Lactic acid

A five-year-old willow (Salix matsudana cv. Zhuliu) was harvested in a forest center in Dingxi, Gansu province, China. The sample of willow trunk (bark and branches removed, a length of 1.0 m, and diameter cross-section of 22 cm, approximately 17 kg) was air-dried for two months in a laboratory. The air-dried willow was cut to 1–1.5 cm in size and ground in a star mill. The fractions obtained by sifting through a 40–80 mesh sieve were used for the subsequent separation of the DES-lignin. The choline chloride and lactic acid used in the synthesis of the deep eutectic solvent were provided by Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China).

Sodium hydroxide, potassium hydrogen phthalate, glucose monohydrate, 99% anhydrous sodium phosphate monohydrate, potassium hexacyanoferrate trihydrate, zinc sulfate heptahydrate, ethanol, phosphoric acid, phenolphthalein indicator, high-performance liquid chromatography grade methanol, boric acid, lithium chloride, lithium hydroxide-monohydrate, 4-methyl-2-pentanol, citric acid, hydrochloric acid, octanoic acid, potassium acetate, sodium acetate trihydrate, acetic acid, sodium chloride, ninhydrin crystal, phenol, and ascorbic acid were purchased from Guoyao Group Chemical Reagent Co., Ltd (Shanghai, China). citric acid (≥99.5% purity), L-pyroglutamic acid (≥99% purity), lactic acid (≥99% purity), acetic acid (≥99% purity), succinic acid (≥99% purity), and malic acid (≥99% purity) were purchased from McLean Biochemical Technology Co., Ltd (Shanghai, China).

Metformin (Met) (Aladdin, Shanghai, China) was dissolved in ddH₂O to prepare a stock solution (10 mM). To assess the toxicity of Met on zebrafish development, continuous soaking treatment was performed using different concentrations of metformin (0 μM, 50 μM, 100 μM, 400 μM, and 800 μM). After evaluating toxicity through the hatching rate, body length, and mobility of the zebrafish, Met (50 μM) was selected for subsequent experiments, while the cells were treated with a concentration of 10 μM for 8 h. To establish a systemic inflammation model, we injected LPS (0.15 mg/mL, 50 nL) (Sigma, L2630) into otic vesicles in zebrafish via microinjection. Cell inflammation was induced by exposure to LPS (2.5 μg/mL) for 2 h. Lactic acid (50 mM) (Macklin, China) was used to treat zebrafish at 5 dpf (days postfertilization) for 10 h, while the cells were treated with Lactic acid (25 mM) for 6 h to increase the level of lactylation. H₂O₂ (200 μM) was used to treat zebrafish at 5 dpf for 2 h to increase the level of reactive oxygen species within the body, while the cells were treated with a concentration of 25 mM for 0.5 h.