Application of sphingomonas exopolysaccharide in preparation of medicine for treating endothelial cell dysfunction

Abstract

The invention discloses an application of sphingomonas exopolysaccharide in treatment of endothelial cell dysfunction. The application range of the sphingomonas exopolysaccharide is expanded, the source is natural and safe, and a new thought is provided for prevention and treatment of cardiovascular diseases.

Description

Technical Field

[0001] The invention relates to the technical field of production of marine active substances, and in particular to application of Sphingomonas exopolysaccharide in preparing a medicine for treating endothelial cell dysfunction. Background Art

[0002] The ocean is the area with the largest number of microorganisms on Earth. It is reported that more than 2 million species of eukaryotes and prokaryotes thrive in the ocean. Marine microorganisms have multiple functions: they can assess the health of marine ecology, maintain the balance of marine ecology, and are an important source of drugs and natural products. Microorganisms constitute a major part of the world's biodiversity, including bacteria, fungi, viruses, archaea and protozoa. Bacteria are an important component of marine microorganisms and are widely distributed in a variety of marine environments. Many evidences show that bioactive components isolated from marine bacteria may have anti-inflammatory, hypoglycemic, hypolipidemic, immunological and antioxidant activities. Although considerable progress has been made in the field of chemical synthesis and synthesis of bioactive compounds, the marine environment is still the richest and most diverse source of various bioactive substances. Marine bacteria can live in harsh marine environments such as deep sea and extreme cold, and have a variety of survival mechanisms to cope with environmental stress, thus producing bioactive substances with unique biological characteristics, which can be used in a variety of industrial and biotechnological applications.

[0003] In nature, Sphingomonas are distributed in a variety of places, such as air, soil, deep sea and polar regions. Studies have shown that the reason why Sphingomonas can survive in such an environment is that in the face of environmental pressure, it can perform efficient metabolic regulation, increase cell metabolism, and thus improve its own survival rate. Sphingomonas is not only the main role in the fermentation and production of welan gum, but also has great application potential in biodegradation and helping plant remediation. Sphingomonas echinoides is a Sphingomonas isolated from deep seawater. Therefore, Sphingomonasechinoides has great research value.

[0004] The functionality of exopolysaccharides in food is related to their ability to act as hydrocolloids, bind water and retain moisture. Exopolysaccharides are used in many food sectors, mainly in the dairy and cereal sectors. There is a growing awareness of their health-related benefits and their use in the production of functional products with added value, meeting consumer demand for natural and healthy products with fewer additives. In the dairy fermentation industry, exopolysaccharides of lactic acid bacteria are very important for the rheological properties of the products. For example, they have been used in low-fat (RF) and semi-fat cheeses to improve the texture and rheological properties of these products. In 2005, Awad et al. studied the effects of using L. lactis subsp. cremoris strain (JFR1) on the properties of RF cheddar cheese. RF cheese produced using this exopolysaccharide had a higher moisture content and similar texture and sensory properties compared to the control group. The moisture content was higher than the control group and the textural properties were similar to those of full-fat cheese. In fermented milk, such as yogurt, exopolysaccharides can act as an influence on the formation of casein-gel structures. For example, the interaction between exopolysaccharides or between exopolysaccharides and proteins is responsible for the formation of sour taste in yogurt. The degree of interaction is responsible for the porosity in yogurt. The intrinsic viscosity of exopolysaccharides depends on their molecular weight and backbone structure, while the chemical composition has no significant effect. Generally speaking, exopolysaccharides with high molecular weight, hard backbone and few branches will produce a high viscosity milky texture, while low molecular weight, highly branched and flexible exopolysaccharides will produce a thin product with low viscosity. Purified components of exopolysaccharides also have specific effects. For example, the addition of dextran obtained by purification of exopolysaccharides to a formula containing low-protein wheat flour significantly increased the volume, hardness and shelf life of the low-protein bread produced.

[0005] Endothelial cell dysfunction is considered to be one of the main causes of cardiovascular diseases such as coronary heart disease. Endothelial cell dysfunction may be related to a variety of factors such as inflammation, oxidative stress, and genetic variation. These factors lead to endothelial cell dysfunction or damage, affecting the normal physiological function of blood vessels. Western medicine is often used as the main treatment in clinical practice, but the treatment effect of Western medicine alone on patients needs to be improved. At present, there are no reports on the effects of Sphingomonas exopolysaccharides on endothelial dysfunction. Summary of the invention

[0006] The purpose of the present invention is to provide an application of Sphingomonas exopolysaccharide in the preparation of a drug for treating endothelial cell dysfunction, which is green and safe and provides a new idea for the prevention and treatment of cardiovascular diseases.

[0007] The technical solution adopted by the present invention to solve the technical problem is: Application of Sphingomonas exopolysaccharide in the preparation of medicine for treating endothelial cell dysfunction. The present invention firstly discovers that Sphingomonas exopolysaccharide has the function of effectively alleviating endothelial cell dysfunction, has natural source, is safe, and has little toxic and side effects.

[0008] The Sphingomonas is specifically Sphingomonas echinoidesh.

[0009] The endothelial cell dysfunction is lipopolysaccharide-induced endothelial cell dysfunction.

[0010] The Sphingomonas exopolysaccharide is prepared by the following steps: (1) inoculating Sphingomonas echinoides into 2216E liquid culture medium, shaking and culturing in a shaker at 20° C. for 5-10 hours, ultrasonically treating the fermentation liquid of Sphingomonas echinoides, and continuing to shake and culture for 30 hours after the ultrasonic treatment to obtain a fermentation liquid of Sphingomonas echinoides; (2) filtering the fermentation broth of Sphingomonas echinoides, centrifuging to obtain the fermentation broth supernatant, and concentrating by rotary evaporation to obtain a concentrate; (3) Sevage reagent (chloroform: n-butanol = 5:1 volume ratio) and the concentrate were mixed and shaken in a volume ratio of 1:4, and the precipitate was removed by centrifugation. This was repeated three times to obtain a deproteinized solution; (4) The deproteinized solution treated in step (3) is precipitated with anhydrous ethanol at 4° C. overnight, the precipitate is collected by centrifugation, and the precipitate is freeze-dried in vacuum to obtain crude extracellular polysaccharide; (5) The crude exopolysaccharide is dissolved in distilled water to form a crude exopolysaccharide solution, which is purified on a DEAE-Sepharose Fast Flow column and eluted with a NaCl solution. The eluate is collected, concentrated, dialyzed, and freeze-dried to obtain purified exopolysaccharide.

[0011] The invention improves the extraction method of sphingomonas extracellular polysaccharide, and ultrasound intervention at a specific time can significantly increase the yield of sphingomonas extracellular polysaccharide. The method is simple and efficient, and can extract the best extracellular polysaccharide component that effectively alleviates endothelial cell dysfunction.

[0012] In step (1), the ultrasonic treatment parameters are: ultrasonic power 30-90W, ultrasonic frequency 25kHz, and ultrasonic time 20-40s.

[0013] In step (2), the concentrated solution is concentrated by rotary evaporation at 50° C. to obtain a concentrated solution of 1 / 5 of its original volume.

[0014] In step (4), the volume ratio of the deproteinized liquid to anhydrous ethanol is 1:3-1:5.

[0015] In step (5), the concentration of the NaCl solution is 0.1 M.

[0016] The beneficial effects of the present invention are: 1. The present invention improves the method for extracting extracellular polysaccharides of Sphingomonas, which is simple and efficient and can extract the best extracellular polysaccharide components that effectively alleviate endothelial cell dysfunction.

[0017] 2. It expands the application scope of Sphingomonas extracellular polysaccharides, which are natural and safe, and provide new ideas for the prevention and treatment of cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results are shown as mean ± SD (n = 3). The values ​​indicated by different letters are significantly different (p < 0.05).

[0019] Figure 2 These are SEM micrographs of Sphingomonas exopolysaccharide. (a) Exopolysaccharide morphology before ultrasound, ×1000; (b) Exopolysaccharide morphology after ultrasound, magnification ×1000.

[0020] Figure 3 This is the elution curve of EPS after DEAE-Sepharose FF ion exchange chromatography.

[0021] Figure 4 The results are shown as mean ± SD (n = 3). Different letters indicate significant differences (p < 0.05).

[0022] Figure 5 The effects of crude polysaccharides and three EPS components on the secretion levels of inflammatory factors (TNF-α, INF-β) and NO in HUVEC cells induced by LPS. (a) The effects of crude polysaccharides and three EPS components on the TNF-α content of HUVEC cells induced by LPS; (b) The effects of crude polysaccharides and three EPS components on the IFN-β content of HUVEC cells induced by LPS; (c) The effects of crude polysaccharides and three EPS components on the NO content of HUVEC cells induced by LPS; The results are expressed as mean ± standard deviation (n = 3). The values ​​represented by different letters are significantly different (p < 0.05).

[0023] Figure 6 The results are shown as mean ± SD (n = 3). The values ​​indicated by different letters are significantly different (p < 0.05).

[0024] Figure 7 The results are expressed as mean ± standard deviation (n = 4). The values ​​indicated by different letters are significantly different (p < 0.05).

[0025] Figure 8 The effect of EPS-2 on LPS-induced HUVEC cell hyperpermeability. (a) Cell migration and invasion were detected by wound healing, transwell migration and invasion assays; (b) Relative quantitative analysis of migration area was performed using ImageJ software; (c) and (d) Relative quantitative analysis of migration and invasion cell numbers was performed using ImageJ software, and the results were expressed as mean ± standard deviation (n = 3). The values ​​represented by different letters were significantly different (p < 0.05).

[0026] Fig. 9 The effect of EPS-2 on the parameters related to inflammation and anti-inflammatory ability of HUVEC cells. (a) and (b) The effect of EPS-2 on the content and mRNA level of inflammatory factors in HUVEC cells; the results are expressed as mean ± standard deviation (n = 3). The values ​​represented by different letters are significantly different (p < 0.05).

[0027] Fig.10 The effects of EPS-2 on parameters related to oxidative damage and antioxidant capacity of HUVEC cells. (a) and (b) The effects of EPS-2 on the relative level of ROS induced by LPS; (c) and (d) The effects of EPS-2 on the content and mRNA levels of antioxidant enzymes Cu / Zn SOD, MnSOD, CAT, GPx, GST, and GR; (e) and (f) The effects of EPS-2 on the relative protein levels of N-Nrf2, T-Nrf2, and HO-1; The results are expressed as mean ± standard deviation (n = 3). The values ​​represented by different letters are significantly different (p < 0.05). DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below through specific embodiments.

[0029] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0030] The Sphingomonas echinoides strain was purchased from the China Marine Microbial Culture Collection Center, and the strain was Sphingomonas echinoides isolated from deep sea water.

[0031] Embodiment 1: Application of Sphingomonas exopolysaccharide in the preparation of medicine for treating lipopolysaccharide-induced endothelial cell dysfunction.

[0032] The Sphingomonas exopolysaccharide is prepared by the following steps: (1) Sphingomonas echinoides stored in -80℃ glycerol tubes was inoculated into 2216E solid culture medium (Qingdao Haibo Biotechnology Co., Ltd.) for activation twice. A single colony was picked and inoculated into 100mL 2216E liquid culture medium (Qingdao Haibo Biotechnology Co., Ltd.), and cultured in a 20℃ shaker at 120rpm for 12h as a seed fermentation liquid. The seed fermentation liquid was inoculated into 500mL 2216E liquid culture medium at a ratio of 2% (v / v), and cultured in a 20℃ shaker at 160rpm. The Sphingomonas echinoides fermentation liquid was ultrasonically treated at 0, 5, 10, 15, 20, and 25h of culture. The ultrasonic probe was disinfected with alcohol and cleaned with sterile water, and inserted 1cm below the liquid level. The sample was placed in an ice bath during the treatment process. The ultrasonic frequency was 25kHz. After ultrasonic treatment, the shaking culture was continued for 30 hours to obtain the Sphingomonas echinoides fermentation liquid; (2) The fermentation broth of Sphingomonas echinoides was filtered and centrifuged at 5000 rpm for 15 min to obtain the fermentation broth supernatant, which was concentrated to 1 / 5 of the original volume by rotary evaporation at 50° C. to obtain a concentrate; (3) Sevage reagent and concentrate were mixed and shaken in a volume ratio of 1:4, and centrifuged at 10,000 rpm and 4°C for 20 min to remove the precipitate. This was repeated three times to obtain a deproteinized solution. (4) The deproteinized solution treated in step (3) was precipitated with anhydrous ethanol at 4°C overnight, centrifuged at 8000 rpm for 15 min to collect the precipitate, and the precipitate was freeze-dried in vacuum to obtain crude extracellular polysaccharide; (5) The crude exopolysaccharide was dissolved in distilled water to form a crude exopolysaccharide solution with a concentration of 0.5 g / mL, and then purified on a DEAE-Sepharose Fast Flow column. First, the chromatography column (2.6×50 cm) was cleaned with distilled water, and after the ion column was loaded, it was fully rinsed with distilled water to fully balance the anion column. NaCl solution was used for elution, and the eluate was collected, concentrated, dialyzed (300 Da), and freeze-dried to obtain purified exopolysaccharide.

[0033] Using the conditions of Example 1, the parameters were changed to conduct a single factor experiment: The ultrasonic power can be selected as 0, 30, 60, 90, 120, or 150 W; the ultrasonic time can be selected as 0, 20, 40, 60, 80, 100, or 120 s; the volume ratio of the deproteinizing solution to anhydrous ethanol can be selected as 1:1, 1:2, 1:3, 1:4, 1:5, or 1:6; In step (5), distilled water and (0.1, 0.2, 0.3, 0.4, 0.5 M) NaCl solution were used for elution at a flow rate of 1 mL / min. 5 mL of eluate was collected from each tube, and the polysaccharide content was detected by the phenol-sulfuric acid method. The elution curve was drawn with the absorbance value as the ordinate and the number of tubes as the abscissa.

[0034] Under different ultrasound-assisted conditions, the amount of Sphingomonas exopolysaccharide extracted changes as follows Figure 1 As shown. Figure 1 It can be seen that when the ultrasonic power increases from 0W to 30W, the EPS (extracellular polysaccharide) extraction amount increases significantly. When it increases from 30W to 60W, the EPS extraction amount increases significantly and the increase is greater than 0-30W. When the ultrasonic power is 60-120W, the EPS extraction amount begins to drop sharply. When the ultrasonic power is 120-150W, the EPS extraction amount tends to be stable. Figure 1 As can be seen from b, when the ultrasonic time increases from 0s to 40s, the EPS extraction amount increases significantly, while when the ultrasonic time is 40-80s, the EPS extraction amount decreases significantly, and when the ultrasonic time is 80-120s, the EPS extraction amount tends to be stable. Figure 1 c It can be seen that when the alcohol precipitation ratio (volume ratio of deproteinized liquid to anhydrous ethanol) increases from 1:1 to 1:3, the EPS extraction amount increases significantly. When the alcohol precipitation ratio is between 1:3 and 1:6, the EPS extraction amount is basically at the same level. Figure 1 It can be seen that when the ultrasonic stage was 0-10h, the EPS extraction amount increased significantly, when the ultrasonic stage was 10-15h, the EPS extraction amount decreased significantly, and when the ultrasonic stage was 15-25h, the EPS extraction amount tended to be stable.

[0035] The extraction parameters were adjusted as follows: ultrasound intervention for 10 hours of culture, ultrasound power of 60W, ultrasound time of 40s, volume ratio of deproteinized solution to anhydrous ethanol of 1:3, elution with 0.1M sodium chloride, and the rest was the same as in Example 1, with no ultrasound-assisted extraction as a control. SEM observations showed that the microstructure of the EPS of Sphingomonas echinoides extracted without ultrasound-assisted extraction was granular, and the EPS particles treated with ultrasound were finer and more uniform ( Figure 2 ).

[0036] The extraction parameters were adjusted as follows: ultrasound intervention for 10 hours of culture, ultrasound power of 60W, ultrasound time of 40s, volume ratio of deproteinized solution to anhydrous ethanol of 1:3, elution with distilled water and NaCl solution (0.1, 0.2, 0.3, 0.4, 0.5M), flow rate of 1mL / min. 5mL of eluate was collected in each tube, and the polysaccharide content was detected by phenol-sulfuric acid method. Since the polysaccharide content was positively correlated with the absorbance value, the absorbance value was used to represent the polysaccharide content. The absorbance value was used as the ordinate and the number of tubes as the abscissa to draw the elution curve ( Figure 3 ), the crude polysaccharide EPS was separated by anion column to obtain three components, named EPS-1 (eluted with distilled water), EPS-2 (eluted with 0.1 M NaCl solution) and EPS-3 (eluted with 0.2 M NaCl solution).

[0037] Effects of EPS fractions on the proliferation activity of HUVEC cells Before the biological activity evaluation, the effects of crude polysaccharide and three EPS fractions on the proliferation activity of HUVEC cells (purchased from the Chinese Academy of Sciences Cell Bank) in the concentration range of 100-300 μg / mL were first determined. Figure 4 As shown. It was found that 200 μg / mL was the best concentration for each component to affect the proliferation activity of HUVEC cells (p<0.05). Therefore, 200 μg / mL of each component was selected as the concentration for preliminary activity screening.

[0038] The present invention utilizes lipopolysaccharide (LPS) to treat and construct an endothelial injury cell model to study the role of EPS in regulating endothelial cell dysfunction and its related mechanisms.

[0039] Control group: untreated cells; LPS group: HUVEC cells were treated with LPS (1.0 μg / mL) for 24 h; LPS+EPS group: HUVEC cells were treated with EPS (200 μg / mL) for 1 h and then with LPS (1.0 μg / mL) for 24 h.

[0040] Effects of EPS fractions on the secretion levels of inflammatory factors and NO in HUVEC cells The optimal EPS fractions for inhibiting LPS-induced inflammatory response in HUVEC cells were preliminarily screened by detecting the effects of crude polysaccharides and three EPS fractions (200 μg / L) on the secretion levels of inflammatory factors (TNF-α, IFN-β) and NO in HUVEC cells induced by LPS. Effects of crude polysaccharides (EPS group) and three EPS fractions on the secretion levels of inflammatory factors (TNF-α, IFN-β) and NO in HUVEC cells induced by LPS Figure 5As shown, crude polysaccharides and three EPS separation components significantly reduced the increase of TNF-α, INF-β and NO induced by LPS (p<0.05), and the secretion levels of TNF-α, INF-β and NO after EPS-2 treatment were significantly lower than those after EPS-1 and EPS-3 treatments (p<0.05).

[0041] Effects of EPS fractions on antioxidant enzyme activities in HUVEC cells induced by LPS The optimal EPS component for improving LPS-induced oxidative stress in HUVEC cells was preliminarily screened by detecting the effects of crude polysaccharides and three EPS fractions (200 μg / L) on the antioxidant enzyme activities of HUVEC cells induced by LPS. Figure 6 As shown, crude polysaccharides and three EPS separation components significantly reversed the decrease in T-SOD, CAT, GPx, GST and GR activities caused by LPS (p<0.05), and the T-SOD, CAT, GPx, GST and GR enzyme activities after EPS-2 treatment were significantly higher than those after EPS-1 and EPS-3 treatments (p<0.05).

[0042] Three extracellular polysaccharides, EPS-1, EPS-2 and EPS-3, were isolated from the fermentation broth of Sphingomonas. After activity screening, the best component for alleviating endothelial cell dysfunction was EPS-2. After testing, the basic physical and chemical properties of EPS-2: the total sugar content of EPS-2 accounted for as high as 74.3%, the protein content was 1.3%, the uronic acid content was low at 1.2%, and the ash content was 23.2%, indicating that EPS-2 had a high purity.

[0043] EPS-2 alleviates lipopolysaccharide-induced endothelial cell dysfunction The present invention uses lipopolysaccharide (LPS) to treat and construct an endothelial injury cell model to study the role of EPS-2 in regulating endothelial cell dysfunction and its related mechanisms. Control group: untreated cells; LPS group: treated with LPS (1.0 μg / mL) for 24 hours; LPS+EPS-2 group: HUVEC cells were treated with EPS-2 (200 μg / mL) for 1 hour and then treated with LPS (1.0 μg / mL) for 24 hours.

[0044] like Figure 7 As shown, compared with the control group, LPS treatment alone significantly reduced the viability of HUVEC cells (p<0.05). Compared with the LPS treatment group alone, the viability of HUVEC cells in the LPS+EPS-2 group was significantly increased (p<0.05), indicating that EPS-2 helps to reverse the negative effect of LPS on HUVEC cell viability.

[0045] Effect of EPS-2 on LPS-induced HUVEC cell hyperpermeability The scratch test was used to evaluate the migration ability of HUVEC cells. HUVEC cells from each group in the logarithmic growth phase were inoculated in a six-well plate. When the cells were confluent to 90%, a vertical line was made along the midpoint with a 200 μL pipette tip, the old culture medium was discarded, and the cells were washed twice with PBS and cultured for 24 hours. The images were observed using an inverted fluorescence microscope. ImageJ software was used for quantitative analysis.

[0046] Transwell migration and invasion assays were used to evaluate the migration and invasion abilities of the cells. 5 Cells were seeded at a density of 10 cells / well in a 12-well plate. After incubation, the non-migrated cells were removed from the upper chamber, and the cells on the surface of the lower chamber were fixed with 4% paraformaldehyde and then stained with 0.25% crystal violet for 10 min. For the invasion assay, 20 μL 1.2 mg / mL matrix gel was pre-coated at 37°C for 2 h before inoculating the cells, and the subsequent operations were the same as the Transwell migration assay.

[0047] Effects of EPS-2 on LPS-induced HUVEC cell hyperpermeability Figure 8 As shown. In the scratch, migration and invasion assays, the total number of potential migratory and invasive cells decreased significantly after LPS treatment compared with the control group; compared with the LPS treatment group alone, the total number of migratory and invasive cells in the LPS+EPS-2 group increased significantly. The experimental results show that EPS-2 can reduce the hyperpermeability of HUVEC cells induced by LPS.

[0048] Effects of EPS-2 on parameters related to inflammation and anti-inflammatory ability of HUVEC cells like Fig. 9 As shown in a, compared with the control group, the contents of IL-1β, IL-6, IL-8, TNF-α, VCAM-1, ICAM-1 and MCP-1 in HUVEC cells of the LPS treatment group alone were significantly increased (p<0.05); compared with the LPS treatment group alone, the contents of IL-1β, IL-6, TNF-α, VCAM-1, ICAM-1 and MCP-1 in HUVEC cells of the LPS+EPS-2 group were significantly decreased (p<0.05), while the IL-8 content did not change significantly (p>0.05).

[0049] like Fig. 9As shown in b, compared with the control group, the mRNA levels of IL-1β, IL-6, IL-8, TNF-α, VCAM-1, ICAM-1 and MCP-1 in the LPS treatment group alone were significantly increased (p<0.05); compared with the LPS treatment group alone, the mRNA levels of IL-1β, IL-6, TNF-α, VCAM-1, ICAM-1 and MCP-1 in HUVEC cells in the LPS+EPS-2 group were significantly decreased (p<0.05), but there was no significant difference in IL-8 (p>0.05).

[0050] Effects of EPS-2 on oxidative damage and antioxidant capacity-related parameters of HUVEC cells Effects of EPS-2 on parameters related to oxidative damage and antioxidant capacity of HUVEC cells Fig.10 As shown. Fig.10 As shown in a, b, compared with the control group, LPS treatment alone significantly increased the relative ROS level of HUVEC cells (p<0.05); compared with the LPS treatment group alone, the relative ROS level in HUVEC cells of the LPS+EPS-2 group was significantly reduced (p<0.05).

[0051] like Fig.10 As shown in Figure c, compared with the control group, LPS treatment significantly reduced the activities of Cu / Zn-SOD, CAT, GPx, GST and GR (p<0.05); compared with the LPS alone treatment group, the activities of Cu / Zn-SOD, CAT, GPx, GST and GR in HUVEC cells in the LPS+EPS-2 group were significantly increased (p<0.05). There was no significant difference in MnSOD activity among the groups (p>0.05).

[0052] like Fig.10 As shown in Figure d, compared with the control group, LPS treatment significantly reduced the mRNA levels of Cu / Zn-SOD, GPx, GST and GR (p<0.05); compared with the LPS alone treatment group, the mRNA levels of Cu / Zn-SOD, GPx, GST and GR in HUVEC cells in the LPS+EPS-2 group were significantly increased (p<0.05). There was no significant difference in the mRNA levels of MnSOD and CAT among the groups (p>0.05).

[0053] like Fig.10 As shown in e and f, compared with the control group, LPS treatment significantly reduced the levels of N-Nrf2 and HO-1 proteins (p<0.05); compared with the LPS alone treatment group, the levels of N-Nrf2 and HO-1 proteins in the LPS+EPS-2 group were significantly increased. However, there was no significant difference in the level of T-Nrf2 protein among the groups (p>0.05).

[0054] In summary, EPS-2 alleviated LPS-induced endothelial dysfunction in HUVEC cells. First, in HUCVC cells, EPS-2 reversed the negative effect of LPS on HUVEC cell viability. Secondly, EPS-2 alleviated LPS-induced HUVEC cell hyperpermeability and reduced the content and mRNA levels of inflammatory factors, which may be related to the MAPK (ERK, JNK) / NF-κB / STAT3 signaling pathway. Furthermore, EPS-2 reduced the content of ROS and increased the activity and mRNA levels of antioxidant enzymes, which may be related to the activation of the Nrf2 / HO-1 signaling pathway. This study provides new ideas and theoretical basis for EPS-2 as an effective ingredient for potentially improving endothelial dysfunction and preventing and treating cardiovascular diseases.

[0055] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. Application of Sphingomonas exopolysaccharide in the preparation of drugs for treating endothelial cell dysfunction.

2. The use according to claim 1, characterized in that: The Sphingomonas is specifically Sphingomonas echinoidesh.

3. The use according to claim 1, characterized in that: The endothelial cell dysfunction is lipopolysaccharide-induced endothelial cell dysfunction.

4. The use according to claim 1, characterized in that: The Sphingomonas exopolysaccharide is prepared by the following steps: (1) inoculating Sphingomonas echinoides into 2216E liquid culture medium, shaking and culturing in a shaker at 20° C. for 5-10 hours, ultrasonically treating the fermentation liquid of Sphingomonas echinoides, and continuing to shake and culture for 30 hours after the ultrasonic treatment to obtain a fermentation liquid of Sphingomonas echinoides; (2) filtering the fermentation broth of Sphingomonas echinoides, centrifuging to obtain the fermentation broth supernatant, and concentrating by rotary evaporation to obtain a concentrate; (3) Sevage reagent and concentrate were mixed and shaken in a volume ratio of 1:4, and the precipitate was removed by centrifugation. This was repeated three times to obtain a deproteinized solution. (4) The deproteinized solution treated in step (3) is precipitated with anhydrous ethanol at 4° C. overnight, the precipitate is collected by centrifugation, and the precipitate is freeze-dried in vacuum to obtain crude extracellular polysaccharide; (5) The crude exopolysaccharide is dissolved in distilled water to form a crude exopolysaccharide solution, which is purified on a DEAE-Sepharose FastFlow column and eluted with a NaCl solution. The eluate is collected, concentrated, dialyzed, and freeze-dried to obtain purified exopolysaccharide.

5. The use according to claim 1, characterized in that: In step (1), the ultrasonic treatment parameters are: ultrasonic power 30-90W, ultrasonic frequency 25kHz, and ultrasonic time 20-40s.

6. The use according to claim 1, characterized in that: In step (2), the concentrated solution is concentrated by rotary evaporation at 50° C. to obtain a concentrated solution of 1 / 5 of its original volume.

7. The use according to claim 1, characterized in that: In step (4), the volume ratio of the deproteinized liquid to anhydrous ethanol is 1:3-1:

5.

8. The use according to claim 1, characterized in that: In step (5), the concentration of the NaCl solution is 0.1 M.

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