3D biological scaffold delivers Bergenin to reduce neuroinflammation in rats with cerebral hemorrhage | Journal of Translational Medicine | Full Text

Journal of Translational Medicine volume 22, Article number: 946 (2024) Cite this article

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Intracerebral hemorrhage (ICH) is a severe form of stroke characterized by high incidence and mortality rates. Currently, there is a significant lack of effective treatments aimed at improving clinical outcomes. Our research team has developed a three-dimensional (3D) biological scaffold that incorporates Bergenin, allowing for the sustained release of the compound.

This 3D biological scaffold was fabricated using a combination of photoinitiator, GEMA, silk fibroin, and decellularized brain matrix (dECM) to encapsulate Bergenin through advanced 3D bioprinting techniques. The kinetics of drug release were evaluated through both in vivo and in vitro studies. A cerebral hemorrhage model was established, and a 3D biological scaffold containing Bergenin was transplanted in situ. Levels of inflammatory response, oxidative stress, and apoptosis were quantified. The neurological function of rats with cerebral hemorrhage was assessed on days 1, 3, and 5 using the turning test, forelimb placement test, Longa score, and Bederson score.

The 3D biological scaffold incorporating Bergenin significantly enhances the maintenance of drug concentration in the bloodstream, leading to a marked reduction in inflammatory markers such as IL-6, iNOS, and COX-2 levels in a cerebral hemorrhage model, primarily through the inhibition of the NF-κB pathway. Additionally, the scaffold effectively reduces the expression of hypoxia-inducible factor 1-alpha (HIF-1α) in primary cultured astrocytes, which in turn decreases the production of reactive oxygen species (ROS) and inhibits IL-6 production induced by hemin. Subsequent experiments reveal that the 3D biological scaffold containing Bergenin promotes the activation of the Nrf-2/HO-1 signaling pathway, both in vivo and in vitro, thereby preventing cell death. Moreover, the application of this 3D biological scaffold has been demonstrated to improve drug retention in the bloodstream.

This strategy effectively mitigates inflammation, oxidative stress, and cell death in rats with cerebral hemorrhage by inhibiting the NF-κB pathway while concurrently activating the Nrf-2/HO-1 pathway.

ICH is a severe form of stroke characterized by a high incidence and mortality rate. Despite this, effective treatments to improve clinical outcomes have yet to be established [1]. The mechanisms underlying the injury are complex and not fully understood; however, an increasing body of evidence suggests that the inflammatory cascade response significantly contributes to the progression of damage. This includes the activation of glial cells, the release of inflammatory cytokines, and subsequent neuronal loss. Therefore, targeting inflammation may be a critical factor in enhancing the overall prognosis of ICH [2].

Conventional routes of drug administration in clinical practice, such as oral, intravenous, and in situ delivery, often face challenges in maintaining effective drug concentrations. This has created a pressing need for the development of innovative delivery vehicles. Recently, photocurable hydrogels have garnered significant research attention due to their minimally invasive implantation capabilities [3]. In the context of stroke, these hydrogels can be transplanted into the brain using a stereotactic procedure with a needle. Studies have demonstrated that photocurable hydrogels can facilitate host cell infiltration, promote endogenous brain tissue repair [4], guide drug and growth factor delivery to enhance angiogenesis and neural circuit recovery [5], and assist in the transplantation of stem cells to restore lost neurons [6]. However, immune reactions, influenced by factors such as implant biocompatibility, play a critical role in the interactions between the hydrogel and surrounding host tissues [7]. Glial cells, particularly microglia and macrophages, are key innate immune cells in the brain. The polarization of these cells into pro-inflammatory (M1) and anti-inflammatory (M2) subtypes is essential for promoting brain injury repair and neurorestoration [8]. Nevertheless, the challenge remains in understanding how to effectively regulate immune responses and neuroinflammation using biomaterials like photocurable hydrogels [9].

Over the past decade, numerous studies have demonstrated that biomaterials can enhance cell tissue adhesion and entrapment by mimicking natural tissues, improving the environment for angiogenesis, and promoting cell survival. This ultimately facilitates central nervous system tissue regeneration [10,11,12,13,14]. While individual extracellular matrix (ECM) components like collagen, hyaluronic acid, and fibrin scaffolds have been created for central nervous system (CNS) applications, these solutions often fall short of fully replicating natural tissues due to the intricate composition of the ECM. As a result, dECM has emerged as a promising approach to closely mimic natural tissues.

Decellularization involves the removal of cellular and nuclear components from tissues or organs to prevent initial immune responses, while maintaining the natural ECM’s structure and composition [15, 16]. Decellularized tissues can take the form of porous solids, be ground into a powder, or be gelatinized into hydrogels. The preserved intact decellularized ECM contains similar concentrations and ratios of GAGs, fibronectin, and adhesion proteins, which can stimulate tissue- or organ-specific regenerative responses [17]. Additionally, dECM is well-tolerated by xenogeneic recipients due to the conservation of molecules across species [18]. Injectable forms of dECM are particularly suitable for central nervous system applications, such as neuroregeneration repair [19].

Decellularization involves the removal of cellular and nuclear components from tissues or organs to mitigate initial immune responses, while preserving the natural structure and composition of the ECM [15, 16]. Decellularized tissues can exist as porous solids, be processed into powders, or be transformed into hydrogels. The intact dECM retains similar concentrations and ratios of glycosaminoglycans, fibronectin, and adhesion proteins, which can elicit tissue- or organ-specific regenerative responses [17]. Furthermore, dECM is generally well-tolerated by xenogeneic recipients due to the conservation of molecules across species [18]. Injectable forms of dECM are particularly advantageous for applications in the central nervous system, such as neuroregeneration repair [19].

Silk fibroin, a natural protein derived from the silk glands of insects, is a fundamental component of silkworm silk and other insect threads. Due to its exceptional biocompatibility and biodegradability, silk fibroin holds considerable promise for medical and bioengineering applications. It can be utilized to create a variety of biomaterials, including tissue engineering scaffolds, drug slow-release carriers, and wound dressings [20]. The structural stability and adjustability of silk fibroin enhance its versatility to meet diverse application needs. Notably, silk fibroin possesses high tensile strength and elastic modulus, and it also exhibits good water solubility and plasticity, allowing for manipulation through temperature, pH, and solvent conditions during processing [21]. Furthermore, silk fibroin demonstrates low immunogenicity and outstanding biocompatibility, facilitating cell interaction, adhesion, and growth. These attributes underscore the significant potential of silk fibroin in the fields of tissue engineering and regenerative medicine.

Bergenin, also known as arbutin, is a natural compound found in plants such as Ardisia japonica and those in the Magnoliaceae family. It possesses various biological activities and is commonly utilized in traditional herbal medicine for its anti-inflammatory, antioxidant, antibacterial, and antitumor properties [22]. Research indicates that Bergenin can inhibit inflammatory responses and oxidative stress, potentially alleviating pain and tissue damage associated with inflammation [23]. Furthermore, Bergenin has exhibited antibacterial and antimicrobial effects, proving effective against certain bacteria and fungi [24].

Although dECM therapy has the advantages of low immunogenicity and adaptability to the ICH microenvironment, some challenges still need to be addressed [17]. Given that ICH forms within a closed structure, in situ injection becomes the best method for ICH treatment [3]. However, direct injection of drugs does not last long enough to achieve long-term effects, and multiple injections are impractical for clinical applications. To overcome these challenges, suitable vectors are needed. Traditional hydrogel carriers have problems such as poor biocompatibility and difficulty in degradation [19], and dECM carriers cannot play a good role in sustained-release drugs. Based on this, we found that combining dECM, photocurable hydrogel and silk fibroin through 3D printing to synthesize 3D biological scaffolds with its the excellent spatial structure, biocompatibility and low cytotoxicity can become an ideal carrier. In addition, the 3D biological scaffold has good drug adsorption properties, allowing Bergenin to easily adhere to it.

In this study, we developed a 3D biological scaffold composed of a photocurable hydrogel, dECM, and silk fibroin. The scaffold was fabricated using 3D printing techniques and subsequently encapsulated with Bergenin. Employing stereotactic technology, the scaffold was transplanted into the striatum of rats suffering from ICH, with the aim of reducing neuroinflammation and promoting the recovery of neural function (Scheme 1).

The mechanism of 3D biological scaffold synthesis and encapsulation of Bergenin in the treatment of ICH

Adult male Sprague-Dawley rats aged 10–12 weeks were utilized in the study and housed in controlled conditions at 25℃ with a 12-hour light-dark cycle. The rats had ad libitum access to food and water and weighed between 250 and 300 g. Brain tissue was extracted through careful decellularization steps, including the use of low-osmotic treatment (0.1mM PMSF; 24 h), ionic detergent (0.1% SDS; 24 h), nonionic detergent (1% TritonX-100; 48 h), various solutions including nuclease solution (DNAse and RNase; 12 h) and 5% peracetic acid solution (2 h), and then lyophilized and sterilized by gamma irradiation before storage. The dECM solution was prepared by dissolving dECM powder and pepsin in concentrated hydrochloric acid, with subsequent adjustments to pH and addition of gelatin, photoinitiator, and silk fibroin to create a bioink for 3D printing. The bioink underwent three cycles of sterilization before being loaded into exosomes and solidified under blue light exposure. Utilizing a 3D printer with specific settings, a circular porous mesh scaffold was successfully printed with precision and accuracy for potential applications in tissue engineering. After printing, the structures were cross-linked with 5% calcium chloride solution for 5 min and washed three times with sterile PBS.

Histological analysis was conducted on both natural and decellularized rat brain tissue samples by fixing them in 4% polyformaldehyde and staining with hematoxylin and eosin (HE), Masson’s trichrome stain, and Sirius Red stain using commercial kits from Servicebio, China.

The freeze-dried dECM is processed into a powder using liquid nitrogen. To prepare the dECM solution, 300 mg of dECM powder and 120 mg of pepsin (from porcine source, Sigma, USA) are dissolved in 3 ml of concentrated hydrochloric acid (pH = 2.0). The mixture is stirred and shaken at 37℃ and 80×g for 12 hours to ensure complete digestion. Following the primary digestion, the pH of the dECM solution is adjusted to 7.35–7.45 by adding 10 M sodium hydroxide to inactivate the pepsin. Subsequently, a mixture consisting of 300 mg of gelatin (Sigma, USA), 15 mg of photoinitiator (Sigma, USA), and 300 mg of silk fibroin is prepared by stirring in 3 ml of triple-distilled water at 55℃ for 30 minutes. This mixture is then combined with the dECM solution to create the dECM ‘bio-ink’. The dECM ‘bio-ink’ undergoes three cycles of sterilization treatment before being loaded into a syringe equipped with 20 NMR and FT-IR Bergenin. Exposure to blue light for 15 minutes initiates gelation. Using a 3D printer (Bio-Architect®-WS; Hangzhou Regenovo Biotechnology Co., Ltd., China), the dECM ‘bio-ink’ is printed through a cooled stainless steel nozzle with a diameter of 100 micrometers. The printing platform is maintained at 4℃, and extrusion pressure settings are adjusted based on the ink flow rate. Circular porous mesh scaffolds with dimensions of 8 × 8 × 3 mm³ are printed at a scanning speed of 6 mm/s. Post-printing, the structures are crosslinked with a 5% calcium chloride solution for 5 min and subsequently washed three times with sterile PBS.

Following gold plating, the cross-section morphology of the striatum, dECM, photocurable hydrogel, silk fibroin, and 3D biological scaffold was examined using a scanning electron microscope (Hitachi, Japan).

1H NMR was used for the characterization of 3D biological scaffold. dECM, Photocurable hydrogel and 3D biological scaffold in D2O, place them in a nuclear magnetic measurement tube, and measure the spectrum of hydrogen atoms in the relevant structural formulas with an AVANCE 600 nuclear magnetic resonance spectrometer. dECM, photocurable hydrogel, and 3D biological scaffold were dried and ground into fine powders and pressed into discs. An IRTracer-100 Fourier transform infrared spectrometer was used with a scanning range of 400 to 4000 cm− 1 to measure the vibration spectrum of each atom.

An in vitro standard curve was established to investigate the release behavior of Bergenin drug in simulated blood (pH = 7.4) through the dialysis bag diffusion method. Drug concentration was assessed at a wavelength of 430 nm using ultraviolet spectrophotometry. The release rate was determined using a formula that incorporated drug concentrations at various sampling times, volumes of release medium, and drug weight. In the animal study, distinct drug formulations were administered to individual groups, and blood samples were collected at specific time points for drug concentration analysis.

Newborn rats were anesthetized with isoflurane for brain removal under sterile conditions. The extracted brains were placed in D-Hank solution, followed by dissection of the cerebellum, brainstem, and hippocampus under a dissecting microscope. Meninges and vascular tissues covering the cerebral cortex were removed, and the remaining brain tissue was cut into small 1 mm³ pieces. These tissue fragments were then immersed in a solution containing trypsin and DNase, incubated at 37 °C for 5 min with repeated pipetting until most tissue dissolved. The resulting cell suspension was filtered, cells were centrifuged at 1000 g for 2 min, resuspended in a cell culture medium, and seeded into culture flasks. After 24 h, the medium was replaced.

Hemin (20µM; Sigma-Aldrich) was utilized to induce hemorrhagic stroke in primary glial cell cultures. The control group cells were exposed to DMEM culture medium lacking FBS. Bergenin (MCE, USA, purity: 99.75%, C14H16O9) was introduced at varying concentrations to the DMEM culture medium, with or without a 3D biological scaffold and Hemin (20µM; Sigma-Aldrich). Cell viability was assessed using a Cell Counting Kit-8 (CCK8) (Sigma, USA) following the manufacturer’s guidelines.

ROS were measured using the ROS Assay Kit - Highly Sensitive DCFH-DA Kit (Do Jindo, Japan). Following removal of the culture medium, cells from each group underwent two washes with HBSS. Subsequently, highly sensitive DCFH-DA were added to the cells, which were then incubated in a CO2 incubator at 37℃ for 30 min. After discarding the working solution, the cells were washed with HBSS and examined under a fluorescence microscope (DMI3000B, Leica, Germany). Quantitative analysis of intracellular ROS was performed using flow cytometry (BD, USA), with three replicates for each group.

A total of 120 male adult Sprague-Dawley (SD) rats aged 10–12 weeks were kept in consistent environmental conditions (temperature 25 °C, 12-hour light-dark cycle) with ad libitum access to food and water. The rats weighed between 250 and 300 g. They were randomly assigned to five groups: Sham group, ICH group, ICH + Bergenin group, ICH + Bergenin + hydrogel group, and ICH + Bergenin + 3D biological scaffold group. The study was conducted in accordance with the Animal Experimental Ethics Committee of Hebei Medical University Second Hospital and adhered to the National Institutes of Health guidelines for the Care and Use of Laboratory Animals.

Following intraperitoneal injection of 2% sodium pentobarbital (40 mg/kg) to anesthetize the rats, they were positioned in a prone stance within a stereotaxic frame (NEUROSTAR, Germany). Collagenase IV (Sigma-Aldrich, St. Louis, MO) was then injected into the right striatum to induce ICH. The sham operation group was injected with normal saline. 2 h after injection of collagenase IV, Bergenin (0.5ul/kg) was injected in situ with or without 3D biological scaffold. On the 5th postoperative day, SD rats were euthanized and brain tissues were collected.

The study utilized the modified Longa score and Bederson score to quantitatively assess the neurological damage resulting from ICH on days 1, 3, and 5 post-establishment of an in vivo model. The Longa score method assigns scores from 0 to 4, with 0 indicating no neurological deficit, 1 representing incomplete extension of the left forelimb, 2 denoting left circling behavior, 3 indicating falling to the left side, and 4 signifying no spontaneous walking and decreased level of consciousness. Rats with a Longa score of 0 exhibit no neurological defects, while those with a score of 4 display severe brain injury and are therefore excluded from the study. On the other hand, the Bederson score method ranges from 0 to 5, with 0 denoting no deficit, 1 representing forelimb flexion, 2 indicating reduced resistance to forelimb flexion, 3 denoting circling into the paralyzed side during free movement, 4 signifying vertical rotation or accompanied by seizure activity, and 5 indicating no movement. A higher Bederson score correlates with more severe damage.

The Forelimb Placing Test (FPT) and Corner Turn Test (CTT) are utilized for assessing sensory-motor function in rats. In the Whisker-induced FPT, rats extend their forelimbs and grasp freely. Each forelimb is individually tested by touching the corresponding whiskers to the edge of a tabletop corner. Healthy rats typically place their forelimb onto the tabletop on the same side as the stimulated whisker. In cases of injury, rats may place the contralateral forelimb onto the tabletop where the stimulated whisker is located. The forelimbs of each rat undergo 10 tests, and the correct forelimb placement rate is determined based on the proportion of responses to whisker stimulation. In the CTT, rats are allowed to enter a corner at a 30° angle and can choose to turn left or right. The direction of the turn is recorded over 6 repetitions, and the percentage of right turns is calculated.

After establishing an ICH model in SD rats for 5 days, the rats were anesthetized with 2% pentobarbital sodium at a dose of 40 mg/kg, and brain tissues were extracted. The brain was then immediately divided into the left hemisphere, right hemisphere, and cerebellum. Subsequently, the brain samples were weighed using an electronic analytical balance (BS210S, SARTORIUS, Germany) to obtain the wet weight (WW). Following this, the brain samples were dried at 100℃ for 24 h to obtain the dry weight (DW). The brain water content was calculated using the formula (WW - DW) / WW × 100%.

SD rats were sacrificed for hematoma evaluation 5 days after establishing the ICH model. One rat was randomly selected from each group, and its brain was promptly removed after perfusion with 4% PFA (Solarbio, Beijing, China). The brain was then sliced into 1 mm thick sections, which were imaged using an Olympus stereo microscope (SZX7) to record the volume of hematoma in each slice.

Brain rats were perfused with 4% formaldehyde via cardiac injection, followed by rapid collection of brain tissue. The tissue was then fixed in 4% PFA at 4℃ for 6 h before being embedded in paraffin. Subsequently, the embedded brain tissue was sectioned into 5micron slices and baked overnight at 60℃.

TEM from Japan was utilized to examine the morphological characteristics of mitochondria in rat brains 5 days post ICH.

Denature-modifying neurons were identified using the FJC kit (TR-100-FJ, Biosensis, Australia). Initially, paraffin-embedded sections were dewaxed with xylene and subsequently hydrated using a gradient of ethanol. The sections were then rinsed with distilled water and incubated in potassium permanganate for 15 min. Following this, the sections were immersed in Fluoro-Jade C and incubated with a DAPI solution. Finally, DPX was applied for mounting. An inverted fluorescent microscope (DMI3000B, Leica, Germany) was utilized by independent observers to conduct three separate counts of FJC-positive neurons in each brain section. Quantitative analysis was performed using ImageJ software (ImageJ 1.4, NIH, USA), and data were expressed as cells per mm² based on the average number of FJC-positive neurons observed within the field.

Approximately 1 × 106 glial cells from each group and 10 mg of brain tissue surrounding the hematoma perfused with pre-cooled PBS are utilized for the subsequent tests. Iron, MDA, and GSH levels in the brain tissue or glial cells surrounding the hematoma in rats are assessed using the Iron Assay Kit (AB83366, ABCAM, USA), Lipid Peroxidation Assay Kit (AB118970, ABCAM, USA), and GSH Assay Kit (Nanjing Jiancheng, China).

CCK-8 (Cell Counting Kit-8) experiment was conducted following the protocol outlined in the CCK kit (ZOMANBIO, ZP328, Beijing, China) to assess cell proliferation or inhibition rate. Cells were cultivated in a 96-well plate and subjected to individual treatments for each group. A blank group served as the control. Post-treatment, the culture medium was substituted with a 10% CCK-8 solution and incubated for 2 h. Subsequently, absorbance at 450 nm was recorded, and the cellular survival rates for each group were determined.

We used an RNA extraction kit to extract total RNA from the samples and measure the concentration of RNA by measuring the absorbance of the RNA solution at 260 nm with a UV spectrophotometer. Then we used the PCR kit to reverse transcribe RNA into cDNA. RT-qPCR was performed according to the instructions provided with SYBR Premix Ex Tap™ II Kit (Takara). GAPDH was used as the internal control for the experimental samples, and the relative gene expression of the samples was calculated using the 2 − ΔΔCt method.

Cell supernatants or animal serum were collected and centrifuged to eliminate any precipitate, and ELISA assay kits (Abclone, RK00020, China) were used to detect the expression levels.

To quantitatively assess neuronal apoptosis, double staining with GFAP (green) and TUNEL (red) was conducted 24 h post ICH, following the manufacturer’s guidelines of the In Situ Apoptosis Detection Kit (Roche, Indianapolis, USA). The count of TUNEL-positive neurons in six sections surrounding the hematoma region of each brain tissue was manually performed at a magnification of ×200 and analyzed using Image J software (Image J 1.4, NIH). The results are expressed as the percentage (%) of TUNEL-positive neurons.

Annexin V/PI double staining is conducted for flow cytometric apoptosis analysis. Cells are incubated with 0.5 µg/mL Annexin V-FITC and 50 µg/mL PI in the dark for 5 min, followed by analysis using FACS scan flow cytometry.

Nuclear and cytoplasmic fractions were isolated from cells using MinuteTM cytoplasmic and nuclear extraction kits (SC-003, Invent, USA). Brain tissues and glial cells were lysed with RIPA lysis buffer (Beyotime, China) and the resulting samples were collected. Protein concentrations were determined using a BCA kit (23225, Thermo Fisher Scientific, USA), followed by separation of equal protein samples through SDS-PAGE and transfer to PVDF membranes (Sigma-Aldrich, USA). Subsequently, membranes were blocked with 5% skim milk for 2 h and then incubated overnight with primary antibodies at 4℃. Visualization analyses were conducted using the Odyssey Imaging System (Li-COR, USA), and band densities on blots were quantified using Image J Software (1.4, NIH, USA).

Data were analyzed and plotted using Graph Pad Prism 7. Summary data are presented as mean ± SEM. Neurological scores were analyzed using the Kruskal-Wallis test and Dunn’s multiple comparison test. Other values were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. P-values less than 0.05 were considered statistically significant.

In this study, SD rat brain tissue was selected for decellularization due to its ability to undergo physical and chemical processes that effectively eliminate cellular components while preserving the tissue’s structure and proteins (Fig. 1A). This preservation supports the restoration of specific tissue functions [26]. Analysis of the DNA content indicated that over 99% of the DNA from the dECM had been successfully eliminated (Fig. 1B). Additionally, the realtive collagen content in the dECM surpassed that found in fresh CNS tissue (Fig. 1C). Histological examinations were carried out on both the original and decellularized tissues to evaluate the efficacy of the process. HE staining confirmed the presence of cellular components in the original tissue and their absence in the decellularized tissue. Masson’s and Sirius Red staining were used to validate the retention of collagen proteins in the decellularized tissue (Fig. 1D). These findings indicate that the decellularized matrix maintains the original tissue’s structure and components, making it a suitable ‘bioink’ for 3D bioprinting applications that can guide cell positioning, growth, and migration.

Preparation and characterization of 3D biological scaffold. (A) Image of brain decellularization and hydrogel preparation. (B) Quantitative determination of DNA content (n = 3). (C) Quantitative determination of collagen retention (n = 3). (D) HE and Masson staining demonstrate decellularization, and Sirius red staining demonstrates retention of collagen fibers (n = 3). (E) Scanning electron microscopy (SEM) was used to observe the microstructure of the 3D biological scaffold and its components. (F) FTIR evaluation of the microstructure of 3D biological scaffolds and their components. (G) NMR evaluation of the microstructure of the 3D biological scaffold and its components. Scale bars: D, 200 μm; E, 100 μm. Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Bioprinting technology is used to fabricate 3D biological scaffold by magnetically stirring dECM, photocurable hydrogel, and silk fibroin solutions to create bioinks. Scanning electron microscopy (SEM) was used to observe the microstructure of dECM, photocurable hydrogel, silk fibroin, and 3D biological scaffold after freeze-drying, revealing an open and interconnected porous morphology (Fig. 1E). Subsequent analysis of the assembly of the scaffold was conducted using NMR and FT-IR techniques. The chemical structures of the hydrogel, Silk Fibroin, Brain Acellular Matrix, and 3D biological scaffold were characterized through 1 H NMR spectroscopy, as illustrated in Fig. 1G. The 1 H NMR spectrum of the hydrogel displayed signals at 5.78 ppm (d, 2 H, J = 8.0 Hz) and 5.89 ppm (d, 2 H, J = 8.0 Hz), corresponding to the aromatic protons of the sulfonamide ring. Signals from the hydrogen atoms in the chitosan ring were observed around 3.1–3.84 ppm. The characteristic peaks of the hydrogen atoms in the 3D biological scaffold matched those of the Brain Acellular Matrix, appearing between 2.07 and 2.17 ppm. Peaks at 7.08 and 7.57 ppm confirmed the presence of the 3D biological scaffold, validating the successful synthesis of the scaffold.

The FT-IR spectra of the 3D biological scaffold containing Bergenin are shown in Fig. 1F-F. In the FT-IR spectrum, a distinctive absorption peak of C = O stretching vibration was observed at 1625 cm− 1. The peak at 709 cm− 1 indicated the out-of-plane bending vibration of aromatic hydrogens in the scaffold. Peaks around 3000 cm− 1 were attributed to the stretching vibration of aromatic hydrogens, while peaks between 1455 and 1600 cm− 1 suggested the presence of the benzene ring skeleton, and the peak at 1248 cm− 1 represented the C-C stretching vibration of the aromatic ketone. The FT-IR spectrum of the hydrogel (Fig. 1F-C) displayed a characteristic peak at 1752 cm− 1, corresponding to C = O stretching, and another peak at 932 cm− 1, indicating the presence of C-O bonds in the hydrogel. No significant characteristic peaks of the hydrogel or the 3D biological scaffold were detected in the microparticles, but peaks around 1600 cm− 1 were present in the physical mixture, signifying the presence of characteristic peaks of the 3D biological scaffold. These findings confirmed the encapsulation of Bergenin within the 3D biological scaffold. Furthermore, the characteristic peaks of Bergenin in the 3D biological scaffold were weakened, possibly due to the adsorption of a small amount of Bergenin on the surface of the scaffold.

Bergenin and 3D biological scaffold were combined in a volumetric flask and dissolved in methanol using ultrasound, followed by dilution with methanol to the mark. A full-wavelength scan ranging from 200 to 600 nm was conducted using UV spectrophotometry. Bergenin displayed maximum absorption at 229 nm, establishing 229 nm as its detection wavelength (Fig. 2A). Drug-loaded microspheres containing Bergenin were prepared at concentrations of 0.5, 1, 2, 5, 8, and 10 mg/ml. 3 mL of acetic acid and stirred for 3 h to rupture the microspheres and dissolve the drug. Following centrifugation at 3500 rpm for 15 min to remove the microspheres, the absorbance of the supernatant was measured at 229 nm using a UV spectrophotometer. The study revealed an increase in drug encapsulation efficiency with rising drug concentration. The relationship between microsphere particle size and PDI at varying drug concentrations was illustrated in Fig. 2B. Notably, for drug loading content of 5, 8, and 10 mg/mL in hydrogel and 3D biological scaffold, optimal encapsulation efficiency was observed at 8 mg/mL (Fig. 2C).

Evaluation of 3D biological scaffold-encapsulated Bergenin. (A) Bergenin shows an absorption maximum at 229 nm. (B) Examination of the relationship between microsphere particle size and polydispersity index (PDI) at different drug concentrations (n = 3). (C, D) In ​​vitro release assay (n = 3). (E) Perform pharmacokinetic testing (n = 3). Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

The release of Bergenin at 8 mg/mL follows a first-order release model, occurring in two stages with an initial burst release within the first 12 h (Fig. 2D). Specifically, 79.3%, 63.4%, and 55.4% of Bergenin was released within the first 12 h, highlighting the sustained-release effect of Bergenin when encapsulated in the 3D biological scaffold. The release of 20µM Bergenin from hydrogel and the 3D biological scaffold, both encapsulated with 20µM Bergenin solution, was monitored at various time points (0.1, 0.2, 0.4, 0.6, 1, 2, 4, 8, 12, 16, 24, 30, and 48 h) using liquid measurement (Fig. 2E). While Bergenin in solution was rapidly released, with only 10% remaining after 2 h, Bergenin encapsulated in the 3D biological scaffold exhibited a slower release rate, with 90% released within 24 h. The release profile of Bergenin encapsulated in the 3D biological scaffold is depicted in Fig. 2E, affirming the successful construction of a 3D biological scaffold for the sustained release of Bergenin.

An in vitro cultured rat primary astrocyte model of cerebral hemorrhage was established for this study. Immunofluorescence staining of the primary cultured astrocytes revealed positive expression of GFAP, confirming their glial characteristics (Fig. 3A). Cell viability was assessed using CCK-8 assays on primary astrocytes cultured for 8 h with varying concentrations of Bergenin encapsulated in a 3D biological scaffold, as well as different concentrations of Hemin. Treatment with Bergenin encapsulated in the 3D biological scaffold did not exhibit significant toxicity compared to the control group (Fig. 3B), while Hemin resulted in a dose-dependent decrease in cell viability (Fig. 3C). Among the various concentrations of Bergenin encapsulated in the 3D biological scaffold, 50 μm Bergenin demonstrated the most notable protective effect on cell viability (Fig. 3D and E). Furthermore, exposure of cells to 20 µM Hemin for 8 h led to a significant increase in the expression of IL-6, iNOS, and COX-2 (Fig. 3F). For further study, cell viability was measured with Bergenin and Bergenin encapsulated in 3D biological scaffold co-cultured with 20 µM Hemin for 8 h. This evaluation aimed to determine whether Bergenin encapsulated in a 3D biological scaffold inhibits Hemin-induced cytotoxicity better than Bergenin alone.

Bergenin encapsulated in 3D biological scaffold improves the in vitro survival rate of primary glial cells, reduces the expression levels of pro-inflammatory cytokines and mediators, and inhibits NF-κB activity. (A) Identification of primary glial cells. (B-E) CCK8 was used to evaluate cell viability and determine the optimal concentration of Hemin to establish an in vitro cerebral hemorrhage model (n = 3). (F) Hemin was found to induce the expression of pro-inflammatory cytokines in a dose-dependent manner (n = 3). (G-M) Measurement of IL-6, iNOS and COX-2 mRNA or protein expression levels (n = 3). (N-Q) Measurement of protein expression levels of p-IKB and p-NF-KB p65 (n = 3). Scale bars: A, 200 μm. Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

IL-6, iNOS, and COX-2 are key regulators of immune responses and inflammation control, influencing the balance between pro-inflammatory and anti-inflammatory signals in the immune system. This study investigates the impact of Bergenin encapsulated in a 3D biological scaffold on the expression of inflammatory cytokines in primary astrocytes stimulated by Hemin. Analysis of IL-6, iNOS, and COX-2 mRNA or protein levels in primary astrocytes treated with 20µM Hemin for 8 h in the presence of 50µM Bergenin encapsulated in the 3D biological scaffold was conducted using RT-PCR or Western blot analysis (Fig. 3G and M). Cells exposed to Hemin exhibited a significant upregulation in IL-6, iNOS, and COX-2 mRNA expression, which was notably attenuated by Bergenin encapsulated in the 3D biological scaffold. Additionally, Bergenin encapsulated in the 3D biological scaffold demonstrated superior inhibition of inflammatory cytokine production in primary astrocytes treated with Hemin when compared to Bergenin alone and hydrogel.

NF-κB p65 is a crucial transcription factor responsible for pro-inflammatory responses and gene expression regulation in the presence of inflammatory signals [27]. In this study, we examined the impact of Bergenin encapsulated in a 3D biological scaffold on the activation of NF-κB p65 and the phosphorylation of IκB and NF-κB p65. Western blot analysis was conducted on primary astrocytes treated with 20µM Hemin for 8 h along with 50µM Bergenin encapsulated in the 3D biological scaffold. Treatment with Bergenin significantly inhibited the increased activity of NF-κB p65 induced by Hemin (Fig. 3N and P). In addition, primary astrocytes were treated with 50µM Bergenin encapsulated in 3D biological scaffold or 30µM JSH-23 (an NF-κB nuclear translocation inhibitor) and 20µM Hemin. Both Bergenin encapsulated in 3D biological scaffold and JSH-23 effectively inhibited Hemin-induced enhancement of NF-κB transcriptional activity (Fig. 3Q). These findings indicate that the anti-inflammatory properties of Bergenin are related to the regulation of the NF-κB pathway in primary astrocytes exposed to Hemin, and that Bergenin encapsulated in 3D biological scaffold exhibits superior anti-inflammatory effects.

HIF-1α, a crucial factor in cellular response to low oxygen levels [28], Analysis through PCR or Western blot revealed a significant increase in mRNA and protein levels of HIF-1α with Hemin treatment, which was notably reduced with Bergenin encapsulation in the 3D biological scaffold (Fig. 4A and C). Inflammation-induced ROS accumulation and oxidative stress were assessed using H2DCFDA [29]. The 3D biological scaffold with Bergenin inhibited Hemin-induced ROS production in astrocytes (Fig. 4D and E), akin to the ROS scavenger N-acetyl-cysteine (NAC). Co-treatment with NAC or the HIF-1α inhibitor YC-1 effectively mitigated ROS elevation by Hemin (Fig. 4F and G). Notably, NAC did not inhibit HIF-1α protein levels, highlighting the role of HIF-1α in ROS regulation in astrocytes. YC-1 also reversed the increased IL-6 production induced by Hemin (Fig. 4H), similar to NAC’s effect. These findings suggest that Bergenin’s anti-inflammatory properties involve suppressing ROS and HIF-1α expression under Hemin conditions, with enhanced efficacy when encapsulated in the 3D biological scaffold.

Bergenin encapsulated in 3D biological scaffold can reduce HIF-1α expression and ROS production in Hemin-stimulated primary glial cells and activate the Nrf-2 signaling pathway. (A-C) Measurement of HIF-1α mRNA or protein expression levels (n = 3). (D, E) ROS production measured in primary glial cells (n = 3). (F-H) Measurement of HIF-1α and IL-6 expression levels (n = 3). (I-K) Measurement of GSH, MDA and Fe [2+] expression levels (n = 3). (L-O) Measurement of HO-1 and Nrf-2 protein expression levels (n = 3). Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Primary astrocytes treated with Hemin demonstrated notable cellular toxicity, characterized by elevated levels of intracellular malondialdehyde (MDA) and Fe2+, along with decreased glutathione (GSH) levels. Application of Bergenin at a concentration of 50µM effectively reversed the alterations in intracellular MDA, Fe2+, and GSH caused by Hemin. Moreover, Bergenin delivered via a 3D biological scaffold exhibited improved efficacy in attenuating these effects (Fig. 4I, J and K).

The antioxidant-related protein HO-1 can counteract the oxidative effects of ROS generation induced by Lipopolysaccharide (LPS) stimulation through the activation of Nrf-2-mediated transcription [30]. To investigate the impact of Bergenin on the Nrf-2/HO-1 pathway under Hemin conditions, primary astrocytes were co-exposed to 50µM Bergenin encapsulated in the 3D biological scaffold or 5 mM NAC with 20µM Hemin for 8 h. Western blot analysis showed that Bergenin increased HO-1 expression when combined with Hemin, and NAC also increased HO-1 expression in the presence of Hemin. Moreover, Hemin led to an increase in cytoplasmic Nrf-2 levels compared to the control group, but Bergenin reversed this effect and also increased nuclear Nrf-2 levels. In contrast, NAC did not impact the nuclear translocation of Nrf-2 (Fig. 4L and O). These findings suggest that Bergenin’s anti-inflammatory properties in Hemin-activated primary astrocytes may be linked to enhanced HO-1 expression through the Nrf-2 pathway, with Bergenin in the 3D biological scaffold showing improved efficacy.

FACS analysis was conducted to evaluate the protective effects of Bergenin encapsulated in a 3D biological scaffold against Hemin-induced cell death in primary astrocytes. Cells were exposed to 20µM Hemin along with 50µM Bergenin encapsulated in the 3D biological scaffold or 5 mM NAC. Treatment with either Bergenin or NAC resulted in a decrease in Hemin-induced apoptosis (Fig. 5A and B). JC-1 staining (Fig. 5C and D) and Western blot analysis (Fig. 5E and H) were used to assess the protein levels of BAX, BCL-2, and cleaved caspase-3. Following Hemin exposure, elevated levels of BAX and cleaved caspase-3 were observed, while BCL-2 expression decreased in primary astrocytes. However, treatment with Bergenin in the 3D biological scaffold or NAC led to a reduction in BAX and cleaved caspase-3 levels, and an increase in BCL-2 expression. Notably, Bergenin encapsulated in the 3D biological scaffold demonstrated superior efficacy in mitigating Hemin-induced apoptosis in primary astrocytes.

3D biological scaffold encapsulation of Bergenin can eliminate Hemin-induced apoptosis in primary glial cells. (A-D) Apoptosis flow cytometry and JC-1 show that 3D biological scaffold encapsulation of Bergenin or NAC treatment can reduce the apoptosis rate induced by Hemin treatment (n = 3). (E-H) Determination and statistical analysis of BAX, BCL-2 and CASP3 protein expression levels (n = 3). Scale bars: C, 50 μm. Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Brain imaging scans were conducted to assess the effectiveness of Bergenin encapsulated in a 3D biological scaffold in reducing hematoma volume. The ICH group displayed a substantial hematoma in brain tissue sections (Fig. 6A). Following ICH injury, the treatment groups exhibited a decrease in hematoma volume. Histological analysis using HE and Masson’s staining revealed marked edema and necrosis in the brain tissue surrounding the hematoma in the ICH group, characterized by irregular cellular arrangement, increased extracellular space, and infiltration of inflammatory cells. Nevertheless, compared to the ICH group, the treatment groups showed significantly reduced pathological damage (Fig. 6B and C). TEM studies illustrated that the treatment groups effectively protected against brain tissue damage induced by ICH, preventing mitochondrial ridge loss and outer membrane rupture (Fig. 6D). On the fifth day post-ICH, changes in Nissl bodies were evaluated and the water content of various brain anatomical structures was measured to assess the treatment’s impact on brain edema. The results indicated an increase in the number of Nissl bodies in the treatment groups compared to the ICH group (Fig. 6E) and a notable decrease in brain water content (Fig. 6F), with the treatment utilizing Bergenin encapsulated in the 3D biological scaffold demonstrating the most significant effect.

Bergenin encapsulated in 3D biological scaffold can reduce collagenase-induced brain damage in ICH rats. (A-C) Representative images of hemorrhagic lesions, HE staining, and Masson staining results in rats from different groups. (D) TEM was used to examine the mitochondrial morphology of rats in different groups. (E) Nissl staining results of rats in different groups (n = 4). (F) Brain water content was measured to assess the effect of treatment on cerebral edema (n = 4). Scale bars: B and C, 200 μm /50µm; D, 4 nm /1nm. Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Neurobehavioral experiments were conducted on days 1, 3, and 5 post-ICH to evaluate the impact of Bergenin on neurofunctional deficits resulting from ICH. Longa and Bederson scores assessed neurological damage, while foot-fault and corridor task tests measured sensorimotor function. The ICH group initially showed decreased neurological scores post-surgery compared to the SHAM group, but improved significantly after Bergenin treatment (Fig. 7A and B). Bergenin also improved forelimb placement in ICH rats (Fig. 7C) and performance in the corridor task test (Fig. 7D), especially when used in a 3D biological scaffold.

Bergenin encapsulated in 3D biological scaffold can reduce collagenase-induced brain damage in ICH rats. (A) Corner point testing was performed on days 1 to 5 after ICH (n = 12). (B) Forelimb placement test (n = 12). (C) Longa score and (D) Pedersen score were used to evaluate neurological recovery (n = 12). (E, F) Representative images of FJC staining results of rat brain sections from different groups (n = 4). (G-I) Fe2+, GSH, and MDA levels in peribleeding tissue were assessed on day 5 after ICH (n = 4). Scale bars: E, 200 μm. Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FJC staining was utilized to detect neurodegeneration in neuronal cells, including dendrites, axons, and terminals. The staining results indicated a notable presence of neurodegeneration surrounding the hematoma in rat brain tissue post-ICH injury. Treatment with Bergenin notably reduced the number of Fluoro-Jade C-positive cells, with the most significant effect observed when Bergenin was utilized within the 3D biological scaffold (Fig. 7E and F). To assess the impact of Bergenin on oxidative stress in the rat ICH model, the accumulation of malondialdehyde (MDA), glutathione (GSH), and Fe2 + was evaluated. Rats with cerebral hemorrhage displayed elevated MDA levels, increased Fe2 + concentrations, and decreased GSH levels. As depicted in Fig. 7G and I, treatment with Bergenin markedly improved the levels of MDA, GSH, and Fe2 + in comparison to the ICH group, with the most significant effect observed when Bergenin was encapsulated in the 3D biological scaffold.

TUNEL staining (Fig. 8A and B) and Western blot analysis were employed to evaluate the expression of key proteins, such as BAX, BCL-2, and CASP3. The results indicate that the Bergenin treatment group displayed notably elevated levels of BCL-2 and reduced levels of BAX and CASP3 compared to the ICH group. Particularly, the treatment utilizing Bergenin encapsulated in the 3D biological scaffold exhibited the most pronounced effect (Fig. 8C and F).

Bergenin encapsulated in 3D biological scaffold reduces collagenase-induced brain damage in ICH rats. (A, B) Representative images of TUNEL staining results of rat brain sections from different groups (n = 4). (C-F) Determination and statistical analysis of BAX, BCL-2 and CASP3 protein expression levels (n = 4). Scale bars: A, 200 μm. Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

In this study, a novel photosensitive injectable 3D biological scaffold was developed for the treatment of neuroinflammation in ICH. This innovative 3D biological scaffold combines the therapeutic advantages of dECM and hydrogel to achieve higher efficacy than traditional biological scaffolds. Through the synergistic effect of dECM and hydrogel, this 3D biological scaffold is particularly suitable for the ICH microenvironment [32,33,34,35,36]. In ICH models, Bergenin has been shown to effectively reduce inflammation and oxidative stress by regulating NF-κB and Nrf-2/HO-1 pathways, while the 3D biological scaffold slows down its breakdown, allowing Bergenin to be retained for 3 days. The storage of Bergenin in 3D biological scaffold also enables it to inactivate the inflammasome and inhibit the release of inflammatory factors, ultimately suppressing inflammation and promoting the survival of nerve cells. The collaboration of 3D biological scaffold and Bergenin in the ICH microenvironment enhances the resilience of neural cells to changes in the early stages of ICH, reducing cell apoptosis and even supporting their proliferation. This innovative approach provides promising results in modulating neuroinflammation and improving ICH treatment outcomes.

The 3D biological scaffold shows excellent compatibility with Bergenin and does not cause immune reactions. A key requirement for successful decellularization is a dry weight DNA content below 50 ng/mg, DNA fragments shorter than 200 bp, or the absence of visible nuclear material in DAPI or HE staining [37]. Test results show that nearly all cellular components in dECM have been eliminated, reducing the risk of dECM inducing an immune response. Recent studies have demonstrated the regenerative properties of rat brain decellularization [38, 39]. This approach can address matrix degradation caused by disorders of matrix metabolism and counteract the deleterious effects of metalloproteinases. Furthermore, dECM strengthens the extracellular matrix while retaining its original structure, enhancing structural integrity, promoting the growth of glial cells, and mitigating the effects of inflammatory factors [39]. Most importantly, the 3D biological scaffold can effectively slow down Bergenin release while exhibiting high Bergenin loading capacity.

Bergenin exerts neuroprotective and immunoprotective effects by regulating neuroinflammation [40]. In a mouse model of Parkinsons disease, Bergenin treatment effectively reduced the expression of serum inflammatory factors such as TNF-α and IL-6 [40]. In addition, Bergenin reduced the expression levels of TNF-α and IL-1β while increasing the expression of IL-6 in lipopolysaccharide-stimulated human dental pulp stem cells. In addition, Bergenin can also inhibit the release of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 from macrophages, which are involved in hypothalamic inflammation induced by high-fat stimulation [41]. A study using heme to induce ICH-like in primary glial cells and SD rats showed a dose-dependent increase in the expression of inflammatory cytokines IL-6, NOS and COX-2 [42]. We found that treatment with Bergenin-containing 3D biological scaffold significantly reduced the expression of IL-6, iNOS and COX-2 induced by Hemin exposure. These findings indicate that the 3D biological scaffold encapsulating Bergenin has anti-inflammatory effects on primary glial cells under Hemin conditions.

Glial cells are the main defense and immune response cells in the CNS [43]. When faced with an inflammatory response, damaged glial cells produce inflammatory mediators and cytokines by regulating the NF-κB pathway [44, 45]. NF-κB p65 is a nuclear transcription factor that plays a key role in regulating gene transcription and triggering inflammatory responses [46]. Normally, NF-κB forms a complex with IκB in the cytoplasm. However, conditions such as ICH lead to IκB degradation and translocation of NF-κB p65 from the cytoplasm to the nucleus. Several studies have highlighted the connection between the NF-κB pathway and inflammation-related neurological diseases. Inhibiting NF-κB transcription in glial cells has been shown to reduce the expression of inflammatory mediators such as iNOS and COX-2, as well as pro-inflammatory cytokines such as IL-6 [39, 40]. Previous studies have highlighted the important role of NF-κB and HIF in the activation of genes related to angiogenesis and inflammation, indicating a close biochemical and functional link between inflammation and bleeding [47,48,49,50]. This study aimed to explore the protective mechanism of Bergenin-containing 3D biological scaffold against Hemin-induced inflammatory response in primary glial cells. Our results showed that the 3D biological scaffold encapsulating Bergenin effectively inhibited the phosphorylation of NF-κB p65 in Hemin-stimulated primary glial cells, resulting in reduced expression levels of iNOS, COX-2, and IL-6.

In ICH models, accumulation of HIF-1α can activate genes related to oxidative stress and regulate cellular oxygen sensors. Hemin is a commonly used hypoxia mimetic that can increase HIF-1α levels [51, 52]. This study shows that exposure to Hemin enhances HIF-1α expression, while treatment with a 3D biological scaffold containing Bergenin attenuates this expression. Hemin also induces the generation of reactive oxygen species (ROS) and oxidative stress, which are associated with various clinical conditions [53]. Our results showed that Hemin-induced ROS production was significantly reduced in primary glial cells treated with Bergenin-containing 3D biological scaffold. In addition, both the HIF-1α inhibitor YC-1 and the ROS scavenger NAC can effectively reduce ROS levels, and NAC does not affect the expression of HIF-1α in primary glial cells after Hemin treatment. These findings indicate that the 3D biological scaffold encapsulating Bergenin has antioxidant properties in Hemin-induced primary glial cells by inhibiting the HIF-1α pathway and reducing ROS generation [54, 55]. In addition, oxidative stress can amplify the inflammatory response of primary glial cells, leading to neuronal damage. In the ICH model, pro-inflammatory factors such as HIF-1α and ROS play a crucial role in the inflammatory process caused by ICH [56,57,58]. To study the effects of HIF-1α and ROS on the production of pro-inflammatory factors in the ICH model, primary glial cells were exposed to Hemin after pretreatment with NAC and YC-1. NAC and YC-1 can attenuate Hemin-induced IL-6 upregulation, which is similar to that observed with Bergenin. Our results show that Bergenin can inhibit the inflammatory response of primary glial cells triggered by Hemin by regulating HIF-1α expression and inhibiting ROS production, which is consistent with previous studies [59,60,61].

Under normal conditions, Nrf-2 is normally found in the cytoplasm and is blocked from entering the nucleus by Keap1. However, during oxidative stress, Nrf-2 dissociates from Keap1 and moves to the nucleus, where it controls the production of antioxidant proteins such as HO-1 [61,62,63]. Studies link chronic inflammation to HO-1 in ICH models. Previous studies have shown that Bergenin can prevent inflammation and oxidative damage by affecting the Nrf-2/HO-1 signaling pathway [64]. Our study showed that Hemin treatment reduced nuclear Nrf-2 levels, but using 3D biological scaffold containing Bergenin increased the movement of Nrf-2 into the nucleus and enhanced the expression of HO-1 after Hemin treatment [65]. These results suggest that activation of the Nrf-2/HO-1 pathway plays a role in the anti-inflammatory effects of Bergenin-containing 3D biological scaffolds on primary glial cells during Hemin-induced injury. This reflects the previously observed neuroprotective effects of activation of the Nrf-2/HO-1 pathway in microglia [66, 67].

dECM and 3D biological scaffold treatment represent two innovative directions in the field of ICH treatment. Their combination enhances the ability to stimulate neural cell proliferation and growth and regulate extracellular matrix metabolism. Our experimental results show that this combination can effectively suppress ICH, while dECM or Bergenin alone have limited effects. Compared to cell therapies, both dECM and 3D biological scaffold exhibit minimal immunogenicity, which is a key advantage of 3D biological scaffolds in clinical settings. In terms of material selection, we chose biomaterials with low possibility of body rejection, which reduces the risk of cell rejection and ensures biocompatibility. Furthermore, we implemented an efficient method to enrich the dECM, thereby reducing the required time to only 1 day compared with previous studies. Our ultimate goal is to advance the clinical application of 3D biological scaffold.

In our study, a Bergenin-rich injectable 3D biological scaffold was developed to effectively reduce inflammation and oxidative stress by regulating NF-κB and Nrf-2/HO-1 pathways in ICH models. These findings highlight the biosafety of Bergenin-containing 3D biological scaffold and its regulatory effect on neuroinflammation, promoting the recovery of neurological function. Therefore, Bergenin-rich injectable 3D biological scaffold provides a new strategy for the development of biotherapeutics for ICH.

The data used to support the findings of this study are included in the article.

Intracerebral hemorrhage

Three-dimensional (3D) biological scaffold

Decellularized Brain matrix

Hypoxia-inducible factor 1-alpha

Reactive oxygen species

Pro-inflammatory

Anti-inflammatory

Extracellular matrix

Central nervous system

Hematoxylin and eosin

Scanning Electron Microscopy

1H Nuclear Magnetic Resonance Hydrogen Spectrum

Fourier-transform infrared

Cell Counting Kit-8

The Forelimb Placing Test

Corner Turn Test

Transmission Electron Microscopy

Fluoro-Jade C

Enzyme-linked immunosorbent assay

N-acetyl-cysteine

Malondialdehyde

Glutathione

Lipopolysaccharide

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Central Guiding Local Science and Technology Development Fund Projects (236Z7752G); the Medical Research Project of Hebei Provincial Health Commission (20230031); Special Project for the Construction of Hebei Province International Science and Technology Cooperation Base (193977143D).

Department of Neurosurgery, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, 050000, China

Aobo Zhang, Lulu Cong, Chengrui Nan, Zongmao Zhao & Liqiang Liu

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Aobo Zhang and Liqiang Liu designed the study and wrote the manuscript. Aobo Zhang, Lulu Cong, Chengrui Nan, Zongmao Zhao and Liqiang Liu performed the behavioral testing and experiments and analyzed the data. Aobo Zhang and Liqiang Liu contributed to revising the manuscript. All authors read and approved the final manuscript.

Correspondence to Liqiang Liu.

This study was approved by the Medical Ethics Committee of the Second Hospital of Hebei Medical University (Approval Number: 2024-R190).

All authors have read the manuscript and provided their consent for the submission.

The authors declare no competing interests.

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Zhang, A., Cong, L., Nan, C. et al. 3D biological scaffold delivers Bergenin to reduce neuroinflammation in rats with cerebral hemorrhage. J Transl Med 22, 946 (2024). https://doi.org/10.1186/s12967-024-05735-1

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Received: 01 July 2024

Accepted: 03 October 2024

Published: 17 October 2024

DOI: https://doi.org/10.1186/s12967-024-05735-1

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