A comprehensive study on the effects of PBM combined with hUCMSCs on microglial polarization in inflammatory states was conducted. Microglia differentiate into either M1 or M2 phenotype and perform distinct roles upon detecting potentially injurious or anomalous signals via crucial signalling pathways. In response central nervous system injury, microglia activate the neuroprotective M2 phenotype to release neurotrophic factors and phagocytose. However, sustained activation results in changes in microglial function [5], an excessive release of inflammatory mediators resulting in neuronal death, and an enhanced neurotoxic M1 phenotype, which inhibits M2 phenotype expression. This inhibition plays a key role in neurodegenerative diseases [28].
First, we investigated the polarization status of microglia and discovered that both treatment with hUCMSCs and PBM combined with hUCMSCs decreased CD86 expression in inflamed BV2 cells. There was no significant difference in CD86 expression between the control and 808-CM groups, indicating that 808-CM inhibited M1 phenotype polarization more efficiently. In addition, 808-CM promoted microglial differentiation into the M2 phenotype under inflammatory conditions. Administration of hUCMSCs at 808 nm, or in combination with inflamed mice, resulted in the promotion of M2 phenotype polarization of microglia, as well as enhanced learning and memory in mice. The findings indicate that the combination treatment promotes microglial polarization and has a protective effects in both cells and animals. Consistent with experimentally findings, previous studies have demonstrated that modulation of microglial polarization could enhance the protective effects of microglia. Light with a wavelength of 1070 nm alters microglial polarization, reduces M1-like microglia around cortical blood vessels, and improves cognition and memory [29]. Melatonin can reduce apoptosis and modulate microglial polarization towards the M2 phenotype, which has a neuroprotective role in brain injury caused by ischaemic stroke [30]. Electroacupuncture enhances the expression of M2 microglia, regulates neuronal excitability, and produces analgesic effects [31]. Exosomes originating from bone marrow MSCs modulate microglial polarization and ameliorate cerebral ischaemia/reperfusion injury [32]. Additionally, hUCMSC therapy inhibits M1 microglia and apoptosis, promotes microglial M2 polarization, and improves ataxia in inflamed mice [33].
The persistent release of pro-inflammatory cytokines by hyperactivated microglia leads to heightened neuroinflammation and exacerbates neurodegenerative diseases [34]. Factors such as LPS, TNF-α, and cellular debris stimulate microglia to secrete inflammatory factors, further exacerbating neuroinflammation [35]. We used LPS at different concentrations to induce microglia and found that it promoted the secretion of pro-inflammatory cytokines. Memory behaviour is impaired in LPS-treated mice, and accumulation of pro-inflammatory factors is induced in the hippocampus [36]. Our findings are consistent with previous research demonstrating that inflammatory conditions in mice lead to increased secretion of inflammatory factors in brain tissues and serum. Additionally, mice with inflammation exhibit impaired learning and memory abilities in a water maze test.
Investigating strategies to reduce neuroinflammation may enable the treatment of patients with neurodegenerative diseases [37]. Bioactive factors secreted in MSCs have immunomodulatory effects [38]. In MSC therapy, AD patients show attenuated neuroinflammation, restored blood-brain barrier function, and improved cognition [39], while stroke patients exhibit reduced inflammation in the brain and periphery [40]. Exosomes derived from atorvastatin-pretreated MSCs can promote cardiac function recovery, reduce apoptosis, and inhibit inflammatory factor secretion in the peri-infarct area [41]. Additionally, PBM has a modulatory effect on inflammatory microglia and in animal models. The application of 810 nm light promoted the recovery of motor function in spinal cord-injured mice, reduced apoptosis, inhibited neurotoxic microglial activation, and alleviated neuroinflammation [18]. Near infrared light enhances learning and memory in both humans and animal models [42, 43]. Moreover, treatment with coenzyme Q10 and 810 nm light alone or together can boost cognitive function, decrease levels of TNF-α and IL-1β, and alleviate neuroinflammation in mice with cerebral ischaemia [44].
However, there is limited research regarding the use of PBM pre-treated MSCs in therapy. Our findings show that treatment with 808-CM did not significantly alter the expression of pro-inflammatory and anti-inflammatory cytokines in inflammatory microglia compared to the control group. Nevertheless, it effectively regulated the levels of inflammatory cytokines in activated microglia. These findings suggest that 808-CM has a better therapeutic effect on microglia in the inflammatory state. A more significant difference in the expression of inflammatory factors in inflammatory mice with the 808 nm light combination treatment compared to the LPS group was observed. Different microglial phenotypes release various factors and have different functions [28, 45]. Treatment with 808-CM in cells and 808 nm light combined with hUCMSCs in inflammatory mice significantly inhibited TNF-α and IL-6 expression while promoting M2 phenotype polarization, promoted IL-10 expression, and improved learning and memory ability in inflammatory mice. Hence, stem cell therapy along with non-invasive PBM appears to have broad prospects, and can produce superior therapeutic effects than stem cells.
The Notch pathway regulates gliogenesis and neuronal differentiation. In addition, it is involved in inflammatory response processes [46] and pathological events [47] in the central nervous system. Furthermore, the Notch signalling pathway plays a crucial role in microglial activation and inflammatory processes in neuroinflammatory diseases. Our study indicates that LPS upregulates the expression of the Notch pathway, thereby inducing microglia to adopt the M1 phenotype and promoting neuroinflammation. Consistent with previous research, Notch signalling orchestrates the activation of microglia, contributing to neuroinflammation [48]. Our research shows that treatment with either hUCMSCs or PBM combined with hUCMSCs within BV2 inflammatory cells leads to a significant reduction in Notch pathway expression and M1 phenotype polarization, with 808 nm light pre-treatment of hUCMSCs having a more significant effect. This phenomenon may be attributed to the reduced expression of the Notch pathway in hUCMSCs induced by 808 nm light. Furthermore, many medicines can modulate macrophage polarization by targeting the Notch signalling pathway [49]. Irisin attenuates post-ischaemic inflammation and neuronal apoptosis, and ameliorates neurological dysfunction by modulating the Notch pathway [50]. Lipoxin A4 inhibits the expressions of Notch-1, Hes1, induced nitric oxide synthase, and CD32, and enhances M2 microglial cell expression, which has a protective effect against ischaemic stroke [51]. Extracellular vesicles from stem cells in adipose tissue effectively decrease macrophage polarization towards M1 and reduce inflammation by modulating Notch-miR148a-3p signalling [52]. Therefore, regulation of the Notch signalling pathway is a promising subject for research into the prevention and treatment of neuroinflammatory diseases. Nevertheless, our study discovered that 635 nm light and 808 nm light had distinct effects on the Notch signalling pathway. The specific differences between the two wavelengths have not been investigated in depth. To gain a more efficient understanding of its relationship with neuroinflammation, it may be worthwhile to investigate the molecular mechanisms involved in Notch distinctions at different wavelengths.