The mechanism of N-acetylserotonin regulating microglial polarization via NOD1/Rip2 pathway in rats after retinal ischemia reperfusion_Chinese Journal of Ocular Fundus Diseases

Authors：

Xu Ying ¹ , Liu Jianliang ¹ , Zhao Yuze ¹ , Wang Chenxu ¹ , Fu Xinhao ² , Li Xiaoshuang ³ , Wang Xiaoli ² ,  Zhao Yansong ¹

1. Department of Ophthalmology, The Affiliated Hospital of Weifang Medical University, Weifang 261031, China;
2. School of Medical Imaging, Weifang Medical University, Weifang 261053, China;
3. School of Basic Medicine, Weifang Medical University, Weifang 261053, China;

Corresponding author：

Zhao Yansong, Email: zhaoyansong74@163.com

Keywords：

Retina ischemia-reperfusion injury; N-acetylserotonin; Retinal microglia; Nucleotide-binding and oligomerization domain; Animal experiment

DOI：

10.3760/cma.j.cn511434-20231007-00408

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Objective To investigate the effect of N-acetylserotonin (NAS) on the retinal microglia polarization in retinal ischemia-reperfusion injury (RIRI) rats and explore its mechanism via nucleotide-bound oligomeric domain 1 (NOD1)/receptor interacting protein 2 (Rip2) pathway. Methods Healthy male Sprague Dawley rats were randomly divided into Sham (n=21), RIRI (n=21) and NAS (injected intraperitoneally 30 min before and after modeling with NAS, 10 mg/kg, n=18) groups, using random number table. And the right eye was used experimental eye. The RIRI model of rats in RIRI group and NAS group was established by anterior chamber high intraocular pressure method. Rats in NAS group were intraperitoneally injected with 10 mg/kg NAS before and 30 min after modeling, respectively. The retinal morphology and the number of retinal ganglion cell (RGC) in each group were detected by hematoxylin-eosin staining and immunohistochemical staining. The effect of NAS on polarization of retinal microglia was detected by immunofluorescence staining. Transcriptome sequencing technology was used to screen out the differentially expressed genes between Sham and RIRI groups. Western blot and real-time quantitative polymerase chain reaction (RT-PCR) were used to examine the differentially expressed genes. Immunohistochemical staining, Western blot and RT-PCR were used to investigate the effect of NAS on the expression of NOD1 and Rip2 protein and mRNA in retinal tissue and microglia of rats. General linear regression analysis was performed to determine the correlation between the number difference of NOD1⁺ cells and the number difference of M1 and M2 microglia in retinal tissues of rats in NAS group and RIRI group. Results A large number of RGC were observed in the retina of rats in Sham group. 24 h after modeling, compared with Sham group, the inner retinal thickness of rats in RIRI group was significantly increased and the number of RGC was significantly decreased. The thickness of inner retina in NAS group was significantly thinner and the number of RGC was significantly increased. Compared with Sham group, the number of retinal microglia of M1 and M2 in RIRI group was significantly increased. Compared with RIRI group, the number of M1 microglia decreased significantly and the number of M2 microglia increased significantly in NAS group. There was statistical significance in the number of M1 and M2 microglia in the retina of the three groups (P＜0.05). Transcriptome sequencing results showed that retinal NOD1 and Rip2 were important differential genes 24 h after modeling. The mRNA and protein relative expressions of NOD1 and Rip2 in retina of RIRI group were significantly higher than those of Sham group, with statistical significance (P＜0.05). The number of NOD1⁺ and Rip2⁺ cells and the relative expression of mRNA and protein in retinal microglia in RIRI group were significantly higher than those in Sham group, and NAS group was also significantly higher than that in Sham group, but lower than that in RIRI group, with statistical significance (P＜0.05). The number of Iba-1⁺/NOD1⁺ and Iba-1⁺/Rip2⁺ cells in retinal microglia in RIRI group was significantly increased compared with that in Sham group, and the number of Iba-1⁺/Rip2⁺ cells in NAS group was significantly decreased compared with that in RIRI group, but still significantly higher than that in Sham group, with statistical significance (P＜0.05). Correlation analysis results showed that the difference of retinal NOD1⁺ and Rip2⁺ cells in NAS group and RIRI group was positively correlated with that of M1 microglia (r=0.851, 0.895), and negatively correlated with that of M2 microglia (r=−0.797, −0.819). The differences were statistically significant (P＜0.05). Conclusion NAS can regulate the microglial polarization from M1 to M2 phenotype, the mechanism is correlated with the NOD1/Rip2 pathway.

Citation： Xu Ying, Liu Jianliang, Zhao Yuze, Wang Chenxu, Fu Xinhao, Li Xiaoshuang, Wang Xiaoli, Zhao Yansong. The mechanism of N-acetylserotonin regulating microglial polarization via NOD1/Rip2 pathway in rats after retinal ischemia reperfusion. Chinese Journal of Ocular Fundus Diseases, 2024, 40(4): 287-295. doi: 10.3760/cma.j.cn511434-20231007-00408 Copy

1.	Chronopoulos P, Manicam C, Zadeh JK, et al. Effects of resveratrol on vascular function in retinal ischemia-reperfusion injury[J]. Antioxidants (Basel), 2023, 12(4): 853. DOI: 10.3390/antiox12040853.
2.	Shi Y, Liu Y, Wu C, et al. N, N-Dimethyl-3beta-hydroxycholenamide attenuates neuronal death and retinal inflammation in retinal ischemia/reperfusion injury by inhibiting Ninjurin 1[J]. J Neuroinflammation, 2023, 20(1): 91. DOI: 10.1186/s12974-023-02754-5.
3.	Chen X, Wang X, Cui Z, et al. M1 microglia-derived exosomes promote activation of resting microglia and amplifies proangiogenic effects through Irf1/miR-155-5p/Socs1 axis in the retina[J]. Int J Biol Sci, 2023, 19(6): 1791-812. DOI: 10.7150/ijbs.79784.
4.	Chen D, Peng C, Ding XM, et al. Interleukin-4 promotes microglial polarization toward a neuroprotective phenotype after retinal ischemia/reperfusion injury[J]. Neural Regen Res, 2022, 17(12): 2755-2760. DOI: 10.4103/1673-5374.339500.
5.	Chen Y, Lin J, Schlotterer A, et al. MicroRNA-124 alleviates retinal vasoregression via regulating microglial polarization[J/OL]. Int J Mol Sci, 2021, 22(20): 11068[2021-10-14]. https://pubmed.ncbi.nlm.nih.gov/34681723/. DOI: 10.3390/ijms222011068.
6.	Sun X, Ma L, Li X, et al. Ferulic acid alleviates retinal neovascularization by modulating microglia/macrophage polarization through the ROS/NF-kappaB axis[J/OL]. Front Immunol, 2022, 13: 976729[2022-09-02]. https://pubmed.ncbi.nlm.nih.gov/36119027/. DOI: 10.3389/fimmu.2022.976729.
7.	Aparicio-Soto M, Alarcón-de-la-Lastra C, Cárdeno A, et al. Melatonin modulates microsomal PGE synthase 1 and NF-E2-related factor-2-regulated antioxidant enzyme expression in LPS-induced murine peritoneal macrophages[J]. Br J Pharmacol, 2014, 171(1): 134-144. DOI: 10.1111/bph.12428.
8.	Caruso R, Warner N, Inohara N, et al. NOD1 and NOD2: signaling, host defense, and inflammatory disease[J]. Immunity, 2014, 41(6): 898-908. DOI: 10.1016/j.immuni.2014.12.010.
9.	Trindade BC, Chen GY. NOD1 and NOD2 in inflammatory and infectious diseases[J]. Immunol Rev, 2020, 297(1): 139-161. DOI: 10.1111/imr.12902.
10.	Lin HB, Naito K, Oh Y, et al. Innate immune Nod1/RIP2 signaling is essential for cardiac hypertrophy but requires mitochondrial antiviral signaling protein for signal transductions and energy balance[J]. Circulation, 2020, 142(23): 2240-2258. DOI: 10.1161/CIRCULATIONAHA.119.041213.
11.	Liu J, Zhang N, Zhang M, et al. N-acetylserotonin alleviated the expression of interleukin-1beta in retinal ischemia-reperfusion rats via the TLR4/NF-kappaB/NLRP3 pathway[J/OL]. Exp Eye Res, 2021, 208: 108595[2021-07-07]. https://pubmed.ncbi.nlm.nih.gov/34243961/. DOI: 10.1016/j.exer.2021.108595.
12.	Zhou T, Yang Z, Ni B, et al. IL-4 induces reparative phenotype of RPE cells and protects against retinal neurodegeneration via Nrf2 activation[J]. Cell Death Dis, 2022, 13(12): 1056. DOI: 10.1038/s41419-022-05433-0.
13.	Scheid S, Goeller M, Baar W, et al. Inhalative as well as intravenous administration of H(2)S provides neuroprotection after ischemia and reperfusion injury in the rats' retina[J/OL]. Int J Mol Sci, 2022, 23(10): 5519[2022-05-15]. https://pubmed.ncbi.nlm.nih.gov/35628328/. DOI: 10.3390/ijms23105519.
14.	Shosha E, Fouda AY, Lemtalsi T, et al. Endothelial arginase 2 mediates retinal ischemia/reperfusion injury by inducing mitochondrial dysfunction[J/OL]. Mol Metab, 2021, 53: 101273[2021-06-15]. https://pubmed.ncbi.nlm.nih.gov/34139341/. DOI: 10.1016/j.molmet.2021.101273.
15.	Qin Q, Yu N, Gu Y, et al. Inhibiting multiple forms of cell death optimizes ganglion cells survival after retinal ischemia reperfusion injury[J]. Cell Death Dis, 2022, 13(5): 507. DOI: 10.1038/s41419-022-04911-9.
16.	Jiang N, Li Z, Li Z, et al. Laquinimod exerts anti-inflammatory and antiapoptotic effects in retinal ischemia/reperfusion injury[J/OL]. Int Immunopharmacol, 2020, 88: 106989[2020-09-18]. https://pubmed.ncbi.nlm.nih.gov/33182069/. DOI: 10.1016/j.intimp.2020.106989.
17.	Yun-Jia L, Xi C, Jie-Qiong Z, et al. Semaphorin3A increases M1-like microglia and retinal ganglion cell apoptosis after optic nerve injury[J]. Cell Biosci, 2021, 11(1): 97. DOI: 10.1186/s13578-021-00603-7.
18.	Colonna M, Butovsky O. Microglia function in the central nervous system during health and neurodegeneration[J]. Annu Rev Immunol, 2017, 35: 441-468. DOI: 10.1146/annurev-immunol-051116-052358.
19.	Wang M, Ye X, Hu J, et al. NOD1/RIP2 signalling enhances the microglia-driven inflammatory response and undergoes crosstalk with inflammatory cytokines to exacerbate brain damage following intracerebral haemorrhage in mice[J]. J Neuroinflammation, 2020, 17(1): 364. DOI: 10.1186/s12974-020-02015-9.
20.	Liu Y, Li S, Zhang G, et al. NOD1 induces pyroptotic cell death to aggravate liver ischemia-reperfusion injury in mice[J/OL]. MedComm (2020), 2022, 3(3): e170[2022-08-31]. https://pubmed.ncbi.nlm.nih.gov/36092860/. DOI: 10.1002/mco2.170.
21.	Yang H, Li N, Song LN, et al. Activation of NOD1 by DAP contributes to myocardial ischemia/reperfusion injury via multiple signaling pathways[J]. Apoptosis, 2015, 20(4): 512-522. DOI: 10.1007/s10495-015-1089-1.
22.	Liu Y, Guo Y. Activation of nucleotide-binding oligomerization domain-containing protein 1 by diaminopimelic acid contributes to cerebral ischemia-induced cognitive impairment[J/OL]. Neurosci Lett, 2021, 743: 135547[2021-01-19]. https://pubmed.ncbi.nlm.nih.gov/33352290/. DOI: 10.1016/j.neulet.2020.135547.

1. Chronopoulos P, Manicam C, Zadeh JK, et al. Effects of resveratrol on vascular function in retinal ischemia-reperfusion injury[J]. Antioxidants (Basel), 2023, 12(4): 853. DOI: 10.3390/antiox12040853.
2. Shi Y, Liu Y, Wu C, et al. N, N-Dimethyl-3beta-hydroxycholenamide attenuates neuronal death and retinal inflammation in retinal ischemia/reperfusion injury by inhibiting Ninjurin 1[J]. J Neuroinflammation, 2023, 20(1): 91. DOI: 10.1186/s12974-023-02754-5.
3. Chen X, Wang X, Cui Z, et al. M1 microglia-derived exosomes promote activation of resting microglia and amplifies proangiogenic effects through Irf1/miR-155-5p/Socs1 axis in the retina[J]. Int J Biol Sci, 2023, 19(6): 1791-812. DOI: 10.7150/ijbs.79784.
4. Chen D, Peng C, Ding XM, et al. Interleukin-4 promotes microglial polarization toward a neuroprotective phenotype after retinal ischemia/reperfusion injury[J]. Neural Regen Res, 2022, 17(12): 2755-2760. DOI: 10.4103/1673-5374.339500.
5. Chen Y, Lin J, Schlotterer A, et al. MicroRNA-124 alleviates retinal vasoregression via regulating microglial polarization[J/OL]. Int J Mol Sci, 2021, 22(20): 11068[2021-10-14]. https://pubmed.ncbi.nlm.nih.gov/34681723/. DOI: 10.3390/ijms222011068.
6. Sun X, Ma L, Li X, et al. Ferulic acid alleviates retinal neovascularization by modulating microglia/macrophage polarization through the ROS/NF-kappaB axis[J/OL]. Front Immunol, 2022, 13: 976729[2022-09-02]. https://pubmed.ncbi.nlm.nih.gov/36119027/. DOI: 10.3389/fimmu.2022.976729.
7. Aparicio-Soto M, Alarcón-de-la-Lastra C, Cárdeno A, et al. Melatonin modulates microsomal PGE synthase 1 and NF-E2-related factor-2-regulated antioxidant enzyme expression in LPS-induced murine peritoneal macrophages[J]. Br J Pharmacol, 2014, 171(1): 134-144. DOI: 10.1111/bph.12428.
8. Caruso R, Warner N, Inohara N, et al. NOD1 and NOD2: signaling, host defense, and inflammatory disease[J]. Immunity, 2014, 41(6): 898-908. DOI: 10.1016/j.immuni.2014.12.010.
9. Trindade BC, Chen GY. NOD1 and NOD2 in inflammatory and infectious diseases[J]. Immunol Rev, 2020, 297(1): 139-161. DOI: 10.1111/imr.12902.
10. Lin HB, Naito K, Oh Y, et al. Innate immune Nod1/RIP2 signaling is essential for cardiac hypertrophy but requires mitochondrial antiviral signaling protein for signal transductions and energy balance[J]. Circulation, 2020, 142(23): 2240-2258. DOI: 10.1161/CIRCULATIONAHA.119.041213.
11. Liu J, Zhang N, Zhang M, et al. N-acetylserotonin alleviated the expression of interleukin-1beta in retinal ischemia-reperfusion rats via the TLR4/NF-kappaB/NLRP3 pathway[J/OL]. Exp Eye Res, 2021, 208: 108595[2021-07-07]. https://pubmed.ncbi.nlm.nih.gov/34243961/. DOI: 10.1016/j.exer.2021.108595.
12. Zhou T, Yang Z, Ni B, et al. IL-4 induces reparative phenotype of RPE cells and protects against retinal neurodegeneration via Nrf2 activation[J]. Cell Death Dis, 2022, 13(12): 1056. DOI: 10.1038/s41419-022-05433-0.
13. Scheid S, Goeller M, Baar W, et al. Inhalative as well as intravenous administration of H(2)S provides neuroprotection after ischemia and reperfusion injury in the rats' retina[J/OL]. Int J Mol Sci, 2022, 23(10): 5519[2022-05-15]. https://pubmed.ncbi.nlm.nih.gov/35628328/. DOI: 10.3390/ijms23105519.
14. Shosha E, Fouda AY, Lemtalsi T, et al. Endothelial arginase 2 mediates retinal ischemia/reperfusion injury by inducing mitochondrial dysfunction[J/OL]. Mol Metab, 2021, 53: 101273[2021-06-15]. https://pubmed.ncbi.nlm.nih.gov/34139341/. DOI: 10.1016/j.molmet.2021.101273.
15. Qin Q, Yu N, Gu Y, et al. Inhibiting multiple forms of cell death optimizes ganglion cells survival after retinal ischemia reperfusion injury[J]. Cell Death Dis, 2022, 13(5): 507. DOI: 10.1038/s41419-022-04911-9.
16. Jiang N, Li Z, Li Z, et al. Laquinimod exerts anti-inflammatory and antiapoptotic effects in retinal ischemia/reperfusion injury[J/OL]. Int Immunopharmacol, 2020, 88: 106989[2020-09-18]. https://pubmed.ncbi.nlm.nih.gov/33182069/. DOI: 10.1016/j.intimp.2020.106989.
17. Yun-Jia L, Xi C, Jie-Qiong Z, et al. Semaphorin3A increases M1-like microglia and retinal ganglion cell apoptosis after optic nerve injury[J]. Cell Biosci, 2021, 11(1): 97. DOI: 10.1186/s13578-021-00603-7.
18. Colonna M, Butovsky O. Microglia function in the central nervous system during health and neurodegeneration[J]. Annu Rev Immunol, 2017, 35: 441-468. DOI: 10.1146/annurev-immunol-051116-052358.
19. Wang M, Ye X, Hu J, et al. NOD1/RIP2 signalling enhances the microglia-driven inflammatory response and undergoes crosstalk with inflammatory cytokines to exacerbate brain damage following intracerebral haemorrhage in mice[J]. J Neuroinflammation, 2020, 17(1): 364. DOI: 10.1186/s12974-020-02015-9.
20. Liu Y, Li S, Zhang G, et al. NOD1 induces pyroptotic cell death to aggravate liver ischemia-reperfusion injury in mice[J/OL]. MedComm (2020), 2022, 3(3): e170[2022-08-31]. https://pubmed.ncbi.nlm.nih.gov/36092860/. DOI: 10.1002/mco2.170.
21. Yang H, Li N, Song LN, et al. Activation of NOD1 by DAP contributes to myocardial ischemia/reperfusion injury via multiple signaling pathways[J]. Apoptosis, 2015, 20(4): 512-522. DOI: 10.1007/s10495-015-1089-1.
22. Liu Y, Guo Y. Activation of nucleotide-binding oligomerization domain-containing protein 1 by diaminopimelic acid contributes to cerebral ischemia-induced cognitive impairment[J/OL]. Neurosci Lett, 2021, 743: 135547[2021-01-19]. https://pubmed.ncbi.nlm.nih.gov/33352290/. DOI: 10.1016/j.neulet.2020.135547.

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The mechanism of N-acetylserotonin regulating microglial polarization via NOD1/Rip2 pathway in rats after retinal ischemia reperfusion

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