Expression and role of ABIN1 in sepsis: In vitro and in vivo studies
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Open Medicine 2021; 16: 33–40
Research Article
Haolan Li, Aichen Sun*, Taocheng Meng, Yan Zhu
Expression and role of ABIN1 in sepsis: In vitro
and in vivo studies
https://doi.org/10.1515/med-2021-0008
received March 19, 2020; accepted October 20, 2020
1 Introduction
Abstract: In this research, we attempted to explain the Sepsis is defined as severe systemic inflammatory reac-
effect and the related molecular mechanisms of ABIN1 in tion, it contributes to multiple tissues and organ dysfunc-
lipopolysaccharide (LPS)-induced septic mice or RAW264.7 tion, and the process is intricate [1,2]. Currently, the
macrophages. LPS was adopted to treat RAW264.7 macro- morbidity and mortality of sepsis remain high around
phages for 4 h, and the levels of inflammatory factors were the world. Although great advancements have been
assessed by ELISA. Besides, ABIN1 expression was made to decrease the mortality, there are still rare effec-
measured by quantitative reverse transcription polymerase tively apply in sepsis treatment. Certainly, understanding
chain reaction. Apparently, LPS enhanced immuno- the mechanism of sepsis will be a benefit for the therapy
reaction, suggested by increased expression of IL-1β, tumor of sepsis.
necrosis factor (TNF)-α, and IL-6. ABIN1 levels were Macrophages, an important part of innate immune
obviously reduced compared to the control. Furthermore, system, are related to various diseases and play a vital
we evaluated the roles of ABIN1-plasmid in immuno- role in sepsis by regulating immune responses and inflam-
reaction and nuclear factor-κB (NF-κB) pathway. We found matory factors secretion, such as tumor necrosis factor
that ABIN1-plasmid significantly reduced the expression of (TNF)-α, IL-10, and IFN-g [3,4]. Recent research has indi-
IL-1β, TNF-α, and IL-6 in LPS-treated cells and inhibited cated that excessive production of inflammatory cytokines
NF-κB pathway activation. Meanwhile, a septic mouse was observed during sepsis [5,6]. Therefore, controlling
mode was conducted to validate the role of ABIN1 in macrophages activity and preventing abnormal activation
inflammatory response and organ damage in vivo. These were potentially powerful strategies for sepsis treatment.
data suggested that ABIN1-plasmid significantly inhi- Lipopolysaccharide (LPS) is widely used to explore inflam-
bited the secretion of inflammatory cytokines and Cr, mation in vivo and in vitro models, where it stimulates
BUN, AST, and ALT levels in the serum of LPS-stimulated many inflammatory factors [7]. For example, Araya
mice compared to LPS + control-plasmid group, reflecting et al. revealed that LPS induced TNF-α, IL-1β, IL-6, and
the relieved inflammation and organ injury. In summary, PGE2 secretion in the whole blood of patients with Type 1
the present findings indicated that ABIN1 alleviated sepsis diabetes mellitus [8].
by repressing inflammatory response through NF-κB Accumulating evidences have shown that numerous
signaling pathway, emphasizing the potential value of factors induce inflammation, including nuclear factor-κB
ABIN1 as therapeutic strategy for sepsis. (NF-κB) [9]. NF-κB pathway, a regulator in inflammation
Keywords: ABIN1, inflammation, macrophages, sepsis process, is associated with many diseases. NF-κB family
includes five members, such as p65 (RelA), RelB, c-Rel,
p50/105 (NF-κB1), and p52/p100 (NF-vB2), which exist in
unstimulated cells as homo- or heterodimers bound to
inhibitory κB (IκB) family proteins [10]. The released
p65/p50 NF-κB dimer is transferred from the cytoplasm
* Corresponding author: Aichen Sun, Department of Orthopaedics, to the nucleus, binds to specific DNA sequences, and
Zaozhuang Municipal Hospital, No. 47 Longtou Road, Zaozhuang, promotes the transcription of target genes [11,12]. In the
Shandong 277102, China, e-mail: sunaichen1445@163.com classical pathway, NF-κB activation triggers the produc-
Haolan Li: Department of Infectious Diseases, Zaozhuang Municipal
tion of pro-inflammatory cytokines and NO [13,14].
Hospital, Zaozhuang, 277102, China
Taocheng Meng, Yan Zhu: Department of ICU, Zaozhuang Municipal Researches have revealed that classic NF-κB pathway inhi-
Hospital, Zaozhuang 277102, China bition can prevent the production of pro-inflammatory
Open Access. © 2021 Haolan Li et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0
International License.34 Haolan Li et al.
cytokines [15,16]. ABIN1, a homologue of ABIN proteins, approved by the Institutional Animal Care and Use
was defined as a suppressor of immune system and plays Committee (IACUC) of Zaozhuang Municipal Hospital.
important roles in immunity regulation as well as tissue
homeostasis [17]. ABINs have been described as three
different proteins (ABIN-1, ABIN-2, and ABIN-3), which
bind to the ubiquitin-edited NF-κB inhibitor protein A20, 2.2 Animal model
and show limited sequence homology [18,19]. Upregulation
of ABINs inhibits the activation of NF-κB through TNF and The mice were adapted to the standard condition for 2
several other stimuli [18]. Similar to A20, the expression of weeks before experiment. Then we grouped the mice into
ABIN1 is also dependent on NF-κB, which suggests a the control and the sepsis group (n = 10 for each group).
potential role of the A20/ABIN complex in the negative LPS (10 mg/kg; Santa Cruz, CA) was adopted to establish
feedback regulation of the activation of NF-κB [18,19]. sepsis model, and the control group received normal
Previous study suggests that upregulation of A20/ABIN1 saline. At 24 h after LPS treatment following the last
injection, ABIN1-plasmid (80 mg/kg/day) or control-
leads to macrophage polarization during infection [20].
plasmid (80 mg/kg/day) was injected by tail intrave-
However, the roles of ABIN1 in the development of sepsis
nous injection for three consecutive days. After that,
need to be further explored.
the mice were anesthetized using pentobarbital (30 mg/kg,
In this research, the enhanced inflammatory factors
intraperitoneal injection), and the blood was subsequently
levels and decreased ABIN1 mRNA and protein expres-
harvested and centrifuged to extract serum for histopatho-
sion were observed in LPS-treated macrophages. It was
logical evaluation at 24 h after LPS treatment as detailed
subsequently determined that ABIN1-plasmid signifi-
earlier. The mice health and behavior were observed every
cantly increased the levels of ABIN1 and suppressed
2 days. During our experiment, no mice died. Throughout
the inflammatory cytokines compared to LPS treatment
the whole experiment, we did our best to alleviate the pain
group. Furthermore, ABIN1-plasmid was indicated to
of the mice, and experiments were ended when the mice lost
depress the p-p65 levels and reduced the ratio of p-p65/
more than 15% of their body weight.
p65. All the data above indicated that ABIN1-plasmid
protects against the secretion of LPS-stimulated pro-in-
flammatory cytokines by inhibiting NF-κB signal activa-
tion in macrophages. Further study indicated the anti-
inflammation and organ-protective effects of ABIN1-plasmid
2.3 Cell culture and LPS stimulation
on in vivo model.
The RAW264.7 macrophage was cultivated in DMEM (Gibco,
In conclusion, our study provided novel evidence that
USA), including 10% FBS and 1% penicillin/streptomycin.
ABIN1 is an important regulator and participated in the
Then macrophage was incubated in an incubator with 5%
progression of sepsis by activating the NF-κB pathway.
CO2. In this study, macrophage was treated with 1 μg/mL
LPS for 4 h and harvested for further research.
2 Materials and methods
2.4 Cytokines and organ injury markers’
2.1 Animals
detection
C57BL/6 mice (20–24 g) were purchased from the Model After LPS treatment for 24 h, blood was collected from
Animal Center of Shanghai and were adopted in this each group, centrifuged (1,600 × g, 15 min), and the
study at 6–8 weeks age. The mice were kept in a tempera- supernatant was gathered for analysis. IL-1β, IL-6, and
ture- and humidity-controlled room with free-issue diet TNF-α levels from serum were quantitatively detected
and drinking. All animal procedures were conducted using ELISA (BD biosciences). Serum ALT, AST, Cr, and
referring to the National Institutes of Health Guide for BUN were measured to assess organ damage, referring to
the Care and Use of Laboratory Animals and were the manufacturer’s instructions.ABIN1 in sepsis 35
The expression levels of IL-1β, IL-6, and TNF-α in the Scientific) and the membranes were incubated with
cell supernatant were also detected using ELISA kits blocking solution for 1 h. Anti-GAPDH (1:1,000; Abcam),
(Beyotime, Shanghai, China), according to the manufac- anti-ABIN1 (1:1,000; Abcam), anti-p65 (1:1,000; Abcam), and
turer’s protocols. anti-p-p65 (1:1,000; Abcam) were applied as primary anti-
bodies. After washing with PBST for four times, membranes
were incubated with secondary antibody for 1 h. The
proteins were quantified using ECL detection reagent
2.5 Cell transfection and reagents (Pierce Biotechnology).
Macrophage cells were planted into a 6-well plate and
cultured for 24 h, with cell transfection performed when
the cell density was up to 50–60%. For transfection, control- 2.8 Statistical analysis
plasmid (cat no. sc-437275; Santa Cruz Biotechnology) or
ABIN1-plasmid (cat no. sc-425441-ACT; Santa Cruz SPSS 20.0 statistical software (IBM Corp.) was adopted to
Biotechnology) was transfected into macrophages using analyze the statistics. All the results were represented as
Lipofectamine® 2000 reagent (Invitrogen, Carlsbad, CA), mean ± SD from three independent experiments. Student’s
referring the manufacturer’s protocol. After that, RAW t-test and ANOVA followed by Student–Newman–Keuls
264.7 macrophages were induced by LPS for 4 h and tests were applied for evaluating differences between the
the transfection efficiency was conducted by quantitative groups. We considered statistically significant as p < 0.05.
reverse transcription polymerase chain reaction (qRT-PCR)
and western blot analysis.
3 Results
2.6 qRT-PCR
3.1 LPS treatment reduced ABIN1 levels and
TRIzol reagent (Thermo Fisher Scientific, MA) was adopted decreased inflammatory factors’
to isolate total RNA from serum and macrophages, following secretion in RAW264.7 macrophages
the manufacturer’s protocol. PrimeScript™ RT reagent kit
(TaKaRa) was adopted to synthesize cDNAs. Then we detect As is well acknowledged that sepsis is related to immu-
ABIN1 levels by real-time PCR system, referring to the noreaction. First, macrophages were stimulated by
manufacturer’s protocol. GAPDH was regarded as control. LPS for 4 h, and the IL-1β, IL-6, and TNF-α levels
Primer sequences were obtained from Sangon Biological were evaluated using ELISA to assess the immuno-
and listed as follows: reaction. We observed that expression of inflammatory
GAPDH-forward, 5′-AAGGTTCGGAGTCAACGGA-3′; factors was obviously higher in LPS-treated group than
reverse, 5′-TTAAAAGCAGCCCTGGTGA-3′; that in control (Figure 1a–c). Moreover, we analyze the
ABIN1-forward, 5′-GTGTTCCGATGTGGCTCT-3′; ABIN1 levels by qRT-PCR and western blot assays, respec-
reverse, 5′-ACCCACTCCTCCTTGGC-3′. tively. Our data showed that ABIN1 was downregulated in
Amplification conditions were as follows: after 5 min LPS-stimulated macrophages (Figure 1d and e). These
denaturation at 95°C, 40 cycles were conducted including data indicated that ABIN1 may be involved in sepsis
95°C for 15 s, 60°C for 1 min, 55°C for 1 min, and final step at progression.
72°C for 10 min. The target genes expression was assessed
by 2−ΔΔCq method.
3.2 ABIN1-plasmid suppressed the
expression of inflammatory factors
2.7 Western blot analysis in LPS-stimulated macrophages
Cells’ proteins were extracted by RIPA lysis buffer (Beyotime) The above results that we obtained indicate that ABIN1
and each sample was subjected to 10% SDS–PAGE. Then participated in the development of sepsis. Then we explored
they were transferred onto PVDF membranes (Owl the potential pathogenesis of ABIN1 in LPS-induced36 Haolan Li et al. Figure 1: Expression of ABIN1 was suppressed and inflammatory cytokines were enhanced in LPS-induced macrophages. Macrophages were stimulated by 1 μg/mL LPS for 4 h. (a) TNF-α, (b) IL-1β, and (c) IL-6 levels in RAW264.7 macrophages. (d) ABIN1 mRNA and (e) protein expression in macrophages were assessed by qRT-PCR and western blot assay, respectively. **p < 0.01 vs control. Figure 2: ABIN1-plasmid reversed the effects of LPS on inflammatory factors in macrophages. (a) mRNA and (b) protein levels of ABIN1 in macrophages after ABIN1-plasmid transfection. After transfection with control-plasmid or ABIN1-plasmid, the levels of (c) TNF-α, (d) IL-1β, and (e) IL-6 in different groups were determined. &&p < 0.01 vs control-plasmid; **p < 0.01 vs control, and ##p < 0.01 vs LPS + control- plasmid.
ABIN1 in sepsis 37
macrophages. Macrophages were transfected with control- 3.4 ABIN1-plasmid protects against the
plasmid or ABIN1-plasmid for 48 h, followed by 4 h LPS effects of LPS on liver injury in septic mice
treatment. As shown in Figure 2a, ABIN1-plasmid promi-
nently enhanced the ABIN1 mRNA (Figure 2a) and protein To assess the effects of ABIN1 on immunoreaction and
levels (Figure 2b) in macrophages, compared with control. LPS-stimulated liver injury in vivo, LPS was applied to
Multiple relative researches indicated that excessive inflam- conduct sepsis model followed by control-plasmid or
matory response often appeared during the progression of ABIN1-plasmid stimulation. qRT-PCR was conducted to
sepsis; thus we detected the inflammatory cytokines by measure ABIN mRNA levels in LPS-induced mice serum.
ELISA in different groups. We observed upregulation of The results indicated that ABIN1 mRNA was remarkably
inflammatory factors in LPS-treated macrophages. On the suppressed in LPS-treated mice serum compared to
contrary, compared to LPS group, secretion of LPS-inducible control group (Figure 4a). However, the effects of LPS
cytokines was remarkably inhibited after ABIN1-plasmid on ABIN1 mRNA were alleviated by ABIN1-plasmid
treatment (Figure 2c–e). The aforementioned further (Figure 4a). Furthermore, ELISA assay was applied to
revealed that ABIN1 regulated the inflammatory reac- assess the release of inflammatory factors in mice serum
tion in macrophages. in different groups (control, LPS, LPS + control-plasmid,
and LPS + ABIN1-plasmid). As shown in Figure 4b–d, we
found increased inflammatory factor levels in the LPS-
induced mice serum. However, the secretion of inflam-
3.3 ABIN1-plasmid inhibited NF-κB pathway matory factors was remarkably inhibited in the serum of
activation in LPS-stimulated LPS + ABIN1-plasmid-treated mice compared with LPS +
macrophages control-plasmid group (Figure 4b–d).
To further verify the positive impact of ABIN1-plasmid
Western blot assay was adopted to evaluate the related on LPS-stimulated liver injury in vivo, ELISA assay was
protein expression. Results from Figure 3a showed that carried out to analyze the liver damage alterations. Our
LPS increased the p-p65 levels compared with control, results suggested that the levels of serum kidney and liver
whereas the effects were reversed in ABIN1-plasmid + injury markers (Cr, BUN, ALT, and AST) were increased
LPS group. Moreover, the same variation trend of p-p65/ after LPS treatment compared to control (Figure 4e–h).
p65 was observed in different groups, and we found that These effects of kidney and liver injury markers on LPS-
p-p65/p65 were enhanced after LPS treatment and decreased induced mice were significantly reduced in ABIN1-plasmid
by ABIN1-plasmid (Figure 3b). The aforementioned combination treatment group (Figure 4e–h). The above
suggested that ABIN1 exerted an important role in inflam- data showed that ABIN1 prevented the inflammatory
matory development by activating NF-κB pathway in vitro. response progression in septic mice.
Figure 3: ABIN1-plasmid suppressed LPS-stimulated NF-κB activation in macrophages. Control-plasmid or ABIN1-plasmid was transfected
into macrophages for 48 h, followed by LPS treatment. (a) Western blot analysis was adopted to evaluate p-p65 and p65 expression in four
groups (control, LPS, LPS + control-plasmid, and LPS + ABIN1-plasmid). (b) Ratio of p-p65/p65. **p < 0.01 vs control, and ##p < 0.01 vs LPS +
control-plasmid.38 Haolan Li et al.
Figure 4: LPS affected the inflammatory factors’ secretion and liver damage. (a) qRT-PCR analysis was carried out to evaluate ABIN1
expression. Secretion of (b) TNF-α, (c) IL-1β, and (d) IL-6 in serum were verified using ELISA assay in control and LPS groups. (e) Serum Cr,
(f) BUN, (g) ALT, and (h) AST levels were determined in control and LPS groups. **p < 0.01 vs control, and ##p < 0.01 vs LPS + control-
plasmid.
4 Discussion and the mechanism of it remain unclear. Based on the
aforementioned research, this study aimed to elaborate
Sepsis remains the main cause of morbidity and mortality the role of ABIN1 in sepsis. qRT-PCR and western blot
in clinic worldwide, mainly caused by immune response analysis were conducted to evaluate ABIN1 mRNA and
[21–23]. Various reports have highlighted that immune protein levels in RAW264.7 macrophages treated with or
cells are involved in sepsis, including macrophage. without LPS. In support of this result, previous studies
Macrophage has been reported to mediate inflamma- have confirmed that ABIN1 was significantly downregu-
tion-related diseases by releasing inflammatory factors lated in LPS-treated RAW264.7 macrophages. Therefore,
[24,25]. Moreover, it is reported that LPS is often used to we imagined that upregulation of ABIN1 expression might
stimulate macrophages in in vivo or in vitro models. exert anti-inflammatory functions in sepsis. We further
Excessive accumulation of inflammatory factors is the transfected control-plasmid or ABIN1-plasmid into RAW
main cause of organ dysfunction syndrome in sepsis 264.7 macrophages for 48 h, and then the cells were
[26]. Based on these, in our research, we explored treated with 1 μg/mL LPS for further 4 h. We found that
deeply that the effects of macrophages on sepsis are ABIN1-plasmid significantly increased ABIN1 levels and
essential to cure sepsis. First, we provided evidence alleviated the secretion of inflammatory factors in LPS-
that the secretion of inflammatory cytokines was promoted induced macrophages. Taking these data into considera-
in LPS-induced macrophages. In accordance with our tion, our results indicated that ABIN1 regulated immuno-
findings, Bian et al. reported that LPS significantly reaction in sepsis.
enhanced the production of inflammatory factors in As we all know, the inflammatory response is resulted
macrophages [27]. from many signaling pathways, among which NF-κB
ABIN1 is an A20-binding protein, which functions as pathway is the major regulator to the inflammatory
an inhibitor in NF-κB pathway [28]. Various investiga- genes [31]. In addition, several reports have revealed
tions have revealed that ABIN1 was participated in that suppression of NF-κB promoted LPS-stimulated
many diseases. For example, Zhou et al. found that sepsis [32]. We subsequently detected the expression
ABIN1 controls beta protein activation and protects of NF-κB-related proteins by western blot assay. Our
from inflammatory disease [29]. In addition, Korte et al. findings showed that the protein expression of p-p65
showed that ABIN1 determines glomerulonephritis severity and p-p65/p65 ratio was enhanced after LPS treatment,
by activating intrinsic glomerular inflammation [30]. How- whereas these changes were reduced by ABIN1-plasmid.
ever, whether ABIN1 is involved in the formation of sepsis The above results revealed that ABIN1 regulatedABIN1 in sepsis 39
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