- Academic Editors
†These authors contributed equally.
Background: Current studies have demonstrated that disintegrin and
metalloproteinase 17 (ADAM17) plays a critical role in the pathogenesis of
sepsis. MicroRNA (miR)-145 is known to control immune responses as an
anti-inflammatory modulatory molecule. However, a fundamental understanding of
how miR-145 regulates ADAM17 and, more broadly, sepsis-induced inflammatory
response remains unknown. Methods: We used western blotting and
quantitative real-time PCR (qRT-PCR) to measure expression levels of ADAM17 and
miR-145. Enzyme-linked immunosorbent assays (ELISA) were performed to measure
cytokine production. To determine if ADAM17 is a target gene of miR-145,
bioinformatics analyses and luciferase reporter assays were conducted. The
impacts of ADAM17 and miR-145 on sepsis-induced inflammatory responses were
accessed in vitro using human umbilical endothelial cells (HUVECs)
treated with lipopolysaccharide (LPS). Sepsis-induced inflammatory response was
measured in vivo using a polymicrobial septic mouse model induced by
cecal ligation and puncture (CLP) with pre-injection of a miR-145 agomir.
Results: In HUVECs treated with LPS, miR-145 expression was
downregulated and miR-145 negatively regulated ADAM17 expression through direct
binding to the ADAM17 transcript 3
Sepsis-3 describes a sepsis condition of organ dysfunction brought on by a multifaceted host response to an infection, with a complex pathogenesis identified to engage early activation of both pro- and anti-inflammatory responses [1]. Sepsis continues to be the primary cause of mortality in intensive care units (ICU) globally, claiming millions of lives annually and placing a heavy burden on health care systems [2, 3].
A disintegrin and metalloproteinase 17 (ADAM17), is one of the major shedding
enzymes within the ADAM family, and is widely expressed in endothelial cells,
leukocytes, and platelets [4]. ADAM17 is engaged in the shedding of over 80
cellular substrates, including inflammatory cytokines (e.g., TNF-
MicroRNAs (miRNAs) are small endogenous RNAs that generally target the
3
Recently, the effects of miR-145 on sepsis and other inflammatory responses in vivo and in vitro have been investigated. Cao et al. [20] identified a significant decrease of miR-145 in exosomes from blood samples of septic patients and in lung tissues from LPS-treated mice. Additionally, miR-145 was found to attenuate sepsis-induced acute lung injury. Huang et al. [21] found that miR-145 regulated macrophage polarization by targeting IL-16 mRNA, while the function of miR-145 in improving macrophage-mediated inflammation through Arf6 was observed in other studies [22].
Bioinformatic analysis predicted an extraordinarily well-conserved binding
between 3
American Type Culture Collection (ATCC; Manassas, VA, USA) provided human
umbilical vein endothelial cells (HUVECs) and the human embryonic kidney cell
line 293T. All cell lines were validated by STR profiling and tested negative for
mycoplasma. Cells were all cultured in a humidified incubator at 37 °C
and 5% CO
GenePharma, Inc. (Shanghai, China) designed and constructed the LV-miR-145
plasmid consisting of the green fluorescence protein (GFP) reporter gene that
overexpressed miR-145, and the LV-negative control (LV-NC) plasmid which does not
target any recognized human genes. To establish cell lines that stably
overexpress miR-145, HUVECs were infected with LV-miR-145 and control cells were
infected with LV-NC. The related sequences were as followed: miR-145,
5
C57BL/6 male mice aged 5–8 weeks were utilized in this study. The experimental animal ethics committee of Guangdong Medical University reviewed and approved the animal experimentation protocols outlined in this manuscript. Per a previous study [23], the cecal ligation and puncture (CLP) operation was performed on mice to construct the polymicrobial sepsis model. Briefly, the mice were anesthetized using ketamine (100 mg/kg) and xylazine (5 mg/kg) by intraperitoneal injections. A surgical incision was subsequently made in the middle of abdomen, cecum was uncovered, ligated beneath the ileocaecal valve, punctured twice using a 20-gauge needle, and a tiny amount of feces from both sides of the perforation site was gently extruded. After the surgery, the cecum was placed back into the abdominal cavity and the incision was sutured. In sham-operation mice, the same surgical operation was carried out without conducting the CLP operation. All mice were given a subcutaneous injection of normal saline (50 mL/kg) immediately after the procedure to sustain hemodynamic conditions. Throughout the experiments, mice were provided water and food ad libitum.
The miR-145-5p agomir (5
Following the manufacturer’s protocol, total RNA was extracted from cells using
TRIzol reagent (Beyotime Biotechnology, Shanghai, China). The complementary DNA
(cDNA) was synthesized for qRT-PCR analysis of ADAM17 by using PrimeScript™
RT reagent Kit with gDNA Eraser (Takara, Shiga, Japan). An
Applied Biosystems 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) was used
for qRT-PCR using a SYBR Green RT-PCR Kit (Takara). Hairpin-it™ microRNA
RT-PCR Quantitation Kit (GenePharma, Inc., Shanghai, China) was used for qRT-PCR
analysis of miR-145. Sangon Biotech (Shanghai, China) Co., Ltd. designed the
primers of qRT-PCR according to the Primer3Plus software, available online,
version 3.3.0 (http://www.bioinformatics.nl/primer3plus), as followed: ADAM17,
5
RIPA lysis buffer (Beyotime Biotechnology) was used to solubilize cells or
tissue homogenates. Total protein concentrations were measured using a BCA assay
(Beyotime Biotechnology). A total of 30 µg Proteins were
electrophoresed on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel (NCM
Biotech, Suzhou, China). And 1.5 µL of Prestained Color Protein Ladder
samples (P0076, Beyotime Biotechnology) was also loaded into each lane. The SDS
polyacrylamide gels were subsequently transferred to polyvinylidene difluoride
(PVDF) membranes. The membranes were incubated with antibodies against ADAM17
(sc-390859, mouse monoclonal antibody, 1:800, Santa Cruz Biotechnology, Santa Cruz, CA, USA;
A00604, rabbit polyclonal antibody, 1:800, Boster Biological Technology, China)
or
We used several bioinformatic tools in this investigation, including Starbase
(https://starbase.sysu.edu.cn) and TargetScan (http://www.targetscan.org), to
predict the regulatory mechanism between ADAM17 and miR-145. The wild-type
3
With the use of commercial ELISA kits from Boster Biological Technology (Wuhan,
China), the expression levels of IL-6, TNF-
Kidney and lung tissues were removed from sacrificed mice challenged with either sham or CLP surgery and subsequently fixed in formaldehyde solution for 48 hours. Per conventional methodology, the fixed tissues were embedded in paraffin and sectioned (4.5 µm thickness) for immunohistochemical (IHC) staining, immunofluorescence (IF) staining and hematoxylin-eosin (H&E) staining.
To permeabilize the sections for IHC, immunostaining permeabilization solution with Triton X-100 (Beyotime Biotechnology) was used following deparaffinization and hydration. The slices were then subjected to steam in improved citrate antigen retrieval solution (Beyotime Biotechnology) for 30 minutes and cooled at room temperature for more than 30 minutes. Endogenous peroxidase activity was quenched with blocking buffer. Then, an anti-ADAM17 antibody (A00604, rabbit polyclonal antibody, 1:200, Boster Biological Technology) diluted to 1:300 was added overnight at 4 °C. After that, sections were incubated with polymer helper and polyperoxidase-anti-rabbit IgG (Boster Biological Technology, Wuhan, China) for 1 h at 37 °C. Diaminobenzidine (DAB; Boster Biological Technology) was used as the chromogen and following counterstaining with hematoxylin, specimens were dehydrated, mounted, and observed under a microscope. The staining intensity of ADAM17 was quantified using Image J software with the estimation of average optical density obtained.
The lung and kidney sections were permeabilized as described above and incubated with QuickBlock™ blocking buffer (Beyotime Biotechnology). After this, tissue slices were incubated with anti-ADAM17 antibody (A00604, rabbit polyclonal antibody, 1:300, Boster Biological Technology). Following a PBS rinses, the sections were stained in the dark with Alexa Fluor 488-labeled Goat Anti-Rabbit IgG (Beyotime Biotechnology). Subsequently, tissue slices were stained using DAPI in the dark and were observed under a fluorescence microscope. Captured images were then processed and analyzed using Image J software.
To evaluate the pathological change in lung and kidney tissues, the sections
were stained with a hematoxylin-eosin (H&E) staining kit (Beyotime
Biotechnology) according to the manufacturer’s guidelines. As previously
described [23, 25, 26], the pathological changes, specifically inflammatory cell
infiltration, alveoli wall edema, pulmonary alveoli congestion, and hemorrhage in
lung tissues, were graded using a scoring scheme from 0 to 3 and used to estimate
sepsis-related lung damage. Histological scoring of kidney injury was scored as
0, no injury; 1,
GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA) and SPSS
software version 25.0 (SPSS Inc., Chicago, IL, USA) were used for statistical
analyses. The difference between two independent samples was evaluated using
either Student’s t test or a non-parametric Mann-Whitney U test. For
comparison of multiple groups, a one-way ANOVA followed by Tukey’s multiple
comparison post-hoc test was used. The log-rank test was used to assess the
difference of Kaplan-Meier survival analyses. p
After stimulating with LPS (500 ng/mL) for 6 h, miR-145 expression in HUVECs was detected by using qRT-PCR. As shown in Fig. 1A, miR-145 expression in LPS-stimulated HUVECs was significantly decreased compared with HUVECs treated with PBS as control. For the purpose of assessing the role of miR-145 in regulation of ADAM17 expression in HUVECs, we used LV-miR-145 to infect HUVECs and establish stably miR-145 overexpressed cell lines. Results of qRT-PCR validated overexpression of miR-145 in HUVECs infected with LV-miR-145 (Fig. 1B). As presented in Fig. 1C,D, both qRT-PCR and western blot analysis indicated that miR-145 overexpression significantly decreased ADAM17 mRNA expression and protein production by 62.3% and 32.6%, respectively.
Lipopolysaccharide (LPS) reduces miR-145 expression, overexpression of miR-145
downregulated ADAM17 expression by directly binding to the 3
Bioinformatic analyses predicted a binding site for miR-145 within the
3
To produce stable cell lines and test the impact of miR-145 on LPS-stimulated
ADAM17 expression as well as inflammatory reactions in HUVECs, we used lentivirus
engineered from the LV-ADAM17-siRNA and LV-miR-145 plasmid to transfect HUVECs
and GFP fluorescence was observed in an inverted fluorescent microscope (Fig. 2A). As presented in Fig. 2B, the protein production of ADAM17 was significantly
decreased by 33.3% in HUVECs transfected with LV-miR-145 compared with cells
transfected with LV-NC following LPS stimulation. Similar results were observed
in HUVECs transfected with LV-ADAM17-siRNA. These results indicated that
overexpression of miR-145 produced a similar effect to siRNA-induced knockdown of
ADAM17 expression. In addition, ADAM17 knockdown significantly downregulated the
expression levels of IL-6, TNF-
miR-145 alleviated LPS-induced endothelial inflammation by
targeting ADAM17 in HUVECs. HUVECs were infected by LV-NC, LV-miR-145,
LV-ADAM17-siRNA or the combined of LV-miR-145 and LV-ADAM17-siRNA. (A) Green
fluorescence protein (GFP) fluorescence was observed in an inverted fluorescent
microscope to track transfection efficiency of LV-NC, LV-miR-145 and
LV-ADAM17-siRNA. (B) After 6 hours of LPS (500 ng/mL) stimulation, the expression
of ADAM17 in HUVECs was detected by western blot. (C) After 6 hours of LPS (500
ng/mL) stimulation, ELISA assay was performed to detect the expression levels of
IL-6, TNF-
Given the role of miR-145 in the downregulation of ADAM17 expression in vitro, we next investigated whether miR-145 overexpression is associated with diminished inflammatory response in vivo. Mice were injected with a miR-145 agomir or control (NC) agomir 12 h prior to CLP or sham surgery. Subsequently, mice were sacrificed and serum, kidney, and lung tissues obtained 12 h after surgery (Fig. 3A). Western blotting indicated that ADAM17 expression was significantly decreased by 58.5% in lung tissues taken from polymicrobial septic animals treated with miR-145 agomir compared to those treated with the NC agomir (Fig. 3B).
Overexpression of miR-145 reduces expression of ADAM17,
attenuates sepsis-induced inflammatory responses and acute lung injury. (A)
Schematic of experimental design and time line. Polymicrobial sepsis model of
mice was induced in C57BL/6 mice by cecal ligation and puncture (CLP) operation, and miR-145 agomir or miR-145
agomir NC (30 mg/kg) were delivered into the mice by intraperitoneal injection 12
h before CLP. The same surgical procedure but without CLP was performed in
sham-treated mice. Lung tissues, kidney tissues and blood were harvested 12 h
after the operation. (B) Expression of ADAM17 protein in lung tissues was
detected by western blot. (C,D) Immunofluorescence (IF) staining with antibody
against ADAM17 (green) was performed to observe ADAM17 localization in lung
tissue sections, and cell nucleus was stained by DAPI (blue). Results of relative
fluorescence intensity reflecting the level of ADAM17 expression. (E,F)
Representative images showing ADAM17 immunohistochemical (IHC) staining in lung tissue sections. Scale
bars, 100 µm and 50 µm. Results of average optical
density reflecting the level of ADAM17 expression. (G,H) Representative images of
H&E staining of lung tissue sections. Scale bars, 100 µm, 50
µm. The severity of lung injury was evaluated by lung injury score.
(I) Cytokines (IL-1
Next, we conducted IHC and IF staining with an antibody against ADAM17 to locate the protein in lung tissues. As shown in Fig. 3C,D, results of immunofluorescence staining showed that ADAM17 expression in lung tissues of mice following CLP was increased relative to sham mice, while significant downregulation of ADAM17 was observed in mice treated with the miR-145 agomir when compared to controls. Results of IHC analysis also indicated ADAM17 production in lung tissues of septic mice treated with miR-145 agomir was significantly reduced (Fig. 3E,F).
To further explore the severity of lung injury, we performed histopathological
analysis of lung tissues. The results of H&E staining (Fig. 3G,H) showed obvious
alveolar tissue damage and neutrophil accumulation induced by CLP within the
lungs. Furthermore, evaluation of lung injury score indicated a significant
reduction in mice injected with the miR-145 agomir compared to controls. These
findings suggest that miR-145 protects against sepsis-induced inflammation and
acute lung injury by suppressing ADAM17 expression. The serum levels of ICAM-1,
VCAM-1, IL-1
As shown in Fig. 4A, ADAM17 protein production was significantly decreased by 44.8% in kidney tissues of mice treated with miR-145 agomir when compared to NC agomir-treated mice upon CLP operation. The results of IF analysis indicated that ADAM17 abundance was more concentrated in the renal cortex than in the medulla, and overall ADAM17 abundance was slightly reduced in kidney sections from miR-145 agomir-treated septic mice compared with the NC agomir-treated mice upon CLP operation (Fig. 4B,C). As presented in Fig. 4D,E, IHC indicated decreased production of ADAM17 in the kidney tissue from miR-145 agomir treated mice relative to NC agomir treated mice upon CLP operation. H&E staining indicated that miR-145 agomir significantly alleviated intracellular vacuolization, tubular cell necrosis, tubule dilation, and improved kidney histological scores compared to septic mice treated with control agomir (Fig. 4F,G). Taken together, these results suggest a protective function of miR-145 against sepsis-induced acute kidney injury by reducing the expression of ADAM17.
MiR-145 agomir reduces expression of ADAM17, attenuates
sepsis-induced acute kidney injury and offers survival benefit. (A) Expression
of ADAM17 protein in kidney tissues was detected by western blot. (B,C) IF
staining with antibody against ADAM17 (green) was performed to observe ADAM17
localization in kidney tissue sections, and cell nucleus was stained by DAPI
(blue). Results of relative fluorescence intensity reflecting the level of ADAM17
expression. (D,E) Representative images showing ADAM17 IHC staining in kidney
tissue sections. Scale bars, 100 µm and 50 µm. Results
of average optical density reflecting the level of ADAM17 expression. (F,G)
Representative images of H&E staining of kidney tissue sections. Scale bars, 100
µm and 50 µm. Semiquantitative analysis of tubular
injury: tubular cell necrosis, loss of the brush border, vacuolization, tubule
dilation, or cast formation. (H) The survival curves of septic mice injected with
miR-145 agomir and miR-145 agomir NC respectively (n = 13 per group). Data were
analyzed by log-rank test. Error bar represent standard deviation of the mean
(SD). *p
As shown in Fig. 4H, Kaplan-Meier survival analysis demonstrated that miR-145 agomir injection significantly (p = 0.0195) improved septic mouse survival during a four-day window relative to septic mice injected with NC agomir. These findings clearly indicate a protective impact of miR-145 agomir against sepsis-induced mortality.
Sepsis is a detrimental response to pathogenic microorganism infection inducing systemic inflammation. It has been demonstrated that dysregulated inflammatory and immunological responses, as well as dysfunction within vascular endothelium play crucial roles in the pathogenesis of sepsis [3]. LPS and other pathogen-associated molecular patterns (PAMPs) activate both immune cells and endothelium cells, which can generate a variety of inflammatory cytokines that seriously harm cells and disrupt microcirculation, ultimately leading to organ dysfunction. In the current study, we examined the molecular functions of the miR-145/ADAM17 axis on acute organ damage and inflammatory responses induced by sepsis. These results provide evidence for a mechanism by which miR-145 limits sepsis-induced organ injury and inflammation by downregulating ADAM17.
miRNAs, as posttranscriptional regulators by targeting 3
ADAM17, also known as tumor necrosis factor alpha-converting enzyme (TACE),
mediates inflammatory reactions by cleaving the extracellular domains of
proinflammatory substates at specific transmembrane sites [8, 9]. The shedding of
numerous substrates, such as VCAM-1, ICAM-1, IL-6R, and TNF-
To further elucidate the in vivo pathological mechanisms of miR-145 and
ADAM17 we used a septic mouse model. CLP, a widely used model for establishing
polymicrobial sepsis, markedly increased ADAM17 expression in lung and kidney
tissue. Although substantial residual amount of ADAM17 remained, miR-145
treatment dramatically decreased ADAM17 expressions in CLP mice to a similar
level as those in mice without CLP operation, indicating an antagonistic effect
of miR-145 against upregulation of ADAM17 following sepsis. Evidence indicates
that ADAM17-dependent shedding/modulation events are involved in the development
and progression of inflammation via activation of several key inflammatory
signaling including NF-
Sepsis is a life-threatening organ dysfunction due to dysregulated host response to infection, leading to inflammatory damage to nearly every organ system. Thus, we further evaluate the role of miR-145 agomir in sepsis-induced acute lung injury (ALI) and acute kidney injury (AKI). The expression of ADAM17 in lung tissue as judged by IHC and IF staining was decreased in septic mice treated with miR-145 agomir when compared to controls. Moreover, the histopathological analysis of lung tissues showed that miR-145 agomir improved lung histology and limited leukocyte infiltration. In accordance with these findings, previous studies demonstrated a role of miR-145 in mitigating LPS-induced acute lung injury [37, 38]. In related studies, COVID-19-related lung inflammation and damage in a preclinical mouse model were observed to be exacerbated by ADAM17 activation and ameliorated by its silencing [39]. Hence, it is reasonable to hypothesize that miR-145 limits CLP-induced acute lung injury by reducing ADAM17 expression. Similar results obtained in the analysis of kidney tissues further validated a proinflammatory role for ADAM17 in acute kidney injury, and this is consistent with other studies [40, 41].
Overall, miRNA-145, as an upstream mediator of ADAM17, functions in limiting LPS-induced endothelium inflammatory response in HUVECs, and constrains organ injury in mice during response to sepsis. ADAM17 is not the only target gene of miR-145. The miRNAs-associated off-target adverse effects due to the pleiotropic nature of miRNAs are causing concern. Targeting miR-145 also potentially knockdown the expression of other irrelevant genes in different tissues and organs, which may be challenging and cause unexpected side effects, as shown in the recent studies of the unexpected miR-145’s toxicity to podocytes [42, 43]. Our results provided evidence that miR-145 was potentially effective in anti-inflammatory therapy for sepsis. However, the network of miR-145 target genes and cellular signaling still lacks a full understanding. And optimal delivery of miR-145 specifically to damaged vascular endothelial cells still remains challenge.
In summary, we have found that miR-145 directly binds to the 3
The authors declare that all data supporting the findings of this study are available within the paper and its Additional file.
QL, JH and MX conceived and designed the experiments, and participated in its design and coordination and helped to draft the manuscript and revise the manuscript. YYL and LL performed the experiment and statistical analysis, and drafted and revised the manuscript. YL, RY and SL helped to perform the experiments, statistical analysis and draft the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the experimental animal ethics committee of Guangdong Medical University (GDY2202700).
Not applicable.
This work was supported by the Medical Scientific Research Foundation of Guangdong Province (A2022477), the Science and Technology Innovation Leading talents Project of Jieyang City (2022SRC004), the Natural Science Foundation of Guangdong Province (2021A1515010871), and Science and Technology Project of Jieyang City (skjcx062).
The authors declare no conflict of interest.
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