Traditional Chinese medicine active ingredients-based selenium nanoparticles regulate antioxidant selenoproteins for spinal cord injury treatment

Abstract

Background: As Traditional Chinese Medicine (TCM) drugs, Huangqi and Danshen are always applied in combination for spinal cord injury (SCI) treatment based on the compatibility theory of TCM. Astragalus Polysaccharidesis (APS) and Tanshinone IIA (TSIIA) are the main active ingredients of Huangqi and Danshen, and they both possess neuroprotective effects through antioxidant activities. However, low solubility and poor bioavailability have greatly limited their application. In recent years, selenium nanoparticles (SeNPs) have drawn enormous attention as potential delivery carrier for antioxidant drugs.

Results: In this study, TCM active ingredients-based SeNPs surface decorated with APS and loaded with TSIIA (TSIIA@SeNPs-APS) were successfully synthesized under the guidance of the compatibility theory of TCM. Such design improved the bioavailability of APS and TSIIA with the benefits of high stability, efficient delivery and highly therapeutic efficacy for SCI treatment illustrated by an improvement of the antioxidant protective effects of APS and TSIIA. The in vivo experiments indicated that TSIIA@SeNPs-APS displayed high efficiency of cellular uptake and long retention time in PC12 cells. Furthermore, TSIIA@SeNPs-APS had a satisfactory protective effect against oxidative stress-induced cytotoxicity in PC12 cells by inhibiting excessive reactive oxygen species (ROS) production, so as to alleviate mitochondrial dysfunction to reduce cell apoptosis and S phase cell cycle arrest, and finally promote cell survival. The in vivo experiments indicated that TSIIA@SeNPs-APS can protect spinal cord neurons of SCI rats by enhancing GSH-Px activity and decreasing MDA content, which was possibly via the metabolism of TSIIA@SeNPs-APS to SeCys₂ and regulating antioxidant selenoproteins to resist oxidative stress-induced damage.

Conclusions: TSIIA@SeNPs-APS exhibited promising therapeutic effects in the anti-oxidation therapy of SCI, which paved the way for developing the synergistic effect of TCM active ingredients by nanotechnology to improve the efficacy as well as establishing novel treatments for oxidative stress-related diseases associated with Se metabolism and selenoproteins regulation.

Keywords: Antioxidant selenoproteins; Selenium nanoparticles; Spinal cord injury; Traditional Chinese Medicine active ingredients.

Fig. 1

Design of TSIIA@SeNPs-APS based on compatibility theory of TCM. A Synthetic schematic diagram of TSIIA@SeNPs-APS and its regulation of antioxidant selenoproteins for SCI treatment. B TEM images of (a) SeNPs, (b) SeNPs-APS and (c) TSIIA@SeNPs-APS. The scale bar is 200 nm. C AFM image of TSIIA@SeNPs-APS. D Corresponding thickness profile of TSIIA@SeNPs-APS in C. E Size distribution and F Surface charge of SeNPs, SeNPs-APS and TSIIA@SeNPs-APS in aqueous solutions. G The particle size distribution changes of SeNPs, SeNPs-APS and TSIIA@SeNPs-APS as time progresses. H Se 3d XPS pattern of SeNPs, SeNPs-APS and TSIIA@SeNPs-APS. I FT-IR spectra of (a) SeNPs, (b) APS, (c) SeNPs-APS, (d) TSIIA and (e) TSIIA@SeNPs-APS. J–K UV–Vis spectra of SeNPs, APS, SeNPs-APS, TSIIA and TSIIA@SeNPs-APS

Fig. 2

Cellular uptake and intracellular trafficking of TSIIA@SeNPs-APS. A Fluorescence intensity of couramin-6-labelled TSIIA@SeNPs-APS in PC12 cells for different time assessed by flow cytometry. B Cellular uptake of couramin-6-labelled TSIIA@SeNPs-APS in PC12 cells after pretreated with different inhibitors. Significant difference between control group and other groups is indicated by *P < 0.05, **P < 0.01, ***P < 0.001. C Intracellular trafficking of couramin-6-labelled TSIIA@SeNPs-APS in PC12 cells, which were stained by DAPI (blue, nucleus) and Lysotracker (red, lysosome), and visualized using a fluorescence microscopy. The scale bar is 10 nm. D Schematic illustration of cellular uptake and intracellular trafficking of TSIIA@SeNPs-APS

Fig. 3

Effects of t-BOOH on cell viability, apoptosis and cell cycle of PC12 cells reversed by TSIIA@SeNPs-APS. A The viability of PC12 cells under different concentrations of TSIIA@SeNPs-APS for 24 h (calculated by Se concentration). B The viability of PC12 cells pretreated with t-BOOH and then treated with SeNPs, SeNPs-APS and TSIIA@SeNPs-APS for 24 h, respectively, at the concentration of 1.25 and 2.5 μM (calculated by Se concentration). Significant difference between the 2.5 μM TSIIA@SeNPs-APS group and the other groups is indicated by *P < 0.05, **P < 0.01, ***P < 0.001. C The viability of PC12 cells pretreated with t-BOOH and then treated with APS and TSIIA@SeNPs-APS for 24 h, respectively, at the concentration of 0.32 and 0.64 μg/ml (calculated by APS concentration). **P < 0.01. D The viability of PC12 cells pretreated with 100 μM t-BOOH and then treated with TSIIA and TSIIA@SeNPs-APS for 24 h, respectively, at the concentration of 0.22 and 0.45 ng/ml (calculated by TSIIA concentration). *P < 0.05, **P < 0.01. E Flow cytometry analysis of the effects of SeNPs, SeNPs-APS and TSIIA@SeNPs-APS on the cell cycle distribution of PC12 cells pretreated with t-BOOH. F Proportion of the cell cycle in (E). G Western blot analysis of CDK2. Lane 1–5: groups of control, t-BOOH, t-BOOH + SeNPs, t-BOOH + SeNPs-APS and t-BOOH + TSIIA@SeNPs-APS, respectively. H Annexin V-FITC/PI double-staining to evaluate the effects of SeNPs, SeNPs-APS and TSIIA@SeNPs-APS on the apoptosis of PC12 cells pretreated with t-BOOH. I Quantitative analysis of the proportion of apoptosis in (H). J Western blot analysis of pro-Caspase-3 and pro-Caspase-9. Lane 1–5: the same as groups in G

Fig. 4

Protective effects of TSIIA@SeNPs-APS against oxidative stress-induced mitochondrial dysfunction in PC12 cells. A The ΔΨm of PC12 cells, which were pretreated with 100 μM t-BOOH for 2 h and then treated with SeNPs, SeNPs-APS and TSIIA@SeNPs-APS for 24 h, examined by JC-1 staining and flow cytometry. B JC-1 fluorescence images of PC12 cells in A. C Western blot analysis of Bcl-2 family proteins. Lane 1–5: groups of control, t-BOOH, t-BOOH + SeNPs, t-BOOH + SeNPs-APS and t-BOOH + TSIIA@SeNPs-APS, respectively. D–F ROS scavenging efficiency of (1) SeNPs, (2) SeNPs-APS and (3) TSIIA@SeNPs-APS for ABTS^•+, H₂O₂ and •OH, respectively. G Changes in intracellular total ROS level in PC12 cells, which were examined by DCFH-DA staining in group 1–5: groups of control, t-BOOH, t-BOOH + SeNPs, t-BOOH + SeNPs-APS and t-BOOH + TSIIA@SeNPs-APS, respectively. H The fluorescence intensity and representative images of DCFH-DA in PC12 cells at 120 min after treatment

Fig. 5

Improvement of locomotor recovery and neurons survival by TSIIA@SeNPs-APS in SCI rats. A BBB scores and B Inclined plane test of rats at different times. Significant difference between TSIIA@SeNPs-APS group and SCI group is indicated by *P < 0.05, **P < 0.01 and ***P < 0.001. C Footprint analysis of rats in each group. D Snapshots extracted from video recordings showing a sequence of hindlimb movements rats in each group while walking. Iliac crest, knee, and ankle joints were showed by dots and lines. The moving direction of hindlimb was showed by arrow. Representative images of E H&E staining, F Nissl staining, and G TUNEL/DAPI double-staining of spinal cord tissues in each group. Black arrows: motor neurons in E and Nissl bodies in F

Fig. 6

The metabolism and bioactivity mechanism of TSIIA@SeNPs-APS in vivo. A TEM images revealing the morphological and structural changes of TSIIA@SeNPs-APS incubated in injured spinal cord homogenates within 24 h. B Chromatograms of Se speciation in the spinal cord tissue by HPLC-ICP-MS. Corresponding quantitative analysis of C SeCys₂ and (D) SeIV in (B). The changes of E GSH-Px activity and F MDA level in the spinal cord tissue of each group. Significant difference between the SCI group and the other groups is indicated by **P < 0.01 and ***P < 0.001. G Expression of GPX2, GPX4, TRXR1, TRXR2, SelK and SelT protein in the spinal cord tissue detected by Western blot. H Relative gene expression of GPX2, GPX4, TRXR1, TRXR2, SelK and SelT detected by qPCR. I H&E staining of major organs in different groups for toxicity study in vivo