Naringin Ameliorates IL-6 Mediated mitochondrial dysfunction and skeletal muscle degeneratio via AMPK/Nrf-2 pathway

Naringin Ameliorates IL-6 Mediated mitochondrial dysfunction and skeletal muscle degeneratio via AMPK/Nrf-2 pathway | 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 Research Article Naringin Ameliorates IL-6 Mediated mitochondrial dysfunction and skeletal muscle degeneratio via AMPK/Nrf-2 pathway Shang Gao, Tianhao Wang, Lu Zhang, Shang Chen, Shu Jia, Chunyang Meng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3892612/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 Ethnopharmacological relevance : Paraspinal muscle degeneration often arises as a hazardous consequence of intervertebral disc degeneration (IVDD). This degeneration correlates with oxidative stress and mitochondrial dysfunction. This study investigated the therapeutic potential of naringin in managing paraspinal muscle degeneration associated with disc degeneration. Materials and methods : C2C12 cells were stimulated with IL-6 to establish an in vitro model of skeletal muscle degeneration for assessing the protective impact of naringin on skeletal muscle. The most effective concentration of naringin in C2C12 cells was identified through a CCK8 assay. The antioxidant prowess of naringin was evaluated via biochemical methods and Elisa. The influences of naringin and IL-6 on apoptosis, mitochondrial function, and associated signaling pathways were examined using cytometry, ROS detection, western blot, and transmission electron microscopy. Results : Our findings demonstrated a significant reduction in discogenic paraspinal degeneration with naringin therapy. Naringin glycosides notably enhanced the expression of key proteins involved in both muscle anabolism and catabolism, including MAFbx, MuRF1, MyoD, and MyoG.Moreover, naringin contributed to maintaining redox homeostasis by augmenting antioxidant activity and preventing excessive ROS peroxide accumulation. To impede paraspinal muscle degeneration, naringin upregulated MyoD and MyoG expression while downregulating MAFbx and MuRF1 through the activation of AMPK/Nrf-2 signaling pathway. Conclusion : These findings underscore naringin's robust therapeutic potential in enhancing mitochondrial activity, regulating oxidative stress, and halting paraspinal muscle degeneration. Naringin Paravertebral muscle degeneration Oxidative stress Mitochondrial dysfunction AMPK/Nrf-2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Low back pain (LBP) stands as a paramount public health concern globally, frequently manifesting as a prevalent symptom of intervertebral disc degeneration (IVDD). IVDD stands as the leading cause of disability and diminished quality of life, exerting a substantial financial toll on families and society [ 1 – 3 ]. Among the various types of LBP, discogenic low back pain (DLBP) emerges as the most common, affecting approximately 39% of individuals grappling with chronic LBP [ 4 – 6 ]. The root cause of DLBP primarily stems from IVDD, prompting the continuous increase of inflammatory factors, including IL-6, TNF-α, IL-1β, in the local microenvironment. This leads to inflammatory infiltration in the surrounding skeletal muscles, resulting in a decline in the quality and functionality of paravertebral muscle degeneration (PMD) [ 7 , 8 ]. The paravertebral muscles, chiefly consisting of the multifidus, erector spinae, and psoas, serve as pivotal components supporting posture and facilitating physical movement. They play a critical role in minimizing intervertebral hypermobility and ensuring lumbar segmental stability [ 9 , 10 ]. These muscles, vital for lumbar spine mobility, endure constant impact from PMD, a prevalent and substantial contributor to DLBP [ 11 – 13 ]. In PMD, a persistent imbalance between protein synthesis and degradation leads to a decline in skeletal muscle mass and function, significantly impacting morbidity and mortality rates. Unfortunately, there are currently no established pharmaceutical treatments for PMD-induced skeletal muscle dysfunction [ 14 – 16 ]. Numerous studies have emphasized that inflammatory infiltration, mitochondrial dysfunction and oxidative stress play an important role in PMD [ 17 , 18 ]. Inflammation, a crucial pathogenic factor, plays a pivotal role in triggering skeletal muscle degeneration. Inflammatory processes stimulate the release of pro-inflammatory cytokines and trigger the activation of genes linked to the proteolytic pathway during the degeneration of skeletal muscle. Interleukin-6 (IL-6), serving as a pro-inflammatory cytokine, exerts regulatory control over the metabolic processes of muscle. It upregulates the expression of pro-atrophy genes, namely Atrophy Protein 1 and MuRF1, in the context of skeletal muscle degeneration, consequently fostering the degradation of skeletal muscle tissue. However, further research is imperative to precisely delineate the impact of oxidative stress resulting from inflammatory responses on the impairment of muscle function. Mitochondria, being the principal organelles in cells, are pivotal for generating the energy required for bodily activities [ 19 ]. They notably contribute to maintaining cellular energy homeostasis and orchestrating various biological processes [ 20 ]. Despite their essential role, mitochondria also serve as significant sources of reactive oxygen species (ROS) and release cytochrome c, a pro-apoptotic factor [ 21 , 22 ]. Dysfunctional mitochondria can excessively generate ROS, release cytochrome c, and trigger an overabundance of pro-apoptotic and inflammatory factors. This cascade leads to cell apoptosis, disrupting normal muscle metabolism [ 23 ]. Amp-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis, responds to diverse physiological, hormonal, and dietary signals [ 24 , 25 ]. AMPK plays a pivotal role in modulating ATP activity and preserving energy homeostasis [ 26 ]. It's noteworthy that AMPK predominantly governs the energy balance of skeletal muscle and the heart, thereby upholding their fundamental cellular activities [ 27 ]. In oxidative stress (OS) response, Nrf-2 becomes a key regulator of endogenous antioxidant defense [ 28 ]. Under normal circumstances, Nrf2 binds to its negative regulatory protein Keap1 (Kelch-like ECH-associated protein 1) and is in a cellular negative regulatory state. [ 29 , 30 ]. Under oxidative stress, Nrf2 can dissociate from Keap1, enter the nucleus, and bind to antioxidant stress response elements (ARE) to promote the expression of antioxidant genes [ 31 , 32 ]. In the realm of oxidative stress, a sophisticated interplay exists between AMP-activated protein kinase (AMPK) and Nuclear factor erythroid 2-related factor 2 (Nrf2). Through its modulation of cellular energy metabolism, AMPK exerts an influential impact on the activity of Nrf2. Furthermore, AMPK possesses the capability to directly govern the translation and transcription activity of Nrf2. This intricate cross-regulation serves to uphold the cellular redox balance, fortifying cells against damage stemming from oxidative stress. In essence, the collaborative interplay between AMPK and Nrf2 orchestrates a synergistic response within cells, ensuring adept adaptation and robust defense mechanisms against oxidative stress. Extensive research has highlighted the multifaceted pharmacological properties of naringin, the primary flavonoid found in grapefruit. Its antioxidant, anti-inflammatory, and anti-apoptotic actions are intricately associated with redox regulation [ 33 , 34 ]. Naringin has shown promising potential in improving mitochondrial function, enhancing the antioxidant capacity of C2C12 cells, and preventing paraspinal muscle degeneration. Moreover, oral administration of naringin has been found to increase aerobic enzyme activity, elevate MyHC I expression, and augment the percentage of slow-twitch fibers in mouse skeletal muscle. Simultaneously, it reduces α-GPDH enzyme activity, MyHC II expression, and the proportion of fast-twitch fibers. Additionally, naringin boosts serum adiponectin levels, activating the expression of AdipoR1, APPL1, AMPK, and PGC-1α, thereby facilitating the transition of skeletal muscle fibers from fast-twitch to slow-twitch states [ 35 – 37 ]. C2C12 cells are commonly used to study muscle development, muscle nutrient metabolism, and other muscle-related biological processes. Through the C2C12 cell model, we can further explore the regulatory role of key cell targets and signaling pathways, and help to clarify the specific biological mechanism of naringin in the process of reversing skeletal muscle degeneration. As part of this study, our objective is to explore the effects of naringin on C2C12 myoblasts previously exposed to IL-6, aiming to elucidate its impact on these cells. Materials and Methods 2.1 Cell viability. In adherence to the CCK-8 kit guidelines, the levels of IL-6 and naringin within C2C12 cells were quantified. Specifically, 96 wells, each containing 6 × 10 3 C2C12 cells, were utilized. Notably, IL-6 is a well-recognized factor contributing to myotube atrophy. The experimental protocol involved a 24-hour treatment of C2C12 cells with 10 ng/ml of IL-6, followed by subsequent exposure to varying doses of naringin for an additional 24 hours. Subsequently, 10 µl of the CCK-8 solution was added to each well and incubated for 4 hours at 37°C, shielded from light. The optical density (OD) at 450 nm was then assessed to determine cellular viability. 2.2 Real-Time Quantitative PCR Detection. A combination of IL-6 (10 ng/ml) and Naringin (20 µM) was administered to C2C12 cells. Total mRNA from C2C12 cells was isolated using the over-column technique (AC0203, Spark Jade). Subsequent reverse transcription followed precise stages in accordance with the kit instructions (AG0304, Spark Jade). The mixture was then incubated at 94°C for 3 minutes post-combination with 2 SYBR Green qPCR mix (AH0104, Spark Jade). To quantify the relative expression levels of the target and reference genes, the 2-CT approach was employed. For internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was utilized. The primers employed are listed in Table 1 . 2.3 Western Blotting. C2C12 cells underwent various treatments followed by a rinse in pre-cooled PBS. Subsequently, they were lysed on ice for 30 minutes using protein lysis buffer (Solarbio) supplemented with protease inhibitors and then centrifuged at 300×g. The protein concentration of the isolated proteins was determined using the BCA Protein Concentration Assay Kit. For protein analysis, 30 µg of protein was loaded onto a 15% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis. A wet transfer technique was employed for the film transfer onto polyvinylidene difluoride (PVDF) membranes. Following transfer, the membranes, carrying the proteins, were incubated in 5% milk for blocking. Subsequently, the membranes were probed overnight at 4°C with primary antibodies: anti-AMPK(ab32047), anti-Nrf-2(ab54481), anti-Bcl-2 (ab221661) from Abcam; anti-MAFbx (JE41-27), anti-Bax(SZ3-07) from HUABIO; anti-p-AMPK(AP1002), anti-cleaved-caspase-3(A22672), anti-MyoD(A0671), anti-MuRF-1(A3101), anti-MyoG (DF8273), anti-cleaved caspase-9 (AF8470) from ABclonal. On the following day, PVDF membranes were treated with an HRP-conjugated secondary antibody (1:50,000, HUABIO) for 1 hour. The immunoblots were then captured using a ChemDocTM XRS + system (Bio-Rad, USA) and visualized using an enhanced chemiluminescence (ECL) HRP substrate Kit (Bio–Rad, USA). 2.4 Immunofluorescence Staining. The C2C12 cells were rinsed with PBS and subsequently fixed in 4% paraformaldehyde for 30 minutes. Following fixation, the cells were incubated in a PBS solution containing 0.3% Triton X-100 for 30 minutes and then sealed with 5% BSA. Primary antibodies for MyoD, Myogenin, MuRF1, and Fbx32 were applied to the cells and left to incubate overnight at 4°C. Subsequently, the cells were treated with the Alexa Fluor® 488 goat anti-rabbit secondary antibody (ab150077) for two hours. DAPI dye (ab104139) was used to stain the nuclei. Fluorescence microscopy was employed to visualize and quantify fluorescence. 2.5 Transmission Electron Microscopy (TEM). The treated C2C12 cells underwent fixation in 5% glutaraldehyde for 24 hours, followed by treatment with 1% osmium tetroxide for 1 hour, and subsequent dark staining in 2% uranyl acetate for 1 hour. Gradual dehydration was performed using graded ethanol for 15 minutes, followed by embedding the cells in an epoxy propane embedding mixture overnight. Post-embedding, ultrathin slices were acquired and treated with uranyl acetate and lead citrate for staining. Subsequently, the slices were examined using TEM (Transmission Electron Microscopy) for analysis. 2.6 Transfection of siRNA. The non-specific control siRNA (NC siRNA) and Nrf-2-targeting siRNA were obtained from GenePharma. C2C12 cells were cultured in 6-well plates. Upon reaching 70% confluency, the cells were transfected with either NC-siRNA or Nrf-2-siRNA, following the manufacturer's instructions. After transfection, the cells were collected for subsequent analysis. 2.7 Superoxide Dismutase (SOD) Activity and ATP Levels Measurement. The Total Superoxide Dismutase (T-SOD) Colorimetric Assay Kit (WST-1 technique) (Elabscience: E-BC-K020-M) and Adenosine Triphosphate (ATP) Colorimetric Assay Kit (E-BC-K157-M) were employed following the manufacturer's protocols to assess SOD enzymatic activity and ATP levels in C2C12 cells. Post-treatment, C2C12 cells were maintained at 37°C in a 5% CO2 environment. After rapid detachment using a scraper, the cells were collected via centrifugation at 1000×g for 10 minutes at 4°C. Subsequently, the lysed cells (10 6 cells) were centrifuged and homogenized using 300–500 ml of homogenate medium. The supernatant obtained was then quantified using the appropriate assay working solutions. 2.8 Detection of ROS Level. Following cell treatment, we utilized a ROS Assay Kit (Beyotime, SOO33S) to quantify the levels of reactive oxygen species (ROS). Briefly, the treated cells were incubated according to the provided instructions, followed by a 25-minute incubation with 10 µM 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA). The intracellular ROS fluorescence intensity was measured using cell flow cytometry. 2.9 TUNEL Staining. As per the manufacturer's protocol, C2C12 cells were analyzed using a TUNEL apoptosis assay kit (Beyotime, C1086). The cells, cultured in 12-well plates, were fixed with 4% paraformaldehyde and permeabilized using prepared PBS containing 0.3% Triton X-100. Subsequently, the cells were treated with TUNEL detection solution and incubated at 37°C in the dark for 60 minutes. Finally, the cells were visualized using a confocal microscope. 2.10 Analysis of Apoptosis Using Flow Cytometry. We evaluated the apoptosis percentage in C2C12 cells using an Annexin V FITC/PI Apoptosis Detection kit (Yeasen, 40302ES20), following the kit's guidelines. Post-digestion with 0.25% pancreatic enzyme (without EDTA) and centrifugation at 300 g, the cells were collected. After rinsing with PBS and subsequent centrifugation for 5 minutes, the C2C12 cells were incubated in the dark for 15 minutes. This involved treating the cells with 5 µl of Annexin V-FITC and 10 µl of PI staining solution in 100 µl of 1 binding buffer. Later, the cells were suspended in 400 µl of 1 binding buffer for analysis using flow cytometry. 2.11 Mitochondrial membrane potential (ΔΨm). We assessed the mitochondrial membrane potential (ΔΨm) using the Mitochondrial Membrane Potential Test Kit (C2003S, Beyotime), following the manufacturer's instructions. The measurement was conducted in isolated muscle mitochondria or myoblasts (6 x 10 3 cells/well). Red fluorescence (Ex 525 nm/Em 590 nm) denotes JC-1 aggregates, while green fluorescence (Ex 490 nm/Em 530 nm) represents JC-1 monomers. The ΔΨm in each group was determined via cell flow cytometry, calculating the ratio of JC-1 aggregates to monomers. 2.12 Determination of the mitochondrial permeability transition pore (mPTP). After trypsinization of the C2C12 cells, they were resuspended in culture media and centrifuged at 1000g for 5 minutes at room temperature. Supernatant was discarded, and cells were resuspended in assay buffer to achieve a single-cell suspension at a density of 1 x 10 6 cells/ml, using Calcein AM staining solution, fluorescence quenching workup solution, or Ionomycin control. The suspension was incubated at 37°C in the absence of light for 30 minutes. Post-incubation, cells were collected by centrifugation at 1000g for 5 minutes at room temperature. Further, each sample was supplemented with 1 ml of assay buffer, gently resuspended, and centrifuged at 1000g for 5 minutes at room temperature to remove excess solution. Following this, 400 µl of assay buffer was utilized for final cell resuspension. Subsequently, stained samples were kept on ice for immediate identification and analysis using flow cytometry. 2.13 Assay using enzyme-linked immunosorbent. ELISA kits were used to assess the impact of PF on blood levels of inflammatory cytokines in rats, including interleukin TNF-ɑ, IL-1β, IL-6, and IL-10. Using a microplate reader, the optical density (OD) at 450 nm was eventually determined. 2.14 Oxidative Stress Assessment. The total antioxidant capacity (T-AOC) and the activity of the enzymes catalase (CAT), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), lactate dehydrogenase (LDH), and succinate dehydrogenase (SDH) were estimated by Nanjing Jiancheng. 2.15 Statistics Analysis. The experimental outcomes were depicted as mean ± SD, derived from independent repetition of each experiment at least three times. Statistical analysis for significant differences was conducted employing one-way analysis of variance (ANOVA), where statistical significance was considered at P < 0.05. Results 3.1 Effect of Naringin on C2C12 Cell Viability. Figure 1 (a) showcases the molecular structure of naringin. In our experimental setup, we primed the cells with IL-6 (10 ng/ml) to ascertain the optimal concentration of naringin. Subsequently, various naringin concentrations were administered to the cells to evaluate C2C12 cell proliferation. After a 24-hour treatment period, the CCK-8 test indicated that the optimal concentration was 20 µM, as illustrated in Figs. 1 (b) and 1(c). 3.2 Naringin prevents the damage in C2C12 cells caused by IL-6. The dysregulated anabolic and catabolic metabolism within myoblasts significantly contributes to muscular dysfunction. Key metabolic regulators within C2C12 cells, including MyoD, MyoG, MuRF1, and Fbx32, were assessed using techniques such as RT-qPCR, immunofluorescence, and western blotting. Immunofluorescence staining unveiled intriguing insights: after exposure to naringin, there was a noticeable increase in the expression of MyoD and MyoG in C2C12 cells. Conversely, IL-6 intervention amplified the presence of MuRF1 and Fbx32 in myocytes, an effect that was counteracted by naringin treatment. Additionally, IL-6 intervention spurred myocyte catabolism. The RT-qPCR results corroborated these observations, showcasing that naringin boosted MyoD and MyoG mRNA expression while attenuating the IL-6-induced increase in MuRF1 and Fbx32 mRNA expression (Figs. 2 (i)-(j) and Figs. 2 (k)-(l)). Western blot analysis further supported these findings by illustrating that IL-6 intervention led to heightened MuRF1 and Fbx32 protein levels while simultaneously reducing the expression levels of MyoD and MyoG proteins. However, following naringin treatment, an opposite trend emerged (Fig. 2 (m)-(q)). 3.3 Naringin prevents IL-6-induced apoptosis in C2C12 cells. Various methods including RT-qPCR, flow cytometry, western blot, and Tunel analysis were employed to evaluate the impact of Naringin on C2C12 cells. Tunel and flow cytometry analyses (Figs. 3 (a)-(b) and (g)) indicated a significant increase in apoptosis of C2C12 cells upon exposure to IL-6, whereas Naringin treatment notably reduced apoptosis levels. Examining mRNA expression via RT-qPCR revealed that IL-6 elevated the levels of Bax, c-caspase-3, and c-caspase-9 mRNA (Figs. 3 (c)-(f)). Conversely, naringin treatment led to a decrease in these mRNA levels, while exhibiting an opposite behavior for Bcl-2 expression. Western blot analysis unveiled that IL-6 triggered an upregulation of Bax, c-caspase-3, and c-caspase-9 expression while concurrently downregulating Bcl-2. After naringin treatment, it was observed that naringin elevated the levels of Bcl-2 protein while suppressing the expression of Bax, caspase-3, and caspase-9. 3.4 The overproduction of inflammatory factors is prevented by naringin. Elisa testing uncovered how IL-6 and naringin impacted the cell's capability to secrete inflammatory factors. The results showcased that IL-6 heightened the discharge of IL-6, IL-1, and TNF- from C2C12 cells, concurrently reducing the secretion of IL-10. However, subsequent naringin intervention prompted an upsurge in IL-10 secretion while simultaneously curbing the release of IL-6, IL-1β, and TNF-ɑ (Figs. 4(a)–(d)). 3.5 Naringin enhances mitochondrial performance. The ROS, JC-1, and mPTP examinations highlighted the impact of IL-6 on excessive ROS accumulation, leading to compromised mitochondrial activity (Fig. 5 (b)). Additionally, IL-6 disrupted mitochondrial membrane potential and permeability transition pore function, resulting in reduced mitochondrial activity and cell apoptosis. Subsequently, naringin's intervention alleviated this ongoing deterioration (Figs. 5 (a) and (c)). Upon IL-6 intervention, transmission electron microscopy revealed mitochondrial enlargement, fusion of inner and outer mitochondrial membranes, and cristae reduction. Naringin played a role in ameliorating mitochondrial morphology degradation (Figs. 5 (d)). In the assessment of mitochondrial antioxidant capacity, IL-6 decreased GSH-px, T-AOC, CAT, ATP, SOD, and SDH production, while increasing MDA and LDH damage. Remarkably, naringin reversed these trends (Figs. 5 (e)–(l)). 3.6 Naringin elevated the Expression of p-AMPK in IL-6-induced C2C12 cells. We investigated the impact of Naringin on p-AMPK stimulation and compared metabolic protein expression in C2C12 cells under conditions of p-AMPK activation versus p-AMPK inhibition. Notably, AMPK levels remained constant, but a slight expression of p-AMPK was observed under IL-6 influence. Consequently, MyoD, MyoG, and Bcl-2 expression markedly decreased, while MuRF1, Fbx32, Bax, c-caspase-3, and c-caspase-9 expression levels significantly increased. Conversely, upon Naringin intervention, the expression of p-AMPK, MyoD, MyoG, and Bcl-2 decreased, accompanied by reduced levels of MuRF1, Fbx32, Bax, c-caspase-3, and c-caspase-9. Notably, administration of AMPK inhibitors led to opposite reactions compared to Naringin treatment (Figs. 6 (a)-(i)). 3.7 Naringin's knockdown of Nrf-2 effect. Through protein blot analysis, we confirmed Nrf-2's function in C2C12 cells and the influence of naringin on it. Naringin significantly elevated Nrf-2 protein expression, a effect that was reversed upon Nrf-2 knockdown. Additionally, the simultaneous use of an AMPK inhibitor resulted in decreased Nrf-2 expression levels. These findings suggest that naringin might safeguard Nrf-2 via AMPK (Fig. 7 (a)-(b)). 3.8 Naringin increased the expression of Nrf-2 in C2C12 cells that had been exposed to IL-6. In our previous work, we found Naringin's effect on Nrf-2. To explore its influence on proteins related to C2C12 cell metabolism via the AMPK/Nrf-2 pathway, we conducted protein blotting. IL-6 notably increased MuRF1, Fbx32, Bax, c-caspase-3, and c-caspase-9 expression while decreasing Nrf-2, MyoD, MyoG, and Bcl-2 proteins. However, upon Naringin treatment, there was an elevation in Nrf-2, MyoD, MyoG, and Bcl-2 expression along with decreased MuRF1, Fbx32, Bax, c-caspase-3, and c-caspase-9 levels. Interestingly, this reversal was negated upon AMPK inhibition (Fig. 8 (a)-(j)). Discussion Naringin has demonstrated its efficacy in enhancing skeletal muscle mass through the amelioration of insulin resistance, reduction of oxidative stress, and modulation of Atrogin-1 and MuRF-1 expression. Moreover, naringenin, a derivative of naringin, exhibits the ability to elevate antioxidant enzyme levels, thereby mitigating oxidative stress and fostering improved muscle health and function. This underscores the potential of naringin compounds as a viable adjunctive therapy for conditions such as hyperlipidemia and hyperglycemia, which have adverse effects on muscle physiology. Emerging evidence suggests that dietary supplementation with 0.04% naringin facilitates the conversion of muscle fiber types from fast to slow. This shift is particularly noteworthy as slow-twitch fibers harbor a higher abundance of robust mitochondria, offering enhanced resilience against oxidative stress induced by external factors. Such findings contribute to the growing body of knowledge supporting the therapeutic promise of naringin in optimizing muscle fiber composition and bolstering muscular resilience[ 34 , 36 – 37 ]. This study reaffirmed naringin's therapeutic potential in exploring the probable involvement of AMPK in paraspinal muscle degeneration associated with discogenic low back pain [ 44 , 45 ]. In the context of paraspinal muscle degeneration, crucial factors include inflammation, oxidative stress, and mitochondrial dysfunction. Our investigation also revealed a up-regulation of Nrf-2, which plays a pivotal role in amplifying the cellular response to oxidative stress and shielding cells from its damage [ 46 , 47 ]. Notably, our findings demonstrated that reducing Nrf-2 levels amplified IL-6-induced oxidative stress and mitochondrial dysfunction in C2C12 cells, thereby counteracting naringin's protective effect against paraspinal muscle degeneration. These outcomes align with our foundational hypothesis. Our experiments revealed that inflammatory factors like IL-6 triggered an increase in ROS levels, leading to dysfunctional mitochondria and apoptosis. IL-6 stimulation in C2C12 cells notably elevated MuRF1 and Fbx32 levels while downregulating MyoD and MyoG expression. The heightened ROS levels in IL-6-treated cells significantly upregulated Bax, c-caspase-3, and c-caspase-9, while reducing Bcl-2 expression, ultimately promoting apoptosis. Prior research suggests that elevated intracellular ROS levels might also drive MuRF1 and Fbx32 expression. Decreased ATP and SOD levels observed in the IL-6 group further contribute to apoptosis and muscle catabolism. These findings highlight the pivotal role of apoptosis and muscle catabolism in the pathophysiological mechanisms underlying paraspinal muscle degeneration. Inflammatory cytokines activate muscle growth inhibitors, leading to skeletal muscle degeneration by impeding muscle cell proliferation and inducing protein breakdown in phenotypic muscle cells. Naringin, as recent studies indicate, can effectively curb the excessive production of pro-inflammatory cytokines like IL-6, IL-1β, and TNF-α. Skeletal muscle degeneration escalates due to an overwhelming accumulation of ROS, disrupting the redox balance, diminishing antioxidant capacity, and elevating peroxide production. This imbalance with increased MDA and reduced CAT, GSH-Px, SOD, and T-AOC levels potentially expedites muscle protein breakdown. Our investigation discovered that IL-6-stimulated C2C12 cells exhibited decreased levels of CAT, GSH-Px, SOD, and T-AOC, while showing elevated MDA and LDH levels. Mitochondria, being the site for ATP production crucial for maintaining bodily functions and powering skeletal muscle, were notably affected in our findings. Reduced SDH levels and diminished mitochondrial ATP production were observed during skeletal muscle degeneration. Furthermore, IL-6 intervention led to decreased mPTP stability, increased ROS production, and mitochondrial loss in C2C12 cells. These findings strongly support the potential of naringin to alleviate oxidative stress and enhance mitochondrial function amid paraspinal muscle degeneration. AMPK, a pivotal energy sensor, plays a fundamental role in maintaining cellular balance by regulating energy levels[ 53 ]. Through the AMPK signaling pathway, essential functions like ATP synthesis, upkeep of redox potential, and ensuring optimal mitochondrial function are facilitated[ 54 ]. Nrf-2, renowned for its antioxidant properties, serves as a key regulator of mitochondrial biogenesis and function. Its influence on mitochondrial dynamics, bioenergetics, and the enhancement of mitochondrial mass and functionality collectively contribute to elevated cellular energy levels[ 55 , 56 ]. AMPK and Nrf2 usually maintain the organism's oxidative capacity and energy balance, respectively. Increased p-AMPK activity can rectify disruptions in energy regulation caused by harmful stimuli by regulating mitochondrial function, while heightened Nrf2 activity bolsters cellular antioxidant defenses, elevates antioxidant levels, and scavenges accumulated peroxides to maintain the body's redox equilibrium. Factors such as inflammation, trophic damage, and oxidative stress co-regulate AMPK/Nrf2 and are directly associated with paravertebral muscle degeneration. When AMPK/Nrf2 signaling was activated, levels of p-AMPK and Nrf2 were notably higher in the naringin-treated group compared to the IL-6-induced intervention group. However, the protective effect of naringin was nullified in the presence of the AMPK inhibitor dorsomorphin. Conclusions Studies have shown that naringin treatment modulates oxidative stress and restores mitochondrial function to impair apoptosis and increase muscle metabolic synthesis and decrease its catabolism via AMPK.Activation of AMPK/Nrf-2 protects cells from inflammatory responses and oxidative stress leading to apoptosis and muscle catabolic degradation. Declarations Data Availability All data generated during this study are available from the corresponding author on reasonable request. There was no data published previously. Conflicts of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Authors’ Contributions S.Gao wrote this article completely; L. Zhang and T. H. Wang measured and recorded all of the data for this article. S. Chen and S. Jia analyzed and interpreted the data. Prof. Z. F. Zhang, C. Y. Meng and Z. Wang designed and supervised this study. All authors approved this final manuscript. Funding This study was sponsored by the postdoctoral foundation of affiliated hospital of Jining medical university (JYFY321210), the Young and middle-aged experts with outstanding contributions in Shandong Province (2020SDTG01), and the Youth Science Fund of National Natural Science Foundation of China (82102184).This study was conducted with approval from the Affiliated hospital of Jining Medical University (No. 2023-03-B045) and was performed in accordance with the Declaration of Helsinki. Acknowledgments We thank the participants who generously agreed to be interviewed for this current study. Z. F. Zhang appreciate the instructions given by the professor Y. Wang, working in the Chinese PLA general hospital, during his PhD. References Maher C, Underwood M, Buchbinder R. Non-specific low back pain. Lancet. 2017 Feb 18;389(10070):736-747. Kadow T, Sowa G, Vo N, et cl. 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Orientin downregulating oxidative stress-mediated endoplasmic reticulum stress and mitochondrial dysfunction through AMPK/SIRT1 pathway in rat nucleus pulposus cells in vitro and attenuated intervertebral disc degeneration in vivo. Apoptosis. 2022 Dec;27(11-12):1031-1048. Zhao Y, Jiang Q, Zhang X, et cl. l-Arginine Alleviates LPS-Induced Oxidative Stress and Apoptosis via Activating SIRT1-AKT-Nrf2 and SIRT1-FOXO3a Signaling Pathways in C2C12 Myotube Cells. Antioxidants (Basel). 2021 Dec 7;10(12):1957. Li W, Trieu J, Blazev R, et cl. Sulforaphane attenuates cancer cell-induced atrophy of C2C12 myotubes. Am J Physiol Cell Physiol. 2023 Feb 1;324(2):C205-C221. Wang L, Jiao XF, Wu C, et cl. Trimetazidine attenuates dexamethasone-induced muscle atrophy via inhibiting NLRP3/GSDMD pathway-mediated pyroptosis. Cell Death Discov. 2021 Sep 18;7(1):251. Pyun DH, Kim TJ, Park SY, et cl. Patchouli alcohol ameliorates skeletal muscle insulin resistance and NAFLD via AMPK/SIRT1-mediated suppression of inflammation. Mol Cell Endocrinol. 2021 Dec 1;538:111464. Wang DT, Yin Y, Yang YJ, et cl. Resveratrol prevents TNF-α-induced muscle atrophy via regulation of Akt/mTOR/FoxO1 signaling in C2C12 myotubes. Int Immunopharmacol. 2014 Apr;19(2):206-13. Zanders L, Kny M, Hahn A, et cl. Sepsis induces interleukin 6, gp130/JAK2/STAT3, and muscle wasting. J Cachexia Sarcopenia Muscle. 2022 Feb;13(1):713-727. Alvarez AM, DeOcesano-Pereira C, Teixeira C, et cl. IL-1β and TNF-α Modulation of Proliferated and Committed Myoblasts: IL-6 and COX-2-Derived Prostaglandins as Key Actors in the Mechanisms Involved. Cells. 2020 Sep 1;9(9):2005. Zhang H, Chi M, Chen L, et cl. Daidzein alleviates cisplatin-induced muscle atrophy by regulating Glut4/AMPK/FoxO pathway. Phytother Res. 2021 Aug;35(8):4363-4376. Kim TJ, Pyun DH, Kim MJ, et cl. Ginsenoside compound K ameliorates palmitate-induced atrophy in C2C12 myotubes via promyogenic effects and AMPK/autophagy-mediated suppression of endoplasmic reticulum stress. J Ginseng Res. 2022 May;46(3):444-453. Boran T, Zengin OS, Seker Z, et cl. Ripretinib induced skeletal muscle toxicity through mitochondrial impairment in C2C12 myotubes. Toxicology. 2023 May 1;489:153489. Moreno-Ulloa A, Miranda-Cervantes A, Licea-Navarro A, et cl. (-)-Epicatechin stimulates mitochondrial biogenesis and cell growth in C2C12 myotubes via the G-protein coupled estrogen receptor. Eur J Pharmacol. 2018 Mar 5;822:95-107 Table Table 1 Primers used in qRT-PCR. Additional Declarations No competing interests reported. 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-3892612","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":268984892,"identity":"4ce79005-02f3-4592-8128-cab5a8af3ff0","order_by":0,"name":"Shang Gao","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shang","middleName":"","lastName":"Gao","suffix":""},{"id":268984893,"identity":"6f80bad0-0053-4145-a58e-4f4728c0b833","order_by":1,"name":"Tianhao Wang","email":"","orcid":"","institution":"The fourth medical center of the Chinese PLA general hospital","correspondingAuthor":false,"prefix":"","firstName":"Tianhao","middleName":"","lastName":"Wang","suffix":""},{"id":268984894,"identity":"81ee85a0-d6dc-47f3-bfb4-43ee6ab25bfc","order_by":2,"name":"Lu Zhang","email":"","orcid":"","institution":"Affiliated hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Zhang","suffix":""},{"id":268984895,"identity":"7a4db991-d0ec-49ee-93c8-d4327bad9fa7","order_by":3,"name":"Shang Chen","email":"","orcid":"","institution":"Affiliated hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shang","middleName":"","lastName":"Chen","suffix":""},{"id":268984896,"identity":"abc06b7a-f7c1-4a89-a471-466b8d3bde46","order_by":4,"name":"Shu Jia","email":"","orcid":"","institution":"Affiliated hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shu","middleName":"","lastName":"Jia","suffix":""},{"id":268984897,"identity":"b7f5dc6a-6e6f-4609-bd10-f1be35385f24","order_by":5,"name":"Chunyang Meng","email":"","orcid":"","institution":"Affiliated hospital of Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chunyang","middleName":"","lastName":"Meng","suffix":""},{"id":268984898,"identity":"a167f294-6e44-4947-a159-184323757cdb","order_by":6,"name":"Zheng Wang","email":"","orcid":"","institution":"The fourth medical center of the Chinese PLA general hospital","correspondingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Wang","suffix":""},{"id":268984899,"identity":"60ecf43d-8c9a-4efd-9b76-57e88c9504f9","order_by":7,"name":"Zifang Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIie3RP0vDQBjH8QuB65LY9RdSmrdwJVAdHHwpjwjekoKTZCoW4bL4AgIVX0bnhILTDY4BHeKiawaRDAU928EpaUfB+w7PLfeB+8OYzfYXw24OzXBqEqdjPsiKg0hww5grmvQyPvI0HULYD+FBrtfnDzgTvSJa3r69fmxOMFxmT6GvXKnAiLXpqpM494/H8cgD8KKvYl/xmQoXhXOnnzuJC5qGBsxZldCFr7yZGhXkOqqbcMjPEAKIDFn7CpKDRC/xkEyDhgBRyXKRa0F7CZBch6wAJlXisialiTKPXPbdJcrlKmg3c4wr+d6S+IqiLCvrNu0m2yfwdmf8/Y6id7/JabfLoN630Waz2f5p3+yvVYbxP0CKAAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated hospital of Jining Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zifang","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-01-24 02:29:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3892612/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3892612/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50299705,"identity":"51695d8f-2c60-4f8a-9247-11c551380b6a","added_by":"auto","created_at":"2024-01-29 11:34:23","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55670,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePositive effect of Naringin on IL-6-induced C2C12 cells.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Chemical structure of naringin. \u003cstrong\u003e(b)\u003c/strong\u003e C2C12 cells were treated with Naringin (0, 5, 10, 20 and 40 μM) for 24. \u003cstrong\u003e(c) \u003c/strong\u003eC2C12 cells were treated with 10 ng/ml IL-6 and 0, 10, 20, and 40 μM Naringin for 24 h. Cell viability was measured with a CCK-8 assay. The group treated with Naringin, *P\u0026lt;0.05, **P\u0026lt;0.01; the group treated with IL-6,#P\u0026lt;0.05, ##P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3892612/v1/2766757cfbaf1a4009dfac11.jpg"},{"id":50299706,"identity":"1208ae30-fe77-410f-9057-99540c4be399","added_by":"auto","created_at":"2024-01-29 11:34:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":861646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNaringin regulates metabolism of C2C12 cells. (a)-(h) \u003c/strong\u003eThe expression of MyoD, MyoG, MuRF-1, MAFbx was determined by immunofluorescence.\u003cstrong\u003e (i)-(l) \u003c/strong\u003eThe expression of MyoD, MyoG, MuRF-1 and MAFbx was determined by RT-qPCR. \u003cstrong\u003e(m)-(q) \u003c/strong\u003eThe expression of MyoD, MyoG, MuRF-1 and MAFbx was determined by western blot. The group treated with Naringin, \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01; the group treated with IL-6,\u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3892612/v1/3c294d354c551ae90fe65284.jpg"},{"id":50299707,"identity":"d1c22a80-ec41-436f-9ba4-4660df6e85a3","added_by":"auto","created_at":"2024-01-29 11:34:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":478931,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNaringin suppressed apoptosis of C2C12 cells. (a)-(b) \u003c/strong\u003eThe apoptosis of C2C12 cells was observed by TUNEL.\u003cstrong\u003e (c)-(f) \u003c/strong\u003eThe expression of Bax, Bcl-3, c-caspase-3 and c-caspase-9 was determined by RT-qPCR. \u003cstrong\u003e(g) \u003c/strong\u003eThe apoptosis rate of C2C12 cells was determined by flow cytometry. \u003cstrong\u003e(h)-(l)\u003c/strong\u003e The expression of Bax, Bcl-3, c-caspase-3 and c-caspase-9 was determined by western blot. The group treated with Naringin, \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01; the group treated with IL-6,\u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3892612/v1/7fa935711f5c81bffdacee7c.jpg"},{"id":50299708,"identity":"6e0be0cc-cb13-4f05-afd6-4a7fe9c4fb8b","added_by":"auto","created_at":"2024-01-29 11:34:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNaringin regulated inflammation level of C2C12 cells. (a)-(d) \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003eexpression level of IL-1β, IL-6, TNF-α and IL-10. The group treated with Naringin, \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01; the group treated with IL-6,\u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3892612/v1/95f1b0d703881608afc15de3.jpg"},{"id":50299991,"identity":"3822c1ab-ac20-4f7b-aa14-3d3f6c52d3c5","added_by":"auto","created_at":"2024-01-29 11:42:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":571661,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNaringin regulated mitochondrial function of C2C12 cells. (a) \u003c/strong\u003eMitochondrial membrane potential (ΔΨm) was detected by flow cytometry. \u003cstrong\u003e(b) \u003c/strong\u003eThe expression of intracellular ROS generation in different groups.\u003cstrong\u003e (c) \u003c/strong\u003eThe mitochondrial permeability transition pore was detected by flow cytometry. \u003cstrong\u003e(d) \u003c/strong\u003eThe mitochondrial morphology was observed by transmission electron microscopy. \u003cstrong\u003e(e)-(l) \u003c/strong\u003eThe expression level of GSH-px, T-AOC, CAT, ATP, SOD, SDH, MDA and LDH was detected. The group treated with Naringin, \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01; the group treated with IL-6,\u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3892612/v1/8b0ca2e575ca1123df082583.jpg"},{"id":50299709,"identity":"bb6ef784-0396-49f5-9ae3-6466b51bef47","added_by":"auto","created_at":"2024-01-29 11:34:23","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":471476,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNaringin regulated AMPK and p-AMPK in C2C12 cells. (a)-(j) \u003c/strong\u003eThe protein expression level of AMPK, p-AMPK, MyoD, MyoG, MuRF1, Fbx32, Bax, Bcl-2 c-casapse-3, c-casapse-9 was detected by western blot. The group treated with Naringin, \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01; the group treated with IL-6,\u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01; the group treated with Drosomorphin, \u003csup\u003e∆\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e∆∆\u003c/sup\u003eP\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3892612/v1/653826eafb22a45adef4ac2f.jpg"},{"id":50299712,"identity":"efd44416-4bda-49f7-8634-1a24e52bc1c4","added_by":"auto","created_at":"2024-01-29 11:34:23","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":43208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNaringin regulated Nrf-2 in C2C12 cells. (a)-(b) \u003c/strong\u003eThe protein expression level of Nrf-2 was detected by western blot. The group treated with Naringin, \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01; the group treated with IL-6,\u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01; the group treated with Drosomorphin, \u003csup\u003e∆\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e∆∆\u003c/sup\u003eP\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3892612/v1/b51597502ba3a5c27a653e89.jpg"},{"id":50299711,"identity":"23a24e95-acac-4459-a255-c271d83ee694","added_by":"auto","created_at":"2024-01-29 11:34:23","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":435381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNaringin regulated AMPK/Nrf-2 in C2C12 cells. (a)-(j) \u003c/strong\u003eThe protein expression level of Nrf-2, MyoD, MyoG, MuRF1, Fbx32, Bax, Bcl-2 c-casapse-3, c-casapse-9 was detected by western blot. The group treated with Naringin, \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01; the group treated with IL-6,\u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01; the group treated with Drosomorphin, \u003csup\u003e∆\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e∆∆\u003c/sup\u003eP\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3892612/v1/5ba82d15bb00256208e102ce.jpg"},{"id":50300817,"identity":"1e785354-c687-4064-a472-96379b53ab58","added_by":"auto","created_at":"2024-01-29 11:58:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1304386,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3892612/v1/cb0fba45-0f3d-481a-b294-ed749722ea0a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Naringin Ameliorates IL-6 Mediated mitochondrial dysfunction and skeletal muscle degeneratio via AMPK/Nrf-2 pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLow back pain (LBP) stands as a paramount public health concern globally, frequently manifesting as a prevalent symptom of intervertebral disc degeneration (IVDD). IVDD stands as the leading cause of disability and diminished quality of life, exerting a substantial financial toll on families and society [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among the various types of LBP, discogenic low back pain (DLBP) emerges as the most common, affecting approximately 39% of individuals grappling with chronic LBP [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The root cause of DLBP primarily stems from IVDD, prompting the continuous increase of inflammatory factors, including IL-6, TNF-α, IL-1β, in the local microenvironment. This leads to inflammatory infiltration in the surrounding skeletal muscles, resulting in a decline in the quality and functionality of paravertebral muscle degeneration (PMD) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The paravertebral muscles, chiefly consisting of the multifidus, erector spinae, and psoas, serve as pivotal components supporting posture and facilitating physical movement. They play a critical role in minimizing intervertebral hypermobility and ensuring lumbar segmental stability [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese muscles, vital for lumbar spine mobility, endure constant impact from PMD, a prevalent and substantial contributor to DLBP [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In PMD, a persistent imbalance between protein synthesis and degradation leads to a decline in skeletal muscle mass and function, significantly impacting morbidity and mortality rates. Unfortunately, there are currently no established pharmaceutical treatments for PMD-induced skeletal muscle dysfunction [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Numerous studies have emphasized that inflammatory infiltration, mitochondrial dysfunction and oxidative stress play an important role in PMD [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Inflammation, a crucial pathogenic factor, plays a pivotal role in triggering skeletal muscle degeneration. Inflammatory processes stimulate the release of pro-inflammatory cytokines and trigger the activation of genes linked to the proteolytic pathway during the degeneration of skeletal muscle. Interleukin-6 (IL-6), serving as a pro-inflammatory cytokine, exerts regulatory control over the metabolic processes of muscle. It upregulates the expression of pro-atrophy genes, namely Atrophy Protein 1 and MuRF1, in the context of skeletal muscle degeneration, consequently fostering the degradation of skeletal muscle tissue. However, further research is imperative to precisely delineate the impact of oxidative stress resulting from inflammatory responses on the impairment of muscle function. Mitochondria, being the principal organelles in cells, are pivotal for generating the energy required for bodily activities [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. They notably contribute to maintaining cellular energy homeostasis and orchestrating various biological processes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Despite their essential role, mitochondria also serve as significant sources of reactive oxygen species (ROS) and release cytochrome c, a pro-apoptotic factor [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Dysfunctional mitochondria can excessively generate ROS, release cytochrome c, and trigger an overabundance of pro-apoptotic and inflammatory factors. This cascade leads to cell apoptosis, disrupting normal muscle metabolism [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Amp-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis, responds to diverse physiological, hormonal, and dietary signals [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. AMPK plays a pivotal role in modulating ATP activity and preserving energy homeostasis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. It's noteworthy that AMPK predominantly governs the energy balance of skeletal muscle and the heart, thereby upholding their fundamental cellular activities [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn oxidative stress (OS) response, Nrf-2 becomes a key regulator of endogenous antioxidant defense [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Under normal circumstances, Nrf2 binds to its negative regulatory protein Keap1 (Kelch-like ECH-associated protein 1) and is in a cellular negative regulatory state. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Under oxidative stress, Nrf2 can dissociate from Keap1, enter the nucleus, and bind to antioxidant stress response elements (ARE) to promote the expression of antioxidant genes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the realm of oxidative stress, a sophisticated interplay exists between AMP-activated protein kinase (AMPK) and Nuclear factor erythroid 2-related factor 2 (Nrf2). Through its modulation of cellular energy metabolism, AMPK exerts an influential impact on the activity of Nrf2. Furthermore, AMPK possesses the capability to directly govern the translation and transcription activity of Nrf2. This intricate cross-regulation serves to uphold the cellular redox balance, fortifying cells against damage stemming from oxidative stress. In essence, the collaborative interplay between AMPK and Nrf2 orchestrates a synergistic response within cells, ensuring adept adaptation and robust defense mechanisms against oxidative stress.\u003c/p\u003e \u003cp\u003eExtensive research has highlighted the multifaceted pharmacological properties of naringin, the primary flavonoid found in grapefruit. Its antioxidant, anti-inflammatory, and anti-apoptotic actions are intricately associated with redox regulation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Naringin has shown promising potential in improving mitochondrial function, enhancing the antioxidant capacity of C2C12 cells, and preventing paraspinal muscle degeneration. Moreover, oral administration of naringin has been found to increase aerobic enzyme activity, elevate MyHC I expression, and augment the percentage of slow-twitch fibers in mouse skeletal muscle. Simultaneously, it reduces α-GPDH enzyme activity, MyHC II expression, and the proportion of fast-twitch fibers. Additionally, naringin boosts serum adiponectin levels, activating the expression of AdipoR1, APPL1, AMPK, and PGC-1α, thereby facilitating the transition of skeletal muscle fibers from fast-twitch to slow-twitch states [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. C2C12 cells are commonly used to study muscle development, muscle nutrient metabolism, and other muscle-related biological processes. Through the C2C12 cell model, we can further explore the regulatory role of key cell targets and signaling pathways, and help to clarify the specific biological mechanism of naringin in the process of reversing skeletal muscle degeneration. As part of this study, our objective is to explore the effects of naringin on C2C12 myoblasts previously exposed to IL-6, aiming to elucidate its impact on these cells.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Cell viability.\u003c/strong\u003e In adherence to the CCK-8 kit guidelines, the levels of IL-6 and naringin within C2C12 cells were quantified. Specifically, 96 wells, each containing 6 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e C2C12 cells, were utilized. Notably, IL-6 is a well-recognized factor contributing to myotube atrophy. The experimental protocol involved a 24-hour treatment of C2C12 cells with 10 ng/ml of IL-6, followed by subsequent exposure to varying doses of naringin for an additional 24 hours. Subsequently, 10 \u0026micro;l of the CCK-8 solution was added to each well and incubated for 4 hours at 37\u0026deg;C, shielded from light. The optical density (OD) at 450 nm was then assessed to determine cellular viability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Real-Time Quantitative PCR Detection.\u003c/strong\u003e A combination of IL-6 (10 ng/ml) and Naringin (20 \u0026micro;M) was administered to C2C12 cells. Total mRNA from C2C12 cells was isolated using the over-column technique (AC0203, Spark Jade). Subsequent reverse transcription followed precise stages in accordance with the kit instructions (AG0304, Spark Jade). The mixture was then incubated at 94\u0026deg;C for 3 minutes post-combination with 2 SYBR Green qPCR mix (AH0104, Spark Jade). To quantify the relative expression levels of the target and reference genes, the 2-CT approach was employed. For internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was utilized. The primers employed are listed in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Western Blotting.\u003c/strong\u003e C2C12 cells underwent various treatments followed by a rinse in pre-cooled PBS. Subsequently, they were lysed on ice for 30 minutes using protein lysis buffer (Solarbio) supplemented with protease inhibitors and then centrifuged at 300\u0026times;g. The protein concentration of the isolated proteins was determined using the BCA Protein Concentration Assay Kit. For protein analysis, 30 \u0026micro;g of protein was loaded onto a 15% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis. A wet transfer technique was employed for the film transfer onto polyvinylidene difluoride (PVDF) membranes. Following transfer, the membranes, carrying the proteins, were incubated in 5% milk for blocking. Subsequently, the membranes were probed overnight at 4\u0026deg;C with primary antibodies: anti-AMPK(ab32047), anti-Nrf-2(ab54481), anti-Bcl-2 (ab221661) from Abcam; anti-MAFbx (JE41-27), anti-Bax(SZ3-07) from HUABIO; anti-p-AMPK(AP1002), anti-cleaved-caspase-3(A22672), anti-MyoD(A0671), anti-MuRF-1(A3101), anti-MyoG (DF8273), anti-cleaved caspase-9 (AF8470) from ABclonal. On the following day, PVDF membranes were treated with an HRP-conjugated secondary antibody (1:50,000, HUABIO) for 1 hour. The immunoblots were then captured using a ChemDocTM XRS\u0026thinsp;+\u0026thinsp;system (Bio-Rad, USA) and visualized using an enhanced chemiluminescence (ECL) HRP substrate Kit (Bio\u0026ndash;Rad, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Immunofluorescence Staining.\u003c/strong\u003e The C2C12 cells were rinsed with PBS and subsequently fixed in 4% paraformaldehyde for 30 minutes. Following fixation, the cells were incubated in a PBS solution containing 0.3% Triton X-100 for 30 minutes and then sealed with 5% BSA. Primary antibodies for MyoD, Myogenin, MuRF1, and Fbx32 were applied to the cells and left to incubate overnight at 4\u0026deg;C. Subsequently, the cells were treated with the Alexa Fluor\u0026reg; 488 goat anti-rabbit secondary antibody (ab150077) for two hours. DAPI dye (ab104139) was used to stain the nuclei. Fluorescence microscopy was employed to visualize and quantify fluorescence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Transmission Electron Microscopy (TEM).\u003c/strong\u003e The treated C2C12 cells underwent fixation in 5% glutaraldehyde for 24 hours, followed by treatment with 1% osmium tetroxide for 1 hour, and subsequent dark staining in 2% uranyl acetate for 1 hour. Gradual dehydration was performed using graded ethanol for 15 minutes, followed by embedding the cells in an epoxy propane embedding mixture overnight. Post-embedding, ultrathin slices were acquired and treated with uranyl acetate and lead citrate for staining. Subsequently, the slices were examined using TEM (Transmission Electron Microscopy) for analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Transfection of siRNA.\u003c/strong\u003e The non-specific control siRNA (NC siRNA) and Nrf-2-targeting siRNA were obtained from GenePharma. C2C12 cells were cultured in 6-well plates. Upon reaching 70% confluency, the cells were transfected with either NC-siRNA or Nrf-2-siRNA, following the manufacturer\u0026apos;s instructions. After transfection, the cells were collected for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Superoxide Dismutase (SOD) Activity and ATP Levels Measurement.\u003c/strong\u003e The Total Superoxide Dismutase (T-SOD) Colorimetric Assay Kit (WST-1 technique) (Elabscience: E-BC-K020-M) and Adenosine Triphosphate (ATP) Colorimetric Assay Kit (E-BC-K157-M) were employed following the manufacturer\u0026apos;s protocols to assess SOD enzymatic activity and ATP levels in C2C12 cells. Post-treatment, C2C12 cells were maintained at 37\u0026deg;C in a 5% CO2 environment. After rapid detachment using a scraper, the cells were collected via centrifugation at 1000\u0026times;g for 10 minutes at 4\u0026deg;C. Subsequently, the lysed cells (10\u003csup\u003e6\u003c/sup\u003e cells) were centrifuged and homogenized using 300\u0026ndash;500 ml of homogenate medium. The supernatant obtained was then quantified using the appropriate assay working solutions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Detection of ROS Level.\u003c/strong\u003e Following cell treatment, we utilized a ROS Assay Kit (Beyotime, SOO33S) to quantify the levels of reactive oxygen species (ROS). Briefly, the treated cells were incubated according to the provided instructions, followed by a 25-minute incubation with 10 \u0026micro;M 2\u0026prime;-7\u0026prime;-dichlorodihydrofluorescein diacetate (DCFH-DA). The intracellular ROS fluorescence intensity was measured using cell flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 TUNEL Staining.\u003c/strong\u003e As per the manufacturer\u0026apos;s protocol, C2C12 cells were analyzed using a TUNEL apoptosis assay kit (Beyotime, C1086). The cells, cultured in 12-well plates, were fixed with 4% paraformaldehyde and permeabilized using prepared PBS containing 0.3% Triton X-100. Subsequently, the cells were treated with TUNEL detection solution and incubated at 37\u0026deg;C in the dark for 60 minutes. Finally, the cells were visualized using a confocal microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 Analysis of Apoptosis Using Flow Cytometry.\u003c/strong\u003e We evaluated the apoptosis percentage in C2C12 cells using an Annexin V FITC/PI Apoptosis Detection kit (Yeasen, 40302ES20), following the kit\u0026apos;s guidelines. Post-digestion with 0.25% pancreatic enzyme (without EDTA) and centrifugation at 300 g, the cells were collected. After rinsing with PBS and subsequent centrifugation for 5 minutes, the C2C12 cells were incubated in the dark for 15 minutes. This involved treating the cells with 5 \u0026micro;l of Annexin V-FITC and 10 \u0026micro;l of PI staining solution in 100 \u0026micro;l of 1 binding buffer. Later, the cells were suspended in 400 \u0026micro;l of 1 binding buffer for analysis using flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Mitochondrial membrane potential (\u0026Delta;\u0026Psi;m).\u003c/strong\u003e We assessed the mitochondrial membrane potential (\u0026Delta;\u0026Psi;m) using the Mitochondrial Membrane Potential Test Kit (C2003S, Beyotime), following the manufacturer\u0026apos;s instructions. The measurement was conducted in isolated muscle mitochondria or myoblasts (6 x 10\u003csup\u003e3\u003c/sup\u003e cells/well). Red fluorescence (Ex 525 nm/Em 590 nm) denotes JC-1 aggregates, while green fluorescence (Ex 490 nm/Em 530 nm) represents JC-1 monomers. The \u0026Delta;\u0026Psi;m in each group was determined via cell flow cytometry, calculating the ratio of JC-1 aggregates to monomers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12 Determination of the mitochondrial permeability transition pore (mPTP).\u003c/strong\u003e After trypsinization of the C2C12 cells, they were resuspended in culture media and centrifuged at 1000g for 5 minutes at room temperature. Supernatant was discarded, and cells were resuspended in assay buffer to achieve a single-cell suspension at a density of 1 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml, using Calcein AM staining solution, fluorescence quenching workup solution, or Ionomycin control. The suspension was incubated at 37\u0026deg;C in the absence of light for 30 minutes. Post-incubation, cells were collected by centrifugation at 1000g for 5 minutes at room temperature. Further, each sample was supplemented with 1 ml of assay buffer, gently resuspended, and centrifuged at 1000g for 5 minutes at room temperature to remove excess solution. Following this, 400 \u0026micro;l of assay buffer was utilized for final cell resuspension. Subsequently, stained samples were kept on ice for immediate identification and analysis using flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.13 Assay using enzyme-linked immunosorbent.\u003c/strong\u003e ELISA kits were used to assess the impact of PF on blood levels of inflammatory cytokines in rats, including interleukin TNF-ɑ, IL-1\u0026beta;, IL-6, and IL-10. Using a microplate reader, the optical density (OD) at 450 nm was eventually determined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.14 Oxidative Stress Assessment.\u003c/strong\u003e The total antioxidant capacity (T-AOC) and the activity of the enzymes catalase (CAT), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), lactate dehydrogenase (LDH), and succinate dehydrogenase (SDH) were estimated by Nanjing Jiancheng.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.15 Statistics Analysis.\u003c/strong\u003e The experimental outcomes were depicted as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, derived from independent repetition of each experiment at least three times. Statistical analysis for significant differences was conducted employing one-way analysis of variance (ANOVA), where statistical significance was considered at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Effect of Naringin on C2C12 Cell Viability.\u003c/strong\u003e Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(a) showcases the molecular structure of naringin. In our experimental setup, we primed the cells with IL-6 (10 ng/ml) to ascertain the optimal concentration of naringin. Subsequently, various naringin concentrations were administered to the cells to evaluate C2C12 cell proliferation. After a 24-hour treatment period, the CCK-8 test indicated that the optimal concentration was 20 \u0026micro;M, as illustrated in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(b) and 1(c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Naringin prevents the damage in C2C12 cells caused by IL-6.\u003c/strong\u003e The dysregulated anabolic and catabolic metabolism within myoblasts significantly contributes to muscular dysfunction. Key metabolic regulators within C2C12 cells, including MyoD, MyoG, MuRF1, and Fbx32, were assessed using techniques such as RT-qPCR, immunofluorescence, and western blotting. Immunofluorescence staining unveiled intriguing insights: after exposure to naringin, there was a noticeable increase in the expression of MyoD and MyoG in C2C12 cells. Conversely, IL-6 intervention amplified the presence of MuRF1 and Fbx32 in myocytes, an effect that was counteracted by naringin treatment. Additionally, IL-6 intervention spurred myocyte catabolism. The RT-qPCR results corroborated these observations, showcasing that naringin boosted MyoD and MyoG mRNA expression while attenuating the IL-6-induced increase in MuRF1 and Fbx32 mRNA expression (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(i)-(j) and Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(k)-(l)). Western blot analysis further supported these findings by illustrating that IL-6 intervention led to heightened MuRF1 and Fbx32 protein levels while simultaneously reducing the expression levels of MyoD and MyoG proteins. However, following naringin treatment, an opposite trend emerged (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(m)-(q)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Naringin prevents IL-6-induced apoptosis in C2C12 cells.\u003c/strong\u003e Various methods including RT-qPCR, flow cytometry, western blot, and Tunel analysis were employed to evaluate the impact of Naringin on C2C12 cells. Tunel and flow cytometry analyses (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e(a)-(b) and (g)) indicated a significant increase in apoptosis of C2C12 cells upon exposure to IL-6, whereas Naringin treatment notably reduced apoptosis levels. Examining mRNA expression via RT-qPCR revealed that IL-6 elevated the levels of Bax, c-caspase-3, and c-caspase-9 mRNA (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e(c)-(f)). Conversely, naringin treatment led to a decrease in these mRNA levels, while exhibiting an opposite behavior for Bcl-2 expression. Western blot analysis unveiled that IL-6 triggered an upregulation of Bax, c-caspase-3, and c-caspase-9 expression while concurrently downregulating Bcl-2. After naringin treatment, it was observed that naringin elevated the levels of Bcl-2 protein while suppressing the expression of Bax, caspase-3, and caspase-9.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 The overproduction of inflammatory factors is prevented by naringin.\u003c/strong\u003e Elisa testing uncovered how IL-6 and naringin impacted the cell's capability to secrete inflammatory factors. The results showcased that IL-6 heightened the discharge of IL-6, IL-1, and TNF- from C2C12 cells, concurrently reducing the secretion of IL-10. However, subsequent naringin intervention prompted an upsurge in IL-10 secretion while simultaneously curbing the release of IL-6, IL-1\u0026beta;, and TNF-ɑ (Figs.\u0026nbsp;4(a)\u0026ndash;(d)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Naringin enhances mitochondrial performance.\u003c/strong\u003e The ROS, JC-1, and mPTP examinations highlighted the impact of IL-6 on excessive ROS accumulation, leading to compromised mitochondrial activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e(b)). Additionally, IL-6 disrupted mitochondrial membrane potential and permeability transition pore function, resulting in reduced mitochondrial activity and cell apoptosis. Subsequently, naringin's intervention alleviated this ongoing deterioration (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e(a) and (c)). Upon IL-6 intervention, transmission electron microscopy revealed mitochondrial enlargement, fusion of inner and outer mitochondrial membranes, and cristae reduction. Naringin played a role in ameliorating mitochondrial morphology degradation (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e(d)). In the assessment of mitochondrial antioxidant capacity, IL-6 decreased GSH-px, T-AOC, CAT, ATP, SOD, and SDH production, while increasing MDA and LDH damage. Remarkably, naringin reversed these trends (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e(e)\u0026ndash;(l)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Naringin elevated the Expression of p-AMPK in IL-6-induced C2C12 cells.\u003c/strong\u003e We investigated the impact of Naringin on p-AMPK stimulation and compared metabolic protein expression in C2C12 cells under conditions of p-AMPK activation versus p-AMPK inhibition. Notably, AMPK levels remained constant, but a slight expression of p-AMPK was observed under IL-6 influence. Consequently, MyoD, MyoG, and Bcl-2 expression markedly decreased, while MuRF1, Fbx32, Bax, c-caspase-3, and c-caspase-9 expression levels significantly increased. Conversely, upon Naringin intervention, the expression of p-AMPK, MyoD, MyoG, and Bcl-2 decreased, accompanied by reduced levels of MuRF1, Fbx32, Bax, c-caspase-3, and c-caspase-9. Notably, administration of AMPK inhibitors led to opposite reactions compared to Naringin treatment (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e(a)-(i)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Naringin's knockdown of Nrf-2 effect.\u003c/strong\u003e Through protein blot analysis, we confirmed Nrf-2's function in C2C12 cells and the influence of naringin on it. Naringin significantly elevated Nrf-2 protein expression, a effect that was reversed upon Nrf-2 knockdown. Additionally, the simultaneous use of an AMPK inhibitor resulted in decreased Nrf-2 expression levels. These findings suggest that naringin might safeguard Nrf-2 via AMPK (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e(a)-(b)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8 Naringin increased the expression of Nrf-2 in C2C12 cells that had been exposed to IL-6.\u003c/strong\u003e In our previous work, we found Naringin's effect on Nrf-2. To explore its influence on proteins related to C2C12 cell metabolism via the AMPK/Nrf-2 pathway, we conducted protein blotting. IL-6 notably increased MuRF1, Fbx32, Bax, c-caspase-3, and c-caspase-9 expression while decreasing Nrf-2, MyoD, MyoG, and Bcl-2 proteins. However, upon Naringin treatment, there was an elevation in Nrf-2, MyoD, MyoG, and Bcl-2 expression along with decreased MuRF1, Fbx32, Bax, c-caspase-3, and c-caspase-9 levels. Interestingly, this reversal was negated upon AMPK inhibition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e(a)-(j)).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNaringin has demonstrated its efficacy in enhancing skeletal muscle mass through the amelioration of insulin resistance, reduction of oxidative stress, and modulation of Atrogin-1 and MuRF-1 expression. Moreover, naringenin, a derivative of naringin, exhibits the ability to elevate antioxidant enzyme levels, thereby mitigating oxidative stress and fostering improved muscle health and function. This underscores the potential of naringin compounds as a viable adjunctive therapy for conditions such as hyperlipidemia and hyperglycemia, which have adverse effects on muscle physiology. Emerging evidence suggests that dietary supplementation with 0.04% naringin facilitates the conversion of muscle fiber types from fast to slow. This shift is particularly noteworthy as slow-twitch fibers harbor a higher abundance of robust mitochondria, offering enhanced resilience against oxidative stress induced by external factors. Such findings contribute to the growing body of knowledge supporting the therapeutic promise of naringin in optimizing muscle fiber composition and bolstering muscular resilience[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThis study reaffirmed naringin\u0026apos;s therapeutic potential in exploring the probable involvement of AMPK in paraspinal muscle degeneration associated with discogenic low back pain [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. In the context of paraspinal muscle degeneration, crucial factors include inflammation, oxidative stress, and mitochondrial dysfunction. Our investigation also revealed a up-regulation of Nrf-2, which plays a pivotal role in amplifying the cellular response to oxidative stress and shielding cells from its damage [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]. Notably, our findings demonstrated that reducing Nrf-2 levels amplified IL-6-induced oxidative stress and mitochondrial dysfunction in C2C12 cells, thereby counteracting naringin\u0026apos;s protective effect against paraspinal muscle degeneration. These outcomes align with our foundational hypothesis.\u003c/p\u003e\n\u003cp\u003eOur experiments revealed that inflammatory factors like IL-6 triggered an increase in ROS levels, leading to dysfunctional mitochondria and apoptosis. IL-6 stimulation in C2C12 cells notably elevated MuRF1 and Fbx32 levels while downregulating MyoD and MyoG expression. The heightened ROS levels in IL-6-treated cells significantly upregulated Bax, c-caspase-3, and c-caspase-9, while reducing Bcl-2 expression, ultimately promoting apoptosis. Prior research suggests that elevated intracellular ROS levels might also drive MuRF1 and Fbx32 expression. Decreased ATP and SOD levels observed in the IL-6 group further contribute to apoptosis and muscle catabolism. These findings highlight the pivotal role of apoptosis and muscle catabolism in the pathophysiological mechanisms underlying paraspinal muscle degeneration.\u003c/p\u003e\n\u003cp\u003eInflammatory cytokines activate muscle growth inhibitors, leading to skeletal muscle degeneration by impeding muscle cell proliferation and inducing protein breakdown in phenotypic muscle cells. Naringin, as recent studies indicate, can effectively curb the excessive production of pro-inflammatory cytokines like IL-6, IL-1\u0026beta;, and TNF-\u0026alpha;. Skeletal muscle degeneration escalates due to an overwhelming accumulation of ROS, disrupting the redox balance, diminishing antioxidant capacity, and elevating peroxide production. This imbalance with increased MDA and reduced CAT, GSH-Px, SOD, and T-AOC levels potentially expedites muscle protein breakdown. Our investigation discovered that IL-6-stimulated C2C12 cells exhibited decreased levels of CAT, GSH-Px, SOD, and T-AOC, while showing elevated MDA and LDH levels. Mitochondria, being the site for ATP production crucial for maintaining bodily functions and powering skeletal muscle, were notably affected in our findings. Reduced SDH levels and diminished mitochondrial ATP production were observed during skeletal muscle degeneration. Furthermore, IL-6 intervention led to decreased mPTP stability, increased ROS production, and mitochondrial loss in C2C12 cells. These findings strongly support the potential of naringin to alleviate oxidative stress and enhance mitochondrial function amid paraspinal muscle degeneration.\u003c/p\u003e\n\u003cp\u003eAMPK, a pivotal energy sensor, plays a fundamental role in maintaining cellular balance by regulating energy levels[\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]. Through the AMPK signaling pathway, essential functions like ATP synthesis, upkeep of redox potential, and ensuring optimal mitochondrial function are facilitated[\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]. Nrf-2, renowned for its antioxidant properties, serves as a key regulator of mitochondrial biogenesis and function. Its influence on mitochondrial dynamics, bioenergetics, and the enhancement of mitochondrial mass and functionality collectively contribute to elevated cellular energy levels[\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eAMPK and Nrf2 usually maintain the organism\u0026apos;s oxidative capacity and energy balance, respectively. Increased p-AMPK activity can rectify disruptions in energy regulation caused by harmful stimuli by regulating mitochondrial function, while heightened Nrf2 activity bolsters cellular antioxidant defenses, elevates antioxidant levels, and scavenges accumulated peroxides to maintain the body\u0026apos;s redox equilibrium. Factors such as inflammation, trophic damage, and oxidative stress co-regulate AMPK/Nrf2 and are directly associated with paravertebral muscle degeneration. When AMPK/Nrf2 signaling was activated, levels of p-AMPK and Nrf2 were notably higher in the naringin-treated group compared to the IL-6-induced intervention group. However, the protective effect of naringin was nullified in the presence of the AMPK inhibitor dorsomorphin.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eStudies have shown that naringin treatment modulates oxidative stress and restores mitochondrial function to impair apoptosis and increase muscle metabolic synthesis and decrease its catabolism via AMPK.Activation of AMPK/Nrf-2 protects cells from inflammatory responses and oxidative stress leading to apoptosis and muscle catabolic degradation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated during this study are available from the corresponding author on reasonable request. There was no data published previously.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.Gao wrote this article completely; L. Zhang and T. H. Wang measured and recorded all of the data for this article. S. Chen and S. Jia analyzed and interpreted the data. Prof. Z. F. Zhang, C. Y. Meng and Z. Wang designed and supervised this study. All authors approved this final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was sponsored by the postdoctoral foundation of affiliated hospital of Jining medical university (JYFY321210), the Young and middle-aged experts with outstanding contributions in Shandong Province (2020SDTG01), and the Youth Science Fund of National Natural Science Foundation of China (82102184).This study was conducted with approval from the Affiliated hospital of Jining Medical University (No. 2023-03-B045) and was performed in accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the participants who generously agreed to be interviewed for this current study. Z. F. Zhang appreciate the instructions given by the professor Y. Wang, working in the Chinese PLA general hospital, during his PhD.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMaher C, Underwood M, Buchbinder R. Non-specific low back pain. Lancet. 2017 Feb 18;389(10070):736-747.\u003c/li\u003e\n \u003cli\u003eKadow T, Sowa G, Vo N, et cl. Molecular basis of intervertebral disc degeneration and herniations: what are the important translational questions? Clin Orthop Relat Res. 2015 Jun;473(6):1903-12.\u003c/li\u003e\n \u003cli\u003ePriyadarshani P, Li Y, Yao L. Advances in biological therapy for nucleus pulposus regeneration. Osteoarthritis Cartilage. 2016 Feb;24(2):206-12.\u003c/li\u003e\n \u003cli\u003eMohd Isa IL, Teoh SL, Mohd Nor NH, et cl. Discogenic Low Back Pain: Anatomy, Pathophysiology and Treatments of Intervertebral Disc Degeneration. Int J Mol Sci. 2022 Dec 22;24(1):208.\u003c/li\u003e\n \u003cli\u003eZhao L, Manchikanti L, Kaye AD, et cl. 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Eur J Pharmacol. 2018 Mar 5;822:95-107\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003ePrimers used in qRT-PCR.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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Mitochondrial dysfunction, AMPK/Nrf-2","lastPublishedDoi":"10.21203/rs.3.rs-3892612/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3892612/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eEthnopharmacological relevance\u003c/b\u003e: Paraspinal muscle degeneration often arises as a hazardous consequence of intervertebral disc degeneration (IVDD). This degeneration correlates with oxidative stress and mitochondrial dysfunction. This study investigated the therapeutic potential of naringin in managing paraspinal muscle degeneration associated with disc degeneration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMaterials and methods\u003c/b\u003e: C2C12 cells were stimulated with IL-6 to establish an in vitro model of skeletal muscle degeneration for assessing the protective impact of naringin on skeletal muscle. The most effective concentration of naringin in C2C12 cells was identified through a CCK8 assay. The antioxidant prowess of naringin was evaluated via biochemical methods and Elisa. The influences of naringin and IL-6 on apoptosis, mitochondrial function, and associated signaling pathways were examined using cytometry, ROS detection, western blot, and transmission electron microscopy.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e: Our findings demonstrated a significant reduction in discogenic paraspinal degeneration with naringin therapy. Naringin glycosides notably enhanced the expression of key proteins involved in both muscle anabolism and catabolism, including MAFbx, MuRF1, MyoD, and MyoG.Moreover, naringin contributed to maintaining redox homeostasis by augmenting antioxidant activity and preventing excessive ROS peroxide accumulation. To impede paraspinal muscle degeneration, naringin upregulated MyoD and MyoG expression while downregulating MAFbx and MuRF1 through the activation of AMPK/Nrf-2 signaling pathway.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusion\u003c/b\u003e: These findings underscore naringin's robust therapeutic potential in enhancing mitochondrial activity, regulating oxidative stress, and halting paraspinal muscle degeneration.\u003c/p\u003e","manuscriptTitle":"Naringin Ameliorates IL-6 Mediated mitochondrial dysfunction and skeletal muscle degeneratio via AMPK/Nrf-2 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-29 11:34:19","doi":"10.21203/rs.3.rs-3892612/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":"10d57e42-9296-44cd-9e85-efa9a83e23ee","owner":[],"postedDate":"January 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-01T08:51:57+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-29 11:34:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3892612","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3892612","identity":"rs-3892612","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}