Lycium barbarum glycopeptide alleviates neuroinflammation in spinal cord injury via modulating docosahexaenoic acid to inhibiting MAPKs/NF-kB and pyroptosis pathways

Abstract

Background: Lycium barbarum polysaccharide (LBP) is an active ingredient extracted from Lycium barbarum that inhibits neuroinflammation, and Lycium barbarum glycopeptide (LbGp) is a glycoprotein with immunological activity that was purified and isolated from LBP. Previous studies have shown that LbGp can regulate the immune microenvironment, but its specific mechanism of action remains unclear.

Aims: In this study, we aimed to explore the mechanism of action of LbGp in the treatment of spinal cord injury through metabolomics and molecular experiments.

Methods: SD male rats were randomly assigned to three experimental groups, and after establishing the spinal cord hemisection model, LbGp was administered orally. Spinal cord tissue was sampled on the seventh day after surgery for molecular and metabolomic experiments. In vitro, LbGp was administered to mimic the inflammatory microenvironment by activating microglia, and its mechanism of action in suppressing neuroinflammation was further elaborated using metabolomics and molecular biology techniques such as western blotting and q-PCR.

Results: In vivo and in vitro experiments found that LbGp can improve the inflammatory microenvironment by inhibiting the NF-kB and pyroptosis pathways. Furthermore, LbGp induced the secretion of docosahexaenoic acid (DHA) by microglia, and DHA inhibited neuroinflammation through the MAPK/NF-κB and pyroptosis pathways.

Conclusions: In summary, we hypothesize that LbGp improves the inflammatory microenvironment by regulating the secretion of DHA by microglia and thereby inhibiting the MAPK/NF-κB and pyroptosis pathways and promoting nerve repair and motor function recovery. This study provides a new direction for the treatment of spinal cord injury and elucidates the potential mechanism of action of LbGp.

Keywords: Docosahexaenoic acid; Lycium barbarum glycopeptide; Neuroinflammation; Spinal cord injury.

Fig. 1

Lycium barbarum glycopeptide promotes nerve regeneration and benefits motor function recovery. A The motor function of each group was evaluated based on the BBB score. The score of the LbGp treatment group was higher than that of the SCI group after the second week and did not significantly differ from that of the sham group. n = 5 for each group;***p < 0.001, SCI vs. LbGp treatment group. B–D The expression levels of BDNF and GDNF in spinal cord tissue were detected by western blotting. E, F Fluorescence staining micrographs of NeuN protein in spinal cord tissues of the different groups. The data are presented as the means ± SEMs of at least 3 independent experiments, Scale bar, 200 µm; n = 3 per group;*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus each group

Fig. 2

Lycium barbarum glycopeptide inhibits NF-kB-and pyroptosis-related proteins in vivo. A SCI treatment with different doses (10 mg/kg-100 mg/kg) of LbGp reduced the pro-IL-18 protein expression levels. B–D Fluorescence staining micrographs of NLRP3, ASC,andcaspase-1 protein on the 7th day in spinal cord tissues of the different groups. E–M On day 7, the protein expression levels of NLRP3, ASC, caspase-1, P-p65, P-65, GSDMD, GSDMD-N, IL-18, and IL-1β in spinal cord tissue from each group were detected by western blotting. The data are presented as the means ± SEMs of at least 3 independent experiments. Scale bar, 200 µm; n = 3 per group;*p < 0.05,**p < 0.01, ***p < 0.001, and ****p < 0.0001 versus each group

Fig. 3

Lycium barbarum glycopeptide inhibits microglial MAPKs/NF-kB and pyroptosis-related pathways. A mRNA expression level of IL-18 in ATP + LPS-stimulated microglia treated with different doses(100 µg/kg–400 µg/ml) of LbGp. B–L The expression levels of P-p38, P38, p-JNK, JNK, P-p65, P-65, NLRP3, caspase-1-p20, ASC, GSDMD, GSDMD-N, IL-18, and IL-1β protein in each group of activated microglia were detected by western blotting. The data are presented as the means ± SEMs of at least 3 independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus each group

Fig. 4

Lycium barbarum glycopeptide induces microglia to secrete DHA. A Heat map of differential metabolic substances between the SCI and LbGp groups in spinal cord tissue(In vivo). B Heat map of differential metabolic substances between the ATP + LPS and LbGp treatment groups in microglial supernatants (In vitro). C Venn diagram of elevated metabolites in spinal cord tissues from the LbGp-treated group and microglia from the LbGp-treated group. D Microglia were activated with ATP + LPS for 4 hand incubated with LbGp for 24 h, and the DHA levels in the cell supernatants were assayed. E, F mRNA expression levels of FADS1 and FADS2 in ATP + LPS-stimulated microglia treated with LbGp. The data are presented as the means ± SEMs of at least 3 independent experiments; n = 6per group;*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus each group

Fig. 5

DHA inhibits microglial MAPKs/NF-kB and pyroptosis-related pathways. A‒K The expression levels of P-p38, P38, P-JNK,JNK, P-p65, P-65, NLRP3, caspase-1-p20, ASC, GSDMD, GSDMD-N, IL-18, and IL-1β protein in each group of activated microglia were detected by western blotting. The data are presented as the means ± SEMs of at least 3 independent experiments;*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus each group

Fig. 6

Model illustrating the role of LbGp in the treatment of SCI by inducing microglia to secrete DHA to inhibit the MAPK/NF-κB and pyroptosis pathways. Activation of microglia and release of inflammatory factors following SCI. LbGp stimulates microglia to produce DHA by regulating the key enzymes FADS1 and FADS2 in microglia, and thus, DHA can improve neuro inflammation by inhibiting the MAPK/NF-kB and pyroptosis pathways group.