Application of Pinellia polysaccharide in the preparation of drugs for relieving atopic dermatitis

Abstract

The present invention belongs to the technical field of new medical applications, and specifically discloses the application of Pinellia polysaccharide (PTP) in the preparation of a drug for relieving atopic dermatitis. The PTP of the present invention is a pure Chinese medicine extract, and the extraction method is simple and nontoxic, the yield is high, and the effective component is high; it has a good effect of treating and relieving atopic dermatitis, acts on reducing serum IgE levels, reduces the protein expression levels of TSLP, IFN‑γ, and IL‑4, and suppresses the mRNA expression levels of IFN‑γ, IL‑4, TSLP, TNF‑α, MDC, and TARC; there is no cytotoxic effect, no obvious side effects on the body, and no obvious inflammation and damage are caused in various organs; and the effective dose is small, easy to use, wide in scope, definite in efficacy, safe and reliable, no rogue drug properties, no side effects, and can play the effect of treating both the symptoms and the root causes.

Description

Technical Field

[0001] The invention belongs to the technical field of new medical applications, and particularly relates to application of pinellia polysaccharide in preparing a drug for alleviating atopic dermatitis. Background Art

[0002] Atopic dermatitis (AD) is a chronic inflammatory skin disease that is usually characterized by intense itching and redness of the skin, and is prone to recurrence. This disease not only affects the patient's physical health, but may also have a negative impact on their mental health.

[0003] Existing treatments for AD can be divided into two categories: topical and systemic. Topical treatments include topical hormones, immunosuppressants, and antibiotics. Currently, widely used glucocorticoids are effective in rapidly relieving itching and redness caused by dermatitis, but their long-term use may lead to adverse reactions, including skin pigmentation, thinning of the skin, and suppression of the immune system, thereby increasing the risk of infection and disease. In contrast, immunosuppressants such as calmodulin inhibitors have strong anti-inflammatory activity and do not cause skin atrophy, but require complex dose monitoring and control. Excessive use of antibiotics may lead to drug resistance and disrupt the balance of the flora, thereby reducing immunity. Systemic treatment mainly uses phototherapy, including narrow-band ultraviolet B (NB-UVB) phototherapy and long-wave ultraviolet (UVA-1) phototherapy. NB-UVB exerts anti-inflammatory effects by reducing the production of proinflammatory cytokines, but long-term use may increase the risk of actinic keratosis. UVA-1 produces anti-inflammatory activity through multiple pathways, but may induce acute adverse reactions, such as skin pigmentation and erythema, and even chronic reactions, such as photocarcinogenesis.

[0004] The number of AD patients continues to increase worldwide. Although AD has been widely studied, there is still no drug that can cure atopic dermatitis, and its pathogenesis has not been fully elucidated, which may involve multiple factors such as immune system signal imbalance, genetic factors, skin dysfunction, psychological state, etc. Therefore, the development of AD-related therapeutic drugs and the exploration of their mechanism of action are of great significance for the treatment of AD and improving the quality of life of patients.

[0005] Pinellia ternata is the tuber of the plant Pinellia ternata (Thunb.) Breit. of the Araceae family. As one of the traditional Chinese medicines in my country, it was first recorded in Shennong's Herbal Classic and has thousands of years of clinical practice. It is warm in nature, pungent in taste, and slightly toxic. It has the effects of drying dampness and resolving phlegm, lowering adverse qi and stopping vomiting, and eliminating lumps and dispersing nodules. Pinellia ternata is mainly produced in central and southern my country, such as Sichuan, Hubei, and Anhui. Its chemical composition is complex, including alkaloids, organic acids, volatile oils, proteins, flavonoids, sterols, amino acids, and rich inorganic elements and polysaccharides. Among them, polysaccharides are important active ingredients of Pinellia ternata and one of the material bases for its efficacy.

[0006] Polysaccharides, also known as polysaccharides, are natural high molecular polymers, usually composed of more than 10 monosaccharides connected by glycosidic bonds. Polysaccharides are widely present in animals, plants and microorganisms. According to their sources, polysaccharides can be divided into five categories: animal polysaccharides, plant polysaccharides, algae polysaccharides, bacterial polysaccharides and fungal polysaccharides. The synthesis of polysaccharides from different biological sources is affected by many external factors, resulting in complex and diverse structures, which are reflected in the differences in monosaccharide composition, configuration, molecular weight, degree of branching and chain conformation. At the same time, polysaccharides are also very rich in biological activities, such as antioxidant, antiviral, antitumor, and immunomodulatory activities. With the continuous development of modern medical technology, the research and development of polysaccharides from traditional Chinese medicines with strong activity and high safety has received more and more attention.

[0007] There are relatively few studies on the immunomodulatory effects of Pinellia polysaccharides in China, especially in the treatment of AD. Therefore, it is necessary to further explore the therapeutic effect of Pinellia polysaccharides on AD and its mechanism of action.

[0008] There is no related report in the prior art, and this application will fill this gap. Summary of the invention

[0009] The present invention aims to target the pathogenesis of atopic dermatitis (AD), which is currently unclear and difficult to cure, and to avoid the problem of multiple adverse reactions caused by commonly used glucocorticoid drugs. We aim to conduct in-depth research on the effect and mechanism of action of Pinellia polysaccharide in the treatment of AD, and provide a scientific basis for finding new candidate drugs for the treatment of AD.

[0010] To this end, the present invention provides the use of pinellia polysaccharide in preparing a drug for alleviating atopic dermatitis.

[0011] The pinellia polysaccharide (PTP) is an extract or powder of processed pinellia such as French pinellia and / or raw pinellia;

[0012] The preparation method of the pinellia polysaccharide powder comprises the following steps:

[0013] Wash and dry processed Pinellia ternata such as French Pinellia ternata and / or Raw Pinellia ternata, and then crush them with a grinder, and then pass through a 60-mesh sieve to obtain Pinellia ternata powder; weigh 80g-100g of Pinellia ternata powder, add anhydrous ethanol and reflux for 1h, the ratio of Pinellia ternata powder to anhydrous ethanol is 100g: (650-800)mL, filter to obtain a precipitate, discard the supernatant, repeat three times, and the precipitate obtained after three ethanol refluxes is defatted Pinellia ternata powder; add distilled water to the obtained defatted Pinellia ternata powder and place it in a water bath at 85℃-95℃, reflux for 1h, filter with gauze to obtain the supernatant, discard the precipitate, repeat 3-5 times. The supernatant is combined to obtain a preliminary extracted polysaccharide solution; the preliminary extracted polysaccharide solution is placed in a 4°C refrigerator for 10h-14h, anhydrous ethanol is added while stirring, and after standing at a low temperature of 4°C for 10h-14h, it is centrifuged at 7500rpm for 15min, the precipitate is collected, and the supernatant is discarded to obtain a crude polysaccharide precipitate; distilled water is added to dissolve the precipitate, 6M hydrochloric acid is used to adjust the pH of the solution to 2, and it is allowed to stand at 4°C for 10h-14h to obtain a crude polysaccharide solution; the crude polysaccharide solution is centrifuged at 7500rpm for 10min-15min, the supernatant is collected, the precipitate is discarded, the pH of the solution is adjusted to 7 with 6M NaOH solution, 95% ethanol is added while stirring to make the ethanol concentration reach 60%, it is allowed to stand at 4°C for 10h-14h, and the precipitate is collected by centrifugation to obtain a wet polysaccharide precipitate, and the wet polysaccharide precipitate is dried in a freeze vacuum oven to obtain a crude polysaccharide powder of Pinellia polysaccharide, which is stored in a desiccator at room temperature.

[0014] In this application document, all percentages in ethanol concentration are by volume.

[0015] For specific experimental operations, result recording and conclusion analysis, please see the specific implementation method.

[0016] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0017] The pinellia polysaccharide used in the present invention shows a significant therapeutic effect on atopic dermatitis (AD). The results of the determination of the degree of skin lesions in the mouse ears showed that pinellia polysaccharide can significantly alleviate the inflammatory symptoms such as erythema, edema, roughness and hardening of the mouse ears, and reduce the mouse ear dermatitis score. Further observations by H&E staining and TB staining results showed that pinellia polysaccharide reduced the thickness of the epidermis of the mouse ear skin and inhibited the mast cell infiltration in the skin inflammation area. Through a comprehensive analysis of mouse serum ELISA, IHC staining and RT-qPCR results, the high-dose group of pinellia polysaccharide (BH) can significantly reduce the IgE level of serum, while reducing the protein expression levels of TSLP, IFN-γ and IL-4. In addition, pinellia polysaccharide can also inhibit the mRNA expression levels of IFN-γ, IL-4, TSLP, TNF-α, MDC and TARC in mice. The above series of results show that pinellia polysaccharide effectively relieves inflammation by reducing the expression of Th2 inflammatory factors. Analysis of H&E staining results of mouse heart, liver, spleen, lung and kidney sections showed that Pinellia polysaccharide had no obvious side effects on the body while treating AD, and did not cause obvious inflammation and damage to various organs. Therefore, it is expected to become a safe drug for treating AD.

[0018] Pinellia polysaccharide has shown significant therapeutic effects in the treatment of AD, and its therapeutic effect is concentration-dependent to a certain extent. In the pathogenesis of AD, the immune regulation function is destroyed, leading to the imbalance of Th1 / Th2 in the body. Th2-type cytokines induce immunoglobulin class switching in B lymphocytes, promoting increased IgE synthesis. Pinellia polysaccharide inhibits the abnormal increase of Th2 cytokines, significantly reduces the content of IgE in serum, exerts anti-inflammatory effects and regulates immune balance. In addition, Pinellia polysaccharide has good biosafety and has no obvious side effects on the body. Pinellia polysaccharide exhibits good anti-inflammatory and immunomodulatory activity, and is expected to become a drug for the treatment of AD, providing a theoretical basis for the clinical application of Pinellia polysaccharide as an anti-inflammatory and immune adjuvant in the treatment of AD.

[0019] The applicant will further explain the present invention in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a process flow chart of extracting polysaccharides from Pinellia ternata in Example 2;

[0021] Figure 2 This is the HPLC spectrum of Pinellia polysaccharide after acid hydrolysis and derivatization;

[0022] Figure 3 This is a picture showing the skin inflammation and evaluation results of the left ear of a medium-sized mouse;

[0023] Among them, (A) Photos of the ear skin of each group of mice on the last day of the experiment; (B) Thickness of the ear skin of each group of mice; (C) Inflammation score of the ear skin of each group of mice. Data are expressed as mean ± standard deviation (Mean ± SD, n = 6). Compared with group N, ###p < 0.001, compared with group M, ***p < 0.001.

[0024] Figure 4 This is a graph showing the results of testing the serum IgE level in mice;

[0025] Data are expressed as mean±SD (n=3). Compared with group N, ###p<0.001, compared with group M, ***p<0.001, **p<0.01.

[0026] Figure 5 This is the test result of the skin histological indicators of the left ear of mice;

[0027] Among them, (A) H&E staining of the ear skin of mice in each group (scale bar = 100 μm); (B) TB staining of the ear skin of mice in each group (scale bar = 100 μm); (C) Epidermal thickness of the ear skin of mice in each group (green arrow); (D) Mast cell count of the ear skin of mice in each group (red arrow). Data are expressed as mean ± standard deviation (Mean ± SD, n = 3). Compared with group N, ###p < 0.001, compared with group M, ***p < 0.001, **p < 0.01.

[0028] Figure 6 This is the result of immunohistochemical staining of the skin tissue of the left ear of mice;

[0029] Among them, (A) Representative images of TSLP, IFN-γ, and IL-4 expression in the ear skin of mice in each group by immunohistochemical staining (scale bar = 50 μm); (B) Average optical density analysis; Data are expressed as mean ± standard deviation (Mean ± SD, n = 3). Data are expressed as mean ± standard deviation (Mean ± SD, n = 3). Compared with group N, ###p<0.001, compared with group M, ***p<0.001, **p<0.01.

[0030] Figure 7 Representative images of H&E staining of mouse heart, liver, spleen, lung, and kidney (scale bar = 100 μm).

[0031] Figure 8 This is the qPCR result of mouse ear tissue;

[0032] Data are expressed as mean±SD (n=3). Compared with group N, ###p<0.001, compared with group M, ***p<0.001. DETAILED DESCRIPTION

[0033] Below, the applicant will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1: Preparation method of Pinellia polysaccharide: materials and instruments

[0035] 1. Medicinal Materials

[0036] French Pinellia, provided by Qianbanxia Pharmaceutical Co., Ltd., is used to extract Pinellia Tuber Polysaccharides, referred to as PTP.

[0037] 2. Reagents

[0038] Calcipotriol (MC903) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; dexamethasone cream (DEX) was purchased from Sanjiu Pharmaceutical; anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd.; dimethyl sulfoxide was purchased from Sinopharm Chemical Reagent Co., Ltd.; normal saline was purchased from Wuhan Binhu Shuanghe Pharmaceutical Co., Ltd.; anhydrous ethanol and methanol were purchased from Beijing Chemical Plant; Tris was purchased from Beijing Solebow Technology Co., Ltd.; fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; trypsin and DMEM were purchased from Hyclone, USA; MTT was purchased from Sigma; total RNA extraction kit was purchased from Bao-Riyi Biotechnology Co., Ltd. (Takara 9108); RNA reverse transcription kit was purchased from Bao-Riyi Biotechnology Co., Ltd. (Takara, RR047A); qPCR kit was purchased from Bao-Riyi Biotechnology Co., Ltd. (Takara, RR820A)

[0039] 3. Instruments

[0040] Multifunctional microplate reader (Tecan, USA), three-hole electric constant temperature water bath (Shanghai Yiheng), high pressure sterilizer (Shanghai Boxun), ice maker (Panasonic, Japan), constant temperature oscillator (Qilin Bell), electronic balance (Sartorius), refrigerator (Haier, China), ultrapure water system (Elga), high-speed low-temperature tissue grinder (Wuhan Sewell Biotechnology Co., Ltd.), micro-volume UV spectrophotometer Nanodrop (ThermoFisher, USA), PCR instrument (BIORAD, USA), Step OnePlus Applied Biosystems (ABI, USA).

[0041] Example 2: Preparation method, content determination and composition analysis of Pinellia polysaccharide

[0042] 1. Extraction and determination of Pinellia polysaccharides

[0043] 1.1 Extraction of Pinellia polysaccharides

[0044] Wash and dry the French pinellia, grind it with a grinder, and sieve it with a mesh size of 60 to obtain pinellia powder; weigh 100g of pinellia powder, add 720mL of anhydrous ethanol, heat and reflux for 1h, filter to obtain a precipitate, discard the supernatant, repeat three times, and the obtained precipitate is defatted pinellia powder; add 710mL of distilled water to the obtained defatted pinellia powder and place it in a 90℃ water bath, reflux for 1h, filter with gauze to obtain a supernatant, discard the precipitate, repeat 3 times, combine the supernatants to obtain 1L of supernatant, which is the preliminary extracted polysaccharide solution; The polysaccharide solution was placed in a 4°C refrigerator for 12 hours, and then 600 mL of anhydrous ethanol was added while stirring. After standing at 4°C for 12 hours, the filtrate was centrifuged at 7500 rpm for 15 minutes, and then the precipitate was collected and the supernatant was discarded to obtain a crude polysaccharide precipitate; 300 mL of distilled water was added to dissolve the crude polysaccharide precipitate, and 6M hydrochloric acid was used to adjust the pH to 2, and then the solution was allowed to stand at 4°C for 12 hours to obtain a crude polysaccharide solution; the crude polysaccharide solution was centrifuged at 7500 rpm for 15 minutes, and then the supernatant was collected and the precipitate was discarded. The pH of the supernatant was adjusted to 7 with 6M NaOH solution, and 95% ethanol was added while stirring to make the ethanol concentration reach 60%, and then the solution was allowed to stand at 4°C for 12 hours, and the precipitate was collected by centrifugation to obtain a wet polysaccharide precipitate. The wet polysaccharide precipitate was freeze-dried in a vacuum to obtain a crude polysaccharide powder of Pinellia polysaccharide, which was stored in a dryer at room temperature. The experimental flow chart is as follows Figure 1 The weight of the obtained powder was weighed on a balance, and then the yield of Pinellia polysaccharide was calculated according to the formula:

[0045] Pinellia polysaccharide yield % = (mass of crude polysaccharide powder / mass of Pinellia powder) × 100%

[0046] 1.2 Determination of total content of polysaccharides in Pinellia ternata

[0047] The total sugar content in Pinellia polysaccharide was detected by sulfuric acid-phenol method: (1) Drawing of glucose standard curve: Accurately weigh 2 mg of glucose standard in a 50 mL volumetric flask, quantify to scale, and shake well. Pipette 0.1, 0.2, 0.4, 0.6, 0.8 mL of the above glucose standard solution into test tubes, respectively, make up to 1 mL with deionized water, add 0.25 mL of 6% phenol and 2.5 mL of concentrated sulfuric acid (wherein the sulfuric acid content is 95%-98%, the same below, not repeated), shake well and cool. Take two other test tubes, add 1 mL of deionized water, add equal amounts of concentrated sulfuric acid and phenol, shake well and cool. After leaving at room temperature for 20 minutes, measure the absorbance on a UV spectrophotometer at a wavelength of 482 nm. Draw a standard curve with concentration as the horizontal axis and wavelength as the vertical axis, and fit the standard curve regression equation. (2) Determination of polysaccharide content in samples: Accurately weigh 1 mg of Pinellia polysaccharide extracted by ultrasonication and freeze-dried, dissolve it in 1 mL of deionized water, take 50 μL and make up to 1 mL with deionized water, add 0.25 mL of 6% phenol and 2.5 mL of concentrated sulfuric acid, shake well and cool. After standing at room temperature for 20 minutes, use the corresponding reagent as a blank control, use a UV-visible spectrophotometer to measure the absorbance at 482 nm, and insert it into the glucose standard curve to obtain the sample solution concentration X, and calculate the total sugar content of Pinellia polysaccharide according to the formula.

[0048] Polysaccharide content (%) = (XD) / M×100%

[0049] Where: X is the polysaccharide concentration of the sample solution to be tested (mg / mL), D is the dilution factor of the sample solution, and M is the initial concentration of the test solution (1 mg / mL)

[0050] 1.3 Analysis of monosaccharide composition of Pinellia polysaccharide

[0051] The monosaccharide composition of Pinellia polysaccharides was analyzed by complete acid hydrolysis combined with PMP (1-phenyl-3-methyl-5-pyrazolone) derivatization: (1) Complete acid hydrolysis: Weigh 2 mg of Pinellia into a screw-top test tube, add 2 mL of 2M trifluoroacetic acid (TFA) to a concentration of 1 mg / mL, seal the tube, mix well, and place in a 110°C oven for hydrolysis reaction for 4 hours. Take out the hydrolyzate, cool to room temperature, place in a 70°C water bath to evaporate the TFA solution, add methanol, and evaporate to dryness. Repeat five times to add 2 mL of methanol and remove the excess TFA solution. After the sample is evaporated, add 200 μL of water to dissolve it as the test solution. (2) PMP derivatization: Take 100 μL of the test solution, add 100 μL of 0.6 M sodium hydroxide solution and mix thoroughly, then add 200 μL of 0.5 M PMP-methanol solution and vortex to mix; place in a 70°C oven and add, and derivatize for 30 minutes. After the reaction is completed, take out the sample and cool it to room temperature. Add 0.3M HCl solution to adjust the pH to neutral, and then add chloroform to the reaction solution for five times to extract and remove the excess PMP. (3) HPLC analysis: ZORBAX Eclipse Plus C18 (column parameters: 4.6mm×250mm, 5μm); 30℃. Ammonium acetate (pH6.5)-acetonitrile (83:17) as the mobile phase; the flow rate is 1mL / min.

[0052] Example 3: Verification of the therapeutic effect of the Pinellia polysaccharide powder obtained in step 1.1 of Example 2 on atopic dermatitis

[0053] 1AD model establishment and drug administration method

[0054] This method aims to evaluate the therapeutic effect of Pinellia polysaccharide on AD. The experiment established a mouse model that reproduces the characteristics of human AD by topically applying calcipotriol (MC903) to the inner ear of mice. The treatment of dermatitis in mice with high and low doses of Pinellia polysaccharide was observed to explore the effect of Pinellia polysaccharide on the treatment of AD.

[0055] BALB / c mice (female, weight 18-20g, 5 weeks old) were raised in a pathogen-free environment, with free access to water and food, and a 12h light-dark cycle was strictly implemented to avoid animal interference. After one week of adaptation, the experimental mice were randomly divided into five groups (n=6): normal control group (N), AD model group (M), dexamethasone cream positive drug group (DEX, 2g / kg), low-dose group of Pinellia polysaccharide (BL, 125mg / kg), and high-dose group of Pinellia polysaccharide (BH, 500mg / kg). Except for the normal control group, the other four groups of mice were all treated with 2nmol MC903 (dissolved in anhydrous ethanol) applied to each ear every morning for modeling, and applied continuously for 17 days. The normal control group (N) mice were smeared with an equal amount of anhydrous ethanol on the left and right ears as a control. The DEX positive drug group and Pinellia polysaccharide treatment group (BH, BL) mice were smeared daily from the afternoon of the 7th day, and the administration lasted for 11 days.

[0056] 2. Detection of related indicators

[0057] 2.1 Detection of mouse serum IgE levels

[0058] On the 17th day of the experiment, blood was collected from each group of mice after anesthesia, placed in a venous blood sample collection tube, left at room temperature for 1 hour, centrifuged at 4000×g, 4℃ for 10 minutes, and the supernatant was frozen in a -80℃ refrigerator. The enzyme-linked immunosorbent assay (ELISA) was used to detect the IgE content in the serum of each group of mice. The specific experimental method was performed according to the instructions of the kit. After the incubation was completed, the reaction was terminated and the absorbance at 450nm (OD1) was detected using an enzyme marker. A standard curve was made, the equation was fitted, and the concentration of each sample was calculated according to the OD value to obtain the total IgE content in the serum of each group of mice.

[0059] 2.2 Evaluation of the severity of ear lesions in mice

[0060] The ear thickness and skin lesion degree of mice were recorded before each use of MC903 modeling (recorded and scored every three days, refer to Table 1 for the scoring criteria). On the 16th day of the experiment, the scratching behavior was manually recorded 10 minutes after the administration of MC903 (scratching behavior definition: intentionally scratching the modeling skin with the front or hind paws).

[0061] Table 1. Mouse ear skin dermatitis degree score table

[0062]

[0063] 2.3 H&E staining of mouse ear tissues and major organs

[0064] This study aims to observe the effects of atopic dermatitis on the skin tissue level and to explore the changes that occur in the tissue under atopic dermatitis conditions. By comparing the skin slice models of the model group and the normal group, the potential role of Pinellia polysaccharide in improving skin inflammation was analyzed, and the main organs were collected to test the biosafety of Pinellia polysaccharide.

[0065] On the 17th day of the experiment, the mice were euthanized after anesthesia and blood was taken. The left ear tissue and major organs were collected and fixed in 4% neutral formalin for 24 hours. Subsequently, the mice were dehydrated and waxed using a fully automatic dehydrator. The treated mouse ear tissue was immersed in the wax liquid, the tissue was filled in the mold, and the mold was removed after the wax block solidified to obtain the embedded wax block. The wax block was fixed on the slicer and trimmed into 5μm thick slices. The slices were unfolded in warm water, the flattened wax film was placed on the glass slide, and the slices were baked in the baking machine. The slices were placed in the slice rack in sequence, dewaxed with xylene, dehydrated with gradient ethanol, and finally soaked in distilled water for dewaxing. The nuclei were stained with hematoxylin staining solution, the floating color was rinsed, 1% ammonia water was reversed and then rinsed again, and 1% hydrochloric acid ethanol was differentiated and then the unstained part was rinsed. The slices were immersed in eosin solution and then rinsed again. Dehydrated with different concentrations of ethanol and soaked in xylene twice. After checking the section number, the sections were observed under an optical microscope and the epidermal thickness of the mouse ear skin was measured.

[0066] 2.4 Toluidine blue (TB) staining of mouse ear tissue

[0067] The improvement of mast cell infiltration by Pinellia polysaccharide was evaluated by comparing mast cell counts in the model group and the normal group.

[0068] The methods for fixing, making wax blocks and tissue sections of mouse ear skin tissue are the same as those for embedding and dewaxing of sections in "2.3 H&E staining of mouse ear tissue". Immerse the sections in toluidine blue dye and rinse slightly. Then, differentiate them in glacial acetic acid and stop differentiation after observing until the cell nuclei and intracellular particles are clearly visible. Rinse in running water and blow dry with cold air. Use alcohol for gradient dehydration, then make it transparent in xylene, and finally seal the sections with neutral gum. After checking the numbers, use an optical microscope to observe and count the number of mast cells in the mouse ear skin tissue, and perform statistical analysis.

[0069] 2.5 Immunohistochemical staining of mouse ear tissue (IHC staining)

[0070] Immunohistochemistry was used to detect the expression of IL-4, IFN-γ and TSLP proteins in the left ear tissues of mice in each group. ImageJ software was used to analyze the immunohistochemical staining results and determine the average optical density (AOD).

[0071] The methods for sampling, fixing, making wax blocks and tissue sections of mouse ear skin tissue are the same as those for sampling, embedding and sectioning in "2.3 H&E staining of mouse ear tissue". After the paraffin tissue sections are completed, they are rinsed with phosphate-buffered saline, then immersed in citric acid buffer, boiled and cooled to room temperature. After antigen retrieval, the tissue sections are rinsed 3 times with PBS, incubated with 2% H2O2, rinsed 3 times with PBS, and endogenous peroxidase is removed with 5% BSA. Then, the sections are immersed in the primary antibodies of IFN-γ, IL-4 and TSLP and incubated overnight at 4°C. The incubated sections are taken out the next day, rinsed with PBS after rewarming at room temperature for 45 minutes, and then the diluted secondary antibody is added dropwise, followed by the addition of DAB for color development. After color development, nuclear counterstaining is performed with hematoxylin, the sections are sealed and photographed. Check the sections that have been prepared, affix numbers, and observe the staining using an optical microscope and analyze them.

[0072] 2.6 Real-time quantitative PCR (RT-qPCR)

[0073] By observing the changes in mRNA in mouse tissues, we explored the expression levels of genes that induce atopic dermatitis and the effect of Pinellia polysaccharides in inhibiting inflammatory factors at the genetic level.

[0074] 2.6.1 Total RNA extraction

[0075] (1) Weigh 50-100 mg of mouse ear skin tissue sample and add 1 mL of RNAiso Plus. Use a pre-cooled high-speed cryogenic tissue grinder to thoroughly homogenize it. Transfer the homogenate to a centrifuge tube and let it stand at room temperature for 5 minutes. Centrifuge at 12,000 × g at 4°C for 5 minutes, carefully aspirate the supernatant, and transfer it to a new centrifuge tube. (2) Add chloroform (1 / 5 volume of RNAisoPlus) to the homogenate lysate and mix until the solution is emulsified and milky white; let it stand at room temperature for 5 minutes, and centrifuge at 12,000 × g at 4°C for 15 minutes. The liquid in the centrifuge tube is divided into three layers: the top layer is a colorless supernatant, the middle layer is a white protein, and the bottom layer is a colored organic phase. Aspirate the supernatant and transfer it to another new centrifuge tube. (3) Add 0.5-1 times the volume of RNAiso Plus to the supernatant, invert the centrifuge tube to mix it thoroughly, let it stand at room temperature for 10 minutes, and centrifuge at 12,000 × g at 4°C for 10 minutes. After centrifugation, the white precipitate at the bottom of the test tube is the total RNA. Carefully discard the supernatant, wash the precipitate 2-3 times with an equal amount of 75% ethanol as RNAiso Plus, centrifuge at 7500×g for 5 minutes at 4℃ and carefully discard the supernatant. (4) After drying the precipitate at room temperature, add 50μL of RNase-free water to dissolve the precipitate. The resulting RNA solution is measured by NanoDrop OD 260 With OD 280, and calculate the ratio of the two, screen samples with a ratio between 1.8 and 2.2, and record the RNA concentration for subsequent experiments.

[0076] 2.6.2 Reverse transcription to cDNA

[0077] According to PrimeScript TM RT reagent Kit with gDNAEraser (Perfect Real Time) Kit (Takara, RR047A) was used. Total RNA, gDNA Eraser, and 5×gDNA Eraser Buffer were prepared on ice with Master Mix I and allowed to stand at room temperature for 30 min to remove genomic DNA. Then PrimeScript RT Enzyme Mix I, RT Primer Mix, 5×PrimeScript Buffer 2, and RNase Free dH2O were mixed on ice and prepared with Master MixI to prepare a 20 μL reaction system. Reverse transcription was performed on a PCR instrument with an incubation condition of 37°C for 15 min and an annealing condition of 85°C for 5 s. The reverse transcription product was stored at -80°C for future use.

[0078] 2.6.3qPCR reaction

[0079] According to TB Premix Ex Taq TM II (Tli RNaseH Plus) kit (Takara, RR820A) method for qPCR reaction. First, thaw TB Green Premix Ex Taq II and ROX Reference Dye at room temperature, mix them upside down, add TB Green Premix Ex Taq II, ROX Reference Dye, PCR primers and enzyme-free water according to 20μL reaction system and mix quickly, cover the EP tube cap and centrifuge. The whole process needs to be kept at low temperature and away from light. Perform PCR reaction according to standard cycling conditions. Use StepOne Plus AppliedBiosystems two-step method for amplification reaction. After obtaining the amplification CT value, use 2 -ΔΔCT Method for data processing.

[0080] Table 2. qPCR primer sequence list

[0081]

[0082]

[0083] Experimental results of Examples 1-3

[0084] 1. Extraction and determination of polysaccharides from Pinellia ternata

[0085] 1.1 Yield of Pinellia polysaccharides

[0086] The freeze-dried powder of Pinellia ternata polysaccharide (crude polysaccharide powder) was successfully extracted from Pinellia ternata by water extraction and alcohol precipitation. The weight of the obtained powder was accurately weighed to obtain 14.45 g of freeze-dried Pinellia ternata polysaccharide powder, with a yield of 14.45%.

[0087] 1.2 Total sugar content of Pinellia polysaccharides

[0088] The total sugar content in Pinellia polysaccharide was measured by sulfuric acid phenol method and calculated to be 3.61%.

[0089] 1.3 Analysis of monosaccharide composition of Pinellia polysaccharide

[0090] like Figure 2 As shown, the obtained Pinellia polysaccharide is mainly composed of aminogalactose (GalN), gluconic acid (GlcA), iduronic acid (IdoA), galacturonic acid (GalA) and glucose (Glc), wherein the molar ratio of GalN:GlcA:IdoA:GalA:Glc is 1:3.06:5.29:136.75:2.01.

[0091] 2. Comparison of ear skin lesions in each group of mice

[0092] like Figure 3 As shown in the figure, no obvious inflammatory damage was observed in the ear skin of mice in the normal control group. The ear skin of mice in the AD model group began to become red, swollen, rough, and hard on the 7th day of the experiment. With the increase in the number of MC903 applications, the mice gradually developed epidermal dryness and desquamation, and the ear skin of some mice ulcerated and bled. Compared with the normal group, the swelling degree of the auricle and dermatitis score of mice in the AD model group increased significantly (###p<0.001), that is, the AD model was successfully established. Compared with the AD model group, after 11 days of treatment with Pinellia polysaccharide, the dermatitis score of AD mice was significantly reduced (***p<0.001), and the swelling degree of the auricle was significantly reduced (***p<0.001). The experimental results show that Pinellia polysaccharide can effectively alleviate the symptoms of redness, swelling, roughness, ulceration and bleeding of the ear skin of AD mice due to inflammation.

[0093] 3. Serum IgE expression levels of mice in each group

[0094] IgE is the primary clinical indicator of AD patients, and its upregulation indicates inflammation. The ELISA method was used to detect the serum IgE levels of each group of mice. Figure 4As shown in the figure, compared with the normal control group, the serum IgE level of mice in the AD model group was significantly increased (###p<0.001). Compared with the AD model group, the serum IgE level of mice was decreased after treatment with Pinellia polysaccharide, and the difference was statistically significant (***p<0.001, **p<0.01), and it was dose-dependent. The experimental results showed that Pinellia polysaccharide can effectively reduce serum IgE levels, thereby alleviating IgE-mediated hypersensitivity reactions in AD mice.

[0095] 4. H&E and TB staining results of ear tissues of mice in each group

[0096] Figure 5 The results of H&E staining in the figure showed that the skin structure of mice in the normal control group was regular, the cell morphology was normal, and the epithelial layer was clear and complete. The epidermis (green arrow), dermis and subcutaneous tissue of the ear skin of mice in the AD model group proliferated, and obvious inflammatory cell infiltration was observed. Compared with the normal group, the epidermal thickness of the AD model group increased significantly (###p<0.001). Compared with the AD model group, after treatment with Pinellia polysaccharide, the inflammatory infiltration was significantly reduced, and the epidermal thickness was significantly reduced (***p<0.001). The results of TB staining showed that compared with the normal control group, the number of mast cells (red arrows) in the ear skin of mice in the AD model group increased abnormally, and the infiltration was obvious, and the difference was statistically significant (###p<0.001). Compared with the AD model group, after treatment with Pinellia polysaccharide, the mast cell infiltration was alleviated, and the number of mast cells was significantly reduced (***p<0.001, **p<0.01). The experimental results show that Pinellia polysaccharide can effectively reduce the local inflammatory response of the skin of AD mice and alleviate AD pathological damage.

[0097] 5. IHC staining results of ear tissues of mice in each group

[0098] TSLP is a cytokine secreted by epithelial cells that promotes Th2-type immune responses. IL-4 and IFN-γ are cytokines closely related to atopic dermatitis (AD). IL-4 is usually closely linked to Th2-type immune responses, and its overexpression may trigger allergic symptoms and inflammation, thus playing a key role in the pathogenesis of AD. In contrast, IFN-γ is usually associated with Th1-type immune responses, and its overexpression may cause abnormal activation of the immune system. IHC staining was used to detect the expression of TSLP, IFN-γ, and IL-4 proteins in the ear skin of each group of mice. Figure 6As shown in the figure, compared with the normal control group, the protein expression levels of TSLP, IFN-γ, and IL-4 in the AD model group mice were significantly increased (###p<0.001). Compared with the AD model group, the protein expression levels of TSLP, IFN-γ, and IL-4 in the pinellia polysaccharide treatment group mice were significantly decreased (***p<0.001). The experimental results show that pinellia polysaccharide can inhibit Th1 and Th2 cytokines, thereby exerting anti-inflammatory effects and slowing down the progression of AD.

[0099] 6. H&E staining results of the main organs of mice in each group

[0100] like Figure 7 As shown, the H&E staining results of various organ tissues showed no obvious pathological changes. The experimental results showed that there was no obvious inflammatory response when Pinellia polysaccharide was used to treat atopic dermatitis, and it had good biosafety.

[0101] 7. RT-qPCR analysis of the therapeutic effect of Pinellia polysaccharide on AD at the gene level

[0102] MDC and TARC are chemokines for Th2 cells, and both can promote the occurrence of Th2 immune response. The expression levels of Th2 cytokine IL-4, Th1 cytokine IFN-γ, TNF-α, and three types of mRNA, TSLP, MDC, and TARC, in the ear tissues of each group of mice were detected. Figure 8 As shown in the figure, compared with the normal control group, the expression levels of IL-4, IFN-γ, TNF-α and TSLP, MDC, and TARC mRNA in the ear tissue of the mice in the AD model group were significantly upregulated (###p<0.001); compared with the AD model group, the expression levels of IL-4, IFN-γ, and TSLP mRNA in the Pinellia polysaccharide treatment group were significantly downregulated (***p<0.001), and the mRNA levels of TNF-α, MDC, and TARC in the high-dose Pinellia treatment group were significantly reduced (***p<0.001). The experimental results show that Pinellia polysaccharide can inhibit the gene expression levels of Th1 and Th2 cytokines and TSLP, MDC, and TARC mRNA, regulate Th1 / Th2 balance, and alleviate Th2-induced inflammatory response.

Claims

1. Application of Pinellia polysaccharide in the preparation of drugs for relieving atopic dermatitis, It is characterized in that The preparation method of the pinellia polysaccharide comprises the following steps: Wash and dry the French pinellia, then crush and sieve; weigh the obtained French pinellia powder, add anhydrous ethanol, reflux for a period of time, filter to obtain a precipitate, discard the supernatant, and repeat three times; the obtained precipitate is the defatted pinellia powder; add distilled water to the obtained defatted pinellia powder, place it in a water bath at 85°C-95°C, reflux for a period of time, filter with gauze to obtain a supernatant, discard the precipitate, and repeat 3-5 times; combine the supernatants to obtain a preliminary extracted polysaccharide solution; place the preliminary extracted polysaccharide solution in a refrigerator and let it stand for 10h-14h, add anhydrous ethanol while stirring, let it stand at a low temperature of 4°C for 10h-14h, centrifuge for 15min, collect the precipitate, discard the supernatant, and obtain a crude polysaccharide precipitate; add distilled water to dissolve the precipitate, adjust the pH of the solution to 2, and let it stand at 4°C for 10h-14h; Obtaining a crude polysaccharide solution; The crude polysaccharide solution is centrifuged for 10-15 minutes, the supernatant is collected, the precipitate is discarded, the pH of the solution is adjusted to 7, 95% ethanol is added while stirring to make the ethanol concentration reach 60%, and then it is allowed to stand at 4°C for 10-14 hours, and the precipitate is collected by centrifugation to obtain a wet polysaccharide precipitate. The wet polysaccharide precipitate is freeze-dried in a vacuum to make Pinellia polysaccharide powder, which is stored in a desiccator at room temperature.

2. The use according to claim 1, Features: The pinellia polysaccharide is used for reducing serum IgE level, reducing protein expression levels of TSLP, IFN-γ and IL-4, and inhibiting mRNA expression levels of IFN-γ, IL-4, TSLP, TNF-α, MDC and TARC.

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