Re: 성상교세포에서 Nrf2 활성 매개, 타우린의 신경세포 보호기전 탐구 2023 논문

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Open AccessArticle

Taurine Chloramine-Mediated Nrf2 Activation and HO-1 Induction Confer Protective Effects in Astrocytes

by 

Song-I Seol

 1,

In Soon Kang

 2,

Ji Seok Lee

 2,3,

Ja-Kyeong Lee

 1,* and

Chaekyun Kim

 2,3,*

1

Department of Anatomy, Inha University School of Medicine, Incheon 22212, Republic of Korea

2

Laboratory of Leukocyte Signaling Research, Department of Pharmacology, Inha University School of Medicine, Incheon 22212, Republic of Korea

3

BK21, Program in Biomedical Science & Engineering, Inha University, Incheon 22212, Republic of Korea

*

Authors to whom correspondence should be addressed.

Antioxidants 2024, 13(2), 169; https://doi.org/10.3390/antiox13020169

Submission received: 16 December 2023 / Revised: 23 January 2024 / Accepted: 25 January 2024 / Published: 29 January 2024

(This article belongs to the Special Issue Natural Bioactive Compounds Exerting Health Promoting Effects through Ameliorating Oxidative Stress)

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Abstract

Taurine is ubiquitously distributed in mammalian tissues, with the highest levels in the brain, heart, and leukocytes. Taurine reacts with hypochlorous acid (HOCl) to produce taurine chloramine (Tau-Cl) via the myeloperoxidase (MPO) system. In this study, we elucidated the antioxidative and protective effects of Tau-Cl in astrocytes. Tau-Cl increased the expression‎ and nuclear translocation of nuclear factor E2-related factor (Nrf2) and the expression‎ of Nrf2-regulated antioxidant genes, including heme oxygenase 1 (HO-1). Nrf2 activity is negatively regulated by Kelch-like ECH-associated protein 1 (Keap1). Tau-Cl decreased the level of the reduced thiol groups of Keap1, resulting in the disruption of the Keap1-Nrf2 complex. Consequently, Tau-Cl rescued the H2O2-induced cell death by enhancing HO-1 expression‎ and suppressing reactive oxygen species. In conclusion, Tau-Cl confers protective effects in astrocytes by disrupting the Keap1-Nrf2 complex, thereby promoting Nrf2 translocation to the nucleus, wherein it binds to the antioxidant response element (ARE) and accelerates the transcription of antioxidant genes. Therefore, in astrocytes, the activation of the Keap1-Nrf2-ARE pathway by Tau-Cl may increase antioxidants and anti-inflammatory mediators as well as other cytoprotective proteins, conferring protection against brain infection and injury.

타우린은 

포유류 조직에 어디에나 분포하며 

뇌(신경), 심장, 백혈구에서 가장 높은 수치를 보입니다. 

타우린은 

미엘로퍼옥시다아제(MPO) 시스템을 통해 

차아염소산(HOCl)과 반응하여 타우린 클로라민(Tau-Cl)을 생성합니다. 

이 연구에서는 

성상교세포에서 

Tau-Cl의 항산화 및 보호 효과를 규명했습니다. 

Tau-Cl은 

핵 인자 E2 관련 인자(Nrf2)의 발현과 핵 전위를 증가시키고 

헴 옥시게나제 1(HO-1)을 포함한 Nrf2 조절 항산화 유전자의 발현을 증가시켰습니다. 

Nrf2 활성은 켈치 유사 ECH 관련 단백질 1(Keap1)에 의해 음성적으로 조절됩니다. 

Tau-Cl은 Keap1의 환원 티올 그룹의 수준을 감소시켜 

Keap1-Nrf2 복합체의 파괴를 초래했습니다. 

결과적으로 Tau-Cl은 

HO-1 발현을 강화하고 

활성 산소 종을 억제하여 

H2O2에 의한 세포 사멸을 구제했습니다. 

결론적으로, 

Tau-Cl은 

성상세포에서 Keap1-Nrf2 복합체를 파괴하여

 Nrf2의 핵으로의 전위를 촉진함으로써 

항산화 반응 요소(ARE)와 결합하여 

항산화 유전자의 전사를 촉진함으로써 보호 효과를 부여합니다. 

따라서 

성상교세포에서 

Tau-Cl에 의한 Keap1-Nrf2-ARE 경로의 활성화는 

항산화제와 항염증 매개체 및 기타 세포 보호 단백질을 증가시켜 

뇌 감염 및 손상에 대한 보호 기능을 부여할 수 있습니다.

Keywords: 

astrocytes; myeloperoxidase (MPO); nuclear factor E2-related factor (Nrf2); heme oxygenase 1 (HO-1); Kelch-like ECH-associated protein 1 (Keap1)

Graphical Abstract

1. Introduction

Taurine (2-aminoethansulfolic acid) is one of the most abundant nonproteinogenic amino acids; it is ubiquitously distributed in mammalian tissues, with the highest levels found in the heart, brain, and leukocytes [1]. Taurine concentrations range from 1 to 20 μmol/g in the brain and from 20 to 50 mM in human neutrophils [2,3,4]. It plays a vital role in many biological processes, including central nervous system (CNS) and retinal development, membrane stabilization, calcium mobilization, neurotransmission, reproduction, and detoxification [1,5,6]. Furthermore, taurine confers protective effects on infections and inflammation [6,7]. It reacts with the highly toxic hypochlorous acid (HOCl), which is released by the myeloperoxidase (MPO) system of leukocytes, to generate the less toxic taurine chloramine (Tau-Cl) [8]. Tau-Cl inhibits the production of proinflammatory mediators and increases the expression‎ of several antioxidant enzymes. Therefore, taurine ameliorates inflammation by eliminating the highly toxic HOCl as well as modulating inflammatory mediators via Tau-Cl action [5,6].

The brain contains high concentrations of taurine, which functions as an osmoregulator and a neuromodulator [9,10]. Taurine exhibits neuroprotective effects in neurons and astrocytes and animal models of neurological disorders such as ischemic stroke and inflammation [11,12,13,14,15,16]. Furthermore, it decreases the expression‎ of apoptotic protein during ischemic injury [12,17] and maintains intracellular Ca2+ homeostasis [18], thereby attenuating apoptotic neuronal death [12,19]. Under pathological conditions, neutrophils infiltrate the regions of inflammatory areas or infected tissues. Neutrophils are the most abundant leukocytes and contain high concentrations of MPO and taurine; therefore, activated neutrophils produce considerable amounts of Tau-Cl. In our previous study, we demonstrated that neutrophils infiltrate ischemic injury areas and that Tau-Cl exerts robust neuroprotective effects using a rat middle cerebral artery occlusion (MCAO) model [20]. Tau-Cl significantly decreased the infarct volume and neurological deficits and promoted motor function. Furthermore, it significantly increased the levels of antioxidant enzymes such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), glutamate–cysteine ligase catalytic (GCLC), and peroxiredoxin-1 (Prx-1) in the post-ischemic brain and in BV2 cells, a microglial cell line [20].

Astrocytes are the most abundant glial cell type in the CNS that outnumbers neurons [21]. Astrocytes perform various functions in the normal brain, including maintaining a stable extracellular environment by regulating the balance of ions and fluid, which are essential for neurons [22]. Furthermore, they control the energy supply to the neurons [23,24] and regulate synapse formation and neurotransmitter turnover [25,26]. Astrocytes are the primary protective cells in the brain. Under pathological conditions, astrocytes confer neuroprotection by regulating ionic balance and energy metabolism and by inducing scar formation, which protects adjacent neural tissues via separating brain injury-induced lesions [21].

Nuclear factor E2-related factor (Nrf2) is a basic-region leucine zipper transcription factor that plays a vital role in oxidative stress response. Nrf2 binds to the antioxidant response element (ARE) located in the promoter regions of several antioxidant and detoxifying genes, including HO-1, NQO1, glutathione S-transferases (GST), and GCL modifier (GCLM) [27,28,29,30,31]. Under basal conditions, Nrf2 activity is tightly regulated by Kelch-like ECH-associated protein 1 (Keap1), an E3 ubiquitin ligase substrate adaptor [32,33]. Keap1 targets Nrf2 for proteasomal degradation. However, during oxidative or electrophilic stress, Keap1 is inactivated by the modification of its highly reactive cysteine residues; as a result, Nrf2 escapes ubiquitination, accumulates within the cell, and translocates to the nucleus, where it promotes its antioxidant transcription program [34,35,36]. Tau-Cl increases the cytosolic accumulation and nuclear translocation of Nrf2, resulting in the high expression‎ of several Nrf2-regulated antioxidant enzymes [20,37,38,39,40]. However, to the best of our knowledge, the mechanism by which Tau-Cl regulates Nrf2 activation remains unclear.

In this study, we investigated the antioxidant effects of Tau-Cl in astrocytes C6 cells. We determined that Tau-Cl regulates the expression‎ and nuclear translocation of Nrf2 and the subsequent expression‎ of Nrf2-induced genes in C6 cells. Furthermore, we examined the molecular mechanism by which Tau-Cl triggers Nrf2 activation. Ultimately, we explored the protective effects exerted by Tau-Cl on astrocytes.

1. 소개
타우린(2- 아미노에탄설폴릭산)은

가장 풍부한 비단백질 생성 아미노산 중 하나로

포유류 조직에 편재적으로 분포하며

심장, 뇌, 백혈구에서 가장 높은 수치가 발견됩니다 [1].

타우린 농도는

뇌에서 1 ~ 20 μmol/g,

인간 호중구에서 20 ~ 50mM입니다[2,3,4].

타우린은

중추신경계(CNS) 및 망막 발달,

막 안정화,

칼슘 동원,

신경 전달,

생식 및 해독을 포함한 많은 생물학적 과정에서 중요한 역할을 합니다[1,5,6].

또한

타우린은

감염과 염증에 대한 보호 효과를 부여합니다 [6,7].

타우린은

백혈구의 미엘로퍼옥시다제(MPO) 시스템에 의해 방출되는

독성이 강한 차아염소산(HOCl)과 반응하여

독성이 적은 타우린 클로라민(Tau-Cl)을 생성합니다[8].

타우-Cl은

전 염증 매개체의 생성을 억제하고

여러 항산화 효소의 발현을 증가시킵니다.

따라서

타우린은

독성이 강한 HOCl을 제거하고

Tau-Cl 작용을 통해 염증 매개체를 조절함으로써 염증을 개선합니다 [5,6].

뇌에는 고농도의 타우린이 함유되어 있으며,

이는 산화질소 조절제와

신경 조절제로 기능합니다 [9,10].

타우린은

뉴런과 성상세포 및 허혈성 뇌졸중 및 염증과 같은

신경 장애의 동물 모델에서 신경 보호 효과를 나타냅니다 [11,12,13,14,15,16]16]

타우린은 신경세포와 성상세포에서 신경 보호 효과를 나타냅니다.

또한

허혈성 손상 시 세포사멸 단백질의 발현을 감소시키고[12,17],

세포 내 Ca2+ 항상성을 유지하여[18],

세포사멸성 신경세포 사멸을 약화시킵니다[12,19].

병리학적인 조건에서

호중구는 염증 부위나 감염된 조직 부위에 침투합니다.

호중구는

가장 풍부한 백혈구이며

고농도의 MPO와 타우린을 함유하고 있으므로

활성화된 호중구는 상당한 양의 Tau-Cl을 생성합니다.

이전 연구에서 우리는 쥐 중대뇌동맥 폐색(MCAO) 모델을 사용하여 호중구가 허혈성 손상 부위에 침윤하고 Tau-Cl이 강력한 신경 보호 효과를 발휘한다는 것을 입증했습니다[20]. Tau-Cl은 경색 부피와 신경학적 결손을 현저히 감소시키고 운동 기능을 촉진했습니다. 또한 허혈 후 뇌와 미세아교세포주인 BV2 세포에서 헴 산소화 효소-1(HO-1), NAD(P)H:퀴논 산화 환원 효소 1(NQO1), 글루타메이트-시스테인 리가제 촉매(GCLC), 퍼옥시레독신-1(Prx-1) 같은 항산화 효소의 수준을 크게 증가시켰다[20]는 것을 확인할 수 있었습니다.

성상교세포는

중추신경계에서 가장 풍부한

신경교세포 유형으로 뉴런보다 더 많습니다[21].

성상교세포는

뉴런에 필수적인 이온과 체액의 균형을 조절하여

세포 외 환경을 안정적으로 유지하는 등

정상적인 뇌에서 다양한 기능을 수행합니다 [22].

또한

뉴런으로의 에너지 공급을 조절하고[23,24]

시냅스 형성과 신경전달물질 전환을 조절합니다[25,26].

성상교세포는

뇌의 주요 보호 세포입니다.

병리학적인 조건에서

성상 세포는

이온 균형과 에너지 대사를 조절하고

흉터 형성을 유도하여

뇌 손상으로 인한 병변을 분리하여

인접한 신경 조직을 보호함으로써 신경 보호 기능을 수행합니다 [21].

핵 인자 E2 관련 인자(Nrf2)는

산화 스트레스 반응에 중요한 역할을 하는

염기 영역 류신 지퍼 전사인자입니다.

Nrf2는

여러 항산화 및 해독 유전자의 프로모터 영역에 위치한 항산화 반응 요소(ARE)에 결합하며,

여기에는 HO-1, NQO1, 글루타치온 S-전달효소(GST), GCL 조절인자(GCLM) 등이 포함됩니다[27,28,29,30,31].

기저 조건에서 Nrf2 활성은

E3 유비퀴틴 리가제 기질 어댑터인 켈치 유사 ECH 관련 단백질 1(Keap1)에 의해

엄격하게 조절됩니다 [32,33].

Keap1은

프로테아좀 분해를 위해

Nrf2를 표적으로 삼습니다.

그러나 산화적 또는 친유전적 스트레스 동안 Keap1은 반응성이 높은 시스테인 잔기의 변형에 의해 비활성화되고, 그 결과 Nrf2는 유비퀴틴화를 벗어나 세포 내에 축적되어 핵으로 이동하여 항산화 전사 프로그램을 촉진합니다 [34,35,36].

Tau-Cl은

Nrf2의 세포질 축적과 핵 전위를 증가시켜

여러 Nrf2 조절 항산화 효소의 높은 발현을 초래합니다 [20,37,38,39,40].

그러나

우리가 아는 한,

Tau-Cl이 Nrf2 활성화를 조절하는 메커니즘은 아직 명확하지 않습니다.

2. Materials and Methods2.1. C6 Cell Culture and Tau-Cl Treatment

C6 astroglioma cells (American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 1% penicillin, and streptomycin at 37 °C in a 5% CO2 incubator. Cells were prepared a day before Tau-Cl treatment. Tau-Cl was synthesized by adding equimolar amounts of NaOCl (Aldrich Chemical, Milwaukee, MI, USA) to taurine (Sigma), and the formation of Tau-Cl was monitored by measuring UV absorption (200~400 nm) [41]. Tau-Cl was diluted to 200 μM in DMEM containing 10% FBS. C6 cells were treated with Tau-Cl for the indicated time points.

2.2. Nuclear and Cytoplasmic Extract Preparation

C6 cells were lysed with solution A (0.5% Triton X-100, 0.5% NP-40, 10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, and 1 mM DTT) containing complete Mini Protease Inhibitor Cocktail tablet (Roche diagnostics, Basel, Switzerland). Lysates were centrifuged at 17,500× g for 5 min at 4 °C. The supernatant containing cytoplasmic protein was collected and stored at −80 °C. Pellets were lysed with solution B (10% glycerol, 20 mM HEPES [pH 7.9], 0.4 M NaCl, 1 mM EDTA, and 1 mM DTT) containing complete Mini Protease Inhibitor Cocktail tablet. Lysates were centrifuged at 17,500× g for 10 min at 4 °C. The supernatant containing the nuclear protein was collected and stored at −80 °C.

2.3. Immunoblotting

Cells were lysed with RIPA buffer (0.25% sodium-deoxycholate, 150 mM NaCl, 50 mM Tris-HCl [pH 7.4], and 1% NP-40) containing complete Mini Protease Inhibitor Cocktail tablet. Lysates were centrifuged at 12,000× g for 10 min at 4 °C and the supernatants were loaded into 12% SDS-PAGE gels. The primary antibodies used were as follows: anti-Nrf2 (sc-722, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-α-tubulin (GTX112141, 1:10,000; GeneTex, Irvine, CA, USA), anti-lamin B1 (12987-1-AP, 1:2000; Proteintech, Rosemont, IL, USA), anti-HO-1 (43966S, 1:2000; Cell Signaling, Danvers, MA, USA), anti-NQO1 (ab34173, 1:5000; Abcam, Cambridge, UK), anti-GCLC (ab207777, 1:2000; Abcam), and anti-GCLM (ab153967, 1:1000; Abcam). Horseradish peroxidase-conjugated secondary antibodies (AP132P, 1:4000; Merck Millipore, Darmstadt, Germany) were used. Immunoblots were detected using a chemiluminescence kit (Merck Millipore).

2.4. Immunocytochemistry

C6 cells (4 × 104) were treated with Tau-Cl (200 μM) for 3 or 9 h or with taurine (200 μM) for 9 h, and then fixed with 4% paraformaldehyde for 20 min. The anti-Nrf2 antibody (16396-1-AP, 1:200; Proteintech) was incubated overnight, followed by incubation with FITC-labeled anti-IgG antibody (AP132F, 1:200; Merck Millipore). Cells were counterstained with 4′,-6-diamidino-2-phenylindole (DAPI; H-1200; Vector Laboratories, Newark, CA, USA) to visualize the nuclei. Images were observed under a Zeiss LSM 510 META microscope (Carl Zeiss Meditec AG, Jena, Germany).

2.5. Modification of the Thiol Groups of Keap1 by Tau-Cl

C6 cells were incubated with 200 or 500 μM Tau-Cl for 5, 10, and 20 min, and then washed three times with PBS. Thereafter, the cells were lysed with RIPA buffer containing 2 mM PMSF and 20 μg/mL chymostatin. The lysate was incubated with 1 μg/mL biotin polyethyleneoxide iodoacetamide (Biotin-IAA) (Sigma) for 18 h. The mixtures were incubated with 20 μL of streptavidin–agarose beads (50% slurry) (Pierce, Rockland, IL, USA) for 1 h at 4 °C with rotation and centrifuged at 10,000× g for 1 min. The pellets containing reduced Keap1 were washed three times with PBS and subjected to 10% SDS-PAGE, followed by immunoblotting with an anti-Keap1 antibody (SC-15246, 1:500; Santa Cruz Biotechnology). The supernatants containing oxidized Keap1 were incubated with 1 μg/mL biotin maleimide (Sigma) or Biotin-IAA for 18 h. Then, the mixtures were incubated with streptavidin–agarose beads, and the pellets were analyzed by immunoblotting as with reduced Keap1 samples.

2.6. Reaction of the Thiol Groups of Keap1 with Tau-Cl

Titrants of thiol groups, including 2.2′-dipyridyl disulfide and 4,4′-dipyridyl disulfide, react with the thiols of Keap1 [42]. To determine the potency with which Tau-Cl reacts with thiol groups, 0.5 and 2 μM Tau-Cl or 4,4′-dipyridyl disulfide (Sigma) were added to a solution of 0.5 μM Keap1 (Sino Biological, Beijing, China) solution dissolved in PBS. Immediately after mixing, the changes in the absorbance at 325 nm were monitored for 20 min at intervals of 5 min.

2.7. Cell Viability Assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to measure cell viability. C6 cells were pre-treated with 50, 100, or 200 μM of Tau-Cl or 200 μM of taurine for 9 h, followed by treatment with 300 μM of H2O2 for 1 h in DMEM containing 10% FBS. After 24 h, the cells were incubated with 1 mg/mL MTT (Sigma) for 1 h. The medium was removed and 500 μL of DMSO was added to solubilize the formazan product. The mixture (100 μL) was added to a 96-well plate and the absorbance was measured at 540 nm using a microplate reader.

2.8. siRNA Transfection

C6 cells (4 × 104) were seeded in 24-well plates at 24 h before transfection. Rat HO-1-specific siRNA (siHO-1; 5′-GUC AUG GCC ACU UUG AUA UCA GUG T-3′ and 5′-ACA CUG AUA UCA AAG UGG CCA UGA CGC-3′) and a nonspecific siRNA (siCon; 5′-CGU UAA UCG CGU AUA AUA CGC GUA T-3′ and 5′-AUA CGC GUA UUA UAC GCG AUU AAC GAC-3′) were purchased from Integrated DNA Technologies, Inc. (Coralville, IA, USA). The siHO-1 or siCon (both 40 pM) was mixed with 1 μL/well of Oligofectamine reagent (Invitrogen, Carlsbad, CA, USA) in Opti-MEM (Invitrogen) according to the manufacturer’s instructions. The siRNA-Oligofectamine complexes were added to C6 cells. After incubation for 15 h, the cells were treated with 200 μM Tau-Cl.

2.9. Reactive Oxygen Species (ROS) Quantification

C6 cells (4 × 104) were seeded into 24-well plates and cultured for 24 h. C6 cells were pre-treated with 200 μM of Tau-Cl or 200 μM of taurine for 9 h, followed by treatment with 300 μM of H2O2 for 1 h. Cells were then incubated in DMEM containing 10 μM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Thermo Fisher Scientific, Waltham, MA, USA) for 30 min. After washing cells with PBS, fluorescence and differential interference contrast images were obtained using a Zeiss microscope. Quantified fluorescence changes were obtained using ImageJ (http://rsbweb.nih.gov/ij/).

2.10. Statistical Analysis

The statistical analyses were performed using analysis of variance (ANOVA) followed by the Newman–Keuls test. Results are presented as the mean ± standard error of the mean (SEM). Statistical significance was accepted for p value < 0.05. The analyses were performed using PRISM software 5.0 (Graph Pad Software, Boston, MA, USA).

3. Results3.1. Tau-Cl Enhances the Expression‎ and Nuclear Translocation of Nrf2 in C6 Cells

To investigate whether Tau-Cl induces Nrf2 activation in C6 cells, the cells were treated with 200 μM Tau-Cl for 3, 6, 9, 12, or 24 h. The total amount of Nrf2 gradually increased and reached the maximum level after 9 h of treatment (Figure 1A,B). The elevated Nrf2 levels persisted until 24 h (Figure 1A,B). Next, we investigated whether Tau-Cl induces Nrf2 translocation from the cytoplasm to the nucleus. Nuclear Nrf2 levels significantly increased after 9 h of Tau-Cl treatment and the enhanced level was maintained until 12 h (Figure 1C,D); in contrast, cytoplasmic Nrf2 levels gradually decreased during the same period (Figure 1C,D). In the case of taurine (200 μM for 9 h), while total Nrf2 levels moderately increased (Figure 1A,B), the nuclear translocation of Nrf2 remained inconclusive (Figure 1C,D). To confirm‎ the translocation of Nrf2 from the cytoplasm to the nucleus by Tau-Cl, double immunofluorescence staining was performed using an anti-Nrf2 antibody and DAPI. Under normal conditions, Nrf2 was detected in the cytoplasm of C6 cells (Figure 1E). However, it was detected in the nucleus as early as 3 h after Tau-Cl treatment, with a significant increase in nuclear accumulation at 9 h, when co-localization of Nrf2 and DAPI was evident (Figure 1F,G). Consistent with the results obtained from immunoblotting, the nuclear translocation of Nrf2 was not evident after taurine treatment (200 μM for 9 h) (Figure 1H). Together, these results suggest that Tau-Cl increases the total amount of Nrf2 and induces its translocation from the cytoplasm to the nucleus in astrocytes.

Figure 1. The upregulation and cytoplasmic-to-nuclear translocation of Nrf2 by Tau-Cl in C6 cells. C6 cells were treated with 200 μM of Tau-Cl for 3, 6, 9, 12, or 24 h or with 200 μM of taurine for 9 h. Nrf2 levels in total cell lysates (A,B) or in nuclear and cytosolic fractions (C,D) were determined by immunoblotting. Representative images are presented in A and C, and quantified results are presented as mean ± SEM (n = 3) in (B,D). (E–H) C6 cells were treated with 200 μM of Tau-Cl for 3 or 9 h or with 200 μM of taurine for 9 h. Double immunofluorescence staining was performed using anti-Nrf2 antibody and DAPI. Arrowheads indicate Nrf2 in the nucleus. The scale bar represents 50 μm. * p < 0.05 versus untreated controls.

3.2. Tau-Cl Induces the Upregulation of Various Antioxidant Enzymes Downstream of Nrf2

Because Nrf2 induces the expression‎ of various antioxidant genes, we investigated whether Tau-Cl upregulates the expression‎ of antioxidant genes in C6 cells. C6 cells were treated with 200 μM Tau-Cl for 3, 6, 9, 12, or 24 h. HO-1 expression‎ significantly increased after 6 h of 200 μM Tau-Cl treatment, reached the maximum level at 12 h, and subsequently decreased (Figure 2A,B). Similarly, Tau-Cl significantly induced NQO1, GCLC, and GCLM in C6 cells; however, the timing of induction differed for each gene (Figure 2). Collectively, these results suggest that Tau-Cl activates Nrf2 and induces the upregulation of various antioxidant genes in astrocytes.

Figure 2. Induction of various antioxidant genes by Tau-Cl in C6 cells. C6 cells were treated with Tau-Cl (200 μM) for 3, 6, 9, 12, or 24 h and protein levels of HO-1, NQO1, GCLM, GCLC, and β-actin were determined by immunoblotting. Representative images of the protein levels are in (A) and quantified results are presented in (B–E) as mean ± SEM (n = 3). * p < 0.05 and ** <0.01 versus untreated controls.

3.3. Tau-Cl Induces the Disulfide Bond Formation of Keap1

Keap1 binding leads to the anchoring of Nrf2 in the cytoplasm, which is attached to the actin cytoskeleton. Inducers and electrophiles disrupt the Keap1-Nrf2 complex, and Nrf2 translocates to the nucleus, where it binds to the ARE region and initiates transcription [42]. Because Tau-Cl activates Nrf2 and increases the expression‎ of downstream genes including HO-1, NQO-1, and GCLM (Figure 1 and Figure 2), we elucidated whether Tau-Cl disrupts the binding between Keap1 and Nrf2. Maleimide and iodoacetamide (IAA) are known to bind to free thiol (-SH) groups [43]. Tau-Cl treatment decreased IAA-bound Keap1 at 5 and 10 min (Figure 3A,B), suggesting that Tau-Cl increases the oxidation of the thiol groups of Keap1 to generate disulfide bonds. Next, we attempted to identify oxidized Keap1; however, it remained undetectable within our experimental system. In addition, Tau-Cl decreased IAA-bound beta-actin (Figure 3B,C; β-actin (R)), suggesting its ability to oxidize thiol groups.

Figure 3. Modification of the thiol groups of Keap1 by Tau-Cl. (A–C) C6 cells were incubated with 200 or 500 μM Tau-Cl and the cell lysates were reacted with Biotin-iodoacetamide (IAA). IAA-bound Keap1 was precipitated, subjected to SDS-PAGE, and determined by immunoblotting with anti-Keap1 antibody. The quantified results are presented as mean ± SEM (n = 5 for (A,B), n = 4 for (B)). * p < 0.05 and ** <0.01 versus untreated controls. (D,E) 0.5 and 2 μM Tau-Cl (D) or 4,4′-dipyridyl disulfide (E) were added to 0.5 μM Keap1 solution, and the changes in the absorbance at 325 nm were monitored for 20 min at intervals of 5 min. The quantified results are presented as mean ± SEM (n = 3 for (D) and n = 1 for (E)). * p < 0.05 versus 0.5 μM Keap1.

The thiol groups of Keap1 react with 4,4′-dipyridyl disulfide and reveal distinct UV absorptions; therefore, 4,4′-dipyridyl disulfide is used as a spectroscopic titration reagent for the thiol groups [42]. The addition of Tau-Cl to Keap1 solution decreased its UV absorption at 325 nm in a manner similar to 4,4′-dipyridyl disulfide (Figure 3D,E), suggesting that Tau-Cl modifies the thiol groups of Keap1. Nevertheless, this method has experimental limitations owing to the difficulties associated with obtaining a sufficiently high concentration of Keap1 to reveal significant absorbance changes. Moreover, Keap1 undergoes rapid oxidation under normal laboratory conditions.

3.4. Tau-Cl Suppresses H2O2-Induced Cell Death in C6 Cells

To determine whether Tau-Cl confers protective effects in C6 cells, we examined the cell viability of C6 cells after H2O2 treatment in the presence or absence of Tau-Cl (Figure 4A). The viability of C6 cells was decreased to 49.0 ± 2.2% after treatment with 300 μM H2O2 for 1 h (Figure 4B). However, the pretreatment of C6 cells with 100 and 200 μM Tau-Cl for 9 h significantly improved cell survival to 59.2 ± 2.0% and 64.2 ± 3.0%, respectively (Figure 4B). In contrast, pretreatment with 200 μM taurine for 9 h did not improve the cell viability (Figure 4B). Moreover, cell survival was increased after treatment of 200 μM Tau-Cl for 6, 9, and 12 h (Figure 4C). These findings suggest that Tau-Cl inhibits astrocyte cell death induced by H2O2.

Figure 4. Protection of H2O2-treated C6 cells by Tau-Cl. (A) Schematic diagram of time points for treatment of Tau-Cl and H2O2 and cell viability assay. (B) C6 cells were pre-treated with Tau-Cl (50, 100, or 200 μM) or taurine (200 μM) for 9 h and then treated with H2O2 (300 μM) for 1 h. (C) C6 cells were pre-treated with 200 μM of Tau-Cl for 3, 6, 9, or 12 h and then treated with H2O2 (300 μM) for 1 h. For all experiments, MTT assays were performed 24 h after H2O2 treatment and cell viabilities are presented as mean ± SEM (n = 8). *** p < 0.001 versus untreated controls, # p < 0.05 and ### p < 0.001 between indicated groups.

3.5. Tau-Cl-Mediated HO-1 Induction Is Responsible for the Protective Effects in C6 Cells

We examined whether the Tau-Cl-mediated protective effect is related to the upregulation of antioxidant genes in C6 cells, particularly HO-1, using siRNA-mediated HO-1 knockdown (Figure 5A). HO-1 was significantly induced in C6 cells after H2O2 treatment (100 μM, 1 h) (2.2-fold) (Figure 5B,C). Furthermore, preincubation with 200 μM Tau-Cl for 9 h further enhanced H2O2-induced HO-1 upregulation by 6.8-fold (Figure 5B,C), indicating that Tau-Cl augmented H2O2-induced HO-1 upregulation. Moreover, Tau-Cl preincubation enhanced H2O2-induced Nrf2 expression‎ and other Nrf2-regulated antioxidant enzymes such as GCLM, GCLC, and NQO1 (Figure S1). When C6 cells were transfected with HO-1 siRNA, Tau-Cl-induced HO-1 expression‎ decreased to 43.5 ± 0.3% compared with siRNA-non-transfected control cells (Figure 5D). However, this reduction was not observed in cells transfected with nonspecific siRNA (siCon) (Figure 5D). Importantly, the increased viability observed for Tau-Cl-pretreated/H2O2-treated cells was significantly reduced in HO-1 siRNA-transfected cells, that is, it decreased from 75.1 ± 4.2% to 54.7 ± 2.4% (Figure 5E). However, a similar level of protective effect was observed in siCon-transfected cells (Figure 5E), indicating that HO-1 is responsible for the protective effect of Tau-Cl. Taken together, these results suggest that Tau-Cl-mediated HO-1 upregulation is responsible for the protective effects of Tau-Cl in H2O2-treated astrocytes.

Figure 5. Suppression of Tau-Cl-mediated protective effects in H2O2-treated C6 cells by HO-1 knockdown. (A) Schematic diagram of time points for HO-1 knockdown, treatment of Tau-Cl and H2O2, and cell viability assay. (B,C) C6 cells were pre-treated with Tau-Cl (200 μM) for 9 h and then treated with H2O2 (100 μM) for 1 h. Protein levels of HO-1 were determined by immunoblotting at 6 h after H2O2 treatment. (D) C6 cells were transfected with HO-1 siRNA or non-specific siRNA (siCon). After 15 h, cells were treated with Tau-Cl (200 μM) for 9 h. HO-1 levels were determined by immunoblotting. Representative images of the protein levels are in B and D and quantified results are presented D as mean ± SEM (n = 3). (E) Cell viabilities of C6 cells after Tau-Cl pretreatment/H2O2 treatment with and without HO-1 siRNA transfection were examined using MTT assays at 24 h after H2O2 treatment. Results are presented as mean ± SEM (n = 3). ** p < 0.01 versus untreated controls, ## p < 0.01, # p < 0.05 and $$ p < 0.01 between indicated groups.

3.6. Tau-Cl Inhibits ROS Production in H2O2-Treated C6 Cells

We then investigated whether Tau-Cl suppresses ROS induction in H2O2-treated C6 cells (Figure 6A). When C6 cells were treated with H2O2 (300 μM, 1 h) and stained with CM-H2DCFDA, an intracellular ROS indicator, induction of DCF fluorescence was observed (Figure 6B,C). Interestingly, pretreatment of C6 cells with 200 μM of Tau-Cl for 9 h significantly suppressed the induction of DCF to 69.8 ± 2.9% of that in treatment-naïve H2O2 control cells (Figure 6B,C). In contrast, pretreatment with 200 μM of taurine for 9 h failed to suppress DCF induction (Figure 6B,C), demonstrating that Tau-Cl suppressed H2O2-induced ROS generation in H2O2-treated C6 cells. Importantly, suppression of ROS induction by Tau-Cl was not detected in HO-1 siRNA-transfected C6 cells, however, ROS induction was suppressed in siCon-transfected C6 cells (Figure 6D,E), suggesting that HO-1 is responsible for this suppression. Taken together, these results indicate that Tau-Cl suppresses ROS induction in H2O2-treated C6 cells and HO-1 upregulation is responsible for this effect.

Figure 6. Suppression of H2O2-induced ROS production by Tau-Cl in C6 cells. (A) Schematic diagram of time points for HO-1 knockdown, treatment of Tau-Cl and H2O2, and ROS assay. (B,C) C6 cells were pre-treated with Tau-Cl (200 μM) for 9 h and then treated with H2O2 (300 μM) for 1 h. Intracellular ROS levels were measured using CM-H2DCFDA at 1 h after H2O2 treatment. (D,E) C6 cells were transfected with HO-1 siRNA or non-specific siRNA (siCon), pre-treated with Tau-Cl (200 μM) for 9 h, and then treated with H2O2 (300 μM) for 1 h. ROS levels in C6 cells with and without HO-1 siRNA transfection were examined using CM-H2DCFDA at 1 h after H2O2 treatment. Results are presented as mean ± SEM (n = 3). The scale bar represents 100 μm. ** p < 0.01, * p < 0.05 versus untreated controls, # p < 0.05 and ## p < 0.01 versus H2O2 controls, $ p < 0.05 and & p < 0.05 between indicated groups.

4. Discussion

Astrocytes exert protective effects on neurons via their antioxidant activity [44]. They produce antioxidant molecules in the brain, playing a vital role in preventing ROS elevation and neuronal cell death in various CNS disorders [45]. HO-1 expression‎ is weak in the brain and is limited to small groups of neurons and neuroglia [46]. However, the protective effects of HO-1 in the brain have been reported under several pathological conditions [47,48,49]. In an animal model of intracerebral hemorrhage, the strong neuroprotective effects of HO-1 overexpression‎ in astrocytes have been reported [50]. In the present study, we demonstrated the autocrine function of Tau-Cl-mediated HO-1 induction, i.e., its ability to suppress H2O2-induced astrocyte cell death. However, astrocyte HO-1 may confer paracrine effects on neighboring cells, including neurons and microglia. In previous studies, we demonstrated the neuroprotective effect of HO-1 in astrocytes using conditioned media [47,48] and in animal models of MCAO [49]. Recently, Zhang et al. [51] reported that astrocyte-derived exosomes protect hippocampal neurons after traumatic brain injury by activating Nrf2 signaling in both rat and mouse models. In addition to neurons, studies have reported the beneficial effects of astrocyte HO-1 on microvascular function after various acute injuries, including post-ischemic myocardial injury [52], hemorrhagic shock [53], and seizures [54]. Alfieri et al. [55] reported that preconditioning stimuli increase HO-1 expression‎, primarily in perivascular astrocytes, which are responsible for preserving the barrier function in a transient rat MCAO model. Nevertheless, additional studies are warranted to investigate the protective effects of enhanced astrocyte HO-1 levels in other brain cell types.

Taurine is one of the most abundant free amino acids in the brain. However, its concentration in the brain decreases with age. Astrocytes are the primary taurine producers in the CNS [56]. Astrocytes release taurine as a gliotransmitter and provide neurons with hypotaurine as a substrate for taurine production [56]. As mentioned above, astrocytes play vital roles in maintaining normal brain function as well as protecting against inflammatory responses in the brain. Reactive astrocytes release inflammatory cytokines, produce various antioxidant molecules, including GSH, and activate ROS-detoxifying enzymes such as GST, GSH peroxidase, thioredoxin reductase, and catalase to improve neuronal survival [57,58,59]. In our previous study, we observed that neutrophils infiltrate into the ischemic region and that Tau-Cl exerts neuroprotective effects in the post-ischemic brain after MCAO and BV2 cells via increasing the levels of antioxidant enzymes, including HO-1 [20]. In the present study, we elucidated the effects of Tau-Cl in astrocytes. Tau-Cl-induced upregulation and nuclear translocation of Nrf2 as well as upregulated Nrf2-regulated antioxidant genes (Figure 1 and Figure 2), suggesting that Tau-Cl-derived antioxidants protect astrocytes and ameliorate neuronal injury. Tau-Cl recovered cell survival against oxidative stress (H2O2)-induced cell death (Figure 4), which was associated with increased HO-1 expression‎ and decreased ROS production (Figure 5 and Figure 6). Therefore, the induction of HO-1 by Tau-Cl mitigates H2O2-induced astrocyte cell death through its anti-oxidative properties.

Under homeostatic conditions, the levels of Nrf2 protein are maintained at a relatively low level owing to constitutive ubiquitin-mediated proteasomal degradation of Nrf2 by Keap1 [27,60]. Nrf2 is primarily localized in the cytoplasm with interaction with Keap1. In response to oxidative stress or electrophilic attacks, Keap1 undergoes oxidation at specific cysteine residues, leading to the disruption of its interaction with Nrf2. The disruption results in the stabilization of Nrf2, a critical step in initiating its translocation into the nucleus to regulate transcriptional activities. The translocation of Nrf2 into the nucleus occurs by interacting with the importins, facilitated by the three nuclear localization sequences (NLS) located in Neh1, Neh2, and Neh3 domains [61,62]. Importins recognize the NLS of Nrf2, allowing Nrf2-importin complexes to traverse the nuclear pore complex, comprising various nucleoporins that regulate the transport of molecules between the cytoplasm and the nucleus.

There are two major mechanisms by which Nrf2 dissociates from Keap1: one is a conformational change in Keap1 and the other is the phosphorylation of the serine residues of Nrf2 [63,64]. For the phosphorylation of the serine residues of Nrf2, various protein kinases, particularly mitogen-activated protein kinases (MAPKs), can trigger transcriptional activity via Nrf2 phosphorylation. We observed that Tau-Cl exhibited no stimulatory effects on various protein kinases, including MAPKs. Moreover, Tau-Cl inhibited lipopolysaccharide-induced MAPK activation in RAW 264.7 cells [41]. Therefore, we determined the effect of Tau-Cl on the conformational changes of Keap1, with particular attention on determining whether Tau-Cl modifies the reactive cysteine residues of Keap1, which play a pivotal role in its association with Nrf2. Human Keap1 contains 27 cysteines with thiol groups that form covalent bonds with other cysteine thiols, bind to metals and metalloids, and react with oxidants [65]. The chemical modification of the cysteine residues of Keap1 results in the loss of Nrf2 repressor function and changes the expression‎ of Nrf2 target genes, which collectively restore redox balance and resolve inflammation, thereby ensuring a comprehensive cytoprotection. Tau-Cl decreased IAA-bound Keap1 (Figure 3), suggesting that Tau-Cl converts the thiol groups into disulfide bonds. Most Nrf2 inducers are electrophiles that readily react with the cysteine thiol groups of Keap1 [42]. As a titrant for thiols, 4,4′-dipyridyl disulfide exhibits unique UV spectra; its absorbance changes when it reacts with thiols. Tau-Cl decreased the UV absorption of Keap1 similarly to 4,4′-dipyridyl disulfide, suggesting that Tau-Cl changes the thiol groups of Keap1 (Figure 3). Although we could not identify the cysteine residues targeted by Tau-Cl, we provided tentative evidence that Tau-Cl changes the thiol groups of Keap1.

5. Conclusions

In the present study, we investigated the antioxidative effects of Tau-Cl in astrocytes. Tau-Cl augmented Nrf2 expression‎ and activation and upregulated Nrf2-regulated antioxidants such as HO-1, NQO1, GCLC, and GCLM. The activation of Nrf2 by Tau-Cl occurs by disrupting the Keap1-Nrf2 complex via the oxidation of the thiol groups of Keap1. Consequently, Tau-Cl rescued the cells from H2O2-induced death by further enhancing HO-1 expression‎ and suppressing ROS production. Nevertheless, additional studies are required to comprehensively understand the role of Tau-Cl not only in astrocytes but also in other glial cells and neurons. The neuroprotective effects of Tau-Cl in astrocytes can be a promising therapeutic target for various neuroinflammatory and neurodegenerative diseases.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox13020169/s1, Figure S1: Up-regulations of genes downstream of Nrf2 by Tau-Cl-pre-treatment in H2O2-treated C6 cells.

Author Contributions

Conceptualization, J.-K.L. and C.K.; Methodology, S.-I.S., I.S.K., J.S.L., and C.K.; Validation, J.-K.L. and C.K.; Writing—Original Draft Preparation, J.-K.L. and C.K.; Writing—Review and Editing, J.-K.L. and C.K.; Funding Acquisition, J.-K.L. and C.K. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Mid-carrier Research Grant (2021R1A2C2010920 to J.-K.L.) and Basic Science Research Program (2022R1I1A1A01055015 to I.S.K) funded by the National Research Foundation of Korea.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are included in the article and are also available on request from the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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