Cell viability assay using CCK-8 showed that treatment of Human Gingival Fibroblasts (HGFs) with (-)-Aspartic acid (D-Asp) at concentrations ≤ 30 mM for 24, 48, and 72 hours had no cytotoxic effects. Concentrations of 60 mM and 120 mM significantly inhibited HGF proliferation. [1]
Pretreatment of HGFs with 1, 10, or 30 mM (-)-Aspartic acid (D-Asp) for 24 hours significantly reduced the increased membrane permeability induced by HMGB1 (10 µg/mL). This was evidenced by decreased Propidium Iodide (PI) staining, a reduction in Annexin V-FITC+/PI+ double-positive cells in flow cytometry, and lowered Lactate Dehydrogenase (LDH) release into the culture supernatant compared to the HMGB1-only group. The protective effect was dose-dependent, with 30 mM showing the strongest effect. [1]
Pretreatment with 1, 10, or 30 mM (-)-Aspartic acid (D-Asp) significantly inhibited the HMGB1-induced upregulation of pyroptosis-related mRNA expression, including NLRP3, caspase-1, GSDMD, IL-1β, and IL-18, as measured by qRT-PCR. The inhibition was dose-dependent. [1]
ELISA analysis showed that pretreatment with 10 mM and 30 mM (-)-Aspartic acid (D-Asp) significantly reduced the HMGB1-induced release of IL-1β and IL-18 proteins into the cell culture supernatant. The 1 mM dose reduced IL-18 release but did not significantly affect IL-1β release. [1]
Western blot analysis demonstrated that pretreatment with 30 mM (-)-Aspartic acid (D-Asp) notably downregulated the HMGB1-induced protein expression levels of caspase-1, NLRP3, and GSDMD. [1]

Cell Viability Assay: Human Gingival Fibroblasts (HGFs) were seeded into 96-well plates at a density of 5×10³ cells per well and incubated overnight. Cells were then treated with varying concentrations of (-)-Aspartic acid (D-Asp) (1, 10, 30, 60, or 120 mM) for 24, 48, and 72 hours. After treatment, Cell Counting Kit-8 (CCK-8) reagent was added to each well, and the plates were incubated. Absorbance was measured at 450 nm using a microplate reader to determine cell viability. [1]
Membrane Permeability/Pyroptosis Assessment (PI/Hoechst Staining): After drug and HMGB1 treatments, HGFs were stained with Propidium Iodide (PI, 2 µg/mL) and Hoechst 33342 (5 µg/mL) for 5 minutes at 37°C. Stained cells were immediately observed and imaged using a fluorescence inverted microscope. PI-positive (red) cells indicating membrane permeability were quantified. [1]
Flow Cytometry for Cell Death: HGFs were seeded in 6-well plates at 1×10⁶ cells per well. After interventions, cells were collected, washed, and resuspended in buffer. Cell staining was performed using an Annexin V-FITC/PI apoptosis detection kit according to the manufacturer's instructions. A flow cytometer was used to acquire at least 1×10⁴ events per sample, and the percentage of Annexin V-FITC+/PI+ cells (indicative of late apoptosis/pyroptosis) was analyzed. [1]
Lactate Dehydrogenase (LDH) Release Assay: After drug and HMGB1 treatments, the cell culture supernatant was collected. The level of LDH released from damaged cells into the supernatant was measured using an LDH assay kit. The reaction mixture's absorbance was measured at 450 nm using a microplate reader. Higher absorbance indicates greater membrane damage and LDH release. [1]
Quantitative Real-Time PCR (qRT-PCR): Total RNA was extracted from HGFs using TRIzol reagent. cDNA was synthesized from the RNA using a reverse transcription kit. Gene expression levels of NLRP3, caspase-1, GSDMD, IL-1β, and IL-18 were quantified using SYBR Green-based real-time PCR master mix on a real-time PCR detection system. The PCR cycling conditions included an initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. GAPDH was used as the internal reference gene, and relative gene expression was calculated using the 2^(-ΔΔCt) method. [1]
Enzyme-Linked Immunosorbent Assay (ELISA): After treatments, the cell culture supernatant was collected and centrifuged. The levels of secreted IL-1β and IL-18 proteins in the supernatant were measured using specific human ELISA kits according to the manufacturer's protocols. Absorbance was read at 450 nm. [1]
Western Blot Analysis: Total cellular proteins were extracted from HGFs using RIPA lysis buffer. Protein concentration was determined using a BCA protein assay kit. Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked and then incubated overnight at 4°C with primary antibodies against caspase-1, NLRP3, GSDMD, and β-actin (loading control). After washing, membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence (ECL) reagents and analyzed with imaging software. [1]

In vitro cytotoxicity assessment of human gingival fibroblasts (HGF) showed that concentrations of (-)-aspartic acid (D-Asp) at 60 mM and 120 mM significantly inhibited cell proliferation/viability after treatment for 24, 48, and 72 hours, indicating that these high concentrations had cytotoxic effects. Under the test conditions, concentrations ≤ 30 mM were considered non-cytotoxic. [1]

[1]. D-aspartic acid protects against gingival fbroblasts infammation by suppressing pyroptosis. Mol Biol Rep. 2022 Jul;49(7):5821-5829.

[2]. D-Aspartic acid stimulates steroidogenesis through the delay of LH receptor internalization in a mammalian Leydig cell line. J Endocrinol Invest. 2016 Feb;39(2):207-213.

[3]. Involvement of D-aspartic acid in the synthesis of testosterone in rat testes. Life Sci. 1996;59(2):97-104.

[4]. Occurrence of D-aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release. FASEB J. 2000 Apr;14(5):699-714.

[5]. D'Aniello A. D-Aspartic acid: an endogenous amino acid with an important neuroendocrine role. Brain Res Rev. 2007 Feb;53(2):215-34.

D-Aspartic acid is the D-enantiomer of aspartic acid and a metabolite in mice. It is an aspartic acid, a D-α-amino acid, the conjugate acid of D-aspartic acid (1-), and an enantiomer of L-aspartic acid. D-Aspartic acid has been reported to be found in Pinus densiflora, Cyperus aromaticus, and other organisms with relevant data. D-Aspartic acid is a D-isomer. (-)-Aspartic acid (D-Asp) is a D-amino acid. This study investigated its protective effect against HMGB1-induced pyroptosis in human gingival fibroblasts (HGFs), a model associated with peri-implantitis inflammation. [1]
The proposed mechanism of action is that (-)-aspartate (D-Asp) competitively antagonizes the binding of HMGB1 to Toll-like receptors (TLRs) on fibroblasts. This interference inhibits the activation of the downstream classical pyroptosis pathway (TLR-NLRP3-caspase-1-GSDMD), leading to decreased expression of inflammasome components (NLRP3, caspase-1), porogen (GSDMD), and pro-inflammatory cytokines (IL-1β, IL-18). [1]
This study shows that (-)-aspartate (D-Asp) has the potential to be used as a therapeutic agent to prevent inflammation caused by pyroptosis in diseases such as peri-implantitis, providing a new strategic perspective for treatment. However, this study is limited to an in vitro model. [1]

C4H7NO4

133.1027

133.037

1783-96-6

27881-01-2

83887

White to off-white solid powder

1.5±0.1 g/cm3

264.1±30.0 °C at 760 mmHg

300ºC

113.5±24.6 °C

0.0±1.1 mmHg at 25°C

1.531

-0.67

3

5

3

9

133

1

C([C@H](C(=O)O)N)C(=O)O

CKLJMWTZIZZHCS-UWTATZPHSA-N

InChI=1S/C4H7NO4/c5-2(4(8)9)1-3(6)7/h2H,1,5H2,(H,6,7)(H,8,9)/t2-/m1/s1

(2R)-2-aminobutanedioic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~7.69 mg/mL (~57.78 mM)
DMSO : ~1 mg/mL (~7.51 mM)

Solubility in Formulation 1: 2 mg/mL (15.03 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)