Midkine Ameliorates the LPS-Induced Apoptosis of Airway Smooth Muscle Cells Through the Notch2 Pathway

Background: Airway smooth muscle cells (ASMCs) produce several cytokines during in�ammation, causing changes in extracellular matrix components, leading to airway remodeling. Midkine (MK) promotes the chemotaxis of in�ammatory cells and releases proin�ammatory factors. Whether Notch and Midkine jointly affect the proliferation and apoptosis of ASMCs is unknown. This research aimed to study the role of MK in ASMCs using an LPS-induced acute lung injury model. Methods: ASMCs were cultured in vitro and divided into �ve groups according to treatment: control, lipopolysaccharide (LPS), non-target siRNA, MK siRNA, and g-secretase inhibitor LY411575. Cell proliferation was assessed using the Cell Counting Kit-8 assay. Apoptosis was measured by �ow cytometry. Changes in the levels of cytokines related to the MK/Notch2 signaling pathway were detected by Western blotting, qPCR, and immuno�uorescence. Results: LPS increased the mRNA and protein expression of MK and Notch2. MK silencing and LY411575 reduced this effect. LPS reduced the viability and increased the rate of apoptosis of ASMCs. This effect was attenuated by exogenous MK and enhanced by MK silencing and LY411575 treatment. Conclusions: The MK/Notch2 signaling pathway plays a regulatory role in ASMC proliferation and apoptosis in airway in�ammation.


Background
Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is characterized by acute and progressive respiratory failure caused by various factors [1]. ARDS may occur at the end-stage of several diseases. The occurrence and development of ALI/ARDS are mainly related to microbial infections and other factors, including severe trauma, non-cardiogenic shock, poisoning, long-term cardiopulmonary resuscitation, surgery, and acute pancreatitis [2]. Midkine (MK) is a heparin-binding growth factor that attracts in ammatory cells, releases cytokines, and participates in in ammation [3,4]. MK activates Notch signaling [5,6,7]. MK induces the structural recombination of actin by binding to the Notch-2 receptor, resulting in the phosphorylation and activation of STAT3, promoting cell growth and differentiation. MK activates other pathways by inducing changes in the Notch-2 receptor and stimulating the expression of Hes1 and NF-κB [8]. MK is highly expressed in cystic pulmonary brosis and ARDS [9,10]. However, the mechanisms underlying MK actions are incompletely understood.
Airway smooth muscle cells (ASMCs) have contractile activity and produce in ammatory cytokines and growth factors, and these cells are regulated by extracellular matrix components [11,12]. The abnormal proliferation and hypertrophy of ASMCs can lead to airway wall thickening and, ultimately, airway remodeling [13]. However, it is unclear whether the MK-Notch pathway participates in ASMC proliferation. MK may inhibit apoptosis and promote ASMC proliferation through the Notch pathway, and these effects can be reversed by MK inhibition. This study assesses the effect of MK on rat ASMCs in vitro.

Materials
Rat airway smooth muscle cells were purchased from Guangzhou Gennio Biological Technology Co., Ltd. All experiments were approved by the Research Ethics Committee of our institution.

Cell culture
Airway smooth muscle cells were cultured in DMEM containing 10% FBS in a humidi ed incubator with 5% CO 2 at 37 ℃, and the medium was changed every 2 days for subcultivation. Mid-logarithmic-phase ASMC cells were divided into ve groups according to treatment, as follows: (a) Control: untreated.

Western blotting
The total protein of ASMC cells from each group was extracted and prepared according to standard procedures and quanti ed using the BCA method. Proteins were separated on a 12% SDS polyacrylamide gel and transferred to a PVDF membrane. The membrane was incubated with primary antibodies (anti-MK, anti-Notch2, and anti-β-actin) (Abcam) at 4 ℃ overnight and then incubated with goat anti-rabbit IgG at room temperature for 1 h. The membrane was washed three times with TBST. Immunoreactive bands were visualized using the Tanon 5200 enhanced chemiluminescence detection system (Tanon, China) and quanti ed by densitometry using ImageJ. The relative expression of MK and Notch2 was measured in the four study groups.
Real-time quantitative PCR was carried out using a SYBR green PCR kit. The ampli cation conditions included a denaturation step at 95 ℃ for 5 min, followed by 40 cycles at 95 ℃ for 10 s and 60 ℃ for 30 s, and an extension step at 60 ℃ for 30 s. The reaction mixture contained 10 µL of the Hieff® qPCR SYBR® Green Master Mix, 0.5 µL of each primer (10 µM), 1 µL of template DNA, and 8 µL of sterile ultrapure water. Gene expression was normalized to GAPDH using the 2 − ΔΔCt method. The mRNA expression of MK and Notch2 was determined in ASMCs from each group (Fig. 3).
Immuno uorescence microscopy ASMCs (10 5 cells/mL) from each group were inoculated in dish (NEST 801002) which is used for confocal laser microscopy. Cells were washed in PBS three times (3 min each time), xed in 4% paraformaldehyde for 15 min, and washed in PBS as described above. Cells were permeabilized with 0.5% Triton X-100 in PBS at 4°C for 15 min and washed in PBS. The slides were incubated with anti-MK and anti-Notch2 primary antibodies at 4°C overnight, washed with PBST three times (3 min each time), and incubated with uorescein-conjugated goat anti-rabbit IgG for 1 h at room temperature. Non-speci c reactions were blocked with normal goat serum for 30 min at room temperature. Nuclei were counterstained with DAPI. Sections were observed under a uorescence microscope. The protein expression of MK and Notch2 in ASMCs of each group was evaluated by immuno uorescence microscopy.
Assessment of apoptosis by ow cytometry ASMCs (10 5 cells/mL) were transferred to six-well plates and digested with trypsin without EDTA. Cells were resuspended in 1X binding buffer (BD Pharmingen™), incubated with 5 µL of annexin V-FITC (BD Pharmingen™) in the dark for 15 min at room temperature, and then incubated with 5 µL of propidium iodide for 15 min at room temperature.

Statistical analysis
Data were expressed as means and standard deviations and analyzed using SPSS software version 20.0. Inter-group and intra-group differences were evaluated by one-way analysis of variance and independentsample t-test, respectively. P-values of less than 0.05 were considered statistically signi cant.

Half maximal inhibitory concentration of LPS and LY411575
The effects of LPS and LY411575 on the viability of ASMC cells were evaluated using the CCK-8 assay. The IC50 of LPS and LY411575 was 0.2 mg/mL and 0.03 mg/mL, respectively (Fig. 1).
Effect of LPS and MK-siRNA on cell viability LPS signi cantly decreased the viability of ASMCs (P < 0.05), and this effect was potentiated by MK siRNA transfection and LY411575 treatment (Fig. 5).

Effect of rMK on cell viability
Recombinant midkine(rMK) did not signi cantly affect the survival of ASMCs (Fig. 6).
Effect of rMK on cell viability in the presence of LPS rMK signi cantly increased the survival of ASMCs treated with LPS for 48 and 72 h, and this increase was dose-independent (Fig. 7).

Effect of LPS and rMK on the rate of apoptosis
The apoptosis rate of ASMCs was determined by ow cytometry (Fig. 8A). LPS signi cantly increased the rate of apoptosis, and this effect was enhanced by MK silencing and reduced by rMK treatment (Fig. 8B).
Effects of LPS, MK silencing, and LY411575 on the rate of apoptosis The apoptosis rate of ASMC cells was detected by ow cytometry (Fig. 9A). LPS increased the rate of apoptosis, and MK silencing and LY411575 potentiated this effect (Fig. 9B).

Discussion
ALI is a clinical disease with high mortality and high treatment burden. Despite great progress in diagnosis and treatment, disease prognosis is poor. ALI is characterized by the apoptosis and necrosis of vascular endothelial cells and impaired vascular integrity, leading to increased permeability and pulmonary edema. LPS is a glycolipid of the outermost membrane of Gram-negative bacteria. This glycolipid induces in ammatory reactions and cytokine secretion and has been shown to trigger ALI in animal models [14,15]. In our study, the effect of LPS-induced ALI was assessed using a cell model. Some studies have found that TNF-α expression is increased in LPS-induced ALI. TNF-α reduces vascular permeability, leading to pulmonary vascular endothelial cell injury and pulmonary edema [16,17]. MK promotes the release of MCP-1, MIP-2, and other chemokines, and the aggregation of macrophages and neutrophils [18]. MK expression is low in healthy organs and increases during oxidative stress, in ammation, and tissue repair [19,20]. In this study, LPS increased the protein expression of MK in rat airway smooth muscle cells. However, the role of MK and related signaling pathways in ASMCs in LPSinduced ALI is incompletely understood.
MK promotes cell proliferation [21]. MK is expressed in many tumors and in ammatory diseases and has anti-apoptotic effects [22]. The present study assessed the effect of rMK on the proliferation and apoptosis of ASMCs. In a mouse model of acid-and ventilator-induced lung injury, an increase in collagen deposition and hydroxyproline levels and a decrease in lung compliance were attenuated in MK −/− mice vs. wild-type mice [25]. The inhibition of Nox1, MK, and Notch2 attenuated epithelial-tomesenchymal transition (EMT), demonstrating that MK plays an important role in airway remodeling [10,23]. MK enhances the hypoxia-induced proliferation and differentiation of human lung epithelial cells [24]. Our results showed that ASMC survival was lower in the MK siRNA group than in the LPS group, indicating that MK reduced LPS-induced ALI. In addition, LPS decreased the viability and rate of apoptosis of ASMCs, whereas rMK attenuated these effects. These results show that MK participates in the repair of in ammatory tissues, increases ASMC survival, and inhibits apoptosis, leading to airway remodeling, and these effects are reversed by MK inhibition.
Notch2 is an MK receptor and an evolutionarily conserved pathway that regulates many cell-fate decisions during development. Notch signaling activity is regulated by receptor proteolysis. Upon proteolysis, the Notch intracellular domain travels to the nucleus and interacts with a transcription factor complex to regulate gene expression [25,26]. The γ-secretase inhibitor of Notch signaling LY411575 inhibits osteoclast differentiation and bone destruction through the Notch/HES1/MAPK/Akt pathway [27]. Notch2 causes tumor invasion and metastasis by regulating EMT and is downregulated by Notch inhibitors and endogenous compounds, resulting in mesenchymal-to-epithelial transition [28]. Notch2 interacts with MK in human lung epithelial cells, and MK-induced EMT is dictated by mechanical stretching. Notch2 signaling inhibition blocks EMT more effectively than endogenous MK. Silencing MK or Notch2 reduces pulmonary brosis [29]. Notch2 is required for in ammatory cytokine-driven goblet cell metaplasia in the human lung, and Notch2 inhibition can be a therapeutic strategy for preventing this pathological process in airway diseases [30]. Antisense oligonucleotides (ASOs) downregulated Jag1 and Notch2 in goblet cell metaplasia associated with allergen-induced asthma and upregulated the ciliated cell marker FoxJ1. Moreover, the ASO-mediated decrease in Jag1 and Notch 2 expression inhibited goblet cell metaplasia, mucus production, and airway remodeling [31]. Our results showed that LPS increased the mRNA and protein expression of MK and Notch2, consistent with previous studies. Tian et al. found that MK promoted the proliferation of gastric cancer cells through Notch signaling, whereas the cisplatininduced apoptosis of these cells was reduced by rMK and enhanced by MK silencing. Apoptosis may be induced in MK siRNA-transfected cells by an apoptosome-dependent mechanism, with the downregulation of Bcl-2 expression, upregulation of Bax expression, reduction in mitochondrial membrane potential, release of cytochrome c, and the activation of caspases 3, 8, and 9 [32,33]. This study found similar results in airway smooth muscle cells. The inhibition of MK and Notch2 expression repressed the proliferation of ASMCs and promoted apoptosis, demonstrating that MK and Notch2 jointly participate in lung tissue remodeling.

Conclusion
Our results demonstrate that MK has a role in ALI. MK promotes the proliferation of ASMCs and reduces cell apoptosis through Notch2 signaling. This study helped elucidate the role of ASMCs in a rat model of airway remodeling. The results con rmed that MK plays a role in ASMC proliferation in vitro in an LPSinduced ALI model through Notch2 signaling. However, the functions of proteins downstream of the MK/Notch2 signaling pathway are unclear, and the role of MK can be further investigated using other gene-delivery technologies, such as lentiviral vectors.

Declarations Declaration of interest
The authors declare that they have no con ict of interest related to the present study.