Klebsiella sp.S22, exopolysaccharide EPSK22 and application of Klebsiella sp.S22
By screening Klebsiella Klebsiella sp.S22 and extracting the extracellular polysaccharide EPSK22, the problem of cisplatin-induced renal injury was solved, and the effect of significantly reducing renal injury was achieved, and the application value of medicinal and functional foods was achieved.
Patent Information
- Application Number
- CN202510188578.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-20
- Publication Date
- 2025-07-25
AI Technical Summary
In the prior art, the mechanism of renal injury induced by cisplatin is complex, existing drugs have side effects, and plant polysaccharides are difficult to extract, which limits their clinical application. Microbial polysaccharides have the advantages of simple extraction and are not affected by source and season. It is necessary to develop effective microbial polysaccharides to prevent cisplatin-induced nephrotoxicity.
Klebsiella Klebsiella sp.S22 was screened and the extracellular polysaccharide EPSK22 was extracted from its fermentation broth. The extracellular polysaccharide EPSK22 is composed of mannose, rhamnoseng, glucuronic acid, and galactose. It alleviates oxidative damage by regulating the Nrf2 pathway, alleviates inflammatory response, reduces apoptosis, and significantly alleviates cisplatin-induced renal injury.
Exocellular polysaccharide EPSK22 significantly alleviates renal injury induced by cisplatin, alleviates oxidative damage by regulating the Nrf2 pathway, alleviates inflammatory responses, and reduces apoptosis. It has medicinal and functional food value, and protects renal injury caused by chemotherapy drugs.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microbiology, and particularly to a Klebsiella sp. S22, an extracellular polysaccharide EPSK22 and their applications. Background Art
[0002] Cisplatin is an effective anti-tumor drug that can target rapidly dividing cells and is used to treat various solid tumors. However, its main clinical side effect is nephrotoxicity, and about 30% of patients receiving cisplatin treatment will develop acute kidney injury (AKI). The exact mechanism of cisplatin-induced acute kidney injury is not clear, but it may involve factors such as apoptosis, oxidative stress and inflammatory responses.
[0003] Cisplatin-induced nephrotoxicity has a complex pathological mechanism. Currently, combination treatment strategies for various pathways may enhance the protection of the kidneys. Several drugs used clinically to treat cisplatin-induced kidney injury show different side effects.
[0004] Literature reports that some polysaccharides have the function of protecting cisplatin-induced kidney injury and have no toxic side effects; for example, the polysaccharide from the leaves of L. europaeum L. has anti-inflammatory and antioxidant properties, as well as significant kidney protection effects; ginseng polysaccharide can inhibit cisplatin-induced kidney inflammation and apoptosis by inhibiting endoplasmic reticulum stress and reducing the expression of NF-κB p65 and TNF-α. However, there are few reports on polysaccharides preventing cisplatin-induced nephrotoxicity at present, and most of them are plant polysaccharides. The clinical application of plant polysaccharides is limited due to the complex extraction and purification processes and characteristics such as the seasonality of the sources. Microbial polysaccharides have the advantages of simple extraction and being unaffected by sources, seasons, etc. Therefore, developing microbial polysaccharides has important clinical application value in preventing cisplatin-induced nephrotoxicity. Summary of the Invention
[0005] Based on the technical problems existing in the background art, the present invention proposes a Klebsiella sp. S22, an extracellular polysaccharide EPSK22 and their applications. The present invention screens out a Klebsiella sp. S22, and extracts a new extracellular polysaccharide EPSK22 from its fermentation broth. The extracellular polysaccharide EPSK22 can significantly reduce cisplatin-induced kidney injury and has medicinal and functional food values.
[0006] The present invention proposes a Klebsiella sp. S22, and its preservation number is CCTCC NO: M20241932.
[0007] A strain was screened from the farmland soil in Hefei, Anhui of the present invention, named Klebsiella sp. S22, and the strain was deposited in the China Center for Type Culture Collection (CCTCC) on September 6, 2024. The deposit address is Wuchang District, Bayi Road, Luojia Mountain, Wuhan University, Hubei Province, China, and the deposit number is CCTCC NO: M 20241932.
[0008] The present invention also provides an extracellular polysaccharide EPSK22, which is composed of mannose, rhamnose, glucuronic acid, and galactose in a molar ratio of 2:2:1:1.
[0009] Preferably, the average molecular weight of the extracellular polysaccharide EPSK22 is 60,000 - 70,000 Da; more preferably 60,000 - 65,000 Da.
[0010] Preferably, the structural formula of the extracellular polysaccharide EPSK22 is shown in formula (I):
[0011] →2)[α-L-Rha-(1→3)-β-D-Galp-(1→2)-α-L-Rhap-(1→3)]-α-D-Manp-(1→4)-β-D-GlcAp-(1→2)-α-D-Manp-(1→; Formula (I).
[0012] Preferably, the extracellular polysaccharide EPSK22 is produced using the above-mentioned Klebsiella sp. S22.
[0013] The present invention also provides a production method of the above extracellular polysaccharide EPSK22, including the following steps: taking the fermentation broth of Klebsiella sp. S22 as described in claim 1, performing alcohol precipitation, and taking the precipitate to obtain the extracellular polysaccharide EPSK22.
[0014] Preferably, Klebsiella sp. S22 is fermented in a fermentation medium to obtain a fermentation broth.
[0015] Preferably, 1 L of the fermentation medium contains: 28 - 32 g / L of sucrose, 3 - 5 g / L of yeast powder, 0.04 - 0.06 g / L of CaCl2, 0.8 - 1.2 g / L of NaH2PO4, 0.002 - 0.003 g / L of ZnCl2, 0.3 - 0.5 g / L of MgSO4, 0.012 - 0.013 g / L of FeSO4, 0.002 - 0.004 g / L of MnSO4.
[0016] More preferably, the 1L fermentation medium contains: 30 g / L sucrose, 4 g / L yeast powder, 0.05 g / L CaCl2, 1.0 g / L NaH2PO4, 0.0025 g / L ZnCl2, 0.4 g / L MgSO4, 0.0125 g / L FeSO4, 0.003 g / L MnSO4.
[0017] Preferably, the pH of the fermentation medium is 6.8 - 7.2; more preferably, the pH is 7.0.
[0018] The solvent of the above fermentation medium is water; the above fermentation medium is sterilized by high temperature before use.
[0019] Preferably, the temperature of fermentation culture is 28 - 32 °C, and the time of fermentation culture is 46 - 50 h.
[0020] Preferably, the fermentation culture is carried out at a rotation speed of 200 - 250 rpm.
[0021] Preferably, the alcohol precipitation solvent is at least one of ethanol and isopropanol.
[0022] Preferably, the volume ratio of the fermentation broth to the alcohol precipitation solvent is 1:2 - 4.
[0023] Preferably, the precipitate is taken for purification to obtain the extracellular polysaccharide EPSK22.
[0024] Preferably, the purification step includes: a. Extract the aqueous solution containing the precipitate with Sevag solution, let it stand and separate into three layers, take the upper aqueous phase, and then centrifuge to take the supernatant; b. Use the supernatant to replace the aqueous solution containing the precipitate in a, repeat step a until it stands and separates into two layers, take the upper aqueous phase, and then centrifuge to take the supernatant, carry out alcohol precipitation treatment, take the precipitate to obtain the extracellular polysaccharide EPSK22.
[0025] Preferably, in the purification step, the Sevag solution is a mixed solution composed of chloroform and n-butanol at a volume ratio of 3 - 5:1.
[0026] Preferably, in the purification step, the volume ratio of the aqueous solution containing the precipitate to the Sevag solution is 1:3 - 5.
[0027] Preferably, in the aqueous solution containing the precipitate, the concentration of the precipitate is 0.008 - 0.012 g / mL.
[0028] Preferably, in the purification step, the alcohol precipitation solvent is at least one of ethanol and isopropanol.
[0029] Preferably, in the purification step, the volume ratio of the supernatant to the alcohol precipitation solvent is 1:2 - 4.
[0030] The present invention also provides the use of the above-mentioned Klebsiella sp. S22 or its fermentation product, and the above-mentioned extracellular polysaccharide EPSK22 in the preparation of a drug or health product for preventing or treating kidney injury.
[0031] Preferably, the kidney injury is drug-induced kidney injury.
[0032] Preferably, the drug is a chemotherapeutic drug.
[0033] Preferably, the drug is cisplatin.
[0034] Beneficial effects:
[0035] In the present invention, a strain of Klebsiella sp. S22 was screened from the farmland soil in Hefei, Anhui, and a new extracellular polysaccharide EPSK22 was obtained by extracting from the fermentation broth of Klebsiella sp. S22, which is composed of mannose, rhamnose, glucuronic acid, and galactose in a molar ratio of 2:2:1:1.
[0036] The inventors found through research that the extracellular polysaccharide EPSK22 can significantly alleviate cisplatin-induced kidney injury, can reduce oxidative damage caused by cisplatin by regulating the Nrf2 pathway, relieve cisplatin-induced renal oxidative stress, can relieve the inflammatory response induced by cisplatin by reducing inflammatory cell infiltration and downregulating the gene expression of the NF-κB pathway, and alleviate the inflammatory response of cisplatin-induced acute kidney injury; it can reduce cisplatin-induced renal cell apoptosis through the Bcl-2 / Bax / Caspase-3 signaling pathway. The extracellular polysaccharide EPSK22 has medicinal and functional food value in preventing or treating cisplatin-induced kidney injury, and has good characteristics for protecting against kidney injury caused by chemotherapeutic drugs. Description of the drawings
[0037] Figure 1 It is a colony photograph of Klebsiella sp. S22.
[0038] Figure 2 It is a phylogenetic tree.
[0039] Figure 3 It is a monosaccharide composition map, where a is the standard monosaccharide and b is the extracellular polysaccharide EPSK22.
[0040] Figure 4 It is a one-dimensional NMR spectrum of the extracellular polysaccharide EPSK22. Among them, Figure A is 1 1H NMR, Figure B is 13 13C NMR, and Figure C is DEPT135 NMR.
[0041] Figure 5Two-dimensional NMR spectra of extracellular polysaccharide EPSK22, where Figure A is COSY, Figure B is HSQC, Figure C is TOCSY, Figure D is NOESY, and Figure E is HMBC.
[0042] Figure 6 Shows the results of the effect of extracellular polysaccharide EPSK22 on cisplatin-induced kidney injury. Among them, A is the treatment timeline of extracellular polysaccharide EPSK22; B is the detection result of Cre, n = 6; C is the detection result of BUN level, n = 6; D-E are the relative mRNA expression levels of NGAL and KIM-1 in sequence, n = 6; F is the typical picture detected by WB, n = 3; G-H are the relative protein expression levels of NAGL and Kim-1 in sequence, n = 3; I is the photo of the kidney; J is the typical H&E staining image of kidney sections, n = 3.
[0043] Figure 7 Shows the results of the effect of extracellular polysaccharide EPSK22 on oxidative stress in cisplatin-induced kidney injury. Among them, A-D are the levels of GSH, CAT, SOD, and MDA in each group of mice in sequence, n = 6; E-L are the relative mRNA expression levels of GSH-Px, CAT, SOD1, SOD2, Nrf2, Gpx1, HO-1, and NQO1 in sequence, n = 6; M is the typical picture detected by WB, n = 3; N-Q are the relative protein expression levels of Nfr2, Gpx1, HO-1, and NQO1 in sequence, n = 3.
[0044] Figure 8 Shows the results of the effect of extracellular polysaccharide EPSK22 on the inflammatory response in cisplatin-induced kidney injury. Among them, A-B are the representative immunohistochemical staining results of F4 / 80+ macrophages and CD3+ T cells in the kidney in sequence; C-D are the quantitative analyses of F4 / 80+ and CD3+ in sequence (n = 3, scale bar 200 μm); E-G are the relative mRNA expression levels of IL-6, TNF-α, and COX-2 in sequence, n = 6; H is the typical picture detected by WB; I-L are the relative protein expression levels of p-p65 / p65, IL-6, TNF-α, and COX-2 in sequence, n = 3.
[0045] Figure 9 Shows the results of the effect of extracellular polysaccharide EPSK22 on cisplatin-induced renal cell apoptosis. Among them, A is the representative image of TUNEL staining of kidney sections (n = 3, scale bar 200 μm); B is the quantitative analysis of TNUEL staining; C-E are the relative mRNA expression levels of Casp3, BAX, and Bcl-2 in sequence, n = 6; F is the typical picture detected by WB; G-I are the relative protein expression levels of Casp3, BAX, and Bcl-2 in sequence, n = 3. Detailed implementation methods
[0046] Next, the technical solutions of the present invention will be described in detail through specific embodiments. However, it should be clearly stated that these embodiments are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.
[0047] Example 1
[0048] Isolation and identification of Klebsiella sp. S22 strain
[0049] Take 0.5 g of farmland soil from Hefei, Anhui, and mix it with 10 ml of sterile normal saline to obtain a mixed solution. Take 100 μl of the mixed solution for dilution and inoculate it into a solid medium (containing 15 g / L agar, 30 g / L sucrose, 1 g / L KNO3, 0.1 g / L MgSO4, 1.0 g / L NaH2PO4, 0.0125 g / L FeSO4 in 1 L of water, pH = 7.0). After culturing at 28 °C for 48 - 72 h, select the bacteria producing extracellular polysaccharide on the colony surface and perform further streak isolation until a pure single colony is obtained;
[0050] The morphological characteristics of this colony are as Figure 1 shown. It can be seen that this strain is round and convex, with a smooth surface and is white;
[0051] Take this single colony and use a kit to extract the colony DNA, and use primers 27F / 1492R to amplify and obtain the 16S rRNA sequence of the strain. Submit it to NCBI (the sequence number of Klebsiella sp. S22 strain is PQ269771), and construct a phylogenetic tree as Figure 2 . It is found that this strain clusters with the genus Klebsiella, indicating that it is Klebsiella. It is named Klebsiella sp. S22, and this strain was deposited in the China Center for Type Culture Collection (CCTCC) on September 6, 2024. The deposit address is Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan, Hubei Province, China, and the deposit number is CCTCC NO: M 20241932.
[0052] Example 2
[0053] A method for producing extracellular polysaccharide EPSK22, comprising the following steps:
[0054] Inoculate Klebsiella sp. S22 into a seed medium and culture it at 30 °C and 230 rpm for 18 - 24 h to obtain a seed solution. Then inoculate it into a fermentation medium at an inoculation amount of 5% and culture it at 30 °C and 230 rpm for 48 h to obtain a fermentation broth; add three volumes of ethanol to the fermentation broth for alcohol precipitation, then centrifuge at 8000 g for 10 min, collect the precipitate, and dry it in an oven at 40 °C to obtain a crude polysaccharide, with a yield of 16.4 g / L;
[0055] Purify the crude polysaccharide. The purification steps include:
[0056] a. Prepare an aqueous solution of the crude polysaccharide at a concentration of 0.01 g / mL, add Sevag solution (Sevag solution is a mixture of chloroform and n-butanol in a volume ratio of 4:1), such that the volume ratio of the crude polysaccharide aqueous solution to the Sevag solution is 1:4. Vigorously shake for 20 min, and let the solution stand. The solution is divided into three layers (the middle layer is the denatured protein layer, the upper layer is the aqueous phase, and the lower layer is the organic phase). Take the upper aqueous phase, centrifuge at 8000 g for 10 min, and collect the supernatant.
[0057] b. Use the supernatant in step a to replace the crude polysaccharide aqueous solution in step a, and repeat step a three times until the standing solution is divided into two layers (without the middle protein layer). Take the upper aqueous phase, centrifuge at 8000 g for 10 min, take the supernatant, add three volumes of ethanol for alcohol precipitation, then centrifuge at 8000 g for 10 min, collect the precipitate, and dry it in an oven at 40 °C to obtain the extracellular polysaccharide EPSK22.
[0058] The formulas of the above seed medium and fermentation medium are the same, and both are: in 1 L of water, containing 30 g / L sucrose, 4 g / L yeast powder, 0.05 g / L CaCl2, 1.0 g / L NaH2PO4, 0.0025 g / L ZnCl2, 0.4 g / L MgSO4, 0.0125 g / L FeSO4, 0.003 g / L MnSO4, pH = 7.0. The medium is sterilized at 121 °C for 20 min.
[0059] Example 3
[0060] Take the extracellular polysaccharide EPSK22 obtained in Example 2 for structural analysis, as follows:
[0061] (1) Detection of molecular weight
[0062] Using high performance gel permeation chromatography (HPGPC) method, with pullulan polysaccharide as the standard, the average molecular weight of the extracellular polysaccharide EPSK22 is determined to be 63800 Da.
[0063] (2) Monosaccharide composition analysis
[0064] Using PMP derivatization combined with high performance liquid chromatography to analyze the monosaccharide composition of the extracellular polysaccharide EPSK22, the results are as Figure 3 shown. Figure 3 is the monosaccharide composition map of the extracellular polysaccharide EPSK22, where a is the standard monosaccharide and b is the extracellular polysaccharide EPSK22.
[0065] From Figure 3It can be seen that the exopolysaccharide EPSK22 is composed of mannose, rhamnose, glucuronic acid, and galactose in a molar ratio of 2:2:1:1.
[0066] (3) Methylation analysis
[0067] The exopolysaccharide EPSK22 was subjected to methylation analysis, and the results are shown in Table 1.
[0068] Table 1 Methylation analysis results of exopolysaccharide EPSK22
[0069]
[0070] As can be seen from Table 1, the exopolysaccharide EPSK22 is mainly composed of T-Rhap, 1,2-linked Rhap, 1,2-linked-Manp, 1,4-linked GlcpA, 1,3-Galp, and 1,2,3-linked Manp in a molar ratio of 15.86:16.33:17.61:14.26:16.27:17.28. The molar ratio of each glycoside is consistent with the monosaccharide composition result.
[0071] (4) NMR analysis
[0072] The exopolysaccharide EPSK22 was depolymerized with trifluoroacetic acid, dissolved in deuterated water to a concentration of 50 mg / ml, and subjected to NMR analysis using deuterated acetone as an internal standard. The results are as Figures 4 - 5 shown. Figure 4 Figure 1 is the one-dimensional NMR spectrum of the exopolysaccharide EPSK22. Among them, Figure A is 1 1H NMR, Figure B is 13 13C NMR, and Figure C is DEPT135 NMR.
[0073] Figure 5 Figure 2 is the two-dimensional NMR spectrum of the exopolysaccharide EPSK22. Among them, Figure A is COSY, Figure B is HSQC, Figure C is TOCSY, Figure D is NOESY, and Figure E is HMBC.
[0074] It can be seen from Figures 4 - 5 that in the 13 13C NMR spectrum, the peaks at δ 216.15 ppm and multiple peaks at δ 30 ppm are the signal peaks of the internal standard deuterated acetone (CD3COCD3); 1 in the 1H spectrum, the anomeric proton peaks are located at δ 4.5 - 5.5 ppm, indicating the presence of sugar residues with α and β configurations in the exopolysaccharide EPSK22; 13The peak of the anomeric carbon in the 13C spectrum is located at δ95 - 106 ppm. The signals at δ94.36 and δ93.87 are the signals of free anomeric carbons exposed after the depolymerization of the polysaccharide. According to the HSQC spectrum, six anomeric signals were obtained and labeled with A - F. The configurations were determined by chemical shifts. The chemical shifts of each glycoside were determined by the cross - peaks of the 2D NMR spectrum and are summarized in Table 2.
[0075] Briefly, the COSY spectrum was used to determine the second proton signal on the glycoside through the anomeric proton, and the relevant cross - signals were marked on the COSY spectrum; the proton signals at other positions on the glycoside were assigned through the COSY, TOCSY, and NOESY spectra; the HSQC spectrum was analyzed to identify the carbon signals corresponding to the cross - peaks, thereby labeling all the signals related to the six glycosides; some characteristic peaks were marked on the 1 1D NMR, for example: in the 13 13C spectrum, the carboxyl group is located at δ171.84 ppm, and the carbon signal of CH3 in Rha is located at δ16.93 ppm; the proton signal of CH3 in Rha is located at 1 δ1.24 in the 1H spectrum; the two inverted peaks near δ60 ppm are the signals of C6 in Man and Gal.
[0076] Table 2 NMR analysis results of extracellular polysaccharide EPSK22
[0077]
[0078] According to the NMR analysis, combined with the monosaccharide composition and methylation analysis, the structure of extracellular polysaccharide EPSK22 is shown in formula (I). Extracellular polysaccharide EPSK22 is composed of the following hexasaccharide repeating unit:
[0079] →2)[α - L - Rha-(1→3)-β - D - Galp-(1→2)-α - L - Rhap-(1→3)]-α - D - Manp-(1→4)-β - D - GlcAp-(1→2)-α - D - Manp-(1→; Formula (I).
[0080] Example 4
[0081] Study on the alleviation of cisplatin - induced kidney injury by extracellular polysaccharide EPSK22
[0082] (1) Construction of acute kidney injury (AKI) model
[0083] The mice were randomly divided into 5 groups, with 6 mice in each group;
[0084] One group of mice was first orally administered normal saline for three days, once a day; then intraperitoneally injected with normal saline, denoted as the control group (CK);
[0085] A group of mice were orally administered normal saline once a day for three days; then cisplatin was intraperitoneally injected at a dose of 20 mg / kg, denoted as the model group (MOD).
[0086] A group of mice were orally administered extracellular polysaccharide EPSK22 once a day for three days, with a dosage of 20 mg / kg each time; then cisplatin was intraperitoneally injected at a dose of 20 mg / kg, denoted as the low-dose group (LE).
[0087] A group of mice were orally administered extracellular polysaccharide EPSK22 once a day for three days, with a dosage of 40 mg / kg each time; then cisplatin was intraperitoneally injected at a dose of 20 mg / kg, denoted as the medium-dose group (ME).
[0088] A group of mice were orally administered extracellular polysaccharide EPSK22 once a day for three days, with a dosage of 80 mg / kg each time; then cisplatin was intraperitoneally injected at a dose of 20 mg / kg, denoted as the high-dose group (HE).
[0089] 72 h after intraperitoneal injection of normal saline or cisplatin, all mice in each group were euthanized by cervical dislocation; serum and kidney samples were collected.
[0090] (2) Histopathological examination
[0091] The kidneys were fixed with 4% paraformaldehyde for 24 h, then dehydrated, paraffin-embedded and cut into 7-μm sections. The tissue sections were stained with hematoxylin and eosin (H&E) and imaged using a microscope system.
[0092] (3) Serological and urine analysis
[0093] Blood samples were collected in tubes containing EDTA, centrifuged at 3500 rpm for 10 min at 4 °C, and the supernatant was extracted; kits from Nanjing Jiancheng Bioengineering Institute, China were used to evaluate blood urea nitrogen (BUN) and serum creatinine (Cre) levels.
[0094] (4) Examination of oxidative stress markers
[0095] 50 mg of kidney samples were homogenized in 0.45 mL of normal saline and centrifuged at 4000 rpm for 10 min at 4 °C to obtain the supernatant; kits from Nanjing Jiancheng Bioengineering Institute, China were used to measure malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) levels.
[0096] (5) RNA extraction and quantitative PCR
[0097] Total RNA was isolated from renal tissues using TRIzol (Karroten science, Nanjing, China). Reverse transcription was performed using an M-MLV reverse transcription kit (Invitrogen). Real-time PCR was carried out using SYBR Premix Ex Taq (Toyobo) on a LightCycle480 system according to the manufacturer's guidelines. The β-actin expression level was used as an internal reference. Relative expression levels were analyzed according to the 2 -ΔΔCT method. The primer information used is listed in Table 3.
[0098] Table 3 Primer Information
[0099]
[0100] (6) WB analysis
[0101] Kidney samples were homogenized in RIPA buffer and then electrophoresed on a 10 - 15% SDS gel; the proteins were transferred to a PVDF membrane (Millipore, Bedford, MA, USA) and blocked with 5% BSA in PBST for 1 h at room temperature; then the blocked PVDF membrane was incubated overnight at 4°C with specific primary antibodies diluted in PBST with 5% non-fat milk (the primary antibodies used were as follows: anti-β-actin, anti-Kim, anti-Ngal, anti-NRF2, anti-Gpx-1, anti-NQO1, anti-HO-1, anti-Bax, anti-Bcl-2, anti-caspase-3, anti-COX-2, anti-IL-6, anti-TNF-α, anti-NF-κB, anti-p-NF-κB), the PVDF membrane after primary antibody incubation was washed with PBST and incubated with a 1:10000 diluted HRP-conjugated secondary antibody; after washing, the PVDF membrane after secondary antibody incubation was treated with a chemiluminescence kit. Image J software was used to quantify the protein band intensity.
[0102] (7) TUNEL staining
[0103] Apoptosis in renal tissues was detected using an ApopTag Plus peroxidase in situ apoptosis detection kit (Millipore, Billerica, MA). The cell nuclei were counterstained with DAB and hematoxylin.
[0104] (8) Immunohistochemistry
[0105] The sections (4 μm) were dewaxed and hydrated, then incubated with proteinase K; then the sections were treated with 3% H2O2 for 10 min, washed, and incubated in serum diluted with PBS for 1 h, followed by incubation with biotin and avidin D for 15 min; then the sections were incubated with anti-CD3 antibody and anti-F4 / 80 antibody overnight at 4 °C; then the sections were incubated with an HPR-conjugated secondary antibody for 1 h at room temperature, washed, and then treated with DAB reagent to produce brown staining at the epitope sites. Hematoxylin was used to counterstain the cell nuclei. The kidney sections were examined using an optical microscope. The percentage of positive staining area was quantified using Image J software.
[0106] The experimental results are as Figures 6 - 9 shown.
[0107] Figure 6 For the effects of extracellular polysaccharide EPSK22 on cisplatin-induced kidney injury, where A is the treatment timeline of extracellular polysaccharide EPSK22; B is the detection result of Cre, n = 6; C is the detection result of BUN level, n = 6; D-E are the relative mRNA expression levels of NGAL and KIM-1 in sequence, n = 6; F is a typical picture detected by WB, n = 3; G-H are the relative protein expression levels of NAGL and Kim-1 in sequence, n = 3; I is a photo of the kidney; J is a typical H&E staining image of kidney sections, n = 3, the scale bar is 100 μm, black arrows indicate vacuolar degeneration of renal tubular epithelial cells, red arrows indicate exfoliation of renal tubular epithelial cells and cell debris, and green arrows indicate necrosis of renal tubular epithelial cells. Data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001.
[0108] As Figure 6 shown in B-C, cisplatin significantly caused renal insufficiency, cisplatin administration increased the levels of BUN and Cre, while extracellular polysaccharide EPSK22 treatment significantly reduced these levels in a dose-dependent manner;
[0109] As Figure 6 shown in D-E, Figure 6 G-H, NGAL and KIM-1 are well-recognized biomarkers of kidney injury, NGAL shows high sensitivity, and KIM-1 shows specificity; administration of extracellular polysaccharide EPSK22 significantly reduced the increase in mRNA and protein levels of NGAL and KIM-1 induced by cisplatin;
[0110] As Figure 6As shown in I-J, in the MOD group, a significant decrease in renal blood flow was clearly observed in the kidneys of mice treated with cisplatin; in the extracellular polysaccharide EPSK22 group, especially in the medium and high-dose groups, the kidneys were filled with blood and had clear outlines, similar to those in the CK group; HE staining analysis showed that after cisplatin injection, the renal tubules of mice were significantly damaged, manifested as vacuolar degeneration, exfoliation, and necrosis of renal tubular epithelial cells, while in the extracellular polysaccharide EPSK22 group, this phenomenon decreased in a dose-dependent manner;
[0111] It can be seen from Figure 6 that the pretreatment of extracellular polysaccharide EPSK22 can significantly alleviate cisplatin-induced renal injury.
[0112] Figure 7 The results show the effects of extracellular polysaccharide EPSK22 on oxidative stress in cisplatin-induced renal injury. Among them, A-D are the levels of GSH, CAT, SOD, and MDA in mice of each group, n = 6; E-L are the relative mRNA expression levels of GSH-Px, CAT, SOD1, SOD2, Nrf2, Gpx1, HO-1, and NQO1, n = 6; M is a typical picture detected by WB, n = 3; N-Q are the relative protein expression levels of Nfr2, Gpx1, HO-1, and NQO1, n = 3; the data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001.
[0113] As Figure 7 shown in A-D, cisplatin-induced renal injury is closely related to oxidative stress; the levels of GSH, CAT, SOD, and MDA are important indicators reflecting antioxidant capacity; cisplatin treatment significantly reduces antioxidant capacity, manifested as decreased levels of GSH, CAT, and SOD, and increased levels of MDA at the same time; while extracellular polysaccharide EPSK22 increases the levels of GSH, CAT, and SOD, and decreases the level of MDA at the same time.
[0114] As Figure 7 shown in E-H, after using cisplatin in the model group, the expression levels of key antioxidant enzymes SOD1, SOD2, CAT, and GSH-Px were significantly decreased; while extracellular polysaccharide EPSK22 could counteract this effect;
[0115] As Figure 7As shown in I-L, Nrf2 and its target genes, such as Gpx1, HO-1, and NQO1, play crucial roles in the antioxidant pathway; the inventors investigated the effect of extracellular polysaccharide EPSK22 on the Nrf2 antioxidant signaling pathway; compared with the control group, cisplatin treatment in the model group significantly reduced the mRNA levels of Nrf2, Gpx1, NQO1, and HO-1, leading to oxidative damage; in contrast, extracellular polysaccharide EPSK22 treatment significantly increased their expression levels; WB results showed a similar trend at the protein level (such as Figure 7 M-Q).
[0116] As Figure 7 can be seen: extracellular polysaccharide EPSK22 can alleviate cisplatin-induced oxidative damage and relieve cisplatin-induced renal oxidative stress by regulating the Nrf2 pathway.
[0117] Figure 8 The results of the effect of extracellular polysaccharide EPSK22 on the inflammatory response of cisplatin-induced kidney injury are shown. Among them, A-B are the representative immunohistochemical staining results of F4 / 80+ macrophages and CD3+ T cells in the kidney in turn; C-D are the quantitative analysis of F4 / 80+ and CD3+ in turn (n = 3, scale bar 200 μm); E-G are the relative mRNA expression levels of IL-6, TNF-α, and COX-2 in turn, n = 6; H is a typical picture detected by WB; I-L are the relative protein expression levels of p-p65 / p65, IL-6, TNF-α, and COX-2 in turn, n = 3; the data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001.
[0118] As Figure 8 shown in A-D, inflammatory cell infiltration is associated with cisplatin-induced acute kidney injury; immunohistochemical results showed that in the model group, cisplatin significantly increased the infiltration of T cells and macrophages in the mouse kidney, while the extracellular polysaccharide EPSK22 treatment group showed a significant downregulation;
[0119] Figure 8 As shown in E-L, the inflammatory response is closely related to cisplatin-induced kidney injury; the effect of extracellular polysaccharide EPSK22 on the NF-κB signaling pathway was investigated by measuring the expression levels of NF-κB and its downstream genes and proteins; in the model group, phosphorylated NF-κB P65 and its downstream targets TNF-α, IL-6, and COX-2 showed a significant upregulation, while after treatment with extracellular polysaccharide EPSK22, their expression was significantly reduced in a dose-dependent manner.
[0120] As Figure 8It can be seen that extracellular polysaccharide EPSK22 can alleviate cisplatin-induced inflammatory response by reducing inflammatory cell infiltration and downregulating the gene expression of NF-κB pathway, and mitigate the inflammatory response of cisplatin-induced acute kidney injury.
[0121] Figure 9 The results of the effect of extracellular polysaccharide EPSK22 on cisplatin-induced renal cell apoptosis are shown as follows. Among them, A is the representative image of TUNEL staining of kidney sections (n = 3, scale bar 200 μm); B is the quantitative analysis of TNUEL staining; C-E are the relative mRNA expression levels of Casp3, BAX, and Bcl-2 in sequence, n = 6; F is the typical picture detected by WB; G-I are the relative protein expression levels of Casp3, BAX, and Bcl-2 in sequence, n = 3. Data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001.
[0122] As Figure 9 shown in A-B, cisplatin can significantly increase the number of TUNEL-positive cells, indicating induction of cell apoptosis; while after treatment with extracellular polysaccharide EPSK22, the number of TUNEL-positive cells decreased in a dose-dependent manner;
[0123] As Figure 9 shown in C-E, Bax, Bcl-2, and Caspase-3 are key proteins involved in regulating cell apoptosis. Bax promotes cell apoptosis, Bcl-2 supports cell survival, and Caspase-3 executes the apoptotic process; cisplatin increases the expression of Bax and Caspase-3 and decreases the expression of Bcl-2, thereby inducing renal cell apoptosis; treatment with extracellular polysaccharide EPSK22 significantly improved this phenomenon by reducing the levels of Bax and Caspase-3 and increasing the level of Bcl-2 at the same time; the WB results showed a similar trend at the protein level (as Figure 9 shown in F-I).
[0124] It can be Figure 9 seen that extracellular polysaccharide EPSK22 can alleviate cisplatin-induced renal cell apoptosis through the Bcl-2 / Bax / Caspase-3 signaling pathway.
[0125] In summary, extracellular polysaccharide EPSK22 may alleviate cisplatin-induced acute kidney injury by reducing oxidative stress, inhibiting inflammation, and preventing renal cell apoptosis; extracellular polysaccharide EPSK22 has medicinal and functional food value in preventing kidney injury caused by cisplatin chemotherapy.
[0126] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, shall be covered by the protection scope of the present invention.
Claims
1. A Klebsiella sp. S22 with a preservation number of CCTCC NO: M20241932.
2. An extracellular polysaccharide EPSK22, characterized in that, The extracellular polysaccharide EPSK22 is composed of mannose, rhamnose, glucuronic acid, and galactose in a molar ratio of 2:2:1:1; preferably, the average molecular weight of the extracellular polysaccharide EPSK22 is 60,000 - 70,000 Da; preferably, the structural formula of the extracellular polysaccharide EPSK22 is shown in Formula (I): →2)[α-L-Rha-(1→3)-β-D-Galp-(1→2)-α-L-Rhap-(1→3)]-α-D-Manp-(1→4)-β-D-GlcAp-(1→2)-α-D-Manp-(1→; Formula (I).
3. The exopolysaccharide EPSK22 according to claim 2, wherein, The extracellular polysaccharide EPSK22 is produced using the Klebsiella sp. S22 described in Claim 1.
4. A production method of extracellular polysaccharide EPSK22 as described in claim 2 or 3, characterized in that, It includes the following steps: Take the fermentation broth of the Klebsiella sp. S22 described in Claim 1, perform alcohol precipitation, and take the precipitate to obtain the extracellular polysaccharide EPSK22.
5. The production method of extracellular polysaccharide EPSK22 according to claim 4, characterized in that, The Klebsiella sp. S22 is fermented and cultured in a fermentation medium to obtain a fermentation broth; preferably, 1 L of the fermentation medium contains: 28 - 32 g / L of sucrose, 3 - 5 g / L of yeast powder, 0.04 - 0.06 g / L of CaCl2, 0.8 - 1.2 g / L of NaH2PO4, 0.002 - 0.003 g / L of ZnCl2, 0.3 - 0.5 g / L of MgSO4, 0.012 - 0.013 g / L of FeSO4, 0.002 - 0.004 g / L of MnSO4; preferably, the pH of the fermentation medium is 6.8 - 7.
2.
6. The method for producing extracellular polysaccharide EPSK22 according to claim 4 or 5, characterized in that, The temperature of the fermentation culture is 28 - 32 °C, and the time of the fermentation culture is 46 - 50 h; preferably, the fermentation culture is carried out at a rotation speed of 200 - 250 rpm.
7. The production method of extracellular polysaccharide EPSK22 according to any one of claims 4-6, characterized in that, The alcohol precipitation solvent is at least one of ethanol and isopropanol; preferably, the volume ratio of the fermentation broth to the alcohol precipitation solvent is 1:2 - 4.
8. The production method of extracellular polysaccharide EPSK22 according to any one of claims 4-7, characterized in that, Take the precipitate for purification to obtain the extracellular polysaccharide EPSK22; preferably, the purification step includes: a. Extract the aqueous solution containing the precipitate with Sevag solution, let it stand and divide into three layers, take the upper aqueous phase, and then centrifuge to take the supernatant; b. Use the supernatant to replace the aqueous solution containing the precipitate in a, repeat step a until it stands and divides into two layers, take the upper aqueous phase, and then centrifuge to take the supernatant, perform alcohol precipitation treatment, and take the precipitate to obtain the extracellular polysaccharide EPSK22.
9. The production method of extracellular polysaccharide EPSK22 according to claim 8, wherein, In the purification step, the Sevag solution is a mixed solution composed of chloroform and n-butanol in a volume ratio of 3 - 5:1; preferably, in the purification step, the volume ratio of the aqueous solution containing the precipitate to the Sevag solution is 1:3 - 5; preferably, in the aqueous solution containing the precipitate, the concentration of the precipitate is 0.008 - 0.012 g / mL; preferably, in the purification step, the alcohol precipitation solvent is at least one of ethanol and isopropanol; preferably, in the purification step, the volume ratio of the supernatant to the alcohol precipitation solvent is 1:2 - 4.
10. Use of Klebsiella sp. S22 or its ferment, or extracellular polysaccharide EPSK22 as described in claim 2 or 3 in the preparation of a medicament or health product for preventing or treating kidney injury; preferably, the kidney injury is drug-induced kidney injury; preferably, the drug is a chemotherapeutic drug; preferably, the drug is cisplatin.