The HBECs were purchased from the American Type Culture Collection (ATCC; https://www. atcc.org/) and stored in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich Chemical Company, St Louis, MO) containing 10% FBS (Sigma-Aldrich Chemical Company) and penicillin-streptomycin solution (100×; Thermo Fisher Scientific Inc, Waltham, MA). Puerarin was provided by Shanghai McLean Biochemical Technology Company, batch number: C11030370, purity > 98%. The suspension was prepared with sterilized saline at the concentration of 50 µg/mL, 100 µg/mL and 200 µg/mL and stored in a refrigerator at 4℃ in the dark for subsequent experiments.
Preparation Of 100% Cigarette Smoke Extract
The cigarettes used in this experiment were all Nanjing cigarettes of a brand. The cigarette collection system was assembled according to the cigarette support tube, gas collection bottle, buffer bottle and peristaltic pump. During gas collection, after adding about 10 mL DMEM culture medium into the gas collection bottle, start the peristaltic pump, adjust the speed about 160 r/min, at this time the gas flow rate is about 600 mL/min, ignite 1.5 cigarettes in turn, and shake the gas collection bottle continuously to dissolve the cigarette smoke as soon as possible. After the cigarette is burned out, the liquid obtained is recorded as 100% CSE, placed on the ultra-clean table for filtration and sterilization. After that, the CSE solution was added into DMEM to prepare the CSE mixture, which held a concentration of 20% and used as soon as possible within 30 minutes.
Rna Extraction And Rt-qpcr
Total RNA was isolated from HEBCs by using the TRIzol (Invitrogen, Carlsbad, CA, USA)). Single-stranded cDNA was synthesized with the PrimeScript Reagent Kit (Promega, USA). Real-time PCR was conducted by using SYBR Premix Ex TaqTM Kit (Applied Biosystems, Foster City, CA, USA). The reaction was run in ABI7500 Real-time PCR system (Applied Biosystems, Carlsbad, CA). GAPDH was used as an endogenous control. Briefly, 2 µL of cDNA was added to 10 µL of the 1 × SYBR green PCR master mix with 0.4 µL of Taq polymerase enzyme (RiboBio, China), 0.8 µL of each primer and 6 µL ddH2O to a final volume of 20 µL. The RT-PCR cycling conditions consisted of: 95 ˚C for 10 min; then 35 cycle amplification for 20 s at 95 ˚C, 30 s at 55 ˚C, 15 s at 72 ˚C; followed by 1 min at 72 ˚C. The primers used in this study were synthesized from Sangon Biotech (Shanghai, China). The level of mRNA was normalized to β-actin expression using the 2−ΔΔCt method.
Western Blot Analysis
The cells were lysed for 20 min on ice in ice-cold lysis buffer (Roche). The lysates were centrifuged at 12,000 × g for 20 min at 4℃ to obtain a clear lysate. The protein content of each sample was determined using the BCA Protein Assay Kit (Thermo Scientific). Then, equal amounts of proteins (15 µg/lane) were separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidenedifluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% (w/v) nonfat dry milk in TBST (Tris-buffered saline-0.1%Tween) at 25 °C for 3 h and then incubated with the following primary antibodies: rabbit monoclonal anti-β-actin antibody (1:900, Abcam, ab179467), rabbit monoclonal anti-PINK1 antibody (1:1000, Abcam, ab216144), mouse monoclonal anti-Parkin antibody (1:1500, Abcam, ab77924), rabbit polyclonal anti-Cleaved Casepase3 antibody (1:1100, Abcam, ab2302), rabbit monoclonal anti-Bax antibody (1:2000, Abcam, ab32503), rabbit monoclonal anti-DRP1 antibody (1:1000, Abcam, ab184247), rabbit polyclonal anti-FUNDC1 antibody (1:500, Abcam, ab224722), rabbit monoclonal anti-PI3K antibody (1:1000, Abcam, ab32089), rabbit polyclonal anti-AKT antibody (1:1500, Abcam, ab179463), rabbit polyclonal anti-mTOR antibody (1:2000, Abcam, ab2732). The bands were visualized using horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (1:15000, Boster) prior to the ECL protocol (Amersham Biosciences, Piscataway, NJ, USA).
Detection Of Mmp Level By Flow Cytometry
Cells were collected by 0.25% trypsin (containing EDTA) digestion method; 200 mesh screen filtration, cell counting, 1.5 × 105 cells were collected; cells were resuspended by adding l mL JC-1 working solution (l×) under dark conditions, incubated at 37 ℃ for 20 min in dark, shaken once at 2ཞ3 min intervals; and centrifuged at 4 ℃ for 1500 rmp; the hearts were washed twice with JC-1 staining buffer (l×) for 5 min, and the cells were resuspended with 300 µL JC-1 staining buffer (l×) for flow cytometry detection.
Detection Of Ros Content By Flow Cytometry
Cells were collected by 0.25% trypsin (containing EDTA) digestion method; cells were filtered through 200 mesh screen, counted, and 1.5 × 105 cells were collected; preparation of DCFH-DA working solution: incomplete cell culture solution is 1:1000; cells were resuspended by adding DCFH-DA working solution under dark conditions, and incubated at 37 degrees in the dark for 20 min; cells were washed three times with incomplete cell culture medium and resuspended with 300 µL of incomplete cell culture medium for flow cytometry detection.
Atp Content Detection
Cells were collected by digestion with 0.25% trypsin (including EDTA); ATP lysate was added, mixed with shaking, ice bath for 10 min, 4 ℃ freezing centrifuge for 12000 × g Centrifuge for 5 min, extract the supernatant; prepare ATP detection working solution with ATP detection reagent: ATP detection diluent = 1:4 in dark condition; 100 µL working fluid was added into 96-well plate, stand at room temperature for 2ཞ5 minutes, then add standard diluent and sample 20 µL, and quickly mixed with a pipette gun; RUL was measured on a microplate reader and ATP concentration was calculated; protein concentration was measured at the same time, and finally ATP concentration: protein concentration was used as the final result, unit was nmoL/mg.
All statistical analyses were performed using the SPSS software (ver. 13.0; SPSS, Chicago, IL). The quantitative data derived from three independent experiments are expressed as mean ± SEM. Significance was determined by one-way ANOVA or t-test. Values of P < 0.05 were considered statistically significant.