WO2023217179A1 - 甘露糖抑制细胞焦亡或减轻化疗药物毒副作用的应用 - Google Patents

甘露糖抑制细胞焦亡或减轻化疗药物毒副作用的应用 Download PDF

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WO2023217179A1
WO2023217179A1 PCT/CN2023/093225 CN2023093225W WO2023217179A1 WO 2023217179 A1 WO2023217179 A1 WO 2023217179A1 CN 2023093225 W CN2023093225 W CN 2023093225W WO 2023217179 A1 WO2023217179 A1 WO 2023217179A1
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Prior art keywords
mannose
cisplatin
mice
chemotherapy
side effects
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English (en)
French (fr)
Inventor
吴乔
王维嘉
艾元立
陈航姿
刘凡健
洪雪辉
方伟
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Xiamen University
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Xiamen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents

Definitions

  • the invention relates to the field of medical science and technology, and in particular to the application of mannose in inhibiting pyroptosis or reducing the toxic and side effects of chemotherapy drugs.
  • Chemotherapy is a method that uses chemically synthesized drugs to kill tumor cells or inhibit the growth of tumor cells. It is the most common method in the clinical treatment of malignant tumors.
  • the scope of chemotherapy is systemic. While killing tumor cells, it also kills a large number of normal tissue cells, resulting in toxic side effects of chemotherapy.
  • Common toxic side effects during chemotherapy include: hair loss, oral and throat ulcers, nausea, vomiting, diarrhea, constipation, reduced bone marrow hematopoietic function, damage to the nerve and muscle systems, etc., which affect the function of reproductive organs, kidneys, heart and other organs.
  • Mannose is a drug used to treat urinary tract infections ( B, D,ALTARAC SD-mannose powder for prophylaxis of recurrent urinary tract infections in women:a rand omized clinical trial[J].World J Urol,2014,32(1):79-84), can also inhibit tumor growth and enhance chemotherapy
  • the effect of drugs in treating tumors (GONZALEZ P S, O'PREY J, CARDACI S, et al. Mannose impairs tumor growth and enhances chemoth erapy[J]. Nature, 2018, 563(7733):719-723), but about mannose Other functions have been rarely studied.
  • the present invention provides a kind of mannose for use in the preparation of drugs, kits, pharmaceutical compositions or health care products that inhibit pyroptosis or reduce the toxic and side effects of chemotherapy drugs.
  • the mannose can effectively inhibit cell pyroptosis or reduce the toxic and side effects of chemotherapy drugs, which are organ damage, and has unexpected beneficial effects.
  • the purpose of the present invention is to provide a new use of mannose for inhibiting cell pyroptosis or reducing the toxic and side effects of chemotherapy drugs.
  • Mannose is a monosaccharide.
  • Mannose can inhibit normal cell pyroptosis caused by clinical chemotherapy drugs cisplatin or oxaliplatin (including the gold standard of pyroptosis such as detection of cell pyroptosis morphology, GSDME shearing and LDH release).
  • Mouse experiments further show that mannose inhibits cisplatin or oxaliplatin-induced pyroptosis of tissue cells and can significantly alleviate the toxic side effects caused by cisplatin or oxaliplatin (including alleviating the damage to the small intestinal villi and colorectum of mice caused by cisplatin or oxaliplatin). shortening and decreased renal function), thus providing important experimental basis for reducing the toxic and side effects of chemotherapy drugs.
  • the present invention provides the use of mannose in the preparation of drugs, kits, pharmaceutical compositions or health care products that inhibit cell pyroptosis or reduce the toxic and side effects of chemotherapy drugs, which are organ damage.
  • the organ includes the intestine or kidneys.
  • the chemotherapeutic drug is cisplatin, carboplatin, oxaliplatin, cyclophosphamide, ifosfamide, melphalan, busulfan, mechlorethamine, chlorambucil, thiothelide Pi, carmustine, nimustine, lomustine, semustine, pemetrexed, methotrexate, fluorouracil, capecitabine, cytarabine, gemcitabine, 6- Mercaptopurine, thioguanine, daunorubicin, doxorubicin, epirubicin, arubicin, idarubicin, bleomycin, mitomycin, vincristine, vindesine, Vinorelbine, camptothecin, irinotecan, topotecan, paclitaxel, docetaxel, albumin-bound paclitaxel, paclitaxel liposome, etoposide, ten
  • the pharmaceutical composition includes mannose, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the kit includes (i) an agent comprising mannose, and (ii) an agent comprising a chemotherapeutic agent.
  • the present invention provides the use of mannose in the preparation of a medicament for inhibiting pyroptosis induced by chemotherapy drugs in normal intestinal and renal cells.
  • the present invention provides the use of mannose in the preparation of drugs for alleviating intestinal toxic side effects caused by chemotherapy drugs.
  • the present invention provides the use of mannose in the preparation of drugs for alleviating renal toxic side effects caused by chemotherapy drugs.
  • the drug further includes an effective amount of a chemotherapeutic agent.
  • the medicine can be made into the following dosage forms: tablets, capsules, granules, injections, etc.
  • the mannose can be in powder, solution or other forms and can be metabolized to produce mannose, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the mannose can inhibit chemotherapeutic drug-induced enterocyte pyroptosis.
  • the mannose can inhibit chemotherapeutic drug-induced renal cell pyroptosis.
  • the mannose can alleviate the toxic side effects of chemotherapy drugs in the small intestine and colorectum of mice.
  • the mannose can alleviate the toxic side effects of chemotherapy drugs on mouse kidneys.
  • the mannose can reduce the toxic side effects of chemotherapy drugs without affecting the efficacy.
  • the present invention provides a method for inhibiting chemotherapeutic drug-induced pyroptosis of normal intestinal cells and renal cells.
  • a method comprising contacting normal cells with (i) an agent comprising an effective amount of mannose and (ii) an agent comprising an effective amount of a chemotherapeutic drug, the agent may be contacted with the cells simultaneously or sequentially touch.
  • the present invention provides a method for alleviating intestinal toxic side effects caused by chemotherapy drugs.
  • the method includes administering an effective amount of the drug of the present invention to a subject, and the agent can An agent comprising an effective amount of mannose and (ii) a chemotherapeutic agent is administered simultaneously or sequentially to the small intestine and the colorectum.
  • the present invention provides a method for alleviating renal toxic side effects caused by chemotherapy drugs.
  • the method includes administering an effective amount of the drug of the present invention to a subject, and the agent can be (i) an agent containing an effective amount of mannose and (ii) an agent containing an effective amount of a chemotherapeutic agent are administered simultaneously or sequentially.
  • the present invention provides a method for reducing the toxic and side effects of chemotherapy drugs without affecting the efficacy.
  • the method includes administering an effective amount of the drug of the present invention to a subject, and the reagent can is the simultaneous or sequential administration of (i) an agent comprising an effective amount of mannose and (ii) an effective amount of a chemotherapeutic agent.
  • the present invention provides the use of a mannose-containing reagent in the preparation of a drug for inhibiting pyroptosis of normal intestinal and renal cells induced by chemotherapy drugs, and the drug can inhibit pyroptosis of normal intestinal and renal cells. .
  • the present invention provides the use of a reagent containing mannose in the preparation of a drug for alleviating the intestinal toxic side effects caused by chemotherapy drugs.
  • the drug can alleviate the intestinal toxic side effects caused by chemotherapy drugs. .
  • the present invention provides the use of a mannose-containing reagent in the preparation of a drug for alleviating the renal toxic side effects caused by chemotherapy drugs.
  • the drug can alleviate the toxic side effects of renal tissue and its related renal function.
  • Indicators such as blood urea nitrogen (BUN), creatinine and cystatin C.
  • the present invention provides the use of a reagent containing mannose in the preparation of a drug for alleviating the toxic side effects of chemotherapy drugs without affecting the efficacy.
  • the drug can alleviate the toxic side effects caused by chemotherapy drugs and Does not affect efficacy.
  • the mannose can be in powder, solution or other forms and can be metabolized to produce mannose, or a pharmaceutically acceptable salt thereof.
  • mannose has the function of inhibiting normal cell pyroptosis.
  • pyroptosis inhibited by mannose can protect normal tissues and organs and greatly reduce the toxic side effects caused by chemotherapy drugs.
  • Existing drugs that alleviate the toxic and side effects of chemotherapy drugs usually alleviate some symptoms caused by chemotherapy or enhance organ functions. The therapeutic effects are limited and treat the symptoms but not the root cause.
  • mannose can fundamentally inhibit the pyroptosis of intestinal and renal cells, thereby further Fundamentally, the occurrence of toxic side effects is reduced, and the therapeutic effect is better.
  • the use of mannose combined with chemotherapy drugs as auxiliary treatment is especially suitable for patients with low kidney function or intestinal problems. It can be used as a complementary medicine in clinical practice.
  • Figure 1 is a comparison chart of the results of pyroptosis induced by the chemotherapy drug cisplatin in normal intestinal cells (IEC-6 and FHs 74 cells) and renal cells (HK-2 cells) in different treatment groups in Example 1; wherein, Figure a is a morphological observation of pyroptosis of normal intestinal cells (IEC-6 and FHs 74 cells) and kidney cells (HK-2 cells) induced by the chemotherapy drug Cisplatin in different treatment groups in Example 1; Figure b is a protein electrophoresis comparison of GSDME protein shearing after cisplatin induces normal cells in different treatment groups in Example 1; Figure c shows the LDH release after cisplatin induces normal cells in different treatment groups in Example 1 summary graph.
  • Figure a is a morphological observation of pyroptosis of normal intestinal cells (IEC-6 and FHs 74 cells) and kidney cells (HK-2 cells) induced by the chemotherapy drug Cisplatin in different treatment groups in Example 1
  • Figure b
  • Figure 2 is a comparison chart of the intestinal toxic and side effects results of mice treated with the chemotherapy drug cisplatin (Cisplatin) in different treatment groups in Example 2;
  • Figure a is a comparison of the chemotherapy drug Cisplatin in different treatment groups in Example 2 Comparison of the changes in the colorectal length of mice caused by (Cisplatin) treatment;
  • Figure b is a comparison of changes in the villi or crypts of the small intestine of mice caused by treatment with the chemotherapy drug cisplatin (Cisplatin) in different treatment groups in Example 2;
  • Figure c is a comparison of changes in the villi or crypts of the small intestine in mice Protein electrophoresis comparison chart of the shearing of GSDME protein in mouse small intestine after treatment with the chemotherapy drug cisplatin in different treatment groups in Example 2.
  • Figure 3 is a comparison chart of the renal toxicity and side effects of mice caused by the treatment of the chemotherapy drug cisplatin (Cisplatin) in different treatment groups in Example 2;
  • Figure a is a comparison of the chemotherapy drug cisplatin (Cisplatin) in the different treatment groups in Example 2 The results of renal PAS staining of mice treated with Cisplatin;
  • Figure b shows the blood urea nitrogen (BUN), creatinine (creatinine), and Statistical chart of changes in cystatin C levels;
  • Figure c is a protein electrophoresis comparison chart of the shearing of GSDME protein in mouse kidneys after treatment with the chemotherapy drug cisplatin (Cisplatin) in different treatment groups in Example 2.
  • Figure 4 is a comparison chart of the changes in the volume of transplanted tumors in nude mice after treatment with the chemotherapy drug Cisplatin in different treatment groups in Example 3;
  • Figure a shows nude mice after treatment with the chemotherapy drug Cisplatin in different treatment groups Statistical chart of the volume changes of transplanted tumors in the figure;
  • Figure b shows pictures of transplanted tumors in nude mice after treatment with the chemotherapy drug Cisplatin in different treatment groups.
  • Figure 5 is a comparison chart of the intestinal toxic side effects of mice treated with the chemotherapy drug cisplatin in different treatment groups in the transplanted tumor-bearing mouse model in Example 3;
  • Figure a shows the results of different treatments in Example 3 Comparison of changes in colorectal length of mice caused by treatment with the chemotherapy drug Cisplatin in the groups;
  • Figure b shows the results of changes in the villi or crypts of the small intestine of mice caused by treatment with the chemotherapy drug Cisplatin in different treatment groups in Example 3 Figure;
  • Figure c is a protein electrophoresis comparison chart of the shearing of GSDME protein in the mouse small intestine after treatment with the chemotherapy drug cisplatin in different treatment groups in Example 3.
  • Figure 6 is a comparison chart of the toxic and side effects on the kidneys of mice caused by the chemotherapy drug cisplatin (Cisplatin) in different treatment groups in the transplanted tumor-bearing mouse model in Example 3;
  • Figure a shows the results of the different treatment groups in Example 3
  • Figure b shows the blood urea nitrogen (BUN) and blood creatinine (BUN) and blood creatinine ( creatinine) and blood cystatin C level change statistical chart;
  • Figure c is a protein electrophoresis comparison chart of the shearing of GSDME protein in mouse kidneys after treatment with the chemotherapy drug cisplatin (Cisplatin) in different treatment groups in Example 3.
  • Figure 7 is a comparison chart of the volume changes of primary liver cancer after treatment with the chemotherapy drug cisplatin in different treatment groups in Example 4.
  • Figure 8 is a comparison chart of the intestinal toxic side effects of mice treated with the chemotherapy drug cisplatin in different treatment groups in the primary liver cancer mouse model in Example 4;
  • Figure a shows the results of different treatments in Example 4 Comparison of changes in the colorectal length of mice caused by treatment with the chemotherapy drug Cisplatin in the group;
  • Figure b shows a comparison of changes in the villi or crypts of the small intestine of mice caused by treatment with the chemotherapy drug Cisplatin in different treatment groups in Example 4 Figure;
  • Figure c is a protein electrophoresis comparison chart of the shearing of GSDME protein in the mouse small intestine after treatment with the chemotherapy drug cisplatin in different treatment groups in Example 4.
  • Figure 9 is a comparison chart of the toxic and side effects on the kidneys of mice caused by the chemotherapy drug cisplatin (Cisplatin) in different treatment groups in the primary liver cancer mouse model in Example 4;
  • Figure a shows the results of the different treatment groups in Example 4 Comparison of PAS staining of mouse kidneys after treatment with the chemotherapy drug Cisplatin;
  • Figure b shows the blood urea nitrogen (BUN), blood creatinine ( creatinine) and blood cystatin C level changes;
  • Figure c is a protein electrophoresis comparison chart of the shearing conditions of several batches of mouse kidney GSDME proteins treated with the chemotherapy drug cisplatin (Cisplatin) in different treatment groups in Example 4.
  • Figure 10 is a comparison chart of the volume change of primary liver cancer after treatment with the chemotherapy drug oxaliplatin in different treatment groups in Example 5.
  • Figure 11 is a comparison chart of the intestinal toxic and side effects of mice treated with the chemotherapy drug oxaliplatin in different treatment groups in Example 5 in the primary liver cancer mouse model;
  • Figure a shows the results in Example 5 Picture of changes in the colorectal length of mice caused by treatment with the chemotherapy drug Oxaliplatin in different treatment groups;
  • Figure b shows changes in the villi in the small intestine of mice caused by treatment with the chemotherapy drug Oxaliplatin in different treatment groups in Example 5 Or a comparative picture of crypt changes;
  • Figure c is a comparative picture of the protein electrophoresis of the GSDME protein in the mouse small intestine after treatment with the chemotherapy drug oxaliplatin in different treatment groups in Example 5.
  • Figure 12 is a comparative chart of the volume changes of primary gastric cancer after treatment with the chemotherapy drug oxaliplatin in different treatment groups in Example 6.
  • Figure 13 is a comparison chart of the intestinal toxic and side effects of mice treated with the chemotherapy drug oxaliplatin in different treatment groups in the primary gastric cancer mouse model in Example 6;
  • Figure a shows the different results in Example 6 Comparison of changes in the colorectal length of mice caused by treatment with the chemotherapy drug Oxaliplatin in the treatment groups;
  • Figure b shows changes in the villi in the small intestine of mice caused by treatment with the chemotherapy drug Oxaliplatin in different treatment groups in Example 6 Or a comparative picture of crypt changes;
  • Figure c is a comparative picture of the protein electrophoresis of the GSDME protein in the mouse small intestine after treatment with the chemotherapy drug oxaliplatin in different treatment groups in Example 6.
  • Figure 14 is a comparison chart of the pyroptosis of melanoma cells A375 cells under the treatment conditions of cell pyroptosis inducer CCCP/FeSO 4 in Example 7;
  • Figure a is an observation picture of cell morphology in different treatment groups;
  • Figure b is a comparison of cell morphology in different treatment groups Protein electrophoresis comparison chart of GSDME protein shearing in the treatment groups;
  • Figure c is a statistical chart of LDH release from cells in different treatment groups.
  • Figure 15 is a diagram showing the metabolic pattern of mannose (Mannose) in the hexosamine-biosynthesis pathway (HBP) in Example 7 and a comparison diagram of cell pyroptosis after knocking out or inhibiting key genes;
  • Figure a shows Mannose Diagram of the metabolic pattern of sugar after it enters cells;
  • Figure b shows the comparison of CCCP/FeSO 4- induced cell pyroptosis in different groups after knocking out GFAT1 and GFAT2 in A375 cells;
  • Figure c shows the use of GFAT1 to inhibit cell death in A375 cells Comparison of cell pyroptosis induced by CCCP/FeSO 4 in different groups after treatment with DON;
  • Panel a in Figure 16 is a comparative protein electrophoresis diagram showing that mannose activates AMPK in Example 7, causing an increase in the phosphorylation level of AMPK protein and its downstream protein ACC;
  • Panel b in Figure 16 shows the use of AMPK activity inhibitors in A375 cells Comparison of CCCP/FeSO 4- induced cell pyroptosis in different groups after compound C treatment;
  • Figure 16 (c) shows the CCCP/FeSO 4 -induced cell pyroptosis in different groups after AMPK ⁇ 1 and AMPK ⁇ 2 were knocked out in A375 cells. Comparison chart of pyrolysis conditions.
  • Figure 17 is a comparison chart of the effect of changes in metabolite levels in the hexosamine biosynthetic pathway on mannose (Mannose) activating AMPK protein in Example 7;
  • Figure a shows the results in different groups after knocking out GFAT1 and GFAT2 in A375 cells. Comparison of changes in AMPK phosphorylation levels induced by mannose;
  • Figure b shows the changes in the levels of metabolites GlcNAc-6P and GlcNAc-1P in cells under mannose treatment conditions;
  • Figure c shows A375 cells treated with different metabolites , Comparison of changes in AMPK phosphorylation levels in different groups.
  • Figure a in Figure 18 shows the phase detection of the metabolite GlcNAc-6P and protein AMPK ⁇ 1 using isothermal titration calorimetry (ITC) in Example 7.
  • Figure b in Figure 18 is a diagram of the effect of the metabolite GlcNAc-6P on the interaction between protein AMPK ⁇ 1 and protein LKB1 in Example 7.
  • Figure 19 is an interaction diagram between protein AMPK ⁇ 1 and protein GSDME in Example 7;
  • Figure a shows the interaction between protein AMPK ⁇ 1 and protein GSDME detected using co-IP technology in A375 cells;
  • Figure b shows the use of in vitro phosphorylation Analytical experimental technology to detect the effect of AMPK complex on GSDME phosphorylation level of protein electrophoresis;
  • Figure c shows the results of mass spectrometry analysis of protein phosphorylation sites of GSDME.
  • Figure 20 is a comparison chart of cell pyroptosis induced by CCCP/FeSO 4 in different groups in A375 cells in which GSDME protein was knocked out in Example 7, GSDME-Flag protein and phosphorylation mimic protein GSDME T6E- Flag were supplemented respectively. .
  • Figure 21 is a comparison chart of the intestinal toxic side effects of mice treated with the chemotherapy drug cisplatin (Cisplatin) in different treatment groups in the intestinal-specific AMPK knockout mouse model (AMPK-DKO) in Example 8; wherein, Figure a shows the changes in the length of the colorectum of mice caused by treatment with the chemotherapy drug Cisplatin in different treatment groups in Example 8; Figure b shows the changes in the small intestine of mice caused by treatment with the chemotherapy drug Cisplatin in different treatment groups in Example 8 Comparison of villus changes and crypt changes; Figure c is a protein electrophoresis comparison of the shearing of GSDME protein in the mouse small intestine after treatment with the chemotherapeutic drug cisplatin (Cisplatin) in different treatment groups in Example 8.
  • AMPK-DKO intestinal-specific AMPK knockout mouse model
  • Figure 22 is a comparison chart of the intestinal toxic and side effects of mice treated with the chemotherapy drug cisplatin (Cisplatin) in different treatment groups of systemic knock-in GSDME T6E point mutation mice in Example 8;
  • Figure a is Example 8
  • Figure b shows the changes in the colorectal length of mice caused by treatment with the chemotherapy drug Cisplatin in different treatment groups in Example 8;
  • Figure b shows changes in the villi and crypts of the small intestine of mice caused by treatment with the chemotherapy drug Cisplatin in different treatment groups in Example 8 Comparison of changes;
  • Figure c is a protein electrophoresis comparison of the shearing of GSDME protein in the mouse small intestine after treatment with the chemotherapy drug cisplatin in different treatment groups in Example 8.
  • Figure 23 is a comparison chart of the renal toxicity and side effects of mice treated with the chemotherapy drug cisplatin (Cisplatin) in different treatment groups of systemic knock-in GSDME T6E point mutation mice in Example 8;
  • Figure a shows the results in Example 8 Comparison of PAS staining and damage statistics of mouse kidneys after treatment with the chemotherapy drug Cisplatin in different treatment groups;
  • Figure b shows the blood urea in mice caused by treatment with the chemotherapy drug Cisplatin in different treatment groups in Example 8
  • Statistical chart comparing changes in nitrogen (BUN), creatinine (creatinine), and cystatin C levels;
  • Figure c shows the shearing of GSDME protein in the kidneys of several groups of mice treated with the chemotherapy drug cisplatin (Cisplatin) in different treatment groups in Example 8 Protein electrophoresis comparison chart.
  • Figure 24 is a statistical chart of the effect of Mannose on relieving intestinal toxic side effects in chemotherapy patients in Example 9;
  • Figure a is a statistical chart of the number of daily diarrheas in different groups of patients after receiving the XELOX chemotherapy regimen;
  • Figure b is a statistical chart of the number of diarrhea in different groups of patients Statistical chart of white blood cell test results in the feces of different patients.
  • room temperature means ambient temperature, which can be 20°C-30°C; in some embodiments, it is 22°C-28°C; in some embodiments, it is 24°C-26°C; in some embodiments, it is 24°C-26°C; , is 25°C.
  • optional means that the subsequently described event or circumstance may, but need not, occur.
  • optional surfactant means that the surfactant may or may not be present.
  • weight percent or “percent by weight” or “wt%” is defined as the weight of an individual component in a composition divided by the total weight of all components of the composition and multiplied by 100.
  • wt% means mass percentage
  • references to the terms “one embodiment,” “some embodiments,” “an example,” “specific examples,” or “some examples” or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.
  • the reagents used in the present invention can be purchased from the market or can be prepared by the method described in the present invention.
  • the present invention provides a monosaccharide substance mannose, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the mannose inhibits chemotherapeutic drug-induced enterocyte pyroptosis.
  • the mannose inhibits chemotherapeutic drug-induced renal cell pyroptosis.
  • the mannose alleviates the toxic side effects of chemotherapy drugs in the small intestine and colorectum of mice.
  • the mannose alleviates the toxic side effects of chemotherapy drugs on mouse kidneys.
  • the mannose alleviates the toxic side effects of chemotherapy drugs without affecting the efficacy of chemotherapy drugs.
  • the invention provides a kit comprising (i) a reagent comprising mannose, and (ii) a reagent comprising a chemotherapeutic drug.
  • a kit comprising (i) a reagent comprising mannose, and (ii) a reagent comprising a chemotherapeutic drug.
  • the ratio of mannose to chemotherapy drugs can be 20 to 50:1.
  • the present invention provides a method of inhibiting chemotherapeutic drug-induced pyroptosis in normal intestinal cells and renal cells, the method comprising contacting normal cells with (i) an agent comprising an effective amount of mannose and (ii) The cells are contacted with a reagent containing an effective amount of a chemotherapeutic agent, which may be contacted with the cells simultaneously or sequentially.
  • the present invention provides a method for alleviating intestinal toxic side effects caused by chemotherapy drugs.
  • the method includes administering an effective amount of the drug of the present invention to a subject.
  • the reagents can be administered simultaneously or sequentially.
  • An agent comprising (i) an effective amount of mannose and (ii) an effective amount of a chemotherapeutic agent is administered to the small intestine and colorectum.
  • the present invention provides a method for alleviating renal toxic side effects caused by chemotherapy drugs.
  • the method includes administering an effective amount of the drug of the present invention to a subject.
  • the agents can be administered simultaneously or sequentially.
  • an agent comprising an effective amount of a chemotherapeutic agent is administered simultaneously or sequentially.
  • the present invention provides a method for reducing the toxic and side effects of chemotherapy drugs without affecting the efficacy.
  • the method includes administering an effective amount of the drug of the present invention to a subject.
  • the reagents can be administered simultaneously or sequentially.
  • An agent is administered that (i) contains an effective amount of mannose and (ii) contains an effective amount of a chemotherapeutic agent.
  • the present invention provides the use of a mannose-containing reagent in the preparation of a medicament for inhibiting the pyroptosis of normal intestinal and renal cells induced by chemotherapeutic drugs.
  • the medicament can inhibit the pyroptosis of normal intestinal and renal cells induced by chemotherapeutic drugs. Scorched to death.
  • the present invention provides the use of a mannose-containing agent in the preparation of a medicament for alleviating intestinal toxic side effects caused by chemotherapeutic drugs.
  • the medicament can alleviate the small intestinal toxic side effects caused by chemotherapeutic drugs.
  • the present invention provides the use of a mannose-containing reagent in the preparation of a drug for alleviating the renal toxicity and side effects caused by chemotherapy drugs.
  • the drug can alleviate the renal tissue toxicity and side effects caused by chemotherapy drugs and its related renal function indicators. , such as blood urea nitrogen (BUN), creatinine and cystatin C.
  • the present invention provides the use of a mannose-containing reagent in the preparation of a medicament for alleviating the toxic side effects of chemotherapy drugs without affecting the therapeutic efficacy.
  • the medicament can alleviate the toxic side effects caused by chemotherapy drugs without affecting the therapeutic efficacy.
  • the mannose may be in powder, solution or other forms and may be metabolized to produce mannose, or a pharmaceutically acceptable salt thereof.
  • the chemotherapeutic drug may be cisplatin, carboplatin, oxaliplatin, cyclophosphamide, ifosfamide, melphalan, busulfan, mechlorethamine, chlorambucil, Tepa, carmustine, nimustine, lomustine, semustine, pemetrexed, methotrexate, fluorouracil, capecitabine, cytarabine, gemcitabine, 6 -Mercaptopurine, thioguanine, daunorubicin, doxorubicin, epirubicin, arubicin, idarubicin, bleomycin, mitomycin, vincristine, vindesine , vinorelbine, camptothecin, irinotecan, topotecan, paclitaxel, docetaxel, albumin-bound paclitaxel, paclitaxel liposome, etoposide, teni
  • the medicines involved in the present invention can also be formulated into other pharmaceutically acceptable dosage forms for enteral or parenteral administration, such as tablets, capsules, granules, and injections according to methods known in the art. wait.
  • an effective amount refers to the amount that produces the technical effects of the present invention such as inhibiting normal cell pyroptosis or alleviating the toxic and side effects caused by chemotherapy drugs, or achieving the treatment, prevention, alleviation and/or alleviation of the diseases or conditions described in the present invention in a subject.
  • Amount of inventive compound Such amounts generally vary depending on several factors that are within the ability of one of ordinary skill to determine and calculate given the description provided herein. These factors include, but are not limited to: the specific individual and his or her age, weight, height, general medical condition, and medical history, the specific compound used, the vehicle in which the compound is formulated, the route of administration of the compound selected, and the treatment being treated the nature and severity of the illness.
  • composition refers to a composition comprising mannose and, depending on the mode of administration and the nature of the dosage form, at least one pharmaceutically acceptable ingredient selected from the following, including: carriers, diluents, adjuvants, excipients , such as preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants and dispersants.
  • carriers diluents, adjuvants, excipients , such as preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants and dispersants.
  • suspending agents examples include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, or mixtures of these substances.
  • Prevention of microorganisms is ensured by a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It is best to include isotonic agents such as sugar, sodium chloride, etc.
  • Prolonged absorption of injectable dosage forms can be brought about by the use of agents that delay absorption, such as aluminum monostearate and gelatin.
  • suitable carriers, diluents, solvents or excipients include water, ethanol, polyols, suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • excipients include lactose, sodium citrate, calcium carbonate, dicalcium phosphate.
  • disintegrants include starch, alginic acid, and some complex silicates.
  • lubricants include magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol.
  • pharmaceutically acceptable means, within the scope of reasonable medical judgment, suitable for contact with human and lower animal cells without undue toxicity, irritation, allergic reactions, etc., and corresponding to a reasonable benefit/risk ratio .
  • pharmaceutically acceptable dosage form refers to a dosage form of a compound of the invention, including, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations (including suspensions, sprays, inhalation tablets, lozenges , emulsions, solutions, granules, capsules and suppositories) as well as liquid preparations for injection, including liposome preparations.
  • liquid preparations including suspensions, sprays, inhalation tablets, lozenges , emulsions, solutions, granules, capsules and suppositories
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are compatible with the administration of the drug. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference in the field. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% of human serum albumin. Liposomes and non-aqueous carriers, such as immobilized oils, may also be used. The use of such media and reagents for pharmaceutical actives is well known in the art. Remove any conventional media or reagents that are incompatible with the antibody. In addition, its use in compositions is contemplated.
  • the term "remission” generally refers to obtaining a symptomatic and/or pathological alleviating effect.
  • This effect can be the reduction or severity of toxic side effects of tissues and organs during chemotherapy; it can also be the reduction of symptoms, such as vomiting, diarrhea, etc.; it can also be the mental relief of patients during chemotherapy, such as the reduction of pain. , the mood becomes more relaxed and pleasant, etc.
  • salts refers to relatively nontoxic inorganic and organic acid addition salts, base addition salts, and the like of the compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compound. In particular, acid addition salts can be prepared by independently reacting the purified free base form of the compound with a suitable organic or inorganic acid and isolating the salt so formed.
  • Organic acid adduct salts formed from organic acids that form pharmaceutically acceptable anions including but not limited to formates, acetates, propionates, benzoates, maleates, fumarates, Succinate, tartrate, citrate, ascorbate, ⁇ -ketoglutarate, ⁇ -glycerophosphate, alkyl sulfonate or aryl sulfonate; preferably, the alkyl sulfonate It is methylsulfonate or ethylsulfonate; the arylsulfonate is benzenesulfonate or p-toluenesulfonate.
  • Suitable inorganic salts may also be formed including, but not limited to, hydrochlorides, hydrobromides, hydroiodates, nitrates, bicarbonates and carbonates, sulfuric acid salt or phosphate, etc.
  • Exemplary acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, hard Fatty acid salt, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, methanesulfonate, glucoheptonate, lactiobionate, sulfamate, malonate, salicylate, propionate, methylene-bis-b-hydroxynaphthylate acid salt, gentisate, isothiosulfate, di-p-toluoy
  • Base addition salts can also be prepared by independently reacting the purified acid form of the compound with a suitable organic or inorganic base and isolating the salt so formed.
  • Base addition salts include pharmaceutically acceptable metal salts and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium and aluminum salts. Sodium and potassium salts are preferred.
  • Suitable inorganic base addition salts are prepared from metal bases including sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide and zinc hydroxide.
  • Suitable amine base addition salts are prepared from amines that are sufficiently basic to form stable salts and preferably include those commonly used in medicinal chemistry due to their low toxicity and acceptability for pharmaceutical use.
  • examples of the amine include ammonia, ethylenediamine, N-methyl-glucosamine, lysine, arginine, ornithine, choline, N,N′-dibenzylethylenediamine, chlorine Procaine, diethanolamine, procaine, N-benzylphenylethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, di Benzylamine, diphenylhydroxymethylamine, dehydrorosinamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine
  • the experimental method is as follows:
  • cell lysis buffer A 50mM Tris, pH 7.4, 300mM NaCl, 1% NP-40, 1mM PMSF and full enzyme inhibitor Cocktail before use
  • cell lysis buffer A 50mM Tris, pH 7.4, 300mM NaCl, 1% NP-40, 1mM PMSF and full enzyme inhibitor Cocktail before use
  • Protein electrophoresis Take 2 to 10 ⁇ g of protein sample and run it in a discontinuous SDS-PAGE gel at a voltage of 100V. After the sample enters the separation gel, adjust the voltage to 150V.
  • Electrotransfer The electrotransfer solution is pre-cooled at 4°C. After cutting the glue, immerse the methanol-pretreated PVDF membrane and filter paper of the same size in the electrotransfer solution. After the PVDF membrane is attached to the glue, cover both sides with filter paper to eliminate air bubbles. , install in the electroporation tank in the order of the membrane facing the positive electrode, and electrorotate on ice (100V, 60min).
  • Blocking Block with blocking solution at room temperature for 1 hour;
  • Primary antibody reaction After blocking, the membrane is incubated with the corresponding primary antibody at room temperature for 1 to 3 hours;
  • Secondary antibody reaction wash 3 times with TBST, 5 minutes each time, then add the corresponding secondary antibody and incubate at room temperature for 1 to 3 hours.
  • Exposure imaging wash 3 times with TBST, 10 minutes each time. Prepare ECL solution A and solution B, mix them at 1:1 (V/V), and use a developing machine for exposure and imaging.
  • Figure 1 shows the results of mannose inhibiting pyroptosis of normal intestinal cells and renal cells induced by the chemotherapy drug cisplatin.
  • mannose can inhibit the pyroptosis morphology of normal cells induced by cisplatin.
  • Cisplatin significantly induced swelling and cell membrane rupture in rat intestinal crypt epithelial cells (IEC-6), human small intestinal epithelial cells (FHs 74) and human renal tubular epithelial cells (HK-2).
  • IEC-6 rat intestinal crypt epithelial cells
  • FHs 74 human small intestinal epithelial cells
  • HK-2 human renal tubular epithelial cells
  • the experimental method is as follows:
  • SPF grade C57BL/6 mice ( ⁇ 20g) were randomly divided into four groups, namely control group (Ctrl), mannose group (Mannose), cisplatin group (Cisplatin), and cisplatin + mannose.
  • the treatment group (Cisplatin+Mannose) was kept in a 12h light and 12h dark environment.
  • mice in the control group were intragastrically treated with 500 ⁇ L of sterile water every day, and the mice in the mannose group (Mannose) and the cisplatin + mannose co-treatment group (Cisplatin + Mannose) were treated with mannose (Mannose). (in H 2 O), each mouse was administered 500 ⁇ L of 20% mannose once a day for a total of 5 days.
  • mice After the mice were treated with drugs, the mice were killed by cervical dissection, and the colorectum of the mice was taken out for photography and length measurement.
  • Kidney tissue was fixed with 4% paraformaldehyde overnight.
  • PAS staining Periodic Acid-Schiff stain
  • Periodic acid oxidizes the hydroxyl groups on two adjacent carbons of sugars into aldehyde groups, and then uses Schiff's reagent to react with the aldehyde groups to make them appear purple-red.
  • Uromodulin increases dramatically and is stained by PAS. Therefore, PAS staining can reflect the degree of kidney damage.
  • mice After the mice were treated with drugs, blood was collected by eyeball blood collection. After the blood was coagulated at 37°C, the serum was collected by centrifugation at 3000 r/min for 5 min. The serum was sent to the hospital for testing of renal function-related indicators such as blood urea nitrogen (BUN), creatinine (creatinine) and cystatin C. If blood urea nitrogen (BUN), creatinine (creatinine) and cystatin C levels are significantly elevated, it indicates impaired renal function.
  • BUN blood urea nitrogen
  • creatinine creatinine
  • cystatin C cystatin C
  • Graphpad prism 9 statistical software was used to perform statistical analysis on the experimental data.
  • the experimental data obtained are expressed as mean ⁇ standard error; the difference between the control group and the experimental group was analyzed using two-factor analysis of variance and fisher's test, * indicates p ⁇ 0.05, the difference is significant; ** means p ⁇ 0.01, the difference is highly significant; *** means p ⁇ 0.001, the difference is extremely significant; ns means p>0.05, there is no significant difference.
  • Statistical analysis showed that mannose treatment did not affect the colorectal length of mice (p>0.05), and could alleviate the toxic side effects of the chemotherapy drug cisplatin in the colorectum of mice (p ⁇ 0.01).
  • mice in the mannose group (Mannose) had no changes in the villi and crypts of the small intestine.
  • the mice in the cisplatin group (Cisplatin) had a reduced number of small intestinal villi, a shortened length, and small intestinal crypts.
  • the small intestinal villi and small intestinal crypts of mice in the cisplatin + mannose co-treated group (Cisplatin + Mannose) returned to normal significantly.
  • the mannose treatment according to the invention does not affect the villi and crypts of the small intestine of mice, and can alleviate the damage to the villi and crypts of the small intestine of mice caused by the chemotherapy drug cisplatin.
  • Graphpad prism 9 statistical software was used to perform statistical analysis on the experimental data.
  • the experimental data obtained are expressed as mean ⁇ standard error; the difference between the control group and the experimental group was analyzed using two-factor analysis of variance and fisher's test, * indicates p ⁇ 0.05, the difference is significant; ** means p ⁇ 0.01, the difference is highly significant; *** means p ⁇ 0.001, the difference is extremely significant; ns means p>0.05, there is no significant difference.
  • Statistical analysis showed that mannose treatment did not affect mouse kidneys (p>0.05) and could alleviate mouse kidney tissue damage caused by the chemotherapy drug cisplatin (p ⁇ 0.001).
  • the experimental method is as follows:
  • MKN45 cells were inoculated into immunodeficient nude mice at a dose of 2 ⁇ 10 6 cells per animal, and administration began on the 8th day after inoculation.
  • mice SPF grade immunodeficient mice ( ⁇ 20g) were randomly divided into four groups, namely control group (Ctrl), mannose group (Mannose), cisplatin group (Cisplatin), and cisplatin + mannose co-treatment.
  • mice in the control group were intragastrically treated with 500 ⁇ L of sterile water every day, and the mice in the mannose group (Mannose) and the cisplatin + mannose co-treatment group (Cisplatin + Mannose) were treated with mannose (Mannose). (in H 2 O), each mouse was administered 500 ⁇ L of 20% mannose once a day for a total of 7 days.
  • Cisplatin was injected intraperitoneally at a dose of 12 mg/ kg, injected once on the first and fourth days after mannose treatment.
  • mice After the mice began to receive drug treatment, the volume of the tumors was measured every day. After the mice were treated with drugs, the mice were killed by breaking their necks, and the transplanted tumors were taken out, photographed and weighed.
  • Graphpad prism 9 statistical software was used to perform statistical analysis on the experimental data.
  • the experimental data obtained are expressed as mean ⁇ standard error; the difference between the control group and the experimental group was analyzed using two-factor analysis of variance and fisher's test, * indicates p ⁇ 0.05, the difference is significant; ** means p ⁇ 0.01, the difference is highly significant; *** means p ⁇ 0.001, the difference is extremely significant; ns means p>0.05, there is no significant difference.
  • Statistical analysis showed that mannose did not affect the inhibitory effect of the chemotherapy drug cisplatin on the volume of transplanted tumors in nude mice (p>0.05).
  • Graphpad prism 9 statistical software was used to perform statistical analysis on the experimental data.
  • the experimental data obtained are expressed as mean ⁇ standard error; the difference between the control group and the experimental group was analyzed using two-factor analysis of variance and fisher's test, * indicates p ⁇ 0.05, the difference is significant; ** means p ⁇ 0.01, the difference is highly significant; *** means p ⁇ 0.001, the difference is extremely significant; ns means p>0.05, there is no significant difference.
  • Statistical analysis showed that without affecting the inhibitory effect of the chemotherapy drug cisplatin on transplanted tumors, mannose could alleviate the colorectal toxic side effects of the chemotherapy drug cisplatin in mice (p ⁇ 0.05).
  • mannose can alleviate the damage to small intestinal villi and crypts caused by cisplatin without affecting the inhibitory effect of cisplatin on transplanted tumors.
  • mice in the cisplatin group had fewer intestinal villi, shorter lengths, and fewer intestinal crypts.
  • the cisplatin+mannose co-treatment group Cisplatin mice's small intestinal villi and intestinal crypts were significantly restored to normal.
  • the mannose of the invention can alleviate the damage to the villi and crypts of the small intestine of mice caused by the chemotherapy drug cisplatin.
  • the mannose treatment described in the invention can inhibit the shearing of the GSDME protein in the small intestine of mice caused by the chemotherapy drug cisplatin.
  • the experimental method is as follows:
  • mice induced with liver cancer were randomly divided into four groups, namely the control group, mannose group, cisplatin group, and cisplatin + mannose co-treatment group, and were raised in a 12-hour light and 12-hour dark environment.
  • mice in the control group were intragastrically treated with 500 ⁇ L of sterile water every day.
  • the mice in the mannose group and the cisplatin + mannose co-treatment group were intragastrically treated with mannose (Mannose, dissolved in H 2 O) every day.
  • Mannose Mannose, dissolved in H 2 O
  • Each mouse was orally administered 500 ⁇ L of 20% mannose once a day for a total of 4 weeks.
  • mice were treated with drugs, the mice were killed by cervical dissection, the mouse livers were removed, and the number of tumors was recorded.
  • Figure 8 shows the test results of mannose alleviating the intestinal toxic side effects of cisplatin in mice without affecting the inhibitory effect of cisplatin on primary liver cancer.
  • mannose can alleviate the damage to small intestinal villi and crypts caused by cisplatin without affecting the inhibitory effect of cisplatin on primary liver cancer.
  • mice in the cisplatin group had fewer intestinal villi, shorter lengths, and fewer intestinal crypts.
  • the cisplatin+mannose co-treatment group Cisplatin mice's small intestinal villi and intestinal crypts were significantly restored to normal.
  • the mannose of the invention can alleviate the damage to the villi and crypts of the small intestine of mice caused by the chemotherapy drug cisplatin.
  • Graphpad prism 9 statistical software was used to perform statistical analysis on the experimental data. The experimental data were expressed as mean ⁇ standard error; the difference between the control group and the experimental group was analyzed using two-factor analysis of variance and fisher's test. * indicates p ⁇ 0.05, with significant difference. ; ** means p ⁇ 0.01, the difference is highly significant; *** means p ⁇ 0.001, the difference is extremely significant; ns means p>0.05, there is no significant difference.
  • mannose can alleviate the renal tissue damage caused by cisplatin without affecting the inhibitory effect of cisplatin on primary liver cancer.
  • the PAS staining in the kidneys of the cisplatin group (Cisplatin) was significantly stronger, and the kidney injury score (Tubular injury score) also showed significant kidney damage.
  • the kidney tissue of mice in the cisplatin+mannose co-treatment group (Cisplatin+Mannose) significantly returned to normal, and the kidney damage score was also significantly lower. It shows that without affecting the inhibitory effect of the chemotherapy drug cisplatin on primary liver cancer, the mannose of the invention can alleviate the kidney tissue damage in mice caused by the chemotherapy drug cisplatin.
  • Graphpad prism 9 statistical software was used to perform statistical analysis on the experimental data.
  • the experimental data obtained are expressed as mean ⁇ standard error; the difference between the control group and the experimental group was analyzed using two-factor analysis of variance and fisher's test, * indicates p ⁇ 0.05, the difference is significant; ** means p ⁇ 0.01, the difference is highly significant; *** means p ⁇ 0.001, the difference is extremely significant; ns means p>0.05, there is no significant difference.
  • Statistical analysis showed that without affecting the inhibitory effect of the chemotherapy drug cisplatin on primary liver cancer, mannose could alleviate the renal tissue damage in mice caused by the chemotherapy drug cisplatin (p ⁇ 0.001).
  • Mannose affects the chemotherapy drug oxaliplatin in inhibiting the development of primary liver cancer and the toxic side effects caused by oxaliplatin.
  • the experimental method is as follows:
  • mice induced with liver cancer were randomly divided into four groups, namely the control group (Ctrl), the mannose group (Mannose), the oxaliplatin group (Oxaliplatin), and the oxaliplatin + mannose co-treatment group. (Oxaliplatin+Mannose), kept in a 12h light, 12h dark environment.
  • mice in the control group were intragastrically treated with 500 ⁇ L of sterile water every day, and the mice in the mannose group (Mannose) and the oxaliplatin + mannose co-treatment group (Oxaliplatin + Mannose) were given mannose (Mannose). , dissolved in H 2 O), each mouse was administered 500 ⁇ L of 20% mannose once a day for a total of 4 weeks.
  • Oxaliplatin group (Oxaliplatin) and oxaliplatin+mannose co-treatment group (Oxaliplatin+Mannose) mice were treated with oxaliplatin (Oxaliplatin, dissolved in 5% glucose solution). Platinum was injected intraperitoneally at a dose of 10 mg/kg, once a week for a total of 4 weeks.
  • mice were treated with drugs, the mice were killed by cervical dissection, the mouse livers were removed, and the number of tumors was recorded.
  • the experimental data obtained are expressed as mean ⁇ standard error; the difference between the control group and the experimental group was analyzed using two-factor analysis of variance and fisher's test, * indicates p ⁇ 0.05, The difference is significant; ** means p ⁇ 0.01, the difference is highly significant; *** means p ⁇ 0.001, the difference is extremely significant; ns means p>0.05, there is no significant difference.
  • Statistical analysis showed that mannose did not affect the inhibitory effect of the chemotherapy drug oxaliplatin on primary liver cancer (p>0.05).
  • mannose can inhibit the shearing of small intestinal GSDME protein caused by oxaliplatin without affecting the inhibitory effect of oxaliplatin on primary liver cancer.
  • the GSDME in the small intestine of mice in the oxaliplatin group (Oxaliplatin) was significantly sheared, and the oxaliplatin + mannose co-treatment group (Oxaliplatin + Mannose) was compared with the oxaliplatin group (Oxaliplatin).
  • GSDME shearing was significantly reduced in the mouse small intestine.
  • the mannose treatment described in the invention can inhibit the shearing of the GSDME protein in the small intestine of mice caused by the chemotherapy drug oxaliplatin.
  • the experimental method is as follows:
  • mice with primary gastric cancer were randomly divided into four groups, namely the control group (Ctrl), the mannose group (Mannose), the oxaliplatin group (Oxaliplatin), and the oxaliplatin + mannose co-treatment group. (Oxaliplatin+Mannose), kept in a 12h light, 12h dark environment.
  • mice in the control group (Ctrl) were intragastrically treated with 500 ⁇ L of sterile water every day, and mice in the mannose group and oxaliplatin + mannose co-treatment group were intragastrically treated with mannose (dissolved in H 2 O)
  • each mouse was orally administered 500 ⁇ L of 20% mannose once a day for a total of 26 days.
  • mice in the oxaliplatin group and the oxaliplatin + mannose co-treatment group were treated with oxaliplatin (dissolved in 5% glucose solution).
  • Oxaliplatin was injected intraperitoneally and mixed with mannose. Treatment was started one day after treatment, with a dose of 3 mg/kg, once a day, and a total of 15 times of treatment, with 5 days of treatment and 5 days of non-treatment, for a total of 25 days.
  • mice were treated with drugs, the mice were killed by cervical dissection, their stomachs were removed, and the tumor volume was recorded.
  • the gastric tumor volume of mice in the oxaliplatin group was significantly smaller than that of the control group. Compared with the mice in the oxaliplatin group, the gastric tumor volume of the oxaliplatin + mannose co-treatment group was not. Variety. It shows that the mannose of the present invention will not affect the inhibitory effect of the chemotherapy drug oxaliplatin on primary gastric cancer.
  • Graphpad prism 9 statistical software was used to perform statistical analysis on the experimental data.
  • the experimental data obtained are expressed as mean ⁇ standard error; the difference between the control group and the experimental group was analyzed using two-factor analysis of variance and fisher's test. * indicates p ⁇ 0.05. The difference is significant; ** indicates p ⁇ 0.01, the difference is highly significant *** means p ⁇ 0.001, the difference is extremely significant; ns means p>0.05, there is no significant difference.
  • Statistical analysis showed that mannose did not affect the inhibitory effect of the chemotherapy drug oxaliplatin on primary gastric cancer (p>0.05).
  • Figure 13 shows the test results of mannose alleviating the intestinal toxic side effects of oxaliplatin in mice without affecting the inhibitory effect of oxaliplatin on primary gastric cancer.
  • the experimental data obtained are expressed as mean ⁇ standard error; the difference between the control group and the experimental group was analyzed using two-factor analysis of variance and fisher's test, * indicates p ⁇ 0.05, the difference is significant; ** means p ⁇ 0.01, the difference is highly significant; *** means p ⁇ 0.001, the difference is extremely significant; ns means p>0.05, there is no significant difference.
  • Statistical analysis showed that without affecting the inhibitory effect of the chemotherapy drug oxaliplatin on primary gastric cancer, mannose could alleviate the colorectal toxic side effects of the chemotherapy drug oxaliplatin in mice (p ⁇ 0.05).
  • mannose can inhibit the shearing of small intestinal GSDME protein caused by oxaliplatin without affecting the inhibitory effect of oxaliplatin on primary gastric cancer.
  • the GSDME in the small intestine of mice in the oxaliplatin group (Oxaliplatin) was significantly sheared, and the oxaliplatin + mannose co-treatment group (Oxaliplatin + Mannose) was compared with the oxaliplatin group (Oxaliplatin).
  • GSDME shearing was significantly reduced in the mouse small intestine.
  • the mannose treatment described in the invention can inhibit the shearing of the GSDME protein in the small intestine of mice caused by the chemotherapy drug oxaliplatin.
  • the experimental method is as follows:
  • CCCP/FeSO 4 or CCCP/FeSO 4 +Mannose in Figure 14, Figure 15, Figure 16 and Figure 20 means the simultaneous use of CCCP (carbonyl cyanide).
  • Chlorophenylhydrazone (20 ⁇ g/mL, dissolved in DMSO) and FeSO 4 (100 ⁇ g/mL, dissolved in H 2 O) were used to treat cells, in which CCCP/FeSO 4 + Mannose also used Mannose (20 mM, dissolved in H 2 O ) to treat cells;
  • DON in Figure 15 c indicates that cells were treated with DON (6-diazo-5-oxo-L-norleucine) (40 ⁇ M, dissolved in H 2 O);
  • Figure 16 Compound C in the middle figure b indicates that cells were treated with Compound C (dimorphin hydrochloride) (12.5 ⁇ M, dissolved in 1M HCl);
  • GlcN-6P in figure c in Figure 17 and figure b in Figure 18 indicates the use of GlcN-6P (Glucosamine 6-phosphate) (20mM, dissolved in H 2 O) was used to treat cells.
  • GlcNAc-6P in panel c in Figure 17 and panel b in Figure 18 represents the use of GlcNAc-6P (6-phosphate-N-acetyl). Glucosamine) (20mM, dissolved in H 2 O) was used to treat the cells.
  • the GlcNAc-1P in Figure 17 c and Figure 18 b represents the use of GlcNAc-1P (N-acetylglucosamine-1-phosphate diphosphate).
  • UDP-GlcNAc in Figure 17 c and Figure 18 b indicates the use of UDP-GlcNAc (uridine-5'-diphosphate-N- Cells were treated with acetyl-glucosamine sodium salt) (20 mM, dissolved in H 2 O).
  • GlcN-6P, GlcNAc-6P, GlcNAc-1P and UDP-GlcNAc should be pretreated with streptococcal hemolysin O (SLO, 200ng/mL) for 10 minutes in advance before processing cells to increase the permeability of the cell membrane and accelerate the Metabolite uptake.
  • SLO streptococcal hemolysin O
  • the target cells were infected with lentivirus (the cells used in Figure 15 b, Figure 17 a and Figure 20 were all A375), and 10 ⁇ g/ml polypropylene (Sigma) was added. After 24 hours, cells were treated with puromycin (InvivoGen) for 2-3 days. Knockout efficiency was determined by immunoblotting. Puromycin-resistant cell lines with higher knockout efficiency (more than 80%) were maintained for further experiments.
  • the DNA sequence used to construct the sgRNA plasmid is as follows:
  • Control plasmid NTC sgRNA sequence AAATGTCAGGCCGCGCCGTT
  • the GFAT2 sgRNA sequence used in Figure 15, Figure 15 and Figure 17, Figure 1a is TTCAAGAGTGTCCACTACCC.
  • Figure 20 used GSDME sgRNA sequence AGTCTTCATTTGGAACCCTG
  • the "GFAT1/2 sgRNA” described in Figure 15 b and Figure 17 a represents the simultaneous knockout of proteins GFAT1 and GFAT2 in cells; the "AMPK ⁇ 1/ ⁇ 2 sgRNA” described in Figure 16 c represents the simultaneous deletion of proteins GFAT1 and GFAT2 in cells. Knockout proteins AMPK ⁇ 1 and AMPK ⁇ 2.
  • Lentiviral transfection method for protein expression Use calcium phosphate transfection method to transfect the overexpression plasmid containing the target gene (4 ⁇ g, 6cm culture dish) into HEK293T cells to produce lentivirus. After collecting the generated lentivirus, infect the above The corresponding gene knockout cell line constructed in "2. Constructing a gene knockout cell line using CRISPR/Cas9 technology" is used to obtain a complementing cell line after protein knockout.
  • the cell line described in Figure 20 is a cell line infected with the GSDME sgRNA virus and then re-infected with a GSDME point mutation lentivirus carrying a mutation of the sixth threonine to glutamic acid (GSDME T6E ).
  • Flag-tagged AMPK ⁇ 1 FL-AMPK ⁇ 1
  • HA-AMPK ⁇ 1 HA-AMPK ⁇ 1
  • Myc-AMPK ⁇ 1 Myc-AMPK ⁇ 1 proteins
  • HEPES 4-hydroxyethylpiperazinethanesulfonic acid
  • pH 7.4 pH 7.4
  • LC-MS Liquid mass spectrometry
  • the change detection method of GlcNAc-6P and GlcNAc-1P metabolite levels in cells after mannose treatment with different time gradients as described in Figure 17 b after treating cells with mannose for different times, the cells after treatment ( 5 ⁇ 10 6 ) were washed three times with PBS (4°C), and the metabolites in each sample were extracted with 80% methanol solution (1.6 mL, -80°C) to obtain the extract. Collect the extract into a 2 ml tube, vortex for 1 min and centrifuge at 14,000 ⁇ g and 4 °C for 10 min. The supernatant was completely evaporated using a vacuum centrifuge (Labconco Corporation). Resuspend the sample in 200 ⁇ l 50% acetonitrile.
  • Each sample (2 ⁇ l) was injected and analyzed using a QTRAP (SCIEX, QTRAP 6500plus) mass spectrometer connected to a UPLC system (Waters, ACQUITY UPLC System).
  • the bar is Column (5 ⁇ m, 50 ⁇ 2 mm, Phenomenex).
  • mobile phase buffer B is 100% acetonitrile.
  • the column and sample temperatures were 40°C and 10°C, respectively.
  • the QTRAP mass spectrometer was operated in MRM mode in negative mode and analytical criteria were used to optimize the deaggregation potential and collision energy.
  • the binding affinity between AMPK ⁇ 1 and GlcNAc-6P was measured using a MicroCal iTC200 instrument (GE Healthcare) at 25°C. After AMPK ⁇ 1 was dissolved in a solution containing 25mM Tris (pH 8.0), 300mM NaCl, 5mM MgCl2, 1mM EDTA, 10% glycerol, 2mM DTT and 10mM GSH, the protein was dialyzed against PBS. Meanwhile prepare the substrate in PBS. During the titration experiment, the AMPK ⁇ 1 protein concentration in the cells was 10 ⁇ M, while the concentration of GlcNAc-6P as substrate in the syringe was 1 mM.
  • Cells were lysed in cell lysis buffer A (50mM Tris, pH 7.4, 300mM NaCl, 1% NP-40, 1mM PMSF and holoenzyme inhibitor Cocktail added before use). Cell lysates were incubated with specific antibodies and protein G-Sepharose beads (Millipore) for 3 hours at 4°C with rotation. The agarose beads were collected by centrifugation and washed three times with cell lysis buffer A, and the results were subsequently analyzed by immunoblotting.
  • cell lysis buffer A 50mM Tris, pH 7.4, 300mM NaCl, 1% NP-40, 1mM PMSF and holoenzyme inhibitor Cocktail added before use. Cell lysates were incubated with specific antibodies and protein G-Sepharose beads (Millipore) for 3 hours at 4°C with rotation. The agarose beads were collected by centrifugation and washed three times with cell lysis buffer A, and the results were subsequently analyzed by immunoblotting.
  • Flag-AMPK ⁇ 1 and HA-LKB1 were first transfected into A375 cells by lentivirus, and GlcNAc-6P (6-phosphate Cells were treated with -N-acetylglucosamine) (20mM, dissolved in H2O ) for 6 hours, followed by immunoprecipitation analysis with HA-specific antibodies.
  • cell lysates were incubated with anti-GSDME antibody (Proteintech) overnight at 4°C, followed by addition of protein G-Sepharose beads at 4°C. 30 minutes.
  • Mannose can inhibit cell pyroptosis induced by carbonyl cyanide chlorophenylhydrazone (CCCP) and FeSO 4 (an induction method) in melanoma cells A375, which is specifically manifested as cell blebbing ( Figure 14 Figure a), the cleavage activation of GSDME protein in cells ( Figure 14, Figure b), and the release of lactate dehydrogenase (LDH) in cells ( Figure 14, Figure c) can be inhibited by mannose.
  • CCCP carbonyl cyanide chlorophenylhydrazone
  • FeSO 4 an induction method
  • the next step is to explore the mechanism of inhibiting pyroptosis after activation of AMPK.
  • AMPK can directly bind to GSDME, a key protein of cell pyroptosis (Figure 19, panel a), because the main enzymatic activity of AMPK is its kinase activity (i.e., the ability to phosphorylate other proteins).
  • AMPK can enhance the phosphorylation level of GSDME ( Figure 19, b).
  • the experimental method is as follows:
  • mice were constructed by the Experimental Animal Center of Xiamen University.
  • AMPK-DKO mice were donated by Professor Lin Shengcai (State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University).
  • Wild-type mice (WT) and mice with a simulated point mutation corresponding to the sixth threonine of GSDME (GSDME T6E) were randomly divided into 4 groups, namely the control group (Ctrl) and the mannose group (Mannose).
  • cisplatin group (Cisplatin), cisplatin + mannose co-treatment group (Cisplatin + Mannose); wild-type mice (WT) and corresponding intestinal-specific AMPK knockout mice (AMPK-DKO) were randomly divided into There are 2 groups, namely the cisplatin group (Cisplatin) and the cisplatin + mannose co-treatment group (Cisplatin + Mannose). They were kept in a 12h light and 12h dark environment.
  • mice in the control group (Ctrl) were intragastrically treated with 500 ⁇ L of sterile water every day, and the mice in the mannose group and cisplatin + mannose co-treatment group were intragastrically treated with mannose (dissolved in H 2 O). Each mouse was orally administered 500 ⁇ L of 20% mannose once a day for a total of 6 days.
  • mice in the cisplatin group and cisplatin + mannose co-treatment group were treated with cisplatin (dissolved in 5% glucose solution).
  • Cisplatin was injected intraperitoneally, and treatment began one day after mannose treatment.
  • the dosage It is 18mg/kg, and it can be administered once after 1 day of pretreatment with mannose.
  • mice and GSDME T6E mice were constructed and compared them with WT mice.
  • the performance of mice and GSDME T6E mice in the cisplatin group (Cisplatin) showed that after phosphorylation modification of the sixth threonine, the damage caused by chemotherapy drugs to the normal tissues of mice was greatly reduced ( Figure 22 and Figure 23), then it is proved that mannose-induced phosphorylation of the sixth threonine of mouse GSDME is the key to its function of inhibiting side effects.
  • the experimental method is as follows:
  • Results and result analysis The results are shown in Figure 24 a and Figure 24 b.
  • side effects such as diarrhea (increased number of bowel movements per day), enteritis (feces The number of white blood cells in the patient increases), and taking mannose at the same time as chemotherapy can significantly reduce the side effects of chemotherapy drugs (picture a of Figure 24, picture b of Figure 24).

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Abstract

甘露糖抑制细胞焦亡或减轻化疗药物毒副作用的应用，涉及医药科技领域。甘露糖通过抑制化疗药物诱导的细胞焦亡，进而缓解化疗药物导致的肠道和肾脏等毒副作用，同时不会影响化疗药物对肿瘤的治疗效果。甘露糖可在制备抑制细胞焦亡或减轻化疗药物毒副作用的药物、试剂盒、药物组合物或保健品中应用。可减轻化疗药物引起肾脏和肠道的副作用。可作为补充药物用于临床，在保证化疗药物疗效的同时，尽可能减轻化疗药物的毒副作用，提升患者的身心健康，同时不影响化疗药物抑制肿瘤的效果；为癌症患者的临床用药提供新选择。

Description

甘露糖抑制细胞焦亡或减轻化疗药物毒副作用的应用 技术领域
本发明涉及医药科技领域，尤其是涉及甘露糖抑制细胞焦亡或减轻化疗药物毒副作用的应用。
背景技术
癌症是一类严重威胁人类健康的疾病。目前对癌症治疗的方法仍有很大的局限性，特别是癌症治疗过程中患者承受着巨大的痛苦。化学治疗(简称化疗)是利用化学合成药物杀伤肿瘤细胞或抑制肿瘤细胞生长的方法，是临床恶性肿瘤治疗中最常见的一种手段。化疗的作用范围是全身性，在杀死肿瘤细胞的同时也会杀死大量的正常组织细胞，由此造成化疗的毒副作用。化疗过程中常见的毒副作用主要有：脱发、口腔及喉咙溃疡、恶心、呕吐、腹泻、便秘、骨髓造血功能降低、神经及肌肉系统损伤等，影响生殖器官的功能、对肾脏、心脏等器官也会造成损伤。尽管在化疗前或化疗中会给予止吐、护胃、抗生素干预，物理防护等措施，进行饮食调整，一定程度上减轻化疗过程带给患者的痛苦，但患者受到化疗药物的毒副作用依然很严重。因此，寻找和发现能够减轻化疗带给患者痛苦的临床药物意义重大。
研究发现，化疗药物引起机体的毒副作用与正常组织器官中GSDME介导的细胞焦亡密切相关，阻断细胞焦亡可以有效缓解化疗药物引起的毒副作用(WANG Y,GAO W,SHI X,et al.Chemotherapy drugs induce pyroptosis through caspase-3cleavage of a gasdermin[J].Nature,2017,547(7661):99-103)。
甘露糖是一种治疗尿道感染的药物(B,D,ALTARAC S.D-mannose powder for prophylaxis of recurrent urinary tract infections in women:a rand omized clinical trial[J].World J Urol,2014,32(1):79-84)，也可以抑制肿瘤生长并且增强化疗药物治疗肿瘤的效果(GONZALEZ P S,O”PREY J,CARDACI S,et al.Mannose impairs tumour growth and enhances chemoth erapy[J].Nature,2018,563(7733):719-723)，但是关于甘露糖的其他功能研究很少。
因此，仍亟需一种降低化疗药物的毒副作用，减轻化疗过程带给患者的痛苦的方法。
发明内容
发明概述
为解决上述技术问题，本发明提供一种甘露糖在制备抑制细胞焦亡或减轻化疗药物毒副作用的药物、试剂盒、药物组合物或保健品中应用。所述甘露糖能有效抑制细胞焦亡或减轻化疗药物毒副作用，所述毒副作用为器官损伤，具有预料不到的有益效果。
发明详述
本发明的目的在于提供甘露糖抑制细胞焦亡或减轻化疗药物毒副作用的新用途。甘露糖为一种单糖类物质。
细胞实验发现，甘露糖可以抑制临床化疗药物顺铂或者奥沙利铂(oxaliplatin)引起的正常细胞焦亡(包括检测细胞焦亡形态、GSDME剪切和LDH释放等焦亡金标准)。小鼠实验进一步显示，甘露糖通过抑制顺铂或者奥沙利铂诱导组织细胞焦亡，能够显著缓解其引起的毒副作用(包括缓解顺铂或者奥沙利铂导致的小鼠小肠绒毛以及结直肠的缩短和肾功能降低)，从而为减轻化疗药物的毒副作用提供重要的实验依据。
本发明提供甘露糖在制备抑制细胞焦亡或减轻化疗药物毒副作用的药物、试剂盒、药物组合物或保健品中的应用，所述毒副作用为器官损伤。
在一些实施例中，所述器官包括肠道或肾脏。
在一些实施例中，所述化疗药物为顺铂、卡铂、奥沙利铂、环磷酰胺、异环磷酰胺、美法仑、白消安、氮芥、苯丁酸氮芥、塞替派、卡莫司汀、尼莫司汀、洛莫司汀、司莫司汀、培美曲塞、甲氨蝶呤、氟脲嘧啶、卡培他滨、阿糖胞苷、吉西他滨、6-巯基嘌呤、硫鸟嘌呤、柔红霉素、多柔比星、表柔比星、阿柔比星、伊达比星、博来霉素、丝裂霉素、长春新碱、长春地辛、长春瑞滨、喜树碱、伊立替康、托泊替康、紫杉醇、多西紫杉醇、白蛋白结合型紫杉醇、紫杉醇脂质体、依托泊苷、替尼泊苷、丙卡巴肼、达卡巴嗪、替莫唑胺、L-门冬酰胺酶、去甲斑蝥素、甲基斑蝥素等常见的临床化疗药物或其药学上可接受的盐中的至少一种。
由于上述化疗药物共同特征都是诱导凋亡，甘露糖对这些药物引起的毒副作用都具有缓解效果。
在一些实施例中，所述药物组合物包含甘露糖，或其药学上可接受的盐，以及药学上可接受的载体。
在一些实施例中，所述试剂盒包括(i)包含甘露糖的试剂，和(ii)包含化疗药物的试剂。
在一些实施例中，本发明提供甘露糖在制备用于抑制化疗药物诱导正常肠和肾细胞焦亡的药物中应用。
在一些实施例中，本发明提供甘露糖在制备用于缓解化疗药物导致肠道毒副作用的药物中应用。
在一些实施例中，本发明提供甘露糖在制备用于缓解化疗药物导致肾脏毒副作用的药物中应用。
在一些实施例中，所述药物还包含有效量的化疗药物。
在一些实施例中，所述药物可制成以下剂型：片剂、胶囊、粒剂、注射液等。
在一些实施例中，所述甘露糖可以是粉末状、溶液状或其他形式以及可以代谢产生甘露糖的物质，或其药学上可接受的盐，以及药学上可接受的载体。
在一些实施例中，所述甘露糖可抑制化疗药物诱导的肠细胞焦亡。
在一些实施例中，所述甘露糖可抑制化疗药物诱导的肾细胞焦亡。
在一些实施例中，所述甘露糖可缓解化疗药物引起的小鼠小肠和结直肠的毒副作用。
在一些实施例中，所述甘露糖可缓解化疗药物引起的小鼠肾的毒副作用。
在一些实施例中，所述甘露糖可减轻化疗药物毒副作用且不影响疗效。
在一些实施例中，根据前述任一方面，本发明提供一种抑制化疗药物诱导的正常肠细胞和肾细胞焦亡 的方法，所述方法包括使正常细胞与(i)包含有效量的甘露糖的试剂和(ii)包含有效量的化疗药物的试剂接触，所述试剂可以是同时或者序贯地与所述细胞接触。
在一些实施例中，根据前述任一方面，本发明提供一种减轻化疗药物引起的肠道毒副作用的方法，所述方法包括对受试者施用有效量的本发明的药物，所述试剂可以是同时或序贯地施用(i)包含有效量的甘露糖的试剂和(ii)包含有效量的化疗药物，所述肠道是小肠和结直肠。
在一些实施例中，根据前述任一方面，本发明提供一种减轻化疗药物引起的肾脏毒副作用的方法，所述方法包括对受试者施用有效量的本发明的药物，所述试剂可以是同时或序贯地施用(i)包含有效量的甘露糖的试剂和(ii)包含有效量的化疗药物。
在一些实施例中，根据前述任一方面，本发明提供一种减轻化疗药物毒副作用且不影响疗效的方法，所述方法包括对受试者施用有效量的本发明的药物，所述试剂可以是同时或序贯地施用(i)包含有效量的甘露糖的试剂和(ii)包含有效量的化疗药物。
在一些实施例中，根据前述任一方面，本发明提供包含甘露糖的试剂在制备用于抑制化疗药物诱导正常肠和肾细胞焦亡药物的用途，该药物可以抑制正常肠和肾细胞焦亡。
在一些实施例中，根据前述任一方面，本发明提供包含甘露糖的试剂在制备用于缓解化疗药物引起肠道毒副作用的药物中的用途，该药物可以缓解化疗药物导致的肠道毒副作用。
在一些实施例中，根据前述任一方面，本发明提供包含甘露糖的试剂在制备用于缓解化疗药物引起肾脏毒副作用的药物中的用途，该药物可以缓解肾脏组织毒副作用及其相关肾功能指标，如血尿素氮(BUN)、肌酐(creatinine)和胱抑素C。
在一些实施例中，根据前述任一方面，本发明提供包含甘露糖的试剂在制备用于缓解化疗药物毒副作用且不影响疗效的药物中的用途，该药物可以缓解化疗药物导致的毒副作用且不影响疗效。
在一些实施例中，根据前述任一方面，所述甘露糖可以是粉末状、溶液状或其他形式以及可以代谢产生甘露糖的物质，或其药学上可接受的盐。
有益效果
本发明研究发现，甘露糖具有抑制正常细胞焦亡的功能。重要的是，通过甘露糖抑制的细胞焦亡能够对正常组织器官产生保护功能，大大减轻化疗药物引起的毒副作用。现有缓解化疗药物毒副作用的药物通常是减轻化疗引起的一些症状或增强器官的功能，治疗效果有限，治标不治本；而甘露糖可以从根本上抑制肠道和肾脏细胞的焦亡，进而从根本上减轻毒副作用的发生，治疗效果更佳，采用甘露糖配合化疗药物辅助治疗尤其适用于肾功能低下或有肠道问题的患者。可作为补充药物用于临床，在保证化疗药物疗效的同时，尽可能减轻化疗药物的毒副作用，提升患者的身心健康，同时不影响化疗药物抑制肿瘤的效果；为癌症患者的临床用药提供新选择。
附图说明
图1为实施例1中不同处理组别中化疗药物顺铂(Cisplatin)诱导正常肠细胞(IEC-6和FHs 74细胞)和肾细胞(HK-2细胞)焦亡的结果对比图；其中，图a为实施例1中不同处理组别中化疗药物顺铂(Cisplatin)诱导正常肠细胞(IEC-6和FHs 74细胞)和肾细胞(HK-2细胞)焦亡后的形态观察图；图b为实施例1中不同处理组别中顺铂诱导正常细胞后的GSDME蛋白剪切情况的蛋白电泳对比图；图c为实施例1中不同处理组别中顺铂诱导正常细胞后的LDH释放统计图。
图2为实施例2中不同处理组别中化疗药物顺铂(Cisplatin)处理引起的小鼠肠道毒副作用结果对比图；其中，图a为实施例2中不同处理组别中化疗药物顺铂(Cisplatin)处理引起小鼠结直肠长度变化对比图；图b为实施例2中不同处理组别中化疗药物顺铂(Cisplatin)处理引起小鼠小肠绒毛变化或隐窝变化对比图；图c为实施例2中不同处理组别中化疗药物顺铂(Cisplatin)处理后小鼠小肠GSDME蛋白的剪切情况的蛋白电泳对比图。
图3为实施例2中不同处理组别中化疗药物顺铂(Cisplatin)处理引起的小鼠肾脏毒副作用结果对比图；其中，图a为实施例2中不同处理组别中化疗药物顺铂(Cisplatin)处理后的小鼠的肾PAS染色结果图；图b为实施例2中不同处理组别中化疗药物顺铂(Cisplatin)处理引起的小鼠血尿素氮(BUN)、肌酐(creatinine)、胱抑素C水平变化统计图；图c为实施例2中不同处理组别中化疗药物顺铂(Cisplatin)处理后小鼠肾脏GSDME蛋白的剪切情况的蛋白电泳对比图。
图4为实施例3中不同处理组中化疗药物顺铂(Cisplatin)处理后裸鼠中移植瘤体积变化结果对比图；其中图a为不同处理组中化疗药物顺铂(Cisplatin)处理后裸鼠中移植瘤体积变化统计图；图b为不同处理组中化疗药物顺铂(Cisplatin)处理后裸鼠中移植瘤的图片。
图5为实施例3中在移植瘤荷载小鼠模型中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠肠道毒副作用的结果对比图；其中，图a为实施例3中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠结直肠长度变化对比图；图b为实施例3中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠小肠绒毛变化或隐窝变化结果图；图c为实施例3中不同处理组中化疗药物顺铂(Cisplatin)处理后小鼠小肠GSDME蛋白的剪切情况的蛋白电泳对比图。
图6为实施例3中在移植瘤荷载小鼠模型中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠肾脏毒副作用结果对比图；其中，图a为实施例3中不同处理组中化疗药物顺铂(Cisplatin)处理后小鼠的肾PAS染色图；图b为实施例3中不同处理组中化疗药物顺铂(Cisplatin)处理后小鼠的血尿素氮(BUN)、血肌酐(creatinine)、血胱抑素C水平变化统计图；图c为实施例3中不同处理组中化疗药物顺铂(Cisplatin)处理后小鼠肾脏GSDME蛋白的剪切情况的蛋白电泳对比图。
图7为实施例4中不同处理组中化疗药物顺铂(Cisplatin)处理后原发肝癌体积变化对比图。
图8为实施例4中在原发肝癌小鼠模型中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠肠道毒副作用的结果对比图；其中，图a为实施例4中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠结直肠长度变化对比图；图b为实施例4中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠小肠绒毛变化或隐窝变化对比图；图c为实施例4中不同处理组中化疗药物顺铂(Cisplatin)处理后小鼠小肠GSDME蛋白的剪切情况的蛋白电泳对比图。
图9为实施例4中在原发肝癌小鼠模型中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠肾脏毒副作用结果对比图；其中，图a为实施例4中不同处理组中化疗药物顺铂(Cisplatin)处理后的小鼠肾PAS染色对比图；图b为实施例4中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠血尿素氮(BUN)、血肌酐(creatinine)、血胱抑素C水平变化对比统计图；图c为实施例4中不同处理组中化疗药物顺铂(Cisplatin)处理几批小鼠肾脏GSDME蛋白的剪切情况的蛋白电泳对比图。
图10为实施例5中不同处理组中化疗药物奥沙利铂(Oxaliplatin)处理后原发肝癌体积变化对比图。
图11为在原发肝癌小鼠模型中，实施例5中不同处理组中化疗药物奥沙利铂(Oxaliplatin)处理引起小鼠肠道毒副作用结果对比图；其中，图a为实施例5中不同处理组中化疗药物奥沙利铂(Oxaliplatin)处理引起小鼠结直肠长度变化图；图b为实施例5中不同处理组中化疗药物奥沙利铂(Oxaliplatin)处理引起小鼠小肠绒毛变化或隐窝变化的对比图；图c为实施例5中不同处理组中化疗药物奥沙利铂(Oxaliplatin)处理后小鼠小肠GSDME蛋白的剪切情况的蛋白电泳对比图。
图12为实施例6中不同处理组中化疗药物奥沙利铂(Oxaliplatin)处理后原发胃癌的体积变化对比图。
图13为实施例6中在原发胃癌小鼠模型中不同处理组中化疗药物奥沙利铂(Oxaliplatin)处理引起小鼠肠道毒副作用结果对比图；其中，图a为实施例6中不同处理组中化疗药物奥沙利铂(Oxaliplatin)处理引起小鼠结直肠长度变化对比图；图b为实施例6中不同处理组中化疗药物奥沙利铂(Oxaliplatin)处理引起小鼠小肠绒毛变化或隐窝变化的对比图；图c为实施例6中不同处理组中化疗药物奥沙利铂(Oxaliplatin)处理后小鼠小肠GSDME蛋白的剪切情况的蛋白电泳对比图。
图14为实施例7中在细胞焦亡诱导剂CCCP/FeSO4处理条件下，黑色素瘤细胞A375细胞焦亡情况对比图；其中图a为不同处理组中的细胞形态观察图；图b为不同处理组中GSDME蛋白的剪切情况的蛋白电泳对比图；图c为不同处理组别中细胞的LDH释放统计图。
图15为实施例7中己糖胺生物合成途径(Hexosamine-biosynthesis pathway,HBP)中甘露糖(Mannose)的代谢模式图及敲除或抑制关键基因后细胞焦亡情况对比图；图a为甘露糖进入细胞后的代谢模式图；图b为在A375细胞中敲除GFAT1和GFAT2后，不同组别中CCCP/FeSO4诱导的细胞焦亡情况对比图；图c为在A375细胞中利用GFAT1抑制剂DON处理后，不同组别中CCCP/FeSO4诱导的细胞焦亡情况对比图；
图16中图a为实施例7中甘露糖激活AMPK，引起AMPK蛋白及其下游蛋白ACC的磷酸化水平升高的蛋白电泳对比图；图16中图b为在A375细胞中利用AMPK活性抑制剂compound C处理后，不同组别中CCCP/FeSO4诱导的细胞焦亡情况对比图；图16中图c为在A375细胞中敲除AMPKα1和AMPKα2后，不同组别中CCCP/FeSO4诱导的细胞焦亡情况对比图。
图17为实施例7中己糖胺生物合成途径中代谢物水平变化对甘露糖(Mannose)激活AMPK蛋白的效果对比图；图a为在A375细胞中敲除GFAT1和GFAT2后，不同组别中甘露糖诱导的AMPK磷酸化水平变化的对比图；图b为在甘露糖处理条件下，细胞中代谢物GlcNAc-6P和GlcNAc-1P的水平变化图；图c为在不同代谢物处理A375细胞后，不同组别中AMPK磷酸化水平变化的对比图。
图18中图a为实施例7中利用等温滴定量热法(ITC)检测代谢物GlcNAc-6P与蛋白AMPKα1的相 互作用图；图18中图b为实施例7中代谢物GlcNAc-6P对蛋白AMPKα1与蛋白LKB1之间相互作用的影响效果图。
图19为实施例7中蛋白AMPKα1与蛋白GSDME相互作用图；其中，图a为在A375细胞中，利用co-IP技术检测蛋白AMPKα1与蛋白GSDME之间相互作用图；图b为利用体外磷酸化分析实验技术，检测AMPK复合物对GSDME磷酸化水平影响的蛋白电泳效果图；图c为GSDME的蛋白磷酸化位点质谱分析结果。
图20为实施例7中在敲除了GSDME蛋白的A375细胞中，分别回补GSDME-Flag蛋白和磷酸化模拟蛋白GSDMET6E-Flag，不同组别中CCCP/FeSO4诱导的细胞焦亡情况对比图。
图21为实施例8中在肠道特异性敲除AMPK小鼠模型(AMPK-DKO)中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠肠道毒副作用结果对比图；其中，图a为实施例8中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠结直肠长度变化图；图b为实施例8中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠小肠绒毛变化和隐窝变化的对比图；图c为实施例8中不同处理组中化疗药物顺铂(Cisplatin)处理后小鼠小肠GSDME蛋白的剪切情况的蛋白电泳对比图。
图22为实施例8中在全身性敲入GSDMET6E点突变小鼠中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠肠道毒副作用结果对比图；其中，图a为实施例8中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠结直肠长度变化图；图b为实施例8中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠小肠绒毛变化和隐窝变化的对比图；图c为实施例8中不同处理组中化疗药物顺铂(Cisplatin)处理后小鼠小肠GSDME蛋白的剪切情况的蛋白电泳对比图。
图23为实施例8中在全身性敲入GSDMET6E点突变小鼠中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠肾脏毒副作用结果对比图；其中，图a为实施例8中不同处理组中化疗药物顺铂(Cisplatin)处理后的小鼠肾PAS染色对比图及损伤统计图；图b为实施例8中不同处理组中化疗药物顺铂(Cisplatin)处理引起小鼠血液尿素氮(BUN)、肌酐(creatinine)、胱抑素C水平变化对比统计图；图c为实施例8中不同处理组中化疗药物顺铂(Cisplatin)处理几组小鼠肾脏GSDME蛋白的剪切情况的蛋白电泳对比图。
图24为实施例9中甘露糖(Mannose)缓解化疗患者肠道毒副作用的效果统计图；图a为不同组别患者在接收XELOX化疗方案治疗后每日腹泻次数统计图；图b为不同组别患者粪便中白细胞检测结果统计图。
术语定义：
本发明中，“室温”表示环境温度，可以为20℃-30℃；在一些实施例中，为22℃-28℃；在一些实施例中，为24℃-26℃；在一些实施例中，为25℃。
在本发明的上文中，无论是否使用“大约”或“约”等字眼，所有在此公开了的数字均为近似值。基于公开的数字，每一个数字的数值有可能会出现±10％以下的差异或者本领域人员认为的合理的差异，如±1％、±2％、±3％、±4％或±5％的差异。
术语“任选”、“任选的”或“任选地”是指随后描述的事件或情形可以但不一定出现。例如，“任选的表面活性剂”是指表面活性剂可以存在或可以不存在。
术语“重量百分比“或“以重量计的百分比”或“wt％””定义为组合物中单个组分的重量除以组合物所有组分的总重量然后乘以100。
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或多项的组合。
术语“wt％”表示质量百分比。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
具体实施方式
为了使本领域的技术人员更好地理解本发明的技术方案，下面进一步披露一些非限制实施例以对本发明作进一步的详细说明。
本发明所使用的试剂均可以从市场上购得或者可以通过本发明所描述的方法制备而得。
在一个实施方案中，本发明提供一种单糖类物质甘露糖,或其药学上可接受的盐，以及药学上可接受的载体。
任选地，所述甘露糖抑制化疗药物诱导的肠细胞焦亡。
任选地，所述甘露糖抑制化疗药物诱导的肾细胞焦亡。
任选地，所述甘露糖缓解化疗药物引起的小鼠小肠和结直肠的毒副作用。
任选地，所述甘露糖缓解化疗药物引起的小鼠肾脏的毒副作用。
任选地，所述甘露糖缓解化疗药物毒副作用且不影响化疗药物的疗效。
在一个实施方案中，本发明提供一种试剂盒，所述试剂盒包括(i)包含甘露糖的试剂，(ii)包含化疗药物的试剂。仅作为参考，甘露糖与化疗药物的比例可为20～50︰1。
在一个实施方案中，本发明提供一种抑制化疗药物诱导的正常肠细胞和肾细胞焦亡的方法，所述方法包括使正常细胞与(i)包含有效量的甘露糖的试剂和(ii)包含有效量的化疗药物的试剂接触，所述试剂可以是同时或者序贯地与所述细胞接触。
在一个实施方案中，本发明提供一种减轻化疗药物引起的肠道毒副作用的方法，所述方法包括对受试者施用有效量的本发明的药物，所述试剂可以是同时或序贯地施用(i)包含有效量的甘露糖的试剂和(ii)包含有效量的化疗药物，所述肠道是小肠和结直肠。
在一个实施方案中，本发明提供一种减轻化疗药物引起的肾脏毒副作用的方法，所述方法包括对受试者施用有效量的本发明的药物，所述试剂可以是同时或序贯地施用(i)包含有效量的甘露糖的试剂和(ii)包含有效量的化疗药物。
在一个实施方案中，本发明提供一种减轻化疗药物毒副作用且不影响疗效的方法，所述方法包括对受试者施用有效量的本发明的药物，所述试剂可以是同时或序贯地施用(i)包含有效量的甘露糖的试剂和(ii)包含有效量的化疗药物。
在一个实施方案中，本发明提供了包含甘露糖的试剂在制备用于抑制化疗药物诱导正常肠和肾细胞焦亡的药物中的用途，该药物可以抑制化疗药物引起的正常肠和肾细胞的焦亡。
在一个实施方案中，本发明提供包含甘露糖的试剂在制备用于缓解化疗药物引起的肠道毒副作用的药物中的用途，该药物可以缓解化疗药物引起的小肠毒副作用。
在一个实施方案中，本发明提供包含甘露糖的试剂在制备用于缓解化疗药物引起的肾脏毒副作用的药物中的用途，该药物可以缓解化疗药物引起的肾脏组织毒副作用及其相关肾功能指标，如血尿素氮(BUN)、肌酐(creatinine)和胱抑素C。
在一个实施方案中，本发明提供包含甘露糖的试剂在制备用于缓解化疗药物毒副作用且不影响疗效的药物中的用途，该药物可以缓解化疗药物导致的毒副作用且不影响疗效。
在一个实施方案中，所述甘露糖可以是粉末状，溶液状或其他形式以及可以代谢产生甘露糖的物质，或其药学上可接受的盐。
在一个实施方案中，所述化疗药物可为顺铂，卡铂、奥沙利铂、环磷酰胺、异环磷酰胺、美法仑、白消安、氮芥、苯丁酸氮芥、塞替派、卡莫司汀、尼莫司汀、洛莫司汀、司莫司汀、培美曲塞、甲氨蝶呤、氟脲嘧啶、卡培他滨、阿糖胞苷、吉西他滨、6-巯基嘌呤、硫鸟嘌呤、柔红霉素、多柔比星、表柔比星、阿柔比星、伊达比星、博来霉素、丝裂霉素、长春新碱、长春地辛、长春瑞滨、喜树碱、伊立替康、托泊替康、紫杉醇、多西紫杉醇、白蛋白结合型紫杉醇、紫杉醇脂质体、依托泊苷、替尼泊苷、丙卡巴肼、达卡巴嗪、替莫唑胺、L-门冬酰胺酶、去甲斑蝥素、甲基斑蝥素等常见的临床化疗药物或其药学上可接受的盐中的至少一种。
在一个实施方案中，本发明所涉及的药物还可按本领域已知方法配成肠道或非肠道给药的其它药学上可接受的剂型，如片剂、胶囊、粒剂、注射液等。
术语“有效量”指产生本发明抑制正常细胞焦亡或减轻化疗药物引起的毒副作用等技术效果或在受试者中实现治疗、预防、减轻和/或缓解本发明所述疾病或病症的本发明化合物的量。这样的量通常依若干因素变化，而所述变化是在已知本文提供的描述的普通技术人员能够确定和计算的范围内。这些因素包括，但不限于：特定的个体及其年龄、体重、身高、一般身体状况和医疗经历，所使用的特定化合物，化合物配制于其中的载体，所选择的化合物的施用途径，以及被治疗的病症的性质和严重性。
术语“药物组合物”指包含甘露糖以及依施用方式和剂型的性质而定的至少一种选自以下药学上可接受的成分的组合物，包括：载体、稀释剂、佐剂、赋形剂，例如防腐剂、填充剂、崩解剂、润湿剂、乳化剂、悬浮剂、甜味剂、矫味剂、香味剂、抗菌剂、抗真菌剂、润滑剂和分散剂。悬浮剂的实例包括乙氧化异硬脂醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、偏氢氧化铝、膨润土、琼脂和黄蓍胶、或这些物质的混合物。通过多种抗菌剂和抗真菌剂，例如对羟基苯甲酸酯、氯代丁醇、苯酚、山梨酸等，能够确保预防微生物的作用。最好包括等渗剂，例如糖、氯化钠等。通过使用延迟吸收剂，例如单硬脂酸铝和明胶，能够使注射剂型延长吸收。适宜的载体、稀释剂、溶剂或赋形剂的实例包括水、乙醇、多元醇、它们的适宜的混合物、植物油(例如橄榄油)、和注射有机酯例如油酸乙酯。赋形剂的实例包括乳糖、枸橼酸钠、碳酸钙、磷酸二钙。崩解剂的实例包括淀粉、藻酸和一些复合硅酸盐。润滑剂的实例包括硬脂酸镁、十二烷基硫酸钠、滑石以及高分子量聚乙二醇。
术语“药学上可接受”指在合理的医疗判断范围内，适用于与人和低等动物细胞接触而没有过度的毒性、刺激性、变应性反应等，且与合理的益处/风险比相应。
术语“药学上可接受的剂型”指本发明化合物的剂型，包括例如片剂、糖锭剂、散剂、酏剂、糖浆剂、液体制剂(包括混悬剂、喷雾剂、吸入片剂、锭剂、乳剂、溶液剂、颗粒剂、胶囊剂和栓剂)以及用于注射的液体制剂，包括脂质体制剂。可在Remington’s Pharmaceutical Science、Mack Publishing Co.、Easton、PA等中发现配制技术和制剂。
术语“药学上可接受的载体”旨在包括与药物给药相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延缓剂等。合适载体描述于最新版的Remington”s Pharmaceutical Sciences中，这是本领域的标准参考书目。此类载体或稀释剂的优选示例包括但不限于水、盐水、林格氏溶液、葡萄糖溶液和5％的人血清白蛋白。也可以使用脂质体和非水性载体，例如固定化油。将此类介质和试剂用于药物活性物质是本领域熟知的。除去任何常规的介质或试剂与抗体不相容之外，设想其在组合物中的用途。
术语“缓解”一般是指获得症状上和/或病理上的减轻效应。该效应可是化疗过程中组织器官的毒副作用的减少或严重程度降低；也可是症状上的减轻，例如呕吐、腹泻等症状上的减轻；也可是病人化疗过程中精神上的宽慰，例如疼痛感降低、心情更加放松愉悦等效果。
术语“药学上可接受的盐”表示本发明化合物的相对无毒的无机和有机酸加成盐、碱加成盐等。这些盐可以在化合物的最终分离和纯化过程中原位制备。特别是，可以通过独立使纯化的游离碱形式的化合物与适宜的有机或无机酸反应，和将如此形成的盐分离，来制备酸加成盐。由形成药学上可以接受的阴离子的有机酸形成的有机酸加合盐，包括但不限于甲酸盐、乙酸盐、丙酸盐、苯甲酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、柠檬酸盐、抗坏血酸盐、α-酮戊二酸盐、α-甘油磷酸盐、烷基磺酸盐或芳基磺酸盐；优选地，所述烷基磺酸盐为甲基磺酸盐或乙基磺酸盐；所述芳基磺酸盐为苯磺酸盐或对甲苯磺酸盐。也可形成合适的无机盐，包括但不限于盐酸盐、氢溴酸盐、氢碘酸盐、硝酸盐、碳酸氢盐和碳酸盐、硫酸 盐或磷酸盐等。示例性酸加成盐包括氢溴酸盐、盐酸盐、硫酸盐、硫酸氢盐、磷酸盐、硝酸盐、乙酸盐、草酸盐、戊酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月桂酸盐、硼酸盐、苯甲酸盐、乳酸盐、磷酸盐、甲苯磺酸盐、柠檬酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、naphthylate、甲磺酸盐、葡庚糖酸盐、乳糖酸盐(lactiobionate)、氨基磺酸盐、丙二酸盐、水杨酸盐、丙酸盐、亚甲基-双-b-羟基萘甲酸盐、龙胆酸盐、异硫代硫酸盐、二对甲苯酰酒石酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐、环己基氨基磺酸盐和奎尼酸盐月桂基磺酸盐(quinateslaurylsulphonate)等(参见Berge等人，PharmaceuticalSalts，J.Pharm.Sci.，66：1-9(1977)和Remington′s Pharmaceutical Sciences，第17版，MackPublishing Company，Easton，Pa.，1985，第1418页)。也可以通过独立进行使纯化的酸形式的化合物与适宜的有机或无机碱反应，和分离如此形成的盐，来制备碱加成盐。碱加成盐包括药学上可接受的金属盐和胺盐。适宜的金属盐包括钠、钾、钙、钡、锌、镁和铝盐。钠和钾盐为优选。适宜的无机碱加成盐是由金属碱制备的，所述金属碱包括氢化钠、氢氧化钠、氢氧化钾、氢氧化钙、氢氧化铝、氢氧化锂、氢氧化镁和氢氧化锌。适宜的胺碱加成盐是由胺制备的，所述胺具有足够的碱性以形成稳定的盐，并且优选包括由于其药用的低毒性和可接受性而在医药化学中常用的那些胺，所述胺的实例包括氨、乙二胺、N-甲基-葡糖胺、赖氨酸、精氨酸、鸟氨酸、胆碱、N，N′-二苄基乙二胺、氯普鲁卡因、二乙醇胺、普鲁卡因、N-苄基苯乙胺、二乙胺、哌嗪、三(羟基甲基)-氨基甲烷、四甲基氢氧化铵、三乙胺、二苄胺、二苯羟甲胺、脱氢松香胺、N-乙基哌啶、苄胺、四甲基铵、四乙基铵、甲胺、二甲胺、三甲胺、乙胺、碱性氨基酸例如赖氨酸和精氨酸、二环己基胺等。
下面将结合附图和具体实施例对本发明的实施方案进行详细描述，本领域技术人员将会理解。下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
甘露糖(Mannose)对化疗药物顺铂(Cisplatin)诱导正常细胞焦亡的影响。
实验方法如下：
1.细胞处理：
(1)取对数生长期的正常细胞株大鼠小肠隐窝上皮细胞(IEC-6)、人小肠上皮细胞(FHs 74)和人肾小管上皮细胞(HK-2)，以4×105细胞/mL密度接种于6孔培养板上。
(2)培养过夜后，将培养液更换成含0.5％血清的DMEM培养基，分别用Cisplatin(顺铂)(10μg/mL，溶解在DMF)、Mannose(甘露糖)(20mM，溶解在H2O)、Cisplatin+Mannose组合物处理细胞。
2.显微镜形态观察：
细胞处理24h后，显微镜观察细胞形态并拍照。细胞发生焦亡过程中细胞吸水胀大，细胞膜破裂，细胞呈现鼓泡形态。
3.免疫印迹(Westernblotting)检测：
(1)收集培养液，于离心机中3000r/min离心5min，收集部分悬浮的死亡细胞，离心后弃掉上清置于冰上。
(2)根据细胞量在6孔培养板中每孔加入适量细胞裂解缓冲液A(50mM Tris，pH 7.4，300mM NaCl，1％NP-40，临用前加1mM PMSF和全酶抑制剂Cocktail)，然后用超声波破碎仪破碎细胞，并于冷冻离心机中13000r/min，4℃离心30min；
(3)将上清吸出，加入等体积的2×SDS样品缓冲液，95℃煮15min，进行如下Westernblotting检测。
(4)蛋白电泳：取2～10μg蛋白样品，于不连续SDS-PAGE胶中电泳，电压100V，待样品进入分离胶后将电压调为150V。
(5)电转移：电转液于4℃预冷，切好胶后将同样大小的甲醇预处理的PVDF膜以及滤纸浸于电转液中；PVDF膜贴于胶后，两面覆盖滤纸，赶尽气泡，按膜朝正极的顺序装于电转槽中，于冰上电转(100V，60min)。
(6)抗原抗体反应：
封闭：封闭液室温封闭1h；一抗反应：封闭后膜与相应一抗于室温孵育1～3h；
二抗反应：TBST洗3次，每次5min，之后加入相应的二抗，于室温孵育1～3h。
(7)曝光成像：TBST洗3次，每次10min。配制ECL的A液和B液以1︰1(V/V)混和，用显影机进行曝光成像。
4.乳酸脱氢酶(LDH)释放检测：
细胞处理24h后，按promega公司细胞毒性检测试剂盒的方法和步骤检测细胞外乳酸脱氢酶的含量。细胞凋亡或坏死(包括细胞焦亡)造成的细胞膜结构的破坏会导致细胞浆内的酶释放到培养液，其中包括酶活性较为稳定的乳酸脱氢酶(lactatedehydroge-nase，LDH)。通过检测从细胞膜破裂释放到培养液中的LDH活性，就可以实现对细胞毒性的定量分析。
结果及结果分析：图1给出甘露糖抑制化疗药物顺铂诱导的正常肠细胞和肾细胞焦亡的结果。
由图1的图a可知，甘露糖能抑制顺铂诱导正常细胞的焦亡形态。顺铂显著诱导大鼠小肠隐窝上皮细胞(IEC-6)、人小肠上皮细胞(FHs 74)和人肾小管上皮细胞(HK-2)发生肿胀，细胞膜破裂。本发明所述甘露糖与顺铂共处理组相对于顺铂组细胞肿胀、细胞膜破裂的细胞比例显著减少，表明本发明所述甘露糖能够抑制顺铂诱导的细胞焦亡。
由图1的图b可知，甘露糖能抑制顺铂诱导正常细胞的GSDME蛋白剪切。顺铂显著诱导大鼠小肠隐窝上皮细胞(IEC-6)、人小肠上皮细胞(FHs 74)和人肾小管上皮细胞(HK-2)发生GSDME蛋白剪切。本发明 所述甘露糖与顺铂共处理组相对于顺铂组GSDME蛋白剪切的比例显著减少，表明本发明所述甘露糖能够抑制顺铂诱导的GSDME蛋白剪切。
由图1的图c可知，甘露糖能抑制顺铂诱导正常细胞的LDH释放。顺铂显著诱导大鼠小肠隐窝上皮细胞(IEC-6)、人小肠上皮细胞(FHs 74)和人肾小管上皮细胞(HK-2)发生细胞膜破裂以及乳酸脱氢酶的释放。本发明所述甘露糖与顺铂共处理组相对于顺铂组LDH释放的比例显著减少，表明本发明所述甘露糖能够抑制顺铂诱导的细胞质膜破裂，抑制细胞焦亡。
实验结果表明，本发明所述甘露糖与顺铂共处理组相对于顺铂组LDH释放的比例显著减少。说明本发明所述甘露糖能够抑制顺铂诱导的细胞膜破裂，抑制细胞焦亡。
每组实验设置3次独立重复，然后应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；具差异极其显著性。统计分析表明甘露糖可以抑制顺铂诱导的细胞膜破裂和细胞焦亡(p<0.001)。
实施例2
甘露糖对化疗药物顺铂引起的小鼠肠道和肾脏毒副作用的影响。
实验方法如下：
1.小鼠处理：
(1)将SPF级的C57BL/6小鼠(～20g)随机分为四组，分别为对照组(Ctrl)、甘露糖组(Mannose)、顺铂组(Cisplatin)、顺铂+甘露糖共处理组(Cisplatin+Mannose)，饲养在12h光照，12h黑暗环境中。
(2)对照组(Ctrl)小鼠每天用500μL无菌水进行灌胃处理，甘露糖组(Mannose)和顺铂+甘露糖共处理组(Cisplatin+Mannose)小鼠进行甘露糖(Mannose，溶解于H2O中)灌胃预处理，每只小鼠灌胃20％的甘露糖500μL，每天处理一次，共处理5天。
(3)顺铂组(Cisplatin)和顺铂+甘露糖共处理组(Cisplatin+Mannose)小鼠进行顺铂(Cisplatin，溶于PBS中)处理，顺铂采用腹腔注射的方式，剂量为18mg/kg，在甘露糖处理一天后注射一次。
2.小鼠结直肠长度测量：
小鼠经过药物处理结束后，断颈处死小鼠，取出小鼠的结直肠进行拍照和长度测量。
3.组织化学染色分析：
取出小鼠的小肠进行H&E组织化学染色分析：
(1)用PBS将小肠内容物冲洗干净。取5cm左右长度的小肠，沿纵轴剖开，然后将小肠卷成“瑞士卷”的形状。
(2)卷好的小肠组织用4％的多聚甲醛进行固定过夜。
(3)固定好的组织随后进行脱水、石蜡包埋。
(4)包埋好的组织进行石蜡切片。
(5)切好的组织进行脱蜡复水，之后进行苏木精-伊红染色。
(6)封片后进行镜检。
取出小鼠肾脏进行PAS组织化学染色分析：
(1)将小鼠肾脏用PBS洗净。
(2)肾脏组织用4％的多聚甲醛进行固定过夜。
(3)固定好的组织随后进行脱水、石蜡包埋。
(4)包埋好的组织进行石蜡切片。
(5)切好的组织进行脱蜡复水，进行PAS染色。PAS染色法(Periodic Acid-Schiff stain)在组织学上主要用来检测组织中的糖类。过碘酸把糖类相邻两个碳上的羟基氧化成醛基，再用Schiff试剂和醛基反应使其呈现紫红色。当肾脏受到损伤后，尿调节蛋白(Uromodulin)剧烈增加，并被PAS染色。因此PAS染色能够反映肾脏的损伤程度。
(6)封片后进行镜检。
4.免疫印迹(Westernblotting)检测：
取出小鼠小肠和小鼠肾脏，用PBS冲洗干净。剪取5mm小肠段或剪取0.1g的肾脏组织，加1.5mL 2×SDS样品缓冲液置于组织破碎仪中进行裂解。95℃煮15min，待进行Western blotting检测(检测方法参考实施例1)。
5.血尿素氮(BUN)、肌酐(creatinine)和胱抑素C(Cystatin C)含量检测：
小鼠经过药物处理后，采用眼球取血的方法进行取血，血液在37℃凝固后，3000r/min离心5min收集血清。血清送医院检测血尿素氮(BUN)、肌酐(creatinine)和胱抑素C等肾功能相关指标。如果血尿素氮(BUN)、肌酐(creatinine)和胱抑素C含量显著升高，表示肾功能受损。
结果及结果分析：甘露糖能缓解顺铂引起的小鼠肠道毒副作用，试验结果参见图2。
由图2的图a可知，甘露糖可以缓解顺铂引起的结直肠缩短。甘露糖组(Mannose)相较于对照组(Ctrl)小鼠结直肠长度没有变化，顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠结直肠长度显著缩短，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠结直肠长度显著恢复正常。说明发明所述甘露糖处理不会影响小鼠结直肠长度，且可以缓解化疗药物顺铂引起的小鼠结直肠的毒副作用。
图2的图a中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明甘露糖处理不会影响小鼠结直肠长度(p>0.05)，且可以缓解化疗药物顺铂引起的小鼠结直肠的毒副作用(p<0.01)。
由图2的图b可知，甘露糖可以缓解顺铂引起的小肠绒毛和隐窝损伤。甘露糖组(Mannose)相较于对照组(Ctrl)小鼠小肠绒毛和隐窝没有变化，顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠小肠绒毛数量减少、长度缩短以及小肠隐窝的减少，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠小肠绒毛和小肠隐窝显著恢复正常。说明发明所述甘露糖处理不会影响小鼠小肠绒毛和小肠隐窝，且可以缓解化疗药物顺铂引起的小鼠小肠绒毛和隐窝的损伤。
图2的图b中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明甘露糖处理不会影响小鼠小肠绒毛和隐窝(p>0.05)，且可以缓解化疗药物顺铂引起的小鼠小肠绒毛和隐窝的损伤(p<0.001)。
由图2的图c可知，甘露糖可以抑制顺铂引起的小肠GSDME蛋白剪切。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠小肠GSDME显著剪切，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠小肠GSDME剪切显著减少。说明发明所述甘露糖处理可以抑制化疗药物顺铂引起小鼠小肠GSDME的剪切。
甘露糖缓解顺铂引起的小鼠肾脏毒副作用试验结果参见图3。
由图3的图a可知，甘露糖可以缓解顺铂引起的肾脏毒副作用。甘露糖组(Mannose)相较于对照组(Ctrl)小鼠肾脏组织情况没有变化，顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠肾脏PAS染色显著更强，对肾脏损伤程度打分(Tubular injury score)也显示肾脏显著损伤，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠肾脏组织显著恢复正常，肾脏损伤程度打分也显著更低。说明发明所述甘露糖处理不会影响小鼠肾脏，且可以缓解化疗药物顺铂引起的小鼠肾脏组织损伤。
图3的图a中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明甘露糖处理不会影响小鼠肾脏(p>0.05)，且可以缓解化疗药物顺铂引起的小鼠肾脏组织损伤(p<0.001)。
由图3的图b可知，甘露糖可以缓解顺铂引起的肾功能损伤。甘露糖组(Mannose)相较于对照组(Ctrl)小鼠血尿素氮(BUN)、肌酐(creatinine)和胱抑素C含量没有变化，顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠血尿素氮(BUN)、肌酐(creatinine)和胱抑素C含量显著升高，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠血尿素氮(BUN)、肌酐(creatinine)和胱抑素C显著恢复正常。说明发明所述甘露糖处理不会影响小鼠肾脏，且可以缓解化疗药物顺铂引起的小鼠肾功能损伤。
图3的图b中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明甘露糖处理不会影响小鼠的肾功能(p>0.05)，且可以缓解化疗药物顺铂引起的小鼠肾功能损伤(p<0.001)。
由图3的图c可知，甘露糖可以抑制顺铂引起的肾脏GSDME蛋白剪切。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠肾脏GSDME显著剪切，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠肾脏GSDME剪切显著减少。说明发明所述甘露糖处理可以抑制化疗药物顺铂引起小鼠肾脏GSDME的剪切。
实施例3
甘露糖对化疗药物顺铂抑制裸鼠移植瘤生长以及对顺铂引起毒副作用的影响。
实验方法如下：
1.小鼠处理：
(1)将MKN45细胞按每只2×106细胞量接种到免疫缺陷型裸鼠，接种后第8天开始给药处理。
(2)将SPF级的免疫缺陷小鼠(～20g)随机分为四组，分别为对照组(Ctrl)、甘露糖组(Mannose)、顺铂组(Cisplatin)、顺铂+甘露糖共处理组(Cisplatin+Mannose)，饲养在12h光照，12h黑暗环境中。
(3)对照组(Ctrl)小鼠每天用500μL无菌水进行灌胃处理，甘露糖组(Mannose)和顺铂+甘露糖共处理组(Cisplatin+Mannose)小鼠进行甘露糖(Mannose，溶解于H2O中)灌胃预处理，每只小鼠灌胃20％的甘露糖500μL，每天处理一次，共处理7天。
(4)顺铂组(Cisplatin)和顺铂+甘露糖共处理组(Cisplatin+Mannose)小鼠进行顺铂(Cisplatin，溶于PBS中)处理，顺铂采用腹腔注射的方式，剂量为12mg/kg，在甘露糖处理后第一天和第四天分别注射一次。
2.移植瘤体积和重量测量：
小鼠接受药物处理开始后，每天测量肿瘤的体积，小鼠经过药物处理结束后，断颈处死小鼠，将移植瘤取出进行拍照和称重。
3.化疗药物副作用检测：
结直肠长度测量，小肠H&E组织化学染色分析，小肠和肾脏GSDME蛋白剪切检测，肾脏PAS组织化学染色分析，血尿素氮(BUN)、肌酐(creatinine)、胱抑素C含量检测(检测方法参考实施例2)。
结果及结果分析：甘露糖不会影响化疗药物顺铂对裸鼠移植瘤的抑制效果，试验结果参见图4。
由图4的图a可知，甘露糖不会影响顺铂对裸鼠移植瘤体积的抑制效果。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠肿瘤体积显著减小，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠肿瘤体积没有变化。表明本发明所述甘露糖不会影响化疗药物顺铂对裸鼠移植瘤体积的抑制效果。
图4的图a中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明甘露糖不会影响化疗药物顺铂对裸鼠移植瘤体积的抑制效果(p>0.05)。
由图4的图b可知，甘露糖不会影响顺铂对裸鼠移植瘤重量的抑制效果。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠肿瘤重量显著减小，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠肿瘤重量没有变化。表明本发明所述甘露糖不会影响化疗药物顺铂对裸鼠移植瘤重量的抑制效果。
图4的图b中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明甘露糖不会影响化疗药物顺铂对裸鼠移植瘤重量的抑制效果(p>0.05)。
在不影响顺铂对移植瘤抑制效果的情况下，甘露糖缓解顺铂引起的小鼠肠道毒副作用试验，结果参见图5。
由图5的图a可知，在不影响顺铂对移植瘤抑制效果的同时，甘露糖可以缓解顺铂引起的结直肠的毒副作用。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠结直肠长度显著缩短，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠结直肠长度显著恢复正常。表明在不影响化疗药物顺铂对移植瘤抑制效果的同时，发明所述甘露糖可以缓解化疗药物顺铂引起的小鼠结直肠的毒副作用。
图5的图a中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明在不影响化疗药物顺铂对移植瘤抑制效果的同时，甘露糖可以缓解化疗药物顺铂引起的小鼠结直肠的毒副作用(p<0.05)。
由图5的图b可知，在不影响顺铂对移植瘤抑制效果的同时，甘露糖可以缓解顺铂引起的小肠绒毛和隐窝的损伤。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠小肠绒毛数量减少、长度缩短以及小肠隐窝的减少，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠小肠绒毛和小肠隐窝显著恢复正常。表明在不影响化疗药物顺铂对移植瘤抑制效果的同时，发明所述甘露糖可以缓解化疗药物顺铂引起的小鼠小肠绒毛和隐窝的损伤。
图5的图b中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。 统计分析表明在不影响化疗药物顺铂对移植瘤抑制效果的同时，甘露糖可以缓解化疗药物顺铂引起的小鼠小肠绒毛和隐窝的损伤(p<0.001)。
由图5的图c可知，在不影响顺铂对移植瘤抑制效果的同时，甘露糖可以抑制顺铂引起小肠GSDME蛋白的剪切。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠小肠GSDME显著剪切，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠小肠GSDME剪切显著减少。表明在不影响化疗药物顺铂对移植瘤抑制效果的同时，发明所述甘露糖处理可以抑制化疗药物顺铂引起小鼠小肠GSDME蛋白的剪切。
在不影响顺铂对移植瘤抑制效果的情况下，甘露糖缓解顺铂引起的小鼠肾脏毒副作用试验，结果参见图6。
由图6的图a可知，在不影响顺铂对移植瘤抑制效果的同时，甘露糖可以缓解顺铂引起的肾脏组织损伤。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠肾脏PAS染色显著更强，对肾脏损伤程度打分(Tubular injury score)也显示肾脏显著损伤，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠肾脏组织显著恢复正常，肾脏损伤程度打分也显著更低。表明在不影响化疗药物顺铂对移植瘤抑制效果的同时，发明所述甘露糖可以缓解化疗药物顺铂引起的小鼠肾脏组织损伤。
图6的图a中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明在不影响化疗药物顺铂对移植瘤抑制效果的同时，甘露糖(Mannose)可以缓解化疗药物顺铂引起的小鼠肾脏组织损伤(p<0.001)。
由图6的图b可知，在不影响顺铂对移植瘤抑制效果的同时，甘露糖可以缓解顺铂引起的肾功能损伤。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠血尿素氮(BUN)、肌酐(creatinine)和胱抑素C含量显著升高，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠血尿素氮(BUN)、肌酐和胱抑素C显著恢复正常。表明在不影响化疗药物顺铂对移植瘤抑制效果的同时，发明所述甘露糖可以缓解化疗药物顺铂引起的小鼠肾功能损伤。图6的图b中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明在不影响化疗药物顺铂对移植瘤抑制效果的同时，甘露糖可以缓解化疗药物顺铂引起的小鼠肾功能损伤(p<0.001)。
由图6的图c可知，在不影响顺铂对移植瘤抑制效果的同时，甘露糖可以抑制顺铂引起的肾脏GSDME蛋白剪切。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠肾脏GSDME显著剪切，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠肾脏GSDME剪切显著减少。表明在不影响化疗药物顺铂对移植瘤抑制效果的同时，发明所述甘露糖处理可以抑制化疗药物顺铂引起小鼠肾脏GSDME的剪切。
实施例4
甘露糖对化疗药物顺铂抑制原发肝癌发生发展以及对顺铂引起毒副作用的影响。
实验方法如下：
1.小鼠处理：
(1)构建DEN/CCl4诱导小鼠原发肝癌模型，具体方法如下：将2周大的雄鼠按25mg/kg的剂量腹腔注射一针二乙基亚硝胺(DEN，溶于PBS中)，一周后开始每周注射一次10％的四氯化碳(CCl4，溶于玉米油中)，四氯化碳的剂量为0.5mL/kg，持续4～5个月。
(2)将诱导出肝癌的小鼠随机分为四组，分别为对照组、甘露糖组、顺铂组、顺铂+甘露糖共处理组，饲养在12h光照，12h黑暗环境中。
(3)对照组小鼠每天用500μL无菌水进行灌胃处理，甘露糖组和顺铂+甘露糖共处理组小鼠给予甘露糖(Mannose，溶解于H2O中)灌胃处理，每只小鼠灌胃20％的甘露糖500μL，每天处理一次，共处理4周。
(4)顺铂组和顺铂+甘露糖共处理组小鼠进行顺铂(Cisplatin，溶于PBS中)处理，顺铂采用腹腔注射的方式，剂量为8mg/kg，每周处理一次，共处理4周。
2.原发肝癌体积和数量测量：
小鼠经过药物处理结束后，断颈处死小鼠，取出小鼠肝脏，记录肿瘤数量。
3.化疗药物副作用检测：
结直肠长度测量，小肠H&E组织化学染色分析，小肠和肾脏GSDME蛋白剪切检测，肾脏PAS组织化学染色分析，血尿素氮、肌酐、胱抑素C含量检测(检测方法参考实施例2)。
结果及结果分析：甘露糖不会影响化疗药物顺铂对原发肝癌的抑制效果，结果参见图7。
由图7可知，顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠肝脏肿瘤体积和数量显著减小/少，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠肝脏肿瘤的体积和数量没有变化。表明本发明所述甘露糖不会影响化疗药物顺铂对原发肝癌的抑制效果。图7中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明甘露糖不会影响化疗药物顺铂对原发肝癌的抑制效果(p>0.05)。
图8给出在不影响顺铂对原发肝癌抑制效果的情况下，甘露糖缓解顺铂引起的小鼠肠道毒副作用试验结果。
由图8的图a可知，在不影响顺铂对原发肝癌抑制效果的同时，甘露糖可以缓解顺铂引起的结直肠的毒副作用。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠结直肠长度显著缩短，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠结直肠长度显著恢复正常。表明在不影响化疗药物顺铂对 原发肝癌抑制效果的同时，发明所述甘露糖可以缓解化疗药物顺铂引起的小鼠结直肠的毒副作用。图8的图a中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明在不影响化疗药物顺铂对原发肝癌抑制效果的同时，甘露糖可以缓解化疗药物顺铂引起的小鼠结直肠的毒副作用(p<0.001)。
由图8的图b可知，在不影响顺铂对原发肝癌抑制效果的同时，甘露糖可缓解顺铂引起的小肠绒毛和隐窝的损伤。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠小肠绒毛数量减少、长度缩短以及小肠隐窝的减少，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠小肠绒毛和小肠隐窝显著恢复正常。表明在不影响化疗药物顺铂对原发肝癌抑制效果的同时，发明所述甘露糖可以缓解化疗药物顺铂引起的小鼠小肠绒毛和隐窝的损伤。应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明在不影响化疗药物顺铂对原发肝癌抑制效果的同时，甘露糖可以缓解化疗药物顺铂引起的小鼠小肠绒毛和隐窝的损伤(p<0.001)。
由图8的图c可知，在不影响顺铂对原发肝癌抑制效果的同时，甘露糖可以抑制顺铂引起小肠GSDME蛋白的剪切。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠小肠GSDME显著剪切，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠小肠GSDME剪切显著减少。表明在不影响化疗药物顺铂对原发肝癌抑制效果的同时，发明所述甘露糖处理可以抑制化疗药物顺铂引起小鼠小肠GSDME蛋白的剪切。
在不影响顺铂对原发肝癌抑制效果的情况下，甘露糖缓解顺铂引起的小鼠肾脏毒副作用试验，结果参见图9。
由图9的图a可知，在不影响顺铂对原发肝癌抑制效果的同时，甘露糖可以缓解顺铂引起的肾脏组织损伤。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠肾脏PAS染色显著更强，对肾脏损伤程度打分(Tubular injury score)也显示肾脏显著损伤。顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠肾脏组织显著恢复正常，肾脏损伤程度打分也显著更低。表明在不影响化疗药物顺铂对原发肝癌抑制效果的同时，发明所述甘露糖可以缓解化疗药物顺铂引起的小鼠肾脏组织损伤。
图9的图a中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。 统计分析表明在不影响化疗药物顺铂对原发肝癌抑制效果的同时，甘露糖可以缓解化疗药物顺铂引起的小鼠肾脏组织损伤(p<0.001)。
由图9的图b可知，在不影响顺铂对原发肝癌抑制效果的同时，甘露糖可缓解顺铂引起的肾功能损伤。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠血尿素氮(BUN)、肌酐(creatinine)和胱抑素C含量显著升高，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠血尿素氮(BUN)、肌酐(creatinine)和胱抑素C显著恢复正常。表明在不影响化疗药物顺铂对原发肝癌抑制效果的同时，发明所述甘露糖可以缓解化疗药物顺铂引起的小鼠肾功能损伤。图9的图b中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明在不影响化疗药物顺铂对原发肝癌抑制效果的同时，甘露糖可缓解化疗药物顺铂引起的小鼠肾功能损伤(p<0.001)。
由图9的图c可知，在不影响顺铂对原发肝癌抑制效果的同时，甘露糖可以抑制顺铂引起的肾脏GSDME蛋白剪切。顺铂组(Cisplatin)相较于对照组(Ctrl)小鼠肾脏GSDME显著剪切，顺铂+甘露糖共处理组(Cisplatin+Mannose)相较于顺铂组(Cisplatin)小鼠肾脏GSDME剪切显著减少。表明在不影响化疗药物顺铂对原发肝癌抑制效果的同时，发明所述甘露糖处理可抑制化疗药物顺铂引起小鼠肾脏GSDME的剪切。
实施例5
甘露糖对化疗药物奥沙利铂(Oxaliplatin)抑制原发肝癌发生发展以及对奥沙利铂引起毒副作用的影响。
实验方法如下：
1.小鼠处理：
(1)构建DEN/CCl4诱导小鼠原发肝癌模型，具体方法如下：将2周大的雄鼠按25mg/kg的剂量腹腔注射一针二乙基亚硝胺(DEN，溶于PBS中)，一周后开始每周注射一次10％的四氯化碳(CCl4，溶于玉米油中)，四氯化碳的剂量为0.5mL/kg，持续4～5个月。
(2)将诱导出肝癌的小鼠随机分为四组，分别为对照组(Ctrl)、甘露糖组(Mannose)、奥沙利铂组(Oxaliplatin)、奥沙利铂+甘露糖共处理组(Oxaliplatin+Mannose)，饲养在12h光照，12h黑暗环境中。
(3)对照组(Ctrl)小鼠每天用500μL无菌水进行灌胃处理，甘露糖组(Mannose)和奥沙利铂+甘露糖共处理组(Oxaliplatin+Mannose)小鼠给予甘露糖(Mannose，溶解于H2O中)灌胃处理，每只小鼠灌胃20％的甘露糖500μL，每天处理一次，共处理4周。
(4)奥沙利铂组(Oxaliplatin)和奥沙利铂+甘露糖共处理组(Oxaliplatin+Mannose)小鼠进行奥沙利铂(Oxaliplatin，溶于5％葡萄糖溶液中)处理，奥沙利铂采用腹腔注射的方式，剂量为10mg/kg，每周处理一次，共处理4周。
2.原发肝癌体积和数量测量：
小鼠经过药物处理结束后，断颈处死小鼠，取出小鼠肝脏，记录肿瘤数量。
3.化疗药物副作用检测：
结直肠长度测量，小肠H&E组织化学染色分析，小肠和肾脏GSDME蛋白剪切检测(检测方法参考实施例2)。
结果及结果分析：甘露糖不会影响化疗药物奥沙利铂对原发肝癌的抑制效果，结果参见图10。
由图10可知，奥沙利铂组(Oxaliplatin)相较于对照组(Ctrl)小鼠肝脏肿瘤体积和数量显著减小/少，奥沙利铂+甘露糖共处理组(Oxaliplatin+Mannose)相较于奥沙利铂组(Oxaliplatin)小鼠肝脏肿瘤的体积和数量没有变化。表明本发明所述甘露糖不会影响化疗药物奥沙利铂(Oxaliplatin)对原发肝癌的抑制效果。图10中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明甘露糖不会影响化疗药物奥沙利铂对原发肝癌的抑制效果(p>0.05)。
在不影响奥沙利铂对原发肝癌抑制效果的情况下，甘露糖缓解奥沙利铂引起的小鼠肠道毒副作用试验，结果参见图11。
由图11的图a可知，在不影响奥沙利铂对原发肝癌抑制效果的同时，甘露糖可以缓解奥沙利铂引起的结直肠的毒副作用。奥沙利铂组(Oxaliplatin)相较于对照组(Ctrl)小鼠结直肠长度显著缩短，奥沙利铂+甘露糖共处理组(Oxaliplatin+Mannose)相较于奥沙利铂组(Oxaliplatin)小鼠结直肠长度显著恢复正常。表明在不影响化疗药物奥沙利铂对原发肝癌抑制效果的同时，发明所述甘露糖可以缓解化疗药物奥沙利铂引起的小鼠结直肠的毒副作用。图11的图a中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明在不影响化疗药物奥沙利铂对原发肝癌抑制效果的同时，甘露糖可以缓解化疗药物奥沙利铂引起的小鼠结直肠的毒副作用(p<0.05)。
由图11的图b可知，在不影响奥沙利铂对原发肝癌抑制效果的同时，甘露糖可以缓解奥沙利铂引起的小肠绒毛和隐窝的损伤。奥沙利铂组(Oxaliplatin)相较于对照组(Ctrl)小鼠小肠绒毛数量减少、长度缩短以及小肠隐窝的减少，奥沙利铂+甘露糖共处理组(Oxaliplatin+Mannose)相较于奥沙利铂组(Oxaliplatin)小鼠小肠绒毛和小肠隐窝显著恢复正常。表明在不影响化疗药物奥沙利铂对原发肝癌抑制效果的同时，甘露糖可缓解化疗药物奥沙利铂引起的小鼠小肠绒毛和隐窝的损伤。图11的图b中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001， 具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明在不影响化疗药物奥沙利铂对原发肝癌抑制效果的同时，甘露糖可缓解化疗药物奥沙利铂引起的小鼠小肠绒毛和隐窝的损伤(p<0.001)。
由图11的图c可知，在不影响奥沙利铂对原发肝癌抑制效果的同时，甘露糖可以抑制奥沙利铂引起小肠GSDME蛋白的剪切。奥沙利铂组(Oxaliplatin)相较于对照组(Ctrl)小鼠小肠GSDME显著剪切，奥沙利铂+甘露糖共处理组(Oxaliplatin+Mannose)相较于奥沙利铂组(Oxaliplatin)小鼠小肠GSDME剪切显著减少。表明在不影响化疗药物奥沙利铂对原发肝癌抑制效果的同时，发明所述甘露糖处理可抑制化疗药物奥沙利铂引起小鼠小肠GSDME蛋白的剪切。
实施例6
甘露糖对化疗药物奥沙利铂抑制原发胃癌发生发展以及对奥沙利铂引起毒副作用的影响。
实验方法如下：
1.小鼠处理：
(1)原发胃癌模型：采用在胃上皮细胞持续表达COX-2/mPGES-1诱导自发胃癌，48周大的小鼠用于后续实验。
(2)将原发胃癌的小鼠随机分为四组，分别为对照组(Ctrl)、甘露糖组(Mannose)、奥沙利铂组(Oxaliplatin)、奥沙利铂+甘露糖共处理组(Oxaliplatin+Mannose)，饲养在12h光照，12h黑暗环境中。
(3)对照组(Ctrl)小鼠每天用500μL无菌水进行灌胃处理，甘露糖组和奥沙利铂+甘露糖共处理组小鼠给予甘露糖(溶解于H2O中)灌胃处理，每只小鼠灌胃20％的甘露糖500μL，每天处理一次，共处理26天。
(4)奥沙利铂组和奥沙利铂+甘露糖共处理组小鼠进行奥沙利铂(溶于5％葡萄糖溶液中)处理，奥沙利铂采用腹腔注射的方式，在甘露糖处理一天后开始进行处理，剂量为3mg/kg，每天一次，按处理5天停药5天的方式共给药处理15次，共25天。
2.原发胃癌体积测量：
小鼠经过药物处理结束后，断颈处死小鼠，取出小鼠的胃，记录肿瘤体积。
3.化疗药物副作用检测：
结直肠长度测量，小肠H&E组织化学染色分析，小肠和肾脏GSDME蛋白剪切检测(检测方法参考实施例2)。
结果及结果分析：甘露糖不会影响化疗药物奥沙利铂对原发胃癌的抑制效果，实验结果参见图12。
由图12可知，奥沙利铂组较于对照组小鼠胃部肿瘤体积显著减小，奥沙利铂+甘露糖共处理组相较于奥沙利铂组小鼠胃部肿瘤的体积没有变化。表明本发明所述甘露糖不会影响化疗药物奥沙利铂对原发胃癌的抑制效果。
图12中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示 p<0.01，具差异高度显著性***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明甘露糖不会影响化疗药物奥沙利铂对原发胃癌的抑制效果(p>0.05)。
图13给出在不影响奥沙利铂对原发胃癌抑制效果的情况下，甘露糖缓解奥沙利铂引起的小鼠肠道毒副作用试验结果。
由图13的图a可知，在不影响奥沙利铂对原发胃癌抑制效果的同时，甘露糖可以缓解奥沙利铂引起的结直肠的毒副作用。奥沙利铂组(Oxaliplatin)相较于对照组(Ctrl)小鼠结直肠长度显著缩短，奥沙利铂+甘露糖共处理组(Oxaliplatin+Mannose)相较于奥沙利铂组小鼠结直肠长度显著恢复正常。表明在不影响化疗药物奥沙利铂对原发胃癌抑制效果的同时，发明所述甘露糖可缓解化疗药物奥沙利铂引起的小鼠结直肠的毒副作用。图13的图a中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明在不影响化疗药物奥沙利铂对原发胃癌抑制效果的同时，甘露糖可以缓解化疗药物奥沙利铂引起的小鼠结直肠的毒副作用(p<0.05)。
由图13的图b可知，在不影响奥沙利铂对原发胃癌抑制效果的同时，甘露糖可以缓解奥沙利铂引起的小肠绒毛和隐窝的损伤。奥沙利铂组(Oxaliplatin)相较于对照组(Ctrl)小鼠小肠绒毛数量减少、长度缩短以及小肠隐窝的减少，奥沙利铂+甘露糖共处理组(Oxaliplatin+Mannose)相较于奥沙利铂组(Oxaliplatin)小鼠小肠绒毛和小肠隐窝显著恢复正常。表明在不影响化疗药物奥沙利铂对原发胃癌抑制效果的同时，发明所述甘露糖可以缓解化疗药物奥沙利铂引起的小鼠小肠绒毛和隐窝的损伤。图13的图b中，应用Graphpad prism 9统计软件对实验数据进行统计分析，所得实验数据以均数±标准误差表示；对照组与实验组差异分析采用两因素方差分析及fisher检验，*表示p<0.05，具差异显著性；**表示p<0.01，具差异高度显著性；***表示p<0.001，具差异极其显著性；ns表示p>0.05，没有显著差异。统计分析表明在不影响化疗药物奥沙利铂对原发胃癌抑制效果的同时，甘露糖可以缓解化疗药物奥沙利铂引起的小鼠小肠绒毛和隐窝的损伤(p<0.001)。
由图13的图c可知，在不影响奥沙利铂对原发胃癌抑制效果的同时，甘露糖可以抑制奥沙利铂引起小肠GSDME蛋白的剪切。奥沙利铂组(Oxaliplatin)相较于对照组(Ctrl)小鼠小肠GSDME显著剪切，奥沙利铂+甘露糖共处理组(Oxaliplatin+Mannose)相较于奥沙利铂组(Oxaliplatin)小鼠小肠GSDME剪切显著减少。表明在不影响化疗药物奥沙利铂对原发胃癌抑制效果的同时，发明所述甘露糖处理可以抑制化疗药物奥沙利铂引起小鼠小肠GSDME蛋白的剪切。
实施例7：
甘露糖抑制细胞内GSDME介导的细胞焦亡信号通路机制研究
实验方法如下：
1.细胞处理：
除特别描述外，细胞培养方式与药物处理方式同实施例1，其中图14、图15、图16与图20中的CCCP/FeSO4或CCCP/FeSO4+Mannose表示同时使用CCCP(羰基氰化氯苯腙)(20μg/mL，溶解在DMSO)和FeSO4(100μg/mL，溶解在H2O)对细胞进行处理，其中CCCP/FeSO4+Mannose还采用Mannose(20mM，溶解在H2O)对细胞进行处理；图15中图c的DON表示使用DON(6-重氮-5-氧代-L-正亮氨酸)(40μM，溶解在H2O)对细胞进行处理；图16中图b的CompoundC表示使用Compound C(盐酸双吗啡肽)(12.5μM，溶解在1M HCl)对细胞进行处理；图17中图c与图18中图b中的GlcN-6P表示使用GlcN-6P(6-磷酸氨基葡萄糖)(20mM，溶解在H2O)对细胞进行处理，图17中图c与图18中图b中的GlcNAc-6P表示使用GlcNAc-6P(6-磷酸-N-乙酰氨基葡萄糖)(20mM，溶解在H2O)对细胞进行处理，图17中图c与图18中图b中的GlcNAc-1P表示使用GlcNAc-1P(N-乙酰基葡萄糖胺-1-磷酸二钠盐)(20mM，溶解在H2O)对细胞进行处理，图17中图c与图18中图b中的UDP-GlcNAc表示使用UDP-GlcNAc(尿苷-5′-二磷酸-N-乙酰基-葡糖胺钠盐)(20mM，溶解在H2O)对细胞进行处理。其中GlcN-6P、GlcNAc-6P、GlcNAc-1P和UDP-GlcNAc在处理细胞前应使用链球菌溶血素O(SLO，200ng/mL)提前预处理细胞10分钟，使细胞膜通透性增加，加快细胞对代谢物摄取。
2.利用CRISPR/Cas9技术构建基因敲除细胞系
首先通过分子克隆技术构建慢病毒骨架sgRNA质粒，然后通过磷酸钙转染的方式进行病毒包装：sgRNA质粒：包装质粒psPAX2(Addgene)：包装质粒pMD2.G(Addgene)＝4μg:3μg:1μg(6cm培养皿)，然后转染HEK293T细胞来产生慢病毒；转染8-12小时后，除去培养基，并加入含有NEAA的新鲜温热培养基，转染48小时后收集慢病毒上清液。用慢病毒感染目的细胞(图15中图b，图17中图a与图20所使用细胞均为A375)，并添加10μg/ml聚丙烯(Sigma)。24小时后，将细胞用嘌呤霉素(InvivoGen)处理2-3天。通过免疫印迹法确定敲除效率。维持具有较高敲除效率(超过80％)的嘌呤霉素抗性细胞系用于进一步实验。
其中用于构建sgRNA质粒的DNA序列如下所示：
对照用质粒：NTC sgRNA序列AAATGTCAGGCCGCGCCGTT
图15中图b与图17中图a所使用GFAT1 sgRNA序列CTCCGTACACCAATCAACAG
图15中图b与图17中图a所使用GFAT2 sgRNA序列TTCAAGAGTGTCCACTACCC
图16中图c所使用AMPKα1 sgRNA序列TCCTGTTACAGATTGTATGC
图16中图c所使用AMPKα2 sgRNA序列ACGTTATTTAAGAAGATCCG
图20所使用GSDME sgRNA序列AGTCTTCATTTGGAACCCTG
其中图15中图b与图17中图a所描述“GFAT1/2 sgRNA”表示同时在细胞中敲除蛋白GFAT1和GFAT2；其中16中图c所描述“AMPKα1/α2 sgRNA”表示同时在细胞中敲除蛋白AMPKα1和AMPKα2。
3.蛋白质敲除后回补分析
慢病毒转染法表达蛋白：利用磷酸钙转染法将带有目的基因的过表达质粒(4μg，6cm培养皿)转染进HEK293T细胞来产生慢病毒，将产生的慢病毒收集后，感染上述“2.利用CRISPR/Cas9技术构建基因敲除细胞系”中构建完成的对应基因敲除细胞系，获取蛋白质敲除后回补细胞系。图20中所描述细胞系为在感染了GSDME sgRNA病毒后的细胞系中，再次感染带有第六位苏氨酸突变成谷氨酸(GSDMET6E)的GSDME点突变慢病毒所得细胞系。
4.体外磷酸化分析
在HEK293T细胞中共表达AMPK的三个亚基：带有Flag标签的AMPKα1(Flag-AMPKα1)，带有HA标签的AMPKβ1(HA-AMPKβ1)和带有Myc标签的AMPKγ1(Myc-AMPKγ1)蛋白，其中质粒Flag-AMPKα1、HA-AMPKβ1和Myc-AMPKγ1的比例为4.5μg:3μg:3.6μg(10cm培养皿)，并用抗HA抗体免疫沉淀AMPK复合物(AMPK complex)。将免疫沉淀纯化的AMPK复合物与细菌表达的带有GST标签的GSDME蛋白(GST-GSDME)在100μl激酶缓冲液(20mM HEPES(4-羟乙基哌嗪乙磺酸)，pH 7.4；5mM MgCl2；1mM EGTA(乙二醇双(2-氨基乙基醚)四乙酸)；1mM DTT(二硫苏糖醇)；1％蛋白酶和磷酸酶抑制剂混合物；200μMATP(腺嘌呤核苷三磷酸))在37℃下孵育30分钟。通过加入SDS上样缓冲液终止反应，得到磷酸化的GST-GSDME样品。然后将反应混合物通过蛋白质印迹确定磷酸化水平变化。
5.质谱分析蛋白磷酸化位点
同上述“3.体外磷酸化分析”实验方法制备磷酸化的GST-GSDME样品，将磷酸化的GST-GSDME样品(4μg)进行SDS-PAGE并用考马斯蓝染色。切下80kDa左右特异性条带并用胰凝乳蛋白酶消化，然后通过磷酸化质谱分析。
6.液质联用(LC-MS)检测细胞中代谢物水平变化
图17中图b所描述的不同时间梯度的甘露糖处理完成后的细胞中GlcNAc-6P与GlcNAc-1P代谢物水平的变化检测方式：用甘露糖处理细胞不同时间后，处理完成后的细胞(5×106)用PBS(4℃)洗涤三次，每个样品中的代谢物用80％甲醇溶液(1.6mL，-80℃)萃取，得到萃取物。将萃取物收集到2ml管中，涡旋1分钟并在14000×g和4℃条件下离心10分钟。用真空离心机(Labconco Corporation)将上清液蒸发完全。将样品重悬于200μl 50％乙腈中。注入每个样品(2μl)并用与UPLC系统(Waters，ACQUITY UPLC系统)连接的QTRAP(SCIEX，QTRAP 6500plus)质谱仪。该柱是柱(5μm，50×2mm，Phenomenex)。流动相缓冲液A(pH＝9.7)为20mMNH 4Ac，流动相缓冲液B为100％乙腈。柱和样品的温度分别为40℃和10℃。使用0.3mL/min的恒定流速，梯度如下：t＝0-1分钟，90％B；t＝15-17分钟，30％B；t＝17-20分钟，90％B。QTRAP质谱仪以MRM模式以负模式运行，并使用分析标准优化去聚电位和碰撞能量。
7.等温滴定量热法(ITC)分析
使用MicroCal iTC200仪器(GE Healthcare)在25℃下测量AMPKα1与GlcNAc-6P之间的结合亲和力。将AMPKα1在含有25mM Tris(pH 8.0)，300mMNaCl，5mM MgCl 2，1mM EDTA，10％甘油，2mM DTT和10mM GSH溶解之后，用PBS透析蛋白质。同时在PBS中制备底物。在滴定实验期间，细胞中AMPKα1蛋白浓度为10μM，而作为注射器中底物的GlcNAc-6P浓度为1mM。
8.免疫共沉淀技术(co-IP)分析
在细胞裂解缓冲液A(50mM Tris，pH 7.4，300mM NaCl，1％NP-40，临用前加1mM PMSF和全酶抑制剂Cocktail)中裂解细胞。将细胞裂解物与特异性抗体和蛋白G-琼脂糖珠(Millipore)在4℃转动孵育3小时。离心收集琼脂糖珠并用细胞裂解缓冲液A洗涤三次，随后通过免疫印迹技术对结果进行分析。对于图18中图b所描述的Flag-AMPKα1与HA-LKB1之间免疫共沉淀的分析，首先通过慢病毒转染Flag-AMPKα1和HA-LKB1进入A375细胞内，用GlcNAc-6P(6-磷酸-N-乙酰氨基葡萄糖)(20mM，溶解在H2O)处理细胞6小时，随后用HA特异性抗体进行免疫沉淀分析。对于图19中图a所描述的AMPKα1和GSDME之间免疫共沉淀的分析，将细胞裂解物与抗GSDME抗体(Proteintech)在4℃温育过夜，然后在4℃下加入蛋白G-琼脂糖珠30分钟。
结果及结果分析：结果见图14～图20。
甘露糖(Mannose)可以抑制黑色素瘤细胞A375中，由羰基氰化氯苯腙(CCCP)和FeSO4(一种诱导方式)诱导的细胞焦亡，具体表现为细胞鼓泡的现象(图14的图a)，细胞中GSDME蛋白的剪切激活(图14的图b)，细胞中乳酸脱氢酶(LDH)的释放(图14的图c)能够被甘露糖抑制住。随后我们对机制展开了研究，甘露糖进入细胞后的代谢方式如(图15的图a)所示，其中甘露糖会进入到己糖胺代谢通路中，我们在细胞中同时敲除了GFAT1和GFAT2(这两个蛋白酶是甘露糖进入己糖胺代谢通路的第一个酶，也是最关键的酶)，发现甘露糖就无法继续抑制CCCP和FeSO4诱导的细胞焦亡(图15的图b)，同时如果加DON(一种能够抑制GFAT1的蛋白酶活性的药物)就发现在这种条件下，甘露糖也不能抑制CCCP和FeSO4诱导的细胞焦亡(图15的图c)，这就说明甘露糖进入细胞后，需要进入到己糖胺代谢通路中进行代谢反应才能发挥抑制细胞焦亡的作用。
甘露糖在进入到细胞后，会引起细胞内AMPK酶的激活，体现在AMPK的磷酸化水平的升高，以及其下游底物ACC的磷酸化水平的升高(图16的图a)，我们发现加入Compound C(AMPK的蛋白酶活性抑制剂)后，甘露糖不能继续抑制CCCP和FeSO4诱导的细胞焦亡(图16的图b)，同时在细胞中敲除AMPKα1和AMPKα2后，发现甘露糖也不能继续抑制CCCP和FeSO4诱导的细胞焦亡(图16的图c)，那就说明甘露糖引起的AMPK的激活在它发挥抑制细胞焦亡的功能上是必不可少的。同时发现敲除了GFAT后，甘露糖激活AMPK的能力也变弱了(图17的图a)，证明己糖胺代谢通路也是甘露糖激活AMPK的必经之路。
我们通过对甘露糖加入后细胞内的代谢物水平变化进行分析，发现在细胞内加入甘露糖之后，会引起 细胞内代谢物GlcNAc-6P的浓度显著升高，而作为对照GlcNAc-1P的浓度则没有什么变化(图17的图b)，GlcNAc-6P是甘露糖进入到己糖胺代谢通路后产生的一个代谢物，同时我们直接向细胞中加入GlcNAc-6P后，也可以引起细胞内AMPK的磷酸化水平升高，激活AMPK，达到加入甘露糖类似的效果(图17的图c)。同时我们还发现GlcNAc-6P能够直接与AMPK的α亚基AMPKα1结合(图18的图a)，同时通过免疫共沉淀技术(co-IP)分析发现这种结合还能够增强AMPK与其上游激活蛋白LKB1的结合(图18的图b)，那就证明甘露糖进入细胞后代谢成为GlcNAc-6P，GlcNAc-6P可以直接和AMPK结合，然后可以增强AMPK与其上游蛋白LKB1的结合能力，最终增强AMPK的激活。
接下来就是对AMPK的激活后抑制细胞焦亡的机制进行了探究。我们首先发现AMPK与细胞焦亡的关键蛋白GSDME能够直接结合(图19的图a)，因为AMPK最主要的酶活性是其所具有的激酶活性(即磷酸化其他蛋白的能力)，我们随后发现AMPK能够增强GSDME的磷酸化水平(图19的图b)，同时我们通过磷酸化质谱的方式检测到AMPK会引起GSDME的第六位苏氨酸发生较强的磷酸化修饰水平升高(图19的图c)，为了探究这种磷酸化修饰的功能，我们发现将GSDME的第六位苏氨酸突变成谷氨酸(即T6E突变体，是一种在该位点模拟磷酸化的突变体)后，CCCP和FeSO4就无法继续诱导细胞焦亡(图20)，那么就证明甘露糖引起的AMPK的激活，会进一步引起GSDME蛋白的第六位苏氨酸的磷酸化水平增加，该位点磷酸化水平增加后就会抑制CCCP和FeSO4诱导其剪切激活的能力。
实施例8：
甘露糖抑制化疗药物顺铂毒副作用机制的验证
实验方法如下：
1.小鼠处理：
(1)基因修饰小鼠获取：GSDMET6E小鼠由厦门大学实验动物中心构建。AMPK-DKO小鼠由林圣彩教授(厦门大学生命科学学院细胞应激生物学国家重点实验室)赠送。
(2)将野生型小鼠(WT)及对应GSDME第六位苏氨酸模拟点突变小鼠(GSDMET6E)分别随机分为4组，分别为对照组(Ctrl)、甘露糖组(Mannose)、顺铂组(Cisplatin)、顺铂+甘露糖共处理组(Cisplatin+Mannose)；将野生型小鼠(WT)及对应肠道特异性敲除AMPK小鼠(AMPK-DKO)分别随机分为2组，分别为顺铂组(Cisplatin)、顺铂+甘露糖共处理组(Cisplatin+Mannose)。饲养在12h光照，12h黑暗环境中。
(3)对照组(Ctrl)小鼠每天用500μL无菌水进行灌胃处理，甘露糖组和顺铂+甘露糖共处理组小鼠给予甘露糖(溶解于H2O中)灌胃处理，每只小鼠灌胃20％的甘露糖500μL，每天处理一次，共处理6天。
(4)顺铂组和顺铂+甘露糖共处理组小鼠进行顺铂(溶于5％葡萄糖溶液中)处理，顺铂采用腹腔注射的方式，在甘露糖处理一天后开始进行处理，剂量为18mg/kg，甘露糖预处理1天后给药1次即可。
2.化疗药物副作用检测：
结直肠长度测量，小肠H&E组织化学染色分析，小肠和肾脏GSDME蛋白剪切检测(检测方法参考实施例2)。
结果及结果分析：
1.为了证明甘露糖在发挥抑制正常组织化疗副作用的过程中AMPK所发挥的重要作用，我们在小鼠的小肠上皮细胞中敲除了AMPK蛋白(AMPK-DKO)，通过对比WT小鼠与AMPK-DKO小鼠在顺铂+甘露糖共处理组(Cisplatin+Mannose)的表现可以发现敲除AMPK后，甘露糖无法继续抑制化疗药物顺铂对小鼠的结肠(图21的图a)，小肠产生的损伤(图21的图b和图c)。
2.为了证明甘露糖在发挥抑制正常组织化疗副作用的过程中GSDME蛋白的第六位苏氨酸磷酸化水平的升高所发挥的重要作用，我们构建了GSDMET6E的小鼠，通过对比WT小鼠与GSDMET6E小鼠在顺铂组(Cisplatin)的表现可以发现将第六位苏氨酸带上磷酸化修饰之后，化疗药物对小鼠的正常组织造成的损伤大大降低了(图22和图23)，那么就证明甘露糖引起的小鼠GSDME的第六位苏氨酸磷酸化是其发挥抑制副作用功能的关键。
实施例9：
甘露糖对XELOX治疗方案引起患者肠道损伤的影响
实验方法如下：
1.患者分组和实验设计：
2022年4月至2022年9月，哈尔滨医科大学附属第一医院收治8例诊断为胃肠癌并在术后辅助化疗(XELOX方案)中出现严重腹泻的患者。所有患者均获得书面知情同意书。该研究得到伦理委员会委员会(2022167)的批准，并根据赫尔辛基宣言进行。患者包括年龄在18至65岁之间，均具有接受XELOX方案后腹泻的阳性病史。如果患者接受靶向治疗或免疫治疗，改变化疗方案，未能完成化疗过程，则排除患者。符合条件的患者在两个随机但连续的XELOX方案周期(每个周期21天)中入组。在前一个周期中，患者接受常规XELOX方案(作为对照)；在后一个周期中，相同的患者从接受XELOX方案的第1天开始每天一次口服500mg甘露糖(NOWFoods)，直到化疗周期完成的第21天。在这些连续的化疗周期中每天记录排便次数。在第21天收集并评估患者血液和粪便。
结果及结果分析：结果见图24的图a和图24的图b。通过对8名患有胃癌或结直肠癌且正在接受化疗的患者进行临床试验，我们发现当患者正常接受化疗时，会产生较为强烈的副作用，如腹泻(每日排便次数增加)，肠炎(粪便中白细胞的数目增加)，化疗的同时服用甘露糖可以明显减弱化疗药物产生的副作用(图24的图a，图24的图b)。
本发明的方法已经通过较佳实施例进行了描述，相关人员明显能在本发明内容、精神和范围内对本文 所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明内。

Claims (10)

  1. 甘露糖在制备抑制细胞焦亡或减轻化疗药物毒副作用的药物、试剂盒、药物组合物或保健品中应用，所述毒副作用为器官损伤。
  2. 如权利要求1所述应用，其特征在于所述器官为肠道或肾脏。
  3. 如权利要求1所述应用，其特征在于所述化疗药物为顺铂、卡铂、奥沙利铂、环磷酰胺、异环磷酰胺、美法仑、白消安、氮芥、苯丁酸氮芥、塞替派、卡莫司汀、尼莫司汀、洛莫司汀、司莫司汀、培美曲塞、甲氨蝶呤、氟脲嘧啶、卡培他滨、阿糖胞苷、吉西他滨、6-巯基嘌呤、硫鸟嘌呤、柔红霉素、多柔比星、表柔比星、阿柔比星、伊达比星、博来霉素、丝裂霉素、长春新碱、长春地辛、长春瑞滨、喜树碱、伊立替康、托泊替康、紫杉醇、多西紫杉醇、白蛋白结合型紫杉醇、紫杉醇脂质体、依托泊苷、替尼泊苷、丙卡巴肼、达卡巴嗪、替莫唑胺、L-门冬酰胺酶、去甲斑蝥素、甲基斑蝥素，或其药学上可接受的盐中的至少一种。
  4. 如权利要求1所述应用，其特征在于所述药物制成以下剂型：片剂、胶囊、粒剂或注射液。
  5. 如权利要求1所述应用，其特征在于所述药物组合物包含甘露糖，或其药学上可接受的盐，以及药学上可接受的载体。
  6. 如权利要求1所述应用，其特征在于所述试剂盒包括：(i)包含甘露糖的试剂，和(ii)包含化疗药物的试剂。
  7. 甘露糖在制备用于抑制化疗药物诱导正常肠和肾细胞焦亡的药物中应用。
  8. 甘露糖在制备用于缓解化疗药物导致肠道毒副作用的药物中应用。
  9. 甘露糖在制备用于缓解化疗药物导致肾脏毒副作用的药物中应用。
  10. 如权利要求7～9中任一项所述应用，其特征在于所述药物还包含有效量的化疗药物。
PCT/CN2023/093225 2022-05-11 2023-05-10 甘露糖抑制细胞焦亡或减轻化疗药物毒副作用的应用 Ceased WO2023217179A1 (zh)

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