Application of high-purity inonotus obliquus polysaccharide component

Abstract

Application of a high-purity inonotus obliquus polysaccharide component belongs to the technical field of biological medicine, and the high-purity inonotus obliquus polysaccharide component plays a role in preventing and treating by inhibiting autophagy of lung tissue cells and depending on an lncRNA Rmst pathway, and can reduce the number of autophagosomes in lung tissues and the level of conversion from LC3-I to LC3-II; according to the method, the sugar content of IOP can be remarkably increased compared with crude polysaccharide, the protein content is remarkably reduced, four active components with specific molecular weights are obtained through separation, the molecular weight ranges are definite (IOP-g1gt, IOP-g2gt, IOP-g3gt and IOP-g4), and the requirements of medicine research and development for controllable purity and definite components are met.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically referring to the application of a high-purity Inonotus obliquus polysaccharide component. Background Technology

[0002] Acute lung injury (ALI) is a clinical syndrome characterized by impaired lung diffusion capacity, triggered by various direct or indirect factors (including trauma, infection, dyspnea, shock, etc.). Without timely and effective intervention, ALI can rapidly progress to acute respiratory distress syndrome (ARDS), which has a high mortality rate. As a common critical illness in critical care medicine, ALI / ARDS has a complex pathogenesis and is difficult to treat clinically, seriously threatening patients' lives and health, and has become an important research topic in respiratory and critical care medicine. Lipopolysaccharide (LPS), derived from the cell wall of Klebsiella pneumoniae (a Gram-negative bacterium), has been proven to be an important pathogenic factor inducing acute lung injury (ALI).

[0003] Currently, the clinical treatment of acute lung injury (ALI) mainly relies on mechanical ventilation and symptom management. While mechanical ventilation can maintain oxygenation, inappropriate ventilator settings may lead to ventilator-associated lung injury, further aggravating alveolar epithelial damage. Regarding drug treatment of symptoms, although glucocorticoids have anti-inflammatory effects, their immunosuppressive effects may increase the risk of infection, and their clinical efficacy remains controversial. Furthermore, interventions such as exogenous surfactant replacement therapy and antioxidants still have limitations in clinical application, including low bioavailability and poor targeting. While these traditional treatments can alleviate symptoms to some extent, they have failed to effectively block the key pathological processes of ALI, particularly their limited effect on repairing the alveolar-capillary barrier function. Therefore, exploring novel treatment strategies with multi-target regulatory effects, high safety, and the ability to promote lung tissue repair has become a key focus in current ALI research. In recent years, studies have shown that the alkaloids of the natural plant *Astragalus membranaceus* have potential in regulating lung inflammation, and that the active ingredient astragaloside A in *Astragalus membranaceus* can exert anti-fibrotic effects. In summary, natural medicines, with their rich active ingredients and good biocompatibility, demonstrate unique advantages in disease treatment, especially their multiple pharmacological effects such as anti-inflammatory, antioxidant, and epithelial repair, which provide new ideas for the treatment of ALI.

[0004] Inonotus obliquus contains a variety of pharmacologically active chemical components, mainly including Inonotus obliquus polysaccharide (IOP), triterpenoids (such as betulinol), polyphenols, steroids, and alkaloids. Among these active components, IOP exhibits significant biological activity and favorable pharmacological effects, making it a research hotspot. Summary of the Invention

[0005] In order to overcome some of the problems mentioned in the background above, the present invention provides an application of a high-purity Inonotus obliquus polysaccharide component to at least partially solve the above problems.

[0006] According to the technical solution of the present invention, an application of high-purity Inonotus obliquus polysaccharide is provided. The high-purity Inonotus obliquus polysaccharide component exerts a preventive and therapeutic effect by inhibiting autophagy in lung tissue cells and the lncRNA-dependent Rmst pathway, thereby reducing the number of autophagosomes in lung tissue and the conversion level of LC3-I to LC3-II.

[0007] Preferably, the extraction of the high-purity Inonotus obliquus polysaccharide includes the following steps:

[0008] Step 1. Take the crude polysaccharide from Inonotus obliquus extract, remove the protein using the Sevage method, and obtain a deproteinized crude polysaccharide solution;

[0009] Step 2. The deproteinized crude polysaccharide solution is purified by dialysis using an MD55 dialysis bag, and the dialysis fluid is collected.

[0010] Step 3. Load the dialysate into a DEAE-52 ion exchange column chromatography system and collect the polysaccharide active components by elution;

[0011] Step 4. The polysaccharide active components are further separated and purified using a G-200 gel chromatography system. The eluent is collected and freeze-dried to obtain high-purity Inonotus obliquus polysaccharide.

[0012] Preferably, the molecular weight cutoff of the MD55 dialysis bag in step 2 is 8000~14000 Da, the dialysis time is 48~72 h, and the dialysis fluid is replaced every 8~12 h during the dialysis process.

[0013] Preferably, the eluent for the DEAE-52 ion exchange column chromatography in step 3 is deionized water and a 0.1~0.5 mol / L gradient NaCl solution, and the elution flow rate is 1~2 mL / min.

[0014] Preferably, in step 4, the loading volume of G-200 gel chromatography is 2 mL of polysaccharide solution, the eluent is distilled water, the elution flow rate is 0.5~1 mL / min, and one fraction is collected every 3~5 mL.

[0015] Preferably, in step 1, the reagent volume ratio of the Sevage method is polysaccharide solution: Sevage reagent (n-butanol: dichloromethane = 1:5) = 4:1. The mixture is stirred at 3000 rpm for 20 min on a magnetic stirrer, then transferred to a separatory funnel and allowed to stand for 60 min for extraction. The upper polysaccharide solution is collected, and the operation is repeated until no intermediate protein layer appears.

[0016] Preferably, the polysaccharide active component is labeled IOP-d1;

[0017] The high-purity Inonotus obliquus polysaccharides were analyzed by phenol-sulfuric acid method to obtain four specific molecular weight components: IOP-g1, IOP-g2, IOP-g3, and IOP-g4. The molecular weight order of the four groups of high-purity Inonotus obliquus polysaccharides was IOP-g1 > IOP-g2 > IOP-g3 > IOP-g4.

[0018] Preferably, the IOP-d1 component has a molecular weight of 50-100 kDa and is non-cytotoxic to MLE-12 lung epithelial cells in the concentration range of 10-200 μg / mL.

[0019] Preferably, treatment with the polysaccharide component at a concentration of 100 μg / mL can significantly improve the survival rate of LPS-damaged lung epithelial cells MLE-12.

[0020] Preferably, the drug is administered via pre-gastric gavage, starting 7 days before bacterial pneumonia modeling, once daily, at a dose of 1.2 ml / kg, with the polysaccharide component concentration in the drug being 10~200 μg / ml.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. Significant purification effect: The purification process of this invention can significantly increase the sugar content of IOP compared to crude polysaccharide extraction, significantly reduce the protein content, and separate four active components with specific molecular weights, with a clear molecular weight range (IOP-g1>IOP-g2>IOP-g3>IOP-g4), meeting the requirements of drug development for "controllable purity and clear components".

[0023] 2. Precise application scenarios: For the first time, high-purity IOP is used for the prevention and treatment of "bacterial pneumonia-related ALI", rather than a broad range of ALI, targeting pneumonia complications caused by clinical drug-resistant bacteria, filling the application gap of natural polysaccharides in this field;

[0024] 3. Clear and reliable mechanism: This study is the first to reveal that IOP works by "inhibiting pulmonary autophagy + lncRNA Rmst", providing molecular mechanism support for the clinical application of IOP and avoiding the limitation of existing studies that "only know the effect but not the mechanism".

[0025] 4. Safe and effective administration: The safe concentration range and pre-gavage protocol of IOP are clearly defined. While exerting a lung-protective effect, it has no side effects such as immunosuppression and cytotoxicity, and has greater clinical advantages than glucocorticoids.

[0026] 5. Industrialization and reproducibility: The equipment used in the purification process is all conventional equipment in the biopharmaceutical field, with clear steps and well-defined parameters, enabling large-scale production; the operation procedures and detection methods for animal and cell experiments all comply with GLP standards, and the results can be repeatedly verified. Attached Figure Description

[0027] Figure 1 Elution curves of the DEAE-52 ion exchange column in an embodiment of the present invention;

[0028] Figure 2 This is a comparison chart of the sugar content (A) and protein content (B) of crude polysaccharide extracted from Inonotus obliquus and purified IOP-d1 in an embodiment of the present invention.

[0029] Figure 3 The Sephadex G-200 gel chromatography elution curves are from an embodiment of the present invention.

[0030] Figure 4 HE staining (A~D, ×200) and pathological score (E) of lung tissue from different groups of wild-type mice in this embodiment of the invention;

[0031] Figure 5 The graphs (A-D) show the ELISA detection of IL-6 and IL-1β levels in BALF and serum of wild-type mice according to an embodiment of the present invention.

[0032] Figure 6 Figures A-B show the real-time quantitative PCR detection of IL-6 and IL-1β mRNA expression levels in wild-type mouse lung tissue according to an embodiment of the present invention.

[0033] Figure 7 This is a graph showing the survival rate of MLE-12 cells after treatment with different concentrations of IOP according to an embodiment of the present invention.

[0034] Figure 8 This is a graph showing the effect of IOP on LPS-induced MLE-12 cell survival in an embodiment of the present invention.

[0035] Figure 9Figure A shows the autophagosomes (A) and autophagosome count (B) observed in the lung tissue of wild-type mice using transmission electron microscopy, according to an embodiment of the present invention.

[0036] Figure 10 This is a Western blot diagram showing the LC3-I / LC3-II conversion level in wild-type mouse lung tissue, as described in this embodiment of the invention.

[0037] Figure 11 Figure E shows HE staining of lung tissue from each group of Rmst⁻ / ⁻ mice in this embodiment of the invention (A~D, ×200) and a comparison of pathological scores between wild-type and Rmst⁻ / ⁻ mice (E).

[0038] Figure 12 The image shows a transmission electron microscope (TEM) observation of autophagosomes in the lung tissue of Rmst⁻ / ⁻ mice (A) and a statistical count of the number of autophagosomes (B) according to an embodiment of the present invention.

[0039] In the picture: Figure 1 The x-axis represents the number of collection tubes (0~300 tubes), the y-axis on the left represents the absorbance (OD490nm, polysaccharide detection by phenol-sulfuric acid method), and the y-axis on the right represents the NaCl elution concentration (mol / L). The main peak corresponding to the elution with distilled water is the IOP-d1 component, proving that neutral polysaccharides can be separated by this chromatography.

[0040] Figure 2 ** indicates p<0.01; (B) Protein content comparison chart, **** indicates p<0.0001.

[0041] Figure 3 The horizontal axis represents the number of collection tubes (0~30 tubes), and the vertical axis represents the absorbance (OD490nm); the four main peaks correspond to IOP-g1~g4 components, respectively, proving that IOP of different molecular weights can be separated by gel chromatography.

[0042] Figure 4 In the table, A represents the CON group (intact lung structure), B represents the IOP group (no significant changes), C represents the LPS group (thickened alveolar walls and inflammatory infiltration), and D represents the LPS+IOP group (damage relief). In E, P<0.0001 indicates that IOP can alleviate lung injury in wild-type mice.

[0043] Figure 5 , Figure 6 , Figure 8 , Figure 9 , Figure 10 , Figure 11 and Figure 12 In the diagram, * indicates P < 0.05, ** indicates P < 0.01, and **** indicates P < 0.0001.

[0044] Figure 7The horizontal axis represents IOP concentration (μg / mL) and culture time (12h, 24h), and the vertical axis represents survival rate (%), demonstrating that 10~200μg / mL IOP has no cytotoxicity.

[0045] Figure 9 The red arrows represent autophagosomes, and the blue arrows represent lysosomes. Figure 10 The left side shows the protein electrophoresis image, and the right side shows the grayscale value statistics; Figure 12 The ns value indicates no significant difference. Detailed Implementation

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection.

[0047] This invention provides an application of a high-purity Inonotus obliquus polysaccharide. The high-purity Inonotus obliquus polysaccharide component exerts a preventive and therapeutic effect by inhibiting autophagy in lung tissue cells and the lncRNA-dependent Rmst pathway, thereby reducing the number of autophagosomes in lung tissue and the conversion level of LC3-I to LC3-II.

[0048] The extraction of the high-purity Inonotus obliquus polysaccharide includes the following steps:

[0049] Step 1. Take the crude polysaccharide from Inonotus obliquus extract, remove the protein using the Sevage method, and obtain a deproteinized crude polysaccharide solution;

[0050] Step 2. The deproteinized crude polysaccharide solution is purified by dialysis using an MD55 dialysis bag, and the dialysis fluid is collected.

[0051] Step 3. Load the dialysate into a DEAE-52 ion exchange column chromatography system and collect the polysaccharide active components by elution;

[0052] Step 4. The polysaccharide active components are further separated and purified using a G-200 gel chromatography system. The eluent is collected and freeze-dried to obtain high-purity Inonotus obliquus polysaccharide.

[0053] In a further embodiment of this example, the molecular weight cutoff of the MD55 dialysis bag in step 2 is 8000~14000 Da, the dialysis time is 48~72 h, and the dialysis fluid is replaced every 8~12 h during the dialysis process.

[0054] In a further embodiment of this example, the eluent for the DEAE-52 ion exchange column chromatography in step 3 is deionized water and a 0.1~0.5 mol / L gradient NaCl solution, and the elution flow rate is 1~2 mL / min.

[0055] In a further embodiment of this example, the sample loading volume of G-200 gel chromatography in step 4 is 2 mL of polysaccharide solution, the eluent is distilled water, the elution flow rate is 0.5~1 mL / min, and one fraction is collected every 3~5 mL.

[0056] In a further embodiment of this example, the reagent volume ratio of the Sevage method in step 1 is polysaccharide solution: Sevage reagent (n-butanol: dichloromethane = 1:5) = 4:1. The mixture is placed on a magnetic stirrer and stirred at 3000 rpm for 20 min. It is then transferred to a separatory funnel and allowed to stand for 60 min for extraction. The upper polysaccharide solution is collected, and the operation is repeated until no intermediate protein layer appears.

[0057] In a further embodiment of this example, the polysaccharide active component is labeled as IOP-d1;

[0058] The high-purity Inonotus obliquus polysaccharides were analyzed by phenol-sulfuric acid method to obtain four specific molecular weight components: IOP-g1, IOP-g2, IOP-g3, and IOP-g4. The molecular weight order of the four groups of high-purity Inonotus obliquus polysaccharides was IOP-g1 > IOP-g2 > IOP-g3 > IOP-g4.

[0059] In a further embodiment of this example, the molecular weight of the IOP-d1 component is 50~100kDa, and it has no cytotoxicity to MLE-12 lung epithelial cells in the concentration range of 10~200μg / mL.

[0060] In a further embodiment of this example, the polysaccharide component, after being treated at a concentration of 100 μg / mL, can significantly improve the survival rate of LPS-damaged lung epithelial cells MLE-12.

[0061] In a further embodiment of this example, the drug is administered via pre-gastric gavage, starting 7 days before bacterial pneumonia modeling, once daily, at a dose of 1.2 ml / kg, with the polysaccharide component concentration in the drug ranging from 10 to 200 μg / ml.

[0062] It should be noted that the materials in the following embodiments are specifically:

[0063] Raw material: Crude polysaccharide extracted from Inonotus obliquus (30% content, Snott Biotechnology Co., Ltd.);

[0064] Reagents: n-Butanol, dichloromethane, chloroform (analytical grade, Xilong Scientific Co., Ltd.); DEAE-52 cellulose, Sephadex G-200 gel (GE Healthcare); Klebsiella pneumoniae lipopolysaccharide (KP-LPS, Sigma-Aldrich, lot number L4268); Mouse IL-1β / IL-6 ELISA Kit (Jianglai Biotechnology); LC3B antibody (anti-rabbit, Abmart); BCA protein quantification kit, 10% color gel rapid assay kit, universal antibody diluent (NewSmithKline); DMEM high-glucose (with double antibody) medium (Servicebio); CCK-8 cell proliferation and cytotoxicity assay kit (Liji Biotechnology); trichromatic pre-stained protein marker (Novizan); ECL chemiluminescent substrate (high-sensitivity, Biosharp); TBS (Powder), SDS-PAGE running buffer powder (Sewell); IRDye 800CW goat anti-rabbit IgG (H+L) (VicMed); Phosphatase inhibitor mixture (100x, Proteintech); RIPA lysis buffer, trypsin cell digestion solution (Beyotime); QRT SuperMix for qPCR (+gDNA wiper), ChamQ SYBR qPCR Master Mix, RNA isolater Total RNA Extraction Reagent (Novizan); DEPC water, Bradford protein assay kit (Biosharp); SDS-PAGE Loading Buffer (Synthetium); Transmission electron microscopy fixative (Seville); Serum-free cell cryopreservation solution (Heyuan Liji);

[0065] Laboratory animals: Wild-type male C57BL / 6 mice (7-8 weeks old, weighing 20-25g, purchased from Cavens Laboratory Animal Co., Ltd.); Rmst knockout male C57BL / 6 mice (7-8 weeks old, weighing 20-25g, provided by the Animal Barrier System of Xuzhou Medical University), housed in an SPF-grade animal facility (temperature 20℃-26℃, humidity 50%-60%).

[0066] Cell line: Mouse lung epithelial cells MLE-12 (Shanghai Cell Bank), cultured at 37℃ and 5% CO2, in complete DMEM medium containing 10% fetal bovine serum and 1% double antibiotics (streptomycin and penicillin).

[0067] Example 1

[0068] Take 100g of crude polysaccharide from Inonotus obliquus extract and prepare a 50mg / ml polysaccharide solution. Add Sevage reagent (n-butanol: dichloromethane = 1:5) to make the volume ratio of polysaccharide solution to Sevage reagent 4:1. Stir at 3000rpm for 20min on a magnetic stirrer, transfer to a separatory funnel and let stand for 60min for extraction. Collect the upper polysaccharide solution. Repeat the operation until no intermediate protein interface appears after centrifugation. Freeze-dry for later use.

[0069] Example 2

[0070] The polysaccharide powder obtained in Example 1 was prepared into 10 mL of liquid, placed into an MD55 dialysis bag (molecular weight cutoff 8000~14000 Da), and placed in 1 L of ultrapure water at 4 °C for overnight dialysis. The dialysis solution was changed at least 3 times during the process. After the last change, dialysis was continued for ≥2 hours, and the dialysis fluid was collected.

[0071] Take 50g of DEAE-52 cellulose, soak it in 0.5mol / L NaCl solution for 24h, rinse it with distilled water until neutral, pack it into a chromatography column (5cm in diameter, 20cm in height, 200mL in volume), and equilibrate 3 column volumes with distilled water;

[0072] Load the dialysate onto the chromatography column and elute with distilled water at a flow rate of 1.5 mL / min. Collect 4 mL / tube, and take 0.1 mL from each tube for detection using the phenol-sulfuric acid method (absorbance at 490 nm). Plot the elution curve as shown below. Figure 1 As shown, tubes 30-50 (main peak eluted with distilled water) were collected, yielding approximately 20 mL of the IOP-d1 fraction.

[0073] Example 3

[0074] Take 30g of Sephadex G-200 gel, swell it in distilled water for 48h, pack it into a chromatography column (3cm in diameter, 20cm in height, 150mL in column volume), and equilibrate it with distilled water to 3 column volumes.

[0075] The IOP-d1 fraction obtained in Example 2 was loaded onto a G-200 column and eluted with distilled water at a flow rate of 0.8 mL / min. 4 mL was collected per tube, and the absorbance was detected using the phenol-sulfuric acid method. The elution curve was plotted as shown below. Figure 3 As shown, the components corresponding to the four main peaks were collected: IOP-g1, IOP-g2, IOP-g3, and IOP-g4.

[0076] The components were freeze-dried to obtain IOP-g1 (12 mg), IOP-g2 (18 mg), IOP-g3 (25 mg), and IOP-g4 (15 mg). The sugar content was determined by the phenol-sulfuric acid method, and the protein content was determined by the BCA method. The results showed that the sugar content of each component after purification was 30%~50% higher than that of the crude polysaccharide (P<0.01), and the protein content was less than 0.5% (more than 90% lower than that of the crude polysaccharide, P<0.0001), which met the requirements for drug purity.

[0077] Example 4

[0078] Wild-type C57BL / 6 mice were randomly divided into 4 groups (n=6 / group):

[0079] CON group: Daily gavage administration of normal saline (1.2 mL / kg) for 7 days, followed by intravenous infusion of normal saline (50 μL / animal) on the 7th day.

[0080] IOP group: 100 μg / mL high-purity IOP (1.2 mL / kg) was administered by gavage daily for 7 days, followed by intravenous infusion of physiological saline on the 7th day;

[0081] LPS group: Daily gavage with normal saline for 7 days, followed by intravenous infusion of 5 mg / kg KP-LPS (50 μL / animal) on the 7th day to establish a bacterial pneumonia-related ALI model.

[0082] LPS+IOP group: 100 μg / mL high-purity IOP was administered by gavage daily for 7 days, followed by 5 mg / kg KP-LPS via intravenous drip on the 7th day.

[0083] An additional Rmst⁻ / ⁻ mouse control group (Rmst⁻ / ⁻LPS group, Rmst⁻ / ⁻LPS+IOP group) was established, with the same grouping and treatment as wild-type mice, except for the genotype of the mice.

[0084] HE staining and pathological scoring: Mice were sacrificed 12 hours after modeling, and right lung tissue was taken, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5μm), and stained with HE. According to Table 1 (Lung Tissue Pathological Scoring Table), the scores were given from 5 dimensions: "focal alveolar wall thickening, interstitial edema, hyaline membrane formation, inflammatory cell infiltration, and hemorrhage" (0~3 points / item, total score 0~15 points).

[0085] Table 1 Lung Tissue Pathology Scoring Table

[0086] Rating Items none Mild moderate Severe Focal alveolar wall thickening 0 1 2 3 Pulmonary interstitial edema 0 1 2 3 Transparent film formation 0 1 2 3 Inflammatory cell infiltration in alveoli and interstitium 0 1 2 3 Alveolar and capillary hemorrhage 0 1 2 3

[0087] Results: In the LPS group, the alveolar walls of the lung tissue of mice showed diffuse thickening and significant inflammatory cell infiltration, with a total score of 12.5±1.2. The total score in the LPS+IOP group decreased to 5.2±0.8 (P<0.0001), while the total score in the Rmst⁻ / ⁻LPS+IOP group remained at 11.8±1.0 (no significant difference from the Rmst⁻ / ⁻LPS group), demonstrating that IOP can alleviate lung injury in reliance on Rmst. Figure 4 and Figure 11 As shown.

[0088] Detection of pro-inflammatory factors: Bronchoalveolar lavage fluid (BALF) and serum from mice were collected, and the levels of IL-6 and IL-1β were detected using an ELISA kit; RNA was extracted from lung tissue, and the expression levels of IL-6 and IL-1β mRNA were detected by real-time quantitative PCR.

[0089] Results: The IL-6 content in BALF of the LPS group was 3 times that of the CON group (P<0.0001), and the serum IL-6 content was 12 times that of the CON group; the IL-6 and IL-1β content in BALF and serum of the LPS+IOP group decreased by 40%~60% compared with the LPS group (P<0.01), and the mRNA expression level was significantly reduced (P<0.0001), demonstrating that IOP can inhibit the inflammatory response, such as Figure 5 and Figure 6 As shown.

[0090] Transmission electron microscopy: A small piece of left lung tissue (1 mm³) was taken, fixed with 2.5% glutaraldehyde, stained with osmium tetroxide, dehydrated in a gradient manner, embedded in resin, and ultrathinly sectioned (70 nm). The number of autophagosomes was observed by transmission electron microscopy (marked with red arrows).

[0091] Results: The number of autophagosomes in lung tissue was (18.5±2.3) per field of view in the LPS group; it decreased to (6.2±1.5) per field of view in the LPS+IOP group (P<0.01); while the number of autophagosomes in the Rmst⁻ / ⁻LPS+IOP group remained at (17.8±2.1) per field of view, demonstrating that IOP-dependent Rmst-dependent inhibition of autophagy, as Figure 9 and Figure 12 As shown.

[0092] Western Blot detection: Total protein was extracted from lung tissue, quantified, and then subjected to SDS-PAGE gel electrophoresis (60V stacking gel, 80V running gel). The membrane was transferred (300mA, 60min), blocked, and then incubated overnight at 4℃ with LC3 antibody (1:1000). Secondary antibody (1:10000) was incubated at room temperature for 1h, and then detected by a two-color laser imaging system.

[0093] Results: The LC3-II / LC3-I ratio in the LPS group was 2.8 times that in the CON group; in the LPS+IOP group, this ratio decreased to 1.5 times (P<0.01), demonstrating that IOP inhibits the LC3-I→LC3-II conversion. Figure 10 As shown.

[0094] Cytotoxicity screening: MLE-12 cells were seeded in 96-well plates (1×10⁻⁶ cells / well). 4 (10, 50, 100, and 200 μg / mL of high-purity IOP were added to each well, and the cells were cultured for 12 h and 24 h, respectively. Then, CCK-8 reagent (10 μL / well) was added, and the cells were incubated at 37 °C for 2 h. The absorbance at 450 nm was measured, and the survival rate was calculated.

[0095] Results: Cell viability was >95% after treatment with all IOP concentrations (no significant difference compared to the blank control group), demonstrating that 10–200 μg / mL IOP is non-toxic to MLE-12. Figure 7 As shown.

[0096] Verification of cell protection effect: MLE-12 cells were divided into CON group, IOP group (100μg / mL), LPS group (5μg / mL) and IOP+LPS group. After 24h of culture, the survival rate was detected by CCK-8 assay.

[0097] Results: The cell survival rate in the LPS group was 68.7% ± 3.5%; the survival rate in the IOP + LPS group increased to 86.2% ± 4.1% (P < 0.05), demonstrating that IOP can protect LPS-damaged lung epithelial cells. Figure 8 As shown.

[0098] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of high purity Inonotus obliquus polysaccharide, characterized in that, The high-purity inonotus obliquus polysaccharide component plays a preventive and control role by inhibiting autophagy of lung tissue cells and depending on the lncRNA Rmst channel, and can reduce the number of autophagosomes in lung tissue and the conversion level of LC3-I to LC3-II.

2. The use of high-purity Inonotus obliquus polysaccharides according to claim 1, characterized in that, The extraction of the high-purity inonotus obliquus polysaccharide includes the following steps: Step 1. Take the inonotus obliquus extract crude polysaccharide, remove the protein by the Sevage method to obtain a deproteinized crude polysaccharide liquid; Step 2. The deproteinized crude polysaccharide liquid is purified by MD55 dialysis bag, and the inner dialysis liquid is collected; Step 3. The inner dialysis liquid is loaded onto a DEAE-52 ion exchange column chromatography system, and the polysaccharide active component is collected by elution; Step 4. The polysaccharide active component is further separated and purified by a G-200 gel chromatography system, the eluate is collected and freeze-dried to obtain high-purity inonotus obliquus polysaccharide.

3. The use of high-purity Inonotus obliquus polysaccharides according to claim 1, characterized in that, The molecular weight cut-off of the MD55 dialysis bag in step 2 is 8000-14000 Da, and the dialysis time is 48-72 h. The dialysis external liquid is replaced every 8-12 h during the dialysis process.

4. The use of high-purity Inonotus obliquus polysaccharides according to claim 1, characterized in that, The eluent of the DEAE-52 ion exchange column chromatography in step 3 is deionized water and 0.1-0.5 mol / L gradient NaCl solution, and the elution flow rate is 1-2 mL / min.

5. The use of high-purity Inonotus obliquus polysaccharides according to claim 1, characterized in that, The loading amount of the G-200 gel chromatography in step 4 is 2 mL of polysaccharide solution, the eluent is distilled water, and the elution flow rate is 0.5-1 mL / min. Every 3-5 mL is collected as a component.

6. The use of high-purity Inonotus obliquus polysaccharides according to claim 1, characterized in that, The reagent volume ratio of the Sevage method in step 1 is polysaccharide liquid: Sevage reagent (n-butanol: dichloromethane = 1:5) = 4:1, placed on a magnetic stirrer at 3000 rpm for 20 min, transferred to a separatory funnel for 60 min of static extraction, the upper polysaccharide solution was collected, and the operation was repeated until no intermediate protein layer appeared.

7. The use of high-purity Inonotus obliquus polysaccharides according to claim 1, characterized in that, The polysaccharide active component is labeled as IOP-d1; The high-purity inonotus obliquus polysaccharide is detected by the phenol-sulfuric acid method to obtain four specific molecular weight components IOP-g1, IOP-g2, IOP-g3 and IOP-g4, wherein the molecular weight of the four high-purity inonotus obliquus polysaccharides is IOP-g1>IOP-g2>IOP-g3>IOP-g4.

8. The use of high-purity Inonotus obliquus polysaccharides according to claim 6, characterized by the fact that, The molecular weight of the IOP-d1 component is 50-100 kDa, and it has no cytotoxicity to lung epithelial cells MLE-12 in the concentration range of 10-200 μg / mL.

9. The use of high-purity Inonotus obliquus polysaccharides according to claim 6, characterized by the fact that, After the polysaccharide component is treated at a concentration of 100 μg / mL, the survival rate of LPS-damaged lung epithelial cells MLE-12 can be significantly improved.

10. The use of high-purity Inonotus obliquus polysaccharides according to claim 9, characterized in that, The drug is administered by pre-gastric administration, and the administration time is 7 days before bacterial pneumonia modeling. The drug is administered once a day, the administration dose is 1.2 ml / kg, and the polysaccharide component concentration in the drug is 10-200 μg / ml.

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