Nardostachys jatamansi Extract and Nardosinone Exert Neuroprotective Effects by Suppressing Glucose Metabolic Reprogramming and Modulating T Cell Infiltration

Abstract

Background: Nardostachys jatamansi DC. (Gansong), a widely utilized herb in traditional Chinese medicine, has been historically employed in the management of various neuropsychiatric disorders. Nardosinone (Nar), a sesquiterpenoid compound, has been identified as one of the principal bioactive constituents of N. jatamansi. This study investigated the effects of ethyl acetate extract (NJ-1A) from N. jatamansi and its active constituent nardosinone on neuroinflammatory mediator release, glucose metabolic reprogramming, and T cell migration using both in vitro and in vivo experimental models.

Methods: Lipopolysaccharide(LPS)-induced BV-2 microglial cells and a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid (MPTP/p)-induced male C57BL/6N mouse chronic model of Parkinson's disease were applied.

Results: Both NJ-1A and Nar could significantly suppress LPS-induced production of M1 pro-inflammatory factors or markers in microglia and could inhibit the glycolytic process and promote oxidative phosphorylation via the AKT/mTOR signaling pathway. Furthermore, they exhibited the capacity to attenuate chemokine release from activated microglia, consequently reducing T cell migration. In vivo experiments revealed that NJ-1A and Nar effectively inhibited microglial activation, diminished T cell infiltration, and mitigated the loss of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the substantia nigra of MPTP-induced mice.

Conclusions: NJ-1A and nardosinone exert neuroprotective effects through the modulation of microglial polarization states, regulation of metabolic reprogramming, and suppression of T cell infiltration.

Keywords: Nardostachys jatamansi; Parkinson’s disease; T cell infiltration; metabolic reprogramming; microglia; nardosinone; neuroinflammation.

Figure 1

Nar is the main component of NJ-1A. Analysis by high-performance liquid chromatography (HPLC) reveals that Nar is the predominant component of NJ-1A, accounting for approximately 10.2% based on the relative peak area. The chromatographic method and the other compounds identified in NJ-1A are shown in File S1.

Figure 2

NJ-1A and Nar can inhibit the production of pro-inflammatory factors in microglia induced by LPS. (A–C) NJ-1A and Nar can inhibit the production of NO, IL-6, and TNF-α in LPS-induced BV-2 cells without affecting cell viability, n = 5; (D) NJ-1A and Nar can inhibit the expression of iNOS protein, n = 3; (E,F) NJ-1A and Nar can inhibit the mRNA expression of pro-inflammatory factors but do not inhibit the mRNA expression of anti-inflammatory cytokines, n = 4. Data represent the mean ± SD; vs. Control, # p < 0.05, ### p < 0.001; vs. LPS, * p < 0.05, *** p < 0.001. NJ-1A: Ethyl acetate extract of N. jatamansi, Nar: nardosinone, LPS: Lipopolysaccharide, MINO: Minocycline.

Figure 3

NJ-1A and Nar can inhibit the generation of ROS in BV-2 cells induced by LPS. (A,B) After BV-2 cells were stimulated with LPS for 24 h, the content of ROS inside the cells was detected by flow cytometry, n = 4. (C) Fluorescence microscopic images of BV-2 cells treated with LPS and/or NJ-1A and Nar after incubation with DCFH probe for 24 h. Data represent the mean ± SD; vs. Control, # p < 0.05; vs. LPS, ** p < 0.01, *** p < 0.001.

Figure 4

NJ-1A and Nar can inhibit the phosphorylation levels of IκB-α and NF-κB p65 in BV-2 cells after LPS stimulation. (A,B) The results of Western blot showed that NJ-1A and Nar could inhibit the phosphorylation of IκB-α and NF-κB p65, n = 4. (C) The results of fluorescence microscopy of the cells showed that NJ-1A and Nar could inhibit the phosphorylation of NF-κB p65. Data represent the mean ± SD; vs. Control, ### p < 0.001; vs. LPS, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

NJ-1A and Nar can reverse the glucose metabolic reprogramming of LPS-induced BV-2 cells. (A,C) NJ-1A and Nar can inhibit the increase of ECAR in BV-2 cells, n = 5, (B,D) and can reverse the decrease of OCR in BV-2 cells, n = 4. (E) NJ-1A and Nar inhibited the mRNA expression of key glycolytic enzymes, n = 4. Data represent the mean ± SD; vs. Control, # p < 0.05, ## p < 0.01; vs. LPS, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

NJ-1A and Nar can inhibit the production of chemokines and the migration of T lymphocyte line CTLL-2. (A) The results of the chemokine protein chip showed that NJ-1A and Nar could inhibit the secretion of several chemokines. (B) NJ-1A and Nar inhibited the mRNA expression of CXCL10, CCL2, CCL12, and CCL5 chemokines, n = 4. (C) Schematic diagram of the Transwell experiment. (D) The results of flow cytometry showed that NJ-1A and Nar could inhibit the migration of CTLL-2 cells, n = 3. (E) RT-PCR results showed that CTLL-2 cells expressed CXCR3 and CCR5. Data represent the mean ± SD; vs. Control, # p < 0.05, ## p < 0.01, ### p < 0.001; vs. LPS, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

NJ-1A and Nar could inhibit the expression of MHCII in BV-2 cells induced by LPS, n = 4. Data represent the mean ± SD; vs. Control, ### p < 0.001; vs. LPS, ** p < 0.01.

Figure 8

NJ-1A and Nar inhibit the secretion of chemokines and inflammatory factors in activated BV-2 cells through the AKT/mTOR signaling pathway. (A–C) NJ-1A and Nar inhibited the phosphorylation of AKT and mTOR, n = 3. (D,E) The mTOR agonist MHY1485 abolished the inhibitory effects of NJ-1A and Nar on NO, CCL5, and CXCL10, n = 5. (F) The mTOR agonist MHY1485 eliminated the effect of NJ-1A and Nar on the phosphorylation of mTOR, n = 3. Data represent the mean ± SD; vs. Control, # p < 0.05, ## p < 0.01, ### p < 0.001; vs. LPS, * p < 0.05, ** p < 0.01, *** p < 0.001, & p < 0.05, && p < 0.01, &&& p < 0.001.

Figure 9

NJ-1A and Nar can ameliorate the damage of dopaminergic neurons in the MPTP-induced mouse model of Parkinson’s disease. (A) In vivo experimental flow chart. (B) The weight gain of mice at the end of the experiment, n = 10. (C,D) Western blot results showed that NJ-1A and Nar can reverse the reduction of TH expression in the ventral midbrain of the mouse model of Parkinson’s disease, n = 4. (E–G) Immunohistochemistry results showed that NJ-1A and Nar can reverse the reduction of TH-positive cells in the substantia nigra and striatum of the mouse model of Parkinson’s disease, n = 4. (40x magnification) (H) The rotarod test. Data represent the mean ± SD; vs. Control, ## p < 0.01, ### p < 0.001; vs. MPTP, * p < 0.05, ** p < 0.01.

Figure 10

NJ-1A and Nar can inhibit the activation of microglia in the brains of mice in models of Parkinson’s disease induced by MPTP. (A–C) Western blot results showed that NJ-1A and Nar can inhibit the expression of activated microglial markers IBA-1, iNOS, and MHCII in the ventral midbrain, n = 4; (D,E) Immunofluorescence results demonstrated that NJ-1A and Nar can suppress the expression of the activated microglial marker Iba1 in the substantia nigra and striatum, n = 4. Data represent the mean ± SD; vs. Control, # p < 0.05, ## p < 0.01, ### p < 0.001; vs. MPTP, * p < 0.05, ** p < 0.01. Scale bar, 100 μm.

Figure 11

NJ-1A and Nar can reduce the number of CD4⁺ T (A) and CD8⁺ T (B) cells in the substantia nigra, n = 4. The black arrows indicate the positive cells, and the frames indicate the magnified positive cells. Data represent the mean ± SD; vs. Control, ## p < 0.01; vs. MPTP, ** p < 0.01, *** p < 0.001.