The effect of Wogonoside on the expression of pulmonary brosis factor in Mycoplasma pneumoniae pneumonia

an with the


RESULTS
The results showed the bound effect of Scutellaria baicalensis and TGF-β1 was effective. The active ingredients which can be bound with TGF-β1 in the solution of Scutellaria baicalensis and Qinbai were obtained and analyzed to investigate the mechanism of Qinbai inhibiting the expression of TGF-β1.
UPLC-Q-TOF-MS results showed that the active ingredients was Wogonoside. SPR a nity analysis showed that the a nity constant was 21.71 µM. Pharmacological experiments revealed that Wogonoside strongly inhibited the expression of TGF-β1 and Smad3 in A549 cells infected by M.pneumoniae. CONCLUSION Wogonoside in Qinbai can be bound with TGF-β1 and down-regulate the expression of lung brosis factors TGF-β1 and Smad3. The nding may improve our understanding the molecular mechanism of Qinbai mediating MPP and provide new sights into the future pharmacological investigation of Qinbai.

Background
Mycoplasma pneumoniae (M.pneumoniae), a gram-stained negative microorganism, is a major cause for respiratory tract infections and community-acquired pneumonia [1] , accounting for approximately 10%-30% of all cases and as much as to 50% in outbreak years [2] . M.pneumoniae is spherical or silk without cell wall, which makes it instinctually resistant to antimicrobials, such as β-lactams [3] , azithromycin and glucocorticoid [4] . M.pneumoniae could attach to epithelial members, produce reactive oxygen species and injure epithelial cells. Mycoplasma pneumoniae pneumonia (MPP) is an acute respiratory infectious pneumonia with pulmonary interstitial brosis. M.pneumoniae invades the bronchioles, connective tissue around the bronchi and the alveolar septum, and eventually leads to interstitial brosis. Due to lung hypoplasia in children, M.pneumoniae is prone to cause interstitial brosis after repeated infections [5] . TGF-β1 is well accepted as the central mediator for brosis [6] . It promotes the formation of tissue brosis factor in the aspect of strongest expression, increases in brosis diseases, promotes brosis foci in ammatory cells and broblast aggregation [7,8] Qinbaiqingfei pellet (Qinbai) has already been approved as the rst effective new traditional Chinese medicine which can delay activities of M.pneumoniae and protect lung epithelial cells in clinical trials. The previous studies have shown Qinbai can inhibit the expression of M.pneumoniae adhesion proteins P1 and P30 and promote the repair of airway tissues and epithelial cells [9][10][11] . Yao et al. pointed that Qinbai inhibited epithelial-mesenchymal transition (EMT) of alveolar type epithelial cells by reducing the content of TGF-β and restore the normal morphology and function of the lung by increasing the expression of SP-A [12] . Wang et al. found that Qinbai decreased the expression of cytokines Wnt5a and α-SMA, and inhibit the cellulose deposition after MPP [13] .
The traditional Chinese medicine Scutellaria baicalensis, the dry root of Scutellaria baicalensis Georgi, is the most important herb in Qinbai. Scutellaria baicalensis has been used for the treatment of "Lung-Heat" syndrome in China for a long time. In Xiaochaihu Decoction and Chaigejieji Decoction which are the classical prescriptions for the treatment of pulmonary infectious diseases and initially recorded in Han Dynasty (20-220 AD), Scutellaria baicalensis is the main herb [14] . In the traditional Chinese medicine practice, there have reported several researches on the pulmonary in ammation treatment by Scutellaria baicalensis [15][16][17] . Xu et al. [14] pointed that Scutellaria baicalensis may inhibit excessive release of proin ammatory cytokines, thereby inhibiting the systemic in ammatory response syndrome and promoting lung repair of tissue in ammatory lesions. Scutellaria baicalensis can suppress different tissue and organ brosis occurrence and development via various mechanisms, including down-regulating expression of promote-brosis cytokines, inhibiting pro-brogenic signaling pathways, anti-in ammatory and anti-oxidant effects [19][20][21] . It's con rmed that the active components of Scutellaria baicalensis are Wogonoside, Baicalin, Baicalein and Wogonin [22,23] . Wogonoside is a avonoid ingredient with various biological activities, such as anti-tumor, anti-in ammatory and anti-oxidation [24][25][26] . Previous studies showed that Wogonoside inhibited the TLR4 expression and the phosphorylation of NF-kappa B p65 and I kappa B induced by LPS, exerting anti-in ammatory effects [23,27] . However, there are few reports on the treatment of MPP with Wogonoside and the mechanism of Wogonoside inhibiting the deposition of pulmonary interstitial cellulose to treat MPP needs to be further studied.
The surface plasmon resonance (SPR) biosensor is a refractometer that measures changes in the optical re ectivity of a thin metal lm when species adsorb or bind to its surface or to any material coated onto its surface [18,28] . SPR has been applied in numerous elds including the researches of Chinese herbal medicine targets, medical diagnostics and pharmaceutical analysis [29] . Based on SPR, Cao et al. rstly discovered that physcion-8-O-β-D-monoglucoside was a bioactive ingredient and the a nity to TNF-R1 was 376 nM [30] . Tu et al. con rmed that protocatechualdehyde could control cardiovascular remodeling by binding with collagen I [13] . In present study, it was found that Scutellaria baicalensis extract in Qinbai showed the best binding with TGF-β1. On this foundation, SPR was applied to identify the active ingredient in Scutellaria baicalensis and reveal the mechanism of Qinbai in the treatment of MPP.

Preparation of six herbal extracts of Qinbai
Qinbai is composed of six Chinese herbal medicines including Scutellaria baicalensis, Platycodon grandi orus, 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 ltrate 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 ne powder at low temperature.
Pheretima aspergillum extract: 63g Pheretima aspergillum was crushed properly and extracted twice with 70% ethanol solution, the rst time for 2 hours, the second time for 1.5 hours. After ltration, the ltrate was combined.Platycodon grandi orus extract: 42g Platycodon grandi orum was decocted twice with water for 1.5h each time, and the ltrate was ltered and combined. At 60 ℃, the ltrate 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 grandi orus.

SDS-PAGE
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 ow 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 Nhydroxysuccinimide 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 ow rate of 10 μL/min. Finally, the CM5 sensor chip was blocked with ethanolamine. The rst 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 ow 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 nal concentration of 6 μg/mL. Similarly, 20 mg/mL Scutellaria baicalensis extract was injected into the chip surface at a ow 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 ow cells and incubated for 20s to allow the bound ingredient to be dissociated into the recovery solution. The ow 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 su cient samples, the total number of cycles was set to 20. 20 mg/mL Qinbai solution was also processed and recovered as the procedures above.

UPLC-QTOF/MS analysis
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 ACQUITY TM UPLC system. The chromatographic columns were a Waters ACQUITY UPLC BEH C 18 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 ow 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, N 2 ; 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 a nity 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 tted a steady-state a nity model to obtain the a nity constant (K D ). Equilibrium dissociation constant (K D ) was derived by tting 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% CO 2 and subcultured every 7 days. A549 cells were maintained in RPMI 1640 medium with 10% FBS at 37 ℃ and 5% CO 2 , and subcultured every 3 days. 1×10 5 A549 cells were divided into infected cell group (infected by 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 quanti ed by densitometry using the Image J software.

Statistical analysis
All experiments were repeated at least twice. Data were presented as mean±SD and analyzed statistically with Dunnett's test. Differences were considered signi cant at P<0.05.

SDS-PAGE
As shown in Fig.1, TGF-β1 protein showed a clear band in the molecular weight range of 48 kDa and 35 kDa, which was in accordance with the experimental standard.
The bound results of Scutellaria baicalensis and other herbal extracts with TGF-β1 by SPR analysis According to Fig.2, TGF-β1 protein solution diluted in pH 4.0 sodium acetate showed the best bound effect and the bound value reached 8894.9 RU. According to the results in Fig.3, the binding value of Scutellaria baicalensis extract to TGF-β1 protein was signi cantly higher than that of other ve herbs.
The results in Fig.4 showed that the bound curve of the second channel gradually increased and was higher than the rst channel. The bound value of the 2-1 channel reached 159.2 RU in the steady state, indicating that the Scutellaria baicalensis extract showed a good binding with the target protein in vitro.
Recognition and recovery of the TGF-β1 bound ingredients TGF-β1 protein solution was connected to four channels of CM5 chip for shing ligand. The bound values in Fig.5 were 8192.9 RU, 6679.6 RU, 7687.1 RU, 5939.9 RU, indicating the protein was successfully bound with CM5 chip. Wogonoside was indenti ed as a recovery ingredient based on the retention time and m/z information in both positive and negative modes (Fig.6, Fig.7, Fig.8 and Table 1). The negative mode was employed as it showed better sensitivity of ion response than the positive mode. Fig.6A and  (Fig.6B and Fig.7B), the fragment ion was m/z 283 due to loss of 176(C 6 H 8 O 6 ) and m/z 268 was from the loss of 15(CH 3 ). The retention time of Wogonoside was consistent with recovered ingredients and the error were less than 10 ppm. Table 1 UPLC-Q-TOF-MS data in both positive and negative ion mode of Wogonoside and recovered samples.
The bound results of Wogonoside with TGF-β1 protein by SPR analysis SPR assay was conducted to verify the direct interaction of Wogonoside with TGF-β1 protein. As shown in Fig.9A, serial concentrations of Wogonoside ranging from 17.5 nM/mL to 280 nM/mL were injected and bound with TGF-β1 protein. It was found that the binding value increased gradually with the increase of the concentration. SPR analysis revealed that the dissociation constant (K D ) was calculated as 21.71 μM (Fig.9B), indicating that Wogonoside was a potent bound ingredient with TGF-β1 protein. The two curves were very close at 70 nM/mL which showed that the experiment was reproducible.
The decreased expression of TGF-β1 and Smad3 in M.pneumoniae-infected A549 cells by Wogonoside As shown in Fig.10, the mRNA levels of TGF-β1 and Smad3 in the M.pneumoniae group were higher than those of the blank group (P<0.01). Meanwhile, it was found that the downregulation of TGF-β1 and Smad3 was observed after treated with 40 μM Wogonoside. Western blotting analysis (Fig.11) revealed that Wogonoside could decrease the protein expression of TGF-β1 (P<0.01) and Smad3 (P<0.05). In conclusion, the experimental results indicated that Wogonoside could reduce the expression of TGF-β1 and Smad3, thereby inhibiting the activity of M.pneumoniae.

Discussion
As we all know, it's a very di cult process to nd the active ingredients from complex herbal medicines. SPR biosensors have become an important tool for characterizing and quantifying biomolecular interactions. In addition, considerable progress has been made in the development of SPR sensors for detecting chemical and biological species. Based on the SPR technology, the six herbal extracts of Qinbai were bound with TGF-β1 protein respectively, and then the active ingredients which could be bound with TGF-β1 protein were screened preliminarily. Nevertheless, SPR does not provide information for structural identi cation of protein-bound ingredients in an unknown mixture. UPLC-Q-TOF-MS was adopted to analyze the active ingredients. In this study, we described a method for screening the active ingredient from Chinese herbal medicines by SPR biosensor and UPLC-Q-TOF-MS technology.
The result showed that the bound value of Scutellaria baicalensis extract with TGF-β1 protein reached 2881.9 RU, which was signi cantly higher than the other ve herbs in Qinbai. The result laid a foundation for the study of the effective components of Qinbai. The bound result of Scutellaria baicalensis extract and TGF-β1 protein (Fig.4) showed that the bound value gradually increased with the prolongation of injection time and was signi cantly higher than that of the reference channel. In addition, the difference of bound values at stable state reaches 159.2RU, indicating certain binding of Scutellaria baicalensis extract with TGF-β1 protein. UPLC-Q-TOF-MS results showed that the molecular formula, retention time, and fragmentation information of active ingredients that could be bound with TGF-β1 in the solution of Scutellaria baicalensis and Qinbai were consistent with those of Wogonoside standard, indicating that Wogonoside in Scutellaria baicalensis is one of the major active ingredients in Qinbai. The a nity of Wogonoside and TGF-β1 protein was determined based on SPR technology. 1: 1 bound model of small molecules and proteins was used for kinetic analysis. The a nity constant range of small molecules and proteins was 10 -6 M 10 -3 M. The bound values of Wogonoside with TGF-β1 at different concentrations in Fig.9B showed that the K D was 21.71 μM which was consistent with the a nity characteristics of small molecules and proteins, indicating that there is a speci c binding between Wogonoside and TGF-β1.
Respiratory mucosal epithelial cells are particularly susceptible to pathogens. Repairing damaged respiratory mucosal epithelium is very important for the treatment of MPP. Therefore, A549 cells were selected for this study. M.pneumoniae stain assay was used to evaluate the cells with the infection of M.pneumoniae. It has been demonstrated that 10 6 CCU M.pneumoniae could lead to the presence of cloud-like stains around A549 cells [31] . The result suggested that M.pneumoniae had been successfully adhered to A549 cells. In present study, 10 6 CCU M.pneumoniae was selected to build M.pneumoniae models.
The former studies indicated that A549 cells were obviously inhibited after the treatment with different doses of Wogonoside. The half-inhibitory concentration (IC 50 ) value was identi ed to be 46.1 μM [32] .
Wogonoside of 40 μM was selected in this study to detect the ability to inhibit the expression of TGF-β1 and Smad3 after M.pneumoniae infection. Previous studies revealed that the effects of TGF-β are performed by the activation of downstream Smads, including Smad3 and Smad4 [33] . In addition, Smad3 mediated TGF-β1-augmented contraction in HFL-1 cells [34] . In present study, the results of PCR and Western blotting showed that 40 μM Wogonoside markedly inhibited the impression of TGF-β1 and Smad3 in M.pneumoniae models

Conclusion
In conclusion, the present study indicated Wogonoside in Qinbai can be bound with TGF-β1 and downregulate the expression of lung brosis factors TGF-β1 and Smad3. The nding may improve our understanding the molecular mechanism of Qinbai mediating MPP and provide new sights into the future pharmacological investigation of Qinbai.  The bound result of TGF-β1 protein with CM5 chip