(2023) 24:22
Li et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-023-00485-2

BMC Molecular andCell Biology

Open Access

RESEARCH

Knockdown of ELF4 aggravates renal
injury in ischemia/reperfusion micethrough promotion of pyroptosis, inflammation,oxidative stress, and endoplasmic reticulum
stress
Li Li1* , Shunying Wang2 and Wenming Wang2

Abstract
Background Renal ischemia/reperfusion (I/R) injury is a major cause of acute kidney injury (AKI). Dysfunction of E74like ETS transcription factor 4 (ELF4) leads to inflammation. This research intended to look into the function and mechanisms of ELF4 in I/R and oxygen–glucose deprivation/reperfusion (OGD/R) model.
Results In I/R and OGD/R model, ELF4 expression was downregulated. ELF4 knockout aggravated I/R-induced kidney
injury, oxidative stress (OS), endoplasmic reticulum stress (ERS), apoptosis, inflammation, and pyroptosis in mice. In
HK-2 cells treated with OGD/R, suppression of ELF4 expression inhibited cell proliferation and promoted cell apoptosis, OS, ERS, inflammation, and pyroptosis. Moreover, ELF4 overexpression led to the opposite results.
Conclusion ELF4 deficiency aggravated I/R induced AKI, which was involved in apoptosis, OS, ERS, inflammation,and pyroptosis. Targeting ELF4 may be a promising new therapeutic strategy for preventing inflammation after IR-AKI.
Keywords Acute kidney injury, ELF4, Inflammation, Pyroptosis, Oxidative stress
BackgroundAs a major kidney disease, acute kidney injury (AKI) is aclinical syndrome characterized by sudden loss of renal
function with sublethal renal tubular injury [1]. Clinically, renal ischemia/reperfusion (I/R) injury is a major
cause of AKI, usually occurring in hypovolemic shock,surgery, sepsis, trauma, and kidney transplantation [2–4].
*Correspondence:
Li Liwangshyingg@163.com
1
Department of Nephrology, Jinan City People’s Hospital, No. 001,
Changshao North Road, Laiwu District, Jinan, Shandong 271199, People’s
Republic of China
2
Department of Cadre Health Section, Jinan City People’s Hospital, Jinan,
Shandong 271199, People’s Republic of China

Although AKI is paid close attention for the higher morbidity and mortality rates, AKI is still a difficult problem
in diagnosis and treatment in clinic [5]. AKI pathogenesis is complicated, which is related to abnormal apoptosis, oxidative stress (OS), endoplasmic reticulum stress(ERS), inflammatory responses, and pyroptosis [6–10].
However, the exact mechanisms of AKI remains poorly
understood. Therefore, exploring the potential mechanism of AKI is of great significance for treatment of AKI.
The E-Twenty-Six (ETS) transcription factor family iscomposed of 29 members in human and 28 members inmouse, which participates in various signaling pathways[11–13]. E74 like ETS transcription factor 4 (ELF4), originally called "myeloid Elf1-like factor", has a hand in tumorigenesis, regulating immune responses, DNA damage© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Li et al. BMC Molecular and Cell Biology(2023) 24:22

response, and cell cycle regulation [14–17]. Lee et al.
have indicated that ELF4 knockout (KO) in mice contributes to the increased disease severity after experimental
autoimmune encephalomyelitis induction [18]. Du et al.
have shown that ELF4 KO mice are sensitive to dextran
sulfate sodium-induced salt colitis [19]. Moreover, ELF4
is reported to restrain inflammation and protecte against
mucosal disease [20]. However, the influence of ELF4 isunclear in AKI. Therefore, studying the influence of deficiency of ELF4 on renal injury is necessary.
At the present study, in vivo model induced by I/R andin vitro model induced by oxygen–glucose deprivation/
reperfusion (OGD/R) were used for determining theinfluences and mechanisms of ELF4 in AKI. Mice lacking ELF4 showed worsened kidney structure and function after I/R. Subsequent studies revealed a direct effectof ELF4 in protecting HK-2 cells from apoptosis, OS,
ERS, inflammation, and pyroptosis. A new target for AKI
treatment may obtain from our findings.

Results
In kidney tissues of I/R mice and cells of OGD/R, ELF4
expression is decreased

To explore whether ELF4 involved in kidney injury, we
detected the levels of ELF4. Figure 1A showed that ELF4mRNA expression was markedly decreased in kidney tissues of I/R mice. Furthermore, ELF4 protein expression
was significantly reduced, which was indicated by western blot and immunohistochemical analysis (Fig. 1B andC). ­ELF4−/− mice were applied to investigate the function of ELF4 in I/R mice (Fig. 1D). In vitro, HK-2 cells
were simulated with OGD/R to mimic I/R. To look into
the influence of ELF4 in cell model of renal injury, ELF4
siRNA and overexpression plasmid were transfected into
HK-2 cells. OGD/R significantly reduced ELF4 expression, transfection of ELF4 siRNA further markedly
reduced ELF4 expression, and transfection of ELF4 overexpression plasmid significantly induced ELF4 expression(Fig. 1E-H).
In mice, ELF4 knockout aggravates I/R‑induced kidney
injuryFigure 2A and B suggested that serum Cr and BUN levels
were low in both E­ LF4−/− and WT mice of sham group,
while serum Cr and BUN levels were markedly increased
in both E­ LF4−/− and WT mice of I/R group. Moreover, inPage 2 of 11

I/R group, ­ELF4−/− further obviously increased serum Cr
and BUN levels. H&E staining showed that both ­ELF4−/−and WT mice of sham group had normal kidney structure, after renal I/R, both E­ LF4−/− and WT mice showedswollen, extensive expansion and deformation of renal
tubules. In I/R group, ­ELF4−/− further aggravated tubular
injury (Fig. 2C). It has shown in rats of I/R injury modelthat KIM-1 expression in the proximal tubule is inducedby ischemia [21]. I/R significantly enhanced KIM-1
expression, and E­ LF4−/− further enhanced KIM-1 expression in I/R group (Fig. 2D).
ELF4 deficiency aggravates apoptosis

TUNEL staining was used to evaluate apoptosis in I/Rmouse model. TUNEL staining showed that cell apoptosis was markedly increased by I/R, and E­ LF4−/− further
increased cell apoptosis (Fig. 3A). Bax, a proapoptotic
member of the Bcl-2 family. Bcl-2 exerts a death-sparing activity against apoptosis induced by I/R [22]. I/R
obviously promoted Bax expression and significantly
inhibited Bcl-2 expression in renal tissues, and E­ LF4−/−
exacerbated these changes (Fig. 3B). As indicated inFig. 3C and D, OGD/R significantly inhibited cell viability and markedly promoted cell apoptosis, suppression of
ELF4 expression further aggravated these effects inducedby OGD/R. Overexpression of ELF4 markedly eliminatedthe influence of OGD/R on cell viability, apoptosis.
ELF4 deficiency aggravates OS and ERS

In I/R induced kidney injury, to understand the involvement of ELF4 and whether this influences were related to
OS and ERS, we assessed the change of OS index (SOD,
CAT, and GSH-PX), OS related proteins (Nrf2, HO-1,
and NQO-1), and ERS related proteins (GRP78, CHOP,
and caspase-12). Figure 4A-C presented that I/R markedly reduced serum SOD, CAT, and GSH-PX levels. In
I/R group, E­ LF4−/− further reduced serum SOD, CAT,and GSH-PX levels. A obvious reduction of protein levelsof Nrf2, HO-1, and NQO-1 was showed in renal tissuesof I/R group, after renal I/R, ­ELF4−/− further aggravatedthese protein change (Fig. 4D). In addition, I/R caused an
obvious increase of GRP78, CHOP, and caspase-12 protein in renal tissue, and E­ LF4−/− further increased thesechanges of proteins (Fig. 4E). Figure 4F showed that ROS
level was obviously increased by OGD/R, suppression of
ELF4 further enhanced ROS level, and overexpression(See figure on next page.)
Fig. 1 In kidney tissues of I/R mice and cells of OGD/R, ELF4 expression was decreased. A ELF4 mRNA expression was detected by way of qRT-PCR
in kidney tissues of sham and I/R mice. ELF4 protein expression in kidney tissues of sham and I/R mice was surveyed using western blot (B)and immunohistochemistry (C). D In kidney tissues of WT and ­ELF4−/− mice, ELF4 protein change was ascertained using western blot. E ELF4 mRNA
expression was surveyed by way of qRT-PCR in HK-2 cells. F In HK-2 cells, western blot was applied to survey ELF4 protein expression. G ELF4 mRNA
expression was surveyed by way of qRT-PCR in HK-2 cells. H In HK-2 cells, ELF4 protein expression was checked by way of western blot. Mouse n = 5.
Cell n = 3. * p < 0.05 vs sham, WT, or control group; # p < 0.05 vs OGD/R groupLi et al. BMC Molecular and Cell Biology

Fig. 1 (See legend on previous page.)(2023) 24:22

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Li et al. BMC Molecular and Cell Biology(2023) 24:22

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Fig. 2 In I/R mice, ELF4 deficiency aggravated kidney injury. Serum Cr (A) and BUN levels (B) in WT and ­ELF4−/− mice. C In WT and ­ELF4−/− mice,the renal histological injury was estimated by way of H&E staining. D KIM-1 protein change was ascertained in kidney tissues of WT and ­ELF4−/−
mice using western blot. Mouse n = 5. * p < 0.05 WT sham group, # p < 0.05 ­ELF4−/− sham or WT I/R groupof ELF4 significantly decreased ROS level. Consistently,
OGD/R markedly inhibited Nrf2, HO-1, and NQO-1
protein levels, and suppression of ELF4 further aggravated the inhibition role (Fig. 4G). In addition, OGD/R
obviously increased the levels of GRP78, CHOP, caspase-12, and suppression of ELF4 further aggravated theinhibition role (Fig. 4H). Moreover, western blot analysis(Fig. 4G-H) showed that overexpression of ELF4 significantly increased Nrf2, HO-1, and NQO-1 protein levels,and markedly decreased GRP78, CHOP, caspase-12.
ELF4 deficiency aggravates inflammation and pyroptosis

Subsequently, the influence of ELF4 on inflammatory
cytokines was investigated. Figure 5A showed that I/Rmarkedly increased the levels of IL-6, TNF-α, IL-18, and
IL-1β, and these inflammatory cytokines were further
increased by ELF4 deficiency in I/R groups. Furthermore,in renal tissues, I/R significantly enhanced pyroptosis
related proteins (GSDMD, N-GSDMD, and caspase-11)

protein levels, and these proteins in renal tissues of I/Rgroup was further increased following ELF4 deficiency(Fig. 5B). Moreover, in OGD/R-treated cells, western blot
analysis showed that ELF4 knockdown or overexpression
significantly increased or decreased IL-6, TNF-α, IL-18,
IL-1β, GSDMD, N-GSDMD, and caspase-4 protein levels(Fig. 5C-D).

Discussion
In clinic, renal ischemia that occurs in kidney surgery,transplantation, hemorrhagic, cardiogenic, septic shock,and other clinical environments is the most common
cause of AKI [4, 23]. Renal I/R is generally acknowledge
as an important cause of AKI mortality, especially among
patients in ICU [24–26]. At present, there is still no available treatment to prevent ischemic injury, and there is notherapy or drug that can completely reduce I/R induced
mortality [27–29]. Thus, searching for effective therapeutic approaches for renal I/R injury is an urgent need.

Li et al. BMC Molecular and Cell Biology(2023) 24:22

Page 5 of 11

Fig. 3 ELF4 deficiency aggravated apoptosis. A In WT and ELF4-/- mice, TUNEL staining was applied to survey renal cell apoptosis. B Bax and Bcl-2
protein levels were detected by way of western blot in kidney tissues of WT and ELF4-/- mice. C HK-2 cell viability was ascertained by way of CCK-8
assay. D Flow cytometry was carried out to assess cell apoptosis in HK-2 cells. Mouse n = 5. Cell n = 3. * p < 0.05 WT sham or control group, # p < 0.05
­ELF4−/− sham, WT I/R, or OGD/R group

In this study, I/R and OGD/R induced AKI model were
established. We found that ELF4 expression was markedly downregulated in I/R and OGD/R induced model,
which indicated that ELF4 might take part in the pathological process of AKI.
In I/R and OGD/R model, we restrained ELF4 expression to explore the effects of ELF4 in AKI. The mostcommonly used markers for renal function assessment

including Cr and BUN [30]. The levels of serum Cr and
BUN were found to be increased in I/R mice [31–33]. Asa transmembrane glycoprotein, KIM-1 can be split intosoluble fragments and eventually excreted into urine[34, 35]. Therefore, KIM-1 is often used as a biomarker
of renal injury [36]. Our data demonstrated that inhibition of ELF4 exacerbated kidney damage, as evidenced
by the increase of serum Cr, serum BUN, KIM-1 protein

Li et al. BMC Molecular and Cell Biology(2023) 24:22

Page 6 of 11

Fig. 4 ELF4 deficiency aggravated OS and ERS. Serum SOD (A), CAT (B), and GSH-PX (C) levels in WT and ­ELF4−/− mice. D In kidney tissues of WT
and ­ELF4−/− mice, Nrf2, HO-1, and NQO-1 protein levels were surveyed by way of western blot. E GRP78, CHOP, and caspase-12 protein levels weredetected by way of western blot in kidney tissues of WT and ­ELF4−/− mice. F Flow cytometry was carried out to assess ROS levels in HK-2 cells. The
levels of OS related proteins (Nrf2, HO-1, and NQO-1; G), ER stress related proteins (GRP78, CHOP, and caspase-12; H) were surveyed using western
blot in HK-2 cells. Mouse n = 5. Cell n = 3. * p < 0.05 WT sham or control group, # p < 0.05 ­ELF4−/− sham, WT I/R, or OGD/R groupLi et al. BMC Molecular and Cell Biology(2023) 24:22

Page 7 of 11

Fig. 5 ELF4 deficiency aggravated inflammation and pyroptosis. A In kidney tissues of WT and ­ELF4−/− mice, IL-6, TNF-α, IL-18, and IL-1β protein
levels were detected by way of western blot. B GSDMD, N-GSDMD, and caspase-11 protein levels were surveyed in kidney tissues of WT and ­ELF4−/−
mice using western blot. The levels of inflammatory cytokines (IL-6, TNF-α, IL-18, and IL-1β; C), and pyroptosis-related proteins (GSDMD, N-GSDMD,
and caspase-11; D) were detected using western blot in HK-2 cells. Mouse n = 5. Cell n = 3. * p < 0.05 WT sham or control group, # p < 0.05 ­ELF4−/−
sham, WT I/R, or OGD/R groupexpression in renal tissue. In AKI, the main pathwaysof cell death are apoptosis and necrosis [37, 38]. In thisstudy, in I/R mice and HK-2 cells treated with OGD/R,
inhibition of ELF4 expression markedly increased cell
apoptosis. Taken together, inhibition of ELF4 expression
aggravated renal injury.
The pathogenesis of AKI associated with I/R is involvedin tubular damage and inflammatory response [23]. Moreand more evidence showed that I/R promoted the releaseof inflammatory factors in renal tissue, which leads tosevere renal cell apoptosis, thereby causing AKI [39–41].
For ameliorating AKI and facilitating recovery, inhibition of inflammatory response is a promising therapeutic approach [42]. As an important inflammatory factor,
IL-1β takes part in the process of hosts against pathogens[43]. IL-18 is mainly produced by activated mononuclear
macrophages, which participates in ischemic AKI [44].

Moreover, other inflammatory cytokines, for instance,
IL-6 and TNF-α can be elevated by IL-1β and IL-18[45]. At the present work, I/R and OGD/R markedly
increased the levels of IL-6, TNF-α, IL-1β, and IL-18.
Meanwhile, in I/R and OGD/R model, inhibition of ELF4
further increased IL-6, TNF-α, IL-18, and IL-1β levels,
but overexpression of ELF4 led to the opposite results.
Doitsh et al. have reported that pyroptosis generated proinflammatory mediators, thereby triggering inflammatory reactions [46]. After that, we detected the changesof pyroptosis-related proteins, and found that inhibitionof ELF4 further increased GSDMD, N-GSDMD, and caspase-11 levels in I/R and OGD/R model. Furthermore,in OGD/R model, GSDMD, N-GSDMD, and caspase-11
levels were markedly reduced by overexpression of ELF4.
In short, in pathogenesis of AKI, inhibition of ELF4 promoted inflammatory response and pyroptosis.

Li et al. BMC Molecular and Cell Biology(2023) 24:22

The accumulation of ROS during I/R process is vital in
the course of AKI [47]. After reperfusion, the first injury
event is that mitochondria produce ROS, secondary tissue damage and subsequent inflammation caused by nonmitochondrial ROS [48]. Common biochemical markersof oxidative damage include SOD, CAT, and GSH-PX[49]. In OGD/R model, inhibition of ELF4 significantly
increased ROS levels, but overexpression of ELF4 dramatically decreased ROS levels. In I/R model, inhibitionof ELF4 markedly reduced SOD, CAT, and GSH-PX levels. Moreover, in I/R and OGD/R model, inhibition of
ELF4 significantly inhibited the expression of OS related
proteins (Nrf2, HO-1, and NQO-1). Apart from regulating OS, it was indicated that Nrf2/HO-1 pathway had a
hand in regulating apoptosis, ERS, and inflammation[50–52]. To further investigate the mechanism of ELF4
in AKI, ERS related proteins (GRP78, CHOP, and Caspase-12) were detected. Our findings indicated that ELF4
inhibition significantly promoted ERS in I/R and OGD/R
model, as evidenced by increasing GRP78, CHOP, andcaspase-12 levels. Based on these findings, inhibition of
ELF4 aggravated renal injury, which was associated with
the OS and ERS.
In summary, inhibition of ELF4 aggravated renal injury
in I/R treated mice by promoting OS, ERS, inflammation,and pyroptosis. In future studies, ELF4 supported thetranscription of OS, ERS, inflammation, and pyroptosisrelated gene will be studied in I/R-induced AKI. Ourfindings may provide a novel mechanistic insight into
I/R-induced AKI.

Methods
Mouse model of renal I/R injury

The male ELF4 knockout ­(ELF4−/−) and wild-type (WT)C57BL/6 mice were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). In a 12 h
light/12 h dark cycle, eating and drinking could be freely
obtained by mice. The animal care and use committee ofour hospital authorized all experiments.
ELF4−/− mice and WT mice were randomly assigned
to sham and I/R group, respectively. AKI model was
induced by I/R surgery [53]. In brief, after anesthesia with
1% sodium pentobarbital solution, mice were underwent
a midline laparotomy. Next, a microaneurysm clamp(Fine Science Tools, USA) was used to tighten both renal
vessels for 30 min to induce ischemia. After removing theclamp for 6 h, the serum was collected from mice before
sacrificing. In sham group, mice were exposed to thesame procedure, except using the microaneurysm clamp.
Cell culture and treatments

Human proximal tubular epithelial cells (HK-2) were
provided by Procell Life Science&Technology Co.,Ltd

Page 8 of 11(CL-0109, Wuhan, China). Under condition of 37 °C and
5% ­CO2, cells were maintained in DMEM (11885084,
Invitrogen, USA) with 1% penicillin/streptomycin and
10% foetal bovine serum.
Small interference RNA targeting ELF4 (si-ELF4),
overexpression plasmid of ELF4 (ov-ELF4), and negative control (si-NC and ov-NC) were transfected into
HK-2 cells by way of Lipofectamine 2000 reagents(11668030, Invitrogen). After transfection for 48 h, cells
in OGD/R + si-NC, OGD/R + si-ELF4, OGD/R + ov-NC,
and OGD/R + ov-ELF4 group were treated with OGD/R.
To establish OGD/R model, HK-2 cells were exposed
to glucose-free medium supplemented with 1% ­O2, 5%­CO2, and 94% ­N2 for 4 h at 37 °C [10]. Next, the complete
medium with 21% ­O2 was used for maintaining cells for
6 h. In control group, cells were maintained in complete
medium with 21% ­O2.
Quantitative real‑time polymerase chain reaction(qRT‑PCR)In kidney tissues and HK-2 cells, total RNA was extracted
by means of TRIzol reagent (15596026, Invitrogen, USA).
The PrimeScript™ RT Reagent Kit (RR037Q, Takara,
Japan) was made use of synthesizing complementaryDNA. The SYBR Premix Ex Tag Kit (RR820A, Takara)
was made use of performing qRT-PCR. The sequences ofprimers were listed in Supplementary table 1.
Western blot

In kidney tissues and HK-2 cells, RIPA lysis buffer (20–
188, Sigma-Aldrich) was used for extracting proteins.
An equal quantity of protein was electrophoresed using
SDS-PAGE and then transferred onto PVDF membranes. At 4 °C, primary antibodies against ELF4, Nrf2,
NQO-1, GRP78, KIM-1, and IL-6 (1:1000, ab96075,
ab137550, ab34173, ab21685, ab302932, and ab259341,
Abcam, USA); IL-1β (1:1000, sc-12742, Santa Cruz Biotechnology); IL-18 and N-GSDMD (1:1000, A1115 andA22523, ABclonal, USA); Bax, Bcl-2, TNF-α, caspase-4,
caspase-11, Caspase-12, CHOP, HO-1, and GSDMD(1:1000, 2772, 3498, 3707, 4450, 14340, 35965, 2895,
43966, and 39754, Cell signaling Technology, USA), and
GAPDH (1:5000, 5174, Cell signaling Technology, USA)
were deal with membranes overnight. After that, secondary antibodies (1:50000, 7074, Cell signaling Technology)
were handled with membranes for 1 h. At last, protein
bands were visualized by way of an enhanced chemiluminescence detection kit (Beyotime Biotechnology, China).
Image J software (USA) was used to quantify protein
expression.

Li et al. BMC Molecular and Cell Biology(2023) 24:22

Page 9 of 11

Immunohistochemistry analysis

Detection of cell apoptosis

The kidney tissues of mice were fixed in 10% formalin (HT501128, Sigma-Aldrich). After that, paraffin
was taken to embed kidney samples. After dewaxing
and rehydrating, Sects. (4 μm-thick) were incubatedwith 3% hydrogen peroxide (H1009, Sigma-Aldrich)for 10 min. Next, sections were blocked with 5%
bovine serum albumin (A1933, Sigma-Aldrich) for 1 h
and incubated with antibody against ELF4 (1:200, bs14563R, Bioss) 4˚C. Nexy day, sections were incubatedwith secondary antibody (8114, Cell signaling Technology). At last, sections were developed color using
3,3’-diaminobenzidine (P0202, Beyotime Biotechnology). Under a microscopy (BX53, Olympus, Japan),
ELF4 positive cells were quantified.

After indicated treatment, 200 µl Annexin V-FITC(C1062S-1, Beyotime Biotechnology) and 5 µl PI(C1062S-3, Beyotime Biotechnology) were handled with
HK-2 cells for 15 min. Finally, the apoptotic cells were
gauged using FACS Calibre flow cytometry (Accuri C6Plus, BD Biosciences, USA).

Haematoxylin and Eosin (H&E) staining

Statistical analysis

Afterdewaxing
andrehydrating,
the
renal
Sects. (4 μm-thick) were stained with haematoxylin (C0107100 ml, Beyotime Biotechnology) for 5 min and stained witheosin (C0109, Beyotime Biotechnology) for 1 min. Under
an optical microscope (Olympus), renal tissue damage wasassessed in a blinded manner.

Data analyses were conducted by means of GraphPad
Prism software (USA). P < 0.05 was supposed to statistical significant. All data were statistically comparedusing t-test or one-way analysis of variance (ANOVA).

Terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL) staining

In kidney tissues, cell apoptosis was ascertained by wayof the TUNEL apoptosis detection kit (C1089, Beyotime
Biotechnology). Briefly, after dewaxing and rehydrating,the renal sections were treated with proteinase K without
DNase (20 µg/ml) for 20 min. Next, sections were stainedwith the TUNEL reaction mixture, followed by staining with DAPI. Eventually, under an optical microscope,
apoptotic cells were observed and counted.
Biochemical assays

For the detection of serum creatinine (Cr, C011-2–1),
blood urea nitrogen (BUN, C013-2–1), superoxide dismutase (SOD, A001-1–2), catalase (CAT, A007-1–1), andglutathione peroxidase (GSH-PX, A005-1–2), the serum
was collected from mice blood through centrifuging at
3,000 rpm for 10 min. Nanjing jiancheng bioengineering
institute (China) provided corresponding kits.
Detection of cell viability

After indicated treatment, cell counting kit 8 (CCK-8)
reagents (C0037, Beyotime Biotechnology) were applied
to handle HK-2 cells. After handling for 2 h, at 450 nm, a
BioTek ELX-800 microplate reader (USA) was utilized togauge the optical density (OD) value.

Detection of reactive oxygen species (ROS)

After indicated treatment, DCFH-DA (S0033S, Beyotime
Biotechnology) was applied to handle with HK-2 cells
for 20 min. Next, serum free medium was used to washcells for three times, and then cells were analyzed by wayof flow cytometry at 480 nm excitation wavelength and525 nm emission wavelength.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12860-​023-​00485-2.
Additional file 1.
Acknowledgements
Not applicable.
Authors’ contributions
L L designed the study; L L and SY W performed the research; WM W analyzed
data, and wrote the paper.
Funding
This work was supported by grants from science and technology development plan of Laiwu Science and Technology Bureau (WSCG2018-006). The
funding bodies played no role in the design of the study and collection,
analysis, interpretation of data, and in writing the manuscript.
Availability of data and materials
The datasets used and analyzed during the current study are available fromthe corresponding author.

Declarations
Ethics approval and consent to participate
All experimental protocol was approved by ethics committee of the Jinan City
People’s Hospital. All methods were carried out in accordance with relevant
guidelines and regulations; all methods are reported in accordance with
ARRIVE guidelines (https://​arriv​eguid​elines.​org) for the reporting of animal
experiments.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.

Li et al. BMC Molecular and Cell Biology(2023) 24:22

Page 10 of 11

Received: 17 January 2023 Accepted: 13 July 2023
24.

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