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Cationic Polymer Brush-Modified Carbon Nanotube-Meditated eRNA LINC02569 Silencing Attenuates Nucleus Pulposus Degeneration by Blocking NF-κB Signaling Pathway and Alleviate Cell Senescence

ABSTRACT: Enhancer RNAs (eRNAs) are noncoding RNAs that synthesized at active enhancers. eRNAs have important regulatory characteristics and appear to be significant for maintenance of cell identity and information processing. Series of functional eRNAs have been identified as potential therapeutic targets for multiple diseases. Nevertheless, the role of eRNAs on intervertebral disc degeneration (IDD) is still unknown yet. Herein, we utilized the nucleus pulposus samples of patients and identified a key eRNA (LINC02569) with the Arraystar eRNA Microarray. LINC02569 mostly locates in nucleus and plays an important role in the progress of IDD by activating nuclear factor kappa-B (NF-κB) signaling pathway. We used a cationic polymer brush coated carbon nanotube (oCNT-pb)-based siRNA delivery platform that we previously designed, to transport LINC02569 siRNA (si-02569) to nucleus pulposus cells. The siRNA loaded oCNT-pb accumulated in nucleus pulposus cells with lower toxicity and higher transfection efficiency, compared with the traditional siRNA delivery system. Moreover, the results showed that the delivery of si-02569 significantly alleviated the inflammatory response in the nucleus pulposus cells via inhibiting P65 phosphorylation and preventing its transfer into the nucleus, and meanwhile alleviated cell senescence by decreasing the expression of P21. Altogether, our results highlight that eRNA (LINC02569) plays important role in the progression of IDD and could be a potential therapeutic target for alleviation of IDD.

SUBMITTER: Huang Y

PROVIDER: S-EPMC8802762 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAccelerated cellular senescence within the nucleus pulposus (NP) region is a common feature of disc degeneration. Our previous work indicated that TNF-α promoted NP cell senescence. Although the intervertebral disc has been reported to be an estrogen-sensitive tissue, it is unclear whether estrogen can inhibit premature senescence of NP cells.ObjectiveTo investigate whether 17beta-estradiol (E2) can attenuate TNF-α-induced premature senescence of NP cells and the potential mechanism behind this regulatory process.MethodsIsolated NP cells and intact intervertebral discs from healthy rats were cultured with or without TNF-α, E2 or their combination. The pan estrogen receptor (ER) antagonist ICI 182780 was used to investigate the role of ER. Direct and indirect indicators including cell proliferation, SA-β-Gal activity, telomerase activity, cell cycle, and the expression of matrix macromolecules (aggrecan and collagen II) and senescence markers (p16 and p53) were used to evaluate the premature senescence of NP cells. Additionally, intracellular reactive oxygen species (ROS) and NF-κB/p65 activity were also detected in the NP cell cultures.ResultsIn the NP cell cultures, E2 significantly increased cell proliferation potency, telomerase activity and the expression of matrix macromolecules but attenuated SA-β-Gal activity, senescence marker (p53 and p16) expression and G1 cycle arrest in TNF-α-treated NP cells. Furthermore, E2 inhibited ROS generation and phospho-NF-κB/p65 expression in the TNF-α-treated NP cells. However, the ER antagonist ICI 182780 abolished the effects of E2 on TNF-α-treated NP cells. In the disc organ cultures, E2 also significantly increased matrix synthesis, whereas it decreased senescence marker (p53 and p16) expression, which could be abolished by the ER antagonist ICI 182780.ConclusionThe interaction between E2 and ER can attenuate TNF-α-induced premature senescence of rat NP cells through interfering with the ROS/NF-κB pathway.

Project description:Luteolin (3',4',5,7-tetrahydroxy flavone) is a flavonoid, which is widely distributed in various plants including flowers, vegetables, and medicinal herbs and spices. Luteolin can be applied in the treatment of various diseases due to its multiple biological activities, such as anti-inflammatory, anticancer, and antioxidative activity. However, its role in intervertebral disc degeneration has not been previously reported. Therefore, the purpose of the present study was to explore the effects of luteolin on Tumor necrosis factor (TNF)-α-induced inflammatory injury and senescence of human nucleus pulposus cells (HNPCs), as well as the underlying mechanisms of action of this compound. Cell viability and apoptosis were assessed by MTT assay and TUNEL staining, respectively. ELISA kits were applied to detect the levels of inflammatory cytokines and the activity of telomerase. Senescence β-galactosidase staining was used to detect the activity levels of β-galactosidase in the cells. Cell transfection was performed to achieve interference of sirtuin 6 (Sirt6). The protein expression levels were detected by western blot analysis. TUNEL staining and western blot analysis were performed to assess the expression levels of apoptosis-related proteins. The results indicated that TNF-α induced a significant decrease in HNPC viability and an increase in inflammatory factor levels, while the application of luteolin effectively increased cell viability and decreased intracellular interleukin (IL)-1β and IL-6 expression levels. Furthermore, luteolin decreased apoptosis compared with the TNF-α groups in a dose-dependent manner. In addition, the results of the detection kits suggested that luteolin reversed TNF-α-induced senescence. Notably, interference with Sirt6 partially reduced the protective effect of luteolin on TNF-α-induced HNPC senescence via the Sirt6/NF-κB pathway. In summary, the data indicated that luteolin suppresses TNF-α-induced inflammatory injury and senescence of HNPCs via the Sirt6/NF-κB pathway.

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Cationic Polymer Brush-Modified Carbon Nanotube-Meditated eRNA LINC02569 Silencing Attenuates Nucleus Pulposus Degeneration by Blocking NF-κB Signaling Pathway and Alleviate Cell Senescence

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