Experimental Study on Muscone Reducing NF-κB Pathway Activity by Targeting IL-1R1 to Attenuate Oxidative Stress Injury in Schwann Cells

Summary

This paper studies the effect of muscone, the main component in musk, on the proliferation and apoptosis of Schwann cells. The results preliminarily illustrate the potential mechanism of musk reducing NF-κB pathway activity by inhibiting IL-1R1 to attenuate oxidative stress injury in Bell's palsy.

Abstract

The underlying mechanism of how natural musk treats Bell's palsy is not clear. This study aims to reveal the potential mechanism of musk in treating peripheral nerve injury in vitro. To achieve this, an oxidative stress injury model was established in Schwann cells (SCs) using cobalt chloride (CoCl₂). To assess the protective effects of Muscone (Mus) on this injury and explore its mechanism, cell viability assays, siRNA transfection, enzyme-linked immunosorbent assays (ELISA), quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, immunofluorescence staining, and ultrastructural analysis of cells were employed. As a result, it is indicated that Mus intervention enhanced SC proliferation and S-100b mRNA under hypoxia, while also reducing reactive oxygen species (ROS) and elevating superoxide dismutase (SOD) levels. Mus ameliorated oxidative stress and inflammation in hypoxic SCs by reducing IL-1β and TNF-α. Furthermore, Mus modulated key indicators involved in oxygen metabolism, cell repair, and neurometabolism, including C-JUN and GDNF. IL-1R1, localized to the SC membrane, was upregulated by hypoxia but significantly downregulated following Mus intervention. Mus also inhibited the expression of NF-κB pathway components (IRAK1, IKK, p65, p50) and increased IκB-α. Critically, abolishing IL-1R1 expression via siRNA knockdown showed a consistent effect with Mus. It is concluded that by suppressing IL-1R1, Mus attenuated inflammation in hypoxic SCs through inhibition of the NF-κB pathway. This study indicated IL-1R1's role in hypoxia-induced SC dysfunction. Furthermore, Mus might be a potentially effective medicinal for treating Bell's palsy.

Introduction

Muscone (Mus), 3-methylcyclopentadecanone, was isolated from the natural extract of deer musk as early as 1906¹. Musk, a valuable and effective traditional Chinese medicine (TCM), has extensive curative effects such as detumescence, anti-inflammation, resuscitation, and blood circulation-promotion². Musk-containing TCM has been utilized for a variety of common diseases, including central nervous system disease³, detoxification⁴, gastrointestinal tumors⁵, viral hepatitis⁶, and coronary heart disease⁷, etc. As the active pharmaceutical ingredient of Musk, Mus has shown excellent biological effects involving anti-inflammation⁸, anti-oxidation^8,9, neuronal damage repair, and myocardial infarction-relieving¹⁰, etc. Mechanistically, studies have exhibited that Mus can reduce the release of inflammation cytokines such as Interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, reactive oxygen species (ROS)^1,8, and regulate the mitogen-activated protein kinase (MAPK), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), AKT/mechanistic target of rapamycin (mTOR), and CaN/Erk1/2 pathway^1,8,11, etc.

In China, it has been reported that some doctors have successfully treated Bell's palsy with natural musk according to earlier literature records and clinical experience^12,13,14,15. Mus is not a recommended drug in the diagnosis and treatment guidelines, but in underdeveloped medical areas or for some people who cannot use steroids or antiviral drugs, Mus can be another preferred option. Literature analysis in recent years also shows that musk is the most frequently used Chinese medicine in the published Chinese medicine patch prescriptions and acupuncture point studies for the treatment of peripheral facial palsy¹⁶. Although there are many pharmacological and medical studies on Mus, how musk cured Bell's palsy exactly, and what the underlying neurochemical and cellular biological mechanisms are, is still unknown.

Bell's palsy, an acute idiopathic facial paralysis, results from dysfunction of the peripheral facial nerve, which innervates all facial muscles. The most noticeable sign of Bell's palsy symptoms is the weakness and drooping of facial muscles on one side. Although Bell's facial paralysis is not life-threatening, it can affect the patient's quality of life and even cause anxiety and depression. What is particularly worrying is that up to 30% of the patients suffer long-term sequelae, including synkinesis, contracture, permanent facial paresis, and facial asymmetry, even with appropriate treatment. Therefore, finding the resolution of Bell's palsy and decreasing the risk of paralysis are our major aims. Current knowledge on the pathogenic mechanism of Bell's palsy remains unclear. Recent research has demonstrated that the reactivation of viruses may be the major cause of Bell's palsy, and these viruses can cause a neuronal edema and inflammation to trigger the attack of Bell's palsy.

Building on recent evidence, we hypothesized that Mus alleviates the symptoms of Bell's palsy by improving oxidative stress damage to the facial nerve. This study experimentally indicated Mus's therapeutic effects, revealing that upregulated IL-1R1 in SCs activates the NF-κB pathway, causing cellular damage. Crucially, we demonstrated that Mus inhibits NF-κB signaling by downregulating IL-1R1. Our findings identified the role of IL-1R1 upregulation in SCs exposed to oxidative stress in vitro and positioned Mus administration as a novel therapeutic strategy.

Protocol

This experimental research was conducted with the approval of the Institutional Animal Care and Use Committee (Approval Number: LL-240077).

Cell culturing and drug intervention
Rat Schwann cells (SCs) were cultured in Schwann Cell Medium, consisting of basal medium, 5% fetal bovine serum, 1% Schwann cell growth supplement, 1% penicillin, and 1% streptomycin. All cells were kept in a 5% CO₂ atmosphere at 37 °C. When allowed to grow up to a density of 70-80%, SCs were divided into four groups (n = 5 per group): (1) NC group; (2) NC+CoCl₂ group; (3) NC+CoCl₂+Mus group; (4) NC+CoCl₂+siIL-1R1+Mus group.

Cobalt chloride (CoCl₂), a frequently employed agent to mimic hypoxia, is known to trigger responses similar to those of hypoxia, such as the impeding degradation and stabilization of hypoxia-inducible factors and accumulation of HIF-1α protein¹⁵. According to the modified method based on Osuru et al.¹⁷, in the NC+CoCl₂ group and NC+CoCl₂+Mus group, SCs were cultured in Schwann Cell Medium supplemented with 240 µM CoCl₂ for 8 h, at atmospheric conditions of 95% air and 5% CO₂ at 37 °C in a humidified incubator (21% O₂). Then, in the Mus groups, the intervention of 1.25 µg/mL Mus for an additional 24 h was applied. Meanwhile, in the NC+CoCl₂+siIL-1R1+Mus group, the SCs were firstly treated with 24 h of siRNA infection, then continually cultured with 240 µM CoCl₂ for 8 h, followed by the intervention of 1.25 µg/mL Mus for an additional 24 h, as in the former groups. The flowchart of the experimental process is shown in Figure 1.

Cell viability assay
The viability of SCs was determined using the cell counting kit-8 (CCK-8) assay reagent according to the manufacturer's instructions. Cultured SCs at a density of 5 × 10⁵ cells/well were seeded in a 96-well cell culture plate with 100 µL of complete growth medium at 37 °C in a 5% CO₂ humidified atmosphere. When the confluency reached 70-80%, 10 µL of CCK-8 solution was added to each well, and the plates were incubated for 1 h at 37 °C. The optical density (OD) of the 96-well plates was measured at 450 nm using a microplate reader, and the cell viability was calculated. The cells were divided into four groups: NC group, NC+CoCl₂ group, NC+CoCl₂+Mus group, and NC+CoCl₂+siIL-1R1+Mus group. The experimental results were corrected using a blank well (medium+CCK8 solution). Cell survival rate = [(experimental group - blank well)/(NC group - blank well)] × 100%.

siRNA transfection
Transfection was conducted according to the manufacturer's instructions of a commercial in vitro siRNA/miRNA transfection reagent using synthetic siRNA duplexes of siIL-1R1 (AUAGUCUUGGAUUUUCUCCAA, GGAGAAAAUCCAAGACUAUGA) with a concentration of 100 ng/µL for 24 h. After siIL-1R1 transfection, the siIL-1R1 expressions were assessed by performing RT-qPCR analysis after 24 h of transfection¹⁸, and the efficiency must be ensured to be higher than 80%

Enzyme-linked immunosorbent assay (ELISA)
Post-culture supernatants were collected and centrifuged under group-specific conditions (2950 g, 20 min, 25 °C). Extracellular levels of IL-1β, TNF-α, ROS, and SOD were assayed via ELISA following kit instructions. Final absorbance readings at 450 nm were obtained using a microplate reader.

RNA isolation and quantitative real-time PCR (RT-qPCR)
Following manufacturer instructions, total RNA was extracted from cultured cells using TRizol reagent. Target genes (IL-1β, TNF-α, S-100b, IL-1R1) were amplified in 20 µL of SYBR qPCR mix reactions for 40 cycles on a PCR instrument. Data normalization used β-actin, with PCR specificity verified by melting curve analysis. The 2^−ΔΔCt method quantified relative expression from five independent samples assayed in triplicate. Primers are listed in Table 1.

Western blot
Following treatment, SCs from all experimental groups were incubated for 24 h and harvested. Total protein was extracted using a total protein extraction kit. Protein lysates were combined with sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer denatured at 98 °C for 5 min, and separated (20 µg per lane) on 10-12% SDS-polyacrylamide gels. Resolved proteins were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% non-fat dry milk for 60 min at room temperature, then incubated overnight at 4 °C in a volume of 6 mL with the following primary antibodies: anti-HIF-1α antibody (1:1000), anti-GAPDH antibody (1:1000), anti-IL-1R1 antibody (1:1000), anti-C-JUN antibody (1:1000), anti-GDNF antibody (1:1000), anti-IRAK1 antibody (1:1000), anti-IKK antibody (1:1000), anti-p65 antibody (1:800), anti-p50 antibody (1:900), anti-IκB-α antibody (1:900) and anti-β-actin antibody (1:1000). After washing, membranes were probed with horseradish peroxidase (HRP)-conjugated secondary antibodies for 60 min at 25 °C. Protein signals were detected using Image Studio Digits Ver 4.0. Band intensities were quantified with ImageJ software and normalized to β-actin and GAPDH expression. Three independent experiments were performed.

Immunofluorescence
IL-1R1 localization in SCs was assessed following Zhao et al.¹⁹. Harvest cells during the logarithmic growth phase by trypsinization, resuspend them in fresh complete medium, and triturate thoroughly to obtain a uniform single-cell suspension. Seed the cells onto culture plates containing sterile cover slips. Once the cells have reached full confluence, rinse them 1-2 times with phosphate-buffered saline (PBS), carefully remove the cover slips, and fix the adherent cells with 4% paraformaldehyde in PBS for 10 min, permeabilize with 0.05% Triton X-100 (10 min), and block with 5% goat serum (1 h). Primary incubation used rabbit anti-rat IL-1R1 mAb. After PBS washes, samples were incubated with FITC-conjugated goat anti-rabbit IgG for 2 h at room temperature. Add water-soluble mounting agents to the cover slip for mounting. Immunolabeled cells were visualized and quantified using a fluorescence microscope with the excitation wavelength 492 nm and the emission wavelength 520 nm.

Ultrastructure analysis
Following Zhao et al.'s protocol¹⁹, SCs were fixed in 2.5% glutaraldehyde and post-fixed overnight at 4°C in 1% osmium tetroxide. Samples underwent an ethanol/acetone dehydration series. Ultrathin sections (80 nm) were stained with uranyl acetate and lead citrate prior to ultrastructural examination using a transmission electron microscope.

Disposal of hazardous chemical reagents
The medium and solutions containing CoCl₂ are collected into a designated heavy metal waste container. All disposable materials that have come into contact with the aforementioned liquids, including pipette tips, centrifuge tubes, and gloves are classified as heavy metal hazardous waste and are transferred to a licensed hazardous waste management company approved by the relevant authorities for proper disposal. Discard waste paraformaldehyde solution into a dedicated aldehyde waste container. Used consumables should be collected as aldehyde waste and processed by the same licensed hazardous waste management company.

Statistical analysis
Multi-group comparisons were analyzed by one-way ANOVA with Tukey's post-hoc test in GraphPad Prism 8.0. Statistical significance was defined as P < 0.05. Data are presented as mean ±± standard deviation (SD).

Results

Protective effects of Mus on oxidative stress injury of Schwann cells in vitro
The expression of HIF-1α exhibited changes in response to variations in hypoxic circumstances²⁰. To model hypoxia, SCs were exposed to CoCl₂, which successfully induced hypoxia as confirmed by elevated HIF-1α protein levels, a canonical hypoxia marker, via western blotting (Figure 2A). Subsequent Mus intervention of hypoxic SCs asses...

Discussion

Bell's palsy is an idiopathic peripheral facial nerve palsy characterized by unilateral facial weakness. The myelin sheath performs various crucial functions in the effective functioning of neurons²³. It ensures the smooth conduction of nerve impulses, supports neurons by supplying nourishing factors, and aids in the process of regeneration²⁴ . Hence, loss of myelin in the peripheral nervous system can result in demyelinating neuropathies, which can cause Muscular atrophy, ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% trypsin digestion solution (without EDTA)	Servicebio	G4002
10x TBS	Eubiotech	BF0153
10-180 KDPrestained Color Protein Marker	Eubiotech	P0069
5% non-fat dry milk	Beyotime Biotechnology, China	P0216-300g
anti-C-JUN antibody	Cell Signaling, USA	#9165T
anti-GAPDH antibody	abcam, UK	ab245355
anti-GDNF antibody	Cell Signaling, USA	#47808
anti-HIF-1α antibody	Cell Signaling, USA	#14179
anti-IKK antibody	Cell Signaling, USA	#2682
anti-IL-1R1 antibody	Cell Signaling, USA	#6775	for WB
anti-IL-1R1 antibody	proteintech	84094-6-RR	for immunofluorescence
anti-IRAK1 antibody	Cell Signaling, USA	#4395
anti-IκB-α antibody	Cell Signaling, USA	#8943
anti-p50 antibody	abcam, UK	ab305263
anti-p65 antibody	abcam, UK	ab16502
anti-rat IL-1R1 mAb	Beyotime Biotechnology, China	AF7212
anti-β-actin antibody	abcam	ab179467
BCA Protein Quantification Kit	Eubiotech	WB2123
Cell counting kit-8 (CCK-8) assay reagent	Beyotime Biotechnology, China	C0048M
Cobalt chloride (CoCl2)	Macklin, China	C804815
EuECL Star	Eubiotech	WB2131
Fetal bovine serum (FBS)	Bergolin,China	PWL001
Fitc-labeled goat anti-rabbit IgG	Servicebio	GB22303
Fluorescence microscope	Nikon
Hieff Trans in vitro siRNA/miRNA transfection reagent	YEASEN, China	40806ES01
Horseradish peroxidase (HRP)-conjugated secondary antibodies Hrp-labeled goat anti-mouse IgG	Servicebio, China	GB23301
IL-1β, TNF-α, ROS, and SOD ELISA Kit	BOSTER, China	BES1762K, BES0481K, BES5018K, BES2920K
Muscone (Mus)	Aladdin, China	M141496
PBS buffer	Servicebio	G4202
Penicillin-Streptomycin Solution, 100x	Procell	PB180120
Polyvinylidene fluoride (PVDF) membranes	Millipore, USA	IPFL00010
Rat Schwann cells (SCs)	Procell Life Science & Technology China	CM-M111
RIPA Lysis Buffer	Eubiotech	WB2116
Schwann Cell Medium	ScienCell, USA	1701
SDS-PAGE loading buffer	Beyotime Biotechnology, China	P0015
SDS-PAGE Protein l Loading Buffer, 5x	Eubiotech	WB2122
si-IL-1R1	Gene Universal	RX0114371
Sodium dodecyl sulfate polyacrylamide gelelectrophoresis(SDS-PAGE) Preparation kit	Eubiotech	WB2118
SYBR qPCR Mix	Beyotime Biotechnology, China	D7260
Total Protein Extraction Kit	Beyotime Biotechnology, China	PP123-01
Transmission electron microscope	Hitachi	HT7800
Trizol	Beyotime Biotechnology, China	R0011
Tween-20	Eubiotech	WB2114
Water-soluble mounting agents	Absin, China	abs9240
Western Transfer Buffer	Eubiotech	WB2129