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American Journal of Translational Research logoLink to American Journal of Translational Research
. 2021 Jul 15;13(7):7492–7507.

Shiquan Yuzhen Decoction inhibits angiogenesis and tumor apoptosis caused by non-small cell lung cancer and promotes immune response

Zhengda Sun 1,*, Keping Shen 1,*, Yage Xie 1, Bing Hu 1, Ping He 1, Yanlin Lu 1, Haiyan Xue 2
PMCID: PMC8340251  PMID: 34377231

Abstract

Background: TCM treatment for lung carcinoma has been reported by many researches. Shiquan Yuzhen Decoction can be used in the clinical treatment of lung carcinoma, but its specific mechanism is still under exploration at present. Methods: The active ingredients and mechanism of Shiquan Yuzhen Decoction on non-small cell lung carcinoma were discussed by network pharmacology. The main active ingredients, targets and disease genes of non-small cell lung carcinoma of Shiquan Yuzhen Decoction were screened through relevant databases. Lewis lung carcinoma bearing mice model was established by inoculating Lewis lung carcinoma cells to C57BL/6 mice under the right armpit. Different doses of Shiquan Yuzhen Decoction were used to observe the apoptosis and angiogenesis changes of tumor tissues in mice. Results: A total of 26 key active compounds meeting the evaluation of generic properties and 182 main targets were screened out. The multi-level network model shows that Shiquan Yuzhen Decoction can regulate the target gene network of non-small cell lung carcinoma. And it can inhibit tumor growth in tumor-bearing mice, induce apoptosis of tumor cells, and evidently increase the activities of Caspase-3, 8 and 9. The dose of 17.4 g/kg can evidently inhibit the formation of microvessels in transplanted tumor tissues, improve the sensitivity of mice’s diet and activities, increase the spleen index of tumor-bearing mice, and inhibit inflammatory factors. Conclusion: Shiquan Yuzhen Decoction can evidently improve the quality of life of Lewis lung carcinoma-bearing mice and inhibit tumor growth in mice, which is a potential clinical treatment plan.

Keywords: Non small cell lung carcinoma, Shiquan Yuzhen Decoction, KEGG, GO, PPI, angiogenesis, immunoprophylaxis

Introduction

Lung carcinoma (LC) ranks first in male malignant tumors in China and second in female malignant tumors, and it is the main cause of carcinoma death, ranking first in malignant tumors [1]. The incidence and mortality of LC in China are increasing year by year. It is estimated that by 2025, LC patients will reach 1 million, and China will have the highest incidence rate of LC in the world [2]. Non-small cell lung carcinoma (NSCLC) is most common in LC, accounting for more than 85% of LC cases [3]. Although modern scientific and medical technology is constantly developing in a breakthrough way, and modern clinical diagnosis and treatment of LC disease are increasingly rich, the morbidity and mortality of LC are still high, especially the overall treatment effect of middle and late LC is not satisfactory, which poses great danger to human life safety [4,5].

In recent years, the role of Chinese medicine in preventing and treating LC has been attracted more attention [6,7]. Chinese medicine acts in preventing and treating LC, improving the quality of life of LC patients, preventing metastasis and recurrence, and acts in palliative treatment, which fully embodies the characteristics and advantages of the holistic view of Chinese medicine. In particular, the complex components and multi-target effects of Chinese herbal medicine have many advantages in LC prevention and treatment [8,9]. Many researches revealed that Chinese medicine acts in regulating immune function, inhibiting tumor angiogenesis, inducing tumor cell apoptosis, directly killing tumor cells and inhibiting tumor cell proliferation [10,11]. However, there are still different views on the treatment of Chinese medicine. It is considered that the composition of Chinese medicine is complex, and it is impossible to judge the way of the drugs which can improve the occurrence of tumor.

Network pharmacology is put forward on the basis of the theoretical development of multidirectional pharmacology, network biology, system biology, and big data analysis technology. It uses the holistic concept to explain the disease development process and the mechanism of drugs acting on the body, and at the same time, it provides new ideas and methods for new drug research and development [12]. Through big data technology, we analyzed the existing information including high-throughput sequencing, proteome, drugs, diseases and other related databases, and combined them with various data reports to establish a network of “drug-target-disease”. Then, we applied related software tools to comprehensively display the relationship of drugs-targets, targets- diseases, diseases-diseases, and drugs-drugs. Therefore, we can observe the intervention and influence of drugs on diseases from multi-dimensional, all-round and deep-seated aspects, and can also find the possible targets and signal pathways of drugs for target diseases [13-15].

In this research, we explored the latent targets and signal pathways of Shiquan Yuzhen Decoction with the reference of network pharmacology, and verified it by animal models, which provided a basis for clinical use of Shiquan Yuzhen Decoction.

Methods and data

Screening of active ingredients

TCM chemical composition database TCMID (http://www.megabionet.org/tcmid) [16], TCMSP (http://tcmspw.com/tcmsp.php) [17], and HIT (http://lifecenter.sgst.cn/hit) were applied [18]. Referring to the articles on the research of ginseng, Astragalus membranaceus, raw dioscorea, rhizoma anemarrhenae, radix scrophulariae, Os Draconis, raw oyster, salvia, rhizoma sparganii, and curcuma zedoary in PubMed, CNKI and Wanfang websites, the chemical constituents of these ten herbs were searched, and the repeated parts were summarized and deleted as the chemical components of Shiquan Yuzhen decoction. The OB value and DL value of each chemical component of Shiquan Yuzhen Decoction were obtained by TCMID and TCMSP, and the chemical components with OB≥30% and DL≥0.18 were taken as the active components of Shiquan Yuzhen Decoction.

Target selection of active ingredients

Selected active ingredients were screened through uniprot database (http://www.uniprot.org/) [19] and Pubchem platform (https://pubchem.ncbi.nlm.nih.gov/) [20]. The “reviewed (Swiss prot)” was defined as “human” search, and the results were standardized to obtain the potential target of Shiquan Yuzhen decoction.

Query of NSCLC target gene

Through Genecards (https://www.genecards.org/) [21], OMIM (https://www.omim.org/) [22], DisGeNET (http://www.disgenet.org/) [23] and TTD (http://bidd.nus.edu.sg/group/cjttd/) [24] database, “NSCLC” and “Non-small-cell carcinoma” were applied as search terms, the target genes of NSCLC were obtained respectively, and then the differentially expressed genes obtained in all databases were merged and the duplicate parts were deleted, thus obtaining the genes related to NSCLC.

Screening and network construction of disease target of drug action

Gene mapping was carried out on the target points corresponding to the active ingredients in Shiquan Yuzhen Decoction and the disease gene information of NSCLC. Venny2.1 (http://bioinfogp.cnb.csic.es/tools/venny/index.html) [25] was used to visualize the Venn diagrams of the active ingredient target points of Shiquan Yuzhen Decoction and the disease gene of NSCLC, and the target points of Shiquan Yuzhen Decoction acting on NSCLC were obtained. The network analyzer plug-in in Cytoscape3.7.2 was used. According to the topological feature of degree of freedom, the main active ingredients with degree more than twice of the median were selected as the key active ingredients [26], and the target corresponding to the key active components was called the core target. Based on this, the network diagram of “drug target disease” was constructed and visualized.

Analysis of biological pathways and biological targets

Enrichment analysis was performed using DAVID (https://david.ncifcrf.gov/) [27] online database. The key active components and core targets were introduced to the system, and the enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were carried out. GO enrichment analysis (confidence value P < 0.01), Biological Process (BP), Cellular Component (CC) and Molecular Function (MF) were selected for core target enrichment and function annotation. In KEGG enrichment analysis, FDR < 0.01 was selected, and combined with the related literature of NSCLC, the signal pathway of Shiquan Yuzhen Decoction on NSCLC was screened out.

Analysis of protein co-expression

The functional protein association platform STRING V11.0 (https://string-db.org/) was used for protein co-expression analysis. The core target proteins of the key active components of Shiquan Yuzhen Decoction acting on NSCLC were uploaded to string. The target protein PPI network of Shiquan Yuzhen decoction was constructed by selecting “Homo sapiens” and setting minimum required interaction score > 0.9, so as to screen high confidence data.

Source of cell and animal

Altogether 40 healthy and clean C57BL/6 mice (with 20 males and 20 females) aged 4-6 weeks and weighed 17-20 g were obtained from Hunan SJA Laboratory Animal Co., Ltd, Changsha, Hunan, China. The mice were raised in the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine, with four mice in a cage, which was circulated day and night, and the suitable activity space of mice was fully ensured. The indoor temperature was 17-24°C, and the mice were free to eat common standard feed and drink pure water. The mice were raised adaptively for one week, and the mice were grouped after they were applied to the environment. Lewis lung carcinoma cells were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.

Compatibility and preparation of prescriptions

Shiquan Yuzhen Decoction consisted of ginseng (12 g), Astragalus membranaceus (12 g), raw dioscorea (12 g), rhizoma anemarrhenae (12 g), radix scrophulariae (12 g), Os Draconis (12 g), raw oyster (12 g), Salvia miltiorrhiza (6 g), rhizoma anemarrhenae (4.5 g) and Curcuma zedoariae (4.5 g). All the above medicines were purchased from the outpatient Chinese medicine pharmacy of Pudong Branch of Longhua Hospital affiliated to Shanghai University of Traditional Chinese Medicine. The above medicinal herbs were obtained according to the prescribed amount. The Os Draconis and raw oyster were bathed in 10 times of water for 30 min, then decocted for 1 h in a medicine pot, then other medicines were bathed in 10 times of water for 30 min, then combined with the first decocted medicines and decocted for 1 h, filtered, the filtrate was stored, then 8 times of water was added and the medicines were decocted for 1 h, filtered, and the filtrates of the two times were combined, concentrated to contain 2 g/ml of crude drug, and stored in a refrigerator at 4°C for later use.

Animal model establishment and treatment

Lewis lung carcinoma cell line was cultivated in a cell culture dish with a radius of 5 cm. RPMI1640 culture medium including 10% FBS, 1% 100 mg/L streptomycin and 1% 100 mg/L penicillin was cultivated with constant temperature, constant humidity and with 5% CO2, and the culture medium was changed every other day. After adherent growth, the cells were subcultured when reaching 75% of the bottom area of the culture dish. After a certain amount, Lewis lung carcinoma cells in logarithmic growth stage were digested, centrifuged and counted, and then re-suspended to prepare cell suspension with 5 × 106 cells/ml. Then, 0.2 ml Lewis lung carcinoma cell suspension was injected into the right armpit of the mice in model group and Shiquan Yuzhen Decoction group to establish Lewis lung carcinoma bearing mouse model. Nude mice in blank group were not injected with cells. Sixty-five mice were divided into two experimental samples (the first 40 mice were observed for weight changes and tumor tissues were collected, and the remaining 25 mice were observed for organ index changes), which were divided into blank control group, model group and low, medium and high dose groups of Shiquan Yuzhen Decoction, with 8 or 5 mice in each group. Mice in Shiquan Yuzhen Decoction group and model group were inoculated with Lewis lung carcinoma cells in armpit, and were administered by gavage after successful model establishment. According to the “Equivalent dose ratio table converted by body surface area between human and animal”, the model group was given 0.2 ml/of normal saline, the low dose group was given 8.7 g/kg daily, the middle dose group was given 17.4 g/kg daily, and the high dose group was given 34.8 g/kg daily. The mice in the model group and Shiquan Yuzhen Decoction groups applied intragastric administration once a day for 17 consecutive days, while the blank control group was not intervened. After 24 hours of the last administration, the mice were executed, and the subcutaneous transplanted tumor of the mice was quickly cut out with ophthalmic scissors, weighed and the tumor volume and tumor inhibition rate were obtained (tumor volume formula: V = 1/2 × a2 × b, tumor inhibition rate calculation formula: (average tumor weight of model group-average tumor weight of Shiquan Yuzhen Decoction group)/average tumor weight of model group × 100). Hair glossiness and softness, mental state, activity, diet, sleep and aggregation of mice in each group were observed every day, and the weight of mice was recorded every other day, and the curve of body mass change was visualized. The mice were executed 24 h after the last administration. The spleen and thymus tissues were cut out with ophthalmic scissors and weighed on an electronic microbalance. The spleen index and thymus index were calculated according to the formula of organ index (mg/g) = organ weight (mg)/body weight (g). This study was approved by the welfare and ethics committee of experimental animals in Shanghai University of Traditional Chinese Medicine.

Elisa

In this study, IL-6, TNF-α, t-PA, PAI-1 and FVii in tumor tissue or serum were detected by Elisa, and the detection steps were carried out according to the kit instructions.

TUNEL

In this study, TUNEL method was used to detect the apoptosis of mouse tumor tissues, which was briefly described as follows. The collected tumor tissues were fixed with 4% paraformaldehyde for 24 hours and embedded in paraffin to make sections. Paraffin sections were taken out and dewaxed in xylene. Gradient dehydration was carried out, 20 μg/ml protease K working solution and endogenous peroxidase strong blocking solution were cultivated for 20 min at room temperature. A 50 ul of biotin labeled solution was incubated at 37°C for 1 h in the dark. Labeled reaction stop solution was added and placed at room temperature for 10 min. A 50 ul of Streptavidin-HRP working solution was cultivated at room temperature for 30 min. Color developing solution was applied to observe the tissues under a microscope. Ten fields of vision were obtained under high magnification (× 400), and the number of apoptotic cells per unit was calculated.

Western blot

Total protein was extracted from tumor tissues of nude mice by RIP analysis lysis buffer, and the protein concentration was detected by BCA kit. A 50 μg of the protein was moved on sodium dodecyl sulfate polyacrylamide gel, and then moved into polyvinylidene fluoride membrane by wet transfer method. The membrane was sealed with 5% skimmed milk at room temperature for 1 h, and then sealed with the target (VEGFA 1:500, HIF-1α 1:500) at 4°C overnight. β-actin was applied as an internal reference. Next, the membrane was incubated with horseradish peroxidase labeled goat anti-rabbit secondary antibody (1:3000) for IgG H&L for 1 h. ECL kit was used for development, and Image J software was used for analysis.

Detection of apoptosis-related protein activity

Caspase-3, -8, -9 protein activity detection kit was used for detection. The collected subcutaneous transplanted tumor tissues were frozen in liquid nitrogen and placed in a refrigerator at -80°C for later use. RIP was used to crack the tissues, followed by homogenization. The BCA kit was used to adjust the protein concentration. In a 96-well plate, 50 ul of sample to be detected, 40 ul of detection buffer and 10 ul of Ac-DEVD-pNA (2 mm) were added to each well, mixed evenly, and incubated overnight at 37°C in the dark. Blank control was set according to the instructions. The absorbance at 405 nm was determined by spectrophotometer.

Immunohistochemical method

Paraffin sections were put into xylene and absolute ethanol for dewaxing, antigen repair and sealing. After blocking, BSA blocking solution was gently shaken off. CD31 (CD31 diluted 1:1000), CD8 (CD8 diluted 1:1000) and Anti-FOXP3 (FOXP3 diluted 1:1000) were added on the slice, placed at 4°C overnight. Secondary antibody (1:400) was put in and reacted for 1 h at room temperature in dark environment. DAPI dye solution was added dropwise, and placed for 10 min at room temperature away from light. And finally, these were sealed. The slices were counted according to Weidner evaluation standard, The microvessel density of each immunofluorescence section was observed under a low power microscope (× 40). After randomly selecting the most representative range of 10 microvessels, the number of microvessels in each range was observed and counted under the high power microscope (× 200 or × 400), and the average value was taken as the MVD value.

Statistical methods

The experimental results were expressed in (x̅ ± s) and processed by SPSS23.0. One-way ANOVA analysis was applied between groups. Under the condition of satisfying normal distribution and homogeneous variance, independent sample t test was adopted between any two groups. If the variance was uneven, the corrected variance analysis was adopted. If it did not conform to the normal distribution, the Kruskal-Wallis H method of nonparametric test was adopted, and P < 0.05 means that the difference is statistically significant.

Results

Main activity formation and target prediction of Shiquan Yuzhen Decoction

Through database search and literature search, the duplicate items and chemical components without target points were removed, and 117 kinds of active components of Shiquan Yuzhen Decoction with OB > 30% and DL > 0.18 were found (Table S1), and 269 corresponding target points were obtained (Table S2), among which calcium carbonate and calcium phosphate, the main components of Os Draconis and Oyster, were eliminated because there were no effective targets. Then, we searched in Genecards, TTD, OMIM and DisGenet databases, and found 4082 expressed genes related to NSCLC diseases. We visualized Venn diagrams from 269 targets corresponding to the active ingredients of Shiquan Yuzhen Decoction, and found 188 intersecting differences with NSCLC related expressed genes (Figure 1A). Then, in order to further analyze the relationship between targets and main active ingredients, we selected the main active ingredients with a Degree more than twice the median for network topology, and screened out 26 active ingredients (Table S3). Then, according to the screened active ingredients and targets, a network diagram of “drug-target-disease” was constructed (Figure 1B).

Figure 1.

Figure 1

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Activity formation and potential target prediction of Shiquan Yuzhen Decoction. A. Venn diagrams were visualized to analyze the common potential targets of NSCLC disease-related expressed genes and Shiquan Yuzhen Decoction. B. The network diagram of “drug-target-disease” was visualized by cytoscape software (orange diamond represents NSCLC, green hexagon represents effective composition of Shiquan Yuzhen Decoction, pink hexagon represents 26 key active components of Shiquan Yuzhen Decoction, and blue triangle represents potential target).

Enrichment analysis of potential targets of Shiquan Yuzhen Decoction

In the above exploration, we identified the latent targets of Shiquan Yuzhen Decoction. In order to further analyze the mechanism of Shiquan Yuzhen Decoction in lung carcinoma, we respectively analyzed the biological function and signal pathway enrichment of 182 core target genes corresponding to 26 selected key active ingredients, and obtained 134 entries of biological processes by GO analysis, including 98 entries related to biological processes. The top 10 entries of biological processes according to the total number of genes mainly involved the positive regulation of RNA polymerase promoter II transcription, the negative regulation of DNA transcription template and apoptosis process (Figure 2A-C). KEGG analysis showed that 62 qualified KEGG pathways were obtained. Combined with the literature, 15 signaling pathways related to NSCLC diseases were screened out. Enriched signal pathways were mostly related to apoptosis, inflammation and angiogenesis (Figure 2D). It was suggested that Shiquan Yuzhen Decoction may treat non-small cell lung carcinoma by improving immunity, inhibiting angiogenesis and promoting apoptosis. At the end of the study, we visualized PPI network according to the predicted potential target genes (Figure 3A), in which 182 nodes contained 844 edges, and the top 30 key nodes were screened for display (Figure 3B). Among them, protein kinase (AKT1), signal transduction and transcription activator (STAT3), vascular endothelial growth factor (VEGFA, EGFR) and interleukin (IL-2, IL-4, IL-6) of Shiquan Yuzhen Decoction acting in NSCLC targets had a high effect, suggesting that these target proteins act in the treatment of NSCLC.

Figure 2.

Figure 2

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Analysis results of GO and KEGG. A-C. Functional analysis of 182 potential target genes by GO enrichment, including Biological Process (BP), Cellular Component (CC) and Molecular Function (MF). D. KEGG enrichment was used to analyze the potential signal pathways of 182 potential target genes.

Figure 3.

Figure 3

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Analysis results of PPI. A. PPI was applied to filter the top 30 key nodes, and the numbers in the figure represent the number of intersecting edges. B. PPI network diagram.

Shiquan Yuzhen Decoction can hinder tumor forming in tumor-bearing mice

Through the above research, we identified the potential mechanism of Shiquan Yuzhen Decoction in the treatment of lung carcinoma. In order to further prove the role of Shiquan Yuzhen Decoction in LC treatment, we established C57BL/6 mouse model and used different doses of Shiquan Yuzhen Decoction for treatment. Through experiments, we observed that, except the blank control group, the model group and Shiquan Yuzhen Decoction groups could touch the subcutaneous tumor on the third day after subcutaneous inoculation, and the tumor volume gradually increased with time. In the first 9 days after modeling, the growth rate of tumor volume of tumor-bearing mice in model group and Shiquan Yuzhen Decoction groups was decreased, and there was no obvious difference among the groups. After modeling for 9 days, the growth rate of tumor volume of mice in each group was faster than before (Figure 4A). After the last administration, the mice were executed, and the tumor tissues were obtained and detected. Compared with the model group, the weight of subcutaneous transplanted tumor in the middle and high dose groups of Shiquan Yuzhen Decoction was lighter, but there was no difference compared with the low dose group (Figure 4B). In addition, the calculation showed that the tumor inhibition rates of low, middle and high dose groups of Shiquan Yuzhen Decoction were 2.43%, 8.43% and 11.74% respectively, indicating that Shiquan Yuzhen Decoction had a good effect on inhibiting tumor growth.

Figure 4.

Figure 4

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Shiquan Yuzhen Decoction inhibits tumor growth in tumor-bearing mice. A. Tumor growth of tumor-bearing mice after modeling. B. Detection of tumor quality in tumor bearing mice after the last Administration * indicates P < 0.05.

Shiquan Yuzhen Decoction promotes apoptosis of tumor tissue

Through observation and measurement, we found that Shiquan Yuzhen Decoction had a good effect on inhibiting tumor growth. Combined with network pharmacology, we analyzed the medicines and found that the effective components of Shiquan Yuzhen Decoction can directly act on apoptosis-related pathways of NSCLC, thus inducing tumor cells to produce apoptosis. In order to verify our hypothesis, the apoptosis of tumor tissue was detected by Tunel method. The results revealed that compared with the model group, the apoptosis of subcutaneous transplanted tumor tissues of tumor-bearing mice in the low Shiquan Yuzhen Decoction group increased slightly, and that in the middle and high-dose groups was evidently higher than that in the model group (Figure 5A). In addition, we also detected the expression of apoptosis-related proteins in tumor tissues. Compared with the model group, the activities of Caspase-3, -8 and -9 in subcutaneously transplanted tumor cells of tumor-bearing mice in the middle and high dose groups of Shiquan Yuzhen Decoction were evidently enhanced, and the activities of Caspase-3 and -9 in subcutaneously transplanted tumor cells of tumor-bearing mice in the low dose group of Shiquan Yuzhen Decoction were evidently increased (Figure 5B), suggesting that Shiquan Yuzhen Decoction could induce apoptosis of lung carcinoma tissues and play a role in inhibiting carcinoma.

Figure 5.

Figure 5

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Shiquan Yuzhen Decoction promotes tumor apoptosis. A. Apoptosis of tumor tissue was detected by Tunel method. B. Caspase protein activity detection kit was used to detect Caspase-3, -8 and -9 activities in tissues. * indicates P < 0.05; ** indicates P < 0.01.

Shiquan Yuzhen Decoction can inhibit tumor angiogenesis

Angiogenesis, as a difficult issue in LC treatment, puzzles clinicians. We found that Shiquan Yuzhen Decoction could regulate HIF-1 signal pathway and VEGF signal pathway, so we speculated that Shiquan Yuzhen Decoction may inhibit the expression of angiogenesis-related proteins to achieve the purpose of inhibiting tumor angiogenesis. At first, we detected the expression of CD31 in tumor tissues of mice. CD31 in subcutaneous transplanted tumor tissues of model group was evidently higher than that of Shiquan Yuzhen Decoction in middle and high dose groups, while the expression of CD31 in tumor tissues of low dose group was slightly lower than that of model group, but higher than that of middle and high dose groups (Figure 6A). In addition, MVD count showed that compared with the model group, the MVD in the middle and high dose groups of Shiquan Yuzhen Decoction was evidently lower (Figure 6B). Compared with the model group, the Western Blot indicated that Shiquan Yuzhen Decoction could evidently inhibit the expression of VEGFA and HIF-1a protein in subcutaneous transplanted tumor (Figure 6C), which suggested that Shiquan Yuzhen Decoction could effectively inhibit tumor angiogenesis.

Figure 6.

Figure 6

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Effect of Shiquan Yuzhen Decoction on tumor angiogenesis. A. Effect of Shiquan Yuzhen Decoction on CD31 in tumor tissue was detected by immunofluorescence staining. B. Effect of Shiquan Yuzhen Decoction on MVD in tumor tissue was detected by MVD counting. C, D. Effect of Shiquan Yuzhen Decoction on VEGFA, HIF-1A in tumor tissue was detected by WB. * indicates P < 0.05.

Shiquan Yuzhen Decoction can improve the immunity of tumor-bearing mice and prevent the metastasis of lung malignant tumor

At the end of the study, in order to further understand the immunity of Shiquan Yuzhen Decoction on tumor-bearing mice and prevent the metastasis of lung malignant tumor, we observed the behavior and weight of mice, and found that the blank control group mice were in good mental state, active and normal diet, and the weight showed a slow growth trend. On the third day after planting tumor, mice in other groups could touch subcutaneous tumor, and had low spirits, decreased food intake, sluggish action, lacked of luster of hair, and were conditioned to curling and gathering. In addition, the weight of tumor-bearing mice in the model group and Shiquan Yuzhen Decoction groups decreased evidently on the fourth day after modeling, but gradually returned to normal with time (Figure 7A). We also measured the immune organ index of mice. We found that compared with the blank group, the spleen index and thymus number of the tumor-bearing mice in the model group and Shiquan Yuzhen Decoction group were evidently higher. Compared with the model group, the spleen index of tumor-bearing mice in the middle and high dose groups of Shiquan Yuzhen Decoction was evidently increased (Figure 7B). Moreover, we found by Elisa that the expression of IL-6 in subcutaneous transplanted tumor tissues of tumor-bearing mice in the middle and high dose groups of Shiquan Yuzhen Decoction was evidently decreased, while the expression of TNF-α in serum of tumor-bearing mice in the middle and high dose groups of Shiquan Yuzhen Decoction was increased compared with the model group (Figure 7C). And we also found through comparison that the middle and high concentration of Shiquan Yuzhen Decoction significantly inhibited tumor growth in mice Effect (Figure 7D). In addition, we detected the expression of CD8+T and Treg (FOXP3) cells in mouse tumor tissues by IHC experiment. The results showed that CD8+T positive rate in mice increased significantly with the increase of drug concentration, while FOXP3 positive rate in mice decreased gradually with the increase of drug concentration (Figure 7E). Finally, we also detected the factors related to coagulation and fibrinolysis in tumor-bearing mice. Compared with the blank group, PAI-1 was increased in the model group and the middle and low dose groups of Shiquan Yuzhen Decoction, and the expression of FVii was higher in the model group and the high and low dose groups of Shiquan Yuzhen Decoction. Compared with the model group, PAI-1 and FVii in plasma of mice in the middle and high dose groups of Shiquan Yuzhen Decoction decreased evidently (Table 1).

Figure 7.

Figure 7

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Effect of Shiquan Yuzhen Decoction on immunity and metastasis of malignant tumor in tumor-bearing mice. A. Effect of Shiquan Yuzhen Decoction on the body weight of tumor-bearing mice. B. Effect of Shiquan Yuzhen Decoction on the weight of immune organs in tumor-bearing mice. C. Comparison of tumor volumes collected after the mice were sacrificed. D. Effect of Shiquan Yuzhen Decoction on IL-6 in tumor tissue and TNF-α in serum of tumor-bearing mice. E. The expression of CD8+T and Treg(FOXP3) cells in mouse tumor tissues was detected by IHC test (400×). * indicates compared with the blank group, P < 0.05, and ▲ indicates compared with the model group, P < 0.05.

Table 1.

Effect of SQYZT on the expression of t-PA, PAI-1 and FVii in plasma of tumor-bearing mice (x ± s, n = 8)

Group t-PA (ng/ml) PAI-1 (ng/ml) FVii (IU/ml)
Blank group 14.20±0.713 1.501±0.267▲ 3.2290±0.5696▲
Model group 15.904±1.216 2.249±0.416* 4.6983±0.7120*
SQYZT low dose group 16.095±0.927 2.218±0.399* 4.3936±0.4950*
SQYZT medium dose group 17.586±2.197 1.869±0.145*,▲ 3.8491±0.4348▲
SQYZT high dose group 17.544±1.323 1.806±0.284▲ 4.0054±0.6033*,▲
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*

indicates compared with the blank group, P < 0.05;

▲

indicates compared with the model group, P < 0.05.

Discussion

The characteristic medical theory of Chinese medicine has been formulated and developed for thousands of years, and a variety of traditional Chinese medicines are combined with each other in a complex way to produce synergistic effect to improve the clinical curative effect [28]. The holistic view of TCM focuses on the recovery of overall function and the elimination of etiology. In recent years, there are many similarities between the proposal of network pharmacology and the basic theory of Chinese medicine [29]. Therefore, the network pharmacology can be applied to the research of Chinese medicine, and its compound prescription can be applied to explore the main components and possible mechanism of action of Chinese medicine and its compound prescription on diseases.

In this study, 26 key active ingredients and their corresponding 182 active targets were screened out, and the target network of active ingredients of Shiquan Yuzhen Decoction was constructed. The results showed that most of the single active ingredients of Shiquan Yuzhen Decoction can affect multiple targets, such as quercetin [30], luteolin [31] and kaempferol [32], which all played an ideal role in anti-tumor. Although putative targets in each single active ingredient are different, there are many overlapping targets in different active ingredients, and their coordinated action has produced immunomodulation, carcinoma cell apoptosis promotion, tumor cell metastasis inhibition and tumor angiogenesis inhibition characteristics [33]. In addition, we enriched the predicted 182 targets, and found that Shiquan Yuzhen Decoction was involved in regulating the biological processes of cell proliferation, angiogenesis, gene expression and apoptosis [34]. The treatment of NSCLC may also influence some cell components and molecular functions, including nuclear matter, nucleus, cytoplasm, protein binding, enzyme binding and DNA binding [35]. Through KEGG enrichment analysis, a total of 15 NSCLC-related KEGG pathways containing PI3K-Akt signal pathway [36] and HIF-1 signal pathway [37] were evidently enriched. These signal pathways were mostly related to apoptosis, inflammation, proliferation, metastasis and angiogenesis. Finally, through PPI network analysis, we found that the core targets of Shiquan Yuzhen Decoction in treating NSCLC mainly focused on apoptosis, inflammation, angiogenesis, proliferation and cell cycle [38]. This suggested that Shiquan Yuzhen Decoction may improve the occurrence of NSCLC by regulating apoptosis and angiogenesis in related ways.

Over proliferation of cells and obstruction of apoptosis are important reasons for tumor formation. The formation of tumor microvessels provides a channel for nutrient transport of tumor cells, which greatly accelerates the proliferation and metastasis of tumor cells. In conclusion, tumor apoptosis and tumor angiogenesis act in judging malignant tumors [39,40]. In order to verify the results of biological information analysis, we established the LC tumor-bearing mouse model. Through observation, we found that the medium and high doses of Shiquan Yuzhen Decoction could evidently hinder the growth of subcutaneous transplanted tumor in tumor-bearing mice, increase the incidence of apoptosis of tumor cells, and evidently increase the active expression of Caspase-3, Caspase-8 and Caspase-9. In addition, our experiment also found that Shiquan Yuzhen Decoction with medium and high dose concentration could evidently hinder the formation of blood vessels in transplanted tumor tissues, and reduce the expression of HIF-1a and downstream VEGFA in tissues. It showed that Shiquan Yuzhen Decoction had a good effect on inhibiting the formation of tumor microvessels. It was suggested that Shiquan Yuzhen Decoction could inhibit angiogenesis by promoting apoptosis of tumor cells, thus improving the occurrence of LC.

Tumor patients, whose physique is weak, suffer some damage to their immune function. Therefore, in the course of treatment, they should not only obtain anti-carcinoma effect, but also take care of the improvement of patients’ quality of life and immune function [41]. In network pharmacology analysis, we predicted that TNF [42] and NF-kappa B [43] were the two main signal pathways of Shiquan Yuzhen Decoction. Therefore, at the end of the study, we analyzed the effects of Shiquan Yuzhen Decoction on immune function of tumor-bearing mice. Thymus is the place where T cells differentiate, develop and mature, and it is an important lymphoid organ [44]. Spleen is mainly involved in humoral immunity, which is the center of cellular immunity and humoral immunity and the largest immune organ of the body [45]. Both of them act in phagocytosis of tumor cells and clearance of necrotic tissues [46]. Through behavioral observation and immune organ index measurement, we found that Shiquan Yuzhen Decoction could improve the mental state and diet of tumor-bearing mice, maintain the body weight of mice in a state of continuous growth, and improve the spleen index of mice. Elisa revealed that Shiquan Yuzhen Decoction could reduce the expression of IL-6 in tumor tissue and increase the expression of TNF-α in serum. We speculated that Shiquan Yuzhen Decoction could improve the immune function, promote the release of TNF-α from NK cells and T cells, and play an anti-tumor role. As HIF-1a protein in tumor tissue was inhibited, IL-6 downstream of HIF-1a protein gene was also inhibited, so the content and expression of IL-6 in subcutaneous transplanted tumor tissue of tumor bearing mice in medium and high dose Shiquan Yuzhen Decoction groups were lower than those in model group.

Patients with malignant tumors often suffer from vascular endothelial cell damage due to stimulation of tumor cells, radiotherapy and chemotherapy, surgery and other factors, thus leading to hypercoagulable state [47]. Once the blood hypercoagulable state appears in malignant tumor, it can further promote the metastasis of tumor cells and thrombosis of the body, and further aggravate the occurrence of hypercoagulable state [48]. Therefore, improving the coagulation status of malignant tumor patients can effectively prevent the metastasis of lung malignant tumor and prolong the life of tumor patients. In this study, the coagulation-related factors of tumor-bearing mice were also tested. The results revealed that t-PA in plasma of tumor-bearing mice in model group was no different from that in blank group, but FVI and PAI-1 were higher than those in blank group, indicating that tumor cells still had some influence on the coagulation of mice, while the expressions of FVI and PAI-1 in plasma of tumor-bearing mice in middle and high doses of Shiquan Yuzhen Decoction were evidently lower than those in model group. Therefore, we considered that Shiquan Yuzhen Decoction with a certain concentration can improve the hypercoagulability caused by tumor factors.

In the above research, we have basically determined the relevant mechanism of Shiquan Yuzhen Decoction in LC treatment, but there are still some shortcomings in this study. First, as a basic experiment, the application of Shiquan Yuzhen Decoction in clinic is less, which lacks accurate and sufficient clinical data support. Secondly, this study failed to make an in-depth study on the prevention and treatment of different stages of NSCLC with different concentrations of Shiquan Yuzhen Decoction. The interaction among immune function, angiogenesis, apoptosis and other related proteins in the progression of non-small cell lung carcinoma has not been discussed in depth. Therefore, we hope to carry out clinical research and related basic research in future to improve our experimental conclusions.

To sum up, Shiquan Yuzhen Decoction can evidently improve the quality of life of Lewis lung carcinoma-bearing mice and inhibit tumor growth in mice, which is a potential clinical treatment plan.

Acknowledgements

National Thirteenth Five-Year Science and Technology Major Special Project for New Drug Innovation and Development: The construction of a demonstration technology platform for the clinical evaluation of new drugs for malignant tumor and other diseases (2017ZX09304001); Long-Yi Scholars and Research Team Program fromState Clinical Research Center of TCM in Longhua Hospital (LYTD-65).

Disclosure of conflict of interest

None.

Supporting Information

ajtr0013-7492-f8.pdf (705.6KB, pdf)

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