63. Rutin attenuates distraction spinal cord injury by inhibiting microglial inflammation through downregulation of P38 MAPK/NF-?B/STAT3 pathway

Abstract

BACKGROUND CONTEXT

Distraction spinal cord injury (DSCI) is a severe complication following scoliosis correction surgery, for which there are currently no effective clinical treatments.

PURPOSE

This study aims to evaluate the inhibitory effects of rutin, a natural product, on inflammation in DSCI and to investigate the underlying mechanisms.

STUDY DESIGN/SETTING

This study employed a combination of in vitro and in vivo experimental designs to investigate the anti-inflammatory and neuroprotective effects of rutin in the context of DSCI. The in vitro component utilized a cell culture model to assess the direct effects of rutin on LPS-induced inflammation in microglial cells. The in vivo component involved a randomized controlled animal model of DSCI to evaluate the therapeutic potential of rutin. Molecular studies, including mRNA sequencing, KEGG pathway analysis, and molecular docking, were conducted to elucidate the underlying mechanisms of rutin's action. The study design ensured a comprehensive approach to understanding the role of rutin in mitigating inflammation and promoting recovery following spinal cord injury.

PATIENT SAMPLE

N/A

OUTCOME MEASURES

N/A

METHODS

In Vitro Studies: Microglial cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Cells were seeded in 6-well plates at a density of 1 × 10^6 cells per well and allowed to adhere overnight. To induce inflammation, cells were treated with 1 µg/mL lipopolysaccharide (LPS) for 24 hours. Concurrently, cells were exposed to varying concentrations of rutin (10 µM, 20 µM, and 50 µM) to assess its anti-inflammatory effects. After treatment, cell supernatants were collected, and the levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) were measured using enzyme-linked immunosorbent assays (ELISA). Cell viability was assessed using the MTT assay to ensure that rutin concentrations used did not induce cytotoxicity. In Vivo Studies: Adult male Sprague-Dawley rats (weight 250-300 g) were used for the dorsal spinal cord injury (DSCI) model. Rats were anesthetized with intraperitoneal injections of ketamine (80 mg/kg) and xylazine (10 mg/kg). A laminectomy was performed at the T10 vertebral level, and the dorsal spinal cord was exposed. DSCI was induced using a weight-drop impactor (10 g weight dropped from a height of 25 mm). Post-surgery, rats were randomly divided into three groups: sham-operated (control), DSCI + vehicle (saline), and DSCI + rutin (50 mg/kg/day, intraperitoneal injection). Behavioral assessments were conducted using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale at 1, 3, 7, 14, and 21 days postinjury. Rats were sacrificed at 21 days postinjury, and spinal cord tissues were harvested for further analysis. Molecular Studies: Total RNA was extracted from spinal cord tissues using TRIzol reagent, and mRNA sequencing was performed using the Illumina HiSeq 4000 platform. Differentially expressed genes (DEGs) were identified using the DESeq2 package in R, with a threshold of |log2 fold change| > 1 and adjusted p-value < 0.05. Functional enrichment analysis of DEGs was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. The top DEGs were validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Molecular docking studies were performed to investigate the interaction between rutin and P38 MAPK using AutoDock Vina. The crystal structure of P38 MAPK (PDB ID: 1P38) was obtained from the Protein Data Bank, and the binding affinity was calculated.

RESULTS

In vitro, microglial cells were exposed directly to rutin to assess its ability to inhibit lipopolysaccharide (LPS)-induced inflammation. In rats with DSCI, the inhibitory effect of rutin on DSCI was evaluated using behavioral tests. mRNA sequencing was performed on spinal cord tissues to elucidate the mechanism of rutin's action. Rutin significantly suppressed the LPS-induced increase in inflammatory factors in microglial cells. In DSCI rats treated with rutin, scores in the Basso-Beattie-Bresnahan (BBB) were significantly improved. The mechanism of rutin's action was found to be related to its ability to reduce inflammatory infiltration in spinal cord tissue, protecting neurons from apoptosis and microstructural demyelination. Through assays of transcriptomic differentially expressed genes (DEGs), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and RT-qPCR validation of the top DEGs, MAPK13 (also known as P38 MAPK) was finally identified as the key target gene in promoting DSCI development. Further molecular docking analysis indicated an interaction between rutin and P38 MAPK, supporting the rutin's action and the underlying mechanism in anti-inflammation.

CONCLUSIONS

In conclusion, rutin effectively inhibited the development of DSCI in rats. The mechanism of rutin's action was associated with its activity in blocking the P38 MAPK/NF-κB/STAT3 pathway in the microglial cells of spinal cord. Rutin could be developed as a potential anti-DSCI drug for clinical applications.

FDA Device/Drug Status

Rutin (Approved for this indication)