Protective effects of Berberine on hippocampal neurons after ischemia in vivo and in vitro

Abstract

Background

Apoptosis plays a pivotal role in neuronal death following ischemic brain injury. Berberine (BBR), a natural isoquinoline alkaloid extracted from Coptis chinensis, exhibits multiple biological activities and has shown neuroprotective effects in experimental ischemia models. However, its direct protective effects on neurons and the underlying apoptosis-related mechanisms remain to be fully clarified.

Methods

A transient forebrain ischemia model was established in mice to evaluate the neuroprotective effects of BBR in vivo. Cognitive function was assessed using the Y-maze test, and hippocampal neuronal injury was examined by H&E staining. In vitro, primary hippocampal neurons were subjected to oxygen–glucose deprivation (OGD) to mimic ischemic injury. Neuronal viability, mitochondrial function, intracellular calcium levels, apoptosis, and related signaling pathways were analyzed using CCK-8, LDH, JC-1, Fluo-4 AM, Hoechst staining, caspase-3 activity assay, and Western blotting.

Results

BBR significantly reduced hippocampal neuronal loss and improved cognitive deficits in mice after transient forebrain ischemia. In primary hippocampal neurons exposed to OGD, BBR markedly enhanced cell viability, alleviated mitochondrial dysfunction, attenuated intracellular calcium overload, and suppressed neuronal apoptosis. Mechanistically, BBR may increase the expression of phospho-Akt and Bcl2 while reducing the expression of Bax and the activity of cleaved caspase-3.

Conclusions

These findings demonstrate that berberine exerts significant neuroprotective effects against ischemia-induced neuronal injury by targeting apoptosis-related signaling pathways, particularly the Akt/caspase-3 axis, and support its potential as a therapeutic candidate for ischemic stroke.

Introduction

Stroke is a disease that brings great challenges to human health worldwide. It is the second largest cause of death and burden to humans among all diseases after ischemic heart diseases [1, 2]. Ischemic stroke constitutes most (87%) stroke cases [3]. The primary cause of ischemic stroke leading to patient death or disability is neuronal death in the central nervous system (CNS) due to interruption of blood flow via a transient or permanent vascular obstruction. Neurons are believed to be the cells that are most vulnerable to ischemia. A short period of blood flow interruption could induce irreversible neuron degeneration even if blood begins to circulate shortly thereafter. This circumstance results in many restrictions to the use of thrombolysis therapy (using recombinant tissue plasminogen activator) and mechanical thrombectomy to restore the blood supply, which are currently carried out in clinics. Neuron protection is therefore urgently needed in the area of stroke treatment, and many studies have attempted to identify agents that protect neurons when ischemic stroke occurs [4].

Many years of research have led to the conclusion that glutamate excitotoxicity is the main cause of neuronal death in ischemia. The excessive release of glutamate from presynaptic membranes after ischemia that overworks the glutamate receptors is a key component in neurochemical event sequences including intracellular calcium overloading, reactive oxygen species (ROS) production, and mitochondrial stress, which all lead to neuronal death [5, 6]. Glutamate receptor inhibitors have therefore been applied to treat ischemia. Although some of the inhibitors could relieve the ischemic insult in animal experiments [7, 8], all of the clinical trials have unfortunately failed [9].

Many kinds of substances have been found to have neuron protective properties [10, 11], but none of them were found to have definitive clinical effects. Berberine (BBR) is a natural active ingredient extracted from the rhizome of Coptis chinensis. It is a promising therapeutic agent for myocardial ischemia and reperfusion injury [12], so it is reasonable to assume that BBR may also exert a protective effect against ischemia injury in the CNS. BBR is neuroprotective in CNS diseases such as Alzheimer’s disease (AD) [13, 14].

Previous experimental studies have demonstrated the antioxidative effect of BBR in diverse injury models such as AD [13, 14], and its anti-inflammatory effects in diseases such as diabetes [15]. Some studies also found that BBR had antitumor properties [16, 17]. Most importantly, many studies have found BBR to have protective effects on ganglion cells, a type of neuron, in a diabetes model [18], and on cytomegalovirus-induced apoptosis [19]. Another study found that BBR can prevent neuronal death after exposure to ischemia/reperfusion-induced testicular injury by eliminating ROS [20]. Some in vivo studies also demonstrated that BBR can protect brain tissue against middle cerebral artery occlusion (MCAO) via an antiapoptosis pathway in mice and rats [21–23].

In this study, we evaluated whether BBR can affect neuronal apoptosis, and attempted to provide evidence for the clinic application of BBR in treating stroke.

Experimental procedures

Animals

Adult male C57BL/6 mice (22–26 g, 10–12 weeks old) were supplied from the Animal Experiment Center of Southern Medical University (Guangzhou, China). All animal studies were conducted by the ethical committee of Jinan University.

Anesthesia and euthanasia

All animals were rendered fully unconscious by an overdose of inhaled isoflurane prior to sacrifice. Adequate depth of anesthesia was confirmed by the absence of the pedal withdrawal reflex. While remaining unconscious, euthanasia was performed by carbon dioxide (CO₂) inhalation, and exposure was maintained after respiratory arrest to ensure irreversible death. Cervical dislocation was subsequently applied as a secondary physical method to confirm death before tissue collection. This procedure was conducted in accordance with institutional guidelines for animal care and use and was designed to minimize pain, distress, and suffering.

Transient forebrain ischemia model

Mice were randomly divided into 4 groups, namely sham group, sham + BBR group, ischemia group and ischemia + BBR group. All mice were initially anesthetized with 5% isoflurane, then were placed on an operating board and anesthetized continuously with 2% isoflurane using a nasal mask. The bilateral common carotid arteries were then bluntly dissected using curved forceps and clipped for 20 min. During the operation, the body temperature was maintained at 37 °C using heating pads. Subsequently, the skin was sutured and disinfected. The sham and sham + BBR groups undergo surgery without artery clipping, while the ischemia and ischemia + BBR groups undergo 20-min bilateral carotid artery occlusion.

H&E staining and quantitative analysis of hippocampal CA1 neuron damage

The animals were deeply anesthetized using 5% isoflurane and perfused with 4% paraformaldehyde for about 5 min, following phosphate-buffered saline perfusion (PBS; 0.01 mol/L, pH 7.4) for about 3 min. At 1 day post fixation, 40-μm-thick coronal sections containing the hippocampus were cut using a Vibratome (VT1000 S, Leica, Germany). The sections were stained using hematoxylin and eosin (H&E; Thermo Fisher Scientific, USA) according to the manufacturer's instruction.

Five animals in each group were examined for neuronal damage with the method previously established [24, 25]. In brief, four to six slices of typical hippocampal tissues in each animal were photographed using a microscope (CTR 6000, Leica, Germany) under 100 × magnifications. The level of neuronal injury in the area of CA1 was assessed with the method previously published by our team [25], which was based on extent of neuron death: level 0 (no damage), level 0.5 (< 10%), level 1.0 (10–30%, mild damage), level 1.5 (30–50%, mild-to-moderate damage), level2.0 (50–70%, moderate damage), level 2.5 (70–90%, moderate-to-severe damage) and level 3.0 (> 90%, severe damage).

Y-maze test

The Y-maze experiment was used to evaluate the spatial memory of mice. Briefly, A Y maze is equally divided into three arms. All of the arms were opened and the mice were allowed to move freely among the three arms for 10 min. A video-based software tracking system was used to track the duration and number of times that the mice enter into each arm to evaluate their spatial recognition.

Primary hippocampal culture

For primary hippocampal culture, female mice at E18 gestation were anesthetized with 5% isoflurane. The brains were removed from the embryos. The hippocampi were dissected from the brains and treated using 0.125% (w/v) trypsin (Gibco, USA) at 37 °C for 30 min. Fetal bovine serum (Gibco, USA) was added to a 10% concentration to stop digestion. After suspension, centrifugation, and filtration, cells were resuspended in Neurobasal Medium (Gibco, USA). Neurons were then seeded on multiwell plates (Corning, USA) at a density of 10⁵ cells/cm², coated with 0.01% (w/v) poly-L-lysine (Sigma, USA), and put into a standard incubator (Heal Force, China) that was maintained at 37 °C in 95% air with a 5% CO2 humidified incubator. For morphology tracing, cells were placed in 24-well plates at the low concentration of 10⁴ cells/well, and half of the medium was changed every week.

Oxygen glucose deprivation and reperfusion

Primary neurons were placed in an anaerobic chamber (Coy Lab, USA) after 2-week culture and rinsed three times using deoxygenated (bubbled using N₂ for 30 min to remove the residual O₂), glucose-free balanced salt solution (BSS) consisting of 116 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, 26.2 mM NaHCO₃, and 1.0 mM NaH₂PO₄. The incubator was humidified and maintained at 5% CO₂ and 95% N₂ at 37 °C. Cells were exposed to the OGD condition for a designated period of 4 h, which was terminated by replacing the glucose-free BSS with a normal culture medium and by returning the cultures to a standard maintained incubator. Control cultures were cultured within BSS that contained 4500 mg/L D-glucose and maintained in the standard incubator.

CCK-8 assay

Neuronal viability was quantitatively evaluated using CCK-8 kit (Dojindo, Japan). In brief, primary neurons culture were treated using OGD or drugs for 24 h, and 10 μL of CCK-8 solution was added to each of the 96-well plates that contained 90 μL of medium, which were incubated for 4 h at 37 °C. The absorbance was then measured using a microplate reader at 450 nm.

LDH assay

Neuron injury was quantitatively evaluated by measuring the LDH released from dead or damaged neurons. The LDH activity in the medium and total activity were measured using an LDH assay kit (Dojindo, Japan) at 450 nm following the instructions of the manufacturer. The level of LDH activities was calculated as (LDH activity in medium/total activity) × 100.

Western blotting

Western blotting analysis was performed on neuron lysates exposed to different treatments. Neurons were lysed in an ice-cold lysis buffer with the protease inhibitors (Merck, USA) and phosphatase inhibitors (Merck, USA) for 3 min, collected in Eppendorf tube, immediately frozen in liquid nitrogen and transferred into −80 °C refrigerator for storage. Then samples were centrifuged (12,000 rpm, 15 min, and 4 °C) to obtain the supernatant, and their protein concentrations were determined using bicinchoninic acid (Pierce, USA). Equivalent amounts of protein (20 μg) from each sample were subjected to sodium dodecyl sulfate–polyacrylamide electrophoresis using 12% Bis–Tris Gels (Beyotime, China) under reducing conditions, and the proteins were then transferred onto a polyvinylidene difluoride membrane. After a blocking step (0.1% Tween 20 and 5% nonfat milk in TBS for 1 h at room temperature), membranes were incubated overnight using primary antibodies at 4 °C with gentle shaking. The following primary antibodies were used: phospho-Akt (p-Akt; Ser-473; 1:1000; CST, USA; molecular weight, 60 kDa), Akt (1:1000; CST, USA, molecular weight, 60 kDa), cleaved caspase-3 (1:1000; CST, USA, molecular weight, 17 and 19 kDa), Bcl2 (1:5000; Proteintech, USA, molecular weight, 26 kDa), Bax (1:5000; Proteintech, USA, molecular weight, 21 kDa). Membranes were washed for 10 min three times, and incubated using HRP-conjugated antirabbit IgG or HRP-conjugated antimouse IgG (1:5000, Abcam, UK). The membranes were then incubated using ECL liquid, and the blots were detected using an imaging system (GE, USA). Signal intensities were analyzed using ImageJ software.

Caspase-3 activity assay

Caspase-3 activity was measured using a commercially available kit. The procedure followed the introductions of the manufacturer. Briefly, all cell proteins were extracted and quantified following the protocol described in the Western blot method. In a blank plate, 50 μL of protein and 200 μL of substrate were mixed, and the mixture was incubated in the dark for 1 h at 37 °C. The relative fluorescence units were observed on a fluorescence plate reader with excitation at 380 nm and emission at 420–460 nm, and then analyzed.

Ca2+ measurement in cultured neurons

We used Fluo-4 AM (Dojindo, Japan) to measure the intracellular calcium level. The primary hippocampal neurons were cultured on Petri dishes with a coverslip. Cells were washed using Hanks’ solution three times and were then loaded in the dark with 10 μmol/L Fluo-4 AM for 30 min at 37 °C. After incubation, the cells were rinsed three times (3 min/time) using Hanks’ solution to remove the extracellular Fluo-4 AM. Images were obtained using fluorescence microscopy, and their fluorescence intensities were analyzed using ImageJ software.

Hoechst 33342

Hoechst 33342 was used to measure neuron apoptosis. Briefly, the medium was removed and the neurons were rinsed three times using PBS, and Hoechst was then added for 5 min and then removed, and the cells were then rinsed three times to remove the surplus dye. A fluorescent microscope was then used to take photographs for the analysis. Neurons with condensed or fragmented chromatin were considered apoptotic. These results were quantitated by double-blind counting of apoptotic and total neurons in each visual field and expressed as the percentage apoptosis.

JC-1 staining

JC-1 staining was used to evaluate the mitochondrial membrane potential (MMP) following the manufacturer's instruction (Beyotime, China) [26].

Drugs and reagents

In vivo, mice received 40 mg/kg/day BBR dissolved in distilled water intragastrically in line with previous study [16]. The treatment started 7 days before ischemia until the mice were sacrificed. As a control, mice were treated with the same volume distilled water and time schedule. In vitro, BBR was dissolved in distilled water at a concentration of 1 mM to prepare a stock solution, and the BBR working solution was diluted to 1 μM referring to a previous report [27].

Statistics

All results were presented as mean ± SEM values except where specified otherwise. Analysis of variance (ANOVA) was used for multiple comparisons followed by post-hoc analysis with Tukey’s LSD using SPSS 13.0 (Abacus concepts Inc., Chicago, IL, USA). Differences were considered statistically significant at p < 0.05.

Results

BBR reduced neuron damage and lessened cognitive dysfunction after ischemia in vivo

To test the effects of BBR on ischemia-induced neuronal loss, we first established a transient global ischemia model that used mice. We intragastrically fed the mice with BBR daily for the 7 days before ischemia onset and continued after surgery until the mice were sacrificed 14 days post ischemia. H&E staining of the coronal brain sections indicated that remarked neurons died in the hippocampus in the ischemia group at 14 days after ischemia compared with the sham group. However, with BBR treatment, neuronal damage was significantly reduced compared with the vehicle-treated group (ischemia group) in the hippocampus (Fig. 1A & B). We then examined the effects of BBR on ischemia-induced short-memory impairment using the Y-maze test (Fig. 1C), and the spontaneous alternation was measured and compared among the four groups. As shown in Fig. 1D, the alternation rate was significantly lower in the ischemia group than the sham group at 14 days after ischemia (43.39 ± 2.67% vs 62.33 ± 3.10%); however, after BBR treatment, this value was significantly elevated comparing with the ischemia group (55.44 ± 2.79% vs 43.39 ± 2.67%).

Fig. 1.

Berberine (BBR) decreased neuronal death and improved the cognitive function of mice. A Hematoxylin–eosin staining showing the effects of BBR on neuron survival in the hippocampus of mice subjected to ischemia and dead neurons were indicated with arrows in the ischemia group; scale bar, 100 μm. B Quantitative analysis of neuronal damage in the CA1 area of the hippocampus (n = 5 per group, **p < 0.01, ****p < 0.0001, analysis of variance [ANOVA]). C Schematic diagram of the Y maze. D Behavioral analysis in the Y maze showing the effects of BBR on ischemia-induced cognitive dysfunction (8–10 mice per group; *p < 0.05, ***p < 0.001, ANOVA)

BBR enhanced the viability of primary hippocampal neurons exposed to OGD

The protective effect of BBR on neurons in an animal model of ischemia may be attributable to its direct action on neurons or the effect on other supportive cells such as glial cells [28]. BBR improves the short-memory function of mice, which is related to the hippocampus [29]. An in vitro ischemia model (OGD) was established with primary hippocampal neurons to directly examine the effect of BBR on neurons. BBR (1 μM) was added to the neuron medium 24 h before OGD and was removed during and after applying an ischemic insult. When treated with BBR, the cell viability increased to 73.98 ± 5.28%, compared with the 53.92 ± 5.16% in the OGD group as tested using the CCK8 assay at 24 h after OGD exposure (Fig. 2A). After 24 h of OGD, the LDH leakage in neurons also increased to 297.99 ± 26.34% relative to that in the control group, but after BBR administration, the LDH leakage significantly decreased to 185.52 ± 27.25% (Fig. 2B). In addition, BBR did not affect cell viability in normal culture condition.

Fig. 2.

Effects of BBR on neuronal viability and death. A. Quantitative results showing the effects of BBR treatment on neuronal viability 24 h post oxygen glucose deprivation (OGD) exposure with a duration of 4 h (n = 5 per group, *p < 0.05, ****p < 0.0001, ANOVA).B. Quantitative results showing the effects of BBR treatment on neuronal death when exposed to OGD for 4 h (n = 5 per group, **p < 0.01, ****p < 0.0001, ANOVA).C. Representative images showing the morphology of primary neurons before and 24 h after OGD with or without BBR; scale bar, 20 μm

To further confirm the neuroprotection from BBR, we traced the morphology of neurons using a phase-contrast microscope in situ before and 24 h after OGD treatment. When primary hippocampus neurons were cultured for 2 weeks, they displayed smooth and pyramidal cell bodies surrounded with a bright circle. The nuclei had a normal shape and no obvious contrast with the cell membrane. BBR treatment had no detectable effects in normal neurons. But after the neurons were exposed to OGD, the soma became round-shaped with pyknosis in their nuclei (Fig. 2C, red arrow). Most of the dendrites were separated from the soma and broken, showing bead-like morphology (red triangle), but the neuron damage was reversed by BBR administration.

Effects of BBR on mitochondria stress and calcium influx

Using JC-1 to test the MMP, we found that 4 h of OGD could cause MMP dysfunction, as shown by the green/red fluorescence intensities increasing to 182.32 ± 7.93% of that in the control, which suggested mitochondrial stress. BBR treatment could also significantly change this value to 90.61 ± 8.43% of the control value (Fig. 3A & B). We then used Fluo 4-AM to detect the intracellular free calcium in primary neurons. After OGD, the Fluo 4-AM fluorescent density increased by approximately twofold (209.34 ± 7.63%) at 24 h after OGD. However, BBR dramatically reduced the neuronal intracellular concentration of calcium to 114.01 ± 7.22% of the control (Fig. 3C & D).

Fig. 3.

BBR reduced mitochondrial injury and intracellular calcium levels. A. Representative images of JC-1 staining. Red fluorescence indicates JC-1 aggregates in cells with relatively intact mitochondrial membrane potential, whereas green fluorescence indicates JC-1 monomers in cells with reduced mitochondrial membrane potential. Scale bar, 20 μm. B. Statistical analysis of JC-1 staining (n = 5 per group, ****p < 0.0001, ANOVA). C. Representative image of Fluo-4 AM staining; scale bar, 20 μm. D. Statistical analysis of Fluo-4 AM staining (n = 5 per group, ****p < 0.0001, ANOVA)

OGD-induced neuronal apoptosis was alleviated by BBR administration

We used Hoechst 33342 to stain the neuron nuclei. In the control and control + BBR groups, the nuclei were normal with very few (3.34 ± 0.98%) abnormal condensed ones that presented apoptosis. But when the neurons were exposed to OGD, the apoptotic neurons with condensed nucleus significantly increased to 54.24 ± 5.06% of total cells. Although it had no effect on neuronal apoptosis in normal condition, BBR administration markedly reduced the percentage of dead neurons (25.15 ± 5.18% vs 54.24 ± 5.06%, Fig. 4) in the circumstance of OGD insult. These results indicated not only the morphology of somas and dendrites but also that the nuclei of cultured neurons could be restored by applying BBR.

Fig. 4.

Neuronal apoptosis was inhibited by BBR. A Representative image of Hoechst 33342 staining; scale bar, 50 μm. B Statistical analysis of apoptotic neurons (n = 5 per group, ***p < 0.001, ****p < 0.0001, ANOVA)

BBR inhibits neuronal apoptosis via activation of Akt phosphorylation

To further confirm whether the neuroprotection of BBR was mediated by an antiapoptotic pathway, a caspase-3 substrate probe was used to measure the activity of caspase-3, which increased significantly to 2.03 ± 0.19 times at 24 h after OGD. However, when treated using BBR, the caspase-3 activity decreased significantly to 1.50 ± 0.12 times that of the control (Fig. 5A). Cleaved caspase-3 expression was then further detected using Western blotting. The cleaved caspase-3 increased to 2.25 ± 0.29 times that of the control. BBR significantly reduced the cleaved caspase-3 expression to 1.48 ± 0.13 times that of the control (Fig. 5B & C). BBR activates phosphoinositide 3-kinase (PI3K)/Akt signaling in NSC34 motor-neuron-like cells [30]. We also examined Akt phosphorylation in primary neurons, and after OGD the p-Akt level was significantly reduced, while BBR could increase the expression of p-Akt (Fig. 5B & D). In addition, OGD increased the pro-apoptotic protein Bax and decreased the anti-apoptotic protein Bcl-2, while treatment with BBR reduced Bax expression and restored Bcl-2 levels (Fig. 5E-G). In the in vivo study, we further observed that BBR increased p-Akt expression (Fig. 5H & I), and BBR administration reversed the changes in Bax and Bcl-2 levels (Fig. 5K-M). In summary, our findings suggest that BBR inhibits neuronal apoptosis, potentially through the activation of Akt phosphorylation.

Fig. 5.

Phospho-Akt (p-Akt)–caspase-3 pathway was inhibited by BBR in primary neurons. A Caspase-3 activity was reduced by BBR incubation, as measured using a caspase-3 assay kit (n = 6 per group, *p < 0.05, ****p < 0.0001, ANOVA). B Western blot analysis of the expression levels of cleaved caspase-3 and p-Akt in vitro.C-D. Quantification of the expression of cleaved caspase-3 (C) and p-Akt (D) in vitro (n = 4 per group, *p < 0.05, ***p < 0.001, ****p < 0.0001, ANOVA). E. Western blot analysis of the expression levels of Bax and Bcl2 in vitro. F-G Quantification analysis of the expression of Bax (F) and Bcl2 (G) in vitro. (n = 3 per group, *p < 0.05, **p < 0.01 ANOVA).H Western blot analysis of the expression levels of cleaved caspase-3 and p-Akt in vivo. I-J Quantification analysis of the expression of p-Akt (I) and cleaved caspase-3 (J) in vivo. (n = 3 per group, *p < 0.05, ***p < 0.001, ANOVA). K Western blot analysis of the expression levels of Bax and Bcl2 in vivo. L-M Quantification analysis of the expression of Bax (L) and Bcl2 (M) in vivo. (n = 3 per group, *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA)

Discussion

Stroke is a serious global problem that needs to be solved. No effective therapy has been identified despite continuing research. Although the protective effects of BBR have been reported in MCAO and transient global forebrain ischemia models [31, 32], the direct effect of BBR on neurons still needs to be clarified. The present study focused on the role of BBR in ischemia-induced apoptosis. Consistent with another study [28], we confirmed that BBR promoted the survival of neurons in the hippocampus in vivo. Previous study found that BBR improved gerbil memory in step-down avoidance task evaluations [28], while we found that BBR improved the cognitive function of mice in the Y-maze task. In vitro, we found that BBR can increase the viability of primary cultured hippocampus neurons subjected to OGD through two classic methods (CCK8 and LDH assays). To directly examine the effect of BBR, we traced the neurons before and after OGD and confirmed that BBR improved the survival of neurons subjected to OGD.

A previous study found that ischemia induced intracellular calcium production [33]. BBR extract reduced neuron damage in the hippocampi of gerbils after transient forebrain ischemia by down-regulating the N-methyl-D-aspartate receptor, which is a major calcium channel in the brain [32]. BBR also had protective effects on oligodendrocyte cell line after OGD/reperfusion and reduced intracellular calcium in this cell line [34]. The application of a calcium probe in the present study revealed that BBR could block intracellular calcium after an ischemic insult. BBR can also restore mitochondria function. The expression levels of proapoptotic proteins such as cleaved caspase-3 were reduced. When neurons were challenged by OGD, the levels of prosurvival protein p-Akt decreased. This finding suggests that activation of the PI3K/Akt signaling pathway may contribute to the neuroprotective effects of BBR in primary hippocampal neurons. However, it should be noted that other well-established anti-apoptotic signaling pathways, such as ERK1/2 and p38 MAPK, have also been implicated in neuronal survival following ischemic injury. Since these pathways were not directly examined in the present study, we cannot exclude their potential involvement in the observed protective effects. BBR enhanced the PI3K/protein kinase B signaling pathway in gerbil brain tissue [28]. In the present study, we further confirmed that BBR activated this survival-promoting pathway in primary hippocampus neurons.

The currently recommended therapies for stroke are thrombolysis and mechanical thrombectomy, which aim to restore blood circulation. If thrombolysis or mechanical thrombectomy therapy is carried out beyond the therapeutic window after the ischemic stroke event, the risk of hemorrhage increases markedly, which causes even worse results. Due to the short therapeutic window, these methods should not be used in any patient to avoid the risk of hemorrhage. Many researchers have therefore investigated how to expand the therapeutic window. Paradoxically, tissue plasminogen activator has been found to potentiate N-methyl-D-aspartate receptor-mediated calcium influx and neuronal death. Rescuing the neurons is another important aspect when treating stroke. It is difficult for neurons to regenerate in the CNS, so reducing neuron loss after stroke or increasing the resistance of neurons to stroke is meaningful. Many drugs that have neuron-protective potential were being tested in clinics, and traditional Chinese medicine could be a good source of such drugs. Our finding that BBR could target the ischemia-unique cascade to protect the primary cultured neurons represents new evidence that BBR can be used to treat stroke or as an adjuvant therapy.

Conclusions

Berberine exerts its protective effects by targeting apoptosis-related signaling pathways and provide important experimental evidence supporting its potential application in the treatment of ischemic injury.

Abbreviations

AD

Alzheimer’s disease

Akt

Protein kinase B

ANOVA

Analysis of variance

BBR

Berberine

BSS

Balanced salt solution

Ca²⁺

Calcium ion

CCK-8

Cell Counting Kit-8

cleaved caspase-3

Cleaved cysteine-aspartic acid protease-3

CNS

Central nervous system

ECL

Enhanced chemiluminescence

H&E

Hematoxylin and eosin

LDH

Lactate dehydrogenase

MCAO

Middle cerebral artery occlusion

MMP

Mitochondrial membrane potential

OGD

Oxygen-glucose deprivation

PBS

Phosphate-buffered saline

PI3K

Phosphoinositide 3-kinase

p-Akt

Phosphorylated Akt

ROS

Reactive oxygen species

rt-PA

Recombinant tissue plasminogen activator

SEM

Standard error of the mean

tPA

Tissue plasminogen activator

Y-maze

Y-shaped maze

Authors’ contributions

DD, XL performed the investigation, formal analysis and wrote the original draft of the manuscript. ZL carried out the investigation, performed formal analysis, and contributed to the conceptualization of the study. QZ, YW, YY, and LL conducted data curation. ZZ provided supervision. ZS contributed to writing—review and editing and performed validation. All authors read and approved the final manuscript.

Funding

This work was supported by the Project of Administration of Traditional Chinese Medicine of Guangdong Province (20232023), Medical Joint Fund of Jinan University (YXZY2024015), Medical Research Project of Foshan City (20260261), Scientific Research and Cultivation Fund of the Affiliated Shunde Hospital of Jinan University (202401003, 202401015) and the Guangdong Basic and Applied Basic Research Foundation (2024A1515013181).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All methods were carried out in accordance with relevant guidelines and regulations. The study protocol was confirmed by the ethical committee of the Affiliated Shunde Hospital of Jinan University (JDSY-LL-2024203).

Competing interests

The authors declare no competing interests.