Honokiol attenuates mitochondrial fission and cell apoptosis by activating Sirt3 in intracerebral hemorrhage

Abstract

Background: 

Sirtuin-3 (Sirt3) has been documented to protect against mitochondrial dysfunction and apoptosis. Honokiol (HKL) is a Sirt3 pharmacological activator with reported neuroprotective effects in multiple neurological disorders. The present study aimed to explore the neuroprotective effects of HKL and the role of Sirt3 following intracerebral hemorrhage (ICH).

Methods: 

An in vivo ICH model in rats was established by injecting autologous blood into the right basal ganglia. PC12 cells were stimulated with hemin. For the in vivo investigation, the modified Neurological Severity Scores and the Morris water maze test were performed to assess neurological deficits. Hematoxylin–Eosin and Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining were employed to evaluate the histopathology and apoptosis. Immunohistochemical staining was used to investigate the expression of Sirt3. Adenosine triphosphate (ATP) levels were quantified to assess mitochondrial dysfunction. Cell counting kit-8, lactate dehydrogenase assay, and flow cytometry were used to analyze cell vitality and apoptosis in vitro. Immunofluorescence staining was performed to observe mitochondrial morphology and dynamin-related protein 1 (Drp1) localization to mitochondria. Western blot was applied to quantify the expression of Sirt3, Bax, Bcl-2, cleaved-caspase-3, Drp1, phosphorylation of Drp1 at serine-616, and phosphorylation of Drp1 at serine-637 in vivo and in vitro.

Results: 

HKL treatment alleviated neurological deficits, attenuated the histopathological damage and cell apoptosis, and restored the decreased ATP levels in ICH rats. HKL improved cell survival rate, reduced cell apoptosis, and inhibited mitochondrial fission in PC12 cells. Moreover, both in vivo and in vitro models showed increased phosphorylation of Drp1 at Ser616, and reduced phosphorylation of Drp1 at Ser637. Meanwhile, immunofluorescence co-localization analysis revealed that hemin increased the overlap of Drp1 and mitochondria in PC12 cells. The phosphorylation and mitochondrial translocation of Drp1 were effectively reversed by HKL treatment. Importantly, the selective Sirt3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine suppressed these effects.

Conclusion: 

Our findings demonstrated that HKL ameliorated ICH-induced apoptosis and mitochondrial fission by Sirt3, suggesting that HKL has immense prospects for the treatment of ICH.

Introduction

Intracerebral hemorrhage (ICH) is a devastating cerebrovascular disease associated with high mortality and morbidity, with no breakthroughs in definitive treatment in recent years.^[1] The pathophysiological processes following ICH are broadly classified into two stages: primary brain injury, which refers to mechanical damage disruption caused by the hematoma expansion, and secondary brain injury (SBI), which includes oxidative stress, inflammatory reaction, and mitochondrial dysfunction.^[2,3] SBI can further exacerbate primary lesions, eventually resulting in neuronal apoptosis and severe neurological deficits. Although numerous studies have explored therapeutic strategies targeting SBI, ICH outcomes are still unsatisfactory. Accordingly, the quest for an effective means to reduce SBI has become essential to improve ICH patient outcomes.

Mitochondria are highly dynamic organelles that constantly fuse and divide in response to changes in energy demand and supply and maintain cellular function.^[4,5]An increasing body of evidence suggests that disruption of this dynamic balance is manifested by increased mitochondrial fission and fragmentation in brain injury after ICH.^[6,7] Dynamin-related protein 1 (Drp1) is a GTPase considered an essential modulator for mitochondrial fission. The balance between Drp1 and the Ser-616/Ser-637 phosphorylation ratio reflects Drp1 activity. It is generally accepted that phosphorylation at Ser616 promotes DRP1 recruitment around the outer membrane and induces mitochondrial fission, while phosphorylation at Ser-637 has the opposite effect of impairing Drp1 recruitment and inhibiting mitochondrial fission.^[8] Increased mitochondrial fission results in the release of cytochrome c and the long-term opening of mitochondrial permeability transition pore. These cellular changes ultimately result in cell apoptosis.^[9,10] In contrast, inhibiting mitochondrial fission can reduce ICH-induced brain damage.^[11,12] Sirtuin-3 (Sirt3) is a member of the sirtuin family and a NAD⁺-dependent protein deacetylase mainly located in mitochondria. It has been reported that Sirt3 could inhibit mitochondrial fission and neuron apoptosis in t-BHP-induced hepatocytes injury,^[13] post-infarction cardiac injury,^[14] and cerebral ischemia-reperfusion injury.^[15] However, the effects of Sirt3 on ICH-induced injury remain unclear.

Honokiol (HKL) is a bioactive polyphenolic compound isolated from Magnolia grandiflora, which exerts various pharmacological properties such as anti-oxidation, anti-depressant, anti-inflammatory, and anti-apoptosis, with great clinical potential.^[16-19] HKL possessed a neuroprotective property, mainly due to its ability to cross the blood–brain barrier as a small molecule.^[20,21] It is reported that HKL could promote mitochondrial fusion and neural survival via the Sirt3/AMPK pathway in subarachnoid hemorrhage.^[22] Moreover, HKL protected the brain against ischemia-reperfusion injury in mice by inhibiting reactive oxygen species (ROS) production and enhancing mitochondrial function.^[23] Meanwhile, a recent study indicated that HKL could protect against hyperglycemic ICH-induced neuronal injury.^[13] Nevertheless, the effect of HKL in pure ICH and whether HKL could work on mitochondrial fission remain unclear. This study excluded the harmful effects of hyperglycemia on ICH, and present study mainly aimed to explore the effects and the mechanisms of HKL on apoptosis and mitochondrial fission in ICH.

Methods

Animals

Adult male Sprague–Dawley rats (250–300 g) were housed under controlled conditions with a 12 h light/dark cycle and ad-libitum access to water and a maintenance diet. All animal experiments were approved by the Animal Ethics Committee of North China University of Science and Technology and were carried out in accordance with the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Researchers were requested to fully master injection and fixation methods. After adequate anesthesia, the operation was performed quickly and gently. All these efforts were aimed at minimizing animal suffering in the study.

ICH model in vivo

The ICH animal model was established as previously described.^[24] Briefly, rats were anesthetized with chloral hydrate (100 mg/kg) by intraperitoneal injection and were fixed on a stereotaxic apparatus. The skull was exposed, and a 1.0 mm burr hole was drilled in the skull (0.2 mm anterior to the bregma, 3.5 mm lateral to the midline). Then, 50 μL of autologous blood from the tail artery was infused into the right basal ganglia using a micro-injection pump at a speed of 3.3 μL/min and a depth of 5.5 mm below the skull surface. After the injection, the needle was held in place for 5 min and then slowly withdrawn. The sham group rats were injected with an equal volume of saline.

Animal grouping and treatment

In this part of the experiment, 80 rats were randomly divided into four groups: sham group (n = 20), ICH group (n = 20), ICH + vehicle group (n = 20), and ICH + HKL group (n = 20). During the experiment, two rats died in the ICH group and one rat died in the ICH + vehicle group. In order to ensure the experiment scale, the rats that died during the experiment were removed and randomly supplemented. HKL (purity ≥98%, MedchemExpress, Monmouth Junction, NJ, USA) was dissolved in dimethyl sulfoxide (DMSO) and diluted in an equal volume of phosphate-buffered saline (PBS). Then the drug was injected intraperitoneally at a dose of 10 mg/kg 15 min before ICH and 60 min after ICH. This dose was chosen based on previous studies showing that 10 mg/kg HKL has neuroprotective effects from hemorrhagic damage.^[16,22] Rats in the vehicle group were administered an equal volume of DMSO: PBS (1:1) intraperitoneally.

Modified neurological severity score (mNSS)

As previously reported,^[25] the mNSS test was performed to assess the neurological function of rats (n = 6, per group) before injury and 1, 3, 7, and 14 days after ICH by an experienced investigator who was blinded to the treatment groups. Briefly, this test included the results from the motor, sensory, reflex, and balance tests. The scores ranged from 0 to 18; the higher score, the more severe the neurological deficit.

Morris water maze (MWM) test

The spatial learning and memory of the rats (n = 6, per group) were evaluated by the MWM as previously described.^[26] In short, MWM was performed in a black pool (diameter: 180 cm, height: 50 cm) filled with water (temperature: 22–24°C). The pool was divided into four quadrants of equal sizes. Then, a platform (diameter: 10 cm) was positioned in a target quadrant 2 cm below the water surface. The experiment included a place and a spatial exploration trial. Before the trial, the rats were allowed to swim freely in the pool for 1 min without the platform for pre-adaptation. For the first 4 days (16–19 days after ICH), all rats were placed in the water facing the wall in each of the four quadrants, and the rats were provided with a maximum of 90 s to find the submerged platform. If the rats did not find the platform within 90 s, they were guided to it and allowed to remain there for 20 s. Each training was separated by at least 15 min. The time the rats spent reaching the platform (escape latency) and the swimming speed were ascertained based on video recordings made by a camera placed above the maze. On day 5 (20 days after ICH), the spatial memory ability of the rats was evaluated by the spatial navigation trial. The platform was removed from the pool, and the rats were placed into the quadrant opposite the target quadrant and allowed to swim freely for 60 s. The percentage time that the rats spent in the target quadrant and the number of times that the rats passed through the platform location were recorded systematically.

Hematoxylin and Eosin (HE) staining

HE staining was conducted according to standard histological protocols. Briefly, rats were euthanized with an overdose of anesthetic and perfused intracardially with PBS and followed by 250 mL of 4% paraformaldehyde (PFA). The brains were removed and kept in 4% PFA for 24 h, and then embedded in paraffin after gradient alcohol dehydration. Brain tissues were cut into 5 μm-thick coronal sections for further analysis. After dewaxing, tissue sections were stained with hematoxylin for 5 min and differentiated by 1% hydrochloric alcohol for 10 s. Then the sections were stained with eosin for 3 min followed by dehydration with graded alcohol and clearing in xylene. Finally, the stained sections were visualized under an optical microscope (Olympus Optical Co., Ltd., Tokyo, Japan).

Immunohistochemistry staining

Immunohistochemistry was performed 24 h following ICH using a universal streptavidin-perosidase (SP) kit (Zhongshanjinqiao Biotechnology, Beijing, China) according to the manufacturer's instructions. Briefly, after deparaffinization and rehydration, sections were incubated overnight at 4°C with anti-Sirt3 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Samples without primary antibodies were used as a negative control. Subsequently, the sections were incubated with a secondary antibody conjugated with horseradish peroxidase (HRP) for 1 h. Finally, the sections were stained with diaminobenzidine and counterstained with hematoxylin. Three microscope fields for each section were randomly selected for photographing under an optical microscope (Olympus). All images were examined in a blinded manner.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay

The apoptosis rate in brain tissues was determined with a TUNEL assay (Beyotime, Shanghai, China). Briefly, paraffin-embedded brain sections were deparaffinized and rehydrated, and incubated with proteinase K solution (20 μg/mL) at 37°C for 30 min. After washing, sections were incubated with a TUNEL working solution and subsequently stained with 4,6-diamidino-2-phenylindole (DAPI) solution. Finally, stained sections were observed under a fluorescence microscope (Olympus), and the apoptosis rate was calculated as the number of apoptotic cells (red)/total number of cells (blue) × 100%.

Adenosine triphosphate (ATP) assay

At 24 h after ICH, the total ATP content in perihematomal brain tissues of rats (n = 3, per group) was detected using an ATP assay kit (Sigma, St Louis, Missouri, USA). All the procedures were performed according to the manufacturer's instructions. In brief, 10 mg tissue was lysed in 100 μL of ATP assay buffer. Then the ATP reaction mix and background control were added to the wells and incubated for 30 min at 37°C. Finally, the absorbance of the solution was measured at 570 nm. The ATP concentration of the samples was expressed as nmol/mg protein. Triplicate wells were used for each sample and the experiment was repeated thrice.

Cell culture and treatment

Highly differentiated PC12 cells were obtained from the Chinese Academy of Sciences (Shanghai, China). PC12 cells were cultured in RPMI-1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Bovogen, Melbourne, VIC, Australia) and 1% penicillin-streptomycin (Gibco BRL). The cells were maintained in an incubator at 37°C with 5% CO₂.

PC12 cells were exposed to 40 μmol/L hemin (a breakdown product of hemoglobin which plays a key role in ICH; Sigma) for 12 h to simulate the ICH in vitro model. Gradient concentrations of HKL were added to the cells for 12 h, and the optimal concentration was determined by detecting the viability of PC12 cells using CCK-8 assay. Subsequently, to assess the neuroprotective effects of HKL against hemin-induced injury, PC12 cells were divided into three groups: control group, hemin group, and hemin + HKL group (the cells were pretreated with 10 μmol HKL for 6 h and then exposed to hemin for another 12 h). To investigate the protective mechanisms of HKL, a Sirt3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) (Selleck Chemicals, Houston, TX, USA) was used to silence the expression of Sirt3 and the cells were divided into four groups: control group, hemin group, hemin + HKL group, and hemin + HKL + 3-TYP group (the cells were treated with 10 μmol/L HKL and 50 μmol/L 3-TYP for 6 h before hemin treatment). The 3-TYP concentrations were chosen based on previous studies.^[27,28] The control group received the same final volume of the solvent.

CCK-8 assay

Cell viability was determined by CCK-8 Assay Kit (Beyotime) according to the manufacturer's protocol. PC12 cells (10⁴ cells/well) were seeded onto 96-well culture plates and incubated for 24 h. After the treatments, the culture medium was removed and replaced with a serum-free medium containing 10% CCK-8 reagent, followed by incubation for 1.5 h at 37°C. Finally, the absorbance was measured at 450 nm wavelength with a microplate reader (BioTek Instruments, VT, USA).

Flow cytometry (FCM) analysis

Cell apoptosis was detected using an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (Beyotime) according to the manufacturer's instructions. After treatment, PC12 cells were collected and centrifuged at 1000 rounds/min for 5 min. Cells were resuspended with 195 μL binding buffer and subsequently incubated with a mixture of 5 μL Annexin V-FITC and 5 μL PI for 15 min at room temperature in the dark. The cell apoptosis was detected using a flow cytometer (Beckman Coulter Inc., Miami, FL, USA).

Lactate dehydrogenase (LDH)

It is widely acknowledged that damaged cells release LDH into the medium. Therefore, the LDH leakage was measured to assess the hemin-induced cytotoxicity by an LDH assay kit (Jiancheng Biotech, Nanjing, Jiangsu, China), following the manufacturer's instructions. PC12 cells were seeded onto 96-well plates (10⁴ cells/well). After treatment, the culture supernatants were collected, and the absorbance at 490 nm was tested by a microplate reader (BioTek Instruments).

Mitochondrial morphology and Immunohistochemistry

PC12 cells were seeded onto coverslips and incubated for 24 h. After treatment, the cells were incubated with 100 nmol/L MitoTracker Red (ThermoFisher Scientific, Waltham, MA, USA) at 37°C for 30 min. Next, the cells were fixed in 4% PFA for 30 min and permeabilized with 0.3% Triton X-100 in PBS for 10 min at room temperature. The cells were blocked with 10% donkey serum (Biological Industries, Cromwell, CT, USA) in PBS for 30 min at 37°C and then incubated with anti-Drp1 antibody (1:50, Santa Cruz Biotechnology) overnight at 4°C. Subsequently, the cells were incubated with Alexa Fluor 488-conjugated donkey anti-mouse secondary antibody (1:200, Invitrogen, Carlsbad, CA, USA) for 1 h at 37°C, and then counterstained with DAPI; (Beyotime) and visualized under a laser confocal microscope (Leica, Wetzlar, Germany). Mitochondrial morpholo₂gical characteristics, including form factor (FF, perimeter²/[4π×area]) and aspect ratio (AR, major axis/minor axis), were quantified using ImageJ software (NIH, Bethesda, MD, USA) as described before.^[29]

Western blot

Western blot analysis was performed 4 h after ICH in vivo and 12 h after hemin treatment in vitro (n = 6, per group). The protein was extracted from the brain tissues around the hematoma or PC12 cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime), and the concentration was determined by a BCA protein assay kit (Beyotime). Each protein sample was mixed with 5× sodium dodecyl sulfate (SDS) sample buffer and boiled at 100°C for 10 min. Equal amounts of protein sample (40 μg) were carefully added to each well of 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels for electrophoresis and then transferred onto polyvinylidene-difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). After blocking with 5% non-fat dry milk for 2 h, the PVDF membranes were incubated with diluted primary antibodies at 4°C overnight, including anti-Sirt3 antibody (1:500, Santa Cruz Biotechnology), anti-Drp1 antibody (1:250, Santa Cruz Biotechnology), anti-phospho-Drp1 (Ser616) antibody (1:1000, Affinity Biosciences, Changzhou, Jiangsu, China), anti-phospho-Drp1(Ser637) antibody (1:1000, Affinity Biosciences), anti-Bax antibody (1:1000, GeneTex, Inc., Irvine, CA, USA), anti-Bcl-2 antibody (1:500, arigo, Taiwan, China), anti-caspase3 antibody (1:1000, GeneTex, Inc.), and anti-β-actin antibody (1:100000, ABclonal Technology, Wuhan, Hubei, China). Subsequently, the membranes were washed and incubated with the HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (1:5000, KPL, Gaithersburg, MD, USA) at 37°C for 1 h. Finally, the blots were revealed with an enhanced chemiluminescence (ECL) Plus reagent (Zoman Biotechnology, Beijing, China), and the relative intensity of the bands was analyzed by ImageJ software (NIH, Washington, USA).

Statistical analysis

All results were expressed as the mean ± standard deviation. The mNSS and MWM test data (escape latency) were analyzed by two-way repeated-measures analysis of variance (ANOVA) followed by the least significant differences (LSD) post hoc test or Tukey's post hoc test. Statistical significance among multiple groups was calculated using one-way ANOVA with Tukey's post hoc test. Graphs were generated with GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). All data analyses were performed using SPSS v25 software (SPSS Inc., Chicago, IL, USA). P values <0.05 were statistically significant.

Results

HKL attenuated neurological deficits in ICH rats

The mNSS and MWM tests were used to detect the effect of HKL on the neurological function of ICH rats. The mNSS test of rats in the four groups was conducted 1 day before and 1, 3, 7, 14 days after ICH. As shown in Figure 1A, on days 1, 3, 7, and 14 post-ICH, the mNSS score in the ICH group was significantly higher than that in the sham group (P < 0.05). Meanwhile, the mNSS score in the ICH + HKL group was significantly lower than that in the ICH group on all 4 days (P < 0.05 on days 1, 3, 7, and 14). Moreover, there was no significant difference between the ICH + HKL group and the ICH + vehicle group (P > 0.05).

Figure 1: HKL promoted the recovery of neurological function in rats after ICH. Rats were treated with HKL (10 mg/kg) or vehicle and injected with 50 μL autologous blood. (A) The mNSS score of each group (n = 6). (B–F) Effects of HKL on cognitive-behavioral impairments of ICH rats were tested by the MWM (n = 6). (B) The average speed of each group on the first day of the experiment. (C) Representative swimming-path traces of each group in the place and spatial navigation trial. Bule point indicates the start position, and red point indicates the end position. (D) Average escape latency of each group per day in the place navigation task. (E) The percentage of duration in the target quadrant for each group in the spatial exploratory task. (F) The number of times each rat passed through the platform in the spatial navigation task. Data are expressed as the mean ± SD. ∗P < 0.05 vs. Sham group; ⁺ P < 0.05 vs. ICH group; NS: non-significant difference compared to the ICH group. HKL: Honokiol; ICH: Intracerebral hemorrhage; mNSS: Modified Neurological Severity Score; MWM: Morris water maze; SD: Standard deviation.

After ICH induction, the effect on long-term neurological function was assessed using the MWM test from day 16 to day 20. The swimming speed was not significantly different among these groups on the first day of the experiment (P > 0.05, Figure 1B). The representative swimming-path traces for each group in the place and spatial navigation trials are displayed in Figure 1C. During the place navigation trial, the escape latency was recorded to assess the learning ability of ICH rats (Figure 1C, D). Compared with the sham group, the escape latency of rats in the ICH group was significantly longer (P < 0.05 for days 16 to 19). However, the escape latency was significantly shortened in the ICH + HKL group compared to the ICH groups on days 18 and 19 (P < 0.05). Besides, no significant differences in latency were observed between the ICH + HKL group and the ICH + vehicle group. During the space navigation trial, the percentage duration in the target quadrant and the frequency of swimming across the platform were representative of the cognitive functions associated with memory (Figure 1C, E, F). The results showed that the rats in the ICH group spent remarkably less time in the target quadrant than those in the sham and ICH + HKL groups (P < 0.05). In addition, there were significant reductions in the number of times the rats crossed the platform in the ICH group compared with the sham group (P < 0.05), while rats in the ICH + HKL group crossed more frequently than those in the ICH group (P < 0.05). No differences were observed between the ICH and ICH + vehicle groups (P > 0.05). The above findings substantiated that HKL treatment could promote neurological functional recovery in ICH rats.

HKL attenuated cerebral injury around the hematoma and cellular apoptosis in ICH rats

HE staining was conducted to evaluate morphological changes following ICH, as shown in Figure 2A. The results showed that the neuronal cells were intact, regularly arranged, and evenly distributed in the sham group. In contrast, the neurons in the ICH group and ICH + vehicle group exhibited an intricate arrangement with loosened cytoplasm and karyopyknosis. Administration of HKL mitigated the pathological damages in the perihematomal regions of cerebral tissues.

Figure 2: HKL alleviated pathological damage and cell apoptosis in rats after ICH. (A) Histopathological changes were assessed by HE staining (40× original magnification and 200× original magnification). Scale bar = 200 μm. (B) The apoptotic cells were detected by TUNEL (red) staining (40× original magnification and 200 × original magnification). Scale bar = 200 μm. (C) Percentage of TUNEL-positive cells (n = 5). (D) Representative Western blot images of Bax, Bcl-2, caspase-3 and cleaved-caspase-3. (E–G) Western blot quantification of Bax, Bcl-2 and cleaved-caspase-3/ caspase-3 ratio (n = 6). Data are expressed as the mean ± SD. ^∗ P < 0.05 vs. Sham group; ⁺ P < 0.05 vs. ICH group; NS: non-significant difference compared to the ICH group. HE: Hematoxylin–Eosin; HKL: Honokiol; ICH: Intracerebral hemorrhage; SD: Standard deviation; TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Apoptotic cells in the perihematomal region were evaluated by TUNEL assays after ICH (Figure 2B, C). The results showed that the cell apoptotic rate in the ICH group was significantly higher than that in the sham group (P < 0.05). In contrast, the number of apoptotic cells in the ICH + HKL group was significantly decreased in comparison with that in the ICH group (P < 0.05). Furthermore, we examined the expression of apoptosis-associated proteins by Western blot (Figure 2D–G). We found that the expression of Bax and the ratio of cleaved caspase-3/caspase-3 in the ICH group was significantly increased compared to the ICH group (P < 0.05), while the Bcl-2 expression was significantly decreased (P < 0.05). However, HKL treatment significantly reduced the expression of Bax and the ratio of cleaved caspase-3/caspase-3, and simultaneously increased the expression of Bcl-2 compared to the ICH group (P < 0.05). There was no significant difference between the ICH group and the ICH + vehicle group (P > 0.05). These results revealed that HKL exerted protective effects against cell apoptosis in ICH.

HKL upregulated the expression of Sirt3 and inhibited mitochondria fission after ICH in rats

Immunohistochemistry assays and Western blot were conducted to investigate the expression of Sirt3 in brain tissues at 24 h post-ICH in rats (Figure 3A–D); brownish staining in the cytoplasm indicated a positive immune reaction. Moreover, the expression of Sirt3 in the ICH group was significantly downregulated compared with the sham group (P < 0.05). In contrast, the ICH + HKL group exhibited significant upregulation of Sirt3 protein levels compared to the ICH group (P < 0.05). These findings supported that Sirt3 might play a crucial role in the protective effects exerted by HKL against ICH.

Figure 3: HKL upregulates Sirt3 expression and inhibits mitochondrial fission in rats after ICH. (A) Representative immunohistochemical staining images of Sirt3 in each group. Positive cells were stained brown (200× original magnification). Scale bar = 200 μm. (B) Results of the relative intensity analysis of Sirt3 immunohistochemistry (n = 5). (C) Western blot images of Sirt3, Drp1, p-Drp1(Ser616) and p-Drp1(Ser637) in each group. (D–G) Western blot quantification for Sirt3, Drp1, p-Drp1(Ser616) and p-Drp1(Ser637) in each group (n = 6). (H) ATP content in perihematomal brain tissues of the rats (n = 3). Data are expressed as the mean ± SD. ∗P < 0.05 vs. Sham group; ⁺ P < 0.05 vs. ICH group; NS: non-significant difference compared to the ICH group. ATP: Adenosine triphosphate; Drp1: Dynamin-related protein 1; HKL: Honokiol; ICH: Intracerebral hemorrhage; p-Drp1(Ser616): Phosphorylation of Drp1 at serine-616; p-Drp1 (Ser637): Phosphorylation of Drp1 at serine-637; Sirt3: Sirtuin-3; SD: Standard deviation.

To verify whether HKL participated in regulating mitochondrial fission and mitochondrial function, we examined the expression of fission protein and ATP content in the perihematomal brain tissues. As shown in Figure 3C–G, the expression of Drp1 and phosphorylation of Drp1 at serine-616 (p-Drp1(Ser616)) was significantly increased, but that of phosphorylation of Drp1 at serine-637 (p-Drp1(Ser637)) was decreased after ICH (P < 0.05); however, after the treatment with HKL, the expression of Drp1 and p-Drp1(Ser616) was down-regulated perceptibly, while that of p-Drp1(Ser637) was up-regulated (P < 0.05). Meanwhile, the ATP content was measured in each group to evaluate the mitochondrial damage after ICH. As shown in Figure 3H, the ATP levels were significantly decreased in ICH rats, while HKL treatment significantly increased the ATP content in brain tissues compared to the ICH group (P < 0.05). In addition, no significant differences were found between the ICH group and the ICH + vehicle group (P > 0.05). Thus, these findings suggest that HKL could inhibit mitochondrial fission and restore mitochondrial function after ICH in vivo.

HKL increased cell viability and decreased cell apoptosis in hemin-treated PC12 cells

The protective effects of HKL against hemin-induced toxicity were detected by CCK-8 and LDH assay. To determine the safe concentrations of HKL in vitro, PC12 cells were treated with HKL at different concentrations (from 0 to 40 μmol/L) for 12 h. As can be seen from Figure 4A, the results indicated that HKL exerted no toxicity effects on PC12 cells at a concentration of 1 to10 μmol/L. However, HKL showed significant cytotoxicity when the concentrations are >20 μmol/L. Furthermore, HKL treatment effectively enhanced the viability of hemin-stimulated PC12 cells in a dose-dependent manner. Exposure to 1, 5, and 10 μmol/L HKL increased the cell survival rate to 50.6%, 56.7% and 67.7%, respectively (Figure 4B). Due to the most significant effect observed above, 10 μmol/L HKL was chosen for the subsequent experiments.

Figure 4: HKL increased the viability and decreased apoptosis of hemin-induced PC12 cells. (A) PC12 cells were treated with HKL at different concentrations (1, 5, 10, 20 and 40 μmol/L), and the cell viability was detected at 12 h by the CCK-8 assay (n = 3). (B) PC12 cells were pretreated with different concentrations of HKL (1, 5 and 10 μmol/L) for 12 h and then exposed to 40 μmol/L hemin for another 12 h. Subsequently, the cell viability was detected (n = 3). (C) PC12 cells were divided into three groups, and the effects of HKL (10 μmol/L) on the release of LDH were detected (n = 3). (D) Apoptosis of PC12 cells was assessed by flow cytometric, and representative FCM plots are shown. (E) Quantified results of the flow cytometric analysis (n = 3). The cell viability results were presented as the percentage of control cell viability. Data are expressed as the mean ± SD. ∗P < 0.05 vs. Control group; ⁺ P < 0.05 vs. Hemin group; mol/L. CCK-8: Cell counting kit-8; FCM: Flow cytometry; FITC: Fluorescein isothiocyanate; HKL: Honokiol; LDH: Lactate dehydrogenase; SD: Standard deviation.

The cellular injury was assessed by quantifying the LDH released from PC12 cells. As shown in Figure 4C, HKL significantly reversed the hemin-induced increase in LDH release in PC12 cells (P < 0.05). To explore whether HKL could affect cell apoptosis, FCM analysis was performed. We found that 10 μmol/L HKL could significantly inhibit hemin-induced apoptosis, compared to the hemin group (P < 0.05) (Figures 4D, E). These findings demonstrated that HKL exerted a protective effect against hemin-induced damage in vitro.

HKL protects against hemin-induced apoptosis by Sirt3 in vitro

To verify the role of Sirt3 in HKL-mediated protection against hemin-induced toxicity, the PC12 cells were incubated with HKL (10 μmol/L) and 3-TYP (50 μmol/L, a Sirt3 inhibitor) for 6 h and subsequently treated with hemin (40 μmol/L) for 12 h. Cell survival was determined by CCK-8 assay. As indicated in Figure 5A, the cells pretreated with HKL exhibited a significant increase in cell survival compared with hemin alone (P < 0.05). However, HKL-mediated cell survival was significantly inhibited by 3-TYP treatment (P < 0.05), indicating that Sirt3 played an important role in HKL-mediated cell protection against hemin-induced cytotoxicity. To further investigate the mechanism of protection of HKL against hemin-induced injury in PC12 cells, the expression of Sirt3 and apoptosis-associated proteins were quantified by Western blot (Figure 5B–F). As shown in Figure 5C, the expression of Sirt3 was decreased in the hemin group but increased in HKL-pretreated cells. Furthermore, this HKL-mediated enhancement in Sirt3 expression was significantly reversed by Sirt3 inhibitor 3-TYP (P < 0.05). In addition, HKL significantly downregulated the expression of Bax and the ratio of cleaved caspase-3/caspase-3 but upregulated Bcl-2 levels in hemin-injury cells (Figure 5D, E). In contrast, 3-TYP effectively reversed the HKL anti-apoptotic effect. Overall, our results corroborated that the anti-apoptotic effect of HKL is mediated by the Sirt3 signaling pathway.

Figure 5: HKL protects against hemin-induced apoptosis by Sirt3 in vitro. (A) After adding 3-TYP, the cell viability was detected (n = 3). (B) Representative protein expression bands of Sirt3, Bax, Bcl-2, caspase-3 and cleaved-caspase-3 as measured by Western blot. (C–F) Western blot quantification of Bax, Bcl-2 and cleaved-caspase-3/ caspase-3 ratio (n = 6). Data are expressed as the mean ± SD. ∗P < 0.05 vs. Control group; ^† P < 0.05 vs. Hemin group; ^‡ P < 0.05 vs. Hemin + HKL group. HKL: Honokiol; Sirt3: Sirtuin-3; SD: Standard deviation; 3-TYP: 3-(1H-1,2,3-triazol-4-yl) pyridine.

HKL protects against hemin-induced mitochondrial fission by Sirt3 in vitro

Previous research has shown the fragmented mitochondria and impaired mitochondrial function in ICH.^[11] To detect the effects of HKL on mitochondria fission in PC12 cells, immunofluorescent staining of mitochondria was performed using MitoTracker Red probe. As shown in Figure 6A–C, we observed that PC12 cells treated with hemin had more fragmented mitochondria with decreased AR and FF ratios compared with the untreated cells (P < 0.05). After treatment with HKL, the mitochondrial AR and FF ratios were increased significantly (P < 0.05), and the mitochondria might have more branches. On the other hand, 3-TYP increased mitochondrial fragmentation in PC12 cells. Next, to investigate the mechanisms underlying mitochondrial fission regulated by HKL, the proteins that directly regulate mitochondrial dynamics, including Drp1, p-Drp1(Ser616), and p-Drp1(Ser637), were examined. The results showed that hemin increased Drp1 expression and Drp1 Ser616 phosphorylation but also reduced Ser637 phosphorylation (Figure 6D–G), which was accompanied by more colocalization of Drp1 in mitochondria (Figure 6H). In contrast, HKL enhanced Ser637 but reduced Ser616 phosphorylation of Drp1, followed by the lower Drp1-mitochondria colocalization. Moreover, the effects of HKL on Drp1 phosphorylation modification were abolished by 3-TYP. These results proved that HKL could prevent Drp1-mediated mitochondrial fission by Sirt3 in hemin-exposed PC12 cells.

Figure 6: HKL protects against hemin-induced mitochondrial fission by Sirt3 in vitro. (A) Mitochondrial morphology was evaluated by Mitotracker Red staining (1000× original magnification, scale bar = 10 μm). (B, C) FF and AR of mitochondrial. (D) Representative protein expression bands of Drp1, p-Drp1(Ser616) and p-Drp1(Ser637) as measured by Western blot. (E–G) Western blot quantification for Drp1, p-Drp1(Ser616) and p-Drp1(Ser637) (n = 6). (H) Mitochondrial localization of DRP1 assessed under confocal a laser scanning microscope (green). Mitochondrial morphology was stained with MitoTracker (red) (1000× magnification, scale bar = 10 μm). Data are expressed as the mean ± SD. ^∗ P < 0.05 vs. Control group; ^† P < 0.05 vs. Hemin group; ^‡ P < 0.05 vs. Hemin + HKL group. AR: Aspect ratio; Drp-1: Dynamin-related protein 1; FF: Form factor; HKL: Honokiol; p-Drp1(Ser616): Phosphorylation of Drp1 at serine-616; p-Drp1(Ser637): Phosphorylation of Drp1 at serine-637; Sirt3: Sirtuin-3; SD: Standard deviation; 3-TYP: 3-(1H-1,2,3-triazol-4-yl) pyridine.

Discussion

HKL is a natural compound derived from the magnolia plant and used in traditional Chinese medicine. The therapeutic potential has been reported in various preclinical models.^[20] In the present study, we found that HKL exerted neuroprotective and anti-apoptotic effects on ICH in vitro and in vivo, which may be associated with the upregulation of Sirt3 levels and the inhibition of Drp1-mediated mitochondrial fission. Our results suggest that HKL has immense prospects as a novel therapeutic agent for ICH treatment.

Similar beneficial effects have been documented in animal models of traumatic brain injury^[30] and Alzheimer's disease^[31] in terms of enhanced neurological deficits and improved cognitive function. In the present study, within 14 days after ICH, the mNSS test results indicated that intraperitoneal administration of 10 mg/kg HKL alleviated neurological deficits in the experimental rats. Additionally, treatment with HKL shortened the escape latency, prolonged the exploration time in the targeted quadrant, and increased the number of platform crossings in experimental rats during the MWM test. These results indicated that HKL treatment positively affected the recovery of cognitive deficit induced by ICH in rats. The differentiated neuronlike PC12 cell line is widely acknowledged to exhibit neuronal phenotype and function. The hemin-treated PC12 cell model has been widely used to mimic the neuronal damage process of ICH.^[32] Therefore, this model was used in our study to investigate the effects of HKL in vitro. We found that HKL treatment significantly alleviated the decreased cell viability and the increased release of LDH induced by hemin.

It has been established that a series of intricate mechanisms lead to SBI after ICH, such as oxidative stress, inflammatory response, and cell apoptosis. Importantly, neuronal apoptosis plays a crucial role in the progression of ICH-mediated brain injury. In previous studies, HKL has been demonstrated to exert anti-apoptotic effects in diabetic myocardial ischemia/reperfusion injury^[33] and amyloid-β oligomer-induced neurotoxicity.^[34] Similarly, in the present study, HKL could suppress apoptosis induced by ICH in vivo and in vitro. Notably, Akt is a serine/threonine kinase that promotes cell survival, and HKL is a well-known AKT inhibitor. So, HKL is also used as a novel natural agent against tumors and angiogenesis. Yeh et al^[35] showed that HKL could induce apoptosis of neuroblastoma cells by inhibiting the PI3K/Akt/mTOR signaling pathway. However, it is reported that HKL could prevent mitochondrial damage and apoptosis in cardiac H/R injury accompanied with negative regulation of Akt signaling pathway.^[36] The conflicting results between these studies were probably due to species-specific differences and different methods. In other words, the inhibition of Akt by HKL may exert different effects on cell survival in different situations. We will investigate the effects of HKL on Akt signaling pathway in future experiments.

Mitochondria are dynamic organelles that constantly fuse and divide. However, excessive fission can impair mitochondrial structure, increase the production of ROS, and reduce ATP levels and mitochondrial membrane potential, an early marker of cell apoptosis.^[37] Mitochondrial fission is regulated by Drp1, while several mitochondrial outer membrane proteins, including fission 1 protein (Fis1), mitochondrial fission factor, and mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively), recruit Drp1 from the cytosol to the outer membrane of mitochondria during mitochondrial fission.^[8,38] In addition, phosphorylation of Drp1 at Ser616 can regulate mitochondrial translocation of Drp1 and activate mitochondrial fission, while the phosphorylation of Drp1 at Ser637 has the opposite effect.^[39] Some studies also revealed that phosphorylation of Drp1 at Ser616 was a key regulator of cell apoptosis.^[40,41] Wu et al^[42] found that the upregulated expression of Drp1 and p-Drp1 (Ser616) was associated with excessive mitochondrial fission after subarachnoid hemorrhage in rats; Mdivi-1 (a selective inhibitor of mitochondrial fission protein) reversed this effect and exerted neuroprotective effects against subarachnoid hemorrhage-induced cell apoptosis and oxidative stress. Moreover, mitigating high glucose-induced overexpression of Drp1 or Fis1 genes improved mitochondrial function and protected retinal endothelial cells from apoptosis.^[43] Recently, dynamic mitochondrial network shifts towards mitochondrial fission after ICH have been observed in an increasing number of studies, as evidenced by increased expression of Drp1 and mitochondrial fragmentation, indicating that excessive mitochondrial fission is a major contributor to the development of ICH.^[6,7,11] In the present study, we found HKL could alleviate mitochondrial fission via balancing Drp1 phosphorylation in ICH, as manifested by reduced Ser616 phosphorylation but increased Ser637 phosphorylation. Moreover, HKL inhibited Drp1 recruitment towards mitochondria and increased ATP levels.

It has been reported that HKL is a pharmacological activator of Sirt3 by simultaneously increasing its expression and activity.^[44] Sirt3 is an NAD⁺ -dependent enzyme mainly localized in mitochondria and plays a crucial role in mitochondrial function and energy metabolism.^[45] Liu et al^[14] showed that Sirt3 was downregulated in post-infarction cardiac injury, while Sirt3 overexpression preserved mitochondrial homeostasis and cardiomyocyte viability by attenuating mitochondrial fission. In contrast, Sirt3 deficiency aggravated neuronal apoptosis and neurological deficits after ischemic stroke.^[46] In another study, HKL reversed the decreased expression of Sirt3 in a rat model of subarachnoid hemorrhage to maintain mitochondrial function and promote neural survival.^[22] In the present study, we proved that the protective effects of HKL on mitochondrial fission and apoptosis depending on the activation of Sirt3 signaling pathway.

Our results indicated that HKL is a pleiotropic compound with great clinical potential in ICH. However, HKL shows limitations in clinical application because it has poor aqueous solubility and can be easily degraded by oxidation. Notably, HKL liposome, a novel drug delivery system, has been shown to improve solubility and pharmacokinetics compared with free forms,^[47] and a phase I clinical trial of the HKL liposome in advanced solid tumor malignancies is currently ongoing in Beijing Tiantan Hospital. More researches are needed before HKL can be introduced into clinical applications for treating ICH. In addition, there were several limitations in this study. First, we only focused on the mitochondrial fission protein Drp1 after HKL treatment; other mitochondrial dynamics-related proteins were not evaluated. Moreover, other possible mechanisms of HKL need to be further explored, such as the Akt signaling pathway. Besides, it is unclear whether apoptosis is mediated by the activated Drp1 after ICH, although proven in other models.^[40-42] This potential relationship will be explored in our forthcoming studies.

In conclusion, the present study showed that HKL treatment significantly alleviated short and long-term neurological deficits in ICH rats. Moreover, HKL exerted protective effects against enhanced mitochondrial fission and apoptosis induced by ICH in vivo and in vitro models, mediated partially at least by the Sirt3 signaling pathway. Our study suggests that HKL may be used as a novel therapeutic agent to alleviate brain injury after ICH.

Funding

This work was supported by a grant from the Natural Science Foundation of Hebei Province (No. H2019105137).

Conflicts of interest

None.