Introduction
Its high incidence and mortality rates mean that lung cancer is ranked first among cancer types; there are two main types, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with the latter accounting for 80-85% of lung cancer cases [
1,
2]. There were 715,000 deaths attributed to lung cancer in China in 2020 [
3,
4]. The drugs currently used for the treatment of NSCLC regulate the immune response, target specific molecules, or are directly cytotoxic [
5,
6]. Blockade of programmed cell death (PD)-1 has been extensively used as a standard treatment for NSCLC, but PD-1 blockade has been associated with immune-related adverse events, including pneumonitis, dermatologic adverse events, and gastrointestinal toxicity [
7‐
10]. Natural products from traditional Chinese medicine can be used as multi-targeted cancer therapies, which is a current trend in the treatment of NSCLC [
11,
12], such as cordycepin’s anticancer effect interacting with and activating AMP-activated protein kinase (AMPK) [
13], Emodin inhibited NSCLC proliferation by decreasing the expression of sPLA2-IIa and NF-κB pathways and suppressed mTOR and AKT and activated the AMPK pathway [
14].
Vascular endothelial growth factor (VEGF) is a key regulator of NSCLC development via inhibition of immune cell differentiation, thereby reducing infiltration and promoting immune tumor cell escape [
15].
Cordyceps sinensis, the content of the Bailing capsule, has anti-tumor, enhanced immunity, resistance to oxidation, fibrosis, viruses, and inflammation [
16]. Previous studies showed that
C. sinensis has therapeutic effects on chronic lung and kidney diseases, such as chronic obstructive pulmonary disease, which can improve lung function, arterial blood gas indices, and exercise tolerance [
17,
18]. As reported,
C. sinensis, as an immunosuppressive agent, inhibits the function of antigen-presenting cells, which leads to a state involving a low immune response and affects the ability of dendritic cells (DCs) to stimulate proliferation, then alleviates the toxic and side effects of chemotherapy [
19]. Recently, anti-PD-1 immunotherapy has caused more and more attention in the treatments of NSCLC [
20,
21]. In addition, combination therapy is a commonly used method for cancer treatment [
22]. Thus, in this study, we observed the effect of the single application of
C. sinensis as well as its combination therapy with PD-1inbihitor.
Therefore, the pharmacodynamic characteristics were evaluated and the mechanism underlying C. sinensis on mice with NSCLC was investigated using a combination of transcriptomics, proteomics, and experimental validation.
NSCLC mouse model construction
Lewis lung carcinoma (LLC) cells obtained from Zhong Qiao Xin Zhou Biotechnology (LZQ0009; Shanghai, China) were incubated in DMEM medium (containing 10% fetal bovine serum and 1% penicillin) in 5% CO
2 at 37 ℃. To generate the NSCLC mouse model, LLC cells (80–90% growth) were subjected to trypsin digestion and were then collected and washed with phosphate-buffered saline (PBS), according to a previous study [
23]. Cells were added to Matrigel at a 1:1 ratio after centrifugation, then 1 million cells were injected into each mouse.
C57BL/6J mice (n = 36, weight 20 ± 3 g; Beijing Vital River Laboratory Animal Technology, Beijing, China) were acclimatized under laboratory conditions for 1 week. The temperature of the feeding room was 25 ± 2 ℃, relative humidity of 50–60%, and a 12-h light, 12-h dark cycle. The mice had free access to food and water. Approval from the Care and Use of Laboratory Animals of China Academy of Chinese Medical Sciences (No. 2022B139) was obtained for the animal experiments.
After 7 days of adaptive feeding, 36 male C57BL/6J mice were randomly assigned to six different groups (n = 6 each). Each mouse was anesthetized and a prepared Matrigel mixture was injected vertically into the left lung at a depth of approximately 4 mm. The injection remained for 20 s. The mice were returned to the cage after awakening from anesthesia. The same procedure was performed in the control group, but no Matrigel mixture was injected.
Grouping and administration
The LLC cells used for modeling were stably transfected with luciferase, which emitted fluorescence under substrate excitation. According to a previous study [
23], IVIS lumina Series III (PerkinElmer, Waltham, MA, USA) was used to detect the inoculation results on the 5th day. Each mouse was given an intraperitoneal injection of 150 µL of D-luciferin (Luc-1G, 115144-35-9; Gold Bio-Technology, St. Louis, MO, USA) and observed for 15 min. Then, fluorescent pictures were obtained with a small animal imager and counted. The mice that were successfully modeled were further divided into the following 5 groups (
n = 6 each), including model;
C. sinensis (Bailing-High, BH, 5 g/kg ), PD1 inhibitor (PD1, 2 mg/kg); PD1 inhibitor combined with a low dose of
C. sinensis (2 mg/kg of PD1 inhibitor + 1
g/kg of
C. sinensis, PD1 + BL); PD1 inhibitor combined with a high dose of
C. sinensis (2 mg/kg of PD1 inhibitor + 5 g/kg of
C. sinensis, PD1 + BH).
A C. sinensis solution was prepared in 0.5% sodium carboxymethyl cellulose (10 mL/kg). The PD1 inhibitor (20 mL/kg) was injected intraperitoneally. Distilled water was used as the treatment for mice in the control and model groups. Two weeks later, blood and lung tumor tissues were collected for biochemical analysis, pathologic evaluation, immunohistochemistry, transcriptomics sequencing, proteomics, and Western blot.
Histologic observations of the lung
Mouse lung tissues were obtained from each group and placed in 4% paraformaldehyde and fixed overnight. Paraffin embedding was performed by ethanol dehydration, then the tissue sections were sliced, dewaxed, and rehydrated. For histopathological analysis, tissue sections were stained using hematoxylin and eosin (H&E) and the slides were scanned for examination.
Biochemical analysis in serum by ELISA
ELISA kits for, interleukin (IL)-6, IL-10, tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), erythropoietin (EPO), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were obtained from Shanghai Enzyme-linked Biotechnology (ml002095, ml063159, ml037873, ml016824, ml037757, ml643059, ml002210, and ml037645, respectively; Shanghai, China). Biochemical analyses of sera for the above factors were performed in accordance with instructions supplied by the manufacturer.
Transcriptomics sequencing and data analysis
RNA was extracted from lung tissue from mice (n = 3 per group), an Illumina TruSeq RNA library was constructed, and Illumina NovaSeq 6000 (San Diego, CA, USA) was used for sequencing. Change was defined by setting the cut-off value at 2-fold, and the statistically significant threshold was set at P < 0.05 for differentially-expressed gene screening. Finally, gene ontology (GO) function enrichment analysis and gene set enrichment analysis (GSEA) were conducted using the R programming language.
Proteome detection and mass spectrometry
Proteins were extracted from mouse lung tissues of the control, model, and BH groups. Briefly, 0.1 g of lung tissue from each sample were obtained and rapidly ground by adding appropriate lysis solution (RIPA + 1X protease inhibitor cocktail) for complete lysis of proteins, then the supernatant was obtained after centrifugation (15,000 g for 20 min at 4 ℃). After quantification, 100 µg/100 µL of protein from each group were incubated in 5 mM dithiothreitol for 30 min at 37 ℃ in the dark to allow chemical reduction of disulfide bonds. Next, proteins that had been alkylated with 20 mM iodoacetamide were placed in the dark for 30 min. Samples were washed three times by the following steps: 400 µL of methanol (pre-chilled at − 80 ℃); 100 µL of methylene chloride; and 200 µL of hyper-pure water. The supernatant was discarded and 500 µL of pre-chilled methanol was added to the pellet obtained after centrifugation (15,000 g for 3 min at 4 °C). Then, proteins were dissolved by adding 200 µL of buffer solution of 200 mM 4-(2-hydroxyethyl)-1-piperazine propane sulfonic acid at pH 8.5 to the protein precipitate, and further digested by trypsin at 37 ℃ for 17 h (protein: enzyme = 100 µg: 1 µg). Samples were desalted using a commercial C18 column (Waters, Milford, MA, USA), dissolved in 50 µL of 0.1% formic acid (FA) and centrifuged (15, 000 x g for 30 min). Supernatant aliquots (10 µL) were analyzed via liquid chromatography with tandem mass spectrometry for protein identification.
Database search and proteomic analysis
The Xcalibur analysis system (Thermo Fisher, Waltham, MA, USA) was used to collect MS data. Protein identification was performed using Proteome Discoverer V2.4 aligned to the NCBInr (
http://www.ncbi.nlm.nih.gov/) and UniProt databases (
http://www.uniprot.org/) with the Sequence HT algorithm. Differentially-expressed proteins (DEPs) were screened based on the following standards: upregulated, FC > 1.5 and
P < 0.05; downregulated, FC < 0.67 and
P < 0.05. Then, the DEPs were analyzed by GSEA.
Immunohistochemical staining
The immunohistochemical staining protocol comprised a number of steps, as follows. First, mouse lung tissue from each group was obtained and placed in 4% paraformaldehyde for a 3-h fixation before embedding in paraffin for 24 h at 60 ℃. The paraffin specimens were then dewaxed using xylene and subsequently dehydrated in an ethanol gradient. The dewaxed sections were submerged in water, then in citrate buffer (C1032; Solarbio, Beijing, China) and heated in a microwave oven to allow antigen repair. Each section was then flushed with PBS 3 times and sealed with 3% hydrogen peroxide and 5% bovine serum albumin for 10 min and 1 h, respectively. Next, rabbit anti-mouse Ki-67 (9440 S; CST, Danvers, MA, USA) and rabbit anti-mouse VEGFA polyclonal antibodies (19003-1-AP; Proteintech, Wuhan, China) were added to the samples, which were then incubated overnight at 4 ℃ in a refrigerator. On the following day, samples were washed three times with PBS. Then HRP-labeled goat anti-rabbit IgG (PR30009; Proteintech; 100 drops) was added to each slide, followed by incubation at 25 ℃ for 1 h. DAB staining (DA1010; Solarbio), hematoxylin staining, gradient ethanol dehydration, and neutral gum sealing were then performed.
Western blot analysis
Western blot analysis was performed according to a previously described protocol [
24]. In brief, lung tissue proteins were extracted from each group and the level was measured using a BCA kit (Beyotime, Beijing, China) and adjusted to a final concentration of 1 µg/µL. Next, 5× loading buffer solution was added to the protein at a ratio of 1:4 ratio and heated for denaturation at 96 ℃ for 10 min. A 30-µg protein from each group was separated via SDS-PAGE and then transferred to a PVDF membrane. After a 1-h sealing step with 5% bovine serum albumin (ST2254; Beyotime) at room temperature, the membrane was incubated with primary antibodies overnight in a refrigerator at 4 ℃. The primary antibodies included anti-RhoA (1:1000; Bioss, Beijing, China), anti-raf1 (1:1000; Bioss), and anti-c-fos (1:1000; Bioss). The next day, the membrane was cleaned three times with TBST solution (10 min per time) and incubated with a HRP-linked goat anti-rabbit/mouse IgG, antibody (1:5000; Bioss) at room temperature for 1 h. The membrane was washed a further three times in TBST buffer (10 min per time). Finally, ECL luminescent liquid was added for visualization. The above experiments were repeated three times and image J software was used for calculations.
Routine blood testing
Blood samples were collected from anesthetized mice and stored at 4 ℃. A SYSMEX XN-1000 V instrument (Kobe, Japan) was used to measure platelets (PLTs), white blood cells (WBCs), red blood cells (RBCs), and hemoglobin (HGB).
Flow cytometric analysis of immune cells
Flow cytometry was conducted using a Beckman Coulter counter (CytoFLEX, Brea, CA, USA) in accordance with the instructions supplied by the manufacturer. Blood samples were collected in heparin sodium anticoagulant tubes with red blood cell lysis buffer. Then, after splitting on ice in the dark for 15 min, samples were centrifuged (4 ℃ at 400 × g for 5 min) and incubated with antibodies (4 ℃ for 30 min) to separate DCs, and NK cells, and CD4+ and CD8+ T cells.
Discussion
Among the lung cancer histological subtypes, non-small cell lung cancer (NSCLC) is the most common [
42]. Recently, although the emergence of new targeted drugs has provided more choices for patients, the high price and tolerance and accompanying NSCLC patient unfavorable survival rate limit use [
43,
44]. With accurate therapeutic effects and a minimal side-effect profile, traditional Chinese medicine (TCM) has long been used as a treatment for cancer.
C. sinensis, a widely-used and well known traditional Chinese medicine, was officially classified as a drug in 1964 in the Chinese Pharmacopoeia [
45]. As the most studied and applied specie among Cordyceps,
C. sinensis has already been reported exerting pharmacological activities like antioxidant, anti-cancer, antihyperlipidemic, anti-diabetic, anti-fatigue, anti-aging, anti-depressant, and kidney protection, which presents huge potential for active medical transformation. C.
sinensis powder has been reported to be effective in the inhibition of NSCLC both in the single use and the combination with other drugs in previous studies [
46,
47]. However, the characteristics of pharmacological effects and molecular mechanisms when C.
sinensis was used in the combination with PD-1 inhibitor haven’t been clarified.
Based on the above, we chose an orthotopic cancer model with the subcutaneous injection of lung cancer cells to mimic NSCLC. We successfully constructed an NSCLC mouse model. The alveoli of mice were severely damaged and structurally disrupted, with some alveoli being blocked, ruptured, and collapsed. In addition, a significant increase in the number of lung cancer cells is evident in the in vivo images. Using the NSCLC model, we showed that the anti-inflammatory activities of C. sinensis by significantly inhibiting inflammatory cytokines and oxidative stress indicators in mouse serum. Our study provided direct preclinical evidence that C. sinensis inhibited tumor growth, alleviated the degree of alveolar damage, and had an anti-inflammatory role in mice with NSCLC.
Subsequently, we explored the key genes of
C. sinensis acting on NSCLC in mice through transcriptomics sequencing to reveal the molecular mechanism underlying the development of lung cancer. Our results for transcriptome sequencing prompted us to focus on the RhoA gene, for which a role has highlighted in tumor cell invasion and metastasis [
37] in malignancies such as breast cancer [
48], nasopharyngeal carcinoma [
49], and NSCLC [
50]. Cytokines and chemokines are critical in regulating T-cell recruitment and the overall cellular composition of the tumor microenvironment [
51‐
53]. Activated RhoA leads to the formation of stress fibers within cells and expression of chemokines, cytokines, and growth factors [
54,
55]. The important role of immune function in the incidence and development of lung cancer is well established [
56‐
58]. As reported, fermented cordyceps powder reduces radiation-induced bone marrow suppression, increases the percentage of peripheral blood leukocytes, improves chemotherapy tolerance, and promotes spleen cell proliferation, NK cell activity, and T-helper cytokine secretion in mice with impaired immune function [
59,
60]. Based on the GO (BP) enrichment and GSEA enrichment of DEGs, transcriptome data confirmed that
C. sinensis promoted T cell differentiation and activation, and negatively regulated the inflammatory response. T cells are known to regulate cellular immunity and play an essential role in successful treatment of lung cancer. T cells recognize tumor antigens (proteins expressed only by tumor cells that are induced by genetic mutations in these cells) and trigger strong anti-tumor immune effects, making T cells importan
t targets for tumor treatment.
To systematically clarify the mechanism underlying
C. sinensis action on NSCLC, we further analyzed the protein expression in lung tumor tissues via proteomics. According to the proteomics results, the DEPs of the BH versus model groups were characterized by significant upregulation of MAP kinase activity, and downregulation of MAPK cascade pathways. As described, tumor‑associated macrophages (TAMs) promote the proliferation and invasion of tumor cells by activating the MAPK signaling pathway [
61]. As an autocrine growth factor for NSCLC cells, VEGF has a crucial role in angiogenesis and promotes lung tumor growth [
62,
63]. As a marker of tumor cell proliferation, the Ki-67 index increases to higher levels with tumor development [
64]. Herein, expression levels of VEGF and Ki67 at the protein level in tumor tissues of different treatment groups were detected by immunohistochemistry to indicate decelerating proliferation of tumors in NSCLC mice. Notably, RhoA regulates immune cell differentiation and function, recruits innate immune cells (including neutrophils and macrophages), enhances the antigen presentation ability of immune cells, forms immune synapses, and improves the tumor microenvironment [
65‐
67]. Liu et al. concluded that KLHL17 upregulation in NSCLC promotes tumor cell proliferation and migration via an increase in RhoA expression, as well as activation of the pathway responsible for Ras/MAPK signaling [
68]. Additionally, RhoA Raf is activated by RhoA upstream of the MAPK pathway [
38,
69]. Western blot results showed that
C. sinensis treatment downregulated RhoA, Raf-1, c-fos, phosphorylated ERK1/2 and MEK1/2 in lung tissues of NSCLC mice. Moreover, we showed that EPO and GM-CSF in each administration group were markedly increased. Interestingly, after 14 days of treatment with
C. sinensis, the proportion of immune cells significantly increased, indicating that
C. sinensis mitigated the immune dysfunction due to lung cancer. This finding reflects an important immunostimulatory mechanism in the inhibition of lung cancer cell growth by
C. sinensis. The use of
C. sinensis has been documented in drugs for respiratory infections, as well as promoting activation of the immune response [
70]. Based on previous studies, the active ingredients from
C. sinensis were mostly recognized by Toll-like receptors and C-type lectin receptors during initiation of immunomodulation and more importantly, the following intracellular signaling initiated from the receptors.
C. sinensis can likely defense cancers by recruiting and augmenting immune cells such as natural killer cells and macrophages [
71‐
74]. Although the exact mechanism remains unclear, multiple researches have reported
C. sinensis exerted the effect of recruiting immune cells, such as limiting the expression of cytokines like IL-2 and TNF-α [
75], inhibiting MAPK pathways [
76], and inducing redistributions of peripheral mononuclear T lymphocytes [
77]. Hence,
C. sinensis likely exerted a protective effect against NSCLC by suppressing the RhoA gene, then recruiting immune cells, enhancing immune function, and inhibiting lung cancer via the MAPK pathway.
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