Qihuzha Granule Alleviated Cyclophosphamide-induced Immune Deciency in Mice via IL-6 Related Signaling Pathways


 BackgroundQihuzha granule (QHZG) is a Chinese patent medicine, composed of 11 kinds of edible medicinal plants, which is used to treat dyspepsia and anorexia in children caused by spleen and stomach deficiency syndrome. However, its role and mechanism in immunosuppression induced by cyclophosphamide remained unclear. The purpose of this study is to investigate the effect of QHZG on immunosuppression induced by cyclophosphamide in mice and its possible mechanism.MethodsThe immunosuppression injury model was induced by intraperitoneal injection of cyclophosphamide (100 mg/kg); the mRNA level of cytokines (IL-2/4/6, IFN-γ) and critical targets of signaling pathways related to immune regulation (JNK, ERK, P38, JAK2, SRC and STAT3) were tested by QPCR; related protein levels were detected by western blotting; hematoxylin-eosin (HE) staining was employed to observe the histological alterations; macrophages and neutrophils in the mouse spleen were examined by immunofluorescence analysis. ResultsQHZG significantly increased the spleen index and thymus index of mice with immunodeficiency induced by cyclophosphamide and up-regulated the mRNA expression of cytokines (IL-2/4/6, IFN-γ) and critical targets of signaling pathways related to immune regulation (JNK, ERK, P38, JAK2, SRC and STAT3), which were decreased by cyclophosphamide treatment. The results of immunofluorescence staining and histological analysis showed that QHZG could also protect mice from immunosuppressive injury caused by cyclophosphamide via keeping structural integrity of spleen, and partially restoring the production levels of macrophages and monocytes in the spleen. Further studies indicated that QHZG could significantly counter the decline of phosphorylated protein levels of JAK2/SRC-STAT3 axes (P-JAK2, P-SRC and P-STAT3), and MAPK pathways (P-JNK, P-ERK and P-P38) induced by cyclophosphamide, suggesting that the protective effects of QHZG on immunosuppressive injury triggered by cyclophosphamide were involved in JAK2/SRC-STAT3 axes, and MAPK pathways. Meanwhile, we also found that QHZG could partially restore the vital phosphorylated proteins of PI3K/Akt/mTOR signaling pathway (P-Akt, P-mTOR), which were reduced by cyclophosphamide. The data implied that PI3K signaling pathway was also responsible for the protection of QHZG against the immunosuppression induced by cyclophosphamide in mice. ConclusionsOur study demonstrated that QHZG protected mice from cyclophosphamide-triggered immunosuppressive injury via IL-6 and its downstream signaling pathways including PI3K/Akt/mTOR signal pathway and JAK2-SRC/MAPK/STAT3 axes. These results suggested that QHZG might serve as a new drug for the treatment of the immunosuppression caused by cyclophosphamide therapy.


Introduction
The immune system plays a prominent role in resisting microorganisms and foreign antigens, and involves in the recovery from diseases determined by the host immune status [1]. Cyclophosphamide (CTX) is a broad-spectrum antitumor agent that is effective against both leukemia and many solid tumors. However, CTX has been found to inhibit humoral and cellular immune responses of animals and resulted in immunosuppression of animals [2]. Hence, as an immunosuppressant, it has been widely used in the establishment of immunosuppressive animal models. In this study, the immunosuppressive model of BALB/c mice was established by cyclophosphamide to observe the immunoregulation effect of QHZG on cyclophosphamide-induced immunosuppressive model mice.
Over the years, the discovery of novel immunomodulators has attracted researchers' great attentions and interests [3], and a series of breakthroughs have been made. However, serious adverse reactions of clinical drugs, such as general discomfort, neurotoxicity, etc. [4], were still not enough to meet the treatment requirements of immunosuppressive patients. QHZG is a kind of Chinese patent medicine derived from natural products and mainly composed of 11 traditional edible and medicinal plants [5], such as Dioscorea zingiberensis, Astragalus membranaceus, Poria cocos, roasted licorice, melon, Dendrobium, Atractylodes macrocephala, hawthorn, Codonopsis pilosula, malt, papaya, etc. It was mainly used to treat anorexia and dyspepsia in children with spleen and stomach de ciency syndrome [6]. Due to its good health care effect and high safety, it was also widely used in the adjuvant treatment of children with recurrent respiratory tract infections in clinical [5,7,8]. Moreover, modern pharmacological studies have shown that polysaccharides from Dendrobium, astragalus, Codonopsis pilosula and other major traditional Chinese medicines in QHZG could inhibit the in ammatory reaction caused by a variety of infections, and then have immunomodulatory effect [9,10,11,12]. In addition, some compounds identi ed in QHZG, including Atractyloside II and Atractyloside III in Atractylodes lancea, pachymic acid in Poria cocos and lobeliol in Codonopsis pilosula [13], showed a certain degree of immune enhancing activity. These results suggested that QHZG might has multiple immunomodulatory effects. However, its regulatory effects and the potential mechanisms on immunosuppression have not been reported.
IL-6, a pleiotropic cytokine that acts on a variety of cell types and affects various biological activities, is a key mediator in various in ammatory processes, through which tissues respond to injury and infection, either in acute phase or chronic in ammatory diseases [14], In addition, IL-6 also plays crucial role on the host innate immunity and leads to the transition of acquired immune response [15]. Previous study had shown that IL-6 might contribute to reconstitution of immune system function in immunocompromised mice caused by cyclophosphamide [16]. In summary, IL-6 signaling pathway might serve as potential targets for development of immune modulators. IL-6 utilize gp130 to conduct intracellular signal transduction [17] and gp130 can form complexes with the Janus kinases (JAKs), including JAK2. JAKs were activated through auto-phosphorylation and then phosphorylated gp130, STAT and the tyrosine phosphatase SHP-2. Phosphorylated STAT3 dimerizes is translocated to the nucleus where it acts as a transcription factor. Additionally, phosphorylated SHP-2 and activated gp130 lead to activation of the PI3k/AKT pathway [18]. In the present study, we intended to investigate the immunomodulatory effects of QHZG on cyclophosphamide-induced immunocompromised mice and examined the effects of QHZG on crucial components of IL-6 related pathway to elucidate the possible mechanism.

Establishment of the mice models
Balb/c mice, 16±1g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd, (Liaoning, China). All animal experiments were carried out according to the requirements of animal protection and use Committee of Guizhou Medical University. Brie y, animals were randomly divided into three groups (10 BALB/c mice in each group) and given different treatments: control group: normal saline; Model group: cyclophosphamide 100 mg/kg/d; QHZG group: mice were given QHZG 100 mg/kg/d by gavage.
Except for the control group, CTX (100 mg/kg/d) was injected subcutaneously on the 1 st, 3 rd, 5 th, 7 th, 9 th, 16 th, 18 th, 20 th and 22 nd day. The mice in each group was weighed from the rst day of feeding, and the body weight was recorded every 3 days, a total of 10 times. Thymus index and spleen index were calculated by thymus or spleen weight/body weight.

Histological analysis
In short, the spleen tissue was xed with 4 % paraformaldehyde and stained with hematoxylin eosin. The changes of spleen tissue was observed under inverted microscope (DMi8, Leica, Germany min. After centrifugation at 4 ℃ and 12000 rpm for 10 min, transparent precipitates appeared at the bottom. Discard the supernatant, add 1 ml of precooled 75 % ethanol, and turn it upside down until the precipitate oats in 75 % ethanol. After centrifugation at 4 ℃, 7500 rpm for 5 min, the supernatant was discarded, dried and precipitated at room temperature for 1-2 min, then 20-50 ul RNase free water was added to dissolve and precipitate. The extracted RNA was reverse transcribed according to the instructions of primescript RT reagent kit (TAKALA, Japan). The primers in Table 1 were diluted according to the manufacturer's requirements. QPCR 20 µL system: primer I 0.5 µL, primer II 0.5 µL, SY Green 10 µL, cDNA 1 µL. Gene expression level was calculated by 2 −ΔΔCT method.

Statistical analysis
Results were presented as mean±SD. Data analysis was achieved by using One-way analysis of variance (ANOVA) followed by Tukey post-hoc test (GraphPad Software, San Diego, CA). P < 0.05 was considered of great signi cance.

Results
Immune improvement of QHZG in CTX-induced immunocompromised mice Thymus and spleen are vital immune organs and are accountable for initiating immune reactions in the body [19,20,21]. Thymus and spleen will atrophy when the immune function of the body declines. Therefore, thymus index and spleen index are often used to evaluate the immunity of immunocompromised mice. As shown in Fig. 1, the effects of cyclophosphamide and QHZG on the immune organ weight were tested. Compared to the control group, lower thymus index and spleen index were observed in the CTX treated group, which suggested the mouse immunode ciency model was successfully established by intraperitoneal injection of cyclophosphamide. Whereas QHZG (100 mg/kg) signi cantly raised the thymus index and spleen index of CTX treated mice, suggesting their partial recovery of immune function. Collectively, QHZG might partly contribute to reconstitution of immune system function in CTX induced immunocompromised mice.

Histological analysis of spleen
Since the spleen is the body's largest immune organ [19,20], we further examined the effects of QHZG on the alteration of histomorphology of spleen in CTX treated mice. As shown in Fig. 2, in the control group, the red and white pulp were clearly demarcated and lymphocytes in the white pulp were more densely distributed. However, the spleen tissue structure of the CTX treated group, the boundary between red and white pulp was not clear, the structure of white pulp was more scattered and lymphocytes were sparsely distributed. On the contrary, the white intramedullary lymphocytes were increased and the lymphocytes became dense in the spleen of QHZG treated group, compared with that in the CTX treated group. These results certi ed that QHZG at least protected the spleen from CTX induced immunosuppression injury to a certain extent, thus improving the immune function of immunocompromised mice.

Effects of QHZ on neutrophils in CTX-treated mice
Neutrophils, the rst line of defense to protect the body from infection or in ammation, are key participants in innate immune responses and commonly used to assess nonspeci c immune status [21]. Therefore, Ly-6G immunostaining analysis was used to disclose neutrophil in ltration in cyclophosphamide-induced immunosuppression injury. As shown in Fig. 3, the number of ly-6G positive cells in the cyclophosphamide treated group was markedly decreased compared to that in the control group. Importantly, QHZG treatment led to an increase in the number of ly-6G-positive cells. In a conclusion, in vivo results implied that QHZG treatment could partly restore impaired neutrophils.

Effects of QHZ on macrophage
Macrophage activation plays an important role in innate and adaptive immune responses and is believed to be involved in the pathogenesis of immunosuppression respond [22]. To further explore the possible protective mechanism of QHZG on cyclophosphamide-induced immunosuppressive injury, F4/80 Effects of QHZG on the mRNA of cytokine Immune stimulation is one of the important strategies to improve the body's defense mechanism [23].Activated splenic lymphocytes plays an important role in innate and adaptive immune responses by producing cytokines, such as IL-2, IL-4, IL-6, IFN-γ and other in ammatory mediators. As illustrated in Fig. 5, compared with control group, mRNA levels of IL-2/4/6 and IFN-γ in cyclophosphamide treated group were signi cantly decreased. In contrast, QHZG treatment at 100 mg/kg markedly upregulated the mRNA levels of these cytokines mentioned above, which were reduced by CTX treatment, suggesting that QHZG markedly improved splenocytes function of CTX-treated mice and contributed to reconstitution of immune system function in immunocompromised mice caused by CTX. As is known to all, interleukin-6, a cytokine critical to proin ammatory and immune regulatory cascades, has a broad effect on cells of the immune system and those not of the immune system [24,25] Hence, therapeutic agents targeting the IL-6 axis was effective in immunode ciency disease.
QHZ inhibited PI3k/Akt/mTOR signaling pathway in CTXtreated mice PI3K/Akt signaling pathway is one of crucial downstream events of IL-6 signaling [26]. To further investigate the potential mechanism that QHZG improved splenocytes function and contributed to reconstitution of immune system function in immunocompromised mice caused by CTX, we rstly evaluated the effects of QHZG on PI3K/Akt signaling pathway in CTX-treated mice. As shown in Fig. 6A-6B, the phosphorylation of AKT and mTOR in spleen tissue of CTX treated mice was remarkably decreased without affecting total protein, compared with that in normal group. While, QHZG obviously reversed the alteration of phosphorylated proteins triggered by cyclophosphamide in spleen tissue of mice. Together, QHZG might attenuate immunosuppression induced by cyclophosphamide via the regulation of PI3k/Akt/mTOR signaling pathway.
QHZG prevented mice from CTX-induced immunosuppression injury through JAK2-SRC/MAPK/STAT3 signaling pathway STAT3 is also one of the most important components of the IL-6 signaling pathway. We next investigated whether STAT3 was involved in the splenic protective activities of QHZG. As expected, we observed decreased protein and mRNA levels of STAT3, as well as the p-STAT3 in CTX treated group. Whereas, QHZG treatment signi cantly alleviated the effect of CTX on STAT3 (Fig. 7A and 7D). These ndings suggested that QHZG might exhibit splenic protective activities by regulating the transcription and translation of STAT3.
The activation and expression of cytokines depend on the signal transduction including MAPK [27,28]. In addition, MAPK signaling is also one of the major regulators of the transcription and translation of STAT3 [29]. Based on these ndings, we speculated that QHZG might improve the immunologic function of CTX treated mice through MAPK signaling pathway. To clarify whether QHZG abated cyclophosphamidetriggered immunosuppression via MAPK signaling pathways, we analyzed the alteration of related protein levels of MAPKs pathway. As shown in Fig. 8A-8C, cyclophosphamide treatment decreased the mRNA expression of JNK, ERK and P38 and QHZG could partly reverse the mRNA changes induced by cyclophosphamide (Fig. 8A-8C). Meanwhile, the trend of the phosphorylation of JNK, ERK and P38 in spleen tissue after cyclophosphamide treatment was prominently consistent with the alteration of their total proteins (Fig. 8D).
Additionally, SRC has been shown to mediate the MAPK activation and regulate the STAT3 transcriptional activity through in direct or indirect way, which in turn mediated the production of in ammatory cytokines [30,31]. Compared to the control group, substantially lower levels of the mRNA levels and the protein phosphorylation of SRC in CTX treated group, but not reduced that much in the QHZG co-treatment group. JAK2 is another important mediator of STAT3 and cytokine signal transduction pathway [32]. Therefore, we further investigated the effects of QHZG on the phosphorylation and protein levels of JAK2 in CTX treated mice. Similar to the effects of QHZG on STAT3, CTX treatment signi cantly decreased the mRNA expression and the protein phosphorylation of JAK2 in spleen tissue (Fig. 8A-8D). However, these effects caused by cyclocoagulamide could be signi cantly reversed by pretreatment with QHZG (Fig. 8D). In a conclusion, QHZG might ameliorate immunosuppression caused by CTX treatment via JAK2-SRC/MAPK/STAT3 signaling pathway.

Discussion
CTX is a alkylating agent, which is widely used in the treatment of tumor and immune diseases, due to its good medicinal effect and low price. However, it is also highly cytotoxic and causes a variety of side effects, in particular, reducing the body's normal cellular and humoral immune responses and then leading to immune-de ciency [33,34]. Spleen is the body's most important immune organ, where the immune cells developed and maturated. CTX treatment will lead to atrophy of the spleen and a decline in the spleen index. Therefore, it is of great signi cance to develop a safe and effective immunomodulator to reduce the immunosuppression induced by CTX therapy. Qihuzha granule (QHZG) is rich in trace elements such as zinc, iron, selenium and numerious bioactive components and mainly composed of 11 kinds of traditional edible and medicinal plants. Because of its effectiveness and safety, it is considered to be one of the best-selling drugs for children in China. Modern pharmacological studies have elucidated that many components of QHZG prescription could signi cantly participate in immunostimulant activities [35]. Accumulating evidence displayed major active compounds from the main medicinal materials of QHZG, such as liquiritigenin, isoliquiritigenin and avonoids isolated from Glycyrrhiza uralensis, and astragalosides from Astragalus, have great immunoregulatory activity [8]. Besides, compounds such as Atractyloside II and Atractyloside III in Atractylodes macrocephala, pachymic acid in Poria cocos and Luteolin in Codonopsis pilosula were also found in QHZG and all of these compounds have immune-enhancing effects [13]. Collectively, QHZG might be developed as a promising immunomodulator against CTX-induced immunosuppression. However, the potential molecular mechanisms of QHZG against CTX induced immunosuppressive injury were still unclear.
In the present study, a CTX-induced immunosuppressive mice model was established to investigated the protective effects of QHZG on CTX-induced immunosuppression [36]. The spleen index and immune function of CTX treated mice decreased, compared with that in the untreated mice (Fig. 1). Given the spleen was one of the most important immune organs, the spleen index is often used to evaluate immunity of the tested mice. As shown in Fig. 1, lower thymus index and spleen index were observed in the CTX treated group, whereas QHZG (100 mg/kg) signi cantly raised the thymus index and spleen index of CTX treated mice, suggesting their partial recovery of immune function.
In the immune response, activated splenic lymphocytes produce cytokines including IL-2/4/6 and other in ammatory mediators. IL-2 is an important immune factor secreted by helper T lymphocytes, which has a variety of immune enhancing effects. Among proin ammatory cytokines, IL-6 is one of the most important immune and in ammatory mediators, regulating a variety of cell functions, including the proliferation and differentiation of B cells and T cells [14,15]. IFN-γ is one of the main immune regulatory molecules in inducing immune response to exogenous infectious agents. Compared to the CTX treated group, QHZG signi cantly increased the mRNA levels of IL-2/4/6 and IFN-γ. The data showed that QHZG could signi cantly improve the splenocyte function of the spleen in CTX treated mice.
Macrophages and neutrophils play an important role in resisting microbial infection and maintaining tissue homeostasis, and are activated or inhibited immediately during disease [37]. Consistent with pervious ndings, the monocytes and neutrophils in spleen were lower in immunocompromised mice in the CTX treated group. However, QHZG could signi cantly increase the spleen macrophages and neutrophils, suggesting QHZG could reverse the immunosuppression induced by cyclophosphamide and improve the immune function by promoting the production of macrophages and neutrophils.
PI3K/Akt signaling pathway, one of crucial downstream events of IL-6 signaling, is a central pathway downstream of many cell surface receptors including T cell receptor (TCR), which was involved in autoimmunity [38]. Our results showed that the protein expression of P-Akt and P-mTOR in the model group decreased without affecting the total protein, while QHZG treatment signi cantly increased the protein expression of P-Akt and P-mTOR. These results suggested that PI3K/Akt/mTOR pathway played an important role in QHZG's improvement of immunosuppressive diseases caused by cyclophosphamide.
Extracellular stimulation, such as pathogen infection or chemokines, can rapidly activate MAPKs signaling pathway, and then participate in the synthesis and release of in ammatory factors, and ultimately regulate the immune response [29]. Western blot and qPCR analysis showed cyclophosphamide decreased the phosphorylation of P38, ERK and JNK, and these changes caused by CTX in related protein levels of MAPKs were reversed by QHZG pretreatment. Src is widely believed to have multiple functions in macrophage mediated innate immunity, such as phagocytosis and release of in ammatory cytokines. In addition, Src is also a regulator of MAPK and STAT3 mediated signal transduction, which can activate and stimulate macrophages to produce in ammatory cytokines [30]. Moreover, JAK2 is important for cytokine receptor signal transduction. Once activated, JAK2 kinase phosphorylates STAT3 [30,31,32]. In addition, it has been reported that IL-6 signal interacted with many regulatory factors, such as JAK2/STAT3 [38]. QHZG signi cantly restored the mRNA and phosphorylated protein levels of SRC, JAK2 and STAT3 inhibited by cyclophosphamide, which suggested that QHZG might restore the immune system function in CTX induced immunocompromised mice by regulating JAK2-SRC/MAPK/STAT3 crosstalk signaling pathway (Fig. 9). These results were of great signi cance to understand the molecular mechanism of QHZG in CTX induced immunosuppression. In general, these ndings might provide a basis for the use of QHZG as an effective immunoenhancement therapy or as an alternative strategy to reduce chemotherapy-induced immunosuppression.

Conclusions
In conclusion, QHZG could reverse CTX induced immunosuppression in mice for the rst time. Although the detailed immune enhancement mechanisms of QHZG still needed further exploration, our ndings suggested that QHZG might enhance the innate immune function of mice via IL-6 and its downstream signaling pathways including PI3K/Akt/mTOR signal pathways and JAK2-SRC/MAPK/STAT3 axes. These results suggested that QHZG might serve as a new drug for the treatment of the immunosuppression caused by cyclophosphamide therapy.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The experimental procedures were approved by animal protection and use Committee of Guizhou Medical University.

Consent for publication
Not applicable

Competing interest
The authors declared no conflicts of interest.   Effects of QHZ on neutrophils in CTX-treated mice. Mouse spleens were harvested and xed in 4% buffered formalin. Sections were stained with anti-Ly-6G and DAP in spleen tissues. Nuclei were stained with DAPI (blue). Scale bar, 200 μm.   Proposed working model for QHZG in CTX-induced immunosuppression injury via JAK2/SRC/MAPK/STAT3 and PI3k/Akt/mTOR signaling pathway. QHZG signi cantly alleviated CTXinduced immunosuppression injury and showed protective effects against immunosuppression in mice by JAK2/SRC/MAPK/STAT3 and PI3k/Akt/mTOR signaling pathway.