E004 1 1

ATP levels were measured according to manufacturer’s instructions (A095 Nanjing Jiancheng Bioengineering Research Institute, Nanjing China). Mitochondrial respiratory complex (I, II, III, and IV) activities were determined using kits from Nanjing Jiancheng (A089-1, A089-2, A089-3, and A089-4, Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China), according to manufacturer’s instructions. The mitochondrial membrane potential was determined with JC-1 kit (HY-K0601, MedChem Express, Shanghai, China) via flow cytometry (NovoCyte, ACEA Biosciences). ROS production was measured using a kit from Nanjing Jiancheng Bioengineering Research Institute, China (E004-1-1) according to manufacturer’s instructions.

The concentrations of ROS (E004-1-1, Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) and thiobarbituric acid reactive substances (TBARS) (A003-1-2) and the activities of superoxide dismutase (SOD) (A001-3-2) and glutathione peroxidase (GSH-Px) (A005-1-2) of the impaired CAs and HUVECs were measured as described in the manufacturer's procedures for the detection kits [22 (link)].

According to the instructions (E004-1-1; Nanjing Jiancheng Bioengineering), the cells were cultured with 2,7-DichlorofuorescinDiacetate (DCFH-DA) probe (10 μm) at 37°C in the dark for 30 min, and cultivated with Gemini EM enzyme (Thermo Fisher Scientific Inc., Fremont, CA, United States) to detect the fluorescence intensity of differently treated cells, which represents the relative ROS content. The levels of TNF-α and IL-1β in differently treated OB-6 cells were recorded according to the above-mentioned experimental methods.

SBF broth at 84 h and 168 h were centrifuged at 8085× g and at 4 °C for 10 min to collect Monascus mycelium, then 0.1 g of the collected mycelium were broken in nine-fold volume of ice-cold phosphate buffered solution (PBS) (50 mM, pH = 6.8) to generate the mycelium lysate. Then, the lysate was centrifuged at 8085× g and at 4 °C for 10 min to obtain the supernatant. The activities of peroxidase (POD, EC:1.11.1.7), total kinds of superoxide dismutase (T-SOD, EC1.15.1.1), ETC complex I, II and III IV were determined by using the assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) [20 (link)]. Protein concentrations were measured using the conventional Bradford assay. Reactive oxygen species (ROS) was measured according to the protocol by using the assay kit (E004-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The concentrations of ATP were detected by using the assay kit (A095-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

The levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and protein carbonyl in placentae were analyzed through commercial kits (E004-1-1,A003-1-2, A001-3-2,A006-2-1, A087-1-1,Nanjing Jiancheng Bioengineering Institute, Nanjing, China) respectively.