ZhenQi FuZheng Formula Improves Mouse Hematopoietic Function after Cyclophosphamide-Induced Damage via Enhancing Macrophage Colony-Stimulating Factor Concentrations

Background: Among the hematopoietic system, bone marrow has been recognized as the major source of hematopoietic progenitor cells (HPCs). Immuosuppression can inhibit the growth of bone marrow cells and cyclophosphamide (CTX) has been reported to induce myelosuppression. Regeneration of the immune system and hematopoietic function has become a primary problem in tumor adjuvant therapy. The present study investigated the effects of ZhenQi FuZheng formula (ZQFZ) on hematopoietic function in cells and murine models of cyclophosphamide(CTX)-induced immunosuppression and hematopoietic dysfunction. Methods: According to antibody chip analysis and enzyme-linked immunosorbent assay, ZQFZ regulated seven cytokines in serum and the spleen. Results: In Nrf2-siRNA transfected K562 cells, the regulatory effects of ZQFZ on the expression concentrations of macrophage colony-stimulating factor (M-CSF) and hematopoietic related proteins were strongly abolished. In immunosuppressed mice, ZQFZ enhanced the NK cell activities and regulated the concentrations of cytokines related to immune function. ZQFZ induced proliferation and differentiation, and upregulated the expression of hematopoietic-related proteins, including p90 ribosomal S6 kinases 1 (RSK1p90), c-Myc, and ETS transcription factor in CHRF and/or K562 cells. In mice with hematopoietic dysfunction, ZQFZ contributed to the recovery of blood cell concentrations in peripheral blood to normal values and enhanced the percentages of B lymphocytes (LYs) and juvenile cells in the bone marrow. Conclusions: In spleens of hematopoiesis damaged mice and primary cultured bone marrow cells, ZQFZ upregulated the expression concentrations of NF-E2-related Factor 2 (Nrf2) and its downstream proteins, and the activation of nuclear factor kappa-B. ZQFZ showed remarkably benecial effects on the bone marrow hematopoietic system, at least partially, by enhancing

The hyper-levels of reactive oxygen species (ROS), one of the phenomena and pathological mechanisms that occurs during oxidative stress, is responsible for hematopoietic dysfunction by causing bone marrow failure and hematopoietic malignancies [8]. Increased concentrations of ROS during oxidative stress are toxic to hematopoietic stem/progenitor cells and persturbs the differentiation of hematopoietic cells, and it affects the life span of erythrocytes in mature blood cells [8,9]. Activated nuclear factor-E2-related factor 2 (Nrf2) leads to the suppression on in ammatory factors [10], and promotes the proliferation and differentiation of hematopoietic stem cells (HSCs) by affecting the long-term hematopoietic cell cycle [11].
Thea avins protect HSCs from hematopoietic damage caused by ionizing radiation by reducing ROS concentrations by activating Nrf2 [12].
In the clinic, the injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF) is commonly used to treat myelosuppression and can prevent chemotherapy-induced neutropenia and enhance the function of mature neutrophils [13]. However, adverse effects of rhG-CSF have been noted, such as the proliferation of cancer cells [14]. In contrast, traditional Chinese medicine has been reported to have protective effects on bone marrow [15]. A combination of Astragalus membranaceus and Angelica sinensis stimulate hematopoietic function, as evidenced by its promotion of the recovery of peripheral blood cells and bone marrow nucleated cells (BMNCs), and its acceleration of the proliferation of HSCs and HPCs in mice with hematopoietic dysfunction [16,17]. ZhenQi FuZheng formula (ZQFZ), a compound prescription of astragali radix (Astragalus propinquus Schischkin) and glossy privet fruit (Ligustrum lucidum W.T.Aiton), was rstly put forward on 1974 in China by Professor Yan Sun, and has been used to enhance the immune function and protect against bone marrow and adrenal damage caused by various other diseases, surgery, radiation, and/or chemotherapy in clinics (drug approval number Z20053398).
Astragali radix and glossy privet fruit are recorded as top grade in The Herbal Classic Shen Nong, and both possess immunomodulatory, anti-in ammatory and anti-oxidant effects [18,19]. Based on the previous results, we speculate that ZQFZ improves hematopoietic function, especially hematopoietic suppression induced by chemotherapeutic drugs.
In this experiment, we found that ZQFZ promoted the differentiation of hematopoietic cells, enhanced immune function in immunosuppressed mice, and protected the hematopoietic system in hematopoietic damaged mice. These effects are related to the effects of ZQFZ on the concentration of macrophage colony stimulating factor (M-CSF), which may be modulated via Nrf2 and nuclear factor-kappa B (NF-κB) signaling.
From the femurs and tibias of 6-8 week male BALB/c mice, the bone marrow cells were ushed out and collected with Dulbecco's Modi ed Eagle Medium (DMEM) (HyClone, South Logan, UT, USA) using a 1 mL syringe with a 21-gauge needle. Bone marrow mononuclear cells were collected with the red blood cell lysis buffer (GS3309) (Genview, Pompano Beach, FL, USA) by removing non-nucleated cells. The resultant cells were washed and pelleted with DMEM, seeded into a 6-well plate (2 × 10 6 cells/well) in DMEM coantaining with 10% FBS, 100 µg/mL streptomycin, and100 U/mL penicillin, and cultured at 37 °C in a humidi ed incubator with 5% CO 2 and 95% air.

Evaluation in CTX-injected mice with immunosuppression
The experimental protocol (2017SY0603) has been approved by the Institution Animal Ethics Committee of Jilin University. Seventy 7-week male BALB/c mice weighted 18-22 g (speci c pathogen-free (SPF) grade, SCXK(Liao)-2015-0001) were housed in a controlled environment the same as our previous study [20].
After one-week acclimatization, fty mice were intraperitoneally injected (i.p.) with 75 mg/kg of CTX (AMRESCO, Boise, Idaho, USA) dissolved in normal saline (NS) for 3 days, and then randomly divided into ve groups, gavage with 10 mL/kg of double distilled (D.D.) water serving as model group (n = 10), 4.5 mg/kg of transfer factor oral liquid (TFO) (dissolved in D.D. water) (n = 10), 0.1 g/kg (n = 10), 0.3 g/kg (n = 10) and 0.9 g/kg (n = 10) of ZQFZ (dissolved in D.D. water) once per day for 4 weeks. CTX (60 mg/kg) was injected once per week for maintaining immunosuppression in mice. Another twenty mice were injected (i.p.) with NS for 3 days, and then gavaged with 10 mL/kg of D.D. water serving as the control group (n = 10), and 0.3 g/kg of ZQFZ serving as ZQFZ monotherapy group (n = 10) once per day for 4 weeks. All mice were monitored every day during the whole experiment. 2-h after the last administered, blood was collected from the caudal veins, and then mice were euthanasia by injecting 200 mg/kg of pentobarbital.

Natural killer (NK) cell cytotoxic activity assay
The NK cell cytotoxic activity detection was performed the same as our previous study [22]. Spleen cells of each mouse were obtained by ltering the fresh spleens through the 200 mesh screen. After collection and resuspension, 100 µL of spleen cells (5 × 10 7 cells/mL) and 100 µL of YAC-1 cells (1 × 10 6 cells/mL) (TIB-160TM) (ATCC, Manassas, VA, USA) were suspended with RPMI1640 medium containing 10% FBS were seeded into 96-well plates, and cultured at 37 °C in a 5% CO2 incubator for 4 h. 1% NP-40 treated cells was served as maximum control. The commercial kit (ml002267), obtained from Enzyme-linked Biotechnology (Shanghai, China) was applied to detect the concentration of lactate dehydrogenase (LDH) in 100 µL culture medium. According to the absorbance and the following equation, the NK cell cytotoxic activity was calculated using the same formula as our previous study [22].

Histopathological analysis
Similar as our previous study [20], the pathological alterations of spleen of mice with immunosuppression were detected using the hematoxylin-eosin (H&E) staining under the inverted microscope CKX41 (Olympus, Tokyo, Japan).

The experimental performed in CTX-injected mice with hematopoietic dysfunction
The experimental protocol (2017SY0603) has been approved by the Institution Animal Ethics Committee of Jilin University. Seventy 7-week male BALB/c mice weighted 18-22 g (SPF grade, SCXK(Ji)-2016-0008) were housed in a controlled environment the same as our previous study [20].
After one-week acclimatization, fty mice were injected (i.p.) with 100 mg/kg of CTX dissolved in NS for three days, and then randomly divided into ve groups, gavage with 10 mL/kg of D.D. water serving as model group (n = 10), and 0.1 g/kg (n = 10), 0.3 g/kg (n = 10) and 0.9 g/kg (n = 10) of ZQFZ (dissolved in D.D. water) once per day for 4 weeks, and subcutaneously injecting with 22.5 µg/kg of rhG-CSF (n = 10) (Nanjing Yixun Biotechnology Co., Ltd., Nanjing, China) twice a week for 4 weeks. CTX at dose of 80 mg/kg was injected every Monday to avoid the restoration of hematopoietic function. The control group (n = 10) and ZQFZ monotherapy group (n = 10) were obtained similarly as 2.6. All mice were monitored every day during the whole experiment.

Detection of biochemical indexes in peripheral blood
Blood was sampled 2-h after the last treatement from the caudal veins for peripheral blood analysis immediately with a fully automatic blood analyzer (Drew Scienti c Group, Dallas, TX, USA).

BMMNCs component analysis
200 mg/kg of pentobarbital injection (i.p.) was used for euthanasia to mice. Under the sterile condition, the tibial and femoral section were collected immediately. Similar to our previous research, the ACK lysis buffer was used for the isolation of the bone marrow mononuclear cells (BMMNCs) [20]. The antibodies related to surface markers including FITC-conjugated anti-mouse Lineage Cocktail (133302), APCconjugated anti-mouse CD19 (152410), and PerCP-conjugated anti-mouse CD45 (103129) were applied to stain with cells at 25 °C for 15 min darkly. APC-conjugated anti-rat IgG2b (400611), PerCP-conjugated anti-rat IgG2b (400629), FITC-conjugated anti-rat IgG2b (400605) and FITC-conjugated anti-rat IgG2a (400505) were set as isotype controls. The experimental antibodies were purchased from Biolegend (San Diego, CA, USA). The Cyto ex ow cytometer (Beckman Coulter, California, USA) was applied for analyzing.
The 40 cytokines/chemokines of the spleen in hematopoiesis damaged mice were analyzed using the Mouse Cytokine Array Panel A Kit (R&D Systems, Minneapolis, MN, USA) the same as our previous research [20].

Western blotting
The primary cultured bone marrow cells, the Nrf2-siRNA transfected K652 cells, the CHRF and K562 cells were treated with ZQFZ at doses of 0, 25 and/or 100 µg/mL for 24 h. The treated cells and the spleens of hematopoiesis damaged mice were lyzed using the radioimmunoprecipitation assay lysis buffer containing 1% protease inhibitor cocktail (Sigma-Aldrich) and 2% phenylmethanesulfonyl uoride (Sigma-Aldrich). 30-40 µg of proteins were separated using 10%-12% SDS-PAGE and transferred onto a polyvinylidene di uoride membranes (0.45 µm, Merck Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin (BSA), the membranes were incubated with primary antibodies as Table S1 at 4 °C overnight, following with the exposure to horseradish peroxidase (HRP)-conjugated secondary antibodies (diluted to 1:2000) (NBP2-30347H and NBP2-30348H) (Novus Biologicals, Littleton, Colorado, USA) for 4 h at 4 °C. An enhanced chemiluminescence detection kit (Merck Millipore) combining with an imaging system (BioSpectrum600) were applied to visualize the protein bands. The ImageJ software (Version 1.8.0) (National Institutes of Health, Bethesda, MD) was used to analyze the pixel density of the band.

Statistical analysis
A one-way ANOVA followed by a Tukey's post hoc test comparison was applied for statistical signi cance analysis using SPSS 16.0 software (IBM Corporation, Armonk, NY, USA). Data are expressed as mean ± S.D. and considered signi cant at p < 0.05.

Detection of effective components of ZQFZ
Based on the 2015 version of the Chinese Pharmacopoeia, the speci c composition of ZQFZ was determined by HPLC. Thus, ZQFZ was found to contain 3.934 mg/g of salidroside, 0.138 mg/g of calycosin-7-glucoside, 0.166 mg/g of ligustro avone, 0.026 mg/g of ononin, 0.076 mg/g of quercetin, and 0.017 mg/g formononetin (Fig.S1a-f).
3.2 Immunomodulatory activity of ZQFZ in mice with CTXinduced immunosuppression NK cells exhibit speci c immunomodulatory effects by identifying ligands of target cells and then directly killing these cells [23]. In the spleens of mice with CTX-induced immunosuppression, similar to TFO, ZQFZ enhanced the cytotoxic activities of NK cells by more than 60.4% (p < 0.001) (Fig.S2a). CTX-injection caused narrowed white pulp, enlarged red pulp and an unclear boundary between the white and red pulp in the spleen of mice with CTX-induced immunosuppression; in contrast, ZQFZ repaired these pathological changes (Fig.S2b).

ZQFZ promotes proliferation and differentiation of hematopoietic cells
The CHRF and K562 cell lines are commonly used to study cell proliferation and differentiation related to hematopoietic function [26,27]. Incubation of ZQFZ for 24 h, especially at 100 µg/mL, promoted the proliferation of CHRF (p < 0.01) and K562 cells (p < 0.001) (Fig. 1a) without in uencing their apoptotic rate ( Fig. 1c and d). ZQFZ at 100 µg/mL improved erythroid differentiation of K562 cells, as indicated by an increased benzidine positive cell rate v (p < 0.001) (Fig. 1b). The c-Myc, ETS Transcription Factor ELK1 (ELK1) and RSK1p90 have been recognized to be related to cell growth and differentiation related to hematopoietic function [28]. After 24-h incubation, the enhanced levels of c-Myc, ELK1 and P-RSK1p90 were noted in K562 (p < 0.01) (Fig. 1e) and CHRF cells (p < 0.001) (Fig. 1f) treated with ZQFZ at 100 µg/mL.
The results of H&E staining showed that there were reductions in the numbers of cells and the proportion of vacuoles in the bone marrow cavity (Fig. 2c), multinucleated giant cells in the spleen (arrow in Fig. 2d), and the in ammatory in ltration phenomenon in liver (arrow in Fig. 2f) were noted, which were all strongly reversed by ZQFZ. No signi cant changes in the kidney were seen in any experimental group (Fig. 2e).
ZQFZ alone failed to in uence the production of BMMNCs ( Fig. 2a and b), the bone marrow cavity (Fig. 2c), and the other organ structures (Fig. 2d, e and f) in healthy mice.

Discussion
To the knowledge, we rst report on the effects of ZQFZ on immune and hematopoietic functions in mice.
In mice with CTX-induced immune suppression, ZQFZ enhanced the activities of NK cells, and regulated immune-related factors such as ILs and chemotactic factors. Immunoglobulins protect the body from infections and can enhance humoral immunity and the body's hematopoietic function [32,33]. Excessive production of ROS can affect the activity of in ammation-related proteins and damage the immune system [34]. Among all detected ILs, IL-2, the most comprehensively studied IL, can stimulate the growth of T and B cells, enhance the cytotoxic activities of NK cells, regulate the differentiation of Th1 cells, and further promote the release of IL-6, -10 and − 12 [35,36].
A dynamic balance among the differentiation of red blood cells, the proliferation of HSCs, and the formation of blood cells, is required for regular hematopoiesis [37,38]. The reduced self-renewal on HSCs is responsible for long-term bone marrow damage [39]. As reported, the normal function of the hematopoietic system is partially maintained by HSCs; thus, the protection and improvement of the selfrenewal of HSCs may be key to develop new agents for anti-myelosuppression [39]. It was thus notable that ZQFZ enhanced the proliferation on both of K562 and CHRF cells, promoted the differentiation of marrow [40]. Meanwhile, CD19 controls the differentiation and maturation of B lymphocytes helping to maintain the humoral immunity of the human body [41,42]. Severe autoimmune disease, such as hematopoietic dysfunction can be caused by abnormalities in the immune system [43]. Lin − , a speci c marker for juvenile cells, contributes to cell proliferation and differentiation [31,44]. ZQFZ enhanced the number proportion of bone marrow cells in CTX-damaged bone marrow in mice. The concentration of blood cells in peripheral blood re ects the hematopoietic function of bone marrow [29], and the pathological alterations to peripheral blood caused by CTX were all strongly restored by ZQFZ administration. All of the data con rm the role of ZQFZ in improving hematopoietic function in mice.
Cytokines, such as ILs and IFNs, regulate the proliferation and differentiation of hematopoietic cells [45,46]. IL-2 helps to promote the expression of multiple antigens and antibodies, including IL-5, TNF-β, and CSF [36]. IL-5 regulates the activation of hematopoietic cells [47]. The host cells of a hematopoietic system damaged by long-term chemotherapy/radiotherapy releases proin ammatory cytokines, including TNF-α, that directly inhibit hematopoietic function by suppressing the number of bone marrow precursors and inhibiting the activity of HSCs [48]. It is encouraging to note that ZQFZ enhanced the concentrations of M-  [52,53]. M-CSF can promote the expression of c-Myc [54], RSK, and ETS by indirectly in uencing the activation of ERKs [55,56]. The current data suggest that enhancement of M-CSF is involved in ZQFZ-mediated enhancement on hematopoietic function.
NF-κB signaling acts as the major modulator in in ammatory responses and hematopoiesis, such as the survival of HSCs and HSPCs and the differentiation of hematopoietic precursors [57]. In a classical signaling pathway, phosphor-mTOR promotes the phosphorylation of IKKα + β, leading to the activation of NF-κB [58], which is translocated into the nucleus, where it binds to the enhancer region of M-CSF [59,60]. The reduction of ROS synergistically promotes phosphor-mTOR to further regulate the activation of NF-κB [61]. In contrast, ROS promote the expression of Nrf2 with phosphor-ERK [62]. As a feedback, the activated Nrf2 signaling enhances the expression of SOD1, SOD2, and HO-1, which help to inhibit the over-production of ROS [63]. Consequently, the activated Nrf2 signaling promotes the levels of M-CSF by up-regulating the expression of SOD1 and HO-1 [64,65]. According to a previous study, in K562 cells, the anti-in ammatory avone wogonin inhibited Nrf2 signaling via NF-κB inactivation [66]. We were interested to note that the regulatory effects of ZQFZ on the phosphorylation of NF-κB, the expression of M-CSF and erythroid differentiation related proteins were signi cantly reversed in Nrf2-siRNA-transfected K562 cells. Based on our data, both Nrf2 and NF-κB appear to be involved in regulating the bene cial effect of ZQFZ on hematopoietic function in mice.
There are limitations to this study. A non-dose-dependent effect of ZQFZ, which contains multiple effective components, was noted in some of our experiments, which is common for herbal medicines.
More experiments will be performed to reveal the effective constituents in ZQFZ. In this present study, we reported the improvement induced by ZQFZ on immune and hematopoietic functions; however, the pharmacological mechanisms, especially the link between the two functions, still require deep examination. Furthermore, the nding of several studies suggest that an enhanced level of Nrf2 expression helps to reduce the phosphorylated activation of NF-κB [67], and it was found in another study that the activation of NF-κB was unaffected by Nrf2 [68]. In our study, although we found that low expression of Nrf2 helped to suppress the phosphorylation of NF-κB in Nrf2-siRNA transfected K562 cells, the relationship between NF-κB signaling and Nrf2 signaling still requires further investigation. Due to its complex composition, ZQFZ may not directly in uence the expression and/or activation between Nrf2 and NF-κB during its enhancement of hematopoietic function.