Nrf1 Knock-Down in the Hypothalamic Paraventricular Nucleus Alleviates Hypertension Through Intervention of Superoxide Production-Removal Balance and Mitochondrial Function


Oxidative stress in the hypothalamic paraventricular nucleus (PVN) contributes greatly to the development of hypertension. The recombinant nuclear respiratory factor 1 (Nrf1) regulates the transcription of several genes related to mitochondrial respiratory chain function or antioxidant expression, and thus may be involved in the pathogenesis of hypertension. Here we show that in the two-kidney, one-clip (2K1C) hypertensive rats the transcription level of Nrf1 was elevated comparing to the normotensive controls. Knocking down of Nrf1 in the PVN of 2K1C rats can significantly reduce their blood pressure and level of plasma norepinephrine (NE). Analysis revealed significant reduction of superoxide production level in both whole cell and mitochondria, along with up-regulation of superoxide dismutase 1 (Cu/Zn-SOD), NAD(P)H: quinone oxidoreductase 1 (NQO1), thioredoxin-dependent peroxiredoxin 3 (Prdx3), cytochrome c (Cyt-c) and glutathione synthesis rate-limiting enzyme (glutamyl-cysteine ligase catalytic subunit (Gclc) and modifier subunit (Gclm)), and down-regulation of cytochrome c oxidase subunit VI c (Cox6c) transcription after Nrf1 knock-down. In addition, the reduced ATP production and elevated mitochondrial membrane potential in the PVN of 2K1C rats were reinstated with Nrf1 knock-down, together with restored expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), mitochondrial transcription factor A (Tfam), coiled-coil myosin-like BCL2-interacting protein (Beclin1), and Mitofusin 1 (Mfn1), which are related to the mitochondrial biogenesis, fusion, and autophagy. Together, the results indicate that the PVN Nrf1 is associated with the development of 2K1C-induced hypertension, and Nrf1 knock-down in the PVN can alleviate hypertension through intervention of mitochondrial function and restorement of the production-removal balance of superoxide.

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Data Availability

The data are available upon request to the corresponding authors.



Two-kidney, one-clip


Adeno-associated virus

Beclin1 :

Coiled-coil myosin-like BCL2-interacting protein


Cap-n-collar subfamily of basic leucine zipper

Cox2 :

Mitochondrial-encoded subunit II

Cox6c :

Cytochrome c oxidase subunit VI c


Cu/Zn superoxide dismutase, superoxide dismutase 1

Cyt-c :

Cytochrome c



Drp1 :

Cytosolic GTPase dynamin-related protein 1


Enzyme linked immunosorbent assay

Gclc :

Glutamyl-cysteine ligase catalytic subunit

Gclm :

Glutamyl-cysteine ligase modifier subunit



HE staining:

Hematoxylin–eosin staining




Interleukin-1 beta

Mfn1/2 :

Mitofusin 1/2

Miro1 :

Mitochondrial rho GTPase 1


Mitochondrial DNA



NQO1 :

NAD(P)H: quinone oxidoreductase 1

Nrf1 :

Recombinant nuclear respiratory factor 1

Nrf2 :

Nuclear respiratory factor 2

PGC-1α :

Peroxisome proliferator-activated receptor-γ coactivator 1α


Pro-inflammatory cytokine


Phenylmethanesulfonyl fluoride

Prdx3 :

Thioredoxin-dependent peroxiredoxin 3


Hypothalamic paraventricular nucleus


Reactive oxygen species


Reverse transcript quantitative-polymerase chain reaction


Systolic blood pressure




Spontaneously hypertensive rats

Tfam :

Mitochondrial transcription factor A

Vdac1 :

Voltage-dependent anion-selective channel protein 1


  1. 1.

    Nakshi, S., & Pradeep Kumar, D. (2015). Oxidative stress and antioxidants in hypertension: A current review. Current Hypertension Reviews, 11(2), 132–142.

    CAS  Article  Google Scholar 

  2. 2.

    Bhatti, J. S., Bhatti, G. K., & Reddy, P. H. (2017). Mitochondrial dysfunction and oxidative stress in metabolic disorders: A step towards mitochondria based therapeutic strategies. Biochimica et Biophysica Acta: Molecular Basis of Disease, 1863(5), 1066–1077.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Murphy, M. P. (2009). How mitochondria produce reactive oxygen species. The Biochemical Journal, 417(1), 1–13.

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Dubois-Deruy, E., Peugnet, V., Turkieh, A., & Pinet, F. (2020). Oxidative stress in cardiovascular diseases. Antioxidants, 9(9), 864.

    CAS  Article  PubMed Central  Google Scholar 

  5. 5.

    Togliatto, G., Lombardo, G., & Brizzi, M. F. (2017). The future challenge of reactive oxygen species (ROS) in hypertension: From bench to bed side. International Journal of Molecular Sciences, 18(9), 1988.

    CAS  Article  PubMed Central  Google Scholar 

  6. 6.

    Coleman, C. G., Wang, G., Faraco, G., Marques Lopes, J., Waters, E. M., Milner, T. A., et al. (2013). Membrane trafficking of NADPH oxidase p47(phox) in paraventricular hypothalamic neurons parallels local free radical production in angiotensin II slow-pressor hypertension. Journal of Neuroscience, 33(10), 4308–4316.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Benarroch, E. E. (2005). Paraventricular nucleus, stress response, and cardiovascular disease. Clinical Autonomic Research, 15(4), 254–263.

    Article  PubMed  Google Scholar 

  8. 8.

    Biag, J., Huang, Y., Gou, L., Hintiryan, H., Askarinam, A., Hahn, J. D., et al. (2012). Cyto- and chemoarchitecture of the hypothalamic paraventricular nucleus in the C57BL/6J male mouse: A study of immunostaining and multiple fluorescent tract tracing. The Journal of Comparative Neurology, 520(1), 6–33.

    Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Tian, H., Kang, Y. M., Gao, H. L., Shi, X. L., Fu, L. Y., Li, Y., et al. (2019). Chronic infusion of berberine into the hypothalamic paraventricular nucleus attenuates hypertension and sympathoexcitation via the ROS/Erk1/2/iNOS pathway. Phytomedicine, 52, 216–224.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Han, Y., Zhang, Y., Wang, H. J., Gao, X. Y., Wang, W., & Zhu, G. Q. (2005). Reactive oxygen species in paraventricular nucleus modulates cardiac sympathetic afferent reflex in rats. Brain Research, 1058(1–2), 82–90.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Wang, M. L., Yu, X. J., Li, X. G., Pang, D. Z., Su, Q., Saahene, R. O., et al. (2018). Blockade of TLR4 within the paraventricular nucleus attenuates blood pressure by regulating ROS and inflammatory cytokines in prehypertensive rats. American Journal of Hypertension, 31(9), 1013–1023.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Yu, X. J., Zhao, Y. N., Hou, Y. K., Li, H. B., Xia, W. J., Gao, H. L., et al. (2019). Chronic intracerebroventricular infusion of metformin inhibits salt-sensitive hypertension via attenuation of oxidative stress and neurohormonal excitation in rat paraventricular nucleus. Neuroscience Bulletin, 35(1), 57–66.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Andreyev, A. Y., Kushnareva, Y. E., & Starkov, A. A. (2005). Mitochondrial metabolism of reactive oxygen species. Biochemistry, 70(2), 200–214.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Zorov, D. B., Juhaszova, M., & Sollott, S. J. (2014). Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiological Reviews, 94(3), 909–950.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Scialò, F., Fernández-Ayala, D. J., & Sanz, A. (2017). Role of mitochondrial reverse electron transport in ROS signaling: Potential roles in health and disease. Frontiers in Physiology, 8, 428.

    Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Mazat, J. P., Devin, A., & Ransac, S. (2020). Modelling mitochondrial ROS production by the respiratory chain. Cellular and Molecular Life Sciences, 77(3), 455–465.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Gardner, P. R., Raineri, I., Epstein, L. B., & White, C. W. (1995). Superoxide radical and iron modulate aconitase activity in mammalian cells. Journal of Biological Chemistry, 270(22), 13399–13405.

    CAS  Article  Google Scholar 

  18. 18.

    Pereverzev, M. O., Vygodina, T. V., Konstantinov, A. A., & Skulachev, V. P. (2003). Cytochrome c, an ideal antioxidant. Biochemical Society Transactions, 31(Pt 6), 1312–1315.

    CAS  Article  Google Scholar 

  19. 19.

    Fujii, J., & Ikeda, Y. (2002). Advances in our understanding of peroxiredoxin, a multifunctional, mammalian redox protein. Redox Report, 7(3), 123–130.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Zhang, Y., & Xiang, Y. (2016). Molecular and cellular basis for the unique functioning of Nrf1, an indispensable transcription factor for maintaining cell homoeostasis and organ integrity. Biochemical Journal, 473(8), 961–1000.

    CAS  Article  Google Scholar 

  21. 21.

    Dhar, S. S., Liang, H. L., & Wong-Riley, M. T. (2009). Nuclear respiratory factor 1 co-regulates AMPA glutamate receptor subunit 2 and cytochrome c oxidase: Tight coupling of glutamatergic transmission and energy metabolism in neurons. Journal of Neurochemistry, 108(6), 1595–1606.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Zhao, R., Hou, Y., Xue, P., Woods, C. G., Fu, J., Feng, B., et al. (2011). Long isoforms of NRF1 contribute to arsenic-induced antioxidant response in human keratinocytes. Environmental Health Perspectives, 119(1), 56–62.

    CAS  Article  Google Scholar 

  23. 23.

    Venugopal, R., & Jaiswal, A. K. (1996). Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene. Proceedings of the National Academy of Sciences of the United States of America, 93(25), 14960–14965.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Ohtsuji, M., Katsuoka, F., Kobayashi, A., Aburatani, H., Hayes, J. D., & Yamamoto, M. (2008). Nrf1 and Nrf2 play distinct roles in activation of antioxidant response element-dependent genes. Journal of Biological Chemistry, 283(48), 33554–33562.

    CAS  Article  Google Scholar 

  25. 25.

    Silva, E., & L. F. S., Brito, M. D., Yuzawa, J. M. C., & Rosenstock, T. R.. (2019). Mitochondrial dysfunction and changes in high-energy compounds in different cellular models associated to hypoxia: Implication to schizophrenia. Scientific Reports, 9(1), 18049–18049.

    CAS  Article  Google Scholar 

  26. 26.

    Wiesel, P., Mazzolai, L., Nussberger, J., & Pedrazzini, T. (1997). Two-kidney, one clip and one-kidney, one clip hypertension in mice. Hypertension, 29(4), 1025–1030.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Niu, N., Song, J., Zhao, Y., Wang, H., Zhu, M., Tong, X., et al. (2016). Effect of NRF-1 gene on mitochondrial membrane potential of rats’ cardiomyocytes under the culture of hypoxia. Journal of Ningxia Medical University, 38(006), 648–652.

    Google Scholar 

  28. 28.

    Bai, J., Yu, X. J., Liu, K. L., Wang, F. F., Jing, G. X., Li, H. B., et al. (2017). Central administration of tert-butylhydroquinone attenuates hypertension via regulating Nrf2 signaling in the hypothalamic paraventricular nucleus of hypertensive rats. Toxicology and Applied Pharmacology, 333, 100–109.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Qi, J., Yu, X.-J., Fu, L.-Y., Liu, K.-L., Gao, T.-T., Tu, J.-W., et al. (2019). Exercise training attenuates hypertension through TLR4/MyD88/NF-κB signaling in the hypothalamic paraventricular nucleus. Frontiers in Neuroscience, 13, 1138.

    Article  Google Scholar 

  30. 30.

    Yi, Q. Y., Li, H. B., Qi, J., Yu, X. J., Huo, C. J., Li, X., et al. (2016). Chronic infusion of epigallocatechin-3-O-gallate into the hypothalamic paraventricular nucleus attenuates hypertension and sympathoexcitation by restoring neurotransmitters and cytokines. Toxicology Letters, 262, 105–113.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Su, Q., Qin, D. N., Wang, F. X., Ren, J., Li, H. B., Zhang, M., et al. (2014). Inhibition of reactive oxygen species in hypothalamic paraventricular nucleus attenuates the renin-angiotensin system and proinflammatory cytokines in hypertension. Toxicology and Applied Pharmacology, 276(2), 115–120.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Tsuji, K., Copeland, N. G., Jenkins, N. A., & Obinata, M. (1995). Mammalian antioxidant protein complements alkylhydroperoxide reductase (ahpC) mutation in Escherichia coli. Biochemical Journal, 307(Pt 2), 377–381.

    CAS  Article  PubMed Central  Google Scholar 

  33. 33.

    Cao, Z., Bhella, D., & Lindsay, J. G. (2007). Reconstitution of the mitochondrial PrxIII antioxidant defence pathway: General properties and factors affecting PrxIII activity and oligomeric state. Journal of Molecular Biology, 372(4), 1022–1033.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Dhar, S. S., & Wong-Riley, M. T. (2009). Coupling of energy metabolism and synaptic transmission at the transcriptional level: Role of nuclear respiratory factor 1 in regulating both cytochrome c oxidase and NMDA glutamate receptor subunit genes. Journal of Neuroscience, 29(2), 483–492.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Thinnes, F. P., Walter, G., Hellmann, K. P., Hellmann, T., Merker, R., Kiafard, Z., et al. (2001). Gadolinium as an opener of the outwardly rectifying Cl(−) channel (ORCC). Is there relevance for cystic fibrosis therapy? Pflugers Archiv, 443(Suppl 1), S111–S116.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Blachly-Dyson, E., Zambronicz, E. B., Yu, W. H., Adams, V., McCabe, E. R., Adelman, J., et al. (1993). Cloning and functional expression in yeast of two human isoforms of the outer mitochondrial membrane channel, the voltage-dependent anion channel. Journal of Biological Chemistry, 268(3), 1835–1841.

    CAS  Article  Google Scholar 

  37. 37.

    Wang, F. F., Ba, J., Yu, X. J., Shi, X. L., Liu, J. J., Liu, K. L., et al. (2020). Central blockade of E-prostanoid 3 receptor ameliorated hypertension partially by attenuating oxidative stress and inflammation in the hypothalamic paraventricular nucleus of spontaneously hypertensive rats. Cardiovascular Toxicology.

    Article  PubMed  Google Scholar 

  38. 38.

    Yang, Q., Yu, X. J., Su, Q., Yi, Q. Y., Song, X. A., Shi, X. L., et al. (2020). Blockade of c-Src within the paraventricular nucleus attenuates inflammatory cytokines and oxidative stress in the mechanism of the TLR4 signal pathway in salt-induced hypertension. Neuroscience Bulletin, 36(4), 385–395.

    Article  PubMed  Google Scholar 

  39. 39.

    Yu, X. J., Zhang, D. M., Jia, L. L., Qi, J., Song, X. A., Tan, H., et al. (2015). Inhibition of NF-κB activity in the hypothalamic paraventricular nucleus attenuates hypertension and cardiac hypertrophy by modulating cytokines and attenuating oxidative stress. Toxicology and Applied Pharmacology, 284(3), 315–322.

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Winklewski, P. J., Radkowski, M., & Demkow, U. (2016). Neuroinflammatory mechanisms of hypertension: Potential therapeutic implications. Current Opinion in Nephrology and Hypertension, 25(5), 410–416.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Ott, M., Gogvadze, V., Orrenius, S., & Zhivotovsky, B. (2007). Mitochondria, oxidative stress and cell death. Apoptosis, 12(5), 913–922.

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    de Vries, S. (1986). The pathway of electron transfer in the dimeric QH2: Cytochrome c oxidoreductase. Journal of Bioenergetics and Biomembranes, 18(3), 195–224.

    Article  PubMed  Google Scholar 

  43. 43.

    Gu, J., Wu, M., Guo, R., Yan, K., Lei, J., Gao, N., et al. (2016). The architecture of the mammalian respirasome. Nature, 537(7622), 639–643.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Mailer, K. (1990). Superoxide radical as electron donor for oxidative phosphorylation of ADP. Biochemical and Biophysical Research Communications, 170(1), 59–64.

    CAS  Article  Google Scholar 

  45. 45.

    Will, Y. (1999). Overview of glutathione function and metabolism. Current Protocols in Toxicology.

    Article  Google Scholar 

  46. 46.

    Zelko, I. N., Mariani, T. J., & Folz, R. J. (2002). Superoxide dismutase multigene family: A comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radical Biology and Medicine, 33(3), 337–349.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    McCord, J. M., & Fridovich, I. (1970). The utility of superoxide dismutase in studying free radical reactions. II. The mechanism of the mediation of cytochrome c reduction by a variety of electron carriers. Journal of Biological Chemistry, 245(6), 1374–1377.

    CAS  Article  Google Scholar 

  48. 48.

    Starkov, A. A., & Fiskum, G. (2010). Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state. Journal of Neurochemistry, 86(5), 1101–1107.

    Article  Google Scholar 

  49. 49.

    Ploumi, C., Daskalaki, I., & Tavernarakis, N. (2017). Mitochondrial biogenesis and clearance: A balancing act. The FEBS Journal, 284(2), 183–195.

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Ashkenazi, A., Bento, C. F., Ricketts, T., Vicinanza, M., Siddiqi, F., Pavel, M., et al. (2017). Polyglutamine tracts regulate beclin 1-dependent autophagy. Nature, 545(7652), 108–111.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Cao, Y. L., Meng, S., Chen, Y., Feng, J. X., Gu, D. D., Yu, B., et al. (2017). MFN1 structures reveal nucleotide-triggered dimerization critical for mitochondrial fusion. Nature, 542(7641), 372–376.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Chen, Y., & Dorn, G. W., 2nd. (2013). PINK1-phosphorylated mitofusin 2 is a Parkin receptor for culling damaged mitochondria. Science, 340(6131), 471–475.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Favaro, G., Romanello, V., Varanita, T., Andrea Desbats, M., Morbidoni, V., Tezze, C., et al. (2019). DRP1-mediated mitochondrial shape controls calcium homeostasis and muscle mass. Nature Communications, 10(1), 2576.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Fransson, S., Ruusala, A., & Aspenström, P. (2006). The atypical Rho GTPases Miro-1 and Miro-2 have essential roles in mitochondrial trafficking. Biochemical and Biophysical Research Communications, 344(2), 500–510.

    CAS  Article  PubMed  Google Scholar 

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This study was supported by National Natural Science Foundation of China (Grant Nos. 81800372, 81770426), China Postdoctoral Science Foundation (Grant No. 2017M620457) and the Shaanxi Natural Science Foundation (Grant Nos. 2018JQ8016, 2019JQ-605).

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YL, X-JY and Y-MK contributed to the study conception and design, YL, TX and H-LC performed the experiment and collected the data, YL and TX analyzed the data, YL wrote the first draft of the manuscript and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Yu-Ming Kang.

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Li, Y., Yu, XJ., Xiao, T. et al. Nrf1 Knock-Down in the Hypothalamic Paraventricular Nucleus Alleviates Hypertension Through Intervention of Superoxide Production-Removal Balance and Mitochondrial Function. Cardiovasc Toxicol (2021).

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  • Hypertension
  • Hypothalamic paraventricular nucleus (PVN)
  • Mitochondria
  • Superoxide
  • Recombinant nuclear respiratory factor 1 (Nrf1)
  • Two-kidney, one-clip (2K1C)