Densichek plus densicheck

The determination of the penetration ability of HZQYF on the outer membrane of H. pylori was done by NPN uptake method. H. pylori was scraped from blood agar plates and adjusted to an optical density of 1 McF using DensiCHEK Plus densicheck (BioMerieux, French). Different concentrations of HZQYF extracts (80 and 160 μg/mL) were added, and a growth control was set. After incubation in a tri-gas incubator for 24 h, the samples were centrifuged at 6000 rpm for 10 min and washed twice with PBS buffer. Then, they were resuspended in PBS. The bacterial solution and 40 μM NPN were added into a 96-well plate, and incubated at 37°C for 30 min before conducting fluorescence measurements. The fluorescence measurements are performed using a multifunctional microplate reader with an excitation wavelength of 350 nm, an emission wavelength of 420 nm, and an excitation bandwidth of 5 nm. All measurements are repeated three times.

To collect enough bacteria, H. pylori ATCC 700392 was adjusted to 1 McF using DensiCHEK Plus densicheck (BioMerieux, French) and cultured for 2 days. In a second experiment, 1 mL of bacterial solution (OD₆₀₀ = 0.5–0.6) was diluted 50-fold in BHI broth adding 10% FBS with or without the MIC of HZQYF for 24 h. The sample’s total RNA was extracted using Purelink RNA kit. Their concentrations were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). On a Thermo Scientific 7,500 Fast Real-Time PCR System (Quantitative Real-Time PCR System), RT-qPCR was carried out using the SYBR Premix Ex Tap^™ kit (Takara) and following the manufacturer’s instructions. The 2^−ΔΔCt method was used to examine the data. Table 1 lists the particular primers used (Shu et al., 2016 ) to amplify mRNA fragments. The expression regulation of these genes was assessed in comparison to bacteria from the control group following normalization with the reference gene 16S.