Paeonol

The effect of paeonol on the biofilm formation of C. albicans SC5314 and C. neoformans H99 mono- and dual-species was evaluated by FESEM and CLSM, as previously described (Qian et al., 2020 (link); Qian et al., 2021 (link)). Briefly, mono- and dual-species cultures (1 × 10⁶ CFUs ml⁻¹) of C. albicans SC5314 and C. neoformans H99 were cultured in each well of the 24-well plate containing a sterilized glass coverslip and treated with various concentrations of 0, 1/4 MIC, 1/2 MIC, and MIC paeonol at 30°C for 48 h. For biofilm evaluation by FESEM, 48-h biofilms formed by C. albicans SC5314 and C. neoformans H99 mono- and dual-species on the glass coverslips were fixed in 2.5% glutaraldehyde (v v⁻¹) immediately at −4°C for 2 h followed by treatment and washed gently three times with 10 mM PBS. The fixed cells were then gradually dehydrated by rinsing for 10 min at each concentration using ascending grades of ethanol (30%, 50%, 70%, 90%, and 100%). Finally, the biofilm samples were inspected using FESEM. For examination using CLSM, biofilms were generated as described above. The biofilms were carefully rinsed three times with 10 mM PBS to remove nonadherent fungal cells. The biofilms were incubated with SYTO 9 for 15 min at 25°C. The biofilms were then rinsed twice with 10 mM PBS and observed under CLSM. For biofilm examination using an optical microscope, the biofilms were washed twice with 10 mM PBS to remove the planktonic cells and then stained with 0.1% (w v⁻¹) crystal violet (CV). The samples were then washed three times using 10 mM PBS to remove redundant dyes. Finally, the biofilms were investigated by the optical microscope at ×400 magnification. For determination of the biofilm biomass, 150 μl of overnight cultures (approximately 1 × 10⁶ CFUs ml⁻¹) of mono- and dual-species were added to each well of the 96-well plate and exposed to paeonol at 0, 1/4 MIC, 1/2 MIC, and MIC for 48 h at 30°C. After incubation, the culture medium was removed, and the biofilms were then rinsed twice with 10 mM PBS and stained with 0.1% CV (w v⁻¹) for 20 min at 25°C. Finally, the OD₅₇₀ of each well was measured using a microplate reader.

Male Wistar rats weighing 180–210 g were used after 1 week for proper acclimatization to the animal house conditions (12 h lighting cycle and 25 ± 2°C temperature). Rats were housed three rats per cage, and they had free access to commercial laboratory chow and tap water throughout the experiment. The animal ethical standards were in accordance with EU directive 2010/63/EU. Animals were divided into three groups (n = 6 each): (a) the control nontreated group, (b) the MTX-treated group that received a single intraperitoneal dose of 20 mg/kg MTX [25 (link)] at the end of the fifth day of the experiment, and (c) the MTX/paeonol-treated group treated by a single daily oral dose of 100 mg/kg paeonol suspended in carboxymethyl cellulose [26 (link)] for ten consecutive days and received MTX at the end of the fifth day of the experiment.

Formalin-fixed, paraffin-embedded tissue blocks were cut into 8 μm sections. Sections were deparaffinized, rehydrated, and then underwent haematoxylin and eosin (H&E) staining and were viewed under a microscope (Motic TYPE 102M, Xiamen, China). The histological assessments were conducted by a pathologist who was blinded to the treatment. Each histological characteristic was scored on a scale of 0 (normal) to 5 (maximal). The lung inflammatory score was categorized according to the sum of the score for infiltration cell numbers and for damage level, including thickening of alveolar walls and epithelium, as well as increases in peribronchial and perivascular cuff area.

The paeonol standard was precisely weighed at 5.0 mg, placed in 10 mL volumetric flasks, dissolved with methanol, and shaken well to obtain a paeonol standard solution with a concentration of 0.5 mg/mL. A series of paeonol standard solutions (5, 10, 15, 20, 25 µg/mL) were prepared by removing 0.1, 0.2, 0.3, 0.4, and 0.5 mL of the paeonol standard solution with a pipette and adjusting the volume precisely to 10 mL with a methanol solution. The standard curve was drawn with the peak area y as the vertical coordinate and the paeonol concentration x as the horizontal coordinate. The regression equation of paeonol was y = 84,218x – 10,221, R² = 0.9996.

Male ApoE^−/− mice weighing 17 and 27 g were obtained from Qizhen Laboratory Animal Co., Ltd. (Hangzhou, China) and housed in a room kept at 22 ± 2 °C and relative humidity 50% ± 5%. After a week of random adaptation with food and water, mice were fed a HFD (composed of 40 kcal% fat-derived and 0.15% cholesterol chow) for 12 weeks until atherosclerotic lesions were formed in the arteries, and then the mice were randomly divided into five groups: (1) normal diet (Control), (2) HFD (Model), (3) HFD + paeonol (200 mg/kg body weight), (4) HFD + paeonol (400 mg/kg body weight), (5) HFD+ simvastatin (25 mg/kg body weight, SIM) (n = 8/group). Mice received 0.5% CMC-Na solution containing 200, and 400 mg/kg paeonol by gavage for 4 weeks, while C57BL/6 mice in the control group received 0.5% CMC-Na solution without paeonol by gavage.

The adsorption and desorption static experiments were carried out as follows. A quantity (1.0 g) of pretreated HPD-300 macroporous resin and 25 mL of the sample solution with a concentration of 0.8 mg/mL for paeonol were added into conical flasks (50 mL), and the flasks were continually shaken at 120 rpm for 12 h at a constant temperature (25 °C). During this period, 100 µL of the sample solution was taken every hour, and the paeonol concentrations were determined by HPLC. The adsorption ratios were calculated, and the adsorption kinetic curve was plotted.
When adsorption equilibriums were reached, the macroporous resins were washed with deionized water. A mixture of 25 mL 50% ethanol solution and macroporous resin after adsorption equilibrium were shaken at 120 rpm at 25 °C for 12 h after adsorption equilibrium. The paeonol concentration was measured in 100 µL of the supernatant every hour. The desorption ratios of HPD-300 macroporous resin were measured, and the desorption kinetic curve was plotted. Under the same pH conditions, seven crude MC solutions with different initial paeonol concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.4 mg/mL) were employed to investigate the effect of the paeonol concentration on the adsorption process. The effect of pH on static absorption was researched by adjusting the pH from 2 to 8. Static desorption tests were developed with an ethanol eluent at different concentrations in a range of 20–100% (v/v).