In this study, the neuroinflammatory inhibition of nicotine was clarified using various characterization indexes. Cell co-culture indexes identified the potential neuroprotective phenomenon of nicotine and investigation of molecular mechanisms revealed possible pathways of nicotine action. This study discovered that nicotine reduced the release of several inflammation-related cytokines and chemokines in HMC3 cells and promoted the expression of BDNF and GAND. In addition, in the microglia-neuron co-culture system, this we observed an increase in neuronal survival after nicotine treatment. The study found that nicotine can regulate phosphorylation of PI3K protein through α7 nAChR, which may be a potential mode of action of nicotine.
We selected the human-derived microglia cell line HMC3 as a model system for our experiments. Several murine-derived microglia lines have been studied previously, however, studies using human-derived microglia have been few due to the limitations of the primary source of human microglia and the difficulty in obtaining sufficient amounts[20]. LPS is a component of the outer cell wall of Gram-negative bacteria. It is commonly used to trigger a cascade of immune stimuli and induce inflammation across cells, tissues, and organs. However, there are relatively few studies on the inflammatory response of human-derived microglia, and even fewer have used nicotine to influence the inflammatory response of human-derived microglia. Using human-derived HMC3 cells as experimental models provides more information for future clinical studies of the human nervous system. HMC3 was an ideal candidate for investigation of the effect of LPS on the activation phenotype and inflammatory level. In addition, we performed nicotine intervention to investigate whether nicotine mediates HMC3 inflammatory response through α7 nAChR to better understand its possible mechanism of action.
Previous microglial studies have primarily worked with BV2 cells, focusing on inflammatory indicators in BV2 cells that can be used to describe the level of the physiological activity of microglia[21, 22]. Microglia have two activation phenotypes, a classically activated pro-inflammatory type, and a selectively activated anti-inflammatory type. Therefore, to further understand the functional changes in the cells, many studies have explored the activation phenotype of different types of microglia[23]. Qichun Zhang et al. used acetylcholine to activate α7 nAChR in BV2 cells and observed the transition from the pro-inflammatory type to the anti-inflammatory type[24]. In the present study, we examined a variety of inflammation-related cytokines and chemokines, as well as changes in nitric oxide synthase activity and NO release, to understand the degree of microglial inflammation. We also performed some tests using BV2 cells to facilitate comparison with HMC3 cells.
To detect inflammatory factors, we selected the suspension microarray technique. We screened for the involvement of nicotine in the regulation of inflammatory factors and some chemokines that were not previously noted. Our findings indicated that LPS induced a significant elevation in 12 pro-inflammatory chemokines, suggesting that HMC3 can produce a sensitive response to LPS stimulation. We were also observed that 2 cytokines and 3 chemokines had a weak but significant decrease after nicotine treatment, including IL-6, IL-17, MCP-1 (MCAF), RANTES, and VEGF, while the other factors did not increase. IL-6 and IL-17 are pro-inflammatory factors[25, 26]. Monocyte chemotactic protein-1 activates monocytes and microglia to promote secretion of inflammatory mediators. RANTES regulates expression and T cell-mediated secretion of factors, facilitates leukocyte migration and infiltration, and may regulate cell growth and differentiation. Vascular endothelial growth factor acts with cytokines and promotes inflammatory responses[27, 28]. Overall, nicotine reduced expression of MCP-1, RANTES, and VEGF in HMC3 cells. These factors may regulate the physiological activity of microglia, aggregation of chemotactic proteins, and infiltration of inflammatory cells. Reducing their release inhibits inflammation at the lesion, suggesting that nicotine inhibits the inflammatory response of microglia.
In our experiments, we found that LPS induction did not lead to changes in nitric oxide synthase activity, and it increased NO release in HMC3 and BV2 cells. This is in contrast to the results of several published studies. For example, Marta Garcia-Contreras' team detected a significant upregulation of iNOS expression in HMC3 cells[29], Feng Jiang et al. found an upregulation of iNOS and increased NO release in HMC3 cells[30], and Zhao Shengnan et al. reported that iNOS expression changed dynamically with the time of LPS induction in primary microglia, with the highest expression observed after 24 hours[31]. However, according to Cinzia Dello Russo et al. in a published review, nitrite levels in HMC3 cells were undetectable under basal conditions and remained so after 24 hours of various pro-inflammatory stimuli, including LPS, LPS/IFN-γ, IL-1β, TNF-α alone or in combination with IFN-γ. Moreover, resting HMC3 cells did not express iNOS, nor did they have its expression induced by pro-inflammatory activation[32], which is consistent with our experimental results.
As an immune cell, the most crucial function of microglia is to regulate the immune microenvironment of the brain. Microglia secrete trophic factors which regulate neuronal survival. Our study examined BDNF and GDNF expression in HMC3 cells. Studies have shown that nicotine stimulates microglia, promoting BDNF expression, consistent with our findings in which nicotine increased BDNF expression and decreased inflammation in HMC3 cells[33, 34]. GDNF is not mainly produced by microglia, but other cellular sources may regulate eventual microglial activation[35]. Our results demonstrated that nicotine increased expression of both BDNF and GDNF, which are involved in neuronal growth and proliferation. Nicotine decreases inflammation and promotes neurotrophic factors, providing dual protection for neuronal cells.
In the present study, we focused on the protective role of nicotine with respect to neuroinflammation, and clarified that the target of nicotine is α7 nAChR to exert its anti-inflammatory effect through the α7 nAChR-PI3K signaling pathway. α7 nAChR is a vital target of the cholinergic anti-inflammatory pathway, present on the surface of immune cells and mitochondria, and is capable of being bound by ligands such as acetylcholine and nicotine to regulate the influx of Ca2+[36]. It has been demonstrated across numerous studies that it plays an important role in immunity, its agonists are being intensely investigated[37, 38].
The PI3K/AKT signaling pathway plays a critical role in regulating downstream signaling proteins to reduce neurotoxicity and treat neurodegenerative diseases. It is also involved in regulating immune responses and is implicated in the inflammatory processes of various diseases[39–41]. PI3K has four isoforms, and targeting the PI3Kδ isoform has been shown to reduce the production of pro-inflammatory cytokines[42]. Burdock seeds can also reduce cytokine and chemokine release by modulating the PI3K/Akt pathway[43]. Numerous studies have demonstrated the diverse effects of PI3K in coordinating and protecting against negative responses[44–47]. Furthermore, nicotine has been shown to activate PI3K signaling protein phosphorylation via α7 nAChR, which has been observed in studies of peripheral inflammatory diseases[6, 48]. As such, the α7 nAChR/PI3K pathway may represent a novel therapeutic target for studying neuroinflammation and neurological diseases.
To investigate the indirect neuroprotective effect of nicotine, we established a co-culture system of human-derived microglia and neurons. Co-culture models are often used in the study of neurological diseases to more closely mimic the actual disease state, such as in the study of Alzheimer's disease, where Luchena Celia et al. created a triple co-culture model consisting of mouse astrocytes, neurons, and microglia[49]. To investigate the effect of microglia on cortical neurons, Lopez-Lengowski Kara et al. generated microglia from human induced pluripotent stem cells (iPSCs) and co-cultured them with cortical neurons[50]. In another study, Beaulieu Jimmy et al. constructed a murine microglia-neuron co-culture system using transwell chambers to investigate neuroinflammatory therapeutic effects of oleic acid. This model involved N9 microglia communicating with PC12 neurons in a paracrine manner, similar to our own co-culture system[51]. In our study, we utilized transwell chambers to culture SH-SY5Y neurons in stratification with HMC3 microglia to investigate the indirect effect of nicotine on neurons through microglia. The results demonstrated an increase in neuronal survival, supporting the indirect neuroprotective effect of nicotine.
This study focused on establishment of an in vitro cellular model in which neurons were co-cultured alongside microglia to mimic neuroinflammation to study the potential interactions between microglia and neurons. In the future, we will attempt to select different functional neurons for study of the specific immunomodulatory effects of microglia on neuronal functionality. Moreover, we will continue in vivo animal experiments and construct different neurological disease models for the study of pharmacological effects of nicotine in more depth and detail.
In this study, we utilized the human-derived microglial cell line HMC3 to establish a cellular inflammation model induced by LPS. We then the anti-inflammatory effects of nicotine on HMC3 cells through nicotine intervention. Our results showed that nicotine reduced cellular inflammation levels and increased the release of BDNF and GDNF, which led to a reduction in neuronal apoptosis after co-culturing microglia and neurons. We hypothesize that nicotine exerts its anti-inflammatory effects through the nAChR/PI3K signaling pathway, as it acts directly on the α7 nAChR as an exogenous substance. Overall, our study sheds light on the immunomodulatory effects of nicotine on the central nervous system and its indirect neuroprotective effects, which may have potential medicinal value.