Abstract
Objectives
The present study aimed to characterize the protective role of daphnetin in attenuating HGHF-induced pathological injury in human NK-92 cells.
Methods
The non-toxic concentration of daphnetin was determined using a the Cell Counting Kit-8 (CCK-8) assay. The levels of inflammatory cytokines(TNF-α, IFN-γ, IL-6 and IL-10), in cell supernatants were subsequently detected using the cytometric bead array method. Flow cytometric analysis was then performed to analyze the expression levels of perforin and granzyme B, as well as the cell apoptosis rate.
Results
Daphnetin at a concentration of 10 μg/ml had no significant cytotoxicity on NK-92 cells, and therefore this concentration was selected for subsequent experiments. Compared with the control group, the HGHF group exhibited significantly reduced levels of TNF-α, IFN-γ, IL-6, IL-10, perforin and granzyme B (all P<0.05), along with a significantly increased cell apoptosis rate (P<0.05). Daphnetin treatment led to a marked upregulation of the levels of these inflammatory cytokines and intracellular molecules in both the control and the HGHF groups (all P<0.05); furthermore, the apoptosis rate was appreciably decreased in the HGHF-induced NK-92 cells (P<0.05).
Conclusion
Daphnetin has been shown to alleviate HGHF-induced pathological damage in NK-92 cells both through upregulating the expression of inflammatory cytokines and intracellular molecules (namely, perforin and granzyme B), and via inhibiting cell apoptosis.
Keywords: NK-92 cells, diabetes mellitus, daphnetin, cytokines, intracellular molecules
Graphical Abstract.
1. Introduction
Type 2 diabetes mellitus (T2DM) constitutes a metabolic disorder characterized by dysregulated levels of glucose, lipid, and protein metabolism that is predominantly caused by impaired insulin secretion. Chronic inflammation occupies a pivotal role in the initiation and progression of T2DM pathogenesis.1,2
Natural killer (NK) cells, derived from hematopoietic stem cells and primarily distributed in the liver, spleen, and peripheral blood, are effector lymphocytes characterized by potent cytolytic activity that have the ability to secrete immunoregulatory factors.3,4 Accumulating evidence suggests that NK cells serve as key components of the inflammatory microenvironment, which thereby contribute to the pathogenesis of obesity-associated metabolic diseases, including T2DM.5-7 Preliminary evidence from clinical observations and in vitro cellular experiments has corroborated the role of NK cells in modulating the chronic inflammatory processes that underlie T2DM.8,9 These studies further revealed that NK cells mediate the inflammatory pathological injury that is induced by high glucose and free fatty acids (FFAs) through oxidative stress responses, cytokine secretion and the expression of intracellular molecules (namely, perforin and granzyme B).8,9These findings suggest that NK cells are not merely passive responders but active participants in the inflammatory pathology of T2DM.
Given the functional impairment of NK cells under diabetogenic conditions, strategies aimed at restoring NK cell function may represent a promising therapeutic approach. Daphnetin is a natural coumarin derivative extracted from Chinese herbal medicines that has been shown to exert with multiple pharmacological effects, having roles in diverse processes including anti-inflammation, bacteriostasis, immune regulation, and anti-lipid peroxidation.10,11 Notably, previous studies have demonstrated that daphnetin promotes the secretion of interferon-γ (IFN-γ) through activating the effector functions of human primary NK cells, thereby exerting regulatory effects on the development of diabetes via anti-inflammatory mechanisms. 12 However, it remains unclear whether daphnetin can reverse the functional deficits in NK cells induced by high glucose and high FFAs, and whether such effects contribute to the alleviation of diabetes-associated pathological damage.
To address this research gap, the present study aims to investigate the protective effect of daphnetin against HGHF-induced chronic inflammatory pathological damage in NK-92 cells in vitro. Specifically, we assess its effects on cytokine secretion, expression of key molecules (perforin and granzyme B), and cell apoptosis. The present study therefore aimed to investigate the protective effects of daphnetin against HGHF-induced chronic inflammatory pathological damage in NK-92 cells in vitro. It is anticipated that the findings of the present study will provide novel insights into and perspectives for immunotherapeutic mechanisms.
2. Materials and Methods
The experiments were conducted between March 2025 and August 2025.
2.1. Cell Lines
The human NK cell line (NK-92) was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Wuhan, China) and maintained in alpha-minimum essential medium (α-MEM; C11095500BT, Gibco) supplemented with 100 U/ml recombinant interleukin-2 (IL-2; 200-02, PeproTech Asia), 12.5% horse serum (SBJ-SE-HO014, Nanjing SenBeiJia Biological Technology, China), 12.5% fetal bovine serum (FBS; SH30396.03, HyClone), 0.2 mM inositol, 0.1 mM β-mercaptoethanol, and 0.02 mM folic acid. Cells were cultured at 37°C in a humidified incubator containing 5% CO2. Owing to the specific proliferative characteristics of NK cells, the culture medium was refreshed after every 3 days. All experiments were performed using cells in the logarithmic growth phase.
2.2. Cytotoxicity Assay
Based on a previous study by our research group, 8 the groups were designated as the normal control group (Con) and the HGHF group. Cells in the HGHF group was treated with 44.4 mM glucose and 1.0 mM FFAs. (FFAs; a mixture of palmitic acid and oleic acid at a 1:2 molar ratio, complexed with 1% fatty acid-free bovine serum albumin).
Prior to the formal experiment, cytotoxicity was assessed in NK-92 cells exposed to the same range of daphnetin concentrations; the results indicated that daphnetin exerted no significant cytotoxic effects on cell proliferation.
2.2.1. CCK-8 Assay
NK-92 cells were seeded into 96-well plates at a density of 5 × 103 cells/well, and subsequently treated with various concentrations of daphnetin (0, 5, 10, 20, 30, and 40 μg/ml; purity ≥ 98%, Sigma-Aldrich, USA) prepared in medium containing 1% bovine serum albumin (BSA). Cells were incubated for 4 days at 37°C. At the end of this incubation period, 10 μl of Cell Counting Kit-8 (CCK-8) solution (GK10001, GLPBIO, USA) was added to each well, followed by a 4-hour incubation at 37°C. The absorbance at 450 nm was then measured using a microplate reader (RT-6000, Rayto, Shenzhen, China). Cell viability was calculated as the percentage of absorbance relative to the untreated control group.
2.3. Cell Treatment and Experimental Groups
NK-92 cells were treated for 4 days under the following four conditions, with each condition performed in six independent replicates:
Con group (normal control): 5.5 mM glucose + 1% BSA.
C-Dap group (control + daphnetin): 5.5 mM glucose + 10 μg/ml daphnetin.
HGHF group (high glucose and high FFA): 44.4 mM glucose + 1.0 mM FFA.
H-Dap group (HGHF + daphnetin): 44.4 mM glucose + 1.0 mM FFA + 10 μg/ml daphnetin.
The concentration of daphnetin (10 μg/ml) was selected based on the CCK-8 cytotoxicity assay results, which showed no significant cytotoxic effects at this concentration.
2.4. Detection of Cytokines
NK-92 cells were treated under four distinct experimental conditions in 12-well plates at a density of 1 × 105 cells per well and subjected to the four treatment conditionsfor 4 days. The supernatant was collected by centrifugation for 10 min at 1,000 ×g and 4°C, and the concentrations of TNF-α, IFN-γ, IL-6, and IL-10 were detected using a commercial human cytokine assay kit (Immunofluorescence Assay, Batch No. 20230801, Jiangxi Saiji Biotechnology Co., Ltd., China) according to the manufacturer’s instructions. Cytokines were detected through utilizing a cytometric bead array (CBA)-based technology on a Cytomics FC 500 flow cytometer (Beckman Coulter, Inc.).
Briefly, the principle of the CBA assay is analogous to that of a sandwich enzyme-linked immunosorbent assay (ELISA): target cytokines are captured by specific antibodies conjugated to microspheres (4 μm or 5 μm in diameter) with distinct allophycocyanin (APC) fluorescence intensities. Each cytokine binds to a unique microsphere population, which is further recognized by a biotinylated secondary antibody. After adding 20 μl of streptavidin-phycoerythrin (SA-PE) to each well, the mixture was incubated for 30 minutes at 37°C in the dark. Full details of the experimental procedures that were employed may be found in a previously published study. 13 Data were analyzed using FCAP Array™ software (Version 3.0, BD Biosciences). Standard curves were generated for each cytokine, and concentrations were calculated from the standard curves.
2.5. Intracellular Perforin and Granzyme B Levels
NK-92 cells were subjected to the four treatment conditions described above in 12-well plates at a density of 1 × 105 cells per well for 4 days. Following the incubation period, cells were stained with fluorescent antibody-labeling reagents against CD45 (200259, Beckman), CD3 (200138, Beckman), CD8 (200052, Beckman), CD16 (200109, Beckman), and CD56 (200072, Beckman) for 20 min in the dark. After fixation and permeabilization, cells were labeled with a granzyme/perforin antibody (220202, Ruisikaier Bio, China) for an additional 20 min in the dark. Cells were then washed twice with permeabilization buffer and resuspended in PBS. The expression levels of perforin and granzyme B were analyzed using a flow cytometer (Cytomics FC 500, Beckman Coulter, USA). Data were analyzed with FlowJo software (version 10.5.3, Tree Star, USA). Results are expressed as mean fluorescence intensity (MFI) normalized to the control group.
2.6. Detection of Cell Apoptosis
After 4 days of treatment in 12-well plates at a density of 1 × 105 cells per well, the culture supernatant was discarded, and the adherent cells were washed 2–3 times with pre-cooled phosphate-buffered saline (PBS). Cells were subseqently digested with 0.25% trypsin-EDTA for 2–3 minutes at 37°C, and the digestion reaction was terminated by adding an equal volume of complete cell culture medium. The cell suspension was centrifuged at 500×g for 5 minutes to collect the cell pellet.
Cell apoptosis was detected using an Annexin V-FITC/PI Apoptosis Detection Kit (Immunofluorescence Assay, Batch No. 20230801), purchased from Beijing TransGen Biotech Co., Ltd. Briefly, the collected cell pellet was washed 2–3 times with pre-cooled PBS and centrifuged at 500×g for 5 minutes; the supernatant was discarded after each wash.Subsequently, 100 μl of pre-chilled Binding Buffer was added to resuspend the cells, followed by the addition of 5 μl Annexin V-FITC and 5 μl PI staining solution. The mixture was gently vortexed and incubated at room temperature for 15 min in the dark. Following this incubation, 400 μl of pre-chilled Binding Buffer was added to the cells, and mixed thoroughly. Samples were maintained on ice in the dark and immediately analyzed by flow cytometry: the percentage of Annexin V-FITC-positive cells was defined as the apoptosis rate. A minimum of 10,000 events were acquired per sample.
2.7. Statistical Analysis
To ensure the accuracy and reproducibility of results, all experiments were performed in six independent replicates, and data are presented as mean ± standard deviation (SD). Prior to statistical analysis, data were normalized to the control group (Con) to minimize inter-experimental variability. Normality of data distribution was assessed using the Shapiro-Wilk test. Homogeneity of variances was evaluated using Levene’s test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for pairwise comparisons when significant differences were detected. Statistical analyses were conducted using SPSS software version 21.0 (SPSS, Inc., Chicago, IL, USA). A two-tailed P-value < 0.05 was considered statistically significant.
3. Results
3.1. High Concentrations Daphnetin Treatment Altered Cellular Viability
NK-92 cells were treated with increasing concentrations of daphnetin (0-40μg/ml) for 4 days in the Con group and HGHFs. Whereas treatment with 20 μg/ml daphnetin led to a significant decrease in cellular viability (P<0.05), in the 10 μg/ml or lower exerted only a mild effect on cellular viability (Figure 1). (Figure 1). Based on the above results, a concentration of daphnetin of 10 μg/ml was therefore chosen for further experiments.
Figure 1.
Daphnetin treatment at different concentrations alters cellular viability. NK-92 cells in the normal control (Con) and high-glucose high-free fatty acid (HGHF) groups were treated with various concentrations of daphnetin. Treatment with 20 μg/ml daphnetin resulted in a significant reduction in cell viability (P < 0.05) in both groups. The left panel depicts cell viability in the Con group, whereas the right panel shows cell viability in the HGHF group. *P < 0.05 vs. the corresponding groups without daphnetin treatment. Con, control; HGHF, high glucose and high free fatty acids
3.2. Effects of Daphnetin Treatment on Inflammatory Cytokine Levels in the NK-92 Cells
The levels of inflammatory cytokines (TNF-α, IFN-γ, IL-6 and IL-10) were found to be significantly decreased in the HGHF group (0.85 ± 0.08, 0.86 ± 0.10, 0.86 ± 0.06, 0.72 ± 0.04, respectively) compared with the Con group (1.00 ± 0.00) (P < 0.05). Treatment with daphnetin led to significantly increases in the levels of the inflammatory cytokines in both the Con group and the HGHF group: moreover, the levels of. This means that the inflammatory cytokines in the C-Dap group (1.12 ± 0.07, 1.18 ± 0.11, 1.09 ± 0.11and 1.15 ± 0.12, respectively)were significantly higher than those in the Con group (1.00 ± 0.00) (P < 0.05). In addition, those in the H-Dap group(0.96 ± 0.07, 1.01 ± 0.05, 0.97 ± 0.12 and 0.95 ± 0.05, respectively were significantly higher than those in the HGHF group (P < 0.05) (Figure 2).
Figure 2.
Effects of the Daphnetin treatment on inflammatory cytokine levels in the NK-92 cells. Compared with the control (Con) group (1.00 ± 0.00), the levels of inflammatory cytokines (TNF-α, IFN-γ, IL-6, and IL-10) were significantly decreased in the high-glucose high-free fatty acid (HGHF) group (0.85 ± 0.08, 0.86 ± 0.10, 0.86 ± 0.06, and 0.72 ± 0.04, respectively; P < 0.05).. Daphnetin treatment led to a significant increase in the levels of the inflammatory cytokines in both the Con group and the HGHF group.*P < 0.05 compared with the Con group; #P < 0.05 compared with the HGHF group.TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; IL-6, interleukin-6; IL-10, interleukin-10; Con, control; HGHF, high glucose and high free fatty acids
3.3. Effects of Daphnetin Treatment on Intracellular Molecules (Perforin and Granzyme B) in the NK-92 Cells
In comparison with the Con group (1.00 ± 0.00), the HGHF group exhibited a significant downregulation in the expression of the intracelleular molecules, perforin and granzyme B (0.93 ± 0.02 and 0.92 ± 0.02, respectively) compared with Con group (1.00 ± 0.00) (P < 0.05). Treatment with daphnetin led to a significant increase in the expression of perforin and granzyme B in both the Con group and the HGHF groups. Moreover, the concentrations of perforin and granzyme B in the C-Dap group (1.07 ± 0.02, 1.08 ± 0.02, respectively) were found to be significantly higher than those in the Con group, whereas those in the H-Dap group(0.97 ± 0.02, 0.97 ± 0.02, respectively) were significantly higher than those in the HGHF group. (P < 0.05) (Figure 3).
Figure 3.
Effects of the daphnetin treatment on intracellular molecules (perforin and granzyme B) in the NK-92 cells. Compared with the control (Con) group (1.00 ± 0.00), the HGHF group exhibited a significant downregulation in the expression of perforin and granzyme B (0.93 ± 0.02 and 0.92 ± 0.02, respectively; P < 0.05).. Daphnetin treatment led to a significant increase in the expression of perforin and granzyme B in both the Con group and the HGHF group. *P < 0.05 compared with the Con group; #P < 0.05 compared with the HGHF group.Con, control; HGHF, high glucose and high free fatty acids
3.4. Effects of Daphnetin on Cell Apoptosis in the NK-92 Cells
The cell apoptosis rate in the HGHF group(1.61±0.12) was found to be significantly higher than that in the Con group (1.00 ± 0.00) (P < 0.05). Treatment with daphnetin led to a could significant improvement in cell apoptosis in the HGHF group; specifically, thethe cell apoptosis rate in the H-Dap group(1.22 ±0.05) was found to be significantly lower than that in the HGHF group (Figure 4).
Figure 4.
Effects of the daphnetin treatment on cell apoptosis in the NK-92 cells. The cell apoptosis rate in the HGHF group(1.61±0.12) was significantly higher compared with that in the Con group (1.00 ± 0.00) (P < 0.05). Daphnetin treatment could significantly improve cell apoptosis in the HGHF group. *P < 0.05 compared with the Con group; #P < 0.05 compared with the HGHF group.Con, control; HGHF, high glucose and high free fatty acids
4. Discussion
The rising global prevalence of T2DM has positioned the disease as a major public health challenge, with chronic inflammation widely recognized as a central mediator in its pathogenesis and progression.1,2 Natural Killer (NK) cells serve as pivotal components of the innate immune system. They are able not only to regulate the inflammatory microenvironment but they also undergo functional impairment under the pathological conditions of high glucose (HG) and high free fatty acids (FFA).3-7 Daphnetin, a natural coumarin derivative isolated from traditional Chinese medicinal herbs, has been well-documented to possess multiple pharmacological activities, including those associated with the processes of anti-inflammation, bacteriostasis, immune regulation, and anti-lipid peroxidation.10-12
Consistent with our previous findings,8,9 the present study demonstrated that HGHF treatment significantly reduced the secretion of TNF-α, IFN-γ, IL-6, and IL-10 in NK-92 cells, indicating a state of functional suppression rather than classical pro-inflammatory activation. This pattern aligns with reports by Li et al 6 and Cavalcante-Silva et al, 7 who observed altered NK cell phenotypes and reduced cytokine production under diabetic conditions. A previous study that examined the association between NK cells and T2DM reported on metformin as a drug prescribed to treat T2DM, which was shown to possess antitumor activity via immunity activation. 14 A strong correlation was identified among the activation status of NK cells, exposure to HGHF conditions and immune function, with alterations in cytokine concentrations being closely associated with immune activation.TNF-α and IL-6 are key pro-inflammatory cytokines that mediate the “cytokine storm” in T2DM, whereas IFN-γ regulates the T helper 1 (Th1) immune response and enhances the cytotoxicity of NK cells.15,16 IL-10, as an anti-inflammatory cytokine, is involved in maintaining the balance of the inflammatory microenvironment, which may have a role in further promoting the progression of insulin resistance and pancreatic β-cell dysfunction. 17 In the current study, the levels of TNF-α, IFN-γ, IL-6, and IL-10 in the HGHF group found to be significantly reduced compared with the normal control group (Con). Treatment with daphnetin caused a significant increase in the secretion of TNF-α, IFN-γ, IL-6, and IL-10 in both the Con and HGHF experimental groups. This restoration is consistent with the immunomodulatory effects of daphnetin reported by Yao et al, 12 who found that daphnetin enhanced IFN-γ production and cytotoxicity in human primary NK cells. Notably, this not only constituted a simple restoration of the secretion levels of the inflammatory cytokines, but there was also a a restoration of the balance between pro-inflammatory and anti-inflammatory cytokines. For example, the significant increase in IL-10 concentration may have the effect of suppressing excessive inflammatory responses, whereas the levels of the restoration of TNF-α, IFN-γ and IL-6 may augment the cytotoxicity of NK cells, thereby improving the immune surveillance function.
Natural killer (NK) cells release perforin and granzyme B, where perforin perforates the membrane of target cells to enable the penetration of granzyme B, 18 Acting either directly or via the activation of additional immune cell subsets, perforin and granzyme B trigger apoptosis, hence serving as critical cytotoxic modulators, 19 The results of the present study showed that the expression levels of perforin and granzyme B in the HGHF group were significantly reduced compared with those in the Con group. This finding corroborates previous in vitro studies demonstrating that high-glucose and high-FFA conditions impair the cytotoxic machinery of NK cells.8,9 Daphnetin treatment also significantly upregulated the expression of perforin and granzyme B in NK-92 cells. This observation aligns with the work of Yao et al, 12 who reported that daphnetin enhances the direct cytotoxicity of NK cells. Collectively, these findings indicate daphnetin could effectively rescue the dysfunction of NK cells cytotoxic mediators induced by HGHF stimulation. In addition, these data highlighted the potential of daphnetin as a regulatory agent targeting the perforin-granzyme axis to restore NK cell-mediated immune surveillance.
Perforin forms pores on the surfaces of target cells, allowing granzyme B to enter and induce apoptosis, which is the core mechanism underlying NK cell-mediated cytotoxicity. In cell experiments, detecting apoptosis is crucial in terms of evaluating drug effects induced by inflammatory and other pathological conditions. The results of the present study demonstrated that HGHF treatment led to a significant induction of apoptosis of the NK-92 cells, whereas treatment with daphnetin could significant inhibit the HGHF-induced apoptosis of the NK-92 cells. This anti-apoptotic effect is consistent with previous reports on daphnetin in other cell types. Singh et al 20 demonstrated that daphnetin exerts neuroprotective effects by upregulating the anti-apoptotic protein Bcl-2 and reducing pro-apoptotic Bax and caspase-3 expression.The loss of NK cells not only weakened the immune surveillance function but also disrupted the interaction between NK cells and other immune cells (for example, macrophages and T cells), leading to the persistence of the inflammatory microenvironment in adipose tissue and the pancreas. 21 Our findings extend these observations to NK-92 cells under diabetogenic stress, suggesting that daphnetin’s anti-apoptotic properties may contribute to preserving NK cell numbers and function in the diabetic milieu. Multiple preclinical animal studies and cell line assays have confirmed that daphnetin acts as a potent protective agent that exerts anti-inflammatory and anti-apoptotic effects through targeting various cellular mediators and signaling pathways. 20 Daphnetin was also shown to restore the cellular redox balance via upregulating the anti-apoptotic protein Bcl-2, whereas the levels of proinflammatory cytokines, effector caspase-3 and the pro-apoptotic protein Bax were reduced. 20 In future studies, we will investigate the changes in the mitochondrial membrane potential and the expression of apoptotic proteins in NK-92 cells in order to further clarify the anti-apoptotic mechanism of daphnetin.
The results of the present study have important clinical implications for the immunotherapeutic management of T2DM. First, daphnetin, as a natural component of Chinese herbal medicines, has the advantages of low toxicity and high biocompatibility, making it a potential candidate for the development of T2DM immunotherapies. Secondly, this study has also confirmed that targeting NK cells to both restore their function and inhibit their apoptosis is a feasible strategy for treating T2DM-associated chronic inflammation, thereby providing a novel direction for the development of anti-T2DM drugs.
However,the present study did have a number of limitations. Firstly, it was an in vitro study that only employed only the NK-92 cell line, and the results may not fully reflect the in vivo environment. Future studies should use primary NK cells isolated from patients with T2DM or animal models (for example, db/db mice) to verify the protective effect of daphnetin. Secondly, the specific signaling pathways (for example, NF-κB, JAK-STATand mitochondrial apoptotic signaling pathways) that serve to mediate the protective effects of daphnetin were not explored in depth, and further mechanistic studies are needed to clarify the molecular targets of daphnetin. Thirdly, while our findings demonstrate functional recovery of NK-92 cells following daphnetin treatment, the upstream molecular events—such as receptor engagement or metabolic reprogramming—that initiate these protective effects remain to be elucidated.
5. Conclusions
In conclusion, the present study has demonstrated that daphnetin alleviates HGHF-induced pathological damage in NK-92 cells via upregulating the secretion of inflammatory cytokines, thereby restoring the expression of the intracellular cytotoxic molecules (perforin and granzyme B), and inhibiting cell apoptosis. Taken together, these findings have provided a nove experimental basis for the application of daphnetin in the treatment of T2DM and has been laid a foundation for further exploring the immunomodulatory mechanism of natural coumarin derivatives in metabolic diseases.
Acknowledgements
The authors would like to express their gratitude to Professor Shangdong Liang (Department of Physiology, Medical College of Nanchang University, Nanchang, China) for his valuable guidance provided during the conduct of this study.
Appendix.
Abbreviations
- T2DM
Type 2 Diabetic Mellitus
- NKNatural killerDap
Natural killerDapDaphnetin
- HGHF
High Glucose and High Free Fatty Acids
- TNF
Tumor Necrosis Factor
- IFN
Interferon
- IL
Interleukin.
Author Contribution: Hong Wu and Xiaodong Zhong were responsible for the conception and experimental design of the study. Yijun Nie, Ningrui Wang, Yuhuan Jian, Wenjing Zhou, and Yanan Chen conducted the experiments and collected the data. Xiaodong Zhong drafted the initial manuscript, and Hong Wu provided critical revisions for key intellectual content. All authors participated in the editorial refinement of the manuscript, read it carefully, and gave final approval for submission.
Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the National Natural Science Foundation of China (No. 81660144); Doctoral Research Startup Fund of Nanchang Medical College (No. NYB22005); Fund Project of Jiangxi Provincial Administration of Traditional Chinese Medicine (No. 2023A0192); Fund Project of Jiangxi Provincial Administration of Traditional Chinese Medicine (No. 2024A0139).
The authors declared no potential conflicts of interest with respect to the research, authorship, and/orpublication of this article.
Declaration of AI and AI-assisted Technologies in the Writing Process: During the preparation of this manuscript, the authors used Doubao Software Ver. 3.0 to check spelling and grammar. Following use of this tool, the authors reviewed and revised the content as necessary and accept full responsibility for the published material.
ORCID iD
Data Availability Statement
Raw data are archived in the testing instruments and will be publicly released at the time of publication. All data presented in this paper are available from the lead contact upon request.*
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Raw data are archived in the testing instruments and will be publicly released at the time of publication. All data presented in this paper are available from the lead contact upon request.*