Preparation method of gentian polysaccharide and application of gentian polysaccharide in acute lung injury

Abstract

The invention discloses a preparation method of gentian polysaccharide and application of gentian polysaccharide in acute lung injury, and belongs to the technical field of medicines. Experimental research finds that the gentian polysaccharide can obviously improve the hemolysis state and the hyperemia and edema state of lung tissue by mediating a MyD88 signal channel, the tissue structure is relatively complete, local inflammatory cell infiltration is improved, acute lung injury can be effectively prevented or treated, and the application of the gentian polysaccharide to the preparation of the medicine for treating the lung injury is promoted. A new strategy and a clinical research basis are provided for prevention or treatment of the acute lung injury, a scientific basis is further provided for guiding clinical medication for treating the acute lung injury, and the application prospect is good. Therefore, the gentian polysaccharide can be used for preparing the medicine for treating the lung injury.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a preparation method of gentian polysaccharide and an application thereof in acute lung injury. Background Art

[0002] Acute lung injury (ALI) is a clinical syndrome of capillary endothelial and alveolar epithelial damage and increased vascular permeability caused by various non-cardiogenic factors. Clinically, the definition of ALI has been more than 50 years old, and its clinical manifestations are acute respiratory insufficiency accompanied by shortness of breath, hypoxia-refractory cyanosis, decreased lung compliance, and diffuse alveolar infiltration. In particular, the ongoing pandemic of the new coronavirus (Corona Virus Disease 2019, COVID-19) in recent years has brought new and unique challenges to lung diseases. At present, long-term use of clinical drugs for the treatment of ALI will produce multiple side effects. Therefore, it is urgent to develop a new drug source that is safe, non-toxic and has good therapeutic effects.

[0003] Gentiana Gentiana scabra ) was first recorded in Shennong's Herbal Classic. It is a heat-clearing and dampness-drying medicine with the effects of clearing damp-heat and purging liver fire. Gentian contains a variety of chemical components, mainly including polysaccharides, iridoid glycosides, alkaloids and other effective ingredients. Among them, gentiopicroside and gentian ethanol extract have been proven to have a certain therapeutic effect on ALI. Polysaccharides, as a natural active macromolecular substance, have been widely studied because of their various pharmacological activities. And as a natural and easily available source of drugs, polysaccharides generally have the characteristics of low toxicity and side effects. At present, there are no relevant research reports on the protective effect of gentian polysaccharides on ALI. Summary of the invention

[0004] In view of the above problems, the purpose of the present invention is to provide a preparation method of gentian polysaccharide and its application in acute lung injury. The present invention preliminarily explores the lung protection mechanism of gentian polysaccharide by studying the protective effect of gentian polysaccharide on lipopolysaccharide (LPS)-induced ALI in mice. The conduct of this experiment will provide a scientific basis for the in-depth development of gentian resources and clinical application in the future.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0006] The invention discloses application of gentian polysaccharide in preparing medicine for preventing or treating acute lung injury.

[0007] Furthermore, the drug prevents or treats acute lung injury by regulating the MyD88 pathway.

[0008] Furthermore, the gentian polysaccharide in the medicine exists in the form of an aqueous solution.

[0009] Further, according to any of the above applications, it is characterized in that the preparation of the gentian polysaccharide comprises the following steps: Take 10.0 g of gentian powder, use 10 mL of methanol to extract the gentian three times, evaporate the methanol in the medicinal material, use pure water to extract it three times, combine the three water extracts and add an appropriate volume of ethanol to make the final concentration of ethanol 80%. Put it in a 4°C refrigerator for alcohol precipitation overnight. The precipitate after alcohol precipitation and drying in the shade is the crude gentian polysaccharide.

[0010] The present invention also discloses a medicine for preventing or treating acute lung injury, characterized in that the medicine contains gentian polysaccharide active ingredient.

[0011] Furthermore, the pharmaceutical preparation is a pharmaceutically acceptable dosage form.

[0012] Furthermore, the drug includes pharmaceutically acceptable excipients.

[0013] The invention also discloses an anti-inflammatory drug, characterized in that the drug comprises gentian polysaccharide active ingredient.

[0014] The present invention also discloses a medicine for improving oxidative damage in acute lung injury, characterized in that the medicine comprises the active ingredient of gentian polysaccharide.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0016] The new application of gentian polysaccharide in the present invention has not been reported in existing papers and journals. The present invention provides the protective effect of gentian polysaccharide on acute lung injury in mice by mediating the MyD88 signaling pathway, so the present invention can be used as a scientific basis for new drug development and pharmacological activity research. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The levels of IL-1 and TNF-α in the serum of mice in different treatment groups.

[0018] Figure 2 Figure 2 shows the contents of MPO, MDA and SOD in lung tissues of mice in different treatment groups.

[0019] Figure 3 The following are pathological sections of the lung tissues of mice in different treatment groups. A is the lung tissue of mice in the blank control group; B is the lung tissue of mice in the LPS-induced model group; C is the lung tissue of mice in the dexamethasone positive drug control group; D is the lung tissue of mice in the gentian polysaccharide high-dose group; and E is the lung tissue of mice in the gentian polysaccharide low-dose group.

[0020] Figure 4The expression levels of MyD88, IκBα, Bax and Bcl-2 proteins in the lung tissues of mice in different treatment groups. A is the protein band blot of each protein in different treatment groups; B is the expression level of each protein in different treatment groups. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0022] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0023] The invention provides gentian polysaccharide as a new drug treatment method for acute lung injury and its protective mechanism.

[0024] Experimental methods.

[0025] 1. Extraction of crude gentian polysaccharide: Take 10.0 g of gentian powder, use 10 mL of methanol to heat reflux extract 3 times, 3 h each time, evaporate the methanol in the medicinal material, use pure water to heat reflux extract 3 times, the extraction time is 3 h, 2.5 h and 2 h, respectively, combine the three water extracts and add 95% industrial ethanol, so that the final concentration of ethanol is preferably 80%, put it in a 4 ° C refrigerator for alcohol precipitation overnight for 24 h, and the precipitate after alcohol precipitation and shade drying is the crude gentian polysaccharide ( Gentiana scabra polysaccharide, GSP).

[0026] 2. Experimental grouping and model establishment: 40 mice were randomly divided into a blank control group (NC group), a model group (LPS group), a low-dose and high-dose gentian polysaccharide solution group (GSPL, GSPH), and a positive control group (DXMS group), with 8 mice in each group. For 7 consecutive days, mice in the GSPL group and the GSPH group were gavaged with 8 mg·kg -1 ·d -1 and 32 mg kg -1 ·d -1 Gentiana polysaccharide solution; mice in the DXMS group were intragastrically administered with 2 mg·kg -1 ·d -1 dexamethasone solution; the NC group and LPS group were gavaged with the same volume of distilled water. On the 8th day of administration, mice in all groups except the NC group were intraperitoneally injected with LPS (10 mg kg -1 ) to establish an acute lung injury model.

[0027] 3. Detection of TNF-α and IL-1 in serum: Place the mouse blood sample at 4°C for 30 min, then centrifuge it at 3000 r / min for 15 min, take the upper serum and use the kit to detect the TNF-α and IL-1 levels in the sample. The operation is according to the kit instructions.

[0028] 4. Determination of MPO, MDA and SOD contents in lung tissues: The lung tissues of mice in different treatment groups were removed, rinsed with pre-cooled saline and then homogenized. The homogenized tissues were centrifuged at 3000 r / min for 15 min and the supernatant was taken for later use. The MPO, MDA and SOD contents in the lung tissues were determined using a kit according to the instructions of the kit.

[0029] 5. Pathological changes in mouse lung tissue: The lung tissues of mice in different treatment groups were fixed in 4% paraformaldehyde fixative for 24 h, dehydrated with 75%, 85%, 95%, and 100% ethanol for 5 min each time, and transparentized with xylene. After paraffin embedding overnight, tissue sections were sliced ​​with a thickness of 5 μm; the paraffin sections were dewaxed and then rehydrated with 100%, 95%, 85%, and 75% gradient ethanol. After rehydration, they were immediately washed with phosphate buffered saline (PBS), stained with hematoxylin for 10 min, rinsed, and then stained with eosin stain for 5 min, then the stain on the surface of the sections was cleaned, and then dehydrated with 75% ethanol for 2 s, 85% ethanol for 2 s, 95% ethanol for 2 s, and 100% ethanol for 2 s, and then transparentized in xylene for 2 min. Finally, the sections were sealed with neutral gum to obtain H&E stained sections, which were observed and photographed under an optical microscope.

[0030] 6. Western blot was used to detect the expression levels of myeloid differentiation factor 88 (MyD88), inhibitory subunit of nuclear factor-kappaB α (IκBα), B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-2-associated X (Bax) proteins in mouse lung tissue: The mouse lung tissue was removed and dried with filter paper and weighed. RIPA lysis buffer was added at 10 μL / mg tissue, and the tissue was ground into a homogenate with a tissue homogenizer, lysed on ice for 30 min, and further broken up by ultrasound. The supernatant was taken after centrifugation at 4°C and 13,000 rpm; the protein content was determined by the BCA method. The protein sample was mixed with loading buffer and heated to 95-100°C for denaturation for 5 min. Denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis with 10% separation gel and 5% stacking gel, and then transferred to polyvinylidene fluoride (PVDF) membrane; the membrane was blocked with 5% skim milk powder in Tris-buffered saline (TBST) containing 0.1% Tween-20 at 4°C for 1 h, and the membrane was washed with TBST, and then incubated with specific primary antibodies against MyD88, IκBα, Bax, Bcl-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (internal reference protein) at 4°C overnight, and then washed thoroughly with TBST and incubated with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 h, and developed with ECL chemiluminescence reagent, and the signal was exposed and captured. Image J software was used to analyze the gray value of protein bands; the ratio of the gray value of the target protein (MyD88, IκBα, Bax and Bcl-2) band to the gray value of the internal reference protein (GAPDH) band indicated the relative content of the target protein.

[0031] Experimental results.

[0032] The levels of TNF-α and IL-1 in the serum samples of mice in different treatment groups were detected. Compared with the NC group, the levels of TNF-α and IL-1 in the model group were significantly increased (P<0.01), indicating that the mouse lung injury model induced by LPS was successfully established. Compared with the LPS group, the IL-1 levels in the DEXA group, GSPH group and GSPL group were significantly decreased (P<0.01), while the TNF-α level was significantly decreased in the DEXA group (P<0.01), and significantly decreased in the GSPH group and GSPL group (P<0.05), indicating that GSP has a certain effect on improving inflammation in LPS-induced ALI mice.

[0033] The contents of MPO, MDA and SOD in the lung tissues of mice in different treatment groups were measured. Compared with the NC group, the contents of MPO and MDA in the lung tissues of mice in the LPS group were significantly increased (P<0.01), while the content of SOD was significantly decreased (P<0.01). Compared with the LPS group, the content of SOD in the DEXA group, GSPH group and GSPL group was significantly increased (P<0.01); the content of MDA was significantly decreased in the DEXA group (P<0.01), and significantly decreased in the GSPH group and GSPL group (P<0.05); the content of MPO was significantly decreased in the DEXA group and GSPH group (P<0.01), and significantly decreased in the GSPL group (P<0.05). This indicates that GSP has a certain effect on improving oxidative damage in LPS-induced ALI mice.

[0034] Observation of lung tissue sections of mice in different treatment groups revealed that there was no abnormality in the lung tissue of mice in the NC group and the tissue structure was normal; in the LPS group, there was a large amount of inflammatory cell infiltration, obvious signs of inflammation, hemolysis, congestion and edema, and the tissue structure was destroyed; compared with the LPS group, the above symptoms in the lung tissues of the DEXA group, GSPH group and GSPL group were significantly improved, the tissue structure was relatively intact, and the local inflammatory cell infiltration was improved.

[0035] Compared with the NC group, the expression levels of MyD88, IκBα and Bax proteins in the lung tissue of mice in the LPS group were significantly increased (P<0.01), while the expression level of Bcl-2 protein was significantly decreased (P<0.01); compared with the LPS group, the expression levels of MyD88, IκBα and Bax proteins in the DEXA group, GSPH group and GSPL group showed a downward trend, while the expression level of Bcl-2 protein showed an upward trend, and the differences were statistically significant (P<0.01, P<0.05). (For pharmacological experiments of the compounds of the present invention, please refer to Figure 1 to Figure 4 ).

[0036] In summary, gentian polysaccharide can improve LPS-induced ALI in mice by regulating the MyD88 pathway, and can inhibit the abnormal cell apoptosis phenomenon caused by ALI in tissues through the mitochondria-mediated endogenous cell apoptosis pathway, thereby playing a certain lung protection role. Therefore, the new pharmacological activity of gentian polysaccharide of the present invention can be used as a natural product to develop new Chinese medicines, which has broad prospects.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of gentian polysaccharide in the preparation of drugs for preventing or treating acute lung injury.

2. The use according to claim 1, characterized in that: The drug prevents or treats acute lung injury by regulating the MyD88 pathway.

3. The use according to claim 1, characterized in that: The gentian polysaccharide in the medicine exists in the form of aqueous solution.

4. The use according to any one of claims 1 to 3, characterized in that: The preparation of the gentian polysaccharide comprises the following steps: Take 10.0 g of gentian powder, use 10 mL of methanol to extract the gentian three times, evaporate the methanol in the medicinal material, use pure water to extract it three times, combine the three water extracts and add an appropriate volume of ethanol to make the final concentration of ethanol 80%. Put it in a 4°C refrigerator for alcohol precipitation overnight. The precipitate after alcohol precipitation and drying in the shade is the crude gentian polysaccharide.

5. A drug for preventing or treating acute lung injury, characterized in that: The medicine contains gentian polysaccharide as an active ingredient.

6. The drug according to claim 5, characterized in that The pharmaceutical preparation is a pharmaceutically acceptable dosage form.

7. The drug according to claim 5, characterized in that The drug includes pharmaceutically acceptable excipients.

8. An anti-inflammatory drug, characterized in that: The medicine comprises gentian polysaccharide as an active ingredient.

9. A drug for improving oxidative damage in acute lung injury, characterized in that: The medicine comprises gentian polysaccharide as an active ingredient.

Images