WO2022236814A1 - 一种褐藻寡糖的应用 - Google Patents

一种褐藻寡糖的应用 Download PDF

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WO2022236814A1
WO2022236814A1 PCT/CN2021/093850 CN2021093850W WO2022236814A1 WO 2022236814 A1 WO2022236814 A1 WO 2022236814A1 CN 2021093850 W CN2021093850 W CN 2021093850W WO 2022236814 A1 WO2022236814 A1 WO 2022236814A1
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alginate
renal
group
injury
effect
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PCT/CN2021/093850
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English (en)
French (fr)
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刘振德
高河勇
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海糖（江苏）生物医药科技有限公司
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Priority to PCT/CN2021/093850 priority Critical patent/WO2022236814A1/zh
Priority to EP21941384.6A priority patent/EP4338744A1/en
Priority to JP2023570073A priority patent/JP2024517483A/ja
Publication of WO2022236814A1 publication Critical patent/WO2022236814A1/zh

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • 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
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages

Definitions

  • the invention relates to an application of fucoidan oligosaccharide, which belongs to the technical field of biomedicine.
  • Alginate is mainly found in the cell walls of kelp, sargassum and macroalgae, and is a kind of linear, unbranched, negatively charged polysaccharide compound.
  • Alginate is a binary compound composed of ⁇ -D-(1,4)-mannuronic acid (M) and ⁇ -L-(1,4)-guluronic acid (G). linear block compounds.
  • PM Polymannuronate
  • PG Polyguluronate
  • alginate has been widely used in medical biomaterials and drug sustained and controlled release materials due to its unique physical and chemical properties and good biocompatibility. Studies have also found that alginate has biological activities such as anti-oxidation, immune regulation, and anti-tumor. However, due to its large molecular weight and strong gelation, alginate is not easily absorbed, which greatly limits its application. Oligosaccharides have attracted people's attention because of their clear structure, significant activity, good absorbability, and small side effects.
  • alginate oligosaccharides In recent years, due to the unique structure of alginate oligosaccharides, its activity research has become a hot spot in the research of sugar drugs, and its biological activity research has made important progress. Studies have found that alginate oligosaccharides and their derivatives have a variety of biological activities, such as antioxidant, anti-tumor, anti-coagulation, immune regulation, neuroprotection, anti-inflammatory activity, anti-viral activity, anti-senile dementia, anti-uria Road stones, anti-diabetes, etc.
  • biological activities such as antioxidant, anti-tumor, anti-coagulation, immune regulation, neuroprotection, anti-inflammatory activity, anti-viral activity, anti-senile dementia, anti-uria Road stones, anti-diabetes, etc.
  • Carbohydrates are a class of highly complex and varied biomacromolecules. Unlike oligonucleotides and peptides, carbohydrates are not just linear oligomers but are often branched. The nine common monosaccharides found on mammalian cells can be linked into more diverse structures than the 20 naturally occurring amino acids or the four nucleotides. This complexity in the structure of carbohydrates makes it very difficult to obtain pure carbohydrates from natural sources. Regardless of chemical cleavage or enzymatic cleavage, it is difficult to separate oligosaccharides or polysaccharides with a uniform degree of polymerization. So far, almost all studies have used oligosaccharides or polysaccharides as a mixture of a series of sugars with close polymerization degrees, which brings great difficulties to their activity research, metabolism, toxicology and drug quality research.
  • the fucobiose has two structures of ⁇ G and/or ⁇ M and a combination of any ratio thereof; fucotriose has four structures of ⁇ GG, ⁇ GM, ⁇ MM and ⁇ MG and a combination of any ratio thereof; fucotetraose has two structures of ⁇ GGG, ⁇ GGM, Eight structures of ⁇ GMG, ⁇ GMM, ⁇ MMG, ⁇ MMM, ⁇ MGG and ⁇ MGM and their combinations in any ratio; all oligosaccharides are linked by glycosidic bonds at positions 1 and 4 of monosaccharides; G stands for ⁇ -L-guluronic acid; M stands for ⁇ -D-mannuronic acid; ⁇ means that ⁇ -elimination occurs at the 4,5 positions of ⁇ -L-guluronic acid and/or ⁇ -D-mannuronic acid, and the 4,5 positions of the non-reducing end are generated as common Unsaturated monosaccharides with yoke double bonds; the structure of each monosacchari
  • AKI acute kidney injury
  • CKD long-term chronic kidney disease
  • ESRD end-stage renal disease
  • AKI has become a worldwide public health problem that threatens human health.
  • AKI is a primary lesion of renal parenchyma (glomerulus, renal tubules, interstitium, etc.), with complex etiology (such as ischemia, hypoxia, poisons, drugs, etc.) , infection, etc.), the course of the disease progresses rapidly, and some patients will progress to chronic kidney disease accompanied by complications such as cardiovascular disease.
  • complex etiology such as ischemia, hypoxia, poisons, drugs, etc.
  • ischemia ischemia, hypoxia, poisons, drugs, etc.
  • infection etc.
  • Early diagnosis and timely intervention can minimize renal injury and promote renal function recovery.
  • Early identification and correction of reversible causes, maintenance of internal environment stability, nutritional support, prevention of complications, and renal replacement therapy are still the main treatment strategies for AKI.
  • Hypertensive nephropathy is due to long-term elevation of blood pressure that causes renal vascular lesions, renal capillary thickening, thickening, glomerular fibrosis, vascular lumen narrowing, renal arteriosclerosis, renal parenchymal ischemia, nephron reduction, etc. .
  • Changes in the renal parenchyma will lead to a decrease in the hemofiltration function of the kidney and a decrease in renal function.
  • This is a long-term renal secondary disease caused by continuous increase in blood pressure.
  • the course of the disease is long, and blood pressure control is the basic treatment measure.
  • the current treatment of hypertensive nephropathy mainly adopts the therapy of blood pressure control, however, this therapy is not suitable for the treatment of AKI. Given the complex etiology of AKI, identifying a single therapy that would benefit all AKI patients is challenging.
  • the present invention conducts further research on the fucoidan oligosaccharide, and provides an application of the fucoidan oligosaccharide in the treatment of acute kidney injury.
  • the purpose of the present invention is to provide a kind of application of fucoidan oligosaccharide.
  • the present invention provides a use of a fucoidan oligosaccharide or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating acute kidney injury, wherein the fucoidan oligosaccharide is fucobiose, fucotriose or fucoidan sugar.
  • the fucoidan oligosaccharides are composed of monosaccharides G, M and/or ⁇ linked by glycosidic bonds at positions 1 and 4; wherein, G represents ⁇ -L-guluronic acid , M means ⁇ -D-mannuronic acid, ⁇ means ⁇ -elimination occurs at the 4,5 positions of ⁇ -L-guluronic acid or ⁇ -D-mannuronic acid, and the 4,5 positions are conjugated Unsaturated monosaccharides with double bonds.
  • the fucobiose is selected from ⁇ G, ⁇ M or a combination thereof.
  • the fucoidose is selected from one or more of ⁇ GG, ⁇ GM, ⁇ MM and ⁇ MG.
  • the fucotetraose is selected from one or more of ⁇ GGG, ⁇ GGM, ⁇ GMG, ⁇ GMM, ⁇ MMG, ⁇ MMM, ⁇ MGG and ⁇ MGM.
  • the pharmaceutically acceptable salt is sodium salt, potassium salt, calcium salt, magnesium salt and/or ammonium salt.
  • the acute kidney injury is caused by hypoperfusion, infection, or nephrotoxicity of a drug.
  • the present invention provides a fucoidan oligosaccharide or a pharmaceutically acceptable salt thereof for use in the treatment of acute kidney injury, wherein the fucoidan oligosaccharide is fucobiose, fucotriose or fucotetraose.
  • the present invention provides a method for treating acute kidney injury, comprising administering a therapeutically effective amount of fucoidan oligosaccharide or a pharmaceutically acceptable salt thereof to a patient in need, wherein the fucoidan oligosaccharide is fucobiose, fucoidan sugar or fucoidose.
  • the fucodiose, trisaccharide and tetrasaccharide with a uniform polymerization degree of the present invention have revolutionary progress in the quality control, pharmacology, toxicology and other analysis and research of carbohydrate raw materials.
  • fucobiose trisaccharides
  • tetrasaccharides have very obvious Protective effects.
  • the fucoidan oligosaccharide of the present invention treats animals with acute kidney injury, the serum creatinine level decreases significantly, the urine concentration function of the kidney recovers significantly, the level of renal tubular injury factors (KIM-1, NGAL) is significantly reduced, the expression of inflammatory factors is significantly decreased, and the renal pathology The changes were significantly improved, and the therapeutic effect was enhanced with the dose increasing. Therefore, the fucoidan oligosaccharide of the present invention has a strong effect on treating kidney damage.
  • Figure 1 shows the high performance liquid chromatogram of fucobiose at a wavelength of 230nm
  • Figure 2 shows the hydrogen nuclear magnetic spectrum ( 1 HNMR, solvent D 2 O) of fucobiose
  • FIG. 3 shows the high resolution mass spectrum (HRMS (ESI)) of fucobiose
  • Figure 4 shows the high performance liquid chromatogram of fucoidan at a wavelength of 230nm
  • FIG. 5 shows the hydrogen nuclear magnetic spectrum ( 1 HNMR, the solvent is D 2 O) of fucoidan
  • Figure 6 shows the high resolution mass spectrum (HRMS (ESI)) of fucoidan
  • Figure 7 shows a high performance liquid chromatogram of fucoidose at a wavelength of 230nm
  • Figure 8 shows the hydrogen nuclear magnetic spectrum ( 1 HNMR, the solvent is D 2 O) of fucotetraose
  • Figure 9 shows the high resolution mass spectrum (HRMS (ESI)) of fucotetraose
  • Figure 10 shows the effect of fucobiose on rat serum creatinine levels caused by acute ischemia-reperfusion (I/R) injury;
  • Figure 11 shows the effect of fucobiose on the urine output of rats caused by acute ischemia-reperfusion (I/R) injury
  • Figure 12 shows the effect of fucobiose on the mRNA levels of Kim-1 and NGAL in rat kidney tissue caused by acute ischemia-reperfusion (I/R) injury;
  • Figure 13 shows the effect of fucobiose on the inflammatory indicators in rat kidney tissue caused by acute ischemia-reperfusion (I/R) injury;
  • Figure 14 shows the effect of fucoidan on serum creatinine levels in rats caused by acute ischemia-reperfusion (I/R) injury;
  • Figure 15 shows the effect of fucoidan on the urine output of rats caused by acute ischemia-reperfusion (I/R) injury
  • Figure 16 shows the effect of fucoidan on the mRNA levels of Kim-1 and NGAL in rat kidney tissue caused by acute ischemia-reperfusion (I/R) injury;
  • Figure 17 shows the effect of fucoidan on inflammatory indicators in rat kidney tissue caused by acute ischemia-reperfusion (I/R) injury;
  • Figure 18 shows the effect of fucoidan on pathological sections of rat kidney tissue damage caused by acute ischemia-reperfusion (I/R) injury;
  • Figure 19 shows the effect of fucotetraose and mixed sugars on rat serum creatinine levels caused by acute ischemia-reperfusion (I/R) injury;
  • Figure 20 shows the effect of fucotetraose and mixed sugars on the urine output of rats caused by acute ischemia-reperfusion (I/R) injury;
  • Figure 21 shows the effect of fucotetraose and mixed sugars on the mRNA levels of Kim-1 and NGAL in rat kidney tissue caused by acute ischemia-reperfusion (I/R) injury;
  • Figure 22 shows the effect of fucotetraose on inflammatory indicators in rat kidney tissue caused by acute ischemia-reperfusion (I/R) injury;
  • Figure 23 shows the effect of fucobiose on serum creatinine levels in mice induced by endotoxin phospholipopolysaccharide (LPS);
  • Figure 24 shows the effect of fucobiose on the mRNA levels of Kim-1 and NGAL in mouse kidney tissue caused by endotoxin phospholipopolysaccharide (LPS);
  • Figure 25 shows the effect of fucobiose on the inflammatory indicators in mouse kidney tissue induced by endotoxin phospholipopolysaccharide (LPS);
  • Figure 26 shows the influence of fucobiose and dexamethasone reference substances on the inflammation indicators in mouse kidney tissue caused by endotoxin phospholipopolysaccharide (LPS);
  • Figure 27 shows the effect of fucoidose on serum creatinine levels in mice induced by endotoxin phospholipopolysaccharide (LPS);
  • Figure 28 shows the effect of fucoidan on the mRNA levels of Kim-1 and NGAL in mouse kidney tissue induced by endotoxin phospholipopolysaccharide (LPS);
  • Figure 29 shows the effect of fucoidan on the inflammatory indicators in mouse kidney tissue caused by endotoxin phospholipopolysaccharide (LPS);
  • Figure 30 shows the influence of fucoidan and dexamethasone reference substances on the inflammation indicators in mouse kidney tissue caused by endotoxin phospholipopolysaccharide (LPS);
  • Figure 31 shows the effect of fucoidan on pathological sections of mouse kidney tissue damage caused by endotoxin phospholipopolysaccharide (LPS);
  • Figure 32 shows the effect of fucotetraose and mixed sugars on serum creatinine levels in mice induced by endotoxin phospholipopolysaccharide (LPS);
  • Figure 33 shows the effect of fucotetraose and mixed sugars on the mRNA levels of Kim-1 and NGAL in mouse kidney tissue caused by endotoxin phospholipopolysaccharide (LPS);
  • Figure 34 shows the effect of fucoidose and mixed sugars on the inflammatory indicators in mouse kidney tissue caused by endotoxin phospholipopolysaccharide (LPS);
  • Figure 35 shows the influence of fucoidose, mixed sugar and dexamethasone reference substances on the inflammation indicators in mouse kidney tissue caused by endotoxin phospholipopolysaccharide (LPS);
  • Figure 36 shows the effect of fucoidan on serum creatinine levels in mice induced by cisplatin
  • Figure 37 shows the effect of fucoidan on cisplatin-induced urine output in mice
  • Figure 38 shows the effect of fucoidan on the mRNA levels of Kim-1 and NGAL in the kidney tissue of mice induced by cisplatin;
  • Figure 39 shows the effect of fucoidan on inflammatory indicators in mouse kidney tissue induced by cisplatin
  • Figure 40 shows the mass spectra of the mixed sugars used in Example 4 and Example 5 of the present invention.
  • Fucobiose sodium salt if the two carboxyl groups in the molecule are both sodium salts, the theoretical sodium ion content is 11.58%; the actual ion chromatography test shows that the sodium ion content is 10.3%. If detected by the residue on ignition method, the sodium ion exists in the form of sodium sulfate, and the theoretical residue ratio should be 35.77%; the actual residue on ignition detection, the residue is 34.3%; the results obtained by the two detection methods are relatively close, indicating that the compound carboxylate The acid functionality is indeed in the sodium salt form. However, the measured values are slightly smaller than the theoretical value, probably because the sodium salt is a weak acid and strong base salt, and a small part of carboxylic acid is still in a free state.
  • the obtained sodium salt of fucoidan was tested for purity by high-performance liquid chromatography (HPLC, 230 nm), and its structure was identified by hydrogen nuclear magnetic spectrum ( 1 HNMR) and high-resolution mass spectrometry (HRMS-ESI).
  • Fucotriose sodium salt if the three carboxyl groups in the molecule are all sodium salts, the theoretical sodium ion content is 11.59%; the actual ion chromatography test shows that the sodium ion content is 9.9%. If detected by the residue on ignition method, the sodium ion exists in the form of sodium sulfate, and the theoretical residue ratio should be 35.80%; the measured residue on ignition is 33.01%. The results obtained by the two detection methods are relatively close, indicating that the carboxylic acid functional group of the compound is indeed in the form of a sodium salt. However, the measured values are slightly smaller than the theoretical value, probably because the sodium salt is a weak acid and strong base salt, and a small part of carboxylic acid is still in a free state.
  • the obtained fucotetraose sodium salt was tested for purity by high-performance liquid chromatography (HPLC, 230 nm), and its structure was identified by hydrogen nuclear magnetic spectrum ( 1 HNMR) and high-resolution mass spectrometry (HRMS-ESI).
  • Fucotetraose sodium salt if the four carboxyl groups in the molecule are all sodium salts, the theoretical sodium ion content is 11.59%; the actual sodium ion content is 9.8% as detected by ion chromatography. If detected by the residue on ignition method, the sodium ion exists in the form of sodium sulfate, and the theoretical residue ratio should be 35.80%; the measured residue on ignition, the residue is 32.5%. The results obtained by the two detection methods are relatively close, indicating that the carboxylic acid functional group of the compound is indeed in the form of a sodium salt. However, the measured values are slightly smaller than the theoretical value, probably because the sodium salt is a weak acid and strong base salt, and a small part of carboxylic acid is still in a free state.
  • Example 4 Effect of uniform polymerization degree fucoidan oligosaccharide on acute kidney injury (acute kidney injury, AKI) in rats caused by ischemia-reperfusion (I/R)
  • Kidney injury from ischemia-reperfusion is the standard animal model to mimic acute kidney injury from clinical hypoperfusion.
  • the inventor used the rat ischemia-reperfusion model to administer the fucoidan oligosaccharides with uniform polymerization degree and their mixtures prepared in Examples 1 to 3, respectively, and compared them with the blank group and the model non-administration group to investigate the degree of uniform polymerization.
  • Therapeutic effects of fucoidan oligosaccharides are the standard animal model to mimic acute kidney injury from clinical hypoperfusion.
  • Sprague Dawley rats were selected and purchased from the Experimental Animal Center of Sun Yat-sen University. There were 30 male rats weighing 220-250 grams. The urine volume was collected 24 hours before the operation and there was no abnormality.
  • the model rats were randomly divided into sham operation group, model group group, fucobiose 0.01, 0.05, 0.1 g/kg/day three dose groups (6 rats in each group). In the present invention, fucobiose is abbreviated as "AOS2".
  • Drugs were administered by intragastric administration, and both the model group and the sham operation group were administered with the same volume of normal saline.
  • the rats were anesthetized with 3% pentobarbital sodium in the abdominal cavity, and the skin was routinely disinfected, and the left and right kidneys were exposed from the abdomen.
  • the bilateral renal pedicles were clamped with large arterial clips, and then the kidneys were reset, the wound was covered with gauze, and a small amount of normal saline was dripped for rehydration.
  • the bilateral arterial clips were loosened, and the wound was sutured layer by layer to complete the operation.
  • the rats were placed on a heating pad at 37°C to wait for the rats to recover and then returned to the metabolic cage.
  • the cortex was preserved in trizol, and the mRNA in the cortex was extracted by the trizol method to detect the mRNA expression of AKI biomarkers (KIM-1, NGAL). The results are shown in Figure 12.
  • the total protein was extracted after adding tissue lysate and homogenizing the kidneys by ultrasonic method, and western blotting was used to detect the inflammatory factors (p-NF ⁇ B/NF ⁇ B, pro-IL-1 ⁇ /IL-1 ⁇ ) in the renal cortex tissue. See Figure 13.
  • Figure 10 shows that acute ischemia-reperfusion (I/R) injury causes a significant increase in serum creatinine levels in rats, and different doses of AOS2 reduce serum creatinine to varying degrees, suggesting that AOS2 has a renal protective effect.
  • * means p ⁇ 0.05 compared with sham (sham operation group), # means p ⁇ 0.05 compared with I/R group (model group).
  • Figure 11 shows that acute ischemia-reperfusion (I/R) injury causes increased urine output in rats, and different doses of AOS2 reduces urine output in rats, suggesting that AOS2 has a renal protective effect.
  • * indicates p ⁇ 0.05 compared with sham
  • # indicates p ⁇ 0.05 compared with I/R group.
  • the urine output decreased, indicating that the renal tubular reabsorption function recovered to a certain extent.
  • Figure 12 shows that acute ischemia-reperfusion (I/R) injury causes the mRNA levels of Kim-1 and NGAL to increase in acute kidney injury (AKI) indicators (ie renal tubular injury indicators) in rat kidney tissue, and the dose is 0.1g/ kg/day of AOS2 significantly reduced the expression of the two indicators, suggesting that AOS2 has a renal protective effect.
  • I/R acute ischemia-reperfusion
  • Figure 13 shows that acute ischemia-reperfusion (I/R) injury causes a significant increase in inflammatory indicators in rat kidney tissue, and different doses of AOS2 inhibit renal inflammatory responses to varying degrees, suggesting that AOS2 has a renal protective effect.
  • the dose of I/R+AOS2-L is 0.01g/kg/day; the dose of I/R+AOS2-M is 0.05g/kg/day; the dose of I/R+AOS2-H is 0.1g/kg/day.
  • fucoidan is referred to as "AOS3" for short.
  • the serum creatinine of rats was measured using the creatinine assay kit developed by Nanjing Jiancheng, and the experimental results were statistically processed by the t-value method. The results are shown in Figure 14.
  • the collected 24-hour urine output results are shown in Figure 15.
  • the bilateral kidneys were separated, and the cortex and inner medulla of the kidneys were separated.
  • the cortex was preserved in trizol, and the mRNA in the cortex was extracted by the trizol method to detect the mRNA expression of AKI biomarkers (KIM-1, NGAL).
  • the results are shown in Figure 16.
  • the total protein was extracted after adding tissue lysate and homogenizing the kidneys by ultrasonic method, and western blotting was used to detect the inflammatory factors (p-NF ⁇ B/NF ⁇ B, pro-IL-1 ⁇ /IL-1 ⁇ ) in the renal cortex tissue. See Figure 17.
  • the animals were sacrificed after the last blood collection, and the kidneys were fixed in 4% formaldehyde solution, embedded in paraffin, sectioned, stained with HE, and observed under a light microscope for general renal tissue morphology.
  • the results are shown in Figure 18.
  • Figure 14 shows that acute ischemia-reperfusion (I/R) injury causes a significant increase in serum creatinine levels in rats, and different doses of AOS3 reduce serum creatinine to varying degrees, suggesting that AOS3 has a renal protective effect.
  • * indicates p ⁇ 0.05 compared with sham
  • # indicates p ⁇ 0.05 compared with I/R group.
  • Serum creatinine decreased significantly after treatment with three different doses of AOS3, suggesting a certain recovery of glomerular function.
  • the dosage is 0.1g/kg/day, the serum creatinine can basically return to the normal level, and its therapeutic effect has a certain degree of dose dependence.
  • Figure 15 shows that acute ischemia-reperfusion (I/R) injury causes increased urine output in rats, and different doses of AOS3 reduces urine output in rats, suggesting that AOS3 has a renal protective effect.
  • * indicates p ⁇ 0.05 compared with sham
  • # indicates p ⁇ 0.05 compared with I/R group.
  • the urine output of the three different doses of AOS3 decreased after treatment, especially at the dose of 0.1g/kg/day, the urine output of the rats basically recovered to a near-normal level on the second day, suggesting that the renal tubular function has a certain degree. recovery. Its therapeutic effect is dose-dependent to a certain extent.
  • FIG 16 shows that acute ischemia-reperfusion (I/R) injury causes the mRNA levels of Kim-1 and NGAL to increase in acute kidney injury (AKI) indicators (ie renal tubular injury indicators) in rat kidney tissue, and the dose is 0.1g/ kg/day of AOS significantly decreased the expression of the two indicators, suggesting that AOS3 has a renal protective effect.
  • AKI acute kidney injury
  • * indicates p ⁇ 0.05 compared with sham
  • # indicates p ⁇ 0.05 compared with I/R group.
  • the results showed that the expression of acute kidney injury (AKI) indicators KIM-1 and NGAL in the I/R group rats after ischemia-reperfusion surgery was significantly increased, and 0.1g/kg/day of AOS3 could significantly reduce the two indicators. It is shown that AOS3 has a significant protective effect on acute kidney injury caused by ischemia-reperfusion.
  • Figure 17 shows that acute ischemia-reperfusion (I/R) injury causes a significant increase in inflammatory indicators in rat kidney tissue, and different doses of AOS3 inhibit renal inflammatory responses to varying degrees, suggesting that AOS3 has a renal protective effect.
  • I/R+AOS3-L means the dose is 0.01g/kg/day;
  • I/R+AOS3-M means the dose is 0.05g/kg/day;
  • I/R+AOS3-H means the dose is 0.1g/kg/day .
  • Figure 18 shows the histopathological manifestations of the kidney: sham operation group (SHAM): glomerular morphology, mesangial cells and renal tubules are basically normal; I/R group glomerulus atrophy and shedding, mesangial cells and matrix are reduced to produce cavities, The renal tubules were extensively dilated, the lumen was enlarged, and a large number of epithelial cells appeared edema, necrosis and shedding, and vacuolar degeneration was seen; the glomerular and renal tubule lesions were mild in the I/R+AOS3 (0.1g/kg/day) group.
  • SHAM sham operation group
  • fucobiose and fucotriose The same experimental procedure as for fucobiose and fucotriose was used.
  • the model rats were randomly divided into sham operation group, model group, fucoidose (0.01, 0.05, 0.1g/kg/day three doses) group and mixed sugar (mixed brown algae with a degree of polymerization of 2-8) Oligosaccharides, the mass spectrum of which is shown in Figure 40, was obtained from Ocean University of China, a dose of 0.1 g/kg/day) group (6 animals in each group).
  • fucoidose is abbreviated as "AOS4"
  • AOS mixed saccharides
  • the serum creatinine of rats was measured using the creatinine assay kit developed by Nanjing Jiancheng, and the experimental results were statistically processed by the t-value method. The results are shown in Figure 19.
  • the collected 24-hour urine output results are shown in Figure 20.
  • the bilateral kidneys were separated, and the cortex and inner medulla of the kidneys were separated.
  • the cortex was preserved in trizol, and the mRNA in the cortex was extracted by the trizol method to detect the mRNA expression of AKI biomarkers (KIM-1, NGAL). The results are shown in Figure 21.
  • the total protein was extracted after adding tissue lysate and homogenizing the kidneys by ultrasonic method, and western blotting was used to detect the inflammatory factors (p-NF ⁇ B/NF ⁇ B, pro-IL-1 ⁇ /IL-1 ⁇ ) in the renal cortex tissue. See Figure 22.
  • Figure 19 shows that acute ischemia-reperfusion (I/R) injury causes a significant increase in serum creatinine levels in rats, and different doses of AOS4 and mixed sugars reduce serum creatinine to varying degrees, and AOS4 has a good renal protection effect.
  • * indicates p ⁇ 0.05 compared with sham
  • # indicates p ⁇ 0.05 compared with I/R group.
  • Serum creatinine decreased significantly after treatment with three different doses of AOS4 and mixed sugar (0.1g/kg/day), suggesting that glomerular function has recovered to a certain extent.
  • fucotetraose administered at a dose of 0.1g/kg/day serum creatinine can be restored to near normal levels.
  • the therapeutic effects of different doses of AOS4 are dose-dependent to a certain extent.
  • Figure 20 shows that acute ischemia-reperfusion (I/R) injury causes increased urine output in rats, and different doses of AOS4 and mixed sugar (0.1g/kg/day) reduce the urine output of rats, and AOS4 has a good renal protective effect .
  • * indicates p ⁇ 0.05 compared with sham
  • # indicates p ⁇ 0.05 compared with I/R group.
  • Figure 21 shows that acute ischemia-reperfusion (I/R) injury causes the mRNA levels of Kim-1 and NGAL to increase in acute kidney injury (AKI) indicators (ie renal tubular injury indicators) in rat kidney tissue, and the dose is 0.1g/ kg/day fucotetraose significantly reduced the expression of the two indicators, suggesting that it has better renal protection.
  • AKI acute kidney injury
  • * indicates p ⁇ 0.05 compared with sham
  • # indicates p ⁇ 0.05 compared with I/R group.
  • the results showed that the expression of KIM-1 and NGAL, the indicators of acute kidney injury (AKI) after ischemia-reperfusion in rats in the I/R group was significantly increased, and 0.1g/kg/day of AOS4 could significantly reduce the two indicators. It shows that AOS4 has a good protective effect on acute kidney injury caused by ischemia-reperfusion.
  • Figure 22 shows that acute ischemia-reperfusion (I/R) injury causes a significant increase in inflammatory indicators in rat kidney tissue, and different doses of AOS4 can inhibit renal inflammatory responses to varying degrees, suggesting that AOS4 has a renal protective effect.
  • I/R+AOS4-L means the dose is 0.01g/kg/day;
  • I/R+AOS4-M means the dose is 0.05g/kg/day;
  • I/R+AOS4-H means the dose is 0.1g/kg/day ;
  • I/R+AOS (mixed)-H indicates that the dose is 0.1g/kg/day.
  • Kidney injury from ischemia-reperfusion is the standard animal model to mimic acute kidney injury from clinical hypoperfusion.
  • doses 0.01, 0.05, 0.1g/kg/day
  • fucobiose trisaccharide
  • tetrasaccharide the serum creatinine level of rats
  • serum creatinine level at the dose of 0.1g/kg/day was average It can basically return to the normal value; after administration, the urine concentration function of the kidneys recovered significantly, and the urine output decreased.
  • the 0.1g/kg/day dose group significantly reduced the levels of kidney injury factors (KIM-1, NGAL), inflammation
  • KIM-1, NGAL kidney injury factors
  • inflammation The expression of the factor decreased significantly, the pathological changes of the kidneys were significantly improved, and the therapeutic effect was enhanced with the dose increasing.
  • the detection results of different indicators such as serum creatinine, urine volume, and inflammatory factors all show that fucobiose, triose, and tetrasaccharide have a good protective effect on acute kidney injury in rats caused by ischemia-reperfusion.
  • Kidney injury induced by endotoxin phospholipopolysaccharide treatment is the standard animal model to mimic acute kidney injury from clinical infection.
  • model mice are randomly divided into control group, model group, model+administration group (AOS20. 1g/kg/day) and model + positive control group (dexamethasone acetate 0.1g/kg/day) were 6 in each group, the samples were administered by intragastric administration, and the model group and the control group were all fed with the same volume of normal saline, and the ground Semethasone was administered by intraperitoneal injection.
  • LPS was used for modeling to induce the occurrence of sepsis-type acute kidney injury.
  • each model group was intraperitoneally injected with LPS 15 mg/kg, and the control group was intraperitoneally injected with the same amount of normal saline.
  • the mice were put back into the mouse metabolic cage for observation, during which the body weight, food intake, water intake and urine output of the mice were detected. After 24 hours, the mice were sacrificed, the urine was collected, and the blood samples were collected. Blood was collected from the inferior vena cava of the mice, and the upper serum was collected after centrifugation. Then, the serum creatinine of the mice was measured using the Nanjing Jiancheng creatinine assay kit. The results were statistically processed using the t-value method, and the results are shown in Figure 23.
  • the bilateral kidneys were separated, and the cortex and inner medulla of the kidneys were separated.
  • the cortex was preserved in trizol.
  • the mRNA in the cortex was extracted by the trizol method when used, and the AKI biomarkers (KIM-1, NGAL were detected by Qpcr method, the results are shown in Fig. 24) and the mRNA expression of inflammatory factors (IL-1 ⁇ , IL-18, TNF- ⁇ , MCP-1, the results are shown in Figure 25).
  • the total protein was extracted after adding tissue lysate and homogenizing the kidneys by ultrasonic method, and western blotting was used to detect the inflammatory factors (p-NF ⁇ B/NF ⁇ B, pro-IL-1 ⁇ /IL-1 ⁇ ) in the renal cortex tissue. See Figure 26.
  • Figure 23 shows that LPS treatment causes a significant increase in serum creatinine levels in mice, and AOS2 significantly reduces serum creatinine, which has the same effect on reducing serum creatinine as dexamethasone, suggesting that fucobiose has a renal protective effect.
  • Dex dexamethasone * indicates p ⁇ 0.05 compared with CTL (control group)
  • # indicates p ⁇ 0.05 compared with LPS group (model group).
  • the results showed that compared with the control group, the serum creatinine of the mice was significantly increased after intraperitoneal injection of LPS, and the serum creatinine of the mice in the AOS2 administration group was significantly decreased, and it could basically return to the normal level, indicating that fucobiose has a significant effect on the effects of LPS on mice. Decreased renal function has a significant protective effect.
  • Figure 24 shows that LPS treatment increases the mRNA levels of Kim-1 and NGAL, the indicators of acute kidney injury (AKI) (ie renal tubular injury indicators) in the kidney tissue of mice, suggesting that fucobiose has a renal protective effect.
  • AKI acute kidney injury
  • * indicates p ⁇ 0.05 compared with CTL
  • # indicates p ⁇ 0.05 compared with LPS group.
  • the results show that the AKI indicators (KIM-1, NGAL) in mice were significantly increased after intraperitoneal injection of LPS, and the production of KIM-1 and NGAL was significantly reduced after AOS2 treatment, indicating that AOS2 has a significant effect on LPS-induced kidney injury in mice.
  • Figure 25 shows that LPS treatment caused a significant increase in inflammatory indicators in mouse kidney tissue, and AOS2 significantly inhibited renal inflammatory response, suggesting that fucobiose has a renal protective effect.
  • * indicates p ⁇ 0.05 compared with CTL
  • # indicates p ⁇ 0.05 compared with LPS group.
  • the results showed that the gene expression of inflammatory factors (IL-1 ⁇ , IL-18, TNF- ⁇ , MCP-1) was significantly increased after intraperitoneal injection of LPS in mice, and the production of inflammatory factors was significantly reduced by AOS2 treatment, suggesting that fucobiose plays a role in LPS.
  • the induced acute kidney injury in mice has a good protective effect.
  • Figure 26 shows that LPS treatment caused a significant increase in inflammatory indicators in mouse kidney tissue, and AOS2 significantly inhibited renal inflammatory response, and the effect was close to that of the positive control dexamethasone, suggesting that AOS2 has a renal protective effect.
  • * indicates p ⁇ 0.05 compared with CTL
  • # indicates p ⁇ 0.05 compared with LPS group.
  • the results showed that after LPS treatment, the expression of TLR4, p-NF ⁇ B/NF ⁇ B, and pro-IL-1 ⁇ /IL-1 ⁇ proteins in the mouse kidneys was significantly increased, and AOS2 treatment significantly reduced the production of inflammatory factors, and the effect was similar to that of the ground.
  • the effect of dexamethasone is similar, showing that AOS2 has obvious anti-inflammatory effect.
  • the same experimental method as for fucobiose was used.
  • the serum creatinine of mice was measured using the creatinine assay kit built in Nanjing, and the experimental results were statistically processed using the t-value method.
  • the results are shown in Figure 27.
  • the bilateral kidneys were separated, and the cortex and inner medulla of the kidneys were separated.
  • the cortex was preserved in trizol.
  • the mRNA in the cortex was extracted by the trizol method when used, and the AKI biomarkers (KIM-1, NGAL were detected by Qpcr method, the results are shown in Fig. 28) and the mRNA expression of inflammatory factors (IL-1 ⁇ , IL-18, TNF- ⁇ , MCP-1, the results are shown in Figure 29).
  • Figure 27 shows that LPS treatment causes a significant increase in serum creatinine levels in mice, and AOS3 significantly reduces serum creatinine, which has the same effect on reducing serum creatinine as dexamethasone, suggesting that fucoidan has a renal protective effect.
  • Dex dexamethasone * indicates p ⁇ 0.05 compared with CTL
  • # indicates p ⁇ 0.05 compared with LPS group.
  • the results showed that: compared with the control group, the serum creatinine of the mice was significantly increased after intraperitoneal injection of LPS, and the serum creatinine of the mice in the AOS3 administration group was significantly decreased, and it could basically return to normal levels, suggesting that fucotriose had a significant effect on the mice induced by LPS. Decreased renal function has a significant protective effect.
  • Figure 28 shows that LPS treatment increases the mRNA levels of Kim-1 and NGAL, the indicators of acute kidney injury (AKI) in the kidney tissue of mice (ie renal tubular injury indicators), suggesting that fucoidan has a renal protective effect.
  • * indicates p ⁇ 0.05 compared with CTL
  • # indicates p ⁇ 0.05 compared with LPS group.
  • the results showed that the AKI indicators (KIM-1, NGAL) in mice were significantly increased after intraperitoneal injection of LPS, and the production of KIM-1 and NGAL was significantly reduced after AOS3 treatment, suggesting that AOS3 has a protective effect on the kidney damage caused by LPS in mice .
  • Figure 29 shows that LPS treatment caused a significant increase in inflammatory indicators in mouse kidney tissue, and AOS3 significantly inhibited renal inflammatory response, suggesting that fucoidan has a renal protective effect.
  • * indicates p ⁇ 0.05 compared with CTL
  • # indicates p ⁇ 0.05 compared with LPS group.
  • the results show that the gene expression of inflammatory factors (IL-1 ⁇ , IL-18, TNF- ⁇ , MCP-1) in mice is significantly increased after intraperitoneal injection of LPS, and AOS3 treatment can significantly reduce the production of inflammatory factors, which proves that fucoidan has an important effect on LPS.
  • the induced acute kidney injury in mice has a good protective effect.
  • Figure 30 shows that LPS treatment caused a significant increase in inflammatory indicators in mouse kidney tissue, and AOS3 significantly inhibited renal inflammatory response, and the effect was very close to that of the positive control dexamethasone, suggesting that AOS3 has a better renal protective effect.
  • * indicates p ⁇ 0.05 compared with CTL
  • # indicates p ⁇ 0.05 compared with LPS group.
  • the results showed that after LPS treatment, the expression of TLR4, p-NF ⁇ B/NF ⁇ B, and pro-IL-1 ⁇ /IL-1 ⁇ protein in the mouse kidney significantly increased, and AOS3 treatment significantly reduced the production of inflammatory factors, and its effect was similar to that of dexamethasone.
  • the effects of metasone are very similar, indicating that AOS3 has a better anti-inflammatory effect.
  • Figure 31 Shows the pathological manifestations of renal tissue: control group: glomerular morphology, mesangial cells and renal tubules are basically normal, while in LPS group, glomerular volume increases, inflammatory cell infiltration can be seen in renal interstitium, renal tubules are widely dilated, epithelial Cells appeared edema, necrosis and detachment, and vacuolar degeneration was seen; glomerular and tubular lesions in LPS+AOS3 group were mild. These results show that AOS3 has a significant protective effect on the morphological changes of mouse kidneys induced by endotoxin phospholipopolysaccharide treatment.
  • model mice were randomly divided into control group, model group, model+administration group 1 (AOS4 0.1g/kg/day), model+administration group 2 (mixed brown algae oligosaccharides with a polymerization degree of 2-8 Sugar, whose mass spectrum is shown in Figure 40, obtained from Ocean University of China, 0.1g/kg/day) and model + positive control group (dexamethasone acetate 0.1g/kg/day) each group 6, the samples were given by gavage
  • model group and the control group were fed with the same volume of normal saline, and dexamethasone was administered by intraperitoneal injection.
  • the serum creatinine of mice was measured using the creatinine assay kit developed by Nanjing Jiancheng, and the experimental results were statistically processed by the t-value method.
  • the results are shown in Figure 32.
  • the bilateral kidneys were separated, and the cortex and inner medulla of the kidneys were separated.
  • the cortex was preserved in trizol.
  • the mRNA in the cortex was extracted by the trizol method when used, and the AKI biomarkers (KIM-1, NGAL were detected by Qpcr method, the results are shown in Fig. 33) and the mRNA expression of inflammatory factors (IL-1 ⁇ , IL-18, TNF- ⁇ , MCP-1, the results are shown in Figure 34).
  • Figure 32 shows that LPS treatment causes a significant increase in serum creatinine levels in mice, mixed sugars have a certain effect on reducing serum creatinine, and AOS4 significantly reduces serum creatinine, which is close to the effect of dexamethasone on reducing serum creatinine, suggesting that fucoidan oligosaccharides have renal protection effect.
  • Dex dexamethasone * indicates p ⁇ 0.05 compared with CTL, # indicates p ⁇ 0.05 compared with LPS group.
  • Figure 33 shows that LPS treatment causes acute kidney injury (AKI) indicators (i.e. renal tubular injury indicators) mRNA levels of Kim-1 and NGAL in mouse kidney tissue to increase, fucotetraose and mixed sugars (dose 0.1g/kg/day ) after administration, both indicators decreased, especially fucotetraose, which significantly decreased both indicators, suggesting that fucotetraose had better renal protection.
  • AKI acute kidney injury
  • mRNA levels of Kim-1 and NGAL in mouse kidney tissue to increase
  • fucotetraose and mixed sugars dose 0.1g/kg/day
  • * indicates p ⁇ 0.05 compared with CTL
  • # indicates p ⁇ 0.05 compared with LPS group.
  • mice were significantly increased after intraperitoneal injection of LPS, and the production of KIM-1 and NGAL was significantly reduced after AOS4 administration, suggesting that fucotetraose can play a role in LPS-induced kidney damage in mice. Damage is protective.
  • Figure 34 shows that LPS treatment caused a significant increase in inflammatory indicators in mouse kidney tissue, and AOS4 and mixed sugars significantly inhibited renal inflammatory response, and AOS4 had better renal protection.
  • * indicates p ⁇ 0.05 compared with CTL
  • # indicates p ⁇ 0.05 compared with LPS group.
  • the results showed that the gene expression of inflammatory factors (IL-1 ⁇ , IL-18, TNF- ⁇ , MCP-1) was significantly increased after intraperitoneal injection of LPS in mice, and AOS4 and mixed sugar treatment could significantly reduce the production of inflammatory factors, in which AOS4 It has a good protective effect on acute kidney injury in mice caused by LPS.
  • Figure 35 shows that LPS treatment caused a significant increase in inflammatory indicators in the mouse kidney tissue, and AOS4 and mixed sugars significantly inhibited the renal inflammatory response, and the effect was close to that of the positive control dexamethasone, suggesting that AOS4 has a better renal protective effect.
  • * indicates p ⁇ 0.05 compared with CTL group
  • # indicates p ⁇ 0.05 compared with LPS group.
  • the results showed that after LPS treatment, the expression of inflammatory indicators in mouse kidneys, such as TLR4, p-NF ⁇ B/NF ⁇ B, and pro-IL-1 ⁇ /IL-1 ⁇ , was significantly increased, and the treatment of fucotetraose and mixed sugars significantly reduced the production of inflammatory factors. Its effect is basically consistent with that of dexamethasone, indicating that it has a very effective anti-inflammatory effect.
  • Kidney injury induced by endotoxin phospholipopolysaccharide (LPS) treatment is a standard animal model that mimics acute kidney injury from clinical infection.
  • the serum creatinine level of the mice in the model group treated with LPS was significantly increased, and after the mice were treated with 0.1g/kg/day of fucobiose, triose, and tetrasaccharide, the serum creatinine level was significantly decreased; kidney damage factors (KIM-1, NGAL)
  • KIM-1, NGAL kidney damage factors
  • the level of inflammatory factors and the expression of inflammatory factors were significantly decreased, and the pathological changes of the kidney were significantly improved.
  • Kidney injury induced by cisplatin treatment is a standard animal model of acute kidney injury induced by direct nephrotoxic effects of drugs in clinical practice.
  • model mice are randomly divided into control group, model group, model + low-dose administration group ( AOS3 0.05g/kg/day), model+medium-dose administration group (AOS3 0.1g/kg/day) and model+high-dose administration group (AOS3 0.2g/kg/day), 6 rats in each group, fucotriose
  • the rats in the model group and the control group were given the same volume of normal saline by intragastric administration.
  • Cisplatin was used for modeling to induce the occurrence of drug-toxic acute kidney injury.
  • mice were put back into the mouse metabolic cage for observation, during which the body weight, food intake, drinking water and urine volume of the mice were detected (results shown in Figure 37). After 72 hours, the mice were sacrificed, the urine was collected, and the blood samples were collected. Blood was collected from the inferior vena cava of the mice, and the upper serum was collected after centrifugation. Then, the serum creatinine of the mice was measured using the Nanjing Jiancheng creatinine assay kit.
  • the results were statistically processed using the t-value method, and the results are shown in Figure 36.
  • the bilateral kidneys were separated, and the cortex and inner medulla of the kidneys were separated.
  • the cortex was preserved in trizol, and the mRNA in the cortex was extracted by the trizol method when used, and the AKI biomarkers (KIM-1, NGAL, and the results were shown in Fig. 38) mRNA expression.
  • the results are shown in Figure 39.
  • Figure 36 shows that cisplatin treatment caused a significant increase in serum creatinine levels in mice, and AOS3 significantly decreased serum creatinine levels, suggesting that fucotriose has a renal protective effect.
  • * means p ⁇ 0.05 compared with CTL group (control group)
  • # means p ⁇ 0.05 compared with Cis group (model group).
  • the results showed that compared with the control group, the serum creatinine of the mice was significantly increased after intraperitoneal injection of cisplatin, and the serum creatinine of the mice in the AOS3 administration group decreased significantly, and could basically return to the normal level, indicating that fucotriose had a significant effect on the effects of cisplatin. Decreased kidney function in mice has a significant protective effect.
  • Figure 37 shows that the urine output of mice induced by cisplatin first increased at 24 hours and then decreased at 72 hours to enter the oliguria period.
  • the urine output of mice After administration of different concentrations of AOS3, the urine output of mice first decreased at 24 hours to restore kidney function, and then the urine output gradually tended to normal, suggesting that Fucoidan has a renal protective effect.
  • * indicates p ⁇ 0.05 compared with CTL
  • # indicates p ⁇ 0.05 compared with model group.
  • the urine output After treatment with three different doses of AOS3, the urine output first decreased and then tended to normal, suggesting that the renal tubular function recovered to a certain extent. And showed a certain degree of dose dependence.
  • Figure 38 shows that cisplatin treatment increases the mRNA levels of Kim-1 and NGAL, the indicators of acute kidney injury (AKI) (ie renal tubular injury indicators) in the kidney tissue of mice, suggesting that fucoidan has a renal protective effect.
  • AKI acute kidney injury
  • * indicates p ⁇ 0.05 compared with CTL
  • # indicates p ⁇ 0.05 compared with model group.
  • the results showed that the AKI indicators (KIM-1, NGAL) in mice were significantly increased after intraperitoneal injection of cisplatin, and after AOS3 treatment, the production of KIM-1 and NGAL was significantly reduced, indicating that AOS3 has a role in cisplatin-induced kidney injury in mice. Obvious protective effect.
  • Figure 39 shows that cisplatin treatment caused a significant increase in inflammatory indicators in mouse kidney tissue, and AOS3 significantly inhibited renal inflammatory response, suggesting that AOS3 has a renal protective effect.
  • * indicates p ⁇ 0.05 compared with CTL
  • # indicates p ⁇ 0.05 compared with model group.
  • the results showed that after cisplatin treatment, the expression of inflammatory indicators in mouse kidneys, such as TLR4, p-NF ⁇ B/NF ⁇ B, and IL-1 ⁇ protein, increased significantly, and AOS3 treatment significantly reduced the production of inflammatory factors, showing that AOS3 has obvious anti-inflammatory effects.
  • the serum creatinine level of the mice in the model group treated with cisplatin was significantly increased, and the serum creatinine level was significantly decreased after the mice were treated with 0.1 g/kg/day of fucobiose, triose, and tetrasaccharide; the kidney damage factors (KIM-1, NGAL ) levels and the expression of inflammatory factors decreased significantly, and the therapeutic effect was concentration-dependent.

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Abstract

本发明提供一种褐藻寡糖或其药学上可接受的盐在制备用于治疗急性肾脏损伤的药物中的用途，其中所述褐藻寡糖为褐藻二糖、褐藻三糖或褐藻四糖。经研究发现，在缺血再灌注(I/R)、内毒素磷脂多糖(LPS)和抗肿瘤药物顺铂诱导的急性肾脏损伤动物模型上，褐藻二糖、三糖、四糖都有非常明显的保护作用。本发明的褐藻寡糖治疗急性肾脏损伤动物后，血清肌酐水平显著下降，肾脏的尿浓缩功能恢复明显，明显降低肾小管损伤因子(KIM-1、NGAL)水平，炎症因子表达显著下降，肾脏病理改变显著改善，并随剂量增加，治疗作用增强。因此，本发明的褐藻寡糖具有较强的治疗急性肾脏损伤的功效。

Description

一种褐藻寡糖的应用 技术领域
本发明涉及一种褐藻寡糖的应用，属于生物医药技术领域。
背景技术
糖类(Carbohydrate)与核酸、蛋白质一起并称为三大生命物质。褐藻胶主要存在于海带、马尾藻以及巨藻的细胞壁中，是一类直链、无分枝，具负电荷的多糖类化合物。褐藻胶是由β-D-(1,4)-甘露糖醛酸(Mannuronic acid，M)和α-L-(1,4)-古洛糖醛酸(Guluronic acid，G)组成的二元线型嵌段化合物。其分子中主要存在三种结构片段：β-D-(1,4)-甘露糖醛酸互相连接构成的聚甘露糖醛酸(Polymannuronate，PM)；α-L-(1,4)-古洛糖醛酸互相连接构成的聚古洛糖醛酸(Polyguluronate，PG)；M和G交替共聚形成的PMG片段。
褐藻胶的高粘度和成凝胶等特性使其作为凝固剂、增稠剂、稳定剂等广泛应用于食品、化工、医药、纺织等工业生产中。在医药领域，褐藻胶因其独特的理化性质和良好的生物相容性，在医用生物材料和药物缓控释材料方面具有广泛的应用。研究还发现褐藻胶具有抗氧化、免疫调节、抗肿瘤等生物活性，但褐藻胶由于分子量大，凝胶性强，不容易被吸收，使其应用方面受到很大的限制。而寡糖由于结构明确、活性显著、吸收性好、副作用小等特点，已引起了人们的重视。
近年来，褐藻胶寡糖因结构独特，其活性研究成为了糖类药物研究中的热点，其生物活性研究取得了重要进展。人们研究发现，褐藻胶寡糖及其衍生物具有多种生物活性，例如抗氧化性、抗肿瘤、抗凝血、免疫调节、神经保护、抗炎活性、抗病毒活性、抗老年痴呆、抗尿路结石、抗糖尿病等。
糖类是一类高度复杂同时又变化多端的生物大分子。不像寡核苷酸和多肽，糖类不只是线性的寡聚体，而通常是分支的。在哺乳动物细胞上发现的常见的9种单糖可以连接成比20种天然存在的氨基酸或4种核苷酸相连接更具有多样性的结构。糖类化合物结构的这种复杂性使得从天然来源中获取纯的糖类化合物非常困难。不论化学裂解或酶法裂解，均难以分离得到均一聚合度的寡聚糖或多聚糖。迄今为止，几乎所有的研究采用的寡糖或多聚糖均为聚合度接近的一系列糖的混合物，这为其活性研究、代谢、毒理以及药物的质量研究等带来非常大的困难。
发明人前期的研究针对目前糖类研究的难点，开发了一系列专一性较强的褐藻胶裂解酶，可以分别将褐藻胶分解为纯度较高，非还原性末端具有共轭双键，聚合度均一的褐藻二糖、三糖或四糖；经过酶灭活，离心取上清液，浓缩后，再以凝胶柱或离子交换树脂进一步纯化为均一聚合度的褐藻二糖、三糖或四糖。所述褐藻二糖为ΔG和/或ΔM两种结构及其任意 比例的组合；褐藻三糖为ΔGG、ΔGM、ΔMM和ΔMG四种结构及其任意比例的组合；褐藻四糖为ΔGGG、ΔGGM、ΔGMG、ΔGMM、ΔMMG、ΔMMM、ΔMGG和ΔMGM八种结构及其任意比例的组合；所有寡糖均为单糖1,4位糖苷键连接；G代表α-L-古洛糖醛酸；M代表β-D-甘露糖醛酸；Δ表示α-L-古洛糖醛酸和/或β-D-甘露糖醛酸4,5位发生β-消除，生成非还原端4,5位为共轭双键的不饱和单糖；各单糖的结构如下图所示：
Figure PCTCN2021093850-appb-000001
以ΔGM为例，相应褐藻三糖的结构如下：
Figure PCTCN2021093850-appb-000002
急性肾损伤(acute kidney injury，AKI)，以往称为急性肾衰竭，是指由多种病因引起的短时间内，肾功能快速下降而出现的临床综合征，表现为血清肌酐的快速增加及尿量的减少。据统计，全世界每年约有10％～20％的住院患者被诊断为AKI，ICU患者中甚至超过50％，其中85％的患者来自发展中国家。AKI可增加住院患者死亡率、延长住院时间、增加治疗费用，还可增加心血管事件、远期慢性肾脏病(CKD)和终末期肾脏病(ESRD)的患病风险。尽管肾脏病学界对AKI日趋重视，但目前仍无特异治疗，发病率和死亡率仍然很高，AKI病人心血管事件发病率为38％，如心功能衰竭(风险增高58％)，急性心梗(风险增高40％)、高血压(风险增高22％)、中风(风险增高15％)等，AKI已成为威胁人类健康的世界性公共卫生问题。
当前尚无有效药物可以逆转AKI肾损伤。同一些继发性慢性肾脏疾病如高血压肾病等不同，AKI是肾实质(肾小球、肾小管及间质等)的原发性病变，病因复杂(如缺血、缺氧、毒物、药物、感染等等)，病程进展快速，部分病人会进展到慢性肾脏疾病并伴随如心血管疾病的并发症。早期诊断、及时干预能最大限度地减轻肾损伤、促进肾功能恢复。尽早识别并纠正可逆病因、维持内环境稳定、营养支持、防止并发症及肾脏替代治疗等，仍然是当前针对AKI的主要治疗策略。而高血压肾病是由于血压长期升高引起肾脏血管的病变，肾脏毛细血管变厚、增粗、肾小球纤维化、血管管腔变窄、肾动脉硬化、肾实质缺血、肾单位减少等。肾实质的改变会导致肾脏的血液滤过功能降低，肾功能下降。这是一种长期的、由血压 持续升高所引起的肾脏继发性疾病，病程较长，控制血压是基本的治疗措施。当前治疗高血压肾病主要采用的是控制血压的疗法，然而，这种疗法不适用于治疗AKI。鉴于AKI的发病原因复杂，确定一种能使所有AKI病人受益的单一疗法十分具有挑战性。
本发明针对褐藻寡糖进行了进一步研究，并提供一种褐藻寡糖在治疗急性肾脏损伤中的应用。
发明内容
因此，本发明的目的是提供一种褐藻寡糖的应用。
本发明的目的是通过以下技术方案来实现的：
一方面，本发明提供一种褐藻寡糖或其药学上可接受的盐在制备用于治疗急性肾脏损伤的药物中的用途，其中所述褐藻寡糖为褐藻二糖、褐藻三糖或褐藻四糖。
在本发明的某些实施方案中，所述褐藻寡糖是由单糖G、M和/或Δ通过1,4位糖苷键连接构成的；其中，G表示α-L-古洛糖醛酸，M表示β-D-甘露糖醛酸，Δ表示α-L-古洛糖醛酸或β-D-甘露糖醛酸的4,5位发生β-消除，生成4,5位为共轭双键的不饱和单糖。
在本发明的某些实施方案中，所述褐藻二糖选自ΔG、ΔM或其组合。
在本发明的某些实施方案中，所述褐藻三糖选自ΔGG、ΔGM、ΔMM和ΔMG中的一种或多种。
在本发明的某些实施方案中，所述褐藻四糖选自ΔGGG、ΔGGM、ΔGMG、ΔGMM、ΔMMG、ΔMMM、ΔMGG和ΔMGM中的一种或多种。
在本发明的某些实施方案中，所述药学上可接受的盐为钠盐、钾盐、钙盐、镁盐和/或铵盐。
在本发明的某些实施方案中，所述急性肾脏损伤是由血液灌注不足、感染或药物的肾脏毒性导致的。
另一方面，本发明提供一种褐藻寡糖或其药学上可接受的盐，其用于治疗急性肾脏损伤，其中所述褐藻寡糖为褐藻二糖、褐藻三糖或褐藻四糖。
又一方面，本发明提供一种治疗急性肾脏损伤的方法，包括给予需要的患者治疗有效量的褐藻寡糖或其药学上可接受的盐，其中所述褐藻寡糖为褐藻二糖、褐藻三糖或褐藻四糖。
本发明的均一聚合度的褐藻二糖、三糖和四糖，对于糖类原料药的质量控制、药理、毒理等分析研究，均具有革命性进步。
研究发现，在缺血再灌注(I/R)、内毒素磷脂多糖(LPS)和抗肿瘤药物顺铂诱导的急性肾脏损伤动物模型上，褐藻二糖、三糖、四糖都有非常明显的保护作用。本发明的褐藻寡糖治疗急性肾脏损伤动物后，血清肌酐水平显著下降，肾脏的尿浓缩功能恢复明显，明显降低肾小管损伤因子(KIM-1、NGAL)水平，炎症因子表达显著下降，肾脏病理改变显著改善，并随剂量增加，治疗作用增强。因此，本发明的褐藻寡糖具有较强的 治疗肾脏损伤的功效。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1示出了褐藻二糖在230nm波长下的高效液相色谱图；
图2示出了褐藻二糖的核磁氢谱图( 1HNMR，溶剂D 2O)；
图3示出了褐藻二糖的高分辨质谱图(HRMS(ESI))；
图4示出了褐藻三糖在230nm波长下的高效液相色谱图；
图5示出了褐藻三糖的核磁氢谱图( 1HNMR，溶剂为D 2O)；
图6示出了褐藻三糖的高分辨质谱图(HRMS(ESI))；
图7示出了褐藻四糖在230nm波长下的高效液相色谱图；
图8示出了褐藻四糖的核磁氢谱图( 1HNMR，溶剂为D 2O)；
图9示出了褐藻四糖的高分辨质谱图(HRMS(ESI))；
图10示出了褐藻二糖对急性缺血再灌注(I/R)损伤引起的大鼠血清肌酐水平的影响；
图11示出了褐藻二糖对急性缺血再灌注(I/R)损伤引起的大鼠尿量的影响；
图12示出了褐藻二糖对急性缺血再灌注(I/R)损伤引起的大鼠肾脏组织中Kim-1和NGAL的mRNA水平的影响；
图13示出了褐藻二糖对急性缺血再灌注(I/R)损伤引起的大鼠肾脏组织中炎症指标的影响；
图14示出了褐藻三糖对急性缺血再灌注(I/R)损伤引起的大鼠血清肌酐水平的影响；
图15示出了褐藻三糖对急性缺血再灌注(I/R)损伤引起的大鼠尿量的影响；
图16示出了褐藻三糖对急性缺血再灌注(I/R)损伤引起的大鼠肾脏组织中Kim-1和NGAL的mRNA水平的影响；
图17示出了褐藻三糖对急性缺血再灌注(I/R)损伤引起的大鼠肾脏组织中炎症指标的影响；
图18示出了褐藻三糖对急性缺血再灌注(I/R)损伤引起的大鼠肾脏组织损伤的病理切片的影响；
图19示出了褐藻四糖和混合糖对急性缺血再灌注(I/R)损伤引起的大鼠血清肌酐水平的影响；
图20示出了褐藻四糖和混合糖对急性缺血再灌注(I/R)损伤引起的大鼠尿量的影响；
图21示出了褐藻四糖和和混合糖对急性缺血再灌注(I/R)损伤引起的大鼠肾脏组织中Kim-1和NGAL的mRNA水平的影响；
图22示出了褐藻四糖对急性缺血再灌注(I/R)损伤引起的大鼠肾脏组织中炎症指标的影响；
图23示出了褐藻二糖对内毒素磷脂多糖(LPS)引起的小鼠血清肌酐水平的影响；
图24示出了褐藻二糖对内毒素磷脂多糖(LPS)引起的小鼠肾脏组织中Kim-1和NGAL的mRNA水平的影响；
图25示出了褐藻二糖对内毒素磷脂多糖(LPS)引起的小鼠肾脏组织中炎症指标的影响；
图26示出了褐藻二糖及地塞米松对照品对内毒素磷脂多糖(LPS)引起的小鼠肾脏组织中炎症指标的影响；
图27示出了褐藻三糖对内毒素磷脂多糖(LPS)引起的小鼠血清肌酐水平的影响；
图28示出了褐藻三糖对内毒素磷脂多糖(LPS)引起的小鼠肾脏组织中Kim-1和NGAL的mRNA水平的影响；
图29示出了褐藻三糖对内毒素磷脂多糖(LPS)引起的小鼠肾脏组织中炎症指标的影响；
图30示出了褐藻三糖及地塞米松对照品对内毒素磷脂多糖(LPS)引起的小鼠肾脏组织中炎症指标的影响；
图31示出了褐藻三糖对内毒素磷脂多糖(LPS)引起的小鼠肾脏组织损伤的病理切片的影响；
图32示出了褐藻四糖及混合糖对内毒素磷脂多糖(LPS)引起的小鼠血清肌酐水平的影响；
图33示出了褐藻四糖及混合糖对内毒素磷脂多糖(LPS)引起的小鼠肾脏组织中Kim-1和NGAL的mRNA水平的影响；
图34示出了褐藻四糖及混合糖对内毒素磷脂多糖(LPS)引起的小鼠肾脏组织中炎症指标的影响；
图35示出了褐藻四糖、混合糖及地塞米松对照品对内毒素磷脂多糖(LPS)引起的小鼠肾脏组织中炎症指标的影响；
图36示出了褐藻三糖对顺铂引起的小鼠血清肌酐水平的影响；
图37示出了褐藻三糖对顺铂引起的小鼠尿量的影响；
图38示出了褐藻三糖对顺铂引起的小鼠肾脏组织中Kim-1和NGAL的mRNA水平的影响；
图39示出了褐藻三糖对顺铂引起的小鼠肾脏组织中炎症指标的影响；
图40示出了本发明实施例4和实施例5中所用的混合糖的质谱图。
具体实施方式
下面结合实施例对本发明做进一步的说明，实施例仅为解释和说明性的，绝不意味着以任何方式限制本发明的范围。
实施例1均一聚合度褐藻二糖的制备及其结构鉴定
将100g采购的褐藻胶(购自青岛明月海藻集团有限公司)溶于水，在一定温度下，加入褐藻糖裂解酶(获得自中国海洋大学)，经过一定时间的裂解后，高速离心机离心，取上层清液。将清液再以凝胶柱纯化，去除少量的杂质寡糖和多糖及非糖类杂质，得到纯度95％以上的褐藻二糖钠盐60g。将所得褐藻二糖钠盐用高效液相色谱法(HPLC，230nm)进行纯度检测，并用核磁氢谱( 1HNMR)和高分辨质谱(HRMS-ESI)进行结构鉴定。
HPLC：纯度99.06％，RT＝13.6min(相关谱图见图1)；
1HNMR谱图见图2；
HRMS(ESI)m/z:C 12H 15O 12{(M-H) -}，计算值为351.0569，实测值为351.0572(M-H) -。(相关谱图见图3)；
褐藻二糖钠盐，如果分子中两个羧基均为钠盐，则钠离子理论含量为11.58％；实际离子色谱法检测，钠离子含量为10.3％。如用炽灼残渣法检测，则钠离子以硫酸钠的形式存在，理论残渣比例应该为35.77％；实际炽灼残渣检测，残渣为34.3％；两种检测方法所得结果均比较接近，说明化合物羧酸官能团确为钠盐形式。但实测值均略小于理论值，可能是因为其钠盐为弱酸强碱盐，有少部分羧酸仍呈游离状态。
实施例2均一聚合度褐藻三糖的制备及其结构鉴定
将100g采购的褐藻胶溶于水，在一定温度下，加入褐藻糖裂解酶(获得自中国海洋大学)，经过一定时间的裂解后，高速离心机离心，取上层清液。将清液再以凝胶柱纯化，去除少量的杂质寡糖和多糖及非糖类杂质，得到纯度95％以上的褐藻三糖钠盐70g。将所得褐藻三糖钠盐用高效液相色谱法(HPLC，230nm)进行纯度检测，并用核磁氢谱( 1HNMR)和高分辨质谱(HRMS-ESI)进行结构鉴定。
HPLC：纯度100％，RT＝17.43min(相关谱图见图4)；
1HNMR谱图见图5；
HRMS(ESI)m/z:C 18H 23O 18{(M-H) -}，计算值为527.0890，实测值为527.0891(M-H) -。(相关谱图见图6)；
褐藻三糖钠盐，如果分子中三个羧基均为钠盐，则钠离子理论含量为11.59％；实际离子色谱法检测，钠离子含量为9.9％。如用炽灼残渣法检测，则钠离子以硫酸钠的形式存在，理论残渣比例应该为35.80％；实测炽灼残渣为33.01％。两种检测方式，所得结果均比较接近，说明化合物羧酸官能团确为钠盐形式。但实测值均略小于理论值，可能是因为其钠盐为弱酸强碱盐，有少部分羧酸仍呈游离状态。
实施例3均一聚合度褐藻四糖的制备及其结构鉴定
将100g采购的褐藻胶溶于水，在一定温度下，加入褐藻糖裂解酶(获得自中国海洋大学)，经过一定时间的裂解后，高速离心机离心，取上层 清液。将清液再以凝胶柱纯化，去除少量的杂质寡糖和多糖及非糖类杂质，得到纯度95％以上的褐藻四糖钠盐55g。将所得褐藻四糖钠盐用高效液相色谱法(HPLC，230nm)进行纯度检测，并用核磁氢谱( 1HNMR)和高分辨质谱(HRMS-ESI)进行结构鉴定。
HPLC：纯度99.71％，RT＝18.71min(相关谱图见图7)；
1HNMR谱图见图8；
HRMS(ESI)m/z：C 24H 31O 24{(M-H) -}，计算值为703.1211，实测值为703.1207(M-H) -。(相关谱图见图9)；
褐藻四糖钠盐，如果分子中四个羧基均为钠盐，则钠离子理论含量为11.59％；实测离子色谱法检测，钠离子含量为9.8％。如用炽灼残渣法检测，则钠离子以硫酸钠的形式存在，理论残渣比例应该为35.80％；实测炽灼残渣，残渣为32.5％。两种检测方式，所得结果均比较接近，说明化合物羧酸官能团确为钠盐形式。但实测值均略小于理论值，可能是因为其钠盐为弱酸强碱盐，有少部分羧酸仍呈游离状态。
实施例4均一聚合度褐藻寡糖对缺血再灌注(I/R)引起的大鼠急性肾脏损伤(acute kidney injury,AKI)的影响
缺血再灌注导致的肾脏损伤是模拟临床血液灌注不足导致的急性肾脏损伤的标准动物模型。发明人利用大鼠缺血再灌注模型，分别给药实施例1～3制备的均一聚合度的褐藻寡糖及其混合物，并与空白组和模型不给药组进行对比，考察各均一聚合度褐藻寡糖的治疗效果。
一、褐藻二糖对缺血再灌注引起的大鼠急性肾脏损伤的影响
选取Sprague Dawley大鼠，中山大学实验动物中心购买，220-250克雄性大鼠30只，术前收集24小时尿量测定无异常，将造模大鼠进行随机分组，分为假手术组、模型组、褐藻二糖0.01、0.05、0.1g/kg/天三个剂量组(每组6只)。在本发明中，褐藻二糖简称为“AOS2”。药物采用灌胃给药，模型组、假手术组均灌服同体积生理盐水。手术当天，大鼠以3％戊巴比妥钠腹腔麻醉后，常规消毒皮肤，从腹部开口，暴露左右侧肾脏，假手术组仅对肾脏进行检查，然后逐层缝合创口，结束手术；模型组及造模给药组使用大号动脉夹夹闭双侧的肾脏肾蒂，然后复位肾脏，以纱布覆盖创口并滴加少量生理盐水补液。45min后，松开双侧动脉夹，然后逐层缝合创口，结束手术。将手术后大鼠放置于37℃加热垫上等待大鼠复苏后放回代谢笼，期间检测大鼠体重、进食量、饮水量及尿量。术后常规饲养，24小时后处死大鼠取材。血样收集从大鼠的下腔静脉处取血，离心后收集上层血清，然后使用南京建成的肌酐测定试剂盒使用试剂盒法测定大鼠血清肌酐(Serum creatinine)，实验结果采用t值法进行统计学处理，结果见图10。所收集的24小时尿量结果见图11。取材时分离双侧肾脏，分离肾脏的皮质和内髓，皮质保存于trizol中，使用时采用trizol法提取皮质中 的mRNA，检测AKI生物标记物(KIM-1、NGAL)的mRNA表达，结果见图12。加入组织裂解液使用超声法匀浆肾脏后提取总蛋白，运用免疫印迹技术(western blotting)检测肾脏皮质组织中的炎症因子(p-NFκB/NFκB、pro-IL-1β/IL-1β)，结果见图13。
图10显示急性缺血再灌注(I/R)损伤引起大鼠血清肌酐水平显著升高，不同剂量的AOS2不同程度地降低血清肌酐，提示AOS2具有肾脏保护作用。*表示与sham(假手术组)相比，p<0.05，#表示与I/R组(模型组)相比，p<0.05。
此结果表明：I/R组大鼠在缺血再灌注术后肾脏的滤过功能发生障碍，血清肌酐显著增高。三种不同剂量的AOS2治疗后血清肌酐显著下降，提示肾小球功能有一定程度的恢复。特别是给药剂量为0.1g/kg/天时，血清肌酐基本可恢复至正常水平，说明AOS2对急性肾脏损害具有显著的治疗效果，其治疗效果具有一定程度的剂量依赖性。
图11显示急性缺血再灌注(I/R)损伤引起大鼠尿量增加，不同剂量的AOS2降低大鼠尿量，提示AOS2具有肾脏保护作用。*表示与sham相比，p<0.05，#表示与I/R组相比，p<0.05。此结果表明：I/R组大鼠在缺血再灌注术后肾脏的尿浓缩功能发生障碍，尿量显著增多。给予三种不同剂量的AOS2治疗后尿量有所下降，说明肾小管重吸收功能有一定程度的恢复。
图12显示急性缺血再灌注(I/R)损伤引起大鼠肾脏组织中急性肾脏损伤(AKI)指标(即肾小管损伤指标)Kim-1和NGAL的mRNA水平升高，剂量为0.1g/kg/天的AOS2明显降低了两个指标的表达，提示AOS2具有肾脏保护作用。*表示与sham相比，p<0.05，#表示与I/R组相比，p<0.05。此结果表明：I/R组大鼠在缺血再灌注术后急性肾脏损伤(AKI)指标KIM-1及NGAL表达呈显著性增高，提示肾小管损伤，0.1g/kg/天的AOS2可以明显降低两种指标，说明AOS2对缺血再灌注引起的急性肾脏损伤有显著的保护作用。
图13显示急性缺血再灌注(I/R)损伤引起大鼠肾脏组织中炎症指标显著增加，不同剂量的AOS2不同程度地抑制肾脏炎症反应，提示AOS2具有肾脏保护作用。I/R+AOS2-L剂量为0.01g/kg/天；I/R+AOS2-M剂量为0.05g/kg/天；I/R+AOS2-H剂量为0.1g/kg/天。此结果表明：I/R组大鼠在缺血再灌注术后肾脏炎症指标如TLR4、p-NFκB/NFκB、pro-IL-1β/IL-1β显著上升，AOS2治疗减少炎症因子的产生，并呈一定的剂量依赖性，说明AOS2的抗炎作用显著。
二、褐藻三糖对缺血再灌注引起的大鼠急性肾脏损伤的影响
使用与褐藻二糖相同的实验方法，考察褐藻三糖对缺血再灌注引起的大鼠急性肾脏损伤的影响。在本发明中，褐藻三糖简称为“AOS3”。使用南京建成的肌酐测定试剂盒使用试剂盒法测定大鼠血清肌酐，实验结果采用 t值法进行统计学处理，结果见图14。所收集的24小时尿量结果见图15。取材时分离双侧肾脏，分离肾脏的皮质和内髓，皮质保存于trizol中，使用时采用trizol法提取皮质中的mRNA，检测AKI生物标记物(KIM-1、NGAL)的mRNA表达，结果见图16。加入组织裂解液使用超声法匀浆肾脏后提取总蛋白，运用免疫印迹技术(western blotting)检测肾脏皮质组织中的炎症因子(p-NFκB/NFκB、pro-IL-1β/IL-1β)，结果见图17。此外，在最后一次取血后将动物处死，取肾固定于4％甲醛溶液中，常规石蜡包埋，切片，HE染色，光镜观察一般肾组织形态，结果见图18。
图14显示急性缺血再灌注(I/R)损伤引起大鼠血清肌酐水平显著升高，不同剂量的AOS3不同程度地降低血清肌酐，提示AOS3具有肾脏保护作用。*表示与sham相比，p<0.05，#表示与I/R组相比，p<0.05。此结果表明：I/R组大鼠在缺血再灌注术后肾脏的滤过功能发生障碍，血清肌酐显著增高。三种不同剂量的AOS3治疗后血清肌酐显著下降，提示肾小球功能有一定程度的恢复。特别是给药剂量为0.1g/kg/天时，血清肌酐基本可恢复至正常水平，其治疗效果具有一定程度的剂量依赖性。
图15显示急性缺血再灌注(I/R)损伤引起大鼠尿量增加，不同剂量的AOS3降低大鼠尿量，提示AOS3具有肾脏保护作用。*表示与sham相比，p<0.05，#表示与I/R组相比，p<0.05。此结果表明：I/R组大鼠在缺血再灌注术后肾脏的尿浓缩功能发生障碍，尿量显著增多。三种不同剂量的AOS3治疗后尿量均有所下降，尤其是0.1g/kg/天的剂量给药，大鼠第二天的尿量基本恢复至接近正常水平，提示肾小管功能有一定程度的恢复。其治疗效果具有一定程度的剂量依赖性。
图16显示急性缺血再灌注(I/R)损伤引起大鼠肾脏组织中急性肾脏损伤(AKI)指标(即肾小管损伤指标)Kim-1和NGAL的mRNA水平升高，剂量为0.1g/kg/天的AOS明显降低两个指标的表达，提示AOS3具有肾脏保护作用。*表示与sham相比，p<0.05，#表示与I/R组相比，p<0.05。此结果表明：I/R组大鼠在缺血再灌注术后急性肾脏损伤(AKI)指标KIM-1及NGAL表达呈显著性增高，0.1g/kg/天的AOS3可以明显降低两种指标，显示出AOS3对缺血再灌注引起的急性肾脏损伤有显著的保护作用。
图17显示急性缺血再灌注(I/R)损伤引起大鼠肾脏组织中炎症指标显著增加，不同剂量的AOS3不同程度地抑制肾脏炎症反应，提示AOS3具有肾脏保护作用。I/R+AOS3-L表示剂量为0.01g/kg/天；I/R+AOS3-M表示剂量为0.05g/kg/天；I/R+AOS3-H表示剂量为0.1g/kg/天。此结果表明：I/R组大鼠在缺血再灌注术后，肾脏炎症指标TLR4、p-NFκB/NFκB、pro-IL-1β/IL-1β显著上升，不同剂量的AOS3治疗均可减少炎症因子的产生，并呈一定的剂量依赖性，说明褐藻三糖具有明显的抗炎作用。
图18显示了肾脏组织病理表现：假手术组(SHAM)：肾小球形态、系膜细胞和肾小管基本正常；I/R组肾小球发生萎缩脱落，系膜细胞和基 质减少产生空洞，肾小管广泛扩张，管腔扩大，大量上皮细胞出现水肿、坏死和脱落，可见空泡样变性；I/R+AOS3(0.1g/kg/天)组肾小球和肾小管病变轻微。这些结果提示，I/R组肾小球和肾小管病变非常明显，而同样缺血再灌注后，再给予AOS3(0.1g/kg/天)治疗，肾脏损伤则非常轻微。说明AOS3对于缺血再灌注引起的大鼠肾脏形态学改变具有很好的保护作用。
三、褐藻四糖及混合糖对缺血再灌注引起的大鼠急性肾脏损伤的影响
使用与褐藻二糖和褐藻三糖相同的实验方式。将造模大鼠进行随机分组，分为假手术组、模型组、褐藻四糖(0.01、0.05、0.1g/kg/天三个剂量)组和混合糖(聚合度为2-8的混合褐藻寡糖，其质谱图参见图40，获得自中国海洋大学，0.1g/kg/天一个剂量)组(每组6只)。在本发明中，褐藻四糖简称为“AOS4”，混合糖简称为“AOS(混)”。使用南京建成的肌酐测定试剂盒使用试剂盒法测定大鼠血清肌酐，实验结果采用t值法进行统计学处理，结果见图19。所收集的24小时尿量结果见图20。取材时分离双侧肾脏，分离肾脏的皮质和内髓，皮质保存于trizol中，使用时采用trizol法提取皮质中的mRNA，检测AKI生物标记物(KIM-1、NGAL)的mRNA表达，结果见图21。加入组织裂解液使用超声法匀浆肾脏后提取总蛋白，运用免疫印迹技术(western blotting)检测肾脏皮质组织中的炎症因子(p-NFκB/NFκB、pro-IL-1β/IL-1β)，结果见图22。
图19显示急性缺血再灌注(I/R)损伤引起大鼠血清肌酐水平显著升高，不同剂量的AOS4和混合糖不同程度地降低血清肌酐，AOS4具有很好的肾脏保护作用。*表示与sham相比，p<0.05，#表示与I/R组相比，p<0.05。此结果表明：I/R组大鼠在缺血再灌注术后肾脏的滤过功能发生障碍，血清肌酐显著增高。三种不同剂量的AOS4和混合糖(0.1g/kg/天)治疗后血清肌酐显著下降，提示肾小球功能都有一定程度的恢复。特别是给药剂量为0.1g/kg/天的褐藻四糖，血清肌酐可恢复至接近正常水平。不同剂量的AOS4，其治疗效果具有一定程度的剂量依赖性。
图20显示急性缺血再灌注(I/R)损伤引起大鼠尿量增加，不同剂量的AOS4和混合糖(0.1g/kg/天)降低大鼠尿量，AOS4具有很好的肾脏保护作用。*表示与sham相比，p<0.05，#表示与I/R组相比，p<0.05。此结果表明：I/R组大鼠在缺血再灌注术后肾脏的尿浓缩功能发生障碍，尿量显著增多。三种不同剂量的AOS4和混合糖治疗后尿量有所下降，提示肾小管功能有一定程度的恢复，尤其是AOS4的治疗效果显著好于相同剂量的混合糖，治疗效果呈一定程度的剂量依赖性。
图21显示急性缺血再灌注(I/R)损伤引起大鼠肾脏组织中急性肾脏损伤(AKI)指标(即肾小管损伤指标)Kim-1和NGAL的mRNA水平升高，剂量为0.1g/kg/天的褐藻四糖明显降低两个指标的表达，提示其具有较好的肾脏保护作用。*表示与sham相比，p<0.05，#表示与I/R组相比， p<0.05。此结果表明：I/R组大鼠在缺血再灌注术后急性肾脏损伤(AKI)指标KIM-1及NGAL表达呈显著性增高，0.1g/kg/天的AOS4可以明显降低两种指标，说明AOS4对缺血再灌注引起的急性肾脏损伤有很好的保护作用。
图22显示急性缺血再灌注(I/R)损伤引起大鼠肾脏组织中炎症指标显著增加，不同剂量的AOS4可以不同程度地抑制肾脏炎症反应，提示AOS4具有肾脏保护作用。I/R+AOS4-L表示剂量为0.01g/kg/天；I/R+AOS4-M表示剂量为0.05g/kg/天；I/R+AOS4-H表示剂量为0.1g/kg/天；I/R+AOS(混)-H表示剂量为0.1g/kg/天。此结果表明：I/R组大鼠在缺血再灌注术后肾脏炎症指标如TLR4、p-NFκB/NFκB、pro-IL-1β/IL-1β显著上升，AOS4的治疗能明显减少炎症因子的产生，并呈一定的剂量依赖性，显示AOS4具有抗炎作用。
实验结论
缺血再灌注导致的肾脏损伤是模拟临床血液灌注不足导致的急性肾脏损伤的标准动物模型。褐藻二糖、三糖、四糖在给药不同剂量(0.01、0.05、0.1g/kg/天)治疗后，大鼠血清肌酐水平显著下降，其中0.1g/kg/天剂量时血清肌酐水平均可基本恢复到正常值；给药治疗后，肾脏的尿浓缩功能恢复明显转好，尿量减少，其中0.1g/kg/天剂量组明显降低肾脏损伤因子(KIM-1、NGAL)水平，炎症因子表达显著下降，肾脏病理改变显著改善，并随剂量增加，治疗作用增强。综上所述，血清肌酐、尿量、炎症因子等不同指标的检测结果都显示，褐藻二糖、三糖、四糖均对缺血再灌注引起的大鼠急性肾脏损伤有很好的保护作用，并且与聚合度为2-8的混合褐藻寡糖相比，均一聚合度的褐藻二糖、三糖和四糖的治疗效果显著更好。
实施例5均一聚合度褐藻寡糖对内毒素磷脂多糖(LPS)引起的小鼠急性肾脏损伤的影响
内毒素磷脂多糖处理导致的肾脏损伤是模拟临床感染所致的急性肾脏损伤的标准动物模型。我们利用小鼠内毒素磷脂多糖模型，分别给予不同均一聚合度的褐藻寡糖及其混合物，并与空白组和模型不给药组进行对比，考察各均一聚合度褐藻寡糖的治疗效果。
一、褐藻二糖(AOS2)对LPS引起的小鼠急性肾脏损伤的影响
选取22-28克雄性C57/Bl6小鼠24只，术前收集24小时尿量测定无异常，将造模小鼠进行随机分组，分为对照组、模型组、模型+给药组(AOS20.1g/kg/天)和模型+阳性对照组(醋酸地塞米松0.1g/kg/天)每组6只，样品采用灌胃给药，模型组、对照组均灌服同体积生理盐水，地塞米松采用腹腔注射给药。采用LPS进行造模来诱导脓毒血症型急性肾脏损伤的发生，造模时各模型组腹腔注射LPS 15mg/kg，对照组腹腔注射等量生理盐 水。造模后立刻将小鼠放回小鼠代谢笼进行观察，期间检测小鼠体重、进食量、饮水量及尿量。24小时后处死小鼠取材，收集尿液，血样收集从小鼠的下腔静脉处取血，离心后收集上层血清，然后使用南京建成的肌酐测定试剂盒使用试剂盒法测定小鼠血清肌酐，实验结果采用t值法进行统计学处理，结果见图23。取材时分离双侧肾脏，分离肾脏的皮质和内髓，皮质保存于trizol中，使用时采用trizol法提取皮质中的mRNA，使用Qpcr法检测AKI生物标记物(KIM-1、NGAL，结果见图24)及炎症因子(IL-1β、IL-18、TNF-α、MCP-1，结果见图25)的mRNA表达。加入组织裂解液使用超声法匀浆肾脏后提取总蛋白，运用免疫印迹技术(western blotting)检测肾脏皮质组织中的炎症因子(p-NFκB/NFκB、pro-IL-1β/IL-1β)，结果见图26。
图23显示LPS处理引起小鼠血清肌酐水平显著升高，AOS2明显降低血清肌酐，与地塞米松有同等的降低血清肌酐效果，提示褐藻二糖具有肾脏保护作用。Dex地塞米松，*表示与CTL(对照组)相比，p<0.05，#表示与LPS组(模型组)相比，p<0.05。此结果表明：与对照组相比，小鼠在腹腔注射LPS后血清肌酐显著增高，AOS2给药组小鼠血清肌酐明显下降，基本可恢复至正常水平，说明褐藻二糖对LPS引起的小鼠肾脏功能下降具有显著的保护作用。
图24显示LPS处理引起小鼠肾脏组织中急性肾脏损伤(AKI)指标(即肾小管损伤指标)Kim-1和NGAL的mRNA水平升高，提示褐藻二糖具有肾脏保护作用。*表示与CTL相比，p<0.05，#表示与LPS组相比，p<0.05。此结果表明：小鼠在腹腔注射LPS后AKI指标(KIM-1、NGAL)显著增高，AOS2治疗后，KIM-1及NGAL的产生显著减少，说明AOS2对于LPS引起的小鼠肾脏损伤具有明显的保护作用。
图25显示LPS处理引起小鼠肾脏组织中炎症指标显著增加，AOS2比较明显地抑制肾脏炎症反应，提示褐藻二糖具有肾脏保护作用。*表示与CTL相比，p<0.05，#表示与LPS组相比，p<0.05。此结果表明：小鼠在腹腔注射LPS后炎症因子(IL-1β、IL-18、TNF-α、MCP-1)基因表达显著增高，AOS2治疗明显减低炎症因子的产生，提示褐藻二糖对于LPS引起的小鼠急性肾脏损伤具有良好的保护作用。
图26显示LPS处理引起小鼠肾脏组织中炎症指标显著增加，AOS2明显抑制肾脏炎症反应，效果与阳性对照的地塞米松接近，提示AOS2具有肾脏保护作用。*表示与CTL相比，p<0.05，#表示与LPS组相比，p<0.05。此结果表明：LPS处理后，小鼠肾脏炎症指标如TLR4、p-NFκB/NFκB、pro-IL-1β/IL-1β蛋白表达显著上升，AOS2治疗显著减少了炎症因子的产生，其效果与地塞米松作用接近，显示AOS2具有明显的抗炎作用。
二、褐藻三糖(AOS3)对LPS引起的小鼠急性肾脏损伤的影响
采用与褐藻二糖相同的实验方式。使用南京建成的肌酐测定试剂盒使 用试剂盒法测定小鼠血清肌酐，实验结果采用t值法进行统计学处理，结果见图27。取材时分离双侧肾脏，分离肾脏的皮质和内髓，皮质保存于trizol中，使用时采用trizol法提取皮质中的mRNA，使用Qpcr法检测AKI生物标记物(KIM-1、NGAL，结果见图28)及炎症因子(IL-1β、IL-18、TNF-α、MCP-1，结果见图29)的mRNA表达。加入组织裂解液使用超声法匀浆肾脏后提取总蛋白，运用免疫印迹技术检测肾脏皮质组织中的炎症因子(p-NFκB/NFκB、pro-IL-1β/IL-1β)，结果见图30。此外，在最后一次取血后将动物处死，取肾固定于4％甲醛溶液中，常规石蜡包埋，切片，HE染色，光镜观察一般肾组织形态，结果见图31。
图27显示LPS处理引起小鼠血清肌酐水平显著升高，AOS3明显降低血清肌酐，与地塞米松有同等的降低血清肌酐效果，提示褐藻三糖具有肾脏保护作用。Dex地塞米松，*表示与CTL相比，p<0.05，#表示与LPS组相比，p<0.05。此结果表明：与对照组相比，小鼠在腹腔注射LPS后血清肌酐显著增高，AOS3给药组小鼠血清肌酐明显下降，基本可恢复至正常水平，提示褐藻三糖对LPS引起的小鼠肾脏功能下降具有显著的保护作用。
图28显示LPS处理引起小鼠肾脏组织中急性肾脏损伤(AKI)指标(即肾小管损伤指标)Kim-1和NGAL的mRNA水平升高，提示褐藻三糖具有肾脏保护作用。*表示与CTL相比，p<0.05，#表示与LPS组相比，p<0.05。此结果表明：小鼠在腹腔注射LPS后AKI指标(KIM-1、NGAL)显著增高，AOS3治疗后，KIM-1及NGAL的产生显著减少，提示AOS3对于LPS引起的小鼠肾脏损伤具有保护作用。
图29显示LPS处理引起小鼠肾脏组织中炎症指标显著增加，AOS3比较明显地抑制肾脏炎症反应，提示褐藻三糖具有肾脏保护作用。*表示与CTL相比，p<0.05，#表示与LPS组相比，p<0.05。此结果表明：小鼠在腹腔注射LPS后炎症因子(IL-1β、IL-18、TNF-α、MCP-1)基因表达显著增高，AOS3治疗明显减低炎症因子的产生，证明褐藻三糖对于LPS引起的小鼠急性肾脏损伤具有良好的保护作用。
图30显示LPS处理引起小鼠肾脏组织中炎症指标显著增加，AOS3明显抑制肾脏炎症反应，效果与阳性对照的地塞米松非常接近，提示AOS3具有较好的肾脏保护作用。*表示与CTL相比，p<0.05，#表示与LPS组相比，p<0.05。此结果表明：LPS处理后，小鼠肾脏炎症指标如TLR4、p-NFκB/NFκB、pro-IL-1β/IL-1β蛋白表达显著上升，AOS3治疗显著减少炎症因子的产生，其效果与地塞米松作用非常接近，说明AOS3具有较好的抗炎作用。
图31.显示肾脏组织病理表现：对照组：肾小球形态、系膜细胞和肾小管基本正常，LPS组肾小球体积增大，肾间质可见炎性细胞浸润，肾小管广泛扩张，上皮细胞出现水肿、坏死和脱落，可见空泡样变性；LPS+AOS3 组肾小球和肾小管病变较轻微。这些结果显示AOS3对于内毒素磷脂多糖处理诱导的小鼠肾脏形态学改变具有显著的保护作用。
三、褐藻四糖(AOS4)及混合糖对LPS引起的小鼠急性肾脏损伤的影响
采用与褐藻二糖和褐藻三糖相同的实验方式。将造模小鼠进行随机分组，分为对照组、模型组、模型+给药组1(AOS4 0.1g/kg/天)、模型+给药组2(聚合度为2-8的混合褐藻寡糖，其质谱图参见图40，获得自中国海洋大学，0.1g/kg/天)和模型+阳性对照组(醋酸地塞米松0.1g/kg/天)每组6只，样品采用灌胃给药，模型组、对照组均灌服同体积生理盐水，地塞米松采用腹腔注射给药。使用南京建成的肌酐测定试剂盒使用试剂盒法测定小鼠血清肌酐，实验结果采用t值法进行统计学处理，结果见图32。取材时分离双侧肾脏，分离肾脏的皮质和内髓，皮质保存于trizol中，使用时采用trizol法提取皮质中的mRNA，使用Qpcr法检测AKI生物标记物(KIM-1、NGAL，结果见图33)及炎症因子(IL-1β、IL-18、TNF-α、MCP-1，结果见图34)的mRNA表达。加入组织裂解液使用超声法匀浆肾脏后提取总蛋白，运用免疫印迹技术检测肾脏皮质组织中的炎症因子(p-NFκB/NFκB、pro-IL-1β/IL-1β)，结果见图35。
图32显示LPS处理引起小鼠血清肌酐水平显著升高，混合糖对降低血清肌酐有一定效果，AOS4则明显降低血清肌酐，与地塞米松的降低血清肌酐效果接近，提示褐藻寡糖具有肾脏保护作用。Dex地塞米松，*表示与CTL相比，p<0.05，#表示与LPS组相比，p<0.05。此结果表明：与对照组相比，小鼠在腹腔注射LPS后血清肌酐显著增高，AOS4和地塞米松给药组小鼠血清肌酐明显下降，基本可恢复至正常水平，说明褐藻四糖对LPS引起的小鼠肾脏功能下降具有显著的保护作用，其效果与地塞米松接近。
图33显示LPS处理引起小鼠肾脏组织中急性肾脏损伤(AKI)指标(即肾小管损伤指标)Kim-1和NGAL的mRNA水平升高，褐藻四糖和混合糖(剂量0.1g/kg/天)给药后，两个指标均有所降低，尤其是褐藻四糖，使两个指标均明显降低，提示褐藻四糖具有较好的肾脏保护作用。*表示与CTL相比，p<0.05，#表示与LPS组相比，p<0.05。此结果表明：小鼠在腹腔注射LPS后AKI指标(KIM-1、NGAL)显著增高，AOS4给药治疗后，KIM-1及NGAL的产生显著减少，提示褐藻四糖对于LPS引起的小鼠肾脏损伤具有保护作用。
图34显示LPS处理引起小鼠肾脏组织中炎症指标显著增加，AOS4和混合糖比较明显地抑制肾脏炎症反应，其中AOS4具有较好的肾脏保护作用。*表示与CTL相比，p<0.05，#表示与LPS组相比，p<0.05。此结果表明：小鼠在腹腔注射LPS后炎症因子(IL-1β、IL-18、TNF-α、MCP-1)基因表达显著增高，AOS4和混合糖治疗可以明显减低炎症因子的产生， 其中AOS4对于LPS引起的小鼠急性肾脏损伤具有良好的保护作用。
图35显示LPS处理引起小鼠肾脏组织中炎症指标显著增加，AOS4和混合糖明显抑制肾脏炎症反应，效果与阳性对照的地塞米松接近，提示AOS4具有较好的肾脏保护作用。*表示与CTL组相比，p<0.05，#表示与LPS组相比，p<0.05。此结果表明：LPS处理后，小鼠肾脏炎症指标如TLR4、p-NFκB/NFκB、pro-IL-1β/IL-1β蛋白表达显著上升，褐藻四糖和混合糖治疗显著减少炎症因子的产生，其效果与地塞米松作用基本一致，说明其具有非常有效的抗炎作用。
实验结论
内毒素磷脂多糖(LPS)处理导致的肾脏损伤是模拟临床感染所致的急性肾脏损伤的标准动物模型。LPS处理的模型组小鼠血清肌酐水平显著升高，褐藻二糖、三糖、四糖0.1g/kg/天治疗小鼠后，血清肌酐水平显著下降；肾脏损伤因子(KIM-1、NGAL)水平及炎症因子表达显著下降，肾脏病理改变显著改善。聚合度为2-8的混合糖对以上指标也均有不同程度的改善，但与褐藻二糖、三糖和四糖有一定差距。这些结果表明均一聚合度的褐藻寡糖对内毒素磷脂多糖引起的急性肾脏损伤有很好的保护作用。
实施例6均一聚合度褐藻寡糖对顺铂(Cisplatin)引起的小鼠急性肾脏损伤的影响
顺铂处理导致的肾脏损伤是模拟临床上药物的直接肾脏毒性作用所致的急性肾脏损伤的标准动物模型。我们利用小鼠顺铂模型，分别给予不同均一聚合度的褐藻寡糖及其混合物，并与空白组和模型不给药组进行对比，考察各均一聚合度褐藻寡糖的治疗效果。
一、褐藻三糖(AOS3)对顺铂引起的小鼠急性肾脏损伤的影响
选取22-28克雄性C57BL/J6小鼠30只，术前收集24小时尿量测定无异常，将造模小鼠进行随机分组，分为对照组、模型组、模型+给药低剂量组(AOS3 0.05g/kg/天)、模型+给药中剂量组(AOS3 0.1g/kg/天)和模型+给药高剂量组(AOS3 0.2g/kg/天)每组6只，褐藻三糖采用灌胃给药，模型组、对照组均灌服同体积生理盐水。采用顺铂进行造模来诱导药物毒性型急性肾脏损伤的发生，造模时各模型组腹腔注射顺铂20mg/kg，对照组腹腔注射等量生理盐水。造模后立刻将小鼠放回小鼠代谢笼进行观察，期间检测小鼠体重、进食量、饮水量及尿量(结果见图37)。72小时后处死小鼠取材，收集尿液，血样收集从小鼠的下腔静脉处取血，离心后收集上层血清，然后使用南京建成的肌酐测定试剂盒使用试剂盒法测定小鼠血清肌酐，实验结果采用t值法进行统计学处理，结果见图36。取材时分离双侧肾脏，分离肾脏的皮质和内髓，皮质保存于trizol中，使用时采用trizol法提取皮质中的mRNA，使用Qpcr法检测AKI生物标记物 (KIM-1、NGAL，结果见图38)mRNA表达。加入组织裂解液使用超声法匀浆肾脏后提取总蛋白，运用免疫印迹技术(western blotting)检测肾脏皮质组织中的炎症因子(p-NFκB/NFκB、IL-1β)，结果见图39。
图36显示顺铂处理引起小鼠血清肌酐水平显著升高，AOS3明显降低血清肌酐，提示褐藻三糖具有肾脏保护作用。*表示与CTL组(对照组)相比，p<0.05，#表示与Cis组(模型组)相比，p<0.05。此结果表明：与对照组相比，小鼠在腹腔注射顺铂后血清肌酐显著增高，AOS3给药组小鼠血清肌酐明显下降，基本可恢复至正常水平，说明褐藻三糖对顺铂引起的小鼠肾脏功能下降具有显著的保护作用。
图37显示顺铂引起的小鼠在24h尿量先增加随后72h减少进入少尿期，不同浓度的AOS3给药后在24h先是降低小鼠尿量恢复肾脏功能随后尿量逐渐趋于正常，提示褐藻三糖具有肾脏保护作用。*表示与CTL相比，p<0.05，#表示与模型组相比，p<0.05。此结果表明：模型组小鼠在顺铂注射后肾脏的尿浓缩功能发生障碍，尿量先显著增多随后进入少尿期。三种不同剂量的AOS3治疗后尿量先是有所下降随后趋于正常，提示肾小管功能有一定程度的恢复。并呈一定程度的剂量依赖性。
图38显示顺铂处理引起小鼠肾脏组织中急性肾脏损伤(AKI)指标(即肾小管损伤指标)Kim-1和NGAL的mRNA水平升高，提示褐藻三糖具有肾脏保护作用。*表示与CTL相比，p<0.05，#表示与模型组相比，p<0.05。此结果表明：小鼠在腹腔注射顺铂后AKI指标(KIM-1、NGAL)显著增高，AOS3治疗后，KIM-1及NGAL的产生显著减少，说明AOS3对于顺铂引起的小鼠肾脏损伤具有明显的保护作用。
图39显示顺铂处理引起小鼠肾脏组织中炎症指标显著增加，AOS3明显抑制肾脏炎症反应，提示AOS3具有肾脏保护作用。*表示与CTL相比，p<0.05，#表示与模型组相比，p<0.05。此结果表明：顺铂处理后，小鼠肾脏炎症指标如TLR4、p-NFκB/NFκB、IL-1β蛋白表达显著上升，AOS3治疗显著减少了炎症因子的产生，显示AOS3具有明显的抗炎作用。
同样地，进一步研究发现，褐藻二糖和褐藻四糖对顺铂引起的小鼠急性肾脏损伤也具有与褐藻三糖类似的功效。
实验结论
顺铂处理的模型组小鼠血清肌酐水平显著升高，褐藻二糖、三糖、四糖0.1g/kg/天治疗小鼠后，血清肌酐水平显著下降；肾脏损伤因子(KIM-1、NGAL)水平及炎症因子表达显著下降，治疗效果具有浓度依赖性。这些结果表明褐藻二糖、三糖、四糖对顺铂引起的急性肾脏损伤有很好的保护作用。
最后需要在此指出的是：以上仅是本发明的部分优选实施例，不能理解为对本发明保护范围的限制，本领域的技术人员根据本发明的上述内容做出的一些非本质的改进和调整均属于本发明的保护范围。

Claims (9)

  1. 一种褐藻寡糖或其药学上可接受的盐在制备用于治疗急性肾脏损伤的药物中的用途，其中所述褐藻寡糖为褐藻二糖、褐藻三糖或褐藻四糖。
  2. 根据权利要求1所述的用途，其中所述褐藻寡糖是由单糖G、M和/或Δ通过1,4位糖苷键连接构成的；其中，G表示α-L-古洛糖醛酸，M表示β-D-甘露糖醛酸，Δ表示α-L-古洛糖醛酸或β-D-甘露糖醛酸的4,5位发生β-消除，生成4,5位为共轭双键的不饱和单糖。
  3. 根据权利要求2所述的用途，其中所述褐藻二糖选自ΔG、ΔM或其组合。
  4. 根据权利要求2所述的用途，其中所述褐藻三糖选自ΔGG、ΔGM、ΔMM和ΔMG中的一种或多种。
  5. 根据权利要求2所述的用途，其中所述褐藻四糖选自ΔGGG、ΔGGM、ΔGMG、ΔGMM、ΔMMG、ΔMMM、ΔMGG和ΔMGM中的一种或多种。
  6. 根据权利要求1所述的用途，其中所述药学上可接受的盐为钠盐、钾盐、钙盐、镁盐和/或铵盐。
  7. 根据权利要求1至6中任一项所述的用途，其中所述急性肾脏损伤是由血液灌注不足、感染或药物的肾脏毒性导致的。
  8. 一种褐藻寡糖或其药学上可接受的盐，其用于治疗急性肾脏损伤，其中所述褐藻寡糖为褐藻二糖、褐藻三糖或褐藻四糖。
  9. 一种治疗急性肾脏损伤的方法，包括给予需要的患者治疗有效量的褐藻寡糖或其药学上可接受的盐，其中所述褐藻寡糖为褐藻二糖、褐藻三糖或褐藻四糖。
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