KDM2B regulates inflammation and oxidative stress of sepsis via targeting NF‐κB and AP‐1 pathways

Abstract Backgrounds The kidney is an easily affected organ with sepsis which is a main underlying cause of acute kidney injury (AKI). Histone‐modifying lysine‐specific demethylase 2B (KDM2B) is involved in numerous pathological processes, such as cell senescence and tumor development. However, the role of KDM2B in sepsis‐induced AKI is unclear. Objects To investigate the role of KDM2B on cell viability, inflammation and oxidative stress of sepsis‐associated AKI, and the involved signaling pathways. Methods An AKI model in vitro was established through lipopolysaccharide (LPS)‐induction in HK‐2 cells. Western blots were performed to evaluate the expression of KDM2B, cyclooxygenase 2 (COX2), inducible nitric oxide synthase (iNOS), p65, c‐Jun and c‐Fos, as well as p65 phosphorylation. Cell viability was measured using CCK‐8 kit. ELISA was performed to analyze the production of layered double hydroxide (LDH), tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐18, vascular cell adhesion molecule‐1 (VCAM‐1), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and H2O2. The qPCR was used to evaluate the transcription level of TNF‐α, IL‐1β, IL‐18, and VCAM‐1. Results KDM2B knockdown alleviated LPS‐induced cytotoxicity, decreased LDH release, and improved cell viability. KDM2B knockdown reduced concentration of inflammation‐related molecules including TNF‐α, IL‐1β, IL‐18, and VCAM‐1, and inhibited their transcription. Moreover, KDM2B knockdown promoted the quantity of SOD and GSH, while declined the production of MDA, H2O2, COX2, and iNOS. Further, KDM2B played a role in LPS‐induced HK‐2 cell injury by activating nuclear factor κB (NF‐κB) and activator protein 1 (AP‐1) pathways. Conclusion KDM2B knockdown reduced cytotoxicity, inflammation and oxidative stress in LPS‐induced AKI via inhibiting NF‐κB and AP‐1 pathways, indicating KDM2B may be a promising therapeutic target for the treatment of sepsis‐associated AKI.


| INTRODUCTION
Sepsis commonly occurs in the kidney, which is one kind of organ dysfunction resulting from an adverse host response to infection.It always leads to a condition called sepsis-associated acute kidney injury (AKI), which increases morbidity and mortality from sepsis. 1 The reported incidence of AKI in septic patients ranges from 22% to 53%.Even if AKI patients survive, the likelihood of chronic kidney disease is greatly increased.It has been reported that damage to renal tubular epithelial cells (RTEC) is the main underlying cause of AKI. 2 However, the molecular mechanisms of RTEC injury in AKI are not fully elucidated.
Inflammation plays a crucial role in sepsis development and progression.During infection, the body's immune system responds by releasing various inflammatory molecules, such as cytokines, chemokines, and prostaglandins, to help fight off the invading pathogens. 3In sepsis, however, the immune response becomes dysregulated, leading to excessive and uncontrolled inflammatory responses throughout the body.This widespread inflammation can damage tissues and organs, leading to the progression of sepsis. 4To manage sepsis, it is necessary to address the underlying infection, and control the inflammatory response.
KDM2B belongs to JmjC domain-containing histone demethylase family.KDM2B has been shown to be involved in a variety of fundamental biological and pathological processes, such as cell cycle, senescence, and tumor development. 5KDM2B regulates choline kinase-α at the session of neuronal differentiation and maintains the undifferentiated stage of neuroblasts. 6KDM2B also elevated interleukin (IL-6) production and activated inflammatory responses through gene-specific transcriptional initiation. 7In addition, KDM2B induces activator protein 1 (AP-1) transcriptional activity via SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex, mediating Kaposi sarcoma-associated herpesvirus infection. 8However, the role of KDM2B in sepsis-induced RTEC injury is unclear.
In this study, the cell viability, cytotoxicity, inflammation, and oxidative stress were investigated on lipopolysaccharide (LPS)-induced AKI model with KDM2B knockdown, while the regulation of nuclear factor κB (NF-κB) and AP-1 pathway was also analyzed, which serve to conclude that KDM2B knockdown reduced cytotoxicity, inflammation, and oxidative stress in LPS-induced AKI via inhibiting NF-κB and AP-1 pathways.

| Cell culture
HK-2 cells which are epithelial cell line from normal adult human kidney were maintained in DMEM added with 10% FBS in a 37°C incubator supplied with 5% CO 2 .For the AKI model establishment, the HK-2 cells were treated with 10 μg/mL LPS for 24 h to induce sepsisassociated AKI. 2,9The LPS-induced HK-2 cells were treated with 1 μM SR11302 as an AP-1 inhibitor or JSH-23 as an NF-κB inhibitor, 10 48 h later, the supernatant was used for cytokines analysis and the cells were collected for western blots analysis.

| Cell viability assay
HK-2 cells were cultured in 96-well plates followed by KDM2B knockdown or overexpression, respectively.Twenty-four hours later, the cells were replaced with new medium supplemented with 10 μL CCK-8 per well and cultured for another 5 days.The absorbance was analyzed at 450 nm wavelength, and the cell viability ratio was calculated. 2

| ELISA
LPS-induced HK-2 cells were treated with siKDM2B or with siKDM2B and inhibitors together, 48 h later, the supernatant was collected.The concentration of LDH, TNF-α, IL-1β, IL-18, VCAM-1, SOD, MDA, GSH, and H 2 O 2 in cell medium were evaluated using ELISA kits.In brief, 100 μL sample was added into ELISA well and incubated for 2 h followed by 100 μL detected antibody for 1 h.Then each well was reacted with enzyme working reagent, TMB reagent, and terminated by stop solution.The absorbance value was read at 450 nm. 12

| Western blot
The total protein of HK-2 cells was extracted using RIPA lysis buffer.The lysates were processed for immunoblot with the primary antibodies at 4°C overnight, and then incubated with HRP-conjugated goat anti-mouse IgG at room temperature for 1 h.Finally, the target bands were visualized with ECL reagents (Solarbio) using ChemiDoc Imaging system (Bio-Rad).The relative intensity was measured by ImageJ software and normalized to GAPDH. 13

| Quantitative PCR (qPCR)
Total RNA was isolated using RNeasy kits, and 500 ng RNA was used for reverse transcription (RT) using Universal RT-PCR Kit.ABI 7500 Real-Time PCR System was used to do qRT-PCR with 2 × SYBR Green PCR Mastermix.The copy number (Ct) of target genes was calculated by ∆∆ 2 C − t , and normalized to β-actin level. 13he PCR primers were listed in Table 1.

| Quantification and statistical analysis
Statistical analysis was performed by GraphPad Prism 8.0 (Dotmatics).Data are presented as mean ± standard deviation (SD) from three biological replicates, and the differences between any two groups were calculated by unpaired t tests.Multiple group comparisons were analyzed with analysis of variance.

| KDM2B knockdown alleviates cytotoxicity in sepsis-associated AKI
As is known that AKI induced serious cytotoxicity, to investigate the effect of KDM2B on AKI, HK-2 cells induced by LPS and followed by KDM2B knockdown was used to analyze the cytotoxicity.First, western blots were conducted to evaluate the expression of KDM2B, the data revealed transfection with si-KDM2B#1 or si-KDM2B#2 significantly reduced KDM2B protein level compared with siNC in HK-2 cells, meaning that si-KDM2B#1 or si-KDM2B#2 could be used for KDM2B knockdown (Figure 1A).Next, the cell viability was detected in LPS-induced HK-2 cells, the results showed that LPS induction contributed to cell injury with reduced cell viability, however, si-KDM2B#1 or si-KDM2B#2 transfection significantly increased the cell viability compared with siNC (Figure 1B).Meanwhile, the LDH concentration in medium was measured which demonstrated that LPS elevated LDH production, but si-KDM2B#1 or si-KDM2B#2 transfection significantly decreased the LDH release (Figure 1C).Therefore, KDM2B knockdown alleviates cytotoxicity in HK-2 cells with LPS-induced AKI.

| KDM2B knockdown reduces inflammation in sepsis-associated AKI
To investigate the effect of KDM2B on inflammation in AKI model, four typical inflammation molecules such as TNF-α, IL-1β, IL-18, and VCAM-1 were analyzed in this work.Specifically, ELISA was performed to quantify their concentration in LPSinduced HK-2 cells.The results showed all four molecules were increased in LPS-induced HK-2 cells, however, their concentration could be declined by si-KDM2B#1 or si-KDM2B#2 (Figure 2A).Their mRNA levels were analyzed using qPCR, which revealed that the transcription of TNF-α, IL-1β, IL-18, and VCAM-1 was significantly weakened in HK-2 cells with KDM2B knockdown (Figure 2B).The results above suggested that KDM2B knockdown repressed LPS-induced inflammation in sepsisassociated AKI.

| KDM2B knockdown reduces oxidative stress in sepsis-associated AKI
The effect of KDM2B on oxidative stress was investigated here.5][16] For SOD and GSH, their concentrations were downregulated in LPS-induced HK-2 cells; however, this kind of downregulation could be alleviated by si-KDM2B#1 or si-KDM2B#2, indicating KDM2B knockdown increased the production of SOD and GSH.Correspondingly, the concentration of MDA and H 2 O 2 were increased in LPS-induced AKI model, and si-KDM2B#1 or si-KDM2B#2 transfection could significantly decline the level of MDA and H 2 O 2 (Figure 3A).Another two molecules including iNOS and COX-2 were reported as the typical markers for oxidative stress. 17,18In this study, iNOS and COX-2 were measured using western blot.The data suggested that their expression was elevated in LPS-induced HK-2 cells, however, si-KDM2B#1 or si-KDM2B#2 could decrease significantly the expression of INOS and COX-2 (Figure 3B).In sum, KDM2B knockdown reduces LPSinduced oxidative stress in sepsis-associated AKI.

| KDM2B knockdown inhibits activation of NF-κB and AP-1 pathways
It has been known that inflammation is implemented with NF-κB and AP-1 signaling pathways.In the present study, several key molecules in these pathways were measured using western blot.The p65 phosphorylation levels were elevated in LPS-induced AKI, but p65 phosphorylation was declined in si-KDM2B#1 or si-KDM2B#2 compared with siNC, which demonstrated that KDM2B knockdown repressed NF-κB activation.Furthermore, c-Jun and c-fos in AP-1 pathway were also analyzed, the data showed that their expression levels were also suppressed by si-KDM2B#1 or si-KDM2B#2 (Figure 4).Thus, KDM2B knockdown inhibits activation of NF-κB and AP-1 pathways in LPS-induced AKI.

| KDM2B regulates inflammation and oxidative stress through activating NF-κB and AP-1 pathways
To confirm that KDM2B regulates inflammation and oxidative stress via targeting NF-κB and AP-1 pathways, two inhibitors SR11302 and JSH-23 were used to inhibit NF-κB and AP-1 pathways, respectively.Meanwhile, KDM2B overexpression was established by transfecting KDM2B plasmid into HK-2 cells which increased KDM2B protein level (Figure 5A).KDM2B overexpression declined cell viability, but KDM2B did not affect cell viability once SR11302 or JSH-23 was supplemented, indicating that the effect of KDM2B overexpression declining cell viability was counteracted by SR11302 or JSH-23 inhibitor (Figure 5B).Accordingly, KDM2B overexpression increased LDH production, which was alleviated by SR11302 or JSH-23 inhibitor (Figure 5C).The inflammation factors were also evaluated, the concentration of TNF-α, IL-1β, IL-18, and VCAM-1 were increased in KDM2B overexpression groups.However, their level was no longer regulated by KDM2B overexpression if SR11302 or JSH-23 inhibitor inactivated the NF-κB and AP-1 pathways (Figure 5D).Furthermore, the density of SOD and GSH were decreased in KDM2B overexpression groups, but KDM2B overexpression could not regulate SOD or GSH level in HK-2 cells treated with SR11302 or JSH-23 inhibitors.KDM2B overexpression elevated MDA and H 2 O 2 production in LPS-induced cells, but the activity regulating MDA and H 2 O 2 was suppressed when NF-κB and AP-1 pathways were inactivated by SR11302 or JSH-23 (Supporting Information: Figure S1A).KDM2B overexpression promoted COX2 and iNOS production, while SR11302 or JSH-23 inhibitors reduced their production compared with KDM2B overexpression groups (Supporting Information: Figure S1B).Similarly, in LPS-induced AKI models, the concentrations of COX-2 and iNOS were elevated in KDM2B overexpressed cells, while their concentrations were decreased in SR11302 or JSH-23 inhibitors treatment than KDM2B overexpression (Supporting Information: Figure S1C).All the data above demonstrated that KDM2B regulated inflammation and oxidative stress through activating NF-κB and AP-1 pathways.

| DISCUSSION
The kidney is an easily affected organ with sepsis which is associated with AKI.The reported incidence of AKI in septic patients ranges from 22% to 53%.Previous studies have shown that damage to RTEC is a main underlying cause of AKI. 2 KDM2B is involved in numerous pathological processes, for instance, cell senescence and tumor development. 5However, the role of KDM2B in sepsis-induced AKI is unclear.In the present study, an AKI model was established in vitro through LPS induction in HK-2 cells.LPS induction did reduce cell viability, enhance inflammation and oxidative stress.Meanwhile, two siRNAs (si-KDM2B#1 and si-KDM2B#2) Studies have shown that KDM2B knockdown can alleviate cytotoxicity and improve cell viability in various cell types, including cancer cells, neurons, and cardiomyocytes.For example, KDM2B knockdown in human lung cancer cells led to increased cell viability and decreased cytotoxicity in response to treatment with the chemotherapeutic drug cisplatin. 19Furthermore, KDM2B knockdown has been found to reduce LDH release, indicating a decrease in cell damage or death.Moreover, cardiac ischemia/reperfusion injury in mice demonstrated that KDM2B knockdown reduced LDH release and improved heart function, suggesting a protective effect against ischemic injury. 2,20Consistently, this work revealed that KDM2B knockdown alleviates cytotoxicity, reduced LDH release and promoted cell viability of HK-2 cells with LPS-induced AKI.
KDM2B is also involved in regulating inflammatory responses.KDM2B knockdown resulted in a significant reduction in the expression of several pro-inflammatory genes, including IL-6, TNF-α, and COX-2.Further experiments demonstrated that the reduction in inflammatory gene expression was due to KDM2Bmediated epigenetic modifications of the DNA in their promoters, which reduced the accessibility of transcription factors to these regions. 7However, in nasal epithelial cells, KDM2B exerts an inhibitory effect on inflammatory response via directly acting on the promoter of inflammatory factor genes IL-6 and TNFα. 21Moreover, KDM2B overexpression prevents myocardial injury through reducing the inflammatory response, as KDM2B declines IL-1β, IL-6, and TNF-α in the peripheral blood. 3From above, KDM2B has multiple roles in inflammation regulation, it exerts various effects (promote or inhibit) in the production of inflammatory factors in different diseases.In the present study, four typical inflammation molecules (TNF-α, IL-1β, IL-18, and VCAM-1) were analyzed, which showed all four molecules were increased in LPSinduced HK-2 cells, however, their concentration could be declined by KDM2B knockdown, indicating that KDM2B promotes inflammation in sepsis model.
Knockdown of KDM2B resulted in a significant reduction in oxidative stress levels, as evidenced by decreased levels of reactive oxygen species (ROS) and increased quantity of antioxidant enzymes such as catalase and SOD.The reduction in oxidative stress

F I G U R E 3
KDM2B knockdown reduces oxidative stress in sepsis-associated AKI.(A) ELISA was performed to measure the level of SOD, MDA, GSH, and H 2 O 2 in LPS-induced HK-2 cells.(B) iNOS and COX-2 were measured using western blot in LPS-induced HK-2 cells.Three repeated experiments were used for analysis.Error bar, mean ± SD; *p < .05,**p < .01.AKI, acute kidney injury; LPS, lipopolysaccharide.successfully contributed to KDM2B knockdown in this LPS-induced AKI model.The model and KDM2B knockdown provided effective platforms for studying KDM2B's role in regulating sepsis-associated AKI.

F
I G U R E 4 KDM2B knockdown inhibits activation of NF-κB and AP-1 pathways.Western blots were performed to evaluate the levels of p65 phosphorylation, the protein expression of c-Jun and c-fos in LPS-induced HK-2 cells.The data from three independent replications were used for the analysis.Error bar, mean ± SD; *p < .05,**p < .01.LPS, lipopolysaccharide.F I G U R E 5 KDM2B regulates inflammation through activating NF-κB and AP-1 pathways.(A) Western blot was performed to confirm that KDM2B expression was elevated in HK-2 cells transfected with KDM2B overexpression plasmid.(B) The cell viability was analyzed in HK-2 cells.(C) KDM2B overexpression increased LDH production, which was alleviated by SR11302 or JSH-23 inhibitor.(D) ELISA was used to evaluate the concentration of inflammation factors including TNF-α, IL-1β, IL-18, and VCAM-1.Three repeated experiments were used for the statistical analysis.Error bar, mean ± SD; *p < .05,**p < .01,***p < .001.LDH, layered double hydroxide.