ddH2O 中，超声并用 HCl 调节 pH 至 5）、1 μM DEX（溶于乙醇）和 0.5 mM IBMX（溶于乙醇）的 AM，而 DM II 培养基是无 DEX 和无 IBMX 的 DM I。随后每 2 天对分化的细胞进行一次 AM 变化，直到它们用于诱导脂肪分解。使用前没有支原体污染任何细胞。

Animals  动物

The Model Animal Research Center of Nanjing University provided male BALB/c mice (6–8 weeks old, 20-22 g). Upon approval of the Nanjing University Institutional Animal Care and Use Committee (IACUC) on March 15, 2020, the experimental protocols for this study were compliant with Chinese regulations and National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (United States). Animal experiments are registered with the IACUC under the number 2003124. In a temperature-controlled (21–23 °C) and specific-pathogen-free (SPF) conditional room, mice were kept on a 12:12 light-dark cycle and provided with standard rodent chow and water. It was necessary for the animals to acclimate for a week before the experiment began.
南京大学模式动物研究中心提供了雄性 BALB/c 小鼠 （6-8 周龄，20-22 g）。经南京大学机构动物护理和使用委员会 （IACUC） 于 2020 年 3 月 15 日批准，本研究的实验方案符合中国法规和美国国立卫生研究院实验动物护理和使用指南（美国）。动物实验在 IACUC 注册，编号为 2003124。在温度控制 （21-23 °C） 和无特异性病原体 （SPF） 的条件室中，小鼠保持 12：12 的明暗循环，并提供标准的啮齿动物食物和水。在实验开始前，动物有必要适应一周。

Cancer cachexia-associated adipocyte lipolysis cell model
癌症恶病质相关脂肪细胞脂肪分解细胞模型

To generate C26 tumor cell-derived conditioned medium, the medium was changed to new high-glucose DMEM after 70% confluence had been reached. A differentiation medium was used for 12 days to differentiate 3T3-L1 preadipocytes into 3T3-L1 mature adipocytes. Curcumin-treated 3T3-L1-derived adipocytes were treated with C26 tumor-conditioned medium or CL (100 nm) for 24 h to establish the adipocyte lipolysis cell model. Western blot or morphological analysis was performed after the cells were harvested.
为了生成 C26 肿瘤细胞来源的条件培养基，在达到 70% 汇合后将培养基更换为新的高葡萄糖 DMEM。使用分化培养基 12 天，将 3T3-L1 前脂肪细胞分化为 3T3-L1 成熟脂肪细胞。姜黄素处理的 3T3-L1 衍生脂肪细胞用 C26 肿瘤条件培养基或 CL （100 nm） 处理 24 小时，以建立脂肪细胞脂肪分解细胞模型。收获细胞后进行 Western blot 或形态学分析。

CCK-8 cell viability assay
CCK-8 细胞活力测定

In 96-well plates, 3T3-L1 cells (3*103/ml) were seeded and, after differentiation, treated with 100 μl of fresh medium containing different concentrations of curcumin for 24 h. After addition of CCK-8 (10 mL), the medium was incubated at 37 °C for 2 h. Following the incubation, microplate reader was used to determine the absorbance at 570 nm.
在 96 孔板中，接种 3T3-L1 细胞 （3*103/ml），分化后，用 100 μl 含有不同浓度姜黄素的新鲜培养基处理 24 小时。加入 CCK-8 （10 mL） 后，将培养基在 37 °C 下孵育 2 小时。孵育后，使用酶标仪测定 570 nm 处的吸光度。

Oil Red O staining  油红 O 染色

After three PBS washes, the cells were fixed for 30 min in 4% formalin and subsequently rinsed with cold PBS three times. Cells were stained at room temperature for 30 min in an Oil Red O working solution before being rinsed three times with water. Bright-field microscopy was used to examine the staining. The dye retained in the cells was eluted into isopropanol after the staining solution was removed, and the OD510 was determined.
PBS 洗涤 3 次后，将细胞在 4% 福尔马林中固定 30 分钟，然后用冷 PBS 冲洗 3 次。将细胞在室温下在 Oil Red O 工作溶液中染色 30 分钟，然后用水冲洗 3 次。使用明场显微镜检查染色。去除染色液后，将保留在细胞中的染料洗脱到异丙醇中，测定 OD510。

Cancer cachectic mouse model
癌症恶病质小鼠模型

Two mouse models were established in our study, which included the C26 cancer cachexia mouse model and the β3-adrenoceptor (β3-AR) agonist-induced fat loss model (n =  6 per group). For the C26 cancer cachexia mouse model, male BALB/c mice were implanted subcutaneously with 100 μl (5.0*105) of C26 tumor cells in the right flank. A β3-AR agonist-induced fat loss mouse model was established by treating the mice daily with intraperitoneal injections of CL (1 mg/kg/day, 7 days, i.p.). CL was dissolved in ddH2O. To test the protective role of curcumin in cachexia-associated fat loss, the mice received gavage of curcumin (20 mg/kg or 50 mg/kg) or sterile saline every two days 7 times, and the experiment lasted for approximately 14 days when the mice lost more than 10% of their body weight. Cur was dissolved in a dilute solution (5% DMSO + 40% PEG300 + 5% Tween-80 + 50% ddH2O). Every two days from day 0 to day 14, food intake and body weight were measured. Mice were euthanized under isoflurane anesthesia. Tissues were collected, weighed, and frozen in liquid nitrogen and then stored at −80 °C until ready for further analyses or fixed in 4% paraformaldehyde for histochemistry analysis.
本研究建立了两种小鼠模型，包括 C26 癌症恶病质小鼠模型和 β3-肾上腺素能受体 （β3-AR） 激动剂诱导的脂肪减少模型 （ 每组 n = 6）。对于 C26 癌症恶病质小鼠模型，雄性 BALB/c 小鼠皮下植入右侧 100 μl （5.0*105） 的 C26 肿瘤细胞。通过每天腹膜内注射 CL （1 mg/kg/天，7 天，腹腔注射） 治疗小鼠，建立 β3-AR 激动剂诱导的减脂小鼠模型。CL 溶解在 ddH2O 中。为了测试姜黄素在恶病质相关脂肪减少中的保护作用，小鼠每两天接受姜黄素管饲 （20 mg/kg 或 50 mg/kg） 或无菌盐水 7 次，当小鼠体重减轻 10% 以上时，实验持续约 14 天。将 Cur 溶解在稀溶液（5% DMSO + 40% PEG300 + 5% Tween-80 + 50% ddH 2 O）中。从第 0 天到第 14 天，每两天测量一次食物摄入量和体重。小鼠在异氟醚麻醉下被安乐死。收集组织，称重并在液氮中冷冻，然后储存在 -80 °C 直至准备进一步分析或固定在 4% 多聚甲醛中用于组织化学分析。

Measurement of serum PTHrP levels
血清 PTHrP 水平的测量

The serum levels of PTHrP in mice were measured using ELISA kits (Mouse PTHrP High Sensitivity ELISA Kit, Aviva Systems Biology, San Diego, CA, USA) in accordance with the manufacturer's instructions.
根据制造商的说明，使用 ELISA 试剂盒（小鼠 PTHrP 高灵敏度 ELISA 试剂盒，Aviva Systems Biology，San Diego，CA，USA） 测量小鼠血清中 PTHrP 水平。

Measurement of FFA and TG
FFA 和 TG 的测量

TGs in mouse serum were assessed using a TG Quantification Kit according to the manufacturer's instructions. Using an FFA Quantification Kit, FFA levels in serum of mice were measured for lipolysis experiments following the manufacturer's protocol.
根据制造商的说明，使用 TG 定量试剂盒评估小鼠血清中的 TG 。使用 FFA 定量试剂盒，按照制造商的方案测量小鼠血清中的 FFA 水平以进行脂肪分解实验。

Hematoxylin-eosin (HE) staining
苏木精-伊红 （HE） 染色

Mouse epididymal adipose tissue (eWAT) was fixed in 4% paraformaldehyde, embedded in paraffin, transversely sectioned, and stained with hematoxylin and eosin solutions. An optical microscope was used to acquire morphological images.
将小鼠附睾脂肪组织 （eWAT） 固定在 4% 多聚甲醛中，包埋在石蜡中，横向切片，并用苏木精和伊红溶液染色。使用光学显微镜获取形态学图像。

Western blot analysis  蛋白质印迹分析

In RIPA lysis buffer supplemented with phosphatase inhibitor cocktail I, phosphatase inhibitor cocktail, and protease inhibitor cocktail, the total protein extract was obtained from either eWAT or 3T3-L1 samples. A BCA kit was applied to assess the protein concentration in the supernatants. An electrophoresis was then performed on 25 μg of total protein in 10% SDS-polyacrylamide gel to separate it and transfer it to PVDF membrane.
在补充有磷酸酶抑制剂混合物 I、磷酸酶抑制剂混合物和蛋白酶抑制剂混合物的 RIPA 裂解缓冲液中，从 eWAT 或 3T3-L1 样品中获得总蛋白提取物。应用 BCA 试剂盒评估上清液中的蛋白质浓度。 然后对 10% SDS-聚丙烯酰胺凝胶中的 25 μg 总蛋白进行电泳 ，将其分离并转移到 PVDF 膜上。

Statistical analysis  统计分析

Data are expressed as the mean ± SD. Those values that did not show a normal distribution (as measured by the Kolmogorov–Smirnov test) were tested by Mann-Whitney's rank-sum test. Normally distributed groups were compared using Student's t-test. Analysis of variance (ANOVA) was applied for comparisons between more than two groups. Considered statistically significant was a p value less than 0.05 presented as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. The statistical tests were all two-sided and performed with GraphPad Prism 8.
数据表示为 SD ±平均值。那些未显示正态分布的值（由 Kolmogorov-Smirnov 检验测量）由 Mann-Whitney 秩和检验检验。使用 Student t 检验比较正态分布的组。方差分析 （ANOVA） 用于两组以上之间的比较。被认为具有统计学意义的是小于 0.05 的 p 值，表现为 * p < 0.05、** p < 0.01、*** p < 0.001 和 **** p < 0.0001。统计测试都是双面的，并使用 GraphPad Prism 8 进行。

Results  结果

Treatment with curcumin prevented cachectic body weight loss in mice bearing the C26 tumor
姜黄素治疗可防止携带 C26 肿瘤的小鼠发生恶病质体重减轻

C26 tumor-bearing mice were systematically evaluated for the effect of curcumin on cancer cachexia, and Fig. 1B shows the treatment protocol of this study. In comparison with the control group, the body weight of tumor-bearing mice decreased gradually after inoculation on day eight. On days 9 through 14, in the C26 tumor-bearing group the average rate of weight loss increased, and the weight change in the cachexia group receiving curcumin (20 mg/kg and 50 mg/kg) treatment was less than that seen in the C26 tumor-bearing mouse group (Fig. 1C, Fig. S1A). At the end of the experiment, there was a substantial difference between the C26 and C26 + Cur ( (50 mg/kg) groups in terms of tumor-free body weight (Fig. 1D). Mice in the C26 group had a decrease in total food intake, while curcumin treatment had no discernible effect on food intake (Fig. 1E, Fig. S1B). Tumor size was significantly reduced after curcumin administration (Fig. 1F), and curcumin-treated groups at different dose levels showed no difference in tumor size (Fig. S1C). We tested both the anti-tumor drugs 5-fluorouracil and luteolin for the treatment of C26-tumor bearing mice and found that 5-fluorouracil and luteolin significantly reduced tumor size, but did not improve body weight and adipose weight (Figs. S1 and S2).
系统评价 C26 荷瘤小鼠姜黄素对癌症恶病质的影响， 图 1B 显示了本研究的治疗方案。与对照组相比，荷瘤小鼠的体重在接种后第 8 天逐渐下降。在第 9 天到第 14 天，C26 荷瘤组的平均体重减轻率增加，接受姜黄素 （20 mg/kg 和 50 mg/kg） 治疗的恶病质组的体重变化小于 C26 荷瘤小鼠组（ 图 1C，图 S1A）。在实验结束时，C26 和 C26 + Cur （ （50 mg/kg） 组在无肿瘤体重方面存在显着差异（ 图 1D）。C26 组小鼠的总食物摄入量减少，而姜黄素治疗对食物摄入量没有明显影响（ 图 1E、图 S1B）。姜黄素给药后肿瘤大小显着减小（ 图 1F），不同剂量水平的姜黄素处理组显示肿瘤大小没有差异（图 S1C）。我们测试了抗肿瘤药物 5-氟尿嘧啶和木犀草素治疗荷瘤小鼠的效果，发现 5-氟尿嘧啶和木犀草素显着减小了肿瘤大小，但没有改善体重和脂肪体重（图 S1 和 S2）。

Curcumin attenuated adipose tissue wasting induced by cancer cachexia
姜黄素减轻了癌症恶病质诱导的脂肪组织萎缩

As shown in Fig. 2A, the C26 cachexia model mice had severely atrophied adipose tissue compared to the control group, and HE staining revealed substantial differences in the morphology of the four groups. In the C26 group, we found severe adipose tissue atrophy when the cross-sectional area was quantified using ImageJ, whereas curcumin treatment prevented cachectic fat loss (Fig. 2B). When compared to the C26 cachexia model group, the C26+Cur group (20 mg/kg) exhibited no significant differences in eWAT; however, the mean eWAT weight of the C26+Cur group (50 mg/kg) was significantly higher (Figs. 2C, S1D). In addition, serum FFA and TG levels were also measured in C26 tumor-bearing mice. Curcumin significantly reduced serum FFAs and increased TGs (Fig. 2D and E). These findings indicate that high-dose curcumin treatment protected C26 tumor-bearing mice from cachexia-induced adipocyte atrophy.
如图 2 A 所示 ，与对照组相比，C26 恶病质模型小鼠的脂肪组织严重萎缩，HE 染色显示四组小鼠的形态存在显著差异。在 C26 组中，当使用 ImageJ 量化横截面积时，我们发现严重的脂肪组织萎缩，而姜黄素治疗可防止恶病质脂肪流失（ 图 2B）。与 C26 恶病质模型组相比，C26+Cur 组 （20 mg/kg） 的 eWAT 无显著差异;然而，C26+Cur 组的平均 eWAT 重量 （50 mg/kg） 显着更高（ 图 2C、S1D）。此外， 还测量了 C26 荷瘤小鼠的血清 FFA 和 TG 水平 。姜黄素显着降低血清 FFA 并增加 TG（ 图 2D 和 E）。这些发现表明，高剂量姜黄素治疗可保护 C26 荷瘤小鼠免受恶病质诱导的脂肪细胞萎缩。
Fig 2
  1. Download: Download high-res image (1MB)
    下载： 下载高分辨率图像 （1MB）
  2. Download: Download full-size image
    下载： 下载全尺寸图像

Fig. 2. Curcumin attenuated cancer cachexia-induced adipose tissue wasting. (A). Representative figures of the four groups (control, C26, C26+Cur, and control+ Cur) of epididymal fat and HE staining. (B). A quantitative comparison of adipocyte cross-sectional area (μm2) between the four groups showed that curcumin inhibited the atrophy of adipose tissue. (C). Curcumin significantly increased the epididymal fat weight in C26 tumor bearing cachectic mice. (D). Curcumin significantly increased serum TG levels in cachectic mice. (E). Curcumin significantly reduced FFA content in the serum of cachectic mice. Data are expressed as the mean ± SD (*p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001).
图 2.姜黄素减轻了癌症恶病质诱导的脂肪组织萎缩。附睾脂肪和 HE 染色的四组 （对照、C26、C26+Cur 和对照 + Cur） 的代表图。（B）. 四组脂肪细胞横截面积 （μm2） 的定量比较显示姜黄素抑制脂肪组织的萎缩。姜黄素显着增加了 C26 肿瘤携带恶病质小鼠的附睾脂肪重量。姜黄素显着增加恶病质小鼠的血清 TG 水平。姜黄素显著降低恶病质小鼠血清中的 FFA 含量。数据表示为均值 ± SD （*p < 0.05， ** p < 0.01， ***p < 0.001， **** p < 0.0001）。

Curcumin alleviated cachexia-induced fat loss by affecting the cAMP/PKA/CREB pathway
姜黄素通过影响 cAMP/PKA/CREB 通路减轻恶病质诱导的脂肪流失

The findings of the Western blotting analysis of different groups of eWAT are depicted in Fig. 3. Curcumin at a 50 mg/kg dose improved the upregulation of ATGL, UCP1, and phosphorylated-HSL in the eWAT of cachectic mice (Fig. 3A and B). Furthermore, the protein expression level of FASN, a key enzyme for lipid synthesis, was significantly reduced in eWAT in the cachexia mouse model, and curcumin treatment was able to increase the expression of FASN without significantly affecting the protein expression level of FABP4 (Fig. 3C and D). These findings demonstrate that curcumin ameliorated adipose atrophy in cancer cachexia.
图 3 描述了不同 eWAT 组的 Western blotting 分析结果 。50 mg/kg 剂量的姜黄素改善了恶病质小鼠 eWAT 中 ATGL、UCP1 和磷酸化 HSL 的上调（ 图 3A 和 B）。此外， 在恶病质小鼠模型中，脂质合成的关键酶 FASN 的蛋白表达水平 在 eWAT 中显著降低，姜黄素处理能够增加 FASN 的表达 ，而不会显着影响 FABP4 的蛋白表达水平（ 图 3C 和 D）。这些发现表明姜黄素改善了癌症恶病质中的脂肪萎缩。
Fig 3
  1. Download: Download high-res image (720KB)
    下载： 下载高分辨率图像 （720KB）
  2. Download: Download full-size image
    下载： 下载全尺寸图像

Fig. 3. Curcumin alleviated tumor cachexia lipid degradation by affecting the cAMP/PKA/CREB pathway. (A). Images of Western blot showing the expression of p-HSL (Ser563), ATGL, and UCP1 in the four groups. (B). Quantification of A. (C). Images of Western blot showing the expression of FASN, and FABP4 in the four groups. (D). Quantification of C. (E). Images of Western blot showing the expression of PKA, p-PKA (Tyr197), CREB, and p-CREB (Ser133) in the four groups. (F). Quantification of E. Data are expressed as the mean ± SD (*p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001).
图 3.姜黄素通过影响 cAMP/PKA/CREB 通路减轻肿瘤恶病质脂质降解。Western blot 图像显示四组中 p-HSL （Ser563） 、 ATGL 和 UCP1 的表达。（B）. A. （C） 的定量。Western blot 图像显示 FASN 和 FABP4 在四组中的表达。C. （E） 的定量。Western blot 图像显示四组中 PKA 、 p-PKA （Tyr197） 、 CREB 和 p-CREB （Ser133） 的表达。（F）. E. 数据的量化表示为 SD ±平均值（*p < 0.05，** p < 0.01，***p < 0.001，**** p < 0.0001）。

Notably, the phosphorylation levels of PKA and CREB were considerably elevated in the eWAT of C26 tumor-bearing mice, indicating that the cAMP signaling pathway was activated during adipose tissue degradation in C26 tumor-bearing mice, whereas treatment with curcumin significantly inhibited cAMP pathway activation (Fig. 3E and F). These results suggest that curcumin treatment may ameliorate the loss of eWAT in cancer cachexia by modulating the cAMP/PKA/CREB signaling pathway.
值得注意的是，C26 荷瘤小鼠 eWAT 中 PKA 和 CREB 的磷酸化水平显著升高，表明 cAMP 信号通路在 C26 荷瘤小鼠脂肪组织降解过程中被激活，而姜黄素处理显著抑制 cAMP 通路激活（ 图 3E 和 F）。这些结果表明，姜黄素治疗可能通过调节 cAMP/PKA/CREB 信号通路来改善癌症恶病质中 eWAT 的丢失。

Curcumin treatment attenuated adipose tissue wasting induced by a β3-AR agonist
姜黄素治疗减轻了 β3-AR 激动剂诱导的脂肪组织萎缩

To simulate cachexia fat loss, mice were injected intraperitoneally with CL and assessed for curcumin-protective fat loss. The serum levels of PTHrP were significantly elevated in both models (C26 cachectic mouse model and β3-AR agonist mouse model) (Fig. 4A and B), indicating some similarity in mechanism between the two models. As shown in Fig. 4C, morphologically, the loss of eWAT was very severe in the CL group of mice. Compared with the CL group, the weight of adipose tissue in the epididymis was markedly increased in the CL+Cur group (Fig. 4D). When the cross-sectional area of adipose tissue was quantified by ImageJ, the CL group was found to have severely atrophied adipose tissue, whereas curcumin prevented CL-induced fat loss (Fig. 4E). In addition, serum FFA and triglyceride levels were measured, and the results revealed that treatment with curcumin markedly improved TG levels and prevented the abnormal CL-induced elevation of serum FFA (Fig. 4F and G).
为了模拟恶病质脂肪减少，小鼠腹膜内注射 CL 并评估姜黄素保护性脂肪减少。 两种模型 （C26 恶病质小鼠模型和 β3-AR 激动剂小鼠模型） 的血清 PTHrP 水平均显著升高 （ 图 4A 和 B），表明两种模型之间的机制存在一些相似性。如图 4 C 所示  ，从形态学上讲，CL 组小鼠的 eWAT 丢失非常严重。与 CL 组相比，CL+Cur 组附睾中脂肪组织的重量显著增加（ 图 4D）。当通过 ImageJ 量化脂肪组织的横截面积时，发现 CL 组脂肪组织严重萎缩，而姜黄素阻止了 CL 诱导的脂肪流失（ 图 4E）。此外，测量血清 FFA 和甘油三酯水平，结果显示姜黄素治疗显着改善了 TG 水平，并防止了 CL 诱导的血清 FFA 异常升高（ 图 4F 和 G）。
Fig 4
  1. Download: Download high-res image (1MB)
    下载： 下载高分辨率图像 （1MB）
  2. Download: Download full-size image
    下载： 下载全尺寸图像

Fig. 4. Curcumin attenuated β3-AR agonist-induced adipose tissue wasting. (A) Serum level of PTHrP in a cachectic cancer mouse model. (B) Serum level of PTHrP in the β3-AR agonist mouse model. (C). Representative Figures of the four groups (Control, CL, CL + Cur, Control + Cur) of epididymal fat and HE staining. (D). Curcumin significantly increased epididymal fat weight in model mice. (E). A quantitative comparison of adipocyte cross-sectional area (μm2) between the four groups showed that curcumin inhibited the atrophy of adipose tissue. (F). Curcumin significantly increased serum TG levels in cachectic mice. (G). Curcumin significantly reduced the FFA content in the serum of model mice. Data are expressed as the mean ± SD (*p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001).
图 4.姜黄素减轻 β3-AR 激动剂诱导的脂肪组织萎缩。（A） 恶病质癌小鼠模型中 PTHrP 的血清水平。（B） β3-AR 激动剂小鼠模型中 PTHrP 的血清水平。附睾脂肪和 HE 染色四组 （对照、CL、CL + Cur、对照 + Cur） 的代表图。姜黄素显着增加了模型小鼠的附睾脂肪重量。（E）. 四组脂肪细胞横截面积 （μm2） 的定量比较显示姜黄素抑制脂肪组织的萎缩。姜黄素显着增加恶病质小鼠的血清 TG 水平。姜黄素显著降低模型小鼠血清中 FFA 含量。数据表示为均值± SD （*p < 0.05， ** p < 0.01， ***p < 0.001， **** p < 0.0001）。

Curcumin alleviated β3-AR agonist-induced lipid degradation by affecting the cAMP/PKA/CREB pathway
姜黄素通过影响 cAMP/PKA/CREB 通路减轻 β3-AR 激动剂诱导的脂质降解

The results of Western blot analysis of different groups of eWAT in the CL mouse model are shown in Fig. 5. Curcumin at a 50 mg/kg improved the upregulation of ATGL, UCP1, and phosphorylated HSL in the CL mouse model of eWAT (Fig. 5A and B). In addition, the protein expression levels of FASN and FABP4, key enzymes of lipid synthesis, were not significantly different (Fig. 5C and D).
CL 小鼠模型中不同组 eWAT 的 Western blot 分析结果如图 5 所示 。在 eWAT 的 CL 小鼠模型中，50 mg/kg 的姜黄素改善了 ATGL、UCP1 和磷酸化 HSL 的上调（ 图 5A 和 B）。此外， 脂质合成的关键酶 FASN 和 FABP4 的蛋白表达水平没有显著差异（ 图 5C 和 D）。
Fig 5
  1. Download: Download high-res image (709KB)
    下载： 下载高分辨率图片 （709KB）
  2. Download: Download full-size image
    下载： 下载全尺寸图像

Fig. 5. Curcumin alleviated β3-AR agonist-induced lipid degradation by affecting the cAMP/PKA/CREB pathway. (A). Images of Western blot showing the expression of p-HSL (Ser563), ATGL, and UCP1 in the four groups. (B). Quantification of A. (C). Images of Western-blot showing the expression of FASN, and FABP4 in the four groups. (D). Quantification of C. (E). Images of Western blot showing the expression of PKA, p-PKA (Tyr197), CREB, and p-CREB (Ser133) in the four groups. (F). Quantification of E. Data are expressed as the mean ± SD (*p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001).
图 5.姜黄素通过影响 cAMP/PKA/CREB 通路减轻 β3-AR 激动剂诱导的脂质降解。Western blot 图像显示四组中 p-HSL （Ser563） 、 ATGL 和 UCP1 的表达。（B）. A. （C） 的定量。Western 印迹图像显示 FASN 和 FABP4 在四组中的表达。C. （E） 的定量。Western blot 图像显示四组中 PKA 、 p-PKA （Tyr197） 、 CREB 和 p-CREB （Ser133） 的表达。（F）. E. 数据的量化表示为 SD ±平均值（*p < 0.05，** p < 0.01，***p < 0.001，**** p < 0.0001）。

β3-AR belongs to the G protein-coupled receptor (GPCR) family, and can directly stimulate the cAMP/PKA/CREB signaling pathway. As shown in Fig. 5E and F, in the CL mouse model, PKA and CREB phosphorylation levels were significantly increased, which demonstrated that the cAMP/PKA/CREB signaling pathway was activated, and its activation was substantially inhibited by curcumin treatment in eWAT.
β3-AR 属于 G 蛋白偶联受体 （GPCR） 家族，可直接刺激 cAMP/PKA/CREB 信号通路。如图 5 E 和 F 所示  ，在 CL 小鼠模型中，PKA 和 CREB 磷酸化水平显著增加，这表明 cAMP/PKA/CREB 信号通路被激活，其激活被姜黄素处理在 eWAT 中受到显著抑制。

Curcumin alleviated lipolysis in C26 conditional medium and a β3-AR agonist-induced adipose wasting model in vitro
姜黄素在 C26 条件培养基和 β3-AR 激动剂诱导的体外脂肪消耗模型中减轻脂肪分解

Curcumin did not show cytotoxicity to 3T3-L1 adipocytes at doses less than 25 µM (Fig. 6A). Western blotting analysis showed that treatment with high doses of curcumin (20 or 25 μM) inhibited the activation of ATGL and phosphorylated HSL in C26 medium-treated 3T3-L1 adipocytes; however, 25 μM curcumin significantly downregulated the expression of FASN (Fig. 6A and B). Oil Red O staining of fully differentiated 3T3-L1 adipocytes showed fewer lipid droplets in C26 culture medium and in CL316243-treated adipocytes than in the control group, and curcumin treatment (20 μM) increased the number of lipid droplets (Fig. 6C and D). Phosphorylation of HSL and ATGL protein expression were significantly increased in mature 3T3-L1 adipocytes stimulated by C26 culture medium and the β3-AR agonist CL, and curcumin effectively inhibited this upregulation (Fig. 6E and F). In addition, we observed that FASN expression was significantly downregulated in mature 3T3-L1 adipocytes stimulated with C26 culture medium, whereas curcumin increased FASN expression, which was not observed in the cell groups exposed to CL (Fig. 6E and F). Overall, these results suggested that curcumin inhibits lipolysis and promotes lipid synthesis through key players in the lipolysis and lipid utilization pathways.
姜黄素在剂量小于 25 μM 时对 3T3-L1 脂肪细胞没有显示出细胞毒性（ 图 6A）。Western blotting 分析显示，高剂量姜黄素 （20 或 25 μM） 处理抑制 C26 培养基处理的 3T3-L1 脂肪细胞中 ATGL 和磷酸化 HSL 的激活;然而，25 μM 姜黄素显着下调了 FASN 的表达（ 图 6A 和 B）。完全分化的 3T3-L1 脂肪细胞的油红 O 染色显示 C26 培养基和 CL316243 处理的脂肪细胞中的脂滴比对照组少，姜黄素处理 （20 μM） 增加了脂滴的数量（ 图 6C 和 D）。在 C26 培养基和 β3-AR 激动剂 CL 刺激的成熟 3T3-L1 脂肪细胞中，HSL 和 ATGL 蛋白表达的磷酸化显著增加，姜黄素有效抑制了这种上调（ 图 6E 和 F）。此外，我们观察到 FASN 表达在用 C26 培养基刺激的成熟 3T3-L1 脂肪细胞中显着下调，而姜黄素增加了 FASN 表达，这在暴露于 CL 的细胞组中未观察到（ 图 6E 和 F）。总体而言，这些结果表明姜黄素通过脂肪分解和脂质利用途径中的关键参与者抑制脂肪分解并促进脂质合成。
Fig 6
  1. Download: Download high-res image (2MB)
    下载： 下载高分辨率图片 （2MB）
  2. Download: Download full-size image
    下载： 下载全尺寸图像

Fig. 6. Curcumin alleviated lipolysis in C26 conditional medium and the β3-AR agonist-induced adipose wasting model in vitro. (A). The relative viability of the cells was calculated by CCK-8 reagent. (B). Effect of different curcumin doses on mature 3T3-L1 adipocytes. (C). Curcumin-treated 3T3-L1 cells stained with Oil Red O. (D). Quantification of E. (C). Western-blot images of p-HSL(Ser563), ATGL, and FASN in 3T3-L1 cells treated with C26 medium or CL or C26+Cur or CL+Cur. (F). Quantification of C. Data are expressed as the mean ± SD (*p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001).
图 6.姜黄素在体外减轻 C26 条件培养基和 β3-AR 激动剂诱导的脂肪消耗模型中的脂肪分解。（A）. 通过 CCK-8 试剂计算细胞的相对活力。不同姜黄素剂量对成熟 3T3-L1 脂肪细胞的影响。姜黄素处理的 3T3-L1 细胞用油红 O 染色。E. （C） 的定量。用 C26 培养基或 CL 或 C26+Cur 或 CL+Cur 处理的 3T3-L1 细胞中 p-HSL （Ser563） 、ATGL 和 FASN 的蛋白质印迹图像。C. 数据的定量表示为 SD ±平均值（*p < 0.05，** p < 0.01，***p < 0.001，**** p < 0.0001）。

Discussion  讨论

Patients with cachexia exhibit weight loss and massive muscle and adipose tissue loss, making it a serious challenge in the clinical management of cancer patients (Fearon et al., 2011). The effective treatment of cancer cachexia is important for increasing quality of life, prolonging survival, and improving the response to chemotherapy in cancer patients. However, to date, there are no effective therapeutic agents available in clinical practice, as the cancer cachexia pathogenesis is unknown (Wang et al., 2019). Cancer cachexia can be treated with aggressive nutritional support, but the therapeutic effect is limited due to high metabolism and low energy intake (Muscaritoli et al., 2021). Therefore, screening for effective anticachexia drugs to relieve the symptoms of cachectic patients is imminent. Currently, cancer cachexia is believed to be caused by metabolic abnormalities, including increased energy expenditure and catabolism as well as chronic inflammation (Dev, 2019); therefore, effective anti-inflammatory therapy and rectifying metabolic disorders are the appropriate approaches for the treatment of cachectic weight loss.
恶病质患者表现出体重减轻以及大量肌肉和脂肪组织流失，使其成为癌症患者临床管理的一个严重挑战（Fearon 等 人，2011 年）。癌症恶病质的有效治疗对于提高癌症患者的生活质量、延长生存期和改善对化疗的反应非常重要。然而，迄今为止，临床实践中没有有效的治疗剂，因为癌症恶病质的发病机制尚不清楚（Wang et al.， 2019）。癌症恶病质可以通过积极的营养支持来治疗，但由于高新陈代谢和低能量摄入，治疗效果有限（Muscaritoli 等 人，2021 年 ）。因此，筛选有效的抗恶病质药物以缓解恶病质患者的症状迫在眉睫。目前，癌症恶病质被认为是由代谢异常引起的，包括能量消耗和分解代谢增加以及慢性炎症 （Dev，2019  年 ）;因此，有效的抗炎疗法和纠正代谢紊乱是治疗恶病质性体重减轻的合适方法。
Curcumin, a natural polyphenol derived from turmeric, is a natural nonsteroidal anti-inflammatory agent that has been reported to affect different signaling pathways and molecular targets involved in a variety of cancers, either alone or in combination with other drugs (Giordano and Tommonaro, 2019). It has been reported that curcumin can inhibit cancer cell invasion and proliferation through its growth inhibitory and proapoptotic properties (Gao et al., 2022; Wroński et al., 2021; Zarei et al., 2021); however, the function and application of curcumin in cachexia associated weight loss have yet to be determined. In our study, by using the C26 cancer cachexia model, we observed significant protective effects of curcumin on weight loss in cachexia. In response to curcumin treatment, curcumin significantly attenuated cancer cachexia symptoms. The protective mechanism of curcumin on cachexia is mediated by its metabolite regulatory function, rather than its antitumor function. Although we observed that there was reduced tumor size after curcumin administration, curcumin significantly attenuated weight loss as well as fat atrophy compared to the antitumor agent 5-fluorouracil.
姜黄素是一种源自姜黄的天然多酚，是一种天然非甾体抗炎剂，据报道，单独或与其他药物联合使用，会影响涉及多种癌症的不同信号通路和分子靶标（Giordano 和 Tommonaro，2019  年）。据报道，姜黄素通过其生长抑制和促凋亡特性抑制癌细胞侵袭和增殖（Gao 等人 ，2022 年;Wroński 等 人，2021 年;Zarei et al.， 2021）;然而，姜黄素在恶病质相关体重减轻中的功能和应用尚未确定。在我们的研究中，通过使用 C26 癌症恶病质模型，我们观察到姜黄素对恶病质体重减轻的显着保护作用。对姜黄素治疗的反应，姜黄素显着减轻了癌症恶病质症状。姜黄素对恶病质的保护机制是由其代谢物调节功能介导的，而不是其抗肿瘤功能。尽管我们观察到姜黄素给药后肿瘤大小减小，但与抗肿瘤药物 5-氟尿嘧啶相比，姜黄素显着减轻了体重减轻和脂肪萎缩。
The hallmark pathological feature of cancer cachexia is adipose tissue loss. Our in vivo study confirmed that the weight loss protective effects of curcumin on cancer cachexia are mainly attributed to the inhibition of fat tissue atrophy since we observed a higher fat mass than muscle wasting in cancer cachexia. After curcumin treatment, there was significantly reduced adipose tissue depletion in C26 tumor-bearing mice, accompanied by increased serum TG levels and reduced release of FFAs in C26 tumor-bearing cachectic mice, indicating that curcumin protects lipid tissue.
癌症恶病质的标志性病理特征是脂肪组织丢失。我们的体内研究证实，姜黄素对癌症恶病质的减肥保护作用主要归因于抑制脂肪组织萎缩 ， 因为我们观察到癌症恶病质中的脂肪量高于肌肉萎缩 。姜黄素处理后，C26 荷瘤小鼠脂肪组织耗竭明显减少，C26 荷瘤恶病质小鼠血清 TG 水平升高和 FFA 释放减少，表明姜黄素保护脂质组织。
The pathological process of cachectic fat loss is characterized by altered lipid metabolism and browning of adipose tissue. During cancer cachexia, browning of WAT accompanied by increased heat generation in adipose tissue promotes lipid mobilization (Joshi and Patel, 2022). The overexpression of UCP1 is one of the key features of WAT browning, which is typically expressed in brown adipocyte mitochondrial membranes. UCP1 facilitates the uncoupling of fuel oxidation from ATP production, resulting in heat production (Petruzzelli et al., 2014). Our data revealed that treatment with curcumin significantly inhibited lipolysis and lipid browning in cachectic mice, and the protein expression levels of ATGL and UCP1 proteins were significantly reduced, as well as the phosphorylation levels of HSL. Moreover, the reduced TG levels and elevated FFA levels in the serum of cachectic mice were also improved. These facts demonstrated that curcumin could improve cachexia-induced lipoatrophy.
恶病质性脂肪流失的病理过程以脂质代谢改变和脂肪组织褐变为特征。在癌症恶病质期间，WAT 的褐变伴随着脂肪组织中热量产生的增加会促进脂质动员（Joshi 和 Patel，2022  年 ）。UCP1 的过表达是 WAT 褐变的关键特征之一，通常在棕色脂肪细胞线粒体膜中表达。UCP1 促进燃料氧化与 ATP 生产解耦，从而产生热量 （Petruzzelli 等 人，2014 年）。我们的数据显示，姜黄素处理显著抑制了恶病质小鼠的脂肪分解和脂质褐变，ATGL 和 UCP1 蛋白的蛋白表达水平显著降低，HSL 的磷酸化水平也显著降低。此外，恶病质小鼠血清中 TG 水平降低和 FFA 水平升高也有所改善。这些事实表明，姜黄素可以改善恶病质诱导的脂肪萎缩。
The development of cachexia fat loss is attributed to reduced lipogenesis and increased adipocyte lipolysis. In this study, curcumin markedly ameliorated cachexia-induced lipid degradation both in vitro and in vivo. In in vitro experiments, curcumin ameliorated lipid droplet reduction in fully differentiated 3T3-L1 adipocytes induced by C26 culture medium or CL, and the mechanism was mainly mediated by the inhibition of elevated expression of lipolysis-related regulatory enzymes. Specifically, curcumin-treated C26 tumor-bearing mice showed higher serum TG levels and lower in catabolic FFA. These results suggested that curcumin alleviated adipose tissue loss by inhibiting lipolysis. In cancer cachexia, lipolysis metabolism is highly activated (Fearon et al., 2012). In a clinical trial, ATGL overexpression and high levels of HSL phosphorylation were shown to cause lipolysis in WAT (Das et al., 2011). Interestingly, in another clinical trial, patients with cachexia showed lower levels of low-density lipoprotein (LDL), serum total cholesterol, and TG, while higher levels of glycerol and FFA were observed. Lipolysis in adipose tissue may have contributed to the increase in glycerol and FFA via a variety of cellular pathways (Riccardi et al., 2020). In our study, ATGL expression and phosphorylation HSL levels were significantly reduced in response to curcumin, which fully supports the notion that curcumin could block the pathological process of lipolysis.
恶病质脂肪减少的发展归因于脂肪生成减少和脂肪细胞脂肪分解增加。在这项研究中，姜黄素在体外和体内均显著改善了恶病质诱导的脂质降解。在体外实验中，姜黄素改善了 C26 培养基或 CL 诱导的完全分化 3T3-L1 脂肪细胞的脂滴减少，其机制主要由抑制脂肪分解相关调节酶的表达升高介导。具体来说，姜黄素处理的 C26 荷瘤小鼠表现出较高的血清 TG 水平和较低的分解代谢 FFA 水平。这些结果表明，姜黄素通过抑制脂肪分解来减轻脂肪组织损失。在癌症恶病质中，脂肪分解代谢高度激活（Fearon et al.， 2012）。在一项临床试验中，ATGL 过表达和高水平的 HSL 磷酸化被证明会导致 WAT 中的脂肪分解 （Das et al.， 2011）。有趣的是，在另一项临床试验中 ，恶病质患者的低密度脂蛋白 （LDL） 、血清总胆固醇和 TG 水平较低，而甘油和 FFA 水平较高。脂肪组织中的脂肪分解可能通过多种细胞途径导致甘油和 FFA 的增加（Riccardi 等 人，2020 年）。在我们的研究中，ATGL 表达和磷酸化 HSL 水平在姜黄素的刺激下显著降低，这完全支持姜黄素可以阻断脂肪分解病理过程的观点。
In cachexia-associated lipolysis the increased activity of the cAMP/PKA/CREB pathway contributes to the upregulation of HSL, ATGL, and UCP1. The cachexia mouse model has shown elevated serum parathyroid hormone protein-related peptide (PTHrP), a family B GPCR that activates the PKA signaling pathway, and catecholamines such as epinephrine (EPI) and norepinephrine (NE) signaling through β-AR, which also acts as a GPCR, induces the PKA signaling pathway to promote thermogenic gene expression. The effects of PTHrP on UCP1 and Dio2 transcription are mediated by the PKA pathway and therefore share signaling with β-AR (Kir et al., 2014; Mottillo and Granneman, 2011; Vilardaga et al., 2011). Subsequently, he activation of PKA causes HSL to be phosphorylated with perilipin 1 (PLIN1), releasing comparative gene identification 58 (CGI58), which in turn interacts with ATGL to activate TG hydrolase. CREB is a downstream transcription factor of PKA. When activated, PKA phosphorylates CREB, and phosphorylated CREB interacts with CBP (CREB-binding protein) and p300 to regulate UCP1 expression (Djouder et al., 2010; Lass et al., 2006; Nedergaard et al., 2001).
在恶病质相关脂肪分解中，cAMP/PKA/CREB 通路活性的增加有助于 HSL 、 ATGL 和 UCP1 的上调。恶病质小鼠模型显示血清甲状旁腺激素相关肽 （PTHrP） 升高，PTHrP 是一种激活 PKA 信号通路的 B 家族 GPCR，以及儿茶酚胺如肾上腺素 （EPI） 和去甲肾上腺素 （NE） 信号通过 β-AR 信号传导，也充当 GPCR，诱导 PKA 信号通路促进产热基因表达。PTHrP 对 UCP1 和 Dio2 转录的影响是由 PKA 通路介导的，因此与 β-AR 共享信号传导（Kir 等人 ，2014 年;Mottillo 和 Granneman，2011  年;Vilardaga et al.， 2011）。 随后，PKA 的激活导致 HSL 被 perilipin 1 （PLIN1） 磷酸化，释放比较基因鉴定 58 （CGI58），进而与 ATGL 相互作用以激活 TG 水解酶 。CREB 是 PKA 的下游转录因子。激活后，PKA 磷酸化 CREB，磷酸化的 CREB 与 CBP（CREB 结合蛋白）和 p300 相互作用以调节 UCP1 表达（Djouder 等人 ，2010 年;Lass et al.， 2006;Nedergaard et al.， 2001）。
Curcumin is an acetyltransferase p300/CREB binding protein-specific inhibitor (Balasubramanyam et al., 2004). In our in vivo experiments, elevated levels of PKA and CREB phosphorylation were elevated in the adipose tissue of cachectic mice, and curcumin inhibited the PKA and CREB phosphorylation, suggesting that curcumin inhibited the activation of the cAMP/PKA/CREB pathway, thereby preventing elevated levels of HSL phosphorylation, ATGL, and UCP1. Similarly, in vitro fat degradation experiments using CL simulating norepinephrine-induced fat degradation revealed that curcumin significantly inhibited fat atrophy. These facts suggest that curcumin might ameliorate cachexia-induced fat loss via the cAMP/PKA/CREB signaling pathway. Fig. 7 summarizes and illustrates the mechanism by which curcumin ameliorates cancer cachexia-associated fat atrophy.
姜黄素是一种乙酰转移酶 p300/CREB 结合蛋白特异性抑制剂 （Balasubramanyamet  al.， 2004）。在我们的体内实验中，恶病质小鼠脂肪组织中 PKA 和 CREB 磷酸化水平升高，姜黄素抑制 PKA 和 CREB 磷酸化，表明姜黄素抑制 cAMP/PKA/CREB 通路的激活，从而阻止 HSL 磷酸化、ATGL 和 UCP1 水平升高。同样，使用 CL 模拟去甲肾上腺素诱导的脂肪降解的体外脂肪降解实验表明，姜黄素显着抑制脂肪萎缩。这些事实表明，姜黄素可能通过 cAMP/PKA/CREB 信号通路改善恶病质诱导的脂肪流失。 图 7 总结并说明了姜黄素改善癌症恶病质相关脂肪萎缩的机制。
Fig 7
  1. Download: Download high-res image (755KB)
    下载： 下载高分辨率图像 （755KB）
  2. Download: Download full-size image
    下载： 下载全尺寸图像

Fig. 7. A possible mechanism by which curcumin ameliorates adipose atrophy during cancer cachexia. Different hormones such as PTHrP or EPI activate the cAMP/PKA/CREB pathway, which may result in increased levels of phosphorylation of lipolytic enzymes such as HSL, thus promoting lipolysis. At the same time, an increase in UCP1 leads to white fat browning, causing an energy imbalance. Curcumin mainly inhibits the phosphorylation of PKA and CREB during cancer cachexia thereby ameliorating adipose atrophy.
图 7.姜黄素在癌症恶病质期间改善脂肪萎缩的一种可能机制。PTHrP 或 EPI 等不同激素激活 cAMP/PKA/CREB 通路，这可能导致 HSL 等脂解酶的磷酸化水平增加，从而促进脂肪分解。同时，UCP1 的增加导致白色脂肪褐变，导致能量失衡。姜黄素主要抑制癌症恶病质期间 PKA 和 CREB 的磷酸化，从而改善脂肪萎缩。

Curcumin has been proven to have anti-obesity properties (Lone et al., 2016; Valentine et al., 2019). Furthermore, curcumin has been shown to reduce adiposity and has potential mechanisms involving the cAMP/PKA pathway (Kobori et al., 2018; Wang et al., 2016; Xie et al., 2012b). According to the available articles, curcumin modulates lipid metabolism and inhibits chronic inflammation by interacting with white adipose tissue, where high lipid levels trigger various inflammatory conditions, and in cachexia, various inflammatory responses also follow. Therefore, it cannot be said that curcumin alone can only combat obesity, but it regulates lipid metabolism disorders through its anti-inflammatory effects, and its regulation of lipid metabolism is a double-edged sword.
姜黄素已被证明具有抗肥胖特性（Lone 等 人，2016 年;Valentine et al.， 2019）。此外，姜黄素已被证明可以减少肥胖，并且具有涉及 cAMP/PKA 通路的潜在机制（Kobori 等 人，2018 年;Wang et al.， 2016;Xie et al.， 2012b）。根据现有的文章，姜黄素通过与白色脂肪组织相互作用来调节脂质代谢并抑制慢性炎症，其中高脂质水平会触发各种炎症，而在恶病质中，各种炎症反应也会随之而来。所以，不能说姜黄素单独只能对抗肥胖，但它通过抗炎作用调节脂质代谢紊乱 ，而它对脂质代谢的调节是一把双刃剑。
In addition to observing lipolysis, we also measured the regulatory effects of curcumin on lipogenesis. Although, the increase in lipolysis in cancer cachexia results in lipid atrophy, the enzyme FASN, which promotes lipid synthesis, was also found to be significantly downregulated in cancer cachexia in our experiments, and curcumin reversed this phenomenon, but the mechanism is unclear and needs to be further explored.
除了观察脂肪分解外，我们还测量了姜黄素对脂肪生成的调节作用。虽然癌症恶病质中脂肪分解的增加导致脂质萎缩，但在我们的实验中也发现促进脂质合成的 FASN 酶在癌症恶病质中显着下调，姜黄素逆转了这一现象，但机制尚不清楚，需要进一步探索。

Conclusion  结论

During the development of cancer cachexia, lipid atrophy often precedes weight loss and muscle atrophy, affecting lipid stores and disrupting the body's material and energy stores. The findings of this study demonstrated that curcumin may not only have antitumor activity but also attenuate cancer cachexia-induced adipose atrophy by affecting the cAMP/PKA/CREB signaling pathway. Most importantly, we observed that the anti-cachexia effects of curcumin are largely due to its functions in restricting FFA release and alleviating cachexia-associated adipocyte lipolysis, at the same time promoting lipogenesis, therefore recovering fat mass integrity. The findings of the current study suggested that curcumin could be an effective drug candidate for cancer cachexia treatment.
在癌症恶病质的发展过程中，脂质萎缩通常先于体重减轻和肌肉萎缩，影响脂质储存并破坏身体的物质和能量储存。本研究结果表明，姜黄素不仅可能具有抗肿瘤活性 ，还可以通过影响 cAMP/PKA/CREB 信号通路来减轻癌症恶病质诱导的脂肪萎缩。最重要的是，我们观察到姜黄素的抗恶病质作用主要是由于其在限制 FFA 释放和缓解恶病质相关脂肪细胞脂肪分解方面的功能，同时促进脂肪生成，从而恢复脂肪量完整性。目前的研究结果表明，姜黄素可能是治疗癌症恶病质的有效候选药物。

Funding  资金

This work was supported by grants from the National Natural Science Foundation of China (Nos. 82070912 and 81773326) and the Natural Science Foundation of Jiangsu Province China (No. BE2019676).
这项工作得到了中国国家自然科学基金 （第 82070912 号和第 81773326 号）和中国江苏省自然科学基金 （第 1 号）的资助。BE2019676）。

CRediT authorship contribution statement
CRediT 作者贡献声明

Ranran Wang: Conceptualization, Methodology, Formal analysis, Writing – original draft. Lulu Wei: Methodology, Validation, Formal analysis. Junaid Wazir: Writing – review & editing. Li Li: Validation, Formal analysis. Shiyu Song: Formal analysis. Kai Lin: Methodology. Wenyuan Pu: Validation. Chen Zhao: Validation. Zhonglan Su: Supervision. Quan Zhao: Supervision. Hongwei Wang: Conceptualization, Resources, Writing – review & editing.
王然然： 概念化、方法论、形式分析、写作 - 原稿。 魏璐璐： 方法、验证、形式分析。 朱奈德·瓦齐尔： 写作 - 审查和编辑。 李丽： 验证，形式分析。 宋诗瑜： 形式分析。 林凯： 方法论。 蒲文源： 验证。 陈钊： 验证。 苏钟兰： 监督。 赵全： 监督。 王宏伟： 概念化、资源、写作 – 审查和编辑。

Declaration of Competing Interest
利益争夺声明

The authors declare no conflicts of interest.
作者声明没有利益冲突。

Appendix. Supplementary materials
附录。补充材料

What’s this?  这是什么？

References  引用

Cited by (12)

  • Ndufa8 promotes white fat Browning by improving mitochondrial respiratory chain complex I function to ameliorate obesity by in vitro and in vivo

    2024, Cellular Signalling
    Citation Excerpt :

    After 6 h, the normal medium was replaced. The adipocyte browning model was constructed as described previously [23], differentiated mature 3T3-L1 cells were treated with 10 μM CL316, 243 (CL) for 6 h after transfection. For adipocyte oxidative stress modeling, differentiated mature adipocytes were treated with 200 μM H2O2 for 24 h [24].

  • Exposure to atrazine stimulates progesterone secretion and induces oxidative stress, inflammation, and apoptosis in the ovary of pseudopregnant rats

    2024, Chemosphere
    Citation Excerpt :

    The current ATR-induced increases in StAR, CYP11A1, and HSD3B mRNA and protein levels were consistent with previous studies in rat co-cultured Sertoli-germ cells and Leydig cells (Abarikwu et al., 2012a, 2012b). The PKA/CREB signaling pathway has been involved in various biological processes, including hormone synthesis and cell cycle (Wang et al., 2023). The current ATR treatment increased the mRNA and protein levels of CREB but not PKA.

  • Arctiin, a lignan compound, enhances adipose tissue browning and energy expenditure by activating the adenosine A2A receptor

    2025, Molecular Medicine

  • Global gene expression profiling of perirenal brown adipose tissue whitening in goat kids reveals novel genes linked to adipose remodeling

    2024, Journal of Animal Science and Biotechnology

  • Astaxanthin Ameliorates Skeletal Muscle Atrophy in Mice With Cancer Cachexia

    2024, Nutrition and Cancer

  • Unlocking Prognostic Genes and Multi-Targeted Therapeutic Bioactives from Herbal Medicines to Combat Cancer-Associated Cachexia: A Transcriptomics and Network Pharmacology Approach

    2024, International Journal of Molecular Sciences
View all citing articles on Scopus
View Abstract
© 2022 Elsevier GmbH. All rights reserved.