Abstract
Purpose
Retinal microvascular endothelial cells (RMECs) injury caused by sustained hyperglycemia was the initial factor in diabetic retinopathy (DR). Here, we explored a novel mechanism by which palmitic acid (PA) contributed to RMEC damage in DR.
Methods
Diabetic rats and high glucose (HG)–treated human RMECs (HRMECs) were used for the in vivo and in vitro experiments. The pathological changes of retinal tissues were evaluated by hematoxylin & eosin staining; mannose receptor (CD206) and B7-2 (CD86) expression were detected by immunohistochemical staining and flow cytometry; cell viability was measured using Cell Counting Kit-8; the protein levels were detected by Western blot.
Results
Ferroptosis was induced in the retinal tissues of diabetic rats and HG-treated HRMECs, which was verified by increased kelch-like ECH-associated protein 1 (Keap1), acyl-CoA synthetase long-chain family member 4 (ACSL4), malondialdehyde (MDA), and reactive oxygen species (ROS) levels, whereas glutathione (GSH), nuclear factor erythroid 2-related factor 2 (Nrf2), and glutathione peroxidase 4 (GPX4) levels were decreased. PA exhibited the similar promotion effect on ferroptosis in HRMECs, which was reversed by 2-bromohexadecanoic acid and further enhanced by Palmostatin B. The palmitoylation modification of Keap1 in HRMECs was confirmed, and si-Keap1 reversed the increased Keap1, ACSL4, MDA and ROS, decreased Nrf2, GPX4 and GSH induced by PA. The in vivo experiments further revealed that si-Keap1 alleviated DR and reduced ferroptosis.
Conclusions
These results suggested that PA induced Keap1 palmitoylation to upregulate Keap1 levels to inhibit Nrf2/GPX4 pathway, thus enhancing ferroptosis in HRMECs and aggravating DR.
Keywords: diabetic retinopathy, ferroptosis, Keap1, palmitoylation
Diabetic retinopathy (DR) is one of the most serious complications of diabetic microvessel and can result in retinal detachment and increase the risk of blindness.1 According to the World Health Organization, the number of diabetic patients will exceed 500 million by 2025 worldwide, of which 1/3 will develop DR.2 It is believed that retinal microvascular endothelial cells (RMECs) damage caused by sustained hyperglycemia is the initial factor of vascular pathological changes in DR.3 Despite various researches of RMECs in DR, the underlying molecular mechanisms remain unclear.
More and more studies have revealed that oxidative stress promotes DR occurrence and development by inducing RMEC dysfunction.4,5 Oxidative stress referred to the imbalance between the production and removal of intracellular reactive oxygen species (ROS) resulted in the accumulation of intracellular ROS, and the cells were prone to oxidative damage.6 It has been reported that iron overload promoted DR progression by inducing endothelial cells dysfunction via enhancing the oxidative stress and inflammatory response.7,8 Ferroptosis is a new type of programmed cell death, which is characterized by a large amount of ROS via the Fenton reaction, then ROS induced lipid peroxidation by interacting with polyunsaturated fatty acids on the cell membrane, and released cytotoxic malondialdehyde (MDA) to damage cell protein, DNA, and cell membrane, finally resulting in ferroptosis.9 Ferroptosis plays an important role in tumors, neurodegeneration and other diseases.10–12 Recently, several studies have revealed that ferroptosis also exhibits an important role in the pathogenesis of DR,13,14 but its exact mechanism has not been fully elucidated.
Free fatty acids (FFA) are well-known risk factors in diabetes.15 Palmitic acid (PA) is the most abundant saturated FFA in the human body, which is naturally found in palm oil, coconut oil, and some animal products such as meat, butter, and dairy; thus it is often found in the daily diet. FFA destroyed the REDOX balance of vascular endothelial cells to increase ROS production to cause oxidative stress, thus resulting in endothelial cell dysfunction.16 Also, it has been reported that PA induced oxidative stress to injury retinal ganglion cells, suggesting that PA aggravates diabetic retinopathy.17 However, whether PA can induce ferroptosis of RMECs to promote DR development and the underlying mechanisms remained unclear and were explored in this study.
Material and Methods
The Establishment of Diabetic Rats
All procedures were conducted according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. All animal experimental protocols were approved by the Animal Ethics Committee of Jiangxi Medical College, Nanchang University. Eight-week-old male SD rats (Cavins, Changzhou, China) were fed high-sugar and high-fat diets for eight weeks and then were intraperitoneally injected with streptozocin (dissolved in 0.1 mol/L citrate buffer, 35 mg/kg) two times, once a week. At 72 hours after the last streptozocin injection, animals with a fasting blood glucose concentration ≥13.8 mmol/L for two weeks were considered diabetic.
Experimental Grouping
The diabetic rats were randomly divided into four groups (n = 6): Ctrl, PA, PA + si-NC, and PA + si-Keap1. PA was given by intragastric administration at a dose of 100 mg/kg once a day; si-Keap1 was intravenously administered at a dose of 100 nmol/kg twice a week for two weeks.
Hematoxylin & Eosin (H&E) Staining
SD rats were killed by neck removal, and the right eyeballs were removed. The corneal lens and the vitreous body were removed, and the retinal tissues were obtained. After fixation with 4% formaldehyde, the retinal tissues were embedded in paraffin and cut into 5-µm sections. Then, the sections were stained with H&E kits (Solarbio, Beijing, China). Images were taken under a light microscope (Olympus BX63; Olympus, Tokyo, Japan), and the pathological changes of the retinal tissues were observed.
Immunohistochemistry (IHC) Staining
The retinal tissues were incubated with CD86 (1:100, 19589S; Cell Signaling Technology, Danvers, MA, USA) or CD206 (1:200, 24595S; Cell Signaling Technology) antibodies and then stained with DAB and hematoxylin. Finally, the sections were photographed under an inverted microscope (Olympus BX63).
Western Blot
The extracted proteins were transferred onto a nitrocellulose membrane and then incubated with the following primary antibodies: kelch-like ECH-associated protein 1 (Keap1; 1:2000, 60027-1-Ig; Proteintech, Rosemont, IL, USA), nuclear factor erythroid 2–related factor 2 (Nrf2; 1:1000, GB113808; Servicebio, Wuhan, China), glutathione peroxidase 4 (GPX4; 1:5000, 67763-1-Ig; Proteintech) and acyl-CoA synthetase long-chain family member 4 (ACSL4; 1:6000, 22401-1-AP; Proteintech). The β-actin was the internal control. Horseradish peroxidase–conjugated goat anti-rabbit or mouse IgG served as the secondary antibodies. An enhanced chemiluminescence kit was used to visualize the protein bands.
Cell Culture and Treatment
HRMECs obtained from Yaji Biotechnology (Shanghai, China) were cultured with Dulbecco's modified Eagle's medium (Procell, Wuhan, China) in a 5% CO2 atmosphere at 37°C. Then HRMECs were treated with high glucose (HG, 30 mM) for 24 h, and followed by the treatment with 2-Bromohexadecanoic acid (2-BP, 10 µM) and Palmostatin B (5 µM) for 24 h. Also, HRMECs were transfected with si-FATP1 or si-Keap1 for 24 h, and followed by the treatment with PA (300 µM) for 24 h. The siRNA sequences were listed as follows:
si-FATP1: 5′-CGUCUUUUCUACAUCUACACG-3′
si-Keap1: 5′-GCUACGAUGUGGAAACAGAGA-3′
si-NC: 5′-ACGUGACACGUUCGGAGAATT-3′
Flow Cytometry
THP-1 cells were treated with phorbol-12-myristate-13-acetate (PMA), and the cell surface was stained with anti-mouse CD86 antibody (558703; BD Bioscience, Franklin Lakes, NJ, USA) or anti-mouse CD206 antibody (568273; BD Bioscience ) for 30 minutes at room temperature. Then cells were analyzed using a BD FACSCalibur flow cytometer, and the results were analyzed using Flowjo Software.
Cell Viability
HRMECs were seeded into a 96-well plate and CCK8 regent (Beyotime Institute of Biotechnology, Jiangsu, China) was added to each well and incubated with cells for three hours in the dark. The optical density value of each well was measured at 450 nm using a microplate reader.
Assay of GSH, MDA, and ROS
The levels of glutathione (GSH), MDA, and ROS were examined using GSH test kit (S0053; Beyotime Institute of Biotechnology), ROS test kit (S0033S; Beyotime Institute of Biotechnology), and MDA test kit (A003-4-1; Jiancheng Bioengineering Institute, Nanjing, China), respectively, according to the manufacturer's protocol.
Palmitoylation Detection
According to the Click-iT kit (C10276; Thermo Fisher Scientific, Waltham, MA, USA), HRMECs were treated with Click-iTM palmitate azide (C10265; Thermo Fisher Scientific) for six hours at 37°C, and then cells were lysed using a lysate containing protease and phosphatase inhibitors, followed by treatment with biotin alkyne. Then the cell lysate was incubated with streptavidin beads for two hours at 4°C, and beads with bound protein were removed and tested for palmitoylation modification using Western blot.
Statistical Analysis
The differences among groups were analyzed via unpaired Student's t tests or one-way ANOVA using GraphPad Prism 7.0 after ascertaining normality of distribution via Shapiro-Wilk tests, and followed by Tukey's post hoc test. Significant P value was less than 0.05.
Results
Ferroptosis Is Induced in Retinal Tissues of Diabetic Rats and HG-Treated HRMECs
To evaluate the role of ferroptosis in DR, the diabetic rats were used for the in vivo experiments. As shown in Figures 1A, 1B, H&E staining revealed that the normal group exhibited clear structure and distinct layers in the retinal tissues, whereas the Model group exhibited the thinner retina (green dashed line). IHC staining of CD86 and CD206 further revealed that the Model group exhibited the inflammatory infiltration in the retinal tissues, compared with the normal group (Figs. 1C, 1D). Western blot results revealed that, compared with the normal group, Keap1 and ACSL4 protein levels were significantly upregulated, whereas Nrf2 and GPX4 protein levels were significantly downregulated in the retinal tissues of the Model group (Figs. 1E, 1F). Also, compared with the normal group, the Model group exhibited the markedly decreased GSH (Fig. 1G) and increased MDA (Fig. 1H) and ROS (Fig. 1I) in the retinal tissues. These results suggested that ferroptosis was induced in retinal tissues of diabetic rats, which further motivated the specific mechanistic investigation in vitro. Although the retinal neural cells were major cells in retinal tissues, RMECs played a crucial role in the physiology and pathology of DR; thus, HG-treated HRMECs were used for the in vitro experiments to further assess the role of ferroptosis in DR. Mannitol was used as osmotic controls of HG, and HG markedly reduced cell viability in HRMECs (Fig. 2A). Also, Keap1 and ACSL4 protein levels (Figs. 2B, 2C), MDA (Fig. 2E) and ROS (Fig. 2F) were significantly increased, whereas Nrf2 and GPX4 protein levels (Figs. 2B, 2C) and GSH (Fig. 2D) were significantly decreased in HG-treated HRMECs. Also, HRMECs were co-cultured with PMA-treated THP-1 cells, and HG-treated HRMECs markedly up-regulated CD86 expression (Figs. 2G, 2H) but down-regulated CD206 expression (Figs. 2G, 2I) in THP-1 cells. The above results suggested that ferroptosis participated in the pathological changes in DR.
Figure 1.
The detection of ferroptosis in retinal tissues of diabetes rats. The diabetic rats were established for the in vivo experiments. The retinal tissues of rats (n = 6) were isolated, (A, B) H&E staining was performed to observe the morphological changes of retinal tissues; (C, D) IHC staining was performed to examine CD86 and CD206 expression in retinal tissues; (E, F) Western blot was performed to detect the protein levels of Keap1, Nrf2, GPX4 and ACSL4. The levels of GSH (G), MDA (H), and ROS (I) were detected using different test kits. The difference between two groups was analyzed via unpaired Student's t test, and followed by Tukey's post hoc test. *P < 0.05, **P < 0.01 versus normal group.
Figure 2.
The detection of ferroptosis in HG-treated HRMECs. HRMECs were treated with HG, and mannitol was used as osmotic controls of HG. (A) CCK8 was performed to assess cell viability in HRMECs. (B, C) Western blot was performed to detect the protein levels of Keap1, Nrf2, GPX4 and ACSL4. The levels of GSH (D), MDA (E), and ROS (F) were detected using different test kits. (G–I) HRMECs were co-cultured with PMA-treated THP-1 cells, flow cytometry was performed to detect CD86 and CD206 expression in THP-1 cells. All experiments were repeated three times. The differences among different groups were analyzed via ANOVA, and followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 versus Ctrl group.
PA Enhances Ferroptosis in HRMECs
Next, we found that PA exhibited the similar effect to HG on HRMECs, including reduced cell viability (Fig. 3A), increased Keap1, ACSL4 protein levels (Figs. 3B, 3C), MDA (Fig. 3E) and ROS (Fig. 3F), decreased Nrf2, GPX4 protein levels (Figs. 3B, 3C) and GSH (Fig. 3D), while which were all markedly reversed by si-fatty acid transporter 1 (FATP1), suggesting that PA may induce ferroptosis in HRMECs. Palmitoylation was a common modification of PA, 2-Bromohexadecanoic acid (2-BP) was a palmitoylation inhibitor, whereas Palmostatin B was a palmitoylation inducer. HG reduced cell viability (Fig. 4A), increased Keap1 and ACSL4 protein levels (Figs. 4B, 4C), MDA (Fig. 4E) and ROS (Fig. 4F), decreased Nrf2, GPX4 protein levels (Figs. 4B, 4C) and GSH (Fig. 4D), and which were further enhanced by Palmostatin B, while reversed by 2-BP.
Figure 3.
PA induced ferroptosis in HRMECs. HRMECs were transfected with si-FATP1, and followed by the treatment with PA. (A) CCK8 was performed to assess cell viability in HRMECs. (B, C) Western blot was performed to detect the protein levels of Keap1, Nrf2, GPX4 and ACSL4. The levels of GSH (D), MDA (E), and ROS (F) were detected using different test kits. All experiments were repeated three times. The differences among different groups were analyzed via ANOVA, and followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 versus Ctrl group; #P < 0.05, ##P < 0.01 versus PA + si-NC group.
Figure 4.
Palmitoylation modification affected HG-induced ferroptosis in HRMECs. HRMECs were treated with HG, and followed by the treatment with 2-BP and Palmostatin B, respectively. (A) CCK8 was performed to assess cell viability in HRMECs. (B, C) Western blot was performed to detect the protein levels of Keap1, Nrf2, GPX4 and ACSL4. The levels of GSH (D), MDA (E), and ROS (F) were detected using different test kits. All experiments were repeated three times. The differences among different groups were analyzed via ANOVA, and followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 versus Ctrl group; #P < 0.05, ##P < 0.01 versus HG + DMSO group.
PA Induces Keap1 Palmitoylation To Enhance Ferroptosis in HRMECs
Palmitoylation played an important role in maintaining protein stability, and our preliminary results have revealed the palmitoylation modification of Keap1 in 293T cells, thus, based on the results in Figure 3, we speculate that PA may induced Keap1 palmitoylation to up-regulate Keap1 protein, resulting in ferroptosis in HRMECs. Immunoprecipitation analysis revealed the palmitoylation modification of Keap1 in HRMECs (Fig. 5A). In addition, si-Keap1 significantly reversed the reduced cell viability (Fig. 5B), increased Keap1 and ACSL4 protein levels (Figs. 5C, 5D), MDA (Fig. 5F) and ROS (Fig. 5G), and decreased Nrf2 and GPX4 protein levels (Figs. 5C, 5D) and GSH (Fig. 5E) induced by PA.
Figure 5.
PA induced Keap1 palmitoylation to enhance ferroptosis in HRMECs. (A) HRMECs were transfected with APT1, and immunoprecipitation was performed to assess the palmitoylation modification in HRMECs. HRMECs were transfected with si-Keap1, and followed by the treatment with PA. (B) CCK8 was performed to assess cell viability in HRMECs. (C, D) Western blot was performed to detect the protein levels of Keap1, Nrf2, GPX4 and ACSL4. The levels of GSH (E), MDA (F), and ROS (G) were detected using different test kits. All experiments were repeated for three times. The differences among different groups were analyzed via ANOVA, and followed by Tukey's post hoc test. **P < 0.01, ***P < 0.001 versus Ctrl group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus PA + si-NC group.
Si-Keap1 Alleviates DR and Reduces Ferroptosis In Vivo
Finally, the effect of si-Keap1 on DR was assessed in vivo. As shown in Figures 6A, 6B, H&E staining revealed that the Ctrl group exhibited the reduced ganglion cells (black arrow) and the thinner retina (green dashed line), and the retinopathy in PA group was significantly aggravated in comparison to the Ctrl group; when compared with PA + si-NC group, the retinopathy in the PA + si-Keap1 group was significantly alleviated. IHC staining revealed that the upregulated CD86 expression and downregulated CD206 expression in the retinal tissues in the PA + si-NC group was markedly reversed in the PA + si-Keap1 group (Figs. 6C, 6D). Also, the elevated Keap1 and ACSL4 protein levels (Figs. 6E, 6F), reduced Nrf2 and GPX4 protein levels (Figs. 6E, 6F), decreased GSH (Fig. 6G), increased MDA (Fig. 6H) and ROS (Fig. 6I) in the retinal tissues in PA + si-NC group were all significantly reversed in PA + si-Keap1 group.
Figure 6.
Si-Keap1 alleviated DR and reduces ferroptosis in vivo. Si-Keap1 was injected into DR rats treated with PA (n = 6). (A, B) H&E staining was performed to observe the morphological changes of retinal tissues. (C, D) IHC staining was performed to examine CD86 and CD206 expression in retinal tissues. (E, F) Western blot was performed to detect the protein levels of Keap1, Nrf2, GPX4 and ACSL4. The levels of GSH (G), MDA (H), and ROS (I) were detected using different test kits. The differences among different groups were analyzed via ANOVA, and followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 versus Ctrl group; #P < 0.05, ##P < 0.01 versus PA + si-NC group.
Discussion
Although most majors have supported that vascular endothelial cell damage caused by high glucose was the pathological basis for DR,3 the underlying mechanisms were still unclear. Studies have revealed that the excessive ROS hold an important position in vascular endothelial cell injury and apoptosis.18 Considering the accumulation of ROS in DR, there may be other types of cell death in DR except apoptosis. Ferroptosis was a kind of iron-dependent programmed cell death, when cystine transport protein was inhibited, intracellular GSH was reduced, resulting in the inactivation of GPX4 and the accumulation of lipid peroxidation, and eventually inducing cell death.19 Keap1/Nrf2 signaling pathway was the main defense mechanism against oxidative stress, and Fer-1 (the inhibitor of ferroptosis) reduced ferroptosis via the activation of Nrf2 signaling pathway.20,21 It has been reported that the epigenetic modification of Keap1 regulated its interaction with Nrf2, which further affected DR development.22 Li et al.23 also reported that maresin-1 inhibited high glucose induced ferroptosis in ARPE-19 cells by up-regulating Nrf2 and GPX4 expression, and the in vivo experiments further revealed that maresin-1 reduced MDA and ROS levels in the retinal tissues of diabetic mice. Acyl-CoA synthetase long-chain family (ACSL) participated in the metabolic process of fatty acids, and several studies have revealed that ACSL members were associated with ferroptosis. Zhang et al.24 reported that ACSL1 reduced the level of lipid oxidation and increased the resistance to cell ferroptosis. However, another study revealed that ACSL1 induced ferroptosis by mediating the integration of exogenous conjugated linolenic acid into phospholipids.25 The opposite effect of ACSL1 on ferroptosis revealed by the above two studies might be related to different diseases and cells. Also, Zhao et al.26 reported that inhibiting ACSL4 exhibited a remarkable anti-ferroptosis function, suggesting the promotion effect of ACSL4 on ferroptosis. In this study, increased Keap1 and ACSL4 protein levels, MDA and ROS, while decreased Nrf2 and GPX4 protein levels, and GSH were all observed in the retinal tissues of diabetic rats and HG-treated HRMECs, suggesting that ferroptosis may participate in the development of DR. However, the in vitro experiments of deleting ACSL4 in HRMECs were lack, which was a pity.
PA can be used as a substrate to participate in palmitoylation modification, and the accumulated PA in metabolism can be covalently modified to protein cysteine residues by thioester bonds, thus regulating gene expression. The abnormal palmitoylation modification was involved in various diseases. Zhang et al.27 reported that DHHC9-mediated GLUT1 palmitoylation promoted glycolysis and tumorigenesis in glioblastoma. Bu et al.28 reported that a high-fat diet promoted liver tumorigenesis via palmitoylation and activation of AKT. Also, Zhang et al.29 reported that ZDHHC5 palmitoylated and stabilized SMPDL3B, and silencing SMPDL3B aggravated DR in diabetic mice. Our preliminary results have revealed the palmitoylation modification of Keap1 in 293T cells, and we also observed that PA upregulated Keap1 protein level and induced ferroptosis in HRMECs; thus we speculate that PA may induce Keap1 palmitoylation to upregulate Keap1 protein level, thus resulting in ferroptosis in HRMECs. The 2-BP was a palmitoylation inhibitor, whereas Palmostatin B was a palmitoylation inducer. To our delight, the increased Keap1 and ACSL4 protein levels, MDA and ROS, although decreased Nrf2 and GPX4 protein levels and GSH induced by HG were reversed by 2-BP, were further enhanced by Palmostatin B. Also, in this study, the palmitoylation modification of Keap1 in HRMECs was confirmed via immunoprecipitation analysis, and si-Keap1 significantly reversed the increased Keap1, ACSL4 protein levels, MDA and ROS, decreased Nrf2, GPX4 protein levels and GSH induced by PA. Additionally, we observed that si-Keap1 alleviated DR and reduced ferroptosis in vivo. Thus based on the above results, we concluded that PA induced Keap1 palmitoylation to upregulate Keap1 protein level to inhibit Nrf2/GPX4 pathway, thus enhancing ferroptosis in HRMECs, and aggravating DR.
In addition to ferroptosis, the macrophages were also explored in DR. Ferroptosis released injury-related cytokines and triggered inflammation as a cellular immunogenic death, and cytokines interacted with immune cells to regulate the body's response to disease and infection.30 Macrophages generally functioned through polarization and can be polarized into M1 pro-inflammatory cells and M2 anti-inflammatory cells. In normal retina, M2 was the dominant factor; however, macrophages were mostly polarized toward M1 in DR because of hyperglycemia and systemic metabolic disorders, which induced a large number of inflammatory factors to infiltrate retinal tissues and eventually caused retinopathy.31 It has been reported that optic nerve damage was effectively alleviated by inducing M2 polarization of macrophages.32 As expected, upregulated CD86 expression and downregulated CD206 expression were observed in the retinal tissues of diabetic rats, and the in vitro experiments further revealed that HG-treated HRMECs markedly up-regulated CD86 expression and downregulated CD206 expression in THP-1 cells. Also, PA upregulated CD86 expression and downregulated CD206 expression in the retinal tissues of diabetic rats, which were markedly reversed by si-Keap1, suggesting that PA upregulated Keap1 protein level to induce M1 polarization of macrophages in DR.
In addition, retinal angiogenesis was the main clinical feature of DR. HG induced retinal microvascular dilation and damaged the blood-retinal barrier, then induced angiogenesis.33 Ferroptosis was characterized by a large amount of ROS, and oxidative stress injury in vascular endothelial cells promoted angiogenesis. Silencing ribosome biogenesis regulator 1 homolog (RRS1) inhibited angiogenesis in lung cancer cells by activating ferroptosis mediated by p53 pathway.34 Crocin facilitated angiogenesis by moderating oxidative stress and ferroptosis via Nrf2/GPX4 pathway.35 However, in this study, the relationship between angiogenesis and ferroptosis in HRMECs was unclear, which needed our further exploration.
In summary, this study demonstrated that PA induced Keap1 palmitoylation to up-regulate Keap1 protein level to enhance ferroptosis in HRMECs, thus aggravating DR, which may depend on the inhibition of Nrf2/GPX4 pathway. This study may provide a novel insight into the underlying mechanisms of hyperglycemia-induced retinopathy based on endogenous metabolite modification.
Acknowledgments
Supported by Training Program for Academic and Technical Leaders of Major Disciplines - Young Talents of Jiangxi Province (No. 20232BCJ23044), Natural Science Foundation of Jiangxi Province (No. 20224BAB206050) and Science and Technology Key Program of Education Department of Jiangxi Province (No. GJJ2200125), Science and Technology Program Project of Jiangxi Provincial Administration of Traditional Chinese Medicine (No. 2024B0623).
Disclosure: Z. Mao, None; W. Yang, None; Y. Chao, None; Z. Chen, None; Y. Zou, None
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