Filbertone enhances neurotrophic factors in human neuroblastoma SH-SY5Y cells and in mouse astrocyte C8-D1A cells
The neurotrophic factors, including BDNF, GDNF, and NGF, can be potential mediators for ameliorating neurodegenerative diseases, such as AD and PD [22]. Moreover, in our previous reports, we have shown that filbertone, one of the bioactive compounds, attenuates obesity-induced hypothalamic inflammation [19] and PD murine model [21]. Therefore, we explored whether filbertone affects the induction of neurotrophic factors in neuronal cells. To avoid cytotoxic effects of filbertone on human neuroblastoma SH-SY5Y cells, mouse astrocyte C8-D1A cells, and mouse hypothalamus mHypoE-N1 cells, we first evaluated cell viability using WST-8 assay in these cells treated with various concentrations (5, 10, 20, 40, 80, 160, and 320 µM) of filbertone. No cellular toxicity was observed the treatment with filbertone up to 40 µM in SH-SY5Y cells, up to 80 µM in C8-D1A cells, and up to 20 µM in mHypoE-N1 (Fig. 1A, 1B, Fig. S1A). Therefore, to investigate the effect of filbertone on BDNF transcription in SH-SY5Y cells, C8-D1A cells, and mHypoE-N1 cells, we treated these cells with filbertone at various concentrations (0, 2, 10, and 50 µM). Gene expression of BDNF significantly increased in the presence of filbertone at a concentration of 10 µM in SH-SY5Y cells and C8-D1A cells (Fig. 1C and 1D). Likewise, filbertone increased BDNF mRNA levels in mHypoE-N1 cells at 10 µM (Fig. S1B). GDNF and NGF are known to be expressed in CNS to support neuronal growth and maintain their function [7]. The data showed that filbertone elevated the levels of GDNF and NGF in C8-D1A cells (Fig. 1E and 1F). Taken together, these data indicate that filbertone induces expression of neurotrophic factors in neuronal cells.
Filbertone counteracts the decrease in neurotrophic factors and the activation of Nrf2 caused by HFD in the brain
Studies have shown that a HFD contributes to obesity and also impairs brain function, which is linked to AD and PD [23–26]. To investigate whether BDNF induction by filbertone protects HFD-induced PD, C57BL/6 wild type mice were fed on HFD with 0.2% filbertone (v/w) for 15 weeks. Consistent with previous report [20], the body weight increased by HFD was reduced by filbertone treatment (Fig. S2A and S2B). Reduction of body weight by filbertone was independent of food intake (Fig. S2C). In addition, we found that filbertone reduced weight of epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT), but not brown adipose tissue (BAT) (Fig. S2D). Nevertheless, the data reveals significant variations in body weight based on dietary consumption, but the brain weight did not show significant changes across the different dietary groups (Fig. S2E). Serum triglyceride (TG) levels increased with HFD consumption but decreased with filbertone (Fig. S2F). Similar to our previous study [21], mice fed a HFD showed accumulation of a-synuclein (a-syn) and loss of tyrosine hydroxylase (TH) in the midbrain (Fig. S2G). However, treatment of filbertone reversed expression of a-syn and TH (Fig. S2G). Previously, we reported that filbertone can protect against HFD-induced PD through PERK-TFEB activation [21]. PERK can act as an activator of Nrf2 [27, 28], which induces BDNF expression through transcription activation [8]. To determine whether Nrf2 activation is involved in neuronal protective function of filbertone, we measured the expression of phosphorylated Nrf2, HO-1, and BDNF in midbrain. We found that a HFD reduced Nrf2 phosphorylation and HO-1 protein expression (Fig. 2A). In addition, HFD decreased protein and mRNA levels of BDNF (Fig. 2A and 2B). Filbertone reversed the reduction in Nrf2 phosphorylation and HO-1 protein expression in the midbrain, which was caused by a HFD (Fig. 2A). Furthermore, filbertone led to an increase in protein and mRNA levels of BDNF compared to the group treated with a HFD alone (Fig. 2A and 2B). Several studies suggest that obesity directly impacts the hypothalamus, a crucial brain region for regulating energy homeostasis [29, 30]. This impact leads to inflammatory conditions and activation of glial cells, which in turn contributes to a decrease in BDNF [31]. In addition, an association between PD and hypothalamic dysfunction has been reported [32]. In line with the phenomena observed in the midbrain, a HFD led to a decrease in the mRNA levels of BDNF, NGF, and GDNF mRNA levels in the hypothalamus (Fig. 2C-2E). Filbertone mitigated the decrease in hypothalamic BDNF, NGF, and GDNF mRNA levels induce by a HFD (Fig. 2C-2E). Collectively, these findings indicate that filbertone plays a role in neuronal protection, associated with the upregulation of antioxidant molecules such as Nrf2 and HO-1, along with an increase in neurotropic factors including BDNF, NGF, and GDNF levels.
Filbertone attenuates neuronal inflammation and the activation of glial cells in the brain
Obesity is recognized as a contributing factor to the progression of PD due to elevated systemic inflammation [33]. To assess the anti-inflammatory effect of filbertone on HFD-induced neuronal inflammation, we measured the levels of pro-inflammatory cytokines in the midbrain and hypothalamus using qRT-PCR. Filbertone significantly reduces HFD-induced pro-inflammatory cytokines, such as TNF-a and IL-1b in the midbrain (Fig. 3A and B). Similarly, in the hypothalamus, mRNA levels of pro-inflammatory cytokines such as TNF-a, IL-1b, and MCP-1 were significantly increased by HFD. Filbertone treatment led to a reduction in the expression of pro-inflammatory cytokines (Fig. 3C-3E). Microglia, the resident macrophages of the CNS, along with astrocytes, which are abundant glial cells in the CNS, serve diverse functions including facilitating immune response in the brain [34]. Microglia and astrocytes are known to be responsive to HFD, which leads to their activation and subsequent inflammation [35, 36]. The consumption of HFD partially increased the levels of CD68, CD11b, and Iba-1, which are markers of activated microglia, in the hypothalamus. Filbertone led to a decrease in the levels of these markers (Fig. 3F-3H). Likewise, astrocyte activation was noted in the group fed a HFD; however, filbertone treatment resulted in a decrease in this HFD-induced astrocyte activation, as indicated by GFAP expression levels (Fig. 3I). HSP72, a marker of neuronal injury, was elevated in the hypothalamus due to HFD intake. Conversely, filbertone treatment led to a reduction in the HSP72 mRNA that were increased by the HFD (Fig. 3J). These data clearly suggest that filbertone could potentially serve as an anti-inflammatory agent and as an inhibitor of glial cells activity in HFD-induced brain damage.
Filbertone-mediated Nrf2 activation protects against PA-induced neurotoxicity through the increase of BDNF and HO-1
To confirm the effects of filbertone on HFD-induced damage, we treated SH-SY5Y, C8-D1A, and mHypoE-N1 cells with palmitic acid (PA). PA treatment significantly reduced the expression of BDNF mRNA, while it did not affect the expression of HO-1 mRNA in SH-SY5Y cells (Fig. 4A and 4B). The expression levels of both BDNF and HO-1 were reversed by co-treatment with PA and filbertone (Fig. 4A and 4B). In addition, treatment with filbertone alone increased the levels of BDNF and HO-1 mRNA (Fig. 4A and 4B). We also investigated whether filbertone could reduce pro-inflammatory cytokines, such as TNF-a and IL-6, in the presence of PA. The transcription levels of TNF-a and IL-6 significantly increased with PA treatment, while filbertone decreased PA-induced these cytokines in SH-SY5Y cells (Fig. 4C and 4D). To determine whether filbertone can mitigate PA-induced neurotoxicity, we performed a WST-8 assay. PA treatment reduced the viability of SH-SY5Y cells, whereas filbertone treatment restored cell viability (Fig. 4E). Nrf2/HO-1 signaling pathway is well known for its protective effects against neuroinflammation and neurotoxicity [37]. To verify filbertone’s protective mechanism against neurotoxicity through HO-1 in SH-SY5Y cells, we utilized the HO-1 inhibitor Zn (II)-protoporphyrin IX (ZnPP) in combination with filbertone. Treatment with ZnPP did not counteract the reduction in cell viability induced by PA (Fig. 4F), suggesting that HO-1 plays a crucial role in neuroprotection effects of filbertone. Similarly, the restoration of cell viability by filbertone was diminished by ZnPP in PA-treated C8-D1A and mHypoE-N1 cells (Fig. 4G-4J). To investigate whether filbertone alleviates PA-induced neurotoxicity through the activation of Nrf2 and subsequent upregulation of BDNF and HO-1, we measured the phosphorylation levels of Nrf2. PA treatment did not significantly affect Nrf2 phosphorylation, whereas filbertone treatment induced Nrf2 phosphorylation (Fig. 4K). These results suggest that filbertone protects against PA-induced neurotoxicity by upregulating BDNF and HO-1 levels through the Nrf2 activation.
Filbertone-mediated activation of Nrf2 protects against neurotoxicity induced by MPTP/MPP + through the induction of BDNF and HO-1
To gain insights into the mechanisms underlying PD, it is necessary to reproduce its biochemical, physiological, and morphological features in animal models. Beyond the HFD-induced PD approach [24, 23], the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is classically used to induce PD in mice [38]. Thus, to verify the effect of filbertone on neurotoxicity in PD, we investigated whether filbertone induces the expression of BDNF and HO-1 under MPTP-induced PD conditions.
Consistent with HFD-induced PD model, administration of filbertone with MPTP enhanced TH expression and reduced the accumulation of a-syn (Fig. 5A). To examine whether filbertone protects against MPTP-induced neurotoxicity through BDNF and HO-1 via Nrf2 activation, we measured protein expression of BDNF, HO-1, and phosphorylated Nrf2 in the midbrain. Treatment with MPTP showed decreased levels of phosphorylated Nrf2 and BDNF (Fig. 5B). However, the administration of filbertone led to an increase in the expression of phosphorylated Nrf2, BDNF, and HO-1 expression (Fig. 5B). These results suggest that the protective effects of filbertone on MPTP-induced neuronal damage are related to Nrf2-mediated BDNF and HO-1 expression. To investigate whether filbertone inhibits against neurotoxicity in vitro, we treated SH-SY5Y, C8-D1A, and mHypoE-N1 cells with MPP+. We observed that filbertone reversed the MPP+-induced reduction in cell viability (Fig. 5C). However, in the presence of ZnPP, filbertone failed to rescue the cell viability (Fig. 5D). To confirm the protective effects of filbertone via Nrf2 activation, we assessed Nrf2 phosphorylation. Phosphorylation of Nrf2 was decreased by MPP+ treatment, while filbertone increased Nrf2 phosphorylation (Fig. 5E). Similarly, filbertone mitigated the cells from MPP+-induced cytotoxicity in C8-D1A and mHypoE-N1 cells. However, ZnPP treatment diminished the protective effects of filbertone in C8-D1A and mHypoE-N1 (Fig. 5F-5I). The mRNA levels of BDNF were also reduced in MPP+ treatment, whereas filbertone significantly increased BDNF mRNA levels in SH-SY5Y cells (Fig. 5J). In addition, HO-1 expression was increased by filbertone treatment (Fig. 5K). Furthermore, filbertone successfully reduced the levels of TNF-a and IL-6 in MPP+-treated SH-SY5Y cells (Fig. 5L and 5M). Altogether, these finding suggest that filbertone might protect against MPTP/MPP+-induced neurotoxicity by activating Nrf2-induced BDNF and HO-1 expression and reducing inflammation.
The deficiency of Nrf2 negates the protective effects of filbertone against neurotoxicity in MPTP-treated mice
The aforementioned data suggest that filbertone might ameliorate neurotoxic effects via the activation of Nrf2 in mice treated with MPTP. Initially, we evaluated whether filbertone enhances the expression of antioxidant gene, including HO-1, alongside Nrf2 activation in neuronal cells. Filbertone was observed to augment both the phosphorylation of Nrf2 and the levels of HO-1 protein at a concentration of 20 µM in SH-SY5Y cells (Fig. 6A). Furthermore, filbertone elicited an increase in HO-1 mRNA levels in both C8-D1A cells and mHypoE-N1 cells, demonstrating a dose-responsive effect (Fig. 6B and 6C). A similar dose-dependent elevation was noted in the levels of BDNF expression following filbertone administration (Fig. 6D). To delve into the mechanisms by which filbertone prompts BDNF upregulation through Nrf2, an siRNA approach was employed to suppress Nrf2 in SH-SY5Y cells. This intervention revealed that the levels of BDNF and HO-1 mRNA increased by filbertone treatment in the presence of Nrf2, with no such increase observed in Nrf2-depleted cells (Fig. 6E). At the protein levels, the presence of Nrf2 was essential for the filberton-induced upregulation in BDNF and HO-1 expression (Fig. 6F), along with an increase in Nrf2 phophorylation (Fig. 6F). Nevertheless, in the absence of Nrf2, filbertone failed to upregulate BDNF and HO-1 expression or to activate Nrf2 (Fig. 6F), underscoring the necessity of Nrf2 activation for these processes. To validate these findings, MPTP and either filbertone or a control vehicle were administered to Nrf2-deficient mice. In Nrf2+/+ mice, filbertone treatment with MPTP significantly elevated BDNF and TH levels (Fig. 6G), whereas in Nrf2−/− mice, filbertone was ineffective in reversing the diminished BDNF and TH levels (Fig. 6G), highlighting the critical role of Nrf2 in the protective action of filbertone against PD. Collectively, these findings propose that the elevation of BDNF and HO-1 by filbertone, through Nrf2 activation, may contribute to alleviating the progression of PD.