Gencefe Client Article｜ Engeletin regulates microglial inflammation via TLR4RRP9NF-κB pathway after spinal cord injury

After spinal cord injury (SCI), the injury site blocks the neural connection between the upper and lower segments of the spinal cord, leading to neurological dysfunction, including abnormal reflexes, sensory and motor disorders [1-2]. Reducing secondary neuronal loss or protecting neurons from death is the primary goal of interventional treatment for spinal cord injury. Microglia play their essential anti-inflammatory or pro-inflammatory functions in the injury environment by changing their cell morphology and protein expression profile [3-4]. The natural compound Engeletin (ENG, dihydrokaempferol 3-rhamnoside) has anti-inflammatory, antibacterial and anti-tumor effects [5-7]. The Rrp9 and Sdad1 genes play a role in ribosome biogenesis and are associated with the development of various cancers. [8-10] However, the role of ENG and its hub genes Rrp9 and Sdad1 in regulating SCI microglial polarization and inflammatory response needs further research and verification.

In November 2024, the research team led by Wang Lichun from the Second Affiliated Hospital of Harbin Medical University published an article titled "Regulation of microglia inflammation and oligodendrocyte demyelination by Engeletin via the TLR4/RRP9/NF-κB pathway after spinal cord injury" in the journal Pharmacological Research. They discovered the gene Rrp9/Sdad1 and the small molecule compound ENG that regulate microglial inflammatory response, which has important theoretical significance for further exploring the related mechanisms of oligodendrocyte demyelination. [11]

In this study, the Rrp9 gene synthesized by Cesophy was used and cloned into a eukaryotic expression vector for overexpression of the Rrp9 protein.

Research results:

1.ENG can inhibit the expression of RRP9 under LPS/SCI injury.

The researchers screened out the hub genes Rrp9 and Sdad1 through bioinformatics analysis and verified the PPI (protein-protein interaction) relationship between the two. And through in vivo and in vitro experiments, it was found that the expression of RRP9 and SDAD1 proteins under LPS (lipopolysaccharide)/SCI injury was significantly increased.

The motor function of rats was partially restored after ENG injection. The results of in vitro and in vivo experiments suggested that ENG can reduce the protein expression levels of RRP9 and SDAD1, and reduce M1 polarization and inflammatory response through the NF-κB signaling pathway. After ENG alleviates the inflammatory response, it will reduce the degree of oligodendrocyte demyelination.

2.ENG binds to TLR4 and regulates the NF-κB signaling pathway through RRP9 to affect microglial inflammatory response and oligodendrocyte apoptosis

Through the docking simulation of small molecule ENG-protein TLR4, it was found that ENG has a direct binding site with TLR4, suggesting that ENG may reduce the protein expression levels of RRP9 and SDAD1 through TLR4. ( Figure 1A ) The relationship between ENG and TLR4 was confirmed in in vitro and in vivo experiments. ENG could effectively reduce the expression level of TLR4 protein on the 3rd day after SCI ( Figure 1 C-F ) and the expression level of TLR4 protein

24 h after LPS stimulation ( Figure 1 G-J )

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RNA-seq analysis of Rrp9 knockdown and control groups under LPS stimulation indicated that the Rrp9 gene may be involved in TNF and NF-κB signaling pathways. Detection of changes in related protein expression levels after knockdown of Rrp9 indicated that RRP9 may regulate the inflammatory response of microglia through the NF-κB signaling pathway.

Under LPS stimulation, Rrp9 was knocked down, and the expression level changes of multiple proteins were detected after adding ENG or the specific agonist of TLR1/2 CU-T12–9 (CU), suggesting that ENG binds to TLR4 and regulates the NF-κB signaling pathway through RRP9. (Figure 3 A-K), affecting the inflammatory response of microglia. When microglia were co-cultured with oligodendrocytes, both ENG and Rrp9 knockdown reduced dendrocyte apoptosis, while the addition of CU significantly increased oligodendrocyte apoptosis (Figure 3 L-N).

3. RRP9 and SDAD1 affect microglial M1 polarization and inflammatory response through the NF-κB signaling pathway

After knocking down Sdad1 under LPS stimulation, the changes in the expression levels of multiple proteins were detected. The results suggested that SDAD1 may regulate the inflammatory response of microglia through the NF-κB signaling pathway in vitro (Figure 4 A-L). On this basis, after overexpressing Rrp9 protein, ENG or CU was added, and the expression levels of multiple proteins were analyzed again (Figure 4 M-V). The results showed that ENG/TLR4 can regulate the NF-κB signaling pathway through RRP9/SDAD1, thereby reducing the polarization and inflammatory response of microglia in vitro.

4. ENG can regulate the NF-κB signaling pathway through TLR4/RRP9 to affect the inflammatory response, oligodendrocyte apoptosis and demyelination in vivo.

After injection of Lv-Rrp9KD and ENG, the motor function of rats further recovered, multiple indicators improved, and inflammatory cell infiltration decreased, while CU injection reduced the motor function of rats (Figure 5 A-C). With knockdown of Rrp9, the expression levels of Rrp9, SDAD1 and other proteins were detected to be reduced, and ENG injection further reduced protein expression, while the opposite was true after CU injection (Figure 5D-K).

After ENG injection and Rrp9 knockdown, cell apoptosis was reduced, and the demyelination of oligodendrocytes was also significantly reduced; however, after CU injection, cell apoptosis increased and the demyelination of oligodendrocytes was aggravated ( Figure 5 L-O).

In summary, this study demonstrated that ENG binds to TLR4, mediating the interaction proteins RRP9 and SDAD1 to regulate neuroinflammatory response and oligodendrocyte demyelination after spinal cord injury through the NF-κB signaling pathway. The key gene Rrp9/Sdad1 and the small molecule compound ENG that regulate microglial polarization and inflammatory response were studied, and the related mechanisms of microglial polarization and oligodendrocyte demyelination were further explored, which has important theoretical significance for the study of the related mechanisms of neuroinflammatory response and oligodendrocyte demyelination after SCI.

【References】

[1]????? J.A.G. Crispo, L.K. Kuramoto, J.J. Cragg. Global burden of spinal cord injury: future directions. Lancet Neurol., 22 (11) (2023), pp. 976-978

[2]????? M. Bydon, W. Qu, F.M. Moinuddin, C.L. Hunt, K.L. Garlanger, R.K. Reeves, A.J. Windebank, K.D. Zhao, R. Jarrah, B.C. Trammell, S.El Sammak, G.D. Michalopoulos, K. Katsos, S.P. Graepel, K.L. Seidel-Miller, L.A. Beck, R.S. Laughlin, A.B. Dietz. Intrathecal delivery of adipose-derived mesenchymal stem cells in traumatic spinal cord injury: Phase I trial. Nat. Commun., 15 (1) (2024), p. 2201

[3]????? N. Yin, Y. Zhao, C. Liu, Y. Yang, Z. Wang, W. Yu, K. Zhang, Z. Zhang, J. Liu, Y. Zhang, J. Shi. Engineered nanoerythrocytes alleviate central nervous system inflammation by regulating the polarization of inflammatory microglia. Adv. Mater., 34 (27) (2022), Article e2201322

[4]????? R. Gu, J. Pan, M.U.N. Awan, X. Sun, F. Yan, L. Bai, J. Bai. The major histocompatibility complex participates in Parkinson's disease. Pharmacol. Res., 203 (2024), Article 107168

[5]????? V.P. Pushkala, S.M.P. Sulekha, S. Mathukumar, B. Ragavi, U. Sowmiya. Molecular docking analysis of siddha formulation parangipattai chooranam against vaginal candidiasis. Appl., Biochem. Biotechnol., 194 (3) (2022), pp. 1039-1050

[6]????? H. Bai, H. Yin. Engeletin suppresses cervical carcinogenesis in vitro and in vivo by reducing NF-κB-dependent signaling. Biochem. Biophys. Res. Commun., 526 (2) (2020), pp. 497-504

[7]????? T. Liu, Y. Li, J. Sun, G. Tian, Z. Shi. Engeletin suppresses lung cancer progression by inducing apoptotic cell death through modulating the XIAP signaling pathway: a molecular mechanism involving ER stress. Biomed. Pharmacother., 128 (2020), Article 110221

[8]????? Z. Zhang, H. Yu, W. Yao, N. Zhu, R. Miao, Z. Liu, X. Song, C. Xue, C. Cai, M. Cheng, K. Lin, D. Qi. RRP9 promotes gemcitabine resistance in pancreatic cancer via activating AKT signaling pathway. Cell Commun. Signal., 20 (1) (2022), p. 188

[9]????? Z. Ding, H. Lan, R. Xu, X. Zhou, Y. Pan. LncRNA TP73-AS1 accelerates tumor progression in gastric cancer through regulating miR-194-5p/SDAD1 axis. Pathol. Res. Pract., 214 (12) (2018), pp. 1993-1999

[10]?? M. Zeng, L. Zhu, L. Li, C. Kang. miR-378 suppresses the proliferation, migration and invasion of colon cancer cells by inhibiting SDAD1. Cell Mol. Biol. Lett., 22 (2017), p. 12

[11]?? Chen, W., Zhang, L., Zhong, G., Liu, S., Sun, Y., Zhang, J., ... & Wang, L. (2024). Regulation of microglia inflammation and oligodendrocyte demyelination by Engeletin via the TLR4/RRP9/NF-κB pathway after spinal cord injury.?Pharmacological Research,?209, 107448.