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Olfactory mucosa-mesenchymal stem cells with overexpressed Nrf2 modulate angiogenesis and exert anti-inflammation effect in an in vitro traumatic brain injury model

European Journal of Medical Research volume 30, Article number: 80 (2025) Cite this article

Abstract

Background

Traumatic brain injury (TBI) is a major cause of disability and mortality among children and adults in developed countries. Transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2) has antioxidant, anti-inflammatory and neuroprotective effects and is closely related to TBI. Olfactory mucosa-mesenchymal stem cells (OM-MSCs) could promote neural regeneration. At present, the effects of OM-MSCs with overexpressed Nrf2 in brain diseases remain to be explored.

Methods

The OM-MSCs were prepared and transfected with Nrf2 overexpression plasmid. Those transfected cells were termed as OM-MSCs with Nrf2 overexpression (OM-MSCsNrf2) and co-cultured with rat pheochromocytoma cells PC12 or murine microglia BV2. The effects of OM-MSCsNrf2 on the survival and angiogenesis of PC12 cells were evaluated through cell counting kit-8 (CCK-8) and tube formation assay, and extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were calculated to reflect glycolysis. Immunofluorescence assay was applied to determine the effects of OM-MSCsNrf2 on microglial polarization, and the underlying molecular mechanisms were analyzed based on the quantification tests of RT-qPCR and immunoblotting.

Results

Co-culture of OM-MSCsNrf2 and PC12 cells increased the levels of anti-inflammatory cytokines and pro-angiogenesis factors, enhanced the cell survival and angiogenesis. Moreover, we also observed elevated phosphorylation of PI3K/AKT and suppressed BAX protein expression. Meanwhile, OM-MSCsNrf2 inhibited the levels of pro-inflammatory genes and affected the glycolysis in PC12 cells. In the co-cultured system of OM-MSCsNrf2 and BV2 cells, M2 microglial polarization was observed, and the levels of M2 microglia-relevant genes and the phosphorylation of STAT6/AMPKα/SMAD3 were elevated.

Conclusion

This study proved the effects of OM-MSCsNrf2 on modulating PC12 and BV2 cells in vitro, which, however, necessitates further in vivo validation.

Introduction

Traumatic brain injury (TBI) refers to an acquired insult to the brain from external mechanical force [1,2,3]. As a leading cause of disability and mortality across all age groups in developed countries, TBI leads to over 10 million hospitalizations worldwide at a cost of more than $200 billion [4]. The pathophysiology of TBI includes primary and secondary brain injuries, manifested by tissue damage, neuronal loss, neuroinflammation, oxidative stress (OS), and cell death [5]. Accumulating evidence suggested that enhancing antioxidant response and suppressing inflammation could attenuate brain damage [6,7,8]. This has sparked interest in identifying therapeutic targets capable of exerting both anti-inflammatory and ameliorative effects on TBI.

Nrf2 plays a key role in the defense mechanism that modulates the detoxifying, oxidative, and anti-inflammatory genes. Nrf2 binds to the antioxidant response elements (AREs) of the antioxidant genes, forming Nrf2-binding antioxidant genes and thereby promoting their transcription [9, 10]. Research has shown that Nrf2 possesses could reduce inflammatory responses in various Alzheimer's disease models, showing the potential to serve as a novel therapeutic strategy for treating Alzheimer's disease [11]. Upregulating the Nrf2/ARE pathway can effectively promote neuroprotective effects, which holds significant clinical implications for the treatment of senile cerebral apoplexy [12]. Notably, Nrf2-deficient mice exhibit significant glial cell proliferation [13]. Insufficient activation of Nrf2 is strongly linked to the progression of neurodegenerative diseases, while targeting the Nrf2 signaling pathway can inhibit microglia-mediated neuroinflammation [14]. Furthermore, studies have found that suppressing oxidative stress through the AKT/GSK-3β/Nrf2 pathway mitigates oxidative stress damage in pheochromocytoma cells [15]. The neuroprotective effects of Nrf2 and the involvement of Nrf2 in TBI have been extensively analyzed. For instance, study showed that Nrf2 exerts its neuroprotective effect on mice against TBI-induced ferroptosis [16]. Nrf2 is also implicated in the cerebral protective effect of Hericium erinaceus mycelium and its derivative erinacine C on an animal model with mild TBI in which inflammation and mild TBI-induced deficits were attenuated by upregulating Nrf2-binding antioxidant genes [17]. Pyroptosis is a form of programmed cell death with inflammatory characteristics [18], and Nrf2 has been identified as a pyroptosis-related mediator closely linked to cytokines and disease severity in TBI [19], suggesting that Nrf2 may influence the progression of TBI by modulating inflammatory responses. Based on these findings, this study re-explores the involvement of Nrf2 in TBI using microglia and pheochromocytoma cells.

Current research has demonstrated that though neural stem cells (NSCs) are capable of self-renewal and generating the main phenotypes of cells in central nervous system (CNS), endogenous NSCs sometimes fail to repair the damages caused by severe trauma and some other neurodegenerative diseases [20,21,22,23]. Thus, exogenous cell therapy, such as mesenchymal stem cells (MSCs), have been applied as a potentially effective treatment for neurological diseases [24]. Studies also showed that MSCs could improve the neural networks in chronic refractory epilepsy and emphasized their neuroprotective effects on cerebral hemorrhage models [25, 26]. MSCs exert their therapeutic effects mainly through paracrine mechanisms [27], and their neuroprotective functions have been observed in TBI models [28]. Lu et al. isolated MSCs from olfactory mucosa (OM) [29], and another research confirmed that OM-MSCs possess the ability to promote neural regeneration [30]. Considering the therapeutic potential in neurological diseases and a relatively easy process to collect OM-MSCs, this study employed OM-MSCs to explore their role in brain diseases, aiming to expand the current understanding of OM-MSCs. The graphical abstract of this study can be found in Figure S1.

Materials and methods

Ethics statement

The Ethic Committee of Haikou People’s Hospital (Haikou Affiliated Hospital of Central South University Xiangya School of Medicine) has reviewed and approved the current study to be performed in strict adherence to the Declaration of Helsinki. The patient enrolled has been informed of and provided with written informed consent for the participation.

Isolation of OM-MSCs

The collection of OM and subsequent isolation of OM-MSCs were performed following a previous study [31]. The primary cells used in this study were derived from a 48-year-old female patient who had been clinically diagnosed with TBI but without other illnesses [ZY-IRB-FOM-031]. To collect OM, the patient was administrated with chloramphenicol (#C8050, Solarbio® Life Sciences, Beijing, China) through the nasal cavity for 3 consecutive days. The integrity of the collection tube for OM tissue was verified before use. The gauze was pre-soaked in the solution containing 1.2–1.5% lidocaine (#L8761, Solarbio® Life Sciences, China) and saline and inserted into the nasal cavity of the patient for 8-min local anesthesia. The forceps were used to clamp the mucous tissue and a 3mm × 3mm sample of OM tissue was excised from the posterior 1/3 of the superior turbinate. Subsequently, the OM tissue fragments were transferred to a tube containing Dulbecco’s modified Eagle’s medium/F12 (DMEM/F-12, #C3280, Solarbio® Life Sciences, China) supplemented with GlutaMax supplements (#35,050,061, Gibco, Waltham, MA, USA), 10% fetal bovine serum (#S9020, Solarbio® Life Sciences, China), and 1% penicillin–streptomycin (#P1400, Solarbio® Life Sciences, China). The tissue sections were minced into pieces (~ 0.5 mm3) with a pair of ophthalmic scissor and centrifuged to remove the supernatant. The remaining fragments were incubated under a standard condition (at 37℃ with 5% CO2) for 3–5 days and daily observed to detect OM-MSCs. The OM-MSCs at passages 3 and 4 were utilized for further experiments.

Identification of OM-MSCs

The isolated OM-MSCs were stained with the following antibodies: phycoerythrin (PE)-labeled anti-CD45 antibody (#202207, 1:500, BioLegend, Inc., San Diego, CA, USA), PE/Cy5.5-labeled anti-CD34 antibody (#NBP2-33076PECY55, 1:500, Novus Biologicals, Centennial, CO, USA), fluorescein isothiocyanate (FITC)-labeled anti-CD29 antibody (#sc-9970 FITC, 1:500, Santa Cruz Biotechnology, Dallas, TX, USA), Alexa Fluor® 647-labeled anti-CD90 antibody (#ab317534, 1:500, Abcam, Cambridge, UK), Alexa Fluor® 488-labeled anti-CD73 antibody (#NBP2-71214AF488, 1:500, Novus Biologicals, USA), and allophycocyanin (APC)-labeled anti-CD105 antibody (#NB500-452APC, 1:500, Novus Biologicals, USA) [32]. A total of 1 × 106 dyed OM-MSCs were collected for flow cytometry using FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo software (version 10.5.3, FlowJo, LLC., Ashland, OR, USA).

Cell culture and transfection

Rat pheochromocytoma cell line PC12 (#BNCC100436, BeNa Culture Collection, Xinyang, China), mice microglia cell line BV2 (#BNCC337749, BeNa Culture Collection, China), and human umbilical vein endothelial cells (HUVECs, #CP-H082, Procell, Wuhan, China) were purchased from corresponding suppliers and differently cultured. Specifically, PC12 cells were cultured in Roswell Park Memorial Institute-1640 medium (#C3250, Solarbio® Life Sciences, China) supplemented with 15% horse serum (#S9050, Solarbio® Life Sciences, China) and 5% fetal bovine serum; BV-2 cells were cultured in high-glucose DMEM (#D5194, Solarbio® Life Sciences, China) supplemented with 10% fetal bovine serum; HUVECs were maintained in M199 medium (#11150067, Gibco, USA) including 15% fetal bovine serum, 1% penicillin–streptomycin, 30 μg/mL endothelial cell growth supplement (#211-GS, Sigma, Darmstadt, Germany), and 10 ng/mL epidermal growth factor (#E5036, Sigma, Germany). All the cells were identified via short tandem repeat profiling and tested negative for mycoplasma contamination.

For subsequent studies, OM-MSCs transfected with or without the overexpression plasmid of Nrf2 were termed as OM-MSCs with Nrf2 overexpression (OM-MSCsNrf2) and OM-MSCs. An inverted optical microscope (DP27, Olympus, Tokyo, Japan) was applied to observe the morphology of the cells.

Cell intervention

With the purpose of exploring the efficacy of OM-MSCsNrf2 on PC12 or BV2 cells, OM-MSCs with or without transfection were co-cultured with PC12 cells for 24 h at a ratio of 1:1, while the PC12 cells treated with minocycline (1 μM, #M9190, Solarbio® Life Sciences, China) for 24 h served as a positive control [33].

Similarly, OM-MSCs with or without transfection were co-cultured with BV2 cells for 24 h at a ratio of 1:1, while the BV-2 cells treated with 20 ng/mL Interleukin (IL)−4 (#SRP3211, Sigma, Germany) and IL-13 (#I1896, Sigma, Germany) were used as a positive control [34].

Cell viability assay

The survival of differently treated PC12 cells was measured by CCK-8 assay. In detail, co-cultured PC12 cells were grown in 96-well plates at the density of 5 × 103 cells/well for 12, 24, and 36 h and then added with 10 μL CCK-8 solution (#CA1210, Solarbio® Life Sciences, China) for 4 h. A multi-well microplate reader (Bio-Rad, Hercules, CA, USA) was employed to read the optical density (OD) value at 450 nm, and the cell viability was calculated based on the recorded OD value of experimental group (the PC12 cells co-cultured with OM-MSCsNrf2 and CCK-8 solution), control group (the PC12 cells co-cultured with OM-MSCs and CCK-8 solution), and empty group (without cells and CCK-8 solution). The following formula was applied to calculate cell viability:

$${\text{Cell viability }}\left( \% \right) \, = \, \left( {{\text{OD}}_{{{\text{experimental}}}} - {\text{OD}}_{{{\text{empty}}}} } \right)/\left( {{\text{OD}}_{{{\text{control}}}} - {\text{OD}}_{{{\text{empty}}}} } \right) \, \times { 1}00\% .$$

Cell angiogenesis assay

The conditioned media of PC12 cells in different co-culture system were collected and applied to treat HUVECs (2 × 104 cells) [35]. In detail, HUVECs were cultured in a 24-well plate pre-coated with Matrigel (#M8370, Solarbio® Life Sciences, China) and treated with the condition media for 4 h. The angiogenesis potential of HUVECs influenced by different groups of PC12 cells was observed under an inverted optical microscope. The branch points were counted using the image analysis software (version 1.0, ibidi GmbH, Gräfelfing, Germany) [36].

Cell glycolysis assay

The glycolysis potential of PC12 cells under various co-culture conditions was explored by measuring the ECAR and OCR. Differently co-cultured PC12 cells were seeded in a 96-well culture plate at the density of 1 × 105 cells/well and assayed by Seahorse XF96 instrument (Agilent, Inc., Santa Clara, CA, USA). The corresponding ECAR and OCR of cells were recorded every 10 min within 2 h and analyzed in the Seahorse Wave software (version 2.6, Agilent, Inc., USA) [37].

Cell immunofluorescence assay

To assess the polarization state of BV2 cells after co-culture, immunofluorescence assay was performed to measure the expressions of transmembrane protein 119 (TMEM119), CD86, and CD206 proteins. BV2 cells after the co-culture were fixed in 4% paraformaldehyde (#P1110, Solarbio® Life Sciences, China), treated with 100% pre-chilled methanol (#34885, Sigma, Germany) and incubated with 1% bovine serum albumin (#A8010, Solarbio® Life Sciences, China) for 1 h. Next, the cells were incubated with the following primary antibodies against TMEM119 (label: Alexa Fluor® 647, #ab225494, 1:500, Abcam, UK), CD86 (label: FITC, #NBP2-34569F, 1:500, Novus Biologicals, USA), and CD206 (label: Alexa Fluor® 488, #141709, 1:500, Bio-Legend, Inc., USA) at 4℃ overnight. The secondary antibodies goat anti-rabbit IgG (#ab6702, 1:5000, Abcam, UK) and goat anti-rat IgG (#ab182018, 1:5000, Abcam, UK) were applied for further incubation at room temperature for 1 h. After that, the nuclei of cells were dyed with 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI, #D8200, Solarbio® Life Sciences, China). All the cells were visualized under a laser scanning confocal microscope (LSM710, Carl Zeiss, Oberkochen, Germany) and the mean fluorescence intensity (MFI) of CD86 and CD206 was calculated accordingly.

Enzyme-linked immunosorbent assay (ELISA)

ELISA was applied to calculate the concentration of the following cytokines with the relevant assay kit: IL4 (#SEKR-0004, Solarbio® Life Sciences, China), IL10 (#SEKR-0006, Solarbio® Life Sciences, China), IL13 (#SEKR-0046, Solarbio® Life Sciences, China), vascular endothelial growth factor-A (VEGF-A, #SEKR-0032, Solarbio® Life Sciences, China), and transforming growth factor beta (TGF-β, #SEKR-0012, Solarbio® Life Sciences, China). In detail, the cell culture supernatant was collected following the centrifugation (1000 × g) at 4℃ for 10 min and added to the reaction well for 90-min incubation at 37℃. The biotinylated antibody solution provided by the testing kit and enzyme-labeled working solution were added sequentially to the reaction well for incubation. After the addition of TMB substrate, the reaction was terminated with the termination solution and the OD value was read with a microplate reader. The concentration of the cytokines was determined based on the plotted standard curve.

Reverse-transcription quantitative PCR (RT-qPCR)

Total cellular RNA from cultured cells was extracted with the TriZol assay kit (#15,596,026, Invitrogen, Carlsbad, CA, USA) according to the manuals. After measuring the concentration, cDNA was synthesized from the extracted RNA using a cDNA synthesis assay kit (#K1621, ThermoFisher Scientific, Waltham, MA, USA). The SYBR™ Green PCR Master Mix (#4,344,463, ThermoFisher Scientific, USA) and ABI7500 thermocycler (ThermoFisher Scientific, USA) were used for PCR reaction under the following conditions: at 95℃ for 10 min and 40 cycles at 95℃ for 15 s and at 60℃ for 1 min. The relative mRNA levels were calculated using 2−ΔΔct method with GAPDH as a reference gene [38]. The primers used in the experiment are listed in Table 1 for reference, and these primers were designed for the detection of the target genes in RT-qPCR.

Table 1 Sequences of primers for PCR assay
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Immunoblotting

To investigate the activation status of signaling pathways and functional changes in BV2 cells after co-culture, immunoblotting was performed to assess the phosphorylation status and total protein levels of a series of key signaling pathway proteins in BV2 cells. The total cellular protein was lysed by radio immunoprecipitation assay (RIPA) buffer (#9806, Cell Signaling Technology, Danvers, MA, USA) and the concentration was measured using a bicinchoninic acid protein assay kit (#7780, Cell Signaling Technology, USA). The sample protein was separated on the prepared separation gel and then transferred to the polyvinylidene fluoride membranes (#YA1701, Solarbio® Life Sciences, China). The membranes were blocked in 5% defat milk at ambient temperature for 2 h and incubated with the following primary antibodies against phosphorylated-Phosphoinositide 3-kinase (p-PI3K, #AF5905, 1:1000, Beyotime, Shanghai, China), PI3K (#4292, 1:1000, Cell Signaling Technology, USA), phosphorylated-AKT (p-AKT, #4060, 1:2000, Cell Signaling Technology, USA), protein kinase B (AKT, #4691, 1:1000, Cell Signaling Technology, USA), BCL2 Associated X (BAX, #14796, 1:1000, Cell Signaling Technology, USA), phosphorylated-signal transduction and transcription activator 6 (p-STAT6, #56554, 1:1000, Cell Signaling Technology, USA), STAT6 (#5397, 1:1000, Cell Signaling Technology, USA), phosphorylated-AMP-activated protein kinase alpha (p-AMPKα, #2535, 1:1000, Cell Signaling Technology, USA), AMPKα (#5832, 1:1000, Cell Signaling Technology, USA), phosphorylated-SMAD3 (p-SMAD3, #9520, 1:1000, Cell Signaling Technology, USA), SMAD3 (#9523, 1:1000, Cell Signaling Technology, USA), and GAPDH (#5174, 1:1000, Cell Signaling Technology, USA) at 4℃ overnight. Additionally, horseradish peroxide-conjugated secondary antibody (#SE134, 1:5000, Solarbio® Life Sciences, China) was applied for incubation after rinsing the membrane in TBST thrice. The protein density was measured by enhanced chemiluminescence system (ChemiDoc, Bio-Rad, Hercules, CA, USA) and quantified via ImageJ (version 5.0, Bio-Rad, USA).

Statistical analysis

To ensure the objectivity and accuracy of the data, a blinding procedure was employed. To ensure the accuracy and reliability of the experimental results, all the experiments were conducted in triplicate and the data were expressed as mean ± standard deviation and analyzed via GraphPad Prism software (version 8.0.2, GraphPad, LLC., La Jolla, CA, USA). Two-group data were compared via Student’s t test. For comparisons among three or more groups of data, ordinary one-way ANOVA was conducted, followed by Dunnett's multiple comparisons test. And statistical significance was defined when the p value was less than 0.05.

Results

Effects of OM-MSCsNrf2 on the concentrations of relevant cytokines in PC12 cells

We first characterized the OM-MSCs using flow cytometry assay, and the relevant results showed that the isolated cells were negative for CD45 and CD34 but positive for CD29-, CD90-, CD73-, and CD105 (Fig. 1A), which confirmed the identity of isolated cells were OM-MSCs. RT-qPCR confirmed that the Nrf2 expression in the OM-MSCsNrf2 was significantly higher than that in the OM-MSCs (ctrl) (Fig. 1B, p< 0.0001), while there was no significant difference between OM-MSCs (ctrl) and the negative control group of OM-MSCs (Fig. 1B, p = 0.9606). Observed under the microscope, the OM-MSCs transfected with Nrf2 overexpression plasmid exhibited a fibroblastic or spindle-shaped morphology (Fig. 1C).

Fig. 1
figure 1

Effects of OM-MSCsNrf2 on the levels of relevant cytokines in PC12 cells. A Flow cytometry was used to characterize CD45, CD34, CD29, CD90, CD73, and CD105 of OM-MSCs. B RT-qPCR-based to assess transfection efficiency after overexpression of Nrf2 plasmid. OM-MSCsNrf2: NRF2 overexpression group. OM-MSCs (ctrl): Empty vector transfection group. OM-MSCs: Untransfected OM-MSCs. C Morphology of both OM-MSCs (ctrl) and OM-MSCsNrf2 under the optical microscope. Scale bar: 100 μm. D The changes in cytokine concentrations in PC12 cells measured by ELISA, including IL4, IL10, IL13, VEGF-A, and TGF-β. Results of independent triplicates were expressed as mean ± standard deviation and the statistical difference between two groups was marked with asterisks (“ns” indicates no significant difference, *p < 0.05, and **p < 0.01). OM-MSCs olfactory mucosa-mesenchymal stem cells, Nrf2: Nuclear Factor Erythroid-Derived 2-Like 2, IL interleukin, VEGF-A vascular endothelial growth factor-A, TGF-β transforming growth factor beta

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The effects of OM-MSCsNrf2 on the concentrations of relevant cytokines in PC12 cells were explored. It was found that the concentrations of the cytokines, including IL4 (p = 0.0172), IL10 (p = 0.0371), IL13 (p = 0.0388), VEGF-A (p = 0.0438), and TGF-β (p = 0.0438), were all elevated in PC12 cells following the co-culture with OM-MSCsNrf2 (Fig. 1D).

Effects of OM-MSCsNrf2 on the survival and angiogenesis and proteins related to PI3K/AKT pathway and apoptosis in PC12 cells

Subsequently, the potential impact of OM-MSCsNrf2 on the survival of PC12 cells was assessed by CCK-8 assay. The results showed no significant change in PC12 cells after 12 h of co-culture with OM-MSCsNrf2 (Fig. 2A, p= 0.9962), while the OD values of PC12 cells increased significantly at 24 (Fig. 2A, p= 0.0002) and 48 h (Fig. 2A, p< 0.0001). Moreover, tube formation assay showed that co-culture PC12 cells with OM-MSCsNrf2 in conditioned media increased the number of branch points in HUVECs (Fig. 2B, C, p= 0.0011). Further quantification from immunoblotting assay revealed that the co-culture with OM-MSCsNrf2 promoted the phosphorylated levels of PI3K (Fig. 2D, E ,p= 0.0075) and AKT (Fig. 2D, E, p= 0.0004), and decreased that of BAX (Fig. 2D, E, p= 0.0004). These changes were similar to those induced by minocycline, the positive control in this study, on PI3K (Fig. 2D, E, p=0.0037), AKT (Fig. 2D, E, p=0.0002), and BAX (Fig. 2D, E, p=0.0002).

Fig. 2
figure 2

Effects of OM-MSCsNrf2 on the survival and angiogenesis and proteins related to PI3K/AKT pathway and apoptosis in PC12 cells. A Effects of OM-MSCs (ctrl) and OM-MSCsNrf2 on the survival of PC12 cells based on CCK-8 assay. B, C Angiogenesis assay in evaluating the angiogenesis of PC12 cells following the co-culture with OM-MSCs (ctrl) and OM-MSCsNrf2. scale bar: 200 μm D, E Ratio of p-PI3K to PI3K and p-AKT to AKT as well as BAX protein level in PC12 cells following the co-culture with OM-MSCs (ctrl) and OM-MSCsNrf2, and those cells treated with minocycline were applied as the positive control. Results of independent triplicates were expressed as mean ± standard deviation and the statistical difference between two groups was marked with asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). OM-MSCs olfactory mucosa-mesenchymal stem cells, Nrf2 Nuclear Factor Erythroid-Derived 2-Like 2, OD optical density, p-PI3K phosphorylated-PI3K, PI3K Phosphoinositide 3-kinase, p-AKT phosphorylated-AKT, AKT protein kinase B, BAX BCL2 Associated X, GAPDH Glyceraldehyde-3-phosphate dehydrogenase

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The mechanisms underlying the effect of OM-MSCsNrf2 on the neuroinflammation mediators and glycolysis in PC12 cells

The mRNA expressions of neuroinflammation mediators, such as Nlrp3 (Fig. 3A=, p= 0.0010), Il1b (Fig. 3A, p< 0.0001), Il18 (Fig. 3A, p= 0.0003), and Ccl5 (Fig. 3A=, p= 0.0016) in PC12 cells were all sharply downregulated after co-culture but those of P2ry12 (Fig. 3A, p= 0.0131), and Cx3cr1 (Fig. 3A, p= 0.0032) were upregulated. Measurement of ECAR and OCR showed that the co-culture of PC12 cells with OM-MSCsNrf2 noticeably reduced the ECAR but increase the OCR (Fig. 3B, C).

Fig. 3
figure 3

Relevant mechanisms related to OM-MSCsNrf2 on the neuroinflammation mediators and glycolysis in PC12 cells. A Effects of OM-MSCs and OM-MSCsNrf2 on the mRNA level of Nlrp3, Il1b, Il18, Ccl5, P2ry12, and Cx3cr1 in PC12 cells. B, C Effects of OM-MSCs (ctrl) and OM-MSCsNrf2 on the ECAR (B) and OCR (C) of PC12 cells. Results of independent triplicates were expressed as mean ± standard deviation and the statistical difference between two groups was marked with asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). OM-MSCs olfactory mucosa-mesenchymal stem cells, Nrf2 Nuclear Factor Erythroid-Derived 2-Like 2, Nlrp3 NOD-, LRR- and pyrin domain-containing protein 3; Il1b Interleukin 1 beta; Il18 Interleukin 18, Ccl5 C–C chemokine ligand 5, P2ry12 purinergic receptor P2Y12, Cx3cr1 C-X3-C motif chemokine receptor 1, ECAR extracellular acidification rate, oligo Oligomycin; OCR oxygen consumption rate, 2-DG 2-deoxy-D-glucose, FCCP Trifluoromethoxy carbonylcyanide phenylhydrazone

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Effects of OM-MSCsNrf2 on the microglial polarization in BV2 cells

The second part of this study examined the effects of OM-MSCsNrf2 on the microglial polarization in BV2 cells by observing the MFI of CD86 or CD206 in BV2 cells co-cultured with OM-MSCsNrf2. According to the results from immunofluorescence assay, the co-culture of BV2 cells with OM-MSCsNrf2 led to a decreased MFI of CD86 (Fig. 4A, B, p= 0.0111) but an increased MFI of CD206 (Fig. 4C, D, p= 0.0012). The result was further supported by the detection of the expressions of relevant marker genes. In detail, BV2 cells co-cultured with OM-MSCsNrf2 had low expressions of Cd80 (Fig. 4E, p= 0.0183) and Nos2 (Fig. 4F, p= 0.0008) and high expressions of Arg1 (Fig. 4G, p= 0.0273) and Fizz1 (Fig. 4H, p= 0.0008).

Fig. 4
figure 4

Effects of OM-MSCsNrf2 on the microglial polarization in BV2 cells. A–D CD86 MFI (A, B) and CD206 MFI (C, D) in BV2 cells following the co-culture with OM-MSCs (ctrl) and OM-MSCsNrf2. scale bar: 100 μm. E–H Relative mRNA levels of Cd80 (E), Nos2 (F), Arg1 (G), and Fizz1 (H) in BV2 cells following the co-culture with OM-MSCs (ctrl) and OM-MSCsNrf2. Results of independent triplicates were expressed as mean ± standard deviation and the statistical difference between two groups was marked with asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001). OM-MSCs olfactory mucosa-mesenchymal stem cells, Nrf2 Nuclear Factor Erythroid-Derived 2-Like 2, MFI mean fluorescence intensity, Nos2 nitric oxide synthase 2, Arg1: arginase 1

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Further, we measured the protein levels of mediators related to microglial polarization. It was found that the OM-MSCsNrf2 could enhance the phosphorylation of STAT6 (Fig. 5A, B, p< 0.0001), AMPKα (Fig. 5A and C, p= 0.0270), and SMAD3 (Fig. 5A and D, p= 0.0108), which was consistent with the changes of STAT6 (Fig. 5A, B, p< 0.0001), AMPKα (Fig. 5A and C, p= 0.0242), and SMAD3 (Fig. 5A and D, p= 0.0108) in the positive control IL4 and IL13.

Fig. 5
figure 5

Relevant mechanisms related to OM-MSCsNrf2 on BV2 cells. A After BV2 cells were co-cultured with OM-MSCs (ctrl) and OM-MSCsNrf2 respectively, the protein expression of p-STAT6, STAT6, p-AMPKα, AMPKα, p-SMAD3, and SMAD3 was detected by Western blot technique, and those cells treated with IL4 and IL13 were applied as the positive control. B–D Ratio of p-STAT6/STAT6 (B), p-AMPKα/AMPKα (C), and p-SMAD3/SMAD3 (D) in BV2 cells following the co-culture with OM-MSCs (ctrl) and OM-MSCsNrf2, and those cells treated with IL4 and IL13 were applied as the positive control. Results of independent triplicates were expressed as mean ± standard deviation and the statistical difference between two groups was marked with asterisks (*p < 0.05, and****p < 0.0001). OM-MSCs olfactory mucosa-mesenchymal stem cells, Nrf2 Nuclear Factor Erythroid-Derived 2-Like 2, p-STAT6 phosphorylated-STAT6, STAT6 signal transduction and transcription activator 6, p-AMPKα phosphorylated-AMPKα, AMPKα AMP-activated protein kinase alpha, p-SMAD3 phosphorylated-SMAD3, GAPDH Glyceraldehyde-3-phosphate dehydrogenase, IL4 interleukin 4, IL13 Interleukin 13

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Discussion

Stem cells are clonogenic cells with the capabilities of self-renewing and multilineage differentiation [39]. Recent studies have widely discussed the efficacy of stem cell therapy in the management of TBI [40]. For instance, bone marrow-derived MSCs have an anti-neuroinflammation effect against TBI [41]. Also, the use of neural stem cells delivered via brain-derived tissue-specific bioink can promote the recovery from TBI [42]. Human umbilical cord-derived MSCs effectively protect the brain architecture and neurological function in rat with acute TBI [43]. Currently, although the ideal cell type for transplant-mediated repair has not been determined, autologous transplantation is considered advantageous [44]. The OM is a part of the nasal mucosa and its olfactory neurons has life-long regeneration and repairing capabilities, representing a rich source for cell therapy to improve CNS repair [45, 46]. Nrf2 is a transcriptional gene that has anti-apoptotic and anti-oxidative effects [47, 48]. In this study, we preliminarily explored the role of OM-MSCsNrf2 in TBI based on in vitro cell culture model, and discovered that OM-MSCsNrf2 affected TBI potentially through influencing the survival and angiogenesis of PC12 cells and the polarization of BV2 cells. Such a discovery further supported the protective effects of OM-MSCsNrf2 on CNS injuries.

The anti-inflammatory role of OM-MSCs.Nrf2

Research on moderate and severe TBI showed that inflammation is a crucial secondary physiological response to TBI [49]. Inflammation is characterized by the release of pro-inflammatory cytokines and activation of innate immune response in CNS [50,51,52]. IL4 is a cytokine that suppresses TBI in rats via directing TBI neuroinflammation toward an anti-inflammation state. IL13 is another anti-inflammatory cytokine produced by the immune cells in the brain early after the occurrence of TBI [53, 54]. Study also showed that IL10 is an immunosuppressive cytokine capable of modifying the neuro-inflammatory response after TBI [55]. Following TBI, VEGF is expressed within the brain and acts as a potential angiogenesis and neurogenesis activator. TGF-β can enhance the generation of a new subset of macrophage for tissue repair during the recovery of TBI [56, 57]. The co-culture of OM-MSCsNrf2 increased all these mediators in PC12 cells, and promoted cell survival rate and angiogenesis but reduced glycolysis and pro-inflammatory mediators (Nlrp3, Il1b, Il18, and Ccl5) [58,59,60]. Nlrp3 inflammasome is a multiprotein cytosolic complex closely associated with the activation of inflammatory responses [61], and its downregulation signifies a significant attenuation of the inflammatory storm after brain injury. IL-1β and IL-18 are pro-inflammatory cytokines that can trigger a series of detrimental immune reactions, leading to further tissue damage and neuronal cell death. An increase in CCL5 around the site of axon transection induces leukocyte infiltration to the injured site [62], and by reducing its production, we can potentially limit the influx of harmful immune cells, thereby protecting the vulnerable brain tissue. Collectively, these results proved the anti-inflammatory role of OM-MSCsNrf2 in PC12 cells. Moreover, inflammation in cortices and neuronal dysfunction resulted from TBI is known to be mediated by microglia [63]. Hereafter, we further explored the effects of OM-MSCsNrf2 on the polarization of microglia cell line BV2. We observed the polarization of M2 microglia, which releases anti-inflammatory mediators and triggers neuroprotectivity [64], as evidenced by increased CD206 MFI and upregulated levels of both Arg1 (widely applied for the identification of M2-polarized microglia [65]) and Fizz1 (another M2 microglia indicator [66]). These results supported the anti-inflammatory role of OM-MSCsNrf2 and modulatory effects of OM-MSCsNrf2 on the microglial M2 polarization in cellular model of TBI, suggesting its potential role in reducing post-traumatic brain inflammation in TBI patients.

OM-MSCsNrf2 changed the energy metabolism pattern of PC12 cells

Nrf2 plays a pivotal role in the energy metabolism of PC12 cells. Experimental results indicated that co-culturing PC12 cells with OM-MSCsNrf2 significantly decreased ECAR and increased OCR. This suggested that the glycolytic pathway of the cells was inhibited, while the efficiency of mitochondria in utilizing oxygen to produce energy was enhanced, shifting the cells toward a more efficient energy-producing mode dominated by oxidative phosphorylation. Nrf2 is involved in mitochondrial quality control and structure and regulates mitochondrial redox signaling [67, 68]. Notably, many neurodegenerative diseases are associated with mitochondrial dysfunction [69]. Nrf2 can counteract oxidative stress by activating mitochondrial function and metabolism, thereby influencing the progression of neurodegenerative diseases [70]. This finding indicated that Nrf2 could affect the energy metabolism of PC12 cells by enhancing aerobic respiration, which in turn may impact the occurrence of TBI. However, studies have also found that inhibition of glycolysis significantly impairs the neurite structure of early postnatal mice [71]. Undoubtedly, reduced ATP production is a common hallmark of neurodegenerative diseases [72], but the specific mechanisms by which Nrf2 regulates energy metabolism in TBI require further investigation.

OM-MSCsNrf2 activated PI3K/AKT pathway for neuroprotection

Further, the potential downstream pathway contributing to the effects of OM-MSCsNrf2 was explored based on the cellular model of TBI, and in this study, we focused mainly on PI3K/AKT pathway, which is known to play a protective role in cerebral injuries, including TBI [7, 73, 74]. The PI3K/AKT pathway is a crucial survival pathway in neurons, closely related to the regulation of cellular survival [75, 76]. AKT plays a pivotal role in neuronal survival following TBI, while the phosphorylation of AKT by PI3K promotes cellular survival and prevents neurodegeneration through its intervention on several targets, including apoptotic markers [77]. Previous research also showed that elevated phosphorylation of STAT6, AMPKα and SMAD3 are indicative of microglial M2 polarization [78,79,80]. It is reported that STAT6 is involved in the maintenance of microglial phenotype homeostasis and enhances the release of inflammatory mediators through neurons [81, 82]. In this study, the phosphorylation of PI3K/AKT in PC12 cells and STAT6/AMPKα/SMAD3 in BV2 cells was elevated following the co-culture with OM-MSCsNrf2, which further improved the understanding on the mechanisms underlying the protective effects of OM-MSCsNrf2 in TBI.

The clinical application of OM-MSCs.Nrf2

In summary, Nrf2, as a pivotal transcription factor for cellular antioxidant response, has demonstrated robust neuroprotective effects by modulating the activation of phosphorylation pathways. However, direct therapeutic application of Nrf2 in clinical settings requires further validation. Certain drugs, such as Gracilins, are capable of activating Nrf2, showing the potential to be applied in the treatment of neurodegenerative diseases [83]. Nevertheless, significantly high Nrf2 activity may interfere with normal cellular metabolic processes and may even lead to tumorigenesis [84, 85]. Therefore, further experiments and investigations are needed for the clinical application of Nrf2.

Limitations

However, there were certain limitations in this study to be noted. Our experiments primarily relied on in vitro cellular model, which revealed the biological effects and potential molecular mechanisms of OM-MSCsNrf2 but lacked in vivo validation. This also limited our ability to fully explore the complex physiological environment and intercellular interactions. Therefore, our future study plans to include in vivo validation using animal models of TBI, which will involve behavioral assessment of the animals, histological analysis, and molecular mechanism studies to further confirm the role of Nrf2. Furthermore, although our study provided novel insights for the interactions between Nrf2 and other pathways, longer term analysis to clarify its functional mechanisms is still required.

Conclusion

In summary, the current study demonstrated the protective effects of OM-MSCsNrf2 using both PC12 cells and BV2 cells, evidenced by the improved cell survival and angiogenesis and decreased glycolysis and microglial M2 polarization. The potential involvement of the downstream pathways was additionally explored, providing novel insights into the mechanisms of OM-MSCsNrf2− in the treatment of cerebral diseases.

Data availability

The data is available upon reasonable request from the corresponding authors.

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Acknowledgements

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Funding

Hainan Province Clinical Medical Research Center, LCYX202410; Hainan Health Service Research Project, 22A200114; Hainan Province Clinical Medical Center, 0202068; National Natural Science Foundation of China, 82360259.

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  1. Department of Neurosurgery, Haikou Affiliated Hospital of Central South University Xiangya School of Medicine, Haikou, 570208, China

    Zigui Chen, Jiale Li, Jun Peng & Ying Xia

  2. Department of Neurosurgery, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, 533000, China

    Chunyuan Zhang, Yuhua Fang, He Zhang, Jiawei Luo & Qisheng Luo

  3. Guangxi Engineering Research Center for Biomaterials in Bone and Joint Degenerative Diseases, Baise, 533000, China

    Chunyuan Zhang, Yuhua Fang, He Zhang, Jiawei Luo & Qisheng Luo

  4. Department of Neurosurgery Second Branche, Hunan Provincial People’s Hospital (The First Affiliated Hospital of Hunan Normal University), Changsha, 410005, China

    Changfeng Miao

  5. Department of Clinical Research Center, Haikou Affiliated Hospital of Central South University Xiangya School of Medicine, Haikou, 570208, China

    Yingqi Qiu

Authors
  1. Zigui Chen
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  2. Chunyuan Zhang
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  3. Yuhua Fang
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  4. He Zhang
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  5. Jiawei Luo
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  6. Changfeng Miao
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  7. Jiale Li
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  8. Jun Peng
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  9. Yingqi Qiu
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  10. Ying Xia
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  11. Qisheng Luo
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Contributions

All authors contributed to this present work: [ZGC], [YX] and [QSL] designed the study, [CYZ], [YHF] and [HZ] acquired the data, [JWL], [CFM] and [JLL] interpreted the data. [JP] & [YQQ] improved the figure quality, [ZGC], [CYZ], [YHF], [YX] and [QSL] drafted the manuscript, [YQQ], [HZ], [JWL], [JP] and [CFM] revised the manuscript. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Yingqi Qiu, Ying Xia or Qisheng Luo.

Ethics declarations

Ethics approval and consent to participate

This study was approved by ethic committee of Haikou People’s Hospital (Haikou Affiliated Hospital of Central South University Xiangya School of Medicine) [ZY-IRB-FOM-031].

Consent for publication

The study has obtained the consent from the all patients involved.

Competing interests

The authors declare no competing interests.

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Chen, Z., Zhang, C., Fang, Y. et al. Olfactory mucosa-mesenchymal stem cells with overexpressed Nrf2 modulate angiogenesis and exert anti-inflammation effect in an in vitro traumatic brain injury model. Eur J Med Res 30, 80 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40001-025-02344-6

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40001-025-02344-6

Keywords

European Journal of Medical Research

ISSN: 2047-783X

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