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Introduction

The liver performs and regulates numerous physiological functions, and has the ability to regenerate (1,2). It is an important line of defense against microorganisms and is also a target of the dysregulated inflammation induced by infectious diseases of the liver such as steatohepatitis (3). Based on the International Classification of Diseases, Ninth Revision, Clinical Modification, severe sepsis and septic shock are main causes for hospital patients to be admitted to intensive care unit (4). Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection (5). Among the organ failures involved in sepsis, hepatic dysfunction has a high level of prognostic relevance for the course of sepsis and is a powerful independent predictor of mortality (6).

The Periplaneta americana (PA) extract is found in the American cockroach and comprises polyols, peptides, epidermal growth factors, sticky sugar acid, amino acids and further active components (7). The PA extract notably alleviates swelling of liver tissue and stomach pain in gastric ulcer (8,9). Furthermore, it is widely used in China for its beneficial effects on blood circulation, hematocele clearance and digestion (10). Xinmailong (XML), which is typically made from PA, reverses the cardiomyocyte damage caused by lipopolysaccharide (LPS) by mediating mitophagy and downregulating the expression of inflammatory factors (11). PA extract protected the intestinal mucosal barrier in patients with sepsis, thereby improving the physical condition of the patients and ameliorating prognosis (7). PA extract reduced the level of endotoxin, attenuated gastrointestinal complications, and improved immune function and nutritional status in patients with systemic inflammatory response syndrome (7,12). The results of a previous study demonstrated that XML alleviated intestinal inflammation and improved the intestinal mucosal barrier function (10). By decreasing the expression levels of fibrosis-related genes, the liver function of mice was enhanced, and the pathological fibrosis associated with liver were mitigated or even reversed, resulting in inhibition of liver fibrosis progression (13). Additionally, PA extract accelerated liver regeneration via a complex network of pathways, including AMP-activated protein kinase (AMPK) signaling (14). The activation of AMPK maintained mitochondrial function and regulated autophagy, thus preventing sepsis-induced cell and organ injury (15). A previous study has proposed that inhibition of the AMPK/proliferator-activated receptor γ coactivator-1α (PGC-1α) signaling pathway in rat liver induced diseases associated with mitochondrial dynamics (16).

The present study aimed to investigate the role of XML in LPS-induced liver injury and determined whether the potential underlying mechanism is associated with regulation of the AMPK/PGC-1α signaling pathway.

Materials and methods

Cell culture and treatments

The AML12 cell line (cat. no. 3101MOUSCSP550) was purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and maintained in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Sigma-Aldrich; Merck KGaA), 100 U/ml penicillin and 100 µg/ml streptomycin (Beyotime Institute of Biotechnology). Once cells reached 80% confluence, they were treated with 100 ng/ml LPS for 12 h at 37˚C. Certain cells were also preincubated with 0.25, 0.5, 1, 2 and 4 mg/ml of the PA extract, XML (Yunnan Tengyao Pharmaceutical, Co., Ltd.) for 48 h. Cells were treated with 2 mg/ml AMPK inhibitor, compound C (Sigma-Aldrich; Merck KGaA) for 1 h at 37˚C before model establishment.

Detection of cytochrome c

Cytochrome c content in the mitochondria and cytoplasm was detected using a Quantikine Mouse Cytochrome c Immunoassay kit obtained from R&D Systems, Inc. (cat. no. MCTC0), according to the manufacturer's protocol. The AML12 cells were lysed with lysis buffer (Beyotime Institute of Biotechnology) containing protease inhibitors (Sigma-Aldrich; Merck KGaA). The lysate was centrifuged for 10 min at 750 x g, and the supernatants were collected and subsequently centrifuged at 15,000 x g for 15 min at 4˚C. The supernatants were used as the cytosolic fractions. The pellet containing the mitochondria was combined with PBS plus 0.5% Triton X-100 (Sigma-Aldrich; Merck KGaA) for 10 min at 4˚C. The pellet was centrifuged at 15,000 x g, 4˚C for 10 min, and the supernatant was used as the mitochondrial fraction.

Luciferin-luciferase reaction

Mitochondria were collected from cells by centrifugation at 4˚C, 12,000 x g for 8 min. Then, 100 µl sample was added in a 96-well plate before 100 µl ATP reagent was added for 2 min. ATP concentration was measured using an enhanced ATP assay kit (cat. no. S0027, Beyotime Institute of Biotechnology), according to the manufacturer's protocol.

MTT assay

Cell viability was assessed using an MTT assay. Cells were seeded into 96-well plates at a density of 2x104 cells/well following culture with or without XML at concentrations of 0.25, 0.5, 1, 2 and 4 mg/ml respectively for 48 h at 37˚C. The DMEM containing serum was replaced with MTT (Sigma-Aldrich; Merck KGaA) following the corresponding treatment. Subsequently, 0.5 mg/ml MTT reagent was added to each well and incubated for 4 h at 37˚C. The cell supernatant was discarded and replaced with DMSO (150 µl/well). The absorbance was measured at a wavelength of 570 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

ELISA

AML12 cells were seeded into 96-well plates at a density of 1x105 cells/well and co-treated with 100 ng/ml LPS, 4 mg/ml XML and 5 mM compound C for 12 h. The supernatants of the cultured cells were collected in a centrifuge at 1,000 x g, 4˚C for 15 min to measure the secretion levels of IL-1β (cat. no. F10770), IL-6 (cat. no. F10830) and TNF-α (cat. no. F11630) using commercial ELISA kits, according to the manufacturer's protocol (Shanghai Xitang Biological Technology Co., Ltd.).

Measurement of oxidative stress-related markers

The cell malondialdehyde (MDA; cat. no. A003-4-1) assay kit and total superoxide dismutase (SOD; cat. no. A001-1-2) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute. The measurement of MDA and SOD was performed according to the manufacturer's protocol.

TUNEL assay

Apoptosis of AML12 cells was detected using a TUNEL assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Briefly, 2x105 cells were washed twice with buffer in 24-well plates and then fixed in 0.5 ml 4% paraformaldehyde for 30 min at 37˚C. Subsequently, cells were incubated with 50 µl TUNEL reaction buffer for 1 h at 37˚C. The nuclei were counterstained with 1 µg/ml DAPI in the dark at room temperature for 15 min. Images were visualized and captured using a fluorescence microscope (magnification, x200; Olympus Corporation) and cells were counted in five randomly-selected microscopic fields.

Reverse transcription-quantitative (RT-q)PCR

Total RNA was isolated from AML12 cells using TRIzol® reagent (Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using a SuperScript™ IV First-Strand Synthesis system (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturers' protocol. qPCR was subsequently performed using the SYBR Green PCR system and SYBR Green reagents (Bio-Rad Laboratories, Inc.) in an ABI 7500 thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for qPCR: Initial denaturation at 95˚C for 3 min, followed by 40 cycles of denaturation at 95˚C for 10 sec, annealing at 60˚C for 5 sec and extension at 72˚C for 10 sec. The sequences of the primers were as follows: NRF1 forward, 5'-CGCAGCACCTTTGGAGAA-3' and reverse, 5'-CCCGACCTGTGGAATACTTG-3'; TFAM forward, 5'-GGAATGTGGAGCGTGCTAAA-3' and reverse, 5'-TGCTGGAAAAACACTTCGGAATA-3'; TNF-α forward, 5'-CGTGCTCCTCACCCACAC-3' and reverse, 5'-GGGTTCATACCAGGGTTTGA-3'; IL-6 forward, 5'-ACAACCACGGCCTTCCCTACTT-3' and reverse, 5'-GTGTAATTAAGCCTCCGACT-3'; IL-1β forward, 5'-TCCAGGATGAGGACATGAGCAC-3' and reverse, 5'-GAACGTCACACACCAGCAGGTA-3' and GAPDH forward, 5'-TCACCACCATGGAGAAGGC-3' and reverse, 5'-GCTAAGCAGTTGGTGGTGCA-3'. GAPDH was used for normalization of mRNA levels. Relative mRNA expression levels were calculated using the 2-ΔΔCq method (17).

Western blotting

Following treatment, protein samples were extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) for western blot analysis. A BCA Protein Assay kit (Beyotime Institute of Biotechnology) was used to determine total protein concentrations. Proteins (40 µg/lane) were separated via 10 or 12.5% SDS-PAGE, transferred onto PVDF membranes (MilliporeSigma) and blocked with 5% non-fat milk for 2 h at room temperature. The membranes were washed three times with 1% TBS-Tween-20 and incubated with primary antibodies overnight at 4˚C. Following the primary incubation, membranes were incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (cat. no. 7074S, 1:2,000; Cell Signaling Technology, Inc.) or horse anti-mouse HRP-conjugated secondary antibody (cat. no. 7076S, 1:2,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. Protein bands were developed using an Enhanced Chemiluminescence Western Blotting Substrate kit (Thermo Fisher Scientific, Inc.). The primary antibodies used were as follows: Anti-phosphorylated (p)-AMPK (cat. no. 2535T, 1:1,000; Cell Signaling Technology, Inc.); anti-AMPK (cat. no. 5831T, 1:1,000; Cell Signaling Technology, Inc.); anti-PGC-1α (cat. no. 2178S, 1:1,000; Cell Signaling Technology, Inc.); anti-Bcl-2 (cat. no. 3498T, 1:1,000; Cell Signaling Technology, Inc.); anti-cleaved caspase-3 (cat. no. 9664T, 1:1,000; Cell Signaling Technology, Inc.); anti-caspase-3 (cat. no. 14220T, 1:1,000; Cell Signaling Technology, Inc.); anti-cleaved caspase-9 (cat. no. 20750S, 1:1,000; Cell Signaling Technology, Inc.); anti-caspase-9 (cat. no. 9508T, 1:1,000; Cell Signaling Technology, Inc.); anti-Bax (cat. no. ab182733, 1:2,000; Abcam) and anti-GAPDH (cat. no. 5174T, 1:1,000; Cell Signaling Technology, Inc.). The grayscale values of the membranes were semi-quantified using ImageJ software (version 1.52r; National Institutes of Health). Protein levels were normalized to GAPDH.

Bioinformatics

Kyoto Encyclopedia of Genes and Genomes was used to analyze the signalling pathways involved in AMPK (genome.ad.jp/kegg/).

Statistical analysis

Statistical analysis was performed using GraphPad Prism (version 8.0.1; GraphPad Software, Inc.). All experiments were repeated three times and data are presented as the mean ± standard deviation. An unpaired Student's t-test was used to compare differences between two groups, while one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to compare differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Results

LPS induces mitochondrial dysfunction in AML12 cells

To establish the sepsis-induced liver injury model, LPS was used to induce AML12 cells. As presented in Fig. 1A and B, the level of mitochondrial cytochrome c significantly decreased compared with the control group, while that of cytosolic cytochrome c significantly increased, following LPS induction. Furthermore, ATP synthesis significantly decreased following LPS stimulation compared with the untreated group (Fig. 1C). The relative mRNA expression levels of nuclear respiratory factor 1 (NRF1) and transcription factor A, mitochondrial (TFAM) were detected via RT-qPCR analysis, and their expression levels were significantly decreased following treatment of AML12 cells with LPS compared with the group without LPS (Fig. 1D and E). Oxidative stress and reduction in ATP production are involved in mitochondrial dysfunction, and along with apoptosis, are key factors for the occurrence of acute liver injury caused by sepsis (18). Collectively, these results suggested that LPS induced mitochondrial dysfunction in AML12 cells.

Figure 1 LPS induces mitochondrial dysfunction in AML12 cells. The contents of (A) mitochondrial and (B) cytosolic cytochrome c were measured using corresponding kits. (C) ATP synthesis was evaluated via the luciferin-luciferase reaction. The relative mRNA expression levels of (D) NRF1 and (E) TFAM were detected using reverse transcription-quantitative PCR analysis. Cells were treated with LPS (100 ng/ml). ***P<0.001 vs. control. LPS, lipopolysaccharide; NRF1, nuclear respiratory factor 1; TFAM, transcription factor A, mitochondrial.

XML treatment activates the AMPK/PGC-1α signaling pathway

Kyoto Encyclopedia of Genes and Genomes analysis demonstrated that mitochondrial function can be mediated by the AMPK/PGC-1α signaling pathway; thus, the effect of XML on this pathway was investigated in the present study. AML12 cells were treated with a range of concentrations of XML (0-4 mg/ml), and no significant differences in cell viability were observed (Fig. 2A). Furthermore, the viability of AML12 cells induced by LPS was detected via the MTT assay. The viability of LPS-induced AML12 cells was significantly decreased compared with the untreated group; however, cell viability increased following treatment with XML in a concentration-dependent manner; furthermore, the results showed a significant increase in cell viability compared with the LPS group following 2 and 4 mg/ml XML treatment (Fig. 2B). Therefore, 4 mg/ml XML was used for subsequent experiments. The expression levels of the proteins in the AMPK/PGC-1α signaling pathway were detected using western blot analysis. The expression levels of p-AMPK and PGC-1α were significantly decreased following LPS stimulation compared with the control group, the effects of which were reversed following 4 mg/ml XML intervention (Fig. 2C), suggesting that XML may activate the AMPK/PGC-1α signaling pathway in LPS-induced AML12 cells.

Figure 2 XML treatment activates the AMPK/PGC-1α signaling pathway in LPS-stimulated AML12 cells. (A) MTT assay was used to detect the viability of AML12 cells treated with different concentrations of XML (0.25, 0.5, 1, 2 and 4 mg/ml). (B) MTT assay was used to examine the effect of different concentrations of XML (0.25, 0.5, 1, 2 and 4 mg/ml) on the viability of 100 ng/ml LPS-induced AML12 cells. (C) Western blot analysis was performed to detect the expression levels of proteins in the AMPK/PGC-1α signaling pathway in 100 ng/ml LPS-induced AML12 cells after 4 mg/ml XML treatment. ***P<0.001 vs. control; #P<0.01, ##P<0.01, ###P<0.001 vs. LPS. XML, xinmailong; AMPK, AMP-activated protein kinase; PGC-1α, proliferator-activated receptor γ coactivator-1α; LPS, lipopolysaccharide; p, phosphorylated; t, total.

XML treatment suppresses LPS-induced inflammation, oxidative stress and apoptosis by activating AMPK/PGC-1α signaling in AML12 cells

To determine the potential mechanism via which XML exerts its effects on LPS-induced AML12 cell injury, the AMPK inhibitor compound C (2 mg/ml) was used for pretreatment together with 4 mg/ml XML in LPS-induced AML12 cells. The levels of inflammatory factors were detected using ELISA and RT-qPCR analysis. As presented in Fig. 3A-F, LPS significantly increased the levels of inflammatory factors compared with the control group, including TNF-α, IL-6 and IL-1β, and these levels were significantly decreased following treatment with XML. Notably, combined treatment with XML and compound C increased the expression levels of these factors compared with the LPS+XML group (Fig. 3A-F). In addition, LPS induction significantly elevated the content of MDA, accompanied by significantly decreased SOD activity, compared with the group without LPS; these effects were attenuated following treatment with XML compared with the LPS treatment group. Furthermore, compound C intervention reversed the effect of XML on oxidative stress (Fig. 3G and H). The results of the TUNEL assay further demonstrated the ability of XML to inhibit the apoptosis of LPS-induced AML12 cells, as the apoptosis of AML12 cells triggered by LPS decreased in the LPS+XML group compared with the LPS intervention group (Fig. 4A and B). However, the LPS+XML+compound C group exhibited a higher apoptotic rate compared with the LPS+XML group. The expression of apoptosis-related proteins determined via western blot analysis exhibited a similar trend (Fig. 4C). Collectively, these results suggested that XML may suppress LPS-induced liver injury by activating the AMPK/PGC-1α signaling pathway.

Figure 3 XML treatment suppresses LPS-induced inflammation and oxidative stress in AML12 cells via the activation of AMP-activated protein kinase/proliferator-activated receptor γ coactivator-1α signaling. Levels of (A) TNF-α, (B) IL-6 and (C) IL-β were detected using commercial ELISA kits. mRNA expression levels of (D) TNF-α, (E) IL-6 and (F) IL-β were determined using reverse transcription-quantitative PCR. Levels of (G) MDA and (H) SOD were evaluated using corresponding kits. LPS, 100 ng/ml; XML, 4 mg/ml; compound C, 2 mg/ml. ***P<0.001 vs. control; ###P<0.001 vs. LPS; ∆P<0.05, ∆∆P<0.01, ∆∆∆P<0.001 vs. LPS+XML. XML, xinmailong; LPS, lipopolysaccharide; MDA, malondialdehyde; SOD, superoxide dismutase.

Figure 4 XML treatment attenuates the cell apoptosis of LPS-induced AML12 cells. (A) Apoptosis of AML12 cells was determined using a TUNEL assay. (B) Quantification of cell apoptosis. (C) Western blot analysis was employed to assess the expression of apoptosis-related proteins. LPS, 100 ng/ml; XML, 4 mg/ml; compound C, 2 mg/ml. ***P<0.001 vs. control; #P<0.05, ##P<0.01, ###P<0.001 vs. LPS; ∆P<0.05, ∆∆∆P<0.001 vs. LPS+XML. XML, xinmailong; LPS, lipopolysaccharide.

XML suppresses mitochondrial dysfunction induced by LPS by activating AMPK/PGC-1α signaling in AML12 cells

Liver dysfunction is manifested by mitochondrial dysfunction, and mitochondrial dysfunction and cellular energy depletion enhance acute liver injury induced by sepsis (18,19). Thus, it was hypothesized that XML may suppress mitochondrial dysfunction induced by LPS by activating the AMPK/PGC-1α signaling pathway, thereby resulting in the alleviation of hepatocellular injury induced by LPS. LPS significantly decreased the content of mitochondrial cytochrome c and significantly increased the content of cytosolic cytochrome c compared with the untreated group, and these effects were reversed following treatment with XML (Fig. 5A and B). However, the addition of compound C significantly increased cytosolic cytochrome c content compared with the LPS+XML group and decreased in mitochondrial cytochrome c content, although this was not statistically significant (Fig. 5A and B). The synthesis of ATP, reduced by LPS, was significantly enhanced following treatment with XML compared with the LPS group, which was reversed by compound C (Fig. 5C). The mRNA expression levels of NRF1 and TFAM were detected via RT-qPCR analysis. As presented in Fig. 5D and E, LPS significantly downregulated the expression levels of NRF1 and TFAM, which was abrogated following treatment with XML. Furthermore, compound C markedly downregulated the expression levels of NRF1 and TFAM compared with the LPS+XML group. Collectively, these results indicated that XML may suppress mitochondrial dysfunction induced by LPS by activating the AMPK/PGC-1α signaling pathway.

Figure 5 XML suppresses the mitochondrial dysfunction induced by LPS via the activation of AMP-activated protein kinase/proliferator-activated receptor γ coactivator-1α signaling in AML12 cells. Contents of (A) mitochondrial and (B) cytosolic cytochrome c were measured using corresponding kits. (C) Synthesis of ATP was evaluated using a luciferin-luciferase reaction. The relative mRNA levels of (D) NRF1 and (E) TFAM were detected using reverse transcription-quantitative PCR. LPS, 100 ng/ml; XML, 4 mg/ml; compound C, 2 mg/ml. ***P<0.001 vs. control; ##P<0.01, ###P<0.001 vs. LPS; ∆P<0.05, ∆∆P<0.01, ∆∆∆P<0.001 vs. LPS+XML. XML, xinmailong; LPS, lipopolysaccharide; NRF1, nuclear respiratory factor 1; TFAM, transcription factor A, mitochondrial.

Discussion

PA extract is reported to exert therapeutic effects in various diseases, such as fever, inflammation and chronic heart failure (20). Furthermore, the bioactive compounds of PA extract exert anti-inflammatory, antipyretic, antioxidant and analgesic effects by suppressing the MAPK/NF-κB signaling pathway (20). Previous reports have also indicated the significance of PA extract in immune enhancement, wound healing, anti-fibrosis and protection against viral infections (12,13,21). Additionally, PA extract attenuated gastrointestinal complications, and improved immune function and nutritional status in patients with systemic inflammatory response syndrome (12). Of note, PA extract accelerated liver regeneration in a mouse liver regeneration model following 70% partial hepatectomy and a hepatocyte culture via a complex network of pathways, including the AMPK signaling pathway (14). However, the beneficial effects and mechanisms underlying PA extract in sepsis-induced hepatocyte injury remain to be elucidated. The present study utilized XML and established an LPS-induced liver injury model in vitro to determine the underlying molecular mechanism. The results demonstrated that XML ameliorated LPS-induced hepatocyte damage by improving mitochondrial dysfunction via the AMPK/PGC-1α signaling pathway.

Mitochondrial dysfunction leads to an impairment of fat homeostasis and overproduction of reactive oxygen species in the liver, which play important roles in the promotion of liver injury (22,23). LPS induces ATP deficiency in mitochondria, resulting in cellular dysfunction and cell death (24). The promoter of cytochrome c comprises a recognition site for the transcription factor NRF1, which is responsible for the regulation of the expression of genes involved in the respiratory chain. Furthermore, NRF1 has the ability to activate TFAM (25,26). In the present study, the translocation of mitochondrial cytochrome c to the cytoplasm, a decrease in ATP levels and decreased mitochondrial gene expression demonstrated the presence of mitochondrial dysfunction in AML12 cells.

The degradative products of cells are eliminated by a process referred to as autophagy (27). Autophagy maintains cellular homeostasis, while imbalance in the level of autophagy is associated with tissue injury, cancer and liver-related diseases (28). Mitophagy, a specific type of autophagy that removes damaged mitochondria, restrains mitochondrial dysfunction triggered by oxidative stress and decreases the accumulation of mutations in mitochondrial DNA (29). Defects in mitophagy incur mitochondrial dysfunction under conditions of liver injury induced by ischemic reperfusion (30). The regulation of autophagy is a complex process involving various signaling pathways, including the AMPK-mediated signaling pathway (31,32). AMPK indirectly facilitates mitophagy and cell survival (33). In the present study, the role of XML in the AMPK/PGC-1α signaling pathway was investigated.

AMPK is a key regulator of mitochondrial biogenesis, and the downstream effector of AMPK, PGC-1α, is required to increase the expression of genes associated with oxidative stress following AMPK activation (34). PGC-1α is a potent stimulator that promotes the biogenesis of mitochondria and transcription of genes in the liver, heart and skeletal muscle (35). Under conditions such as fasting and physical exertion, where energy is lacking or in demand, PGC-1α is induced or activated (36). In addition, the regulation of PGC-1α by AMPK is crucial for the modulation of mitochondrial biogenesis (37). In the present study, the suppression of AML12 cell viability by LPS was alleviated by increasing the concentration of XML. The expression levels of p-AMPK and PGC-1α increased following addition of XML to LPS-induced AML12 cells, suggesting that XML activates the AMPK/PGC-1α signaling pathway. Thus, it was hypothesized that XML exerts inhibitory effects on LPS-induced liver injury via the AMPK/PGC-1α signaling pathway. The results of the functional experiments confirmed that XML alleviated the effects on LPS-induced liver injury, as inflammation- and oxidative stress-related factors decreased following treatment of AML12 cells with XML, the effects of which were reversed following treatment with compound C. In addition, compound C promoted the apoptosis of AML12 cells. There is a close association between mitochondrial dysfunction and liver injury, and the results of the present study demonstrated that XML suppressed mitochondrial dysfunction induced by LPS by activating the AMPK/PGC-1α signaling pathway.

In conclusion, the results of the present study demonstrated that XML suppressed mitochondrial dysfunction induced by LPS by activating the AMPK/PGC-1α signaling pathway, which may contribute to understanding of the underlying mechanism via which XML alleviates liver injury.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by Traditional Chinese Medicine Science and Technology Development Program of Jiangsu Province (grant no. YB201991).

Availability of data and materials

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

Authors' contributions

WS and JL were responsible for conceiving and designing the study. WS, LA, JZ and JL performed the experiments. WS, LA and JL analyzed the data and reviewed the manuscript. WS, LA and JZ were responsible for drafting the manuscript. JL interpreted data and revised the final manuscript. All authors have read and approved the final manuscript. WS, LA, JZ and JL confirm the authenticity of all the raw data.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References