Wenshen Yiqi Keli Mitigates the Proliferation and Migration of Cigarette Smoke Extract-Induced Human Airway Smooth Muscle Cells through miR-155/FoxO3a Axis

Some domestic scholars revealed the effectiveness of Wenshen Yiqi Keli (WSYQKL) on chronic obstructive pulmonary disease (COPD). However, the exact mechanism of WSYQKL on COPD is fuzzy and needs further research. We adopted UPLC-Q/TOF-MS to analyze the chemical components of WSYQKL. In in vitro experiments, human airway smooth muscle cells (hASMCs) were intervened with 2.5% cigarette smoke extract (CSE), medicine serum of WSYQKL, miR-155 mimic, and FoxO3a silencing. Cell viability, proliferation, migration, and the expressions of miR-155, PCNA, Ki67, p21, p27, and FoxO3a were examined by cell counting kit-8, EdU staining, Transwell assay, scarification assay, qRT-PCR, immunol cytochemistry, and western blot, respectively. The association between miR-155 and FoxO3a was assessed by database and luciferase reporter gene analysis. We identified 47 kinds of chemical compositions of WSYQKL in ESI+ mode and 42 kinds of components of WSYQKL in ESI− mode. The medicine serum of WSYQKL strongly alleviated the proliferation and migration of hASMCs induced by CSE in a concentration-dependent manner. The medicine serum of WSYQKL enhanced the levels of p21, p27, and FoxO3a and weakened PCNA and Ki67 levels in hASMCs induced by CSE with the increase of concentration. MiR-155 mimic or FoxO3a silencing notably advanced CSE-treated HASMC viability, proliferation, migration, and the levels of PCNA and Ki67 and downregulated the levels of p21, p27, and FoxO3a in CSE-triggered hASMCs, which was reversed by WSYQKL-containing serum. Our results described that WSYQKL alleviated the proliferation and migration of hASMCs induced by CSE by modulating the miR-155/FoxO3a axis.


Introduction
Chronic obstructive pulmonary disease (COPD) is a familiar preventable and treatable disease marked by persistent respiratory symptoms and airfow restriction [1]. Respiratory symptoms and airfow restrictions are caused by airway and/ or alveolar abnormalities caused by toxic particles or gases [2]. Airfow restriction is the major reason for clinical symptoms in COPD patients, and the cardinal pathological feature of airfow restriction is airway remodeling [3,4]. Airway remodeling is the primary pathological basis of COPD and a pivotal factor in the sustainable development of COPD [5]. Te process of airway remodeling involves multiple cells and cytokines, among which airway smooth muscle cells (ASMCs) are particularly considerable and the most in-depth research [6]. Excessive proliferation of ASMCs causes tracheal dysfunction and is participated in airway remodeling and airway hyperresponsiveness [7]. To date, there are no efective COPD treatments for inhibiting cell proliferation of ASMCs and no globally accepted criteria for defning early COPD [8,9]. Terefore, there is an urgent need for new biomarkers for the early detection of COPD to facilitate the timely intervention of the disease.
MicroRNA (miRNA) is an endogenous noncoding RNA that can cleave mRNA to inactivate it and weaken the translation process [10]. Regulation of cellular activities such as proliferation, apoptosis, and diferentiation by altering the expression of miRNAs has been implicated in the development of various diseases including cardiovascular dysfunction, lung disease, and cancer [11,12]. It has been reported that miRNA can modulate the pathological process of COPD [13,14]. Among them, miR-155 was reported to be overexpressed in bronchial epithelial cells mediated by PM2.5 [15]. However, the exact mechanism of miR-155 in COPD is fuzzy. Now, mounting clinicians realize that traditional Chinese medicine (TCM) has its unique advantages in the treatment of COPD. Wenshen Yiqi Keli (WSYQKL) is a commonly used prescription for the clinical treatment of COPD by Professor Xu Zhiying of TCM. WSYQKL is composed of Astragali Radix, Cuscutae Semen, Fluoritum, Paris polyphylla, and Rhizoma Curcumae. Te long-term clinical practice indicated that WSYQKL can ameliorate and maintain the pulmonary function of COPD patients, mitigate clinical symptoms and decrease the number of acute attacks, with good social and economic benefts [16,17]. However, whether WSYQKL afects the proliferation and migration of cigarette smoke extract-induced human airway smooth muscle cells by modulating miR-155 expression has not been reported.
In this study, we used human ASMCs (hASMCs) as target cells to investigate whether WSYQKL-containing serum afects the proliferation and migration of cigarette smoke extract (CSE)-triggered hASMCs by regulating miR-155, and hoped to clarify its specifc regulatory mechanism and provide new ideas for the diagnosis and treatment of COPD.

Preparation of WSYQKL Test Solution.
We took an appropriate amount of granules and grind them to powder, and then precisely weighed 1.00 g of the powder. After adding 25 mL of methanol, the above powder was extracted by ultrasonic for 45 min. After centrifugation (14000 r/min, 20 min), we obtained the supernatant, namely, the test solution. 2.3. Animal Care. Twenty male SD rats of specifc pathogenfree (SPF) grade, weighing 200 ± 20 g, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Te Animal Ethics Committee of Zhejiang Eyong Pharmaceutical Research and Development Center granted the animal experiments. Tey were adapted for 7 days in a breeding room (20 ± 2°C, 55 ± 5%, and 12-h light/dark cycle).

Preparation of WSYQKL-Containing Serum.
WSYQKL was bought from Zhejiang Hospital of TCM. Specifcations: 15 g/pack, 1 pack each time, 2 times a day. A total of 20 rats were randomly distributed into the control group and WSYQKL group, 10 per group. Rats in the WSYQKL group were administered by gavage at a dose of 3.2 g/kg/d, twice a day, for 5 consecutive days. Te drug doses for rats were determined using the following formula: Rats (g/kg) � [human dose (30 g/day)/human weight (60 kg)] × 6.3. Te rats in the control group were given an equal volume of normal saline. Two hours after the last administration, the blood samples of the rats were obtained by eye enucleation. After standing for 1 h, the blood samples were centrifuged and fltered followed by inactivation in a 56°C water bath for 30 min and then stored at −80°C.

Collection of Cigarette Smoke Extract (CSE).
We ignited a cigarette (11 mg tar, 0.9 mg nicotine, and 11 mg carbon monoxide). One end of a rubber tube was connected to a cigarette flter, and the other end was connected to a syringe, to mimic the actual smoking situation of a person. 300 mL of smoke from the syringe was injected into a 10 mL glass bottle of DMEM, which was shaken to dissolve into the culture medium. We obtained 100% stock solution CSE (30 mL of smoke per mL of DMEM).

Cell Viability Analysis of HASMCs.
For the examination of HASMC cell viability, CCK-8 Kit (HY-K0301, MCE, USA) was earmarked. HASMCs (1 × 10 4 /well) were loaded in ninety-six-well plates. Te next day, cells were processed based on the above study design for 24 h. After that, we removed the cell medium and then dripped 10 μL CCK-8 solution into each well. After being reacted at 37°C for 4 h, we recorded the absorbance (450 nm) utilizing a microplate reader (CMaxPlus, MD, USA).

Migration
Analysis of HASMCs. hASMCs were serumstarved for 24 h and then subjected to digestion and centrifugation. Cell precipitation was resuspended in a medium without FBS and appended to the apical chamber (3422, Corning, USA). Te media with 10% FBS was dripped into the basolateral chamber. After 48 h of conventional culture, we removed hASMCs that did not migrate through the inner surface of the apical chamber. Te fxation of hASMCs migrated into the lower compartment was done with methanol. Ten, 0.1% crystal violet (C0775) ofered by Sigma-Aldrich (USA) was applied to stain hASMCs. An optical microscope (IX71, Olympus, Japan) was employed to observe the number of migrated hASMCs. For scarifcation assay, a total of 8000 hASMCs were loaded in six-pore plates. Te next day, the layer of hASMCs was injured with a 10 μL pipette tip. We utilized PBS to rinse the foating hASMCs. Tereafter, MEM medium without serum was added to each well. Te scratched results were taken at 0 h and 48 h and observed by the optical microscope. Te relative migration distance was assessed utilizing Image J (version 1.48).
U6 was validated as the normalizer. Te 2 −ΔΔCt was employed for data evaluation.

Luciferase Reporter Gene Analysis.
We forecasted the binding sites between FoxO3a and miR-155. MiR-155 mimic or miR-155-NC was co-transfected into hASMCs with a pmirGLO vector (E1330) ofered by Promega (USA) Evidence-Based Complementary and Alternative Medicine containing WT or MUT 3′-UTR of FoxO3a utilizing Lipo6000. After 48 h, to assess the luminescence signal, we used Dual-Lucy Assay Kit (D0010, Solarbio, China).

Statistical Analysis.
All analyses were computed utilizing SPSS 16.0 software. Te diferences among multiple groups were compared via one-way ANOVA followed by an SNK test when data satisfed a normal distribution. Kruskal-Wallis H test was performed for those with uneven variance. Data were indicated as mean ± SD. Statistically, P < 0.05 was meaningful.

Te Base Peak Chromatograms of WSYQKL.
UPLC-Q/TOF-MS was employed to qualitatively identify the chemical components of WSYQKL. Te total ion chromatogram of WSYQKL was displayed in Figure 1(a) (positive ion mode) and Figure 1(b) (negative ion mode). Table S1 presented 47 kinds of the chemical composition information of WSYQKL in ESI + mode. Table S2 presented 42 kinds of components of WSYQKL in ESI − mode.

WSYQKL-Containing Serum Inhibited Cell Proliferation on CSE-Mediated HASMCs.
We frst treated hASMCs with 20% FBS and 20% NRS. As displayed in Figure 2(a), hASMCs viability in the 20% FBS group and 20% NRS group was higher relative to the serum-free group, and there was no diference between the two groups. Terefore, 20% NRS can be used instead of 20% FBS (P < 0.01). Next, by performing CCK-8 and EdU staining experiments, 2.5% CSE induced the decrease of hASMCs viability and promoted the EdUpositive cell levels, while the medicine serum of WSYQKL efectively reversed its efect in a concentration-dependent manner (Figures 2(b) and 2(c), P < 0.05).

WSYQKL-Containing Serum Inhibited Migration on CSE-Triggered HASMCs.
To check the role of WSYQKL on the migration of hASMCs induced by CSE, we conducted a Transwell assay and scarifcation assay. Te results demonstrated that 2.5% CSE obviously facilitated the migration of hASMCs (Figures 3(a) and 3(b), P < 0.01). More importantly, we discovered that the medicine serum of WSYQKL strongly alleviated the migration of hASMCs induced by CSE in a concentration-dependent manner (Figures 3(a)and 3(b), P < 0.01).

WSYQKL-Containing Serum Decreased Levels of Proliferation-Related Markers on CSE-Triggered hASMCs.
In this part, immunol cytochemistry was applied to examine the positive expressions of Ki67 and PCNA. We found an upregulation of Ki67 and PCNA in hASMCs induced by CSE (Figure 4(a), P < 0.05). Te medicine serum of WSYQKL down-regulated the expressions of Ki67 and PCNA in hASMCs induced by CSE with the increase of concentration (Figure 4(a), P < 0.05). Te same results were found in the western blot assay (Figure 4(b), P < 0.05). Furthermore, the levels of p21, p27, and FoxO3a in the 2.5% CSE group were lower relative to the control group (Figure 4(b), P < 0.01). Te medicine serum of WSYQKL enhanced the levels of p21, p27, and FoxO3a in HASMCs induced by CSE with the increase in concentration (Figure 4(b), P < 0.05).

WSYQKL-Containing Serum
Reversed the Proproliferative Efect of miR-155 Mimic and si-FoxO3a on CSE-Mediated HASMCs. As presented in Figure 5(a), the qRT-PCR results described that a remarkable increase of miR-155 in CSE-treated hASMCs was found, while the medicine serum of WSYQKL counteracted its efect (P < 0.05). To probe whether WSYQKL afects the efect of miR-155 mimic or si-FoxO3a on CSE-mediated hASMCs, we conducted a series of cell experiments. MiR-155 mimic or si-FoxO3a notably advanced CSE-treated HASMC viability and proliferation, whereas the medicine serum of WSYQKL partially ofset miR-155 mimic or si-FoxO3a-mediated facilitating efect ( Figures 5(b) and 5(c), P < 0.05).

WSYQKL-Containing Serum Reversed miR-155 Mimic and si-FoxO3a Induced Migration on CSE-Mediated
HASMCs. To observe the migration ability of hASMCs in each group, we performed a transwell assay and scarifcation assay. As illustrated in Figures 6(a) and 6(b), miR-155 mimic or FoxO3a silencing extremely enhanced cell migration in hASMCs induced by CSE (P < 0.05). Additionally, we discovered that WSYQKL-containing serum neutralized the facilitation of miR-155 mimic or FoxO3a silencing on CSEtriggered HASMC migration (Figure 6(a) and 6(b), P < 0.05).

Discussion
COPD can increase the risk of cardiovascular disease and has become the third leading reason of human death worldwide [18]. Te occurrence of COPD is afected by external environmental factors and genetic factors; smoking is the commonest risk factor for COPD [19]. At present, the treatment schemes for COPD mostly take drugs, smoking cessation, and psychotherapy; oxygen inhalation and 4 Evidence-Based Complementary and Alternative Medicine        Evidence-Based Complementary and Alternative Medicine hormone therapy are required in severe cases, which afect the quality of life of patients [20,21]. Terefore, it is necessary to fnd an efective way to diagnose COPD. Te proliferation of ASMCs can cause tracheal wall thickening, airway remodeling, and hyperresponsiveness, which is one of the major pathogenesis of COPD [22]. Tus, probing the proliferation of ASMCs exhibits a signifcant efect on airway infammation and airway remodeling of COPD. In the report of ASMCs cultured in vitro, it was uncovered that a certain concentration of CSE advanced the proliferation of ASMCs [23]. Our data were consistent with the previous study. Our study further pointed out that 2.5% of CSE obviously facilitated the migration of hASMCs. Importantly, this research frst proved the modulation of WSYQKL on CSE-mediated hASMCs at the cellular level. Te medicine serum of WSYQKL strongly alleviated the proliferation and migration of hASMCs induced by CSE, indicating that WSYQKL can alleviate the risk of COPD deterioration by weakening the proliferation and migration.
It has been reported that FoxO3a signaling is involved in the modulation of important activities such as cell proliferation, apoptosis, and migration [24,25]. FoxO3a activation can advance the expression of cell cycle inhibitory proteins p21 and p27, and afect cell proliferation and apoptosis [26,27]. By reviewing the literature, it was found that CSE induced the expressions of Ki67, PCNA, and p21 in bronchial epithelial cells and down-regulated the expression of FoxO3a in human lung adenocarcinoma cells [28,29]. Wylam et al. clarifed that there was a high expression of PCNA in CSE-stimulated ASMCs [30]. Impaired FoxO3a activity has been described by some scholars as one of the intracellular responses activated by CSE [31]. Another study pointed out that FoxO3a was underexpressed in the lung tissue of COPD patients [32]. To further probe whether the antiproliferative and antimigratory efects of WSYQKL on CSE were due to the modulation of FoxO3a, we established FoxO3a-silenced hASMCs. Our fndings found that WSYQKL-containing serum weakened the proliferation and migration of CSE-triggered hASMCs by repressing PCNA and Ki67 levels and advancing p21, p27, and FoxO3a levels, while FoxO3a silencing neutralized its efect.
that the antiproliferative and antimigrate efects of WSYQKL on CSE-triggered hASMCs might be associated with an upregulation of FoxO3a by repressing miR-155. Taken together, WSYQKL could suppress CSE-mediated proliferation and migration of hASMCs, and its mechanism might be related to the modulation of the miR-155/FOXO3a axis. Further study on the mechanism of WSYQKL modulating the miR-155/FOXO3a axis might become a novel method for the treatment of COPD.

Data Availability
Te datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.