METTL3 Promotes Nucleus Pulposus Cell Senescence in Intervertebral Disc Degeneration by Regulating TLR2 m6A Methylation and Gut Microbiota.

BACKGROUND: Nucleus pulposus cell (NPC) senescence in intervertebral disc (IVD) tissue is the major pathological cause of intervertebral disc degeneration (IDD). N6-methyladenosine (m6A) methylation and gut microbiota play important roles in the progression of IDD. This study investigated whether methyltransferase-like 3 (METTL3) regulates TLR2 m6A modification and gut microbiota to influence NPC senescence. METHODS: An IDD rat model was established by lumbar IVD puncture and NPCs were challenged with IL-1ß to mimic IVD injury. IDD rats and IL-1ß-exposed NPCs were treated with METTL3-interfering lentivirus and the TLR2 agonist Pam3CSK4. Compositional changes in the rat gut microbiota were analyzed and fecal microbiota transplantation procedures were used. NPC senescence, cell cycle, and the expression of senescence-associated secretory phenotype (SASP) factors were assessed. The m6A enrichment of TLR2 and the binding of IGF2BP1 to TLR2 mRNA were examined. RESULTS: METTL3 and TLR2 were highly expressed in IDD rats. METTL3 silencing attenuated senescent phenotypes and reduced secretion of SASP factors. Pam3CSK4 reversed the beneficial effects of METTL3 silencing on NPC senescence and IVD injury. METTL3 stabilized TLR2 mRNA in an IGF2BP1-dependent manner. METTL3 silencing restored specific gut microbiota levels in IDD rats, which was further reversed by administration of Pam3CSK4. Fecal microbiota from METTL3 silenced IDD rats altered the pathological phenotypes of IDD rats. CONCLUSIONS: These results demonstrate the beneficial effects of METTL3 silencing on NPC senescence and amelioration of IVD injury, involving modulation of TLR2 m6A modification and gut microbiota. These findings support METTL3 silencing as a potential therapeutic target for IDD.

Senescent-like macrophages mediate angiogenesis for endplate sclerosis via IL-10 secretion in male mice.

Endplate sclerosis is a notable aspect of spine degeneration or aging, but the mechanisms remain unclear. Here, we report that senescent macrophages accumulate in the sclerotic endplates of lumbar spine instability (LSI) or aging male mouse model. Specifically, knockout of cdkn2a (p16) in macrophages abrogates LSI or aging-induced angiogenesis and sclerosis in the endplates. Furthermore, both in vivo and in vitro studies indicate that IL-10 is the primary elevated cytokine of senescence-related secretory phenotype (SASP). Mechanistically, IL-10 increases pSTAT3 in endothelial cells, leading to pSTAT3 directly binding to the promoters of Vegfa, Mmp2, and Pdgfb to encourage their production, resulting in angiogenesis. This study provides information on understanding the link between immune senescence and endplate sclerosis, which might be useful for therapeutic approaches.

Ursolic acid molecules dock MAPK1 to modulate gut microbiota diversity to reduce neuropathic pain.

To investigate the efficacy of Ursolic acid in alleviating neuropathic pain in rats with spinal nerve ligation (SNL), the SNL rat model was surgically induced. Different concentrations of Ursolic acid and manipulated target mitogen-activated protein kinase 1 (MAPK1) were administered to the SNL rats. Fecal samples were collected from each group of rats for 16S rDNA analysis to examine the impact of gut microbiota. Molecular docking experiments were conducted to assess the binding energy between Ursolic acid and MAPK1. In vivo studies were carried out to evaluate the expression of inflammatory factors and signaling pathways in spinal cord and colon tissues. Ursolic acid was found to have a beneficial effect on pain reduction in rats by increasing plantar withdrawal latency (PWL) and paw withdrawal threshold (PWT). Comparing the Ursolic acid group with the control group revealed notable differences in the distribution of Staphylococcus, Allobaculum, Clostridium, Blautia, Bifidobacterium, and Prevotella species. Network pharmacology analysis identified MAPK1 and intercellular adhesion molecule-1 (ICAM1) as common targets for Ursolic acid, SNL, and neuropathic pain. Binding sites between Ursolic acid and these targets were identified. Additionally, immunofluorescent staining showed a decrease in GFAP and IBA1 intensity in the spinal cord along with an increase in NeuN following Ursolic acid treatment. Overexpression of MAPK1 in SNL rats led to an increase in inflammatory factors and a decrease in PWL and PWT. Furthermore, MAPK1 counteracted the pain-relieving effects of Ursolic acid in SNL rats. Ursolic acid was found to alleviate neuropathic pain in SNL rats by targeting MAPK1 and influencing gut microbiota homeostasis.