Ursolic acid restores redox homeostasis and proinflammatory cytokines production in denervation-induced skeletal muscle atrophy

doi:10.21203/rs.3.rs-3415310/v1

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Ursolic acid restores redox homeostasis and proinflammatory cytokines production in denervation-induced skeletal muscle atrophy

https://doi.org/10.21203/rs.3.rs-3415310/v1

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published 03 Oct, 2024

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Skeletal muscle (SkM) atrophy results from metabolic disorders causing body and muscle mass loss, affecting morbidity and mortality. Increased oxidative stress, inflammation, and poor prognosis are the leading causes of involuntary weight loss. Ursolic acid (UA), known for its antioxidant and anti-inflammatory properties, can potentially reduce oxidative stress and inflammation in muscles, but its effects on muscle mass regulation are still unknown. Therefore, present study investigated the medicinal efficacy of UA and its mode of action against murine model of SkM atrophy over 7 days of UA supplementation. Denervation-induced SkM atrophy significantly impacts both overall body weight and the weight of individual muscles (p < 0.05). However, supplementation with UA can effectively counteract these effects by promoting the synthesis of the slow-myosin heavy chain, thereby restoring body weight and myotube diameter. Moreover, UA also plays a crucial role in reducing the production levels of reactive oxygen species (ROS), lipid peroxidation (LPO), and caspase-3-like activity in atrophied muscles. UA also, prevents the leakage of creatine kinase (CK) through the upregulation of superoxide dismutase (SOD) and glutathione peroxidase (GPx) expression. Furthermore, the results obtained from qRT-PCR demonstrated a significant decrease in the levels of proinflammatory markers, namely IL-1β, IL-6, TNF-α, and TWEAK, approximately four-fold after the third day of the UA intervention. UA also upregulated PGC-1α, Bcl2, and Akt expression for maintenance of redox homeostasis.

Denervation

Sciatic nerve

Signaling

Skeletal muscle atrophy

Ursolic acid

Skeletal muscle accounts for approximately 40–50% of the total body weight and is the largest organ responsible for regulating protein turnover. In general, skeletal muscles govern the musculature, locomotion, body posture, energy expenditure, and homeostasis of biomolecules. Consequently, the metabolic adaptations of skeletal muscles are intrinsic to the quality and functioning of the muscles, as well as the morbidity and mortality of an individual [1]. Skeletal muscle atrophy, a metabolic disorder, manifests through an elevation in muscle protein catabolism, which stimulates the release of reactive oxygen species and proinflammatory cytokines within the muscles [2]. The enhanced breakdown of proteins is a consequence of various clinical ailments, such as denervation, disuse, immobilization, aging, and cachexia [1]. These circumstances go beyond the deterioration of muscle tissue and lead to the degradation of myofibrillar proteins, particularly myosin heavy chain and actin. Denervation holds a significant role among these conditions, as it is responsible for a 90% decrease in muscle mass, primarily due to the disruption of the integrity of the neuromuscular junction (NMJ) and subsequent decline in protein synthesis [3].

It has been observed that there is a decrease in the number of motor neurons at the junction where nerves meet muscles, known as the neuromuscular junction (NMJ), due to nerve damage. This decrease in motor neurons leads to the commencement of the breakdown of muscle protein through the process of proteolytic degradation [4]. Specifically, after a period of 15 weeks without nerve stimulation, the soleus and tibial anterior muscles only retain 8% and 5% of their capacity to contract, respectively [3]. Alongside the reduction in muscle contraction, there is an increase in the degradation of T-tubulin and desmin, as well as a variation in the functioning of mitochondria [5]. Moreover, the reduced transmission of signals from neurons stimulates the initiation of the STAT-IL6 pathway in muscles, resulting in a significant 18-fold increase in MuRF-1 and an 11-fold increase in Atrogin-1 at the neuromuscular junction (NMJ) [6]. The current investigation's discoveries propose that increased calcium influx after denervation plays a vital role in activating calpains proteases and the proteasomal degradation of proteins via ubiquitin [7]. In contrast, a limited number of studies have also suggested that denervation enhances the phosphorylation of Akt/p70S6K, although the extent of phosphorylation remains unresolved [8, 9].

Currently, there exists a dearth of pharmaceuticals that are accessible to address skeletal muscle atrophy. Consequently, there is an urgent requirement to discover lead compounds that can transform into pharmaceuticals with minimal toxicity levels. UA, a lipophilic triterpenoid with a pentacyclic structure, has been derived from fruits and herbs and has found extensive utilization in traditional Chinese medicine. Recently, UA has been identified as a potent pharmaceutical agent in combatting the production of reactive oxygen species (ROS) and inflammation. An analysis that involved the utilization of microarrays containing human skeletal muscle has provided evidence that UA triggers hypertrophic responses in muscle during spinal cord injury and fasting. Moreover, it augments the strength of the grasp, expenditure of energy, and the presence of brown adipose tissue in the muscle, thereby heightening the synthesis of proteins through the paracrine and autocrine signaling of Akt/IGF-1 [1]. Consistent with these discoveries, UA also alleviates resistance to insulin, obesity, fatty liver disease, and other maladies in an individual. Furthermore, it promotes the regeneration of mitochondria through the signaling pathway of PGC-1α-SIRT1 [10], thus functioning as an additional pharmaceutical agent against a wide range of clinical afflictions.

Therefore, the current investigation aims to determine the consequences of UA supplementation in denervation-induced SMA in mice. In order to achieve this objective, it is hypothesized that damage to the sciatic nerve increases the accumulation of ROS and proinflammatory cytokines in the musculature, subsequently leading to an increase in the expression of proteolytic machinery. This machinery works in conjunction to initiate muscular impairment. The PI3K/Akt signaling pathway is the most significantly affected upstream regulator of protein anabolism. Consequently, the present study reveals how UA stimulates hypertrophy responses in skeletal muscles.

Chemicals and reagents: Saline, betadine, chloroform, mounting media DPX, poly-L-lysine, TRIZOL reagent, triton X-100, and isopentane were obtained from Sigma-Aldrich (St. Louis, USA). Isoflurane IP was purchased from troikaa pharmaceutical Ltd. Ethanol, acetonitrile, acetone, sodium orthovanadate (Na3VO4), sodium fluoride, EDTA, tris HCL, sodium hydroxide, glycine, glacial acetic acid, H2O2, potassium dichromate, sodium carbonate, nitroblue tetrazolium(NBT), hydroxylamine hydrochloride, SDS, n-butanol, pyridine, thiobarbituric acid, reduced GSH, sodium azide, TCA, eosin, hematoxylin, acid alcohol, diastase, periodic acid, Schiff's reagent, xylene, 5,5′dithiobis 2′-nitrobenzoic acid (DTNB), hexadecyltrimethy ammonium bromide (HTAB) was purchased from Merck, Life Sciences, Germany. Optimal cutting temperature embedding medium (OCT), and RNAlater solution were purchased from Thermo Scientific, USA. Polyclonal primary antibody (MyHC: cat no. WJ332704; Laminin-211: cat no. 702201; Fn14: cat no. bs2493R; Bax: cat no. VH313017; LAMP-2A: cat no. W1335835; c-MyHC: cat no WJ332704; Bcl2: cat no. bsm-33047M; MuRF-1: bs-2539R) and anti-mouse secondary antibody (cat no. 516102) were purchased from Genei lab Ltd. India. cDNA synthesis, Maxima SYBR green/ROX qPCR master mix was purchased from Genetix Biotech Asia Pvt. Ltd. Forward and reverse primers (IL-1β, IL-6, HO-1, PGC-α, TNFR1, TNFR2, TNF-α, TWEAK, TGF-β, Smad3, Smad4, myostatin, p38MAPK, Akt, etc) were obtained from Eurofins Pvt. Ltd India. The creatine kinase assay kit was obtained from ERBA Transasia Bio-medical Pvt. Ltd.

Ethical Clearance

Male Swiss albino mice, aged 5 weeks, weighing approximately 25–30 grams, were procured from Lala Lajpat Rai University of Veterinary and Animal Sciences-Hisar, Haryana, India. These mice were then placed in polypropylene cages and subjected to a 12-hour day and night cycle, with a room temperature of 22 ± 3 degrees Celsius, a noise level below 50 decibels, and a humidity level ranging from 50–60% for 9–10 days, to facilitate acclimatization. During this time, the mice had a standard diet and access to water ad libitum. All the experimental procedures were conducted following the guidelines set forth by the Committee for Control and Supervision of Experiments on Animals (CPCSEA), India, as well as the Institutional Animal Ethics Committee (IAEC), Maharshi Dayanand University, Rohtak, Haryana, with reference to letter no. 68–80, dated March 28, 2017.

Sciatic nerve resection

The sample size calculation for the skeletal muscle atrophy induced by sciatic transection was performed using G*Power (v. 3.1.9.4) with a significance level of p < 0.05 and a power of 0.8. For sciatic nerve resection, mice were first anesthetized with isoflurane and then provided with a continuous oxygen supply. Subsequently, the fur on the right leg was removed and a small incision was made behind the knee to expose the nerve. The sciatic nerve was elevated and severed using forceps, resulting in a 6-10mm nerve resection [11].

Grouping of mice and interventions

After the nerve resection, the mice were subjected to random allocation into three groups, namely the control group, the group receiving normal saline to treat sciatic damage, and the group treating sciatic damage with UA at a dose of 25mg/kg intraperitoneally daily [12]. In order to verify the initiation of muscle wasting, the body weight of the mice was regularly monitored throughout the duration of the treatment. After the treatment period, half of the animals were sacrificed on the third day following the injury, while the remaining half were sacrificed on day 7 post-injury to confirm the fluctuations in the levels of proinflammatory markers and the activation of the proteolytic system (Fig. 1). After the sacrifice, the tibial anterior (TA), gastrocnemius, and quadriceps muscles were excised, rapidly frozen in liquid nitrogen, and stored at -80 ^οC for subsequent analysis.

Assessment of morphological parameters and immunohistochemistry

The soleus muscle was fixed in 4% paraformaldehyde and embedded in blocks containing OCT in dry-ice or liquid nitrogen-cooled isopentane for morphological analysis and immunohistochemistry. Subsequently, the blocks underwent cryo-sectioning up to 6–8 µm using a cryostat. The resulting sections were then subjected to staining with hematoxylin & eosin (H&E), periodic-acid Schiff base (PAS) for the evaluation of damage caused by sciatic nerve injury, as well as the determination of glycogen content in the muscle [2]. Sections were subjected to immunofluorescence by being washed with 1X PBS and then incubated in a warm citrate buffer with a pH value of 5 for 30 minutes. Following this incubation, the sections were cooled for 15–20 minutes and washed with PBS thrice. Afterward, the sections were blocked using a 1% concentration of bovine serum albumin for 1 hour. Following this blocking step, the sections were incubated overnight at a temperature of 4^οC with a primary antibody targeting the heavy myosin chain. In order to visualize the primary antibody, the sections were washed three times with PBS for 5 minutes each and then incubated with a secondary antibody for 1 hour. It was followed by two additional washes with PBS and counterstaining with DAPI [13]. The serially stained sections were subsequently observed using a Nikon fluorescence microscope, and images were captured using NIS element 4.30 software (Nikon, US).

Assessment of muscle atrophy markers: Quadriceps muscles were homogenized in lysis buffer as described previously by Yadav et al., 2021. Proteins in aspirated supernatant were quantified using a Bradford assay kit. The measurement of creatine kinase activity was conducted using a commercially accessible kit, and the manufacturer's instructions were adhered to. The tissue homogenate was treated with a detection kit containing R1 reagent (Lot: S101905) and R2 reagent (Lot: S101905; ERBA Transasia Bio-medical Ltd.). Quantification of creatine kinase was accomplished utilizing standard curves.

Evaluation of oxidative stress-specific markers

The utilization of fluorescent protein-based redox indicators, such as DCFH-DA (dichlorodihydrofluorescein diacetate), served the purpose of identifying the entirety of reactive oxygen species (ROS) in muscle extractions. The process involved the observation of a consistent fluorescence signal at wavelengths of 485/535 nm as a result of the oxidation of dyes by ROS, as stated in the study conducted by[14]. Subsequently, the quantification of 4-hydroxynonenal (HNE) and malondialdehyde (MDA) adduct was assessed in both the treated and diseased cohorts, following the methodology outlined by Sharma et al. (2020).

The quantification of hydrogen peroxide production resulting from the presence of free radicals and the decrease of NBT was evaluated following the methodology described by Sharma et al. (2020) using SOD assay. The degree of infiltration by neutrophils was ascertained by quantifying myeloperoxidase (MPO) activity across various cohorts. The MPO activity was quantified using the established methodology outlined by [15].

Hydro-peroxide reduction in muscle homogenate was quantified as described by Yadav et al. (2021). Concisely, the muscle homogenate was incubated with a sodium azide buffer, reduced GSH, and phosphate buffer. Following this, H₂O₂ was added to the mixture and left undisturbed for 10 minutes at 37 ^οC. Subsequently, a 10% trichloroacetic acid solution was added to the mixture and centrifuged for 10 minutes. The resulting supernatant was then subjected to the addition of DTNB and quantified at a wavelength of 412 nm.

Calpain zymography

The gastrocnemius muscles were first subjected to pre-incubation in lysis buffer, followed by a 15-minute homogenization period. Subsequently, the resulting homogenate was again incubated at 4 ^οC and centrifuged at 12,000 rpm for 20 minutes, yielding the supernatant for further analysis. Quantification of protein content was carried out using the Bradford assay kit. The quantified samples were then subjected to electrophoretic separation on a casein gel at 80-90V for 3 hours. The resulting bands were visualized and subjected to analysis using the ImageJ software on white light transilluminators [2].

Caspase-3 activity: The activity of caspase-3 was assessed through a fluorescent assay employing a fluorogenic substrate known as DEVD-AFC (AFC: 7-amino-4- trifluoromethylcoumarin). The DEVD-AFC substrate emits light in the blue spectrum (λmax = 400 nm), and upon being cleaved by caspase 3, the liberated AFC emits fluorescence in the yellow-green range (λmax = 505 nm). This fluorescence was subsequently quantified using either a fluorometer or a fluorescence microtiter plate reader. In order to determine the extent of caspase-3-like activity, a comparison was made between the fluorescence of AFC obtained from a test sample and that of a control sample, allowing for the calculation of the fold increase [16].

RT-PCR

The quantitative real-time polymerase chain reaction (qRT-PCR) was executed utilizing RNA derived from the gastrocnemius muscles employing the TRIZOL technique. The obtained mRNA was reversely transcribed into cDNA using a cDNA synthesis kit, and qRT-PCR was performed using Maxima SYBR Green/ROX qPCR master mix following the manufacturer's instructions [2]. The expression of muscle-specific markers i.e. Atrogin-1, TWEAK, IL-6, TNF-α, TNFR1/2, FoxO1/3, TGF-β, Smad3/4, p-Akt, AMPK, BCL-2, HO-1, IL-1β, mTOR, myostatin, p38MAPK, and PGC-1α were assessed using forward and reverse primers as mentioned in Table S1.

Immunoblotting: The Gastrocnemius muscle was homogenized in lysis buffer, following the method of Yadav et al. (2021). The supernatant was quantified using a Bradford assay kit. Protein (10–15 µg) was mixed with 5X Lamilli buffer and subjected to SDS-PAGE. The bands were transferred onto a nitrocellulose membrane using wet transfer from GE Healthcare Limited. Transferred bands on the nitrocellulose membrane were made visible by Ponceus-S staining. The membrane was then destained using 1X TBST buffer until clear, followed by blocking with 3% skim milk powder dissolved in 1X TBST for 1 hour. After blocking, the membrane was washed 3 times for 5 minutes with 1X TBST buffer and then incubated with primary monoclonal antibody solution (dilution 1:500) for proteins such as MuRF-1, Atrogin, MyHC, Laminin, LAMP2, BCL2, Bax, LC3B, and p-Aktser473. Following primary incubation, the membrane was washed 3 times for 5 minutes with 1X TBST buffer to remove unbound primary antibody. Finally, the membrane was incubated with an anti-rabbit secondary antibody solution (dilution 1:1000) for 35 minutes with gentle shaking. Again, the membrane was subjected to a washing process using 1X TBST three times, each time for five minutes, to eliminate any secondary antibodies that were not bound to the membrane. Following this, the signals were induced by employing a chemiluminescence ECL marker. The resultant activated bands were then observed utilizing the chemiluminescent MPP system provided by Bio-Rad Pvt. Ltd. Finally; the acquired data was analyzed employing the image J software [2].

Statistical analysis

Power analysis conducted using G*Power determined that a group of 18 male Swiss albino mice would yield an 80% statistical power with an alpha level of 0.05 and a beta level of 0.2, thereby evaluating treatment significance. Data obtained from the experiment were expressed as means ± standard deviation (SD) and subjected to analysis using GraphPad prism via one-way ANOVA (p ≤ 0.05).

UA ameliorates the body and muscle weight in denervated animals

Decreased body weight, individual muscle weight, and atrophy are preliminary parameters that determine the onset of denervation-induced SkM atrophy. UA supplementation for 3 and 7 days prevented the loss of body and individual muscle weight in mice. A significant reduction in mass loss has been observed in the whole body weight of mice after UA intervention to 33.43 ± 1.10 gm on day 3 and 37.06 ± 1.11 gm on day 7 compared to 30.83 ± 1.87; 29.5 ± 0.616 gm of the denervated group, respectively (Fig. 2A). Decreased body weight and enhanced rate of atrophy directly affect the soleus, TA, gastrocnemius, and quadriceps muscle weight.

Till day 3, UA treatment prevented the weight loss of the soleus muscle after sciatic nerve resection to some extent, 0.01066 ± 0.00075 gm compared to 0.0086 ± 0.00424 gm of the denervated group, while on day 7, the weight of the soleus muscle increased significantly to 0.01176 ± 0.000321 gm (Fig. 3A). In contrast, the weight of the TA was observed to increase significantly on the 3rd day to 0.008967 ± 0.00651 gm, which gradually increased to 0.01109 ± 0.000201 gm on the 7th day compared to 0.00716 ± 0.000351 gm of denervated and 0.01196 ± 0.000757 gm in healthy animals (Fig. 3B). The TA muscle exhibits a superior degree of motor control, while the soleus muscle demonstrates a high level of reactivity. The TA muscle predominantly consists of fast-twitch muscle fibers, whereas the soleus muscle primarily consists of slow-twitch muscle fibers. The UA-treated mice were unable to improve the gastrocnemius weight on the 3rd day and were 0.0417 ± 0.00175 gm with minor improvement on the 7th day to 0.0537 ± 0.00434 gm compared to the sciatic injured group of 0.0364 ± 0.00011 gm (Fig. 3C). The close homology of gastrocnemius and soleus might be due to their proximity. The weight of quadriceps muscles was found to be decreased to 0.1175 ± 0.00358 gm in the denervated group, while UA significantly prevented/improved the weight on day 3 to 0.169 ± 0.0122 gm, and 0.2164 ± 0.0118 gm on day 7 of treatment, similar to 0.2672 ± 0.0124 gm in healthy control (Fig. 3D).

Overall, treatment with UA did not significantly modify the muscle atrophy rate on day 3 or day 7. The atrophy rates were measured at 0.009 ± 0.0005 and 0.007167 ± 0.001, respectively. In comparison, the denervation control group exhibited a rate of 0.012133 ± 0.0010, while the healthy control group demonstrated a rate of 0.00366 ± 0.000471 (Fig. 2B).

UA restored the damaged muscle fiber and MyHC synthesis

Damage to muscle fibers is the hallmark of the onset of SkM atrophy, characterized by diminished nuclei, increased vacuolization, intracellular space between the fiber, and loss of myofibril protein. The observations revealed that the denervation group demonstrated increased muscle fiber degeneration, resulting in an augmented cross-sectional area, cytoplasmic content breakdown, and necrotic lesions (Fig. 4A). Conversely, on day 7, UA effectively restored fiber regeneration, reduced intracellular space, and increased fiber integrity compared to day 3. Similarly, UA prevented glycogen depletion in the muscle fibers and maintained their structural integrity even after three days. Furthermore, this improvement continued to progress on day 7. The degeneration of muscle fibers is directly associated with the breakdown of myofibril protein, particularly slow-MyHC. The study's findings indicate that removing the sciatic nerve reduced the concentration of slow-MyHC significantly in the muscle fibers compared to a control group of healthy individuals. However, UA successfully restored the levels of slow-MyHC in the muscle fibers, as depicted in Fig. 4D.

UA attenuated the oxidative burden in the sciatic nerve resected group

Leakage of creatine kinase from the muscle is the primary indicator of muscle damage. The present investigation unveiled that the levels of creatine kinase activity were notably reinstated to 17.43 ± 1.10 U/L on the seventh day of UA treatment when compared to 10.67 ± 0.49 U/L in sciatic nerve resected animals (Fig. 5A).

The UA intervention demonstrated a gradual and remarkable reduction in the build-up of ROS levels to 1225.882 ± 44.81 and 933.93 ± 59.49 RFU/mg/protein on days 3 and 7, respectively, as compared to 1588.616 ± 78.10 RFU/mg/protein in the mice that underwent denervation (Fig. 5B). The intervention of the UA resulted in a decrease in the levels of ROS contents, further substantiated by the observed reduction in lipid peroxidation (LPO) activity. The MDA or 4-HNE contents in myofibers decreased to 31.16 ± 1.05 nmol MDA/mg protein on day 3, which further decreased to 23.9 ± 1.65 nmol MDA/mg protein on day 7 after supplementation of UA compared to 25.14 ± 1.94 and 47.16 ± 2.02 nmol MDA/mg protein in sham control and denervated groups, respectively (Fig. 4C).

Similarly, increased inflammation and oxidative burden were measured by leakage of MPO from muscles. The MPO levels were significantly reduced in denervated mice to 10.16 ± 0.95 nmol/min/mg of protein compared to 37.9 ± 2.46 nmol/min/mg of protein healthy control. UA intervention increased the MPO activity to 16.03 ± 2.91 and 22.01 ± 1.58 nmol/min/mg of protein on days 3 and 7 (Fig. 5D).

SOD and GPx triggers the conversion of hydrogen peroxide into water molecules. The intervention of UA exhibited a significant augmentation in the activities of SOD and GPx, resulting in a measurement of 1.63 ± 0.15 U/mg protein and 2.2066 ± 0.10 glutathione consumed/min/mg of protein on the third day (Fig. 5E, F). Subsequently, these activities experienced gradual augmentation, reaching 1.89 ± 0.09 U/mg protein and 2.5 ± 0.10 glutathione consumed/min/mg of protein by day 7. In comparison, the denervated mice displayed activities of 1.2 ± 0.10 U/mg protein and 1.266 ± 0.20 glutathione consumed/min/mg of protein (Fig. 5F). Therefore, the result showed that UA significantly inhibited ROS generation in muscles in a time-dependent manner.

The augmented oxidative load is crucial in activating the proteolytic system and initiating apoptosis. The increased oxidation burden stimulates the proteolytic system and commences programmed cell death. It was confirmed through the observation of a significantly increased level of caspase-3 activity, measuring 8986.767 ± 618.41 AFU/mg of protein, in the skeletal muscle following injury to the sciatic nerve, in comparison to the healthy control, measuring 4135.7 ± 556.94 AFU/mg protein (Fig. 5G). Likewise, substantiating the correlation between different pathways, the administration of UA gradually diminished the activity of caspase-3 in the skeletal muscle, resulting in a reduction of apoptosis to levels of 6406.76 ± 205.93 and 5369.6 ± 151.43 AFU/mg protein on day 3 and 7, respectively.

UA intervention has a differential effect on calpain activity

Previous studies reported that calcium-dependent calpains promote the degradation of myofibrils by cleaving various proteins such as nebulin, desmin, filamin, and troponin. Increased oxidative burden in muscle directly affects calcium homeostasis, thus leading to activation of calpain [17]. The activities of µ-calpain and m-calpain witnessed a four-fold increase on the seventh day after resectioning the sciatic nerve. However, this increase was significantly mitigated by the intervention of UA. In contrast, UA intervention exhibited a twofold augmentation in calpain-3 activity compared to the healthy control and a 1.5-fold increase compared to the denervation control on the seventh day in gastrocnemius muscles (Fig. 6A, B).

Effect of UA intervention on SkM atrophy-related genes

The excision of the sciatic nerve increased the expression of heme oxygenase-1 (HO-1) concentrations by a factor of 2.5, which subsequently diminished to 2-fold on the seventh day as a means of mitigating the oxidative load. The intervention of UA demonstrated ineffectiveness in reducing the expression of HO-1 on day 3. However, on day 7, it exhibited a noteworthy reduction in HO-1 expression, with statistical significance (p < 0.05) and a 2.0-fold decrease (Fig. 7A). Oxidative stress increased the expression of PGC-1α to augment mitochondria biogenesis and antioxidant mechanisms to ameliorate the deleterious impact of free radicals. Hence, the levels of PGC-1α expression exhibited a notable increase of nearly 2.5-fold by day 3 and persisted till day 7 to cope with the oxidative stress generated after denervation. The administration of UA effectively restored the expression of PGC-1α in a gradual and highly significant manner by day 7 (Fig. 7B). Similarly, the oxidative stress induced by denervation also led to a significant increase in the expression of p38MAPK by nearly threefold on the third day, which subsequently decreased to twofold on the seventh day compared to the control group of healthy animals (Fig. 7C). The UA administration gradually restored the elevated p38MAPK levels to a level comparable to that of healthy controls by the seventh day. The activity of p38 MAPK is also essential for the TNF-α gene transcription through TNFR1 signaling.

A significant increase was observed in the expression of the cytokine TNF-α by a factor of seven on the third day following sciatic nerve resection in the skeletal muscle. This elevation subsequently decreased to 4 fold on the seventh day. UA supplementation had no impact on the levels of TNF-α on the third day, whereas a slight reduction was observed on the seventh day (Fig. 7I). The levels of TNFR1 and TNFR2 transcripts, which act as receptors for TNF-α, were observed to experience an approximately 3-fold increase on day three, followed by a reduction to approximately 2-fold on day seven in the skeletal muscles of denervated mice (Fig. 7G, H, I). UA interventions averted the escalation in the expression levels of TNFR1 and TNFR2 on day 3 and maintained it at a comparable level to that of the healthy animals. However, on day 7, UA caused a reduction in these levels below those observed in the healthy animals. Oxidative stress and inflammation are intricately linked phenomena, wherein both phenomena profoundly impact and intensify one another. Therefore, denervation increased myostatin, TWEAK, IL-1β, and IL-6 expression by approximately three times on the third day, which subsequently declined to twice the amount on the seventh day compared to the healthy control group. The administration of UA gradually restored levels of TWEAK, IL-1β, IL-6, and myostatin to a degree comparable to that of the healthy controls by the seventh day (Fig. 7D, E, F, M). The fluctuations in expression levels of myostatin exhibited a similar pattern to those of other proinflammatory cytokines; however, quantitatively, myostatin expression was significantly low.

Chronic oxidative stress and inflammation are implicated in the development of insulin resistance and impede the activation of Akt, perturb glucose metabolism, and induce an alternative signaling pathway via AMPK to uphold energy homeostasis by augmenting catabolism. Denervation increased the expression of Akt to a certain degree, with a fold change of 1.3 on day 3. However, this fold change decreased by 1.4 folds on day 7. On the other hand, supplementation with UA maintained Akt transcription levels similar to those observed in healthy controls (Fig. 8A). Alternatively, the AMPK transcripts exhibited an increase of more than 2.0 folds on both day 3 and day 7 (Fig. 8C). The UA intervention proved ineffective until day 3; however, it maintained slightly higher levels of AMPK on day 7 (RQ = 0.55 and 0.75) than those observed in animals in a healthy state. The mTOR is a downstream target of Akt as well as of AMPK and is involved in the regulation of autophagy and proteolysis. Surprisingly, denervation reduced the mTOR transcript levels by almost 3.0 fold on day 3, which decreased slightly more till day 7 (Fig. 8B). UA intervention restored the expression levels of mTOR by ~ 1.5 fold compared to denervated mice, although it remained significantly low, almost by 1.75 fold on day 7 compared to the healthy control group of mice.

Further, FoxO1 and FoxO3 are also the downstream targets of Akt and AMPK. Akt hinders while AMPK stimulates the FoxO. Therefore, it was observed that the resection of the sciatic nerve resulted in an elevation of FoxO1 and FoxO3 gene expression by a factor of 4 and 16, respectively, on the third day (Fig. 7N, O). These levels experienced a minor decline but remained elevated by a ratio of 3 and 10 in denervated mice. UA intervention resulted in the normalization of Foxo1 levels, yet the levels of FoxO3 remained significantly elevated by approximately six-fold on day 7, which can be correlated with slightly high AMPK levels in denervated mice compared to healthy mice. FoxO3 regulates the transcription of apoptosis, autophagy, and proteolysis-related genes. Hence, there was an observed augmentation of Atrogin-1 transcript quantities by nearly 3.0-fold after the denervation process on the third day, which persistently stayed at 2.0-fold on the seventh day, exhibiting a notable elevation. However, UA intervention normalized the Atrogin-1 levels equal to the healthy animals despite high levels of FoxO3. Bcl-2 is a perceivable indicator of the inhibition of programmed cell death in muscle tissue. The resection of the sciatic nerve resulted in a reduction in the amount of Bcl-2 transcripts by a factor of 7 fold immediately, and this reduction persisted even on day 7. On the other hand, the UA intervention increased the level of Bcl-2 by a factor of ~ 4 fold compared to mice that had undergone denervation but remained two-fold lower compared to animals that were in a healthy state (Fig. 8D).

Moreover, to investigate the fiber switching in atrophied muscles, the expression of ERK1/2 was monitored. The current investigation determined that the levels of ERK1/2 decreased by approximately three times following sciatic damage on the third and seventh days (Fig. 8E).

The TGF-β pathway operates via the cooperation of Smad3 and Smad4 and can either facilitate or impede the process of cellular differentiation, as well as the revival of tissues. The excision of the sciatic nerve resulted in a three-fold elevation in the levels of TGF-β until day 3, which returned to normal by day 7 (Fig. 7J). A similar pattern was observed in the groups that underwent UA intervention. Hence, denervation doesn't seem to affect the TGF-β signaling pathway. However, the levels of Smad3 exhibited an increase ranging from a two-fold to a five-fold increment between day 3 and day 7 (Fig. 8K). Subsequently, following the intervention of UA, these levels displayed a reduction equivalent to that of healthy animals on day 3. However, it is noteworthy that on day 7, the levels of Smad3 were still considerably higher than those of healthy animals, albeit by approximately a two-fold margin, and were also two-fold lower than the levels observed in denervated animals. However, the Smad4 transcripts exhibited a notable increase of 12.5 times on day 3 and maintained a high level of 8.7 times on day 7 in denervated animals compared to the healthy control group. The utilization of UA intervention effectively attenuated the levels of Smad3 transcripts on day 3, although there was a subsequent increase of 2.5 times on day 7 compared to the healthy control group (Fig. 8L).

Effect of UA intervention on protein synthesis

Western blot analysis was conducted to investigate the impact of UA intervention on the expression of the muscle-specific protein in the denervated gastrocnemius muscle. The resection of the sciatic nerve resulted in a significant decrease in the levels of phosphorylated Akt by nearly a factor of six on the third day post-surgery. However, these levels experienced an increase of approximately 4.5-fold by the 7th day. It is worth noting that the intervention of UA did not result in an increase in Akt phosphorylation on the third day, but it did exhibit a significant increase by a factor of almost five on the seventh day (Fig. 9A). The deactivation of Akt leads to the activation of FoxO and NF-ĸB, which subsequently triggers proteolysis primarily by upregulating the expression of Atrogin-1 and MuRF-1. The levels of Atrogin-1 exhibited a nearly threefold increase on day 3, which further escalated to almost sixfold on day 7 following denervation (Fig. 9B). An insignificant decline in the levels of Atrogin-1 was observed after UA intervention on both day 3 and day 7. Similarly, there was an observed elevation in the levels of MuRF-1, with a 1.5-fold increase on day 3, which subsequently reached a 2.5-fold increase on day 7 of denervation in mice compared to the healthy control group (Fig. 9C). UA intervention resulted in a reduction in the levels of MuRF-1 in the denervated models, bringing them close to the levels observed in the healthy control group on both day 3 and day 7.

Moreover, the levels of the proinflammatory cytokine, TWEAK receptor Fn14, exhibited a two-fold decrease on day 3, followed by an increase of two-fold on day 7, in the sciatic nerve resected animals compared to the healthy control group. Intervention utilizing UA exhibited a significant reduction in the levels of Fn14, almost akin to the observed decrease in denervated animals after three days (Fig. 9D). Strikingly, UA effectively reinstated the levels of Fn14 to a state of normalcy, resembling those observed in healthy animals. Therefore, elevated levels of proinflammatory cytokines also increase the expression of genes associated with apoptosis, autophagy, and proteolysis. Sciatic nerve resection increased the levels of LC-3B, an autophagy marker, by fold on day 3 and decreased to less than healthy controls on day 7 (Fig. 10A). The intervention of UA effectively sustained the levels of LC-3B close to those of the healthy control on day 3. Additionally, there was an observed increase in these levels to a certain degree on day 7 compared to the healthy control. Another autophagy marker, Lamp 2A, exhibited a statistically insignificant increase on the third day, succeeded by a twofold reduction on the 7th -day post-denervation, which was consistently upheld close to the healthy control group after intervention with UA (Fig. 10B).

Furthermore, there was a notable reduction of approximately fourfold in the levels of the anti-apoptotic protein Bcl2 in the denervated mice on day 3, which persisted at a consistent and low level on day 7. The UA administration increased the levels of Bcl2 by approximately a factor of 3 compared to denervated animals on both day 3 and day 7 (Fig. 10C). In addition, the pro-apoptotic protein Bax exhibited an approximately 1.5-fold increase, followed by a subsequent 1.5-fold decrease, in denervated animals on day 3 and day 7, respectively, compared to healthy control. The intervention of UA did not yield any observable impact on the expression of Bax on day 3. However, the expression of Bax increased to a level almost comparable to that of the healthy control on day 7 (Fig. 10D). Quantitatively, the Bcl-2 to Bax ratio was normalized by the intervention of UA to maintain the apoptosis in skeletal muscle.

The ultimate consequence of these events is manifested in anabolism or catabolism, thereby impacting the structural proteins of skeletal muscle. The levels of MyHC and laminin-211 exhibited a notable decrease on day 3 in denervated animals compared to the control group (Fig. 11A, B). However, on day 7, these levels experienced a slight increase. The introduction of UA intervention displayed some modest effects in elevating the levels of MyHC and laminin-211 on day 3. Nevertheless, on day 7, UA significantly increased the levels of both proteins, reaching a comparable level as the healthy controls. In conclusion, the data suggests that UA effectively stimulated the protein synthesis machinery on the 7th day, surpassing its impact on the 3rd day.

Skeletal muscle wasting after dissection of the sciatic nerve is distinguishable from cachexia, sarcopenia, and other conditions, owing to disparate initiating events [18]. Following the transection of the sciatic nerve, fibrillation potentials are engendered, thereby leading to membrane depolarization. It, in turn, increased cytosolic concentrations of Na⁺ and Ca²⁺ due to enhanced membrane permeability. The excess Ca²⁺ within the cytoplasm relinquishes its physiological transitory state and molds numerous cellular signaling pathways, enzymes, and biological processes [19].

Elevated levels of calcium in the cytoplasm initiate the activation of the mitochondrial permeability transition pore (mPTP) and the mitochondrial calcium uniporter (MCU) complex, which are dependent on calcium and cyclophilin D. These processes result in the depolarization of the mitochondrial membrane and an increase in the levels of calcium in the mitochondrial matrix. The increased calcium levels within the mitochondria can lead to increased production of reactive oxygen species (ROS), primarily due to electron leakage from the respiratory complexes within the electron transport chain (ETC) [20]. In the present investigation, the overall levels of ROS and LPO experienced an increase on the third day and accentuated further on the seventh day after denervation (Fig. 5B, C). Similarly, MPO, SOD, and GPx activities declined on the third and seventh days (Fig. 5D, E, F). The observations consistently indicate that the initiation of the antioxidant response occurred on the third day and became more pronounced as time progressed after denervation.

The administration of ursolic acid increased muscular mass and volume by suppressing ROS and skeletal muscle gene expression associated with atrophy, specifically by reducing Atrogin-1 and MuRF-1 expression. It can be interpreted through the observations that ursolic acid amplified the IGF-I-induced suppression of FoxO and NF-ĸB transcription factors. Past investigations have demonstrated that elevated expression of Akt activity in skeletal muscle can impede atrophy and foster hypertrophy after sciatic nerve resection [21]. In the current study, the induction of Akt activation through an increase in Akt phosphorylation by UA indicated a crucial intersection in the signaling cascades of insulin and IGF-I. The augmentation in Akt activity in the skeletal muscle has the potential to explain a multitude of UA's effects. These effects include diminished gene expression associated with atrophy, decreased muscle atrophy, augmented muscle hypertrophy, and diminished adiposity [22, 23]. According to previous studies, UA treatment increased Akt phosphorylation [24], but in this study, UA was found to increase Akt phosphorylation on the 3rd and 7th day following treatment (Fig. 9A), not at the outset of treatment. Since UA inhibits protein tyrosine phosphatase 1B (PTP1B), Akt phosphorylation may be related to this inhibition [26]. Akt is phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK-1) and mTORC2 at Thr³⁰⁸ and Ser⁴⁷³ sites, respectively. It was found that Akt was phosphorylated at the Thr³⁰⁸ site and not on the Ser⁴⁷³ site. Hence, the inhibition of PTP1B increases the phosphorylation at the Thr³⁰⁸ site through PI3K/PDK-1 [27, 28].

Protein tyrosine phosphatase (PTP)s dephosphorylates tyrosyl-phosphorylated proteins and regulates insulin receptor signaling [28]. In the current investigation, insulin and IGF-1 levels were not quantified; nevertheless, the suppression of protein tyrosine phosphatase (PTP)1B activity, a counteractive modulator of insulin signaling, was observed. This possibility is supported by previous experiments showing that ursolic acid inhibits PTP1B, a tyrosine phosphatase that inactivates the IGF-I and insulin receptors. However, knockout mice lacking PTP1B did not have increased muscle mass. On the other hand, mice devoid of PTP1B displayed increased responsiveness to insulin due to the elevated phosphorylation of the insulin receptor in muscle [26]. Hence, upregulated Akt signaling may be attributed to the simultaneous inhibition of PTP1B and closely related tyrosine phosphatase TCPTP in vivo by ursolic acid. However, this hypothesis needs to be verified through further studies. Therefore, the activation of Akt and ERK is induced by UA (Fig. 8A, E), leading to the alteration of gene expression in skeletal muscle. This alteration of gene expression reduced the rate of atrophy and promoted the development of hypertrophy.

The current study demonstrates elevated levels of mTORC1 after UA intervention, a crucial cellular regulator that Akt indirectly activated by inhibiting tuberous sclerosis complex 1/2 (TSC1/2) [29]. Activation of mTORC1 subsequently leads to the upregulation of mitochondrial biogenesis mediated by the regulation of PGC-1α [30]. Additionally, the elevation of calcium levels in denervated myofibers triggers the activation of calcium/calmodulin-dependent protein kinase kinase β (CAMKKβ), which phosphorylates AMPK at Thr172, leading to its activation. AMPK is recognized for its ability to enhance the manifestation of PGC-1α, which plays a crucial role in regulating the expression of the slow isoform of MyHC [31]. In denervated mice, all three factors were observed to be elevated, yet the intervention of UA effectively counteracted the levels of expression of these factors.

Conversely, it has been reported that the activation of AMPK by AICAR pretreatment does not result in any alterations in PGC-1α expression [32]. However, our current investigation found that the levels of PGC-1α were upregulated in mice with denervated muscles. Furthermore, PGC-1α has been reported to be associated with increased levels of slow MyHC [33]. In contrast, Western blot analysis revealed that UA significantly increased the abundance of slow-MyHC (Fig. 11A) and laminin-211 (Fig. 11B) proteins while concomitantly decreasing the abundance of fast-MyHC protein and PGC-1α and AMPK levels. Thus, it appears that the impact on the expression of the slow isoform of MyHC is primarily mediated through the trafficking of HDAC4 without the involvement of PGC-1α [34].

Further, AMPK-led increased expression of autophagy-related genes, i.e., LC3B, Lamp2A, Beclin1, Atg5, Atg12, Gabarapl1, and caspase-3, have been reported in the denervated skeletal muscle [35]. In the present study, levels of LC-3B and Lamp2A were found to be increased in the muscles of denervated rats on day 3 (Fig. 10A, B). However, levels of both autophagic proteins reduced dramatically on the 7th day of denervation. UA somewhat decreased the levels of LC-3B and Lamp2A but was insignificant. Hence, more extended studies are required to reach any conclusion. All these autophagy-related proteins were found to be decreased after UA intervention and linked with inhibition of AMPK and activation of Akt. The inhibition of PTP1B in neurons was reported to decrease AMPK activation in peripheral tissues and led to changes in gene expression that promote leanness and increase energy expenditure [36]. The same findings were reported by Zang et al. (2022), who found that knocking down PTP1B can prevent inflammation caused by ox-LDL through the AMPK/SIRT1 signaling pathway [37]. Furthermore, it has been observed that brown adipose tissue and brown adipocytes lacking PTP1B exhibit increased AMPK activity. PTP1B was also found to be involved in cardiac hypertrophy and contractile dysfunction induced by a high-fat diet through the regulation of autophagy-dependent on AMPK [38].

Moreover, the administration of UA had no impact on the manifestation of TGF-β receptors, thus indirectly impeding the Smad2/3 pathway by restraining the inhibitory effect of Akt on Smad3. Markers of TGF-β signaling, specifically ERK1/2, exhibited a significant increase in atrophy compared to the control group. Denervation resulted in the downregulation of the non-canonical TGF-β signaling marker ERK1/2 was mitigated after the administration of UA (Fig. 8E). Also, along with Akt, UA inhibited MAP kinase pathways and NF-κB-mediated transcription. However, Akt also controls the activity of IKK, an activator of NF-κB through mTOR as well as AMPK via control of cellular ATP levels [39].

Consequently, all of the events that occurred in chronological order after the removal of the sciatic nerve were reversed due to the intervention of UA, specifically, the disturbance in the calcium levels succeeded by increased production of ROS and subsequently followed by an escalation in inflammatory response, programmed cell death, cellular self-digestion, and breakdown of proteins. It was demonstrated by increased MyHC (Fig. 11A) and laminin-211 (Fig. 11B) protein levels and mitochondrial biogenesis after UA administration. These observations were further validated by the improvement in myofibers, fiber degeneration, intracellular space, the content of slow-MyHC, and glycogen content in the soleus muscle of animals with resected sciatic nerve after UA administration (Fig. 4). Hence, the effect of denervation was counteracted by UA through the activation of Akt or the reduction of ROS levels via the inhibition of downstream signaling of FoxO, TNF-α, Bcl-2, LC-3B, Lamp2A, Smad2/Smad3, and proinflammatory cytokines. Moreover, the inhibition of AMPK-related downstream signaling, which serves as an alternative signaling pathway to Akt, appears to be associated with the inhibition of PTP1B or the non-specific inhibition of phosphatases.

Nowadays, SMA is a major complication that arises due to various clinical ailments including diabetes, denervation, cachexia, sepsis, etc. Therefore, the present study evaluates the potential of UA in denervation-induced SMA. The short-term UA intervention on days 3 and 7 on skeletal muscle atrophy reverted the body and muscle weight and atrophy rate. After the denervation, increased influx of Ca²⁺, mPTP opening, and ROS accumulation consequences the increased expression of AMPK, p38MAPK, FoxO1, FoxO3a IL-6, and TGF-1β within the muscle, with declined Akt and mTOR activity. The decrease in Akt1 activity was validated by the downregulation of anti-apoptotic Bcl-2 and the upregulation of pro-apoptotic Bax in denervated mice. The decline in Akt activity is further correlated with enhanced expression of Smad3, Atrogin-1, and MuRF-1 in muscle and their upstream regulators AMPK, FoxOs, and NF-ĸB. However, the study also observed that increased AMPK level does not directly control the expression of PGC-1α in mitochondria. Overall, UA intervention led to the suppression of PTP1B and Akt phosphorylation, and Bcl2 expression leading to subsequent inhibition of apoptosis, autophagy, and proteasome activity. Hence, identification of UA receptor(s) and pharmacokinetic studies are crucial for future research to understand metabolic control mechanisms and in vivo effects. Ursolic acid could be explored as a potential therapy for skeletal muscle atrophy due to the lack of current treatments either alone or in combination with other strategies. Further investigation on the derivatives of ursolic acid has the potential to yield novel medical treatments for prevalent metabolic disorders of skeletal muscle.

Acknowledgments

We acknowledge the Indian Council of Medical Research (ICMR), New Delhi, which provided financial support to carry out this work through an extra-mural research project [Ref. no: 52/09/2020-BIO/BMS], and Senior Research Fellowship to Ms. Aarti Yadav (Ref. No 45/12/2020-/BIO/BMS).

Financial & competing interest disclosure

The corresponding author has been financially supported by ICMR-Project, New Delhi.

Authors Information

Authors and Affiliations

Clinical Research Laboratory, Department of Biochemistry, MDU, Rohtak, Haryana, India-124001

Aarti Yadav and Rajesh Dabur

Author contributions

Rajesh Dabur has suggested the idea and conception of this research article. Aarti Yadav carried out the experimental work. Rajesh Dabur interpreted the results and edited the original draft.

Corresponding author: Rajesh Dabur

Ethical Declaration

Ethical approval

All the experimental procedures were conducted following the guidelines set forth by the Committee for Control and Supervision of Experiments on Animals (CPCSEA), India, as well as the Institutional Animal Ethics Committee (IAEC), Maharshi Dayanand University, Rohtak, Haryana, with reference to letter no. 68-80, dated March 28, 2017.

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Ursolic acid restores redox homeostasis and proinflammatory cytokines production in denervation-induced skeletal muscle atrophy

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