Excessive-molecular-weight HA (with a molecular weight of two × 10⁵ g mol⁻¹) and seleninic acid have been procured from Huaxia Chemical Reagent Co., Ltd. (Chengdu, China). Sodium ascorbate was obtained from Aladdin Bio-Chem Expertise Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum have been bought from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; D6883), and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) have been bought from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen peroxide answer (30 wt% in water) was obtained from Sinopharm Chemical Reagent Co., Ltd. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was bought from Shanghai Macklin Biochemical Co., Ltd. Anti-GFAP, anti-NF200, and anti-NeuN major antibodies have been obtained from Abcam (Cambridge, UK). Anti-CD44 and anti-caspase-3 antibodies have been bought from Cell Signaling Expertise (Danvers, MA, USA). The dialysis membrane was obtained from Greenbird Expertise Co., Ltd (Shanghai, China). The remaining compounds have been obtained from industrial suppliers and used with none additional manipulation.

Preparation and characterization of HA-Se NPs

Briefly, 45 mL of aqueous HA answer (1.5 mg/mL) was blended with 600 µL of seleninic acid (0.1 M) in a round-bottom flask. Subsequently, 3 mL of ascorbic acid aqueous answer (0.1 M) was step by step launched into the combination with fixed stirring at ambient temperature (~ 22 °C). The gradual addition of sodium ascorbate answer resulted in a noticeable alteration of the answer’s shade from a transparent hue to a pink tint. Upon completion of the response, the combination was subjected to dialysis for 48 h utilizing ultrapure water and a dialysis bag with a molecular weight cutoff (MWCO) of 300 kDa. Thereafter, the answer was centrifuged at 4,000 rpm for 20 min. Lastly, the HA-Se NPs have been obtained through lyophilization as pink powder (yield: 82.5%).

The scale and zeta potential of HA-Se nanoparticles have been evaluated through dynamic gentle scattering on a Malvern Zetasizer Nano ZS (ZEN3600, Malvern Devices, Worcestershire, UK). The morphology of HA-Se NPs was noticed by means of transmission electron microscopy (TEM) (JOEL-1011, Tokyo, Japan) at an accelerating voltage of 200 kV and emission-scanning electron microscopy (SEM) (Zeiss, Oberkochen, Germany). Utilizing the KBr pellet technique, Fourier rework infrared spectra (FT IR) have been recorded with a Win-IR gadget (Bio-Rad Laboratories, Hercules, CA, USA). The quantity of selenium in HA-Se NPs was decided utilizing an inductively coupled plasma mass spectrometer (ICP-MS, Xseries II, Thermo Scientific, USA). The antioxidant capability of HA-Se NPs was explored by analyzing their affect on DPPH free radicals. Within the management group, a mix of water (2 mL) and DPPH anhydrous ethanol answer (0.4 mM, 2 mL) was utilized. The experimental group, then again, included HA-Se NPs (250/1, 000 µg/mL, 2 mL) and DPPH anhydrous ethanol answer (0.4 mM, 2 mL), whereas the clean group included HA-Se NPs (250/1,000 µg/mL, 2 mL) and absolute ethanol (2 mL). The background was modified by means of the utilization of a mix of two mL of water and a pair of mL of anhydrous ethanol. The aforementioned teams have been uncovered to darkness for a interval of 30 min, after which the absorbance at 517 nm was recorded on a Bio-Rad 680 microplate reader (TECAN Buying and selling AG, Switzerland). The free radical-scavenging charge (%) was decided as per the next system: (1−(A_experimental − A_clean)/A_management) ×100, the place A_experimental, A_clean, and A_management characterize the absorbance values of the experimental, clean, and management group, respectively.

Cytotoxicity and neuroprotective exercise assay in vitro

Astrocytes, pheochromocytoma 12 (PC12) cells, and the BV2 microglia cell line have been procured from the Cell Financial institution of the Chinese language Academy of Science (Shanghai, China). The MTT technique was employed to judge the cytotoxicity of HA-Se NPs in astrocytes and PC12 cells [22]. Briefly, astrocytes or PC12 cells have been seeded onto 96-well plates at a focus of 8,000 cells per properly and incubated for 12 h in DMEM medium. Subsequently, totally different concentrations of HA-Se NPs have been added to the wells, starting from 3.125 to 100 µg/mL. Management group cells have been subjected to remedy with phosphate-buffered saline (PBS). Three parallel checks have been carried out for each focus. After an additional incubation for both 24 or 48 h, answer containing 5 mg/mL MTT was added to every properly at a quantity of 20 µL and allowed to incubate for an additional 4 h. Thereafter, the medium was discarded and exchanged with 150 µL of dimethyl sulfoxide. The absorbance of every properly at 492 nm was decided utilizing an absorbance microplate reader (Infinite M200, Tecan, Switzerland). In an effort to evaluate the potential protecting properties of HA-Se NPs in opposition to H₂O₂-induced oxidative stress [22], astrocytes have been grown in 96-well plates, with every properly containing 8,000 cells. Previous to H₂O₂ publicity, the cells underwent 30-min pre-treatment with both PBS or HA-Se NPs at 50 and 100 µg/mL. Thereafter, the cells have been uncovered to 100 µM H₂O₂ for twenty-four h, and their viability was assessed through MTT assay. To additional consider cell viability, we employed a industrial live-dead cell staining package from Sigma-Aldrich. After staining, cells have been noticed by means of a confocal laser-scanning microscope (CLSM) (LSM 780, Zeiss). Useless cells have been then quantified utilizing the ImageJ software program (1.51k, NIH, Bethesda, MD, USA). DCFH-DA fluorescence depth was measured to quantitatively decide ROS ranges in each astrocytes and PC12 cells.

Evaluation of proinflammatory cytokine ranges in vitro and in vivo

In an effort to study the capability of HA-Se NPs to inhibit irritation, BV2 cells have been first seeded onto 6-well plates at a density of two × 10⁵ cells/properly and permitted to accept 12 h. Cells have been then subjected to pre-treatment with both PBS or HA-Se NPs (20 µg/mL) for two h. Thereafter, the cells have been uncovered to 1 µg/mL lipopolysaccharide (LPS) for twenty-four h. To find out the degrees of interleukin (IL)-1β, tumor necrosis issue α (TNF-α), and IL-6, we employed commercially obtainable enzyme-linked immunosorbent assay kits (Anoric Bio-technology Co., Ltd) and an absorbance microplate reader (Infinite M200, Tecan, Switzerland). The assays have been carried out in triplicate. To verify the in vivo suppressive results of HA-Se NPs on inflammatory cells, which generally exhibit a peak on the lesion website inside 3–7 days post-injury, immunofluorescence evaluation of spinal wire tissue samples was carried out utilizing anti-CD68 and anti-Iba-1 antibodies.

Animal mannequin of SCI

Approval for animal procedures was granted by the Animal Ethics Committee of Jilin College (approval No. SY202103013). Feminine Sprague-Dawley rats, aged 6–8 weeks, have been procured from Liaoning Changsheng Biotechnology Ltd. and supplied with advert libitum entry to meals and water. Sprague-Dawley rats have been anesthetized through intraperitoneal administration of pentobarbital sodium intraperitoneally at a dose of 5 mg per kilogram of physique weight. After disinfecting with iodine volts, a T10 laminectomy process was carried out to disclose the spinal wire, and the harm mannequin was established utilizing a weight-drop gadget (C4P01-001, Shenzhen, China). A 40-g rod was allowed to fall from a top of fifty mm, making contact with the spinal wire’s uncovered dorsal floor, and inflicting an preliminary depth of penetration of two.5 mm. Following the surgical intervention, the fascia, muscle, and pores and skin have been sequentially closed in layers. The Sprague-Dawley rats have been housed in a temperature-regulated setting at 25 ± 2 °C and have been supplied with advert libitum entry to each water and meals. Furthermore, the Sprague-Dawley rats have been handled with cefazolin, given twice every day for a length of 5 days at a dose of 25 mg/kg. The urinary bladders of Sprague-Dawley rats have been manually emptied twice every day till they regained the flexibility to urinate autonomously.

The SCI rats have been randomly divided into 4 teams (n = 9). The rats have been handled with saline, HA-Se NPs (1 mg kg⁻¹), HA-Se NPs (5 mg kg⁻¹), or HA-Se NPs (10 mg kg⁻¹). All rats acquired intravenous injections. The animals underwent anesthesia and have been then perfused with 4% paraformaldehyde and PBS on the 12-week interval post-injury. After being harvested from the middle of harm, the spinal cords, which had a size of roughly 2 cm, have been handled with a 4% paraformaldehyde answer. Thereafter, the specimens have been embedded in paraffin for additional evaluation. These sections have been employed in subsequent experiments.

Evaluation of CD44 expression

CD44 expression within the spinal wire was evaluated seven days after harm. The tissue slices underwent twin labeling utilizing a major anti-CD44 antibody together with major anti-glial fibrillary acidic protein (GFAP), anti-NeuN, anti-CD68, or anti-Iba-1 antibodies. For immunofluorescence staining, the antibody utilized is documented in Further file 1: Desk S1 . CLSM was utilized to acquire photos. Immunofluorescence experiments have been carried out 3 times. To look at the potential for irritation to stimulate CD44 expression in astrocytes, the cells have been grown on 6-well plates and subjected to various concentrations of LPS (10, 20, or 40 µg/mL) or glutamate (100, 200, or 400 µM) for a length of 24 h. CD44 expression was evaluated through western blot and immunofluorescence analyses. Following LPS activation for twenty-four h, astrocytes have been rinsed with PBS and have been then fastened with 4% PFA. Nonspecific binding websites have been obstructed utilizing blocking buffer for a interval of 1.5 h. Thereafter, the cells have been incubated with an anti-CD44 major antibody and incubated in a single day at a temperature of 4 °C. After being washed with PBS, the cells have been uncovered to secondary antibodies and incubated for a length of two h at room temperature. One other PBS wash was then carried out, and the cells have been subsequently uncovered to 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil), a fluorescent probe for cell membranes, for a length of 15 min. In the end, the astrocytes have been examined and visualized through CLSM. This experiment was additionally repeated 3 times.

To establish the potential affiliation between the CD44 receptor and the absorption of HA-Se NPs, a aggressive mobile uptake experiment was carried out. Following a 24-h incubation with LPS, astrocytes have been subjected to a 4-h pretreatment with both HA (1 mg/mL) or PBS. Samples have been then subjected to incubation with 0.1 mg/mL HA-Se NPs for 4 and eight h, adopted by 5 washes with PBS and fixation with 4% PFA for a interval of 20 min. Cell nuclei have been stained with DAPI staining previous to commentary through confocal microscopy (LSM 780, Zeiss, Germany). Imply intracellular fluorescence depth was decided utilizing ImageJ (1.51k, NIH, Bethesda, MD, USA).

Biodistribution of HA-Se NPs

NH₂-Cy5-labeled HA-Se NPs have been synthesized as beforehand described [39]. To visualise the in vivo biodistribution of HA-Se NPs, rats have been administered Cy5-labeled HA-Se NPs at a dose of 10 mg/kg subsequent to the harm. At particular intervals (4, 12, and 24 h), the rats have been humanely sacrificed, and their major organs (coronary heart, lung, liver, spleen, mind, kidney, and spinal wire) have been obtained. Management samples have been obtained by administering Cy5-labeled HA-Se NPs to sham rats, which have been then euthanized 12 h post-injection. The obtained organs have been imaged and analyzed utilizing a Maestro In Vivo Imaging System (IVIS Lumina LT Collection III, PerkinElmer, USA).

Behavioral evaluation of SCI rats

The Basso, Beattie, Bresnahan (BBB) locomotor score scale was employed to judge the motor features of rats. Based mostly on the 21-point BBB open-field grading scale, rats have been individually assessed in an open discipline for five min. The BBB rating assesses the voluntary actions of rat limbs by means of a scale of 0 to 21. A rating of 0 represents paralysis, whereas 21 signifies regular actions. Analysis was carried out by two separate evaluators, and the marks have been ascertained by means of mutual accord.

Histological and immunofluorescence analyses

Mounted spinal wire samples have been embedded in paraffin, adopted by sectioning into 4 μm thick slices within the coronal airplane. By way of H&E staining, pathological adjustments, which included enlargement of cavity space and inflammatory cell infiltration, have been investigated. The extent of demyelination was decided by staining spinal wire slices from every group with 0.1% Luxol quick blue (LFB) on the 12-week mark following harm.

To guage the myelin sheath ultrastructure, we ready tissue for transmission electron microscopy (TEM) as per our beforehand established technique [22]. The spinal wire segments have been fastened in a single day at a temperature of 4 °C utilizing 2.5% glutaraldehyde. Thereafter, the segments have been sliced into 1 mm³ items, osmicated for 90 min, after which dehydrated for a interval of 135 min. Subsequently, the ultrathin sections have been subjected to staining with uranyl acetate and lead citrate, adopted by an intensive examination utilizing TEM. To evaluate the expression of neurofilament 200 (NF200) and NeuN, the slices have been first subjected to permeation with a PBS answer that contained 0.1% Triton X-100 for a interval of 15 min. Thereafter, a 2-h incubation interval with 5% bovine serum albumin (BSA) was utilized for blocking, adopted by thorough washing with PBS containing 0.2% Tween 20. Subsequently, the parts have been subjected to an in a single day incubation at a temperature of 4 °C, using major antibodies in opposition to NF200 and NeuN. The axons and neurons have been distinguished utilizing anti-NF200 and anti-NeuN, respectively. Further file 1: Desk S1 supplies particulars on antibody dilution for immunofluorescence. Pictures have been acquired by means of CLSM. The potential toxicity of HA-Se NPs was assessed 12 weeks post-treatment by means of H&E staining of significant organs.

Western blotting

Spinal wire tissue was lysed utilizing a buffer answer that contained proteinase inhibitors. The lysate was then centrifuged at 13,300 rpm for 15 min. Protein focus was decided utilizing a BCA protein package (Beyotime, Shanghai, China). The process concerned separation of 10 µg protein from every pattern utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent switch of the separated protein onto polyvinylidenedifluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). For blocking, the membranes have been handled with 5% bovine serum albumin for 1.5 h. Thereafter, membranes have been incubated with both anti-CD44 or anti-caspase-3 antibodies at 4 °C in a single day. After washing, the membranes have been incubated with a secondary antibody conjugated to horseradish peroxidase for two h at room temperature. Goal protein expression was normalized based mostly on that of GAPDH or β-actin. Western blotting antibody knowledge is on the market in Further file 1: Desk S2. An enhanced chemiluminescence Western blot detection system (AI600 Imager; GE Healthcare, Chicago, IL, USA) was used to seize protein bands. Densitometric evaluation of the protein bands was carried out utilizing Multi Gauge software program (Fuji, Tokyo, Japan). Western blotting experiments have been repeated 3 times.

Statistical evaluation

To find out the statistical significance of variations, a one-way evaluation of variance or t-tests have been carried out using Prism 8.0.2 (GraphPad Software program, San Diego, CA, USA). *P < 0.05 was deemed statistically vital. **P < 0.01 and ***P < 0.001 have been deemed extremely vital.