Hydrogen sulfide ameliorates high glucose-induced pro-inflammation factors in HT-22 cells: Involvement of SIRT1-mTOR/NF-κB signaling pathway
Introduction
Diabetic encephalopathy (DE) is recognized as a complex and elusive complication of diabetes nowadays [1]. Patients with DE exhibit progressive alterations in brain structures and cognitive decline. These clinical manifestations are primarily caused by hyperglycemia-induced neurotoxicity [2]. Although multiple pathophysiological processes are involved in the progression of DE, accumulating evidence suggests that neuroinflammation plays a critical role in neurodegeneration induced by hyperglycemia [3], [4]. In diabetic animals, the expression of neuroinflammatory cytokines is significantly increased in the hippocampus, an important brain region for cognition [3]. High glucose increases pro-inflammatory responses in the immune cells such as microglia and astrocyte in the central nervous system [5], [6]. However, in neurons, changes triggered by high glucose in the expression of inflammatory cytokines and associated signaling molecules are less reported. The underlying molecular mechanisms about how hyperglycemia activates inflammation in the brain remain further elucidate.
Hydrogen sulfide (H2S) is a noteworthy endogenous gasotransmitter, which participates in regulating various pathophysiological processes [7]. During the progression of diabetes, the H2S levels declined gradually in the patients’ plasma [8], [9]. In line with these clinical findings, preclinical research revealed that hyperglycemia reduced H2S biosynthesis and increased its degradation in rodent models of diabetes [10], [11]. However, supplementation of H2S donor sodium hydrosulfide (NaHS) protected hyperglycemia-induced myocardial tissue damage in vivo and in vitro [12]. In the central nervous system, the production of endogenous H2S is mainly catalyzed by cystathionine-β-synthase (CBS) [13]. Exogenous H2S inhibited the expression of pro-inflammatory factors and decreased the generation of oxidative indicator reactive oxygen species in the mouse model of Alzheimer’s disease [14], [15]. However, it has not been fully investigated the effects and mechanisms of H2S on the inflammation in neurons under hyperglycemia.
Recent research has shown that H2S regulated inflammation-associated signaling molecules, such as silent information regulator-1 (SIRT1) [16], [17], mammalian target of rapamycin (mTOR) [18], [19] and nuclear factor-κB (NF-κB) [20], [21], [22]. H2S upregulated the expression of SIRT1 to prevent homocysteine-induced neurotoxicity in PC12 cells [23]. H2S increased SIRT1 levels in the rat hippocampus, which reduced chronic mild stress-induced depressive-like behavior [24]. However, it is unknown if SIRT1 and its downstream signaling in the neuron are interrupted by high glucose. mTOR is a crucial protein kinase involved in regulating multiple diseases, including cancer, metabolic and neurological diseases. mTOR signaling was dysregulated in type 2 diabetes as well as inflammation [25]. Upregulation of SIRT1 inhibited the expression of mTOR and subsequently attenuated inflammation in bleomycin-induced scleroderma mice [26] and high-fat-diet mice [27], suggesting SIRT1 might be an upstream signaling molecule of mTOR. However, SIRT1-mTOR singling pathway has not been investigated in neurons with high glucose. Activation of mTOR modulated its downstream ubiquitous transcriptional factor NF-κB to facilitate the production of pro-inflammatory cytokines [28], [29]. H2S plays an inhibitory role in lipopolysaccharide (LPS)-triggered inflammatory response via blocking the transactivation of NF-κB in endothelial cells [30]. Collectively, we predicted that H2S might modulate inflammation in neuronal cells by inhibiting the activated SIRT1-mTOR/NF-κB signaling pathway.
In the current study, we applied a neuronal cell line HT-22, which was widely used as an in vitro model of neurodegenerative disease [31], [32], to explore the function of high glucose on H2S and its endogenous synthase CBS, as well as pro-inflammatory cytokines expression and SIRT1-mTOR/NF-κB signaling pathway. Furthermore, we examined whether NaHS or S-adenosylmethionine (SAMe, an allosteric activator of CBS) could attenuate the high glucose-induced inflammatory effect.
Section snippets
Cell line and reagents
The mouse hippocampal neuronal cell line HT-22 cells were kindly gifted from the Research Center for Neurobiology of Xuzhou Medical University (Xuzhou, China). NaHS and SAMe (97% purity) were purchased from Aladdin Reagent Co., Ltd (Shanghai, China) and MedChemExpress (Monmouth Junction, NJ, USA), respectively. Resveratrol and glucose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against CBS and β-actin were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA)
The efficacy of NIR-NP to detect H2S in HT-22 cells
The compound NIR-NP was successfully synthesized and confirmed by 1H NMR (Figure S2) and HRMS (Figure S3). The probe exhibited excellent properties, including an off-on response to H2S (Fig. 1A), high selectivity for H2S over other analytes (Fig. 1B and 1C), concentration- (Fig. 1D and 1E), time- (Fig. 1F and 1G) and pH- (Fig. 1H and 1I) dependent fluorescence intensity.
We then examined the effect of NIP-NP on cell viability using MTT assay. Different concentrations of NIP-NP (5, 10, 20, 40,
Discussion
In this study, the NIR-NP probe was successfully synthesized and showed excellent selectivity and sensitivity to detect endogenous H2S levels in HT-22 cells. Using this probe, we showed that high glucose reduced the H2S levels in neurons. Furthermore, high glucose exposure decreased the level of CBS, SIRT1 and elevated pro-inflammatory cytokines IL-6, IL-1β and TNF-α along with mTOR/NF-κB signaling activation. Importantly, NaHS or SAMe reversed the level of H2S, CBS, pro-inflammatory cytokines
Conclusions
In summary, our results have shown that high glucose activated inflammation through reducing H2S and its synthesizing enzyme CBS in neurons. Conversely, restore H2S levels inhibited inflammation activation. As inflammation is considered to promote neurodegenerative progression in DE, exogenous H2S treatment or enhancing endogenous H2S synthesis might prevent these inflammatory processes and delay neurodegeneration. Our findings laid the groundwork for a profound understanding of DE pathogenesis
Funding
This work was supported by the National Natural Science Foundation of China (81671069), the Open Research Project of Jiangsu Key Laboratory of Brain Disease Bioinformation (JSBL201802), the Open Research Project of Jiangsu Key Laboratory of Immunity and Metabolism (JSKIM201802) and the Science and Technology Planning Project of Xuzhou (KC20168).
CRediT authorship contribution statement
Xinrui Li: Investigation, Project administration, Writing - original draft, Writing - review & editing. Peiquan Yu: Investigation, Project administration, Writing - review & editing. Yinghua Yu: Supervision, Writing - review & editing. Ting Xu: Investigation, Project administration. Jiao Liu: Investigation, Project administration. Yuan Cheng: Investigation, Project administration. Xia Yang: Investigation, Project administration. Xiaoying Cui: Writing - review & editing. Cui Yin: Investigation.
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These authors contributed equally to this work.