Targeting Bradykinin Signaling Pathway: PLGA/BK Microspheres as a Therapeutic Strategy for Delaying Intervertebral Disc Degeneration

Intervertebral disc degeneration(IVDD) is a common spinal condition with limited effective treatments available. This study aims to investigate the impact of poly(lactic-co-glycolic acid)/Bradykinin (PLGA/BK) microspheres on IVDD and its underlying mechanisms. We collected nucleus pulposus samples from both healthy and degenerated human intervertebral discs and conducted immunohistochemical analyses, revealing reduced BK expression in degenerated tissues. Subsequently, we used BK to treat nucleus pulposus cells and conducted Bulk RNA sequencing (RNA-seq), identifying BK's involvement in cellular senescence, extracellular matrix metabolism, and the PI3K signaling pathway. Further experiments using tert-butyl hydroperoxide (TBHP)-induced cell senescence showed that BK treatment reduced senescence, enhanced extracellular matrix synthesis, and inhibited degradation, along with activation of the PI3K pathway. These effects were mediated through B2R (BK receptor 2) and the downstream PI3K pathway. Following this, we developed sustained-release BK microspheres with an optimized manufacturing process. In vitro co-culture experiments showed no observable toxicity. We established an IVDD model in rat tail vertebrae through fine needle puncture, administering local injections of BK sustained-release microspheres. Using various experimental methods, including X-ray, MRI, histopathology, and immunohistochemistry, we found that these microspheres could slow the progression of IVDD. This study highlights the potential of injectable PLGA/BK microspheres to regulate cellular senescence and extracellular matrix metabolism via the B2R and PI3K pathways, ultimately delaying IVDD. Overall design: Experimental Design for Bulk RNA-Seq In this study, bulk RNA sequencing (RNA-seq) was employed to explore the effects of bradykinin (BK) on nucleus pulposus cells (NPCs). The experimental design involved two groups of cultured NPCs: a control group and a BK-intervention group. The goal was to examine how BK affects gene expression, focusing on its potential role in regulating cellular senescence and extracellular matrix metabolism. Grouping and Treatment Control Group: NPCs without any treatment, serving as the baseline. BK Group: NPCs treated with 10 µM BK to assess its influence on gene expression. RNA Extraction and Bulk RNA-Seq Total RNA was extracted from both groups of NPCs using an RNA extraction kit. Bulk RNA-seq was performed on the extracted RNA to capture the complete transcriptome profiles. The workflow included: RNA Extraction: High-quality RNA was extracted and subjected to strict quality control. Library Construction: Libraries were prepared from the extracted RNA and sequenced using the Illumina NovaSeq platform, generating paired-end 150 bp reads. Sequencing: Bulk RNA-seq was conducted to capture the entire transcriptome of NPCs from both the control and BK-treated groups. Bioinformatics Analysis Quality Control: The raw sequencing data underwent stringent quality control, including removal of adapters, low-quality reads, and ambiguous bases using fastp. Reference Genome Alignment: Clean reads were aligned to the reference genome using HISAT2 for precise mapping. Gene Expression Quantification: Gene expression levels were quantified, and differences in expression between the two groups were calculated using RSEM. Differential Expression Analysis: DESeq2 was used to identify differentially expressed genes (DEGs) with significance thresholds of FDR = 0.05 and |log2 fold change| = 1.