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Introduction

At present, the prevalence of diabetes mellitus (DM) is rapidly increasing worldwide, and the number of people living with DM has quadrupled in the past three decades (1,2). DM is a metabolic disorder that primarily manifests as abnormal glucose metabolism with complications that seriously impair the quality of life of patients, including retinopathy, nephropathy and neuropathy (3). Previous studies have demonstrated that diabetes can cause male reproductive dysfunction (4–6). Hyperglycaemic male rats display symptoms of impaired fertility and decreased sperm motility (7,8). A high-glucose (HG) environment associated with diabetes led to impaired testicular Sertoli cell function, which subsequently affected spermatogenesis, resulting in testicular spermatogenesis dysfunction (9). However, the specific mechanism underlying HG-induced impairment of testicular Sertoli cell function is not completely understood.

It has been widely reported that the reproductive function of vertebrates is primarily regulated by the hypothalamus- pituitary-gonadal axis (HPG) (10,11). The hypothalamus regulates reproductive function by synthesizing and secreting gonadotropin-releasing hormone (GnRH), which stimulates the pituitary to secrete two gonadotropins, luteinizing hormone and follicle stimulating hormone (12). Kisspeptins, which are encoded by the KiSS-1 metastasis suppressor (KISS1) gene, serve a role upstream of GnRH and are effective stimulators of the HPG axis in several species (13,14). Increasing evidence indicates that KISS1/KISS1 receptor (KISS1R) serves critical roles in the female reproductive process (15,16), but few studies in the male reproductive system have been conducted. A previous study reported that the level of kisspeptin in serum from infertile men was significantly lower compared with the serum from fertile control individuals (17). Another study implied that KISS1/KISS1R can affect sperm motility, whereas KISS1 receptor antagonists can reduce sperm motility (18). The aforementioned studies suggested that KISS1/KISS1R was closely related to male reproductive function. However, KISS1/KISS1R has not been reported to regulate testicular Sertoli cell viability and apoptosis; therefore, investigating the related mechanisms is of interest.

AKT is a serine/threonine kinase that is recognized as the primary mediator of the downstream effects of PI3K (19). The PI3K/AKT signalling pathway coordinates multiple signals, controlling how cells respond to external stimuli to regulate cell proliferation and survival (20). AKT is activated via phosphorylation within the carboxy terminus at Ser473 (21). KISS1 has been reported to regulate PI3K/AKT (22,23). In porcine ovarian granulosa cells, KISS1 can regulate the cell cycle and inhibit apoptosis by affecting the PI3K signalling pathway (24). The majority of the current research on the PI3K signalling pathway has focused on tumour research as the PI3K signalling pathway serves a critical role in the development of tumours and has been identified as a novel therapeutic target for tumours; however, the function of the PI3K signalling pathway in reproduction is not completely understood.

The aim of the present study was to examine the role of KISS1/KISS1R in Sertoli cells. The mechanism underlying KISS1/KISS1R-regulated Sertoli cell apoptosis under HG conditions was also evaluated.

Materials and methods

Cell culture

The present study was approved by The Animal Ethics Committee of The Second Affiliated Hospital of Nanchang University [approval no. (2018) 031]. A total of five male adult C57BL/6 mice (age, 8 weeks; weight, 25±3 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. Mice were maintained with 12-h light/dark cycles, housed at 60% humidity at 22°C and free water and food. Mice were euthanized by inhalation of 5% isoflurane (Merck Sharp & Dohme-Hoddesdon). Death was verified by monitoring cardiac arrest, respiratory arrest and loss of reflexes. Mouse Sertoli cells were obtained according to the following protocol. Briefly, dealbuginized testes were digested sequentially with trypsin and collagenase. Tissue explants were placed in tissue culture dishes with serum-free minimum essential medium (Sigma-Aldrich; Merck KGaA) supplemented with glutamine. Cells were allowed to adhere and form confluent monolayers for 3 days at 37°C with 5% CO2. Subsequently, the remaining germ cells were removed with hypotonic solution and then 1 mM dibutyryl adenosine 3′:5′ cyclic monophosphate (Sigma-Aldrich; Merck KGaA) was added to the medium. Cells were cultured with RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO2 for 24 h, cells were treated with 5, 25 or 50 mM D-glucose (Sigma-Aldrich; Merck KGaA) for 48 h at 37°C. A dose of 5 µM PI3K/AKT pathway inhibitor (LY294002; cat. no. PZH1144; Invitrogen; Thermo Fisher Scientific, Inc.) was used to treat cells at 37°C for 24 h.

Plasmid constructs and transfection

Plasmids with DNA encoding KISS1 and KISS1R were constructed by inserting the cDNA clone of KISS1 and KISS1R into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.). The pcDNA3.1 empty vector was as a control. Small interfering (si)RNA targeting KISS1 (si-KISS1, 5′-GCAGGAGAGUGAAGAUUAAAU-3′), siRNA targeting KISS1R (si-KISS1R, 5′-CACUUGGUUGAUUAAUCAACU-3′) and the negative control (NC) siRNA (si-NC; non-targeting control; 5′-GUCGAGCUGACCUAUCCGACG-3′) were synthesized by Sangon Biotech Co., Ltd. Cells (1×105 cells/well) were transfected with 1 µg plasmid vector or with 50 nM siRNA using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 6 h. Cells were harvested for further experiments after 24 h.

Reverse transcription-quantitative PCR (qPCR)

Total RNA was extracted from cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using the ReverTra Ace™ qPCR RT Kit (Toyobo Life Science) according to the manufacturer's protocol. Subsequently, qPCR was performed on a qPCR instrument (Eppendorf) using SYBR® Green Real-Time PCR Master Mix (Toyobo Life Science). The program was listed as following: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec, 60°C for 10 sec, and 72°C for 30 sec and a final extension at 72°C for 5 min. The following primers were used for qPCR: KISS1 forward, 5′-TTTCCTCTGTGCCACCCAC-3′ and reverse, 5′-AGGGATTCTAGCTGCTGGCC-3′; KISS1R forward, 5′-CCCACCCTCTGGACATTCAC-3′ and reverse, 5′-CCTAGAAGTGCCTTGAGGCTTG-3′; GAPDH forward, 5′-GAGTCAACGGATTTGGTCGTT-3′ and reverse, 5′-TTGATTTTGGAGGGATCTCG-3′. mRNA expression levels were quantified using the 2−ΔΔCq method (25) and normalized to the internal reference gene GAPDH.

Cell viability assay

Cells were seeded into 96-well plates at a density of 1×104 cells/well. Cell viability was assessed by adding 10 µl Cell Counting Kit-8 (CCK-8) solution (Beijing Solarbio Science & Technology Co., Ltd.) to each well and incubating for 2 h. Absorbance was measured at a wavelength of 450 nm using a microplate spectrophotometer.

Detection of apoptosis by flow cytometry

Cells were seeded into 6-well plates at a density of 1×106 cells/well. Cell apoptosis was assessed using the Annexin V-FITC/PI Apoptosis Detection Kit (Shanghai Yeasen Biotechnology Co., Ltd.) according to the manufacturer's protocol. Cells were digested and harvested. Cells were washed twice with prechilled PBS and then resuspended in 100 µl 1X binding buffer. Subsequently, cells were incubated with 5 µl Annexin V-FITC and 10 µl PI staining solution in the dark at room temperature for 10 min. The data were acquired using a BD FACSCanto™ II (BD Biosciences) flow cytometer. Apoptotic cells (early and late apoptosis) were analysed using FlowJo software (version 7; FlowJo LLC).

TUNEL staining

Cells were fixed with DiffQuik Fixative (Baxter International, Inc.) at 37°C for 30 sec. To conduct TUNEL staining, cells were treated according to the manufacturer's protocol by using DeadEnd™ Fluorometric TUNEL System (cat. no. G3250; Promega Corporation). Cells were permeabilized using 0.1% Triton X-100 solution (Sigma-Aldrich; Merck KGaA) for 30 min at 37°C. A volume of 50 µl TdT reaction mix was then added to the cells at 37°C for 1 h in the dark. Subsequently, 2X saline sodium citrate (300 mM NaCl; 30 mM sodium citrate; pH 7.0) for 15 min to stop reaction. Following washing with PBS, cells were stained with 1 µg/ml DAPI staining solution (cat. no. E607303; Sangon Biotech Co., Ltd.) for 10 min at 37°C. Cells were washed with PBS, then treated with anti-Fade Mounting Medium (cat. no. E675011; Sangon Biotech Co., Ltd.) and observed in five randomly selected fields of view using a fluorescence microscope (Olympus Corporation; magnification ×40).

Western blotting

Total protein was extracted from cells using RIPA buffer (Sigma-Aldrich; Merck KGaA) supplemented with 1% protease inhibitor and phosphorylase inhibitor. Protein concentrations were determined using the BCA Kit (Beyotime Institute of Biotechnology). Cell lysates were mixed with 5X SDS sample buffer and boiled for 10 min. The protein samples (50 µg/lane) were separated by SDS-PAGE on 12% gels and transferred to PVDF membranes. Following blocking with BlockPro blocking solution (Energenesis Biomedical Co., Ltd.) for 1 h at 37°C, the membranes were incubated overnight at 4°C with primary antibodies targeted against: Kisspeptin (cat. no. ab19028; 1:1,000; Abcam), KISS1R (cat. no. 13776; 1:1,000; Cell Signalling Technology, Inc.), phosphorylated (p)-PI3K (cat. no. 17366; 1:1,000; Cell Signalling Technology, Inc.), PI3K (cat. no. 4249; 1:1,000; Cell Signalling Technology, Inc.), p-AKT (cat. no. 4060; 1:1,000; Cell Signalling Technology, Inc.), AKT (cat. no. ab8805; 1:1,000; Abcam), Bad (cat. no. ab32445; 1:1,000; Abcam), Bcl-2 (cat. no. ab182858; 1:1,000; Abcam), Bax (cat. no. ab32503; 1:1,000; Abcam), total caspase-3 (cat. no. ab13847; 1:1,000; Abcam), cleaved caspase-3 (cat. no. ab2302; 1:1,000; Abcam) and GAPDH (cat. no. ABS16; 1:2,000; Sigma-Aldrich; Merck KGaA). After washing with 0.1% Tween-20 in PBS-T, the membranes were incubated with the corresponding goat anti-Rabbit IgG H&L HRP antibody (cat. no. ab205718; 1:2,000; Abcam) at room temperature for 1 h. Protein bands were visualized using ECL Reagents (Beyotime Institute of Biotechnology) and a GEL imaging system (Bio-Rad Laboratories, Inc.). Protein expression was quantified using ImageJ software (V1.8.0; National Institutes of Health) with GAPDH as the loading control.

Statistical analysis

Each experiment was repeated three times. Data are presented as the mean ± SD. Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, Inc.). Comparisons among multiple groups were analysed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

KISS1/KISS1R expression is reduced and cell apoptosis is increased in HG-induced mouse Sertoli cells

Mouse Sertoli cells were treated with 5, 25 or 50 mM D-glucose. KISS1 and KISS1R mRNA expression levels were decreased by glucose treatment in a concentration-dependent manner (Fig. 1A and B). Similar trends were observed for kisspeptin and KISS1R protein expression levels (Fig. 1C). Cell viability was decreased by glucose treatment in a concentration-dependent manner, as determined by performing the CCK-8 assay (Fig. 1D). The flow cytometry results demonstrated that the number of apoptotic cells was increased by glucose treatment in a concentration-dependent manner (Fig. 1E). The results suggested that KISS1 and KISS1R expression levels were decreased and cell apoptosis was increased by glucose treatment in a concentration-dependent manner in mouse Sertoli cells.

Figure 1. KISS1/KISS1R expression is reduced in high glucose-induced mouse Sertoli cells. Mouse Sertoli cells were treated with different concentrations of glucose. (A) KISS1 and (B) KISS1R mRNA expression levels in mouse Sertoli cells were detected via reverse transcription-quantitative PCR. (C) Kisspeptin and KISS1R protein expression levels in mouse Sertoli cells were assessed via western blotting. (D) Mouse Sertoli cell viability was assessed by performing Cell Counting Kit-8 assays. (E) Mouse Sertoli cell apoptosis was evaluated via flow cytometry. *P<0.05 and **P<0.01. KISS1, KiSS-1 metastasis suppressor; KISS1R, KISS1 receptor; D-glu, D-glucose.

KISS1/KISS1R overexpression reduces mouse Sertoli cell apoptosis under HG conditions

Mouse Sertoli cells were transfected with KISS1 and KISS1R overexpression plasmids, and then treated with 50 mM D-glucose. KISS1 and KISS1R mRNA expression levels were significantly decreased in the HG group compared with the control group. Moreover, KISS1 and KISS1R mRNA expression levels were significantly increased by KISS1 or KISS1R overexpression, respectively, compared with the HG + vector group. Compared with the HG + vector group, KISS1 and KISS1R mRNA expression levels were significantly upregulated by KISS1 and KISS1R overexpression (Fig. 2A and B). According to the CCK-8 assay results, compared with the HG + vector group, cell viability was notably increased by KISS1 or KISS1R overexpression alone, whereas cell viability was significantly increased by KISS1 and KISS1R overexpression (Fig. 2C). Cell apoptosis was increased in the HG group compared with the control group. Following KISS1 and KISS1R overexpression, apoptosis significantly decreased compared with the HG + vector group. Consistent trends were observed for cell apoptosis via flow cytometry (Fig. 2D) and TUNEL staining (Fig. 2E). The protein expression levels of Bad, Bcl-2, Bax and caspase-3 were assessed via western blotting. Compared with the control group, Bcl-2 protein expression was significantly downregulated, and Bad, Bax and caspase-3 protein expression levels were significantly upregulated in the HG group. Compared with the HG group, KISS1 or KISS1R overexpression alone slightly upregulated Bcl-2 expression, and slightly downregulated Bad, Bax and caspase-3 protein expression levels. Following KISS1 and KISS1R overexpression, Bcl-2 protein expression was significantly upregulated, and Bad, Bax and caspase-3 protein expression levels were significantly decreased compared with the HG + vector group (Fig. 2F). The results indicated that KISS1/KISS1R overexpression inhibited HG-induced Sertoli cell apoptosis.

Figure 2. KISS1/KISS1R overexpression reduced mouse Sertoli cell apoptosis under HG conditions. Mouse Sertoli cells were transfected with KISS1 and KISS1R overexpression plasmids, and then cultured in 50 mM D-glucose. (A) KISS1 and (B) KISS1R mRNA expression levels in mouse Sertoli cells were detected via reverse transcription-quantitative PCR. (C) Mouse Sertoli cell viability was assessed by performing Cell Counting Kit-8 assays. Mouse Sertoli cell apoptosis was evaluated via (D) flow cytometry and (E) TUNEL staining. Scale bar, 100 µm. (F) Bad, Bcl-2, Bax and caspase-3 protein expression levels in mouse Sertoli cells were assessed via western blotting. *P<0.05, **P<0.01 and ***P<0.001. KISS1, KiSS-1 metastasis suppressor; KISS1R, KISS1 receptor; HG, high glucose.

KISS1/KISS1R regulates the expression of PI3K/AKT signalling pathway-related proteins in mouse Sertoli cells

Mouse Sertoli cells were transfected with KISS1 and KISS1R overexpression plasmids or si-KISS1 and si-KISS1R. Compared with the vector group, KISS1 and KISS1R mRNA expression levels were significantly upregulated by KISS1 or KISS1R overexpression, respectively. KISS1 and KISS1R simultaneous overexpression significantly increased the KISS1 and KISS1R mRNA expression levels compared with the vector group (Fig. 3A and B). However, there were no significant differences observed among the other groups. The protein expression levels of p-PI3K, PI3K, p-AKT and AKT were assessed via western blotting. Compared with the vector group, p-PI3K/PI3K and p-AKT/AKT protein expression levels were slightly increased by KISS1 or KISS1R overexpression alone (Fig. 3C). KISS1 and KISS1R overexpression significantly increased p-PI3K/PI3K and p-AKT/AKT protein expression levels compared with the vector group. Compared with the si-NC group, KISS1 and KISS1R mRNA expression levels were significantly downregulated by KISS1 or KISS1R knockdown, respectively (Fig. 3D and E). Compared with the si-NC group, the protein expression levels of p-PI3K/PI3K and p-AKT/AKT were slightly downregulated by KISS1 or KISS1R knockdown alone. KISS1 and KISS1R knockdown significantly decreased p-PI3K/PI3K and p-AKT/AKT protein expression levels compared with the si-NC group (Fig. 3F). The aforementioned results demonstrated that KISS1 and KISS1R expression affected the expression levels of PI3K/AKT pathway-related proteins.

Figure 3. KISS1/KISS1R regulates the expression of PI3K/AKT signalling pathway-related proteins in mouse Sertoli cells. Mouse Sertoli cells were transfected with KISS1 and KISS1R overexpression plasmids or si-KISS1 and si-KISS1R. (A) KISS1 and (B) KISS1R mRNA expression levels in mouse Sertoli cells following transfection with KISS1 or KISS1R overexpression plasmids were detected via RT-qPCR. (C) PI3K, AKT, p-PI3K and p-AKT protein expression levels in mouse Sertoli cells were assessed via western blotting. (D) KISS1 and (E) KISS1R mRNA expression levels in mouse Sertoli cells following transfection with si-KISS1 or si-KISS1R were detected via RT-qPCR. (F) PI3K, AKT, p-PI3K and p-AKT protein expression levels in mouse Sertoli cells were assessed via western blotting. *P<0.05, **P<0.01 and ***P<0.001. KISS1, KiSS-1 metastasis suppressor; KISS1R, KISS1 receptor; si, small interfering RNA; p, phosphorylated; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control.

PI3K/AKT inhibitor reverses KISS1/KISS1R-mediated protective effects

Mouse Sertoli cells were transfected with KISS1 and KISS1R overexpression plasmids, and then treated with PI3K/AKT pathway inhibitor. Compared with the HG + vector group, KISS1 and KISS1R mRNA expression levels were significantly increased by KISS1 or KISS1R overexpression, respectively (Fig. 4A and B). Compared with the HG + vector group, the protein expression levels of p-PI3K/PI3K and p-AKT/AKT were significantly upregulated by KISS1 and KISS1R overexpression, whereas PI3K/AKT inhibitor LY294002 treatment significantly reversed this effect (Fig. 4C). Compared with the HG + vector group, Bcl-2 expression was significantly upregulated, and Bad, Bax and caspase-3 expression levels were significantly decreased by KISS1 and KISS1R overexpression, which indicated a decrease in apoptosis (Fig. 4D). However, KISS1 and KISS1R overexpression-mediated effects on apoptosis-related protein expression levels were significantly reversed by PI3K/AKT inhibitor treatment. The flow cytometry results demonstrated that compared with the HG + vector group, the number of apoptotic cells was significantly downregulated by KISS1 and KISS1R overexpression, whereas PI3K/AKT inhibitor treatment significantly reversed this effect (Fig. 4E). Similar trends were observed for the analysis of cell apoptosis via TUNEL staining (Fig. 4F). Collectively, the results demonstrated that KISS1/KISS1R regulated HG-induced cell apoptosis by altering the PI3K/AKT signalling pathway.

Figure 4. PI3K/AKT inhibitor reverses KISS1/KISS1R-mediated protective effects. Mouse Sertoli cells were transfected with KISS1 and KISS1R overexpression plasmids, and then treated with the PI3K/AKT signalling pathway inhibitor LY294002. (A) KISS1 and (B) KISS1R mRNA expression levels in mouse Sertoli cells were detected via reverse transcription-quantitative PCR. (C) PI3K, AKT, p-PI3K, p-AKT (D) Bad, Bcl-2, Bax and caspase-3 protein expression levels in mouse Sertoli cells were assessed via western blotting. Mouse Sertoli cell apoptosis was evaluated via (E) flow cytometry and (F) TUNEL staining. Scale bar, 100 µm. *P<0.05, **P<0.01 and ***P<0.001. KISS1, KiSS-1 metastasis suppressor; KISS1R, KISS1 receptor; p, phosphorylated; HG, high glucose.

Discussion

It was estimated that 451 million people lived with DM in 2017, with a prevalence rate of 8.8% (26). Diabetes is associated with diverse clinical complications, including reproductive dysfunction (27–29). Given the multifactorial nature of DM, the mechanisms underlying DM-induced reproductive dysfunction are not completely understood. As hyperglycaemia has a major effect on disease pathophysiology, in vitro approaches have been used to explore the effect of HG on human sperm function (30). The HG environment may cause impaired testicular support cell function, affect spermatogenesis and cause testicular spermatogenesis dysfunction (30). In addition, Liu et al (31) reported that the motility and viability of spermatozoa were markedly reduced after incubation with glucose. In the present study, the results demonstrated that compared with the control group, HG conditions significantly decreased cell viability and significantly increased cell apoptosis in mouse Sertoli cells, which was consistent with a previous study (32). The present study lacked verification of Sertoli cell purity, but this did not have an impact on the conclusions of the present study.

KISS1 and KISS1R are involved in regulating mammalian sexual maturity and development of the reproductive system, and serve an important role in female reproduction (33,34). However, few studies on the role of KISS1/KISS1R in the male reproductive system have been conducted. Previous studies have reported reduced sperm motility and kisspeptin expression in male patients with diabetes (4,35). The results of the present study demonstrated that KISS1/KISS1R expression levels were significantly downregulated in HG-induced mouse Sertoli cells compared with the control group, suggesting that KISS1/KISS1R might serve a significant role in regulating reproductive dysfunction in male patients with diabetes. Therefore, the present study aimed to examine the specific mechanism underlying KISS1/KISS1R-mediated regulation of the reproductive function in male patients with diabetes.

Apoptosis serves a critical role in male reproductive dysfunction (36). Increased testicular cell apoptosis was observed in male mice with reproductive disorders (37). Moreover, increased endoplasmic reticulum stress and apoptosis were observed in murine Leydig tumour cell line 1 cells treated with palmitic acid (38). Yang et al (39) reported that decreased testosterone secretion accompanied by increased apoptosis was observed in mouse Leydig cells after natriuretic peptide receptor 2 inhibition. In the present study, compared with the control group, cell apoptosis was significantly increased in mouse Sertoli cells under HG conditions, which was consistent with a previous study. KISS1 has been reported to regulate PI3K/AKT (22), which was consistent with the results of the present study that demonstrated that KISS1/KISS1R overexpression activated the PI3K/AKT signalling pathway, which regulates apoptosis (40). The typical AKT phosphorylation targets include Bad and caspase-3, which are closely related to apoptosis (41). The present study demonstrated that KISS1/KISS1R mediated Sertoli cell apoptosis via the PI3K/AKT signalling pathway under HG conditions. Although the present study investigated the mechanism underlying KISS1/KISS1R in vitro HG-induced cell models, future studies should investigate the effects of KISS1/KISS1R on Sertoli cells using in vivo diabetic mouse models.

The prevalence of diabetes is high in modern society, and male patients with diabetes are usually affected by reproductive dysfunction (42); therefore, identifying the specific mechanism underlying the regulation of reproductive dysfunction in male patients with diabetes is important. The present study investigated the specific mechanism underlying the regulation of reproductive dysfunction in male patients with diabetes, providing potential targets for the treatment of reproductive dysfunction in male patients with diabetes.

Acknowledgements

Not applicable.

Funding

The present study was supported by The Medical and Health Research Project of Zhejiang Province (grant no. 2019KY620).

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

YL and DMG analysed the data and confirm their authenticity. DMG conceived the study and assisted in drafting the manuscript. JF designed the study. PPZ performed the literature search. JPZ performed the experiments and acquired and analysed the data. SXD assisted in the experimental plan formulation and data analysis, and drafted, edited and reviewed the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by The Animal Ethics Committee of The Second Affiliated Hospital of Nanchang University [approval no. (2018) 031].

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References