Studies On The Improvement On Hematopoietic Function And Anti-Colorectal Cancer Effects Of Zhenqi Fuzheng Formula Via Its Immune Regulation

Zhenqi Fuzheng formula(ZQFZ)is a compound prescription based on the theory of Chinese traditional medicine,which combines the experience of pharmacological research and clinical practice in modern medicine with supporting the healthy and dispelling pathogenic factors.ZQFZ is composed of Radix Astragali and Fructus Ligustri Lucidi.ZQFZ has been used in China for thousands of years and played an important role in adjuvant therapy,In the application of Radix Astragali tonifying qi and Fructus Ligustri Lucidi Nourishing yin and tonifying kidney,it enhances immune system function and protects the bone marrow and adrenal cortex function.Clinically,it is often used to treat deficiency syndrome caused by various diseases.It can be used together with radiotherapy,chemotherapy and surgical treatment to promote the recovery of normal function of the body,so as to correct the syndrome of qi deficiency in patients,with potential anti-tumor activity.At present,the national and international research on ZQFZ focused on the analysis of chemical composition,the efficacy of immunoregulation and the clinical research of synergistic anti-tumor activity.However,studies on ZQFZ's participation in immunoregulation to enhance hematopoietic activity and antitumor activity have not been reported.ZQFZ as the research object,this paper explored its representative components,and preliminarily explored its effect on healthy mice.In combination with in vitro cell experiments and in vivo animal models to explore the immunoregulation,hematopoietic promotion and anti-tumor effect of ZQFZ.The main molecular mechanism of ZQFZ on the immune and hematopoietic system function repair of chemotherapy-damaged mice and the anti-tumor effect of ZQFZ on the immunoregulation of colorectal cancer mice was preliminarily elucidated.The following conclusions were obtained:ZQFZ contained salidroside 3.948 mg/g,Calycosin-7-glucoside 0.145 mg/g,ligustroflavone 0.179 mg/g,ononin 0.032 mg/g,quercetin 0.071 mg/g and formononetin 0.027 mg/g.At first,this paper investigated the effect of ZQFZ on healthy mice.After 4 weeks of intragastric administration of ZQFZ,the body weight,viscera(thymus,spleen,liver and kidney)index,indicators of animal behavior and levels of oxidative stress-related factors were tested to explain the effect of ZQFZ on healthy mice.The results showed that compared with the blank control group ZQFZ had no significant effect on the body weight,organ index,tissue section results and independent activity test of healthy mice.ZQFZ could improve the time of endurance running,bar-turning and exhausting swimming in mice.It can significantly reduce the content of reactive oxygen species(ROS)in serum and liver of healthy mice.It had an antioxidant and anti-fatigue activities.The results showed that ZQFZ had antioxidant and anti-fatigue activities,and no obvious toxic and side effects on healthy mice,suggesting that ZQFZ might have immunomodulatory effects.In this paper,we investigated the effect of ZQFZ on the immunomodulatory ability of immunosuppression mice induced by cyclophosphamide(CTX).After 4 weeks of gavage treatment with ZQFZ,the molecular mechanism by which ZQFZ regulated the immune function of immunosuppressive mice was revealed by detecting the body weight,organ index(spleen and thymus),NK cell killing ability and the expression of immunoregulatory cytokines in the mice.The results showed that after 4 weeks of ZQFZ treatment,the body weight,spleen and liver index of the immunosuppressive mice all recovered significantly compared with the model group.And the killing activity of the NK cells in the mice was significantly enhanced compared with the model group.In addition,ZQFZ promoted the synthesis of immunoglobulin G(Ig G),Ig A,Ig M,interleukin-2(IL-2),IL-6,IL-10,and interferon(IFN)-? and inhibited the expression of IL1-? in the serum of immunosuppressed mice.Pathological examination confirmed that ZQFZ alleviated the phenomenon of multinucleated giant cells in spleen of immunosuppressed mice.Suggesting that ZQFZ could effectively alleviate CTX-induced immunosuppression might be related to the lymphocyte regulation of cytokines,It was suggested that ZQFZ might regulate hematopoietic function and anti-tumor activity.To explore the regulation of ZQFZ on hematopoietic function,we first conducted in vitro cell experiments.XTT and flow cytometry were used to investigate the effect of ZQFZ on the cell viability and apoptosis of two hematopoietic cells(K562 and CHRF).The effect of ZQFZ on the differentiation ability of K562 cells was investigated by benzidine staining.The experimental results showed that ZQFZ could promote the cellular activity of K562 and CHRF cells and had no significant effect on the apoptosis level of the two types of cells.It was suggested that ZQFZ had no obvious cytotoxicity.ZQFZ enhanced the ability of K562 cells to differentiate into erythrocytes.Moreover,ZQFZ could up-regulate the expression of p90 ribosomal S6 kinase(p-rsk1p90),ETS transcription factor(ELK1)and c-Myc phosphorylated by proteins related to hematopoietic cell proliferation and differentiation in K562 and CHRF cells.These results suggested that the proliferation and differentiation of hematopoietic cells promoted by ZQFZ might be related to the activation of transcription factors.Then we built myelosuppression mice model suppression induced by CTX in vivo.It explored whether ZQFZ could promote the reconstruction of bone marrow hematopoietic function in mice.And it revealed the molecular mechanism of ZQFZ in reconstructing hematopoietic function of bone marrow.Experimental results showed that after 4 weeks after treatment of ZQFZ to myelosuppression mice the body weight and organ index of mice affected by CTX significantly were recovered.The swelling of organs(liver and spleen)was alleviated.Lymphocytes(LY),mean hemoglobin content(MCH),hemoglobin(HGB)and neutrophils(NE)in peripheral blood of mice were significantly increased.And the percentage of mononuclear cells(MO)was decreased.In addition,ZQFZ also restored the normal bone marrow cell density,the basic structure of the spleen,the reduction of multinucleated giant cells,the recovery of the inflammatory infiltration of the liver,significantly promoted the generation of B lymphocytes and improved the proliferation of naive cells in myelosuppression mice.It is suggested that the regulation of ZQFZ on hematopoietic function may be related to its immunomodulatory activity.Combined with antibody microarray,ELISA test results and western-blot results,the results showed that the expression levels of IL-2 and IL-5,granulocyte colony stimulating factor(G-CSF)and macrophage colony stimulating factor(M-CSF)in the serum and spleen of myelosuppression mice were significantly increased after treatment of ZQFZ.Moreover,ZQFZ can inhibit the secretion of IFN-?,TNF-?,monocyte chemokine(MCP)-1,MCP-5 and recombinant human chemokine CCL3(MIP-1?)in serum and spleen of myelosuppression mice,and the expression of ROS in spleen of myelosuppression mice.ZQFZ significantly up-regulated the protein expression abundance of M-CSF,superoxide dismutase(SOD)-1,SOD-2,heme oxygenase 1(HO-1),nuclear transcription factor 2(Nrf2)and phosphor nuclear transcription factor B(P-NF-?B)in the spleen of myelosuppression mice.In the generation of bone marrow mononuclear cells(BMMNCs)verify expression level of related protein.ZQFZ significantly reduced the protein expression abundance of P-p38,enhanced the SOD1,SOD2,HO-1,Nrf2,phosphor extracellular regulated protein kinases(P-ERK),phosphor mammalian target of rapamycin(P-m TOR),phosphor inhibitor of nuclear factor kappa-B kinase alpha + beta(P-IKK?+?)and P-NF-?B protein expression abundance.Moreover,the protein expression abundance of PRSK1p90,ELK1,c-Myc,Nrf2,P-NF-?B and M-CSF were significantly up-regulated by ZQFZ in K562 cells with Nrf2-si RNA.It was proved that ZQFZ to promote hematopoietic function reconstruction in mice damaged by chemotherapy may be related to the pathway of Nrf2/NF-?B signaling pathway to enhance M-CSF level.We established colorectal cancer mice model to investigate the effect of ZQFZ on anti-tumor activity in mice with colorectal cancer.It revealed the mechanism of antitumor activity of ZQFZ.The results showed that after 8 weeks treatment of ZQFZ,the body weight,spleen index and thymus index of colorectal cancer mice enhanced significantly,the ratio of colorectal weight to length reduced significantly,and the amount of tumor in the colorectal reduced.In addition,ZQFZ promoted the improvement of white blood cell(WBC),LY and granulocyte(GR)levels,NK cell proliferation and CD3+CD28+ cells production in the blood of colorectal cancer mice.ZQFZ could repair the damaged spleen,reduce the deformation of the hollow vacuoles in the liver,reduce the inflammatory infiltration of the lung and colorectal,and restore the glandular structure in the colorectal.ZQFZ significantly enhanced levels of IL-2,IL-12,TNF-? and IFN-? in the serum and colorectal of mice with colorectal cancer.It was further confirmed that ZQFZ might inhibit colorectal cancer regulation by immunoregulation effects.In conclusion,the above experimental studies showed that:(1)ZQFZ could promote the anti-fatigue and antioxidant activity of healthy mice;(2)ZQFZ significantly promoted the immune regulation ability of immunosuppression mice.And its mechanism might be related to the lymphocyte regulation of immune factors.(3)ZQFZ significantly improved hematopoietic function in myelosuppression mice.And the mechanism may be related to the increased expression of M-CSF in Nrf2/ NF-?B signal transduction pathway.(4)ZQFZ significantly improved the anti-tumor activity of colorectal cancer mice.And the mechanism might be related to regulation by immunoregulation effects by ZQFZ.The proposal of this topic is helpful to further promote the development and promotion of ZQFZ.It will also provide new ideas for the development of drugs that have anti-tumor effects and can alleviate the immunosuppressive and myelosuppressive effects caused by long-term chemotherapy.