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Original Article

Korean J Physiol Pharmacol 2024; 28(1): 11-19

Published online January 1, 2024

Copyright © Korean J Physiol Pharmacol.

Aurantio-obtusin exerts an anti-inflammatory effect on acute kidney injury by inhibiting NF-κB pathway

Haiyan Xiang*, Yun Zhang, Yan Wu, Yaling Xu, and Yuanhao Hong

Department of Nephrology, Wuhan Sixth Hospital, Affiliated Hospital of Jianghan University, Wuhan 430014, Hubei, China

Correspondence to:Haiyan Xiang

Author contributions: H.X. were the main designers of this study. H.X., Y.Z., Y.W., Y.X., and Y.H.performed the experiments and analyzed the data. H.X. drafted the manuscript. All authors read and approved the final manuscript.

Received: June 15, 2023; Revised: September 20, 2023; Accepted: October 4, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Acute kidney injury (AKI) is one of the major complications of sepsis. Aurantio-obtusin (AO) is an anthraquinone compound with antioxidant and anti-inflammatory activities. This study was developed to concentrate on the role and mechanism of AO in sepsis-induced AKI. Lipopolysaccharide (LPS)-stimulated human renal proximal tubular epithelial cells (HK-2) and BALB/c mice receiving cecal ligation and puncture (CLP) surgery were used to establish in vitro cell model and in vivo mouse model. HK-2 cell viability was measured using MTT assays. Histological alterations of mouse renal tissues were analyzed via hematoxylin and eosin staining. Renal function of mice was assessed by measuring the levels of serum creatinine (SCr) and blood urea nitrogen (BUN). The concentrations of pro-inflammatory cytokines in HK-2 cells and serum samples of mice were detected using corresponding ELISA kits. Protein levels of factors associated with nuclear factor kappa-B (NF-κB) pathway were measured in HK-2 cells and renal tissues by Western blotting. AO exerted no cytotoxic effect on HK-2 cells and AO dose-dependently rescued LPS-induced decrease in HK-2 cell viability. The concentrations of pro-inflammatory cytokines were increased in response to LPS or CLP treatment, and the alterations were reversed by AO treatment. For in vivo experiments, AO markedly ameliorated renal injury and reduced high levels of SCr and BUN in mice underwent CLP operation. In addition, AO administration inhibited the activation of NF-κB signaling pathway in vitro and in vivo. In conclusion, AO alleviates septic AKI by suppressing inflammatory responses through inhibiting the NF-κB pathway.

Keywords: Acute kidney injury, Aurantio-obtusin, Inflammation, NF-kappa B

Acute kidney injury (AKI) is one of the most frequent complications of sepsis and septic shock and occurs in nearly 50% of septic patients [1]. It is a devastating syndrome characterized by abnormalities in renal structure and function [2]. Septic AKI is life-threatening for hospitalized patients and critically ill patients [3]. The clinical manifestations of AKI are the increased serum blood urea nitrogen (BUN) and serum creatinine (SCr) levels [4-6]. AKI is related to high risk of cardiovascular disorder, mortality, and progression to end stage renal disease [7-9]. The pathogenesis of septic AKI is not completely understood, and more therapeutic strategies for sepsis-induced AKI are required.

Substantial evidence validates that inflammatory response elicits a crucial role in the pathogenesis of septic AKI [1,10]. Many anti-inflammatory therapies have been reported to mitigate septic AKI [11-13]. Lipopolysaccharide (LPS) can mediate the activation of nuclear factor kappa-B (NF-κB) and contribute to the increased production of inflammatory cytokines [14,15]. Thus, LPS-stimulated human renal proximal tubular epithelial cells (HK-2) were used for exploration of inflammation in in vitro experiments.

Anthraquinone compounds have been validated to elicit essential roles in inflammation alleviation over the past century [16-18]. Aurantio-obtusin (AO) is an anthraquinone compound that can be isolated from the dried seeds of Cassia obtusifolia L. [19]. Emerging evidence demonstrates that AO possesses many biological properties, such as neuroprotective, anti-allergic, anti-genotoxic, anti-mutagenic, anti-hypertension, and anti-oxidative effects [20-23]. A previous study shows that AO decreases the production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-6 via inhibition of the NF-κB signaling pathway in RAW264.7 cells [19]. Oral administration of AO (10 and 100 mg/kg) ameliorates inflammatory responses in lung tissues of LPS-administered mice with acute lung injury [24]. A recent study has reported that anthraquinone compounds can treat chronic renal failure [25]; however, the role of AO in septic AKI has not been clarified yet.

In the present work, the in vitro and in vivo models of sepsis-related AKI were constructed to explore whether AO can mitigate inflammation in AKI and explore its potential mechanism. The study may extend our understanding of AO mechanisms in diseases and might provide solid pharmacological evidence for future clinical applications.

Cell culture and treatment

HK-2 cells were commercially obtained from Shanghai Biological Technology Co., Ltd. enzyme research (C2056-1A). Cells were cultured in Dulbecco's minimal essential medium/Ham's F12 medium (CB003; Epizyme Biotech) containing 10% fetal bovine serum (76294-180, AVANTOR; ZEPING Bioscience & Technologies Co., Ltd.). Cell culture was performed in an incubator with humidified atmosphere of 5.0% carbon dioxide and 37°C. For cell treatment, HK-2 cells were plated in culture plates (96-well, 5 × 105 cells/ml) and incubated with or without increasing concentrations of AO (0, 10, 20, and 50 μM) for 1 day, followed by treatment with or without 1 μg/ml LPS (YT1319; YITA Biotechnology Co., Ltd.) for 1 day. The dose of LPS for cell treatment was selected according to a previous study [26].

3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl tetrazolium bromide (MTT) assay

The viability of HK-2 cells was measured by MTT assays. Cells were plated in culture plates (96-well, 1 × 104 cells/well) for 24 h. Next, cells were incubated with different dosages of AO (0, 10, 20, and 50 μM) for 24 h and then treated with or without 1 μg/ml LPS for 24 h. After that, 20 μl of MTT solution (5 mg/ml, M8180-1; Solarbio) was added to the plates for another 4 h incubation at 37°C. Then, the supernatant was removed, and dimethyl sulfoxide (DMSO, 100 μl) was added for dissolving the reduced formazan. Cell viability was evaluated by measuring the absorbance at 570 nm wavelength utilizing a microplate reader. Untreated cells were set as the control group and regarded as 100% of viable cells. The percentages of viable cells in AO groups or AO + LPS groups were compared to the control group.


BALB/c mice (8 weeks, SPF, C000102) were commercially obtained from Vital River Laboratory Animal Technology. All animals were housed in an environment (23°C–25°C, 50% to 60% humidity) with free access to water and standard diet. All animal experiments were carried out following the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, USA), and the experimental protocols were approved by the Ethics Committee of Wuhan Myhalic Biotechnology Co., Ltd (202203032).

AKI animal model

After acclimation to the environment for 7 days, mice were subjected to cecal ligation and puncture (CLP) operation. Xylazine (10 mg/kg, intramuscular [i.m.]) and ketamine (75 mg/kg, i.m.) were used to anesthetize the mice. The mice were placed in a supine position. The cecum was exposed and isolated by making a midline incision (15 mm) at the cleaned abdomen. The cecum was ligated at 5 mm from the cecal tip using a 4-0 silk suture and then punctured with a 22-gauge needle. Then, the cecum was put back immediately to its original position in the abdomen. The mice received sham operation only underwent peritoneum opening and bowel exposure with neither ligation nor puncture. The incision was closed and the mice were resuscitated using 0.5 ml sterile normal saline solution.

Animal grouping and treatment

Mice were divided into 4 groups (n = 10/group): Sham + DMSO group, Sham + AO group, CLP + DMSO group, and CLP + AO group. Immediately after the surgery, mice in CLP + AO group or sham + AO group were intragastrically given AO (40 mg/kg), and mice in CLP + DMSO group or sham + DMSO group were administered with DMSO (vehicle) of the same volume. AO (Fig. 1A, C17H14O7, purity > 97%) was purchased from Medchem Express and the dosage of AO was in accordance with the usage in a previous study [27]. At 24 h after the CLP surgery, blood samples and kidney tissues were harvested for subsequent experiments.

Figure 1. Aurantio-obtusin (AO) alleviates lipopolysaccharide (LPS)-induced HK-2 cell cytotoxicity.
(A) The chemical structure of AO is shown. (B) HK-2 cells were treated with different concentration of AO (0–50 μM) for 1 d, followed by detection of cell viability using an MTT assay kit. (C) The viability of LPS-stimulated HK-2 cells with or without AO treatment was measured by MTT assays. Values are presented as mean ± SD. ##p < 0.01 vs. control group (without AO or LPS treatment); *p < 0.05, **p < 0.01 vs. LPS (1 μg/ml) group.

Histological examination

The collected kidney tissues were fixed using 10% neutral buffered formalin. After embedded in paraffin, tissue samples were sliced into sections and stained with hematoxylin and eosin (H&E) for morphological examination. The percentage of death area in ten fields of cortical tissues per animal was calculated. Damages such as loss of brush boarder, tubular dilation, and epithelial necrosis were observed. Renal tubular injury was assessed utilizing the previously delineated 0–4 semiquantitative scale [28]. Score 0: normal kidney. Score 1: damage area less than 25%. Score 2: 25%–50% damage area. Score 3: 50%–75% injury percentage. Score 4: damage area more than 75%.

Enzyme-linked immunosorbent assay (ELISA)

The levels of pro-inflammatory cytokines including monocyte chemoattractant protein-1 (MCP-1), IL-6, TNF-α, and IL-1β in the supernatant of HK-2 cells or serum samples of mice were detected using commercial ELISA kits following the product directions.

Western blot analysis

RIPA lysis buffer was used to lyse HK-2 cells and mouse renal tissues on an ice-bath for 0.5 h, and the obtained lysate was subjected to centrifugation at 1 × 104 g at 4°C for 10 min. Prior to separation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, quantification of the proteins was performed utilizing a Bicinchoninic acid protein assay kit (E-BC-K318-M; Elabscience Biotechnology Co., Ltd.). The separated proteins were moved to a PVDF membrane followed by blockage with 5% skimmed milk powder. Next, the membrane was incubated at 4°C with primary antibodies (dilution: 1:1,000) for a night. Primary antibodies were listed as follow: phosphorylated (p)-inhibitor of kappa B alpha (IκBα; #5209), NF-κB p65 (#8242), p-IkappaB kinase (IKK)β (#2078), and β-actin (#4970). After rinsed with tris buffered saline containing 0.5% (v/v) Tween 20 (TBST) four times, the membrane was incubated at 37°C with secondary antibody (#7074, 1:3,000) for 1 h and then washed four times with TBST again. All these antibodies were purchased from Cell Signaling Technology. The bands were visualized using an enhanced chemiluminescence agent (Zen Bioscience) and the signal intensities were analyzed by the ImageJ software (NIH). β-actin served as the loading control.

Renal function measurement

Renal function was evaluated by examining the levels of SCr and BUN which are key index for renal injury. The levels of SCr and BUN in blood samples were evaluated using a biochemical analyzer (BX3010; Sysmex Corporation). The levels of Scr and BUN were measured with the picric acid method and the urease method, respectively.

Statistical analysis

Experimental data from at least three independent trails are expressed as the mean ± standard deviation. GraphPad Prism was employed for statistical analysis. Unpaired Student's t-test was used for the comparison between two groups, and one-way analysis of variance followed by Turkey post-hoc test were applied for the difference comparison among multiple groups. The possibility value less than 0.05 was deemed to be statistically significant.

AO alleviates LPS-induced HK-2 cell cytotoxicity

The chemical structure of AO is shown in Fig. 1A. First, HK-2 cell viability after AO treatment was measured by MTT assays. As shown by Fig. 1B, under normal condition, AO at concentrations of 10 μM, 20 μM, and 50 μM showed no significant effect on the viability of HK-2 cells (Fig. 1B). The results implied that AO ranging from 0 μM to 50 μM displayed no cytotoxic effect on HK-2 cells and the concentration gradient was thereof selected for the following experiments. Afterwards, the impact of AO on the viability of LPS-stimulated HK-2 cells was evaluated. Compared with the viability of HK-2 cells without any treatment (control group), the viability of LPS-stimulated HK-2 cells was significantly reduced (Fig. 1C). In addition, the reduction of cell viability induced by LPS was partly rescued by AO in a dose-dependent manner (Fig. 1C). In summary, AO ranging from 0 μM to 50 μM exerts no injury to HK-2 cells and AO dose-dependently ameliorates LPS-induced cytotoxicity on HK-2 cells.

AO suppresses inflammation in HK-2 cells

As known, LPS can induce inflammatory response in vitro and in vivo. The influence of AO treatment on LPS-induced inflammation in HK-2 cells was explored by measuring levels of proinflammatory cytokines in cells. As expected, the mRNA levels of MCP-1, IL-6, TNF-α, and IL-1β were notably increased in cells after LPS stimulation, and the alterations were gradually reduced by AO treatment in a dose-dependent way (Fig. 2). These results indicated that AO represses the secretion of pro-inflammatory cytokines in LPS-treated HK-2 cells.

Figure 2. Aurantio-obtusin (AO) attenuates inflammatory response in vitro.
HK-2 cells were treated with 0, 10, 20, or 50 μM AO for 1 d and then exposed to 1 μg/ml lipopolysaccharide (LPS) treatment for 1 d. (A–D) The levels of pro-inflammatory cytokines (MCP-1, IL-6, TNF-α, and IL-1β) in HK-2 cells were tested by ELISA. Values are presented as mean ± SD. MCP-1, monocyte chemoattractant protein-1; IL, interleukin; TNF, tumor necrosis factor; NF-κB, nuclear factor kappa-B. ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01 vs. LPS (1 μg/ml) group.

AO inhibits the activation of the NF-κB signaling pathway

Considering the involvement of the NF-κB signaling pathway in the pathogenesis of inflammatory diseases, the effects of AO on protein levels of pathway-related factors were examined. As shown by Fig. 3, LPS significantly increased the protein level of NF-κB p65 in HK-2 cells, and the change was effectively repressed by AO treatment dose-dependently. The finding indicated that the NF-κB-dependent inflammation can be suppressed by AO treatment in LPS-stimulated HK-2 cells. As the downstream kinase of IKK, IκB can be phosphorylated by IKK in the NF-κB signal pathway. It was clearly observed that the phosphorylated levels of IκBα and IKKβ were enhanced in LPS-stimulated HK-2 cells, and the phosphorylation of IκBα and IKKβ was gradually eliminated by AO treatment dose-dependently (Fig. 3). Therefore, it could be concluded that AO blocks LPS-activated NF-κB signal pathway in vitro.

Figure 3. Aurantio-obtusin (AO) inhibits the nuclear factor kappa-B (NF-κB) pathway in lipopolysaccharide (LPS)-stimulated HK-2 cells.
(A–D) The protein expression of NF-κB, p-IκBα, and p-IKKβ were detected in LPS-treated cells with or without AO treatment by Western blotting. Values are presented as mean ± SD. ###p < 0.05 vs. control group; **p < 0.01, ***p < 0.001 vs. LPS (1 μg/ml) group.

AO attenuates renal dysfunction in vivo

Representative images of H&E staining showed that CLP operation significantly changed renal histopathology, such as renal tubule dilation, tubular structure destruction, and renal epithelial cell swollen in CLP + DMSO group relative to that in the Sham + DMSO group (Fig. 4A). Importantly, CLP-induced renal tubular injury was mitigated by AO treatment (Fig. 4A). Renal damage score was determined based on pathological analysis, which revealed that CLP-induced kidney damage was attenuated by AO treatment (Fig. 4B). Levels of SCr and BUN are two indicators of renal function. CLP-operated mice showed higher levels of SCr and BUN than sham-operated mice, while treatment with AO notably reduced SCr and BUN levels in mouse models (Fig. 4C, D). All these results suggested that AO ameliorates CLP surgery-induced renal injury in vivo.

Figure 4. Aurantio-obtusin (AO) attenuates renal injury in vivo.
Mice received CLP surgery or sham operation and administrated with AO or DMSO are divided into four experimental groups: Sham + DMSO, Sham + AO, CLP + DMSO, and CLP + AO. (A) Representative images for H&E staining in renal tissues are provided. Scale bar = 50 μM. (B) Kidney damage score in each group was determined based on pathological analysis. (C, D) The levels of Scr and BUN were measured in blood samples of indicated groups. N = 10/group. Values are presented as mean ± SD. Red arrows: dilation of renal tubules. Blue arrows: swollen renal tubular epithelial cells. Black arrows: destructed tubular structures. CLP, cecal ligation and puncture; DMSO, dimethyl sulfoxide; SCr, serum creatinine; BUN, blood urea nitrogen. **p < 0.01, ***p < 0.001 vs. Sham + DMSO group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. CLP + DMSO group.

AO alleviates CLP-induced inflammation and inactivates NF-κB signaling in vivo

The influences of AO on inflammatory response in CLP-treated mice were also evaluated. The results of ELISA delineated that the levels of MCP-1, IL-6, TNF-α, and IL-1β were markedly elevated in the serum of CLP-operated mice in comparison to those in mice of sham groups. However, the increase in levels of these pro-inflammatory cytokines in CLP-operated mice was notably reduced by AO treatment (Fig. 5A–D). Moreover, protein levels of factors associated with NF-κB pathway were measured in mouse renal tissues. As Fig. 5E–H revealed, protein levels of NF-κB p65, phosphorylated IκBα, and phosphorylated IKKβ were markedly increased in renal tissues of CLP + DMSO group, and the alteration was reversed by AO treatment. Overall, AO inhibits NF-κB-dependent inflammatory response caused by CLP operation in vivo.

Figure 5. Aurantio-obtusin (AO) alleviates CLP-induced inflammation and inactivates NF-κB signaling in vivo.
(A–D) ELISA detected the serum levels of inflammatory cytokines in Sham + DMSO group, Sham + AO group, CLP + DMSO group, and CLP + AO group. (E–H) Protein levels of factors associated with NF-κB pathway in mouse renal tissues were quantified using Western blotting. N = 10/group. Values are presented as mean ± SD. CLP, cecal ligation and puncture; DMSO, dimethyl sulfoxide; MCP-1, monocyte chemoattractant protein-1; IL, interleukin; TNF, tumor necrosis factor; NF-κB, nuclear factor kappa-B. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Sham + DMSO group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. CLP + DMSO group.

AKI has high incidence in intensive care units with characteristics of a rapid increase in SCr and a decrease in urine output [29]. Due to the high mortality of sepsis-associated AKI, it is of great urgency to explore the novel pharmacological interventions for AKI prevention and treatment. This study revealed the role of AO in a cell model and a mouse model of septic AKI. It was found that AO alleviated HK-2 cell injury and renal dysfunction of mice by repressing inflammatory responses via blockage of the NF-κB signaling pathway.

Gram-negative bacteria have been identified as the main etiology of sepsis, and endotoxin LPS is from the cellular wall of the bacteria [30]. LPS can stimulate the release of pro-inflammatory cytokines and result in a transient immune activation, which is characterized by the upregulation of IL-6, IL-1β and TNF-α [31]. The release of excessive pro-inflammatory mediators contributes to fatal systemic inflammatory response syndrome, leading to multi-organ dysfunction, disseminated intravascular coagulation, vascular dysfunction, and shock [32]. In this work, a cell model of AKI was successfully constructed by treating HK-2 cells with 1 μg/ml LPS. AO (0–50 μM) showed no cytotoxicity on HK-2 cells and dose-dependently rescued LPS-induced decrease in cell viability. A previous study validates that AO from 6.25 μM to 50 μM exerts no cytotoxicity to RAW264.7 cells [19].

Since surgical patients, the elderly, and patients with chronic diseases are susceptible to sepsis [26], the findings of in vitro experiments were confirmed in a mouse model of CLP-caused severe polymicrobial sepsis. Histopathology examination in the present work revealed that glomerular structure is damaged, renal tubules were dilated, and renal epithelial cells were swollen in renal tissues of mice received CLP surgery. Importantly, administration of AO attenuated renal injury and lesion severity. Additionally, the levels of SCr and BUN, two index of renal injury, were significantly elevated in CLP + DMSO group compared with those in the Sham group, and AO treatment substantially reduced CLP-induced high concentration of SCr and BUN. Overall, the present study demonstrated that AO protects HK-2 cells and model mice from septic AKI.

According to previous studies, AO plays an essential role in various diseases. AO has an anti-inflammatory role in lung injury [24] and in RAW264.7 cells via inactivation of NF-κB [19]. AO can also mitigate nonalcoholic fatty liver disease by promoting autophagy flux via inducing AMPK phosphorylation and promoting fatty acid oxidation [33]. In addition, AO alleviates obesity by regulating lipid metabolism-related genes and inflammatory cytokines [34] or by activating PPARα-dependent mitochondrial thermogenesis in brown adipose tissue [35]. Moreover, AO has the potential to treat hypertension since it can increase nitric oxide production and regulate endothelium integrity by modulating PI3K/Akt/eNOS pathway [23]. Moreover, AO exerts a neuroprotective role by serving as an antagonist against V1AR [36]. However, AO was reported to exert a hepatotoxic effect on female mice by activating NLRP3 inflammasome partly through KCNN4 upregulation, reactive oxygen species production, and NF-κB inhibition [37]. Therefore, more experiments should be conducted to identify safe dosage of AO for clinical application.

The pathology of AKI in sepsis is multifactorial and complex. Emerging studies highlight the prominent roles of pro-inflammatory cytokines in sepsis-triggered AKI [38]. Excessive release of inflammatory mediators, such as TNF-α, can trigger pathophysiological abnormities and contribute to the pathogenesis of sepsis-related AKI and other systemic dysfunctions [39]. Hence, suppression of inflammatory mediators might become a therapeutic strategy for septic AKI treatment. Our study illuminated that the levels of TNF-α and IL-1β were reduced by AO treatment (0–50 μM) in LPS-stimulated HK-2 cells. IL-1β and TNF-αare the main mediators responsible for the expression of MCP-1 and other chemokines. In HK-2 cells, AO reduced LPS-promoted release of IL-6 and MCP-1, which is in consistence with the results in in vivo studies. According to a recent study, AO with the concentration of 100 and 200 μM significantly weakens the viability of glomerular endothelial cells and induces the secretion of IL-6, TNF-α, TGF-β1, and MCP-1 [40]. Therefore, identifying the safe dosage of AO is important and further investigation should be conducted.

NF-κB is a critical transcription factor and is implicated in LPS-evoked inflammatory responses in renal damage and sepsis pathophysiology [41,42]. NF-κB signaling pathway can be activated by physical interaction with TNF-α, and the activated pathway in turn enhances the expression of other inflammatory cytokines [43], thus aggravating the inflammatory responses in AKI [44]. To validate whether AO affects NF-κB activity, key factors associated with the pathway were measured. It was found that the enhanced phosphorylation of IKKβ and IκBα as well as the activated NF-κB in in vitro and in vivo models were suppressed by AO treatment. NF-κB can be dissociated from IκBα by the phosphorylation effect of IKKs on the inhibitory IκBα protein, which liberates NF-κB to the nucleus to activate targeted inflammatory genes. Consistent with previous studies that revealing the anti-inflammatory role in lung injury and RAW264.7 cells via inhibition of NF-κB signaling [19,24], our study indicated that AO suppressed inflammatory response in sepsis-related AKI by inhibiting NF-κB p65 activation and suppressing phosphorylation levels of IκBα and IKKβ.

The relationship between cytotoxicity and inflammation are complex. Despite the direct upregulation of inflammation mediated by DNA damage response, DNA damage can promote inflammation through cytotoxicity [45]. In the current study, LPS induced the toxicity of HK-2 cells and inflammatory response; to some degree, the cytotoxicity accelerated the inflammation. Therefore, it can be explained that AO ameliorated inflammation in HK-2 cells partly by reducing cytotoxicity.

To summarize, the protective function of AO against septic AKI has been demonstrated in the current study. The underlying mechanism by which AO regulated septic AKI may be correlated with anti-inflammatory functions. AO suppressed the release of pro-inflammatory cytokines by inhibiting the activation of NF-κB via hindering the phosphorylation of the key proteins in this pathway. The study suggested that AO possesses a potential application for the treatment of endotoxemia-induced AKI. Nevertheless, further experiments are necessary to investigate whether therapeutic and prophylactic administration of AO can effectively prevent the occurrence of AKI and improve the clinical outcome of patients.

The authors appreciate all the participants providing supports for this study.

This work was supported by Youth Talent Project of Hubei Provincial Health Commission (No.wj2019H151), Guiding funded proiect of Hubei Provincial Department of Education (No.B2018086) and Doctor Program of Affiliated Hospital of Jianghan University (No.K20230327006).

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