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-deficiency [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 significance 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 significantly participate in immunostimulant activities . Accumulating evidence displayed major active compounds from the main medicinal materials of QHZG, such as liquiritigenin, isoliquiritigenin and flavonoids isolated from Glycyrrhiza uralensis, and astragalosides from Astragalus, have great immunoregulatory activity . 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 . 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 . 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) significantly 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 inflammatory mediators. IL-2 is an important immune factor secreted by helper T lymphocytes, which has a variety of immune enhancing effects. Among proinflammatory cytokines, IL-6 is one of the most important immune and inflammatory 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 significantly increased the mRNA levels of IL-2/4/6 and IFN-γ. The data showed that QHZG could significantly 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 . Consistent with pervious findings, the monocytes and neutrophils in spleen were lower in immunocompromised mice in the CTX treated group. However, QHZG could significantly 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 . 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 significantly 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 inflammatory factors, and ultimately regulate the immune response . 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 inflammatory cytokines. In addition, Src is also a regulator of MAPK and STAT3 mediated signal transduction, which can activate and stimulate macrophages to produce inflammatory cytokines . 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 . QHZG significantly 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 significance to understand the molecular mechanism of QHZG in CTX induced immunosuppression. In general, these findings might provide a basis for the use of QHZG as an effective immunoenhancement therapy or as an alternative strategy to reduce chemotherapy-induced immunosuppression.