Changes in the muscle growth and regulatory factors, TNF-α and Toll-like Receptors 2 and 4 during Exercise, Muscle Atrophy, and Recovery in the Mouse Hind Limb Suspension Model

Changes in the muscle growth and regulatory factors, TNF-α and Toll-like Receptors 2 and 4 during Exercise, Muscle Atrophy, and Recovery in the Mouse Hind Limb Suspension Model | 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 Changes in the muscle growth and regulatory factors, TNF-α and Toll-like Receptors 2 and 4 during Exercise, Muscle Atrophy, and Recovery in the Mouse Hind Limb Suspension Model Mohammad Borhan Al-Zghoul, Saad AL-Nassan, Qusai Mohammad AL-Abedallat, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7180388/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 Background Muscle atrophy, characterized by muscle weakening and a reduction in mass, is primarily caused by inactivity or injury. The molecular mechanisms that drive skeletal muscle growth and development remain poorly understood, hampering the development of novel techniques for treating or preventing muscle atrophy. This study aimed to assess changes in muscle growth and the level of regulatory factors, including TNF-α and Toll-like Receptors 2 and 4, during Exercise, Muscle Atrophy, and Recovery in a Mouse Hind Limb Suspension Model. Methodology Adult male mice were subjected to hind limb unloading to induce muscle wasting for one week. Eighty animals were divided into four main groups: weight-bearing control (Con) group, Hindlimb unloading (HU) group, Hindlimb unloading + exercise training (HU + Ex) group, and exercise training (Exe) group. Total RNA was extracted from the Gastrocnemius muscle, and selected gene expression was evaluated using RT-qPCR analysis. Results Distinct gene expression patterns were observed in response to hindlimb unloading (HU) and hindlimb unloading with exercise (HU + Ex). There was a significant decrease in TLR-2 expression in the HU group, while TLR-4 levels increased compared to those in the other groups. TNF-α expression increased substantially in almost all groups except for the control group. IGF-1 increased with exercise and decreased during HU, showing recovery in the HU + Ex group. Markers of muscle atrophy, MyoG, and MyoD increased during HU and dropped in HU + Ex. Myostatin, MurF, and Atrogin were linked to atrophy, which increased in both the exercised and recovered groups. Exercise increased Irisin expression compared to controls, while HU and HU + Ex groups showed decreased levels but were still elevated compared to controls. Conclusion Understanding the changes in muscle growth and regulatory factors, TNF-α and Toll-like Receptors 2 and 4 during exercise, muscle atrophy, and recovery in the mouse hind limb suspension model can add significant value to the existing data on molecular and cellular mechanisms during and post hind limb recovery from muscle atrophy (hind limb suspension). Biological sciences/Cell biology Biological sciences/Molecular biology Biological sciences/Physiology Toll-like receptors 2 and 4 muscle atrophy Mouse Hind Limb Suspension. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Skeletal muscle is a highly malleable tissue that is vital to whole-body health and metabolism. Skeletal muscles account for approximately 40%-50% of the whole-body mass. The regulation of myogenic precursor cell proliferation and differentiation has been studied in detail [ 1 , 2 ]. In summary, skeletal muscle asynchronously develops in vivo. During this process, the myoblast can proliferate, generating a larger population of myoblasts, or it can irreversibly differentiate and form a myocyte, the building block of individual skeletal muscle cells. The size and strength of a skeletal muscle are determined by its component myocyte's number and cellular structure. However, the majority of these experiments were performed in vitro , where the muscle growth and development assigned to growth factors based solely on their effects on cultured myocytes did not always coincide with their in vivo functions. Muscle mass maintenance is crucial for health and concerns affecting one's quality of life [ 3 , 4 ]. Significant progress has been made in the last decade in understanding the chemicals that govern skeletal muscle hypertrophy. Unsurprisingly, many of these molecules are critical for regulating protein metabolism [ 5 , 6 ]. Muscle atrophy is the weakening of muscles or a reduction in muscle mass and strength triggered by inactivity or injury, and it is found in various disorders, including AIDS, cancer, and congestive heart failure [ 7 – 9 ]. According to this definition, the pathophysiology of atrophy can vary; atrophy is a highly active process mediated by various signaling pathways in which muscle mass is lost due to either a decrease in protein synthesis or increased protein degradation rates [ 10 ]. Healthy skeletal muscle tissue requires physical activity to adjust muscle fibers’ number, size, and properties [ 11 ]. Muscle strength and mass are tightly regulated through protein synthesis and turnover regulatory mechanisms [ 10 , 12 ]. Skeletal muscle has the potential to release myostatin, a member of the transforming growth factor β (TGF- β) family. Myostatin is a negative regulator of muscle development and growth upon release, particularly affecting protein turnover and replacing/distracting pathways [ 13 ]. Conversely, the insulin growth factor 1 (IGF-1) signaling pathway stimulates myofiber size, number, and function positively by promoting the synthesis of new proteins and the distraction of old proteins, leading to an increase in muscle mass [ 10 , 14 ]. Mainly, IGF-1 regulates protein synthesis in the skeletal muscle, increasing muscle mass [ 14 ]. Muscle maintenance is accomplished by the cell's combined action of the two types of proteolytic systems known as the ubiquitin-proteasome pathway (UPP) and the autophagy-lysosome machinery [ 15 , 16 ]. The UPP, MuRF-1, and Atrogin-1 play essential regulatory roles in the development of muscle atrophy by guiding ubiquitinated proteins to the 26S proteasome complex for degradation [ 17 ]. Oxidative stress and inflammation are significant factors in the development of skeletal muscle atrophy [ 18 ]. Atrophic skeletal muscle caused by unloading and immobility is characterized by inflammation and increased levels of oxidative stress. Reducing inflammation and oxidative stress can prevent skeletal muscular atrophy by inhibiting protein breakdown [ 19 – 21 ]. In muscle atrophy, Proinflammatory cytokines such as TNFα, IL-6, and IL-1 are released, subsequently activating STAT3, a transcription factor that up-regulates atrogin-1 [ 22 ]. STAT3 activation can also suppress myogenic differentiation and regulatory factors, including myogenin (MyoG), MyoD, Myogenic Factor 5 (Myf5), and Myogenic Regulator 4 (MRF4) [ 23 ]. This may interfere with myoblast fusion and impair muscle regeneration [ 24 , 25 ]. Toll-like Receptor (TLR) signaling pathways have been identified as inhibitors of muscle mass and can trigger conditions that promote muscle atrophy [ 26 ]. When TLRs, especially TLR-2 and TLR-4, are activated, the NF-KB pathway is triggered, resulting in the expression of the IL-6 gene, which will lead to an inflammation reaction. Moreover, Muscle-specific RING finger protein (MuRF-1) and Atrogin-1 expression decrease muscle mass by degrading myocyte proteins [ 27 ]. However, the expression status of these TLRs during muscle unloading and exercise has never been investigated. Published literature has shown that exercise can decrease the expression of TLRs and inflammatory signaling [ 28 , 29 ]. Exercise-induced myokines play an integral role in regulating metabolism in contracting muscles and distant organs [ 30 ]. Irisin, which is the cleavage product of its precursor fibronectin type III domain-containing 5 (FNDC5) [ 31 ], is currently a renowned myokine for its exercise-induced beneficial effects in browning white adipocytes [ 32 ], improving insulin resistance [ 33 ], promoting skeletal remodeling [ 33 ], and exerting anti-inflammatory effects by suppressing the TLR4 pathway [ 34 , 35 ]. Understanding the crosstalk between irisin, TLR-2, and TLR-4 during muscle atrophy and recovery is the potential we aim to add through our current experimental study. Our long-range goal is to elucidate molecular mechanisms that could be exploited as potential treatments for skeletal muscle atrophy. The results of this study would be important because they could offer potential therapeutic targets for attenuating/preventing muscle atrophy during aging, disuse, and illness. Materials and Methods Experimental design : In this experiment, eighty 15-week-old male BALB/c mice (purchased from Jordan University of Science and Technology’s animal house) were divided into four main groups: weight-bearing control (Con, n = 20), exercise training (Exe, n = 20), hind limb unloading (HU, n = 20), and hind limb unloading + exercise training (HU + Exe, n = 20), for the HU group of mice. The hind limb unloading model was used to induce muscle wasting. Each mouse in the HU group was suspended from its tail to the ceiling of its cage, preventing the hind limbs from weight bearing on the ground but allowing the mouse to move freely inside its individualized cage for one week. Exercise protocol : The exercise protocol included the animals running on an electrically motorized rodent treadmill with a moderate intensity speed of 15m/min, 60 minutes/day, for seven days. Animals in the exercised groups received two familiarization sessions for two days on the treadmill with an intensity of 5-10m/min, 30–40 minutes/session to get accustomed to the treadmill and the training protocol before the initiation of the experiment. Sample collection, RNA extraction, and cDNA Synthesis Mice were anesthetized with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) until the loss of the pedal withdrawal reflex indicated surgical anesthesia. For euthanasia, animals received an overdose of ketamine (200 mg/kg) and xylazine (20 mg/kg). Cervical dislocation was performed to confirm death following the cessation of breathing and pulse. All operations were performed by qualified individuals following the AVMA Guidelines for the Euthanasia of Animals and approved by the Animal Care and Use Committee (ACUC). Gastrocnemius muscle samples (n = 20 animals per group) were collected. Total RNA was extracted using Direct-Zol™ RNA MiniPrep (Zymo Research, Irvine, USA) with TRI Reagent® (Zymo Research, Irvine, USA) according to manufacturer procedure. RNA concentrations were measured using Biotek PowerWave XS2 Spec. From each sample, 2 µg of total RNA was utilized in the reverse transcription reaction using the SuperScript IV VILO Master Mix (Invitrogen, Thermo Fisher Scientific, Wilmington, USA). Relative-Quantitative Real-Time PCR (RT-qPCR) Blastaq™ Green qPCR Master Mix (Applied Biological Materials Inc., Richmond, Canada) was used in a Rotor-Gene Q MDx 5 plex instrument (Qiagen, Hilden, Germany). Briefly, the 20 µl reaction mixture was prepared from 10 µl of the master mix, 2 µl forward primer (2 pmol), 2 µl reveres primer (2 pmol), 2 µl cDNA of the sample, and 4 µl of nuclease-free water. Cycling parameters were 50°C for 2 min, 95°C for 15 min, and 40 cycles of 95°C for 10 s, followed by 30 s at 57°C and 72°C for 10 s, with final melting at 95°C for 20 s. Duplicates from each cDNA were analyzed, fluorescence emission was detected, and relative quantification was calculated automatically. β-Actin and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) were used as internal controls to which the fold changes in gene expression were normalized. The melting curve approved the specificity of a single target amplification. The cDNA sequence for each gene was obtained from the NCBI's Nucleotide database ( https://www.ncbi.nlm.nih.gov/nucleotide/ ). The IDT Primer Quest software ( http://eu.idtdna.com/PrimerQuest/Home/Index ) was used to create all primers. The primer sequences are presented in Table 1. Statistical Analysis All statistical analyses were conducted using IBM SPSS Statistics 26.0 (IBM software, Chicago, USA). One-way analysis of variance (ANOVA) was used to evaluate the existence of differences among the groups. Data are expressed as the mean ± the standard error of the mean (SEM). Values of p < 0.05 were considered statistically significant. Results IGF-1 The mRNA levels of IGF-1 showed a significant increase in the exercised group, indicating an upregulation of IGF-1 gene expression in response to the exercise stimulus. In contrast, during the atrophic state of hind limb unloading (HU), the mRNA levels of IGF-1 demonstrated a notable decrease, indicating a downregulation of IGF-1 gene expression. In the recovered animals (HU + Ex) group, the mRNA levels of IGF-1 exhibited a recovery trend, with an upward shift compared to those in the unloading phase (Fig. 1 ). MyoG Muscle atrophy (HU) showed an increase in the mRNA expression level of MyoG compared to other groups. While there was a significant drop in the expression level of MyoG in the recovered group (HU + Ex) (Fig. 2 ) MyoD The expression levels of MyoD mRNA almost showed the same trend as MyoG expressions (Fig. 3 ). However, the expression level in the recovered group (HU + Ex) did not differ significantly from the control or exercised groups. Myostatin The mRNA level of myostatin showed a significant increase in the exercised group (Ex) compared to the control and HU groups, while it showed a significant increase in the recovered group (HU + Ex) when compared to the other 3 groups (Fig. 4 ) MurF There was a significant increase in the mRNA expression level of MurF in the exercised group (Ex) when compared to control and HU groups and a significant increase in the recovered group (HU + Ex) when compared to other groups (Fig. 5 ) Atrogin The expression level of Atrogin mRNA showed a significant increase in the exercised group when compared to the control, of the hind limb unloading group when compared to the control and exercise groups, and in the recovered group (HU + Ex) when compared to the other 3 groups (Fig. 6 ) TLR-2 The expression level of TLR-2 showed a significant increase in the exercised group compared to the control and a substantial drop in the HU group compared to the control and Ex groups, while there was a significant increase in the recovered group compared to other groups (Fig. 7 ) TLR-4 The expression levels of TLR-4 increased significantly in the HU and HU + Ex groups compared to the control and Ex groups without showing a significant difference between the HU and HU + Ex groups (Fig. 8 ) TNF-α The expression level of TNF-α showed a significant increase in the Ex group compared to the control and a considerable increase in both HU and HU + Ex groups compared to the Control and Ex groups. (Fig. 9 ) Irisin The expression level of Irisin mRNA showed a significant increase in the exercised group (Ex) compared to the control group. While the HU and HU + Ex groups' expression levels dropped significantly compared to the Ex group but were higher considerably from the control group (Fig. 10 ) Discussion Muscle atrophy, a reduction in muscle strength and mass, can have many debilitating effects on one’s health and significantly impair one's quality of life [ 36 ]. The consequences of muscle atrophy, resulting in progressive weakness and diminished physical performance, make daily life challenges more difficult [ 37 – 39 ]. Therefore, it's critical to have interventions and preventative measures to halt or even reverse muscle atrophy so that people may continue to be healthy and perform their jobs well. Our experimental strategy in this work aimed to identify patterns in regulating muscle homeostasis genes during atrophy and exercise-induced recovery. Gaining insight into the molecular and cellular mechanisms involved in these processes is crucial for devising ways to combat muscle loss. Muscle atrophy in the hind limbs was induced using a mouse model of hind limb unloading. Additionally, a regimen of treadmill activities was implemented to investigate the effects of countermeasures during the recovery phase. Insulin growth factor 1 (IGF-1), a compact peptide composed of seventy amino acids, has a molecular weight of 7649 Dalton (Da) [ 40 ]. Like insulin, IGF-1 comprises an Alpha and Beta chain linked by disulfide bonds with 12 amino acids in its C peptide region. The structural resemblance to insulin explains why IGF-1 can interact with little affinity to the insulin receptor[ 41 , 42 ]. The IGF-1 signaling pathway plays a crucial role in regulating protein synthesis in skeletal muscle, which results in increased muscle mass [ 14 ]. This process occurs by binding IGF-1 to the IGF-1 receptor (IGF-1R), which triggers phosphorylation of intracellular components such as insulin receptor substrate-1, an adaptor protein. Following IRS-1 phosphorylation, phosphoinositide 3-kinase (PI3K) is attracted and then phosphorylated, leading to Akt activation [ 14 ]. Activation of Akt suppresses denervation-induced atrophy in rats, demonstrating its significance for myotube growth through the PI3K/Akt pathway [ 14 ]. In our study, a notable increase in the expression of the IGF-1 gene was observed in the recovery group (HU + Ex), which is consistent with its well-established function in promoting muscle growth and repair [ 43 , 44 ]. Conversely, a predictably significant drop was evident in the atrophy group (HU), with various studies demonstrating similar findings. For instance, Kim, Cha [ 45 ] assigned Twelve 7-week-old male rats to either control or hindlimb unloading groups; they reported significant IGF-1 downregulation in the hindlimb group’s soleus and long digital extensor muscles after 2 weeks of treatment. Another study conducted by Li, Feng [ 46 ] showed that there was a notable increase in the expression of IGF-1 mRNA, IGF-1 protein, IGF1R, phosphorylated PI3K, and p-Akt in mice following various exercise regimens, including aerobic exercise, resistance training, whole-body vibration, and electrical stimulation. Myostatin, a member of the transforming growth factor-β (TGF-β) family, once it is released, myostatin will act as a negative regulator of muscle development and growth by inhibiting the proliferation of muscle cells and promoting their breakdown [ 47 , 48 ]. There is an intricate relationship between IGF-1 and Myostatin, with studies suggesting they counteract each other. Myostatin acts as an autocrine/paracrine inhibitor of muscle development in skeletal and cardiac muscles by binding to the activin A receptor type IIB (ACVR2B), which is linked to the type 1 receptors ALK4 and ALK5 [ 49 ]. ACVR2B is expressed in various tissues, including skeletal muscle, adipose tissue, liver, kidney, and heart[ 50 ]. Its downstream effects are mediated through the activation of the canonical SMAD signaling pathway. When activin A binds to ACVR2B, it forms a complex with other receptors leading to various cellular processes [ 51 ]. Furthermore, activation of the NF-κB pathway increases Murf1 expression due to cytokines such as TNF-α, and MuRF1 targets proteins for degradation which leads to muscle atrophy [ 14 ]. However, our gene expression data for myostatin align with the IGF-1 expression results. Although exercises down-regulate the expression of myostatin in skeletal muscles [ 52 , 53 ], our myostatin gene expression levels were significantly higher in the exercised groups. Similar findings regarding myostatin mRNA elevation in response to exercise or repeated muscle contractions were reported in humans, and Okudan 2018, Arrieta, Hervás, et al. 2019) and rodent studies [ 54 ]. Carlson, Booth [ 55 ] reported an increase in myostatin mRNA in both the gastrocnemius-plantaris complex (Gast/PLT) and soleus muscles after the first day of hindlimb unloading in female ICR mice, without a significant difference in comparison to controls after 3 and 7 days of hindlimb unloading. On the other hand, Babcock, Knoblauch [ 56 ] conducted a study with 6-month-old male Wistar rats divided into ambulatory and hindlimb suspension groups for 10 days. They found a significant decrease in myostatin levels in the red tibialis anterior muscle after unloading for 10 days. We believe that these variations in myostatin response to exercises are due to its affection by several factors concerning the exercise parameters: intensity, frequency, and duration, or the inflammatory signaling that might still be persistent in early adaptive responses to exercise and post-atrophy recovery. Myogenin (MyoG) and Myoblast determination protein 1 (MyoD) represent muscle-specific transcription factors belonging to the basic-helix-loop-helix (bHLH) family of DNA-binding proteins [ 57 – 59 ]. Anticipated to play pivotal roles in muscle regeneration in mammalian embryos based on their actions in tissue culture, these proteins have primarily been substantiated through gain-of-function tests in cultured cells, demonstrating their potency as myogenic factors [ 57 – 59 ]. The expression of any of these myogenic factors can transform various tissue culture cells into muscle cells, exhibiting behaviors similar to myoblasts regarding growth factor responses, gene expression, and the capacity to fuse into multinucleated myotubes. Binding to the regulatory regions of muscle-specific genes, myogenic factors activate their expression, thereby facilitating the conversion process [ 57 – 59 ]. In our investigation, both factors exhibited heightened expression during muscle atrophy (HU), with a notable decline in the recovery group (HU + Ex). Macpherson, Wang [ 60 ], whose study involved Conditional MyoG null mice, wherein the soleus muscle was denervated by excising the sciatic nerve to induce muscle atrophy, reveald that Myog expression plays a significant role in reducing muscle mass, force, and cross-sectional area in the denervated muscle [ 60 ]. Due to disuse, the ubiquitin-proteasome system (UPS) plays a crucial role in muscle atrophy [ 61 ]. The UPP, MuRF-1, and Atrogin-1 play essential regulatory roles in muscle atrophy development by guiding ubiquitinated proteins to the 26S proteasome complex [ 17 ]. Ephemeral elevations in MuRF-1 and Atrogin-1 gene expression led to a shift from protein synthesis to degradation, thus causing muscle mass loss [ 17 ]. This mechanism orderly leads proteins to extinction. Additionally, Atrogin-1 controls the degradation of translation initiation factor 3f to inhibit overall protein synthesis [ 17 ]. Examples of sarcomeric proteins regulated by MuRF-1 include myotilin, myosins, troponins, and titin [ 17 ]. Elevated levels of MurF and Atrogin in the exercised and recovery groups indicate increased proteolysis. The increased expression of these factors suggests a transient breakdown of muscle proteins induced by exercise, potentially for remodeling. However, their sustained increase during recovery implies a prolonged impact on protein degradation pathways—a study conducted by Al-Nassan et al. Who employed a 6-week hindlimb unloading model in mice to induce muscle atrophy with daily treadmill running for 1 hour during that period, then measuring gastrocnemius succinate dehydrogenase (SDH) activity, muscle fiber cross-sectional area, and muscle mass in muscle fibers to assess the impact of exercise on muscle atrophy; reported that chronic exercise down-regulate the mRNA expressions of TNF-α and atrogin-1/MAFbx in the atrophied skeletal muscle [ 62 ]. On the other hand, Gomes et al who used cDNA microarrays to compare the gastrocnemius muscle between normal mice and food-deprived mice, Found that atrogin-1, which is explicitly expressed in striated muscles, is induced more than ninefold in muscles of fasted mice and highly expressed during muscle atrophy in various diseases [ 63 ]. Toll-like receptors are crucial for the body's defense against pathogens, recognizing and eliminating pathogens by stimulating the innate immune response. Toll-like receptors can also detect tissue damage and integrity by interacting with danger-associated molecular patterns released by damaged cells [ 64 ]. Twelve mouse TLRs have been identified (TLR1-9, TLR11-13), which are classified based on their expression location; these TLRs may be categorized as intracellular or extracellular receptors [ 64 ]. In vitro studies have demonstrated that TLR activation and suppression significantly influence muscle cell atrophy[ 65 – 67 ]. While most TLRs exhibit similar responses, some ligand-related effects and tissue-specific expression variations are prominent, leading to different outcomes [ 64 ]. TLR-mediated signaling is well-known for its critical role in clearing infections and tissue regeneration. However, it is necessary to tightly control and regulate its function in order to prevent chronic and damaging inflammation resulting from dysregulated interactions with endogenous metabolites [ 64 ]. On the cell surface, TLR (1–6) receptors -especially mouse TLR11 receptors- are expressed [ 68 , 69 ]. TLR1 and TLR6 recognize lipopeptides shared by microbes as well as derivatives produced by damaged cells. Additionally, TLR2/4 can bind to various ligands [ 68 , 69 ], one of which is bacterial lipopolysaccharide, and they also identify a range of endogenous ligands. For example, high mobility group box 1, a ligand for TLR2 and TLR4 that causes inflammation, is released from muscle tissue, leading to tissue damage [ 70 ]. Upon release, HMGB1 transforms into a soluble activator of proinflammatory cytokines [ 71 , 72 ]. Furthermore, research has shown that the interplay between TLR4 and HMGB1 up-regulates the expression of MHCI in mouse muscle tissue and mononuclear cells in peripheral blood [ 73 , 74 ]. MHC-I enables muscle cells to display antigens and plays a role in immune responses, which hastens the removal of injured muscle tissue proteins and leads to the loss of muscle mass [ 75 ]. Heat shock proteins, which function as intracellular molecular chaperones, also become TLR2/4-binding DAMPs when released from damaged cells [ 75 ]. Another ligand for TLR2/4 present in skeletal muscle besides the liver is acute-phase protein serum amyloid A1, whose levels are regulated by producing proinflammatory cytokines [ 75 ]. The expression of TLRs on cell surfaces significantly impacts muscular performance [ 75 ]. In vitro studies have demonstrated that stimulation of muscle cells by TLR4 results in inflammatory C2C12 production [ 76 ]. Due to a lower presence of invasive macrophages, mice lacking TLR2 take longer to clear necrotic tissue after muscle injury [ 77 ]. Activation of TLRs, especially TLR-2 and TLR-4, by creatine ligands triggers the NF-KB pathway, leading to the expression of the IL-6 gene. This results in an inflammatory reaction due to necrotic myocytes as well as MuRF-1 and Atrogin-1, which contribute to decreased muscle mass by degrading myocyte proteins when expressed [ 27 , 78 ]. While most studies focus on explaining the role of up-regulating or downregulating inducers for TLR2 and TLR4 activation, our investigations will directly explore the expression status of these receptors during muscle unloading and exercise. TLR-2 and TLR-4 known inhibitors of muscle mass have varying expression patterns. TLR-4 levels significantly rise during hind limb unloading and remain elevated during recovery, indicating a potential role in atrophy progression and persistence aligning with its known role. Kawanishi, Nozaki [ 79 ] reported an increased TLR4 mRNA expression in the gastrocnemius muscle of wild-type cast immobilized mice; however, no significant difference was noticed between the wild-type and TLR4 knockout mice, thus suggesting a minor role of TLR4 role in muscle atrophy. On the other hand, while TLR-2 shows an increased level with exercise and recovery, there is a surprisingly significant drop in the atrophy group (HU), contradicting earlier reports by Parveen, Bohnert [ 80 ] and Kim, Cha [ 45 ]. Parveen, Bohnert [ 80 ] investigated the expression levels of many TLRs and downstream signaling pathways, the Myeloid Differentiation Primary Response 88 (MyD88). There was a notable increase in the mRNA expression levels of TLR1, TLR2, TLR4, TLR7, TLR8, TLR9, and MyD88 in the Gastrocnemius muscle on days 5 and 14 after sciatic nerve denervation in mice. Additionally, he reported that muscle atrophy is decreased by MyD88 ablation [ 80 ]. Autophagy, FOXO transcription factors, and components of the ubiquitin-proteasome system are all inhibited by the reduction of MyD88. Additionally, this promotes non-canonical NF-κB signaling while decreasing canonical NF-κB pathway activation and inflammatory cytokine receptor expression [ 45 ]. Moreover, without altering mTOR phosphorylation, MyD88 ablation prevents denervation-induced AMPK phosphorylation. Furthermore, during denervation, myofiber-specific XBP1 ablation reduces muscle atrophy. According to Kim, Cha [ 45 ], TLR-MyD88 signaling essentially influences several pathways, such as NF-κB signaling, autophagy, protein degradation, and AMPK, which all play a significant role in skeletal muscle wasting. TNF-α is a homotrimer protein composed of 157 amino acids, secreted mainly by T-lymphocytes, activated macrophages, and natural killer cells [ 81 ]. It has been identified as a critical regulator of inflammatory responses and is thought to be involved in developing specific inflammatory and autoimmune diseases [ 82 ]. In our study, TNF- α shows an expected increase during exercise, potentially indicating muscle stress. However, its further elevation during hind limb unloading and recovery suggests a role in sustained inflammation, contributing to muscle atrophy. Hirose, Nakazato [ 83 ] studied the transcription of type 1 collagen alpha-2 gene in male mice subjected to hindlimb unloading (HU). They reported a significant increase in TNF-a protein levels in the soleus muscle of the HU group on days 3,7, and 14, suggesting a role in muscle atrophy and collagen synthesis. Irisin is a protein derived through the proteolytic processing of fibronectin type III domain-containing 5 (FNDC5), which is a transmembrane protein; it is released from skeletal muscles abundantly in both mice and humans during exercise [ 84 , 85 ]. It is made of 112 amino acids and was shown to play a key role in regulating glucose and energy homeostasis [ 86 , 87 ]. Recent studies suggest a potential role for irisin to act as a mediator in the inflammatory processes within macrophages, further demonstrating its importance in immune regulation [ 34 , 88 ]. One study reported anti-tumor characteristics of irisin by inducing apoptosis of breast malignant cells, thus reinforcing its theory of immune system activation [ 89 ]. Our study showed a significant increase in the mice group undergoing exercise, which is consistent with what was reported by Cho, Jeong [ 90 ]. where FNDC5 mRNA and protein levels were significantly increased in mice subjected to an acute swimming exercise in soleus and gastrocnemius muscles [ 90 ]. Pang, Yang [ 91 ] reported similar findings with significant upregulation of FNDC5 mRNA levels in the mice subjected to 30-min and 1-h treadmill exercise and remained elevated for 24 24-hour recovery period. Conversely, the Irisin level significantly drops during hind limb unloading and recovery, aligning with the findings of Kawao, Moritake [ 92 ]. He studied the role of Irisin in muscle atrophy and bone loss in which he assigned 12 weeks of mice to either hindlimb unloading for 3 weeks or a control group; he reported a significant decrease in the FNDC5 mRNA levels in the soleus and gastrocnemius of the hindlimb group. These findings suggest the role of Irisin's in muscle atrophy and recovery. Conclusion our study provides valuable insights into the intricate molecular mechanisms of muscle atrophy, exercise, and recovery. The divergent expression patterns of key genes and regulators underscore the complexity of these processes, opening avenues for further research and potential therapeutic interventions. Understanding the crosstalk between Toll-like receptors, myokines, and regulatory factors contributes to the broader goal of developing targeted treatments for skeletal muscle atrophy. Declarations Ethics approval and consent to participate All procedures were conducted following applicable guidelines and regulations and received approval from the Institutional Review Board (Animal Care and Use Committee) of Jordan University of Science and Technology (JUST-ACUC) under approval number 368/12/4/16, dated August 2, 2021. These procedures adhered to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH), United States, which follows the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH). The study is reported in accordance with the ARRIVE guidelines, and appropriate measures were taken to minimize animal suffering. All euthanasia procedures followed the American Veterinary Medical Association (AVMA) Guidelines. Conflict of Interest The authors declare that they have no conflict of interest. Consent for publication Not applicable Competing interests The authors declare no conflicts of interest. Funding This research was funded by the Deanship of Research at Jordan University of Science and Technology (Grant numbers: 623/20222 and 623/2022). Author Contribution Mohammad Borhan Al-Zghoul: Supervision, Conceptualization, Methodology, Investigation, Formal analysis, Roles/Writing–original draft. Saad Al-Nassan: Methodology; Writing–review & editing. Qusai Al-Abedallat: Conceptualization, Review & editing. Abdullah Al-Zghoul: Writing–original draft. Mohammad Al-Bdoor: Writing-review & editing of the original draft. Abdel Qader Abu-Salih: Writing-review & editing of the original draft. Acknowledgement The authors would like to express their deep appreciation and thanks to the Deanship of Research, Jordan University of Science & Technology, for its financial support of this work (Grant#: 349/2021 and 623/2022). Data Availability The supplementary tables for the real-time qPCR run files and raw data are available on GitHub at https://github.com/mbalzghoul/Changes-in-the-muscle-growth-and-regulatory-factors-during-Exercise-Muscle-Atrophy-and-Recovery.git. References Olson, E. N. Interplay between proliferation and differentiation within the myogenic lineage. [Review] [94 refs]. Dev. Biology (Orlando) . 154 (2), 261–272 (1992). Zhao, Y. et al. Linc-RAM is required for FGF2 function in regulating myogenic cell differentiation. RNA Biol. 15 (3), 404–412 (2018). Seene, T., Kaasik, P. & Riso, E. M. Review on aging, unloading and reloading: changes in skeletal muscle quantity and quality. Arch. Gerontol. Geriatr. 54 (2), 374–380 (2012). Hughes, D. C. et al. Alterations in the muscle force transfer apparatus in aged rats during unloading and reloading: impact of microRNA-31. J. Physiol. 596 (14), 2883–2900 (2018). Goodman, C. A., Mayhew, D. L. & Hornberger, T. A. 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Cho, E., Jeong, D. Y., Kim, J. G. & Lee, S. The acute effects of swimming exercise on PGC-1α-FNDC5/irisin-UCP1 expression in male C57BL/6J mice. Metabolites 11 (2), 111 (2021). Pang, M. et al. Time-dependent changes in increased levels of plasma irisin and muscle PGC-1α and FNDC5 after exercise in mice. Tohoku J. Exp. Med. 244 (2), 93–103 (2018). Kawao, N., Moritake, A., Tatsumi, K. & Kaji, H. Roles of irisin in the linkage from muscle to bone during mechanical unloading in mice. Calcif. Tissue Int. 103 , 24–34 (2018). Tables Table 1 is not available with this version. 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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0.05).\u003c/p\u003e","description":"","filename":"Onlinefigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7180388/v1/4386ebffd3d44abca5baa2d2.png"},{"id":89931435,"identity":"2f4f479f-1a98-492d-92fd-7a373c3350f3","added_by":"auto","created_at":"2025-08-26 14:20:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":408657,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in mRNA levels of MyoD during muscle exercise, unloading, and exercise post-unloading.\u003c/p\u003e\n\u003cp\u003eThe y-axis represents the relative mRNA expression levels of Myoblast determination protein 1 (MyoD), while the x-axis denotes the conditions of muscle exercise (intensity speed of 15m/min, 60 minutes/day for seven days), unloading (hind limb suspension for seven days), and exercise post-muscle unloading (hind limb suspension for seven days and intensity speed of 15m/min, 60 minutes/day for seven days) and control (weight bearing).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea-b\u003c/sup\u003e means ± SD with different superscripts differ significantly (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7180388/v1/a24c45b298c5e7bc027a680d.png"},{"id":89932712,"identity":"88e4bdbc-5ef3-45e7-a5bc-64df6250f9b2","added_by":"auto","created_at":"2025-08-26 14:28:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":455800,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in mRNA levels of Myostatin during muscle exercise, unloading, and exercise post-unloading.\u003c/p\u003e\n\u003cp\u003eThe y-axis represents the relative mRNA expression levels of Myostatin, while the x-axis denotes the conditions of muscle exercise (intensity speed of 15m/min, 60 minutes/day for seven days), unloading (hind limb suspension for seven days), and exercise post-muscle unloading (hind limb suspension for seven days and intensity speed of 15m/min, 60 minutes/day for seven days) and control (weight bearing).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea-b\u003c/sup\u003e means ± SD with different superscripts differ significantly (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7180388/v1/e90036d8aed0d2f646313500.png"},{"id":89931439,"identity":"e0702f67-ffbd-4ab7-a100-67394f732a23","added_by":"auto","created_at":"2025-08-26 14:20:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":426427,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in mRNA levels of MurF during muscle exercise, unloading, and exercise post-unloading.\u003c/p\u003e\n\u003cp\u003eThe y-axis represents the relative mRNA expression levels of Muscle-specific RING finger protein (MurF), while the x-axis denotes the conditions of muscle exercise (intensity speed of 15m/min, 60 minutes/day for seven days), unloading (hind limb suspension for seven days), and exercise post-muscle unloading (hind limb suspension for seven days and intensity speed of 15m/min, 60 minutes/day for seven days) and control (weight bearing).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea-b\u003c/sup\u003e means ± SD with different superscripts differ significantly (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7180388/v1/e995b7ec04880c8b0e3bd476.png"},{"id":89931445,"identity":"4685fe97-1508-4ee7-806a-e325f0822f9b","added_by":"auto","created_at":"2025-08-26 14:20:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":469369,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in mRNA levels of Atrogin during muscle exercise, unloading, and exercise post-unloading.\u003c/p\u003e\n\u003cp\u003eThe y-axis represents the relative mRNA expression levels of Atrogin, while the x-axis denotes the conditions of muscle exercise (intensity speed of 15m/min, 60 minutes/day for seven days), unloading (hind limb suspension for seven days), and exercise post-muscle unloading (hind limb suspension for seven days and intensity speed of 15m/min, 60 minutes/day for seven days) and control (weight bearing).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea-b\u003c/sup\u003e means ± SD with different superscripts differ significantly (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7180388/v1/283ef2e28338543f46ff33df.png"},{"id":89931449,"identity":"ce6a871b-8bc1-4db0-9490-a77b567446cf","added_by":"auto","created_at":"2025-08-26 14:20:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":421253,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in mRNA levels of TLR-2 during muscle exercise, unloading, and exercise post-unloading.\u003c/p\u003e\n\u003cp\u003eThe y-axis represents the relative mRNA expression levels Toll-like receptor 2 (TLR-2), while the x-axis denotes the conditions of muscle exercise (intensity speed of 15m/min, 60 minutes/day for seven days), unloading (hind limb suspension for seven days), and exercise post-muscle unloading (hind limb suspension for seven days and intensity speed of 15m/min, 60 minutes/day for seven days) and control (weight bearing).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea-b\u003c/sup\u003e means ± SD with different superscripts differ significantly (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7180388/v1/bcc0ef19729fdce701eea553.png"},{"id":89932720,"identity":"4b957166-6056-4791-b319-b0eb27e39518","added_by":"auto","created_at":"2025-08-26 14:28:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":396156,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in mRNA levels of TLR-4 during muscle exercise, unloading, and exercise post-unloading.\u003c/p\u003e\n\u003cp\u003eThe y-axis represents the relative mRNA expression levels of Toll-like receptor 4 (TLR-4), while the x-axis denotes the conditions of muscle exercise (intensity speed of 15m/min, 60 minutes/day for seven days), unloading (hind limb suspension for seven days), and exercise post-muscle unloading (hind limb suspension for seven days and intensity speed of 15m/min, 60 minutes/day for seven days) and control (weight bearing).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003csup\u003ea-b\u003c/sup\u003e means ± SD with different superscripts differ significantly (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"Onlinefigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7180388/v1/04838416655d6e8b27eee433.png"},{"id":89931447,"identity":"7385145d-56c7-4146-a331-25e4a5d092a3","added_by":"auto","created_at":"2025-08-26 14:20:43","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":453222,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in mRNA levels of TNF-α during muscle exercise, unloading, and exercise post-unloading.\u003c/p\u003e\n\u003cp\u003eThe y-axis represents the relative mRNA expression levels of Tumor Necrosis Factor alpha (TNF-α), while the x-axis denotes the conditions of muscle exercise (intensity speed of 15m/min, 60 minutes/day for seven days), unloading (hind limb suspension for seven days), and exercise post-muscle unloading (hind limb suspension for seven days and intensity speed of 15m/min, 60 minutes/day for seven days) and control (weight bearing).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea-b\u003c/sup\u003e means ± SD with different superscripts differ significantly (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefigure9.png","url":"https://assets-eu.researchsquare.com/files/rs-7180388/v1/9535aeeea56e6e78292804ab.png"},{"id":89931454,"identity":"a1f993af-0299-4479-8831-b3cd7803ff50","added_by":"auto","created_at":"2025-08-26 14:20:43","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":409857,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in mRNA levels of Irisin during muscle exercise, unloading, and exercise post-unloading.\u003c/p\u003e\n\u003cp\u003eThe y-axis represents the relative mRNA expression levels of Irisin, while the x-axis denotes the conditions of muscle exercise (intensity speed of 15m/min, 60 minutes/day for seven days), unloading (hind limb suspension for seven days), and exercise post-muscle unloading (hind limb suspension for seven days and intensity speed of 15m/min, 60 minutes/day for seven days) and control (weight bearing).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea-b\u003c/sup\u003e means ± SD with different superscripts differ significantly (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefigure10.png","url":"https://assets-eu.researchsquare.com/files/rs-7180388/v1/47faab8f1a208605604b875d.png"},{"id":90889945,"identity":"c63cae6d-683a-4a08-aaf5-7ceea041be1e","added_by":"auto","created_at":"2025-09-09 10:53:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1126956,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7180388/v1/3d7214e5-7bce-41b4-b814-98429255b1cf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Changes in the muscle growth and regulatory factors, TNF-α and Toll-like Receptors 2 and 4 during Exercise, Muscle Atrophy, and Recovery in the Mouse Hind Limb Suspension Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSkeletal muscle is a highly malleable tissue that is vital to whole-body health and metabolism. Skeletal muscles account for approximately 40%-50% of the whole-body mass. The regulation of myogenic precursor cell proliferation and differentiation has been studied in detail [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In summary, skeletal muscle asynchronously develops in vivo. During this process, the myoblast can proliferate, generating a larger population of myoblasts, or it can irreversibly differentiate and form a myocyte, the building block of individual skeletal muscle cells. The size and strength of a skeletal muscle are determined by its component myocyte's number and cellular structure. However, the majority of these experiments were performed \u003cem\u003ein vitro\u003c/em\u003e, where the muscle growth and development assigned to growth factors based solely on their effects on cultured myocytes did not always coincide with their \u003cem\u003ein vivo\u003c/em\u003e functions. Muscle mass maintenance is crucial for health and concerns affecting one's quality of life [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Significant progress has been made in the last decade in understanding the chemicals that govern skeletal muscle hypertrophy. Unsurprisingly, many of these molecules are critical for regulating protein metabolism [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMuscle atrophy is the weakening of muscles or a reduction in muscle mass and strength triggered by inactivity or injury, and it is found in various disorders, including AIDS, cancer, and congestive heart failure [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. According to this definition, the pathophysiology of atrophy can vary; atrophy is a highly active process mediated by various signaling pathways in which muscle mass is lost due to either a decrease in protein synthesis or increased protein degradation rates [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Healthy skeletal muscle tissue requires physical activity to adjust muscle fibers\u0026rsquo; number, size, and properties [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Muscle strength and mass are tightly regulated through protein synthesis and turnover regulatory mechanisms [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Skeletal muscle has the potential to release myostatin, a member of the transforming growth factor β (TGF- β) family. Myostatin is a negative regulator of muscle development and growth upon release, particularly affecting protein turnover and replacing/distracting pathways [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Conversely, the insulin growth factor 1 (IGF-1) signaling pathway stimulates myofiber size, number, and function positively by promoting the synthesis of new proteins and the distraction of old proteins, leading to an increase in muscle mass [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Mainly, IGF-1 regulates protein synthesis in the skeletal muscle, increasing muscle mass [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMuscle maintenance is accomplished by the cell's combined action of the two types of proteolytic systems known as the ubiquitin-proteasome pathway (UPP) and the autophagy-lysosome machinery [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The UPP, MuRF-1, and Atrogin-1 play essential regulatory roles in the development of muscle atrophy by guiding ubiquitinated proteins to the 26S proteasome complex for degradation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOxidative stress and inflammation are significant factors in the development of skeletal muscle atrophy [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Atrophic skeletal muscle caused by unloading and immobility is characterized by inflammation and increased levels of oxidative stress. Reducing inflammation and oxidative stress can prevent skeletal muscular atrophy by inhibiting protein breakdown [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn muscle atrophy, Proinflammatory cytokines such as TNFα, IL-6, and IL-1 are released, subsequently activating STAT3, a transcription factor that up-regulates atrogin-1 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. STAT3 activation can also suppress myogenic differentiation and regulatory factors, including myogenin (MyoG), MyoD, Myogenic Factor 5 (Myf5), and Myogenic Regulator 4 (MRF4) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This may interfere with myoblast fusion and impair muscle regeneration [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eToll-like Receptor (TLR) signaling pathways have been identified as inhibitors of muscle mass and can trigger conditions that promote muscle atrophy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. When TLRs, especially TLR-2 and TLR-4, are activated, the NF-KB pathway is triggered, resulting in the expression of the IL-6 gene, which will lead to an inflammation reaction. Moreover, Muscle-specific RING finger protein (MuRF-1) and Atrogin-1 expression decrease muscle mass by degrading myocyte proteins [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, the expression status of these TLRs during muscle unloading and exercise has never been investigated. Published literature has shown that exercise can decrease the expression of TLRs and inflammatory signaling [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Exercise-induced myokines play an integral role in regulating metabolism in contracting muscles and distant organs [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIrisin, which is the cleavage product of its precursor fibronectin type III domain-containing 5 (FNDC5) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], is currently a renowned myokine for its exercise-induced beneficial effects in browning white adipocytes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], improving insulin resistance [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], promoting skeletal remodeling [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and exerting anti-inflammatory effects by suppressing the TLR4 pathway [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUnderstanding the crosstalk between irisin, TLR-2, and TLR-4 during muscle atrophy and recovery is the potential we aim to add through our current experimental study. Our long-range goal is to elucidate molecular mechanisms that could be exploited as potential treatments for skeletal muscle atrophy. The results of this study would be important because they could offer potential therapeutic targets for attenuating/preventing muscle atrophy during aging, disuse, and illness.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eExperimental design\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eIn this experiment, eighty 15-week-old male BALB/c mice (purchased from Jordan University of Science and Technology\u0026rsquo;s animal house) were divided into four main groups: weight-bearing control (Con, n\u0026thinsp;=\u0026thinsp;20), exercise training (Exe, n\u0026thinsp;=\u0026thinsp;20), hind limb unloading (HU, n\u0026thinsp;=\u0026thinsp;20), and hind limb unloading\u0026thinsp;+\u0026thinsp;exercise training (HU\u0026thinsp;+\u0026thinsp;Exe, n\u0026thinsp;=\u0026thinsp;20), for the HU group of mice. The hind limb unloading model was used to induce muscle wasting. Each mouse in the HU group was suspended from its tail to the ceiling of its cage, preventing the hind limbs from weight bearing on the ground but allowing the mouse to move freely inside its individualized cage for one week.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExercise protocol\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eThe exercise protocol included the animals running on an electrically motorized rodent treadmill with a moderate intensity speed of 15m/min, 60 minutes/day, for seven days. Animals in the exercised groups received two familiarization sessions for two days on the treadmill with an intensity of 5-10m/min, 30\u0026ndash;40 minutes/session to get accustomed to the treadmill and the training protocol before the initiation of the experiment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSample collection, RNA extraction, and cDNA Synthesis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMice were anesthetized with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) until the loss of the pedal withdrawal reflex indicated surgical anesthesia. For euthanasia, animals received an overdose of ketamine (200 mg/kg) and xylazine (20 mg/kg). Cervical dislocation was performed to confirm death following the cessation of breathing and pulse. All operations were performed by qualified individuals following the AVMA Guidelines for the Euthanasia of Animals and approved by the Animal Care and Use Committee (ACUC).\u003c/p\u003e\u003cp\u003eGastrocnemius muscle samples (n\u0026thinsp;=\u0026thinsp;20 animals per group) were collected. Total RNA was extracted using Direct-Zol\u0026trade; RNA MiniPrep (Zymo Research, Irvine, USA) with TRI Reagent\u0026reg; (Zymo Research, Irvine, USA) according to manufacturer procedure. RNA concentrations were measured using Biotek PowerWave XS2 Spec. From each sample, 2 \u0026micro;g of total RNA was utilized in the reverse transcription reaction using the SuperScript IV VILO Master Mix (Invitrogen, Thermo Fisher Scientific, Wilmington, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRelative-Quantitative Real-Time PCR (RT-qPCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBlastaq\u0026trade; Green qPCR Master Mix (Applied Biological Materials Inc., Richmond, Canada) was used in a Rotor-Gene Q MDx 5 plex instrument (Qiagen, Hilden, Germany). Briefly, the 20 \u0026micro;l reaction mixture was prepared from 10 \u0026micro;l of the master mix, 2 \u0026micro;l forward primer (2 pmol), 2 \u0026micro;l reveres primer (2 pmol), 2 \u0026micro;l cDNA of the sample, and 4 \u0026micro;l of nuclease-free water. Cycling parameters were 50\u0026deg;C for 2 min, 95\u0026deg;C for 15 min, and 40 cycles of 95\u0026deg;C for 10 s, followed by 30 s at 57\u0026deg;C and 72\u0026deg;C for 10 s, with final melting at 95\u0026deg;C for 20 s. Duplicates from each cDNA were analyzed, fluorescence emission was detected, and relative quantification was calculated automatically. β-Actin and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) were used as internal controls to which the fold changes in gene expression were normalized. The melting curve approved the specificity of a single target amplification.\u003c/p\u003e\u003cp\u003eThe cDNA sequence for each gene was obtained from the NCBI's Nucleotide database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/nucleotide/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/nucleotide/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The IDT Primer Quest software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://eu.idtdna.com/PrimerQuest/Home/Index\u003c/span\u003e\u003cspan address=\"http://eu.idtdna.com/PrimerQuest/Home/Index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to create all primers. The primer sequences are presented in Table\u0026nbsp;1.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were conducted using IBM SPSS Statistics 26.0 (IBM software, Chicago, USA). One-way analysis of variance (ANOVA) was used to evaluate the existence of differences among the groups. Data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;the standard error of the mean (SEM). Values of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIGF-1\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe mRNA levels of IGF-1 showed a significant increase in the exercised group, indicating an upregulation of IGF-1 gene expression in response to the exercise stimulus. In contrast, during the atrophic state of hind limb unloading (HU), the mRNA levels of IGF-1 demonstrated a notable decrease, indicating a downregulation of IGF-1 gene expression. In the recovered animals (HU\u0026thinsp;+\u0026thinsp;Ex) group, the mRNA levels of IGF-1 exhibited a recovery trend, with an upward shift compared to those in the unloading phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMyoG\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMuscle atrophy (HU) showed an increase in the mRNA expression level of MyoG compared to other groups. While there was a significant drop in the expression level of MyoG in the recovered group (HU\u0026thinsp;+\u0026thinsp;Ex) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMyoD\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression levels of MyoD mRNA almost showed the same trend as MyoG expressions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, the expression level in the recovered group (HU\u0026thinsp;+\u0026thinsp;Ex) did not differ significantly from the control or exercised groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMyostatin\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe mRNA level of myostatin showed a significant increase in the exercised group (Ex) compared to the control and HU groups, while it showed a significant increase in the recovered group (HU\u0026thinsp;+\u0026thinsp;Ex) when compared to the other 3 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMurF\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThere was a significant increase in the mRNA expression level of MurF in the exercised group (Ex) when compared to control and HU groups and a significant increase in the recovered group (HU\u0026thinsp;+\u0026thinsp;Ex) when compared to other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAtrogin\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression level of Atrogin mRNA showed a significant increase in the exercised group when compared to the control, of the hind limb unloading group when compared to the control and exercise groups, and in the recovered group (HU\u0026thinsp;+\u0026thinsp;Ex) when compared to the other 3 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTLR-2\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression level of TLR-2 showed a significant increase in the exercised group compared to the control and a substantial drop in the HU group compared to the control and Ex groups, while there was a significant increase in the recovered group compared to other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTLR-4\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression levels of TLR-4 increased significantly in the HU and HU\u0026thinsp;+\u0026thinsp;Ex groups compared to the control and Ex groups without showing a significant difference between the HU and HU\u0026thinsp;+\u0026thinsp;Ex groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTNF-α\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression level of TNF-α showed a significant increase in the Ex group compared to the control and a considerable increase in both HU and HU\u0026thinsp;+\u0026thinsp;Ex groups compared to the Control and Ex groups. (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIrisin\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression level of Irisin mRNA showed a significant increase in the exercised group (Ex) compared to the control group. While the HU and HU\u0026thinsp;+\u0026thinsp;Ex groups' expression levels dropped significantly compared to the Ex group but were higher considerably from the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMuscle atrophy, a reduction in muscle strength and mass, can have many debilitating effects on one\u0026rsquo;s health and significantly impair one's quality of life [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The consequences of muscle atrophy, resulting in progressive weakness and diminished physical performance, make daily life challenges more difficult [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, it's critical to have interventions and preventative measures to halt or even reverse muscle atrophy so that people may continue to be healthy and perform their jobs well. Our experimental strategy in this work aimed to identify patterns in regulating muscle homeostasis genes during atrophy and exercise-induced recovery. Gaining insight into the molecular and cellular mechanisms involved in these processes is crucial for devising ways to combat muscle loss. Muscle atrophy in the hind limbs was induced using a mouse model of hind limb unloading. Additionally, a regimen of treadmill activities was implemented to investigate the effects of countermeasures during the recovery phase.\u003c/p\u003e\u003cp\u003eInsulin growth factor 1 (IGF-1), a compact peptide composed of seventy amino acids, has a molecular weight of 7649 Dalton (Da) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Like insulin, IGF-1 comprises an Alpha and Beta chain linked by disulfide bonds with 12 amino acids in its C peptide region. The structural resemblance to insulin explains why IGF-1 can interact with little affinity to the insulin receptor[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The IGF-1 signaling pathway plays a crucial role in regulating protein synthesis in skeletal muscle, which results in increased muscle mass [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This process occurs by binding IGF-1 to the IGF-1 receptor (IGF-1R), which triggers phosphorylation of intracellular components such as insulin receptor substrate-1, an adaptor protein. Following IRS-1 phosphorylation, phosphoinositide 3-kinase (PI3K) is attracted and then phosphorylated, leading to Akt activation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Activation of Akt suppresses denervation-induced atrophy in rats, demonstrating its significance for myotube growth through the PI3K/Akt pathway [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our study, a notable increase in the expression of the IGF-1 gene was observed in the recovery group (HU\u0026thinsp;+\u0026thinsp;Ex), which is consistent with its well-established function in promoting muscle growth and repair [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Conversely, a predictably significant drop was evident in the atrophy group (HU), with various studies demonstrating similar findings. For instance, Kim, Cha [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] assigned Twelve 7-week-old male rats to either control or hindlimb unloading groups; they reported significant IGF-1 downregulation in the hindlimb group\u0026rsquo;s soleus and long digital extensor muscles after 2 weeks of treatment. Another study conducted by Li, Feng [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] showed that there was a notable increase in the expression of IGF-1 mRNA, IGF-1 protein, IGF1R, phosphorylated PI3K, and p-Akt in mice following various exercise regimens, including aerobic exercise, resistance training, whole-body vibration, and electrical stimulation.\u003c/p\u003e\u003cp\u003eMyostatin, a member of the transforming growth factor-β (TGF-β) family, once it is released, myostatin will act as a negative regulator of muscle development and growth by inhibiting the proliferation of muscle cells and promoting their breakdown [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. There is an intricate relationship between IGF-1 and Myostatin, with studies suggesting they counteract each other. Myostatin acts as an autocrine/paracrine inhibitor of muscle development in skeletal and cardiac muscles by binding to the activin A receptor type IIB (ACVR2B), which is linked to the type 1 receptors ALK4 and ALK5 [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. ACVR2B is expressed in various tissues, including skeletal muscle, adipose tissue, liver, kidney, and heart[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Its downstream effects are mediated through the activation of the canonical SMAD signaling pathway. When activin A binds to ACVR2B, it forms a complex with other receptors leading to various cellular processes [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFurthermore, activation of the NF-κB pathway increases Murf1 expression due to cytokines such as TNF-α, and MuRF1 targets proteins for degradation which leads to muscle atrophy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, our gene expression data for myostatin align with the IGF-1 expression results. Although exercises down-regulate the expression of myostatin in skeletal muscles [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], our myostatin gene expression levels were significantly higher in the exercised groups. Similar findings regarding myostatin mRNA elevation in response to exercise or repeated muscle contractions were reported in humans, and Okudan 2018, Arrieta, Herv\u0026aacute;s, et al. 2019) and rodent studies [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCarlson, Booth [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] reported an increase in myostatin mRNA in both the gastrocnemius-plantaris complex (Gast/PLT) and soleus muscles after the first day of hindlimb unloading in female ICR mice, without a significant difference in comparison to controls after 3 and 7 days of hindlimb unloading. On the other hand, Babcock, Knoblauch [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] conducted a study with 6-month-old male Wistar rats divided into ambulatory and hindlimb suspension groups for 10 days. They found a significant decrease in myostatin levels in the red tibialis anterior muscle after unloading for 10 days. We believe that these variations in myostatin response to exercises are due to its affection by several factors concerning the exercise parameters: intensity, frequency, and duration, or the inflammatory signaling that might still be persistent in early adaptive responses to exercise and post-atrophy recovery.\u003c/p\u003e\u003cp\u003eMyogenin (MyoG) and Myoblast determination protein 1 (MyoD) represent muscle-specific transcription factors belonging to the basic-helix-loop-helix (bHLH) family of DNA-binding proteins [\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Anticipated to play pivotal roles in muscle regeneration in mammalian embryos based on their actions in tissue culture, these proteins have primarily been substantiated through gain-of-function tests in cultured cells, demonstrating their potency as myogenic factors [\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The expression of any of these myogenic factors can transform various tissue culture cells into muscle cells, exhibiting behaviors similar to myoblasts regarding growth factor responses, gene expression, and the capacity to fuse into multinucleated myotubes. Binding to the regulatory regions of muscle-specific genes, myogenic factors activate their expression, thereby facilitating the conversion process [\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our investigation, both factors exhibited heightened expression during muscle atrophy (HU), with a notable decline in the recovery group (HU\u0026thinsp;+\u0026thinsp;Ex). Macpherson, Wang [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], whose study involved Conditional MyoG null mice, wherein the soleus muscle was denervated by excising the sciatic nerve to induce muscle atrophy, reveald that Myog expression plays a significant role in reducing muscle mass, force, and cross-sectional area in the denervated muscle [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDue to disuse, the ubiquitin-proteasome system (UPS) plays a crucial role in muscle atrophy [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The UPP, MuRF-1, and Atrogin-1 play essential regulatory roles in muscle atrophy development by guiding ubiquitinated proteins to the 26S proteasome complex [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Ephemeral elevations in MuRF-1 and Atrogin-1 gene expression led to a shift from protein synthesis to degradation, thus causing muscle mass loss [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This mechanism orderly leads proteins to extinction. Additionally, Atrogin-1 controls the degradation of translation initiation factor 3f to inhibit overall protein synthesis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Examples of sarcomeric proteins regulated by MuRF-1 include myotilin, myosins, troponins, and titin [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Elevated levels of MurF and Atrogin in the exercised and recovery groups indicate increased proteolysis. The increased expression of these factors suggests a transient breakdown of muscle proteins induced by exercise, potentially for remodeling. However, their sustained increase during recovery implies a prolonged impact on protein degradation pathways\u0026mdash;a study conducted by Al-Nassan et al. Who employed a 6-week hindlimb unloading model in mice to induce muscle atrophy with daily treadmill running for 1 hour during that period, then measuring gastrocnemius succinate dehydrogenase (SDH) activity, muscle fiber cross-sectional area, and muscle mass in muscle fibers to assess the impact of exercise on muscle atrophy; reported that chronic exercise down-regulate the mRNA expressions of TNF-α and atrogin-1/MAFbx in the atrophied skeletal muscle [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. On the other hand, Gomes et al who used cDNA microarrays to compare the gastrocnemius muscle between normal mice and food-deprived mice, Found that atrogin-1, which is explicitly expressed in striated muscles, is induced more than ninefold in muscles of fasted mice and highly expressed during muscle atrophy in various diseases [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eToll-like receptors are crucial for the body's defense against pathogens, recognizing and eliminating pathogens by stimulating the innate immune response. Toll-like receptors can also detect tissue damage and integrity by interacting with danger-associated molecular patterns released by damaged cells [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Twelve mouse TLRs have been identified (TLR1-9, TLR11-13), which are classified based on their expression location; these TLRs may be categorized as intracellular or extracellular receptors [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. In vitro studies have demonstrated that TLR activation and suppression significantly influence muscle cell atrophy[\u003cspan additionalcitationids=\"CR66\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. While most TLRs exhibit similar responses, some ligand-related effects and tissue-specific expression variations are prominent, leading to different outcomes [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. TLR-mediated signaling is well-known for its critical role in clearing infections and tissue regeneration. However, it is necessary to tightly control and regulate its function in order to prevent chronic and damaging inflammation resulting from dysregulated interactions with endogenous metabolites [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOn the cell surface, TLR (1\u0026ndash;6) receptors -especially mouse TLR11 receptors- are expressed [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. TLR1 and TLR6 recognize lipopeptides shared by microbes as well as derivatives produced by damaged cells. Additionally, TLR2/4 can bind to various ligands [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], one of which is bacterial lipopolysaccharide, and they also identify a range of endogenous ligands. For example, high mobility group box 1, a ligand for TLR2 and TLR4 that causes inflammation, is released from muscle tissue, leading to tissue damage [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Upon release, HMGB1 transforms into a soluble activator of proinflammatory cytokines [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFurthermore, research has shown that the interplay between TLR4 and HMGB1 up-regulates the expression of MHCI in mouse muscle tissue and mononuclear cells in peripheral blood [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. MHC-I enables muscle cells to display antigens and plays a role in immune responses, which hastens the removal of injured muscle tissue proteins and leads to the loss of muscle mass [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Heat shock proteins, which function as intracellular molecular chaperones, also become TLR2/4-binding DAMPs when released from damaged cells [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Another ligand for TLR2/4 present in skeletal muscle besides the liver is acute-phase protein serum amyloid A1, whose levels are regulated by producing proinflammatory cytokines [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. The expression of TLRs on cell surfaces significantly impacts muscular performance [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. In vitro studies have demonstrated that stimulation of muscle cells by TLR4 results in inflammatory C2C12 production [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Due to a lower presence of invasive macrophages, mice lacking TLR2 take longer to clear necrotic tissue after muscle injury [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Activation of TLRs, especially TLR-2 and TLR-4, by creatine ligands triggers the NF-KB pathway, leading to the expression of the IL-6 gene. This results in an inflammatory reaction due to necrotic myocytes as well as MuRF-1 and Atrogin-1, which contribute to decreased muscle mass by degrading myocyte proteins when expressed [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. While most studies focus on explaining the role of up-regulating or downregulating inducers for TLR2 and TLR4 activation, our investigations will directly explore the expression status of these receptors during muscle unloading and exercise.\u003c/p\u003e\u003cp\u003eTLR-2 and TLR-4 known inhibitors of muscle mass have varying expression patterns. TLR-4 levels significantly rise during hind limb unloading and remain elevated during recovery, indicating a potential role in atrophy progression and persistence aligning with its known role. Kawanishi, Nozaki [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e] reported an increased TLR4 mRNA expression in the gastrocnemius muscle of wild-type cast immobilized mice; however, no significant difference was noticed between the wild-type and TLR4 knockout mice, thus suggesting a minor role of TLR4 role in muscle atrophy. On the other hand, while TLR-2 shows an increased level with exercise and recovery, there is a surprisingly significant drop in the atrophy group (HU), contradicting earlier reports by Parveen, Bohnert [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e] and Kim, Cha [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eParveen, Bohnert [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e] investigated the expression levels of many TLRs and downstream signaling pathways, the Myeloid Differentiation Primary Response 88 (MyD88). There was a notable increase in the mRNA expression levels of TLR1, TLR2, TLR4, TLR7, TLR8, TLR9, and MyD88 in the Gastrocnemius muscle on days 5 and 14 after sciatic nerve denervation in mice. Additionally, he reported that muscle atrophy is decreased by MyD88 ablation [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAutophagy, FOXO transcription factors, and components of the ubiquitin-proteasome system are all inhibited by the reduction of MyD88. Additionally, this promotes non-canonical NF-κB signaling while decreasing canonical NF-κB pathway activation and inflammatory cytokine receptor expression [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Moreover, without altering mTOR phosphorylation, MyD88 ablation prevents denervation-induced AMPK phosphorylation. Furthermore, during denervation, myofiber-specific XBP1 ablation reduces muscle atrophy. According to Kim, Cha [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], TLR-MyD88 signaling essentially influences several pathways, such as NF-κB signaling, autophagy, protein degradation, and AMPK, which all play a significant role in skeletal muscle wasting.\u003c/p\u003e\u003cp\u003eTNF-α is a homotrimer protein composed of 157 amino acids, secreted mainly by T-lymphocytes, activated macrophages, and natural killer cells [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. It has been identified as a critical regulator of inflammatory responses and is thought to be involved in developing specific inflammatory and autoimmune diseases [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. In our study, TNF- α shows an expected increase during exercise, potentially indicating muscle stress. However, its further elevation during hind limb unloading and recovery suggests a role in sustained inflammation, contributing to muscle atrophy. Hirose, Nakazato [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e] studied the transcription of type 1 collagen alpha-2 gene in male mice subjected to hindlimb unloading (HU). They reported a significant increase in TNF-a protein levels in the soleus muscle of the HU group on days 3,7, and 14, suggesting a role in muscle atrophy and collagen synthesis.\u003c/p\u003e\u003cp\u003eIrisin is a protein derived through the proteolytic processing of fibronectin type III domain-containing 5 (FNDC5), which is a transmembrane protein; it is released from skeletal muscles abundantly in both mice and humans during exercise [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. It is made of 112 amino acids and was shown to play a key role in regulating glucose and energy homeostasis [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Recent studies suggest a potential role for irisin to act as a mediator in the inflammatory processes within macrophages, further demonstrating its importance in immune regulation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. One study reported anti-tumor characteristics of irisin by inducing apoptosis of breast malignant cells, thus reinforcing its theory of immune system activation [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. Our study showed a significant increase in the mice group undergoing exercise, which is consistent with what was reported by Cho, Jeong [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. where FNDC5 mRNA and protein levels were significantly increased in mice subjected to an acute swimming exercise in soleus and gastrocnemius muscles [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. Pang, Yang [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e] reported similar findings with significant upregulation of FNDC5 mRNA levels in the mice subjected to 30-min and 1-h treadmill exercise and remained elevated for 24 24-hour recovery period.\u003c/p\u003e\u003cp\u003eConversely, the Irisin level significantly drops during hind limb unloading and recovery, aligning with the findings of Kawao, Moritake [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. He studied the role of Irisin in muscle atrophy and bone loss in which he assigned 12 weeks of mice to either hindlimb unloading for 3 weeks or a control group; he reported a significant decrease in the FNDC5 mRNA levels in the soleus and gastrocnemius of the hindlimb group. These findings suggest the role of Irisin's in muscle atrophy and recovery.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eour study provides valuable insights into the intricate molecular mechanisms of muscle atrophy, exercise, and recovery. The divergent expression patterns of key genes and regulators underscore the complexity of these processes, opening avenues for further research and potential therapeutic interventions. Understanding the crosstalk between Toll-like receptors, myokines, and regulatory factors contributes to the broader goal of developing targeted treatments for skeletal muscle atrophy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003e All procedures were conducted following applicable guidelines and regulations and received approval from the Institutional Review Board (Animal Care and Use Committee) of Jordan University of Science and Technology (JUST-ACUC) under approval number 368/12/4/16, dated August 2, 2021. These procedures adhered to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH), United States, which follows the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH). The study is reported in accordance with the ARRIVE guidelines, and appropriate measures were taken to minimize animal suffering. All euthanasia procedures followed the American Veterinary Medical Association (AVMA) Guidelines.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was funded by the Deanship of Research at Jordan University of Science and Technology (Grant numbers: 623/20222 and 623/2022).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMohammad Borhan Al-Zghoul: Supervision, Conceptualization, Methodology, Investigation, Formal analysis, Roles/Writing\u0026ndash;original draft. Saad Al-Nassan: Methodology; Writing\u0026ndash;review \u0026amp; editing. Qusai Al-Abedallat: Conceptualization, Review \u0026amp; editing. Abdullah Al-Zghoul: Writing\u0026ndash;original draft. Mohammad Al-Bdoor: Writing-review \u0026amp; editing of the original draft. Abdel Qader Abu-Salih: Writing-review \u0026amp; editing of the original draft.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to express their deep appreciation and thanks to the Deanship of Research, Jordan University of Science \u0026amp; Technology, for its financial support of this work (Grant#: 349/2021 and 623/2022).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe supplementary tables for the real-time qPCR run files and raw data are available on GitHub at https://github.com/mbalzghoul/Changes-in-the-muscle-growth-and-regulatory-factors-during-Exercise-Muscle-Atrophy-and-Recovery.git.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOlson, E. N. Interplay between proliferation and differentiation within the myogenic lineage. [Review] [94 refs]. \u003cem\u003eDev. 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Tissue Int.\u003c/em\u003e \u003cb\u003e103\u003c/b\u003e, 24\u0026ndash;34 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is not available with this version.\u003c/p\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":"Toll-like receptors 2 and 4, muscle atrophy, Mouse, Hind Limb Suspension.","lastPublishedDoi":"10.21203/rs.3.rs-7180388/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7180388/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eMuscle atrophy, characterized by muscle weakening and a reduction in mass, is primarily caused by inactivity or injury. The molecular mechanisms that drive skeletal muscle growth and development remain poorly understood, hampering the development of novel techniques for treating or preventing muscle atrophy. This study aimed to assess changes in muscle growth and the level of regulatory factors, including TNF-α and Toll-like Receptors 2 and 4, during Exercise, Muscle Atrophy, and Recovery in a Mouse Hind Limb Suspension Model.\u003c/p\u003e\u003ch2\u003eMethodology\u003c/h2\u003e\u003cp\u003eAdult male mice were subjected to hind limb unloading to induce muscle wasting for one week. Eighty animals were divided into four main groups: weight-bearing control (Con) group, Hindlimb unloading (HU) group, Hindlimb unloading\u0026thinsp;+\u0026thinsp;exercise training (HU\u0026thinsp;+\u0026thinsp;Ex) group, and exercise training (Exe) group. Total RNA was extracted from the Gastrocnemius muscle, and selected gene expression was evaluated using RT-qPCR analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eDistinct gene expression patterns were observed in response to hindlimb unloading (HU) and hindlimb unloading with exercise (HU\u0026thinsp;+\u0026thinsp;Ex). There was a significant decrease in TLR-2 expression in the HU group, while TLR-4 levels increased compared to those in the other groups. TNF-α expression increased substantially in almost all groups except for the control group. IGF-1 increased with exercise and decreased during HU, showing recovery in the HU\u0026thinsp;+\u0026thinsp;Ex group. Markers of muscle atrophy, MyoG, and MyoD increased during HU and dropped in HU\u0026thinsp;+\u0026thinsp;Ex. Myostatin, MurF, and Atrogin were linked to atrophy, which increased in both the exercised and recovered groups. Exercise increased Irisin expression compared to controls, while HU and HU\u0026thinsp;+\u0026thinsp;Ex groups showed decreased levels but were still elevated compared to controls.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eUnderstanding the changes in muscle growth and regulatory factors, TNF-α and Toll-like Receptors 2 and 4 during exercise, muscle atrophy, and recovery in the mouse hind limb suspension model can add significant value to the existing data on molecular and cellular mechanisms during and post hind limb recovery from muscle atrophy (hind limb suspension).\u003c/p\u003e","manuscriptTitle":"Changes in the muscle growth and regulatory factors, TNF-α and Toll-like Receptors 2 and 4 during Exercise, Muscle Atrophy, and Recovery in the Mouse Hind Limb Suspension Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-26 14:20:38","doi":"10.21203/rs.3.rs-7180388/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":"0d616206-440f-497e-ab7d-23cdf090e119","owner":[],"postedDate":"August 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53573273,"name":"Biological sciences/Cell biology"},{"id":53573274,"name":"Biological sciences/Molecular biology"},{"id":53573275,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2025-09-09T10:53:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-26 14:20:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7180388","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7180388","identity":"rs-7180388","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}