The Combination of Astragalus membranaceus and Angelica sinensis Inhibits Lung Cancer and Cachexia through Its Immunomodulatory Function

Lung cancer and its related cachexia are the leading cause of cancer death in the world. In this study, we report the inhibitory effect of the combined therapy of Astragalus membranaceus and Angelica sinensis, on tumor growth and cachexia in tumor-bearing mice. Lewis lung carcinoma cells were inoculated into male C57BL/6 and CAnN.Cg-Foxn1nu nude mice. After tumor inoculation, mice were fed orally by the combination of AM and AS in different doses. In C57BL/6 mice, the combination of AM and AS significantly inhibited the growth of cancer tumor and prevented the loss of body weight and skeletal muscle. It also diminished the formation of free radicals and cytokines, stimulated the differentiation of NK and Tc cells, and rebalanced the ratios of Th/Tc cells, Th1/Th2 cytokines, and M1/M2 tumor-associated macrophages. The herbal combination also downregulated the expression of NFκΒ, STAT3, HIF-1α, and VEGF in tumors. In contrast, the findings were not observed in the nude mice. Therefore, the combination of AM and AS is confirmed to inhibit the progression of lung cancer, cancer cachexia, and cancer inflammation through the immunomodulatory function.


Introduction
Lung cancer is the leading cause of cancer death in the world. In United States, 234,030 new cases are estimated to be diagnosed, and 154,050 disease-related deaths are estimated to occur in 2008 [1]. In Taiwan, 12,462 new cases can be diagnosed annually and 9,167 people may die from the disease every year [2]. Although many improvements have been made recently such as minimally invasive operation and immunotherapy, only 18% of the patients live 5 years or more after diagnosis [3].
Cachexia is a common symptom in patients with lung cancer [3]. It is a characteristic of unrecoverable fatigue and involuntary loss of appetite, body weight, skeletal muscle, and fat content [3][4][5]. Cachexia tremendously affects the outcome of patients with lung cancer. In a Japanese study, the median survival of cachectic patients reduced nearly 1.5 years compared to that of noncachectic patients [3]. ese facts indicate a vital and constant need to develop new treatment methods overcoming lung cancer and its cachexia.
TAMs and 1/ 2 cytokines are also correlated to cancer cachexia [9,10,13,14]. Smoldering and continuous inflammation have been observed in patients with cancer cachexia [14]. e increasing serum levels of reactive oxygen species (ROS), nitric oxide (NO), inflammatory cytokines (e.g., IL-1β, IL-4, IL-6, and TNF-α), as well as the upregulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription 3 (STAT3), have been discovered in patients and animals with lung cancers [6,7,9,10]. ese inflammatory cytokines, including IL-6, are generally produced by macrophages. After activation by IL-6, STAT3 can stimulate myeloid cells differentiate into TAMs [14]. In brief, TAMs and these cytokines play an important character in the development and maintenance of cancer cachexia.
Astragalus membranaceus (AM) and Angelica sinensis (AS) are two major herbs applied by Chinese for hundred years. Traditionally, they are used as tonic drugs to treat fatigue, dizziness, anemia, shortness of breath, and gynecologic disorders [15]. Modern research studies have mentioned the potential benefits of AM and AS to patients with lung cancer and cachexia. AM, AS, and their ingredients, such as astragalus polysaccharides (APS), astragalus saponin (AST), astragaloside IV (AS-IV), calycosin, ferulic acid, formononetin, and Z-ligustilide (LIG), were proved to suppress the growth of tumor cells, the release of inflammatory cytokines, the expression of NF-κB and STAT3, and the activation of protumoral macrophages and T cells [8,[16][17][18][19][20][21][22][23]. Recently, the combination therapy of AM and AS, especially in a weight ratio of 5 : 1 (a.k.a. Danggui Buxue Tang (DBT)), has been proved to have anticancer and anticachectic effects [24,25]. erefore, we designed the following experiments to verify our hypothesis that DBT has better anticancer, anti-inflammatory, and anticachectic effects as compared to AM or AS.

Preparation of Herbal Extracts.
e aqueous extracts of AM and AS were kindly provided by Chuang Song Zong Pharmaceutical Co. Ltd. (Kaohsiung, Taiwan), with has a certification of Good Manufacturing Practice. e raw plant materials of AM and AS were obtained from Inner Mongolia and Gansu Province, China, respectively. e identification and authentication of herbs were arranged by qualified experts of the company. e extractions of AM and AS were prepared by boiling in 10-fold (w/w) of water for 90 minutes. All water extractions were concentrated by lyophilization. e combination of AM and AS (DBT) was prepared by mixing the extracts of AM and AS in a 5 : 1 weight ratio.

Phytochemical Analysis of Herbal Extracts.
Ultraperformance liquid chromatography (UPLC) fingerprint was applied to the authentication of AM and AS extracts. Formononetin (purity: 98.43%, Tauto, Shanghai, China), calycosin-7-glucoside (purity: 98.3%, National Institutes for Food and Drug Control, Beijing, China), and AS-IV (purity: 98.3%, Must, Chengdu, China) were used as the reference standards of AM. In addition, ferulic acid (purity: 100%, Sigma-Aldrich, St. Louis, MO, USA) was used as the reference standard of AS. e chromatography was performed in the Acquity UPLC PDA/ELSD eλ detector (Waters, Milford, MA, USA). For the determination of AS-IV, the Acquity UPLC HSS C18 column, 2.1 × 100 mm, 1.8 μm (Waters, Milford, MA, USA), was selected. e column temperature was set at 30°C. e injection volume was 3 μl. e gradient solution was 40% (v/v) acetonitrile in pure water. e flow rate was 0.3 ml/min. e ELSD drift tube temperature was 50°C, and the pressure was set at 20 psi. For the determination of formononetin and calycosin-7-glucoside, the column, the column temperature, and the injection volume were as same as those of AS-IV. e gradient solution A was 0.2% (v/v) formic acid in pure water, and acetonitrile was used as solution B. e gradient program was as follows: 0-23 min, from 95% A and 5% B to 55% A and 45% B. e flow rate was set at 0.4 ml/min. e detection wavelength was 254 nm. For the determination of ferulic acid, the Acquity UPLC HSS T18 column, 2.1 × 100 mm, 1.8 μm (Waters, Milford, MA, USA), was set at a temperature of 35°C. e injection volume was 3 μl. e gradient solution A was 0.1% (v/v) phosphoric acid in pure water. e gradient solution B was 0.1% (v/v) phosphoric acid in acetonitrile/methanol (7 : 3). e gradient program was as follows: 0-28 min, from 90% A and 10% B to 50% A and 50% B. e flow rate was set at 0.3 ml/min. e detection wavelength was 270 nm.

Mice and Animal Experiment.
Male, 4-6-week-old, weighing 18-22 g, C57BL/6J and CAnN.Cg-Foxn1 nu nude mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Mice were housed individually in a climate-controlled room of 12 : 12 dark-light cycle with a constant room temperature of 21.6°C. Before treatment, mice acclimated the new environment and food for ≥4 days. Mice could freely intake water and rodent diet (Young Li Co., New Taipei City, Taiwan). e whole animal study included two subgroups of murine experiments, which used C57BL/6J and nude mice, respectively. Both subgroups have the same protocol as follows. Mice were divided into five weight-matched groups (n � 10 each). In the control (C) group, 0.1 ml of sterile normal saline was injected subcutaneously in the right thigh of each mouse. In tumorbearing mice, 5 × 10 5 LLC cells were diluted in 0.1 ml of sterile normal saline and inoculated into the right thigh of each mouse. e tumor-bearing mice were distributed into four groups: the no treatment (T) group, L group (DBT 1 mg/kg/d, orally administered for 25 days), M group (DBT 2.5 mg/kg/d, orally administered for 25 days), and H group (DBT 5 mg/kg/d, orally administered for 25 days). After 25 days, all mice were sacrificed, and the blood was sampled by cardiac puncture. Meanwhile, the inoculated tumors, gastrocnemius muscle, and spleen were resected for the further examination.

In Vitro Antioxidative Examination.
e ethanolic 2,2diphenyl-1-picrylhydrazyl (DPPH) scavenging, superoxide scavenging, and glutathione (GSH) production assays were used to test the antioxidation ability of herbal extracts in vitro. In the DPPH assay, 100 μl DPPH (0.1 mM) was added to 100 μl herbal extract. e mixing solution was left in the dark at room temperature for 30 min, and then its absorbance was measured at the wavelength of 517 nm. In superoxide scavenging assay, 50 μl herbal extract was treated with 50 μl NBT (300 μM), 50 μl NADH (936 μM), and 50 μl PMS (120 μM). e mixing solution was left at room temperature for 5 min, and its absorbance was measured at the wavelength of 560 nm. In the GSH production assay, RAW264.7 cells (5 × 10 5 cells/ml) were cultured in 6-well plate, treated with herbal extracts in different concentrations, washed, added with trypsin, centrifugated, treated with the GSH assay buffer and iolite Green (AAT Bioquest, Sunnyvale, CA, USA), and left at 37°C for 15 min. e cell accumulation of GSH was observed by flow cytometry at the excitation and emission wavelengths of 488 and 525 nm.

2.7.
In Vitro Anti-Inflammatory Examination. RAW264.7 cells (5 × 10 5 cells/ml) were cultured in 6-well plate, treated with LPS (1 μg/ml) and herbal extract in different concentration, and left at room temperature. After 24 h, the suspension was collected to measure the concentrations of cytokines. e enzyme-linked immunosorbent assay (ELISA) and Quantikine ELISA kits (R&D, Minneapolis, MI, USA) were applied to measure the levels of IL-1β, IL-6, and TNF-α. e absorbance was measured at the wavelength of 450 nm.

In Vitro Phagocytosis
Examination. RAW264.7 cells (5 × 10 5 cells/ml) were cultured in 96-well plate, treated with total 40 μl LPS (1 μg/ml) and herbal extract in different concentrations, and then incubated at 37°C and 5% CO 2 atmosphere for 16 h. After incubation, the mixture was added with 200 μl/well neutral red (0.075%), left in the dark site at 37°C and 5% CO 2 atmosphere for 30 min, treated with 200 μl/well acetic acid/anhydrous alcohol (100 mM), and left in the dark site again for 10 h. e absorbance was measured at the wavelength of 550 nm.

Measurement of Serum Reactive Oxygen Species and NO in Mice.
e dichlorofluorescin diacetate (DCFH-DA), dihydroethidium (DHE), and 4,5-diaminofluorescein diacetate (DAF-2/DA) assays were applied to evaluate the production of ROS (H 2 O 2 and superoxide) and NO in tumor-bearing mice. In the DCFH/DA assay, murine blood was added with 1 ml RBC lysis buffer, centrifuged at 1000 rpm for 5 min, removed the suspension, and then treated with 2 ml PBS and 10 μM DCFH-DA. e mixture was incubated in the dark site for 30 minutes, washed, and then resuspended. e accumulation of oxidized product, 2′,7′-dichlorofluorescein (DCF), was observed by flow cytometry at the excitation and emission wavelengths of 488 and 525 nm. In the DHE assay, murine blood was added with 1 ml RBC lysis buffer, centrifuged at 1000 rpm for 5 min, removed the suspension, and then treated with 2 ml PBS and 10 μM DHE. e mixture was incubated in the dark site for 30 minutes, washed, and then resuspended. e accumulation of oxidized product, 2-hydroxyethidium (2-OH-E+), was observed by flow cytometry at the excitation and emission wavelengths of 488 and 575 nm. In the DAF-2/DA assay, murine blood was added with 1 ml RBC lysis buffer, centrifuged at 1000 rpm for 5 min, removed the suspension, and then treated with 2 ml PBS and 10 μM DAF-2/DA. e mixture was incubated in the dark site for 30 minutes, washed, and then resuspended. e accumulation of the oxidized product, diaminofluorescein-triazole (DAF-2T), was observed by flow cytometry at the excitation and emission wavelengths of 488 and 525 nm.

Measurement of Serum Albumin and Cytokines in Mice.
e inflammation of tumor-bearing mice was observed by measuring serum levels of albumin and inflammatory cytokines. Murine blood was collected and centrifuged at 4°C and 3500 rpm for 30 min, and then the suspension was collected to measure the serum levels of albumin and cytokine. e concentration of albumin was assessed by Spotchemez SP-4430 dry chemical system (Arkray, Kyoto, Japan). e ELISA and Quantikine ELISA kits (R&D, Minneapolis, MI, USA) were applied to measure the levels of IL-1β, IL-4, IL-6, TNF-α, and IFN-c. e absorbance was measured at the wavelength of 450 nm.

Peripheral Blood Cells and Splenocyte Differentiation
Analysis. Murine blood and spleens were collected to evaluate the influence of DBT to peripheral blood cells and splenocyte differentiation. e counts of peripheral blood cells were calculated by using a Sysmex K-1000 automated hematology analyzer (Sysmex American, Lincolnshire, IL, USA). e differentiation of splenocytes was evaluated by flow cytometry. e spleen was treated with 2 ml RBC lysis buffer to isolate splenocytes. e BD FACSCantoTM II system (BD, San Jose, CA, USA) was used for cytometry.
e tumor proteins were also treated with the Pierce BCA protein assay kits ( ermo Fischer, Waltham, MA, USA), and their absorbance was measured at the wavelength of 550 nm.

Immunofluorescence Examination.
e immunofluorescence (IF) technique was used to detect the M1-and M2phenotype TAMs in tumor microenvironment. Rabbit anti-mouse iNOS (Bioss, Woburn, MA, USA) and goat anti-mouse arginase 1 (Santa Cruz, Dallas, TX, USA) were used as primary antibodies in order to label M1 and M2 macrophages, respectively. e secondary antibodies included goat anti-rabbit IgG (Bioss, Woburn, MA, USA) and donkey anti-goat IgG (Santa Cruz, Dallas, TX, USA). In the IF examination, tumor specimens were harvested, fixed in 4% formalin for 48 h, embedded in paraffin, sectioned, deparaffinized in xylene, and rehydrated in ethanol. After antigen retrieval by boiling in 10 mM Tri-EDTA (pH 8.0) for 1 h, the sections were washed with PBST (1 × phosphate-buffered saline (PBS), 0.1% Tween 20), incubated with primary antibodies, washed again with PBST, treated with secondary antibodies, and left in the dark site for 1 h. e slides were observed and pictured by an upright fluorescence microscopy (Olympus BX51, Olympus, Tokyo, Japan). e pictured images were analyzed and merged by ImageJ software (NIH, Bethesda, MD, USA). ImageJ with the colocalization and color deconvolution plugins were also used to quantify immunofluorescence and chromogenic signal intensity on image.
2.14. Statistical Analysis. IBM SPSS Statistics 20 software (IBM, Armonk, NY, USA) was used for statistical analysis. Data were shown as mean ± standard error of mean (SEM). Difference between groups was assessed by one-way analysis of variance (ANOVA). Statistical significance of difference was considered at p < 0.05.

Phytochemical Characteristics of AM and AS.
e HPLC fingerprints of AM and AS are presented in Figure 1. e reference standards of AM included AS-IV, formononetin, and calycosin-7-glucoside. e reference standard of AS was ferulic acid. ese components were confirmed qualitatively and quantitatively in AM and AS. e contents of AS-IV and calycosin-7-glucoside within AM were 0.744 and 0.507 mg/ g, respectively. e contents of ferulic acid within AS were 0.733 mg/g. In addition, the contents of AS-IV, calycosin-7glucoside, and ferulic acid within DBT were 0.62, 0.423, and 0.122 mg/g, respectively.

In Vitro Cytotoxic and Phagocytotic Properties of Herbs.
e cytotoxicity of herbal extracts to BHK and LLC cells is presented in Figures 2(a) and 2(b). AM, AS, and DBT have no significant cytotoxicity to normal BHK cells until the concentration was more than 5 mg/ml. In analogy to BHK cells, all three herbs did not cause LLC cell death until the concentration was more than 10 mg/ml. But provided the herbal concentration at 100 mg/ml, the BHK and LLC viabilities were still more than 50%. Given also the fact that the typical concentration of AM or AS is less than 0.3 mg/ml in traditional Chinese medicine, we consider both AM and AS have no cytotoxicity to normal or malignant cells.  As illustrated in Figure 2(c), the phagocytotic effect of LPS-stimulated RAW264.7 cells was enhanced by the combined treatment of AM and AS. Meanwhile, the ability increased in proportion to the content of AM, and it reached the plateau at the 5 : 1 ratio of AM and AS (aka DBT). As compared with AM and AS, the combination treatment can induce the strongest phagocytotic ability in vitro.

In Vitro Anti-Inflammatory and Antioxidative Abilities of
Herbs. AM, AS, and DBT all exhibited a dose-dependent inhibition to inflammation, as all of them suppressed the generation of IL-1β, IL-6, and TNF-α in RAW264.7 cells (Figures 3(a)-3(c)). Among the three herbs, DBT was presented as the most effective herb to downregulate these cytokines. In addition, AM, AS, and DBT also represented a dose-dependent efficacy to reduce oxidation (Figures 3(d)-3(f )). e production of H 2 O 2 and superoxide declined, but the GSH production increased after herbal treatment. Similar to the results of anti-inflammatory experiments, DBT still appeared as the most effective herb to reduce oxidation. ese findings indicate that DBT contains antiinflammatory and antioxidative functions.

In Vivo Anticancer and Anticachectic Abilities of DBT.
Because DBT appeared as the most effective herb to improve phagocytosis, oxidation, and inflammation in the in vitro experiments, it was chosen for the following study in tumorbearing mice (Figure 4(a)). In tumor-bearing C57/BL6 mice, DBT significantly reduced the growth of inoculated tumor and the loss of body weight and gastrocnemius muscle (Figures 4(b)-4(d)) but also increased the diet intake (C group: 32.2 g/d, T group: 28.1 g/d, H group: 32.7 g/d, p � 0.015). ese effects were presented in a dose-dependent pattern. In contrast, DBT did not improve tumor growth, body weight, weight of gastrocnemius muscle, or diet intake in the nude mice group (Figures 4(b)-4(d)). ese findings suggest that DBT can enhance T cell immunity to inhibit tumor growth and cancer cachexia.

In Vitro Anti-Inflammatory and Antioxidative Properties of DBT.
e serum levels of inflammatory cytokines were elevated in both C57/BL6 and nude mice after tumor inoculation ( Figures 5(a)-5(c)). In C57BL/6J mice, the following DBT caused a dose-dependent reduction in serum levels of IL-1β, IL-6, and TNF-α. By contrast, in nude mice, only IL-1β level was suppressed by treating DBT. e serum albumin level was decreased after tumor inoculation in C57BL/6J mice, but after DBT treatment, it was elevated in a dose-dependent pattern (C group: 2.20 g/dl, T group: 1.38 g/ dl, L group: 1.53 g/dl, M group: 1.75 g/dl, and H group: 1.88 g/dl, p < 0.001). Meanwhile, the levels of H 2 O 2 , superoxide, and NO were elevated after tumor inoculation in C57/BL6 mice, but this phenomenon was not observed in nude mice. In C57/BL6 mice, the levels of ROS and NO were subjected to a dose-dependent reduction under the DBT treatment ( Figures 5(d)-5(f )). Collectively, DBT can modulate T cell immunity to attenuate cancer-related oxidation and inflammation. Table 1, tumorbearing mice suffered from the increase of white blood cells, monocytes, and lymphocytes in peripheral bloods. DBT treatment effectively ameliorates these hematologic abnormalities in C57BL/6J mice, but its effect became modest in nude mice. is finding suggested that DBT depends on a normal T cell immunity to attenuate cancer-related leukocytosis.

Effects of DBT to Murine Peripheral Blood Cells and Splenocyte Differentiation. As illustrated in
e DBT treatment also influenced the differentiation of splenocytes ( Figure 6). In C57BL/6J mice, the percentages of Relative intensity  indicate that DBT can apply the T cell immunity to restore the differentiation of immune cells in the cancer-bearing host.

Effects of DBT on 1/ 2 Cytokines and M1/M2
Polarization. It has been noticed that lung cancer can influence the levels of 1/ 2 cytokines in peripheral blood and the phenotype polarization of M1/M2 macrophages in tumor microenvironment (TAMs). In clinical studies, lung cancer had been associated with the overexpression of 2 cytokines and M2 TAMs; meanwhile, these cytokines and macrophages were correlated to a poor response of chemotherapy and a shorter survival [6,7]. Because DBT had been shown to modulate the host immunity, we continuously explored its characteristic to the differentiation of cytokines and TAMs. In mice with normal immunity, the serum IFN-c (a 1 cytokine) level and the tumoral expression of Arg1+ cells (M2 TAMs) were elevated, but the IL-4 (a 2 cytokine) level and the expression of iNOS+ cells (M1 TAMs) were diminished after tumor inoculation (Figure 7). After DBT treatment, the serum levels of IFN-c and IL-4 and the tumoral expression of iNOS+ and Arg1+ cells were overturned. e effects were in a dose-dependent pattern. ese results demonstrate that DBT can restore the predominance of serum 1 cytokine and M1 TAMs in the host; therefore, it can suppress tumor growth and prolong survival in the host of malignancy.

Effects of DBT to Inflammatory and Angiogenic Pathways in Tumor Cells.
To understand further how DBT interfered in molecular pathways of tumor cells, we examined the expression of NF-κB, STAT3, HIF-1α, and VEGF in tumor tissues. In C57BL/6J mice, the addition of DBT can suppress expression of these given proteins. In terms of protein expression, there was a dose-dependent reduction in the DBT treatment groups when compared with the tumor group ( Figure 8). As a result, DBT can downregulate the inflammation and angiogenesis pathways in the tumor-bearing mice.

Discussion
is study was to research the influence of Astragalus membranaceus (AM) and Angelica sinensis (AS) to cancer growth and cachexia in lung cancer-bearing mice. In literature, AM and AS have been shown to improve anemia, inflammation, fatigue, and the chemotherapy response in patients with lung cancer [15,16,[25][26][27]. However, their direct influence to tumor cells was rarely reported. In this study, we discovered that (1) AM and AS are not toxic to  even at 100 mg/ml of herbal treatment.
is result is consistent to the literatures, in which the LD50 of AM and AS was about 400 mg/ml for normal cell lines, 40 and 1.6 g/ kg, respectively, for normal mice [28,29]. In the following experiments, we found that the combination of AM and AS, in a weight ratio of 5 : 1, can stimulate the strongest phagocytotic function in LPS-stimulated macrophages but also contains the best antioxidative and anti-inflammatory abilities, as compared with the single AM or AS treatment.
is herbal formula is known as Danggui Buxue Tang in traditional Chinese medicine and has been used for patients with weakness or anemia for hundred years. In the modern research, Danggui Buxue Tang and its active components have shown to attenuate the release of ROS, NO, and inflammatory cytokines in animals with nonmalignant diseases [24,26,30,31]. By contrast, our study pointed out its antioxidative and anti-inflammatory functions are also available in the host of malignancy. On the contrary, we also found out that its effects would become indistinct in mice with depleted T cell immunity, which strongly suggests that these functions are associated with the modulation of host immunity.
ere is a variety of immune cells within tumor microenvironment. Among these cells, 1, Tc, and NK cells and M1 TAMs are closely related to antitumoral immunity and cancer inhibition, while 2 and M2 cells play a part in immune suppression and tumor promotion [10,13]. With regard to the polarization of macrophages, 1 cytokines like IFN-c can promote the differentiation of M1 macrophages, while 2 cytokines like IL-4 can stimulate of the expression of M2 phenotype. [7]. In clinic practice, the decreasing ratios of 1/ 2 cytokines and M1/M2 TAMs are closely related to cancer progression and poor prognosis of patients [6,7]. e ingredients of AM and AS, such as astragaloside IV, astragalus, and angelica polysaccharides, contain abilities to improve phagocytotic function of macrophages and cytotoxic activities of NK and CD8+ T cells but inhibit the functions of regulatory T cells [8,18,19,32,33]. In our research, we found that the combination of AM and AS can upregulate the serum IFNc level and the differentiation of Tc, NK cells, and M1 TAMs in tumor-bearing mice. Meanwhile, it also rebalances the ratio of /Tc cell ratio and suppresses the expressions of IL-2 and M2 TAMs. ese findings indicate
During inflammation, the host immune cells typically secrete certain signaling molecules, such as tumor growth factors, angiogenic growth factors, chemokines, or cytokines, in response to a given stimulus. Of these molecules, IL-1β, IL-6, TNF-α, and IL-4 promote the phosphorylation of downstream proteins, e.g., NF-κB and STAT3, which then lead to inflammation, tumor proliferation, and prevention of apoptosis in cancer [34,35]. In our study, the combination of AM and AS enabled to inhibit production of IL-1β, IL-4, IL-6, and TNF-α and thus resulted in the inhibition of phosphorylation of NF-κB and STAT3. Besides, it also enabled to impede the expression of some angiogenic molecules, such as VEGF and HIF-1α. e suppression of these two molecules slows down angiogenesis inside the tumor microenvironment, thus ameliorating tumor progression and distant metastasis. In addition to inhibition of inflammation and angiogenesis in the model of organ fibrosis [36], we further determined that the combined AM and AS therapy can attenuate cancer-related inflammation and tumor angiogenesis.
It has been known that free radicals are mass produced in tumor microenvironment. Bio-free radicals are generally referred to as reactive oxygen species (ROS; e.g., H 2 O 2 , superoxide, and hydroxyl radical) and reactive nitrogen species (RNS; e.g., NO). ey can be produced from endogenous and exogenous sources, such as inflammation, radiation, carcinogen, and hypoxia. e excessive production of free radicals would cause oxidative stresses, which generally activate transcription factors such as NF-κB, STAT3, HIF-1α, and AP-1 [37]. ROS and NF-κB are both involved in 7,8-dihydro-8-oxoguanine (8-oxoG) and KRASmediated inflammation, oncogenesis, and the relative interaction between innate and adaptive immunity [38]. In neoplasms, inordinate NF-κB promote the expression of many vital modulators of cancer progression, such as HIF-1α, AP-1, STAT3, and MUC1 [39]. Furthermore, the hypoxic environment of tumor tissue stimulates the release of HIF-1α, which upregulates the expression of vascular endothelial growth factor (VEGF) and following angiogenesis for cancer progression [40].
ese indicate the close relationship between free radicals, inflammation, and oncogenesis. AM and AS have been confirmed to suppress production of free radicals and inflammation in noncancer models [30,31]. In this study, we identified that the combination of AM and AS can scavenge free radicals in the cancer model. It has been known that persistent inflammation would aggravate carcinogenesis, local invasion, distant metastasis, and drug resistance of tumor [37]. erefore, the strong antioxidative effect of the combination of AM and AS also contributes to its anticancer efficacy.
Tumor-promoting inflammation plays an important role in cancer cachexia. Tumor-released cytokines, such as TNF-α or IL-1, are involved in the NF-κB and MAPK pathways to lead to breakdown of structural muscle proteins and inhibition of protein synthesis. In the murine model of chronic fatigue syndrome, the combination of AM and AS was found to increase body weight and endurance capacity and to decrease mRNA levels of IL-1β, TNF-α, NF-κB, and p38MAPK [24]. In this study, we identified that it favorably modulates the loss of body weight, gastrocnemius muscle, and white adipose tissue (C group: 0.13 g, T group: 0.09 g, L group: 0.1 g, and M and H groups: 0.12 g, p < 0.001) in the cancer model. Consequently, the combination of AM and AS can effectively suppress cancer cachexia.
AM and AS contain numerous effective molecules. e important molecules include ferulic acid, calycosin, formononetin, Z-ligustilide, polysaccharides, and saponins. Each of these molecules features some unique functions. For example, astragalus saponins, astragalus and angelica polysaccharides, ferulic acid, and Z-ligustilide are known to suppress expression of some tumorpromoting cytokines, such as IL-1β, IL-6, and TNF-α [16,17,[41][42][43]. Ferulic acid, formononetin, Z-ligustilide, astragalus saponins, and astragalus and angelica polysaccharides were reported carrying some favorable antioxidation activities [17,30,[44][45][46]. Astragalus saponins and astragalus and angelica polysaccharides reportedly possess some immunomodulation functionalities [17,32,47]. Besides, these molecular discoveries also supported all the current findings of AM and AS which have been found in vitro or using murine models. erefore, we conclude that the combination of AM and AS that synergizes these favorable properties should be recruited in the battle of fighting cancer.

Conclusion
In conclusion, we demonstrate here that the combined therapy of Astragalus membranaceus and Angelica sinensis can suppress oxidative stress, inflammation, tumor proliferation, and angiogenesis. In addition, this herbal combination is able to modulate the host immunity and thus inhibits cancer growth and corresponding cachexia. In the development of new anticancer agents, we consider that this herbal combination stands a good position to be reformulated to the maximum anticancer effect on the basis of the information provided herein as a short-term goal.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.