重组灵芝免疫调节蛋白对环磷酰胺致小鼠白细胞减少症模型治疗及其机制研究
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摘要
本文探讨了重组灵芝免疫调节蛋白（Recombinant Ganoderma LucidumImmunoregulatory Protein，rLZ-8）对环磷酰胺致小鼠白细胞减少症模型治疗及其机制。通过给予不同剂量的环磷酰胺，分别建立单阶段和多阶段环磷酰胺致小鼠白细胞减少症的模型。每日两次腹腔注射rLZ-8治疗白细胞减少症，动物血液分析仪对中性粒细胞、单核细胞、淋巴细胞数量进行分析，发现rLZ-8可以显著治疗由环磷酰胺引起的小鼠白细胞减少症，特别是可以增加体内中性粒细胞、淋巴细胞、单核细胞数量。应用WST-1方法检测rLZ-8对小鼠脾脏细胞的作用，结果显示rLZ-8能够增殖小鼠脾脏细胞的刺激，具有潜在的提高机体免疫系统能力和器官造血功能。利用流式细胞仪检测rLZ-8对淋巴细胞分类的作用，包括B细胞、 CD4~+CD8~+T细胞，结果显示，rLZ-8可以维持CD4~+T和CD8~+T细胞之间的平衡，这对改善环磷酰胺引起的免疫功能损害和免疫系统失衡将起到至关重要的作用。应用酶联免疫吸附法进行小鼠血清IL-3、IL-4细胞因子的测定，发现rLZ-8可以促进IL-3、IL-4在体内的分泌水平。提取小鼠脾脏细胞RNA，通过RNA标记和阵列杂交，获得基因芯片结果。结合统计学分析、聚类分析、GO分析和Pathway分析，得到rLZ-8在治疗白细胞减少症过程中起到关键作用的基因：Akt2、Txk、Plcb2、Nfkb1、Ppp2c、Map2k1。
本实验的创新之处在于：成功建立了两种小鼠白细胞减少症模型，首次证明了rLZ-8可以同时增加各类白细胞数量，具有治疗白细胞减少症的药效学功能。rLZ-8促进免疫细胞增殖作用显著，提升了环磷酰胺诱导的白细胞减少症小鼠的机体免疫能力，对改善环磷酰胺引起的免疫功能损害和免疫系统失衡起到至关重要的作用。在国际上第一次实现了真菌免疫调节蛋白走向临床用药研发的新途径。揭示了rLZ-8治疗白细胞减少症相关的信号转导通路及其分子，为rLZ-8开发为临床用药提供了理论依据。
Leukopenia is characterized by a decrease in the number of circulating whiteblood cells (WBCs), down to a level of approximately4.0×109/L. Leukopenia canbe caused either by primary factors or secondarily, for example, followingradiotherapy or chemotherapy as part of cancer treatment regimens. In fact,approximately90%of cancer patients receiving radiotherapy or chemotherapyexperience symptoms of leukopenia, including bone marrow suppression, frequentfever, and mucosal necrotic ulcers. These symptoms can be life-threatening in casesof severe infection and sepsis. Cyclophosphamide (CY) is one of the mostcommonly used chemotherapy drugs and functions by alkylating DNA to formcross-links, thereby inhibiting tumor cell growth. However, in addition to blockingproliferation of tumor cells, CY also inhibits the growth of normal cells. As a result,CY treatment is associated with a number of side effects, including a decrease in thenumber of circulating white blood cells. Thus, effective treatments for CY-inducedleukopenia are particularly important. Several drugs are capable of increasing whiteblood cell count. These include traditional medicines such as vitamin B6, leucogen,and batyl alcohol, as well as some more recently characterized compounds such ascytokines, including granulocyte colony stimulating factor (G-CSF), granulocytemacrophage colony stimulating factor (GM-CSF), and interleukin-11(IL-11). Whilethese drugs increase leukocyte number, unfortunately, they do not alter the overall immune response. Therefore, finding a compound that both increases leukocytenumber and raises the overall immune response needs to be a priority.
For centuries in Asia, the fungus Ganoderma Lucidum has been used toenhance the body’s adaptive capabilities and promote good health. The GanodermaLucidum immunoregulatory protein (LZ-8), isolated from mycelial extracts, belongsto the fungal immunomodulatory protein (Fip) family. We cloned and expressedrecombinant LZ-8(rLZ-8) in Pichia pastoris and analysed its crystal structure. LZ-8has been reported to be effective in modulating immune function and inhibitingtumor growth. It also induces activation and maturation of human dendritic cells viathe NF-κB and MAPK pathways. Finally, co-treatment of LZ-8and a DNA vaccineagainst MBT-2tumors in mice was significantly more effective than the DNAvaccine alone. Taken together, these studies support an immunomodulatory role forrLZ-8, and, based on these findings, we examined the possibility that rLZ-8could bedeveloped as a drug for the treatment of immune diseases. Given that bone marrowsuppression and subsequent immuno-deficiency are two common side effects ofleukopenia, we examined the effect of rLZ-8as a potential therapy for this disease.
The aim of the study is to investigate the effect and mechanism of recombinantGanoderma Lucidum immunoregulatory protein (rLZ-8) on mouse models ofcyclophosphamide-induced leukopenia, which we have established with bothsingle-phase and multi-phase administration methods. The proliferation of micespleen cells was detected by the WST-1. Mice in rLZ-8group received twice dailyadministration of rLZ-8at160μg/kg. The numbers of WBCs, including neutrophils,lymphocytes, monocytes, eosinophils, and basophils were quantified using anautomated hematology analyzer. We used flow cytometry to determine theproportion of B cells and CD3+T cells, and analyzed secretion of IL-3and IL-4in vivo by commercially available sandwich enzyme-linked immunosorbent assay(ELISA) kits. Total RNA from each sample was extracted and quantified using theNanoDrop1000, RNA integrity was assessed using standard denaturing agarose gelelectrophoresis. About5μg total RNA of each sample was used for labeling andarray hybridization. Differentially expressed genes were identified through VolcanoPlot filtering. Pathway analysis and GO Analysis were performed to reveal thebiological functions of this subset of differentially expressed genes. The resultsshowed that, treatment with rLZ-8had a strong effect on both models ofcyclophosphamide-induced leukopenia. In particular, it increased the number ofneutrophils, lymphocytes, and monocytes. In this study, we find that rLZ-8significantly enhances leukocyte number in a mouse model of CY-inducedleukopenia. By the first day after rLZ-8administration, leukocyte number hadalready started to increase, and by day4, the number of WBCs had alreadyapproached normal levels. Although rLZ-8increased the number of WBCs, itdid not increase the number of WBCs above that of the control. Here, weexamine several classes of WBCs, including neutrophils, lymphocytes,monocytes, eosinophils, and basophils. Neutrophils play a critical role in theblood’s innate immunity. They protect the body against microbial pathogens,particularly as the first line of defense against pyogenic bacteria. Treatmentwith rLZ-8increases the number of neutrophils in the blood, and, subsequently,the body's chemotaxis, phagocytosis, and bactericidal ability. rLZ-8also helpsmaintain a normal level of lymphocytes and monocytes, which helps the body inboth adaptive immunity (in the case of lymphocytes) and in innate immunity (in thecase of monocytes). rLZ-8triggered the proliferation of mouse spleen cells,prompted the increase of mouse spleen volume and the number of spleen cells. rLZ-8treatment also increased the percentage of CD4+T cells and the levels ofsecreted IL-3and IL-4, which contributed to the cyclophosphamide-inducedimmune dysfunction and immune system imbalance. In conclusion, rLZ-8treatmentbenefitted mice with cyclophosphamide-induced leukopenia by improving overallimmune function and by specifically increasing the number of white blood cells.The cytokine IL-4is secreted by T cells and has been shown to enhance theantigen-presenting ability of antigen-presenting cells. IL-3stimulates thedifferentiation of multipotent hematopoietic stem cells into lymphoidprogenitor cells. Additionally, IL-3stimulates proliferation of all cells in themyeloid lineage. rLZ-8promotes proliferation of neutrophils, monocytes, andlymphocytes by increasing the level of IL-3secretion in vivo. The gene chipresults suggested that the co-expression area was stimulated by rLZ-8, which wasthe strongest groups in a dynamic process. This may be genes that play a key role inthe course of drug treatment, such as: Akt2、Txk、Plcb2、Nfkb1、Ppp2c、Map2k1.
In conclusion, we investigate the effect and mechanism of recombinantGanoderma Lucidum immunoregulatory protein (rLZ-8) on mouse models ofcyclophosphamide-induced leukopenia, and concluded that rLZ-8may be apromising remedy for leukopenia after anticancer therapy.
本实验的创新之处在于：成功建立了两种小鼠白细胞减少症模型，首次证明了rLZ-8可以同时增加各类白细胞数量，具有治疗白细胞减少症的药效学功能。rLZ-8促进免疫细胞增殖作用显著，提升了环磷酰胺诱导的白细胞减少症小鼠的机体免疫能力，对改善环磷酰胺引起的免疫功能损害和免疫系统失衡起到至关重要的作用。在国际上第一次实现了真菌免疫调节蛋白走向临床用药研发的新途径。揭示了rLZ-8治疗白细胞减少症相关的信号转导通路及其分子，为rLZ-8开发为临床用药提供了理论依据。
Leukopenia is characterized by a decrease in the number of circulating whiteblood cells (WBCs), down to a level of approximately4.0×109/L. Leukopenia canbe caused either by primary factors or secondarily, for example, followingradiotherapy or chemotherapy as part of cancer treatment regimens. In fact,approximately90%of cancer patients receiving radiotherapy or chemotherapyexperience symptoms of leukopenia, including bone marrow suppression, frequentfever, and mucosal necrotic ulcers. These symptoms can be life-threatening in casesof severe infection and sepsis. Cyclophosphamide (CY) is one of the mostcommonly used chemotherapy drugs and functions by alkylating DNA to formcross-links, thereby inhibiting tumor cell growth. However, in addition to blockingproliferation of tumor cells, CY also inhibits the growth of normal cells. As a result,CY treatment is associated with a number of side effects, including a decrease in thenumber of circulating white blood cells. Thus, effective treatments for CY-inducedleukopenia are particularly important. Several drugs are capable of increasing whiteblood cell count. These include traditional medicines such as vitamin B6, leucogen,and batyl alcohol, as well as some more recently characterized compounds such ascytokines, including granulocyte colony stimulating factor (G-CSF), granulocytemacrophage colony stimulating factor (GM-CSF), and interleukin-11(IL-11). Whilethese drugs increase leukocyte number, unfortunately, they do not alter the overall immune response. Therefore, finding a compound that both increases leukocytenumber and raises the overall immune response needs to be a priority.
For centuries in Asia, the fungus Ganoderma Lucidum has been used toenhance the body’s adaptive capabilities and promote good health. The GanodermaLucidum immunoregulatory protein (LZ-8), isolated from mycelial extracts, belongsto the fungal immunomodulatory protein (Fip) family. We cloned and expressedrecombinant LZ-8(rLZ-8) in Pichia pastoris and analysed its crystal structure. LZ-8has been reported to be effective in modulating immune function and inhibitingtumor growth. It also induces activation and maturation of human dendritic cells viathe NF-κB and MAPK pathways. Finally, co-treatment of LZ-8and a DNA vaccineagainst MBT-2tumors in mice was significantly more effective than the DNAvaccine alone. Taken together, these studies support an immunomodulatory role forrLZ-8, and, based on these findings, we examined the possibility that rLZ-8could bedeveloped as a drug for the treatment of immune diseases. Given that bone marrowsuppression and subsequent immuno-deficiency are two common side effects ofleukopenia, we examined the effect of rLZ-8as a potential therapy for this disease.
The aim of the study is to investigate the effect and mechanism of recombinantGanoderma Lucidum immunoregulatory protein (rLZ-8) on mouse models ofcyclophosphamide-induced leukopenia, which we have established with bothsingle-phase and multi-phase administration methods. The proliferation of micespleen cells was detected by the WST-1. Mice in rLZ-8group received twice dailyadministration of rLZ-8at160μg/kg. The numbers of WBCs, including neutrophils,lymphocytes, monocytes, eosinophils, and basophils were quantified using anautomated hematology analyzer. We used flow cytometry to determine theproportion of B cells and CD3+T cells, and analyzed secretion of IL-3and IL-4in vivo by commercially available sandwich enzyme-linked immunosorbent assay(ELISA) kits. Total RNA from each sample was extracted and quantified using theNanoDrop1000, RNA integrity was assessed using standard denaturing agarose gelelectrophoresis. About5μg total RNA of each sample was used for labeling andarray hybridization. Differentially expressed genes were identified through VolcanoPlot filtering. Pathway analysis and GO Analysis were performed to reveal thebiological functions of this subset of differentially expressed genes. The resultsshowed that, treatment with rLZ-8had a strong effect on both models ofcyclophosphamide-induced leukopenia. In particular, it increased the number ofneutrophils, lymphocytes, and monocytes. In this study, we find that rLZ-8significantly enhances leukocyte number in a mouse model of CY-inducedleukopenia. By the first day after rLZ-8administration, leukocyte number hadalready started to increase, and by day4, the number of WBCs had alreadyapproached normal levels. Although rLZ-8increased the number of WBCs, itdid not increase the number of WBCs above that of the control. Here, weexamine several classes of WBCs, including neutrophils, lymphocytes,monocytes, eosinophils, and basophils. Neutrophils play a critical role in theblood’s innate immunity. They protect the body against microbial pathogens,particularly as the first line of defense against pyogenic bacteria. Treatmentwith rLZ-8increases the number of neutrophils in the blood, and, subsequently,the body's chemotaxis, phagocytosis, and bactericidal ability. rLZ-8also helpsmaintain a normal level of lymphocytes and monocytes, which helps the body inboth adaptive immunity (in the case of lymphocytes) and in innate immunity (in thecase of monocytes). rLZ-8triggered the proliferation of mouse spleen cells,prompted the increase of mouse spleen volume and the number of spleen cells. rLZ-8treatment also increased the percentage of CD4+T cells and the levels ofsecreted IL-3and IL-4, which contributed to the cyclophosphamide-inducedimmune dysfunction and immune system imbalance. In conclusion, rLZ-8treatmentbenefitted mice with cyclophosphamide-induced leukopenia by improving overallimmune function and by specifically increasing the number of white blood cells.The cytokine IL-4is secreted by T cells and has been shown to enhance theantigen-presenting ability of antigen-presenting cells. IL-3stimulates thedifferentiation of multipotent hematopoietic stem cells into lymphoidprogenitor cells. Additionally, IL-3stimulates proliferation of all cells in themyeloid lineage. rLZ-8promotes proliferation of neutrophils, monocytes, andlymphocytes by increasing the level of IL-3secretion in vivo. The gene chipresults suggested that the co-expression area was stimulated by rLZ-8, which wasthe strongest groups in a dynamic process. This may be genes that play a key role inthe course of drug treatment, such as: Akt2、Txk、Plcb2、Nfkb1、Ppp2c、Map2k1.
In conclusion, we investigate the effect and mechanism of recombinantGanoderma Lucidum immunoregulatory protein (rLZ-8) on mouse models ofcyclophosphamide-induced leukopenia, and concluded that rLZ-8may be apromising remedy for leukopenia after anticancer therapy.
引文
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