L hcy

Human hepatocytes (HL-7702) were obtained from the Japanese Collection of Research Bioresources and cultured in RPMI-1640 medium (Thermo, USA) containing 10% fetal bovine serum (FBS) (Hyclone, USA) and 1% penicillin (Thermo, Waltham, MA, USA) humidified with 5% CO₂ air at 37 °C. The cells were transfected with Ad-CFTR when they were 80% confluent. The control cells were transfected with Ad-GFP. After 48 h, the transfection efficiency was detected by fluorescence microscope, and then the cells were treated with L-Hcy (100 μmol/L) (Sigma, USA) for another 24 h for further experiments.

Sixty adult male Sprague-Dawley (SD) rats (160–180 g) (Grade SPF, Certificate Number SCXK (jing) 2016-0006) was purchased from Beijing Vital River Laboratory Co. Ltd (Beijing, China). Use of animals and experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 80-23, revised 1996). The rats were randomly allocated into five groups: sham operation control group (SHAM), MCAO group, MCAO plus Hcy group (MCAO + HCY), MCAO, Hcy plus 3MA group (MCAO + HCY + 3MA), and MCAO plus 3MA group (MCAO + 3MA). The vehicle and d, l-Hcy (1.6 mg/kg/d; Sigma-Aldrich, St. Louis, MO, USA) were administered by tail vein injection for 28 days prior to SHAM or MCAO operation and up to 7 days after surgery. The concentration of Hcy was chosen based on our previous study^{17 (link)}, which showed that the treatment of Hcy (1.6 mg/kg/d) for 3 weeks resulted in a notably higher plasma Hcy concentration (15.05 + 2.66 μM) in MCAO rats, compared with nontreated MCAO animals (7.00 + 1.77 μM). The 3MA (5 mM, 4 mL/kg/d) (Sigma-Aldrich) was administered by tail vein injection for 5 days prior to SHAM or MCAO operation.

The standard stock solutions of 5-PAHSA, 9-PAHSA, 12-PAHSA, 9-PAHPA, 9-SAHSA, 9-POHSA, 9-OAHSA, PA, SA, POA, and OA were purchased from Cayman Chemical (Ann Arbor, MI, USA). The standards of l-Hcy, SAH, l-Car, and TMAO (Sigma Aldrich, St. Louis, MO, USA) were dissolved in double-distilled water (ddH₂O) or HPLC grade methanol (MeOH) with final concentration 0.1 mg/mL for stock solution. The stable isotopes ¹³C₁₆-palmitic acid (¹³C₁₆-PA) and d3, l-methionine (d3-l-Met) were obtained from Sigma Aldrich (St. Louis, MO, USA), and the d9-TMAO was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). All of the stock solutions and stable isotopes solution were stored at −20 °C and the storage time was less than one year. The calibration curves were prepared by using stock dilution and formulated by using the peak area ratio of the analytical standards and the internal standards.