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Lung cancer is one of the most common malignancies in the world. In 2012, a total of 1.8 million new cancer cases and 1.6 million related deaths were reported worldwide [1]. Approximately 10–15% of lung canMeng Wang, Qian-Yu Bi, Ya-Nan Zhang, Xu-Ming Ji 2 Arch Med Sci cers are small cell lung cancers, and about 85% are non-small cell lung cancers, including adenocarcinoma, large cell carcinoma, squamous cell carcinoma, etc. [2]. The 5-year survival rate of lung cancer is currently only 16%, and more than 70% of patients are diagnosed with an advanced stage due to the lack of early clinical symptoms [3]. In traditional Chinese medicine (TCM), the formation of cancer is considered to be caused by the infiltration of a pathogen based on a deficiency of vital-qi [4, 5]. TCM can inhibit tumor growth and metastasis through overall regulation of the tumor microenvironment, thereby regulating and improving the clinical symptoms of patients, improving patients’ quality of life and extending their survival [6]. Therefore, the regulatory effect of TCM on the tumor microenvironment has become one of the main research directions of TCM against tumors. The tumor microenvironment (TME) consists of a  complex network of heterogeneous cell types, including infiltrating immune cells, cancer associated fibroblasts (CAFs), granulocytes, extracellular matrix, endothelial cells, and other stromal components [7]. Among them, CAFs are the main components of stromal cells and can secrete a variety of cytokines to regulate tumor growth and metastasis [8]. Previous study has shown that CAF mediated activation of the Hh-Gli1 pathway is one of the most important molecular mechanisms for promoting the growth, invasion and metastasis of lung cancer [9]. Wenxia Formula (WF) is a TCM formula consisting of four crude herbs: Rheum palmatum L., Aconitum wilsonii Stapf ex Veitch, Panax ginseng C.A. Mey and Angelica sinensis (Oliv.) Diels. The combination of the four herbs utilizes the methods of warming, tonifying, purgation and elimination, and achieves the effect of dispelling cold, restoring yangqi, purgation of stagnation and clearing blood stasis [10]. The results of modern pharmacological studies have shown that the four Chinese herbal medicines have significant antitumor effects [11–14]. Our previous studies have shown that WF can significantly inhibit the proliferation of lung adenocarcinoma cells, induce apoptosis and significantly reduce the expression of PCNA and cyclin D1 [15–18]. However, the specific antitumor components and mechanism of action of WF are not clear. In order to facilitate the study of the material basis of WF, the WF was extracted with ethanol and oil ether, dichloromethane, ethyl acetate and n-butanol. Four extraction sites were obtained, and the effective antitumor extracts of WF were screened by a cell experiment in vitro. We selected human lung adenocarcinoma cells A549 and H1299, human lung squamous cell carcinoma H520, and human large cell lung cancer H460 to investigate their antitumor activities in vitro. By calculating the half-inhibitory concentration (IC50), it was found that the ethyl acetate extract from WF (WFEA extract) had the best antitumor activity. In this study, we aimed to illuminate the possible anti-cancer mechanisms of the WFEA extract. Material and methods Animals BALB/c Nude mice (6-8 weeks, weighing 20 ±2 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). They were housed in groups of 4-6 per cage in laminar flow hoods in a  pathogen-free environment (55 ±10% humidity, 22 ±2°C and 12–12 h/lightdark cycle) with free access to a standard laboratory water and diet. The animals were acclimatized for 1 week before the experiment. The study was approved by the Institutional Animal Care and Use Committee (animal authorization reference number: SCXK2018-0034) at Shandong University of Traditional Chinese Medicine (Shandong, China). Animal welfare and experimental procedures were strictly in accordance with the Guide for the Care and Use of Laboratory Animals (the Ministry of Science and Technology of China, 2006). All efforts were made to minimize the suffering of animals and the number of animals used. Preparation of WFEA extract Rheum palmatum L., Aconitum wilsonii Stapf ex Veitch, Panax ginseng C.A. Mey and Angelica sinensis (Oliv.) Diels were purchased from Shandong Zhonglu hospital. According to the ratio of 9 : 12 : 12 : 6, 9750 g of Panax ginseng C.A. Mey, Rheum palmatum L., Aconitum wilsonii Stapf ex Veitch and Angelica sinensis (Oliv.) Diels dried herbs were weighed. The crushed herbs were extracted ultrasonically twice with 75% ethanol. After being filtered, the extracts were concentrated to have no alcohol taste, and finally the WFEA extract was obtained. Then, it was extracted three times with petroleum ether, dichloromethane, ethyl acetate and n-butanol, respectively. The extracts were combined and concentrated to obtain petroleum ether partial extract, chloroform partial extract, ethyl acetate partial extract and n-butyl alcohol partial extract, respectively. Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) analysis A  XDB-C18 column (4.6 × 100 mm, 1.8 μm) was utilized for separation with the column temperature at 35°C. The standards and samples The anti-lung cancer effect of ethyl acetate extract from Wenxia Formula in vivo via the Hedgehog-Gli1 signaling pathway mediated by cancer associated fibroblasts Arch Med Sci 3 were separated using a  gradient mobile phase consisting of (A) acetonitrile and (B) 0.1% formic acid in water. The flow rate was at 0.5 ml/min. The injection volume of sample was 1 μl. Mass spectrometry was performed on a  Bruker Q-TOF equipped with an electrospray ionization source in a positive ion mode. The temperatures of the electrospray source and desolvation were 200°C and 325°C, respectively. The flow rate of nitrogen was set to 8 l/min. The ESI capillary and cone voltages were set at 3,500 and 30 V, respectively. The MS/MS analysis was performed using a  variable collision energy (15–45 eV), which was optimized for each individual constituent. Cell culture The A549 cell line was purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Luciferase (Luc) gene-labeled human lung adenocarcinoma A549 cells (A549-Luc) were purchased from Keygen Biotech (Jiangsu China). Human Lung Adenocarcinoma CAFs were purchased from iCell-CRO (Shanghai, China). A549 and A549-Luc were grown in RPMI-1640; the CAFs were cultured in DMEM. Medium was supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/ml penicillin and 100 μg/ml streptomycin in an incubator at 37°C with a 5% CO2 atmosphere. Xenograft model procedure Approximately 4 × 106 cells were suspended in FBS-free medium and mixed with Matrigel (200 μl). A549 and A549 plus CAFs (1 : 1) cells were respectively injected subcutaneously into the mouse right flank. When tumor nodules appeared at the injection site, groups of nude mice injected with A549 plus CAFs cells were divided into 5 groups (WFEA high, WFEA middle, WFEA low, cisplatin (CDDP) and control group). The CDDP group was given an intraperitoneal injection of 5 mg/kg cisplatin twice a  week; the WFEA group was given an intragastric administration of WFEA extract at doses of 400 mg/kg, 200 mg/kg and 100 mg/kg, respectively; the control group and A549 group were given intragastric administration of equal volume normal saline. Each group was continuously intervened for 5 weeks. Immunofluorescent staining Tissue sections were dewaxed with xylene and dehydrated with graded alcohol. After washing with distilled water, tissue sections were heated at high pressure in ethylene diamine tetraacetic acid (EDTA) antigen repair buffer (PH 8.0) for antigen retrieval. After the slices were cooled at room temperature, sections were incubated with primary antibodies (1 : 500, 4°C, overnight) and secondary antibodies (37°C, 50 min), respectively. Then paraffin sections were incubated with 4',6-diamidino2-phenylindole (DAPI, Beyotime Biotechnology, China) for 10 min in the dark. Finally, paraffin sections were sealed in anti-fluorescence. Images were acquired on a  laser scanning confocal microscope FV12-IXCOV (OLYMPUS, Japan). Red fluorescence represented the positive expression of genes. The mean optical density was calculated by Image-J to analyze the positive expression. Immunohistochemistry Tissue sections were deparaffinized with xylene and rehydrated with ethanol, followed by antigen retrieval. After the slices were cooled at room temperature, tissue sections were incubated with primary antibodies (37°C, 2 h) and secondary antibodies (37°C, 30 min), respectively. Following a final washing in PBS, a-SMA and FAP signals were visualized with a DAB kit (Sigma, St. Louis, MO, USA), counterstained with hematoxylin and mounted in Kaiser’s glycerol gelatine. For semi-quantitative measurements of a-SMA and FAP density, the slides were photographed and analyzed using a computer-aided image-analyzing system (Motic Images Advanced 3.2). Hematoxylin and eosin (HE) staining Sections were dewaxed in xylene and rehydrated in a series of alcohol solutions. After washing in distilled water for a  short time, the sections were stained in Harris hematoxylin solution for 5 min, washed in tap water and counterstained in eosin-phloxine solution for 2 min. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was isolated using TRIzol reagent (Invitrogen, USA). The cDNA of Ptch1, Smo and Gli1 was prepared using the RevertAid First Strand cDNA Synthesis Kit. The qRT-PCR analysis was performed using the SYBR Green RT-PCR Kit (Takara Bio, Japan) on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, USA). β-Actin was used as the endogenous control to calculate the relative RNA levels. Data were calculated by the comparative c

archives of medical science/archives of medical science (online)

The anti-lung cancer effect of ethyl acetate extract from Wenxia Formula in vivo via the Hedgehog-Gli1 signaling pathway mediated by cancer associated fibroblasts