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. 2026 Aug 6;40(10):5886–5900. doi: 10.1002/ptr.70424

Cordycepin Alleviates Non Alcoholic Fatty Liver Disease Progression by Stabilizing GCH1 to Inhibit Ferroptosis

Chenyang Han 1, Jian Sheng 2, Hongyan Pei 3, Wenyan Li 1, Shasha Wu 1, Yi Yang 1, Chenxi Cao 4,✉, Zhiling Tang 2,✉
PMCID: PMC13636126  PMID: 42560344

ABSTRACT

To investigate the effects and targets of cordycepin (COR) in alleviating non‐alcoholic fatty liver disease (NAFLD). High‐fat diet was used to induce NAFLD in mice, and after COR gavage administration, changes in liver function, glucose and lipid metabolism, and ferroptosis‐related signaling were detected. In vitro, palmitic acid (PA) and oleic acid (PSA) were combined to induce hepatocyte lipid injury. The effect of COR on hepatocyte ferroptosis was examined. GCH1 conditional knockout mice (GCH1‐CKO) and hepatocyte models were constructed, and AAV9‐GCH1 was used to construct GCH1 overexpression mouse models to verify the effect of GCH1 on NAFLD. CETSA‐WB, co‐immunoprecipitation, and DARTS were employed in the COR‐GCH1 interaction and mechanism studies to validate the targeted binding relationship between COR and GCH1. Animal‐level studies showed that COR could improve glucose and lipid metabolism and liver function damage in mouse NAFLD, inhibit ferroptosis occurrence and lipid peroxidation injury. Cell‐level results showed that COR could inhibit hepatocyte ferroptosis. The ferroptosis‐inhibiting effect of GCH1 is independent of GPX4 and other signals; GCH1 directly inhibits reactive oxygen free radicals and suppresses lipid metabolism peroxidation damage. COR's effects are not related to classical anti‐ferroptosis signals such as GPX4 and Nrf2, but only related to GCH1. COR can target the GCH1 K155 site, stabilize protein levels by inhibiting GCH1 ubiquitination degradation, and when GCH1 is knocked out, COR's effects are antagonized. GCH1 has a protective role in NAFLD, and this effect is independent of classical GPX4 signaling. COR can target the GCH1 K155 site, inhibit GCH1 ubiquitination thereby preventing protein degradation and exerting protein stabilization. COR elevates GCH1 levels to inhibit ferroptosis. COR is a small molecule with potential for treating NAFLD.

Keywords: cordycepin, ferroptosis, GCH1, non‐alcoholic fatty liver disease, ubiquitination

COR stabilizes GCH1 and inhibits ferroptosis.

graphic file with name PTR-40-5886-g001.webp

1. Introduction

Non‐alcoholic fatty liver disease (NAFLD) refers to a clinical pathological syndrome in which hepatic steatosis reaches more than 5% through liver biopsy, without heavy alcohol consumption and without other definite liver injury factors such as viral, autoimmune, or drug‐induced liver disease (Younossi et al. 2018; Manne et al. 2018). The mechanism of liver transformation from steatosis to steatohepatitis remains unclear. Some studies have found that inhibiting ferroptosis rather than apoptosis can reduce hepatocyte necrosis, immune cell infiltration, and inflammatory responses (Tsurusaki et al. 2019); this suggests that hepatocyte ferroptosis may serve as a trigger for steatohepatitis inflammation initiation and is another therapeutic target for NAFLD treatment (Stockwell et al. 2017; Jhelum et al. 2020).

Cordycepin is an adenosine derivative isolated from Cordyceps sinensis, with anti‐inflammatory, immunomodulatory, anti‐diabetic, anti‐hyperlipidemic, antioxidant effects, as well as preventive effects on cardiovascular diseases and anti‐cancer effects (Khan and Tania 2023; Lan et al. 2021). In liver disease research, it was found that Cordyceps sinensis mycelium can alleviate liver inflammation by inhibiting Toll‐like receptor 4 (TLR4) expression and suppressing the NF‐κB pathway, thereby reducing circulating TNF‐α levels, improving survival rates, improving liver function, and inhibiting hepatocyte apoptosis and necrosis (Chen et al. 2023). Cordycepin also inhibits lipopolysaccharide (LPS)‐induced production of pro‐inflammatory cytokines in macrophages, exerting anti‐inflammatory effects (Khan and Tania 2020). However, the exact targets and mechanisms of cordycepin in NAFLD have not yet been clarified.

Among classical anti‐ferroptosis signals, GPX4 and Nrf2‐mediated signals are the most studied, but in addition, there are some non‐classical anti‐ferroptosis signals such as FSP‐1, NQO1, DHODH, and GCH1. These antioxidant factors act directly on reactive oxygen species, capturing reactive oxygen free radicals (Reichert et al. 2020). GCH1‐BH4 is a novel endogenous antioxidant signal independent of GPX4/GSH (Hou et al. 2016). GCH1 mainly promotes BH4 synthesis. GCH1 acts as a radical‐trapping antioxidant to protect against lipid peroxidation damage, and GCH1 can also mediate anti‐ferroptosis effects through CoQH2 (Zhang et al. 2025). Overall, GCH1 is an important anti‐ferroptosis signal in vivo, but the role and regulation of GCH1 in NAFLD have not yet been clarified. Through constructing high‐fat diet‐induced NAFLD studies, we showed that GCH1 plays a positive role in NAFLD, inhibiting hepatocyte ferroptosis and lipid metabolism in NAFLD, independent of other anti‐ferroptosis signals, while cordycepin can target and bind to GCH1, increasing protein levels by inhibiting GCH1 ubiquitination degradation, thereby exerting anti‐ferroptosis effects.

2. Material and Methods

2.1. Construction and Treatment of Animal Models

Animal experimental procedures complied with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health and were approved by the Animal Care and Use Committee of Jiaxing. Three‐week‐old male C57BL/6J mice (Charles River Co. Ltd., Beijing, China) were selected and housed under ideal conditions (temperature 21°C ± 1°C, relative humidity 50%), with free access to food and water. After 1 week of acclimatization, mice were used for experiments. Mice were divided into regular diet (RD, Charles River Co. Ltd., Beijing, China) or high‐fat diet (HFD, lipids providing 60% energy; Beijing HFK Biotechnology Co. Ltd., Beijing, China) (Guan et al. 2023; Drummer et al. 2023). After 10 weeks, RD and HFD mice were divided into four groups (Mice were randomly divided into groups using the dice method): RD‐Saline, RD‐COR, HFD‐Saline, and HFD‐COR, with 10 mice per group. COR (Purity ≥ 99%) was administered by gavage at a dose of 25 mg/kg, once daily for four consecutive weeks.

For mechanistic studies, we constructed liver‐specific GCH1 knockout mice (GCH1‐CKO) by crossing GCH1fl/fl mice with liver‐specific Cre recombinase mice. This model was constructed by Nanjing Jiancheng Model Animal Research Institute (Nanjing, China), achieving liver‐specific GCH1 knockout (Xie et al. 2024). Similarly, GCH1‐CKO mice were divided into four groups: RD‐Saline, RD‐COR, HFD‐Saline, and HFD‐COR, with 10 mice per group, using the same treatment methods as described above. In the GCH1 overexpression model, we constructed an adeno‐associated virus serotype 9 (AAV9) liver‐targeting vector carrying GCH1, using AAV9‐NC (EV) as a control (constructed by Shanghai ObiO Company). GCH1 liver‐overexpressing mice (AAV9‐GCH1) were established by tail vein injection (1 × 10^9 vg/mouse) followed by 4 weeks of expression.

2.2. Glucose and Insulin Tolerance Tests (Guan et al. 2023)

For the glucose tolerance test, mice were fasted for 12 h before glucose injection (1 g/kg body weight, Sigma, St. Louis, MO, USA). Blood samples were collected from the tail tip using a portable glucometer at 0, 15, 30, 60, 90, and 120 min after glucose injection. Similarly, after fasting for 6 h, insulin (0.5 IU/kg, Novolin R, Novo Nordisk, Denmark) was administered by intraperitoneal injection, and blood glucose was monitored as previously described. Curves and area under the curve were analyzed.

2.3. Blood Biochemical Analysis

Commercially available kits were used to measure plasma triglyceride (TG), total cholesterol (TC), fasting blood glucose, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels (Nanjing Jiancheng Bioengineering Institute, China). Instructions were strictly followed, and each step was repeated three times.

2.4. Histological Staining

Liver sections were cut using a semi‐automatic rotary microtome and stained with hematoxylin and eosin (H&E). Additionally, modified staining kits (Solarbio Life Science, Beijing, China) were used for Masson's trichrome and Sirius red staining to assess fibrosis. Frozen liver sections were stained with Oil Red O (Solarbio Life Science). All samples were examined and photographed under a microscope (BX51, Olympus, Tokyo, Japan).

2.5. Detection of Lipid Peroxidation and Antioxidant Activity

Liver tissue homogenates were centrifuged (12,000 g, 10 min) to prepare lysates. Protein concentration was determined using a protein assay kit (CW0014, CWBIO, Beijing, China). According to the manufacturer's instructions, commercial kits were used to measure malondialdehyde (MDA, S0131S; Beyotime, Beijing, China) content, glutathione peroxidase (GSH, S0056; Beyotime), and superoxide dismutase (SOD, S0101S; Beyotime) activity in the liver.

3. Immunohistochemical Staining

For GCH1 and Collagen I staining, sections were dewaxed, subjected to antigen retrieval, and treated with 3% hydrogen peroxide, then blocked with BSA. Rabbit anti‐GCH1 and Collagen I monoclonal antibodies (Abcam, USA) were diluted at GCH1 (1:100) and Collagen I (1:100) and incubated overnight at 4°C. Sections were washed with PBS, then horseradish peroxidase‐labeled secondary antibody was added dropwise and incubated at room temperature for 1 h, followed by three PBS washes of 5 min each. DAB chromogenic reaction was performed, nuclei were stained with hematoxylin, and sections were mounted for microscopic examination.

3.1. Real‐Time Quantitative PCR Analysis

Total mRNA was reverse transcribed to generate cDNA using a first‐strand cDNA synthesis kit (R312‐01; Nanjing Vazyme Biotech Co. Ltd.). RT‐PCR amplification was then performed using AceQ qPCR SYBR green master mix (Q111‐02; Nanjing Vazyme Biotech Co. Ltd.) with selected gene primers. GAPDH expression levels were used to normalize relative mRNA levels.

The primers were listed in Table S1 of the Supporting Information.

3.2. Plasmid Construction and Application

In co‐immunoprecipitation experiments, we used recombinant full‐length and mutant plasmids, including pCMV‐GCH1‐WT‐HA, pCMV‐GCH1‐K54‐HA, pCMV‐GCH1‐K93‐HA, pCMV‐GCH1‐K151‐HA, and pCMV‐GCH1‐K160‐HA plasmids, which were constructed by Genma Bio. The four mutants of GCH1 at K54, K93, K151, and K160 were obtained by Lys mutation. HEK 293T tool cells were cultured and transfected using Lipofectamine 6000, then cultured for an additional 24 h before being used for co‐immunoprecipitation experiments.

3.3. Co‐Immunoprecipitation and Western Blot Detection

Tissues and cells were lysed using RIPA lysis buffer (Beyotime, China). Protein concentration was determined and adjusted by the BCA kit (Beyotime, China) using the colorimetric method. Proteins were separated by SDS‐PAGE gel electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk for 1.5 h, membranes were incubated with rabbit anti‐GPX4, Nrf2, FSP‐1, DHODH, GCH1, TFR1, FTH1, and FTL monoclonal antibodies (Abcam, USA). Membranes were then treated with HRP‐labeled secondary antibody for 1 h. Subsequently, ECL fluorescence detection kit mixture was applied to the membrane, and visualization was performed using a gel imaging system. Image visualization and analysis were conducted using the Bio‐Rad image analysis system.

For co‐immunoprecipitation experiments, cell or tissue lysates were incubated with specified antibodies (1:200) overnight at 4°C, with total lysates used as input controls. Subsequently, lysates were immunoprecipitated using Protein A+G Agarose beads at 4°C for 2–4 h. After three washes with PBS, Western blotting was performed on Input or IP proteins.

3.4. Cellular Thermal Shift Assay‐Western Blot (CETSA‐WB)

Heat pulses were performed using total proteins. Cellular protein lysates were transferred to PCR tubes and incubated with COR for 1 h. Then, samples were heated at different temperatures for 3 min, followed by cooling in a thermal cycler at 4°C for 3 min. After centrifugation for 30 min (12,000 rpm, 4°C), soluble protein supernatants were used for Western blotting.

3.5. Drug Affinity Responsive Target Stability (DARTS)

100 μg of cellular proteins were incubated with COR treatment at room temperature for 1 h. Then, different concentrations of thermolysin were used to digest at room temperature for 10 min. Subsequently, proteolysis was terminated with phenylmethylsulfonyl fluoride (PMSF) on ice for 5 min. Finally, proteins were separated by SDS‐PAGE gel electrophoresis. Protein levels were assessed by Western blotting.

3.6. Cell Treatment

Experiments were conducted using mouse primary hepatocytes (Procell, China). Cells were cultured in DMEM (Gibco, Germany) supplemented with 10% FBS (Vazyme, China) and 1% streptomycin and penicillin (NCM, China). Cultures were maintained in a humidified incubator at 37°C with 5% CO2. MPA mixed solution was used to induce cell lipid injury. MPA is a mixed solution of palmitic acid (PA) and oleic acid (OA), with PA 0.25 μmol/L and PSA 0.5 μmol/L. Control cells were treated with PBS. Cells were divided into Ctrl‐PBS, Ctrl‐COR, MPA‐PBS, and MPA‐COR groups, with COR intervention at a concentration of 5 μM.

3.7. Cell Fluorescence Staining

DHE probe was used to detect cellular ROS expression, FerroOrange probe was used to detect Fe2+ expression, and C11‐BODIPY 581/591 probe was used to detect lipid peroxidation levels. Cells were stained according to the instructions, and cell images were observed under an inverted fluorescence microscope. Cells were incubated in 2 μmol/L C11‐BODIPY 581/591 at 37°C in the dark for 30 min. Images were captured under an Olympus DX51 fluorescence microscope (Tokyo, Japan), and fluorescence intensity was measured using Image‐J.

For fluorescence staining of GCH1 and FSP‐1 in cells, after fixation with paraformaldehyde and blocking with horse serum, rabbit anti‐GCH1 and FSP‐1 monoclonal antibodies were added dropwise to cells and incubated overnight at 4°C. Secondary antibody was added and incubated at room temperature in the dark for 2 h. Hoechst dilution (1:5000) was added and incubated at room temperature for 15 min, then mounted. Images were observed under an Olympus DX51 fluorescence microscope (Tokyo, Japan).

3.8. Cell Viability

Cell viability was determined using the CCK‐8 method. Cells were seeded in 96‐well plates at a density of 4 × 10^4 cells/well and cultured for 48 h. Cell viability was detected by the CCK‐8 method, and absorbance at 450 nm was analyzed.

3.9. Small Molecule‐Protein Docking

The 2D structure of the small molecule ligand was obtained from the PubChem database (http://pubchem.ncbi.nlm.nih.gov/), and its 3D structure was created using Chem Office 20.0 software. Protein targets were selected from the RCSB PDB database (http://www.rcsb.org/). Protein structures were processed using PyMOL 2.6.0. Molecular Operating Environment 2019 software was used for energy minimization of compounds. Molecular docking was performed using MOE 2019 with 50 operation cycles.

3.10. Statistical Methods

In our study, results were analyzed using GraphPad Prism 10 and expressed as mean ± standard error of the mean (SEM). The experimental design used randomization and blind analysis to generate equal‐sized groups, and no data points were excluded. Analysis was conducted under blind conditions. Tukey's post hoc test was used for comparisons among multiple groups, while Student's t‐test was used to evaluate comparisons between two groups. p < 0.05 was considered statistically significant.

4. Results

4.1. Cordycepin Improves Liver Injury and Glucose‐Lipid Metabolism in NAFLD Mice

High‐fat diet feeding successfully constructed mouse NAFLD, while causing glucose‐lipid metabolism disorders and liver injury in mice. Mice fed with regular diet given COR intervention showed no obvious pathological changes or glucose‐lipid metabolism disorders. HFD‐Saline mice showed significant body weight increase and liver weight increase, while abdominal fat and subcutaneous fat content increased, and TC and TG levels in peripheral blood increased, showing significant differences compared to RD‐Saline. Compared to HFD‐Saline, HFD‐COR mice showed significant decreases in body weight and liver weight, decreased abdominal and subcutaneous fat content, and decreased TC and TG levels, with significant differences (Figure 1A). In liver tissue pathological staining, H&E staining showed that HFD‐Saline exhibited obvious vacuolar changes and inflammatory injury, with significant hepatocyte damage. Meanwhile, Oil‐Red staining showed obvious lipid deposition, consistent with NAFLD pathological characteristics, showing significant differences compared to RD‐Saline. HFD‐Saline showed fibrotic lesions, and SR and Masson staining showed obvious collagen fiber deposition. Compared to HFD‐Saline, HFD‐COR showed significantly reduced liver injury, lipid deposition, and fibrotic lesions (Figure 1B,C). Fasting blood glucose and AST, ALT analysis results showed that indicator levels were significantly elevated in HFD‐Saline, suggesting liver injury, while HFD‐COR could reduce indicator levels (Figure 1D). Glucose tolerance and insulin tolerance test results also showed that COR could restore high‐fat diet‐impaired glucose tolerance and insulin sensitivity (Figure 1E).

FIGURE 1.

FIGURE 1

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COR improves glucose‐lipid metabolism disorders and liver injury in NAFLD Mice. (A) Mouse body weight, liver weight, subcutaneous fat content, abdominal fat content, and peripheral blood TC and TG levels (n = 10). (B, C) Tissue pathological staining, including H&E, Oil Red O, SR, and Masson, representative images at 200× magnification with 100 μm scale bar and quantitative analysis statistics (n = 10). (D) Fasting blood glucose level analysis, AST and ALT levels (n = 10). (E) Glucose tolerance and insulin tolerance analysis results (n = 10). Data are expressed as mean ± SEM, with each point representing an independent experiment. Inter‐group comparisons:*p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences; ns p > 0.05 indicates no significant difference.

4.2. Cordycepin Inhibits Ferroptosis in NAFLD Mice

We detected ferroptosis‐related indicators in liver tissue. In HFD‐Saline, Fe2+, MDA, LPO, LOX, and SQS levels were upregulated, indicating obvious lipid peroxidation injury, while SOD and GSH levels were downregulated, weakening anti‐ferroptosis ability. COR could reverse these changes and improve anti‐ferroptosis ability (Figure 2A). GCH1 and Collagen I staining showed that COR could increase GCH1 positive rate, reduce Collagen I levels, improve anti‐ferroptosis ability, and inhibit liver fibrosis (Figure 2B,C). DCFH‐DA probe detection of ROS showed that ROS was significantly upregulated in HFD‐Saline, and COR could inhibit ROS production (Figure 2D,E). In iron metabolism detection, it was found that HFD induced elevated protein levels of transferrin receptor 1 (TFR1) and divalent metal transporter 1 (DMT1) in the liver, and reduced ferroportin (FPN) protein levels. In addition to restoring Fpn1 and Dmt1 levels in the liver, COR could also reduce mRNA levels of ferritin heavy chain 1 (Fth1) and acyl‐CoA synthetase long‐chain family member 4 (Acsl4) (Figure 2F). In ferroptosis‐related protein detection, we found that in HFD, COR had no obvious effect on GPX4, and had limited effects on Nrf2, FSP1, and GCH1 expression, but could significantly increase GCH1 expression (Figure 2G,H).

FIGURE 2.

FIGURE 2

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COR inhibits ferroptosis in NAFLD mice. (A) Ferroptosis indicators (n = 10). (B, C) GCH1 and Collagen I immunohistochemical staining and quantitative analysis, representative images at 200× magnification with 100 μm scale bar and quantitative analysis statistics (n = 10). (D, E) DCFH‐DA probe staining and fluorescence intensity quantitative analysis, representative images at 200× magnification with 100 μm scale bar and quantitative analysis statistics (n = 10). (F) Real‐time fluorescence quantitative experiment detection of mRNA quantitative analysis results (n = 10). (G, H) Western blot detection results of relative protein expression and quantitative analysis, including ferroptosis proteins GPX4, Nrf2, FSP‐1, and GCH1 (n = 10). Data are expressed as mean ± SEM, with each point representing an independent experiment. Inter‐group comparisons: *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences; ns p > 0.05 indicates no significant difference.

4.3. Cordycepin Inhibits Hepatocyte Injury and Ferroptosis

In hepatocyte‐level studies, we first tested the toxicity of COR on hepatocytes. Results showed that COR showed no obvious cytotoxicity at 100 μmol/L (Figure 3A). In MPA‐induced cell injury, COR could inhibit cell injury, with the best effect at 5 μmol/L. Therefore, 5 μmol/L was used as the COR intervention concentration in subsequent studies (Figure 3B). DHE probe detection of ROS and FerroOrange probe detection of Fe2+ showed that MPA could increase ROS and Fe2+ levels. To further verify lipid ROS products, C11‐BODIPY was used in the experiment, which changes from red to green upon oxidation. MPA could increase C11 levels, while COR could reverse MPA's effects, reducing ROS, Fe2+, and C11 levels (Figure 3D,E). At the same time, COR could also reduce MDA levels (Figure 3F). GCH1 fluorescence staining showed that MPA reduced GCH1 expression, and COR could reverse this change (Figure 3G,H). COR could also increase GCH1 levels in cells (Figure 3I,J). In addition, MPA can induce the expression of TC and TG in cells, but COR can reduce the expression level of lipids (Figure 3K).

FIGURE 3.

FIGURE 3

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COR inhibits hepatocyte injury and ferroptosis. (A) COR's effect on cell viability detection results (n = 5). (B) Inhibitory effect of COR on MPA‐induced cell injury (n = 5). (C–E) Results and statistical analysis of DHE staining for ROS detection, FerroOrange staining for Fe2+ detection, and C11‐BODIPY 581/591 staining, representative images with 50 μm and 100 μm scale bars and statistical analysis results (n = 5). (F) MDA analysis results (n = 5). (G, H) GCH1 fluorescence staining and quantitative analysis, representative images with 100 μm scale bar and statistical analysis results (n = 5). (I, J) Western blot detection results of relative protein expression and quantitative analysis (n = 5). (K) TC and TG (n = 5). Data are expressed as mean ± SEM, with each point representing an independent experiment. Inter‐group comparisons: *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences; ns p > 0.05 indicates no significant difference.

4.4. GCH1 Is Essential for COR to Exert Its NAFLD Protective Effect

In the GCH1‐specific knockout mouse model, we further clarified the relationship between GCH1 and COR. From the results, after GCH1 knockout, COR's effects were suppressed and could not exert protective effects on NAFLD. There were no significant differences in body weight, liver weight, subcutaneous/abdominal fat, and TC, TG between HFD‐Saline and HFD‐COR (Figure 4A). Tissue staining showed no significant differences in lipid deposition area, SR and Masson staining area between HFD‐Saline and HFD‐COR (Figure 4B,C). There were no significant differences in fasting blood glucose, AST, and ALT (Figure 4D). At the same time, there were no obvious differences in glucose tolerance and insulin tolerance (Figure 4E).

FIGURE 4.

FIGURE 4

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GCH1 knockout eliminates the protective effect of COR on NAFLD glucose‐lipid metabolism and liver injury. (A) Mouse body weight, liver weight, subcutaneous fat content, abdominal fat content, and peripheral blood TC and TG levels (n = 10). (B, C) Tissue pathological staining, including H&E, Oil Red O, SR, and Masson, representative images at 200× magnification with 100 μm scale bar and quantitative analysis statistics (n = 10). (D) Fasting blood glucose level analysis, AST and ALT levels (n = 10). (E) Glucose tolerance and insulin tolerance analysis results (n = 10). Data are expressed as mean ± SEM, with each point representing an independent experiment. Inter‐group comparisons: *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences; ns p > 0.05 indicates no significant difference.

4.5. COR Cannot Inhibit Ferroptosis in NAFLD After GCH1 Knockout

In the GCH1 conditional knockout model, there were no significant differences in ferroptosis indicators Fe2+, MDA, LPO, LOX, SQS, as well as SOD and GSH between HFD‐Saline and HFD‐COR (Figure 5A). There were also no differences in Collagen I positive cells. At the same time, COR could not inhibit ROS levels in tissues (Figure 5D,E). Additionally, COR had no significant effect on mRNA expression of iron metabolism‐related indicators (Figure 5F). In ferroptosis and iron metabolism protein detection, COR had no significant effect on GPX4, FSP‐1, and GCH1, nor on TFR2, FTH1, and FTL. At the same time, GCH1 showed no significant expression (Figure 5G,H).

FIGURE 5.

FIGURE 5

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GCH1 knockout eliminates the ferroptosis‐inhibiting effect of COR. (A) Ferroptosis indicators (n = 10). (B, C) Collagen I immunohistochemical staining and quantitative analysis, representative images at 200× magnification with 100 μm scale bar and quantitative analysis statistics (n = 10). (D, E) DCFH‐DA probe staining and fluorescence intensity quantitative analysis, representative images at 200× magnification with 100 μm scale bar and quantitative analysis statistics (n = 10). (F) Real‐time fluorescence quantitative experiment detection of mRNA quantitative analysis results (n = 10). (G, H) Western blot detection results of relative protein expression and quantitative analysis, including ferroptosis proteins GPX4, FSP‐1, GCH1, and iron metabolism proteins FTH1, FTL, TFR2 (n = 10). Data are expressed as mean ± SEM, with each point representing an independent experiment. Inter‐group comparisons: *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences; ns p > 0.05 indicates no significant difference.

4.6. GCH1 Knockout Eliminates the Inhibitory Effect of COR on Hepatocyte Ferroptosis

In GCH1 knockout hepatocytes, MPA could induce upregulation of ROS and Fe2+ expression, while COR could not reverse this change. At the same time, there was no significant effect on C11‐BODIPY levels, and no significant difference in fluorescence intensity (Figure 6A–C). COR could not reduce MPA‐induced high MDA expression (Figure 6D). After FSP‐1 staining, it was shown that COR did not affect FSP‐1 expression, proving that COR only acts on GCH1 (Figure 6E,F). Protein detection results showed that after GCH1 knockout, COR had no significant effect on ferroptosis proteins GPX4, Nrf, FSP‐1, and GCH1, nor on TFR2, FTH1, and FTL. At the same time, GCH1 showed no significant expression (Figure 6G,H).

FIGURE 6.

FIGURE 6

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GCH1 knockout eliminates the inhibitory effect of COR on hepatocyte ferroptosis. (A–C) Results and statistical analysis of DHE staining for ROS detection, FerroOrange staining for Fe2+ detection, and C11‐BODIPY 581/591 staining, representative images with 50 μm and 100 μm scale bars and statistical analysis results (n = 5). (D) MDA analysis results (n = 5). (E, F) FSP‐1 fluorescence staining and quantitative analysis, representative images with 50 μm scale bar and statistical analysis results (n = 5). (G, H) Western blot detection results of relative protein expression and quantitative analysis (n = 5). Data are expressed as mean ± SEM, with each point representing an independent experiment. Inter‐group comparisons: *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences; ns p > 0.05 indicates no significant difference.

4.7. Determination of COR‐GCH1 Interaction and Binding Sites

The above results showed a direct relationship between COR and GCH1 expression. We performed docking of COR and GCH1. The molecular docking energy was −4.7634 kcal/mol. Residues Val149 and Glu207 on the protein receptor formed hydrogen bond interactions with cordycepin. Residue Lys224 formed electrostatic interactions with cordycepin. Residue Lys151 formed hydrophobic interactions with cordycepin, where Lys151 (K151) is a ubiquitination site of GCH1 (Figure 7A). DARTS detection confirmed that COR and GCH1 can directly bind, and COR protects GCH1 from protease degradation (Figure 7B). CETSA results showed that as temperature increased, COR slowed the decrease of GCH1. After adding COR, the protein's thermal denaturation curve shifted to the right. These results indicate that COR enhances GCH1's thermal stability within a certain temperature range (Figure 6C,D). K54, K93, K151, and K160 are major ubiquitination sites in GCH1. We constructed mutants and tested them with CETSA. Results showed that K151 is COR's main binding site, affecting protein stability, consistent with docking results (Figure 7E,F). Co‐immunoprecipitation detection of COR and GCH1 binding showed that after co‐incubation of Biotin‐COR and recombinant GCH1, binding between the two was observed (Figure 7G–I). Co‐precipitation showed that wild‐type GCH1 binds to COR, but no binding relationship exists in the K151 mutant. This proves that COR binds to GCH1 at the K151 site (Figure 7J).

FIGURE 7.

FIGURE 7

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Validation of the binding relationship between COR and GCH1. (A) Molecular docking simulation results and energy analysis of COR and GCH1. (B) DARTS experimental results (n = 5). (C, D) CETSA experimental results (n = 5). (E, F) Construction schematic of GCH1 mutants and CETSA experimental results (n = 5). (G–I) Co‐immunoprecipitation detection results of COR‐GCH1 (n = 5). (J) Co‐immunoprecipitation detection results of COR‐GCH1 in K155 site mutant (n = 5).

4.8. COR Regulates GCH1 Degradation Through the Ubiquitin‐Proteasome Pathway

Our previous results showed that COR interacts with GCH1, increasing GCH1 protein levels. We detected GCH1 mRNA levels. Results showed that COR does not affect mRNA levels (Figure 8A). COR affects post‐transcriptional protein expression. We found that K151 is a ubiquitination site of GCH1, so the proteasome pathway may be a key regulatory pathway for GCH1. We treated with proteasome inhibitor MG132 and protein biosynthesis inhibitor CHX. Results showed that CHX enhanced GCH1 expression downregulation, which COR reversed. MG132 upregulated GCH1 levels, and combined COR treatment did not affect GCH1 expression. This indicates that COR regulates GCH1 levels through ubiquitination (Figure 8C,D). We detected ubiquitination levels in tissues and cells. Results showed that in tissues, HFD treatment could increase GCH1 ubiquitination levels, which COR reversed (Figure 8B). Similar results were observed in cells (Figure 8E). More importantly, co‐immunoprecipitation experiments strongly confirmed that COR significantly downregulated ubiquitin‐bound GCH1 protein levels, thereby inhibiting the GCH1 ubiquitin‐proteasome degradation pathway (Figure 8F,G). K54 and K151 are important ubiquitination sites in GCH1. We constructed point mutants for research. Results showed that K151 mutation affected COR's ubiquitination effect on GCH1, while K54 showed no change. This indicates that K151 is COR's main target site (Figure 8H,I).

FIGURE 8.

FIGURE 8

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COR regulates GCH1 protein degradation by inhibiting the ubiquitin‐proteasome pathway. (A) mRNA detection results (n = 5). (B) Effect of COR on GCH1 ubiquitination in liver tissue (n = 5). (C, D) Detection results of GCH1 protein relative expression levels (n = 5). (E) Effect of COR on GCH1 ubiquitination in hepatocytes (n = 5). (F, G) Detection of GCH1 ubiquitination (Ub‐GCH1) by immunoprecipitation (n = 5). (H, I) Effect of COR on GCH1 ubiquitination after ubiquitination site mutation (n = 5). (J) Surface diagram of COR‐GCH1 protein binding conformation, with the binding pocket indicated by dashed lines. Data are expressed as mean ± SEM, with each point representing an independent experiment. Inter‐group comparisons: *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences; ns p > 0.05 indicates no significant difference.

4.9. GCH1 Overexpression Can Improve Liver Injury in NAFLD Mice

We performed AAV9‐GCH1 overexpression treatment in wild‐type mice to simulate liver GCH1 overexpression. AAV9 is a liver‐targeting adenovirus that can accumulate in the liver. We used empty vector EV as a control. After high‐fat diet induction, results showed that in AAV9‐GCH1, mouse body weight, liver weight, and abdominal and subcutaneous fat were significantly lower than in EV, while TC and TG levels were also lower than in EV (Figure S1A). Histological staining showed that lipid deposition area, SR and Masson fibrotic lesion tissue area in AAV9‐GCH1 were significantly lower than in EV (Figure S1B,C). Liver function AST and ALT results also showed that indicator levels in AAV9‐GCH1 were lower than in EV (Figure S1D).

We also conducted a Rescue experiment, performing AAV9‐GCH1 overexpression treatment in GCH1 conditional knockout mice to simulate liver GCH1 restoration. Results showed that after GCH1 restoration expression, mouse body weight, liver weight, and abdominal and subcutaneous fat were significantly lower than in EV, while TC and TG levels could also be reduced (Figure S2A). Histological staining showed that lipid deposition area, SR, and Masson fibrotic lesion tissue area in AAV9‐GCH1 were significantly lower than in EV (Figure S2B,C). Liver function AST and ALT results also showed that indicator levels in AAV9‐GCH1 were lower than in EV (Figure S2D).

5. Discussion

Non‐alcoholic fatty liver disease is one of the most common chronic liver diseases, with a disease spectrum ranging from simple steatosis to steatohepatitis, advanced fibrosis, and cirrhosis (Quiles Del Rey and Mancias 2019). Non‐alcoholic steatohepatitis (NASH) is the most severe form of NAFLD and is a critical node for clinical intervention (Li et al. 2020). Studies have shown that programmed cell death triggered by intracellular lipid accumulation in hepatocytes is a possible cause of liver tissue damage and inflammation (Cho et al. 2022). Ferroptosis is the first form of cell death to occur in NASH. In ferroptosis, iron homeostasis imbalance is one of the important inducing factors. When excess free Fe2+ exists in cells, hydroxyl radicals will be formed through the Fenton reaction (Weiland et al. 2019; Killion et al. 2018). This induces DNA, protein, and lipid damage, causing cytotoxicity. In addition to iron pool accumulation, genes affecting iron homeostasis are also involved in ferroptosis, and the occurrence of lipid peroxidation drives the occurrence and progression of ferroptosis (Doll et al. 2017). In 2012, scholars discovered that serum iron > 1.5 times the upper limit of normal in NAFLD patients was closely related to liver iron deposition and histological inflammation, and considered elevated serum iron levels as an independent risk factor for NAFLD progression to NASH and fibrosis (Duan et al. 2022; Wu et al. 2020). It has also been reported that serum ferritin levels are significantly elevated in NASH patients, suggesting some connection between NASH and abnormal liver iron metabolism (Dixon et al. 2012; Stockwell et al. 2017). Taking vitamin E can improve liver iron accumulation and lipid peroxidation in NASH patients, reduce serum transaminase levels, reduce hepatic steatosis, and thereby improve NASH (Jhelum et al. 2020). After administering RSL3 (a ferroptosis inducer) to NASH model mice induced by methionine/choline‐deficient diet (MCD), hepatic steatosis was found to be exacerbated (Kowdley et al. 2012), while treatment with sodium selenite (GPX4 activator) or Fer‐1 significantly reduced NASH severity (Pierantonelli and Svegliati‐Baroni 2019). In the regulation of ferroptosis, the GCH1 signal plays an important role, and this effect is independent of classical signals such as GPX4 (Lin et al. 2025; Yu et al. 2020). GCH1 is the rate‐limiting enzyme for BH4 (antioxidant) synthesis, and BH4 content affects cellular sensitivity to ferroptosis. The GCH1‐BH4 axis can control endogenous BH4 production, CoQ10 abundance, and abnormal phospholipid peroxidation, thereby inhibiting ferroptosis.

Our study overall discovered the mechanism and target of cordycepin in treating NAFLD. Cordycepin is one of the most important components of Cordyceps sinensis. Our results showed that GCH1 is the direct target of COR, and COR can bind to GCH1 to stabilize GCH1 protein levels. A high‐fat diet is a commonly used method for constructing NAFLD models. We first administered COR by gavage in wild‐type mice. COR had no obvious effect on healthy wild‐type mice, and mouse liver function was not affected. However, COR could improve liver injury in NAFLD mice, reduce glucose‐lipid metabolism disorders, effectively reduce subcutaneous and abdominal fat content, improve insulin resistance, and simultaneously inhibit fibrotic lesions in tissues (Yan et al. 2025; Tang et al. 2021).

Further testing found that COR could inhibit ferroptosis occurrence in the liver. This effect was not significantly correlated with activation of classical signals such as GPX4 but was related to GCH1 expression. GCH1 can interact with reactive oxygen species, capture free radicals, and inhibit the lipid peroxidation process. After COR binds to K155 of GCH1, it inhibits the ubiquitination process. Ubiquitination is a way of protein degradation (Mou et al. 2019). When proteins are ubiquitinated, the proteasome system can be activated to degrade proteins and reduce protein levels. However, COR is not correlated with GCH1 mRNA levels, which also indicates that COR's effects are mainly post‐translational modifications. To further explore the specificity of COR's effects on GCH1, we constructed GCH1‐specific knockout mouse models and cell models. We found that when GCH1 is absent, COR's beneficial effects are suppressed. COR no longer improves liver lesions in NAFLD mice, has no obvious effect on glucose‐lipid metabolism disorders, and more importantly, COR cannot further inhibit lipid peroxidation and ferroptosis occurrence. This also proves that GCH1 is the only target of COR, and the two have specificity. We also studied the role of GCH1 in NAFLD. When GCH1 is overexpressed, it can inhibit liver injury and glucose‐lipid metabolism disorders in NAFLD mice, and when GCH1 restoration expression is performed in GCH1 knockout mice, it can still improve the pathology of NAFLD mice, indicating that GCH1 plays a positive role in NAFLD.

Although our study demonstrated the beneficial effects and mechanisms of COR on NAFLD, there are still some limitations. First, we need to further clarify the effective dose and toxic dose of COR in NAFLD. Second, the metabolism of COR in NAFLD needs further study.

6. Conclusion

The results of this study indicate that during the progression of HFD‐induced NAFLD, ferroptosis of hepatocytes in the liver plays an important role, and GCH1, as a key ferroptosis‐inhibiting factor, plays an important role in NAFLD. Cordycepin can exert anti‐ferroptosis effects by binding to GCH1 and stabilizing GCH1 protein expression, thereby alleviating the progression of NAFLD. This is an important effect of cordycepin and has certain significance for drug development for NAFLD.

Author Contributions

Chenyang Han: investigation, conceptualization. Shasha Wu: software, formal analysis, investigation. Wenyan Li: visualization, writing – review and editing. Yi Yang: investigation, resources, project administration. Hongyan Pei: methodology, validation. Zhiling Tang: funding acquisition, writing – original draft. Chenxi Cao: project administration, visualization, writing – review and editing. Jian Sheng: data curation, supervision, resources.

Funding

Jiaxing Science and technology planning project (2024AZ30004), (2023AY31009), (2024AY30019).

Disclosure

Our research paper have not been conducted using artificial intelligence.

Ethics Statement

The requirements considered to be relevant in recent guidelines for best practice in natural products pharmacological research have been taken into account.

Consent

All authors' approval published the article.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: GCH1 Overexpression Can Inhibit Liver Injury in NAFLD

(A) Mouse body weight, liver weight, subcutaneous fat content, abdominal fat content, and peripheral blood TC and TG levels (n = 10)

(B, C) Tissue pathological staining, including H&E, Oil Red O, SR, and Masson, representative images at 200× magnification with 100 μm scale bar and quantitative analysis statistics (n = 10)

(D) AST and ALT levels (n = 10)

Data are expressed as mean ± SEM, with each point representing an independent experiment. Inter‐group comparisons: *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences; ns p > 0.05 indicates no significant difference.

Figure S2: GCH1 Rescue Experiment.

(A) Mouse body weight, liver weight, subcutaneous fat content, abdominal fat content, and peripheral blood TC and TG levels (n = 10)

(B, C) Tissue pathological staining, including H&E, Oil Red O, SR, and Masson, representative images at 200× magnification with 100 μm scale bar and quantitative analysis statistics (n = 10)

(D) AST and ALT levels (n = 10)

Data are expressed as mean ± SEM, with each point representing an independent experiment. Inter‐group comparisons: *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences; ns p > 0.05 indicates no significant difference.

Table S1: Sequences of primers used for RT‐PCR.

PTR-40-5886-s001.docx (373.2KB, docx)

Contributor Information

Chenxi Cao, Email: caochenxi_74@163.com.

Zhiling Tang, Email: tangzl_72@126.com.

Data Availability Statement

The data that supports the findings of this study are available in the Supporting Information of this article.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: GCH1 Overexpression Can Inhibit Liver Injury in NAFLD

(A) Mouse body weight, liver weight, subcutaneous fat content, abdominal fat content, and peripheral blood TC and TG levels (n = 10)

(B, C) Tissue pathological staining, including H&E, Oil Red O, SR, and Masson, representative images at 200× magnification with 100 μm scale bar and quantitative analysis statistics (n = 10)

(D) AST and ALT levels (n = 10)

Data are expressed as mean ± SEM, with each point representing an independent experiment. Inter‐group comparisons: *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences; ns p > 0.05 indicates no significant difference.

Figure S2: GCH1 Rescue Experiment.

(A) Mouse body weight, liver weight, subcutaneous fat content, abdominal fat content, and peripheral blood TC and TG levels (n = 10)

(B, C) Tissue pathological staining, including H&E, Oil Red O, SR, and Masson, representative images at 200× magnification with 100 μm scale bar and quantitative analysis statistics (n = 10)

(D) AST and ALT levels (n = 10)

Data are expressed as mean ± SEM, with each point representing an independent experiment. Inter‐group comparisons: *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences; ns p > 0.05 indicates no significant difference.

Table S1: Sequences of primers used for RT‐PCR.

PTR-40-5886-s001.docx (373.2KB, docx)

Data Availability Statement

The data that supports the findings of this study are available in the Supporting Information of this article.

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