A549 cells were kindly provided by Chinese Academy of Sciences Stem Cell Bank (China). Mycoplasma pneumonia was purchased from DAAN GENE (China). RPMI 1640 medium and fetal bovine serum (FBS) were obtained from Hyclone (USA). PPLO broth was purchased from BD Biosciences (USA). Fresh yeast was purchased from Angel Yeast Company (China). Human TGF-β1 protein was purchased from ACRO-Biosystems (USA). SDS-PAGE Gel Kit was obtained from Solarbio life sciences (China). Amine Couping Kit (BR-1000-50), Acetate 4.0 and NaOH (50 mM) were purchased from GE Healthcare (Sweden). Mouse anti-TGF-β1 antibody (ab64715) and Rabbit anti-Smad3 (ab40854) antibody were purchased from Abcam (USA). Rabbit anti-β-actin (bs-0061R) was purchased from Bioss (UK). Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) and Peroxidase-Conjugated Goat anti-Rabbit IgG (H+L) were purchased from ZSGB-BIO (China). Trizol reagent was purchased from Life Technologies (USA). Go Taq 1-Step RT-qPCR System was purchased from Promega (USA). RIPA reagent was provided by Beyotime (China). Bradford method kit was purchased from Nanjing Jiancheng Biotechnology Co.Ltd (China). Polyvinylidene difluoride (PVDF) membranes were purchased from Biosharp (China). Hypersensitive electrochemiluminescene kit was obtained from Wanleibio (China). Scutellaria baicalensis was purchased from Beijing Tongrentng Pharmacy (China). Qinbai (Batch number: 130701) was obtained from Harbin tianheli Pharmaceutical Co., Ltd. Wogonoside was purchased from Shanghai Yuanye Biotechnology Co.Ltd, (20 mg, B20488, HPLC>98%). Formic acid was purchased from Fisher Scientific (USA). Methanol and acetonitrile were respectively purchased from Kermel (China) and Merck (Germany). ACQUITYTM UPLC system was obtained from Waters (USA). Triple-TOFTM 5600+ mass spectrometer was obtained from AB SCIEX (USA). Acquity UPLC BEH C18 column (2.1 mm×100 mm, 1.7 μM) and AQUITY UPLC BEH C18 VanGuard Pre-Column (2.1 mm×5 mm, 1.7 μM) were purchased from Waters (USA). Fluorescent Quantitative Detection System (Line Gene 9600) was purchased from BIOER (China). Biacore T200 was purchased from GE Healthcare (Sweden). Gel imaging system (Versa Doc MP 4000) was obtained from BIO-RAD (USA).
Preparation of six herbal extracts of Qinbai
Qinbai is composed of six Chinese herbal medicines including Scutellaria baicalensis, Platycodon grandiflorus, Pheretima aspergillum, Stemona japonica, Aster tataricus and Ophiopogon japonicus. According to Chinese Pharmacopoeia (2015 Edition), the six herbal extracts were prepared as follows:
Scutellaria baicalensis extract: 84g Scutellaria baicalensis was extracted with water for 4 h. The filtrate was concentrated to the relative density of 1.05-1.10 (80 ℃), and pH was adjusted to 1.0-2.0 with 2 mol/L HCl at 80 ℃. The solution was allowed to stand for 24 h. The supernatant was washed with water to pH 5.0, and then washed with 70% ethanol until pH reached 7.0. Finally, the solution was pulverized into fine powder at low temperature.
Pheretima aspergillum extract: 63g Pheretima aspergillum was crushed properly and extracted twice with 70% ethanol solution, the first time for 2 hours, the second time for 1.5 hours. After filtration, the filtrate was combined.Platycodon grandiflorus extract: 42g Platycodon grandiflorum was decocted twice with water for 1.5h each time, and the filtrate was filtered and combined. At 60 ℃, the filtrate was concentrated into a transparent paste with a relative density of 1.30-1.35.
The extracts of Stemona japonica, Aster tataricus and Ophiopogon japonicus were prepared according to the extraction process of Platycodon grandiflorus.
TGF-β1 protein solution was diluted from 200 μg/mL to 160 μg/mL with the loading buffer and then heated at 100 ℃ for 5 min. SDS-PAGE was carried out with 12% polyacrylamide gel according to the molecular weight of TGF-β1. The experimental conditions were as follows: the loading volume of TGF-β1 protein was 5 μL, the concentrated gel voltage was 70 V for 30 min and the separated gel voltage was 110 V for 1 h. The protein band was stained with Coomassie brilliant blue for 1 h and then eluted until the background was completely clean. The photograph was taken by the gel imaging system.
The binding of Scutellaria baicalensis and other herbal extracts with TGF-β1 by SPR technology
SPR analysis was performed on the Biacore T200 system. Prior to the analysis, the whole flow path was primed by phosphate buffer saline (PBS) for three times and 70% bianormalizing solution was used for signal normalization. The CM5 sensor chip was activated by injecting the mixture of 100 mM N-hydroxysuccinimide and 400 mM N-ethyl-N'-(diethylaminopropyl)-carbodiimide (BR-1000-50, Uppsala, Sweden). The solution of TGF-β1 protein was diluted in the pH 4.0 sodium acetate to 6 μg/mL and immobilized on the second channel of the CM5 sensor chip for 600 s at a flow rate of 10 μL/min. Finally, the CM5 sensor chip was blocked with ethanolamine. The first channel served as a negative control. The extract of Scutellaria baicalensis and other herbal extracts were dissolved into 20 mg/mL solution with PBS solution respectively.
Every herbal extract was injected to TGF-β1 sensor surface for 60 s at a flow rate of 30 μL/min and then the chip surface was regenerated by glycine hydrochloric acid solution with pH 2.0 for 30s. The sensorgram was recorded and expressed in resonance units (RU).
Recovery of TGF-β1 bound ingredients
TGF-β1 protein was coupled to four channels of another CM5 chip with the final concentration of 6 μg/mL. Similarly, 20 mg/mL Scutellaria baicalensis extract was injected into the chip surface at a flow rate of 5 μL/min for 180 s. The system was then washed with PBS to thoroughly remove the remaining sample solution. Then a small volume of 2 μL recovery solution (0.5% HCOOH) was injected into the flow cells and incubated for 20s to allow the bound ingredient to be dissociated into the recovery solution. The flow direction over the sensor surface was reversed and the recovery solution containing TGF-β1 bound ingredients was deposited in 10 μL ammonium bicarbonate (50 mM). In order to obtain sufficient samples, the total number of cycles was set to 20. 20 mg/mL Qinbai solution was also processed and recovered as the procedures above.
The SPR-recovered samples were dried under nitrogen and dissolved in 100 μL methanol. The analysis of the SPR-recovered samples was performed on Waters ACQUITYTM UPLC system. The chromatographic columns were a Waters ACQUITY UPLC BEH C18 column (2.1×100 mm, 1.7 μM) and a Van Guard Pre-Column (2.1mm×5mm, 1.7 μM). The mobile phase consisted of 0.1% aqueous formic acid (A)-0.1% formic acid acetonitrile (B), using a gradient elution. The flow rate was 0.4 mL/min and the injection volume was 10 μL. Elution procedure is 5% -100% B at 0-13 min, 100%-5% B at 13-13.10 min and 5% B at 13.10-15.00 min for SPR recovered samples. Mass spectrometry detection was performed by the AB SCEIX Triple-TOFTM 5600+ high resolution mass spectrometer. The product ion scan range was 50-1500 Da, enabling dynamic background deduction. The condition of ESI source was as follows: ion source voltage, 5500 V ( positive ion mode), 4500 V (negative ion mode); ion source temperature, 550 ℃; atomizing gas, N2; auxiliary gas pressure, 379.17 kPa; air curtain gas pressure, 241.99 kPa; fragmentor voltage, 80 V; collision energy, ±35 eV; collision energy expansion, 15 eV. IDA was set to respond to the 8 highest peaks of more than 100 cps for secondary mass spectrometry scanning. All data was obtained by Analyst TF 1.6 software and analyzed by Peakview 2.0/masterview 1.0 software.
SPR affinity analysis
The system was primed by 10% methanol before experiments. Wogonoside was diluted in 10% methanol to 5 gradient concentrations (280 nM/mL to 17.5 nM/mL), then injected through the reference and active channels for 60 s at the rate of 30 μL/min, and regenerated with pH 2.0 Glycine-HCL for 60 s. The experiments of 70 nM/mL were performed twice to assess the repeatability. The total number of cycles was 3. Finally, the results were analyzed by Biacore evaluation software (T200 Version 1.0) and fitted a steady-state affinity model to obtain the affinity constant (KD). Equilibrium dissociation constant (KD) was derived by fitting to a 1:1 Langmuir bound model.
M.pneumoniae and cell culture
The M.pneumoniae cells (ATCC 15531) were cultured in PPLO broth (containing 20% fetal bovine serum, 10% yeast extract solution, 1% glucose, and 0.0002% phenol red), incubated at 37°C with 5% CO2 and subcultured every 7 days. A549 cells were maintained in RPMI 1640 medium with 10% FBS at 37 ℃ and 5% CO2, and subcultured every 3 days. 1×105 A549 cells were divided into infected cell group (infected by 106 CCU M.pneumoniae for 4 h), Wogonoside group (infected and given 40 μM Wogonoside for 3 days) and blank group.
Real-time quantitative PCR
Total RNAs were extracted from A549 cells by Trizol. The level of RNAs was amplified by one-step Reverse Transcription-PCR kit and determined by Bioer Technology (China). The ratio for mRNA of interest was normalized to β-actin and presented as the mean ±SD.
The primers used are as follows:
TGF-β1 forward primer: 5´-AGC AAC AAT TCC TGG CGA TAC CTC-3´
TGF-β1 reverse primer: 5´-TCA ACC ACT GCC GCA CAA CTC-3´
Smad3 forward primer: 5´-CAC AGC ATG GAC GCA GGT TCT C-3´
Smad3 reverse primer: 5´-AGG AGA TGG AGC ACC AGA AGG C-3´
β-actin forward primer: 5´-ACA GAG CCT CGC CTT TGC-3´
β-actin reverse primer: 5´-GCG GCG ATA TCA TCA TCC-3´
A549 cells were lysed by RIPA extraction reagent and then centrifuged (4℃) for 10 min at the rate of 12000 rpm/min. Total protein concentration was determined by the Bradford assay method. Samples were subjected to 12% SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk in TBST (200 mM Tris buffer, pH 8.0, containing 150 mM NaCl and 0.1% Tween 20) at room temperature for 1 h. The membranes were incubated with primary antibodies overnight at 4℃. After incubated with HRP-conjuated anti-rabbit IgG or anti-mouse secondary antibodies, protein bands were visualized using enhanced chemiluminescence (ECL) substrate by Versa Doc Imaging Analysis System and quantified by densitometry using the Image J software.
All experiments were repeated at least twice. Data were presented as mean±SD and analyzed statistically with Dunnett's test. Differences were considered significant at P<0.05.