Abstract Bell's palsy is the most common form of facial nerve palsy. This study aimed to explore the pathogenesis of Bell's palsy by investigating the effect of cold-stimulated adipocyte supernatant on adhesion molecule expression in Schwann cell line. Schwann cells were cultured in regular or adipocyte-conditioned medium and analyzed using RNA sequencing. The mRNA expression of Schwann cell adhesion molecules melanoma cell adhesion molecule (MCAM), protocadherin 9 (PCDH9), and intercellular cell adhesion molecule 1 (ICAM1) was determined using real-time reverse-transcription polymerase chain reaction. Differentially expressed genes were identified, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted. Compared with Schwann cells in 37 °C, the expression of MCAM, PCDH9, and ICAM1 was downregulated in Schwann cells treated with cold-stimulated adipocyte supernatant compared with Schwann cells in 37 °C. Adipocytes subjected to cold exposure may weaken the adhesion capacity of Schwann cells and disrupt the local homeostasis of Schwann cell–axon interactions by affecting the expression of MCAM, PCDH9, and ICAM1, ultimately leading to the development of demyelinating lesions. Keywords: Cold exposure, Adipocytes, Schwann cells, Adhesion molecule, RNA-Seq, Bell's palsy Highlights * • Schwann cells treated with cryopreserved adipocytes exhibit reduced adhesion. * • Adipocytes affect the Schwann cell microenvironment by secreting adipokines. * • Microenvironmental changes in Schwann cell function occur via MAPK signaling. Abbreviations ADSC adipose derived mesenchymal stem cell ADIPO CM adipocyte conditioned medium BDNF brain-derived neurotrophic factor BP biological process CAM cell adhesion molecule CC cellular component DEG differentially expressed gene DM differentiation medium FBS fetal bovine serum GO Gene Ontology ICAM1 intercellular cell adhesion molecule 1 IGSF immunoglobulin superfamily KEGG Kyoto Encyclopedia of Genes and Genomes L-DMEM Dulbecco's modified Eagle's medium with low glucose MAPK mitogen-activated protein kinase MAPKK MAPK kinase MAPKKK MAPK kinase kinase MCAM melanoma cell adhesion molecule MF molecular function NGF nerve growth factor PCDH9 protocadherin 9 RNA-Seq RNA sequencing RT-qPCR real-time reverse-transcription polymerase chain reaction 1. Introduction Bell's palsy, also known as acute idiopathic peripheral facial nerve palsy, is the most common form of facial nerve palsy. Bell's palsy generally occurs in young adults, often manifested as the rapid onset of unilateral nerve palsy. Currently, the etiology of Bell's palsy is unknown [[35]1]. Clinical studies have suggested that hypertension, diabetes [[36]2,[37]3], and the adverse cardiovascular and metabolic effects of obesity may increase the risk of Bell's palsy [[38]4]. The epidemiology of Bell's palsy suggests that rapid changes in atmospheric pressure and environmental temperature may be the major risk factors [[39][5], [40][6], [41][7]]. The body is highly susceptible to Bell's palsy when in a state of fatigue and under alternating hot and cold stress [[42]8]. Bell's palsy is an acute demyelinating disease, similar to Guillain–Barré syndrome [[43]9,[44]10]. The myelin sheath, a crucial structure of the peripheral nervous system, is formed by Schwann cells, a type of glial cell. Schwann cells form myelin sheaths by wrapping their plasma membranes in a spiral form around axons. The myelin sheath provides nutritional support to neurons and maintains the homeostasis of the neuronal microenvironment. This structure also enables rapid action potential conduction and facilitates complex Schwann cell–axon interactions [[45]11,[46]12]. The adhesion molecules on the Schwann cell surface play an important role in regulating intercellular interactions, signaling, and axonal regeneration [[47]13]. The process of myelin formation in Schwann cells is regulated by multiple adhesion molecules. Schwann cells promote myelin formation by regulating neuregulin 1 signaling through the cell surface adhesion molecule E-cadherin [[48]14]. Thus, myelin formation in Schwann cells is likely regulated by synergistic signaling events generated by multiple surface adhesion molecules. The facial nerve network branches are mostly covered by subcutaneous fat, which possesses insulating properties that protect the organism from damage caused by hot and cold stresses. White adipocyte tissue, as an active endocrine organ, releases adipocytokines through specific intercellular signaling pathways. This mechanism keeps peripheral nerves in a healthy, repairable state and maintains the innervation of target tissues [[49]15,[50]16]. As facial cold exposure is a risk factor for Bell's palsy, the development of Bell's palsy is associated with metabolic and inflammatory changes in subcutaneous adipose tissue. Cold exposure can cause the upregulation of inflammatory chemokines in adipocytes [[51]17]. However, whether changes in Schwann cell microenvironment resulting from the effects of cold exposure on adipocytes alter Schwann cell function, is not known. In this study, the cells in the mild-hypothermia group were exposed to a temperature of 30 °C. This temperature was selected based on pre-experimental grouping. In addition, 30 °C is a critical point in temperature grading, as this point allows the temperature around the facial nerves to remain relatively stable despite changes in ambient temperature, owing to the protective effects of facial adipose tissue [[52]17]. We investigated how adipocytes subjected to cold exposure affect the expression profile of adhesion molecules in Schwann cells, in order to provide new insights into the etiology of Bell's palsy. 2. Material and methods 2.1. Animals Adipose tissue-derived mesenchymal stem cells (ADSCs) were obtained from 4-week-old male Sprague–Dawley (SD) rats at the Animal Center of Shanxi Medical University. As male SD rats have more adipose tissue than females, males were selected as the experimental animals. The rats were maintained at 25 ± 2 °C under a 12 h light/12 h dark cycle. The experiment involved six male SD rats, each weighing 100–200 g. The experimental design was approved by the Animal Experimentation Committee of the Stomatological Hospital of Shanxi Medical University (No. 2017007). All animal experiments complied with the ARRIVE guidelines. 2.2. Adipocyte culture All instruments were sterilized in a high-temperature autoclave prior to use. SD rats were anesthetized with 150 mg/kg of sodium pentobarbital, and necropsy was performed to ensure the death of all rats. Cardiac and respiratory arrest, muscle relaxation, and lack of reflexes were considered as signs of death. SD rats were submerged in 75% ethanol for 10 min, after which the abdominal skin was cut on ice. White adipose tissue was separated from the groin using ophthalmic scissors and transferred to a Petri dish containing phosphate-buffered saline. To obtain pure white adipose tissue, blood vessels, muscle and other impurities were carefully removed. White adipose tissue was minced and digested in 0.2% collagenase type I solution at 37 °C for 50–60 min. The separated ADSCs were resuspended in Dulbecco's modified Eagle's medium with low glucose (L-DMEM; PYG0072, Boster Bio), supplemented with 10% fetal bovine serum (FBS; 0510, ScienCell) and 1% penicillin/streptomycin/amphotericin B solution. When the third generation of ADSCs reached full confluence, the ADSCs were stimulated with differentiation medium (DM) containing L-DMEM supplemented with 10% FBS, 1% penicillin/streptomycin/amphotericin B sterile solution, 1 μM dexamethasone (D6040-100, Sigma), 200 μM indomethacin (I8280-5, Solarbio), 0.5 mM IBMX (I8450, Solarbio), and 10 μM insulin (I8040-25, Solarbio) for 4 days. The cells were then maintained in DM containing only 10 μM insulin, L-DMEM supplemented with 10% FBS, and 1% penicillin/streptomycin/amphotericin B sterile solution. Mature adipocytes were identified using Oil Red O staining. The adipocytes were then randomly divided into two groups (maintained at 37 °C and 30 °C, respectively). Each group was cultured in serum-free DMEM at 37 °C or 30 °C for 8 h. Thereafter, the cell supernatants were extracted and mixed with DMEM at a 1:1 ratio to produce 37 °C adipose-conditioned medium (37 ADIPO CM) and 30 °C adipose-conditioned medium (30 ADIPO CM), respectively. 2.3. Schwann cell culture Rat Schwann cell line (RSC-96) were obtained from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Schwann cells were cultured in medium with or without adipocyte conditioning. When the cells grew to 80–90% confluence for passaging, all cells were maintained at 37 °C in a humidified 5% CO[2] atmosphere. The number of cells inoculated in each T25 culture medium at the time of passaging was 10^4. The third generation of Schwann cells was used for follow-up experiments. Four groups were established: the 37 DMEM group (37 °C in DMEM medium), 37 ADIPO CM group, 30 ADIPO CM group, and 30 DMEM group (30 °C in DMEM medium). The 37 DMEM and 37 ADIPO CM groups were incubated at 37 °C for 8 h, and the 30 ADIPO CM and 30 DMEM groups were incubated at 30 °C for 8 h. Next, the cells were harvested for RNA extraction and RNA sequencing (RNA-Seq). 2.4. RNA extraction and quality control The cells were collected after 8 h of culture. A total RNA extractor (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) was used for total RNA extraction according to the manufacturer's protocol. The Qubit® 2.0 Fluorometer ([53]Q32866, Invitrogen, MA, USA) was used to measure RNA concentration, and purity was measured by determining the A260/A280 ratio. 2.5. cDNA library construction and RNA-Seq One microgram of RNA per sample was used for mRNA sequencing library preparation. The mRNA in each sample was enriched using oligo (dT) magnetic beads and fragmented with fragmentation buffer (approximately 200 bp). mRNA libraries were created using the Hieff NGS™ MaxUp Dual-mode mRNA Library Prep Kit for Illumina® (12301ES96, Yeasen Biotechnology (Shanghai) Co., Ltd., Shanghai, China) following the manufacturer's protocols. Libraries for paired-end sequencing were prepared (PE150; sequencing reads, 150 bp) at Sangon Biotech (Shanghai) Co., Ltd., using the MGISEQ-2000 platform. 2.6. Identification of differentially expressed genes Data processing was performed using Trimmomatic. Valid data were mapped to the genome using HISAT2, and clean reads were obtained. Differentially expressed genes (DEGs) were identified using the following screening criteria: |log[2] (Fold Change) | > 1 and q-value (adjusted p-value) < 0.05. 2.7. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses Gene Ontology (GO) annotations are divided into three major categories: molecular functions (MFs), biological processes (BPs), and cellular components (CCs). The Kyoto Encyclopedia of Genes and Genomes (KEGG), commonly used for pathway analysis, is a comprehensive database that integrates genomic, chemical, and systemic functional information. All DEGs were subjected to GO functional enrichment analysis, using the public GO database ([54]http://geneontology.org/). DEGs with a q-value of <0.05 were subjected to KEGG pathway enrichment analysis ([55]http://www.kegg.jp). GO enrichment analysis and KEGG pathway enrichment analysis were performed using topGO v2.24.0 software and clusterProfiler v3.0.5, respectively. 2.8. Real-time reverse-transcription polymerase chain reaction Real-time reverse-transcription polymerase chain reaction (RT-qPCR) was used to verify the accuracy of the mRNA expression profiles [[56]18]. Total RNA was extracted from Schwann cells using a total RNA extraction reagent (TRIgent, MF034-01, Mei5bio; Science & Technology Co., Ltd., Beijing, China). The RNA concentration was diluted to 250 ng/μl. Next, 500 ng of each RNA sample was reverse-transcribed into cDNA using a reverse transcription kit (MF166-01, Mei5bio; Science & Technology Co., Ltd.). Reaction conditions were as follows: 42 °C for 15 min, followed by 50 °C for 5 min, 96 °C for 5 min, and a final holding temperature of 4 °C. RT-qPCR analysis was performed via QuantStudio™ (A40426; Thermo Fisher Scientific, MA, USA), using the SYBR Green qPCR Mix (MF797-05, Mei5bio; Science & Technology Co., Ltd.). Primer sequences were designed based on the coding sequence of the target genes obtained from the NCBI database ([57]https://www.ncbi.nlm.nih.gov/) ([58]Table 1). The RT-qPCR program was as follows: an initial 95 °C denaturation step for 30 s, followed by 40 cycles at 95 °C for 15 s, 60.5 °C for 15 s, and 72 °C for 30 s. The experimental RT-qPCR data were normalized using β-actin expression as an internal reference. Three technical replicates and three biological replicates were established for each sample, and the experimental results were quantified using the 2^−ΔΔCt method [[59]18]. RT-qPCR analysis of differentially expressed genes was used to validate the RNA-Seq data. Table 1. RT-qPCR primer sequences. Gene Forward (5ʹ to 3ʹ) Reverse (3ʹ to 5ʹ) MCAM GTCCTCACAGCAGAGCCAACAG ACAGCAAGCACCAGCGTACATAC PCDH9 CCTGGTTCCGTGGTTGCTGAAG TCCTTTGTTGTTCCCGCTCACTATG ICAM1 CCTGGTCCTCCAATGGCTTCAAC TCTGTGGGATGGATGGATACCTGAG ACTB TGTCACCAACTGGGACGATA GGGGTGTTGAAGGTCTCAAA [60]Open in a new tab MCAM: melanoma cell adhesion molecule; PCDH9: protocadherin 9; ICAM1: intercellular cell adhesion molecule-1; ACTB: actin b. 2.9. Statistical analysis The correlations between the RNA-Seq data and RT-qPCR data were analyzed using GraphPad Prism v8.00. The RT-qPCR data are presented as mean ± standard deviation (n = 3). A one-way ANOVA was used for multiple group comparisons, and a t-test was used for two-group comparisons. Dunnett's test was employed as the post hoc test following one-way ANOVA. Results with P < 0.05 were considered statistically significant. 3. Results 3.1. Confirmation of differentiation into mature adipocytes and Schwann cell morphology Inguinal white adipose tissue collected from SD rats was digested to obtain ADSCs. The ADSCs exhibited a spindle-shaped morphology, which is characteristic of fibroblasts ([61]Fig. 1A). After 8 days of induction, the formation of round lipid droplets was clearly visible ([62]Fig. 1B). One hour after Oil Red O staining, the lipid droplets appeared red when 60% isopropanol was added ([63]Fig. 1C), confirming successful differentiation into mature adipocytes. The Schwann cells were small and either round or long and shuttle-shaped ([64]Fig. 1D). Fig. 1. [65]Fig. 1 [66]Open in a new tab Adipose tissue-derived mesenchymal stem cells (ADSCs), adipocytes, and Schwann cells. (A) ADSCs were long, spindle-shaped, and locally fused in a swirling pattern. Scale bar: 500 μm. (B) Differentiation of ADSCs into adipocytes was induced. Adipocytes appeared as round lipid droplets of varying sizes. With time, the lipid droplets slowly fused, and the cell morphology gradually became round. Scale bar: 100 μm. (C) Red staining of round lipid droplets indicated the presence of mature adipocytes. Scale bar: 100 μm. (D) Schwann cells were small and either round or long and shuttle-shaped. Scale bar: 100 μm. (For interpretation of the references to colour in this figure legend, the