Phospholipase a2

Tween^® 20, cholesterol, cetyl alcohol, chloroform, sulforhodamine B, lipopolysaccharide, trypan blue, dexamethasone, trichloroacetic acid, tris (hydroxymethyl)aminomethane buffer, cytochrome C from equine heart (purity ≥ 95%), melittin (purity ≥ 85%, HPLC grade) and phospholipase A2 (activity: 1775 units/mg solid) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Apamin (purity 98.3%) was purchased from CalBiochem (San Diego, CA, USA). Fetal bovine serum, penicillin, streptomycin, trypsin, L-glutamine, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco Invitrogen Life Technologies (Carlsbad, CA, USA). Formic acid (HPLC grade) and acetonitrile (HPLC grade) were obtained from Fisher Scientific (Loughborough, UK). The Griess reagent system kit was bought from Promega (Madison, WI, USA). Ultrapure water was obtained from adequate purification systems (Ellix Essential Millipore^®, Darmstadt, Germany, and TGI Pure Water Systems, Brea, CA, USA).

Trimeric LHCII complexes of spinach were isolated from freshly prepared thylakoid membranes as described earlier61 (link). Monomeric complexes were obtained by incubating LHCII trimers with 1% (w/v) octyl glucoside and 10 μg/mL phospholipase A2 (Sigma)62 (link). Subsequent fast protein liquid chromatography (FPLC) ensured a homogeneous sample preparation. The ensemble fluorescence absorption and emission spectra were measured on a Lambda40 spectro-photometer (Perkin-Elmer) and a FluoroLog Tau-3 (Jobin Yvon), respectively. For SMS experiments, the sample was diluted down to a concentration of ~10 pM in a measuring buffer (20 mM Hepes, pH 8 and 0.03% (w/v) n-Dodecyl β-D-maltoside) and then immobilized on a PLL (poly-L-Lysine, Sigma) coated cover glass. Addition of an oxygen scavenging mix of 750 μg/ml Glucose Oxidase, 100 μg/ml Catalase and 7.5 mg/ml Glucose (all Sigma) to the closed sample chamber inhibited the formation of highly reactive singlet oxygen and improved the photostability of complexes.

HepG2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Fetal bovine serum, DMEM, pancreatin and penicillin were purchased from Gibco (Suzhou, China). Peristaltic pumps were available from MasterFlex (USA). PES membrane filters (0.22 μm, Millex-GV) were purchased from Merck Millipore Ltd.); intravenous indwelling needles (BD Intima II) were from Becton Dickinson medical Devices Co. Ltd. (Suzhou, China). Oleic acid, palmitic acid and sodium deoxycholate were all obtained from Sigma (San Francisco, CA, USA). The protein quantitation kit and the mitochondrial membrane potential detection kit (JC-1) were obtained from Beyotime Biotechnology Co. Ltd. (Shanghai, China). sodium deoxycholate (SDC) and phospholipase A2 were obtained from Sigma company (Beijing, China). Detection kits for triglyceride (TG), malondialdehyde (MDA), reactive oxygen species (ROS), glutathione (GSH) and superoxide dismutase (SOD) were obtained from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The total antioxidant capacity assay kit with the rapid ABTS method and the superoxide anion scavenging capacity test kit were obtained from Solarbio science & technology co. Ltd. (Beijing, China). Baicalin, the main component of Shuganning Injection, was provided by Guizhou Ruihe Pharmaceutical Co. Ltd.

Bee venom from Apis mellifera (L.) bee species was collected in March 2021 by a professional beekeeper from Beni-Mellal, Morocco. The sample was lyophilized and stored until analysis at 4 °C in the dark. The bee venom was harvested by placing a glass plate covered with a food-grade polyethylene film, surmounted by metal wires connected to a potential of 15–20 V. The principle consists of placing the device on the flight board of the bees. After electrical stimulation, the bee stings the glass plate. The food film located above the removable glass bottom allows the bee to withdraw its stinger without dying, and the venom is deposited on the glass plate. The plates were stored in a dry and sterile room for two days, and the food-grade polyethylene film was removed. The glass plates were then scraped with a blade to obtain the venom powder, which was handled with care.
Phospholipase A2 (reference: P9279-1MG) and melittin (reference: M2271-1Mg) from honey bee venom (Apis mellifera L.) were purchased from Sigma Aldrich (St. Louis, MO, USA).

The amount of apamin, phospholipase-A2 and melittin components in BV was analysed by high-performance liquid chromatography (HPLC) variable wavelength detector (VWD) (Agilent 1260 Series). Infinitylab Poroshell C18 EC-C18 (4.6 mm × 50 mm, 2.7 micron) column was used for separation. While the optimum separation temperature was 20 °C, the column flow rate was 1 mL/min. Apamin (Sigma-A1289), Phospholipase A2 (Sigma-P9279) and Melittin (Sigma M2272) standard solutions were prepared at 10 μg/mL, 20 μg/mL, 50 μg/mL and 100 μg/mL concentrations. The buffer A (0.1% trifluoroacetic acid [TFA] in water [H₂O]) and the buffer B (0.1% TFA in acetonitril) were used as mobile phases. The Gradian programme has been optimised for peak holding times. Absorbance measurements were made at 218 nm (Gokmen et al. 2023 (link)).

For phospholipase treatment, 100 µL of EE were mixed with 20 U of Phospholipase D (Sigma-Aldrich, P8398) (PLD) and 20 U of Phospholipase A₂ (Sigma-Aldrich, P6534) (PLA₂) and incubated for 1.5 hr at 30°C followed by incubation at 37°C for another 1.5 hr. For separate phospholipase treatments, 100 µL of EE were mixed with 20 U PLD or PLA2 and incubated for 1.5 hr at 30°C for PLD or 1.5 hr at 37°C for PLA₂. Eight leaves from four plants (two leaves per plant) were treated from the abaxial side of the leaf with a 2 µL drop of the phospholipase-treated EE or an untreated EE which was incubated in the same way as described above. Treated leaves were harvested three days later for analysis and untreated plants served as controls.
Efficacy of PC degradation in phospholipase-treated EE was checked by ³¹P 1D NMR (see above).