Sheng-Yu Jie-Du Tang Lyophilised Extract Reverses Cancer-Induced Myotube Atrophy via PINK1/Parkin-Mediated Mitophagy and Suppression of the NLRP3 Inflammasome

Sheng-Yu Jie-Du Tang Lyophilised Extract Reverses Cancer-Induced Myotube Atrophy via PINK1/Parkin-Mediated Mitophagy and Suppression of the NLRP3 Inflammasome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sheng-Yu Jie-Du Tang Lyophilised Extract Reverses Cancer-Induced Myotube Atrophy via PINK1/Parkin-Mediated Mitophagy and Suppression of the NLRP3 Inflammasome juanjuan peng, Jing Hu, Yang Zhang, Mianhua Wu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8130647/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective : This study aimed to investigate the impact and mechanism of Shengyu Jiedu Tang(SYJDT) lyophilized powder on a cancer-induced muscular atrophy cell model by modulating the mitochondrial autophagy-NLRP3 inflammatory pathway. Methods : Mouse myoblasts C2C12 were exposed to the supernatant of Lewis lung carcinoma (LLC) to establish a carcinogen-induced muscular atrophy cell model. Various parameters including cell proliferation, migration, myotube atrophy, ROS levels, mitochondrial membrane potential, and expression of proteins in the mitochondrial autophagy-NLRP3 signaling pathway were assessed following treatment with different concentrations of Shengyu Jiedu Tang lyophilized powder. Results : The cancer-induced muscular atrophy cell model was successfully developed, demonstrating myotube atrophy, reduced myotube diameter, elevated TNF-α and IL-6 levels in the culture medium. Treatment with Shengyu Jiedu Tang lyophilized powder significantly suppressed cell proliferation and migration, ameliorated myotube atrophy, decreased ROS levels, enhanced mitochondrial membrane potential, upregulated mitochondrial autophagy-related proteins (LC3, Pink1, Parkin), downregulated P62 protein expression, inhibited NLRP3 inflammasome activation, and reduced inflammatory factor expression. Mitophagy inhibitors partially reversed the effects of the lyophilized powder, suggesting a close association with mitophagy. Conclusion : Shengyu Jiedu Tang lyophilized powder can attenuate cancer-induced muscular atrophy by stimulating mitochondrial autophagy, modulating mitochondrial function, suppressing NLRP3 inflammasome activation, and reducing inflammatory factor expression. Biological sciences/Cancer Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Molecular biology mitophagy cancer-associated cachexia skeletal-muscle atrophy Sheng-Yu Jie-Du Tang NLRP3 inflammasome inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Cancer cachexia (CC) is a multifaceted metabolic syndrome typified by weakness, weight loss, muscle and fat atrophy, and systemic inflammation [ 1 – 3 ] . Predominantly manifesting in advanced stages of cancer, CC is a prevalent complication significantly compromising patients' quality of life and survival rates [ 4 , 5 ] . Despite the absence of standardized diagnostic criteria, the detrimental effects of CC are universally acknowledged, underscoring the critical imperative for enhanced interventions to ameliorate its symptoms. The pathogenesis of cancer-related cachexia is closely associated with a systemic inflammatory response, primarily mediated by TNF-α, IL-1, IL-6, and other inflammatory mediators [ 6 ] . Mitochondrial autophagy and NLRP3 inflammasomes play a crucial role in regulating muscle atrophy by facilitating mitochondrial autophagy, eliminating damaged mitochondria, and reducing muscle atrophy through the activation of the Pink1-Parkin signaling pathway [ 7 , 8 ] . In cachexia, NLRP3 inflammasome activation triggers the release of pro-inflammatory cytokines such as IL-1β and IL-18, exacerbating the inflammatory response in muscle cells. This process inhibits muscle protein synthesis, enhances muscle protein degradation, and ultimately contributes to muscle atrophy. Shengyu Jiedu Tang(SYJDT) is a modification of the well-known traditional Chinese medicine formula, Shengyu Tang. Comprising ginseng, astragalus root, angelica root, ligusticum wallichii, white peony root, prepared rehmannia root, and other traditional Chinese medicinal ingredients, Shengyu Tang is recognized for its efficacy in modulating immune function, attenuating inflammatory responses, and enhancing nutritional status. Studies have demonstrated its ability to suppress the expression of inflammatory markers such as TNF-α, IL-1, and IL-6, thereby mitigating systemic inflammation [ 9 ] . Our research team has refined Shengyu Jiedu Tang through extensive clinical practice. While this formulation shows promise in ameliorating muscle atrophy associated with cancer-related cachexia, its intricate composition and precise mechanism of action remain unclear. In this investigation, a cell model simulating carcinogenic muscular atrophy was developed through cell co-culture techniques. Specifically, Lewis cell supernatant was employed to disrupt Mouse myoblasts C2C12. Conditioned medium was prepared using horse serum differentiation medium and LLC culture supernatant, building upon prior research. Following a 96-hour intervention period, the cell model of carcinogenic muscular atrophy was successfully established. Subsequent intervention involved the application of freeze-dried powder of Shengyu Jiedu Tang to explore its effects and underlying mechanisms on the cell model. This preliminary exploration sets the groundwork for further research and potential clinical applications. 2. Materials and Methods 2.1 Cell Culture Lewis lung carcinoma (LLC) (catalog no. TCM-C742) and Mouse myoblasts C2C12 cells (catalog no. TCM-C720) were obtained from Haixing Biotechnology (Suzhou, China). 2.2 Reagents and Compounds Shengyu Jiedu Tang (SYJDT) was obtained from Jiangsu Provincial Hospital of Traditional Chinese Medicine. The composition is as follows (per dose): Honey-fried Astragalus 35g, Ginseng 15g, Prepared Rehmannia 12g, Chinese Angelica 12g, Chuanxiong Rhizome 10g, Achyranthes Root 12g, Psoralea Fruit 12g, Oldenlandia 15g.The crude mixture was decocted with water, vacuum-concentrated to 1 g raw herb per mL, snap-frozen at − 80°C overnight, and lyophilized under vacuum. The resulting dry powder was stored at − 20°C until use.Mitophagy inhibitor: Cyclosporin A (CsA, catalog HYB0579) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).and dissolved in molecular-grade DMSO (Solarbio, Beijing, China; cat. no. D8371) to prepare a 10 mM stock solution. 2.3 Antibodies and Assay Kits Primary antibodies against FBXO32/Atrogin-1 (ab74023), MURF-1/TRIM63 (ab183094), PINK1 (ab216144), Parkin (ab77924), p62/SQSTM1 (ab109012), LC3-Ⅱ (ab192890), NLRP3 (ab263899), caspase-1 (ab207802), ASC (ab283684), and GAPDH (YM3030) were purchased from Abcam (Cambridge, UK). Horseradish-peroxidase (HRP)-conjugated goat anti-rabbit (RS0002) and goat anti-mouse (RS0001) secondary antibodies were obtained from Immunoway (Plano, TX, USA).ELISA kits for murine TNF-α (cat. no. YJ002095-96T) and IL-6 (cat. no. YJ098430-96T) were supplied by Enzyme-linked Biotechnology (Shanghai, China). The reactive oxygen species (ROS) detection kit (cat. no. S0033S) was from Beyotime Biotechnology (Shanghai, China). Special horse serum for myogenic differentiation was purchased from Solarbio (cat. no. S9050-200 mL). 2.4 Cell Culture Media Complete growth media were prepared as follows: LLC cells were cultured in RPMI-1640 supplemented with 10% (v/v) foetal bovine serum and 1% penicillin–streptomycin; Mouse myoblasts C2C12 cells were maintained in high-glucose DMEM containing 20% foetal bovine serum and 1% penicillin–streptomycin. To induce myotube formation, C2C12 cells were switched to differentiation medium (DMEM high glucose plus 2% horse serum) once ≥ 80% confluence was reached.Conditioned media (CM) were generated by collecting supernatants from LLC cultures after 48 h, followed by sequential filtration through 0.45 µm and 0.22 µm membranes. CM was then blended with differentiation medium at volumetric ratios of 1:1, 1:2, 1:4, or 1:8 to generate CM1, CM2, CM3, and CM4, respectively. As shown in Table 1 . Table 1 Culture media formulations Growth medium (LLC) Growth medium (C2C12) Differentiation medium Conditioned medium RPMI-1640 89% High-glucose DMEM 89% High-glucose DMEM 97% LLC cell-conditioned medium FBS10% FBS10% Horse serum 2% Differentiation medium Penicillin–streptomycin 1% Penicillin–streptomycin 1% Penicillin–streptomycin 1% 2.5 Induction of Cancer-Associated Muscle Atrophy In Vitro Mouse myoblasts C2C12 cells and LLC cells were maintained at 37°C in a humidified 5% CO₂ atmosphere and passaged every 48 h. For atrophy induction, LLC-conditioned medium (LLC-CM) was generated by 48-h culture of LLC cells in RPMI-1640 + 10% FBS. The supernatant was cleared by sequential centrifugation (300 × g, 5 min) and filtration (0.22 µm filter membrane). C2C12 myoblasts were differentiated for 96 h in 2% horse-serum/DMEM to form multinucleated myotubes, after which differentiation medium was replaced with LLC-CM diluted 1:1, 1:2, 1:4, or 1:8 in differentiation medium. This regimen reproducibly induced a 30–40% reduction in myotube diameter within 72 h, validating the cachectic milieu. 2.6 Preparation of SYJDT Stock Solution 0.2 g mL⁻¹ SYJDT stock solution: 200 mg lyophilised Sheng-Yu Jie-Du Tang powder was dissolved in 1 mL molecular-grade DMSO, vortex-mixed until complete solubilisation, sterile-filtered, and stored in single-use aliquots at − 80°C. Working dilutions were prepared fresh by diluting the stock in C2C12 growth medium to the indicated concentrations immediately before use. 2.7 Experimental Design and Treatment Schema Group A – Vehicle control: C2C12 growth medium only.Group B – SYJDT lyophilizate: C2C12 medium supplemented with SYJDT freeze-dried powder.Group C – Antagonist control: C2C12 medium plus pharmacological antagonist.Group D – SYJDT + antagonist: C2C12 medium containing both SYJDT lyophilizate and antagonist. 2.8 Real-time monitoring of myotube morphology by inverted microscopy Medium was renewed every 24 h. At the same clock time on days 1, 2, 3 and 4 (24, 48, 72 and 96 h after the onset of treatment) images were captured at 200× magnification. Myotube diameter was randomly measured at three distinct sites per field; data were analyzed with ImageJ. 2.9 ELISA quantification of TNF-α and IL-6 in conditioned medium C2C12 supernatants were collected and assayed in duplicate for TNF-α and IL-6 according to the manufacturer’s instructions. Absorbance was read at 450 nm, standard curves were constructed, and cytokine concentrations were calculated. 2.10 High-Throughput Cytotoxicity and Viability Screen (CCK-8) Cancer-cachexia myotubes exhibiting robust growth were dissociated, counted on an automated cell counter, and reseeded at 5 × 10⁴ cells/mL in 96-well plates (100 µL/well, three replicates per group).Lyophilized Sheng-Yu Jie-Du decoction was reconstituted in complete high-glucose DMEM to final concentrations of 0.25%, 0.5%, 1%, 2% and 3% (w/v); control wells received vehicle medium alone.Plates were returned to a humidified 5% CO₂ incubator at 37°C for 0, 24 or 48 h.At each time point the medium was aspirated and replaced with freshly prepared CCK-8 working solution (nine parts high-glucose DMEM : one part CCK-8 reagent).After 1 h protected from light on a pre-warmed (37°C) orbital shaker, absorbance at 450 nm was recorded on a microplate reader; cell viability and percentage inhibition were calculated from the resulting OD values. 2.11 Wound-healing assay for lateral migration of cancer-cachexia myotubes under Sheng-Yu Jie-Du decoction Based on the CCK-8 dose–response screen, the optimal lyophilized-decoction concentration was selected for migration studies.Myotubes in logarithmic growth were detached, counted, and seeded at 5 × 10⁵ cells per well (or at a density yielding confluent monolayers after 12 h).When cultures reached 80–90% confluence, a sterile 200 µL pipette tip guided by a ruler held above the plate was used to create a straight scratch.After two PBS washes to remove cellular debris, the wound was imaged at 40× magnification (time 0 h).Medium was then replaced with 2 mL of fresh 1% serum-containing high-glucose DMEM (vehicle control) or decoction supplemented medium (experimental group); each condition was run in triplicate.Identical fields were re-imaged at 24 h and 48 h.Wound closure was quantified with ImageJ and expressed as percentage migration relative to the 0-h width. 2.12 Evaluation of Myotube Atrophy Reversal After selection of the optimal lyophilizate concentration in CCK-8 assays, a cancer-cachectic atrophy model was established. Atrophy-model wells were maintained in complete medium for an additional 48 h, whereas SYJDT-treated wells received the pre-determined concentration of freeze-dried powder for the same period. Phase-contrast images (200×) were then acquired on an inverted microscope to evaluate the extent of myotube atrophy and the protective effect of SYJDT. 2.13 Reactive Oxygen Species (ROS) Imaging Six-well plates were used for cell culture. After aspirating the medium, cells were loaded with 10 µM DCFH-DA (1 ml per well, sufficient to completely cover the monolayer) and incubated at 37°C for 20 min. Following incubation, cells were washed three times with serum-free medium and immediately visualized under an inverted microscope; images were captured at 200× magnification using the FITC filter set. 2.14 Mitochondrial Membrane Potential (ΔΨm) After the scheduled interventions, ΔΨm was evaluated using JC-1 staining. A working solution was freshly prepared by vortexing the JC-1 stock vigorously, followed by dilution into 2 ml of JC-1 assay buffer (5×) and thorough mixing. Medium was aspirated from each well of a six-well plate and replaced with 1 ml of complete growth medium; 1 ml of the JC-1 working solution was then added and mixed gently to ensure uniform distribution. Cells were incubated at 37°C for 20 min, after which the supernatant was removed and wells were washed twice with 1× JC-1 assay buffer at 37°C. Finally, 2 ml of warm complete medium was added and three random fields per well were imaged at 200× magnification on a fluorescence microscope. 2.15 Western-blot analysis of mitophagy signaling proteins Cells were lysed in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined with the BCA assay, and 30 µg of each lysate was denatured, resolved on 4–12% Bis-Tris gels, and transferred to 0.22 µm PVDF membranes. After blocking with 5% non-fat dry milk in TBST for 1 h at room temperature, membranes were incubated overnight at 4°C with the following primary antibodies (all validated for murine C2C12): MuRF-1 (1:2 000), FBXO32 (1:1 000), PINK1 (1:1 000), Parkin (1:1 000), p62/SQSTM1 (1:1 000), LC3B (1:2 000) and GAPDH (1:20 000). Following three 10-min washes with TBST, membranes were probed with HRP-conjugated secondary antibodies (1:5 000) for 1 h at room temperature. Immunoreactive bands were visualized with ECL Prime and quantified by densitometry using ImageJ (v1.54). GAPDH served as the loading control. 2.16 RT-qPCR quantification of mitophagy-related transcripts Total RNA was extracted with TRIzol reagent and reverse-transcribed using the High-Capacity cDNA Synthesis Kit (Thermo Fisher) according to the manufacturer’s instructions. Real-time PCR was performed on a QuantStudio 6 Flex system using SYBR Green master mix with gene-specific primers (sequences available upon request). Relative expression was calculated by the 2^(-ΔΔCt) method, normalized to GAPDH, and expressed as fold-change versus the control group. The primers used were showed in Table 2 . Table 2 Target-gene primer sequences for real-time PCR Primer name Sequence (5' → 3') Length (nt) Atrogin/FBX032 Forward primer GGACCCTCCCTAATCCCCTT 20 Atrogin/FBX032 reverse primer CACTGTTGCTGTTCCAGTGC 20 MuRF-1 forward primer CCCTGATCCTCCAGTACCGA 20 MuRF-1 reverse primer GTGTCCTTCCTTACCCTCTGTG 22 pink1 forward primer AGGAAAAGGCCCAGATGTCG 20 pink1 reverse primer CTGTTTGCTGAACCCAAGGC 20 parkin forward primer TGACCAGCTGCGAGTGATTT 20 parkin reverse primer GCAGTCTGGAGATTGGCACT 20 P62 forward primer GTTCCTGAACCCTCTCGTGG 20 P62 reverse primer GACTTTACGGGGTGCCTCAA 20 LC3 forward primer GGCGCTTACAGCTCAATGCT 20 LC3 reverse primer CTCCTGGGAGGCATAGACCA 20 20 NLRP3 forward primer ATTACCCGCCCGAGAAAGG 19 NLRP3 reverse primer CATGAGTGTGGCTAGATCCAAG 22 caspase-1 forward primer ACAAGGCACGGGACCTATG 19 caspase-1 reverse primer TCCCAGTCAGTCCTGGAAATG 22 ASC forward primer GACAGTGCAACTGCGAGAAG 20 ASC reverse primer CGACTCCAGATAGTAGCTGACAA 23 GAPDH forward primer GCGAGATCCCGCTAACATCA 20 GAPDH reverse primer CTCGTGGTTCACACCCATCA 20 2.17 Statistical analysis All experiments were performed in triplicate with a minimum of three biological replicates. Data are expressed as mean ± SD. All analyses and graphs were generated with GraphPad Prism 8.0.1. Inter-group differences were evaluated by one-way ANOVA. A P value < 0.05 was considered statistically significant, and P < 0.01 was regarded as highly significant. 3. Results 3.1 Establishment of a Cancer-Cachectic Myotube Atrophy Model Exposure to increasing concentrations of LLC-conditioned medium (CM) produced a time- and dose-dependent delay in myotube formation, a marked reduction in myotube number, and a significant decrease in cross-sectional diameter (Fig. 1 A, B). The maximal inter-group divergence was observed after 72 h, at which point the mean myotube diameter in condition-1 CM was substantially lower than in normal medium (P < 0.05) and also differed from the other treatment groups (P < 0.05), mirroring previously reported cachectic phenotypes. Consequently, 72-h treatment with condition-1 CM was selected as the standard protocol for generating the cancer-induced atrophy model in all subsequent experiments. 3.2. Cytokine profiling of 72-h conditioned media by ELISA After a 72-h exposure of myotubes to serial dilutions of LLC-conditioned medium (CM), TNF-α and IL-6 levels in the supernatants were quantified. Both cytokines were elevated in every CM group relative to normal growth medium, reaching statistical significance for all except the most dilute condition-4 group (P < 0.01). Among the CM groups, condition-1 generated the highest TNF-α concentration, significantly exceeding each of the other dilutions (P < 0.05), and likewise produced the maximal IL-6 signal, with inter-group differences highly significant (P < 0.01) (Fig. 1 C, D). 3.3 Effects of SYJDT lyophilizate on the cancer-cachectic myotube model 3.3.1 CCK-8 assay C2C12 myotubes exposed to 0.25–3% (v/v) SYJDT or vehicle for 24–48 h showed dose- and time-dependent reductions in viability. Maximal cytostasis without cytotoxicity was achieved with 1% SYJDT at 48 h (Fig. 2 A); this condition was adopted for all subsequent mechanistic studies. 3.3.2 Wound-healing assay Confluent monolayers were scratched and monitored at 0, 24 and 48 h. Vehicle-treated cells closed ≥ 90% of the wound by 48 h, whereas 1% SYJDT markedly attenuated migration (P < 0.01) versus control(Fig. 2 B, C), confirming an anti-migratory effect of the formulation. 3.3.3 Morphometric rescue of atrophic myotubes After 48 h treatment, SYJDT (1%) reversed LLC-CM-induced diameter loss, yielding significantly thicker myotubes than the atrophy model group (P < 0.01; Fig. 2 D, E). 3.4 Modulation of ROS and mitochondrial membrane potential (ΔΨm) Relative to vehicle, 1% SYJDT markedly lowered ROS generation (P < 0.01; Fig. 3 A, B) and elevated ΔΨm (P < 0.01; Fig. 3 C, D). Co-incubation with the mitophagy inhibitor CsA reversed both effects (P < 0.01), while SYJDT + CsA exhibited an intermediate phenotype, indicating that SYJDT’s antioxidant and mitochondrial-protective actions are partly mitophagy-dependent. 3.5 Mitophagy–NLRP3 axis mRNA expression (RT-qPCR) SYJDT up-regulated LC3, PINK1 and Parkin transcripts and down-regulated p62 and NLRP3 compared with vehicle (all P < 0.01). CsA alone reduced PINK1, Parkin and LC3 while increasing p62; these changes were partially rescued in the SYJDT + CsA group (Fig. 4 ), implying that SYJDT activates mitophagy and suppresses NLRP3 inflammasome transcription. 3.6 Mitophagy–NLRP3 axis protein expression (Western blot) SYJDT significantly elevated LC3-II, PINK1 and Parkin, decreased p62, and attenuated NLRP3 protein levels versus vehicle (P < 0.01). CsA antagonised these effects, whereas SYJDT + CsA retained partial rescue (Fig. 5 ). Thus, SYJDT enhances mitophagic flux and dampens NLRP3-driven inflammation in cancer-cachectic myotubes. 4. Discussion Our findings align with a growing body of literature that positions mitochondrial autophagy as a cytoprotective hub in multiple disease models. In neurodegeneration, PINK1/Parkin-mediated mitophagy clears dysfunctional mitochondria, blunts oxidative stress and apoptosis, and slows disease progression [ 10 – 13 ] Geisler et al [ 14 ] first showed in Parkinson models that PINK1/Parkin signalling removes damaged mitochondria and rescues neurons, while Lazarou et al. [ 15 ] established that Parkin-dependent mitophagy is indispensable for neuronal mitochondrial quality control. We now extend this paradigm to muscle: Shenyi Jiedu Tang (SYJDT) up-regulates PINK1, Parkin and LC3, evokes mitophagy, and attenuates cancer-associated muscle atrophy, suggesting a conserved mechanism from brain to skeletal muscle. Parallel evidence implicates chronic low-grade systemic inflammation in cancer cachexia. Pro-inflammatory cytokines (TNF-α, IL-1, IL-6) produced by the tumour micro-environment and host immune cells activate catabolic programmes in liver and skeletal muscle, accelerating protein degradation and weight loss [ 16 , 17 ] . As recently reviewed by Argilés et al. tumor-driven systemic inflammation simultaneously represses PGC-1α/PINK1–Parkin signaling, triggers NLRP3 inflammasome activation, and up-regulates Atrogin-1/MuRF-1, thereby linking mitochondrial dysfunction to accelerated myofibrillar protein degradation in cancer cachexia [ 18 ] . The NLRP3 inflammasome sits at the nexus of this response; its over-activation liberates IL-1β and IL-18 and amplifies tissue injury. In muscle-wasting disorders, NLRP3, ASC and caspase-1 are markedly up-regulated, myofibre cross-sectional area shrinks, and E3 ligases Atrogin-1/MuRF-1 rise—changes reversed by NLRP3 inhibitors [ 19 ] .Likewise, NLRP3-driven pyroptosis and proteolysis have been documented in diabetic myopathy [ 20 ] . NLRP3 inflammasome activation is a key driver of tumor-induced muscle wasting [ 21 ] . Here, SYJDT lyophilised powder dose-dependently suppressed NLRP3, ASC and caspase-1, and lowered circulating TNF-α and IL-6. By restraining inflammasome activation SYJDT dampened inflammation and preserved muscle mass—an anti-atrophy effect that mirrors prior pharmacologic NLRP3 blockade and broadens the anti-inflammatory repertoire of traditional Chinese medicine. Taken together, SYJDT exerts a dual action—promoting mitophagy while braking NLRP3 signalling—thereby coupling mitochondrial quality control to suppression of sterile inflammation. The data resonate with current international priorities (mitophagy-NLRP3 axis) and furnish a mechanistic rationale for deploying SYJDT in cancer cachexia. Limitations and next steps:Although we document SYJDT-mediated modulation of mitophagy and NLRP3, the precise molecular relays remain incomplete. Future work should;Expand sample size and launch multicentre pre-clinical trials;Deploy gene KO/over-expression to establish causal roles for PINK1/Parkin and NLRP3 pathways;Translate findings into early-phase clinical studies evaluating efficacy and safety in patients with muscle wasting;Map additional signalling nodes (e.g., AMPK, mTOR, NF-κB) to capture SYJDT’s poly-pharmacology. In summary, SYJDT engages the mitophagy-NLRP3 axis to restrain muscle atrophy, offering a fresh mechanistic lens for its pharmacology and a theoretical basis for repurposing this herbal formula in neurodegenerative and cachectic conditions. Declarations Competing interests The authors declare that they have no competing interests.All other authors declare no financial relationship with any organization that might have an interest in the submitted work in the previous five years. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding:The author(s) declare that financial support was received for the research and/or publication of this article. The present study was supported by Wu Mian hua Inheritance Studio of National Famous and Old Chinese Medicine Experts (State Administration of Traditional Chinese Medicine No. [2022]75);Academic Experience Inheritance Project for the Seventh Batch of Old Chinese Medicine Experts by State Administration of Traditional Chinese Medicine (State Administration of Traditional Chinese Medicine No. [2022]76); Authors' contributions All of the authors performed the study.PJJ: Conceptualization, Methodology, Writing – Original Draft.HJ: Data Curation, Validation, Formal Analysis.ZY: Investigation, Resources, Writing – Review & Editing.WMH: Supervision, Project Administration, Funding Acquisition All the authors read and approved the final version of the manuscript. T he data availability statement The datasets generated during the current study are not publicly available as they form part of an ongoing research project that has not yet been concluded. 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The role of NLRP3 inflammasome in inflammation-related skeletal muscle atrophy[J]. Front Immunol. 2022, 13: 1035709. Yan X, Fu P, Zhang Y, et al. MCC950 Ameliorates Diabetic Muscle Atrophy in Mice by Inhibition of Pyroptosis and Its Synergistic Effect with Aerobic Exercise[J]. Molecules. 2024, 29(3). Franke M, Bieber M, Kraft P, et al. The NLRP3 inflammasome drives inflammation in ischemia/reperfusion injury after transient middle cerebral artery occlusion in mice[J]. Brain Behav Immun. 2021, 92: 223-233. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformationFulllengthblotsandgelsforFigure5.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8130647","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":555530622,"identity":"a8d5394b-1740-45c0-9562-2c0958f7cd6c","order_by":0,"name":"juanjuan peng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"juanjuan","middleName":"","lastName":"peng","suffix":""},{"id":555530623,"identity":"b8d02b7c-3476-49f3-949a-6759cfbccfde","order_by":1,"name":"Jing 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15:33:25","extension":"html","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89308,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8130647/v1/ea12aa18aaf49b8105923dd9.html"},{"id":97703958,"identity":"11ec4bfe-4143-4b0f-a234-2eb726535066","added_by":"auto","created_at":"2025-12-08 12:44:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7191393,"visible":true,"origin":"","legend":"\u003cp\u003eEstablishment of a cancer-cachectic atrophy model. (A, B) Time- and dose-dependent effects of LLC-conditioned medium on myotube diameter (×200). (C, D) IL-6 and TNF-α levels in supernatants after 72 h exposure to graded CM dilutions.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8130647/v1/c04359f59235fefe516bf2e1.png"},{"id":97895144,"identity":"48df0100-630f-4ee4-963a-b3e791955a1d","added_by":"auto","created_at":"2025-12-10 15:33:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5520134,"visible":true,"origin":"","legend":"\u003cp\u003eSYJDT lyophilizate mitigates cancer-cachectic atrophy in C2C12 myotubes. (A) Viability curve. (B, C) Representative wound-healing images (×40). (D, E) Myotube diameter recovery (×200).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8130647/v1/365f9a9f9055184d10d8cf30.png"},{"id":97894912,"identity":"95338bb9-8331-4419-9311-671e9e2f2753","added_by":"auto","created_at":"2025-12-10 15:33:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4849713,"visible":true,"origin":"","legend":"\u003cp\u003eSYJDT attenuates ROS and preserves ΔΨm (×200). (A, B) DCFH-DA fluorescence. (C, D) JC-1 aggregate/monomer ratio.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8130647/v1/207283a95cdeb0ef5a3c2b86.png"},{"id":97703962,"identity":"02c43402-fa98-40ba-be02-aa6ccfebf10f","added_by":"auto","created_at":"2025-12-08 12:44:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":574021,"visible":true,"origin":"","legend":"\u003cp\u003eMitophagy–NLRP3 pathway mRNA profile.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8130647/v1/78800000253e02bee70e4f9d.png"},{"id":97894529,"identity":"990c8fbc-c7c8-4b5d-8649-34e379ee2b92","added_by":"auto","created_at":"2025-12-10 15:32:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1009744,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative Western blots and quantification of mitophagy–NLRP3 signaling proteins.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8130647/v1/0e8045b7d34005743d52cb6e.png"},{"id":104402260,"identity":"1558dd9e-aec9-46cb-a72e-18857b85857a","added_by":"auto","created_at":"2026-03-11 12:14:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17876546,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8130647/v1/7260a1af-3e08-46ff-af99-0738bec54b9c.pdf"},{"id":97703968,"identity":"74708f7e-dc2f-4858-9588-324aa1ffbf30","added_by":"auto","created_at":"2025-12-08 12:44:29","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":156583,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationFulllengthblotsandgelsforFigure5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8130647/v1/4849741e3f4267c7ec2126b6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSheng-Yu Jie-Du Tang Lyophilised Extract Reverses Cancer-Induced Myotube Atrophy via PINK1/Parkin-Mediated Mitophagy and Suppression of the NLRP3 Inflammasome\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer cachexia (CC) is a multifaceted metabolic syndrome typified by weakness, weight loss, muscle and fat atrophy, and systemic inflammation\u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Predominantly manifesting in advanced stages of cancer, CC is a prevalent complication significantly compromising patients' quality of life and survival rates\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Despite the absence of standardized diagnostic criteria, the detrimental effects of CC are universally acknowledged, underscoring the critical imperative for enhanced interventions to ameliorate its symptoms.\u003c/p\u003e\u003cp\u003eThe pathogenesis of cancer-related cachexia is closely associated with a systemic inflammatory response, primarily mediated by TNF-α, IL-1, IL-6, and other inflammatory mediators\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Mitochondrial autophagy and NLRP3 inflammasomes play a crucial role in regulating muscle atrophy by facilitating mitochondrial autophagy, eliminating damaged mitochondria, and reducing muscle atrophy through the activation of the Pink1-Parkin signaling pathway\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. In cachexia, NLRP3 inflammasome activation triggers the release of pro-inflammatory cytokines such as IL-1β and IL-18, exacerbating the inflammatory response in muscle cells. This process inhibits muscle protein synthesis, enhances muscle protein degradation, and ultimately contributes to muscle atrophy.\u003c/p\u003e\u003cp\u003eShengyu Jiedu Tang(SYJDT) is a modification of the well-known traditional Chinese medicine formula, Shengyu Tang. Comprising ginseng, astragalus root, angelica root, ligusticum wallichii, white peony root, prepared rehmannia root, and other traditional Chinese medicinal ingredients, Shengyu Tang is recognized for its efficacy in modulating immune function, attenuating inflammatory responses, and enhancing nutritional status. Studies have demonstrated its ability to suppress the expression of inflammatory markers such as TNF-α, IL-1, and IL-6, thereby mitigating systemic inflammation\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Our research team has refined Shengyu Jiedu Tang through extensive clinical practice. While this formulation shows promise in ameliorating muscle atrophy associated with cancer-related cachexia, its intricate composition and precise mechanism of action remain unclear.\u003c/p\u003e\u003cp\u003eIn this investigation, a cell model simulating carcinogenic muscular atrophy was developed through cell co-culture techniques. Specifically, Lewis cell supernatant was employed to disrupt Mouse myoblasts C2C12. Conditioned medium was prepared using horse serum differentiation medium and LLC culture supernatant, building upon prior research. Following a 96-hour intervention period, the cell model of carcinogenic muscular atrophy was successfully established. Subsequent intervention involved the application of freeze-dried powder of Shengyu Jiedu Tang to explore its effects and underlying mechanisms on the cell model. This preliminary exploration sets the groundwork for further research and potential clinical applications.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Cell Culture\u003c/h2\u003e\u003cp\u003eLewis lung carcinoma (LLC) (catalog no. TCM-C742) and Mouse myoblasts C2C12 cells (catalog no. TCM-C720) were obtained from Haixing Biotechnology (Suzhou, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Reagents and Compounds\u003c/h2\u003e\u003cp\u003eShengyu Jiedu Tang (SYJDT) was obtained from Jiangsu Provincial Hospital of Traditional Chinese Medicine. The composition is as follows (per dose): Honey-fried Astragalus 35g, Ginseng 15g, Prepared Rehmannia 12g, Chinese Angelica 12g, Chuanxiong Rhizome 10g, Achyranthes Root 12g, Psoralea Fruit 12g, Oldenlandia 15g.The crude mixture was decocted with water, vacuum-concentrated to 1 g raw herb per mL, snap-frozen at \u0026minus;\u0026thinsp;80\u0026deg;C overnight, and lyophilized under vacuum. The resulting dry powder was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use.Mitophagy inhibitor: Cyclosporin A (CsA, catalog HYB0579) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).and dissolved in molecular-grade DMSO (Solarbio, Beijing, China; cat. no. D8371) to prepare a 10 mM stock solution.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Antibodies and Assay Kits\u003c/h2\u003e\u003cp\u003ePrimary antibodies against FBXO32/Atrogin-1 (ab74023), MURF-1/TRIM63 (ab183094), PINK1 (ab216144), Parkin (ab77924), p62/SQSTM1 (ab109012), LC3-Ⅱ (ab192890), NLRP3 (ab263899), caspase-1 (ab207802), ASC (ab283684), and GAPDH (YM3030) were purchased from Abcam (Cambridge, UK). Horseradish-peroxidase (HRP)-conjugated goat anti-rabbit (RS0002) and goat anti-mouse (RS0001) secondary antibodies were obtained from Immunoway (Plano, TX, USA).ELISA kits for murine TNF-α (cat. no. YJ002095-96T) and IL-6 (cat. no. YJ098430-96T) were supplied by Enzyme-linked Biotechnology (Shanghai, China). The reactive oxygen species (ROS) detection kit (cat. no. S0033S) was from Beyotime Biotechnology (Shanghai, China). Special horse serum for myogenic differentiation was purchased from Solarbio (cat. no. S9050-200 mL).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Cell Culture Media\u003c/h2\u003e\u003cp\u003eComplete growth media were prepared as follows: LLC cells were cultured in RPMI-1640 supplemented with 10% (v/v) foetal bovine serum and 1% penicillin\u0026ndash;streptomycin; Mouse myoblasts C2C12 cells were maintained in high-glucose DMEM containing 20% foetal bovine serum and 1% penicillin\u0026ndash;streptomycin. To induce myotube formation, C2C12 cells were switched to differentiation medium (DMEM high glucose plus 2% horse serum) once \u0026ge;\u0026thinsp;80% confluence was reached.Conditioned media (CM) were generated by collecting supernatants from LLC cultures after 48 h, followed by sequential filtration through 0.45 \u0026micro;m and 0.22 \u0026micro;m membranes. CM was then blended with differentiation medium at volumetric ratios of 1:1, 1:2, 1:4, or 1:8 to generate CM1, CM2, CM3, and CM4, respectively. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCulture media formulations\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGrowth medium (LLC)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGrowth medium (C2C12)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDifferentiation medium\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eConditioned medium\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRPMI-1640 89%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHigh-glucose DMEM 89%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHigh-glucose DMEM 97%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLLC cell-conditioned medium\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFBS10%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFBS10%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHorse serum 2%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDifferentiation medium\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePenicillin\u0026ndash;streptomycin 1%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePenicillin\u0026ndash;streptomycin 1%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePenicillin\u0026ndash;streptomycin 1%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Induction of Cancer-Associated Muscle Atrophy In Vitro\u003c/h2\u003e\u003cp\u003eMouse myoblasts C2C12 cells and LLC cells were maintained at 37\u0026deg;C in a humidified 5% CO₂ atmosphere and passaged every 48 h. For atrophy induction, LLC-conditioned medium (LLC-CM) was generated by 48-h culture of LLC cells in RPMI-1640\u0026thinsp;+\u0026thinsp;10% FBS. The supernatant was cleared by sequential centrifugation (300 \u0026times; g, 5 min) and filtration (0.22 \u0026micro;m filter membrane). C2C12 myoblasts were differentiated for 96 h in 2% horse-serum/DMEM to form multinucleated myotubes, after which differentiation medium was replaced with LLC-CM diluted 1:1, 1:2, 1:4, or 1:8 in differentiation medium. This regimen reproducibly induced a 30\u0026ndash;40% reduction in myotube diameter within 72 h, validating the cachectic milieu.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Preparation of SYJDT Stock Solution\u003c/h2\u003e\u003cp\u003e0.2 g mL⁻\u0026sup1; SYJDT stock solution: 200 mg lyophilised Sheng-Yu Jie-Du Tang powder was dissolved in 1 mL molecular-grade DMSO, vortex-mixed until complete solubilisation, sterile-filtered, and stored in single-use aliquots at \u0026minus;\u0026thinsp;80\u0026deg;C. Working dilutions were prepared fresh by diluting the stock in C2C12 growth medium to the indicated concentrations immediately before use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Experimental Design and Treatment Schema\u003c/h2\u003e\u003cp\u003eGroup A \u0026ndash; Vehicle control: C2C12 growth medium only.Group B \u0026ndash; SYJDT lyophilizate: C2C12 medium supplemented with SYJDT freeze-dried powder.Group C \u0026ndash; Antagonist control: C2C12 medium plus pharmacological antagonist.Group D \u0026ndash; SYJDT\u0026thinsp;+\u0026thinsp;antagonist: C2C12 medium containing both SYJDT lyophilizate and antagonist.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Real-time monitoring of myotube morphology by inverted microscopy\u003c/h2\u003e\u003cp\u003eMedium was renewed every 24 h. At the same clock time on days 1, 2, 3 and 4 (24, 48, 72 and 96 h after the onset of treatment) images were captured at 200\u0026times; magnification. Myotube diameter was randomly measured at three distinct sites per field; data were analyzed with ImageJ.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 ELISA quantification of TNF-α and IL-6 in conditioned medium\u003c/h2\u003e\u003cp\u003eC2C12 supernatants were collected and assayed in duplicate for TNF-α and IL-6 according to the manufacturer\u0026rsquo;s instructions. Absorbance was read at 450 nm, standard curves were constructed, and cytokine concentrations were calculated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 High-Throughput Cytotoxicity and Viability Screen (CCK-8)\u003c/h2\u003e\u003cp\u003eCancer-cachexia myotubes exhibiting robust growth were dissociated, counted on an automated cell counter, and reseeded at 5 \u0026times; 10⁴ cells/mL in 96-well plates (100 \u0026micro;L/well, three replicates per group).Lyophilized Sheng-Yu Jie-Du decoction was reconstituted in complete high-glucose DMEM to final concentrations of 0.25%, 0.5%, 1%, 2% and 3% (w/v); control wells received vehicle medium alone.Plates were returned to a humidified 5% CO₂ incubator at 37\u0026deg;C for 0, 24 or 48 h.At each time point the medium was aspirated and replaced with freshly prepared CCK-8 working solution (nine parts high-glucose DMEM : one part CCK-8 reagent).After 1 h protected from light on a pre-warmed (37\u0026deg;C) orbital shaker, absorbance at 450 nm was recorded on a microplate reader; cell viability and percentage inhibition were calculated from the resulting OD values.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Wound-healing assay for lateral migration of cancer-cachexia myotubes under Sheng-Yu Jie-Du decoction\u003c/h2\u003e\u003cp\u003eBased on the CCK-8 dose\u0026ndash;response screen, the optimal lyophilized-decoction concentration was selected for migration studies.Myotubes in logarithmic growth were detached, counted, and seeded at 5 \u0026times; 10⁵ cells per well (or at a density yielding confluent monolayers after 12 h).When cultures reached 80\u0026ndash;90% confluence, a sterile 200 \u0026micro;L pipette tip guided by a ruler held above the plate was used to create a straight scratch.After two PBS washes to remove cellular debris, the wound was imaged at 40\u0026times; magnification (time 0 h).Medium was then replaced with 2 mL of fresh 1% serum-containing high-glucose DMEM (vehicle control) or decoction supplemented medium (experimental group); each condition was run in triplicate.Identical fields were re-imaged at 24 h and 48 h.Wound closure was quantified with ImageJ and expressed as percentage migration relative to the 0-h width.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Evaluation of Myotube Atrophy Reversal\u003c/h2\u003e\u003cp\u003eAfter selection of the optimal lyophilizate concentration in CCK-8 assays, a cancer-cachectic atrophy model was established. Atrophy-model wells were maintained in complete medium for an additional 48 h, whereas SYJDT-treated wells received the pre-determined concentration of freeze-dried powder for the same period. Phase-contrast images (200\u0026times;) were then acquired on an inverted microscope to evaluate the extent of myotube atrophy and the protective effect of SYJDT.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Reactive Oxygen Species (ROS) Imaging\u003c/h2\u003e\u003cp\u003eSix-well plates were used for cell culture. After aspirating the medium, cells were loaded with 10 \u0026micro;M DCFH-DA (1 ml per well, sufficient to completely cover the monolayer) and incubated at 37\u0026deg;C for 20 min. Following incubation, cells were washed three times with serum-free medium and immediately visualized under an inverted microscope; images were captured at 200\u0026times; magnification using the FITC filter set.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Mitochondrial Membrane Potential (ΔΨm)\u003c/h2\u003e\u003cp\u003eAfter the scheduled interventions, ΔΨm was evaluated using JC-1 staining. A working solution was freshly prepared by vortexing the JC-1 stock vigorously, followed by dilution into 2 ml of JC-1 assay buffer (5\u0026times;) and thorough mixing. Medium was aspirated from each well of a six-well plate and replaced with 1 ml of complete growth medium; 1 ml of the JC-1 working solution was then added and mixed gently to ensure uniform distribution. Cells were incubated at 37\u0026deg;C for 20 min, after which the supernatant was removed and wells were washed twice with 1\u0026times; JC-1 assay buffer at 37\u0026deg;C. Finally, 2 ml of warm complete medium was added and three random fields per well were imaged at 200\u0026times; magnification on a fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15 Western-blot analysis of mitophagy signaling proteins\u003c/h2\u003e\u003cp\u003eCells were lysed in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined with the BCA assay, and 30 \u0026micro;g of each lysate was denatured, resolved on 4\u0026ndash;12% Bis-Tris gels, and transferred to 0.22 \u0026micro;m PVDF membranes. After blocking with 5% non-fat dry milk in TBST for 1 h at room temperature, membranes were incubated overnight at 4\u0026deg;C with the following primary antibodies (all validated for murine C2C12): MuRF-1 (1:2 000), FBXO32 (1:1 000), PINK1 (1:1 000), Parkin (1:1 000), p62/SQSTM1 (1:1 000), LC3B (1:2 000) and GAPDH (1:20 000). Following three 10-min washes with TBST, membranes were probed with HRP-conjugated secondary antibodies (1:5 000) for 1 h at room temperature. Immunoreactive bands were visualized with ECL Prime and quantified by densitometry using ImageJ (v1.54). GAPDH served as the loading control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.16 RT-qPCR quantification of mitophagy-related transcripts\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted with TRIzol reagent and reverse-transcribed using the High-Capacity cDNA Synthesis Kit (Thermo Fisher) according to the manufacturer\u0026rsquo;s instructions. Real-time PCR was performed on a QuantStudio 6 Flex system using SYBR Green master mix with gene-specific primers (sequences available upon request). Relative expression was calculated by the 2^(-ΔΔCt) method, normalized to GAPDH, and expressed as fold-change versus the control group. The primers used were showed in Table\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTarget-gene primer sequences for real-time PCR\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequence (5' \u0026rarr; 3')\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLength (nt)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAtrogin/FBX032\u003c/em\u003e\u0026nbsp;Forward primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGGACCCTCCCTAATCCCCTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAtrogin/FBX032\u003c/em\u003ereverse primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCACTGTTGCTGTTCCAGTGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMuRF-1\u003c/em\u003e\u0026nbsp;forward primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCCCTGATCCTCCAGTACCGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMuRF-1\u003c/em\u003e\u0026nbsp;reverse primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGTGTCCTTCCTTACCCTCTGTG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003epink1\u003c/em\u003e\u0026nbsp;forward primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAGGAAAAGGCCCAGATGTCG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003epink1\u003c/em\u003e\u0026nbsp;reverse primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCTGTTTGCTGAACCCAAGGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eparkin\u003c/em\u003e\u0026nbsp;forward primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTGACCAGCTGCGAGTGATTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eparkin\u003c/em\u003e\u0026nbsp;reverse primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGCAGTCTGGAGATTGGCACT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP62\u003c/em\u003e\u0026nbsp;forward primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGTTCCTGAACCCTCTCGTGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP62\u003c/em\u003e reverse primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGACTTTACGGGGTGCCTCAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLC3\u003c/em\u003e forward primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGGCGCTTACAGCTCAATGCT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLC3\u003c/em\u003e\u0026nbsp;reverse primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCTCCTGGGAGGCATAGACCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eNLRP3\u003c/em\u003e\u0026nbsp;forward primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATTACCCGCCCGAGAAAGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eNLRP3\u003c/em\u003e\u0026nbsp;reverse\u0026nbsp;primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCATGAGTGTGGCTAGATCCAAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ecaspase-1\u003c/em\u003e\u0026nbsp;forward\u0026nbsp;primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eACAAGGCACGGGACCTATG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ecaspase-1\u003c/em\u003e\u0026nbsp;reverse\u0026nbsp;primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTCCCAGTCAGTCCTGGAAATG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eASC\u003c/em\u003e\u0026nbsp;forward\u0026nbsp;primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGACAGTGCAACTGCGAGAAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eASC\u003c/em\u003e\u0026nbsp;reverse\u0026nbsp;primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCGACTCCAGATAGTAGCTGACAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u0026nbsp;forward primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGCGAGATCCCGCTAACATCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u0026nbsp;reverse primer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCTCGTGGTTCACACCCATCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.17 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll experiments were performed in triplicate with a minimum of three biological replicates. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. All analyses and graphs were generated with GraphPad Prism 8.0.1. Inter-group differences were evaluated by one-way ANOVA. A P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 was regarded as highly significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Establishment of a Cancer-Cachectic Myotube Atrophy Model\u003c/h2\u003e\u003cp\u003eExposure to increasing concentrations of LLC-conditioned medium (CM) produced a time- and dose-dependent delay in myotube formation, a marked reduction in myotube number, and a significant decrease in cross-sectional diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). The maximal inter-group divergence was observed after 72 h, at which point the mean myotube diameter in condition-1 CM was substantially lower than in normal medium (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and also differed from the other treatment groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), mirroring previously reported cachectic phenotypes. Consequently, 72-h treatment with condition-1 CM was selected as the standard protocol for generating the cancer-induced atrophy model in all subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Cytokine profiling of 72-h conditioned media by ELISA\u003c/h2\u003e\u003cp\u003eAfter a 72-h exposure of myotubes to serial dilutions of LLC-conditioned medium (CM), TNF-α and IL-6 levels in the supernatants were quantified. Both cytokines were elevated in every CM group relative to normal growth medium, reaching statistical significance for all except the most dilute condition-4 group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Among the CM groups, condition-1 generated the highest TNF-α concentration, significantly exceeding each of the other dilutions (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and likewise produced the maximal IL-6 signal, with inter-group differences highly significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Effects of SYJDT lyophilizate on the cancer-cachectic myotube model\u003c/h2\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1 CCK-8 assay\u003c/h2\u003e\u003cp\u003eC2C12 myotubes exposed to 0.25\u0026ndash;3% (v/v) SYJDT or vehicle for 24\u0026ndash;48 h showed dose- and time-dependent reductions in viability. Maximal cytostasis without cytotoxicity was achieved with 1% SYJDT at 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA); this condition was adopted for all subsequent mechanistic studies.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2 Wound-healing assay\u003c/h2\u003e\u003cp\u003eConfluent monolayers were scratched and monitored at 0, 24 and 48 h. Vehicle-treated cells closed\u0026thinsp;\u0026ge;\u0026thinsp;90% of the wound by 48 h, whereas 1% SYJDT markedly attenuated migration (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) versus control(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C), confirming an anti-migratory effect of the formulation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e3.3.3 Morphometric rescue of atrophic myotubes\u003c/h2\u003e\u003cp\u003eAfter 48 h treatment, SYJDT (1%) reversed LLC-CM-induced diameter loss, yielding significantly thicker myotubes than the atrophy model group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Modulation of ROS and mitochondrial membrane potential (ΔΨm)\u003c/h2\u003e\u003cp\u003eRelative to vehicle, 1% SYJDT markedly lowered ROS generation (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B) and elevated ΔΨm (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). Co-incubation with the mitophagy inhibitor CsA reversed both effects (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while SYJDT\u0026thinsp;+\u0026thinsp;CsA exhibited an intermediate phenotype, indicating that SYJDT\u0026rsquo;s antioxidant and mitochondrial-protective actions are partly mitophagy-dependent.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Mitophagy\u0026ndash;NLRP3 axis mRNA expression (RT-qPCR)\u003c/h2\u003e\u003cp\u003eSYJDT up-regulated LC3, PINK1 and Parkin transcripts and down-regulated p62 and NLRP3 compared with vehicle (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). CsA alone reduced PINK1, Parkin and LC3 while increasing p62; these changes were partially rescued in the SYJDT\u0026thinsp;+\u0026thinsp;CsA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), implying that SYJDT activates mitophagy and suppresses NLRP3 inflammasome transcription.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Mitophagy\u0026ndash;NLRP3 axis protein expression (Western blot)\u003c/h2\u003e\u003cp\u003eSYJDT significantly elevated LC3-II, PINK1 and Parkin, decreased p62, and attenuated NLRP3 protein levels versus vehicle (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). CsA antagonised these effects, whereas SYJDT\u0026thinsp;+\u0026thinsp;CsA retained partial rescue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Thus, SYJDT enhances mitophagic flux and dampens NLRP3-driven inflammation in cancer-cachectic myotubes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur findings align with a growing body of literature that positions mitochondrial autophagy as a cytoprotective hub in multiple disease models. In neurodegeneration, PINK1/Parkin-mediated mitophagy clears dysfunctional mitochondria, blunts oxidative stress and apoptosis, and slows disease progression\u003csup\u003e[\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e Geisler et al\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003efirst showed in Parkinson models that PINK1/Parkin signalling removes damaged mitochondria and rescues neurons, while Lazarou et al.\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e established that Parkin-dependent mitophagy is indispensable for neuronal mitochondrial quality control.\u003c/p\u003e\u003cp\u003eWe now extend this paradigm to muscle: Shenyi Jiedu Tang (SYJDT) up-regulates PINK1, Parkin and LC3, evokes mitophagy, and attenuates cancer-associated muscle atrophy, suggesting a conserved mechanism from brain to skeletal muscle.\u003c/p\u003e\u003cp\u003eParallel evidence implicates chronic low-grade systemic inflammation in cancer cachexia. Pro-inflammatory cytokines (TNF-α, IL-1, IL-6) produced by the tumour micro-environment and host immune cells activate catabolic programmes in liver and skeletal muscle, accelerating protein degradation and weight loss \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. As recently reviewed by Argil\u0026eacute;s et al. tumor-driven systemic inflammation simultaneously represses PGC-1α/PINK1\u0026ndash;Parkin signaling, triggers NLRP3 inflammasome activation, and up-regulates Atrogin-1/MuRF-1, thereby linking mitochondrial dysfunction to accelerated myofibrillar protein degradation in cancer cachexia\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe NLRP3 inflammasome sits at the nexus of this response; its over-activation liberates IL-1β and IL-18 and amplifies tissue injury. In muscle-wasting disorders, NLRP3, ASC and caspase-1 are markedly up-regulated, myofibre cross-sectional area shrinks, and E3 ligases Atrogin-1/MuRF-1 rise\u0026mdash;changes reversed by NLRP3 inhibitors\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e .Likewise, NLRP3-driven pyroptosis and proteolysis have been documented in diabetic myopathy\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. NLRP3 inflammasome activation is a key driver of tumor-induced muscle wasting\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHere, SYJDT lyophilised powder dose-dependently suppressed NLRP3, ASC and caspase-1, and lowered circulating TNF-α and IL-6. By restraining inflammasome activation SYJDT dampened inflammation and preserved muscle mass\u0026mdash;an anti-atrophy effect that mirrors prior pharmacologic NLRP3 blockade and broadens the anti-inflammatory repertoire of traditional Chinese medicine.\u003c/p\u003e\u003cp\u003eTaken together, SYJDT exerts a dual action\u0026mdash;promoting mitophagy while braking NLRP3 signalling\u0026mdash;thereby coupling mitochondrial quality control to suppression of sterile inflammation. The data resonate with current international priorities (mitophagy-NLRP3 axis) and furnish a mechanistic rationale for deploying SYJDT in cancer cachexia.\u003c/p\u003e\u003cp\u003eLimitations and next steps:Although we document SYJDT-mediated modulation of mitophagy and NLRP3, the precise molecular relays remain incomplete. Future work should;Expand sample size and launch multicentre pre-clinical trials;Deploy gene KO/over-expression to establish causal roles for PINK1/Parkin and NLRP3 pathways;Translate findings into early-phase clinical studies evaluating efficacy and safety in patients with muscle wasting;Map additional signalling nodes (e.g., AMPK, mTOR, NF-κB) to capture SYJDT\u0026rsquo;s poly-pharmacology.\u003c/p\u003e\u003cp\u003eIn summary, SYJDT engages the mitophagy-NLRP3 axis to restrain muscle atrophy, offering a fresh mechanistic lens for its pharmacology and a theoretical basis for repurposing this herbal formula in neurodegenerative and cachectic conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.All other authors declare no financial relationship with any organization that might have an interest in the submitted work in the previous five years. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003eFunding:The author(s) declare that financial support was received for the research and/or publication of this article. The present study was supported by Wu Mian hua Inheritance Studio of National Famous and Old Chinese Medicine Experts\u0026nbsp;(State Administration of Traditional Chinese Medicine No. [2022]75);Academic Experience Inheritance Project for the Seventh Batch of Old Chinese Medicine Experts by State Administration of Traditional Chinese Medicine (State Administration of Traditional Chinese Medicine No. [2022]76);\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eAll of the authors performed the study.PJJ: Conceptualization, Methodology, Writing \u0026ndash; Original Draft.HJ: Data Curation, Validation, Formal Analysis.ZY: Investigation, Resources, Writing \u0026ndash; Review \u0026amp; Editing.WMH: Supervision, Project Administration, Funding Acquisition All the authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003cstrong\u003ehe data availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are not publicly available as they form part of an ongoing research project that has not yet been concluded. However, they can be obtained from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eThibaut M M, Sboarina M, Roumain M, et al. Inflammation-induced cholestasis in cancer cachexia[J]. J Cachexia Sarcopenia Muscle. 2021, 12(1): 70-90.\u003c/li\u003e\n \u003cli\u003eTanaka K, Nakamura S, Narimatsu H. Nutritional Approach to Cancer Cachexia: A Proposal for Dietitians[J]. Nutrients. 2022, 14(2).\u003c/li\u003e\n \u003cli\u003eBaracos V E, Martin L, Korc M, et al. Cancer-associated cachexia[J]. Nat Rev Dis Primers. 2018, 4: 17105.\u003c/li\u003e\n \u003cli\u003eFearon K, Strasser F, Anker S D, et al. Definition and classification of cancer cachexia: an international consensus[J]. Lancet Oncol. 2011, 12(5): 489-495.\u003c/li\u003e\n \u003cli\u003evon Haehling S, Anker S D. Cachexia as a major underestimated and unmet medical need: facts and numbers[J]. J Cachexia Sarcopenia Muscle. 2010, 1(1): 1-5.\u003c/li\u003e\n \u003cli\u003eBerriel Diaz M, Rohm M, Herzig S. Cancer cachexia: multilevel metabolic dysfunction[J]. Nat Metab. 2024, 6(12): 2222-2245.\u003c/li\u003e\n \u003cli\u003eBeltra M, Pin F, Ballaro R, et al. Mitochondrial Dysfunction in Cancer Cachexia: Impact on Muscle Health and Regeneration[J]. Cells. 2021, 10(11).\u003c/li\u003e\n \u003cli\u003eRahman F A, Graham M Q, Adam A M, et al. Mitophagy is required to protect against excessive skeletal muscle atrophy following hindlimb immobilization[J]. J Biomed Sci. 2025, 32(1): 29.\u003c/li\u003e\n \u003cli\u003eZhao G, Wang Y, Li Y, et al. The neuroprotective effect of modified \u0026quot;Shengyu\u0026quot; decoction is mediated through an anti-inflammatory mechanism in the rat after traumatic brain injury[J]. J Ethnopharmacol. 2014, 151(1): 694-703.\u003c/li\u003e\n \u003cli\u003eKhalil B, El Fissi N, Aouane A, et al. PINK1-induced mitophagy promotes neuroprotection in Huntington\u0026apos;s disease[J]. Cell Death Dis. 2015, 6(1): e1617.\u003c/li\u003e\n \u003cli\u003eDi Rita A, D\u0026apos;Acunzo P, Simula L, et al. AMBRA1-Mediated Mitophagy Counteracts Oxidative Stress and Apoptosis Induced by Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells[J]. Front Cell Neurosci. 2018, 12: 92.\u003c/li\u003e\n \u003cli\u003ePalikaras K, Tavernarakis N. Mitophagy in neurodegeneration and aging[J]. Front Genet. 2012, 3: 297.\u003c/li\u003e\n \u003cli\u003eKamat P K, Kalani A, Kyles P, et al. Autophagy of mitochondria: a promising therapeutic target for neurodegenerative disease[J]. Cell Biochem Biophys. 2014, 70(2): 707-719.\u003c/li\u003e\n \u003cli\u003eGeisler S, Holmstrom K M, Skujat D, et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1[J]. Nat Cell Biol. 2010, 12(2): 119-131.\u003c/li\u003e\n \u003cli\u003eLazarou M, Sliter D A, Kane L A, et al. The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy[J]. Nature. 2015, 524(7565): 309-314.\u003c/li\u003e\n \u003cli\u003eGoncalves D C, Gomes S P, Seelaender M. Metabolic, Inflammatory, and Molecular Impact of Cancer Cachexia on the Liver[J]. Int J Mol Sci. 2024, 25(22).\u003c/li\u003e\n \u003cli\u003eDonohoe C L, Ryan A M, Reynolds J V. Cancer cachexia: mechanisms and clinical implications[J]. Gastroenterol Res Pract. 2011, 2011: 601434.\u003c/li\u003e\n \u003cli\u003eArgiles J M, Busquets S, Stemmler B, et al. Cancer cachexia: understanding the molecular basis[J]. Nat Rev Cancer. 2014, 14(11): 754-762.\u003c/li\u003e\n \u003cli\u003eLiu Y, Wang D, Li T, et al. The role of NLRP3 inflammasome in inflammation-related skeletal muscle atrophy[J]. Front Immunol. 2022, 13: 1035709.\u003c/li\u003e\n \u003cli\u003eYan X, Fu P, Zhang Y, et al. MCC950 Ameliorates Diabetic Muscle Atrophy in Mice by Inhibition of Pyroptosis and Its Synergistic Effect with Aerobic Exercise[J]. Molecules. 2024, 29(3).\u003c/li\u003e\n \u003cli\u003eFranke M, Bieber M, Kraft P, et al. The NLRP3 inflammasome drives inflammation in ischemia/reperfusion injury after transient middle cerebral artery occlusion in mice[J]. Brain Behav Immun. 2021, 92: 223-233.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"mitophagy, cancer-associated cachexia, skeletal-muscle atrophy, Sheng-Yu Jie-Du Tang, NLRP3 inflammasome, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-8130647/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8130647/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e: This study aimed to investigate the impact and mechanism of Shengyu Jiedu Tang(SYJDT) lyophilized powder on a cancer-induced muscular atrophy cell model by modulating the mitochondrial autophagy-NLRP3 inflammatory pathway. \u003cstrong\u003eMethods\u003c/strong\u003e: Mouse myoblasts C2C12 were exposed to the supernatant of Lewis lung carcinoma (LLC) to establish a carcinogen-induced muscular atrophy cell model. Various parameters including cell proliferation, migration, myotube atrophy, ROS levels, mitochondrial membrane potential, and expression of proteins in the mitochondrial autophagy-NLRP3 signaling pathway were assessed following treatment with different concentrations of Shengyu Jiedu Tang lyophilized powder.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The cancer-induced muscular atrophy cell model was successfully developed, demonstrating myotube atrophy, reduced myotube diameter, elevated TNF-α and IL-6 levels in the culture medium. Treatment with Shengyu Jiedu Tang lyophilized powder significantly suppressed cell proliferation and migration, ameliorated myotube atrophy, decreased ROS levels, enhanced mitochondrial membrane potential, upregulated mitochondrial autophagy-related proteins (LC3, Pink1, Parkin), downregulated P62 protein expression, inhibited NLRP3 inflammasome activation, and reduced inflammatory factor expression. Mitophagy inhibitors partially reversed the effects of the lyophilized powder, suggesting a close association with mitophagy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Shengyu Jiedu Tang lyophilized powder can attenuate cancer-induced muscular atrophy by stimulating mitochondrial autophagy, modulating mitochondrial function, suppressing NLRP3 inflammasome activation, and reducing inflammatory factor expression.\u003c/p\u003e","manuscriptTitle":"Sheng-Yu Jie-Du Tang Lyophilised Extract Reverses Cancer-Induced Myotube Atrophy via PINK1/Parkin-Mediated Mitophagy and Suppression of the NLRP3 Inflammasome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 12:44:24","doi":"10.21203/rs.3.rs-8130647/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"15bfac7f-45b1-4731-8a75-3b12508225d3","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59277084,"name":"Biological sciences/Cancer"},{"id":59277085,"name":"Biological sciences/Cell biology"},{"id":59277086,"name":"Health sciences/Diseases"},{"id":59277087,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-03-05T04:40:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 12:44:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8130647","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8130647","identity":"rs-8130647","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}