Coptidis rhizoma and evodiae fructus against lipid droplet deposition in nonalcoholic fatty liver disease‐related liver cancer by AKT

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in the world. NAFLD has become one of the major factors contributing to hepatocellular carcinoma (HCC) development. However, there are no clear targets and therapeutic drugs for NAFLD‐related liver cancer. This study explored the active compounds, target and mechanism of coptidis rhizoma and evodiae fructus in the treatment of NAFLD‐related liver cancer based on the network pharmacology and experimental verification. There were 455 intersection targets of NAFLD‐related liver cancer, and 65 drug‐disease common targets. AKT1 has the highest degree, indicating that it may be a key target of coptidis rhizoma and evodiae fructus in the treatment of NAFLD‐related liver cancer. The expression level of AKT1 was high in high‐risk group, and the overall survival rate was lower than that in low‐risk group. After oleic acid induction, p‐AKT expression and lipid droplet deposition were promoted in HepG2 cells. Quercetin and resveratrol reduced lipid droplet deposition in vivo. Moreover, quercetin inhibited p‐AKT expression, resveratrol both reduced the expression of p‐AKT and AKT. The overall findings suggested that quercetin inhibited AKT in the treatment of NAFLD‐related liver cancer.


| INTRODUCTION
Nonalcoholic fatty liver disease (NAFLD) is a major cause of morbidity and medical burden in the world. Large population-based cohort studies have shown a fourfold increase in the incidence of NAFLD-hepatocellular carcinoma (HCC) and a 2.5-fold increase in the incidence of hepatitis over the past decade, making it the fastestgrowing indication for liver transplantation (Wong et al., 2014;Zhang, Coker, et al., 2021;Zhang, Liu, et al., 2021). Therefore, NAFLD-related HCC cases are often diagnosed late and treatment options are limited. This highlights the need for a clearer understanding of the mechanisms involved in the transition from steatosis to liver cancer, which may help in the discovery of new biomarkers and the development of better screening methods (Rusu et al., 2022).
Also known as protein kinase B (PKB), AKT1 is a member of the AKT kinase family that regulates glycolipid metabolism, proliferation, and cell survival through a range of downstream substrates (Gao et al., 2022). In particular, upregulation of AKT1 subtype in HCC was closely related to the growth and proliferation of HCC cells . AKT regulated fat synthesis factor SREPB-1 through the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway, which increased fatty acid synthesis and lipid content in hepatocytes, accelerating the progression of NAFLD. Moreover, knockdown of glycosylated lysosomal membrane protein increased lipid droplets and differentially altered the PI3K/AKT pathway . Overall, AKT1 played a key role in lipid metabolism and was a promising therapeutic target for metabolic diseases.
At present, traditional Chinese medicine (TCM) has been practiced for many years. More and more attention has been paid to the treatment of metabolic disorders such as NAFLD, because of its remarkable efficacy and few side effects. Coptidis rhizoma (Chinese name: Huang-Lian) and evodiae fructus (Chinese name: Wu-Zhu-Yu) is a combination of classic herbs used in Chinese herbal formulations and is often used for compatibility (Zhang et al., 2022a). The different proportions of coptidis rhizoma and evodiae fructus had a wide range of pharmacological effects, including anti-inflammatory, anticancer, antioxidant, etc. (Wang et al., 2012;Wang, Miao, et al., 2021). coptidis rhizoma and evodiae fructus also had the functions of removing heat from the liver, reducing reflux and stopping vomiting. They were mainly used for stomach vomiting and hypoechoic pain caused by liver fire. Our previous study also showed that coptidis rhizoma and evodiae fructus at 2:1 had a liver protection effect on NAFLD patients (Zhang et al., 2022b). Moreover, a study has showed that Zuojin decoction (coptidis rhizoma-evodiae fructus 6:1) inhibits the growth of liver cancer in mice . Studies also have found that quercetin reduces AKT phosphorylation and improves diet-induced dysregulation of lipid metabolism in NAFLD through PI3K/AKT pathway (Pisonero-Vaquero et al., 2015).
Therefore, this study identified the active compounds and targets of coptidis rhizoma and evodiae fructus in the treatment of NAFLD-related liver cancer based on network pharmacology, providing a basis for in-depth study on the mechanism and clinical application of coptidis rhizoma and evodiae fructus in treating of NAFLD-related liver cancer. The research process is shown in Figure 1.

| Active compounds and potential targets of coptidis rhizoma and evodiae fructus
The active compounds and potential targets of coptidis rhizoma and evodiae fructus were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database (https://tcmspw.com/tcmsp.php) (Shen et al., 2020). Oral bioavailability (OB) and druglikeness (DL) are important indicators to evaluate whether compounds can be developed into drugs according to its absorption, distribution, metabolism and excretion. In TCMSP database, the compounds with OB > 30% and DL >0.18 were preserved . Then the targets names were standardized by UniProt (https://www. unipr ot.org/) (Szklarczyk et al., 2016) database.

NAFLD-associated liver cancer
The GeneCards (https://www.genec ards.org/) database, Therapeutic Target Database (TTD) (http://db.idrbl ab.net/ ttd/) and Online Mendelian Inheritance in Man (OMIM database) (https://omim.org/)  were used to seek the potential targets of NAFLD-related liver cancer. In GeneCards database, score value ≥ median was set as targets for NAFLD and liver cancer. All three databases used the keywords "non-alcoholic fatty liver disease" and "liver cancer." The related targets downloaded from these three databases were merged and then deduplicated to get the potential targets. Venny 2.1 (https://bioin fogp.cnb. csic.es/tools/ venny/ index.html)  online tool was used to draw Venn diagram to realize the core target between "Coptidis Rhizoma", "Evodiae Fructus" and "NAFLD-related liver cancer".

| Construction of intersection target PPI network and hub gene screening
A list of common targets was input into the STRING (https://cn.strin g-db.org/) (Bai et al., 2021) database to construct a PPI network. The cut-off values of some parameters were as follows: species, Homo sapiens; minimum interaction, high confidence (0.9); data type, TSV format. Subsequently, Cytoscape 3.8.2  software was used to visualize the PPI network. CytoHubba, a plug-in for Cytoscape was used to screen the top 10 core genes with higher degree.

| Analysis of GO and KEGG
GO and KEGG analysis of drug-disease common targets was performed using the Database for Annotation, Visualization and Integrated Discover (DAVID, https://david.ncifc rf.gov/) . Visual analysis was performed on bioinformatics tools (Hao et al., 2021) (http://www.bioin forma tics.com.cn/). Three parameters were used as the screening criteria ("Homo sapiens"; p ≤ .05; count ≥10).

| Prognostic analysis of AKT1
The prognostic analysis of AKT1 was performed by an online tool (Assistant for Clinical Bioinformatics, www. aclbi.com), which used a univariate Cox analysis of overall survival (OS) of patients included in The Cancer Genome Atlas (TCGA)-liver hepatocellular carcinoma (LIHC) set by using the "survival" package in the R (version 4.0.3).

| Validation of molecular docking
The compound-target PPI network was calculated by 'Network Analyzer' of Cytoscape software., and the top 4 active compounds were screened according to degree value. The 2D structures of active compounds were obtained from PubChem (https://pubch em.ncbi.nlm. nih.gov) (Xiong et al., 2022) database. Chem3D (Xiong et al., 2022) software was used for energy minimization. The protein ID number corresponding to the key target was searched using Uniport (https://www.unipr ot.org/; The UniProt Consortium, 2017) database. Then, Protein Data Bank (PDB, https://pdbj.org/mine/summa ry/) (Berman et al., 2000) database was used to download the F I G U R E 1 Schematic diagram of research process. CR represents coptidis rhizoma and, EF represents evodiae fructus. 3D structure of target protein. PyMol (Wang, Yuan, Wu, Liu, & Yang, 2021b) software was used to remove small molecule ligands and water molecules. Auto Dock Tools 1.5.6 (Wang, Yuan, et al., 2021) was used to add hydrogen bonds, and the binding energy was used as the evaluation index of molecular dock results. Finally, PyMol software was used to show the results.

| Modeling and oil red O staining
HepG2 cells at logarithmic growth stage were collected by washing and centrifugation to obtain a cell density of 1 × 10 5 cells /mL. The cells were inoculated in 6-well plates at 2 mL/ well and incubated at 37°C with 5% CO 2 .
After cell adhesion, four groups were set up, including control group, oleic acid (OA) group, OA + 50 μM quercetin group and OA + 20 μM resveratrol group. Three replicates were performed in each group. After cell adhesion, OA (800 μM, Cat. #O1008-1G, Sigma) was added to all groups except the control group for 24 h.
Oil red O staining (Cat. #G1262, Solarbio): The oil red O dyeing solution A and B were mixed in a ratio of 3:2 and left for 10 min at room temperature and was filtered before use. After 24 h of drug treatment in each group, 2 mL PBS was added into each well to wash twice, and red O fixing solution was added to fix for 20 min. The fixing solution was discarded, washed twice in distilled water, and rinsed with 60% isopropyl alcohol for 20-30 s. Isopropyl alcohol was discarded and oil red O dye was added for 15 min. Discard the dye solution, add 60% isopropyl alcohol and rinse for 20-30 s until the interstitium is clear, and wash for five times until there is no excess dye solution. Add oil red O buffer for 1 min, discard, add distilled water to cover the cells, and take photos (DMI1, LEICA). Finally, the results were measured by an Image-Pro Plus v6.0 software (Media Cybemetic).

| Statistical analysis
All statistical tests were performed by GraphPad Prism 8 software. Student's t-test was used between two groups, and one-way ANOVA analysis was used when there were three or more groups. All experiments were repeated at least three times, with at least three replicates per sample. All p values <.05 were considered statistically significant.

| Preparation of active compounds and target of coptidis rhizoma and evodiae fructus
According to OB > 30% and DL >0.18, a total of 14 compounds of coptidis rhizoma were obtained, including 11 compounds with targets. Thirty compounds of evodiae fructus were obtained, including 24 compounds with targets in the TCMSP database. Among these compounds, quercetin and berberine were common compounds of coptidis rhizoma and evodiae fructus (Table 1). By combination with UniPort database, a total of 165 targets of coptidis rhizoma and 191 targets of evodiae fructus were obtained. After merging and removing duplicate items, a total of 193 target genes were obtained.

NAFLD-related liver cancer
The target genes of NAFLD-related liver cancer were obtained by TTD, OMIM and GeneCards databases, respectively. After merging the three datasets and removing duplicate items, a total of 1069 target genes related to NAFLD and 3539 target genes related to liver cancer were obtained, respectively ( Figure 2a). Finally, 455 intersection targets of NAFLD and liver cancer were obtained by using Venny, namely the targets of NAFLD-related liver cancer.

| PPI network construction
By integrating 455 core targets into STRING database, a PPI network was obtained (Figure 2b). The minimum required interaction score was set to highest confidence (0.900), and 363 targets. There were 363 nodes and 1749 edges in the graph. Each node represents a different protein; the edges represent their internal relationships, while green to red represent values from small to large.

| Analysis of drug and disease common targets
Then, 65 common targets of coptidis rhizoma and evodiae fructus and NAFLD-related liver cancer were obtained by Venny (Figure 3a). To explore the mechanism of coptidis rhizoma and evodiae fructus in the treatment of NAFLDrelated liver cancer, the PPI network of 65 common targets was constructed using STRING database, and was visualized with Cytoscape software. The minimum required interaction score was set to highest confidence (0.900), and 60 targets were obtained. The PPI network consisted of 60 nodes and 170 edges (Figure 3b). The node size and color were proportional to degree value. The larger the size of node is, the redder the color of node is, the greater is the degree. In addition, core targets with higher degree were calculated by using CytoHubba, the top 10 targets were AKT1, TP53, TNF, IL6, and IL10. Among the top 10 targets, AKT1 has the highest degree (Figure 3c), indicating that it may be a key target of coptidis rhizoma and evodiae fructus in the treatment of NAFLD-related liver cancer. These results showed that the top 10 targets played an important role in the treatment of NAFLD-related liver cancer by coptidis rhizoma and evodiae fructus.

| Prognostic analysis of AKT1
Then, we selected AKT1, the target with the highest degree value, for further analysis. The results showed that the expression level of AKT1 in the high-risk group was higher than those in the low-risk group, and the OS rate in the high-risk group was lower than that in the low-risk group (Figure 4a,b). The 1-, 3-, and 5-year area under the curve (AUC) curves were 0.58, 0.539 and 0.588, respectively (Figure 4c), suggesting the prediction result was reliable. Oil red staining results showed that lipid droplets deposition increased in the OA group compared with the control group (Figure 4d). Further results showed that the expression of p-AKT was significantly increased, but the change of AKT was not obvious in the OA group (Figure 4e), indicating that the increase of p-AKT might be the main factor in NAFLDrelated liver cancer.

| GO and KEGG enrichment analysis
In order to further systematically elucidate the mechanism of drug therapy, gene enrichment analysis was performed using DAVID database. The top eight KEGG pathways and GO items were selected based on gene count and p-value. These 65 intersected genes were mainly enriched in the GO:0010628 (positive regulation of gene expression), GO:0042493 (response to drug), GO:0043066 (negative regulation of apoptotic process), GO:0043065 F I G U R E 2 Acquisition of targets for non-alcoholic fatty liver disease-associated liver cancer (a) Venn diagrams of disease targets. (b) PPI network of disease common targets. Each node represents a different protein; the edges represent their internal relationships, while green to red represent values from small to large.
(positive regulation of apoptotic process), GO:0006954 (inflammatory response), etc. (Figure 5a-c). These results suggested that drug-disease common targets were enriched to insulin resistance, inflammatory response and regulation of apoptotic process, and AKT1 was also involved in these biological processes. The KEGG enrichment analysis revealed that the 65 intersected genes were mainly enriched in several diseases and other pathways, including hsa04933 (AGE-RAGE signaling pathway in diabetic complications), hsa05200 (Pathways in cancer), hsa05417 (Lipid and atherosclerosis), hsa04657 (IL-17 signaling pathway) and hsa04151(PI3K-Akt signaling pathway) (Figures 6 and 7). Interestingly, AKT1 was involved in various processes. Thus, the functions and pathways of the critical related targets in coptidis rhizoma and evodiae fructus in cancer were identified.

F I G U R E 3
Analysis of drug and disease common targets. (a) Venn diagrams of drug-disease targets. (b) PPI network of drug-disease common targets. (c) PPI network of core targets. The circle represents the key targets, and the line represents the interaction between the targets. The color is positively correlated with the degree value, from yellow to red represents the degree value from small to large.

| Molecular docking and experimental verification
In order to verify the accuracy of network analysis, the top four major active compounds (quercetin, betasitosterol, isorhamnetin, and berberine) with higher degree in coptidis rhizoma and evodiae fructus were docked with the AKT1 ( Table 2). The results showed that quercetin (degree = 300), beta-sitosterol (degree = 38), isorhamnetin (degree = 37) and berberine (degree = 34) had high degree. Among them, quercetin and berberine were common compounds of coptidis rhizoma and evodiae fructus. Studies have showed that the binding free energy of receptor and ligand is ≤ −4.25 kcal/mol, indicating that the two had a certain binding activity. When the binding energy was <−5.0 kcal/mol, the binding activity between the two was good. If the binding energy was <−7 kcal/mol, the binding ability was considered strong (Lai et al., 2021;Zhang, Liu, et al., 2021). Molecular docking of major active compounds with key targets was performed using PyMol software. Except beta-sitosterol and AKT1 did not form hydrogen bonds, the other three active compounds formed hydrogen bonds with AKT1. Quercetin formed 6 hydrogen bonds with AKT1 through amino acid residues VAL-83, ARG-15, LYS-20 and GLU-85. Isorhamnetin formed 1 hydrogen bonds with AKT1 through amino acid residues HIS-13. Berberine formed 2 hydrogen bonds with AKT1 through amino acid residues LYS-39 and GLU-40. AKT1 inhibitor resveratrol (Gao et al., 2022) formed 2 hydrogen bonds with AKT1 through amino acid residues LYS-39 and GLU-40 (Figure 8a). The docking energy of 4 active compounds of coptidis rhizoma and evodiae fructus for NAFLD-related liver cancer with AKT1 was shown in Table 2. These results indicated that the active compounds of coptidis rhizoma and evodiae fructus (quercetin, beta-sitosterol, isorhamnetin, and berberine) had a good binding effect on AKTQ in NAFLD-related liver cancer. The docking results of quercetin and AKT1 were the best, indicating that quercetin might be the main compounds.
In order to further verify the inhibitory effect of quercetin on NAFLD-related liver cancer, we conducted in vitro experiments. The results showed that, compared with the control group, the oil red staining area of the OA group was significantly increased, indicating the increase of lipid droplet deposition. Compared with the OA group, the AKT inhibitor resveratrol (Gao et al., 2022) (20 μM) significantly reduced the oil red staining area and inhibited the increase of lipid droplet deposition in HepG2 cells. And quercetin (50 μM) also inhibited the increase of lipid droplet deposition in HepG2 cells (Figure 8b). These results suggested that quercetin, the main compounds of coptidis rhizoma and evodiae fructus, might inhibit the development of NAFLD-related liver cancer.
In addition, these results showed that compared with the control group, the expression of AKT was increased in the OA group. Compared with the OA group, quercetin decreased p-AKT expression. And resveratrol both decreased the expression of p-AKT and AKT (Figure 8c).

| DISCUSSION
Although studies have shown that many cell signaling pathways, genes, and cytokines are involved in the progression and development of NAFLD-related liver cancer. However, the mechanism of NAFLD-related liver cancer F I G U R E 5 Bubble map of GO functional annotation analysis of common targets, including (a) GO-BP (biological process) analysis; (b) GO-CC (cell component) analysis; (c) GO-MF (molecular function) analysis. The bubble from small to large means that the count value from small to large. The color from red to green means that the p value from small to large. pathogenesis remains unclear. In our study, a total of 41 major compounds of coptidis rhizoma and evodiae fructus for NAFLD-related liver cancer were obtained, and 65 common targets for NAFLD-related liver cancer were obtained. In addition, core targets with higher degree were calculated using CytoHubba, including AKT1, TP53, TNF, RXRA, IL1B, IL4, IL6, IL10, MYC and CXCL8, among which AKT1 had the highest degree. TCM has a long history as an important alternative therapy, including coptidis rhizoma and evodiae fructus were mainly used clinically for compatibility (namely formulas) to achieve synergy and detoxification.
AKT was the primary kinase that regulated cell growth and survival. Three subtypes of AKT were identified in mammalian cells, including AKT1, AKT2, and AKT3. AKT1 is a member of the AKT kinase family, which regulates glycolipid metabolism, proliferation, and cell survival through a range of downstream substrates (Gao et al., 2022). In particular, upregulation of AKT1 subtype in HCC was closely related to the growth and proliferation of HCC cells . A study has found that AKT1 deletion completely prevents C-MYC-induced HCC formation in mice (Xu et al., 2019). AKT regulated fat synthesis factor SREPB-1 through the PI3K/AKT/mTOR signaling pathway, increased fatty acid synthesis and lipid content in hepatocytes, accelerating the progression of NAFLD. More and more studies have identified that NAFLD is a complex metabolic disorder mainly characterized by hepatic steatosis, inflammation and fibrosis (Dai et al., 2021). In addition, AKT regulated NF-κB signaling by phosphorylation of IKKα, leading to the degradation of IκB (Tianyu & Liying, 2021). This process released NF-κB from the cytoplasm to the nucleus, which in turn regulated the expression of downstream target genes and ultimately mediates various pathological processes such as liver inflammation and apoptosis (Gao et al., 2022). Some studies have identified that AKT plays an important role in NAFLD-related liver cancer Jeong et al., 2018;Liu et al., 2018;Zhang, 2018). Overall, AKT1 played a key role in lipid metabolism and was a promising therapeutic target for metabolic diseases.
Inflammation was believed to be the main cause behind NAFLD and led to fibrosis and the subsequent progression of cirrhosis (Pettinelli et al., 2011). Since PI3K and AKT activated immune cells by regulating key inflammatory cytokines (Weichhart & Säemann, 2008), changes in the PI3K-Akt signaling pathway contributed to the therapeutic effect of NAFLD (Matsuda et al., 2013) F I G U R E 6 Bubble map of KEGG pathway of intersection target. The bubble from small to large means that the count value from small to large. The color from red to green means that the p value from small to large. and NAFLD-related HCC (Zhang, 2018). PI3K-Akt signaling was also found to be activated in NASH animal models, leading to increased liver damage. Inhibition of this pathway significantly reduced the damage of liver cells and inhibited the activation of hepatic stellate cells. In addition, the PI3K-Akt pathway was one of the most important insulin signaling pathways . Studies have suggested that up-regulation of the PI3K-Akt signaling pathway directly increases new fat production and liver free fatty acids flow through increased lipolysis in adipose tissue (Probst & Reymond, 2018). Similarly, in our study, GO analysis showed that drugdisease common targets were enriched to insulin resistance, inflammatory response and regulation of apoptotic process, and AKT1 was also involved in these biological processes. These results suggested that AKT was involved in the occurrence and development of NAFLD-related liver cancer through these biological processes. KEGG analysis showed that drug-disease common targets were mainly enriched in PI3K-Akt signaling pathway, and key targets such as AKT1, IL-6, TNF, and TP53 regulated these pathways.
Then, the major active compounds of coptidis rhizoma and evodiae fructus were selected for molecular docking with AKT1. Molecular docking results showed that the F I G U R E 7 The PI3K-Akt signaling pathway plays a central role in coptidis rhizoma and evodiae fructus against nonalcoholic fatty liver disease-related liver cancer. The red nodes are the overlapping targets of nonalcoholic fatty liver disease-related liver cancer and coptidis rhizoma and evodiae fructus. White nodes are other targets in the PI3K-Akt signaling pathway. three active compounds (quercetin, isorhamnetin, and berberine) of coptidis rhizoma and evodiae fructus were well docked with AKT1. In addition, the three active compounds (quercetin, isorhamnetin, and berberine) have higher binding energy than the AKT1 inhibitor, resveratrol. These results suggested that they were the most important active compounds, especially quercetin, contributing to the therapeutic effects of the coptidis rhizoma and evodiae fructus on NAFLD-related liver cancer. Quercetin has been found in coptidis rhizoma and evodiae fructus in various studies (Chang et al., 2022;Liu et al., 2016;Luo et al., 2021;Zeng et al., 2011;Zheng et al., 2021). Studies have demonstrated that quercetin reduces AKT phosphorylation and improves diet-induced dysregulation of lipid metabolism genes in mice with NAFLD through PI3K/ AKT pathway (Pisonero-Vaquero et al., 2015). In addition, quercetin reversed lipid accumulation and inflammatory cell infiltration in HFD-induced NASH mice and rats (Hoek-van den Hil et al., 2013). Similarly, we also found that quercetin reduced lipid droplet deposition and decreased the expression of p-AKT. In addition, other major compounds also play an important role in NAFLD-related liver cancer. Isorhamnetin inhibited HCC progression by restoring the TP53/miR-15a/miR-16 axis (Ahmed Youness et al., 2020), and isorhamnetin also reversed longterm fibrosis and liver damage in NASH by reducing systemic inflammation and blocking hematopoietic stem cell activation (Ganbold et al., 2019). Berberine prevented NASH derived HCC in mice by inhibiting inflammation and angiogenesis (Luo et al., 2019). Additionally, berberine induced autophagic death in acute lymphoblastic leukemia cells by inhibiting AKT/mTORC1 signaling pathway . Beta-sitosterol mediated apoptosis of MCA-102 mouse fibrosarcoma cells by downregulating AKT and activating ERK (Moon et al., 2007). Similarly, a study has showed that Ganlu Powder (coptidis rhizoma-evodiae fructus 2:1) reduces liver injury by regulating the release of downstream inflammatory cytokines through AKT1 (Gao et al., 2022).
Unfortunately, the study may have some limitations. Currently, there are few clinical chips or databases available for NAFLD-related liver cancer. In this paper, only the intersection targets of NAFLD and liver cancer can be used as NAFLD-related liver cancer. In future work, we will pay more attention to patients with clinical NAFLDrelated liver cancer, and look forward to further research.
In conclusion, our results showed that the main compounds of coptidis rhizoma and evodiae fructus played an important role in NAFLD-related liver cancer by reducing inflammatory response and promoting apoptosis.

| Conclusion
In conclusion, AKT1 was a key target and quercetin was the main compounds of coptidis rhizoma and evodiae fructus in the treatment of NAFLD-related liver cancer. The increase of p-AKT might be the main factor in NAFLDrelated liver cancer. Quercetin reduced lipid droplet deposition and decreased the expression of p-AKT. Due to the limitations of biological calculation methods, further in vivo experiments are needed to confirm our findings.

ACKNOWLEDGMENTS
Not applicable.

FUNDING INFORMATION
The present study was financially supported by the National Natural Science Foundation of China (No. 81973840 and No. 81273748); National science and Technology major projects of the 13th Five-Year Plan (2018ZX10303502); Science and Technology Program of Hebei (223777156D); Sichuan Provincial Administration of Traditional Chinese Medicine Major science and technology projects (2021XYCZ004).