sensescence

Lactate causes cells in the nucleus pulposus to die, leading to degeneration of the intervertebral disc

Introduction

 

Intervertebral disc degeneration (IVDD) is a leading cause of low back pain and disability worldwide, causing a significant burden on healthcare systems. The intervertebral discs consist of three distinct components: the nucleus pulposus (NP), the annulus fibrosus, and the cartilage endplate. These discs are the largest avascular tissue in the body, and their oxygen and nutrition supply mainly depend on permeation between the NP and the cartilage endplate.

 

Lactate, a common metabolic waste produced during cellular anaerobic respiration, is the main mechanism of energy supply for intervertebral disc cells, especially nucleus pulposus cell (NPC). Under normal conditions, the generation, transport, and clearance of lactate occur in a dynamic balance to maintain intracellular pH and homeostasis of the intervertebral disc. However, under pathological conditions, the concentration of lactate in the intervertebral disc distinctly rises, especially in the center of the degenerative disc.

Intervertebral Disc - Magnified Clear Bone

The disruption of glycolysis and imbalance of lactate transport and clearance are the main causes of lactate accumulation in the intervertebral disc. Injection of lactate into the intervertebral disc of healthy rats elicits degeneration-related symptoms, indicating that lactate can directly aggravate IVDD.

 

High concentrations of lactate can downregulate the expression of extracellular matrix glycosaminoglycan and matrix metalloproteinase 3, regulate apoptosis and autophagy of NPC, and promote NLRP3 inflammasome activation and exacerbate inflammation in NPC. These phenomena are relevant manifestations of cellular senescence and oxidative stress, which are crucial changes in cell biology that occur during IVDD.

 

In this study summary, the researchers use primary cultured rat NPC and a puncture-induced disc degeneration rat model to determine the effect of lactate on NPC senescence and oxidative stress. This study may help better understand the relationship between lactate and NPC senescence and oxidative stress during IVDD and provide a prospective strategy for IVDD treatment.1

 

Methods

 

The methods involved a number of strategies using tissue samples that include measures of:

  • Cell isolation, culture, and treatment
  • Lactate content assays
  • Energy metabolomics analysis
  • Construction and verification of lactate oxidase (LOx)
  • overexpression vector
  • Lentivirus treatment of PIDD rats
  • Magnetic resonance imaging (MRI) analysis
    Microcomputed tomography (μCT) analysis
  • Histological analysis of human nucleus pulposus tissues
  • and rat caudal intervertebral discs
  • Immunofluorescence staining assay
  • Cell counting
  • EdU staining assay
  • Cell cycle assay
  • Senescence-associated β-galactosidase (SA-β-gal) staining
  • assay
  • Reactive oxygen species (ROS) level assay
  • Measurement of mitochondrial membrane potential (MMP)
  • Malondialdehyde (MDA) measurement
  • Transmission electron microscopy (TEM)
  • RNA sequencing and data analysis
  • Reverse transcription-quantitative PCR (RT-qPCR) analysis
  • Akt kinase activity assay
  • Microscale thermophoresis (MST) assay
  • Western blotting
  • Extraction of cytoplasmic and nuclear proteins
  • Molecular docking

Discussion


This study investigates the role of high concentrations of lactate in neuronal senescence and oxidative stress (IVDD) in human and rat intervertebral discs. The researchers found that lactate concentrations significantly increased in the intervertebral discs of human and rats with PIDD, leading to primary rat NPC senescence and oxidative stress. Eliminating lactate accumulation in the rat intervertebral disc alleviated IVDD progression.

 

High concentrations of lactate induced NPC senescence and oxidative stress by inhibiting the PI3K/Akt pathway via binding to Lys39 and Leu52 residues in the PH domain of Akt, then suppressing downstream Akt/p21/p27/cyclin D1 signaling and Akt/Nrf2/HO-1 signaling. This study reveals the role of lactate in NPC senescence and oxidative stress during IVDD and may facilitate the effects of current IVDD therapies.

 

IVDD is characterized by early onset and severe conditions compared to degenerative diseases that occur in other organs and tissues. The intervertebral disc is the largest avascular tissue in the whole body, and it has poor oxygen and nutrition supply. Previous research verified that many metabolites, including amino acids, nucleosides, and bioenergetics metabolites, accumulated in the intervertebral disc. Lactate, pyruvic acid, isocitric acid, α-glutanic acid, UMP, guanine, and UDP-GlcNAc, were the distinctly upregulated metabolites in degenerated intervertebral discs.

 

The researchers then evaluated the changes in the biological function of NPC using a series of cellular senescence and oxidative stress assessment assays. They found that a high concentration of lactate inhibits cell proliferation and promotes NPC senescence. Additionally, a high concentration of lactate promotes the accumulation of ROS and mitochondrial damage in NPC.

 

To determine the potential mechanism by which high concentrations of lactate stimulate NPC senescence and oxidative stress, RNA-seq and bioinformatics analysis were performed. The PI3K/Akt pathway was found to be the most highly enriched signaling pathway, which may underlie lactate-induced NPC senescence and oxidative stress. Activating the PI3K/Akt signaling pathway exerts protective effects in IVDD, and PI3K/Akt activation can ameliorate NPC senescence and oxidative stress.

 

In conclusion, a high concentration of lactate may promote NPC senescence by suppressing the Akt/p21/p27/cyclin D1 pathway and promote NPC oxidative stress by suppressing the Akt/Nrf2/HO-1 pathway. This study provides valuable insights into the role of lactate in NPC senescence and oxidative stress during IVDD and may facilitate the effects of current IVDD therapies.

 

The study investigates the molecular mechanism by which lactate regulates the PI3K/Akt pathway in the degenerative intervertebral disc. It was found that lactate can directly interact with intracellular proteins like SENP1 and PHD2, regulating biological processes. The inhibition of PI3K/Akt pathway by lactate may be associated with lactate-protein interaction. Molecular docking revealed that residues (Lys39 and Leu52) in the PH domain of Akt can form hydrogen bonds with lactate. The Akt protein’s modular structure consists of the PH domain at the N-terminus, the kinase catalytic domain in the middle, and the AGC-kinase C-terminal domain at the C-terminus. Akt activation requires membrane translocation and phosphorylation by the kinase recruited to the membrane. Several small molecule compounds have been proven to inactivate Akt by binding to the PH domain. Genipin and Abrus Agglutinin have been reported to bind to Lys39 and Leu52 in the PH domain, inhibiting Akt activation. This suggests that lactate may suppress Akt phosphoactivation by binding to Lys39 and Leu52 in the PH domain of Akt. Further investigation into potential therapeutics targeting lactate in IVDD treatment is warranted.

 

Conclusion

 

This study explores lactate’s effects on NPC senescence and its underlying mechanism, highlighting its association with IVDD progression. It suggests a new strategy targeting lactate in IVDD treatment, potentially providing a new approach to combat this disease.

At Dynamic Disc Designs, we have worked to highlight the dynamics of the intervertebral disc through accurate modelling to scale to facilitate education and promising solutions pharmacologically, non-surgically and surgically when appropriate.