Oxysophoridine (OSR) is an alkaloid extracted from
Ischemic cerebrovascular disease, which accounts for 70% of all strokes worldwide, is associated with high morbidity and mortality rates (
Oxysophoridine (OSR) is one of the primary alkaloids extracted from
The present study was approved by the Animal Care and Use Committee of Hangzhou Red Cross Hospital (Hangzhou, China; approval no. 20220414) and performed in accordance with Chinese legislation regarding the use of experimental animals. A total of 30 adult male Sprague-Dawley rats (7 weeks old) weighing 280–320 g were purchased from the Laboratory Animal Resources, Chinese Academy of Sciences. The rats were housed at a room temperature of 21±2°C and 55±5% relative humidity with a 12/12-h light/dark cycle and free access to standard chow and tap water. After one week, the cerebral I/R injury model was established using improved thread occlusion of the right middle cerebral artery (MCAO) model as described previously (
The brain tissue from rats in five groups was fixed with 10% neutral formaldehyde buffer at 4°C overnight, dehydrated in a series of graded concentrations of ethanol, embedded in paraffin and cut into 4 µm sections. The sections were stained using hematoxylin for 6 min and 1% eosin for 2 min, both at room temperature. A light microscope was used to assess the pathological changes in the samples following I/R treatment at a magnification of ×400.
Rat brains were dissected into 2 mm coronal slices and incubated in 2% TTC (Beijing Solarbio Science & Technology Co., Ltd.) at 37°C for 10 min. Following TTC staining, the normal brain tissue stained dark red and the infarcted tissue was unstained (white). The tissue was fixed in 4% paraformaldehyde (Beyotime Institute of Biotechnology) for 24 h at 4°C and imaged using a digital camera (Canon, Inc.). The infarcted volume was calculated as follows: Infarcted volume (%)=(volume of white sections/volume of the whole brain) ×100.
ATP levels in brain tissues and HT22 cells were assessed using the CellTiter-Glo Luminescent Assay kit (Promega Corporation) according to the manufacturer's protocol. Fe2+ levels in brain tissue and HT22 cells were evaluated using an Iron Assay kit (cat. no. ab83366; Abcam) according to the manufacturer's protocol. The absorbance at 520 nm was assessed for the determination of iron concentration.
Brain sections (10 µm) that were stored at −18°C were permeabilized using 0.5% Triton X-100 at room temperature for 30 min and blocked using 10% bovine serum albumin (Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Subsequently, brain tissue was incubated with primary antibodies against toll-like receptor (TLR)4 (1:1,000; cat. no. ab22048; Abcam) at 4°C overnight. The cells were exposed to DAPI (BIOSS) at room temperature for 5 min were then cultivated with a secondary Alexa Fluor® 488-conjugated goat anti-mouse antibody (1:1,000; cat. no. ab150113; Abcam) at room temperature in the dark for 1 h. Finally, the sections were rinsed with phosphate-buffered saline (Beyotime Institute of Biotechnology) and imaged using a fluorescence microscope (magnification, ×400; Leica Microsystems GmbH).
The hippocampal HT22 neuronal cell line was purchased from the Ningbo Mingzhou Biotechnology Co., Ltd. The cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (HyClone, Cytiva), 100 U/ml penicillin and 100 µg/ml streptomycin (both Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator with 5% CO2 at 37°C. To establish an
TLR4-specific pcDNA overexpression vector (Oe-TLR4) or pcDNA3.1 empty vector, which served as the negative control (Oe-NC), was purchased from Shanghai Genechem Co., Ltd. A total of 100 nM plasmids were transfected into HT22 cells using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h at 37°C. Following 48 h of transfection, cells were collected for use in subsequent experiments.
Total RNA was extracted from HT22 cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The quality and concentration of RNA were assessed using NanoDrop 2000 (Shanghai Aiyan Biotechnology Co., Ltd.) based on the ratio of absorbance at 260 and 280 nm. A PrimeScript™ RT Reagent kit (Takara Bio, Inc.) was used to reverse-transcribe 2 µg RNA into cDNA using the thermocycling protocol as follows: 25°C for 5 min, 42°C for 30 min, 85°C for 5 min and then held at 4°C for 5 min. Amplification of the cDNA was performed using qPCR using an SYBR Premix Ex Taq™ II kit (Takara Bio, Inc.). The thermocycling protocol was 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 1 min. A final extension step at 72°C for 7 min was performed in each PCR assay. The primer sequences used for PCR were as follows: TLR4 forward (F), 5′-CCCATGCATTTGGCCTTAGC-3′ and reverse (R), 5′-AGAGCACTGAACCTCCTTGC-3′; and GAPDH (F), 5′-GTCGTGGAGTCTACTGGCGTCTTCA-3′ and (R), 5′-TCGTGGTTCACACCCATCACAAACA-3′. The relative mRNA levels were normalized to GAPDH using the 2−ΔΔCq method (
Following various treatments, cells were plated at a density of 5×103 cells/well in 96-well plates and cultured in DMEM with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 24 h. A total of 10 µl CCK-8 solution (Beyotime Institute of Biotechnology) was added to each well and incubated for 2 h. The absorbance at 450 nm was assessed using a microplate reader (Bio-Rad Laboratories, Inc.). All experiments were performed in triplicate with five independent repeats.
IL-1β, TNF-α and IL-10 protein expression levels in HT22 cells were assessed using ELISA kits (cat. nos. EK0393, EK0526 and EK0418 respectively; Wuhan Boster Biological Technology, Ltd.) according to the manufacturer's protocols. The absorbance was assessed at 450 nm using a microplate reader (BioTek Instruments, Inc.).
To assess reactive oxygen species (ROS) generation, DCFH-DA staining (cat. no. D6883; MilliporeSigma) was used. HT22 cells were washed with PBS and incubated with 10 µM DCFH-DA in the dark for 30 min at 37°C. After washing three times with PBS, fluorescence images were obtained using an Axio-Observer-D1 fluorescence microscope (ZEISS AG; magnification, ×400). Furthermore, the activity of superoxide dismutase (SOD) and levels of malondialdehyde (MDA) and catalase (CAT) were assessed using SOD assay kit (cat. no. S0101M), MDA assay kit (cat. no. S0131S) and CAT assay kit (cat. no. S0051), all of which were obtained from Beyotime Institute of Biotechnology. The absorbance at 532 nm was assessed using a Benchmark microplate reader (Bio-Rad Laboratories, Inc.).
TUNEL assay was performed to evaluate the degree of apoptosis in tissue and cells. Following fixation with 4% paraformaldehyde at room temperature for 30 min, tissue sections or cells were incubated with proteinase K at room temperature for 15 min, placed in 3% H2O2 for 15 min at room temperature and treated using a TUNEL detection kit for 60 min at 37°C. Following incubation, the PBS-rinsed cells were co-labeled with 1 µg/ml DAPI working solution for 10 min at 37°C. The positive cells were mounted with fluorescent mounting media (Beijing Solarbio Science & Technology Co., Ltd.) and analyzed using ImageJ 1.8.0 software (National Institutes of Health). More than 10 fields of view/section for each sample were assessed. The labeled cells were visualized using an Olympus BX53 fluorescence microscope (magnification, ×100; Olympus Corporation).
Total protein was extracted from tissue and cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) and then quantified with bicinchoninic acid (BCA) protein assay kit (cat. no. P0012S; Beyotime Institute of Biotechnology). An equal amount of protein (60 µg/lane) was separated on 10% SDS gels and then transferred to a nitrocellulose blotting membrane (Pall Life Sciences). The membranes were blocked with 5% non-fat milk dissolved in 0.1% TBS-T buffer for 2 h at room temperature and probed with primary antibodies as follows: Bax (1:1,000; ab32503), Bcl-2 (1:1,000; ab196495), acyl-CoA synthetase long-chain family member 4 (ACSL4; 1:1,000; ab155282), transferrin 1 (TFR1; 1:1,000; ab269513), ferritin 1 (FTH1; 1:1,000; ab1837810 glutathione peroxidase 4 (GPX4; 1:1,000; ab252833), TLR4 (1:1,000; ab217274), MyD88 (1:1,000; ab219413), phosphorylated (p)-p38 (1:1,000; ab4822), p38 (1:1,000; ab170099), inducible nitric oxide synthase (iNOS; 1:1,000; ab178945), cyclooxygenase 2 (COX-2; 1:1,000; ab179800), p-p65 (1:1,000; ab76302), p65 (1:1,000; ab32536) and GAPDH (1:1,000; ab8245; all Abcam) overnight at 4°C. The membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (1:2,000, ab6789 and ab6721, respectively; Abcam) for 2 h at room temperature. Signals were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Densitometry analysis was performed using ImageJ (Version 1.49; National Institutes of Health).
Statistical analysis was performed using SPSS version 17.0 (SPSS, Inc.) and data are presented as the mean ± SD. Comparisons between multiple groups were performed using one-way ANOVA followed by Bonferroni's post hoc test for multiple comparisons. All cellular experiments were performed ≥3 times from three different cultures. P<0.05 was considered to indicate a statistically significant difference.
The structure was presented in
I/R induction elevated ROS levels in brain tissue of rats; however, OSR treatment markedly decreased accumulation of ROS (
To evaluate the mechanism of action of OSR in I/R-induced rats, the activity of the TLR4/p38MAPK signaling pathway was assessed. Immunofluorescence staining demonstrated that the number of positive cells was markedly higher in the I/R group compared with the control group and that OSR markedly decreased the number of positive cells compared with the I/R group, which indicated that levels of TLR4 decreased following OSR treatment (
To evaluate the role of TLR4/p38MAPK signaling in the regulation of OSR in cerebral I/R injury, an
To study the role of ferroptosis in OGD/R induced cells, the ferroptosis inducer erastin was used. OGD/R significantly decreased cell viability compared with the control, whereas OSR significantly reversed this effect compared with OGD/R group and cell viability was markedly decreased by erastin (
OGD/R led to a significant decrease in SOD activity and CAT levels, but significantly increased MDA levels compared with the control. OSR treatment significantly enhanced the levels of SOD and CAT and significantly reduced the levels of MDA compared with the OGD/R group; however, this was significantly reversed by the addition of erastin compared with the OGD/R + OSR group (
Cerebrovascular disease is one of the primary conditions associated with notable risk to human health and survival (
Studies have reported that Traditional Chinese Medicine can intervene in the pathological process of cerebral I/R injury by affecting multiple targets and/or pathways. For example, galangin represses the ferroptosis in hippocampal tissue of gerbils by activating the SLC7A11/GPX4 axis, thus protecting against cerebral I/R injury (
The activation of innate immune receptors such as TLRs serves an important role in induction of inflammatory responses; TLR4 was the first mammalian TLR recognized (
Ferroptosis is a modulated form of cell death characterized by lethal iron-dependent accumulation of lipid peroxides and is involved in several types of brain disease, including cerebral I/R (
In conclusion, the results of the present study indicated that OSR attenuated brain injury and neuronal apoptosis, oxidative stress and inflammatory response in cerebral I/R injury by inhibiting ferroptosis. Moreover, OSR decreased OGD/R-induced neuronal ferroptosis by inactivation of the TLR4/p38MAPK signaling pathway.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JZ and MM designed the study and drafted and revised the manuscript. LL and GF analyzed the data and reviewed the literature. JZ and MM confirmed the authenticity of all the raw data. All authors performed the experiments. All authors have read and approved the final manuscript.
All procedures using animals were approved by the Animal Care and Use Committee of Hangzhou Red Cross Hospital (approval no. 20220414) and performed in accordance with Chinese legislation regarding experiment animals.
Not applicable.
The authors declare that they have no competing interests.
OSR alleviates brain injury and neuronal apoptosis in I/R-induced rats. (A) Structure of OSR. (B) Pathological changes in rat brain tissue was assessed using hematoxylin and eosin staining. Scale bar, 25 µm. (C) Representative brain sections with the largest volume infarction were assessed using 2,3,5-triphenyltetrazolium chloride staining. (D) Infarct volume. (E) Apoptosis rate was assessed using TUNEL assay. Scale bar, 25 µm. (F) Protein expression levels of Bax and Bcl-2 were semi-quantified using western blotting. Data are presented as mean ± SD. Comparisons between multiple groups were performed using one-way ANOVA followed by Bonferroni's post hoc test for multiple comparisons. ***P<0.001. OSR, oxysophoridine; I/R, ischemia/reperfusion.
OSR decreases ROS accumulation and ferroptosis induced by I/R in rat brain. The levels of (A) ROS, (B) ATP and (C) Fe2+ were assessed in I/R rat brain tissue. The control was set as 100%. Scale bar, 25 µm. (D) Protein expression levels of ACSL4, TFR1, FTH1 and GPX4 were semi-quantified using western blotting. Data are presented as mean ± SD. Comparisons between multiple groups were performed using one-way ANOVA followed by Bonferroni's post hoc test for multiple comparisons. *P<0.05, **P<0.01 and ***P<0.001. ROS, reactive oxygen species; OSR, oxysophoridine; I/R, ischemia/reperfusion; TFR1, transferrin 1; FTH1, ferritin 1; GPX4, glutathione peroxidase 4; ACSL4, acyl-CoA synthetase long-chain family member.
OSR inhibits TLR4/p38MAPK signaling in brain tissue of I/R-induced rats. (A) Protein expression levels of TLR4 in brain tissue of I/R-induced rats were evaluated using immunofluorescence staining. Scale bar, 25 µm. (B) Protein expression levels of TLR4, MyD88, p-p38 and p38 were semi-quantified using western blotting. Data are presented as mean ± SD. Comparisons between multiple groups were performed using one-way ANOVA followed by Bonferroni's post hoc test for multiple comparisons. **P<0.01 and ***P<0.001. OSR, oxysophoridine; I/R, ischemia/reperfusion; p, phosphorylated; TLR, toll-like receptor; IF, immunofluorescence staining.
OSR suppresses OGD/R-induced neuronal ferroptosis by inhibiting TLR4/p38MAPK signaling. (A) Protein expression levels of TLR4, MyD88, p-p38 and p38 were semi-quantified using western blotting. (B) mRNA and (C) protein expression levels of TLR4 in OGD/R-induced cells were assessed using reverse transcription-quantitative PCR and western blotting, respectively. The levels of (D) reactive oxygen species, (E) ATP and (F) Fe2+ were assessed in OGD/R-induced cells. Scale bar, 50 µm. (G) Protein expression levels of ACSL4, TFR1, FTH1 and GPX4 were (H) semi-quantified using western blotting. Data are presented as mean ± SD. Comparisons between multiple groups were performed using one-way ANOVA followed by Bonferroni's post hoc test for multiple comparisons. **P<0.01 and ***P<0.001. OGD/R, oxygen-glucose deprivation/reoxygenation; I/R, ischemia/reperfusion; p, phosphorylated; Oe, overexpression; NC, negative control; OSR, oxysophoridine; TLR, toll-like receptor; TFR1, transferrin 1; FTH1, ferritin 1; GPX4, glutathione peroxidase 4; ACSL4, acyl-CoA synthetase long-chain family member.
Erastin decreases the protective effect of OSR on cellular viability in OGD/R-induced cells. (A) Viability of OGD/R-induced cells treated with or without OSR and/or Erastin were assessed using Cell Counting Kit-8 assay. (B) Apoptosis rate was assessed using TUNEL assay and then (C) quantified. Scale bar, 50 µm. (D) Protein expression levels of Bax and Bcl-2 were semi-quantified using western blotting. Data are presented as mean ± SD. Comparisons between multiple groups were performed using one-way ANOVA followed by Bonferroni's post hoc test for multiple comparisons. *P<0.05, **P<0.01 and ***P<0.001. OGD/R, oxygen-glucose deprivation/reoxygenation; OSR, oxysophoridine.
Erastin decreases the inhibitory effect of OSR on OGD/R-induced oxidative stress and inflammatory response. (A) Levels of SOD, MDA and CAT were assessed using specific detection kits. (B) Protein expression levels of IL-1β, TNF-α and IL-10 were assessed using ELISA. (C) Protein expression levels of iNOS, COX-2, p-p65 and p65 were semi-quantified using western blotting. Data are presented as mean ± SD. Comparisons between multiple groups were performed using one-way ANOVA followed by Bonferroni's post hoc test for multiple comparisons. **P<0.01 and ***P<0.001. OSR, oxysophoridine; OGD-R, oxygen-glucose deprivation/reoxygenation; SOD, superoxide dismutase; MDA, malondialdehyde; CAT, catalase; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase 2; p, phosphorylated.