# Chrysophanol Ameliorates Hemin-Induced Oxidative Stress and Endoplasmic Reticulum Stress by Regulating MicroRNA-320-5p/Wnt3a Pathway in HT22 Cells

Authors: Xu Zhao; Dongge Qiao; Dongsheng Guan; Kun Wang; Yinglin Cui
Journal: Oxidative Medicine and Cellular Longevity
 (2022)

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## Abstract

Oxidative stress, endoplasmic reticulum (ER) stress, and neuronal cell apoptosis have been considered as the main pathogenesis factors of brain injury after intracerebral hemorrhage (ICH). Chrysophanol (CHR) has been proved to have neuroprotective effects, but the role and underlying mechanisms of CHR in ICH remain unclear. HT22 cells were dealt with hemin to mimic an in vitro ICH model and then subjected to treatment with or without CHR. The cell viability, apoptosis, ER stress, and oxidative stress were evaluated by conducting the cell counting kit-8 (CCK-8), TdT-mediated dUTP nick end labeling (TUNEL) staining assays, western blot, and corresponding kit, respectively. Further, microRNA-sequencing, bioinformatic analysis, dual-luciferase reporter method, and rescue experiments were conducted to explore the molecular mechanisms of CHR alleviating hemin-induced ER in HT22 cell. Our data revealed that CHR increased cells viability, antiapoptosis, anti-ER stress, and antioxidative stress under conditions of hemin-induced HT22 cell injury. Mechanically, it was observed that Wnt3a was competitively sponged by miR-320-5p, and CHR activatedβ-catenin pathway by regulating miR-320-5p/Wnt3a molecular axis. Finally, results from the rescue experiment suggested that CHR inhibited hemin-induced cells apoptosis, ER stress, and oxidative stress through regulating the miR-320-5p/Wnt3a axis in HT22 cells. In conclusion, CHR prevented hemin-induced apoptosis, ER stress, and oxidative stress via inhibiting the miR-320-5p/Wnt3a/β-catenin pathway in HT22 cells. Our results certified that CHR could be served as a promising treatment for brain damage following ICH.


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## Body

## 1. Introduction

Intracranial hemorrhage (ICH) is a clinically common type of cerebral apoplexy with high disability rate and mortality, accounting for about 10-15% of all stroke types [1]. The overall survival rate of one year after intracerebral hemorrhage is about 40%, and only 12% to 39% of the survivors have the ability to live independently, which seriously threatens the lives and health of residents and brings heavy burden to families and society [2]. Current research has shown that oxidative stress and endoplasmic reticulum (ER) stress are the prominent contributors in ICH-induced brain injury [3]. Accumulating studies have disclosed that ROS production is enhanced along with the decreasing antioxidant enzyme in the brain following ICH, and the increased oxidative stress aggravates inflammatory reaction, apoptosis, autophagy, and blood-brain barrier destruction, leading to further the aggravation of brain damage, suggesting that antioxidative stress strategies improve ICH-induced brain injury [3, 4]. Deng et al. uncovered that targeted intervention of ERK/Nrf2/HO-1 pathway attenuated neuronal death via attenuating oxidative stress in rats with ICH [5]. Zheng et al. reported that overexpression of Sirt3 inhibited oxidative stress-induced neuronal damage following ICH in rats with hyperglycemia [6]. Wang et al. indicated that the upregulation of SERPINE1 exacerbated cell apoptosis and promoted cell inflammation response in hemin-stimulated HT22 cells, suggesting SERPINE1 as a new mechanism and therapeutic target of brain damage after ICH [7]. Although significant development has been made to explore the mechanisms of brain injury after ICH, there is still no safe and valid clinical therapy method to obviously ameliorate the prognosis of ICH [8]. Therefore, it is important to elucidate the pathophysiology of intracerebral hemorrhage and to discover new therapeutic methods.Chrysophanol (CHR) is an active component found inPolygoni multiflori and Rhamnus alpinus L. and is used to prepare Traditional Chinese Medicine [9, 10]. CHR is an anthraquinone that exhibits excellent pharmacokinetic properties that can be used to achieve a good extent of absorption and slow rate of elimination [11]. It has been recently reported that CHR exhibits numerous beneficial pharmacological properties (such as anticancer, anticardiovascular, hepatoprotective, antioxidative, anti-inflammatory, antiulcer, and antimicrobial properties) [11, 12]. Park et al. disclosed that CHR repressed breast cancer cell formation through regulating mitochondrial apoptosis and ER stress by controlling the AKT/MAPK pathway [13]. Lu et al. revealed that CHR ameliorates doxorubicin-induced cardiotoxicity by inhibiting cellular PARylation [14]. CHR could prevent hepatitis B virus X protein- (HBx-) induced hepatic stellate cell activation through the activation of ER stress [15]. It has been recently reported that CHR exhibits neuroprotective properties. For example, Cui et al. reported that CHR could protect against nerve damage by regulating the process of mitochondrial autophagy in mice suffering from cerebral ischemia/reperfusion [16]. Zhao et al. reported that CHR helped reduce nitrative/oxidative stress and ER stress to alleviate cerebral ischemia/reperfusion-induced neuronal injury in mice [17, 18]. Chu et al. demonstrated that CHR ameliorated cognition dysfunction and neuronal loss by anti-inflammation in mice with diabetic encephalopathy [19]. However, the roles of CHR and the underlying mechanisms associated with treating ICH are still unclear.MicroRNAs (miRNAs) are a member of noncoding RNAs that contain 18~25 nucleotides, which influence numerous biological and pathological processes through the process of posttranscriptional regulation of gene expression [20]. Numerous researchers demonstrated that miRNAs influence the occurrence and development of ICH [21, 22]. For example, upregulation of miR-126 could protect against ICH. The behavioral performance can be improved, and apoptosis can be inhibited via the upregulation of VEGF-A and downregulation of caspase-3 [23]. Modulation of the miR-124/ferroportin pathway inhibited neuronal cell death by decreasing the extent of apoptosis and ferroptosis realized in aged mice with ICH [24]. Isorhynchophylline compactivity sponged TP53 to inhibit its expression by upregulating miR-122-5p. This subsequently prevented the occurrence of ferroptosis-induced nerve injury following ICH [25]. The results revealed that miRNAs exerted critical roles in the occurrence and development of ICH, which could be used as potential therapeutic targets. However, whether CHR function in ICH are still unclear.Hemin, a cytotoxic decomposition product released from hemoglobin, is key mediators of neuronal damage during ICH and is widely used to simulate the ICH injury model [26–28]. For example, Wang et al. demonstrated that TGFβ1/SERPINE1 axis is a novel mechanism promoting the development of ICH using hemin-induced ICH model in vitro [7]. Chu et al. indicated that inhibition of IL-RA protected against neuronal necroptosis by decreasing the necrosome complex levels in hemin-induced ICH model in vitro and vivo, suggesting that targeted inhibition of IL-1R1 may be a promising therapeutic strategy for ICH patients [29]. In addition, HT22 cell is an immortalized mouse hippocampal neuronal cell line subcloned from parent HT4 cells that were originally immortalized from cultures of primary mouse hippocampal neurons [30], and HT22 cell is known to phenotypically resemble neuronal precursor cells, which has been used in cell model to study of hemin-induced injury in neuronal cells [7, 29]. Therefore, we used hemin-induced HT22 cell injury to establish an in vitro ICH model to understand the effect exerted by CHR on the hemin-induced HT22 cell injury and to explore the mechanism following which CHR prevented hemin-induced HT22 cell injury. We conducted comprehensive miRNAs-sequencing, bioinformatics prediction, dual-luciferase reporting, and rescue experiments to arrive at the results.


## 2. Materials and Methods

### 2.1. Reagents

CHR (purity≥98%) was obtained from Solarbio Life Sciences (Beijing, China) and dissolved in dimethyl sulfoxide (DMSO, purity≥99.5.0%, Sigma) to form a stock solution of concentration 40 mM. The solution was stored at -20°C. The prepared stock solution was diluted to form the corresponding working solutions before conducting each set of experiments. Hemin (purity≥96%) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Tunicamycin (TM) was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Dulbecco’s modified eagle’s medium (DMEM), phosphate-buffered saline (PBS), and penicillin/streptomycin were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The cell counting kit-8 (CCK-8), lactate dehydrogenase (LDH) assay kit, malondialdehyde (MDA) assay kit, superoxide dismutase (SOD) assay kit with WST-8, glutathione peroxidase (GPx) assay kit with DTNB, reactive oxygen species (ROS) assay kit, and One Step TdT-mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay kit were bought from Beyotime (Shanghai, China). Fetal bovine serum (FBS) was bought from Gibco (Thermo Fisher Scientific, USA). The caspase-3 assay kit was purchased from Solarbio Life Sciences (Beijing, China). The Hairpin-itTM miRNA real-time PCR quantification kit was obtained from GenePharma (Shanghai, China). The PrimeScript™ RT reagent kit with gDNA eraser and TB Green® Fast qPCR Mix were obtained from Takara (Dalian, China). Antibody against phospho-eukaryotic translation initiation factor 2α (p-Eif2aα, #3398, 1 : 1000), C/EBP homologous protein (CHOP, #2895, 1 : 1000), cleaved caspase-12 (#35965, 1 : 1000), cleaved caspase-3 (#9661, 1 : 1000), Bax (#2772, 1 : 2000), and Bcl-2 (#2870, 1 : 2000) were bought from Cell Signaling Technology (Beverly, MA, USA). Antibody against glucose-regulated protein 78 (GRP78, ab108315, 1 : 1000), β-actin (ab179467, 1 : 5000), Wnt3a (ab219412,1 : 1000), β-catenin (ab108315, 1 : 1000), and Lamin B (ab16048, 0.1 μg/ml) were obtained from Abcam (Cambridge, MA, USA).


### 2.2. Culture and Treatment of HT22 Cells

HT22 cell is an immortalized mouse hippocampal neuronal cell line subcloned from parent HT4 cells that were originally immortalized from cultures of primary mouse hippocampal neurons [30]. HT22 cell is known to phenotypically resemble neuronal precursor cells, which has been used in cell model to study of hemin-induced injury in neuronal cells [7, 29]. HT22 cells (Merck, USA) were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin in an atmosphere with 5% CO2 at 37°C. After growing to 80% confluence, the HT22 cells were used for experiment. The HT22 cells were dealt with various concentrations of hemin (0, 10, 20, 40, 80, and 100 μM) over a period of 6 h to simulate the in vitro ICH model [7]. Subsequent studies were conducted using 20 μM of hemin. To evaluate the security of CHR, HT22 cells were dealt with various concentrations of CHR (0, 5, 10, 20, and 40 μM). To evaluate the neuroprotective effects of CHR, HT22 cells were dealt with CHR after treating them with 20 μM of hemin over a period of 6 h. Following this, they were incubated for 24 h. The negative control groups were dealt with an equal volume of solvent (Figure S1).


### 2.3. CCK-8 Assay

The viability of the HT22 cells was tested by the CCK-8 assay depending on the operating instruction [31]. Briefly, the treated cells were dealt with the CCK-8 reagent at 37°C for 2 hours. Following this, the optical density (OD) of the specimens was detected at 450 nm with a microplate reader (Thermo Fisher Scientific).


### 2.4. Lactate Dehydrogenase (LDH) Activity

The activity of LDH was measured according to the previous report [32]. LDH activity was detected by an LDH Activity Assay Kit (Solarbio, China) on the basis of the operating instruction. Briefly, the obtained supernatants were mixed with various reagents provided in the LDH Activity Assay Kit. Then, the OD of the samples was recorded at 450 nm with a microplate reader (Thermo Fisher Scientific). The LDH activity (U/l) was counted as follows: ODU−ODC×CS×N×1000/ODS−ODB, where ODU indicates the OD value of the specimen pipe, ODC indicates the OD value of the blank pipe, CS indicates the standard concentration (2 mmol/l), N indicates the multiples of dilution of the specimens before testing, ODS indicates the OD value of the standard pipe, and ODB indicates the OD value of the control pipe.


### 2.5. TUNEL Staining

TUNEL staining was performed according to the previous report [33]. Cell apoptosis rates were assessed by the One Step TUNEL Apoptosis Assay Kit (Beyotime, Shanghai, China) on the base of the operating instruction. In brief, the treated cells were immobilized by using 4% paraformaldehyde for 30 min. Following this, they were dealt with the TUNEL detection reagent. Subsequently, these cells were coincubated in the dark at 37°C for 60 min. Finally, the TUNEL-positive cells were observed and counted by the fluorescence microscopy.


### 2.6. Nuclear Protein Extraction

The nuclear protein from the treated cells was obtained using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) [34]. The protocols outlined by the manufacturer were followed to conduct the experiments. The extracted samples were detected using the western blotting technique in the presence of the anti-β-catenin antibody (ab108315, 1 : 1000). LaminB1 (ab16048, 0.1 μg/ml) was served as the internal reference.


### 2.7. Western Blot Analysis

Western blot was operated according to the previous report [35]. The cells were lysed using the precooled radio immune-precipitation assay buffer (RIPA, Beyotime, Shanghai, China) including protease and phosphatase inhibitors (Sigma-Aldrich, USA). The experiments were conducted at 4°C, and the extracted protein specimens were quantified with the BCA Protein Assay Kit (Millipore, US). Equal amounts of protein specimens were dealt with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique. The samples were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Following this, the membranes were sealed with 5% skimmed milk solution at room temperature for 1 h. Subsequently, the specimens were incubated overnight with primary antibodies at 4°C. Finally, the membranes were incubated with the HRP-conjugated secondary antibodies at room temperature (incubation time: 2 h). The samples were visualized using the ECL detection reagent on the ChemiDoc Imaging System (Bio-Rad Laboratories, Inc.). The blot of the membrane was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD) and normalized to β-actin (Abcam, ab179467, 1 : 5000) or LaminB1 (Abcam, ab16048, 0.1 μg/ml). The primary antibodies used to conduct the western blot tests were phospho-eIF2aα (dilution, 1 : 1000, Cell Signaling Technology, #3398), CHOP (dilution, 1 : 1000, Cell Signaling Technology, #2895), cleaved caspase-12 (dilution, 1 : 1000, Cell Signaling Technology, #35965), cleaved caspase-3 (dilution, 1 : 1000, Cell Signaling Technology, #9661), Bax (dilution, 1 : 1000, Cell Signaling Technology, #2772), Bcl-2 (dilution, 1 : 1000, Cell Signaling Technology, #2870), GRP78 (dilution, 1 : 1000, Abcam, ab108315), Wnt3a (dilution, 1 : 1000, Abcam, ab219412), and β-catenin (dilution, 1 : 5000, Abcam, ab108315).


### 2.8. Caspase-3 Activity

The caspase-3 activity was detected following previously reported protocols [33]. The caspase-3 activity was detected with the caspase-3 activity kit (Solarbio, China) according to the manufacturer’s instructions. Briefly, the total protein from the collected cells was extracted with the lysis buffer, and the BCA protein assay kit was used for quantification. The specimens were incubated in the presence of the reaction buffer, and the OD value of the specimens was recorded at 405 nm with a microplate reader (Thermo Fisher Scientific).


### 2.9. Determination of MDA, SOD, and GPx Levels

The levels of MDA, SOD, and GPx in HT22 cells were determined using Lipid Peroxidation MDA Assay Kit (Beyotime Biotechnology, #S0131S), Total Superoxide Dismutase Assay Kit (Beyotime Biotechnology, #S0101S), and Total Glutathione Peroxidase Assay Kit with NADPH (Beyotime Biotechnology, #S0058) following the manufacturer’s instructions, respectively [36].


### 2.10. Measurement of ROS Production

The ROS levels were assessed with Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, #S0063) according to the manufacturer’s operating instruction [37]. Briefly, pretreated cells were incubated with dichlorodihydrofluorescein diacetate (DCFH-DA, 20 μM) in Hanks’ balanced salt buffer at 37°C for 20 min. The ROS levels were detected with a fluorescence microplate reader (BioTek Synergy 2, USA).


### 2.11. MicroRNA-Sequencing

MicroRNA-sequencing was performed according to the previous report [38]. Briefly, total RNAs were extracted from three independent samples of hemin-induced HT22 cells with or without CHR using the TRIzol reagents (Invitrogen) in accordance with the manufacturer’s recommended protocol. Following this, the purified cDNA library was established using an Illumina TruSeq Small RNA Preparation Kit, depending on the manufacturer’s recommendations. Subsequently, the samples were subjected to the process of next-generation sequencing (Illumina HiSeq technology) at the Beijing Genomics Institute (BGI, Shenzhen, China) in accordance with the manufacturer’s instructions. Subsequently, Illumina’s analysis software was used to collect raw sequencing data. Finally, the differentially expressed miRNAs (in hemin-induced HT22 cells and hemin-induced HT22 cells posttreated with CHR) were analyzed using the EBseq R package. The logFC>1 and p value < 0.05 served as the screening criteria to obtain the differentially expressed miRNAs.


### 2.12. Bioinformatics Analysis

It has been proved that the function of miRNAs is closely related to its target mRNA [39]. To explore the potential biological function of differentially expressed miRNAs, the miRWalk software was used to predict the potential target genes for these differentially expressed miRNAs. Then, these target genes of miRNAs were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis. p values < 0.05 were considered significantly.


### 2.13. Quantitative Real-Time PCR (RT-PCR) Analysis

RT-PCR analysis was performed based on previous report [40]. Total RNAs were extracted from the treated cells by the TRIzol reagent (Invitrogen) according to the operating instructions. For miRNAs, the processes of cDNA synthesis and amplification were conducted by the Hairpin-itTM Real-Time PCR miRNAs kit (GenePharma, Shanghai, China). For mRNAs, cDNA synthesis and amplification were conducted using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Dalian, China) and TB Green® Fast qPCR Mix (Takara, Dalian, China), respectively. All RT-PCR tests were performed using the ABI 7500 Fast Real-Time PCR system (Applied Biosystems). GAPDH and U6 were served as the internal reference for mRNAs and miRNAs, respectively. RT-PCR tests were performed in triplicate, and the expressions of miRNAs and mRNAs were computed by the 2-ΔΔCt assay. The primer sequences corresponding to miRNAs or mRNA are shown in Table S1.


### 2.14. Cell Transfection

The miR-320-5p mimics, miR-320-5p inhibitors, and their negative controls (NC inhibitor and NC mimics) were obtained from GenePharma (Suzhou, China). The small hairpin RNA (shRNA) silencing Wnt3a (sh-Wnt3a) and its negative control (sh-NC) were also brought from GenePharma (Shanghai, China). The HT22 cells were transfected by Lipofectamine™ 3000 (Invitrogen) on the base of the operating instructions. Following transfection, the transfection efficiency was confirmed by the RT-PCR technique. The sequences of the miR-320-5p mimics, miR-320-5p inhibitor, and Wnt3a shRNA are listed in TableS1.


### 2.15. Dual-Luciferase Reporter Assay

The dual-luciferase report assay was used to verify the interaction between miR-320-5p and Wnt3a. Dual-luciferase reporter assay was performed according to the previous report [40]. The wild-type or mutant vectors corresponding to Wnt3a 3′UTR were constructed using the pmirGLO dual-luciferase vector. The wild or mutant vectors corresponding to Wnt3a 3′UTR and miR-320-3p mimics (or NC mimics) were cotransfected to HT22 cells using the Lipofectamine™ 3000 reagent (Invitrogen, Carlsbad, CA, USA). Following the process of cotransfection for 48 h, the firefly luciferase and Renilla luciferase activities were detected using the Dual-Luciferase Reporter Assay System. The Renilla luciferase activity was served as an internal reference for the normalization of the firefly luciferase activity.


### 2.16. Statistical Analyses

Data were marked asmean±standarddeviation (S.D.). All data were analyzed and charted by the GraphPad Prism 6.0 software (GraphPad Software, CA, USA). Student’s t-test and the one-way ANOVA tests were conducted to compare the characteristics of two groups and more than two groups, respectively. At least three independent experiments were performed for each set. The p values < 0.05 were considered to be statistically significant.


## 2.1. Reagents

CHR (purity≥98%) was obtained from Solarbio Life Sciences (Beijing, China) and dissolved in dimethyl sulfoxide (DMSO, purity≥99.5.0%, Sigma) to form a stock solution of concentration 40 mM. The solution was stored at -20°C. The prepared stock solution was diluted to form the corresponding working solutions before conducting each set of experiments. Hemin (purity≥96%) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Tunicamycin (TM) was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Dulbecco’s modified eagle’s medium (DMEM), phosphate-buffered saline (PBS), and penicillin/streptomycin were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The cell counting kit-8 (CCK-8), lactate dehydrogenase (LDH) assay kit, malondialdehyde (MDA) assay kit, superoxide dismutase (SOD) assay kit with WST-8, glutathione peroxidase (GPx) assay kit with DTNB, reactive oxygen species (ROS) assay kit, and One Step TdT-mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay kit were bought from Beyotime (Shanghai, China). Fetal bovine serum (FBS) was bought from Gibco (Thermo Fisher Scientific, USA). The caspase-3 assay kit was purchased from Solarbio Life Sciences (Beijing, China). The Hairpin-itTM miRNA real-time PCR quantification kit was obtained from GenePharma (Shanghai, China). The PrimeScript™ RT reagent kit with gDNA eraser and TB Green® Fast qPCR Mix were obtained from Takara (Dalian, China). Antibody against phospho-eukaryotic translation initiation factor 2α (p-Eif2aα, #3398, 1 : 1000), C/EBP homologous protein (CHOP, #2895, 1 : 1000), cleaved caspase-12 (#35965, 1 : 1000), cleaved caspase-3 (#9661, 1 : 1000), Bax (#2772, 1 : 2000), and Bcl-2 (#2870, 1 : 2000) were bought from Cell Signaling Technology (Beverly, MA, USA). Antibody against glucose-regulated protein 78 (GRP78, ab108315, 1 : 1000), β-actin (ab179467, 1 : 5000), Wnt3a (ab219412,1 : 1000), β-catenin (ab108315, 1 : 1000), and Lamin B (ab16048, 0.1 μg/ml) were obtained from Abcam (Cambridge, MA, USA).


## 2.2. Culture and Treatment of HT22 Cells

HT22 cell is an immortalized mouse hippocampal neuronal cell line subcloned from parent HT4 cells that were originally immortalized from cultures of primary mouse hippocampal neurons [30]. HT22 cell is known to phenotypically resemble neuronal precursor cells, which has been used in cell model to study of hemin-induced injury in neuronal cells [7, 29]. HT22 cells (Merck, USA) were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin in an atmosphere with 5% CO2 at 37°C. After growing to 80% confluence, the HT22 cells were used for experiment. The HT22 cells were dealt with various concentrations of hemin (0, 10, 20, 40, 80, and 100 μM) over a period of 6 h to simulate the in vitro ICH model [7]. Subsequent studies were conducted using 20 μM of hemin. To evaluate the security of CHR, HT22 cells were dealt with various concentrations of CHR (0, 5, 10, 20, and 40 μM). To evaluate the neuroprotective effects of CHR, HT22 cells were dealt with CHR after treating them with 20 μM of hemin over a period of 6 h. Following this, they were incubated for 24 h. The negative control groups were dealt with an equal volume of solvent (Figure S1).


## 2.3. CCK-8 Assay

The viability of the HT22 cells was tested by the CCK-8 assay depending on the operating instruction [31]. Briefly, the treated cells were dealt with the CCK-8 reagent at 37°C for 2 hours. Following this, the optical density (OD) of the specimens was detected at 450 nm with a microplate reader (Thermo Fisher Scientific).


## 2.4. Lactate Dehydrogenase (LDH) Activity

The activity of LDH was measured according to the previous report [32]. LDH activity was detected by an LDH Activity Assay Kit (Solarbio, China) on the basis of the operating instruction. Briefly, the obtained supernatants were mixed with various reagents provided in the LDH Activity Assay Kit. Then, the OD of the samples was recorded at 450 nm with a microplate reader (Thermo Fisher Scientific). The LDH activity (U/l) was counted as follows: ODU−ODC×CS×N×1000/ODS−ODB, where ODU indicates the OD value of the specimen pipe, ODC indicates the OD value of the blank pipe, CS indicates the standard concentration (2 mmol/l), N indicates the multiples of dilution of the specimens before testing, ODS indicates the OD value of the standard pipe, and ODB indicates the OD value of the control pipe.


## 2.5. TUNEL Staining

TUNEL staining was performed according to the previous report [33]. Cell apoptosis rates were assessed by the One Step TUNEL Apoptosis Assay Kit (Beyotime, Shanghai, China) on the base of the operating instruction. In brief, the treated cells were immobilized by using 4% paraformaldehyde for 30 min. Following this, they were dealt with the TUNEL detection reagent. Subsequently, these cells were coincubated in the dark at 37°C for 60 min. Finally, the TUNEL-positive cells were observed and counted by the fluorescence microscopy.


## 2.6. Nuclear Protein Extraction

The nuclear protein from the treated cells was obtained using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) [34]. The protocols outlined by the manufacturer were followed to conduct the experiments. The extracted samples were detected using the western blotting technique in the presence of the anti-β-catenin antibody (ab108315, 1 : 1000). LaminB1 (ab16048, 0.1 μg/ml) was served as the internal reference.


## 2.7. Western Blot Analysis

Western blot was operated according to the previous report [35]. The cells were lysed using the precooled radio immune-precipitation assay buffer (RIPA, Beyotime, Shanghai, China) including protease and phosphatase inhibitors (Sigma-Aldrich, USA). The experiments were conducted at 4°C, and the extracted protein specimens were quantified with the BCA Protein Assay Kit (Millipore, US). Equal amounts of protein specimens were dealt with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique. The samples were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Following this, the membranes were sealed with 5% skimmed milk solution at room temperature for 1 h. Subsequently, the specimens were incubated overnight with primary antibodies at 4°C. Finally, the membranes were incubated with the HRP-conjugated secondary antibodies at room temperature (incubation time: 2 h). The samples were visualized using the ECL detection reagent on the ChemiDoc Imaging System (Bio-Rad Laboratories, Inc.). The blot of the membrane was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD) and normalized to β-actin (Abcam, ab179467, 1 : 5000) or LaminB1 (Abcam, ab16048, 0.1 μg/ml). The primary antibodies used to conduct the western blot tests were phospho-eIF2aα (dilution, 1 : 1000, Cell Signaling Technology, #3398), CHOP (dilution, 1 : 1000, Cell Signaling Technology, #2895), cleaved caspase-12 (dilution, 1 : 1000, Cell Signaling Technology, #35965), cleaved caspase-3 (dilution, 1 : 1000, Cell Signaling Technology, #9661), Bax (dilution, 1 : 1000, Cell Signaling Technology, #2772), Bcl-2 (dilution, 1 : 1000, Cell Signaling Technology, #2870), GRP78 (dilution, 1 : 1000, Abcam, ab108315), Wnt3a (dilution, 1 : 1000, Abcam, ab219412), and β-catenin (dilution, 1 : 5000, Abcam, ab108315).


## 2.8. Caspase-3 Activity

The caspase-3 activity was detected following previously reported protocols [33]. The caspase-3 activity was detected with the caspase-3 activity kit (Solarbio, China) according to the manufacturer’s instructions. Briefly, the total protein from the collected cells was extracted with the lysis buffer, and the BCA protein assay kit was used for quantification. The specimens were incubated in the presence of the reaction buffer, and the OD value of the specimens was recorded at 405 nm with a microplate reader (Thermo Fisher Scientific).


## 2.9. Determination of MDA, SOD, and GPx Levels

The levels of MDA, SOD, and GPx in HT22 cells were determined using Lipid Peroxidation MDA Assay Kit (Beyotime Biotechnology, #S0131S), Total Superoxide Dismutase Assay Kit (Beyotime Biotechnology, #S0101S), and Total Glutathione Peroxidase Assay Kit with NADPH (Beyotime Biotechnology, #S0058) following the manufacturer’s instructions, respectively [36].


## 2.10. Measurement of ROS Production

The ROS levels were assessed with Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, #S0063) according to the manufacturer’s operating instruction [37]. Briefly, pretreated cells were incubated with dichlorodihydrofluorescein diacetate (DCFH-DA, 20 μM) in Hanks’ balanced salt buffer at 37°C for 20 min. The ROS levels were detected with a fluorescence microplate reader (BioTek Synergy 2, USA).


## 2.11. MicroRNA-Sequencing

MicroRNA-sequencing was performed according to the previous report [38]. Briefly, total RNAs were extracted from three independent samples of hemin-induced HT22 cells with or without CHR using the TRIzol reagents (Invitrogen) in accordance with the manufacturer’s recommended protocol. Following this, the purified cDNA library was established using an Illumina TruSeq Small RNA Preparation Kit, depending on the manufacturer’s recommendations. Subsequently, the samples were subjected to the process of next-generation sequencing (Illumina HiSeq technology) at the Beijing Genomics Institute (BGI, Shenzhen, China) in accordance with the manufacturer’s instructions. Subsequently, Illumina’s analysis software was used to collect raw sequencing data. Finally, the differentially expressed miRNAs (in hemin-induced HT22 cells and hemin-induced HT22 cells posttreated with CHR) were analyzed using the EBseq R package. The logFC>1 and p value < 0.05 served as the screening criteria to obtain the differentially expressed miRNAs.


## 2.12. Bioinformatics Analysis

It has been proved that the function of miRNAs is closely related to its target mRNA [39]. To explore the potential biological function of differentially expressed miRNAs, the miRWalk software was used to predict the potential target genes for these differentially expressed miRNAs. Then, these target genes of miRNAs were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis. p values < 0.05 were considered significantly.


## 2.13. Quantitative Real-Time PCR (RT-PCR) Analysis

RT-PCR analysis was performed based on previous report [40]. Total RNAs were extracted from the treated cells by the TRIzol reagent (Invitrogen) according to the operating instructions. For miRNAs, the processes of cDNA synthesis and amplification were conducted by the Hairpin-itTM Real-Time PCR miRNAs kit (GenePharma, Shanghai, China). For mRNAs, cDNA synthesis and amplification were conducted using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Dalian, China) and TB Green® Fast qPCR Mix (Takara, Dalian, China), respectively. All RT-PCR tests were performed using the ABI 7500 Fast Real-Time PCR system (Applied Biosystems). GAPDH and U6 were served as the internal reference for mRNAs and miRNAs, respectively. RT-PCR tests were performed in triplicate, and the expressions of miRNAs and mRNAs were computed by the 2-ΔΔCt assay. The primer sequences corresponding to miRNAs or mRNA are shown in Table S1.


## 2.14. Cell Transfection

The miR-320-5p mimics, miR-320-5p inhibitors, and their negative controls (NC inhibitor and NC mimics) were obtained from GenePharma (Suzhou, China). The small hairpin RNA (shRNA) silencing Wnt3a (sh-Wnt3a) and its negative control (sh-NC) were also brought from GenePharma (Shanghai, China). The HT22 cells were transfected by Lipofectamine™ 3000 (Invitrogen) on the base of the operating instructions. Following transfection, the transfection efficiency was confirmed by the RT-PCR technique. The sequences of the miR-320-5p mimics, miR-320-5p inhibitor, and Wnt3a shRNA are listed in TableS1.


## 2.15. Dual-Luciferase Reporter Assay

The dual-luciferase report assay was used to verify the interaction between miR-320-5p and Wnt3a. Dual-luciferase reporter assay was performed according to the previous report [40]. The wild-type or mutant vectors corresponding to Wnt3a 3′UTR were constructed using the pmirGLO dual-luciferase vector. The wild or mutant vectors corresponding to Wnt3a 3′UTR and miR-320-3p mimics (or NC mimics) were cotransfected to HT22 cells using the Lipofectamine™ 3000 reagent (Invitrogen, Carlsbad, CA, USA). Following the process of cotransfection for 48 h, the firefly luciferase and Renilla luciferase activities were detected using the Dual-Luciferase Reporter Assay System. The Renilla luciferase activity was served as an internal reference for the normalization of the firefly luciferase activity.


## 2.16. Statistical Analyses

Data were marked asmean±standarddeviation (S.D.). All data were analyzed and charted by the GraphPad Prism 6.0 software (GraphPad Software, CA, USA). Student’s t-test and the one-way ANOVA tests were conducted to compare the characteristics of two groups and more than two groups, respectively. At least three independent experiments were performed for each set. The p values < 0.05 were considered to be statistically significant.


## 3. Results

### 3.1. The Protective Effect of CHR against Hemin-Induced Apoptosis, ER Stress, and Oxidative Stress in HT22 Cells

It has been reported that hemin is a cytotoxic decomposition product released from hemoglobin during the lysis of erythrocytes, and it mimics the secondary injury observed following ICH [26–28]. To establish an in vitro ICH injury model, HT22 cell was incubated with various concentrations of hemin. Cell viability and cytotoxicity were detected by CCK-8 assay and LDH cytotoxicity assay to confirm an optional condition. The results indicated that HT22 cell viability and cytotoxicity were significantly augmented with increasing hemin concentration in a dose-dependent manner (Figure S2). Dose-response studies showed that 20 μM hemin efficiently induced HT22 cell damage (half maximal effective concentration EC50=20 μM, Figure S2). Therefore, we selected 20 μM of hemin (optimal condition) to treat HT22 cells for 6 h to construct an in vitro ICH injury model for subsequent experiments.To understand the neuroprotective effect of CHR, HT22 cells were dealt with or without 20μM hemin for 6 h followed by CHR (0, 5, 10, 20, and 40 μM) over a period of 24 h. The results of CCK-8 showed that CHR exhibited no cell viability in the absence of hemin but significantly increased HT22 cell viability in hemin-induced cell injury models (Figure 1(a)), indicating that CHR had a concentration-dependent protective effect on hemin-induced damaged cells, while the result of western blot indicated that 10 μM CHR had no effect on the PCNA expression in HT22 cells without hemin but could markedly reduce the PCNA expression in HT22 cells with hemin (Figure 1(b)). In addition, analysis of the LDH activity indicated that CHR could markedly reduce the level of LDH release in HT22 cells with hemin but had no effect on the LDH release level in HT22 cells devoid of hemin (Figure 1(c)). These data suggested that CHR (10 μM) could be efficiently used to treat neuronal cells. Hence, it was used for further studies.Figure 1
Chrysophanol (CHR) prevented HT22 cell survival, antiapoptosis, and anti-ER stress in hemin-induced HT22 cell injury models. (a) Cell viability was studied for nonhemin (0μM) and hemin (20 μM)-treated HT22 cells dealt with varying concentrations of CHR (0, 5, 10, 20, and 40 μM; treatment time: 24 h) by conducting CCK-8 assays. ∗∗∗p<0.001 vs. nonhemin. ##p<0.01 and ###p<0.001 vs. hemin. N=3. (b) Expression of PCNA affected by CHR in hemin-induced HT22 cells was detected using the western blot. N.S.p≥0.05 and ∗∗p<0.01. N=3. (c) LDH release level was analyzed in nonhemin (0 μM) and hemin (20 μM)-treated HT22 cells in the presence and absence of CHR (0, 5, 10, 20, and 40 μM; treatment time: 24 h) by conducting the LDH cytotoxicity assay. N.S.p≥0.05 and ∗∗∗p<0.001. N=3. (d) Caspase-3 activity was studied in hemin-induced cells in the presence and absence of CHR (10 μM). ∗∗∗p<0.001. (e) Rate of cell apoptosis was determined using hemin-induced cells in the presence and absence of CHR (10 μM; treatment time: 24 h) by conducting TUNEL staining assays. Bar=100 μm, ∗∗p<0.01. N=3. (f) The protein expression levels of apoptosis-related genes (cleaved caspase-3, Bax, and Bcl-2) were analyzed in hemin-induced cells in the presence and absence of CHR (10 μM; treatment time: 24 h) using the western blot technique. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001. N=3. (g) Levels of protein expression of ER stress-related genes (p-eIF2α, CHOP, GRP78, and cleaved caspase-12) were analyzed in hemin-induced cells in the presence and absence of CHR (10 μM; treatment time: 24 h) using the western blot technique. ∗p<0.05 and ∗∗p<0.01. N=3.
(a)(b)(c)(d)(e)(f)(g)It has been recently reported by several research groups that apoptosis and ER stress are the major cause of neuronal injury post ICH, and restraint of neuronal apoptosis and ER stress can potentially ameliorate ICH prognosis [41, 42]. To explore the effect of CHR on HT22 cell apoptosis in hemin-induced cell damage, the level of apoptosis was measured by caspase-3 activity, TUNEL staining assay, and western blot assay. It was observed that the caspase-3 activity increased significantly in the hemin-induced HT22 cells under conditions of injury. It was inhibited significantly by CHR posttreatment (Figure 1(d)). The results obtained from the TUNEL staining assays revealed that hemin effectively induced the apoptosis of HT22 cells, which was remarkably mitigated by CHR treatment (Figure 1(e)). In addition, the protein expressions for the apoptosis-related genes were analyzed by the western blot, and the results that the protein expression of Bax and cleaved caspase-3 was remarkably increased and the protein expression of Bcl-2 was significantly decreased in hemin-stimulated HT22 cells, the phenomenon was obviously ameliorated CHR treatment (Figure 1(f)). All the data indicated that CHR could protect neurons from hemin-induced apoptosis and ER stress. Western blot was used to assess the protein expressions of the ER stress-related genes (GRP78, cleaved caspase-12, p-eIF2α, and CHOP), the results demonstrated that the levels of protein expressions of GRP78, cleaved caspase-12, p-eIF2α, and CHOP increased significantly in hemin-induced HT22 cell injury, and the effect was inhibited following the process of CHR treatment (Figure 1(g)). The above results indicated that the process of CHR treatment could reduce hemin-induced ER stress and apoptosis in HT22 cells.Oxidative stress plays a pivotal role in neuronal apoptosis and ER stress, especially brain damage following ICH [5, 43]. We further investigate whether CHR regulates hemin-induced oxidative stress in HT22 cells. As shown in Figure 2, the levels of MDA and ROS were significantly increased in hemin-induced HT22 cell injury, the phenomenon was obviously inhibited by CHR treatment. While SOD activity and GPx activity were significantly decreased in hemin-induced HT22 cell injury, the phenomenon was obviously reversed by CHR treatment. These data indicated that CHR inhibited hemin-induced oxidative stress in HT22 cells.Figure 2
Chrysophanol (CHR) inhibited hemin-induced oxidation stress in HT22 cell. The changes of oxidation stress-related indexes (ROS, MDA, SOD, and GPx) were analyzed in the hemin-induced cells with or without CHR (10μM) (treatment time: 24 h) using the western blot. ∗∗∗p<0.001. N=3.


### 3.2. CHR Protects against Hemin-Induced Apoptosis, ER Stress, and Oxidative Stress by Restraining the Expression of miR-320-5p in HT22 Cells

Recently conducted clinical and preclinical studies have revealed that the levels of expressions of various miRNAs get altered in serum or cerebrospinal fluid following ICH, and these miRNAs could be used as potential therapeutic targets for ICH [22, 44–47], suggesting that dysregulation of miRNAs acts as key roles in development and incidence of ICH. To elucidate the neuroprotective mechanism of CHR against hemin-induced HT22 cell injury, the process of miRNA-sequencing was performed using hemin-induced HT22 cells posttreated with or without CHR. It was observed that the levels of expressions of the three miRNAs were outstandingly upregulated in the CHR treatment group, whereas the levels of expressions of the eight miRNAs were markedly downregulated (Figure 3(a) and Table S2). Then, KEGG and GO analyses were performed for the different miRNAs. The GO enrichment revealed that the differential miRNAs were involved in antioxidative activity, nervous system development, neuron differentiation, and so on (Figure S3A). And KEGG analysis showed that the differential miRNAs were centrally enriched in Wnt signaling pathway, Axon guidance, Rap1 signaling pathway, and so on (Figure S3B). Subsequently, we used the RT-qPCR to verify the six significantly altered miRNAs (three upregulated miRNAs and three downregulated miRNAs). The results of the RT-qPCR demonstrated that the changes in the levels of expression of the six miRNAs were consistent with the results obtained using the miRNAs-sequencing, and the difference of the miR-320-5p was the most significant (Figure 3(b)). And we also observed that CHR treatment significantly inhibited hemin-induced miR-320-5p expression in HT22 cells (Figure 3(c)), manifesting that miR-320-5p could be a key target of CHR to protect hemin-induced HT22 cell injury. To further investigate the effect of miR-320-5p on normal or hemin-induced HT22 cells, HT22 cells transfected with miR-320-5p mimics and NC mimics were dealt with or without hemin. We observed that the miR-320-5p expression significantly increased in normal or hemin-induced HT22 cell transfected with miR-320-5p mimics (Figure S4A). Results from CCK-8 uncovered that the miR-320-5p overexpression inhibited cell viability in hemin-induced HT22 cells but had no effect on HT22 cell viability in the absence of hemin (Figure S4B). The caspase-3 activity assay and TUNEL staining showed that miR-320-5p overexpression remarkably increased hemin-induced apoptosis in HT22 cells but had no effect on HT22 cell apoptosis in the absence of hemin (Figure S4C-S4E). Moreover, we found that overexpression of miR-320-5p promoted the relative levels of MDA and ROS and inhibited the activity of SOD and GPx in hemin-induced HT22 cells; but overexpression of miR-320-5p had no effect on oxidative stress related indexes (MDA, ROS, SOD, and GPx) in HT22 cells without hemin (Figure S4F). These data indicated that overexpression of miR-320-5p enhances hemin-induced cell survival, ER stress, and oxidative stress in HT22 cell.Figure 3
CHR inhibited HT22 cell apoptosis, ER stress, and oxidation stress by downregulating miR-320-5p in hemin-induced HT22 cell injury models. NC mim.: negative control mimics; miR mim.: miR-320-5p mimics. (a) Volcano plot generated for the miRNA expression profiles for the hemin-induced HT22 cell injury and CHR treatment groups. (b) Levels of expression of the top six miRNAs with the most significant difference in expression (identified following the sequencing method) were studied using hemin-induced cells in the presence and absence of CHR (10μM; treatment time: 24 h) using the RT-PCR technique. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001. N=3. (c) Level of expression of miR-320-5p was studied using hemin-induced cells in the presence and absence of CHR (10 μM; treatment time: 24 h) using the RT-PCR technique. ∗p<0.05. N=3. (d) The apoptosis rate in hemin-induced HT22 cells in the presence and absence of CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) was studied by conducting the TUNEL staining assays. ∗∗∗p<0.001. N=3. (e) Protein expression levels of the ER stress-related genes (p-eIF2α, CHOP, GRP78, and cleaved caspase-12) were analyzed in hemin-induced HT22 cells in the presence and absence of CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) using the western blot technique. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001. N=3. (f) The changes of oxidation stress-related indexes (ROS, MDA, SOD, and GPx) were analyzed in hemin-induced HT22 cells in the presence and absence of CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h). ∗∗p<0.01 and ∗∗∗p<0.001. N=3.
(a)(b)(c)(d)(e)(f)Then, we further explored whether the overexpression of miR-320-5p reversed the neuroprotection effect generated by CHR. The results of CCK-8 assays disclosed that CHR treatment outstandingly enhanced cell viability in hemin-induced HT22 cells, and this enhancement was reduced by overexpression of miR-320-5p (FigureS5A). The results obtained using the TUNEL staining assays also indicated that CHR treatment inhibited hemin-induced cell apoptosis in HT22 cells, and the inhibition was alleviated by the overexpression of miR-320-5p (Figure 3(d)). Consistent with the results of TUNEL staining assays, CHR treatment remarkably inhibited caspase-3 activate of HT22 cells in exist of hemin, and the inhibition was weakened by miR-320-5p overexpression (Figure S5B). Moreover, the western blot was used to assess the protein expression of ER stress-related gene (GRP78, cleaved caspase-12, p-eIF2α, and CHOP), the results revealed that CHR inhibited the protein expression of GRP78, cleaved caspase-12, p-eIF2α, and CHOP in the hemin-induced HT22 cells, and the inhibition was alleviated by the overexpression of miR-320-5p (Figure 3(e)). Finally, we examined the changes of oxidative stress-related indicators (MDA, ROS, SOD, and GPx) to evaluate whether CHR affected hemin-induced oxidative stress in HT2 cells through miR-320-5p. As shown in Figure 3(f), CHR inhibited the relative levels of MDA and ROS in the hemin-induced HT22 cells, and the inhibition was obviously alleviated by overexpression of miR-320-5p. While CHR increased the ROS activity and GPx activity in the hemin-induced HT22 cells, the phenomenon was obviously weakened by overexpression of miR-320-5p. Altogether, these data suggested that CHR increased cell survival, antiapoptosis, anti-ER stress, and antioxidative stress by inhibiting miR-320-5p in hemin-induced HT22 cell injury model.


### 3.3. CHR Activates the Wnt3a/β-Catenin Pathway by Restraining miR-320-5p Expression

The miRWalk software was used for predicting the potential target genes of miR-320-5p, and then, the potential target genes of miR-320-5p were performed by KEGG analysis. We found that these genes mainly enriched in the WNT signaling pathway, neuroactive ligand-receptor interaction, cAMP signaling pathway, and so on (FigureS3C). Meanwhile, we found a highly conserved potential binding site between miR-320-5p and WNT3a mRNA 3′UTR in the WNT signaling pathway (Figure 4(a)). A dual-luciferase reporter assay system was used to verify the interaction between miR-320-5p and Wnt3a. Fragments of Wnt3a mRNA 3′-UTR, containing either the binding site (wild type) for miR-320-5p or the binding site of MUT, were designed (Figure 4(a)). The results of dual-luciferase reporter assay system indicated that the cotransfection of Wnt3a-3′-UTR (wild type) with the miR-320-5p mimics resulted in an outstanding reduction in the relative luciferase activity, whereas the cotransfection of the Wnt3a-3′-UTR-mutant with the miR-320-5p mimics did not result in a decrease in the luciferase activity (Figure 4(b)). We further confirmed the regulation of Wnt3a by miR-320-5p in HT22 cells. The miR-320-5p mimics inhibited Wnt3a expression by augmenting the expression of miR-320-5p, while the miR-320-5p inhibitor enhanced the level of expression of Wnt3a by restraining miR-320-5p expression (Figure 4(c)), suggesting that the expression levels of Wnt3a were negatively regulated by miRNA-320-5p present in the HT22 cells. Together, these data revealed that Wnt3a is a direct downstream target of miR-320-5p.Figure 4
CHR activated the Wnt3a/β-catenin signaling pathway by downregulating miR-320-5p in hemin-induced HT22 cell injury models. (a) Potential binding site of miR-320-5p at the 3′-UTR of Wnt3a mRNA is predicted using the miRWalk software. The red color indicates wild-type, and the green color indicates the mutated sequence of Wnt3a at the miR-320-5p-binding site. (b) HT22 cells were cotransfected with miR-320-5p (or NC mimics) and a pmir-GLO vector containing wild-type or mutated miR-320-5p-binding sites (mutated) at Wnt3a 3′-UTR. Luciferase reporter assays were performed 24 h after cotransfection. NC mim.: negative control mimics; miR mim.: miR-320-5p mimics; NC inh.: negative control inhibitor; miR inh.: miR-320-5p inhibitor. N.S.p>0.05, ∗∗∗p<0.001. N=6. (c) HT22 cells were transfected with the miR-320-5p inhibitor or miR-320-5p mimics and their corresponding controls. The expression of miR-320-5p and Wnt3a mRNA was analyzed using the RT-PCR technique. ∗∗∗p<0.001. N=3. (d and e) Expression levels of mRNA and protein of Wnt3a were analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) using the RT-PCR and western blot. ∗∗p<0.05 and ∗∗∗p<0.001. N=3. (f) Expression of miR-320-5p was analyzed in hemin-induced HT22 cells in the presence and absence of CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) using the RT-PCR technique. ∗∗∗p<0.001. N=3. (g) The mRNA expression of Wnt3a was analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) using the RT-PCR technique. ∗∗∗p<0.001. N=3. (h) The protein expression levels of Wnt3a, total β-catenin (T-β-catenin), and nuclear β-catenin (N-β-catenin) were analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) using the western blot technique. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001. N=3.
(a)(b)(c)(d)(e)(f)(g)(h)Wnt3a is a naturally occurring protein that exhibits neuroprotective effects [48]. The mRNA and protein expression of the Wnt3a gene was determined using the RT-qPCR and western blot to explore whether CHR regulated the expression of Wnt3a in the hemin-induced HT22 cell injury group. The results demonstrated that the mRNA and protein expression levels of Wnt3a in hemin-induced HT22 cells were dramatically lower than the levels observed in HT22 cells devoid of hemin. It was also observed that the level of expression of Wnt3a in the hemin-induced HT22 cell injury group could be remarkably improved following the process of CHR treatment (Figures 4(d) and 4(e)). In addition, we carried out rescue experiments to explore whether CHR effected the Wnt3a by inhibiting miR-320-5p. The results of the RT-PCR and western blot revealed that the mRNA and protein expression of Wnt3a in hemin-induced HT22 cells could be significantly upregulated following CHR treatment, and the regulation was weakened by the overexpression of miR-320-5p (Figures 4(f)–4(h)). These data indicated that CHR could enhance Wnt3a expression in the hemin-induced HT22 cell injury through regulating miR-320-5p expression.Wnt3a, one of the most important endogenous ligands of Wnt, participates in the typical Wnt/β-catenin pathway. The Wnt/β-catenin pathway is a central signal transduction pathway that can influence numerous biological processes, such as cell apoptosis, survival, and ER stress [49, 50]. It has been recently revealed that the Wnt/β-catenin pathway significantly influences the incidence of neurological diseases [51, 52]. It has been observed that the activation of the Wnt/β-catenin pathway can help achieve neuroprotection [53–56]. Therefore, we transfected sh-Wnt3a (#1, #2, and #3) and the negative control shRNA (sh-NC) to identify the best sh-Wnt3a in the HT22 cells (Figure S6A) and to explore the effect of Wnt3a on β-catenin pathway, cell survival, apoptosis, and oxidative stress under normal or hemin-induced HT22 cells. In normal or hemin-induced HT22 cells transfected with sh-Wnt3a, the expression levels of Wnt3a and nucleus β-catenin observably decreased, but the expression of total β-catenin was not affected (Figure S6B and S6C). Results from the CCK-8 assays indicated that cell viability was significantly inhibited in normal or hemin-induced cells transfected with sh-Wnt3a (Figure S6D). Treatment with silenced Wnt3a outstandingly increased caspase-3 activity in normal or hemin-induced HT22 cells (Figure S6E). The results from the TUNEL assays revealed that silenced Wnt3a could promote apoptosis in normal or hemin-induced HT22 cells (Figure S6F). In addition, treatment with silenced Wnt3a dramatically increased MDA and ROS levels and decreased the activity of SOD and GPx in hemin-induced HT22 cell damage model, but treatment with silenced Wnt3a had no effect on oxidative stress-related indexes (MDA, ROS, SOD, and GPx) in HT22 cells without hemin (Figure S6G). These data showed that silenced Wnt3a inhibited β-catenin pathway, antiapoptosis, and antioxidative stress in hemin-induced HT22 cells.Based on the above data, we speculated that CHR could regulate theβ-catenin pathway by inhibiting miR-320-5p. The rescue experiments suggested that the protein expression of nucleus β-catenin in hemin-induced HT22 cells could be significantly upregulated following CHR treatment; this enhancement was weakened by the overexpression of miR-320-5p. Differences in the total β-catenin expression were not observed among different groups (Figure 4(h)). These data revealed that CHR activated the Wnt3a/β-catenin pathway by restraining the expression of miR-320-5p in the hemin-induced HT22 cell injury.


### 3.4. CHR Protects against Hemin-Induced Injury by Activating the Wnt3a/β-Catenin Pathway

To further explore whether CHR inhibits hemin-induced cell apoptosis, ER stress, and oxidative stress by activating the Wnt3a/β-catenin pathway, the results of western blot revealed that the levels of protein expression corresponding to Wnt3a and nucleus β-catenin in hemin-induced HT22 cells increased significantly following CHR treatment, and the enhancement was partly inhibited by the suppression of Wnt3a (Figure 5(a)). Subsequently, the results obtained by conducting the CCK-8 assays indicated that the cell viability in hemin-induced HT22 cells increased significantly following the process of CHR treatment, and the extent of the increase in the viability of the cells was reduced by the suppression of Wnt3a (Figure S7A). The caspase-3 activity in hemin-induced HT22 cells decreased significantly following the process of CHR treatment, and the extent of decrease in the activity was alleviated by the suppression of Wnt3a (Figure S7B). In addition, the results obtained by conducting the TUNEL staining assays also indicated that the cell apoptosis rates in hemin-induced HT22 cells were significantly restrained under conditions of CHR treatment, and the restraint was alleviated by the suppression of Wnt3a (Figure 5(b)). Further, the results obtained using the western blot revealed that the protein expressions of the ER stress-related genes (GRP78, cleaved caspase-12, p-eIF2α, and CHOP) in hemin-induced HT22 cells were significantly inhibited under conditions of CHR treatment, and the inhibition was impaired by the suppression of Wnt3a (Figure 5(c)). Finally, we examined the changes of oxidative stress-related indicators (MDA, ROS, SOD, and GPx) to evaluate whether CHR affected hemin-induced oxidative stress in HT2 cells through Wnt3a. As shown in Figure 5(d), CHR inhibited the relative levels of MDA and ROS in the hemin-induced HT22 cells; the inhibition was obviously alleviated by silenced Wnt3a. While CHR increased the ROS activity and GPx activity in the hemin-induced HT22 cells, the phenomenon was obviously weakened by silenced Wnt3a. Altogether, the obtained data suggested that CHR inhibited hemin-induced cell apoptosis, ER stress, and oxidative stress by activating the Wnt3a/β-catenin pathway.Figure 5
CHR inhibited the cell apoptosis, ER stress, and oxidative stress by upregulating Wnt3a in hemin-induced HT22 cell injury models. Sh-NC: negative control shRNA; sh-Wnt3a: Wnt3a shRNA. (a) Protein expression levels of Wnt3a, T-β-catenin, and N-β-catenin were analyzed in hemin-induced HT22 cells (20 μM) with or without CHR (10 μM; treatment time: 24 h) or sh-Wnt3a (treatment time: 24 h) following the western blot technique. ∗∗p<0.01, ∗∗∗p<0.001. N=3. (b) Apoptosis rate in hemin-induced HT22 cells (20 μM) with or without CHR (10 μM; treatment time: 24 h) or sh-Wnt3a (treatment time: 24 h) was studied by conducting the TUNEL staining assays. ∗∗∗p<0.001. N=3. (c) The protein expression levels of ER stress-related genes (p-eIF2α, CHOP, GRP78, and cleaved caspase-12) were analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) or sh-Wnt3a (treatment time: 24 h) using the western blot technique. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001. N=3. (d) The change of oxidative stress-related indexes (MDA, SOD, GPx, and ROS) were analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) or sh-Wnt3a (treatment time: 24 h). ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001. N=3.
(a)(b)(c)(d)


## 3.1. The Protective Effect of CHR against Hemin-Induced Apoptosis, ER Stress, and Oxidative Stress in HT22 Cells

It has been reported that hemin is a cytotoxic decomposition product released from hemoglobin during the lysis of erythrocytes, and it mimics the secondary injury observed following ICH [26–28]. To establish an in vitro ICH injury model, HT22 cell was incubated with various concentrations of hemin. Cell viability and cytotoxicity were detected by CCK-8 assay and LDH cytotoxicity assay to confirm an optional condition. The results indicated that HT22 cell viability and cytotoxicity were significantly augmented with increasing hemin concentration in a dose-dependent manner (Figure S2). Dose-response studies showed that 20 μM hemin efficiently induced HT22 cell damage (half maximal effective concentration EC50=20 μM, Figure S2). Therefore, we selected 20 μM of hemin (optimal condition) to treat HT22 cells for 6 h to construct an in vitro ICH injury model for subsequent experiments.To understand the neuroprotective effect of CHR, HT22 cells were dealt with or without 20μM hemin for 6 h followed by CHR (0, 5, 10, 20, and 40 μM) over a period of 24 h. The results of CCK-8 showed that CHR exhibited no cell viability in the absence of hemin but significantly increased HT22 cell viability in hemin-induced cell injury models (Figure 1(a)), indicating that CHR had a concentration-dependent protective effect on hemin-induced damaged cells, while the result of western blot indicated that 10 μM CHR had no effect on the PCNA expression in HT22 cells without hemin but could markedly reduce the PCNA expression in HT22 cells with hemin (Figure 1(b)). In addition, analysis of the LDH activity indicated that CHR could markedly reduce the level of LDH release in HT22 cells with hemin but had no effect on the LDH release level in HT22 cells devoid of hemin (Figure 1(c)). These data suggested that CHR (10 μM) could be efficiently used to treat neuronal cells. Hence, it was used for further studies.Figure 1
Chrysophanol (CHR) prevented HT22 cell survival, antiapoptosis, and anti-ER stress in hemin-induced HT22 cell injury models. (a) Cell viability was studied for nonhemin (0μM) and hemin (20 μM)-treated HT22 cells dealt with varying concentrations of CHR (0, 5, 10, 20, and 40 μM; treatment time: 24 h) by conducting CCK-8 assays. ∗∗∗p<0.001 vs. nonhemin. ##p<0.01 and ###p<0.001 vs. hemin. N=3. (b) Expression of PCNA affected by CHR in hemin-induced HT22 cells was detected using the western blot. N.S.p≥0.05 and ∗∗p<0.01. N=3. (c) LDH release level was analyzed in nonhemin (0 μM) and hemin (20 μM)-treated HT22 cells in the presence and absence of CHR (0, 5, 10, 20, and 40 μM; treatment time: 24 h) by conducting the LDH cytotoxicity assay. N.S.p≥0.05 and ∗∗∗p<0.001. N=3. (d) Caspase-3 activity was studied in hemin-induced cells in the presence and absence of CHR (10 μM). ∗∗∗p<0.001. (e) Rate of cell apoptosis was determined using hemin-induced cells in the presence and absence of CHR (10 μM; treatment time: 24 h) by conducting TUNEL staining assays. Bar=100 μm, ∗∗p<0.01. N=3. (f) The protein expression levels of apoptosis-related genes (cleaved caspase-3, Bax, and Bcl-2) were analyzed in hemin-induced cells in the presence and absence of CHR (10 μM; treatment time: 24 h) using the western blot technique. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001. N=3. (g) Levels of protein expression of ER stress-related genes (p-eIF2α, CHOP, GRP78, and cleaved caspase-12) were analyzed in hemin-induced cells in the presence and absence of CHR (10 μM; treatment time: 24 h) using the western blot technique. ∗p<0.05 and ∗∗p<0.01. N=3.
(a)(b)(c)(d)(e)(f)(g)It has been recently reported by several research groups that apoptosis and ER stress are the major cause of neuronal injury post ICH, and restraint of neuronal apoptosis and ER stress can potentially ameliorate ICH prognosis [41, 42]. To explore the effect of CHR on HT22 cell apoptosis in hemin-induced cell damage, the level of apoptosis was measured by caspase-3 activity, TUNEL staining assay, and western blot assay. It was observed that the caspase-3 activity increased significantly in the hemin-induced HT22 cells under conditions of injury. It was inhibited significantly by CHR posttreatment (Figure 1(d)). The results obtained from the TUNEL staining assays revealed that hemin effectively induced the apoptosis of HT22 cells, which was remarkably mitigated by CHR treatment (Figure 1(e)). In addition, the protein expressions for the apoptosis-related genes were analyzed by the western blot, and the results that the protein expression of Bax and cleaved caspase-3 was remarkably increased and the protein expression of Bcl-2 was significantly decreased in hemin-stimulated HT22 cells, the phenomenon was obviously ameliorated CHR treatment (Figure 1(f)). All the data indicated that CHR could protect neurons from hemin-induced apoptosis and ER stress. Western blot was used to assess the protein expressions of the ER stress-related genes (GRP78, cleaved caspase-12, p-eIF2α, and CHOP), the results demonstrated that the levels of protein expressions of GRP78, cleaved caspase-12, p-eIF2α, and CHOP increased significantly in hemin-induced HT22 cell injury, and the effect was inhibited following the process of CHR treatment (Figure 1(g)). The above results indicated that the process of CHR treatment could reduce hemin-induced ER stress and apoptosis in HT22 cells.Oxidative stress plays a pivotal role in neuronal apoptosis and ER stress, especially brain damage following ICH [5, 43]. We further investigate whether CHR regulates hemin-induced oxidative stress in HT22 cells. As shown in Figure 2, the levels of MDA and ROS were significantly increased in hemin-induced HT22 cell injury, the phenomenon was obviously inhibited by CHR treatment. While SOD activity and GPx activity were significantly decreased in hemin-induced HT22 cell injury, the phenomenon was obviously reversed by CHR treatment. These data indicated that CHR inhibited hemin-induced oxidative stress in HT22 cells.Figure 2
Chrysophanol (CHR) inhibited hemin-induced oxidation stress in HT22 cell. The changes of oxidation stress-related indexes (ROS, MDA, SOD, and GPx) were analyzed in the hemin-induced cells with or without CHR (10μM) (treatment time: 24 h) using the western blot. ∗∗∗p<0.001. N=3.


## 3.2. CHR Protects against Hemin-Induced Apoptosis, ER Stress, and Oxidative Stress by Restraining the Expression of miR-320-5p in HT22 Cells

Recently conducted clinical and preclinical studies have revealed that the levels of expressions of various miRNAs get altered in serum or cerebrospinal fluid following ICH, and these miRNAs could be used as potential therapeutic targets for ICH [22, 44–47], suggesting that dysregulation of miRNAs acts as key roles in development and incidence of ICH. To elucidate the neuroprotective mechanism of CHR against hemin-induced HT22 cell injury, the process of miRNA-sequencing was performed using hemin-induced HT22 cells posttreated with or without CHR. It was observed that the levels of expressions of the three miRNAs were outstandingly upregulated in the CHR treatment group, whereas the levels of expressions of the eight miRNAs were markedly downregulated (Figure 3(a) and Table S2). Then, KEGG and GO analyses were performed for the different miRNAs. The GO enrichment revealed that the differential miRNAs were involved in antioxidative activity, nervous system development, neuron differentiation, and so on (Figure S3A). And KEGG analysis showed that the differential miRNAs were centrally enriched in Wnt signaling pathway, Axon guidance, Rap1 signaling pathway, and so on (Figure S3B). Subsequently, we used the RT-qPCR to verify the six significantly altered miRNAs (three upregulated miRNAs and three downregulated miRNAs). The results of the RT-qPCR demonstrated that the changes in the levels of expression of the six miRNAs were consistent with the results obtained using the miRNAs-sequencing, and the difference of the miR-320-5p was the most significant (Figure 3(b)). And we also observed that CHR treatment significantly inhibited hemin-induced miR-320-5p expression in HT22 cells (Figure 3(c)), manifesting that miR-320-5p could be a key target of CHR to protect hemin-induced HT22 cell injury. To further investigate the effect of miR-320-5p on normal or hemin-induced HT22 cells, HT22 cells transfected with miR-320-5p mimics and NC mimics were dealt with or without hemin. We observed that the miR-320-5p expression significantly increased in normal or hemin-induced HT22 cell transfected with miR-320-5p mimics (Figure S4A). Results from CCK-8 uncovered that the miR-320-5p overexpression inhibited cell viability in hemin-induced HT22 cells but had no effect on HT22 cell viability in the absence of hemin (Figure S4B). The caspase-3 activity assay and TUNEL staining showed that miR-320-5p overexpression remarkably increased hemin-induced apoptosis in HT22 cells but had no effect on HT22 cell apoptosis in the absence of hemin (Figure S4C-S4E). Moreover, we found that overexpression of miR-320-5p promoted the relative levels of MDA and ROS and inhibited the activity of SOD and GPx in hemin-induced HT22 cells; but overexpression of miR-320-5p had no effect on oxidative stress related indexes (MDA, ROS, SOD, and GPx) in HT22 cells without hemin (Figure S4F). These data indicated that overexpression of miR-320-5p enhances hemin-induced cell survival, ER stress, and oxidative stress in HT22 cell.Figure 3
CHR inhibited HT22 cell apoptosis, ER stress, and oxidation stress by downregulating miR-320-5p in hemin-induced HT22 cell injury models. NC mim.: negative control mimics; miR mim.: miR-320-5p mimics. (a) Volcano plot generated for the miRNA expression profiles for the hemin-induced HT22 cell injury and CHR treatment groups. (b) Levels of expression of the top six miRNAs with the most significant difference in expression (identified following the sequencing method) were studied using hemin-induced cells in the presence and absence of CHR (10μM; treatment time: 24 h) using the RT-PCR technique. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001. N=3. (c) Level of expression of miR-320-5p was studied using hemin-induced cells in the presence and absence of CHR (10 μM; treatment time: 24 h) using the RT-PCR technique. ∗p<0.05. N=3. (d) The apoptosis rate in hemin-induced HT22 cells in the presence and absence of CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) was studied by conducting the TUNEL staining assays. ∗∗∗p<0.001. N=3. (e) Protein expression levels of the ER stress-related genes (p-eIF2α, CHOP, GRP78, and cleaved caspase-12) were analyzed in hemin-induced HT22 cells in the presence and absence of CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) using the western blot technique. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001. N=3. (f) The changes of oxidation stress-related indexes (ROS, MDA, SOD, and GPx) were analyzed in hemin-induced HT22 cells in the presence and absence of CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h). ∗∗p<0.01 and ∗∗∗p<0.001. N=3.
(a)(b)(c)(d)(e)(f)Then, we further explored whether the overexpression of miR-320-5p reversed the neuroprotection effect generated by CHR. The results of CCK-8 assays disclosed that CHR treatment outstandingly enhanced cell viability in hemin-induced HT22 cells, and this enhancement was reduced by overexpression of miR-320-5p (FigureS5A). The results obtained using the TUNEL staining assays also indicated that CHR treatment inhibited hemin-induced cell apoptosis in HT22 cells, and the inhibition was alleviated by the overexpression of miR-320-5p (Figure 3(d)). Consistent with the results of TUNEL staining assays, CHR treatment remarkably inhibited caspase-3 activate of HT22 cells in exist of hemin, and the inhibition was weakened by miR-320-5p overexpression (Figure S5B). Moreover, the western blot was used to assess the protein expression of ER stress-related gene (GRP78, cleaved caspase-12, p-eIF2α, and CHOP), the results revealed that CHR inhibited the protein expression of GRP78, cleaved caspase-12, p-eIF2α, and CHOP in the hemin-induced HT22 cells, and the inhibition was alleviated by the overexpression of miR-320-5p (Figure 3(e)). Finally, we examined the changes of oxidative stress-related indicators (MDA, ROS, SOD, and GPx) to evaluate whether CHR affected hemin-induced oxidative stress in HT2 cells through miR-320-5p. As shown in Figure 3(f), CHR inhibited the relative levels of MDA and ROS in the hemin-induced HT22 cells, and the inhibition was obviously alleviated by overexpression of miR-320-5p. While CHR increased the ROS activity and GPx activity in the hemin-induced HT22 cells, the phenomenon was obviously weakened by overexpression of miR-320-5p. Altogether, these data suggested that CHR increased cell survival, antiapoptosis, anti-ER stress, and antioxidative stress by inhibiting miR-320-5p in hemin-induced HT22 cell injury model.


## 3.3. CHR Activates the Wnt3a/β-Catenin Pathway by Restraining miR-320-5p Expression

The miRWalk software was used for predicting the potential target genes of miR-320-5p, and then, the potential target genes of miR-320-5p were performed by KEGG analysis. We found that these genes mainly enriched in the WNT signaling pathway, neuroactive ligand-receptor interaction, cAMP signaling pathway, and so on (FigureS3C). Meanwhile, we found a highly conserved potential binding site between miR-320-5p and WNT3a mRNA 3′UTR in the WNT signaling pathway (Figure 4(a)). A dual-luciferase reporter assay system was used to verify the interaction between miR-320-5p and Wnt3a. Fragments of Wnt3a mRNA 3′-UTR, containing either the binding site (wild type) for miR-320-5p or the binding site of MUT, were designed (Figure 4(a)). The results of dual-luciferase reporter assay system indicated that the cotransfection of Wnt3a-3′-UTR (wild type) with the miR-320-5p mimics resulted in an outstanding reduction in the relative luciferase activity, whereas the cotransfection of the Wnt3a-3′-UTR-mutant with the miR-320-5p mimics did not result in a decrease in the luciferase activity (Figure 4(b)). We further confirmed the regulation of Wnt3a by miR-320-5p in HT22 cells. The miR-320-5p mimics inhibited Wnt3a expression by augmenting the expression of miR-320-5p, while the miR-320-5p inhibitor enhanced the level of expression of Wnt3a by restraining miR-320-5p expression (Figure 4(c)), suggesting that the expression levels of Wnt3a were negatively regulated by miRNA-320-5p present in the HT22 cells. Together, these data revealed that Wnt3a is a direct downstream target of miR-320-5p.Figure 4
CHR activated the Wnt3a/β-catenin signaling pathway by downregulating miR-320-5p in hemin-induced HT22 cell injury models. (a) Potential binding site of miR-320-5p at the 3′-UTR of Wnt3a mRNA is predicted using the miRWalk software. The red color indicates wild-type, and the green color indicates the mutated sequence of Wnt3a at the miR-320-5p-binding site. (b) HT22 cells were cotransfected with miR-320-5p (or NC mimics) and a pmir-GLO vector containing wild-type or mutated miR-320-5p-binding sites (mutated) at Wnt3a 3′-UTR. Luciferase reporter assays were performed 24 h after cotransfection. NC mim.: negative control mimics; miR mim.: miR-320-5p mimics; NC inh.: negative control inhibitor; miR inh.: miR-320-5p inhibitor. N.S.p>0.05, ∗∗∗p<0.001. N=6. (c) HT22 cells were transfected with the miR-320-5p inhibitor or miR-320-5p mimics and their corresponding controls. The expression of miR-320-5p and Wnt3a mRNA was analyzed using the RT-PCR technique. ∗∗∗p<0.001. N=3. (d and e) Expression levels of mRNA and protein of Wnt3a were analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) using the RT-PCR and western blot. ∗∗p<0.05 and ∗∗∗p<0.001. N=3. (f) Expression of miR-320-5p was analyzed in hemin-induced HT22 cells in the presence and absence of CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) using the RT-PCR technique. ∗∗∗p<0.001. N=3. (g) The mRNA expression of Wnt3a was analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) using the RT-PCR technique. ∗∗∗p<0.001. N=3. (h) The protein expression levels of Wnt3a, total β-catenin (T-β-catenin), and nuclear β-catenin (N-β-catenin) were analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) or miR-320-5p mimics (treatment time: 24 h) using the western blot technique. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001. N=3.
(a)(b)(c)(d)(e)(f)(g)(h)Wnt3a is a naturally occurring protein that exhibits neuroprotective effects [48]. The mRNA and protein expression of the Wnt3a gene was determined using the RT-qPCR and western blot to explore whether CHR regulated the expression of Wnt3a in the hemin-induced HT22 cell injury group. The results demonstrated that the mRNA and protein expression levels of Wnt3a in hemin-induced HT22 cells were dramatically lower than the levels observed in HT22 cells devoid of hemin. It was also observed that the level of expression of Wnt3a in the hemin-induced HT22 cell injury group could be remarkably improved following the process of CHR treatment (Figures 4(d) and 4(e)). In addition, we carried out rescue experiments to explore whether CHR effected the Wnt3a by inhibiting miR-320-5p. The results of the RT-PCR and western blot revealed that the mRNA and protein expression of Wnt3a in hemin-induced HT22 cells could be significantly upregulated following CHR treatment, and the regulation was weakened by the overexpression of miR-320-5p (Figures 4(f)–4(h)). These data indicated that CHR could enhance Wnt3a expression in the hemin-induced HT22 cell injury through regulating miR-320-5p expression.Wnt3a, one of the most important endogenous ligands of Wnt, participates in the typical Wnt/β-catenin pathway. The Wnt/β-catenin pathway is a central signal transduction pathway that can influence numerous biological processes, such as cell apoptosis, survival, and ER stress [49, 50]. It has been recently revealed that the Wnt/β-catenin pathway significantly influences the incidence of neurological diseases [51, 52]. It has been observed that the activation of the Wnt/β-catenin pathway can help achieve neuroprotection [53–56]. Therefore, we transfected sh-Wnt3a (#1, #2, and #3) and the negative control shRNA (sh-NC) to identify the best sh-Wnt3a in the HT22 cells (Figure S6A) and to explore the effect of Wnt3a on β-catenin pathway, cell survival, apoptosis, and oxidative stress under normal or hemin-induced HT22 cells. In normal or hemin-induced HT22 cells transfected with sh-Wnt3a, the expression levels of Wnt3a and nucleus β-catenin observably decreased, but the expression of total β-catenin was not affected (Figure S6B and S6C). Results from the CCK-8 assays indicated that cell viability was significantly inhibited in normal or hemin-induced cells transfected with sh-Wnt3a (Figure S6D). Treatment with silenced Wnt3a outstandingly increased caspase-3 activity in normal or hemin-induced HT22 cells (Figure S6E). The results from the TUNEL assays revealed that silenced Wnt3a could promote apoptosis in normal or hemin-induced HT22 cells (Figure S6F). In addition, treatment with silenced Wnt3a dramatically increased MDA and ROS levels and decreased the activity of SOD and GPx in hemin-induced HT22 cell damage model, but treatment with silenced Wnt3a had no effect on oxidative stress-related indexes (MDA, ROS, SOD, and GPx) in HT22 cells without hemin (Figure S6G). These data showed that silenced Wnt3a inhibited β-catenin pathway, antiapoptosis, and antioxidative stress in hemin-induced HT22 cells.Based on the above data, we speculated that CHR could regulate theβ-catenin pathway by inhibiting miR-320-5p. The rescue experiments suggested that the protein expression of nucleus β-catenin in hemin-induced HT22 cells could be significantly upregulated following CHR treatment; this enhancement was weakened by the overexpression of miR-320-5p. Differences in the total β-catenin expression were not observed among different groups (Figure 4(h)). These data revealed that CHR activated the Wnt3a/β-catenin pathway by restraining the expression of miR-320-5p in the hemin-induced HT22 cell injury.


## 3.4. CHR Protects against Hemin-Induced Injury by Activating the Wnt3a/β-Catenin Pathway

To further explore whether CHR inhibits hemin-induced cell apoptosis, ER stress, and oxidative stress by activating the Wnt3a/β-catenin pathway, the results of western blot revealed that the levels of protein expression corresponding to Wnt3a and nucleus β-catenin in hemin-induced HT22 cells increased significantly following CHR treatment, and the enhancement was partly inhibited by the suppression of Wnt3a (Figure 5(a)). Subsequently, the results obtained by conducting the CCK-8 assays indicated that the cell viability in hemin-induced HT22 cells increased significantly following the process of CHR treatment, and the extent of the increase in the viability of the cells was reduced by the suppression of Wnt3a (Figure S7A). The caspase-3 activity in hemin-induced HT22 cells decreased significantly following the process of CHR treatment, and the extent of decrease in the activity was alleviated by the suppression of Wnt3a (Figure S7B). In addition, the results obtained by conducting the TUNEL staining assays also indicated that the cell apoptosis rates in hemin-induced HT22 cells were significantly restrained under conditions of CHR treatment, and the restraint was alleviated by the suppression of Wnt3a (Figure 5(b)). Further, the results obtained using the western blot revealed that the protein expressions of the ER stress-related genes (GRP78, cleaved caspase-12, p-eIF2α, and CHOP) in hemin-induced HT22 cells were significantly inhibited under conditions of CHR treatment, and the inhibition was impaired by the suppression of Wnt3a (Figure 5(c)). Finally, we examined the changes of oxidative stress-related indicators (MDA, ROS, SOD, and GPx) to evaluate whether CHR affected hemin-induced oxidative stress in HT2 cells through Wnt3a. As shown in Figure 5(d), CHR inhibited the relative levels of MDA and ROS in the hemin-induced HT22 cells; the inhibition was obviously alleviated by silenced Wnt3a. While CHR increased the ROS activity and GPx activity in the hemin-induced HT22 cells, the phenomenon was obviously weakened by silenced Wnt3a. Altogether, the obtained data suggested that CHR inhibited hemin-induced cell apoptosis, ER stress, and oxidative stress by activating the Wnt3a/β-catenin pathway.Figure 5
CHR inhibited the cell apoptosis, ER stress, and oxidative stress by upregulating Wnt3a in hemin-induced HT22 cell injury models. Sh-NC: negative control shRNA; sh-Wnt3a: Wnt3a shRNA. (a) Protein expression levels of Wnt3a, T-β-catenin, and N-β-catenin were analyzed in hemin-induced HT22 cells (20 μM) with or without CHR (10 μM; treatment time: 24 h) or sh-Wnt3a (treatment time: 24 h) following the western blot technique. ∗∗p<0.01, ∗∗∗p<0.001. N=3. (b) Apoptosis rate in hemin-induced HT22 cells (20 μM) with or without CHR (10 μM; treatment time: 24 h) or sh-Wnt3a (treatment time: 24 h) was studied by conducting the TUNEL staining assays. ∗∗∗p<0.001. N=3. (c) The protein expression levels of ER stress-related genes (p-eIF2α, CHOP, GRP78, and cleaved caspase-12) were analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) or sh-Wnt3a (treatment time: 24 h) using the western blot technique. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001. N=3. (d) The change of oxidative stress-related indexes (MDA, SOD, GPx, and ROS) were analyzed in hemin-induced HT22 cells with or without CHR (10 μM; treatment time: 24 h) or sh-Wnt3a (treatment time: 24 h). ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001. N=3.
(a)(b)(c)(d)


## 4. Discussion

ICH accounts for only 10–15% of all strokes. However, the level of mortality and morbidity recorded is extremely high [57]. In recent years, significant advances have been made in the field, and the results can help explain the causes of ICH. The advancements made in the field help in understanding the pathophysiology associated with the disease. The results can help in developing acute therapy and prevention methods. However, specific clinical treatment methods for ICH are not known. It has been previously reported that CHR exhibits neuroprotective effects during brain damage [16, 58]. However, the role and underlying molecular mechanisms of CHR in ICH are unknown. We demonstrated that the process of CHR posttreatment helped protect against hemin-induced neuronal cell injury in HT22 cells by regulating the miR-320-5p/Wnt3a signaling pathway.ICH injury consists of two stages: primary brain injury (PBI) and secondary brain injury (SBI). Typically, PBI is followed by SBI, and it negatively affects human health. The severity of SBI depends on the rate of recovery and location of ICH [42]. Hemin is a neurotoxic product generated during the degradation of free hemoglobin (released following ICH) in the presence of heme oxygenase-1 (HO-1). It significantly affects the incidence of SBI [59]. It has been reported that hemin can mimic secondary injury (in vitro) [26–28]. CHR is one of the most key anthraquinone components extracted from rhubarb, which has broad spectrum of therapeutic potential, such as neuroprotection [12]. Cui et al. reported that CHR suppressed the hippocampus damage by inhibiting the level of mitochondrial autophagy [16]. Chae et al. showed that CHR alleviated hippocampal neuronal cell death via inhibition of Drp1-dependent mitochondrial fission [60]. In addition, our previous studies proved that rhubarb treatments effectively protected nerve cell damage by increasing of the antioxidative stress and anti-inflammatory responses in rat with ICH [61], and studies have shown that CHR is the most abundant-free anthraquinone component in the rhubarb [12]. Therefore, we hypothesized that CHR extracted from rhubarb has a protective effect on ICH following brain injury. Herein, we reported that CHR could protect HT22 cells from hemin-induced neuronal cell injury by improving cell viability and restricting cell apoptosis. Moreover, it has been elucidated that oxidative stress plays critical roles in determining the extent of ICH damage, and oxidative stress after ICH increases the blood-brain barrier, autophagy, apoptosis, and inflammatory response, thus exacerbating brain injury [3]. Our data revealed that CHR could reduce hemin-induced oxidative stress in HT22 cell; this result is similar to the role of CHR in other diseases. For example, Ma et al. reported that CHR relieves cisplatin-induced nephrotoxicity via concomitant inhibition of oxidative stress and apoptosis [62]. Zhao et al. demonstrated that CHR alleviates nitrosation/oxidative stress injury in focal cerebral ischemia/reperfusion [17]. Lin et al. showed that CHR significantly inhibited lipopolysaccharide-induced oxidative stress in microglia [63]. In addition to oxidative stress, ER stress is also involved in brain injury after ICH [64]. And ER stress after ICH causes excess release of ROS, leading to the increase of oxidative stress response [3]. Our data revealed that CHR could reduce hemin-induced ER stress in HT22 cell; this result is similar to the role of CHR in other diseases. For example, CHR was found to protect against I/R-induced nerve injury by restraining the activation of ER stress [18]. CHR hindered the hepatitis B virus X protein- (HBx-) induced activation of hepatic stellate cells by activating ER stress [15].It has been reported that miRNAs can be the potential biomarkers for the diagnosis and prognosis of brain damage [65, 66]. Therefore, it is important to explore miRNAs to develop effective treatment methods for ICH. Herein, we found that the expression of miR-320-5p in the hemin-induced HT22 cell was significantly higher than the expression observed in the NC group. A large number of studies have shown that miR-320-5p is involved in the oxidative stress response of different diseases [67–71]. For example, the inhibition of miR-320 inhibited oxidative stress-induced injury in MC3T3- E1 cells [71]. Xiao et al. demonstrated that aerosol inhalation of edaravone suppressed oxidative stress and improved pulmonary function by downregulating miR-320 in rats with smoke inhalation injury [68]. Zhu et al. proved that the inhibition of miR-320 decreased oxidative stress and cell apoptosis by upregulating Nrf2 expression in myocardial ischemia-reperfusion (I-R) injury [69]. Shen et al. indicated that overexpression of miR-320 hindered the ischemic cerebral-induced oxidative stress injury by inhibiting Nox2/ROS signaling pathway [67]. Fu et al. showed that the expression of miR-320 was downregulated in diabetic retinopathy, and overexpression of miR-320 restrained inflammation and ROS production by directly targeting USP14 in HG-stimulated Müller cells [70]. These studies showed that miR-320 plays a dual role in the regulation of oxidative stress, which may be antioxidative stress or promoting oxidative stress. However, the role of miR-320-5p in brain injury after ICH has not been explored. Our data showed that overexpression of miR-320-5p aggravated hemin-induced oxidative stress and ER stress in HT22 cells, suggesting that miR-320-5p intensifies the oxidative stress response of neurons after ICH. Furthermore, we observed that the overexpression of miR-320-5p in the hemin-induced HT22 cell could be significantly suppressed following CHR treatment, suggesting that the attenuation of miR-320-5p potentially influenced the protective mechanism following which CHR protected against hemin-induced nerve injury. As far as we know, we are the first to report that CHR ameliorated hemin-induced cell damage in HT22 cells by restraining miR-320-5p.It has been recently reported that miRNAs play important roles in regulating gene expression at the transcriptional or posttranscriptional levels by binding to 3′-UTR of diverse target genes [20]. The results obtained by us demonstrated that miR-320-5p could directly bind with Wnt3a to lower its level of expression. Numerous researchers have reported that Wnt3a functioned as a neuroprotective factor under conditions of traumatic brain injury and ischemic stroke. Matei et al. and Zhang et al. reported that intranasal Wnt3a could protect neurons against apoptosis and ameliorate toxic responses of microglia/macrophages and astrocytes. They studied the middle cerebral artery occlusion model [48, 72]. Wei et al. demonstrated that intranasal Wnt3a exhibited a dual neuroprotective and regenerative role during the treatment of focal ischemic stroke [73]. Ruan et al. reported that intranasal Wnt3a inhibited neuronal apoptosis in the early stages of brain damage post subarachnoid hemorrhage [74]. The data obtained by us indicated that the level of expression of Wnt3a in HT22 cells experiencing hemin-induced injury could be increased under conditions of CHR treatment. This indicated that Wnt3a influenced the protective effect generated by CHR. It has been reported that the Wnt/β-catenin pathway dictates the incidence of neurodegenerative and neuropsychiatric disorders. It also influences the occurrence of brain injury [75–78]. Wnt3a is one of the most critical ligands that participate in the typical Wnt/β-catenin pathway. Herein, we reported that CHR activated the Wnt3a/β-catenin pathway by inhibiting miR-320-5p and ameliorated HT22 cell injury by increasing the level of Wnt3a expression.However, this study has several limitations. Firstly, the present study lacks the in vivo animal experiments to prove that administered CHR has a protective effect on ICH. Secondly, the molecular mechanism of CHR inhibiting miR-320-5p expression has not studied in in vivo. Although the hemin-induced ICH model in vitro offers a well-characterized injury mode to evaluate the effect and mechanism of drugs on ICH following neurocyte injury, we acknowledge that this injury model does not completely stimulate the clinical ICH microenvironment and pharmacokinetic process of drugs in vivo [79]. Therefore, the efficacy and mechanism of CHR on brain injury after ICH need to be further verified in vivo. In further study, these parts in our experiment will be designed. Our study suggests that CHR may be a potential drug in clinical treatment of ICH and provides a latest reference for further research and clinical application of CHR in the treatment of ICH.


## 5. Conclusion

The results demonstrated in the present study that posttreatment with CHR prevented hemin-induced cell apoptosis, oxidative stress, and ER stress by modulating the miR-320-5p/Wnt3a pathway in HT22 cells. Considering this, CHR could be used as a potent agent against ICH.



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*Source: 9399658-2022-07-29.xml*