Obtusifolin inhibits podocyte apoptosis by inactivating NF-κB signaling in acute kidney injury

Abstract

Acute kidney injury (AKI) is a common clinical condition and is associated with unacceptable morbidity and mortality. Obtusifolin is an anthraquinone extracted from the seeds of Cassia obtusifolia with anti-inflammatory properties. This study focused on the role and mechanism of obtusifolin in AKI. The mouse podocyte cell line MPC5 was exposed to lipopolysaccharide (LPS) to establish a cell model of AKI. The viability of MPC5 cells treated with obtusifolin and/or LPS was detected by 3-(4, 5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay. Cell apoptosis was analyzed by flow cytometry. The levels of podocyte injury- and apoptosis-related proteins as well as the nuclear factor-kappaB (NF-κB) signaling pathway was examined using western blotting analysis. The renal protective effects of obtusifolin were determined using an LPS-induced mouse model of AKI. Serum creatinine and blood urea nitrogen levels were measured. Hematoxylin–eosin staining of kidney sections was performed to evaluate renal histology. We found that MPC5 cells treated with LPS showed suppressed cell viability (p < 0.01) and increased cell apoptosis (p < 0.001). LPS reduced the protein expression of Bcl-2, nephrin, and synaptopodin as well as increased the protein levels of Bax and Cleaved Caspase-3 in podocytes in a concentration-dependent manner (p < 0.01). In addition, 10 μg/ml LPS-repressed cell viability was rescued by obtusifolin in a concentration-dependent manner (p < 0.01). Moreover, LPS-induced increase in MPC5 cell apoptosis was reversed by obtusifolin treatment (p < 0.01). Obtusifolin administration ameliorated LPS-induced kidney injury and reduced blood urea nitrogen and serum creatinine levels in mice (p < 0.001). Additionally, obtusifolin inhibited LPS-induced activation of NF-κB signaling in vitro and in vivo (p < 0.01). Overall, obtusifolin was effective in protecting renal function against LPS-induced AKI via inactivation of NF-κB signaling, which suggested that obtusifolin may act as a valuable agent for AKI therapy.

Introduction

Sepsis is a heterogenous syndrome caused by a dysregulated host response to fungal, viral, or bacterial infections (Feng et al. 2020; Li et al. 2019). It is well-known that in septic patients, kidney is one of the organs frequently affected (Hellman et al. 2021). According to the statistics, more than 50% of patients with sepsis are accompanied by acute kidney injury (AKI) (Wang 2023). Importantly, the mortality rate of septic AKI patients is higher than those patients with AKI alone, making it a severe threat to human health (Plataki et al. 2011). Therefore, it is needed to explore the mechanisms underlying sepsis-associated AKI and seek practical management strategies to treat this disease.

Podocytes are terminally differentiated glomerular epithelial cells in the kidney and are essential for maintaining the normal structure and function of glomerular filtration barrier (Zhan et al. 2022; Hejazian et al. 2023). The injury of podocytes is considered as the main reason for glomerular diseases (Hanamura et al. 2014; Wang et al. 2014). In recent years, accumulating studies have indicated that podocyte injury is implicated in the process of septic AKI. For instance, lipopolysaccharide (LPS)-caused podocyte injury contributes to AKI pathogenesis by regulating the Wnt/β-catenin pathway (Senouthai et al. 2019). Angptl3 knockout exerts renal protective effects by ameliorating podocyte injury in LPS-induced mouse models of AKI (Ma et al. 2022). Therefore, attenuation of podocyte injury may be beneficial for the treatment of septic AKI.

Apoptosis of podocytes is an important factor involving in the development of sepsis-associated AKI (Zhang et al. 2021a). Apoptosis is a key cellular process, which can timely remove damaged cells to maintain tissue homeostasis. There are three cell apoptosis pathways: death receptor pathway, mitochondrial pathway, and endoplasmic reticulum (ER) pathway (Hu et al. 2018; Jurisić et al. 2004). Bax and Bcl-2 are important proteins in mitochondrial pathway. Bax belongs to the Bcl-2 family and is a core regulator of the intrinsic pathway of apoptosis. Upon apoptotic stimuli, it is activated and oligomerize at the mitochondrial outer membrane to mediate its permeabilization (Popović et al. 2007). Bcl-2 is an anti-apoptotic protein that can repress the function of Bax to restrain apoptotic process (Du 2023). Cleaved Caspase-3 is also closely correlated with activation of mitochondrial pathway (Breckenridge and Xue 2004).

Cassia obtusifolia is a traditional Chinese medicine which possesses a wide range of pharmacological properties, such as neuroprotective, anti-inflammatory, antidiabetic, and antimicrobial properties (Ali et al. 2021). As an anthraquinone compound in the seeds of Cassia obtusifolia, obtusifolin possesses many pharmacological effects, including antiinflammation (Nam, et al. 2021), analgesic effects (He et al. 2014), antioxidant (Tang and Zhong 2014), and antitumor (Hsu et al. 2014). Obtusifolin has been shown to repress mitochondrial apoptosis induced by high glucose in human umbilical vein endothelial cells, and it also reduces capillary cell apoptosis in the retina of streptozotocin-induced diabetic rats (Hou et al. 2014). Moreover, obtusifolin notably ameliorates cognitive impairment induced by scopolamine in mice (Kim et al. 2009), and ameliorates hyperlipidemia and hyperglycemia in diabetic rats by attenuating oxidative damage (Tang and Zhong 2014). However, the effects of obtusifolin against septic AKI have not been investigated yet.

LPS is commonly used for the induction of septic AKI (Hu et al. 2022). In this study, in vitro and in vivo models of septic AKI were established using LPS. We investigated the effects of obtusifolin on kidney injury caused by LPS and explored the possible mechanisms, which might provide an experimental basis for understanding of the beneficial effects of obtusifolin on AKI.

Materials and methods

Cell culture

Mouse podocytes (MPC5) were commercially obtained from iCell Bioscience Inc (Shanghai, China) and incubated in RPMI-1640 medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS; 76,294–180, AVANTOR, Australia) and 20 U/ml recombinant mouse interferon (IFN)-γ at 33 °C. For the differentiation of MPC5 cells, RPMI-1640 medium containing 5% FBS without IFN-γ was used to incubate MPC5 cells at 37 °C for 10 days (Ha and Yu 2021). All the cell culture experiments were performed three independent replicates.

Cell treatment

MPC5 cells were stimulated with 1, 5 and 10 μg/ml LPS (Sigma-Aldrich, Shanghai), 1–10,000 μM obtusifolin (B20858, HPLC ≥ 98%, yuanye Bio-Technology, Shanghai), or 10 μg/ml LPS plus 0, 1, 10, and 100 μM obtusifolin. Cells were treated with obtusifolin for 24 h before LPS induction for 48 h.

3-(4, 5-Dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay

The viability of MPC5 cells was detected using an MTT assay kit (KA1606-E, Abnova, AmyJet Scientific Inc., Wuhan, China) according to the product manuals. MPC5 cells (3 × 10³ cells/well) were plated in 96-well plates and incubated with LPS or/and obtusifolin at 37℃ for 24 h, followed by incubation with 20 μl MTT for 4 h. Each concentration was tested in three independent replicates. Then, the formazan was dissolved by dimethyl sulfoxide. Cell viability was evaluated by measuring the absorbance at 490 nm using a microplate reader as previously described (Scherbakov et al. 2023).

Flow cytometry analysis

Cell apoptosis was tested using an annexin V-FITC/PI apoptosis detection kit (orb322264, biorbyt, booute biotechnology Co., Ltd., Wuhan, China) as per the product directions. Briefly, MPC5 cells were treated 10 μg/ml LPS plus 0, 1, 10, and 100 μM obtusifolin. The collected cells were rinsed twice with phosphate buffer saline and centrifuged (1,200 × g, 4 °C) for 5 min. The supernatant of cells was removed, and the cells were resuspended in binding buffer and stained with Annexin V-FITC and PI in the dark for 15 min. The stained cells were quantified and analyzed using flow cytometer (BD Biosciences, USA) as previously described (Jurisic et al. 2011). The experiment was repeated three times.

Western blotting

Total proteins were extracted using radio immunoprecipitation assay (RIPA) buffer (R0020-100, Solarbio, Beijing, China). A bicinchoninic acid kit (orb219872, biorbyt) was used to determine protein concentration. Prior to transfer to nitrocellulose membranes (N8142, seebio, seebio biotech Co., Ltd., Shanghai, China), protein samples were separated by 10% SDS-PAGE gel. The membranes were blocked with 5% nonfat milk power and then incubated with primary antibodies against p-IκBα (2859, 1:1,000; Cell Signaling Technology), p-IKKβ (2028, 1:1,000), Bax (ab182733, 1:2,000), Cleaved Caspase-3 (ab214430, 1:5,000), nuclear factor-kappaB (NF-κB) p65 (ab32536, 1:1,000), Bcl-2 (ab182858, 1:2,000), nephrin (ab216341, 1:1,000), synaptopodin (ab259976, 1:1,000), and GAPDH (ab9485, 1:2,500) overnight at 4 °C. After washing three times with TBST (5 min/time), the membranes were incubated with appropriate secondary antibody (ab288151, 1:2,000) at 37 °C for 2 h. The signals were visualized using an enhanced chemiluminescence detection kit (orb90506, biorbyt). ImageJ software (NIH, USA) was used to quantify the intensities of target proteins. The experiment was repeated five times.

Animal experiments

Male C57BL/6 mice (8-week-old) were purchased from vital river Co. Ltd. (Beijing, China). Animals had free access to water and were housed in a room with a 12 h light/dark cycle. All protocols involving animals were reviewed and approved by the ethics committee of the hospital. Animals were randomized into four groups: sham, obtusifolin, LPS, LPS + obtusifolin. N = 8/group. For LPS-induced AKI models, LPS (10 mg/kg) was intraperitoneally (i.p.) injected into mice (Liu et al. 2020). For the LPS + obtusifolin group, obtusifolin (100 mg/kg, i.p.) was given 2 h before LPS treatment. The dosage of obtusifolin was selected according to previous study (Nam, et al. 2021). The mice in the sham group were treated with the same amount of 0.9% saline. The mice in the obtusifolin group were injected with obtusifolin (100 mg/kg, i.p.). Mice were sacrificed 24 h after LPS injection. Both kidneys were collected and used for further experiments.

Measurement of serum creatinine (SCr) and blood urea nitrogen (BUN)

Blood samples were centrifuged at 2,000 × g for 10 min at 4 °C and the supernatants were subsequently collected. BUN and SCr levels in murine blood were determined using a Hitachi 7060 automated chemistry analyzer (Diamond Diagnostics, Inc.) as per the manufacturer’s protocols.

Histology evaluation

Both kidneys were used for histology studies. Briefly, kidney samples were fixed with 4% paraformaldehyde overnight at room temperature, embedded in paraffin, and cut into Sects. (4 µm). Sections were then dewaxed, rehydrated, and stained with hematoxylin–eosin stain (Sigma-Aldrich) as per the manufacturer’s protocols.

Statistical analysis

Data are expressed as the mean ± standard deviation (SD) of three independent experiments and were analyzed by GraphPad Prism 7.0 software (San Diego, CA, USA). The distribution of the data was tested by means of the shapiro wilk test. All data in this study conformed to a normal distribution. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc analysis. The p value less than 0.05 was considered statistically significant.

Results

LPS treatment induces MPC5 cell injury

To establish cell models of AKI, varying concentrations of LPS (1, 5 and 10 μg/ml) were used to stimulate MPC5 cells, and the viability of MPC5 cells was tested by an MTT assay kit. The results showed that LPS stimulation significantly reduced MPC5 cell viability in a concentration-dependent manner (p < 0.01, Fig. 1A). Additionally, 10 μg/ml LPS reduced the viability to approximately 50%. Flow cytometry analysis demonstrated that the apoptosis of MPC5 cells was increased after LPS exposure (p < 0.01, Fig. 1 B, C). Moreover, as western blotting showed, LPS stimulation led to a significant increase in Bax and cleaved caspase-3 protein levels as well as a decrease in Bcl-2 protein levels in MPC5 cells (p < 0.01, Fig. 1D–G). The levels of podocyte-specific markers synaptopodin and nephrin in LPS-stimulated podocytes were detected. Western blotting showed that synaptopodin and nephrin protein levels were reduced in the LPS group compared to the control group (p < 0.001, Fig. 1H–J), suggesting that LPS induced MPC5 cell injury.

Fig. 1.

LPS stimulation induces MPC5 cell injury. A MPC5 cells were treated with 1, 5 and 10 μg/ml LPS for 48 h, followed by detection of cell viability using an MTT assay kit. B MPC5 cell apoptosis in the control group, LPS-1, 5, and 10 groups was analyzed using flow cytometry. C Quantification of cell apoptosis in each group. D The protein levels of Bax, Bcl-2, and Cleaved Caspase-3 in each group were measured by western blotting. E–G Quantification of Bax, Bcl-2, and Cleaved Caspase-3 protein expression in each group. H–J The protein levels of synaptopodin and nephrin in each group. All experiments were performed three times and data are expressed as mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs the control group

Obtusifolin alleviates LPS-induced inhibition of MPC5 cell viability

The chemical structure of obtusifolin is shown in Fig. 2A. To determine the cytotoxicity of obtusifolin in vitro, MPC5 cells were treated with different concentrations of obtusifolin for 24 h. It was found that obtusifolin (1–100 μM) did not change the viability of MPC5 cells (Fig. 2B). IC25, IC50, and IC75 values of obtusifolin were 294.49 μM, 1,327.00 μM, and 4,660.80 μM, respectively. Then, MPC5 cells were treated with 10 μg/ml LPS or/and obtusifolin (1, 10, 100 μM). Compared to the control group, LPS stimulation significantly inhibited cell viability (p < 0.01). However, obtusifolin concentration-dependently restored the viability of MPC5 cells under LPS stimulation (p < 0.01, Fig. 2C). Therefore, obtusifolin exerted protective effects against LPS-induced podocyte injury.

Fig. 2.

Obtusifolin alleviates LPS-induced inhibition of MPC5 cell viability. A the chemical structure of obtusifolin. B MPC5 cells were treated with different concentrations of obtusifolin for 24 h. Cell viability was tested using MTT assay. C MPC5 cells were treated with 1, 10, and 100 μM obtusifolin for 24 h and then exposed to 10 μg/ml LPS for 48 h. MTT assay was used for measurement of cell viability. All experiments were performed three times and data are expressed as mean ± SD. ^**p < 0.01 vs the 0 μM obtusifolin group; ^#p < 0.05, ^##p < 0.01 vs the 10 μg/ml LPS group

Obtusifolin attenuates LPS-induced cell apoptosis

To probe into the action of obtusifolin on LPS-induced podocyte damage, MPC5 cells were treated with 1, 10, 100 μM obtusifolin in the presence or absence of LPS exposure. Flow cytometry analysis manifested that increased cell apoptosis caused by 10 μg/ml LPS stimulation was inhibited by obtusifolin treatment in a concentration-dependent way (p < 0.01, Fig. 3A, B). LPS exposure-induced protein expression levels of Bax and cleaved caspase-3 were reduced by obtusifolin (p < 0.01). Additionally, reduced Bcl-2 protein expression caused by LPS was significantly restored upon obtusifolin treatment (p < 0.01, Fig. 3C–F). These results demonstrated that obtusifolin attenuated LPS-induced podocyte apoptosis.

Fig. 3.

Obtusifolin concentration-dependently reduces LPS-induced cell apoptosis. MPC5 cells were divided into five groups: 0 μM obtusifolin group, 10 μg/ml LPS group, and 10 μg/ml LPS + 1, 10, and 100 μM obtusifolin groups. A flow cytometry was performed to evaluate cell apoptosis in each group. B quantification of cell apoptosis in each group. C–F the levels of proteins associated with apoptosis were measured by western blotting. All experiments were performed three times and data are expressed as mean ± SD. ^**p < 0.01, ^***p < 0.001 vs the 0 μM obtusifolin group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs the 10 μg/ml LPS group

Obtusifolin inhibits NF-κB signaling activated by LPS in podocytes

It has been reported that inhibition of NF-κB signaling in mice alleviates LPS-induced AKI (Hu et al. 2022). Obtusifolin treatment reduced the levels of activated NF-κB (He et al. 2014), which also inhibits nuclear translocation of NF-κB p65 in NCI-H292 cells (Nam, et al. 2021). Therefore, we investigated whether obtusifolin regulates NF-κB signaling in podocytes under LPS stimulation. The western blotting results showed that obtusifolin inhibited LPS-induced upregulation of NF-κB p65 expression in a concentration-dependent way (p < 0.01, Fig. 4A, B). Obtusifolin treatment also suppressed IκBα phosphorylation in a concentration-dependent manner (p < 0.01, Fig. 4A–C). Activation of the IkappaB kinases (IKKs) is a key step involved in the activation of NF-κB pathway. Therefore, we investigated whether obtusifolin represses the phosphorylation of IKKβ in LPS-treated MPC5 cells. Immunoblotting analysis showed that LPS-triggered upregulation of IKKβ phosphorylation was reversed by obtusifolin treatment (p < 0.01, Fig. 4A–D). Conclusively, obtusifolin blocked activation of NF-κB signaling in LPS-treated podocytes.

Fig. 4.

Obtusifolin inactivates NF-κB signaling in LPS-challenged podocytes. A the protein expression of NF-κB p65, p-IκBα, and p-IKKβ in different groups was examined by western blotting. B–D quantification of NF-κB p65, p-IκBα, and p-IKKβ protein levels in each group. All experiments were performed three times and data are expressed as mean ± SD. ^**p < 0.01, ^***p < 0.001 vs the 0 μM obtusifolin group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs the 10 μg/ml LPS group

Obtusifolin treatment ameliorates LPS-induced AKI in mice

The renal protective effects of obtusifolin were determined using an LPS-induced mouse model of AKI. SCr and BUN, which are two biomarkers for septic AKI, were examined to evaluate renal function of mice. Compared to the control group, BUN and SCr levels were significantly elevated in the LPS groups (p < 0.001). However, obtusifolin administration significantly reduced LPS-induced BUN and SCr levels (p < 0.001, Fig. 5A, B). As hematoxylin–eosin staining of kidney sections showed, after LPS stimulation, mice exhibited severe kidney injury, including glomeruli abnormality in morphology, loss of brush border, and infiltration of inflammatory cells. However, obtusifolin-treated mice exhibited less changes in morphology and reductions in inflammatory cell infiltration (Fig. 5C). We also examined the levels of apoptosis-related proteins and NF-κB. The results showed that obtusifolin treatment significantly reduced the levels of Bax, Cleaved Caspase-3, and NF-κB p65 in the kidney of LPS-challenged mice (p < 0.001, Fig. 5D–G), suggesting that obtusifolin inhibited apoptosis and inflammation induced by LPS in vivo. Overall, obtusifolin exhibited protective effects against LPS-induced AKI.

Fig. 5.

Obtusifolin treatment ameliorates LPS-induced AKI in mice. Mice were pretreated with obtusifolin and then challenged with LPS. A SCr and B BUN levels were measured in the blood collected 24 h after challenge. C Twenty-four hours after LPS challenge, renal histology was performed in the kidney from mice. D the protein expression of Bax, Cleaved Caspase-3, and NF-κB p65 in the kidney was examined by western blotting. E–G quantification of Bax, Cleaved Caspase-3, and NF-κB p65 protein levels in each group. N = 8/group. Data are expressed as mean ± SD. ^***p < 0.001 vs the shm group; ^###p < 0.001 vs the LPS group

Discussion

Sepsis is a systemic inflammatory response syndrome caused by infection and is the main cause of AKI (Tibi et al. 2023). Obtusifolin is an anthraquinone‑based compound with anti-inflammatory activity, and it has been shown to exert beneficial effects on various inflammatory diseases (He et al. 2014; Hou et al. 2014). LPS is an important part of the outer membrane of gram-negative bacteria (Giordano et al. 2020). In this study, LPS was used to establish a sepsis-induced AKI model. Obtusifolin treatment in vitro inhibited LPS-induced podocyte apoptosis and inflammatory signaling pathway. Scr and BUN levels were measured to evaluate kidney function. These two indicators are widely used to evaluate kidney function in animal experiments (Deng et al. 2024; Qiang et al. 2023). We found that obtusifolin reduced LPS-induced increases in Scr and BUN levels and ameliorated LPS-induced kidney injury, as evidenced by improvements in kidney morphology and reductions in inflammatory cell infiltration. Altogether, these results demonstrated that obtusifolin exerted a protective effect against LPS-induced AKI.

The pathophysiology of sepsis is characterized by the initial severe inflammatory phase and subsequent long-lasting immunosuppression (Liu et al. 2022). It has been reported that injection with LPS in C57BL/6 mice leads to severe kidney injury. Additionally, LPS stimulation increases podocyte apoptosis by increasing the levels of proapoptotic protein Bax and decreasing the levels of antiapoptotic protein Bcl-2 (Ma et al. 2022). The imbalance of body’s immune function caused by apoptosis dysregulation is key contributor to the immunosuppression of septic patients (Brady et al. 2020; Menassa et al. 2020). Apoptosis is a multiple gene-regulated programmed cell death process, and the mitochondria-mediated pathway of apoptosis is a classical apoptosis signaling pathway (Zhang et al. 2021b). Alteration of Bax/Bcl-2 ratio increases cytochrome c release from the mitochondria, which activates the downstream caspase-cascade that ultimately leads to apoptosis (Zaib et al. 2022). Podocytes is a terminally differentiated cell with a limited mitotic ability. Podocyte apoptosis has been considered as an early event that contributes to the initiation of glomerular lesions (Wang et al. 2022). A previous study showed that obtusifolin suppresses mitochondrial apoptosis of human umbilical vein endothelial cells under high glucose conditions (Tang et al. 2018). Obtusifolin administration reduces capillary cell apoptosis in the retina of streptozotocin-induced diabetic rats (Hou et al. 2014). In this study, there was an increase in the protein expression of Bax and Cleaved Caspase-3 and a decrease in the protein expression of Bcl-2 in MPC5 cells after LPS stimulation. However, these proapoptotic effects caused by LPS stimulation were reversed by obtusifolin. Moreover, obtusifolin decreased the Bax and Cleaved Caspase-3 protein levels in LPS-induced AKI mouse models, which was consistent with the findings in vitro.

NF-κB is a nuclear transcription factor that plays a key role in regulating apoptosis, oxidative stress, and inflammation (Wang and Shen 2022). NF-κB expression has been found to be upregulated in the kidney of mice with septic AKI (Ren et al. 2020). In the microenvironment of oxidative damage and inflammation, an activated NF-kappaB dimer translocates to the nucleus and exerts transcriptional effects on downstream inflammatory factors, which aggravates inflammatory response (Balan and Locke 2011). Studies have shown that blocking activation of NF-κB signaling ameliorates renal damage in LPS-induced septic AKI by reducinng apoptosis and renal inflammation (Han et al. 2023; Song et al. 2023). It has been reported that obtusifolin inhibits the nuclear translocation of NF-κB p65 in airway epithelial cells (Choi et al. 2019), and also downregulates the expression of NF-κB in the lumbar spinal cord of complete Freund's adjuvant-induced rats (He et al. 2014). In this study, we found that obtusifolin treatment reduced the phosphorylation of IKKβ and IκBα and the expression of NF-κB p65 in LPS-treated MPC5 cells. Moreover, obtusifolin decreased the expression of NF-κB p65 in LPS-induced AKI mouse models. These findings showed that the protective effects of obtusifolin on AKI might be related to inhibition of NF-κB signaling.

Although we elucidated the potential anti-inflammatory mechanism action of obtusifolin, the exact mechanism how obtusifolin regulates NF-κB signaling remains unknown and needs further investigations, which may provide an experimental basis for understanding of the beneficial effects of obtusifolin on AKI. Additionally, our research's scope was limited to a specific model of AKI induced by LPS, leaving the effectiveness of obtusifolin against alternative causes of AKI yet to be investigated. Furthermore, there is a lack of sufficient drug exposure over time to explain the observed efficacy, and it is needed to determine the long-term benefits resulting from obtusifolin treatment and measure the plasma and tissue levels of obtusifolin to validate the translational potential of obtusifolin for AKI treatment.

In conclusion, we demonstrated that obtusifolin reduces LPS-induced podocyte apoptosis by inhibiting the NF-κB pathway in sepsis-associated AKI, suggesting that obtusifolin might be a promising therapeutic agent for AKI therapy.

Author contributions

Haiyan Xiang was the main designer of this study. Haiyan Xiang, Yan Wu, Yun Zhang and Yuanhao Hong performed the experiments and analyzed the data. Haiyan Xiang, Yan Wu and Yaling Xu drafted the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by Young Talents Project of Health Commission of Hubei Province (wj2019H151).

Data availability

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

All protocols involving animals were reviewed and approved by the Ethics Committee of Wuhan Sixth Hospital, Affiliated Hospital of Jianghan University.