Luteolin and glycyrrhetinic exert cooperative effect on liver cancer by selfassembling into carrier-free nanostructures
Lanlan Zong , Yuxin Dai , Jiahao Xu , Chaofeng Qiao , Yao Qi , Chengyuan Ma , Hong Li , Xiaobin Pang , Xiaohui Pu
Liver cancer is one of the most common cancers worldwide with a high mortality [1-3]. Standard treatments for liver cancer currently include surgery, radiotherapy, and chemotherapy [4,5]. Despite targeted therapy and immunotherapy have led to significant improvement in the treatment of liver cancer, challenges persist, particularly for patients with distant metastases who experience poorer prognoses and a higher likelihood of tumor recurrence due to drug resistance [6-8].
Recent studies have shown that traditional Chinese medicine (TCM) exhibits characteristics such as targeting multiple pathways, having minimal side effects, and demonstrating favorable therapeutic outcomes in cancer treatment [9-11]. Furthermore, the different roles of various TCMs in cancer treatment are gradually being uncovered [11-15]. In clinical practice, TCM has been shown to prolong patient survival and improve their quality of life [16,17]. Therefore, researching and exploring the anti-tumor effects of TCM holds significant practical significance.
Numerous studies have indicated that various small molecules of TCM including artemisinin, triptolide, celastrol, curcumin, and capsaicin could regulate tumor-related signaling pathways, participate in oxidative stress responses within cancer cells, and induce various cell death modes such as apoptosis, autophagy, and ferroptosis [18-24]. Among them, luteolin (LUT) is a common natural flavonoid derived from heat-clearing and detoxifying TCM, and the literature and TCMSP database have emphasized that TCM containing LUT often regulates the liver meridian [25]. Modern pharmacological studies have shown that LUT exhibits anti-inflammatory, antibacterial, antiviral, anti-tumor, and neuroprotective effects [26,27]. The mechanism of LUT in treating liver cancer has been reported, including inhibiting cell proliferation, inducing apoptosis, and inhibiting invasion and migration [28-30]. However, the target of LUT in treating liver cancer has not yet been entirely clearly defined, so if we can find a direct target of LUT in liver cancer, it will help humans find the way to defeat liver cancer. In addition, due to the poor water solubility and low bioavailability, the clinical application of LUT is extremely challenging. Similarly, glycyrrhetinic (GA), another natural compound extracted from the traditional Chinese medicinal plant licorice, has been shown to has anti-inflammatory, antiviral, and anti-tumor proliferative effects in modern pharmacological studies [31]. Interestingly, some studies have shown that the inhibitory effect of GA on liver cancer cells is significantly stronger than that on normal hepatocytes, which indicates that GA exhibits tumor selectivity in liver cancer [32]. Nevertheless, the low water solubility of GA similarly limits its clinical application.
Nanoparticle (NP) drug delivery systems have the potential to enhance drug solubility and improve drug accumulation at target sites [33,34]. In recent years, self-assembled nanodrugs have gained considerable attention due to their high drug-loading capacity, simple preparation, and ability to avoid carrier-induced immune responses and toxicity [35-38]. Meanwhile, Chinese medicine self-assembly nanostrategy (CSAN) is being developed. Active components of TCM that possess self-assembly capabilities include alkaloids, organic acids, flavonoids, terpenoids, polysaccharides, and proteins derived from natural plants, such as curcumin, resveratrol, and artemisinin [39-42]. Compared to traditional nanoformulations, CSAN is easier to manufacture, more efficient and exhibits higher biodegradability and biocompatibility, and does not require any specialized equipment [43]. Additionally, it can improve the solubility and in vivo bioavailability of insoluble TCM active components [39]. CSAN has been shown to exert an antitumor impact, and exhibits a synergistic effect when used in conjunction with other antitumor drugs. It can also be used to construct nanopreparations with metal ions for photothermal treatment or tumor imaging [42]. Therefore, if LUT and GA can be co-delivered to the tumor site in the form of nanodrugs, it will greatly improve their therapeutic effects on tumors and reduce systemic toxicity.
LG-Nanos was formed through self-assembly from nature products LUT and GA in aqueous solution (Fig. 1A). As shown, the LG-Nanos have particle sizes of ~250 nm (Fig. 1B and Table S1 in Supporting information) and appear translucent under opalescent light (Fig. 1C). Their zeta potentials are −26.8 ± 0.30, −28.9 ± 0.52, and −33.7 ± 1.48 mV, respectively (Table S1). Transmission electron microscope (TEM) images (Fig. 1D) demonstrate that the LG-Nanos possessed a claviform morphology, which is more conducive to reducing the viscous resistance of drug transport in blood vessels, quickly passing through the porous blood vessel wall, and penetration and retention in tumor tissues because claviform NPs have a higher aspect ratio than spherical NPs [44,45].
As shown in Fig. 1E, the particle size of LG-Nanos did not change significantly after 14 days of storage at room temperature (RT) and 4 ℃. However, from 14 days to 28 days, the particle size was increased (Fig. 1F). It was found that LG-Nanos-1:2 with 1,2-distearoyl-sn‑glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG2000) was the most stable at RT and 4 ℃, which might be attributed to the fact that more GA provided more surface negative charges for drug NPs as the lowest zeta potential of LG-Nanos-1:2 (Table S1) and produced stronger electrostatic repulsion to prevent particle aggregation. In addition, we found that LG-Nanos with DSPE-PEG2000 had longer-term stability than those without DSPE-PEG2000, possibly because of the strong steric hindrance provided by PEG2000 in DSPE-PEG2000.
As shown in Fig. S1 (Supporting information), results of the transmittance analysis indicated that the LG-Nanos rarely settled and had good kinetic stability at RT and 37 ℃ for 72 h (Figs. S1A and B). At the same time, there was no significant change in the particle size of LG-Nanos at RT and 37 ℃, indicating that LG-Nanos had high thermodynamic stability within 72 h (Figs. S1C and D). The relative transmittance and size changes were within 10% for LG-Nanos (Fig. S1).
The particle size changes of LG-Nanos after diluting them 10, 20, 50, 100, and 200 times are shown in Fig. 1G. The results showed that the particle sizes of all formulations remained at 250–300 nm, indicating that several formulations had good dilution stability. As shown in Fig. 1H, after LG-Nanos-2:1, LG-Nanos-1:1, and LG-Nanos-1:2 were incubated with 10% rat plasma for 48 h, the particle size did not change, indicating good physical stability in the plasma. This suggested that LG-Nanos could remain stable when administered intravenously.
Results of molecular docking demonstrated that there were many hydrogen bond interactions between LUT and GA, and the π-π stacking interaction between the two becomes more obvious with the increase in the proportion of LUT (Fig. 2A).
As shown in Fig. 2B, UV absorption peaks of LUT were at 253, 266, and 348 nm, and the absorption peak of GA was at 259 nm. All the maximum absorption peaks of GA and LUT were observed at the same position in the spectra of LG-1:1. The LG-Nanos UV absorptions also showed the characteristic absorption peaks of GA and LUT. On comparing to the spectra of LG-1:1, we found that there was a red shift to approximately 369 nm, a hypochromic effect with a decrease in peak intensity for the absorption peak of LUT at 348 nm, and a hyperchromic effect at 255 nm, with the merging of fine peaks at 253–266 nm into enhanced single peaks in the spectra of three nanoself-assemblies. The red shift, hypochromic effect, and hyperchromic effect implied that the π-π interaction between the aromatic chromophores of LUT and GA was involved in the self-assembly process, as suggested by the results of molecular docking.
As shown in Fig. 2C, the infrared spectra showed that the stretching vibration of the hydroxyl group was at 3442 cm−1, the C—H stretching vibration peak of the methylene group was observed at 2948 cm−1, the stretching vibration of the carbonyl group in the carboxyl group was observed at 1705 cm−1, and the carbonyl stretching vibration peak in the conjugate structure was observed at 1660 cm−1. In the infrared spectrum of LUT, the peak at 3405 cm−1 indicated the stretching vibration of the hydroxyl group, the absorption peak of the carbonyl group was observed at 1656 cm−1, and the absorption peak of the conjugate system of the benzene ring was observed at 1611 cm−1. The spectrum of LG-1:1 showed all the absorption peaks of LUT and GA at almost the same location. Compared with the LG-1:1 map, the vibration peak of the hydroxyl group of GA shifted from 3440 cm−1 to 3420–3430 cm−1, and the stretching vibration peak of the conjugated carbonyl group of LUT and GA obviously shifted from 1660 cm−1 to approximately 1655 cm−1 in the spectra of the LG-Nanos. All the hydroxyl vibration peaks tended to move towards a low wave number, from 3444 cm−1 to approximately 3420 cm−1. The conjugated carbonyl groups of LUT and GA moved from 1660 cm−1 to approximately 1650 cm−1, and the absorption peak at 1266 cm−1 caused by the coupling of the C—O bond stretching vibration and the O—H bond in LUT, moved to approximately 1260 cm−1. These shifts of the absorption peak to a low wave number region indicated that the π-π stacking of the aromatic ring and the hydrogen bond of the hydroxyl group are involved in the self-assembly process of the two drug molecules, which is consistent with the underlying self-assembly mechanism suggested by the results of UV–vis spectroscopy.
In addition, nuclear magnetic resonance spectroscopy of hydrogen (1H NMR) spectra of DMSO‑d6 were used to analysis the self-assembly mechanism (Fig. 2D). The chemical shifts of H-3 in GA increased from 3.32 ppm to about 3.55 ppm, which indicated GA's H-3 region was affected after self-assembly. Meanwhile, the peak shape of H-8 and H-6 (δ = 6.1–6.5) in the benzene ring of LUT tends to change from being double peaked to being single peaked with the increase in its ratio, which may be caused by the electron delocalization of the π-π conjugate [46]. However, these phenomena were not observed in the 1H NMR spectrum of LG-1:1. These results indicated that the environment of the benzene ring of LUT was also influenced by the formation of nanoself-assemblies.
As shown in Fig. 2E, the characteristic diffraction peaks of the LG-Nanos were significantly reduced compared to the diffraction patterns of the two free drugs and those of LG-1:1, indicating that the drug crystallinity in the LG-Nanos was reduced. This might be attributed to the fact that the intermolecular forces of the drugs during self-assembly destroy the crystal lattice of the single drugs and transform the crystal pattern to some extent. Furthermore, an increase in the LUT ratio of nanoself-assemblies decreased the characteristic diffraction peaks of GA beyond 10°–15°, and there was no significant change in the characteristic diffraction peaks of LUT in the diffraction pattern of nanoself-assemblies, which implied that the increase in the LUT ratio may be conducive to the damage of the intermolecular force of GA itself and the enhancement of the molecular interaction between LUT and GA.
As shown in Table S2 (Supporting information), the calculated binding entropy of LUT-LUT was the largest, indicating that the mutual binding energy between LUT molecules was the weakest. This might be attributed to its rigid conjugated planar structure and strong hydrophobicity, which were not conducive to the interaction of intermolecular hydrogen bonds, even if there were more hydroxyl groups. Owing to the relatively flexible structure of GA acid, which contains carboxyl and hydroxyl groups, the GA-GA binding entropy is the smallest, indicating that the mutual binding energy between its molecules is the strongest. The binding entropy of LUT-GA was intermediate, and it could be inferred that LUT could potentially break the interaction between GA molecules and interact strongly with GA. Fig. 2A also showed that there were many hydrogen bond interactions between LUT and GA, and the π-π stacking interaction between the two becomes more obvious with the increase in the proportion of LUT. These results explained why the X-diffraction peaks of GA decreased and the characteristic diffraction peaks of LUT did not change significantly as the proportion of LUT increased in LG-Nanos. They also explained why LG-Nano-2:1 had good stability. Taken together, we identified that LG-Nanos was formed by electrostatic attraction and π–π stacking (Fig. 2F).
To identify the liver tumor accumulation and the dissolution of LG-Nanos, drug dissolution assay, cellular uptake assay, and tissue distribution assay were performed (Fig. 3A). As shown in Fig. 3B, the cumulative dissolution of LUT and GA in LG-Nanos reached greater than 80% at 15 min. Even at 120 min, the cumulative dissolution of the LG-Nanos was still 1.9 and 1.6 times more than those of the two free drugs, respectively. These results suggested that the LG-Nanos could significantly improve the dissolution rates of the two drugs (Fig. 3A).
As shown in Fig. 3C, in liver cancer cells HepG2, the cellular uptake ratios of LUT and GA were increased significantly by forming LG-Nanos. For LUT, the cellular uptake ratio increased to 34.28%, 27.04%, and 42.74%, respectively (P < 0.001). For GA, the cellular uptake ratio increased to 49.31%, 57.59%, and 44.46%, respectively (P < 0.001). Results above indicated that the LG-Nanos had good target effect on liver cancer cells.
Results of tissue distribution assay demonstrated that, after forming LG-Nanos, LUT and GA were enriched in mouse liver tissues and tumor tissues (Fig. 3D). All the procedures were approved by the Institutional Animal Care and Use Committee of Henan University (No. HUSOM2023–067). It suggested why LG-Nanos exhibited good targeting for liver cancer. Taken together, we proved that LG-Nanos improved liver tumor accumulation and the dissolution compared with free LUT and GA.
Results in Figs. 4A and B showed that LG-Nanos inhibited proliferation of liver cancer cells Huh-7 and HepG2. Among all the groups, the inhibitory effect of LG-Nanos-1:2 was the strongest, and the half maximal inhibitory concentration (IC50) value was 37.05 and 27.12 µmol/L in HepG2 and Huh-7 cell, respectively. The pharmacodynamics of LG-Nanos was evaluated in mouse xenograft model of liver cancer (Fig. 4C). The mice were categorized into the following nine groups: Model, LUT, GA, LG-mixture (1:1, 2:1, and 1:2), and LG-Nanos (1:1, 2:1, and 1:2) groups. Results in Figs. 4D–F showed that LG-Nanos could significantly inhibit the growth of liver cancer (P < 0.01). Consistent with the results in vitro, among all the groups, the inhibitory effect of LG-Nanos-1:2 was the strongest in mouse liver cancer.
As shown in Fig. S2 (Supporting information), gene ontology (GO) analysis results revealed that 20,473 genes exhibited abnormal level with 707 genes level increased while 870 genes level decreased (Fig. S2A). The Venn diagram revealed that there were 23 common targets among LUT, GA and liver cancer (Fig. S2B). As shown in Fig. S2C, results of protein-protein interaction (PPI) analysis suggested that the top 20 target genes including estrogen receptor 1 (ESR1), cytochrome P450-family 2-subfamily E-polypeptide 1 (CYP2E1), ATP-binding cassette-sub-family G-member 2 (ABCG2), and cyclin-dependent kinase 1 (CDK1), etc. Molecular docking was performed to further screen targets. Results showed that LUT bound well to ESR1 with the binding score being −6.33 Å, while GA bound well to CDK1 with the binding score being −8.24 Å (Figs. S2D and E). Subsequent expression analysis (GEPIA2 database) indicated that expression of ESR1 and CDK1 were significantly abnormal in liver cancer (P < 0.05) with increased expression of CDK1 while decreased expression of ESR1 (Fig. S2F). Be consistent with the results of GEPIA2 database analysis, as shown in Fig. S2G, ESR1 was decreased in liver tumor tissues while CDK1 was increased in liver tumor tissues. Survival analysis results suggested that ESR1 was positively correlated with the overall survival rate of patients with liver cancer, while CDK1 was negatively correlated with the overall survival rate of patients with liver cancer (P < 0.001) (Fig. S2H). Taken together, CDK1 and ESR1 were closely related to liver cancer, and they might be the targets targeting by LG-Nanos to inhibit liver cancer.
ESR1 is an important gene transcriptional regulator, known to mediate the effects of estrogen [47]. It was found that up regulating of ESR1 could promote the apoptosis of liver cancer cells, shorten the cell cycle, and inhibit the proliferation and invasion of liver cancer cells [48]. CDK1 is a member of the family of cell cycle regulatory proteins involved in cell cycle maintenance. Several studies have proved that inhibiting CDK1 inhibited proliferation of tumor cells [49]. Meanwhile, genetic interference with CDK1 could cause G2/M arrest in liver cancer [50].
Cell cycle dysregulation is a characteristic hallmark of malignancies, which results in uncontrolled cell proliferation and eventual tumor formation [51]. Taken together, we hypothesized that LUT and GA targeted ESR1 and CDK1 to block the cell cycle and inhibit the proliferation of liver cancer cells, respectively. To further identify our hypothesis, we performed signaling pathway and functional analyses of the 23 common targets. As shown in Fig. S2I, it was proved that the 23 targets were involved in cell cycle-related pathways. At the same time, it was found that the 23 targets were mainly involved in steroid catabolic process, cyclin B1-CDK1 complex, and estrogen response element binding (Fig. S2J). As reported, ESR1 was known to have an important role in steroid catabolic process and estrogen response element binding, which was related to cell cycle in various cancer [52]. Inhibiting of CDK1 could cause G2/M arrest in liver cancer. All the results could well verify our hypothesis: LUT and GA targeted ESR1 and CDK1, respectively, and exerted a synergistic effect to arrest the liver cancer cell cycle at the G2/M phase and inhibit the proliferation of liver cancer cells.
Results of MTT assay showed that LG-Nanos had no significant effect on the proliferation of normal liver cells 7702, and the cell viability was above 85% in LG-Nanos groups (Fig. 5A). The IC50 value was > 300 mmol/L in the three LG-Nanos groups, which demonstrated that LG-Nanos showed good safety in vitro (Fig. 5B). Results also showed that LUT has a certain inhibitory effect on the proliferation of 7702 cells, and the combination of LUT-GA could reduce cytotoxicity. In particular, the three LG-Nanos could significantly reduce cytotoxicity in 7702 cells.
As shown in Fig. 5C, results of hematoxylin and eosin (H&E) staining assay demonstrated that no obvious abnormalities were observed in the heart, liver, spleen, lung, and kidney tissues. Meanwhile, results of hemolytic test showed that LG-Nanos had no significantly effect on hemolysis (Fig. 5D, up). At concentrations below 1.5 µmol/mL, the hemolysis ratio of all the three LG-Nanos was < 5%, indicating that LG-Nanos did not cause hemolysis at concentrations below this level (Fig. 5D, down). The changing trends in the body weight of the mice were shown in Fig. 5E. There was a slight change (P > 0.05) in the body weight of all animals within 20 days of drug administration, which was consistent with the normal growth of the mice. These results suggested that LG-Nanos did not induce systemic toxicity in vivo. Interestingly, as shown in Fig. 5C, in mouse H22 tumor tissues, LG-Nanos could induce nuclear fragmentation, accompanied by vacuoles and inflammatory cell infiltration, especially in the LG-Nanos-1:2 group. At the same time, LG-Nanos inhibited cell proliferation in mouse H22 tumor tissues.
Taken together, LG-Nanos showed good safety in vivo and in vitro. On the other hand, LG-Nanos inhibited proliferation of liver cancer in mice. It might be attributed to the better tumor cell targeting of LG-Nanos (Fig. 5F). We also identified that LUT and GA in LG-Nanos-1:2 have a strong synergistic effect of inhibiting malignant cell proliferation (Fig. 5C).
As reported, there is an increasing number of studies on the self-assembly of TCM, including active components of TCM monomers to decoctions of single or compound TCM [53-55], including terpenoid, organic acids, alkaloids, terpenoids, favonoids, polysaccharides, and proteins derived from natural plants. LUT and GA are favonoid and triterpenoids from natural Chinese medicine, respectively [56,57]. LG-Nanos are self-assembly of TCM with different components. The active components of TCM primarily self-assemble through noncovalent interactions, including hydrogen bonds [58], van der Waals forces, π-π stacking [59], hydrophobic interactions [60], electrostatic interactions [61], and coordination interactions [62]. In our study, it was found that the π-π interaction between the aromatic chromophores of LUT and GA was involved in the self-assembly process of LG-Nanos. Self-assembled nanoformulation based on CSAN can improve the bioavailability and solubility of TCM active components and tumor targeting. They are combined or loaded with chemical drugs to play a combined therapeutic role, inhibit the absorption of toxic components, and reduce side effects. Among them, ginsenoside, urolithin A (UA), and berberine (BBR) exhibit an intervention effect on lung cancer, and their therapeutic efficacy are improved when they are self-assembled to form NPs [63]. In breast cancer, paclitaxel (PTX) and doxorubicin (DOX) show a synergistic effect in chemotherapy with the self-assembly of chemotherapy drugs, and lessen the side effect of chemotherapy drugs [64]. In liver cancer, Vicia ervilia lectin (UEA) NPs shows the ability to target epithelial cell adhesion molecule (EpCAM) [65], OX/ASP-DOCA NPs can precisely target HepG2 solid tumor, glycyrrhizic acid-hydroxycamptothecin (GL-HCPT) micelles can greatly increase HCPT solubility and trigger apoptosis in HepG2 and Huh7 human hepatoma cells [66], CP3-DOX NPs can accelerate DOX release in the acidic pH of the tumor microenvironment [67]. In our study, LUT and GA can be co-delivered to the liver cancer site in the form of nanodrugs, it greatly improves their therapeutic effect on tumor and reduce systemic toxicity.
Synergistic action of both compounds significantly reduced the malignancy of liver cancer cells. Additionally, using network pharmacology analysis and bioinformatics analysis, we identified and validated ESR1 and CDK1 as the direct targets respectively through which LUT and GA induced cell cycle arrest, induced inhibition of cell proliferation in liver cancer. Owing to the enhanced permeability and retention effect and the synergistic action of LUT and GA, LG-Nanos exhibited significant therapeutic efficacy in a mouse model of liver cancer, while also markedly reducing toxic side effect.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Lanlan Zong: Validation, Investigation, Data curation. Yuxin Dai: Writing – original draft, Methodology, Data curation. Jiahao Xu: Visualization, Validation, Conceptualization. Chaofeng Qiao: Software, Methodology. Yao Qi: Software, Data curation. Chengyuan Ma: Software, Methodology. Hong Li: Writing – review & editing, Validation, Investigation, Data curation. Xiaobin Pang: Resources. Xiaohui Pu: Supervision, Resources, Project administration, Funding acquisition.
We are grateful for the financial support from Henan Province Natural Science Foundation (No. 252300420583), Henan Provincial Science and Technology Research Project (Nos. 242102310455, 242102310473, 242102310517), and the Key Project of Science and Technology Research funded by the Henan Provincial Department of Education (No. 24A350002).
Supplementary material associated with this article can be found, in the online version, at doi:
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Figure 1 Characterization of LG-Nanos. (A) Self-assembly structure diagram of LUT and GA. (B) The particle size. (C) The appearance. (D) TEM image. Scale bar: 200 nm. (E, F) The storage stability. (G) The dilution stability. (H) The plasma stability. Data were described as mean ± standard deviation (SD) (n = 3). Statistical analysis was performed using one-way analysis of variance followed by a post hoc test. All analyses were carried out using GraphPad Prism 10.0 software.
Figure 2 The mechanism of LG-Nanos self-assembling. (A) Molecular docking. (B) Ultraviolet-visible spectroscopy (UV–vis) absorption spectroscopy. (C) Fourier transform infrared. (D) 1H NMR. (E) X-ray diffraction pattern. (F) Diagram of LG-Nanos self-assembling mechanism.
Figure 3 The tissue accumulation and dissolution of LG-Nanos. (A) Schematic diagram of the experiment process. (B) Dissolution assay. (C) Cell uptake of LUT and GA in the different formulations. (D) In vivo tissue distribution of LUT and GA in the different formulations at 24 h after administration to mice. Data were described as mean ± SD (n = 3). Statistical analysis was performed using one-way analysis of variance followed by a post hoc test. All analyses were carried out using GraphPad Prism 10.0 software. ***P < 0.001.
Figure 4 The liver cancer therapeutic efficacy of LG-Nanos in vitro and in vivo. (A, B) MTT assay evaluated the effect of LG-Nanos on liver cancer cells Huh-7 and HepG2. (C) Schematic diagram of liver cancer model establishment and treatment process. (D) The tumor images after 20 days of treatment. (E) The tumor volume and (F) the tumor suppression ratio. Data were described as mean ± SD (n = 3). Statistical analysis was performed using one-way analysis of variance followed by a post hoc test. All analyses were carried out using GraphPad Prism 10.0 software. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5 Biocompatibility testing of LG-Nanos. (A, B) MTT assay to evaluate the effect of LG-Nanos on liver normal cells 7702. (C) H&E staining of major organs in mice with various treatments. (D) Hemolytic test. (E) Body weight of mice in different groups were recorded every two days post-treatment. (F) Schematic diagram of the reason for better biological safety. Data were described as mean ± SD (n = 3). Statistical analysis was performed using one-way analysis of variance followed by a post hoc test. All analyses were carried out using GraphPad Prism 10.0 software. ns, P > 0.05.
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