Mechanistic studies of the beneficial effects of Anshen Dingzhi prescription for PTSD treatment: roles of the FKBP5-IKKα-NF-κB-NLRP3 signaling pathway

Abstract

Objective: Anshen Dingzhi prescription (ADP) is an effective remedy for treating post-traumatic stress disorder (PTSD); however, the mechanism underlying its beneficial effects is unclear. This study explores the roles of the neuroinflammation regulated by the FKBP prolyl isomerase 5 (FKBP5)-IκB kinase alpha (IKKα)-nuclear factor kappa-B (NF-κB)-NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) signaling pathway in PTSD.

Methods: The primary components of ADP, including ginsenosides Rg1 and Rb1, were quantified using ultra-performance liquid chromatography. Twelve C57BL/6 mice were allocated to control (D0) and experimental groups on days one, seven, and 14 of single prolonged stress (SPS). Eighteen C57BL/6 mice were allocated to control, SPS, and MCC950, an NLRP3 inhibitor (5 mg/kg) groups. Finally, 24 C57BL/6 mice were allocated to control, SPS, paroxetine hydrochloride (PRX), or ADP (18.4 and 36.8 mg/kg) groups. Mice were administered MCC950, PRX, or ADP for 14 days. The open field test and elevated plus maze were used to evaluate anxiety-like behaviors, whereas fear memory extinction was evaluated using the fear memory test. Western blotting was employed to evaluate the expression levels of the FKBP5-IKKα-NF-κB-NLRP3 signaling pathway, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β. The expression of FKBP5 and NLRP3 was further confirmed by immunofluorescence staining.

Results: The amounts of ginsenosides Rg1 and Rb1 in ADP were (96.85 ± 1.14) and (9.04 ± 0.22) µg/g, respectively. Compared with the D0 group, the levels of the inflammatory cytokine proteins, TNF-α, IL-6, and IL-1β were elevated 1.33- to 1.51-fold and those of FKBP5-IKKα-NF-κB-NLRP3 signaling pathway were increased 1.16- to 1.41-fold in the hippocampus of the D14 group (P < 0.05); the fluorescence intensity of FKBP5 and NLRP3 was also markedly increased (1.33-1.79-fold) in the hippocampus of the D14 group (P < 0.5). Notably, injection of MCC950 (5 mg/kg) reduced the levels of FKBP5-IKKα-NF-κB-NLRP3 (0.80-0.88-fold) and inflammatory cytokines (0.74-0.83-fold), thereby improving the PTSD-like behaviors induced by SPS (P < 0.05). In addition, ADP (36.8 g/kg) significantly improved PTSD-like behaviors and reduced levels of hippocampal inflammatory cytokines (0.70-0.79-fold) and FKBP5-IKKα-NF-κB-NLRP3 (0.50-0.79-fold) (P < 0.05) in SPS mice.

Conclusion: The results suggest a potential therapeutic benefit of ADP in PTSD due to the inhibition of the FKBP5-IKKα-NF-κB-NLRP3 signaling pathway.

Keywords

Anshen Dingzhi prescription / FKBP5-IKKα-NF-κB-NLRP3 signaling pathway / Neuroinflammation / Post-traumatic stress disorder

Conflict of interest statement

Xiaohe Xiao is editorial board members of this journal. The other authors declare no conflict of interest.

Funding

This research was supported by the National Natural Science Foundation of China (82404995, 82404890), Research Funds of Center for Xin’an Medicine and Modernization of Traditional Chinese Medicine of IHM (2023CXMMTCM013), Scientific Research Program of Anhui Provincial Department of Education (2024AH051036, 2024AH040137, 2024AH051044), Excellent Funding for Academic and Scientific Research Activities for Academic and Technological Leaders in Anhui Province (2022D317), and Key Research and Development Plan of Anhui Province (202104j07020004), and Anhui University of Chinese Medicine Talent Support Program (DT2300000173).

Author contributions

Daokang Chen, Jiamin Hu, and Shaojie Yang performed the experiments and analyzed the data. Daokang Chen and Jiamin Hu drafted and revised the manuscript. Manman Ji, Pan Xie, and Zhengrong Zhang contributed to the discussion of this study and the revision of this manuscript. Michel Baudry helped to revise the manuscript. Sheng Zhang and Guoqi Zhu conceived and designed this study, wrote and revised the manuscript, conducted the project administration, and acquired the funding grants.

Ethical approval of studies and informed consent

The animal study was reviewed and approved by the Experimental Animal Ethics Committee of the Anhui University of Chinese Medicine (Approval No. AHUCM-mouse-2022014).

Acknowledgments

None.

Data availability

All data generated or analyzed in this study are included in this published article; further inquiries can be directed to the corresponding authors.

References

[1] He M, Wei JX, Mao M, et al. Synaptic plasticity in PTSD and associated comorbidities: the function and mechanism for diagnostics and therapy. Curr Pharm Des 2018; 24(34):4051-4059.

[2] Chou PH, Ito M, Horikoshi M. Associations between PTSD symptoms and suicide risk: a comparison of 4-factor and 7-factor models. J Psychiatr Res 2020; 129:47-52.

[3] Kessler RC, Aguilar-Gaxiola S, Alonso J, et al. Trauma and PTSD in the WHO World Mental Health Surveys. Eur J Psychotraumatol 2017; 8(5):1353383.

[4] Qu Y, Wu H, Zhu G, et al. The improving effect and mechanisms of Anshen Dingzhi Prescription on Alzheimer’s disease-like behavior induced by D-galactose combined with AβO in mice. Acta Pharm Sin 2024; 59(1):119-134.

[5] Wang J, Zhao P, Cheng P, et al. Exploring the effect of Anshen Dingzhi prescription on hippocampal mitochondrial signals in single prolonged stress mouse model. J Ethnopharmacol 2024; 323:117713.

[6] Yang S, Qu Y, Wang J, et al. Anshen Dingzhi prescription in the treatment of PTSD in mice: Investigation of the underlying mechanism from the perspective of hippocampal synaptic function. Phytomedicine 2022; 101:154139.

[7] Zhang Z, Gao F, Yang S, et al. Mechanism of Anshen Dingzhi prescription intervening against anxiety-like behavior in post-traumatic stress disorder: an analysis based on network pharmacology and molecular docking. J Anhui Univ Chinese Med 2023; 42(4):74-80.

[8] Florido A, Velasco ER, Monari S, et al. Glucocorticoid-based pharmacotherapies preventing PTSD. Neuropharmacology 2023; 224:109344.

[9] Peruzzolo TL, Pinto JV, Roza TH, et al. Inflammatory and oxidative stress markers in post-traumatic stress disorder: a systematic review and meta-analysis. Mol Psychiatry 2022; 27(8):3150-3163.

[10] Holmes SE, Scheinost D, Finnema SJ, et al. Lower synaptic density is associated with depression severity and network alterations. Nat Commun 2019; 10(1):1529.

[11] Ji M, Zhang ZR, Zhu GQ. Mechanism of cuculigoside against PTSD-induced inflammatory response in mouse hippocampus. J Biol 2022; 39(6):14-19.

[12] He W, Hu Z, Zhong Y, et al. The potential of NLRP3 inflammasome as a therapeutic target in neurological diseases. Mol Neurobiol 2023; 60(5):2520-2538.

[13] Yao H, Zhang D, Yu H, et al. Gut microbiota regulates chronic ethanol exposure-induced depressive-like behavior through hippocampal NLRP3-mediated neuroinflammation. Mol Psychiatry 2023; 28(2):919-930.

[14] Dong Y, Li S, Lu Y, et al. Stress-induced NLRP3 inflammasome activation negatively regulates fear memory in mice. J Neuroinflammation 2020; 17(1):205.

[15] Hao W, Wang L, Li S. FKBP5 regulates RIG-I-mediated NF-kappaB activation and influenza a virus infection. Viruses 2020; 12(6):672.

[16] Han L, Yan ZL, Liu CZ. Effects of Anshen Dingzhi prescription on tryptophan metabolic pathway and exercise capacity in rats with chronic fatigue syndrome. Chinese Trad Patent Med 2021; 43(12):3493-3497.

[17] Wang J, Gao F, Cui S, et al. Utility of 7,8-dihydroxyflavone in preventing astrocytic and synaptic deficits in the hippocampus elicited by PTSD. Pharmacol Res 2022; 176:106079.

[18] Gao F, Wang J, Yang S, et al. Fear extinction induced by activation of PKA ameliorates anxiety-like behavior in PTSD mice. Neuropharmacology 2023; 222:109306.

[19] Xie P, Chen L, Wang J, et al. Polysaccharides from Polygonatum cyrtonema Hua prevent post-traumatic stress disorder behaviors in mice: mechanisms from the perspective of synaptic injury, oxidative stress, and neuroinflammation. J Ethnopharmacol 2024; 319(1):117165.

[20] Leary S, Underwood W, Anthony R, et al. AVMA guidelines for the euthanasia of animals: 2013 edition. 2013. Available from: https://www.avma.org/KB/Policies/Pages/Euthanasia-Guidelines.aspx. Accessed July 2023.

[21] Hoffmann J, Luxan G, Abplanalp WT, et al. Post-myocardial infarction heart failure dysregulates the bone vascular niche. Nat Commun 2021; 12(1):3964.

[22] Huang ZL, Liu R, Bai XY, et al. Protective effects of the novel adenosine derivative WS0701 in a mouse model of posttraumatic stress disorder. Acta Pharmacol Sin 2014; 35(1):24-32.

[23] Speer K, Upton D, Semple S, et al. Systemic low-grade inflammation in post-traumatic stress disorder: a systematic review. J Inflamm Res 2018; 11:111-121.

[24] Tursich M, Neufeld RW, Frewen PA, et al. Association of trauma exposure with proinflammatory activity: a transdiagnostic meta-analysis. Transl Psychiatry 2014; 4(7):e413.

[25] Levkovitz Y, Fenchel D, Kaplan Z, et al. Early post-stressor intervention with minocycline, a second-generation tetracycline, attenuates post-traumatic stress response in an animal model of PTSD. Eur Neuropsychopharmacol 2015; 25(1):124-132.

[26] Jones ME, Lebonville CL, Barrus D, et al. The role of brain interleukin-1 in stress-enhanced fear learning. Neuropsychopharmacology 2015; 40(5):1289-1296.

[27] Guan Y, Han F. Key mechanisms and potential targets of the NLRP3 inflammasome in neurodegenerative diseases. Front Integr Neurosci 2020; 14:37.

[28] Nie Y, Huang MY, Yang TY, et al. Ferulic Acid Reduces Inflammatory Response Induced by Radiation through Sirt1-NLRP 3 Pathway. Acupunct Herb Med. 2024; 4(3):367-374.

[29] Guan X, Zhu S, Song J, et al. Microglial CMPK2 promotes neuroinflammation and brain injury after ischemic stroke. Cell Rep Med 2024; 5(5):101522.

[30] Yang L, Xu HH, Hong Q, et al. Crocus sativus L. produces anti-inflammatory effects and regulates NLRP3-NF-κB pathway.. Acupunct Herb Med. 2024; 4(3):375-385.

[31] Wang X, Lin C, Jin S, et al. Cannabidiol alleviates neuroinflammation and attenuates neuropathic pain via targeting FKBP5. Brain Behav Immun 2023; 111:365-375.

[32] Li H, Su P, Lai TK, et al. The glucocorticoid receptor-FKBP51 complex contributes to fear conditioning and posttraumatic stress disorder. J Clin Invest 2020; 130(2):877-889.

[33] Li G, Wang L, Zhang K, et al. FKBP5 genotype linked to combined PTSD-depression symptom in Chinese earthquake survivors. Can J Psychiatry 2019; 64(12):863-871.

[34] Zannas AS, Jia M, Hafner K, et al. Epigenetic upregulation of FKBP5 by aging and stress contributes to NF-kappaB-driven inflammation and cardiovascular risk. Proc Natl Acad Sci U S A 2019; 116(23):11370-11379.

[35] Afonina IS, Zhong Z, Karin M, et al. Limiting inflammation-the negative regulation of NF-kappaB and the NLRP 3 inflammasome. Nat Immunol 2017; 18(8):861-869.

[36] Liu X, Xie DJ, Wang X, et al. Clinical observation on Anshen Dingzhi Pill combined with escitalopram in the treatment of panic disorder. Clin J Trad Chinese Med 2019; 31(12):2281-2284.

[37] Jimenez JC, Berry JE, Lim SC, et al. Contextual fear memory retrieval by correlated ensembles of ventral CA 1 neurons. Nat Commun 2020; 11(1):3492.

[38] Lin J, Gao S, Wang T, et al. Ginsenoside Rb1 improves learning and memory ability through its anti-inflammatory effect in Aβ1-40 induced Alzheimer’s disease of rats. Am J Transl Res 2019; 11(5):2955-2968.

[39] Jiang N, Zhang Y, Yao C, et al. Tenuifolin ameliorates the sleep deprivation-induced cognitive deficits. Phytother Res 2023; 37(2):464-476.

[40] Gegenhuber B, Wu MV, Bronstein R, et al. Gene regulation by gonadal hormone receptors underlies brain sex differences. Nature 2022; 606(7912):153-159.

[41] Krause WC, Rodriguez R, Gegenhuber B, et al. Oestrogen engages brain MC4R signalling to drive physical activity in female mice. Nature 2021; 599(7883):131-135.