Fuzheng Qingjie granules potentiate the anticancer effect of cyclophosphamide by regulating cellular immune function and inducing apoptosis in Hepatoma 22 tumor-bearing mice

  • Authors:
    • Xu‑Zheng Chen
    • Zhi‑Yun Cao
    • You‑Quan Zhang
    • Jin‑Nong Li
    • Lian‑Ming Liao
    • Jian Du
  • View Affiliations

  • Published online on: March 13, 2017     https://doi.org/10.3892/ol.2017.5849
  • Pages: 3261-3268
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Fuzheng Qingjie (FZQJ) is a polyherbal Chinese medicine that has previously been implemented as an adjuvant therapy for gastrointestinal cancer. The present study investigated whether FZQJ is able to potentiate the anticancer effect of cyclophosphamide (CTX). Hepatoma 22 tumor‑bearing mice were randomly divided into a vehicle group, CTX group, FZQJ group and combination (CTX+FZQJ) group. In addition, untreated mice without H22 cells served as blank controls. Seven days post‑treatment, the mice were sacrificed and the tumors were weighed. Blood cells were evaluated using an automatic hemocytometer analyzer and flow cytometer. The expression levels of interleukin (IL)‑2 and tumor necrosis factor (TNF)‑α were evaluated using a radioimmunoassay. Apoptotic cells were observed using a terminal deoxynucleotidyl transferase dUTP nick‑end labeling assay. Alanine transaminase, aspartate aminotransferase, blood urea nitrogen and creatinine were examined using an automatic biochemical analyzer. The results demonstrated that the tumor inhibitory rate and apoptosis index were higher in the combination group, compared with those in the CTX group. Notably, FZQJ was able to alleviate CTX‑induced decreases in the numbers of white blood cells and platelets, CD3+ and CD4+ T lymphocyte subsets, and the concentration of hemoglobin, body weight and thymus index, and increase serum TNF-α and IL-2 levels without overt hepatorenal toxicity. These results suggest that FZQJ granules may enhance the anticancer effect of CTX, in addition to alleviating the side effects.


Hepatocellular carcinoma (HCC) is one of the most frequent causes of cancer-associated mortality globally due to a high incidence and poor prognosis (1). This is particularly high in China due to the 10% hepatitis B virus infection rate in the general population (2). The majority of patients with HCC are diagnosed at intermediate or advanced stages of disease, excluding them from potentially curative treatment options, including resection, local ablation or liver transplantation (3). For intermediate or advanced HCC, chemotherapy is not routinely used as, in addition to severe adverse reactions, including myelosuppression, nausea and vomiting, the response rate is ~10–20% (4). Therefore, there is an urgent requirement to improve the efficacy of chemotherapy and simultaneously decrease the associated adverse reactions.

Traditional Chinese medicine (TCM) emphasizes the importance of inhibiting tumor growth and alleviating the adverse reactions of chemotherapy and/or radiotherapy in order to improve patient quality of life (5). TCM physicians often use Fuzheng Guben herbs and Qingre Jiedu herbs during cancer treatment (6). Fuzheng Guben herbs, including Ganodorma lucidum, Astragalus membranaceus, Ginseng species and Fructus lycii, have been observed to strengthen the immune response through the activation of T and B lymphocytes, macrophages, natural killer (NK) cells and dendritic cells, and promoting the production of cytokines, including interleukins (IL), tumor necrosis factors (TNF) and interferon (7). Additionally, these herbs have been demonstrated to protect bone marrow from cyclophosphamide (CTX) and cytosine arabinoside, and prevent a chemotherapy-induced decrease in white blood cells (WBC), red blood cells (RBC) and platelets (PLT) in the peripheral blood (8). According to TCM, heat is considered an important causative factor in HCC, and Qingre Jiedu has been administered to clear heat and detoxify the body (6). Previous pharmacological studies have demonstrated that Qingre Jiedu herbs (including Hedyotis diffusa Willd, Prunella vulgaris, Lobelia chinensis Lour and Sophora flavescens) contain anthraquinones, polysaccharides, flavonoids, alkaloids and triterpenoids that are able to inhibit tumor cell proliferation, induce cell apoptosis and suppress angiogenesis (911). Matrine from Sophora flavescens is able to induce the apoptosis of HepG2 cells via the upregulation of tumor protein 53, B cell lymphoma-2 (Bcl-2)-associated X protein (Bax) and Fas, and the downregulation of Bcl-2 and c-Myc (12).

Fuzheng Qingjie granules (FZQJ) are composed of Fuzheng Guben and Qingre Jiedu herbs (13). The four Fuzheng Guben herbs are Astragalus membranaceus, Ligustrum lucidum, Ganoderma lucidum and Rhizoma dioscoreae, and the two Qingre Jiedu herbs are Hedyotis diffusa Willd and Prunella vulgaris (13). FZQJ granules are used to treat the symptoms that are commonly observed post-chemotherapy or radiotherapy, including thirst, night sweats, constipation, insomnia, loss of appetite and weakness. A previous study demonstrated that FZQJ may induce apoptosis of HepG2 cells via activating p38 mitogen-activated protein kinases (MAPKs) and inducing mitochondria-dependent apoptosis (13).

CTX is biotransformed principally in the liver to active alkylating metabolites, which cross-link tumor cell DNA (14) in order to interfere with the growth of susceptible rapidly proliferating malignant cells. CTX is used in combination with other antineoplastic drugs to treat a variety of susceptible malignancies, including lymphoma (15) and myeloma (16), ovarian (17), nasopharyngeal (18) and liver cancer (19). However, CTX is also associated with severe toxicities, including diarrhea, nausea, vomiting, bone marrow suppression, haemorrhagic cystitis, fatigue, night sweat, hair loss, immunosuppression and impaired hepatic and renal function (20).

The present study investigated whether FZQJ is able to potentiate the anticancer effects of CTX in hepatoma 22 (H22) tumor-bearing mice, and potentially alleviate CTX-associated toxicities.

Materials and methods

Preparation of FZQJ decoction

FZQJ was manufactured and provided by the Department of Pharmacy, The Second Affiliated Hospital, Fujian University of Traditional Chinese Medicine (Fuzhou, China). FZQJ granules were dissolved in distilled water to produce a solution with a final concentration of 0.3 g/ml, which was stored at 4°C until use.

Mouse xenograft experiments

A total of 50 male specific pathogen free Institute of Cancer Research mice (6-weeks-old, 22–25 g) were purchased from Guangdong Animal Center (Guangzhou, China). All animals lived in a light/dark cycle. Food and tap water were available ad libitum. The room temperature (RT) was maintained at 23±2°C and humidity was approximately 60%. The mice were inoculated subcutaneously on the right side of theirs back with 5×106 H22 cells in Matrigel/Dulbecco's Modified Eagle's medium with gentamycin (BD Biosciences, Franklin Lakes, NJ, USA). H22 cells were purchased from the Shanghai Institute of Life Science (Chinese Academy of Sciences, Shanghai, China) and cultured in RPMI-1640 culture medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C with 5% CO2. Mice were randomly divided into 4 groups (n=10 per group) as follows: Vehicle group (oral distilled water), CTX group (Baxter Oncology GmbH, Halle, Saxony, Germany, intraperitoneally; 40 mg/kg/day), FZQJ group (oral; 6 g/kg/day) and combination group (CTX 40 mg/kg/day intraperitoneally plus FZQJ 6 g/kg/day orally). Mice in the vehicle control group were inoculated with H22 cells and administered distilled water orally. In addition, another ten mice without H22 cells served as the blank group. At day seven, all the mice were sacrificed by cervical dislocation and peripheral blood was collected by ocular enucleation. The tumors were dissected by a surgical operation and weighed.

Thymus index

The thymus was collected from the mice by a surgical operation and weighed. The thymus index was calculated according to the following formula: Thymus index (mg/10 g) = thymus weight (mg)/body weight (g) × 10.

Routine blood analysis

WBC, lymphocyte (LY) and RBC cell numbers as well as PLT and hemoglobin (Hb) concentrations, were examined with EDTA-K2 anticoagulated whole blood using an automatic hemocytometer (Sysmex Corporation, Kobe, Japan). This process was performed ≥3 times for each blood sample.

T and NK immune cells

Anticoagulated whole blood was stained with a fluorescein isothiocyanate-conjugated cluster of differentiation (CD) 3 monoclonal antibody (mAb; cat. no. 100203; dilution, 1:200, BioLegend, Inc., San Diego, CA, USA), in combination with a phycoerythrin (PE)-conjugated CD8a mAb (cat. no. 100707; dilution, 1:80; BioLegend, Inc.), or PE-conjugated CD4 mAb (cat. no. 100509; dilution, 1:200; BioLegend, Inc.) or PE-conjugated CD49b mAb (cat. no., 108907; dilution, 1:80; BioLegend, Inc.), incubated at RT for 15 min in the dark and subsequently lysed using BD FACS™ lysing solution (BD Biosciences). A FACSCalibur™ flow cytometer with CellQuest software (version 5.1; BD Biosciences) was utilized to analyze the percentage of CD3+ T cells, CD4+ T cells, CD8+ T cells and NK cells present. Absolute counts of CD3+ T cells, CD4+ T cells, CD8+ T cells and NK cells were calculated according to the following formula: Absolute cell number = the percentage of cells × the number of LYs. All experiments were performed ≥3 times.

Cytokine assays

The serum levels of IL-2 and TNF-α in H22 tumor-bearing mice were determined using a γ radioimmunoassay counter (Dongya Immunological Technique Institute, Beijing, China), with 125I-IL-2 and 125I-TNF-α radioimmunoassay kits according to the protocol of the manufacturer (Dongya Immunological Technique Institute).

Hepatic and renal functions of H22 tumor-bearing mice

To determine the safety of FZQJ, the serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (CRE) were assessed according to the manufacturer's protocol with a FLEX mode automatic biochemical analyzer (TBA-120FR, Toshiba, Kawasaki, Japan).

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay

The tumor tissues were fixed in 4% paraformaldehyde at RT for 36 h. Following dehydration, the fixed tissues were embedded in paraffin. The samples were sectioned at thickness of 5 µm. Subsequently, the sections were deparaffinized with ≥99% xylene baths at RT, 10 min each, and then rehydrated in graded ethanol solutions of 100, 95, 70, and 50% (v/v). The apoptotic cells of the tumors were detected using a TUNEL assay, according to the manufacturer's protocol (Fuhzou Maixin Biotech Co., Ltd., Fuzhou, China). TUNEL-positivity indicates that the cells exhibit the DNA damage that results from apoptotic cascades (21). These cells possessed a pyknotic nucleus with dark brown staining, and were counted in 10 random fields at ×200 magnification. The apoptotic index was reported as the number of TUNEL-positive cells/total number of cells scored.

Statistical analysis

Statistical analysis was performed using SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA). Data were presented as the mean ± standard deviation. For multiple comparisons, the data were analyzed using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.


FZQJ potentiates the anticancer effect of CTX

To evaluate whether FZQJ is able to potentiate the anticancer effect of CTX, the tumor xenograft weight of each mouse was examined at seven days post-treatment. As presented in Fig. 1A, compared with the vehicle group, tumor weight significantly decreased in all three groups (P<0.01). No significant difference was observed between the CTX group and FZQJ group (P=0.156). Notably, when these drugs were administered simultaneously, the inhibitory rate was higher, compared with CTX alone (P=0.027). Therefore, FZQJ may potentiate the anticancer effect of CTX.

In addition, TUNEL assays were performed in order to assess the apoptotic cells in H22 neoplastic tissue (Fig. 1B), in which the TUNEL-positive cells were stained dark brown. The apoptosis index in all three treated groups was significantly higher, compared with the vehicle group (P<0.01; Fig. 1C). Compared with the CTX group, the apoptosis percentage was higher in the combination group (P=0.049). These data demonstrated that FZQJ may be able to potentiate the anticancer effect of CTX through the induction of apoptosis.

FZQJ alleviates CTX-induced peripheral blood cell and body weight decreases

To evaluate whether FZQJ is able to alleviate the adverse effects of CTX, the numbers of WBCs, RBCs, PLTs, the concentration of Hb in the peripheral blood and the body weight of the mice over the course of treatment were examined. As presented in Fig. 2, PLT counts were higher in the vehicle group, compared with the blank group (P=0.048). The levels of WBCs, RBCs, PLTs and Hb were similar between the vehicle group and the FZQJ group (5.34±1.27) × 109/l vs. (5.90±0.38) × 109/l, (9.07±0.47) × 1012/l vs. (9.83±0.72) × 1012/l, (1594.00±198.66) × 109/l vs. (1665.60±156.87) × 109/l, 14.72±0.74 vs. 15.62±0.83 g/l, respectively, indicating that FZQJ granules are non-toxic to bone marrow. As hypothesized, CTX markedly decreased the WBC and PLT counts, as well as the concentration of Hb, due to its induction of bone marrow suppression. By contrast, when CTX and FZQJ were simultaneously administered, these parameters were all significantly improved (WBCs, P=0.048; PLTs, P=0.047; Hb, P=0.016). Similarly, a significant difference was not observed with respect to the body weight of the mice between the vehicle group and the FZQJ group. And CTX notably induced body weight loss (P<0.01). The CTX-induced body weight decrease was prevented by FZQJ (P=0.050).

FZQJ improves immune function in H22 tumor-bearing mice

To evaluate the effect of FZQJ on the immune function of H22 tumor-bearing mice, the subpopulations of lymphocyte cells, thymus index, serum IL-2 and TNF-α levels were examined. As presented in Fig. 3A, the thymus index in the H22 tumor-bearing mice was significantly decreased, compared with the blank group (P=0.021). The thymus index was further decreased when CTX was administered (P=0.000), whilst FZQJ was able to increase the thymus index of non-treated (P=0.030) and CTX-treated H22 tumor-bearing mice (P=0.049).

In addition, the absolute counts of CD3+ T, CD4+ T and NK cells in the vehicle group were notably lower, compared with those in the blank group (P=0.039, 0.004, 0.045 respectively; Fig. 3B), an effect that tended to be reversed by the administration of FZQJ. As hypothesized, CTX markedly decreased the numbers of LYs, CD3+ T, CD4+ T, CD8+ T and NK cells (Fig. 3B). The addition of FZQJ to CTX increased the numbers of LYs, CD3+ T and CD4+ T cells. As presented in Fig. 3C, it was observed that the levels of IL-2 and TNF-α were highest in the FZQJ groups (P=0.049 and 0.006 respectively, compared with the vehicle group), and IL-2 and TNF-α serve a key role in cellular immunity (2223).

Taken together, these data demonstrate that CTX is able to impede cellular immune function, and that FZQJ granules are able to prevent CTX-induced immune suppression in H22 tumor-bearing mice.

FZQJ exhibits no hepatic and renal toxicity

As presented in Table I, similar levels of ALT, BUN and CRE were observed between the vehicle group and the FZQJ group. CTX induced a marked increase of AST as predicted, and FZQJ was not able to prevent the CTX-induced deterioration of hepatic function. The results demonstrate that FZQJ was unable to alleviate CTX-induced hepatic injury.

Table I.

Hepatic and renal functions of H22-tumor bearing mice.

Table I.

Hepatic and renal functions of H22-tumor bearing mice.

GroupALT, IU/lAST, IU/lBUN, mmol/lCRE, µmol/l
CTX49.20±9.93 429.00±55.84a7.48±1.4116.68±2.91
Combination (CTX+FZQJ)53.00±9.56 433.75±54.99a7.02±0.9918.30±2.79

a P<0.01 vs. the vehicle group. All data presented as mean ± standard deviation. ALT, alanine transaminase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CRE, creatinine; CTX, cyclophosphamide; FZQJ, Fuzheng Qingjie; IU/l, international units per liter.


FZQJ has previously been used as adjuvant treatment during chemotherapy for gastrointestinal malignancies (24). The present study demonstrated that FZQJ is able to potentiate the anticancer efficacy of CTX, and prevent CTX-induced immune suppression and body weight loss without overt hepatorenal toxicity in H22 tumor-bearing mice. The underlying mechanisms for these processes may include FZQJ-induced tumor cell apoptosis and stimulated IL-2 and TNF-α production to enhance cellular immune function.

Numerous studies have demonstrated that Fuzhegn Guben herbs and their ingredients are able to enhance the anticancer effects of chemotherapy and/or radiotherapy whilst reducing certain side effects (5,25). For example, ginsenoside Rg3 combined with CTX decreased cell susceptibility to drug resistance and improved survival time in C57/BL6 mice with Lewis lung carcinoma (26). Shenqi Fuzheng injection has also been demonstrated to improve the immune function of patients with breast cancer receiving neoadjuvant chemotherapy (27). The present study observed that FZQJ produced an antineoplastic effect; however, this was less pronounced, compared with CTX. Notably, FZQJ was able to significantly potentiate the antineoplastic effect of CTX, with the absence of associated and overt side effects. The antineoplastic effect of FZQJ may be associated with the induction of H22 cell apoptosis. However the mechanism underlying the apoptosis-inducing action of FZQJ remains to be elucidated. Previous studies have demonstrated that FZQJ-induced hepatoma cell apoptosis occurs via the regulation of Bcl-2 and Bax expression in vitro (13). In the present study, FZQJ was observed to stimulate IL-2 and TNF-α production. TNF-α is able to induce mitochondrial-mediated apoptosis via the activation of Bcl-2 family proteins, reactive oxygen species, C-Jun, C-Jun N terminal kinases and cathepsin B (2831). IL-2 is able to enhance the antitumor efficacy of TNF-α, despite being less cytotoxic itself (32). In addition, Hedyotis diffusa Willd and Prunella vulgaris in FZQJ granules were reported to induce cell apoptosis via modulation of the IL-6/signal transducer and activator of transcription 3, MAPK and mitochondria-dependent signaling pathways (33,34). The present and previous studies indicated that FZQJ granules were able to induce H22 cell apoptosis via IL-6/stat 3, MAPK and mitochondria-dependent pathway as well as stimulating IL-2 and TNF-α production.

The current study also observed fewer CD3+ T, CD4+ T and NK cells in the peripheral blood of the vehicle group, compared with that of the blank group. The results indicated that cellular immune function was impaired once the blank mice were inoculated subcutaneously with H22 cells. Conversely, mice in the vehicle group exhibited a higher PLT count. This was in accordance with the thrombocytosis observed in patients with hepatic tumors (35). A possible underlying mechanism may be associated with tumor cell-stimulated production of thrombopoietin and IL-6, which promote PLT proliferation and activation (35,36). When CTX alone was administrated for seven days continuously, the WBC, PLT, LY, CD4+ T helper, CD8+ T cytotoxic/suppressor, CD3+ T and NK cells, concentration of Hb, thymus index and mouse body weight all decreased, indicating that CTX induced bone marrow suppression and a gastrointestinal reaction, which are common symptoms in patients receiving this treatment. The present study also demonstrated that FZQJ not only exhibited no toxicity, but that it also significantly increased the numbers of blood cells, the thymus index and body weight. Therefore, FZQJ may combat CTX-induced anemia and the decreased cellular immune function, as well as alleviate certain side effects of this treatment in the gastrointestinal tract.

Pharmacological studies have demonstrated that Astragalus membranaceus, Ligustrum lucidum, Ganoderma lucidum and Rhizoma dioscorea contain potent immune stimulants, for example polysaccharides, which trigger the production of numerous cytokines in vivo, including IL-2, IL-12 and TNF-α, which may activate T and B cells (3741) and modulate the balanced association between Th1 and Th2 cytokines (42,43). Concordantly, the present study demonstrated that FZQJ was able to upregulate the expression of IL-2 and TNF-α, which may be responsible for the observed increase in the T and NK cell counts. In a previous study, Astragalus membranaceus was able to increase serum megakaryocyte colony-stimulating activity and accelerate the recovery of hematopoiesis following bone marrow suppression in anemic mice, which may provide an explanation for the improvement of bone marrow suppression observed following FZQJ administration in CTX-treated mice (44). Finally, Hedyotis diffusa Willd, a component of FZQJ, was also reported to be capable of protecting the gastrointestinal mucosa (45), which may explain the observed improvement in CTX-induced body weight loss.

In conclusion, FZQJ not only improves the anticancer efficacy of CTX, but may also alleviate its adverse effects. Therefore, FZQJ may provide a promising adjuvant treatment during chemotherapy and/or radiotherapy for HCC.


This study was supported by the Natural Science Foundation of Fujian Province (grant nos. 2015J01689 and 2014J01421).





Hepatocellular carcinoma


Traditional Chinese medicine


Fuzheng Qingjie



NK cell

natural killer cell




tumor necrosis factor


reverse transcription-polymerase chain reaction


horseradish peroxidase


terminal deoxynucleotidyl transferase dUTP nick-end labeling


hematoxylin and eosin


white blood cells


red blood cells







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Chen XZ, Cao ZY, Zhang YQ, Li JN, Liao LM and Du J: Fuzheng Qingjie granules potentiate the anticancer effect of cyclophosphamide by regulating cellular immune function and inducing apoptosis in Hepatoma 22 tumor-bearing mice. Oncol Lett 13: 3261-3268, 2017
Chen, X., Cao, Z., Zhang, Y., Li, J., Liao, L., & Du, J. (2017). Fuzheng Qingjie granules potentiate the anticancer effect of cyclophosphamide by regulating cellular immune function and inducing apoptosis in Hepatoma 22 tumor-bearing mice. Oncology Letters, 13, 3261-3268. https://doi.org/10.3892/ol.2017.5849
Chen, X., Cao, Z., Zhang, Y., Li, J., Liao, L., Du, J."Fuzheng Qingjie granules potentiate the anticancer effect of cyclophosphamide by regulating cellular immune function and inducing apoptosis in Hepatoma 22 tumor-bearing mice". Oncology Letters 13.5 (2017): 3261-3268.
Chen, X., Cao, Z., Zhang, Y., Li, J., Liao, L., Du, J."Fuzheng Qingjie granules potentiate the anticancer effect of cyclophosphamide by regulating cellular immune function and inducing apoptosis in Hepatoma 22 tumor-bearing mice". Oncology Letters 13, no. 5 (2017): 3261-3268. https://doi.org/10.3892/ol.2017.5849