research article

Delayed Activation of Sirt1 Induced Autophagy Signaling Promotes Astrogliosis, Infarct in Experimental Stroke

Sireesh Kumar Teertam1, Varalakshmi Manchana2, Prakash Babu Phanithi3*

1Department of Biotechnology & Bioinformatics, school of Life sciences, University of Hyderabad, Hyderabafd-500046 (T.S), India.

2School of Medical Sciences, University of Hyderabad, Hyderabad (T.S), India

3Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad (T.S), India

*Corresponding author: Prakash Babu Phanithi, Department of Biotechnology & Bioinformatics, School of Life Sciences, University of Hyderabad, Prof. C. R. Rao Road, Gachibowli, Hyderabad -500 046 (T.S), India

Received Date: 10 April, 2023

Accepted Date: 24 April, 2023

Published Date: 27 April, 2023

Citation: Teertam SK, Manchana V, Phanithi PB (2023) Delayed Activation of Sirt1 Induced Autophagy Signaling Promotes Astrogliosis, Infarct in Experimental Stroke. Int J Cerebrovasc Dis Stroke 6:148. DOI: https://doi.org/10.29011/2688-8734.100048

Abstract

Curcumin, a polyphenol, is an active constituent derived from Curcuma longa. The protective nature of curcumin against cerebral ischemia-reperfusion (CIR) is well documented. This study aims evaluate the changes in Sirtuin-1 (Sirt1)induced autophagy signaling. We also investigated the neuroprotective effect of curcumin on Sirt1-mediated autophagy and neuronal survival on day7 post-stroke in a rat model of middle cerebral artery occlusion (MCAO). MCAO was performed for 60 min in male Sprague Dawley rats. Immunoblotting and immunohistochemistry were used to detect alteration in Sirt1 and autophagy-related proteins Beclin-1, autophagy-related genes-3, 5, and 7 (Atg), microtubule-associated protein-1A light chain3 (Lc3-I/II), mammalian target of rapamycin (mTOR), p-mTOR, and cleaved caspase-3. CIR, on day1 post-stroke, resulted in significant alteration of Lc3-II/I and p-mTOR. Further, on day7 post-stroke, Sirt1-induced autophagy decreased, while mTOR phosphorylation and cleaved caspase-3 immunoreactivity increased. Curcumin treatment promoted astrogliosis and infarct. Sirt1-mediated autophagy signaling is involved in the promotion of astrogliosis and infarct. EX-527 blocked the increase in Sirt1/ autophagy signaling evoked by curcumin and reduced astrogliosis and infarct. These findings show that curcumin may promote infarct and reactive astrogliosis via activation of Sirt1/autophagy signaling.

Keywords: Curcumin; Sirtuin-1; Autophagy; Astrogliosis; Caspase-3; MCAO

Introduction

Cerebral ischemia (CI) is the leading cause of human mortality and morbidity. Recent studies have demonstrated that some mediators of acute neuronal injury have a protective role in the delayed phase following CI [1]. Failure in neuroprotection may partly be because many neuroprotectants inhibit both the injury mechanisms and those molecular mechanisms required for repair.

In this study, we questioned whether the same phenomenon might also be manifested in neuronal survival pathways.

Curcumin, a polyphenolic compound extracted from Curcuma longa’s rhizomes [2], exhibits protective activity as proven in cardiomyocytes, liver, and lung against ischemiareperfusion injury [3]. Curcumin exhibits anti-inflammatory and anti-apoptotic properties against CIR [4]. It promotes Sirt1 expression and modulates autophagy by regulating several cellular mechanisms [5,6].

Sirt1 is a class-III histone and non-histone deacetylase that depends on NAD+ for its enzymatic activity [7-9]. Sirt1 activates neuronal survival against numerous neurological disorders, like Alzheimer’s and Parkinson’s [10]. Further, Sirt1 activation is shown to reduce neuronal injury in experimental stroke models. Sirt1 protects against CI by regulating p53-mediated neuronal apoptosis and mediates hyperbaric oxygen preconditioning-induced CI tolerance [11-13]. Recent research has linked an increase in Sirt1 expression in the brains of stroke patients [14]. Further, sirt1 can directly or indirectly influence autophagy induction under nutrient starvation/cellular stress [15,16].

Recent studies have shown that some mediators of acute neuronal injury have a protective role in the delayed phase of CIR. Stress-responsive JNK is shown to promote neurovascular remodeling and recovery during delayed stroke [1]. Activation of matrix metalloproteinases (MMP) degrades extracellular matrix during acute ischemia, but inhibition of MMPs in chronic phase prevents neurovascular remodeling and functional recovery [17]. Similarly, delayed inhibition of excitotoxic N-methyl D-aspartate (NMDA) receptors prevent synaptic plasticity and stroke recovery [18]. In the current study looked at the role of Sirt1/autophagy signaling in neuronal death in rats following MCAO. We also examined the neuroprotective effect of curcumin on Sirt1/ autophagy signaling and neuronal survival on day7 post-stroke to understand the effect of curcumin on cellular mechanisms required for neuronal repair during delayed stroke.

Materials and Methods

Reagents and Chemicals

All the antibodies used in this study were purchased from Cell Signaling Technology (CST, US) and drugs were purchased from Sigma (US). Sirt1 (CST; #8649), Autophagy Antibody Sampler Kit (CST; #4445), beta-actin (CST; #8457), EX-527 (Sigma; #E7034-5MG), Curcumin (Sigma; #C1386), GFAP (CST; #12389), Anti-rabbit Ig-G-Alexa fluor-488 (CST; #4412), Antimouse Ig-G Alexaflour-555 (CST; # 4409), Prolong Gold Antifade Reagent with DAPI (CST; #8961), MCAo suture (Ethicon 3-0 non-absorbable; #NW3321), Immunohistochemistry kit (Bio SB, US; #BSB0016), TTC stain (Sigma; #T8877-5G), Ac-DEVDAFC substrate (Sigma; #A0466-1MG).

Transient Middle Cerebral Artery Occlusion (MCAO)

Animal experiments were performed following Institutional and National Animal Ethical Committees (IAEC) guidelines. The National Centre for Laboratory Animal Sciences (NCLAS) in India provided young (3-4 month) male Sprague-Dawley (SD) rats. Animals were housed in a controlled environment with free access to water and food. To avoid sex and age differences, adult male rats were used exclusively. Focal CI was induced by MCAO as per Longa et al. [19]. Rats were anesthetized with ketamine (60mg/Kg) and xylazine (10mg/Kg) administration via the IP route. A middle neck incision was made to identify the left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA). CCA and ICA were clipped with microvascular clips, and the ECA was ligated distally. To block the origin of MCA, a 3-0 nylon monofilament was inserted through an arteriotomy and gently advanced into ICA. Monofilament was inserted into CCA but not MCA in the sham-operated group. After 60 minutes of occlusion, the monofilament was gently removed to allow reperfusion and the animals to recover.

Two different treatment protocols were followed to understand the role of Sirt1-induced autophagy during the delayed stroke. Curcumin alone and curcumin + EX-527 combination (Combination therapy was preferred to minimize the animal number and their suffering). Curcumin activates Sirt1-induced autophagy signaling, while EX-527, a Sirt1-specific inhibitor, inhibits Sir1. We performed all the drug administrations in the early day7.

Animals were sacrificed at the end of the day7. Curcumin (300 mg/kg in 5N NaOH + saline; pH-7.4) was administered via intraperitoneal (IP) route [20], and EX-527 (30 mg/kg in 1% DMSO in PBS) was administered via intracerebroventricular (ICV) route [12]. 5N NaOH in PBS and 1% DMSO in PBS was used as a vehicle for respective groups.

Neurological Deficit Evaluation

We evaluated the neurological deficit for infarct intensity using the 4-point scale described previously [21]. Rats having no abnormality were scored as grade 0, rats who failed to extend forelimb opposite to infarct were scored as grade 1, animals with decreased resistance to lateral push were scored as grade 2, rats which show hemiparesis were scored as grade 3, and the rats who died because of the severe lesion were scored as grade 4. Rats with neurological scores below grade 3 were excluded from the experimental groups.

Immunoblotting

Control and experimental rats (day1, day7, day7+curcumin, and day7curcumin+EX) s were euthanized with an overdose of carbon dioxide (CO2), and ipsilateral brains were excised (n=3). Brains were homogenized in RIPA buffer (150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% NP40, 50 mM Tris-HCl (pH-7.4), and 0.5% Sodium deoxycholate). Tissue homogenates were centrifuged at 10,000 rpm for 20 min at 4°C to separate the supernatant. Protein concentration in the supernatant was determined by Bradford protein assay [22]. Total protein extract was resolved on 10-12% SDS-PAGE gels and transferred overnight onto a nitrocellulose membrane at 4° C using transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol (v/v), adjust pH 8.3). The membranes were blocked for 90 minutes at room temperature with 5% nonfat milk in tris-buffered saline containing Tween-20 (TBS-T) at room temperature (RT) followed by overnight incubation at 4° C with respective primary antibody at 1:1000 dilution. Membranes were then washed and probed with respective horseradish peroxidase (HRP) labelled secondary antibody at 1:3000 dilutions for 90 min at RT. Chemiluminescence was captured using an enhanced chemiluminescent (ECL) detection system (Bio-Rad, USA).

Infarct Volume Measurement

Control and experimental rat brains (day1, day7, day7+curcumin, and day7curcumin+EX) were measured for infarction with standard tetrazolium staining (n=3). Brain tissues were frozen immediately at -20 for 30 min after decapitation. Frozen brain tissues were made into six coronal slices with 2 mm thickness and stained with 1% 2,3,5- triphenyl tetrazolium chloride (TTC) at 37° C for 30 min, followed by 4% paraformaldehyde (PFA) fixation. The infarct area in each slice was evaluated by scanned digital images with ImageJ software (NIH, US). The infarcted areas from each segment were added to derive the total infarct, multiplied by the thickness of the brain section to determine total infarct volume. Correction for edema of infarct was done as described elsewhere [23].

Histology and Immunohistochemistry

Control and experimental rat brains (day7, day7+curcumin, and day7curcumin+EX) (n=3) were fixed by trans cardiac perfusion with phosphate-buffered saline (PBS) and 4% PFA. Paraffin-embedded brain tissues were sectioned into 5-10 mm thick slices using the microtome (Leica, Germany). Brain sections were stained with hematoxylin and eosin (H&E) and then immunostained against cleaved caspase-3 using an IHC kit. Briefly, sections were deparaffinized and hydrated in a decreasing gradient of alcohol followed by endogenous peroxidase blocking with 3% hydrogen peroxide in methanol. After three washes in PBS, antigen retrieval was carried out with Tris-EDTA buffer (pH-9.0). The nonspecific binding was minimized by blocking sections with 0.25% BSA for 30 at RT. The sections were then incubated with primary antibody (1:100 dilution) for 2 h 30 min at RT. Further, brain sections were washed in PBS and incubated in polydetector HRP-labelled secondary antibody for 45 min at RT. Next, staining was visualized by covering the sections in 3’-3’ diaminobenzidine (DAB) buffer for 5 min, followed by counterstaining by hematoxylin. After subsequent dehydration in graded alcohol and xylene, sections were mounted, and images were captured under the light microscope (Olympus, Japan) with 400 X magnification.

Caspase Activity Assay

The caspase activity assay was performed to monitor key effector caspases (caspase-3,6 & 7) involved in apoptosis. Total 100 mg of protein lysates from control and experimental rat brains (day7, day7+curcumin, and day7curcumin+EX) (n=3) were dissolved in 100 ml of analysis buffer (100 mM NaCl, 10 mM DTT, 20 mM HEPES pH 7.4, 10%sucrose, 0.1% CHAPS, 1 mM EDTA) and incubated at 37 C for 6 h. After incubation 5 ml of Caspase Substrate (Ac-DEVD-AFC) was added to the lysates and made up to 1 ml of final concentration with analysis buffer, followed by incubation for 1 h at 37° C. The cleavage of AFC substrate was measured by fluorescence emission using fluorescence spectrophotometer (FluoroMax) at excitation 400 nm and emission 450–505 nm. The intensity of fluorescence signal represents the cumulative activity of effector caspases 3, 6 and 7.

Immunofluorescence

Brain sections from both control and experimental rat brains (day7, day7+curcumin, and day7curcumin+EX) (n=3) were dewaxed and rehydrated through 100-70% graded ethanol. Antigen retrieval was carried out in a microwave with 10 mM citrate buffer with 0.05% tween-20 (pH 6.0). After three washes in PBS, brain sections were blocked by 5% goat serum for 60 min at 37° C followed by incubation in primary antibody (1:100 dilution) for glial fibrillary acidic protein for 18 h at 4°C. After being washed with PBS, sections were covered with Alexa Fluor-485 conjugated goat anti-rabbit IgG (1:3000 dilutions) for 60 min at RT. Sections were mounted with antifade DAPI and Images were captured under the laser scanning confocal microscope (Carl-Zeiss, Germany).

Statistical Analysis

All the experimental data expressed as the mean ± SD and analysed for statistical significance using one-way ANOVA with Tukey’s multiple comparisons test. The experimental data shown are the representative of three independent experiments. Bars represent variation within the experimental samples. Graphs were plotted with GraphPad Prism 7.0 software (GraphPad Inc. US). Significance was shown as asterisks: * indicates p<0.05.

Results

Decrease in Expression of Sirt1/Autophagy Signaling on Day7 Post-Stroke

The sirt1-induced autophagy mechanism confers functional protection against CI. We sought to determine the role of Sirt1induced autophagy signaling on neuronal survival following day seven post-stroke. To explore the changes in Sirt1-induced autophagy signaling in the pathogenesis of ischemia, Sirt1 and autophagy mediators’ expression was assessed using immunoblot analysis. Immunoblotting results showed a significant decrease in the expressions of Sirt1 and most of the autophagy mediators (Beclin-1, Atg-3, Atg-5, and Atg-7) on day1 after MCAo. On the contrary, the ratio of Lc3-II/I was augmented on day1 compared to the sham control (Figure 1A-F, Sirt1<0.02; Beclin-1<0.001; Atg3<0.001; Atg-5<0.003; Atg-7<0.01; Lc3-II/I<0.001).

Further, the expressions of Sirt1 and autophagy mediators was decreased on day7 compared with the sham control (Figure 1A-F, Sirt1<0.001; Beclin-1<0.001; Atg-3<0.001; Atg-5<0.001; Atg-7<0.003). Notably, p-mTOR expression was inversely related to the autophagy mechanism. The expression of p-mTOR was upregulated on day 1 and day 7 when compared to control brains. (Figure 1G, p-mTOR<0.001).

 

Figure 1: Curcumin treatment on day7 post-stroke up-regulates Sirt1 induced autophagy signaling. An equal amount of total protein sample (75ug) was used for immunoblotting. Respective total proteins and beta-actin were served as a loading control (Lc3-II/I, p-mTOR), while Sirt1, Beclin-1, Atg-3, Atg-5, and Atg-7 were normalized to beta-actin. (A-G) Representative graphs show changes in the expression of Sirt1/autophagy/mTOR signaling on day1 and day7 post-stroke. (H-N) The graphs from the brains of the vehicle (day7), curcumin, and curcumin + EX-527 treatment on day7 shows that curcumin treatment augmented Sirt1 induced autophagy on day7 and curcumin + EX-527 treatment show a substantial decrease in Sirt1 induced autophagy. The densitometry values are represented as mean ± SD (n=3). *p<0.05 versus control brain

Decrease in Infarct in Day7 Post-Stroke Rat Brains

Changes in neuronal morphology were confirmed by routine H&E staining (Figure 2A). Typical TTC staining was performed to measure the lesion volume changes, and the results indicate a significant amount of infarction in ipsilateral brains on day1 compared to day7 following MCAo (Figure 2B&C).