Effects of chrysophanol on hippocampal damage and mitochondrial autophagy in mice with cerebral ischemia reperfusion

Abstract

Objective

The cerebral ischemia-reperfusion (I/R) model is crucial for the study of cerebral stroke. Chrysophanol (Chry) can protect nerve damage of mice in cerebral ischemia-reperfusion injury. This study aimed at investigating the neuroprotective effects of chrysophanol through mitochondrial autophagy in mice with ischemia-reperfusion injury.

Materials and methods

Adult mice were stochastically divided into five groups: sham, I/R (solvent), I/R+Chry (dose, 10.0ml/kg), I/R+Chry (dose, 1.0ml/kg), and I/R+Chry (dose, 0.1ml/kg). The cerebral ischemia-reperfusion model was made in I/R and I/R+Chry groups. The changes in hippocampal formation were observed by hematoxylin and eosin (H&E) staining. The expressions of LC3B-II and LC3B-I protein in hippocampus were demonstrated by western blot (WB). The fluorescence intensities of NIX, LC3B, and mitochondria were detected by immunohistochemistry fluorescent (IF).

Results

Comparing with the I/R group, the I/R+Chry groups showed improvements in reducing the damage on the hippocampus, indicated by the reduced ratio of LC3B-II and LC3B-I protein, decreased fluorescence intensity of NIX and LC3B, and increased intensity of mitochondrial fluorescence.

Conclusion

Our study showed that chrysophanol may regulate mitochondrial autophagy through NIX protein and alleviate the damage of hippocampus through decreasing the level of mitochondrial autophagy.

Keywords:

• Chrysophanol
• cerebral ischemia-reperfusion injury
• hippocampus
• mitochondrial autophagy
• LC3B

Introduction

Cerebral stroke is a common and frequently-occurring disease associated with high incidence, mortality, and disability. Globally, every year there are a large number of people suffering from cerebrovascular disease, of which two-thirds of the cases will result in death or permanent disability [1]. Chrysophanol is one of the active constituents identified in traditional Chinese medicine rhubarb. It had been frequently suggested by previous studies that chrysophanol has neuroprotective effects on cerebral ischemia-reperfusion injury through multiple mechanisms including anti-oxidation, anti-inflammation, and inhibition of neuronal apoptosis [2,3].

Cerebral ischemia-reperfusion injury is shown to be responsible for a large number of mitochondrial damages and mitochondria autophagy. Mitochondrial autophagy plays a two-way effect in the regulation of cerebral ischemia-reperfusion injury: with injury of cerebral ischemia and reperfusion leading to lots of mitochondrial damages, appropriate and well-controlled autophagy of mitochondria clears damaged mitochondria and contributes to cell protection; whereas excessive autophagy causes cell death and hippocampal damage [4,5]. In addition, experiments had shown that microtubule-associated protein 1 light chain 3 (LC3B) played a part in mitochondrial autophagy. Free LC3B-I in the cytoplasm can be recruited to the mitochondrial outer membrane and get combined with phosphatidylethanolamine to form LC3B-II when autophagy occurs, then LC3B-I together with LC3B-II lead to mitochondrial autophagy, of which the extent is normally reflected by the ratio of LC3B-II to LC3B-I [6]. Following this pathway, mitochondrial receptor Nip3-like protein X (NIX), which is localized on the mitochondrial outer membrane, was suggested to participate in mitochondrial autophagy through combining its LIR sequence and LC3B [7–9]. This study aims to determine the effect of chrysophanol on hippocampus and mitochondrial autophagy, as well as to investigate the role of NIX protein in the context of chrysophanol on mitochondrial autophagy.

Methods

Animals

All animal experiments were performed according to the Institutional Guidelines for Animal Care and Use Committee (Hebei North University of China). Male healthy Kunming (KM) mice aged 6–8 weeks and weight 18–22 g (National Institutes for Food and Drug Control Co. Ltd, Beijing, China) were previously and stochastically divided into five groups: sham, I/R (solvent), I/R + Chry (dose, 10.0 ml/kg), I/R + Chry (dose, 1.0 ml/kg), and I/R + Chry (dose, 0.1 ml/kg). The mice in groups of I/R + Chry were intraperitoneally administered chrysophanol for 10 days, and the mice in groups of sham and I/R were intraperitoneally administered solvent for 10 days. Then the animals were administered the last chrysophanol before 30 min of ischemia. All mice were kept with commercial rat food and water. They were housed under controlled temperature (22 °C) and relative humidity (50% ± 5%) conditions with a 12-h light/dark cycle.

Establishment of cerebral ischemia-reperfusion injury model

We modified the model from Himori's method [10]. To establish the cerebral ischemia-reperfusion injury model, we administered a 3.5% chloral hydrate solution for anesthesia. After a midline incision at the neck, the bilateral common carotid arteries (CCA) were fully exposed via careful blunt separation. We used a black sterile 3-0 silk soaked with liquid paraffin to pass through the CCA on both sides and placed a loose backhand knot over the trachea. A white sterile 3-0 silk soaked with liquid paraffin was passed through the black silk above each CCA, tied to the black silk, and the incision was sutured by the Hamori method. After the operation, the mice were irradiated with infrared light to keep warm and maintain their body temperature, with care to keep the cages dry and free of water or dust. After 48 h, we tightened the black silk and blocked the CCA on both sides, leading to cerebral ischemia. Then after 5 min, we gently pulled the white silk to make CCA on both sides restore the blood. Sham-operated mice were operated identically except for the process of cerebral ischemia-reperfusion injury. They were housed under controlled temperature (22 °C) and relative humidity (50% ± 5%) conditions with a 12-h light/dark cycle throughout the experiment, to ensure that the mice body states are stable.

Hematoxylin and eosin staining

After 24 h of reperfusion, the mice were anesthetized and decapitated to death. The brain tissues were rapidly excised, cut into 5 mm thick coronal sections, rinsed in 0.9% saline, and fixed in 4% paraformaldehyde solution overnight. We dehydrated the specimen based on the following procedure: 70% alcohol for 2 h, 80% alcohol for 1.5 h, 90% alcohol for 1 h, 95% alcohol for 1 h, anhydrous alcohol I for 30 min, anhydrous alcohol II for 30 min, xylene I for 30 min, xylene II for 30 min, xylene III for 30 min, paraffin I for 30 min, paraffin for II 30 min, and paraffin III for 30 min. Then, the tissues were removed and embedded in paraffin using heated paraffin embedding module (Leica, Wetzlar, Germany). The paraffin blocks were pruned and cut into serial slices of 5-μm-thick using Semi-Automated Rotary Microtome (Leica, Wetzlar, Germany), which were then stained according to the following steps: xylene I for 10 min, xylene II for 10 min, anhydrous alcohol I for 3 min, anhydrous alcohol II for 3 min, 95% alcohol for 3 min, 85% alcohol for 3 min, 75% alcohol for 3 min, running water wash for 1 min, hematoxylin for 15 min, running water wash for 5 s, acid alcohol for 10 s, running water wash for 3 s, bluing reagent for 30 s, running water wash for 3 s, eosin for 1 min, running water wash for 5 s, 70% alcohol for 2 min, 80% alcohol for 2 min, 95% alcohol for 3 min, 100% alcohol for 3 min, 100% alcohol for 3 min, xylene I for 3 min, xylene II for 3 min, and xylene III for 3 min. After the sections were mounted by using neutral resin, hippocampus of pathological changes was observed by Inverted Research Microscope (Nikon, Tokyo Metropolis, Japan).

Western blot

After 24 h of reperfusion, the mice were anesthetized and decapitated to death. The brains of the mice were rapidly excised and the hippocampus was carefully separated. Hippocampus was first gently dissociated in lysis buffer (with protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride (PMSF), Solarbio, Beijing, China). After 1 h, we centrifuged the hippocampus suspension at 12,000 g for 10 min at 4 °C, the resulting supernatant was hippocampal protein. Protein concentrations were determined immediately before WB experiments, using a (BCA) protein assay kit (Boster Biological Technology, Wuhan, China). Proteins were denatured by boiling for 10 min in SDS-PAGE loading buffer (Boster Biological Technology, Wuhan, China), then size-fractionated on 15% Tris-Tricine precast gels and electrophoretically transferred onto 0.2-μm polyvinylidene fluoride membranes at 100 V for 1.5 h by wet blot. Membranes were blocked with blocking bufferI (5% (weight/volume (w/v)) difco skim milk (Solarbio, Beijing, China) in Tris-buffered saline with 0.1% (volume/volume (v/v) Tween 20 (TBS-T)) for 2 h at room temperature, and then incubated overnight at 4 °C in anti-LC3B antibody diluted with blocking bufferI (1:2000; Abcam, Cambridge, UK). Following overnight incubation, we washed membranes for 5 min for 3 times in TBS-T at room temperature and incubated them in Goat anti-Rabbit IgG/HRP antibody diluted with TBS-T (1:8000; Beijing Biosynthesis Biotechnology, Beijing, China) for 1.5 h at room temperature. Then, we washed membranes for 5 min for 3 times in TBS-T. We exposed blots using a chemiluminescence reagent and imaged using multicolor fluorescence and chemiluminescence imaging system (Proteinsimple, California, USA).

Immunohistochemistry fluorescent

The paraffin sections of brain tissues (5-μm thick) were deparaffinized according to the step of deparaffinization by H&E staining, incubated in ultra-pure water for 3 min, and boiled in sodium citrate antigen repair solution for 10 min. We washed the paraffin sections for 5 min for 3 times in phosphate buffer saline (PBS; Boster Biological Technology, Wuhan, China), incubated in blocking bufferII (10% goat serum; Boster Biological Technology, Wuhan, China) for 30 min, then incubated for 5 h at 4 °C in anti-LC3B antibody diluted with blocking buffer II (1:500). After incubation, we removed the anti-LC3B antibody and incubated the paraffin sections for 5 h at 4 °C in anti-NIX antibody diluted with blocking buffer II (1:50; Santa CruzBiotechnology, Santa Cruz, USA). Then, we washed the paraffin sections for 5 min for 3 times in PBS, incubated for 2 h at room temperature in the dark with Alexa Fluor® 647-labeled Goat anti-Rabbit IgG (H + L) diluted with PBS (1:500; Beyotime Biotechnology, Shanghai, China) and Alexa Fluor® 488 - Conjugated Goat anti-Mouse IgG (H + L) diluted with PBS (1:400; Beijing Zhongshan Golden Bridge, Beijing, China). After the incubation, paraffin sections were washed for 5 min for 3 times in PBS in the dark, incubated for 15 min at room temperature in the dark in MitoTracker Red CMXRos (1:500; Solarbio, Beijing, China) diluted with dimethyl sulfoxide (DMSO; Solarbio, Beijing, China), then washed for 5 min for 3 times in PBS in the dark and mounted by using antifading mounting medium. The fluorescence intensity of hippocampus changes was observed by Laser Scanning Confocal Microscopy (Olympus, Tokyo Metropolis, Japan).

Statistical analysis

Experimental data was presented as mean ± SD. The SPSS 17.0 statistical software was utilized for statistical analysis, which was performed one-way ANOVA analysis for comparison between groups. A value of p < 0.05 was defined as statistically significant.

Results

Chrysophanol protected hippocampus in cerebral ischemia-reperfusion injury mice

After 24 h cerebral ischemia-reperfusion, we observed the CA1 region (Figure 1(A)) and DG region (Figure 1(B)) of hippocampus of pathological changes by H & E staining. The results indicated that the hippocampus of the sham group had a complete structure, arranged cells, and clear nuclei; the hippocampus in I/R group had a loose structure, arranged with messy cells, destroyed neuron cell, and unclear nuclei. The hippocampus in I/R + Chry (dose, 10.0 ml/kg) was similar to sham group, as it had a complete structure, a clear hierarchy, clear nuclei, and a relatively close arrangement; The hippocampus in I/R + Chry (dose, 0.1 ml/kg) was similar to I/R group, that is, the structure was loose and hierarchy was unclear.

Figure 1. Chrysophanol alleviated the cerebral ischemia-reperfusion injury of hippocampus in mice. After 24 h of cerebral ischemia-reperfusion, the protective effects of Chry on hippocampus were assessed by hematoxylin and eosin staining. (A) H & E staining results for the histopathological of the CA1 region of the hippocampus indicated the histopathological changes in ischemia-reperfusion model as well as that caused by different concentrations of chrysophanol at a magnification of 200×. (B) H & E staining results for the histopathological of the dentate gyrus of the hippocampus indicated the histopathological changes in ischemia-reperfusion model as well as that caused by different concentrations of chrysophanol at a magnification of 200×.

Chrysophanol inhibited the expression of LC3B in cerebral ischemia-reperfusion mice

The membranes results showed that the ratio of LC3B-II to LC3B-I of I/R group was higher than that of sham group (p < 0.05); The ratio of LC3B-II to LC3B-I decreased in I/R + Chry (dose, 10.0 ml/kg), compared with I/R group (Figure 2(A)) (p < 0.05).

Figure 2. Chrysophanol inhibits LC3B and reduces mitochondrial autophagy of hippocampus in mice. (A) After 24 h of I/R, the result of ratio of LC3BIIto LC3BIby ImageJ analysis of LC3B western blot. (B) Hippocampal total protein was electrophoresed and analyzed by western blot using anti-LC3B antibodies. All Individual experiments were repeated for three times and mean ± SD was calculated. * p < 0.05, ** p < 0.01.

Chrysophanol inhibited the expression of NIX and protected mitochondria in cerebral ischemia-reperfusion mice

The changes of NIX, LC3B, and mitochondria in hippocampus of every group were observed by Laser Scanning Confocal Microscopy. The fluorescence intensity of NIX and LC3B in hippocampus of sham group was low and the fluorescence intensity of mitochondria was high. After cerebral ischemia-reperfusion injury, the fluorescence intensities of NIX and LC3B increased, and fluorescence intensity of mitochondria decreased. The fluorescence intensities of NIX and LC3B in I/R + Chry groups were lower than in I/R group, and the mitochondrial fluorescence intensity was higher than in I/R group (Figure 3(C–E)) (p < 0.05).

Figure 3. (A) The changes of LC3B, NIX, and mitochondrial fluorescence intensity in the different groups of hippocampal CA1 regions were observed by confocal microscopy of 200×. (B) The changes of LC3B, NIX, and mitochondrial fluorescence intensity in the different groups of hippocampal dentate gyrus regions were observed by confocal microscopy of 200×. (C) The result for the mean fluorescence intensity of NIX on the hippocampus in different groups of mice. (D) The result for the mean fluorescence intensity of MitoTracker Red on the hippocampus in different groups of mice. (E) The result for the mean fluorescence intensity of LC3B on the hippocampus in different groups of mice. * p < 0.05, ** p < 0.01 vs. I/R group, ^# p < 0.05, ^## p < 0.01 vs. sham group.

Discussion

H & E staining experiments confirmed severe damages to hippocampal formation after cerebral ischemia-reperfusion injury, and chrysophanol has a protective effect on mice hippocampal damage caused by cerebral ischemia-reperfusion injury. The hippocampal formation is an important component of the limbic system located at the base of the inner side of the temporal lobe, including hippocampus, hippocampal dentate gyrus (DG), and lower support [11]. The hippocampus is associated with a variety of neurological and psychiatric diseases. In particular, the hippocampal CA1 region vertebral system cells are highly susceptible to ischemia and hypoxia injury [12]. The hippocampal formation is a brain area related to learning and memory, with its hippocampal DG region, as the main information afferent region in the hippocampal formation, playing an important role in the rapid acquisition and coding of spatial memory [13]. After cerebral ischemia, the consequent ischemic and hypoxic state of the brain is responsible for great damage to the hippocampus [14]. We speculated that chrysophanol has a protective effect on hippocampal damage caused by cerebral ischemia-reperfusion injury, and our results are consistent with our hypothesis.

We obtained the ratio of LC3B-II to LC3B-I through the data of LC3B-I and LC3B-II in the WB. The results showed that the ratio of LC3B-II to LC3B-I in hippocampus of mice was increased after cerebral ischemia-reperfusion injury, whereas the ratio was decreased in I/R + Chry group (dose, 10.0 ml/kg) (p < 0.05). Mitochondrial autophagy plays a huge role in the complex pathological process of cerebral ischemia-reperfusion injury, in which the protective effect of autophagy during reperfusion can be attributed to the inhibition of mitochondrial autophagy [15]. Meanwhile, microtubule-associated protein 1 light chain 3 (MAP1LC3, LC3), mainly includes five subtypes: LC3A (variant 1: v1; variant 2: v2), LC3B, LC3B-II, and LC3C, among which LC3B and LC3Av1 subtypes have been reported to be involved in the formation and assembly of autophagosomes, and LC3B has been widely used to track the occurrence of autophagy [16]. Physiologically, free LC3B-I in the cytoplasm can be recruited to the mitochondrial outer membrane and combined with phosphatidylethanolamine to form LC3B-II when autophagy occurs. We use the ratio of LC3B-II to LC3B-I to reflect the extent of mitochondrial autophagy [17]. The ratio of LC3B-II to LC3B-I was decreased after administration of chrysophanol, indicating that chrysophanol has an inhibitory effect on the autophagy of hippocampal mitochondria therefore protecting hippocampal cells from damage. Furthermore, the expression of NIX and LC3B protein was reduced and mitochondria autophagy was inhibited after administration of chrysophanol (p < 0.05).

In addition, we found through IF that, comparing with the sham group, the number of mitochondria in the hippocampus of mice decreased, and the expression of NIX and LC3B protein increased after cerebral ischemia-reperfusion injury. The Bcl-2 adenovirus E/B19 kDa interacting protein (BNIP3) and the NIX protein are proteins associated with only the BH3 family, both of which were reported to induce cell death and autophagy. BNIP3 and NIX are regulated by hypoxia, of which the NIX is a necessity for specific autophagy and targeting mitochondrial autophagy [18]. In specific, NIX can promote mitochondrial depolarization and reactive oxygen species production, which inhibits mTOR signaling and activates autophagy [19]. At the same time, NIX can participate in mitochondrial autophagy through the binding of its LIR sequence to the LC3B protein in the Atg8 family of proteins, where the LIR-W35 site is the site of interaction with the Atg8 family of proteins in NIX [20]. The regulatory pathways of mitochondria include PARK1-Parkin, NIX-Bnip3, FUN14, and the like [21–23]. After analysis, NIX and LC3B proteins have a certain linear trend, which illustrates that the chrysophanol of the inhibition on mitochondrial autophagy is at least partially attributed to the regulation of NIX.

In conclusion, chrysophanol has a protective effect on hippocampus after cerebral ischemia-reperfusion injury by inhibiting mitochondrial autophagy, and the inhibition on mitochondrial autophagy is at least partially attributed to the regulation of NIX.

Ethics statement

This study was performed following the recommendations in the Guidance for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of China (permit number: TCM-LAEC2014004). The experimental procedures were based on the Directive 2010/63/EU adopted by the European Union (EU), and the management and euthanasia procedures for all animals were administrated following the guidelines of Ethics Review Committee for Laboratory Animal Welfare of Hebei North University.

Author contributions

WHC, SW, and ZMQ designed the study; WHC collected the data; WHC and ZW performed the H & E Stain and IF experiments; WHC and ZMQ performed the WB experiments; SW provided experimental support; WHC and SW analyzed and interpreted the data; WHC and SW wrote the manuscript and provided financial support.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Program for Science & Technology of Hebei Province (No. 07276166), and the Program for Innovation Talent of Hebei North University (No. 20190414), the Special Fund for graduate of Hebei North University.