Sufentanil enhances M1/M2 phenotypic polarization transition and alleviates LPS-triggered neuroinflammation in BV2 cells

2.1 Cell culture and treatment

The BV2 cells were purchased from the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium (Gbico, 11995065, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, 10099-141, Gbico, Grand Island, NY, USA) at 37 °C with 5% CO₂. To simulate a neuropathic pain cell model, BV2 cells were treated with LPS (100 ng/mg) for 24 hours [8, 9]. Subsequently, BV2 cells were treated with varying concentrations of Suf (0 μM, 10 μM, 20 μM and 40 μM) for 24 hours.

2.2 CCK-8 assay

BV2 cells were seeded in a 96-well plate at a density of 1 × 10⁴ cells per well. After 24 hours of incubation, 10 μL of CCK-8 solution (5 mg/mL, CA1210, Solarbio, Shanghai, China) was added to each well, following which cell viability was assessed using a spectrophotometer (ND-ONE-W, Thermo Fisher Scientific, Waltham, MA, USA) at an absorbance rate of 450 nm.

2.3 Reverse transcription-quantitative PCR (RT-qPCR)

RNA extraction from BV2 cells was performed using TRIzol reagent (Invitrogen, 15596018, Carlsbad, CA, USA). Subsequently, RNA was reverse transcribed into cDNA using the PrimeScript® RT reagent kit (Takara, RR037B, Kusatsu, Japan). Quantitative PCR (qPCR) was conducted using the SYBR® Premix Ex Taq™ II kit (Takara, RR820A, Dalian, China), and the relative mRNA expression was determined using the 2^−ΔΔCt method. The primer sequences used were as follows:

Interleukin-1 beta (IL-1β):

F: 5′-CGACAAAATACCTGTGGCCT-3′,

R: 5′-TTCTTTGGGTATTGCTTGGG-3′;

IL-6:

F: 5′-GAAACCGCTATGAAGTTCCTCTCTG-3′,

R: 5′-TGTTGGGAGTGGTATCCTCTGTGA-3′;

Tumor Necrosis Factor-α (TNF-α):

F: 5′-CATCTTCTCAAAATTCGAGTGACAA-3′,

R: 5′-TGGGAGTAGACAAGGTACAACCC-3′;

IL-10:

F: 5′-GCTCTTACTGACTGGCATGAG-3′,

R: 5′-CGCAGCTCTAGGAGCATGTG-3′;

Glyceraldehyde-3-phosphate dehydrogenase (GADPH):

F: 5′-AGGTCGGTGTGAACGGATTTG-3′,

R: 5′-GGGGTCGTTGATGGCAACA-3′.

2.4 ELISA

Commercial ELISA kits were utilized to quantify the levels of tumor necrosis factor-α (TNF-α, ab208348, Abcam, Shanghai, China), interleukin-1β (IL-1β, ab197742), IL-6 (ab222503), and IL-10 (ab255729).

2.5 Flow cytometry

For cell apoptosis assessment, the fluoresceine isothiocyanate (FITC) Annexin V apoptosis detection Kit (556547, BD Biosciences, San Jose, CA, USA) was used. Briefly, BV2 cells were stained with FITC Annexin V (5 μL) and propidium iodide (5 μL) in the dark, following which cell apoptosis was analyzed using flow cytometry (BD FACSCalibur, BD Biosciences, San Jose, CA, USA).

Regarding changes in macrophage subsets, BV2 cells were incubated with CD16/32 antibody (553141, 1:100, BD Biosciences, San Jose, CA, USA) or anti-CD206 antibody (MAB25351, 1:100, R&D Systems, Inc., Minneapolis, MN, USA) in the dark. The proportions of M1-type macrophages (CD16/32) and M2-type macrophages (CD206) were then determined using flow cytometry.

2.6 Western blot

The proteins were isolated from BV2 cells using radio immunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors (Roche, 4906837001, Beijing, China). Protein separation was achieved through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto polyvinylidene difluoride (PVDF) membranes (Sigma, St Louis, USA). Primary antibodies were incubated with the membranes for 12 hours, followed by a secondary antibody (1/5000; ab6721) for 2 hours. The protein blots were visualized using the enhanced chemiluminescence system (Thermo Fisher Scientific, USA).

The primary antibodies used in these experiments included p-p65 (1/1000; ab76302), p65 (0.5 μg/mL; ab16502), p-inhibitor of NF-κB alpha (IκBa, 1/10,000; ab133462), IκBa (1/1000; ab32518), and β-actin (1 μg/mL; ab8226).

2.7 Statistical analysis

The data are expressed as mean ± standard deviation (SD), and statistical analyses were conducted using SPSS 20.0 (IBM Corp., Armonk, NY, USA). Each experiment was performed at least three times. Comparisons between two groups were assessed using Student’s t-test, and comparisons among multiple groups were evaluated using One-way analysis of variance (ANOVA). A p-value less than 0.05 was considered statistically significant.

3.1 Sufentanil facilitated cell viability and suppressed cell apoptosis in LPS-stimulated BV2 cells

Considering that our initial experiments indicated that cell viability remained unchanged following treatment with 10 μM and 20 μM Suf but was reduced after exposure to 40 μM Suf (Fig. 1A), 10 μM and 20 μM Suf were selected for subsequent investigations. Additionally, while LPS induction led to a decrease in cell viability, this effect was reversed following treatment with Suf (10 μM and 20 μM) (Fig. 1B). Furthermore, it was observed that LPS treatment increased cell apoptosis, which could be mitigated by Suf treatment (10 μM and 20 μM) (Fig. 1C,D). Taken together, these findings indicate that Suf promotes cell viability and suppresses cell apoptosis in LPS-stimulated BV2 cells.

Fig. 1.Sufentanil enhances cell viability and suppresses cell apoptosis in LPS-triggered BV2 cells. (A) Cell viability assessed using the CCK-8 assay with varying concentrations of Sufentanil (Suf) (0 μM, 10 μM, 20 μM and 40 μM). (B) Confirmation of cell viability in Control, LPS, LPS + Suf (10 μM), and LPS + Suf (20 μM) groups using the CCK-8 assay. (C,D) Measurement of cell apoptosis in Control, LPS, LPS + Suf (10 μM), and LPS + Suf (20 μM) groups through flow cytometry. ***p < 0.001. Suf: Sufentanil; LPS: lipopolysaccharide; ns: no significance; PI: propidium iodide; FITC: fluoresceine isothiocyanate.

3.2 Sufentanil attenuated inflammation in LPS-induced BV2 cells

Assessment of inflammation factors revealed that the mRNA expressions of IL-1β, IL-6 and TNF-α were upregulated, while IL-10 was downregulated following LPS induction. However, these effects could be effectively counteracted following treatment with Suf (10 μM and 20 μM) (Fig. 2A). Consistently, ELISA results showed similar alterations in the levels of IL-1β, IL-6, TNF-α and IL-10 (Fig. 2B). Overall, these findings support that Suf can attenuate inflammation in LPS-induced BV2 cells.

Fig. 2.Sufentanil attenuates inflammation in LPS-induced BV2 cells. (A) Assessment of mRNA expressions of IL-1β, IL-6, TNF-α and IL-10 in Control, LPS, LPS + Suf (10 μM), and LPS + Suf (20 μM) groups via RT-qPCR. (B) Analysis of IL-1β, IL-6, TNF-α and IL-10 levels in Control, LPS, LPS + Suf (10 μM), and LPS + Suf (20 μM) groups using ELISA. *p < 0.05, **p < 0.01, ***p < 0.001. Suf: Sufentanil; LPS: lipopolysaccharide; IL-1β: interleukin-1β; TNF-α: tumor necrosis factor-α; ns: no significance.

3.3 Sufentanil enhanced M1/M2 phenotypic polarization transition in LPS-stimulated BV2 cells

The proportions of CD206 (M2 phenotype marker) and CD16/32 (M1 phenotype marker) were assessed to confirm macrophage subtype changes. Following LPS induction, CD206 expression decreased while CD16/32 expression increased. However, these alterations were reversed after treatment with Suf (10 μM and 20 μM) (Fig. 3). Collectively, these findings indicate that Suf enhances the transition from M1 to M2 phenotypic polarization in LPS-stimulated BV2 cells.

Fig. 3.Sufentanil enhances M1/M2 phenotypic polarization transition in LPS-stimulated BV2 cells. Flow cytometry analysis of CD206 and CD16/32 proportions in Control, LPS, LPS + Suf (10 μM), and LPS + Suf (20 μM) groups. *p < 0.05, **p < 0.01, ***p < 0.001. Suf: Sufentanil; LPS: lipopolysaccharide; CD: cluster of differentiation.

3.4 Sufentanil retarded the NF-κB pathway in LPS-mediated BV2 cells

Following LPS induction, the results showed that the expressions of p-p65 and p-IκBa were both increased, while IκBa protein expression was reduced. In addition, these effects could be reversed after treatment with Suf (10 μM and 20 μM) (Fig. 4), suggesting that Suf inhibits the NF-κB pathway in LPS-mediated BV2 cells.

Fig. 4.Sufentanil inhibits the NF-κB pathway in LPS-mediated BV2 cells. Western blot analysis of p-p65, p65, p-IκBa and IκBa protein expressions in Control, LPS, LPS + Suf (10 μM), and LPS + Suf (20 μM) groups. *p < 0.05, ***p < 0.001. Suf: Sufentanil; LPS: lipopolysaccharide; IκBa: inhibitor of NF-κB alpha.