Therapeutic Potential of Polydatin Against Cancer Cachexia by Regulating the STAT3 Signaling Pathway

TL;DR

Polydatin, a natural compound, shows potential in treating cancer cachexia by reducing muscle atrophy and inflammation through the STAT3 signaling pathway.

Contribution

This study demonstrates polydatin's therapeutic potential in cancer cachexia by targeting the STAT3 pathway.

Findings

Polydatin at 100 mg/kg reversed muscle mass reduction and inflammation in CT26-bearing mice.

Polydatin suppressed STAT3 phosphorylation in C2C12 myotubes at 200 µM.

Molecular docking simulations identified polydatin's structural interaction with proteins in the STAT3 pathway.

Abstract

Background/Objectives: Cancer cachexia is a wasting syndrome with significant loss of body weight and muscle mass caused by inflammation and abnormal metabolism in advanced cancers. Despite its detrimental effects on patients, no standard treatment has been established for this syndrome. Thus, finding new treatments will broaden the remedy for cancer cachexia, resulting in increased survival in patients with terminal cancer. Methods: In this study, we assessed the therapeutic effects of the natural compound polydatin on cancer cachexia in vitro and in vivo using C2C12 myoblasts and CT26-bearing mice to elucidate the mechanisms of how it ameliorates muscle atrophy. At the same time, molecular docking analysis of polydatin with the IL6/STAT3 signaling pathway was conducted to demonstrate their interaction. Results: Our data showed that polydatin treatment at 100 mg/kg could attenuate symptoms of cancer cachexia including body weight loss, muscle strength and severe inflammation. Muscle mass reduction—with the shrinking of muscle fibers, an increase in the expression levels of two E3 ubiquitin ligases (MuRF1 and Atrogin-1) and interleukin-6, and a downregulation of MyHC—observed in CT26-bearing mice was reversed by polydatin at 100 mg/kg. On C2C12 myotubes, polydatin also ameliorated muscle atrophy induced by the CT26 conditioned medium and suppressed STAT3 phosphorylation at the concentration of 200 µM. Structural features of polydatin in the proteins in the STAT3 pathway were identified through molecular docking simulations. Conclusions: Taken together, polydatin significantly attenuated muscle atrophy in a cancer cachexia model by inhibiting the STAT3 signaling pathway; thus, it might be a promising compound in the development of drug candidates for cancer cachexia therapy.

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1. Introduction

Cancer cachexia is a complicated condition caused by inflammation and abnormal metabolism in advanced cancers [1]. It is characterized by severe body weight loss, muscle mass reduction, and sometimes adipose tissue depletion, fatigue, and significant loss of appetite. With the acceleration of protein degradation and increased lipolysis due to proinflammatory cytokines, cachexia triggers weakness in patients, reduces their quality of life, reduces their tolerance to cancer therapy, and profoundly affects the survival rates [2]. Up to 80% of patients with cancer suffer from cachexia, and approximately 30% of cancer death is related to this syndrome [3]. Despite considerable research efforts, effective treatments for cancer cachexia remain limited, resulting in the urgent need to find innovative therapeutic strategies.

One of the key features of cancer cachexia is the loss of skeletal muscle mass [4]. Under normal physiological conditions, the balance of protein synthesis and degradation preserves the adult muscle mass. This balance is disrupted during tumor progression; protein degradation is accelerated, and protein synthesis is reduced [5]. Increased expression of genes regulating the ubiquitin–proteasome pathway, such as MuRF1 and Atrogin1, has generally been observed in the muscles of patients with cancer cachexia, leading to a significant decrease in muscle strength.

Among signaling pathways regulating muscle development, signal transducer and activator of transcription 3 (STAT3), which is a member of the STAT family, is associated with muscle atrophy [6]. Upon activation by cytokines, STAT3 is phosphorylated at Y705, dimerized, and translocated into the nucleus where it regulates the expression of target genes [7]. STAT3 activation causes muscle protein degradation by promoting the expression of muscle-specific E3 ubiquitin ligases such as MuRF1 and Atrogin-1 [8].

Polydatin, also known as piceid, is a glucoside derivative of resveratrol found in plants, notably in the roots of the Chinese herb Polygonum cuspidatum (Japanese knotweed) and grapes [9]. Polydatin has received much attention in the medical field owing to its antioxidant, anti-inflammatory, and anticancer properties [10]. The compound shows therapeutic potential in treating diseases including cardiovascular diseases [11], neurodegenerative disorders [10], diabetes [12], and rheumatoid diseases [13]. However, the pharmacological effects and regulatory mechanisms of polydatin on skeletal muscle atrophy in cancer cachexia is barely known.

In this study, we evaluated the efficacy of polydatin on inhibiting the development of cancer cachexia-induced muscle atrophy in Balb/c mice bearing CT-26 colon carcinoma. At the same time, its effects and mechanism of reducing muscle atrophy were assessed on C2C12 cells.

2. Materials and Methods

2.1. Cell Culture

Murine CT26 cells were cultured in an RPMI medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1% Penstrep (PS; Hyclone). C2C12 myoblasts were cultured in DMEM (Hyclone) supplemented with 10% FBS and 1% PS. C2C12 myoblasts were differentiated with a medium containing DMEM with 5% FBS and 1% PS when the cells reached 80% confluency. The differentiation medium was changed every 2 days for 6 days.

2.2. CT26 Conditioned Medium (CM) Preparation

CT26 cells were cultured in a 10 cm cell culture dish. When cell density reached 90% confluency, the old medium was replaced with serum-free DMEM for 48 h. The cell supernatants were collected and centrifuged at 1500 rpm for 10 min to remove the cell debris following by filtration through a 0.2 µm syringe filter. Conditioned medium was prepared for the treatment by diluting the filtered supernatant with DMEM supplemented with 5% FBS (C2C12) at the ratio of 80:20.

2.3. Cell Viability Assay

In total, 10^5^ C2C12 myoblasts were seeded into each well of 96-well plates and allowed to attach overnight in DMEM medium supplemented with 10% FBS and 1% PS. Cells were then treated with different concentrations of polydatin (0, 50, 100, 200, 400, 800, 1600, 3200, and 6400 µM) for 24 h. The cytotoxicity of polydatin to C2C12 myoblasts was determined by adding 10 μL of CCK-8 (Dongin, Seoul, Republic of Korea) to each well and incubating the plate at 37 °C and 5% CO_2_ in humidity for 60 min. The absorbance was measured at 450 nm using an Epoch™ Microplate Spectrophotometer (Biotek, San Francisco, CA, USA).

2.4. LADD Staining

LADD staining was performed to visualize the morphology of the myotubes. After adding CT26 CM and polydatin to C2C12 myotubes and incubating for 24 h, cells were washed with PBS, and EtOH 70% was used to fix cells for 15 min, followed by an addition of LADD solution containing toluidine blue and fuchsin for 5 min. After removing the LADD stain, the cells were washed with distilled water until the washing water became clear. Cell morphology was visualized using a microscope (KCS3-63S, Optinity, Seongnam, Republic of Korea) equipped with Optiview.

2.5. Animal Study

To explore the impact of polydatin on alleviating cancer cachexia symptoms in vivo, CT26-induced cancer cachexia was developed in a murine model. Five-week-old Balb/c male mice were purchased from DBL, Seoul, Korea. For 1 week, mice were allowed to acclimate to the new environment with constant temperature, humidity and 12 h light/dark cycles. CT26 cells (5 × 10^5^ cells/200 µL) were injected intravenously on day 7 to induce cancer cachexia, while those in the control group received DPBS. On the next day, mice were randomly classified into four groups and orally administered with DMSO (control—healthy mice), DMSO (vehicle—CT26-bearing mice), and polydatin at 50 and 100 mg/kg BW (CT26-bearing mice) (Figure 1A). Mice’s body weight and food intake were measured daily, and treatment was stopped after 14 days of administration. Mice were euthanized with 70 mg/kg alfaxalone and 10 mg/kg xylazine. The gastrocnemius muscles were harvested and weighed. All animal experiments were approved by the Animal Care and Use Committee of Sungkyunkwan University (SKKU-2023-04-32-1) and were carried out in accordance with the institutional guidelines. The study design and reporting adhered fully to the ARRIVE guidelines.

2.6. Grip Strength Test

Four-limb grip strength was measured using a digital grip strength meter (Columbus Instruments, Columbus, OH, USA). In brief, each mouse was allowed to hold a metal wire grid connected to a force transducer using four limbs and its tail was gently pulled backward. The maximum force generated by the mouse before releasing its grip was recorded. The test was performed five times for each mouse with a 30 s interval between each trial and the average value was calculated.

2.7. Hematoxylin and Eosin (H&E) Staining

The gastrocnemius muscle tissues were harvested and fixed in 10% neutral buffer formalin for 24 h. Tissues were embedded in paraffin and cut into 5 µm thick sections. Sections were then deparaffinized and stained with H&E. Images were taken randomly for each sample using a microscope (KB-600, Optinity) equipped with Optiview. The average cross-sectional areas of muscle fibers were measured by Image J (ImaheJ ij154).

2.8. Immunohistochemistry (IHC)

The 5 µm thick muscle sections were deparaffinized in xylene and rehydrated in a sequence of ethanol (100%, 95% and 70% ethanol) then distilled water. Antigen retrieval was performed using citrate buffer for 20 min in a water bath at 95 °C, which was then allowed to cool down to room temperature. After that, the slides were washed in PBST (PBS with 0.1% Tween-20) and blocked in BSA 5% for 1 h to prevent non-specific binding. The tissue sections were incubated with laminin (1:1000, ab11575) at 4 °C overnight in a humidified chamber, followed by staining with corresponding fluorescence-conjugated secondary antibodies for 1 h at room temperature in the dark. To stain the nuclei, Vectashield Antifade mounting medium with DAPI (LSbio, Newark, CA, USA) was added to the samples, and visualization was conducted using a Zeiss LSM 900 confocal microscope (v3.4-45914, Zeiss, Jena, Germany).

2.9. Western Blot Analysis

Frozen gastrocnemius tissues and cell samples were lysed with PRO-PREP solution (iNtRON Biotechnology, Gyeonggi, Republic of Korea) following the manufacturer’s instructions for protein extraction. In total, 30 µg of protein was separated on a 10% polyacrylamide gel and protein bands were transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Bedford, MA, USA). After blocking in 5% skim milk at RT for 1 h, membranes were incubated with primary antibodies (Atrogin-1 (1:500, A3193), MuRF (1:500, A3101), MyHC (1:1000, PA5-115216) STAT3 (1:1000, A19566), p-STAT3-Y705 (1:1000, AP0705), GAPDH (1:500, sc-32233)) at 4 °C overnight followed by probing with secondary antibodies (goat anti-rabbit HRP-linked or horse anti-mouse HRP-linked, 1:2000) for 1 h at RT the next day. Protein bands were visualized by adding West-Q Pico Dura ECL Solution (GenDEPOT, Katy, TX, USA) and exposing by Invitrogen iBright 1500 (Waltham, MA, USA).

2.10. Real-Time Quantitative Polymerase Chain Reaction (qPCR)

Total RNA was extracted from tissues and cells using TRI Reagent (MRC, Cincinnati, OH, USA) in accordance with the manufacturer’s instruction. RNA concentration was measured using Epoch™ Microplate Spectrophotometer (Biotek, CA, USA) and qRT-PCR was conducted with one-step AccuPower^®^ GreenStar™ RT-qPCR Master Mix (Bioneer, Daejeon, Republic of Korea) using 100 ng of the RNA template. Temperature control and fluorescent signal were managed by a Rotor Gene Q thermocycler (QIAGEN, Hilden, Germany). GAPDH was used to normalize target gene expressions. Primer sets for qRT-pCR are described in Table 1.

2.11. Molecular Docking Studies

Ligand structures were downloaded from PubChem (Polydatin, CID 5281718; Genistein, CID 5280961; STAT3 inhibitor 1, CID 134130166; Stattic, CID 2779853), and geometry optimization of the ligand 3D structures was performed following ligand preparation, energy minimization, and conformation generation protocols of Discovery Studio 2024 (Biovia, San Diego, CA, USA). The optimized 3D structures are listed in Figure S1.

The crystal structures of STAT3 protein (PDB ID: 1BG1) and IL6 receptor (PDB ID: 1P9M) were obtained from the Protein Data Bank with structural resolutions of 3.65 Å and 2.25 Å, respectively. For molecular docking studies, 3D structures of proteins were prepared following the instruction of Discovery Studio 2024 (Biovia, San Diego, CA, USA). The binding site of the IL6 receptor was defined by Phe229 and Phe 279, called the “hot spot residue” of the IL6–IL6 receptor interaction [14,15,16]. The binding site of STAT3 was defined from the main site of SH2 composed of Lys591, Arg595, and Ile634 [17].

The CDOCKER program, which is based on the CHARMM and Calculate Binding Energies tools in Discovery Studio 2024 (version 24.1, Biovia, San Diego, CA, USA), was used to evaluate the structural effect of the ligand on each protein. Based on the interaction energy of CDOCKER, distinct conformational poses for each molecule were produced and examined. The binding energies of the produced ligand–protein complexes were calculated using the Calculate Binding Energies tool. Two energy values (−CDOCKER interaction energy and binding energy) were generated for the produced complexes. The docking pose of the ligand on the proteins was expressed as two-dimensional diagrams and three-dimensional crystal structures.

2.12. Statistical Analysis

GraphPad Prism software 8 (GraphPad Software Inc., Boston, MA, USA) was used for data processing. The results are presented as means ± standard deviations (SD), and one-way ANOVA was applied, followed by Dunnett multiple-comparison tests. A p value < 0.05 was considered statistically significant.

3. Results

3.1. Polydatin Treatment Attenuated the Symptoms of Cancer Cachexia in CT26-Bearing Mice

To assess the anti-cachexia effects of polydatin, CT26-bearing mice received oral doses of 50 and 100 mg/kg of polydatin for 14 consecutive days. Daily measurements of body weight revealed significant weight loss in the CT26 mice starting on day 10 post-tumor inoculation (Figure 1B,C). Notably, treatment with 100 mg/kg of polydatin led to an improvement in cancer cachexia symptoms. At the end of the experiment, CT26-bearing mice showed an average body weight loss of approximately 11.5% from their initial weight. In contrast, the group treated with 50 mg/kg of Polydatin experienced about 10.6% weight loss, while the polydatin-100 mg/kg group had only a 6% body weight loss (Figure 1C). Food intake was evaluated across four groups, showing no significant differences between the polydatin and diluent groups, suggesting that polydatin did not affect anorexia (Figure 1D). Four-limb grip strength, which showed no significant differences among the four groups at the beginning of the experiment, experienced a remarkable reduction in CT26-bearing mice groups compared to controls; however, treatment of 100 mg/kg polydatin was able to slightly restore muscle strength (Figure 1E).

3.2. Polydatin Alleviated Skeletal Muscle Atrophy in CT26-Bearing Mice

Cancer cachexia is often associated with muscle loss. In this study, the effect of polydatin on the gastrocnemius muscle was tested. In tumor-bearing mice, a remarkable loss of the gastrocnemius muscle was recorded; however, treatment of 100 mg/kg polydatin could attenuate this alteration (Figure 2A,B). Results from laminin and H&E staining exhibited a high population of small muscle fibers in CT26-bearing mice administered with diluent compared with control group, but this was significantly recovered with polydatin treatment (Figure 2C,D). CSA measurements indicated the occurrence of atrophy events in the CT26-bearing mice by the prevalence of shrunken muscle fibers and low average muscle fiber size; however, in response to 100 mg/kg polydatin treatment, the CSA of the skeletal muscle fibers was significantly increased (Figure 2E,F).

3.3. Polydatin Regulated Protein Synthesis and Protein Degradation-Related Genes in Skeletal Muscles

Cachexia-associated muscle atrophy is often associated with the activation of the ubiquitin–proteasome pathway (UPP), which labels the target protein for degradation. The mRNA expressions of two E3 ligase enzymes in the UPP, namely, Atrogin-1 and MuRF1, were evaluated. Here, expression levels of both genes were upregulated in the cachectic mice compared with the control mice. After 100 mg/kg polydatin treatment, the mRNA expressions of Atrogin1 and MuRF1 were significantly decreased. The expression of IL6, a proinflammatory cytokine, was dose-dependently reduced after polydatin treatment, suggesting the function of polydatin in reducing inflammation in muscle samples (Figure 3A). The protein expression level of Atrogin-1 and MuRF1 showed the same pattern as mRNA expression data, in which Atrogin1 and MuRF1 were elevated in CT26-bearing mice; meanwhile, the expression level of MyHC, a myotube biomarker, slightly reduced. However, treatment of 100 mg/kg polydatin could ameliorate the effect of CT26 tumor-induced muscle atrophy by reducing the expression levels of Atrogin-1 and MuRF1 and increasing MyHC compared with CT26-bearing mice administered with DMSO. In addition, the expression level of p-STAT3 was significantly decreased in response to 100 mg/kg polydatin treatment, indicating the involvement of polydatin in this signaling pathway (Figure 3B).

3.4. Polydatin Suppressed C2C12 Myotube Atrophy

Then, we investigated the ability of polydatin in preventing myotube atrophy induced by CT26 CM. Results from the cytotoxicity assay of polydatin using CCK-8 showed that toxicity of polydatin to C2C12 cells was significant from concentrations > 400 µM after 24 h. Therefore, 100 and 200 µM were chosen for the following experiments (Figure 4A). LADD staining was performed to visualize cell morphology, indicating a clear reduction in C2C12 myotube diameter in response to CM addition that was remarkably restored by the intervention of polydatin (Figure 4B,C). An upregulation of Atrogin-1, MuRF, and IL6 was observed at both mRNA (Figure 4D) and protein (Figure 4E) levels; in addition, a decrease in MyHC protein expression in response to CM treatment was inhibited by polydatin treatment. In addition, polydatin treatment considerably downregulated the protein level of p-STAT3 in CM-treated C2C12 cells, demonstrating the involvement of polydatin in facilitating the STAT3 signaling pathway (Figure 4E).

3.5. Binding of Polydatin to Proteins in the IL6/STAT3 Signaling Pathway

Subsequently, a molecular docking analysis of polydatin with the IL6/STAT3 signaling pathway was conducted to elucidate its structural features. The docking pose of polydatin to each IL6 receptor and STAT3 was simulated, and the stabilities of the polydatin–protein complexes were compared using two interaction energies: CDOCKER interaction energies and calculated binding energies. A higher CDOCKER interaction energy and a lower calculated binding energy indicate a greater binding affinity between the protein and the compound [18,19,20].

First, the binding of polydatin to the IL6 receptor was tested and then compared to the isoflavone genistein, a published IL6 receptor inhibitor (Figure 5A and Figure S2, and Table 1). Polydatin docked near the binding site of the IL6 receptor and showed favorable binding interactions. The binding energy of polydatin bound to the IL6 receptor was −287.078 kcal/mol and that of the −CDOCKER interaction energy was 27.7649 kcal/mol. In the IL6 receptor structure, Phe229 and Phe279 residues play a major role in binding IL6 to its receptor [15,16]. As shown in the 3D and 2D interaction diagrams of the polydatin–IL6 receptor complex, the first phenol ring of polydatin formed a Pi–Pi T-shaped interaction with the Phe229 residue, the main amino acid through which IL6 binds to the IL6 receptor. In addition, the second phenol ring of polydatin formed a Pi–Alkyl interaction with the Arg231 residue, and the oxygen ion of the phenol ring formed an attractive charge interaction with the Arg231 residue (Figure 5B). Meanwhile, genistein docked to the binding site of the IL6 receptor by interacting with amino acids including Tyr230 (Pi–Pi stacked), Arg231 (Pi–Alkyl and attractive charge), Glu163 and Glu277 (conventional hydrogen bond) showing −249.748 and 22.7558 kcal/mol of the binding energy and –CDOCKER interaction energy, respectively. However, no non-bond interaction was noted with Phe229 or Phe 279 (Figure S2). These results indicate that polydatin interrupts the binding of IL6 to its receptor through a relatively higher binding affinity than that of genistein.

The interaction of polydatin with STAT3 protein was also tested by comparing to two inhibitor controls, STAT3 inhibitor1 and Stattic (Figure 5C, Figures S3 and S4). STAT3 was activated through the phosphorylation of Tyr705 located in the Src homology 2 (SH2) domain and activated STAT3 monomers interacted via their SH2 domain to form a homodimer of pSTAT3 [21]. Thus, the SH2 domain is an important therapeutic target for STAT3 inhibitor research. Here, polydatin docked near the SH2 domain showing 40.4785 and −450.322 kcal/mol of the binding energy and –CDOCKER interaction energy, respectively. The polydatin–STAT3 complex showed better energy values than STAT3 inhibitor 1 (35.1069 and −197.609 kcal/mol, respectively) and Stattic (22.1701 and −170.22 kcal/mol, respectively) (Table 2). As illustrated in Figure 5D, two phenol rings formed a Pi–cation interaction with Lys591 and Pi–alkyl interaction with Arg595. Moreover, the hydrogen and oxygen ions of the sugar group formed several hydrogen bond interactions with Lys591, Glu594, and Ser636. These results indicate that both the resveratrol and sugar groups in polydatin block the SH2 domain of STAT3 by affecting its interaction with the protein.

Overall, these molecular simulation results reveal that polydatin might inhibit the STAT3 pathway by interrupting both IL-6 receptor and STAT3.

4. Discussion

In this study, a murine colon carcinoma model was employed to investigate cancer cachexia, given its high prevalence in colorectal cancer—affecting approximately 50% of patients during the course of the disease. Establishing a CT26-induced cachexia model is therefore crucial for elucidating the biological, cellular, and molecular mechanisms underlying cachexia, as well as for evaluating potential therapeutic strategies.

Despite the devastating effects on patients with terminal staged cancer, the treatment for cancer cachexia has remained scarce up until now [22]. Thus, finding new treatments will broaden the remedy for cancer cachexia, which could increase the chances of survival of patients with cancer. The usage of natural compounds has emerged recently as a potential approach for alleviating cancer cachexia symptoms owing to their high efficacy and minimal toxicity [23,24,25].

Polydatin, a natural compound, has been used in traditional medicines in many Asian countries. It has anti-inflammatory and anticancer properties; however, its effects on cancer cachexia remains unknown. In this study, polydatin significantly mitigated cancer cachexia symptoms in CT26 cachectic mice and attenuated muscle atrophy in C2C12 myotubes.

In the in vivo model, polydatin showed efficacy in increasing body weight, skeletal muscle mass and strength in a dose-dependent manner. The CSA of the epididymal fat was significantly reduced in the model group; however, treatment attenuated the reductions. In the in vitro experiment, the effects of polydatin on the muscle atrophy of C2C12 myotubes induced by the CT26 CM resulted in an increase in myotube diameter and protein synthesis and a decrease in protein degradation. These results indicated the possibility of using polydatin in treating cancer cachexia.

Muscle wasting is the main feature of cancer cachexia following a decrease in body weight. Among signaling pathways responsible for cancer cachexia, the STAT3 signaling pathway was involved in controlling muscle loss for a long time [6]. Upon the binding of IL6 to its receptor, associated Janus kinases (JAKs) are catalytically activated and subsequently mediate the phosphorylation of STAT3 on tyrosine 705, resulting in their dimerization, nuclear translocation, DNA binding, and gene activation [26]. STAT3 activation induces muscle wasting by increasing the expression of CAAT/enhancer binding protein β (C/EBPβ), which leads to increased expression of E3 ubiquitin ligases MuRF1 and MAFbx/Atrogin-1 [7]. Moreover, it severely worsens inflammation by promoting IL6 production [27]. Thus, STAT3 may serve as a target of cancer cachexia, and inhibiting the STAT3 signaling pathway could support muscle preservation by mitigating systemic inflammation and reducing the expression of muscle-degrading factors such as ubiquitin ligases [7].

In the present study, polydatin significantly inhibited STAT3 activation in both the gastrocnemius muscle of CT26-bearing mice and CM-induced C2C12 myotubes, which eventually downregulated the expression levels of MuRF1 and Atrogin-1. In addition, the expression of IL6, which is considered a cancer cachexia biomarker, was reduced in response to polydatin treatment. Thus, polydatin might exert its protective effects on cachexia by inhibiting the IL-6/STAT3 pathway.

The STAT3 signaling pathway is essential for various physiological processes, including the regulation of cell proliferation, differentiation, and apoptosis. STAT3, a transcription factor, is located in the cytoplasm as a monomer. In response to cytokines or other growth factors, STAT3 is phosphorylated at Y705 within its SH2 domain by upstream kinases; as a result, dimerization and nucleus translocation modulate target gene expression [28].

STAT3 inhibitors can be categorized based on their interaction with direct or indirect target proteins. Direct inhibitors bind STAT3 domains, whereas indirect inhibitors affect STAT3 by interacting with upstream molecules in the STAT3 signaling pathway as IL6 receptors or JAKs [29]. To evaluate the inhibitive effect of polydatin on the STAT3 signaling pathway, molecular docking was performed to determine the target protein. Results demonstrated that polydatin showed binding affinity to both the SH2 domain of STAT3 and the IL6 receptor with a binding energy of −450.322 and −287.078 kcal/mol, respectively. This molecular docking study revealed that polydatin may prevent STAT3 activation, hence alleviating cancer cachexia symptoms.

In summary, this study illustrated that polydatin showed an anti-cancer-cachexia effect by prohibiting STAT3 activation and inhibiting the expression of MuRF1 and Atrogin-1, eventually reducing protein degradation and muscle wasting. The results imply that polydatin could be a potential agent in the treatment of muscle wasting in cancer cachexia. In the future, we will test on multiple cachexia models to validate the generalizability of the observed effects of polydatin across different tumor types and disease stages. In addition, pharmacokinetic analyses and tissue distribution studies will be conducted to elucidate the translational potential of polydatin in cancer cachexia.

5. Conclusions

In conclusion, polydatin alleviated cancer cachexia in CT26-bearing mice and in C2C12 myotubes by improving muscle mass and function and reducing muscle atrophy. These effects were achieved by inhibiting the IL-6/STAT3 signaling pathway, leading to the downregulation of MuRF1 and Atrogin-1 expression, thereby decreasing protein degradation. Our findings suggest that polydatin is a promising natural therapeutic candidate for the treatment of cancer cachexia. Further studies are needed to validate its translational potential.