探讨利拉鲁肽减轻肾纤维化的机制：Fsp1-CoQ10-NAD（P）H通路-动脉网

Exploring Liraglutide’s mechanism in reducing renal fibrosis: the Fsp1-CoQ10-NAD(P)H pathway

Nature 等信源发布 2025-01-12 12:30

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可切换为仅中文

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Abstract

摘要

Studies have confirmed that elevated glucose levels could lead to renal fibrosis through the process of ferroptosis. Liraglutide, a human glucagon-like peptide-1 (GLP-1) analogue, is a potential treatment option for diabetes. This study aimed to examine the potential of liraglutide (LIRA) in inhibiting ferroptosis and reducing high glucose-induced renal fibrotic injury in mice, and whether the Fsp1-CoQ10-NAD(P)H signal pathway is a mechanism for this effect.

研究证实，葡萄糖水平升高可能通过铁浓化过程导致肾纤维化。利拉鲁肽是一种人类胰高血糖素样肽-1（GLP-1）类似物，是糖尿病的潜在治疗选择。本研究旨在探讨利拉鲁肽（LIRA）在抑制小鼠铁浓化和减少高糖诱导的肾纤维化损伤方面的潜力，以及Fsp1-CoQ10-NAD（P）H信号通路是否是这种作用的机制。

In our study, we used db/db mice to simulate Type 2 diabetes mellitus (T2DM). The mice were intraperitoneally injected with LIRA (200 µg/kg/d) daily for 6 weeks. Renal function, pathologic changes, lipid peroxidation levels, iron levels, and ferroptosis were assessed. First, LIRA ameliorated renal dysfunction and fibrosis in db/db mice.

在我们的研究中，我们使用db/db小鼠来模拟2型糖尿病（T2DM）。每天给小鼠腹膜内注射LIRA（200µg/kg/d），持续6周。评估肾功能，病理变化，脂质过氧化水平，铁水平和铁下垂。首先，LIRA改善了db/db小鼠的肾功能不全和纤维化。

Second, LIRA inhibited lipid peroxidation by up-regulating T-SOD, GSH-Px, and GSH activities as well as down-regulating the levels of 8-OHDG, MDA, LPO, 4-HNE, 12-Lox, and NOX4 in db/db mice. In addition, LIRA attenuated iron deposition by decreasing the expression of TfR1 and increasing the expression of FPN1.

其次，LIRA通过上调T-SOD，GSH-Px和GSH活性以及下调db/db小鼠中8-OHDG，MDA，LPO，4-HNE，12-Lox和NOX4的水平来抑制脂质过氧化。此外，LIRA通过降低TfR1的表达和增加FPN1的表达来减弱铁沉积。

Meanwhile, LIRA reduced high levels of high glucose-induced cell viability decline and intracellular lipid peroxidation. Furthermore, LIRA inhibited ferroptosis by adjusting the Fsp1-CoQ10-NAD(P)H pathway in vivo and in vitro. These findings suggested that LIRA attenuated kidney fibrosis injury in db/db mice by inhibiting ferroptosis through the Fsp1-CoQ10-NAD(P)H pathway..

同时，LIRA降低了高水平的高糖诱导的细胞活力下降和细胞内脂质过氧化。此外，LIRA通过在体内和体外调节Fsp1-CoQ10-NAD（P）H途径来抑制ferroptosis。这些发现表明，LIRA通过Fsp1-CoQ10-NAD（P）H途径抑制铁蛋白沉积，从而减轻db/db小鼠的肾纤维化损伤。。

Introduction

导言

Type 2 diabetes mellitus (T2DM) is a prevalent metabolic disorder characterized by disturbances in glucose-lipid metabolism, primarily resulting from insufficient insulin production or resistance to insulin

. The global prevalence of diabetes has escalated significantly, leading to increased mortality rates and reduced life expectancy

。全球糖尿病患病率显着上升，导致死亡率增加和预期寿命缩短

. In patients with T2DM, persistent hyperglycemia plays a direct role in the development of substantial renal impairments, with diabetic kidney disease representing one of its most severe chronic complications

在T2DM患者中，持续性高血糖症在实质性肾损伤的发展中起着直接作用，糖尿病肾病是其最严重的慢性并发症之一

The main features of diabetic nephropathy are proteinuria and a gradual decline in renal function. The underlying pathology is characterized by the accumulation of extracellular matrix (ECM) and damage in the glomerular and tubular interstitium, which ultimately contributes to the development of renal fibrosis and subsequent disruption of renal structure and function.

糖尿病肾病的主要特征是蛋白尿和肾功能逐渐下降。潜在的病理学特征是细胞外基质（ECM）的积累以及肾小球和肾小管间质的损伤，最终导致肾纤维化的发展以及随后肾脏结构和功能的破坏。

. Previous studies on hyperglycemia-mediated activation of renal fibrosis have mostly focused on oxidative stress, neurohumoral mechanisms and pro-inflammatory cascade responses

先前关于高血糖介导的肾纤维化激活的研究主要集中在氧化应激，神经体液机制和促炎级联反应上

. However, recent studies have shown that ferroptosis appeared to play an important role in renal fibrotic injury

然而，最近的研究表明，ferroptosis似乎在肾纤维化损伤中起重要作用

Ferroptosis is a regulated mechanism of cellular demise primarily distinguished by iron-dependent oxidative stress and lipid peroxidation

铁浓化是细胞死亡的调节机制，主要以铁依赖性氧化应激和脂质过氧化为特征

. Oxidative stress could lead to tubular and vascular endothelial cell damage and secretion of pro-fibrotic factors, ultimately leading to fibroblast/pericyte activation and fibroblast/macrophage recruitment

氧化应激可能导致肾小管和血管内皮细胞损伤以及促纤维化因子的分泌，最终导致成纤维细胞/周细胞活化和成纤维细胞/巨噬细胞募集

. Notably, a study has shown that HK-2 cells, which are human renal tubular cells, exhibit both proliferation and fibroblast activation during the process of ferroptosis

值得注意的是，一项研究表明，HK-2细胞是人肾小管细胞，在铁浓化过程中表现出增殖和成纤维细胞活化

. There is empirical evidence that that lipid peroxidation products, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), within the bloodstream of patients afflicted with chronic kidney disease. Additionally, chronic kidney disease patients exhibit a significant decrease in levels of glutathione peroxidase (GSH-Px) and glutathione (GSH).

有经验证据表明，慢性肾脏病患者血液中的脂质过氧化产物，如4-羟基壬烯醛（4-HNE）和丙二醛（MDA）。此外，慢性肾病患者的谷胱甘肽过氧化物酶（GSH-Px）和谷胱甘肽（GSH）水平显着降低。

Furthermore, the presence of fibrosis in these patients amplifies the production of reactive oxygen species (ROS).

此外，这些患者中纤维化的存在放大了活性氧（ROS）的产生。

. Therefore, targeting ferroptosis may may be significant importance in the fight against fibrosis.

因此，针对ferroptosis可能在对抗纤维化方面具有重要意义。

Glucagon-like peptide-1 (GLP-1) is widely used in the treatment of T2DM due to its ability to increase glucagon-dependent insulin release and decrease glucagon secretion

胰高血糖素样肽-1（GLP-1）具有增加胰高血糖素依赖性胰岛素释放和减少胰高血糖素分泌的能力，因此被广泛用于治疗T2DM

. Liraglutide (LIRA), a GLP-1 receptor agonist, has demonstrated a promising prognosis for chronic kidney disease in T2DM, outperforming other GLP-1 receptor agonists in terms of glycaemic control and overall patient tolerability

. Furthermore, previous research has demonstrated that LIRA possessed a beneficial effect on chronic renal failure, particularly in reducing renal fibrosis. However, the precise mechanism, by which LIRA exerts its inhibitory effect on fibrosis remains incompletely understood

此外，先前的研究表明，LIRA对慢性肾衰竭具有有益作用，特别是在减少肾纤维化方面。然而，LIRA对纤维化发挥抑制作用的确切机制尚不完全清楚

. Recent studies have also indicated that LIRA was able to reduce oxidative stress levels in SH-SY5Y cells and inhibit iron overload in the hippocampus of diabetic mice

最近的研究还表明，LIRA能够降低SH-SY5Y细胞的氧化应激水平，并抑制糖尿病小鼠海马的铁超负荷

. Taken together, these findings provide valuable insights into the comprehensive mechanism of action of LIRA.

综上所述，这些发现为LIRA的综合作用机制提供了有价值的见解。

It is well known that mice with genetic defects in the leptin receptor (db/db) or leptin itself (ob/ob) have been widely used as models of T2DM, and although ob/ob and db/db mice are similar in terms of weight gain and obesity, db/db mice have a higher risk of diabetes, with an onset similar to that of T2DM kidney disease.

众所周知，瘦素受体（db/db）或瘦素本身（ob/ob）具有遗传缺陷的小鼠已被广泛用作T2DM模型，尽管ob/ob和db/db小鼠在体重增加和肥胖方面相似，但db/db小鼠患糖尿病的风险较高，其发病类似于T2DM肾病。

. Therefore, we used the db/db mouse model to simulate T2DM to investigate the potential correlation between T2DM-induced kidney injury and ferroptosis, as well as the therapeutic effects of LIRA. The findings of our study could potentially contribute to the existing theoretical framework for managing diabetic kidney injury..

因此，我们使用db/db小鼠模型来模拟T2DM，以研究T2DM诱导的肾损伤与ferroptosis之间的潜在相关性，以及LIRA的治疗效果。我们的研究结果可能有助于管理糖尿病肾损伤的现有理论框架。。

Results

结果

Liraglutide alleviated glycometabolism disorders in db/db mice

利拉鲁肽减轻db/db小鼠的糖代谢紊乱

We used db/db mice to study the pathogenesis of T2DM and its associated complications. At the beginning of the 9th week, blood glucose was monitored in each group. Subsequently, the mice were treated with LIRA for five weeks while blood glucose levels were continuously monitored (Fig.

我们使用db/db小鼠来研究T2DM及其相关并发症的发病机制。在第9周开始时，监测每组的血糖。随后，用LIRA处理小鼠五周，同时连续监测血糖水平（图）。

A). The results showed a statistically significant increase in fasting blood glucose levels in db/db mice compared to db/m mice, with a continuous rise observed starting from the 11th week (Fig.

A）。结果显示，与db/m小鼠相比，db/db小鼠的空腹血糖水平在统计学上显着增加，从第11周开始观察到持续升高（图）。

B). Following LIRA administration, a notable reduction in elevated blood glucose levels was observed in db/db mice starting from the 12th week (Fig.

B）。LIRA给药后，从第12周开始，db/db小鼠的血糖水平显着降低（图）。

B). At the end of the 14th week, tolerance to intraperitoneal pyruvate, a marker of gluconeogenesis, was assessed. Compared to db/m mice, pyruvate tolerance levels were significantly higher after 120 min, but decreased after LIRA treatment (Fig.

B）。在第14周结束时，评估了对腹腔内丙酮酸（糖异生的标志物）的耐受性。与db/m小鼠相比，丙酮酸耐受水平在120分钟后显着升高，但在LIRA治疗后下降（图）。

C). The results also showed a significant increase in HOMA-IR in db/db mice, which was later reduced after LIRA administration (Fig.

C）。结果还显示db/db小鼠的HOMA-IR显着增加，LIRA给药后HOMA-IR随后降低（图）。

D). The db/db mice presented increased diastolic and systolic blood pressure, which was reduced after LIRA treatment (Fig.

D）。db/db小鼠表现出舒张压和收缩压升高，LIRA治疗后血压降低（图）。

E-F). The triglycerides (TG) and cholesterol (CHO) levels were elevated in the db/db mice, and CHO levels were reduced after LIRA treatment (Fig.

E-F）。db/db小鼠的甘油三酯（TG）和胆固醇（CHO）水平升高，LIRA治疗后CHO水平降低（图）。

G). The kidney coefficient (KLW/BW) ratio (Fig.

G）。肾脏系数（KLW/BW）比值（图）。

H) and left kidney weight (Fig.

H）和左肾重量（图）。

I) were all elevated in the db/db group, and the elevated weight was reduced to normal after LIRA treatment. The albumin content was increased (Fig.

一） db/db组均升高，LIRA治疗后体重升高降至正常。白蛋白含量增加（图）。

J), while, the urea nitrogen (BUN) (Fig.

J），而尿素氮（BUN）（图）。

K), creatinine (Scr) (Fig.

K），肌酐（Scr）（图）。

K), beta-2 microglobulin (beta 2-MG) (Fig.

K） β2微球蛋白（β2-MG）（图。

L), and n-acetyl-β-D-glucosidase (NAG) (Fig.

五十）和n-乙酰基-β-D-葡萄糖苷酶（NAG）（图）。

M) levels were elevated in the db/db mice. These indicators returned to almost normal levels after LIRA treatment (Fig.

M） db/db小鼠的水平升高。LIRA治疗后，这些指标恢复到几乎正常水平（图）。

J–M). Based on these findings, we have demonstrated that LIRA effectively improved glucose metabolism and insulin resistance in db/db mice.

。基于这些发现，我们已经证明LIRA有效地改善了db/db小鼠的葡萄糖代谢和胰岛素抵抗。

Fig. 1

图1

LIRA improved the disorders of glycol-metabolism in db/db mice. (

LIRA改善了db/db小鼠的乙二醇代谢紊乱。（笑声）(

) The schematic diagram of the experimental flow. (

)实验流程的示意图。（笑声）(

B类

) The blood glucose of mice from the 9th to 15th week. (The data are shown as the means ± SEM.

)第9至15周小鼠的血糖。（数据显示为平均值±SEM。

= 6. **

< 0.01 vs. db/m group.

与db/m组相比<0.01。

< 0.01 vs. db/db group.

与db/db组相比<0.01。

< 0.01 vs. db/db group 9th week.

第9周与db/db组相比<0.01。

< 0.05,

< 0.01 vs. LIRA group 9th week). (

与LIRA组相比，第9周<0.01）。（笑声）(

C级

)

The intraperitoneal glucose tolerance test of mice with 120 min. (

120分钟小鼠腹腔葡萄糖耐量试验。（笑声）(

) The HOMA-insulin resistance (HOMA-IR) of db/m, db/db and LIRA groups. (

)db/m，db/db和LIRA组的HOMA胰岛素抵抗（HOMA-IR）。（笑声）(

–

F级

) The diastolic and systolic pressure. (

)舒张压和收缩压。（笑声）(

克

) The triglyceride (TG) and cholesterol (CHO) levels in serum. (

)血清中的甘油三酯（TG）和胆固醇（CHO）水平。（笑声）(

小时

–

我

) The ratio of LKW/BW and the left kidney weight of mice in each group. (

)每组小鼠的LKW/BW比率和左肾重量。（笑声）(

–

) The albumin, BUN, Scr, beta-2 microglobulin (beta 2-MG), and n-acetyl-β-D-glucosidase (NAG) content in serum of mice in different groups. The data are shown as the means ± SEM.

)各组小鼠血清白蛋白、BUN、Scr、β-2微球蛋白（β2-MG）和n-乙酰-β-D-葡萄糖苷酶（NAG）含量。数据显示为平均值±SEM。

= 6. **

< 0.05 vs. db/m group.

与db/m组相比<0.05。

< 0.01 vs. db/db group).

与db/db组相比<0.01）。

Full size image

全尺寸图像

Liraglutide significantly attenuated kidney fibrosis damage in db/db mice

利拉鲁肽显着减轻db/db小鼠的肾纤维化损伤

The pathological structure of the kidney was assessed by HE staining. In kidney sections from db/m mice, no discernible structural changes were observed. However, sections from db/db mice showed compensatory dilation of the renal tubules, variable sized vacuoles and partial atrophy of the glomeruli, as shown in Fig. .

通过HE染色评估肾脏的病理结构。在db/m小鼠的肾脏切片中，未观察到明显的结构变化。然而，来自db/db小鼠的切片显示肾小管的代偿性扩张，可变大小的液泡和肾小球的部分萎缩，如图所示。

A-B. We also evaluated renal fibrosis by Masson’s stain (Fig.

A-B.我们还通过Masson染色评估了肾纤维化（图）。

C-D), Sirius red stain (Fig.

。

E-F), reticulocyte fibre stain (Fig.

E-F），网织红细胞纤维染色（图）。

G-H) and elastic fibre stain (Fig.

G-H）和弹性纤维染色（图）。

I-J). These analyses showed a significant increase in the renal fibrosis area in db/db mice. In addition, the renal tissues of the db/db group showed a significant increase in the expression of Collagen-I, Collagen-III and transforming growth factor-β (TGF-β) as observed by western blotting analysis (Fig. .

I-J）。这些分析显示db/db小鼠的肾纤维化面积显着增加。此外，通过蛋白质印迹分析观察到，db/db组的肾组织中胶原蛋白I，胶原蛋白III和转化生长因子-β（TGF-β）的表达显着增加（图。

K-L). The elevated levels of fibrosis were significantly reduced after LIRA administration (Fig.

K-L）。LIRA给药后纤维化水平显着降低（图）。

). The results indicated that db/db mice exhibited kidney damage along with fibrosis and that the use of LIRA resulted in an improvement in renal fibrosis.

)。结果表明，db/db小鼠表现出肾损伤和纤维化，并且使用LIRA导致肾纤维化的改善。

Fig. 2

图2

The kidney dysfunction and fibrosis in db/db mice. (

db/db小鼠的肾功能障碍和纤维化。（笑声）(

–

B类

) HE stain (

)HE染色(

= 3). (

3.(

C级

–

) Masson stain (

)马森染色(

= 3). (

3.(

–

F级

) Sirius red stain (

)天狼星红染色(

= 3). (

3.(

克

–

小时

) Reticulocyte fibre stain (

)网织红细胞纤维染色(

= 3). (

3.(

我

–

) Elastic fibre stain (

)弹性纤维染色(

= 3). (

3.(

–

) The expression and statistics of Collagen-I, Collagen-III and TGF-β proteins in kidney tissue by western blotting (

)免疫印迹法检测肾组织中Ⅰ型胶原、Ⅲ型胶原和转化生长因子β蛋白的表达及统计学意义(

= 6). The results are presented as the mean ± SEM. The scale bar = 25 or 75 μm. *

结果表示为平均值±SEM。比例尺=25或75μm*

< 0.05, **

< 0.01 vs. db/m group.

与db/m组相比<0.01。

< 0.05,

< 0.01 vs. db/db group.

与db/db组相比<0.01。

Full size image

全尺寸图像

Liraglutide markedly improved iron-dependent oxidative stress damage in db/db mice

利拉鲁肽显着改善db/db小鼠的铁依赖性氧化应激损伤

An excess of iron intensifies the production of ROS and leads to lipid peroxidation injury. Therefore, we evaluated iron levels in db/db mice. Perls’ staining revealed iron levels increased in the kidneys of db/db mice compared to the db/m group, suggesting the presence of iron overload in db/db mice (Fig. .

过量的铁会加剧ROS的产生并导致脂质过氧化损伤。因此，我们评估了db/db小鼠的铁水平。Perls染色显示，与db/m组相比，db/db小鼠肾脏中的铁水平升高，表明db/db小鼠存在铁超负荷（图）。

A-B). In addition, the levels of iron-storage proteins, mainly ferritin heavy chain (FTH) and ferritin light chain (FTL), exhibited a significant rise in the kidney tissue of db/db mice (Fig.

A-B）。此外，铁储存蛋白（主要是铁蛋白重链（FTH）和铁蛋白轻链（FTL））的水平在db/db小鼠的肾组织中显着升高（图）。

C-D). Afterwards, we proceeded to examine the expression of proteins involved in the control of iron absorption and release. Western blotting analysis showed that the db/db mice exhibited significantly lower levels of ferroportin 1 (FPN1, an iron export protein) and higher levels of divalent metal transporter 1 (DMT1, the divalent iron uptake protein) and transferrin receptor 1 (TfR1, the ferric iron uptake protein) (Fig. .

C-D）。之后，我们继续检查参与控制铁吸收和释放的蛋白质的表达。。

E-F). Notably, treatment with LIRA resulted in a significant reduction in kidney iron levels.

E-F）。值得注意的是，用LIRA治疗导致肾铁水平显着降低。

Fig. 3

图3

The iron levels in the kidney tissue of db/db mice. (

db/db小鼠肾组织中的铁水平。（笑声）(

) Perls’ staining of kidney tissue (scale bar = 75 or 50 μm,

)肾组织的Perls染色（比例尺=75或50μm，

= 3). (

3.(

B类

) The mean density of Fe content is shown in panel A. (

)Fe含量的平均密度如图A所示(

C级

–

) The expression and statistics of FTL and FTH proteins (

)FTL和FTH蛋白的表达和统计(

= 6). (

6.(

–

F级

) The expression and statistics of FPN1, DMT1, and TfR1 proteins by western blotting (

)western blotting检测FPN1、DMT1和TfR1蛋白的表达和统计(

= 6). The results are presented as the mean ± SEM. *

结果表示为平均值±SEM*

< 0.05, **

< 0.01 vs. db/m group.

与db/m组相比<0.01。

< 0.05,

< 0.01 vs. db/db group.

与db/db组相比<0.01。

Full size image

全尺寸图像

At the same time, LIRA therapy effectively ameliorated iron-dependent oxidative stress damage in db/db mice. This was demonstrated by the lower levels of total superoxide dismutase (T-SOD) and catalase (CAT) activities in the renal tissue of db/db mice compared to db/m mice (Fig.

同时，LIRA治疗有效改善了db/db小鼠的铁依赖性氧化应激损伤。与db/m小鼠相比，db/db小鼠肾组织中总超氧化物歧化酶（T-SOD）和过氧化氢酶（CAT）活性水平较低（图）。

A-B). In addition, the higher levels of serum 8-hydroxy-2-deoxyguanosine (8-OHdG), and elevated malonaldehyde (MDA) and lipid peroxidation (LPO) levels in renal tissue were also found in db/db mice (Fig.

A-B）。此外，在db/db小鼠中也发现血清8-羟基-2-脱氧鸟苷（8-OHdG）水平较高，肾组织中丙二醛（MDA）和脂质过氧化（LPO）水平升高（图）。

C-E). At the same time, NAD(P)H Oxidase 4 (NOX-4), 4-HNE and 12-Lox protein levels were all increased in the renal tissues of db/db mice as detected by western blotting (Fig.

C-E）。同时，通过蛋白质印迹检测，db/db小鼠肾组织中NAD（P）H氧化酶4（NOX-4），4-HNE和12-Lox蛋白水平均升高（图）。

F-G). However, LIRA treatment increased T-SOD and CAT activities, while decreasing 8-OHdG, MDA, LPO, NOX-4, 4-HNE and 12-Lox levels compared to the db/db group (Fig.

F-G）。然而，与db/db组相比，LIRA处理增加了T-SOD和CAT活性，同时降低了8-OHdG，MDA，LPO，NOX-4，4-HNE和12-Lox水平（图）。

). These observations indicated that db/db mice exhibited elevated levels of iron and oxidative stress, which could be ameliorated by LIRA.

)。这些观察结果表明db/db小鼠表现出升高的铁水平和氧化应激，这可以通过LIRA改善。

Fig. 4

图4

The oxidative stress levels in the kidney tissue of db/db mice. (

db/db小鼠肾组织中的氧化应激水平。（笑声）(

–

B类

) The T-SOD and CAT activities in kidney tissue (

)肾组织中T-SOD和CAT活性(

= 5). (

5.(

C级

) The 8-hydroxy-2 deoxyguanosine (8-OHDG) in serum (

)血清中的8-羟基-2-脱氧鸟苷（8-OHDG）(

= 6). (

6.(

–

) The MDA and LPO content in kidney tissue (

)肾组织中MDA和LPO含量(

= 5). (

5.(

F级

–

克

) The expression and statistics of NOX-4, 4-HNE and 12-Lox proteins in kidney tissues (

)NOX-4、4-HNE和12-Lox蛋白在肾组织中的表达及统计学分析(

= 6). The results are presented as the mean ± SEM. *

结果表示为平均值±SEM*

< 0.05, **

< 0.01 vs. db/m group.

与db/m组相比<0.01。

< 0 05,

< 0.01 vs. db/db group.

与db/db组相比，小于0.01。

Full size image

全尺寸图像

Liraglutide prevented ferroptosis in the kidneys of db/db mice

利拉鲁肽预防db/db小鼠肾脏铁垂

Studies have shown that ferroptosis is associated with increased levels of iron and lipid peroxidation. Electron microscopy revealed that the mitochondrial membranes of the db/db mice were wrinkled, the cristae were reduced or disappeared and the outer membrane ruptured (Fig.

研究表明，铁浓化与铁和脂质过氧化水平升高有关。电子显微镜显示db/db小鼠的线粒体膜起皱，嵴减少或消失，外膜破裂（图）。

A). We then detected the activity of GSH, an indicator of ferroptosis, and observed a notable reduction in the levels of GSH and GSH-Px in db/db mice (Fig.

A）。然后，我们检测了GSH的活性，GSH是铁浓化的指标，并观察到db/db小鼠中GSH和GSH-Px水平的显着降低（图）。

B-C). In the db/db group, western blotting analysis revealed a significant decrease in the levels of GPX-4 and Fsp1 compared to the db/m group (Fig.

B-C）。在db/db组中，蛋白质印迹分析显示，与db/m组相比，GPX-4和Fsp1水平显着降低（图）。

D). In the meantime, immunohistochemical analysis also revealed a decrease in GPX-4 levels in the renal tissues of db/db mice (Fig.

D）。同时，免疫组织化学分析还显示db/db小鼠肾组织中GPX-4水平降低（图）。

E). Furthermore, the ratio of NAD

E）。此外，NAD的比例

/NADH and CoQ10(H

/NADH和辅酶Q10（H）

)/CoQ10 were respectively down-regulated in the kidney tissue of the db/db mice (Fig.

)/辅酶Q10在db/db小鼠的肾组织中分别下调（图）。

F-G). However, LIRA treatment reversed all the trends (Fig.

F-G）。然而，LIRA治疗逆转了所有趋势（图）。

). These results suggested that LIRA could potentially reduce ferroptosis in the kidneys of db/db mice via the Fsp1-CoQ10-NADH pathway.

)。这些结果表明，LIRA可能通过Fsp1-CoQ10-NADH途径减少db/db小鼠肾脏的铁浓化。

Fig. 5

图5

The ferroptosis in kidney tissue of db/db mice. (

db/db小鼠肾组织中的铁蛋白沉积。（笑声）(

) The ultrastructure of mitochondria observed by TEM. (

)透射电镜观察线粒体超微结构。（笑声）(

B类

–

C级

) The activity of GSH and GSH-Px in the kidney tissue (

)肾组织中GSH和GSH-Px的活性(

= 5). (

5.(

) The expression and statistics of GPX-4 and Fsp1 proteins (

)GPX-4和Fsp1蛋白的表达和统计(

= 6). (

6.(

) The immunohistochemical staining of GPX-4 protein (scale bar = 100 μm,

)GPX-4蛋白的免疫组织化学染色（比例尺=100μm，

= 3). (

3.(

F级

–

克

) The ratio of NAD

)NAD的比例

/NADH and CoQ10 (H

/NADH和辅酶Q10（H

)/CoQ10 (

/辅酶Q10

= 4). The results are presented as the mean ± SEM. *

结果表示为平均值±SEM*

< 0.05, **

< 0.01 vs. db/m group.

与db/m组相比<0.01。

< 0.05,

< 0.01 vs. db/db group.

与db/db组相比<0.01。

Full size image

全尺寸图像

Liraglutide inhibited the HG-induced ferroptosis in HK-2 cells

利拉鲁肽抑制汞诱导的HK-2细胞铁浓化

To further verify whether LIRA suppresses ferroptosis, we selected HK-2 cells with high glucose (HG) to mimic hyperglycaemia in vitro. HK-2 cells were treated with different concentrations of HG for 24 and 48 h (Fig.

为了进一步验证LIRA是否抑制ferroptosis，我们选择了具有高糖（HG）的HK-2细胞来模拟体外高血糖。用不同浓度的HG处理HK-2细胞24和48小时（图）。

A-B). Therefore, we chose a concentration of 30 mM HG for 48 h as the condition for later cell culture. Then, cells were divided into: Con, HG, RSL-3 (5 µM), LIRA (HG + 500 nM LIRA), and Fer-1 (HG + 5 µM Fer-1) groups. Cell viability was significantly reduced in the HG and RSL-3 groups compared to the control group, as shown in Fig. .

A-B）。因此，我们选择浓度为30毫米汞柱48小时作为以后细胞培养的条件。然后，将细胞分为：Con，HG，RSL-3（5μM），LIRA（HG +500 nM LIRA）和Fer-1（HG +5μMFER-1）组。与对照组相比，HG和RSL-3组的细胞活力显着降低，如图所示。

C. Furthermore, the cell viability recovered in the LIRA group was comparable to that in the Fer-1 group (Fig.

C、此外，LIRA组恢复的细胞活力与Fer-1组相当（图）。

C-E). Next, we observed the ultrastructural characteristics of mitochondria and mitochondrial membrane potential (MMP) in each group. In the HG group, the mitochondria were smaller with fragmented ridges and localized fat infiltration (Fig.

C-E）。接下来，我们观察了每组线粒体和线粒体膜电位（MMP）的超微结构特征。在HG组中，线粒体较小，脊碎裂，局部脂肪浸润（图）。

F), accompanying with the decline of MMP levels (Fig.

F），伴随着MMP水平的下降（图）。

G), which was similarly to the results in the RSL-3 group. The administration of LIRA and Fer-1 improved the mitochondrial ultrastructure and MMP, as shown in Fig.

G），这与RSL-3组的结果相似。LIRA和Fer-1的给药改善了线粒体超微结构和MMP，如图所示。

F-G. In addition, the MFI of BODIPY 581/591 C11 was elevated in the HG group, and the alterations were reversed by LIRA treatment (Fig.

此外，HG组BODIPY 581/591 C11的MFI升高，LIRA治疗逆转了这种改变（图）。

H-I). Western blotting analysis revealed that GPX-4 and Fsp1 expression levels were decreased in the HG group, aligning with the in vivo findings (Fig.

H-I）。蛋白质印迹分析显示，HG组GPX-4和Fsp1表达水平降低，与体内发现一致（图）。

J). In contrast, the LIRA and Fer-1 groups displayed contrasting outcomes (Fig.

J）。相反，LIRA和Fer-1组显示出相反的结果（图）。

J). Meanwhile, the immunofluorescence double-label staining revealed a notable rise in the expression of 4-HNE protein and a decline in the expression of Fsp1 protein in HK-2 cells following exposure to HG, which could be improved by LIRA and Fer-1 (Fig.

J）。同时，免疫荧光双标染色显示，暴露于HG后，HK-2细胞中4-HNE蛋白的表达显着增加，Fsp1蛋白的表达下降，这可以通过LIRA和Fer-1得到改善（图1）。

K). Furthermore, the ratio of NAD

K）。此外，NAD的比例

/NADH and CoQ10(H

/NADH和辅酶Q10（H）

)/CoQ10 were respectively down-regulated in the HG group, and it was reversed by LIRA and Fer-1 treatment (Fig.

)/HG组辅酶Q10分别下调，LIRA和Fer-1治疗可逆转辅酶Q10（图）。

L-M). These results further confirmed that LIRA could inhibit ferroptosis induced by HG.

L-M）。这些结果进一步证实，LIRA可以抑制HG诱导的铁浓化。

Fig. 6

图6

Liraglutide inhibited HG-induced ferroptosis in HK-2 cells. (

利拉鲁肽抑制HG诱导的HK-2细胞铁浓化。（笑声）(

–

B类

) The cell viability of HK-2 cells treated with HG with 0, 10, 20, 30, and 50 mM for 24 h and 48 h respectively (

)分别用0、10、20、30和50 mM的HG处理HK-2细胞24小时和48小时(

= 5, **

< 0.01 vs. Con group). (

与对照组相比，<0.01）。(

C级

) The cell viability of HK-2 cells treated HG (30 mM), RSL3 (5 µM), liraglutide (LIRA, 500 nM), and Fer-1 (5 µM) (

)经HG（30 mM），RSL3（5µM），利拉鲁肽（LIRA，500 nM）和Fer-1（5µM）处理的HK-2细胞的细胞活力(

= 6). (

6.(

–

) The cell viability of HK-2 cells detected by flow cytometry (

)流式细胞仪检测HK-2细胞活力(

= 3). (

3.(

F级

) The ultrastructure of HK-2 cells mitochondria was observed by TEM (scale bar = 1.0 μm,

)透射电镜观察HK-2细胞线粒体的超微结构（比例尺= 1.0μm，

= 3). (

3.(

克

) The JC-1 staining of HK-2 cells (scale bar = 100 μm,

)HK-2细胞的JC-1染色（比例尺=100μm，

= 3). (

3.(

小时

–

我

) The fluorescence absorption spectrum of BODIPY 581/591 C11 (scale bar = 100 μm,

)BODIPY 581/591 C11的荧光吸收光谱（比例尺= 100μm，

= 5). (

5.(

) The expression and statistics of GPX-4 and Fsp1 proteins (

)GPX-4和Fsp1蛋白的表达和统计(

= 3). (

3.(

) The immunofluorescence double label staining of Fsp1 and 4-HNE (scale bar = 50 μm,

)Fsp1和4-HNE的免疫荧光双标记染色（比例尺= 50μm，

= 3). (

3.(

–

) The ratio of NAD

)NAD的比例

/NADH and CoQ10 (H

/NADH和辅酶Q10（H

)/CoQ10 (

/辅酶Q10

= 4). The results are presented as the mean ± SEM. *

结果表示为平均值±SEM*

< 0.05, **

< 0.01 vs. Con group.

与对照组相比，<0.01。

< 0.05,

< 0.01 vs. HG group.

与HG组相比<0.01。

Full size image

全尺寸图像

Liraglutide inhibited HG-induced ferroptosis in HK-2 cells via Fsp1-CoQ10

利拉鲁肽通过Fsp1-CoQ10抑制汞诱导的HK-2细胞铁浓化

To determine the inhibitory effect of LIRA on ferroptosis via the Fsp1-CoQ10 signaling pathway, we selected iFsp1, an inhibitor of Fsp1, for validation in vitro. The cells were divided into HG, HG + iFsp1, and HG + iFsp1 + Fer-1 groups. The CCK8 results showed a decrease in cell viability with the addition of iFsp1 compared to the HG group.

为了确定LIRA通过Fsp1-CoQ10信号通路对铁浓化的抑制作用，我们选择了Fsp1抑制剂iFsp1进行体外验证。将细胞分为HG，HG+ iFsp1和HG+ iFsp1+ Fer-1组。与HG组相比，CCK8结果显示添加iFsp1后细胞活力降低。

However, the addition of Fer-1 was able to restore cell viability (Fig. .

然而，添加Fer-1能够恢复细胞活力（图）。

A). The results obtained from the BODIPY 581/591 C11 probe indicated a significant decrease in lipid peroxidation levels in the HG + iFsp1 + Fer-1 group compared to the HG + iFsp1 group (Fig.

A）。从BODIPY 581/591 C11探针获得的结果表明，与HG+iFsp1组相比，HG+iFsp1+Fer-1组的脂质过氧化水平显着降低（图1）。

B-C).

B-C）。

Fig. 7

图7

Liraglutide inhibited HG-induced ferroptosis via Fsp1-CoQ10. (

利拉鲁肽通过Fsp1-CoQ10抑制HG诱导的ferroptosis。（笑声）(

) The cell viability of HK-2 cells (

)HK-2细胞的细胞活力(

= 6, **

< 0.01 vs. HG group,

与HG组相比<0.01，

< 0.05 vs. HG + iFsp1 group). (

与HG带++iFsp1组相比，<0.05）。(

B类

–

C级

) The fluorescence absorption spectrum of BODIPY 581/591 C11 (scale bar = 100 μm,

)BODIPY 581/591 C11的荧光吸收光谱（比例尺= 100μm，

= 3, **

< 0.01 vs. HG group,

与HG组相比<0.01，

< 0.01 vs. HG + iFsp1 group). (

与HG Song++iFsp1组相比，<0.01）。(

) The cell viability of HK-2 cells (

)HK-2细胞的细胞活力(

= 6, **

< 0.01 vs. HG + LIRA group). (

与HG+LIRA组相比<0.01）。（笑声）(

–

F级

) The fluorescence absorption spectrum of BODIPY 581/591 C11 (scale bar = 100 μm,

)BODIPY 581/591 C11的荧光吸收光谱（比例尺= 100μm，

= 3, **

< 0.01 vs. HG + LIRA group). (

与HG+LIRA组相比<0.01）。（笑声）(

克

) Flow cytometry results of BODIPY 581/591 C11 probe. (H) The immunofluorescence double label staining of Fsp1 and 4-HNE (scale bar = 50 μm,

)BODIPY 581/591 C11探针的流式细胞术结果。（H），

= 3). (

3.(

我

–

) The expression and statistics of GPX-4 and Fsp1 proteins (

)GPX-4和Fsp1蛋白的表达和统计(

= 3, **

< 0.01 vs. HG + LIRA group). (

与HG+LIRA组相比<0.01）。（笑声）(

–

) The ratio of NAD

)NAD的比例

/NADH and CoQ10 (H

/NADH和辅酶Q10（H

)/CoQ10 (

/辅酶Q10

= 4, *

< 0.05 vs. HG + LIRA group). The results are presented as the mean ± SEM.

与HG+LIRA组相比<0.05）。结果表示为平均值±SEM。

Full size image

全尺寸图像

To further verify whether the Fsp1-CoQ10 pathway was involved in the protection of LIRA, the cells were divided into the HG + LIRA and HG + iFsp1 + LIRA groups. The CCK8 results demonstrated a decrease in cell viability in the HG + iFsp1 + LIRA in comparison to the HG + LIRA group (Fig.

为了进一步验证Fsp1-CoQ10途径是否参与LIRA的保护，将细胞分为HG+LIRA和HG+iFsp1+LIRA组。CCK8结果显示，与HG+LIRA组相比，HG+iFsp1+LIRA的细胞活力降低（图）。

D). Meanwhile, there was a significant elevation in lipid peroxidation levels within the HG + iFsp1 + LIRA group compared to the HG + LIRA group (Fig.

D）。同时，与HG+LIRA组相比，HG+iFsp1+LIRA组脂质过氧化水平显着升高（图）。

E-G). Immunofluorescence double label staining indicated Fsp1 levels decreased and 4-HNE levels increased in the HG + iFsp1 + LIRA group compared to the HG + LIRA group (Fig.

E-G）。免疫荧光双标记染色显示，与HG+LIRA组相比，HG+iFsp1+LIRA组Fsp1水平降低，4-HNE水平升高（图）。

H). Western blotting analysis results further validated that GPX-4 and Fsp1 expression levels were all decreased in the HG + iFsp1 + LIRA group in comparison to the HG + LIRA group (Fig.

H）。Western blotting分析结果进一步证实，与HG+LIRA组相比，HG+iFsp1+LIRA组GPX-4和Fsp1表达水平均降低（图1）。

I-J). Furthermore, the ratio of NAD

I-J）。此外，NAD的比例

/NADH and CoQ10(H

/NADH和辅酶Q10（H）

)/CoQ10 were respectively down-regulated in the HG + iFsp1 + LIRA group (Fig.

)/辅酶Q10分别在HG+iFsp1+LIRA组下调（图）。

K-L). These findings suggested that LIRA might have a role in inhibiting ferroptosis by upregulating Fsp1 expression, and this inhibitory function of LIRA was partially hindered by the administration of iFsp1.

K-L）。这些发现表明，LIRA可能通过上调Fsp1表达来抑制ferroptosis，并且LIRA的这种抑制功能部分受到iFsp1给药的阻碍。

Discussion

讨论

The global prevalence of T2DM is increasing significantly due to rapid economic development and urbanisation

由于经济的快速发展和城市化，2型糖尿病的全球患病率正在显著增加

. The primary pathological features of T2DM are insulin resistance and hyperinsulinemia, with excessive release of glucose into the bloodstream being a major contributor to fasting hyperglycemia

T2DM的主要病理特征是胰岛素抵抗和高胰岛素血症，葡萄糖过度释放到血液中是空腹高血糖的主要原因

. In normal physiology, the kidneys exhibit both glucose consumption and release, constituting approximately 20% of the overall endogenous glucose release. There is a notable deviation in renal glucose release, which is linked to heightened renal glycogenolysis in T2DM

在正常生理学中，肾脏表现出葡萄糖消耗和释放，约占内源性葡萄糖释放总量的20%。

. Organismal blood glucose levels are consistently regulated, with gluconeogenesis serving as a crucial metabolic pathway for synthesizing glucose from non-hexose precursors to maintain glucose homeostasis

生物体的血糖水平一直受到调节，糖异生是从非己糖前体合成葡萄糖以维持葡萄糖稳态的关键代谢途径

. Renal gluconeogenesis contributes to 40% of endogenous gluconeogenesis and is regulated by insulin, cellular glucose levels, and stress hormones, highlighting the kidney’s significant role in preserving glucose levels during diverse stress conditions

肾脏糖异生占内源性糖异生的40%，受胰岛素，细胞葡萄糖水平和应激激素的调节，突出了肾脏在不同应激条件下保持葡萄糖水平的重要作用

Research has demonstrated that kidney disease is a prevalent chronic complication of T2DM, with a morbidity of approximately 30–40%

研究表明，肾脏疾病是T2DM普遍存在的慢性并发症，发病率约为30-40%

. Several studies have provided evidence that elevated glucose levels could induce oxidative stress and pathological alterations, including diffuse glomerulosclerosis, tubulointerstitial fibrosis, and fibrosis in renal mesangial cells and tubular cells

几项研究提供的证据表明，葡萄糖水平升高可诱导氧化应激和病理改变，包括弥漫性肾小球硬化，肾小管间质纤维化以及肾系膜细胞和肾小管细胞纤维化

. Furthermore, sustained hyperglycemia results in the buildup of advanced glycation end-product precursors, ultimately leading to apoptosis of glomerular and tubular cells

此外，持续的高血糖会导致晚期糖基化终产物前体的积累，最终导致肾小球和肾小管细胞凋亡

. GLP-1 is the body’s main incretin hormone and its receptor agonist can stimulate the ability of patients with T2DM to secrete insulin, reduce the expression of inflammatory markers such as CD68 in the kidney, and reduce the risk of kidney damage and worsening of diabetes-induced albuminuria. Glomerular endothelial cells are protected by inhibition of Ang II in the signaling pathway at phosphorylated c-Raf (Ser338).

GLP-1是人体主要的肠促胰岛素激素，其受体激动剂可以刺激T2DM患者分泌胰岛素的能力，降低肾脏中CD68等炎症标志物的表达，降低肾脏损伤和糖尿病诱导的白蛋白尿恶化的风险。肾小球内皮细胞受到磷酸化c-Raf（Ser338）信号通路中Ang II抑制的保护。

. In addition, there is increasing evidence that GLP-1 also plays a role in renal protection during acute kidney injury (AKI), including alleviation of apoptosis and inhibition of renal interstitial fibrosis

此外，越来越多的证据表明，GLP-1在急性肾损伤（AKI）期间也在肾脏保护中发挥作用，包括减轻细胞凋亡和抑制肾间质纤维化

. When the kidney is damaged, excessive connective tissue accumulation during repair or reaction leads to renal interstitial fibrosis, and activated myofibroblasts are the main matrix-secreting cell type

当肾脏受损时，修复或反应过程中结缔组织过度积聚会导致肾间质纤维化，活化的肌成纤维细胞是主要的基质分泌细胞类型

, with TGF-β as its major regulator

，以TGF-β为主要调节因子

. Several studies have confirmed the suppressive effect of LIRA on the progression of fibrosis in the liver of db/db mice

几项研究证实了LIRA对db/db小鼠肝脏纤维化进展的抑制作用

and the renal fibrosis induced by unilateral ureteral obstruction (UUO) by inhibiting the TGF-β/Smad3 signaling pathway

通过抑制TGF-β/Smad3信号通路诱导单侧输尿管梗阻（UUO）引起的肾纤维化

. Our findings demonstrated that db/db mice exhibited significantly heightened levels of blood creatinine and urea nitrogen, accompanied by renal fibrosis, elevated TGF-β, and collagen fibres. However, the administration of LIRA significantly alleviated these manifestations. Studies have shown that ferroptosis plays a crucial part in the advancement of renal fibrosis.

我们的研究结果表明，db/db小鼠的血肌酐和尿素氮水平显着升高，并伴有肾纤维化，TGF-β和胶原纤维升高。然而，LIRA的给药显着缓解了这些表现。研究表明，ferroptosis在肾纤维化的进展中起着至关重要的作用。

. This association is supported by the shared metabolic pathways of ferroptosis and fibrosis, such as glycolysis and glutamine catabolism. Importantly, enhanced glutamine catabolism is pivotal in the activation of fibroblasts, and the subsequent facilitation of the tricarboxylic acid cycle further contributes to the initiation of ferroptosis.

这种关联得到了铁浓化和纤维化的共同代谢途径的支持，例如糖酵解和谷氨酰胺分解代谢。重要的是，增强的谷氨酰胺分解代谢在成纤维细胞的活化中至关重要，随后三羧酸循环的促进进一步有助于铁浓化的发生。

. Renal tubular epithelial cells are highly susceptible to ferroptosis, leading to the release of pro-fibrotic mediators, specifically TGF-β and connective tissue growth factor (CTGF), and these mediators then regulate the proliferation and differentiation of mesenchymal fibroblasts through paracrine signaling.

肾小管上皮细胞对铁浓化高度敏感，导致促纤维化介质的释放，特别是TGF-β和结缔组织生长因子（CTGF），然后这些介质通过旁分泌信号调节间充质成纤维细胞的增殖和分化。

Additionally, ferroptosis leads to a decline in the intracellular GSH level and a rise in lipid peroxidation

此外，ferroptosis导致细胞内GSH水平下降和脂质过氧化增加

. Conversely, the application of ferroptosis blockers, like Fer-1 or desferrioxamine (DFO), has been found to impede the paracrine impacts of pro-fibrotic factors in epithelial cells

相反，已发现应用ferroptosis阻滞剂（如Fer-1或去铁胺（DFO））会阻碍促纤维化因子对上皮细胞的旁分泌影响

. Moreover, the study also has reported that the hypoxia inducible factor-1α/heme oxygenase 1 (HIF-1α/HO-1) pathway exacerbated the progression of renal tubular injury and fibrosis induced by renal ferroptosis in a mouse model of diabetes

此外，该研究还报道了缺氧诱导因子-1α/血红素加氧酶1（HIF-1α/HO-1）途径加剧了糖尿病小鼠模型中肾铁浓化引起的肾小管损伤和纤维化的进展

. Moreover, the administration of an xCT inhibitor has been demonstrated to exert a protective effect against renal fibrosis in rats with UUO by efficiently decreasing the quantities of TGF-β, ROS, and lipid peroxides

此外，xCT抑制剂的给药已被证明通过有效降低TGF-β，ROS和脂质过氧化物的量，对UUO大鼠的肾纤维化具有保护作用

. The protective mechanism is accomplished by the facilitation of ferritin degradation and modulation of the xCT/GPX-4 axis

保护机制是通过促进铁蛋白降解和调节xCT/GPX-4轴来实现的

. Consequently, the suppression of ferroptosis presents a promising approach for alleviating renal fibrotic damage.

。

The occurrence of ferroptosis in the renal system is believed to be primarily driven by impaired iron metabolism, accumulation of lipid peroxides, and an imbalance in antioxidant capacity

据信，肾系统铁浓化的发生主要是由铁代谢受损，脂质过氧化物积累和抗氧化能力失衡引起的

. Iron overload has been identified as a contributing factor to ferroptosis, playing a secondary role in both T2DM patients

铁超负荷已被确定为导致铁浓化的因素，在两名T2DM患者中均起次要作用

and db/db mice

和db/db小鼠

. The elevated cellular iron levels may lead to pancreatic β-cell dysfunction

.细胞铁水平升高可能导致胰腺β细胞功能障碍

and kidney damage

和肾脏损伤

, attributed to the exacerbation of oxidative stress through the Fenton reaction

，归因于芬顿反应加剧了氧化应激

. Furthermore, prolonged episodes of elevated blood glucose levels possess the capability to stimulate an increase in the utilization of oxygen by mitochondria, hinder mitochondrial functionality, activate NAD(P)H activity, and subsequently intensify the production of ROS. This process further exacerbates the dysfunction of β-cells and insulin resistance.

此外，血糖水平升高的长时间发作具有刺激线粒体利用氧气的增加，阻碍线粒体功能，激活NAD（P）H活性并随后增强ROS产生的能力。这一过程进一步加剧了β细胞的功能障碍和胰岛素抵抗。

. A previous study has demonstrated the GSH-GPX-4 pathway of ferroptosis, with phospholipid peroxides (PLOOHs), a lipid derived from ROS, playing a pivotal role in this inhibition

先前的一项研究已经证明了铁浓化的GSH-GPX-4途径，其中磷脂过氧化物（PLOOHs）是一种源自ROS的脂质，在这种抑制中起着关键作用

The cystine/glutamate reverse transporter (system Xc

胱氨酸/谷氨酸反向转运蛋白（系统Xc

−

) facilitates the entry of cystine into the cell, where it undergoes reduction to cysteine through either the GSH-dependent or thioredoxin reductase 1 (TXNRD1)-dependent cystine reduction pathway. This pathway facilitates the synthesis of GSH, a highly efficient reductant that acts as a coenzyme for GPX-4.

)促进胱氨酸进入细胞，在那里它通过GSH依赖性或硫氧还蛋白还原酶1（TXNRD1）依赖性胱氨酸还原途径还原为半胱氨酸。该途径促进GSH的合成，GSH是一种高效的还原剂，可作为GPX-4的辅酶。

GSH plays an essential part in the elimination of lipid oxides by converting them into non-harmful alcohols, which is crucial for scavenging free radicals and safeguarding cells from oxidative stress.

谷胱甘肽通过将脂质氧化物转化为无害的醇，在消除脂质氧化物中起着至关重要的作用，这对于清除自由基和保护细胞免受氧化应激至关重要。

. In our study, we observed a significant decrease in the levels of GSH and GPX-4 and an increase in the levels of LPO in the kidney tissue of db/db mice. These observations suggest an imbalance in the Xc

在我们的研究中，我们观察到db/db小鼠肾组织中GSH和GPX-4水平显着降低，LPO水平升高。这些观察结果表明Xc不平衡

−

system in hyperglycaemia (Fig.

高血糖系统（图）。

Fig. 8

图8

The schematic diagram illustrates the involvement of ferroptosis in the kidneys of db/db mice. HG has been found to induce renal fibrosis and ferroptosis in diabetic mice. This is attributed to the increased levels of TfR1 and DMT1, as well as the decreased level of FPN1, which results in elevated LIP in the kidney.

该示意图说明了db/db小鼠肾脏中铁蛋白沉积的参与。已经发现HG在糖尿病小鼠中诱导肾纤维化和铁浓化。这归因于TfR1和DMT1水平的升高，以及FPN1水平的降低，这导致肾脏LIP升高。

The excess of labile iron pool (LIP), primarily consisting of Fe.

过量的不稳定铁池（LIP），主要由Fe组成。

, exacerbates the generation of lipid ROS in response to the Fenton reaction. Furthermore, the reduced expression of Fsp1 hinders the CoQ10 to its reduced form, CoQ10(H

，加剧了响应芬顿反应的脂质ROS的产生。此外，Fsp1的表达降低阻碍了辅酶Q10的还原形式辅酶Q10（H

), thereby impeding its antioxidant effects. Additionally, the imbalance in the Xc

)，从而阻碍了其抗氧化作用。此外，Xc中的不平衡

−

system disrupts the exchange and synthesis of GSH, leading to the inhibition of GSH-dependent antioxidant defences and ultimately resulting in ferroptosis. Liraglutide has the potential to enhance the expression of Fsp1 and ameliorate the Xc

该系统破坏GSH的交换和合成，导致GSH依赖性抗氧化防御的抑制，并最终导致铁浓化。利拉鲁肽有可能增强Fsp1的表达并改善Xc

−

system, thereby mitigating the ferroptosis response in the renal tissue of db/db mice.

系统，从而减轻db/db小鼠肾组织中的铁浓化反应。

Full size image

全尺寸图像

To date, advancements concerning experimental pharmaceuticals targeting ferroptosis in animal or cellular models of diabetes, various interventions such as hypoglycemic agents, antihypertensive agents, and traditional Chinese herbal medicines have shown efficacy in inhibiting ferroptosis

迄今为止，在糖尿病的动物或细胞模型中，针对铁浓化的实验药物的进展，各种干预措施（例如降糖药，抗高血压药和中草药）已显示出抑制铁浓化的功效

. Among these interventions, hypoglycemic drugs have been widely utilized, with dapagliflozin being identified as a mitigating agent for diabetic kidney disease (DKD) through ferroptosis inhibition. Furthermore, empagliflozin has the potential to improve cardiac function by reducing ferroptosis. Additionally, empagliflozin has demonstrated the ability to enhance cardiac function by decreasing ferroptosis through NLRP3-MyD88 pathways in mice treated with doxorubicin.

在这些干预措施中，降糖药物已被广泛使用，达格列净通过抑制铁浓化被确定为糖尿病肾病（DKD）的缓解剂。此外，empagliflozin有可能通过减少ferroptosis来改善心脏功能。此外，empagliflozin已证明通过用多柔比星治疗的小鼠中的NLRP3-MyD88途径减少铁浓化来增强心脏功能的能力。

. LIRA was approved for managing T2DM and obesity

LIRA被批准用于治疗T2DM和肥胖症

. LIRA has demonstrated its ability to reduce non-alcoholic fatty liver disease linked to T2DM by activating the AMPK/ACC pathway and inhibiting ferroptosis

LIRA已证明其通过激活AMPK/ACC途径和抑制铁浓化来减少与T2DM相关的非酒精性脂肪肝疾病的能力

. In addition, LIRA could attach to the GLP-1 receptor, leading to the enhancement of hepatic lipid homeostasis by activating hepatic fibroblast growth factor 21 signaling pathway

此外，LIRA可以附着于GLP-1受体，通过激活肝成纤维细胞生长因子21信号通路增强肝脏脂质稳态

. According to our previous investigation, LIRA triggered the Nrf2/HO-1 pathway, resulting in decreased levels of oxidative stress in the liver and brain of db/db mice, thereby inhibiting ferroptosis

根据我们之前的研究，LIRA触发了Nrf2/HO-1途径，导致db/db小鼠肝脏和大脑中的氧化应激水平降低，从而抑制了铁浓化

. It’s clear that the main regulators of ferroptosis in well-known metabolic pathways have been extensively studied, but there is an urgent need to investigate other novel mechanisms involved in ferroptosis.

很明显，众所周知的代谢途径中的铁浓化的主要调节因子已被广泛研究，但迫切需要研究与铁浓化有关的其他新机制。

Undoubtedly, GPX plays a crucial role in the regulation of ferroptosis. However, recent research has identified three GPX-4-independent pathways, namely Fsp1/CoQ10, GCH1, and DHODH, which are involved in this process

毫无疑问，GPX在调节ferroptosis中起着至关重要的作用。然而，最近的研究已经确定了三种与GPX-4无关的途径，即Fsp1/CoQ10，GCH1和DHODH，它们参与了这一过程

. Extensive research has been carried out to investigate key regulators of ferroptosis within established pathways, leading to the need for further exploration of innovative mechanisms contributing to ferroptosis. Recent genetic screening studies have successfully identified the Fsp1/CoQ10 system as a comprehensive safeguard against ferroptosis induced by pharmacological inhibition of GPX-4 or GPX-4 knockdown.

。已经进行了广泛的研究，以在既定途径内调查铁浓化的关键调节因子，因此需要进一步探索有助于铁浓化的创新机制。最近的基因筛选研究已经成功地将Fsp1/CoQ10系统鉴定为防止由GPX-4或GPX-4敲低的药理学抑制引起的铁浓化的综合保护措施。

This discovery implies the presence of regulatory mechanisms that operate independently of the GPX-4/GSH axis, thereby offering protection against ferroptosis within the organism.

这一发现意味着存在独立于GPX-4/GSH轴运行的调节机制，从而提供对生物体内铁浓化的保护。

. Our results also found the inhibition of the GPX-4/GSH axis as showed by LIRA (Fig.

我们的结果还发现抑制GPX-4/GSH轴，如LIRA所示（图）。

Fsp1, a highly conserved NAD(P)H-dependent ubiquinone oxidoreductase, is found in both bacterial and mammalian systems

Fsp1是一种高度保守的NAD（P）H依赖性泛醌氧化还原酶，存在于细菌和哺乳动物系统中

. Following translation, Fsp1 undergoes carbamylation modification, which serves to anchor it to the cellular membrane

翻译后，Fsp1进行氨甲酰化修饰，将其锚定在细胞膜上

. This modification enables the oxidation of NAD(P)H to NAD(P)+, utilizing flavin adenine dinucleotide (FAD) acting as a cofactor. Furthermore, Fsp1 facilitates the conversion of reducing CoQ10 at the plasma membrane to CoQ10(H

这种修饰能够利用黄素腺嘌呤二核苷酸（FAD）作为辅因子将NAD（P）H氧化为NAD（P）+。此外，Fsp1有助于将质膜上的辅酶Q10还原为辅酶Q10（H

), leading to the reduction of toxic lipid peroxides to non-toxic lipids and exhibiting anti-ferroptosis effects

)，导致有毒脂质过氧化物减少为无毒脂质，并表现出抗铁浓化作用

. Evidence suggests that Fsp1 plays a role in maintaining the normal structure and function of renal tissues. Specifically, the lack of Fsp1 in renal tubular epithelial cells has been linked to tubular necrosis

。具体来说，肾小管上皮细胞缺乏Fsp1与肾小管坏死有关

, whereas an upregulation of Fsp1 expression in these cells has been observed to impede cellular ferroptosis and alleviate acute kidney injury

，而已观察到这些细胞中Fsp1表达的上调可阻止细胞铁浓化并减轻急性肾损伤

. Moreover, the ubiquitination of Fsp1 appeared to have a role in the occurrence of tubular ferroptosis during episodes of acute kidney injury

此外，在急性肾损伤发作期间，Fsp1的泛素化似乎在肾小管上睑下垂的发生中起作用

. Our study revealed that LIRA enhanced the Fsp1 expression while enhancing the CoQ10(H

我们的研究表明，LIRA增强了Fsp1的表达，同时增强了辅酶Q10（H

)/CoQ10 levels in the kidney tissue of db/db mice (Fig.

)/db/db小鼠肾组织中辅酶Q10水平（图）。

). Similarly, these observations were replicated in HK-2 cells cultured under HG conditions, indicating the suppression of LIRA by the Fsp1-CoQ10 pathway.

)。类似地，这些观察结果在HG条件下培养的HK-2细胞中复制，表明Fsp1-CoQ10途径抑制LIRA。

Collectively, these combined results indicated that ferroptosis played a role in renal fibrosis and contributed to kidney injury in db/db mice, possibly because of the increased levels of ROS, lipid peroxidation, and iron accumulation. Notably, our research suggested that LIRA might offer a protective effect against ferroptosis, which was not solely reliant on GPX-4 but also involved the Fsp1-CoQ10 pathway.

总的来说，这些综合结果表明，ferroptosis在肾纤维化中起作用，并导致db/db小鼠的肾损伤，可能是由于ROS水平升高，脂质过氧化和铁积累。值得注意的是，我们的研究表明，LIRA可能对ferroptosis具有保护作用，这不仅依赖于GPX-4，而且还涉及Fsp1-CoQ10途径。

However, it was important to note that these initial findings might serve as a foundation for future investigations into the therapeutic potential of LIRA in the management of renal ferroptosis and fibrotic injury..

然而，重要的是要注意，这些初步发现可能为未来研究LIRA在治疗肾性上睑下垂和纤维化损伤方面的治疗潜力奠定基础。。

Methods

方法

Ethical approval

道德认可

All the animal experiments were performed in compliance with the guidelines of ARRIVE and Committee of Experimental Animal Welfare Ethics of the Hebei University of Chinese Medicine. The Committee of Experimental Animal Welfare Ethics of the Hebei University of Chinese Medicine approved all the experimental protocols (DWLL2020089)..

所有动物实验均按照ARRIVE和河北中医药大学实验动物福利伦理委员会的指导进行。河北中医药大学实验动物福利伦理委员会批准了所有实验方案（DWLL2020089）。。

Animals

动物

The non-diabetic littermate db/m mice (C57BLKS, SPF grade, male, 8 weeks) and the db/db mice (C57BLKS, SPF grade, male, 8 weeks) were obtained from Changzhou Cavens Experimental Animal Co., Ltd (License number: SCXK (Su) 2021-0013). All the mice were housed in the animal center that maintained a controlled temperature range of 22–24 °C and humidity level of 55-60%.

非糖尿病同窝db/m小鼠（C57BLKS，SPF级，雄性，8周）和db/db小鼠（C57BLKS，SPF级，雄性，8周）获自常州卡文斯实验动物有限公司（许可证号：SCXK（Su）2021-0013）。所有小鼠都被安置在动物中心，保持22-24°C的受控温度范围和55-60%的湿度水平。

Following a week of adaptive feeding, the db/db mice (.

适应性喂养一周后，db/db小鼠（。

= 18) were randomly allocated into two groups: the db/db group and the LIRA group. The mice in the LIRA group received intraperitoneal injections of LIRA (200 mg/kg/d, MCE, HY-P0014) from the 9th to the 15th week, while the control group (db/m) group was administered equivalent quantities of saline.

==18）被随机分为两组：db/db组和LIRA组。LIRA组小鼠在第9周至第15周接受腹腔注射LIRA（200 mg/kg/d，MCE，HY-P0014），而对照组（db/m）组给予等量的盐水。

Once a week, the blood glucose levels were examined using a blood glucose meter (S59400839789, Sinocare). Finally, the mice were anesthetized and sacrificed by intraperitoneal injection of sodium pentobarbital and then blood and kidney tissue was collected to further analyses. The body weight and kidney weight of the mice in each group were counted, and the kidney coefficient (kidney coefficient = kidney weight/body weight, KLW/BW) was calculated..

每周一次，使用血糖仪（S59400839789，Sinocare）检查血糖水平。最后，将小鼠麻醉并通过腹膜内注射戊巴比妥钠处死，然后收集血液和肾组织以进一步分析。计算各组小鼠的体重和肾脏重量，并计算肾脏系数（肾脏系数=肾脏重量/体重，KLW/BW）。。

Intraperitoneal pyruvic acid tolerance test

腹腔丙酮酸耐受试验

On the penultimate day before the culmination of the modelling process, an intraperitoneal pyruvate tolerance test was administered. Following a period of 14 h of fasting, the mice were subjected to an intraperitoneal injection of sodium pyruvate at a dosage of 2 g/kg. Utilizing the tail-blood collection technique, blood glucose levels were assessed at intervals of 15, 30, 60, and 120 min.

在建模过程达到高潮前的倒数第二天，进行了腹膜内丙酮酸耐受性测试。禁食14小时后，小鼠腹腔注射丙酮酸钠，剂量为2 g/kg。利用尾血采集技术，每隔15、30、60和120分钟评估血糖水平。

Afterwards, the calculation of the area under the dynamic glucose curve (AUC) was performed..

然后，计算动态葡萄糖曲线下面积（AUC）。。

Blood pressure

血压

Tail artery pressure was measured in each group using the non-invasive tail sleeve method. The experimental platform was preheated for 30 min, awake mice were fixed to the experimental platform with a magnetic fixator, the mouse tail was passed through the sleeve and fixed to the experimental platform with tape, the rubber sleeve was pressed onto the root of the mouse tail and the measurement began after 5–10 min of adaptation..

使用无创尾袖法测量每组的尾动脉压力。将实验平台预热30分钟，用磁力固定器将清醒的小鼠固定在实验平台上，将小鼠尾巴穿过套筒并用胶带固定在实验平台上，将橡胶套筒压在小鼠尾巴的根部，并在适应5-10分钟后开始测量。。

Pathological staining

病理染色

Pathological changes in the kidney were analyzed using HE stain. The paraffin sections of the kidneys were deparaffinized by a gradient of alcohol. Subsequently, the sections were subjected to hematoxylin stain solution, followed by ethanol-hydrochloric acid differentiation, eosin re-staining, another round of gradient alcohol dehydration, and xylene for clarification.

使用HE染色分析肾脏的病理变化。肾脏的石蜡切片用梯度酒精脱蜡。随后，将切片进行苏木精染色溶液，然后进行乙醇-盐酸分化，曙红再染色，另一轮梯度酒精脱水和二甲苯澄清。

Finally, the slices were sealed with neutral resin. Renal fibrosis was evaluated by Masson stain, Sirius red stain solution, reticular fibre stain solution, and elastic fibre stain solution..

最后，切片用中性树脂密封。通过Masson染色，天狼星红染色溶液，网状纤维染色溶液和弹性纤维染色溶液评估肾纤维化。。

ELISA

酶联免疫吸附试验

beta 2-MG (E-EL-M2411, Elabscience), 8-OHdG (E-EL-0028), and NAG (E-BC-K064-M, Elabscience) in serum were measured by ELISA. Mice serum was collected and measured according to the product instructions. Finally, the absorbance of beta 2-MG, 8-OHdG and NAG were detected separately using a multifunctional microplate reader (Varioskan LUX, Thermo Fisher Scientific) at the wavelength of 450 nm, 450 nm and 400 nm.

通过ELISA测量血清中的β2-MG（E-EL-M2411，Elabscience），8-OHdG（E-EL-0028）和NAG（E-BC-K064-M，Elabscience）。。最后，使用多功能酶标仪（Varioskan LUX，Thermo Fisher Scientific）在450 nm，450 nm和400 nm的波长下分别检测β2-MG，8-OHdG和NAG的吸光度。

The corresponding activity or concentration was calculated according to the standard curve..

根据标准曲线计算相应的活性或浓度。。

Biochemical analysis

生化分析

Serum levels of BUN (S03036, Shenzhen Leidu Technology, China), SCr (S03076, Shenzhen Leidu Technology), albumin (ALB, S03043, Shenzhen Leidu Technology), TG (S03027, Shenzhen Leidu Technology) and CHO (S03042, Shenzhen Leidu Technology) were determined using a biochemical analyser (Chemray 800, Shenzhen Leidu Technology).

使用生化分析仪（Chemray 800，深圳雷都科技）测定血清BUN（S03036，深圳雷都科技，中国），SCr（S03076，深圳雷都科技），白蛋白（ALB，S03043，深圳雷都科技），TG（S03027，深圳雷都科技）和CHO（S03042，深圳雷都科技）的水平。

Mice serum was collected and the appropriate volume was added to be measured by fully automated detection. The concentration was calculated the according to their formulas..

收集小鼠血清并加入适当的体积以通过全自动检测进行测量。根据他们的公式计算浓度。。

Transmission electron microscope

透射电子显微镜

The ultrastructure of mitochondria in HK-2 cells and mouse kidney tissues was observed by TEM. After centrifugation to collect cells or tissue precipitates for fixation, samples were embedded, polymerised and ultrathin sectioned at a thickness of 60–80 nm. Sections were stained in the dark with a 2% uranyl acetate saturated alcohol solution to enhance contrast.

透射电镜观察HK-2细胞和小鼠肾组织线粒体的超微结构。离心收集细胞或组织沉淀物进行固定后，将样品包埋，聚合并以60-80 nm的厚度超薄切片。切片在黑暗中用2%乙酸铀酰饱和醇溶液染色以增强对比度。

Images were examined using a HITACHI HT7700 electron microscope..

使用日立HT7700电子显微镜检查图像。。

Antioxidant and lipid peroxidation products

抗氧化剂和脂质过氧化产物

The mouse kidney tissues were subjected to homogenization at a concentration of 10% (g/V), and subsequent measurements were conducted following the guidelines provided by the kit manufacturer. The activities of T-SOD (A001-1-2, Nanjing Jiancheng Bioengineering Institute), GSH (A006-2-1, Nanjing Jiancheng Bioengineering Institute), GSH-Px (A005-1-2, Nanjing Jiancheng Bioengineering Institute), and CAT (A007-1-1, Nanjing Jiancheng Bioengineering Institute) were assessed to evaluate the antioxidant capacity.

将小鼠肾脏组织以10%（g/V）的浓度进行匀浆，并按照试剂盒制造商提供的指南进行后续测量。评估T-SOD（A001-1-2，南京建成生物工程研究所），GSH（A006-2-1，南京建成生物工程研究所），GSH-Px（A005-1-2，南京建成生物工程研究所）和CAT（A007-1-1，南京建成生物工程研究所）的活性以评估抗氧化能力。

MDA (A003-1-2, Nanjing Jiancheng Bioengineering Institute) and LPO (A106-1-2, Nanjing Jiancheng Bioengineering Institute) were used as by-products of lipid peroxidation..

MDA（南京建成生物工程研究所A003-1-2）和LPO（南京建成生物工程研究所A106-1-2）被用作脂质过氧化的副产物。。

Perls’ staining

Perls染色

Perls’ staining was achieved by combining potassium ferricyanide and hydrochloric acid, which reacts with Fe

Perls的染色是通过将铁氰化钾和与铁反应的盐酸结合起来实现的

to generate soluble colouring. The sections went through dewaxing and were washed three times in phosphate buffer (PBS, 0.01 M, pH 7.4). Subsequently, they were exposed to 3% H

产生可溶性着色。切片脱蜡并在磷酸盐缓冲液（PBS，0.01 M，pH 7.4）中洗涤三次。随后，他们暴露于3%的H

for 20 min at room temperature (RT). Subsequently, the staining solution was immersed for six hours at RT. After staining, the sections were DAB enhanced, re-stained with hematoxylin, and sealed using neutral glue. Positive cells were observed under the microscope.

在室温（RT）下放置20分钟。随后，将染色溶液在室温下浸泡6小时。染色后，将切片DAB增强，用苏木精重新染色，并使用中性胶密封。显微镜下观察阳性细胞。

Immunohistochemistry

免疫组织化学

After deparaffinisation and rehydration, the sections were incubated with 3% H

脱石蜡和再水化后，切片与3%H孵育

to remove endogenous peroxidase activity. Subsequently, antigen retrieval at high temperature was carried out, and the sections were incubated with 10% goat serum at 37 °C for 1 h. Following this, GPX-4 (ET1706-45, Huabio) and Fsp1 (20886-1-AP, Proteintech) were incubated overnight at 4 °C. Subsequently, the sections were incubated with secondary antibodies labelled with HRP label for 1 h at 37 °C.

去除内源性过氧化物酶活性。随后，在高温下进行抗原修复，并将切片与10%山羊血清在37℃下孵育1小时。之后，将GPX-4（ET1706-45，Huabio）和Fsp1（20886-1-AP，Proteintech）在4°C下孵育过夜。随后，将切片与用HRP标记的二抗在37℃孵育1小时。

Finally, the sections were stained using a DAB kit and sealed with a sealer. The average density of positive proteins was calculated using IPP 6.0 software..

最后，使用DAB试剂盒对切片进行染色，并用密封剂密封。使用IPP 6.0软件计算阳性蛋白的平均密度。。

Cell culture

细胞培养

Human Renal Tubular Epithelial Cells, HK-2 Cells, was purchased from Wuhan Pricella Biotechnology Co., Ltd (CL-0109). HK-2 cells were cultured in penicillin, streptomycin, 10% fetal bovine serum and MEM medium at 37 °C and 5% CO

人肾小管上皮细胞HK-2细胞购自武汉普氏生物技术有限公司（CL-0109）。HK-2细胞在青霉素、链霉素、10%胎牛血清和MEM培养基中于37°C和5%CO培养

. To simulate diabetes, the cells were exposed to HG conditions. HK-2 cells were cultured with different concentrations of HG for 48 h to evaluate the protective effect of LIRA. At the same time, the cells were treated individually with liraglutide, RSL-3 (a ferroptosis activator, MCE, HY-100218 A), Ferrostatin-1 (Fer-1, an inhibitor of ferroptosis, MCE, HY-100579), and iFsp1 (an inhibitor of Fsp1, MCE, HY-136057).

为了模拟糖尿病，将细胞暴露于汞条件下。将HK-2细胞与不同浓度的HG一起培养48小时以评估LIRA的保护作用。同时，用利拉鲁肽，RSL-3（ferroptosis activator，MCE，HY-100218 a），Ferrostatin-1（Ferrostatin-1，ferroptosis抑制剂，MCE，HY-100579）和iFsp1（Fsp1，MCE，HY-136057的抑制剂）。

Following the treatment, cell viability and western blotting analysis were conducted for assessment..

治疗后，进行细胞活力和蛋白质印迹分析以进行评估。。

Cell viability

细胞活力

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8). HK-2 cells were seeded into 96-well culture plates with a density of 1 × 10

使用细胞计数试剂盒-8（CCK-8）评估细胞活力。将HK-2细胞接种到密度为1×10的96孔培养板中

cells per well. After treatment with or without HG, CCK-8 reagents were added to the plates and incubated at 37 °C for 2 h. Absorbance was measured at a wavelength of 450 nm using a multifunctional microplate reader (Varioskan LUX, Thermo Fisher Scientific).

每孔细胞数。用或不用HG处理后，将CCK-8试剂加入平板中，并在37℃下孵育2小时。使用多功能酶标仪（Varioskan LUX，Thermo Fisher Scientific）在450 nm的波长下测量吸光度。

After the treatment of cells in each group, the adherent cells were digested with EDTA-free trypsin, washed twice with pre-cooled PBS, and centrifuged to collect the precipitates. The cells were resuspended with 500 µL binding buffer, then 5 µL annexin V and PI were added and incubated for 15 min in the dark.

每组细胞处理后，将贴壁细胞用不含EDTA的胰蛋白酶消化，用预冷的PBS洗涤两次，然后离心以收集沉淀物。将细胞用500µL结合缓冲液重悬，然后加入5µL膜联蛋白V和PI，并在黑暗中孵育15分钟。

Finally, apoptosis was detected by flow cytometry (FC 500 MCL, Beckman Coulter)..

最后，通过流式细胞术（FC 500 MCL，Beckman Coulter）检测细胞凋亡。。

JC-1 staining

JC-1染色

JC-1 staining was used to measure the MMP levels. Cells were seeded on 6 cm cell culture at a density of 2 × 10

JC-1染色用于测量MMP水平。将细胞以2×10的密度接种在6 cm细胞培养物上

/per well. Before staining, the cells were prepared by removing the supernatant and washing them three times with PBS. JC-1 staining solution was then added according to the instructions and the cells were incubated for 30 min at RT in the dark. Finally, the fluorescence signals were observed under a fluorescence microscope..

/。染色前，通过去除上清液并用PBS洗涤三次来制备细胞。然后根据说明加入JC-1染色溶液，并将细胞在室温下在黑暗中孵育30分钟。最后，在荧光显微镜下观察荧光信号。。

Lipid peroxidation assay

脂质过氧化测定

After inoculation the HK-2 cells into a 24-well plate, the lipid peroxidation sensor BODIPY 581/591 C11 (10 µM, D3861, Invitrogen) was added and incubated for 30 min at 37 °C. Afterwards,

将HK-2细胞接种到24孔板中后，加入脂质过氧化传感器BODIPY 581/591 C11（10µM，D3861，Invitrogen），并在37°C下孵育30分钟。之后，

the culture medium was removed and the cells were rinsed three times with PBS. Fluorescence measurements were performed at two different wavelengths: one with an excitation/emission of 581/591 nm and the other at 488/510 nm. The ratio of the fluorescence intensities emitted at 590 nm and 510 nm provides a quantitative assessment of lipid peroxidation within the cells..

除去培养基，用PBS冲洗细胞三次。在两种不同的波长下进行荧光测量：一种激发/发射为581/591 nm，另一种为488/510 nm。在590 nm和510 nm处发射的荧光强度之比提供了细胞内脂质过氧化的定量评估。。

NAD/NADH assay

NAD/NADH测定

NAD/NADH levels in mouse kidney tissue and HK-2 cells were measured using the NAD/NADH Assay Kit (MM-44925M1/MM-45162M2, MEIMIAN). Tissues or cells were washed with cold PBS, homogenised in NAD/NADH extraction buffer and assayed according to the kit instructions. The concentrations of NAD/NADH were calculated from a standard curve..

使用NAD/NADH测定试剂盒（MM-44925M1/MM-45162M2，MEIMIAN）测量小鼠肾组织和HK-2细胞中的NAD/NADH水平。用冷PBS洗涤组织或细胞，在NAD/NADH提取缓冲液中匀浆，并根据试剂盒说明进行测定。NAD/NADH的浓度由标准曲线计算。。

CoQ10 (H2)/CoQ10 assay

辅酶Q10（H2）/CoQ10测定

CoQ10(H

辅酶Q10（H）

)/CoQ10 levels in mouse kidney tissue and HK-2 cells were detected using the CoQ10/CoQ10(H

)/用辅酶Q10/辅酶Q10（H

) Assay Kit (MM45967M2/MM-45496M2, MEIMIAN). Tissues or cells were washed with cold PBS and homogenised in extraction buffer, and then the assay procedure was performed according to the kit instructions. The CoQ10(H

)测定试剂盒（MM45967M2/MM-45496M2，MEIMIAN）。用冷PBS洗涤组织或细胞，并在提取缓冲液中匀浆，然后根据试剂盒说明进行测定程序。辅酶Q10（H

)/CoQ10 levels were calculated from the standard curve.

)/从标准曲线计算辅酶Q10水平。

Western blotting analysis

蛋白质印迹分析

Kidney tissue or HK-2 cells were prepared separately for homogenisation with cooled PIRA lysate. The supernatant was collected and the amount of total protein was measured. The proteins were then separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. The blots were incubated with 5% skimmed milk at RT for 2 h and then washed with TBST.

分别制备肾组织或HK-2细胞，用冷却的PIRA裂解物进行均化。收集上清液并测量总蛋白的量。然后通过SDS-PAGE电泳分离蛋白质并转移到PVDF膜上。将印迹与5%脱脂奶在室温下孵育2小时，然后用TBST洗涤。

The blots were then incubated with primary antibodies overnight at 4 °C, including Collagen-I (GB112543, Servicebio), Collagen-III (GB11023, Servicebio), TGF-β(bs-0086R, Bioss), NOX-4 (A00403, Boster), 4-HNE (ARG23717, Arigo), MDA (ab243066, Abcam), TfR1(3-6800, Invitrogen), DMT1(abs112967, Absin), FPN1(MTP11-A, Alpha Diagnostic International), FTH(ab183781, Abcam), FTL(ab218400, Abcam), GPX-4, Fsp1, GAPDH (GB15002, Servicebio), β-actin (GB15001, Servicebio), α-Tubulin (GTX628802, GeneTex).

然后将印迹与一抗在4°C孵育过夜，包括胶原蛋白I（GB112543，Servicebio），胶原蛋白III（GB11023，Servicebio），TGF-β（bs-0086R，Bioss），NOX-4（A00403，Boster），4-HNE（ARG23717，Arigo），MDA（ab243066，Abcam），TfR1（3-6800，Invitrogen），DMT1（ab112967，Absin），FPN1（MTP11-A，Alpha Diagnostic International），FTH（ab183781，Abcam），FTL（ab218400，Abcam），GPX-4，Fsp1，GAPDH（GB15002，Servicebio），β-肌动蛋白（GB15001，Servicebio），α-微管蛋白（GTX628802，GeneTex）。

On the second day, blots were incubated with secondary antibodies conjugated with HRP at RT. The ECL method was used to determine the immunoreactive protein, while the mean grey value was determined using Image J software..

第二天，将印迹与与HRP偶联的二抗在室温下孵育。使用ECL方法测定免疫反应蛋白，同时使用Image J软件测定平均灰度值。。

Statistical analysis

统计分析

The data were analyzed using SPSS 23.0 statistical software and presented as mean ± SEM. The data was analyzed using a one-way ANOVA, followed by a

使用SPSS 23.0统计软件对数据进行分析，并以平均值±SEM表示。数据使用单因素方差分析进行分析，然后进行方差分析

post hoc

事后

LSD test. Significance was determined at

LSD测试。重要性确定于

< 0.05. Graphs were generated using Prism 9.0 software.

<0.05。使用Prism 9.0软件生成图形。

Data availability

数据可用性

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Further inquiries can be directed to the corresponding author.

。进一步的询问可以直接联系通讯作者。

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Acknowledgements

致谢

Thanks to all authors for their contributions. This work was supported by the Hebei Province medical science research project (NO. 20231577, 20241868).

感谢所有作者的贡献。这项工作得到了河北省医学科学研究项目（编号2023157720241868）的支持。

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Author notes

作者笔记

Qi Chen, Ji-Xian Song and Zhi Zhang have contributed equally to this work.

齐晨、宋季贤和张志对这项工作做出了同样的贡献。

Authors and Affiliations

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Hebei Technology Innovation Center of TCM Combined Hydrogen Medicine, Hebei University of Chinese Medicine, NO.3, Luqian Xingyuan Road, Shijiazhuang, 050200, Hebei Province, China

河北中医药大学河北中医药联合氢医学技术创新中心，河北省石家庄市路前兴源路3号，050200

Qi Chen, Ji-Xian Song, Zhi Zhang, Ji-Ren An, Yu-Jing Gou & Yashuo Zhao

陈琦，宋季贤，张志，纪仁安，郭玉晶，赵亚硕

The Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang, 050011, China

河北医科大学第四附属医院，石家庄，050011

Miao Tan

苗潭（Miao Tan）

College of Basic Medicine, Chengde Medical University, Chengde, 067000, China

承德医科大学基础医学院，承德067000

Qi Chen & Yu-Jing Gou

齐晨与余静沟

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Qi Chen

齐晨（音译）

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Ji-Xian Song

宋季贤

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Zhi Zhang

张志（Zhi Zhang）

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Ji-Ren An

纪仁安

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Yu-Jing Gou

余景沟

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苗潭（Miao Tan）

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Yashuo Zhao

赵雅硕

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Contributions

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Conceptualization: Yashuo Zhao, Qi Chen, Ji-Xian Song, Miao Tan; Methodology: Qi Chen, Ji-Xian Song, Yu-Jing Gou and Zhi Zhang; Resources: Qi Chen, Ji Xian Song, Ji-Ren An, Miao Tan; Data Curation: Ji-Xian Song, Ji-Ren An, Qi Chen, and Zhi Zhang; Writing-Original Draft: Ji-Xian Song, Qi Chen; Writing- Review & Editing: Yashuo Zhao and Miao Tan; Visualization: Ji-Xian Song, Ji-Ren An, Qi Chen, Yu-Jing Gou; Supervision: Yashuo Zhao, and Miao Tan; Funding acquisition: Yashuo Zhao and Miao Tan.

概念化：赵亚硕，陈琦，宋季贤，谭淼；方法论：祁晨，宋季贤，苟余静，张志；；数据管理：宋季贤，安季仁，陈琦，张志；撰写原稿：宋季贤、陈琦；写作-评论和编辑：赵亚硕和谭淼；可视化：宋季贤、安季仁、陈琦、余静沟；监督：赵亚硕，谭淼；资金收购：赵亚硕和谭淼。

All authors contributed to the article and approved the submitted version..

所有作者都为这篇文章做出了贡献，并批准了提交的版本。。

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Miao Tan

苗潭（Miao Tan）

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Yashuo Zhao

赵雅硕

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Chen, Q., Song, JX., Zhang, Z.

陈Q.，宋JX。，张，Z。

et al.