CN113209111A

CN113209111A - Application of brown algae oligosaccharide

Info

Publication number: CN113209111A
Application number: CN202110528901.8A
Authority: CN
Inventors: 刘振德; 高河勇
Original assignee: Shanghai Haitang Biomedical Technology Co ltd
Current assignee: Shanghai Haitang Biomedical Technology Co ltd
Priority date: 2021-05-14
Filing date: 2021-05-14
Publication date: 2021-08-06
Also published as: CN113440532B; CN113440532A

Abstract

The invention provides an application of brown algae oligosaccharide or pharmaceutically acceptable salt thereof in preparing a medicament for treating acute kidney injury, wherein the brown algae oligosaccharide is brown algae disaccharide, brown algae trisaccharide or brown algae tetrasaccharide. Researches show that the brown algae disaccharide, trisaccharide and tetrasaccharide have very obvious protective effects on acute kidney injury animal models induced by ischemia reperfusion (I/R), endotoxin phospholipid polysaccharide (LPS) and antitumor drug cisplatin. After the brown algae oligosaccharide disclosed by the invention is used for treating an acute kidney injury animal, the serum creatinine level is obviously reduced, the urine concentration function of the kidney is obviously recovered, the renal tubular injury factor (KIM-1, NGAL) level is obviously reduced, the inflammatory factor expression is obviously reduced, the pathological change of the kidney is obviously improved, and the treatment effect is enhanced along with the increase of the dosage. Therefore, the brown algae oligosaccharide has a strong effect of treating acute kidney injury.

Description

Application of brown algae oligosaccharide

Technical Field

The invention relates to an application of brown algae oligosaccharide, belonging to the technical field of biological medicines.

Background

Carbohydrates (carbohydrates) are combined with nucleic acids, proteins and are called three major life substances. Algin is mainly present in the cell walls of kelp, gulfweed and kelp, and is a straight-chain, unbranched and negatively charged polysaccharide compound. Algin is a binary linear block compound consisting of β -D- (1,4) -Mannuronic acid (M) and α -L- (1,4) -Guluronic acid (Guluronic acid, G). Three structural fragments mainly exist in the molecule: polymannuronate (PM) composed of β -D- (1,4) -mannuronic acid linked to each other; polyguluronic acid (PG) consisting of α -L- (1,4) -guluronic acid linked to each other; m and G are copolymerized alternately to form a PMG fragment.

The characteristics of high viscosity, gel formation and the like of the algin enable the algin to be widely used in industrial production of food, chemical industry, medicine, textile and the like as a coagulating agent, a thickening agent, a stabilizing agent and the like. In the field of medicine, the algin has wide application in medical biomaterials and drug sustained and controlled release materials due to unique physicochemical properties and good biocompatibility. Research also finds that the algin has biological activities of oxidation resistance, immunoregulation, tumor resistance and the like, but the algin is greatly limited in application because of large molecular weight and strong gelling property and is not easy to absorb. The oligosaccharide has attracted attention due to the characteristics of definite structure, remarkable activity, good absorbability, small side effect and the like.

In recent years, due to the unique structure of alginate-derived oligosaccharides, the activity research becomes a hotspot in the research of carbohydrate drugs, and the research on the biological activity of alginate-derived oligosaccharides has made an important progress. It has been found that algin oligosaccharide and its derivatives have various biological activities, such as antioxidant, anti-tumor, anticoagulant, immunoregulatory, neuroprotective, anti-inflammatory, antiviral, anti-senile dementia, anti-lithangiuria, anti-diabetic, etc.

Carbohydrates are a highly complex class of biological macromolecules with simultaneously varying multiple ends. Unlike oligonucleotides and polypeptides, carbohydrates are not only linear oligomers, but are often branched. The common 9 monosaccharides found on mammalian cells can be linked into a more diverse structure than 20 naturally occurring amino acids or 4 nucleotides. This complexity of the carbohydrate structure makes it very difficult to obtain pure carbohydrates from natural sources. It is difficult to separate oligosaccharides or polysaccharides having a uniform degree of polymerization regardless of chemical cleavage or enzymatic cleavage. To date, almost all of the studies have used a mixture of a series of sugars with close degrees of polymerization, which has made it very difficult to study the activity, metabolism, toxicology, and drug quality.

Aiming at the difficulty of the current saccharide research, the inventor develops a series of alginate lyase with strong specificity, and can decompose the alginate into the fucoidan, trisaccharide or tetrasaccharide with high purity, conjugated double bond at the non-reducing end and uniform polymerization degree; inactivating enzyme, centrifuging to obtain supernatant, concentrating, and purifying with gel column or ion exchange resin to obtain brown algae disaccharide, trisaccharide or tetrasaccharide with uniform polymerization degree. The brown algae disaccharide is a combination of two structures of delta G and/or delta M and any proportion thereof; the brown algae trisaccharide is a combination of four structures of delta GG, delta GM, delta MM and delta MG and any proportion thereof; the brown algae tetrasaccharide is a combination of eight structures of delta GGG, delta GGM, delta GMG, delta GMM, delta MMG, delta MMM, delta MGG and delta MGM and any proportion thereof; all oligosaccharides are connected by

monosaccharide

1,4 glycosidic bonds; g represents alpha-L-guluronic acid; m represents beta-D-mannuronic acid; delta represents alpha-L-guluronic acid and/or beta-D-

mannuronic acid

4,5 position to generate beta-elimination, and unsaturated monosaccharide with

non-reducing end

4,5 position as conjugated double bond is generated; the structure of each monosaccharide is shown below:

taking Δ GM as an example, the structure of the corresponding fucoidan is as follows:

acute renal injury (AKI), which has been known as acute renal failure, is a clinical syndrome in which renal function rapidly decreases in a short period of time due to various causes, and is manifested by a rapid increase in serum creatinine and a decrease in urine volume. Statistically, about 10% to 20% of hospitalized patients are diagnosed with AKI worldwide each year, with even more than 50% of ICU patients, 85% of them from developing countries. AKI can increase mortality, prolong hospitalization, increase treatment costs, and also increase the risk of cardiovascular events, advanced Chronic Kidney Disease (CKD), and end-stage kidney disease (ESRD). Although the importance of AKI is increasing in the kidney disease community, no specific treatment is available at present, the morbidity and mortality are still high, the incidence rate of cardiovascular events of AKI patients is 38%, such as heart failure (58% of risk), acute myocardial infarction (40% of risk), hypertension (22% of risk), stroke (15% of risk) and the like, and the AKI becomes a worldwide public health problem threatening the health of human beings.

Currently there are no effective drugs that can reverse AKI kidney injury. Unlike some secondary chronic kidney diseases such as hypertensive nephropathy, AKI is a primary lesion of renal parenchyma (glomeruli, tubules, interstitium, etc.), has a complex etiology (such as ischemia, hypoxia, toxicants, drugs, infection, etc.), progresses rapidly, and some patients progress to chronic kidney diseases with complications such as cardiovascular diseases. Early diagnosis and timely intervention can reduce kidney injury to the maximum extent and promote renal function recovery. Early identification and correction of reversible etiology, maintenance of homeostasis, nutritional support, prevention of complications, and renal replacement therapy remain the current primary treatment strategies for AKI. Hypertensive nephropathy is characterized by the development of renal blood vessels due to prolonged elevation of blood pressure, thickening and thickening of renal capillaries, glomerular fibrosis, narrowing of vascular lumens, renal arteriosclerosis, renal parenchymal ischemia, and nephron reduction. Changes in renal parenchyma can lead to reduced renal hemofiltration and reduced renal function. The medicine is a long-term secondary disease of the kidney caused by continuous rise of blood pressure, has a long disease course, and is a basic treatment measure for controlling the blood pressure. Currently, the treatment of hypertensive nephropathy is primarily a blood pressure control therapy, however, this therapy is not suitable for treating AKI. Given the complex etiology of AKI, it is challenging to identify a monotherapy that would benefit all patients with AKI.

The invention further researches brown algae oligosaccharide and provides an application of brown algae oligosaccharide in treating acute kidney injury.

Disclosure of Invention

Therefore, the invention aims to provide the application of the brown algae oligosaccharide.

The purpose of the invention is realized by the following technical scheme:

the invention provides an application of brown algae oligosaccharide or pharmaceutically acceptable salt thereof in preparing a medicament for treating acute kidney injury, wherein the brown algae oligosaccharide is brown algae disaccharide, brown algae trisaccharide or brown algae tetrasaccharide.

In certain embodiments of the invention, the brown algae oligosaccharide is composed of monosaccharides G, M and/or Δ linked through glycosidic linkages at 1,4 positions; wherein G represents alpha-L-guluronic acid, M represents beta-D-mannuronic acid, and Delta represents alpha-L-guluronic acid or beta-D-mannuronic acid, and beta-elimination occurs at the 4,5 positions to generate unsaturated monosaccharide with conjugated double bonds at the 4,5 positions.

In certain embodiments of the invention, the fucoidan is selected from ag, am, or a combination thereof.

In certain embodiments of the invention, the fucoidan is selected from one or more of Δ GG, Δ GM, Δ MM, and Δ MG.

In certain embodiments of the invention, the brown algae tetrasaccharide is selected from one or more of Δ GGG, Δ GGM, Δ GMG, Δ GMM, Δ MMG, Δ MMM, Δ MGG and Δ MGM.

In certain embodiments of the invention, the pharmaceutically acceptable salt is a sodium, potassium, calcium, magnesium and/or ammonium salt.

In certain embodiments of the invention, the acute kidney injury is caused by hypoperfusion of blood, infection, or renal toxicity of the drug.

The brown algae disaccharide, trisaccharide and tetrasaccharide with uniform polymerization degree provided by the invention have revolutionary progress on analytical researches such as quality control, pharmacology and toxicology of saccharide bulk drugs.

Researches find that the brown algae disaccharide, trisaccharide and tetrasaccharide have very obvious protective effects on acute kidney injury animal models induced by ischemia reperfusion (I/R), endotoxin phospholipid polysaccharide (LPS) and antitumor drug cisplatin. After the brown algae oligosaccharide disclosed by the invention is used for treating an acute kidney injury animal, the serum creatinine level is obviously reduced, the urine concentration function of the kidney is obviously recovered, the renal tubular injury factor (KIM-1, NGAL) level is obviously reduced, the inflammatory factor expression is obviously reduced, the pathological change of the kidney is obviously improved, and the treatment effect is enhanced along with the increase of the dosage. Therefore, the brown algae oligosaccharide has a strong effect of treating kidney injury.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows a high performance liquid chromatogram of fucoidan at a wavelength of 230 nm;

FIG. 2 shows nuclear magnetic hydrogen spectrum of brown algae disaccharide (A)¹HNMR, solvent D₂O)；

FIG. 3 shows a high resolution mass spectrum (HRMS (ESI)) of fucoidan;

FIG. 4 shows a high performance liquid chromatogram of fucoidan at a wavelength of 230 nm;

FIG. 5 shows nuclear magnetic hydrogen spectrum of fucoidan (A)¹HNMR with solvent D₂O)；

FIG. 6 shows a high resolution mass spectrum (HRMS (ESI)) of fucoidan;

FIG. 7 shows a high performance liquid chromatogram of fucoidan at a wavelength of 230 nm;

FIG. 8 shows the nuclear magnetic hydrogen spectrum of fucoidan (M)¹HNMR with solvent D₂O)；

FIG. 9 shows a high resolution mass spectrum (HRMS (ESI)) of fucoidan;

FIG. 10 shows the effect of fucoidan on serum creatinine levels in rats induced by acute ischemia reperfusion (I/R) injury;

FIG. 11 shows the effect of fucoidan on urine volume in rats induced by acute ischemia-reperfusion (I/R) injury;

FIG. 12 shows the effect of fucoidan on mRNA levels of Kim-1 and NGAL in rat kidney tissue caused by acute ischemia-reperfusion (I/R) injury;

FIG. 13 shows the effect of fucoidan on the inflammatory marker in rat kidney tissue caused by acute ischemia-reperfusion (I/R) injury;

FIG. 14 shows the effect of fucotriose on serum creatinine levels in rats induced by acute ischemia reperfusion (I/R) injury;

FIG. 15 shows the effect of fucotriose on the urine volume in rats induced by acute ischemia-reperfusion (I/R) injury;

FIG. 16 shows the effect of fucoidan on mRNA levels of Kim-1 and NGAL in rat kidney tissue caused by acute ischemia-reperfusion (I/R) injury;

FIG. 17 shows the effect of fucoidan on inflammatory markers in rat kidney tissue caused by acute ischemia-reperfusion (I/R) injury;

FIG. 18 shows the effect of fucoidan on pathological sections of rat kidney tissue injury caused by acute ischemia-reperfusion (I/R) injury;

FIG. 19 shows the effect of fucoidan and mixed saccharides on serum creatinine levels in rats induced by acute ischemia reperfusion (I/R) injury;

FIG. 20 shows the effect of fucoidan and mixed sugars on urine volume in rats induced by acute ischemia-reperfusion (I/R) injury;

FIG. 21 shows the effect of fucoidan and mixed sugars on mRNA levels of Kim-1 and NGAL in rat kidney tissue caused by acute ischemia-reperfusion (I/R) injury;

FIG. 22 shows the effect of fucoidan on the inflammatory marker in rat kidney tissue caused by acute ischemia reperfusion (I/R) injury;

FIG. 23 shows the effect of fucoidan on serum creatinine levels in mice induced by endotoxin phospholipid polysaccharides (LPS);

FIG. 24 shows the effect of fucoidan on levels of mRNA for Kim-1 and NGAL in mouse kidney tissue induced by endotoxin phospholipid polysaccharide (LPS);

FIG. 25 shows the effect of fucoidan on the inflammatory marker in mouse kidney tissue induced by endotoxin phospholipid polysaccharide (LPS);

FIG. 26 shows the effect of fucoidan and dexamethasone controls on the inflammatory markers in mouse kidney tissue induced by endotoxin phospholipid polysaccharide (LPS);

FIG. 27 shows the effect of fucoidan on serum creatinine levels in mice induced by endotoxin phospholipid polysaccharide (LPS);

FIG. 28 shows the effect of fucoidan trisaccharide on mRNA levels of Kim-1 and NGAL in mouse kidney tissue induced by endotoxin phospholipid polysaccharide (LPS);

FIG. 29 shows the effect of fucoidan on the inflammatory marker in mouse kidney tissue induced by endotoxin phospholipid polysaccharide (LPS);

FIG. 30 shows the effect of fucoidan trisaccharide and dexamethasone control on the inflammatory marker in mouse kidney tissue induced by endotoxin phospholipid polysaccharide (LPS);

FIG. 31 shows the effect of fucoidan on pathological sections of endotoxin phospholipid polysaccharide (LPS) -induced kidney tissue damage in mice;

FIG. 32 shows the effect of fucoidan and mixed sugars on serum creatinine levels in mice induced by endotoxin phospholipid polysaccharides (LPS);

FIG. 33 shows the effect of fucoidan and mixed sugars on mRNA levels of Kim-1 and NGAL in mouse kidney tissue induced by endotoxin phospholipid polysaccharide (LPS);

FIG. 34 shows the effect of fucoidan and mixed sugars on the inflammation index in mouse kidney tissue induced by endotoxin phospholipid polysaccharide (LPS);

FIG. 35 shows the effect of fucoidan, mixed sugar and dexamethasone control on the inflammatory markers in mouse kidney tissue induced by endotoxin phospholipid polysaccharide (LPS);

FIG. 36 shows the effect of fucotriose on cisplatin-induced serum creatinine levels in mice;

FIG. 37 shows the effect of fucotriose on cisplatin-induced urine output in mice;

FIG. 38 shows the effect of fucotriose on mRNA levels of Kim-1 and NGAL in mouse kidney tissue caused by cisplatin;

FIG. 39 shows the effect of fucoidan on cisplatin-induced inflammation in mouse kidney tissue;

FIG. 40 shows the mass spectra of the mixed sugars used in examples 4 and 5 of the present invention.

Detailed Description

The present invention is further illustrated by the following examples, which are illustrative and explanatory only and are not meant to limit the scope of the invention in any way.

Example 1Preparation of fucoidan with uniform polymerization degree and structural identification thereof

Dissolving 100g of purchased algin (purchased from Qingdao Mingyue algae group, Ltd.) in water, adding fucoidan lyase (obtained from China oceanic university) at a certain temperature, performing pyrolysis for a certain period of time, centrifuging with a high-speed centrifuge, and collecting the supernatant. And purifying the clear liquid by using a gel column to remove a small amount of impurity oligosaccharide, polysaccharide and non-saccharide impurities, thereby obtaining 60g of the fucoidan sodium salt with the purity of more than 95%. The purity of the brown algae disaccharide sodium salt is detected by high performance liquid chromatography (HPLC, 230nm), and nuclear magnetic hydrogen spectrum is used (¹HNMR) and high resolution mass spectrometry (HRMS-ESI).

HPLC: purity 99.06%, RT 13.6min (see fig. 1 for the relevant spectrum);

¹the HNMR spectrogram is shown in figure 2;

HRMS(ESI)m/z:C₁₂H₁₅O₁₂{(M-H)^-}, calculated 351.0569, found 351.0572(M-H)^-. (the correlation spectrum is shown in FIG. 3);

the theoretical content of sodium ions in the sodium alginate disaccharide sodium salt is 11.58 percent if two carboxyl groups in a molecule are sodium salts; the content of sodium ions is 10.3 percent by actual ion chromatography detection. If the residue is detected by a residue on ignition method, sodium ions exist in the form of sodium sulfate, and the theoretical residue proportion is 35.77 percent; detecting actual residues on ignition, wherein the residue is 34.3%; the results obtained by the two detection methods are relatively close, which indicates that the carboxylic acid functional group of the compound is in a sodium salt form. However, the observed values are slightly smaller than the theoretical values, probably because the sodium salt is a weak acid strong base salt and a small part of the carboxylic acid is still in a free state.

Example 2Preparation of brown algae trisaccharide with uniform polymerization degree and structure identification thereof

Dissolving 100g of purchased algin in water, adding fucoidan lyase (obtained from China university of oceans) at a certain temperature, performing pyrolysis for a certain time, centrifuging with a high-speed centrifuge, and collecting supernatant. Mixing the clear liquid withPurifying by gel column to remove a small amount of oligosaccharide, polysaccharide and non-saccharide impurities, and obtaining 70g of brown algae trisaccharide sodium salt with the purity of more than 95%. The purity of the brown algae trisaccharide sodium salt is detected by high performance liquid chromatography (HPLC, 230nm), and nuclear magnetic hydrogen spectrum is used (¹HNMR) and high resolution mass spectrometry (HRMS-ESI).

HPLC: purity 100%, RT 17.43min (see fig. 4 for the relevant spectrum);

¹the HNMR spectrum is shown in FIG. 5;

HRMS(ESI)m/z:C₁₈H₂₃O₁₈{(M-H)^-}, calculated 527.0890, found 527.0891(M-H)^-. (see FIG. 6 for a correlation spectrum);

the sodium content of sodium ions is 11.59% if three carboxyl groups in the molecule are sodium salts; the content of sodium ions is 9.9 percent by actual ion chromatography detection. If the residue is detected by a residue burning method, sodium ions exist in the form of sodium sulfate, and the theoretical residue proportion is 35.80%; the residue on ignition was found to be 33.01%. The results obtained by the two detection modes are relatively close, which indicates that the carboxylic acid functional group of the compound is in a sodium salt form. However, the observed values are slightly smaller than the theoretical values, probably because the sodium salt is a weak acid strong base salt and a small part of the carboxylic acid is still in a free state.

Example 3Preparation of brown algae tetrasaccharide with uniform polymerization degree and structure identification thereof

Dissolving 100g of purchased algin in water, adding fucoidan lyase (obtained from China university of oceans) at a certain temperature, performing pyrolysis for a certain time, centrifuging with a high-speed centrifuge, and collecting supernatant. And purifying the clear liquid by using a gel column to remove a small amount of impurity oligosaccharide, polysaccharide and non-saccharide impurities, thereby obtaining 55g of the sodium alginate tetrasaccharide with the purity of more than 95%. The brown algae tetrasaccharide sodium salt is subjected to purity detection by high performance liquid chromatography (HPLC, 230nm), and nuclear magnetic hydrogen spectrum (A), (B), (C) and (D)¹HNMR) and high resolution mass spectrometry (HRMS-ESI).

HPLC: purity 99.71%, RT 18.71min (see fig. 7 for the relevant spectrum);

¹HNMR spectrogram8；

HRMS(ESI)m/z：C₂₄H₃₁O₂₄{(M-H)^-}, calculated 703.1211, found 703.1207(M-H)^-. (the correlation spectrum is shown in FIG. 9);

the sodium alginate tetrasaccharide salt has sodium ion content of 11.59% if four carboxyl groups in the molecule are sodium salts; the content of sodium ions is 9.8 percent by the detection of an actual ion chromatography. If the residue is detected by a residue burning method, sodium ions exist in the form of sodium sulfate, and the theoretical residue proportion is 35.80%; residue on ignition was found to be 32.5%. The results obtained by the two detection modes are relatively close, which indicates that the carboxylic acid functional group of the compound is in a sodium salt form. However, the observed values are slightly smaller than the theoretical values, probably because the sodium salt is a weak acid strong base salt and a small part of the carboxylic acid is still in a free state.

Example 4Effect of alginate oligosaccharide with uniform polymerization degree on Acute Kidney Injury (AKI) of rats caused by ischemia-reperfusion (I/R)

Renal injury from ischemia reperfusion is a standard animal model that mimics clinical acute renal injury from hypoperfusion. The inventor utilizes a rat ischemia reperfusion model, respectively administers the brown alga oligosaccharides with uniform polymerization degrees prepared in examples 1-3 and a mixture thereof, compares the brown alga oligosaccharides with uniform polymerization degrees with a blank group and a model non-administrated group, and inspects the treatment effect of the brown alga oligosaccharides with uniform polymerization degrees.

First, brown algae disaccharide influence on rat acute kidney injury caused by ischemia reperfusion

Sprague Dawley rats, purchased from the Experimental animal center of Zhongshan university, 30 male rats with 220-. In the present invention, the brown algae disaccharide is simply referred to as "AOS 2". The medicine is administrated by gastric perfusion, and the normal saline with the same volume is administrated by both the model group and the sham operation group. On the day of operation, after the rats are subjected to abdominal anesthesia by 3% sodium pentobarbital, the skin is disinfected conventionally, the left and right kidneys are exposed from an abdominal opening, only the kidneys are inspected by a sham operation group, then the wounds are sutured layer by layer, and the operation is finished; the model group and the model-making administration group use a large-sized artery clamp to clamp and close renal pedicles of the kidney on both sides, then the kidney is reset, a wound is covered by gauze, and a small amount of physiological saline solution is dripped for fluid infusion. After 45min, loosening the bilateral artery clamps, then suturing the wound layer by layer, and ending the operation. After the operation, the rats are placed on a heating pad at 37 ℃ and put back to the metabolism cage after the rats are revived, and the weight, the food intake, the water intake and the urine output of the rats are detected in the period. After the operation, the rats are bred in a routine way, and the materials are sacrificed after 24 hours. Blood sample collection blood is taken from the inferior vena cava of a rat, upper Serum is collected after centrifugation, then rat Serum creatinine (Serum creatinine) is measured by using a kit method by using a Nanjing-built creatinine measurement kit, the experimental result is statistically processed by using a t value method, and the result is shown in figure 10. The results of the 24-hour urine collected are shown in FIG. 11. Bilateral kidneys were isolated at the time of material collection, cortex and medulla of the kidneys were separated, the cortex was stored in trizol, mRNA in the cortex was extracted by trizol method at the time of use, and mRNA expression of AKI biomarkers (KIM-1, NGAL) was detected, and the results are shown in fig. 12. Adding tissue lysate, homogenizing kidney with ultrasound, extracting total protein, and detecting inflammatory factor (p-NFkB/NFk B, pro-IL-1 beta/IL-1 beta) in kidney cortex tissue with western blotting technique (see FIG. 13).

FIG. 10 shows that acute ischemia reperfusion (I/R) injury causes significant elevation of serum creatinine levels in rats and different doses of AOS2 reduce serum creatinine to varying degrees, suggesting that AOS2 has renal protective effects. Denotes p <0.05 compared to sham (sham group) and # denotes p <0.05 compared to I/R group (model group).

This result shows that: the filtration function of the kidney of the rats in the I/R group after ischemia-reperfusion is disordered, and the serum creatinine is obviously increased. Serum creatinine was significantly reduced following treatment with three different doses of AOS2, suggesting some degree of recovery of glomerular function. Particularly, the serum creatinine can be basically recovered to a normal level when the administration dosage is 0.1 g/kg/day, which shows that AOS2 has a remarkable treatment effect on acute kidney injury, and the treatment effect has a certain degree of dose dependence.

FIG. 11 shows that acute ischemia reperfusion (I/R) injury causes increased urine volume in rats, and different doses of AOS2 decreased urine volume in rats, suggesting that AOS2 has a renal protective effect. Denotes p <0.05 compared to sham and # denotes p <0.05 compared to the I/R group. This result shows that: the I/R group rats were impaired in the function of urinary concentration in the kidneys after ischemia-reperfusion, and significantly increased in urine volume. There was a decrease in urine volume following treatment with three different doses of AOS2, indicating some degree of restoration of tubular reabsorption function.

FIG. 12 shows that acute ischemia-reperfusion (I/R) injury causes elevated mRNA levels of Kim-1 and NGAL as indicators of acute renal injury (AKI) (i.e., tubular injury indicators) in rat kidney tissue, and AOS2 at a dose of 0.1 g/kg/day significantly reduced the expression of both indicators, suggesting that AOS2 has renal protective effects. Denotes p <0.05 compared to sham and # denotes p <0.05 compared to the I/R group. This result shows that: the I/R group rats show significant increase in the expression of KIM-1 and NGAL indexes of Acute Kidney Injury (AKI) after ischemia-reperfusion operation, which indicates that the renal tubular injury is caused, and 0.1 g/kg/day AOS2 can obviously reduce the two indexes, which indicates that AOS2 has significant protection effect on acute kidney injury caused by ischemia-reperfusion.

FIG. 13 shows that acute ischemia-reperfusion (I/R) injury causes a significant increase in inflammatory markers in rat kidney tissue, and different doses of AOS2 inhibited the renal inflammatory response to varying degrees, suggesting that AOS2 has a renal protective effect. The dosage of I/R + AOS2-L is 0.01 g/kg/day; the dosage of I/R + AOS2-M is 0.05 g/kg/day; the dose of I/R + AOS2-H was 0.1 g/kg/day. This result shows that: the kidney inflammation indexes of I/R group rats after ischemia-reperfusion operation, such as TLR4, p-NF kappa B/NF kappa B, pro-IL-1 beta/IL-1 beta, are obviously increased, and AOS2 treatment reduces the generation of inflammatory factors and shows certain dose dependence, which shows that the anti-inflammatory effect of AOS2 is obvious.

Effect of brown algae trisaccharide on acute kidney injury of rat caused by ischemia reperfusion

The same experimental method as for fucoidan was used to examine the effect of fucoidan on acute renal injury in rats induced by ischemia-reperfusion. In the present invention, fucoidan is abbreviated as "AOS 3". The rat serum creatinine was measured by using the kit method using the Nanjing-constructed creatinine assay kit, and the experimental results were statistically processed by using the t-value method, and the results are shown in FIG. 14. The results of the 24-hour urine collected are shown in FIG. 15. The bilateral kidney is separated when the material is taken, cortex and medulla of the kidney are separated, the cortex is preserved in trizol, mRNA in the cortex is extracted by the trizol method when in use, and the mRNA expression of AKI biomarkers (KIM-1, NGAL) is detected, and the result is shown in figure 16. Adding tissue lysate, homogenizing kidney with ultrasound method, extracting total protein, and detecting inflammatory factor (p-NFkB/NFk B, pro-IL-1 beta/IL-1 beta) in kidney cortex tissue with western blotting technique (see FIG. 17). In addition, after the last bleeding, the animals were sacrificed and kidneys were fixed in 4% formaldehyde solution, embedded in paraffin, sectioned, HE stained, and the general renal tissue morphology was observed with a light microscope, and the results are shown in fig. 18.

FIG. 14 shows that acute ischemia reperfusion (I/R) injury causes significant elevation of serum creatinine levels in rats and different doses of AOS3 reduce serum creatinine to varying degrees, suggesting that AOS3 has renal protective effects. Denotes p <0.05 compared to sham and # denotes p <0.05 compared to the I/R group. This result shows that: the filtration function of the kidney of the rats in the I/R group after ischemia-reperfusion is disordered, and the serum creatinine is obviously increased. Serum creatinine was significantly reduced following treatment with three different doses of AOS3, suggesting some degree of recovery of glomerular function. Particularly, when the dosage of the medicine is 0.1 g/kg/day, the serum creatinine can be basically recovered to a normal level, and the treatment effect has certain degree of dose dependence.

FIG. 15 shows that acute ischemia reperfusion (I/R) injury causes increased urine volume in rats, and different doses of AOS3 decreased urine volume in rats, suggesting that AOS3 has a renal protective effect. Denotes p <0.05 compared to sham and # denotes p <0.05 compared to the I/R group. This result shows that: the I/R group rats were impaired in the function of urinary concentration in the kidneys after ischemia-reperfusion, and significantly increased in urine volume. The urine volume decreased after treatment with AOS3 at all three different doses, especially at the dose of 0.1 g/kg/day, and the urine volume returned to approximately normal levels the next day in the rats, indicating some degree of restoration of tubular function. The therapeutic effect is dose-dependent to some extent.

FIG. 16 shows that acute ischemia-reperfusion (I/R) injury causes an increase in mRNA levels of Kim-1 and NGAL, which are indicators of acute renal injury (AKI) (i.e., renal tubular injury indicators), in rat kidney tissue, and that AOS at a dose of 0.1 g/kg/day significantly decreases the expression of both indicators, suggesting that AOS3 has a renal protective effect. Denotes p <0.05 compared to sham and # denotes p <0.05 compared to the I/R group. This result shows that: the expression of KIM-1 and NGAL indexes of the I/R group rats after ischemia-reperfusion surgery is obviously increased, and 0.1 g/kg/day of AOS3 can obviously reduce the two indexes, which shows that AOS3 has obvious protective effect on acute kidney injury caused by ischemia-reperfusion.

FIG. 17 shows that acute ischemia-reperfusion (I/R) injury causes a significant increase in inflammatory markers in rat kidney tissue, and different doses of AOS3 inhibited the renal inflammatory response to varying degrees, suggesting that AOS3 has a renal protective effect. I/R + AOS3-L represents a dose of 0.01 g/kg/day; I/R + AOS3-M expressed the dose was 0.05 g/kg/day; I/R + AOS3-H represents a dose of 0.1 g/kg/day. This result shows that: after ischemia-reperfusion operation, kidney inflammation indexes of TLR4, p-NF kappa B/NF kappa B, pro-IL-1 beta/IL-1 beta of rats in the I/R group are obviously increased, the generation of inflammatory factors can be reduced by treating the rats with different doses of AOS3, and certain dose dependence is shown, which indicates that the brown algae trisaccharide has obvious anti-inflammatory effect.

Fig. 18 shows the pathological manifestations of the kidney tissue: SHAM group (SHAM): glomerular morphology, mesangial cells and renal tubules are essentially normal; the glomerulus of the I/R group is atrophied and shed, mesangial cells and stroma are reduced to generate cavities, renal tubules are expanded widely, lumen is enlarged, a large number of epithelial cells are subjected to edema, necrosis and shedding, and vacuole-like degeneration can be seen; the glomeruli and the tubules of the group I/R + AOS3(0.1 g/kg/day) were slightly diseased. These results suggest that glomerular and tubular disease was very evident in group I/R, while renal injury was very mild when AOS3(0.1 g/kg/day) was given after the same ischemia-reperfusion. The AOS3 shows that the compound has good protective effect on the morphological change of the rat kidney caused by ischemia-reperfusion.

Influence of fucoidan and fucoidan on acute kidney injury of rat caused by ischemia-reperfusion

The same experimental protocol as for fucoidan and fucoidan was used. The model-making rats were randomly grouped into a sham operation group, a model group, a fucoidan tetrasaccharide (three doses of 0.01, 0.05 and 0.1 g/kg/day) group and a mixed saccharide (mixed fucoidan oligosaccharide with a polymerization degree of 2-8, the mass spectrum thereof is shown in fig. 40, and is obtained from the university of oceans in china, one dose of 0.1 g/kg/day) group (6 per group). In the present invention, fucoidan is abbreviated as "AOS 4", and the mixed sugar is abbreviated as "AOS (mixed)". The rat serum creatinine was measured by using the kit method using the Nanjing-constructed creatinine assay kit, and the experimental results were statistically processed by using the t-value method, and the results are shown in FIG. 19. The results of the 24-hour urine collected are shown in FIG. 20. Bilateral kidneys were isolated at the time of material collection, cortex and medulla of the kidneys were separated, the cortex was stored in trizol, mRNA in the cortex was extracted by trizol method at the time of use, and mRNA expression of AKI biomarkers (KIM-1, NGAL) was detected, and the results are shown in fig. 21. Adding tissue lysate, homogenizing kidney with ultrasound, extracting total protein, and detecting inflammatory factor (p-NFkB/NFk B, pro-IL-1 beta/IL-1 beta) in kidney cortex tissue with western blotting technique (see FIG. 22).

FIG. 19 shows that acute ischemia-reperfusion (I/R) injury causes significant elevation of serum creatinine levels in rats, different doses of AOS4 and mixed sugar reduce serum creatinine to different extents, and AOS4 has excellent renal protection. Denotes p <0.05 compared to sham and # denotes p <0.05 compared to the I/R group. This result shows that: the filtration function of the kidney of the rats in the I/R group after ischemia-reperfusion is disordered, and the serum creatinine is obviously increased. There was a significant drop in serum creatinine following treatment with three different doses of AOS4 and mixed sugars (0.1 g/kg/day), indicating a degree of recovery in glomerular function. In particular, serum creatinine can be restored to a level close to normal when the fucoidan is administered at a dose of 0.1 g/kg/day. The therapeutic effect of AOS4 at various doses was somewhat dose-dependent.

FIG. 20 shows that acute ischemia-reperfusion (I/R) injury causes increased urine output in rats, and different doses of AOS4 and mixed sugar (0.1 g/kg/day) reduced urine output in rats, AOS4 has excellent renal protection effect. Denotes p <0.05 compared to sham and # denotes p <0.05 compared to the I/R group. This result shows that: the I/R group rats were impaired in the function of urinary concentration in the kidneys after ischemia-reperfusion, and significantly increased in urine volume. The urine volume is reduced after the treatment of the AOS4 and the mixed sugar with three different doses, which indicates that the renal tubular function is recovered to a certain degree, and particularly, the treatment effect of the AOS4 is obviously better than that of the mixed sugar with the same dose, and the treatment effect is dose-dependent to a certain degree.

FIG. 21 shows that acute ischemia-reperfusion (I/R) injury causes the increase of mRNA levels of Kim-1 and NGAL in the acute renal injury (AKI) index (i.e., renal tubular injury index) in rat kidney tissue, and that fucoidan at a dose of 0.1 g/kg/day significantly reduces the expression of both indices, suggesting that it has a better renal protection effect. Denotes p <0.05 compared to sham and # denotes p <0.05 compared to the I/R group. This result shows that: the expression of KIM-1 and NGAL indexes of the I/R group rats after ischemia-reperfusion surgery is obviously increased, and 0.1 g/kg/day of AOS4 can obviously reduce the two indexes, which shows that AOS4 has good protection effect on acute kidney injury caused by ischemia-reperfusion.

FIG. 22 shows that acute ischemia-reperfusion (I/R) injury causes a significant increase in inflammatory markers in rat kidney tissue, and different doses of AOS4 can inhibit the renal inflammatory response to different degrees, suggesting that AOS4 has a renal protective effect. I/R + AOS4-L represents a dose of 0.01 g/kg/day; I/R + AOS4-M expressed the dose was 0.05 g/kg/day; I/R + AOS4-H represents a dose of 0.1 g/kg/day; I/R + AOS (mixed) -H represents a dose of 0.1 g/kg/day. This result shows that: the kidney inflammation indexes of I/R group rats after ischemia reperfusion operation, such as TLR4, p-NF kappa B/NF kappa B, pro-IL-1 beta/IL-1 beta, are obviously increased, the generation of inflammatory factors can be obviously reduced by the treatment of AOS4, and certain dose dependence is shown, and the AOS4 has anti-inflammatory effect.

Conclusion of the experiment

Renal injury from ischemia reperfusion is a standard animal model that mimics clinical acute renal injury from hypoperfusion. The serum creatinine level of rats is remarkably reduced after the brown algae disaccharide, the brown algae trisaccharide and the brown algae tetrasaccharide are treated by different dosages (0.01, 0.05 and 0.1 g/kg/day), wherein the serum creatinine level can be basically recovered to a normal value at the dosage of 0.1 g/kg/day; after the administration treatment, the urine concentration function of the kidney is obviously recovered, the urine volume is reduced, wherein the 0.1 g/kg/day dose group obviously reduces the level of kidney injury factors (KIM-1, NGAL), the expression of inflammatory factors is obviously reduced, the pathological change of the kidney is obviously improved, and the treatment effect is enhanced along with the increase of the dose. In conclusion, the detection results of different indexes such as serum creatinine, urine volume, inflammatory factors and the like all show that the fucoidan, the trisaccharide and the tetrasaccharide all have good protection effect on acute kidney injury of rats caused by ischemia reperfusion, and compared with the mixed fucoidan oligosaccharide with the polymerization degree of 2-8, the therapeutic effect of the fucoidan, the trisaccharide and the tetrasaccharide with uniform polymerization degree is remarkably better.

Example 5Effect of alginate oligosaccharides with uniform polymerization degree on acute kidney injury of mice caused by endotoxin phospholipid polysaccharide (LPS)

Kidney damage caused by endotoxin phospholipidosis treatment is a standard animal model to mimic acute kidney damage caused by clinical infection. The therapeutic effect of the brown alga oligosaccharides with different uniform polymerization degrees is investigated by respectively administering the brown alga oligosaccharides with different uniform polymerization degrees and the mixture thereof by using a mouse endotoxin phospholipid polysaccharide model and comparing the model with a blank group and a model non-administration group.

First, brown algae disaccharide (AOS2) influences LPS-induced acute kidney injury of mice

24 male C57/Bl6 mice of 22-28 g are selected, 24 hours of urine volume is collected before operation, no abnormality is detected, the molded mice are randomly grouped into 6 mice in each group, namely a control group, a model + administration group (AOS20.1g/kg/day) and a model + positive control group (dexamethasone acetate 0.1 g/kg/day), samples are administrated by gastric lavage, the model group and the control group are both administrated by normal saline with the same volume, and dexamethasone is administrated by intraperitoneal injection. LPS is adopted for modeling to induce the generation of sepsis acute kidney injury, and each model group is injected with LPS 15mg/kg in the abdominal cavity, and a control group is injected with normal saline with the same quantity in the abdominal cavity. Immediately after molding, the mice were returned to the mouse metabolism cage for observation, during which the weight, food intake, water intake, and urine output of the mice were measured. The mice are sacrificed after 24 hours to take materials, urine is collected, blood samples are collected, blood is taken from the inferior vena cava of the mice, upper serum is collected after centrifugation, then the serum creatinine of the mice is measured by using a kit method by using a creatinine measurement kit established by Nanjing, the experimental result is statistically processed by using a t value method, and the result is shown in figure 23. The bilateral kidneys were isolated when the material was taken, the cortex and medulla of the kidneys were isolated, the cortex was stored in trizol, mRNA in the cortex was extracted using the trizol method at the time of use, and mRNA expression of AKI biomarkers (KIM-1, NGAL, see FIG. 24) and inflammatory factors (IL-1. beta., IL-18, TNF-. alpha., MCP-1, see FIG. 25) was detected using the Qpcr method. Adding tissue lysate, homogenizing kidney with ultrasound, extracting total protein, and detecting inflammatory factor (p-NFkB/NFk B, pro-IL-1 beta/IL-1 beta) in kidney cortex tissue with western blotting technique (see FIG. 26).

FIG. 23 shows that the serum creatinine level of mice is significantly increased by LPS treatment, AOS2 significantly reduces serum creatinine, and has the same effect of reducing serum creatinine as dexamethasone, suggesting that fucoidan has kidney protection effect. Dex dexamethasone indicates p <0.05 compared to CTL (control group) and # indicates p <0.05 compared to LPS group (model group). This result shows that: compared with the control group, the serum creatinine of the mice is obviously increased after the mice are injected with LPS in the abdominal cavity, and the serum creatinine of the mice of the AOS2 administration group is obviously reduced and basically can be restored to the normal level, which shows that the fucoidan has obvious protective effect on the reduction of the kidney function of the mice caused by the LPS.

FIG. 24 shows that LPS treatment caused elevated mRNA levels of Kim-1 and NGAL, an indicator of acute renal injury (AKI), an indicator of tubular injury, in mouse kidney tissue, suggesting that fucoidan has a renal protective effect. Indicates p <0.05 compared to CTL and # indicates p <0.05 compared to LPS group. This result shows that: after the mice are injected with LPS in the abdominal cavity, AKI indexes (KIM-1 and NGAL) are obviously increased, and after AOS2 treatment, the production of KIM-1 and NGAL is obviously reduced, which shows that AOS2 has obvious protective effect on the kidney injury of the mice caused by LPS.

FIG. 25 shows that the inflammatory index in the kidney tissue of mice is significantly increased by LPS treatment, and AOS2 significantly inhibits the inflammatory response of kidney, suggesting that fucoidan has kidney protection effect. Indicates p <0.05 compared to CTL and # indicates p <0.05 compared to LPS group. This result shows that: after the mice are injected with LPS in the abdominal cavity, the gene expression of inflammatory factors (IL-1 beta, IL-18, TNF-alpha and MCP-1) is obviously increased, and the AOS2 treatment obviously reduces the generation of the inflammatory factors, which indicates that the fucoidan has good protective effect on the acute kidney injury of the mice caused by the LPS.

FIG. 26 shows that the inflammatory index in the kidney tissue of mice is significantly increased by LPS treatment, AOS2 significantly inhibits the renal inflammatory response, and the effect is close to that of positive control dexamethasone, suggesting that AOS2 has a renal protection effect. Indicates p <0.05 compared to CTL and # indicates p <0.05 compared to LPS group. This result shows that: after LPS treatment, the mouse kidney inflammation indexes such as TLR4, p-NF kappa B/NF kappa B, pro-IL-1 beta/IL-1 beta protein expression are obviously increased, the generation of inflammatory factors is obviously reduced by AOS2 treatment, the effect is close to that of dexamethasone, and the AOS2 has an obvious anti-inflammatory effect.

Effect of Brown algae trisaccharide (AOS3) on LPS-induced acute kidney injury in mice

The same experimental protocol as for fucoidan was used. The mouse serum creatinine is measured by using a kit method by using the built creatinine measurement kit of Nanjing, the experimental result is statistically processed by a t value method, and the result is shown in figure 27. The bilateral kidneys were isolated at the time of material collection, the cortex and medulla of the kidneys were isolated, the cortex was stored in trizol, mRNA in the cortex was extracted using the trizol method at the time of use, and mRNA expression of AKI biomarkers (KIM-1, NGAL, see FIG. 28) and inflammatory factors (IL-1. beta., IL-18, TNF-. alpha., MCP-1, see FIG. 29) was detected using the Qpcr method. Total protein was extracted by adding tissue lysate to the kidney and homogenizing the kidney using ultrasound, and inflammatory factors (p-NFkB/NFk B, pro-IL-1. beta./IL-1. beta.) were detected in renal cortical tissue using immunoblotting technique, the results are shown in FIG. 30. In addition, after the last bleeding, the animals were sacrificed and kidneys were fixed in 4% formaldehyde solution, embedded in paraffin, sectioned, HE stained, and the general renal tissue morphology was observed with a light microscope, and the results are shown in fig. 31.

FIG. 27 shows that the serum creatinine level of mice is significantly increased by LPS treatment, AOS3 significantly reduces serum creatinine, and has the same effect of reducing serum creatinine as dexamethasone, suggesting that fucoidan has kidney protection effect. Dex dexamethasone, # indicates p <0.05 compared to CTL and # indicates p <0.05 compared to LPS group. This result shows that: compared with a control group, the serum creatinine of the mice is remarkably increased after the mice are injected with LPS in an abdominal cavity, the serum creatinine of the mice in the AOS3 administration group is remarkably reduced and can be basically recovered to a normal level, and the brown algae trisaccharide is prompted to have a remarkable protective effect on the reduction of the kidney function of the mice caused by the LPS.

FIG. 28 shows that LPS treatment caused elevated mRNA levels of Kim-1 and NGAL, the indicators of acute renal injury (AKI), the indicator of tubular injury, in mouse kidney tissue, suggesting that fucotriose has a renal protective effect. Indicates p <0.05 compared to CTL and # indicates p <0.05 compared to LPS group. This result shows that: after the mice are injected with LPS in the abdominal cavity, AKI indexes (KIM-1 and NGAL) are obviously increased, and after AOS3 treatment, the production of KIM-1 and NGAL is obviously reduced, which indicates that AOS3 has a protective effect on the kidney injury of the mice caused by LPS.

FIG. 29 shows that the inflammatory index in mouse kidney tissue is significantly increased by LPS treatment, and AOS3 significantly inhibits the renal inflammatory response, suggesting that fucotriose has a renal protective effect. Indicates p <0.05 compared to CTL and # indicates p <0.05 compared to LPS group. This result shows that: after mice are injected with LPS in the abdominal cavity, the gene expression of inflammatory factors (IL-1 beta, IL-18, TNF-alpha and MCP-1) is obviously increased, and AOS3 treatment obviously reduces the generation of the inflammatory factors, so that the brown algae trisaccharide has a good protective effect on acute kidney injury of the mice caused by the LPS.

FIG. 30 shows that the inflammatory index in the kidney tissue of mice is significantly increased by LPS treatment, AOS3 significantly inhibits the renal inflammatory response, and the effect is very close to that of positive control dexamethasone, suggesting that AOS3 has a better renal protection effect. Indicates p <0.05 compared to CTL and # indicates p <0.05 compared to LPS group. This result shows that: after LPS treatment, the mouse kidney inflammation indexes such as TLR4, p-NF kappa B/NF kappa B, pro-IL-1 beta/IL-1 beta protein expression are obviously increased, the generation of inflammatory factors is obviously reduced by AOS3 treatment, the effect is very close to that of dexamethasone, and the AOS3 has a better anti-inflammatory effect.

Figure 31 shows the pathological manifestations of kidney tissue: control group: glomerular morphology, mesangial cells and renal tubules are basically normal, the volume of glomeruli in an LPS group is increased, inflammatory cell infiltration can be seen in renal interstitium, renal tubules are widely expanded, epithelial cells are subjected to edema, necrosis and shedding, and vacuolar degeneration can be seen; glomerular and tubular renal lesions were mild in the LPS + AOS3 group. These results show that AOS3 has a significant protective effect on mouse renal morphological changes induced by endotoxin phospholipidosome treatment.

Influence of fucoidan tetrasaccharide (AOS4) and mixed saccharides on acute kidney injury of mice caused by LPS

The same experimental procedure was used as for fucoidan and fucoidan. The modeling mice are randomly grouped into 6 groups, namely a control group, a model + administration group 1 (AOS40.1g/kg/day), a model + administration group 2 (mixed alginate-derived oligosaccharide with the polymerization degree of 2-8, the mass spectrogram of the mixed alginate-derived oligosaccharide is shown in a figure 40 and is obtained from China oceanic university, 0.1 g/kg/day) and a model + positive control group (dexamethasone acetate, 0.1 g/kg/day), wherein samples are administrated by gastric perfusion, the model group and the control group are both administrated by normal saline with the same volume, and dexamethasone is administrated by intraperitoneal injection. The mouse serum creatinine is measured by using a kit method by using the built creatinine measurement kit of Nanjing, the experimental result is statistically processed by a t value method, and the result is shown in figure 32. The bilateral kidneys were isolated at the time of material collection, the cortex and medulla of the kidneys were isolated, the cortex was stored in trizol, mRNA in the cortex was extracted using the trizol method at the time of use, and mRNA expression of AKI biomarkers (KIM-1, NGAL, see the results in FIG. 33) and inflammatory factors (IL-1. beta., IL-18, TNF-. alpha., MCP-1, see the results in FIG. 34) was detected using the Qpcr method. Total protein was extracted by adding tissue lysate to the kidney and homogenizing the kidney using ultrasound, and inflammatory factors (p-NFkB/NFk B, pro-IL-1. beta./IL-1. beta.) were detected in renal cortical tissue using immunoblotting technique, the results are shown in FIG. 35.

FIG. 32 shows that the serum creatinine level of mice is significantly increased by LPS treatment, the mixed sugar has a certain effect on reducing serum creatinine, and AOS4 significantly reduces serum creatinine, and the effect is close to that of dexamethasone, thus indicating that the alginate oligosaccharide has kidney protection effect. Dex dexamethasone, # indicates p <0.05 compared to CTL and # indicates p <0.05 compared to LPS group. This result shows that: compared with a control group, the serum creatinine of the mice is obviously increased after the mice are injected with LPS in the abdominal cavity, the serum creatinine of the mice of the AOS4 and dexamethasone administration group is obviously reduced and can be basically recovered to a normal level, which shows that the fucoidan tetrasaccharide has obvious protective effect on the reduction of the mouse kidney function caused by the LPS, and the effect of the fucoidan tetrasaccharide is close to that of dexamethasone.

FIG. 33 shows that LPS treatment caused an increase in mRNA levels of Kim-1 and NGAL, which are indicators of acute renal injury (AKI) (i.e., indicators of tubular injury) in mouse kidney tissue, and that fucoidan and mixed saccharide (at a dose of 0.1 g/kg/day) were both reduced, especially fucoidan, which significantly reduced both indicators, suggesting that fucoidan has a better renal protective effect. Indicates p <0.05 compared to CTL and # indicates p <0.05 compared to LPS group. This result shows that: after the mice are injected with LPS in the abdominal cavity, AKI indexes (KIM-1 and NGAL) are obviously increased, and after AOS4 is administrated for treatment, the production of KIM-1 and NGAL is obviously reduced, which indicates that the brown algae tetrasaccharide has a protective effect on the kidney injury of the mice caused by LPS.

FIG. 34 shows that LPS treatment significantly increased the inflammation index in mouse kidney tissue, and AOS4 and mixed sugar significantly inhibited the kidney inflammation reaction, wherein AOS4 has better kidney protection effect. Indicates p <0.05 compared to CTL and # indicates p <0.05 compared to LPS group. This result shows that: after mice are injected with LPS in the abdominal cavity, the gene expression of inflammatory factors (IL-1 beta, IL-18, TNF-alpha and MCP-1) is obviously increased, the generation of the inflammatory factors can be obviously reduced by the treatment of AOS4 and mixed sugar, wherein AOS4 has good protective effect on the acute kidney injury of the mice caused by the LPS.

FIG. 35 shows that the inflammatory index in the kidney tissue of mice is significantly increased by LPS treatment, AOS4 and mixed sugar significantly inhibit the renal inflammatory response, and the effect is close to that of positive control dexamethasone, suggesting that AOS4 has a better renal protection effect. Indicates p <0.05 compared to the CTL group and # indicates p <0.05 compared to the LPS group. This result shows that: after LPS treatment, the mouse kidney inflammation indexes such as TLR4, p-NF kappa B/NF kappa B, pro-IL-1 beta/IL-1 beta protein expression are obviously increased, the brown algae tetrasaccharide and mixed sugar treatment obviously reduce the generation of inflammatory factors, the effect of the brown algae tetrasaccharide and mixed sugar treatment is basically consistent with that of dexamethasone, and the effect of the brown algae tetrasaccharide and mixed sugar treatment is proved to have very effective anti-inflammatory effect.

Conclusion of the experiment

Kidney damage caused by endotoxin phospholipid polysaccharide (LPS) treatment is a standard animal model that mimics acute kidney damage caused by clinical infection. The serum creatinine level of the model group mice treated by LPS is obviously increased, and the serum creatinine level is obviously reduced after the mice are treated by 0.1 g/kg/day of fucoidan disaccharide, trisaccharide and tetrasaccharide; the kidney injury factor (KIM-1, NGAL) level and the inflammatory factor expression are obviously reduced, and the pathological change of the kidney is obviously improved. The mixed sugar with the polymerization degree of 2-8 also improves the indexes to different degrees, but has a certain difference with the brown algae disaccharide, trisaccharide and tetrasaccharide. These results indicate that the alginate oligosaccharides with uniform polymerization degree have good protection effect on acute kidney injury caused by endotoxin phospholipid polysaccharides.

Example 6 Effect of alginate oligosaccharides with homogeneous degree of polymerization on Cisplatin (Cisplatin) -induced acute Kidney injury in mice

Cisplatin-treated renal injury is a standard animal model that mimics acute renal injury caused by the direct nephrotoxic effects of clinical drugs. The mouse cisplatin model is utilized to respectively administer the brown alga oligosaccharides with different uniform polymerization degrees and the mixture thereof, and the therapeutic effect of the brown alga oligosaccharides with the uniform polymerization degrees is investigated by comparing the model with a blank group and a model non-administration group.

Effect of brown algae trisaccharide (AOS3) on cisplatin-induced acute kidney injury in mice

Selecting 30 male C57BL/J6 mice 22-28 g, collecting urine volume for 24 hours before operation without abnormality, randomly grouping the molded mice into a control group, a model + administration low dose group (AOS30.05g/kg/day), a model + administration medium dose group (AOS30.1g/kg/day) and a model + administration high dose group (AOS30.2g/kg/day) which are 6 mice in each group, wherein the fucoidan is administrated by gastric perfusion, and the model group and the control group are both administrated by normal saline with the same volume. Cisplatin is adopted for modeling to induce the occurrence of drug-toxicity acute kidney injury, and during modeling, 20mg/kg of cisplatin is injected into the abdominal cavity of each model group, and the same amount of normal saline is injected into the abdominal cavity of a control group. Immediately after the model was made, the mice were returned to the mouse metabolism cage for observation, during which the weight, feed amount, water intake amount and urine amount of the mice were measured (see the results in FIG. 37). The mice are sacrificed after 72 hours to take materials, urine is collected, blood samples are collected, blood is taken from the inferior vena cava of the mice, upper serum is collected after centrifugation, then the serum creatinine of the mice is measured by using a kit method by using a built creatinine measurement kit of Nanjing, the experimental result is statistically processed by using a t value method, and the result is shown in figure 36. Bilateral kidneys were isolated from the material, cortex and medulla of the kidneys were isolated, the cortex was stored in trizol, mRNA in the cortex was extracted using trizol method and mRNA expression of AKI biomarkers (KIM-1, NGAL, see FIG. 38) was detected using Qpcr method. Adding tissue lysate, homogenizing kidney with ultrasound, extracting total protein, and detecting inflammatory factor (p-NF kappa B/NF kappa B, IL-1 beta) in kidney cortex tissue with western blotting technique (see FIG. 39).

FIG. 36 shows that cisplatin treatment significantly increased serum creatinine levels in mice and AOS3 significantly decreased serum creatinine, suggesting that fucotriose has a renal protective effect. Denotes p <0.05 compared to the CTL group (control group) and # denotes p <0.05 compared to the Cis group (model group). This result shows that: compared with a control group, the serum creatinine of the mice is remarkably increased after the mice are injected with the cisplatin in the abdominal cavity, and the serum creatinine of the mice in the AOS3 administration group is remarkably reduced and basically can be restored to a normal level, so that the fucoidan trisaccharide has a remarkable protective effect on the reduction of the kidney function of the mice caused by the cisplatin.

FIG. 37 shows that cisplatin-induced mice increased urine volume first and then decreased 72h into the oliguric phase, and that different concentrations of AOS3 administered before 24h reduced urine volume to restore kidney function and gradually normalized urine volume, suggesting that fucotriose has a renoprotective effect. Denotes p <0.05 compared to CTL and # denotes p <0.05 compared to model group. This result shows that: the urine concentration function of the kidney of the model group mice is disordered after cisplatin injection, and the urine volume is firstly increased remarkably and then enters the oliguria period. The decrease in urine volume followed by normalization after treatment with three different doses of AOS3 suggests some degree of restoration of tubular function. And to some extent dose-dependent.

FIG. 38 shows that cisplatin treatment caused elevated mRNA levels of Kim-1 and NGAL as indicators of acute renal injury (AKI) (i.e., a renal tubular injury indicator) in mouse renal tissue, suggesting that fucotriose has a renal protective effect. Denotes p <0.05 compared to CTL and # denotes p <0.05 compared to model group. This result shows that: after the mice are injected with cisplatin in the abdominal cavity, AKI indexes (KIM-1 and NGAL) are remarkably increased, and after AOS3 treatment, the production of KIM-1 and NGAL is remarkably reduced, which shows that AOS3 has obvious protective effect on kidney injury of the mice caused by cisplatin.

FIG. 39 shows that cisplatin treatment significantly increased inflammation markers in mouse kidney tissue, and AOS3 significantly inhibited the renal inflammatory response, suggesting that AOS3 has a renal protective effect. Denotes p <0.05 compared to CTL and # denotes p <0.05 compared to model group. This result shows that: after cisplatin treatment, the mouse kidney inflammation indexes such as TLR4 and p-NF kappa B/NF kappa B, IL-1 beta protein expression are obviously improved, AOS3 treatment obviously reduces the generation of inflammatory factors, and AOS3 has an obvious anti-inflammatory effect.

Similarly, further research shows that the fucoidan and fucoidan have similar effects on cisplatin-induced acute kidney injury in mice.

Conclusion of the experiment

The serum creatinine level of the cisplatin-treated model group mice is remarkably increased, and the serum creatinine level is remarkably reduced after the mice are treated by 0.1 g/kg/day of fucoidan disaccharide, trisaccharide and tetrasaccharide; the kidney injury factor (KIM-1, NGAL) level and the inflammatory factor expression are obviously reduced, and the treatment effect has concentration dependence. These results indicate that the brown algae disaccharide, trisaccharide and tetrasaccharide have good protection effect on acute kidney injury caused by cisplatin.

Finally, it should be pointed out here that: the above is only a part of the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention, and the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above description are intended to be covered by the present invention.

Claims

1. Use of a brown algae oligosaccharide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating acute kidney injury, wherein the brown algae oligosaccharide is brown algae disaccharide, brown algae trisaccharide or brown algae tetrasaccharide.

2. The use of claim 1, wherein the brown algae oligosaccharide is composed of monosaccharides G, M and/or Δ linked through a glycosidic linkage in position 1, 4; wherein G represents alpha-L-guluronic acid, M represents beta-D-mannuronic acid, and Delta represents alpha-L-guluronic acid or beta-D-mannuronic acid, and beta-elimination occurs at the 4,5 positions to generate unsaturated monosaccharide with conjugated double bonds at the 4,5 positions.

3. The use of claim 2, wherein the fucoidan is selected from Δ G, Δ M, or a combination thereof.

4. The use of claim 2, wherein the brown alginate triose is selected from one or more of Δ GG, Δ GM, Δ MM, and Δ MG.

5. The use of claim 2, wherein the fucoidan is selected from one or more of Δ GGG, Δ GGM, Δ GMG, Δ GMM, Δ MMG, Δ MMM, Δ MGG and Δ MGM.

6. The use of claim 1, wherein the pharmaceutically acceptable salt is a sodium, potassium, calcium, magnesium and/or ammonium salt.

7. The use according to any one of claims 1 to 6, wherein the acute kidney injury is caused by hypoperfusion of blood, infection or renal toxicity of the drug.