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Abstract

Background

Death-associated protein kinase (DAPK1) is one of the positive regulators of apoptosis, and it is widely involved in apoptosis induced by multiple pathways. We examined that the function of DAPK1 in Clinical treatment of arterial aneurysm and its underlying mechanisms. Arterial aneurysm is a common cerebrovascular disease with high disability and fatality rate.

Objectives

Male C57BL/6 mice or DAPK1−/− mice were injected with 50 mg/kg pentobarbital sodium and then were injected with angiotensin II (AngII) infusion for vivo model. hASMCs (Human artery smooth muscle cell) were treated with murine recombinant IL-6 (20 ng ml−1; Cell Signaling) for vitro model.

Results

DAPK1 gene, mRNA expression, and protein expression were induced in mice of arterial aneurysm. DAPK1 mRNA expression was increased and Area Under Curve was 0.9075 in patients with arterial aneurysm. Knockout of DAPK1 decreased inflammation and vascular injury in mice model of arterial aneurysm. Beclin1/NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) signal pathway is a critical downstream effector of DAPK1 by TAP production. The regulation of Beclin1 participated in the effects of DAPK1 on inflammation of arterial aneurysm by ATP-dependent NLRP3 inflammasome. The regulation of NLRP3 participated in the effects of DAPK1 on inflammation of arterial aneurysm.

Conclusion

Collectively, our data indicated that DAPK1 may be a potential biomarker for arterial aneurysm in clinical treatment and activated inflammation levels in arterial aneurysm through NLRP3 inflammasome by Beclin1. DAPK1 might be a key pathogenic event underlying excess inflammation of arterial aneurysm.

Keywords

DAPK1 NLRP3 Beclin1 arterial aneurysm

Introduction

Arterial aneurysm is a common cerebrovascular disease with high disability and fatality rate.^1,2 It is the primary cause of subarachnoid hemorrhage and is also the third leading cause of cerebrovascular accidents after cerebrovascular disease and hypertension, which seriously threatens the life and health of patients.^1,2 Studies have shown that the localized expansion and bulging of the wall caused by local congenital defects of the intracranial arterial wall, increased intraluminal pressure, etc., are the main causes of arterial aneurysm, which is also associated with vasculitis, hypertension, and cerebral arteriosclerosis. Epidemiological studies have demonstrated that arterial aneurysm can occur at any age, which is most common in people aged 40–60 and is rare in adolescents.^3,4

At present, accumulative studies have confirmed the presence of macrophages and lymphocyte infiltration in the tumor wall of intracranial tumors.⁵ It is speculated that when cerebral blood vessels are damaged, inflammatory cells, such as macrophages and lymphocytes, can infiltrate into the blood vessel wall, secret inflammatory cytokines and proteolytic enzymes, causing secondary damage to the blood vessel wall, thereby accelerating apoptosis of vascular stromal cells and smooth muscle cells and causing tumor-like bulging.^6,7

NLRP3 inflammasome is involved in the inflammation and apoptosis of various central nervous system diseases, such as cerebral hemorrhage, cerebral infarction, brain trauma, Alzheimer’s disease and meningitis.⁸ Stimuli including lipid phosphorylation, tissue damage, and microbial infection can promote the generation of massive oxygen free radicals, which in turn activate the expression of NLRP3 inflammasome.^9,10 Arterial aneurysm and tissue damage promote the release of a large amount of oxygen free radicals, thereby leading to increased expression of NLRP3 inflammasome complex.¹¹ Animal experiments have also confirmed that the early application of oxygen free radical inhibitors can attenuate the inflammatory response in arterial aneurysm by inhibiting the expression of NLRP3 inflammasome, further improving prognosis.¹²

Inflammasome receptors can also directly interact with Beclin1.¹³ Some inflammasome receptors, such as NLRC4, NLRP3, NLRP4, and NLRP10, have a strong affinity for the evolutionary conserved domain of Beclin1.¹⁴ The combination of NLRP3 and Beclin1 complex can inhibit the maturation of autophagosome. In contrast, knockout of NLRP3 and NLRC4 genes can promote autophagy in vitro. However, autophagy is likely to activate inflammasome through the negative feedback mechanism.¹⁵

Death-associated protein kinase-1 is a serine/threonine protein kinase regulated by calmodulin (CaM) and is involved in a variety of pathophysiological processes in the body.¹⁶ Previous studies have shown that DAPK1 is one of the positive regulators of apoptosis, and it is widely involved in apoptosis induced by multiple pathways.^17,18 Recent studies have found that in addition to regulating apoptosis and autophagy, DAPK1 also plays an important role in a series of inflammationory regulation.^17,18 We examined that the function of DAPK1 in Clinical treatment of arterial aneurysm and its underlying mechanisms.

Materials and methods

Patients with arterial aneurysm

Normal volunteers (n = 20, age = 56.23±11.76, man = 8, female = 12) and patients with arterial aneurysm (n = 20, age = 54.14 ± 12.55, man = 9, female = 11) were collected by People’s Hospital of Quzhou from May 2018 to August 2019. All the experiments were conducted under the guidelines issued by the Institutional Care and Use Committee of People’s Hospital of Quzhou. The protocols for the experiments in this study were reviewed and approved by University Research Committee of People’s Hospital of Quzhou. Informed consent of all subjects was subscribed by all selected personnel. All serum (2–3 mL) of normal volunteers and patients with arterial aneurysm were collected, saved at −80°C and used to measure DAPK1 mRNA expression using quantitative Real-Time PCR.

Animals and vivo model of arterial aneurysm

Male C57BL/6 mice (6 weeks, 18–20 g) or DAPK1^−/− mice (5 weeks, 15–17 g) were housed with 1%–2% isoflurane in a closed chamber for 2–5 min until immobile. Mice were removed and taped with 1.0 ± 1.5% isoflurane at 37 ± 2°C using nosecone during minor surgery. Then, mice were subcutaneously implanted at Mini‐osmotic pumps (Model 2004; ALZET, Cupertino, CA, USA) with (1000 ng/kg/min, 05-23-0101, sigma) in the neck region of anaesthetized mice.

Arterial tissue sample were collected, washed with PBS, and used for other experiments. All the animal experiments were conducted under the guidelines issued by the Institutional Animal Care and Use Committee of People’s Hospital of Quzhou. All experiments have been approved by the Ethics Committee of People ‘s Hospital of Quzhou (No.2018061801).

Quantitative real-time PCR detection and gene microarray hybridization

Total RNA was extracted from arterial tissue sample or cells samples by using Trizol reagent (Life Technologies Corporation, Carlsbad, CA, USA). Taqman MicroRNA Reverse Transcription Kit and Taqman Universal Master Mix II with TaqMan McroRNA Assay (Applied Biosystems, Foster City, CA, USA) were used for testing the gene expression level.

Total RNA was labeled using cyanine-5-cTP and hybridized to the SurePrint and G3 Mouse Whole Genome GE 8x60 K Microarray G4852 A platform. Images were quantified and feature-extracted using Agilent Feature Extraction Software (A.10.7.3.1; Agilent Technologies, Inc.).

Histological examination

Arterial samples after mice sacrificed were collected and fixed with 4% paraformaldehyde for 24 h at room temperature. Lung tissue samples fixed with paraformaldehyde were paraffin-embedded. Blood vessel tissue samples were cut into 5 μm sections using a paraffin slicing machine and stained with hematoxylin and eosin. Lung tissues were observed under light microscopy (magnification, ×200; BH3-MJL; Olympus Corporation, Tokyo, Japan).

Cell culture and vitro model

Human aortic SMC (hASMCs) were propagated in growth media (SmGM-2) with 5% fetal bovine serum (FBS). DAPK1 plasmid, siDAPK1 mimics, Beclin1 plasmid, si-Beclin1, NLRP3 plasmid, si-NLRP3 mimics, negative plasmid, or negative mimics were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). DAPK1 plasmid (100 ng), si-DAPK1 (60 ng), Beclin1 plasmid (100 ng), si-Beclin1 (60 ng), NLRP3 plasmid (100 ng), si-NLRP3 (60 ng), negative plasmid (100 ng), or negative mimics (60 ng) were transfected into hASMCs using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) for 48 h. hASMCs were treated with murine recombinant IL-6 (20 ng ml−1; Cell Signaling) for 12 h.

ELISA assay

Cell samples were collected at 2000 g at 4°C, proteins were extracted by using radioimmunoassay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China) and were quantified by the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). Then, cells were measured the concentrations of TNF-α (H052), IL-6 (H007), IL-1β (H002), and IL-18 (H015) levels using ELISA kits (Nanjing Jiancheng Institute of Biological Engineering).

Serum samples were collected and then the concentrations of TNF-α (H052), IL-6 (H007), IL-1β (H002) and IL-18 (H015) levels were measured using ELISA kits (Nanjing Jiancheng Institute of Biological Engineering).

ATP production level using kit

Cell samples were collected at 2000 g at 4°C, proteins were extracted by using radioimmunoassay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China). ATP production level was measured using ATP production kit (S0026, Beyotime Biotechnology, Shanghai, China).

Western blot analysis

Proteins were extracted by using radioimmunoassay (RIPA) lysis buffer (P0013 B, Beyotime Biotechnology, Shanghai, China) with protease inhibitor (Phenylmethanesulfonyl fluoride, PMSF, ST506, Beyotime Biotechnology, Shanghai, China). Proteins were quantified by the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). Total proteins (50 μg) were loaded onto 10% SDS-PAGE, separated by electrophoresis and transferred to PVDF membranes. Membranes were blocked by 5% skimmed milk in TBS/Tween 20 (0.1%) for 1 h with shaking and then incubated overnight at 4°C with the specific primary antibodies Dapk1 (1:1000, 3008, Cell Signaling Technology), NLRP3 (1:1000, 15101, Cell Signaling Technology), Beclin1 (1:500, sc-48341, Santa Cruz Biotechnology), and β-Actin (1:5000, sc-47778, Santa Cruz Biotechnology). The membranes were incubated with the secondary antibodies conjugated with HRP for 1 h after washing with TBS/Tween 20 (0.1%) for 15 min. Finally, images were captured using Fujifilm LAS-4000 mini (Fujifilm, Tokyo, Japan).

Immunofluorescent staining

After experiment, hASMCs were washed with PBS, fixed with 4% paraformaldehyde (P0099, Beyotime Biotechnology, Shanghai, China) for 15 min at room temperature and then incubated with using 0.25% Triton X-100 (ST677, Beyotime Biotechnology, Shanghai, China) for 15 min hASMCs was incubated with Beclin1 (1:100, sc-48341, Santa Cruz Biotechnology) at 4°C overnight after blocking with 5% BSA for 1 h. hASMCs was incubated with goat anti-rabbit IgG-cFL 555 antibody (1:100) for 2 h at room temperature and stained with DAPI for 15 min and washed with PBS for 15 min. The images of hASMCs obtained using a Zeiss Axioplan two fluorescent microscope (carl Zeiss AG, Oberkochen, Germany).

Statistical Analysis

All experiments were repeated at least three times as the means ± SD. p-values were calculated using two-tailed Student’s t test between two groups, orone-way analysis of variance for more than two groups. Statistical analyses were performed by using SPSS 19.0 statistical software. p-value of < .05 was considered to indicate a statistically significant result.

Results

Death-associated protein kinase-1 expression in patients with arterial aneurysm or mice model of arterial aneurysm

To investigate the function of DAPK1 in arterial aneurysm, we analyzed the expression of DAPK1 in mice with arterial aneurysm using gene chip (Figure 1(a)). We found that DAPK1 gene, mRNA expression and protein expression were induced in mice of arterial aneurysm (Figure 1(a)–(d)). Then, we analyzed the gene expression of DAPK1 in patients with arterial aneurysm. We found that DAPK1 mRNA expression was increased and AUC (Area Under Curve) was 0.9075 in patients with arterial aneurysm (Figure 1(e)-(f)). These results showed that DAPK1 may participate in the occurrence and progress of diseases of arterial aneurysm.

Figure 1.

DAPK1 expression in patients with arterial aneurysm or mice model of arterial aneurysm. Hierarchical clustering analysis of (old change >2 or <-2, p < .05, A), Dapk1 mRNA (B) and protein (C and D) expressions in mice of arterial aneurysm; serum Dapk1 mRNA (E) and sensitivity analyze (F) in patients with arterial aneurysm. Sham, sham control group; AAA, mice model of arterial aneurysm; Normal, normal volunteers group; Patients-AAA, patients with arterial aneurysm group. ##p < .01 compared with sham control group or normal volunteers group. All experiments were repeated at least three times as the means ± SD.

Knockout of death-associated protein kinase 1 showed decreased inflammation in mice model of arterial aneurysm

To investigate the effects of DAPK1 in mice model of arterial aneurysm, we used DAPK1^−/− mice to confirm the effects of DAPK1 in mice model of arterial aneurysm. Knockout DAPK1 reduced incidence of AngII-induced abdominal aortic aneurysm (AAA), total aortic weight, maximal abdominal aortic diameter, grade of elastin degradation and collagen deposition, and inhibited TNF-α, IL-6, IL-1β, and IL-18 levels in mice model of arterial aneurysm (Figure 2). The above results indicated that knock out of DAPK1 reduced inflammation reactions in arterial aneurysm model.

Figure 2.

Knockout of DAPK1 showed decreased inflammation in mice model of arterial aneurysm. Incidence of AngII-induced AAA (A), HE staining (B), total aortic weight (C), maximal abdominal aortic diameter (D), grade of elastin degradation (E), collagen deposition (F), TNF-α, IL-6, IL-1β and IL-18 levels (G, H, I and J). AAA, mice model of arterial aneurysm; AAA+DAPK1^−/−, DAPK1^−/− mice model of arterial aneurysm. ##p < .01 compared with mice model of arterial aneurysm group. All experiments were repeated at least three times as the means ± SD.

Beclin1/NLRP3 signal pathway is a critical downstream effector of DAPK1 by ATP production

To investigate that the critical downstream of DAPK1 in arterial aneurysm, we analyzed the gene expression of crucial downstream in vitro model by over-expression of DAPK1 (Figure 3(a)–(b)). Online bioinformatics tools (TargetScan, RegRNA, CircNet) observed that Beclin1/NLRP3 signal pathway may be a critical downstream effector of DAPK1 (Figure 3(c)). DAPK1 was pulled down by Beclin1 and in a mutual pulldown experiment using an anti-Flag antibody, Beclin1 could also pulldown DAPK1 (Figure 3(d)–(e)). Next, over-expression of DAPK1 induced Beclin1 expressions in vitro model (Figure 4(a)). Over-expression of DAPK1 induced DAPK1, Beclin1, and NLRP3 protein expressions in vitro model (Figure 4(b)–(e)). Down-regulation of DAPK1 inactivated DAPK1, Beclin1, and NLRP3 protein expressions in vitro model (Figure 4(c),(f),(h)). Then, over-expression of DAPK1 elevated ATP production levels, and down-regulation of DAPK1 reduced ATP production levels in vitro model (Figure 4(i)-(j)). Meanwhile, over-expression of DAPK1 accelerated the release of IL-1β and IL-18 levels, and down-regulation of DAPK1 reduced the release of IL-1β and IL-18 levels in vitro model (Figure 4(k)–(n)). Taken together, these results suggested that Beclin1/NLRP3 signal pathway is a critical downstream effector of DAPK1 by ATP production.

Figure 3.

Beclin1/NLRP3 signal pathway is a critical downstream effector of DAPK1. Hierarchical clustering analysis of (old change >2 or <-2, p < .05, (A) and microarray data (B), interpretation of result (C), DAPK1 pulldown Beclin1 (D), Beclin1 pulldown DAPK1 (E). Negative, negative mimics group; DAPK1, over-expression of DAPK1 group. All experiments were repeated at least three times as the means ± SD.

Figure 4.

DAPK1 induced NLRP3 signal pathway via ATP production by Beclin1. Beclin1 expression (Immunofluorescent staining, A), DAPK1, Beclin1 and NLRP3 protein expression by over-expression of DAPK1 (B, C, D and E); DAPK1, Beclin1 and NLRP3 protein expression by down-regulation of DAPK1 (C, F, G and H); ATP production levels by over-expression of DAPK1 and down-regulation of DAPK1 (I and J); IL-1β and IL-18 levels by over-expression of DAPK1 and down-regulation of DAPK1 (K, L, M and N). Negative, negative mimics group; DAPK1, over-expression of DAPK1 group; siDAPK1, down-regulation of DAPK1 group. ##p < .01 compared with negative mimics group. All experiments were repeated at least three times as the means ± SD.

The regulation of Beclin1 in the effects of DAPK1 on inflammation of arterial aneurysm

We next examined that role of Beclin1 in the effects of DAPK1 on inflammation of arterial aneurysm. Only si-Beclin1 suppressed Beclin1 mRNA expression in vitro model, compared with si-negative group (Figure 5(a)). Si-Beclin1 suppressed the protein expressions of Beclin1 and NLRP3 in vitro model of arterial aneurysm by over-expression of DAPK1, contrasted with over-expression of DAPK1 group (Figure 5(b),(d),(e)). Beclin1 plasmid induced Beclin1 and NLRP3 protein expressions in vitro model of arterial aneurysm by down-regulation of DAPK1 compared with down-regulation of DAPK1 group (Figure C, 5E-5 F). Next, si-Beclin1 reduced ATP production levels, and inhibited IL-1β and IL-18 release levels in vitro model of arterial aneurysm by over-expression of DAPK1 (Figure 5(g)-(i)). Lastly, Beclin1 plasmid stimulated ATP production levels, and increased IL-1β and IL-18 release levels in vitro model of arterial aneurysm by down-regulation of DAPK1 (Figure 5(j)–(l)).

Figure 5.

The regulation of Beclin1 in the effects of DAPK1 on inflammation of arterial aneurysm Beclin1 mRNA expression in vitro mode by si-Beclin1 (A), Beclin1 and NLRP3 protein expression by over-expression of DAPK1 and down-regulation of Beclin1 (D and E); Beclin1 and NLRP3 protein expression by down-regulation of DAPK1 and over-expression of Beclin1 (C, E, and F); ATP production levels, IL-1β and IL-18 levels by over-expression of DAPK1 and down-regulation of Beclin1 (G, H, and I); ATP production levels, IL-1β and IL-18 levels by down-regulation of DAPK1 and over-expression of Beclin1 (J, K, and L). Negative, negative mimics group; DAPK1, over-expression of DAPK1 group; siDAPK1, down-regulation of DAPK1 group; DAPK1+si-Beclin1, over-expression of DAPK1 group+ down-regulation of Beclin1 group; siDAPK1+ Beclin1, down-regulation of DAPK1 and over-expression of Beclin1 group. ##p < .01 compared with negative mimics group, **p < .01 compared with over-expression of DAPK1 group or down-regulation of DAPK1 group. All experiments were repeated at least three times as the means ± SD.

The regulation of NLRP3 in the effects of DAPK1 on inflammation of arterial aneurysm

This study explored that the role of NLRP3 in the effects of DAPK1 on inflammation of arterial aneurysm. Only si-NLRP3 suppressed Beclin1 mRNA expression in vitro model, compared with si-negative group (Figure 6(a)). Si-NLRP3 suppressed NLRP3 protein expression in vitro model of arterial aneurysm by over-expression of DAPK1, and NLRP3 plasmid induced NLRP3 protein expression in vitro model of arterial aneurysm by down-regulation of DAPK1 (Figure 6(b)-(f)). Si-NLRP3 lowered IL-1β and IL-18 release levels in vitro model of arterial aneurysm by over-expression of DAPK1, and NLRP3 plasmid increased IL-1β and IL-18 release levels in vitro model of arterial aneurysm down-regulation of DAPK1 (Figure 6(e)-(h)).

Figure 6.

The regulation of NLRP3 in the effects of DAPK1 on inflammation of arterial aneurysm. NLRP3 mRNA expression (A), NLRP3 protein expression by over-expression of DAPK1 and down-regulation of NLRP3 (B and D); NLRP3 protein expression by down-regulation of DAPK1 (C and D); IL-1β and IL-18 levels by over-expression of DAPK1 and down-regulation of NLRP3 (E and F); IL-1β and IL-18 levels by down-regulation of DAPK1 and over-expression of Beclin1 (G and H). Negative, negative mimics group; DAPK1, over-expression of DAPK1 group; siDAPK1, down-regulation of DAPK1 group; DAPK1+si-NLRP3, over-expression of DAPK1 group+ down-regulation of NLRP3 group; siDAPK1+ DAPK1, down-regulation of DAPK1 and over-expression of NLRP3 group. ##p < .01 compared with negative mimics group, **p < .01 compared with over-expression of DAPK1 group or down-regulation of DAPK1 group. All experiments were repeated at least three times as the means ± SD.

Discussion

Arterial aneurysm can compress the surrounding brain tissue with its increasing volume.¹⁹ The compression of the oculomotor nerve can cause strabismus and eye movement disorders.¹⁹ Compression of the trigeminal nerve can cause forehead pain, and severe compression of the language center can cause aphasia.²⁰ When the volume is too large, the intracranial pressure would increase to cause headaches, vomiting, etc among patients.²¹ In recent years, the occurrence and development of arterial aneurysm have been reported to be possibly associated with immune inflammatory response.²¹ In addition, inflammatory cells may participate in the occurrence and development of arterial aneurysm by secreting inflammatory cytokines.²¹ In the present study, DAPK1 mRNA expression was increased in patients with arterial aneurysm or mice model of arterial aneurysm. Knockout of DAPK1 showed decreased inflammation in mice model of arterial aneurysm. Li et al.²² showed that Dapk1 improves inflammation in LPS-induced acute lung injury via p38MAPK/NF-κB signaling pathway. These results showed that DAPK1 may induced inflammation in arterial aneurysm. This study only analyzed the expression of DAPK1 in patients with arterial aneurysm; however, this study not analyze the effects of DAPK1 on inflammation of patients with arterial aneurysm. It is an insufficient of this study, we will explain the effects of DAPK1 on inflammation of patients with arterial aneurysm.

A large number of studies have shown that NLRP3 inflammasome plays an important role in the pathophysiological mechanism of cerebral ischemia.^2,23 Therefore, blocking or inhibiting the activation of NLRP3 may become a novel therapeutic target for ischemic cerebrovascular disease.²³ This data, Beclin1/NLRP3 signal pathway is a critical downstream effector of DAPK1 by ATP production. Oikonomou et al.²⁴ showed that noncanonical fungal autophagy inhibits inflammation in response to IFN-γ via DAPK1/NLRP3 activation.

In severe stress response, autophagy dysfunction can activate inflammasomes, which are receptors for damage-associated molecular patterns (DAMPs) in the body.²⁵ Inflammasome activation can promote the secretion of IL-1β and IL-18 in tissue, change the function of the immune system, and cause acute tissue damage.^16,17 Numerous experiments have shown that the anti-apoptotic protein Bcl-2 family members can increase apoptotic tolerance and inhibit autophagy, which may be mediated by the formation of Beclin1 inhibitory complexes.^16,17 Therefore, autophagy, apoptosis, and inflammatory response are closely associated with the pathophysiological process of the occurrence and development of certain diseases.²⁶ Notably, Beclin1 is located in the center of the complex cellular response network and is the hub of the three factors. The results of this study show the regulation of Beclin1 participated in the effects of DAPK1 on inflammation of arterial aneurysm by ATP-dependent NLRP3 inflammasome. Thongchot et al.²⁷ reported that Dihydroartemisinin promoted apoptosis in cholangiocarcinoma through a DAPK1-BECLIN1 pathway. Furthermore, we have shown that DAPK1 promoted inflammation of arterial aneurysm via by ATP-dependent NLRP3 inflammasome by Beclin1. In further experiment, we will analyze the effects of DAPK1 affect the function and differentiation of leukocytes or lymphocytes and macrophages at the site of aneurism.

In conclusion, the present study has demonstrated that DAPK1 mRNA expression was increased in arterial aneurysm. DAPK1 promoted inflammation of arterial aneurysm via by ATP-dependent NLRP3 inflammasome by Beclin1. Based on these results, DAPK1 is promising therapeutic targets for arterial aneurysm associated with inflammatory disorders.

Footnotes

Author contributions

Senyan Wu, Wei Lu: guarantor of integrity of the entire study, study concepts and design, definition of intellectual content, clinical studies, manuscript review; Guobing Cheng, Jiawen Wu: study design, literature research, experimental studies, data acquisition, and analysis, statistical analysis, manuscript preparation; Sheng Liao: experimental studies; Qiang Hu: experimental studies; Xiaoyang Li, Buping Jiang: data acquisition and analysis; Guobing Cheng, Jiawen Wu: manuscript preparation, and editing.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical approval

This study was approved by the Ethics committee of the People’s Hospital of Quzhou(No.2018061801)

Informed Consent

All Patients have given their written informed consent.

ORCID iD

Wei Lu

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