Application of isoliquiritigenin in relieving toxicity of pinellia ternate and pinellia ternate detoxification medicine

Abstract

The invention provides application of isoliquiritigenin in relieving toxicity of pinellia ternate and a pinellia ternate detoxification medicine, and belongs to the technical field of biology. The invention proves that the ISL can inhibit the expression increase of TNF-alpha and IL-1beta mRNA caused by pinellia ternate poison needle crystals and PTL, inhibit the increase of ROS in macrophages caused by pinellia ternate poison needle crystals, and weaken inflammatory response caused by pinellia ternate poison needle crystals. According to the present invention, the ISL can inhibit the oxidative stress reaction caused by PTL by inhibiting the combination of PTL and the macrophage membrane, and inhibit the excessive generation of ROS in the macrophage so as to block the MAPK inflammation pathway activation, such that the activation of the JAK-STAT signal pathway is inhibited, the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 is reduced, the inflammatory cascade reaction process is blocked, and the inflammatory reaction degree is significantly weakened;

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to application of isoliquiritigenin in relieving the toxicity of pinellia tuber and a pinellia tuber detoxifying drug. Background Art

[0002] Pinellia ternata (Thunb.) Ten.ex Breitenb. is a perennial herbaceous plant of the genus Pinellia in the Araceae family. It is also known as "Three-Step Jump" and "Field Guard." It grows primarily in shaded, damp, and sheltered locations such as hillsides and grassy areas. Toxicity studies on Pinellia ternata include the following: ① Irritation toxicity: Raw Pinellia ternata is highly irritating to various mucous membranes, including the tongue, throat, eyes, stomach, and intestines. Improper use can easily cause aphonia, conjunctival edema, and even severe throat edema and death from asphyxiation. ② Cardiotoxicity: Raw Pinellia ternata can cause abnormalities in related biochemical parameters and activate the TGF-β signaling pathway, but without causing any structural changes. ③ Hepatotoxicity and renal toxicity: Long-term use of Pinellia ternata can lead to liver and kidney lesions. Studies have shown that alkaloids extracted from Pinellia ternata can cause significant damage to liver tissue 2 hours after administration. Reproductive toxicity: A study using LC / MS and GC / MS metabolomics to evaluate the effects of Pinellia ternata on pregnant rats found no significant effect on placental weight or histological changes. However, it interfered with glycerophospholipid, amino acid, and carbohydrate metabolism in rats, causing disturbances in intermediate metabolism. Pinellia ternata can also affect embryonic development, leading to severe fetal malformations, pregnancy termination, and fetal death.

[0003] Currently, research on the toxicity of Pinellia ternata (Pinellia ternata) generally agrees that calcium oxalate raphides are one of its primary toxic components. Studies have found that the irritant toxicity of calcium oxalate raphides is related to their unique crystal structure. Administration of these raphides to the eyes of rabbits has been found to cause edema and congestion. This is due to their unique crystal structure—extremely slender, pointed at both ends, and with barbs and grooves on their surface—hence the name "Pinellia ternata raphides." In addition to calcium oxalate, Pinellia ternata raphides also contain 6.084% protein. Modern pharmacological studies have revealed that Pinellia ternata protein exhibits a degree of reproductive toxicity. Current research suggests that the primary mechanism of toxicity in raw Pinellia ternata is that after the raphides pierce mucous membranes, such as the oral cavity, the lectin proteins carried by the raphides enter the tissues along with the raphides, inducing macrophages to release large amounts of inflammatory factors and neutrophil chemotactic factors, exacerbating the migration of neutrophils to the inflamed tissues and triggering a series of inflammatory reactions.

[0004] Licorice is the dried rhizome of the licorice plant, which has the effects of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, among which detoxification is also one of the most important effects of licorice. However, the detoxification mechanism of licorice on pinellia is still unclear. Isoliquiritigenin (ISL) is a flavonoid compound with a chalcone structure isolated from the rhizome of licorice. Studies have shown that isoliquiritigenin has a very wide range of biological and pharmacological activities, such as anti-inflammatory, antioxidant, antiviral and antibacterial, antidepressant, and anti-tumor effects, but whether isoliquiritigenin has a detoxification effect on the toxicity of pinellia has not been disclosed. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of isoliquiritigenin in relieving the toxicity of Pinellia ternata and a Pinellia ternata detoxifying drug, which can be used to inhibit inflammation caused by Pinellia ternata nephrite crystals and Pinellia ternata lectin protein.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides application of isoliquiritigenin in relieving the toxicity of pinellia tuber.

[0008] Furthermore, the isoliquiritigenin can be used to inhibit inflammation caused by Pinellia ternata raphide crystals and Pinellia ternata lectin protein.

[0009] Furthermore, the isoliquiritigenin can weaken the inflammatory response caused by Pinellia ternata raphide crystals and Pinellia ternata lectin protein, reduce the secretion of inflammatory factors TNF-α, IL-1β, and IL-6, and reduce the increase of ROS in macrophages caused by Pinellia ternata raphide crystals; inhibit the decrease of oxidative stress indicators SOD and the increase of MDA caused by PTL; inhibit the early apoptosis of macrophages; block the MAPK inflammatory pathway, and inhibit the activation of the JAK-STAT signaling pathway.

[0010] The present invention also provides a pinellia ternata detoxifying drug, which comprises isoliquiritigenin.

[0011] Application of a pinellia ternata detoxifying drug in relieving the toxicity of pinellia ternata.

[0012] The beneficial effects of the present invention compared with the prior art are:

[0013] (1) The present invention extracts Pinellia ternata raphide crystals from raw Pinellia ternata, and the purity of the Pinellia ternata raphide crystals is as high as 80% or more as determined by high performance liquid chromatography. Scanning electron microscopy reveals that stimulation with Pinellia ternata raphide crystals can change the morphological structure of macrophages. PTL stimulation with Pinellia ternata raphide crystals changes the morphological structure of macrophages, increasing the mRNA expression of inflammatory factors TNF-α and IL-1β. PTL increases the mRNA expression of inflammatory factors TNF-α and IL-1β. The above results indicate that Pinellia ternata raphide crystals and PTL have inflammatory functions, can change the structure of macrophages, and cause macrophages to produce an inflammatory response.

[0014] (2) The present invention demonstrates that ISL can inhibit the increased expression of TNF-α and IL-1β mRNA caused by Pinellia ternata raphide crystals and PTL, inhibit the increase of ROS in macrophages caused by Pinellia ternata raphide crystals, and weaken the inflammatory response caused by Pinellia ternata raphide crystals.

[0015] (3) ISL can inhibit the oxidative stress response caused by PTL by inhibiting the binding of PTL to the macrophage membrane, and inhibit the excessive production of ROS in macrophages, resulting in the blocking of the activation of the MAPK inflammatory pathway, thereby inhibiting the activation of the JAK-STAT signaling pathway, reducing the secretion of inflammatory factors TNF-α, IL-1β, and IL-6, blocking the inflammatory cascade reaction process, and significantly reducing the degree of inflammatory response.

[0016] (4) In vivo efficacy experiments found that when Pinellia ternata lanceolate crystals and PTL act together, the inflammatory response will be aggravated; ISL can weaken the inflammatory response caused by the combined action of Pinellia ternata lanceolate crystals and PTL. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 The morphological structure diagram of the Pinellia ternata raphide crystals in the embodiment of the present invention, wherein a is the morphology of the Pinellia ternata raphide crystals under an optical microscope at 20X; b is the structure of the Pinellia ternata raphide crystals under a scanning electron microscope at 3500 times;

[0019] Figure 2 The method for detecting the content of Pinellia ternata needle crystals by high performance liquid chromatography in the embodiment of the present invention is as follows;

[0020] Figure 3 Purification and verification of Pinellia ternata lectin in the examples of the present invention;

[0021] Figure 4 Schematic diagram of the growth of Pinellia ternata raphide colonies after 7 days in the embodiment of the present invention;

[0022] Figure 5 The effect of the Pinellia ternata lectin of the present invention on mouse and rabbit red blood cells, wherein a is mouse blood and b is rabbit blood;

[0023] Figure 6 The scanning electron microscope observation of the effects of Pinellia ternata raphide crystals on macrophages at different times in the embodiment of the present invention;

[0024] Figure 7 The effect of ISL on the activity of RAW 264.7 cells in the examples of the present invention;

[0025] Figure 8 The PCR reaction procedure in the embodiment of the present invention is as follows;

[0026] Figure 9 : The expression results of Pinellia ternata raphide crystals on macrophage-related mRNA genes in the embodiment of the present invention, wherein a is TNF-α mRNA gene; b is IL-1β mRNA gene; c is IL-6 mRNA gene;

[0027] Figure 10 The expression of macrophage-related inflammatory factor genes by Pinellia ternata lectin in the embodiment of the present invention, wherein a is the expression result of TNF-α mRNA gene and b is the expression result of IL-1β mRNA gene;

[0028] Figure 11 The expression of inflammatory factor gene mRNA of macrophages by isoliquiritigenin in the embodiment of the present invention is shown in FIG. 1 , wherein a is the expression result of TNF-α mRNA gene, and b is the expression result of IL-1β mRNA gene;

[0029] Figure 12 The expression of inflammatory factor genes in the present invention, wherein a is the expression result of TNF-α mRNA gene, and b is the expression result of IL-1β mRNA gene;

[0030] Figure 13 The effect of ISL on macrophage ROS in the embodiment of the present invention, wherein a is the change of macrophage ROS detected by flow cytometry, b is the statistical analysis chart of ROS change, and c is the fluorescence value of ISL on cell ROS detected by fluorescence inverted microscope;

[0031] Figure 14 is the effect of ISL incubation time on cellular ROS in the embodiment of the present invention, where a is incubating cells for 3 hours, b is incubating cells for 6 hours, c is incubating cells with ISL for 6 hours, and d is incubating cells with ISL for 6 hours;

[0032] Figure 15 The change of cell MDA is detected by using a microplate reader in the embodiment of the present invention;

[0033] Figure 16 The changes of cell SOD were detected by using a microplate reader in the embodiment of the present invention;

[0034] Figure 17 Flow cytometry was used to detect cell apoptosis in the embodiment of the present invention;

[0035] Figure 18 Figure 2 shows the binding of FITC-labeled PTL to the macrophage cell membrane surface in an embodiment of the present invention, where a and b are the binding of different concentrations of FITC-labeled PTL to the macrophage cell membrane surface detected by flow cytometry and treated with Flow Jo, respectively, and c is the flow cytometry analysis result;

[0036] Figure 19 FITC-labeled PTL binds to the cell membrane surface of macrophages in the embodiment of the present invention, wherein a is the fluorescence intensity of cells incubated with FITC-labeled PTL and macrophages for different times detected by flow cytometry, b is the fluorescence intensity of cells incubated with FITC-labeled PTL and macrophages for different times treated with Flow Jo, and c is the analysis result of flow cytometry detection;

[0037] Figure 20 The laser confocal microscopy was used to detect the binding of FITC-labeled PTL to the macrophage cell membrane surface;

[0038] Figure 21 The flow cytometry method of the present invention is to detect the binding of FITC-labeled PTL to the macrophage cell membrane surface;

[0039] Figure 22 The FITC-labeled PTL and ISL of the present invention are co-incubated and bound to the macrophage cell membrane surface;

[0040] Figure 23 The expression of cellular inflammation-related genes in the examples of the present invention, wherein a is the expression result of STAT3 mRNA gene, and b is the expression result of JAK2 mRNA gene;

[0041] Figure 24 : The effect of ISL on the expression of macrophage-related proteins in the PTL model in Example 1 of the present invention, wherein a is the Western blot band of P-38, P-P38, ERK, P-ERK, JNK, and P-JNK, b is P-P38, c is P-ERK, and d is the protein level of P-ERK;

[0042] Figure 25Figure 3 shows the effect of ISL on the expression of macrophage-related proteins in the PTL model in the examples of the present invention, where a is the Western blot band of STAT3, P-STAT3, JAK2, and P-JAK2, b is the protein level of P-STAT3, c is P-JAK2, and d is the protein level of P-ERK;

[0043] Figure 26 This is a photo of a rabbit conjunctival irritation experiment in an embodiment of the present invention;

[0044] Figure 27 In the embodiment of the present invention, flow cytometry is used to quantitatively analyze cells in mouse blood;

[0045] Figure 28 This is the HE staining result of rabbit conjunctiva in the embodiment of the present invention;

[0046] Figure 29 This is the HE staining result of mouse peritoneal membrane in the examples of the present invention. DETAILED DESCRIPTION

[0047] The following are detailed descriptions of the embodiments of the present invention. The embodiments are intended to explain the present invention and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature within the art or the product specifications are used. Reagents or instruments used without manufacturer's indication are commercially available conventional products.

[0048] Example 1

[0049] Example 1 of the present invention studies the effect of ISL on inflammation caused by Pinellia ternata raphide crystals and PTL, and the specific steps are as follows:

[0050] 1. Extraction of Pinellia ternata needle crystals

[0051] Weigh an appropriate amount of Pinellia powder, put it into a mortar, add an appropriate amount of petroleum ether and grind it into a homogenous slurry. Use three layers of medical gauze to filter the filtrate. Filter the filtrate through a 0.22μm microporous filter membrane to obtain a white powder, which is the Pinellia thorn crystal. Observe it under a microscope. The results are as follows Figure 1 shown. Figure 1 It shows that the Pinellia ternata poison needle crystals have an overall slender structure with relatively sharp ends.

[0052] 2. Determination of Pinellia ternata needle crystal content

[0053] 1. Chromatographic Conditions: Column: C18 5μm 4.6×250mm; Flowability: 0.25% aqueous phosphoric acid; Flow rate: 0.3 mL / min; Detection wavelength: 210 nm; Column temperature: 28°C; Injection volume: 10 μL. 2. Preparation of Standard Solution: Accurately weigh 9.61 mg of oxalic acid reference solution, dissolve in ultrapure water, and dilute to 2 mL. Dilute the standard solution to various concentrations by serial dilution. 3. Preparation of sample solution: Weigh about 3.62 mg of Pinellia ternata needle crystals, add 0.2 mL of HCl (concentrated hydrochloric acid: water = 1:1) solution, mix with 1 mL of pure water, heat in a 70°C water bath, and ultrasonicate for 10 min. Centrifuge at 5000 rpm for 8 min, collect the supernatant, and continue to add 1 mL of 0.1 mol / L HCl solution to the precipitate. Treat the precipitate three times in the same way as above, combine the supernatants, transfer the combined supernatants to a 5 mL volumetric flask, make up to volume with ultrapure water, shake well, and filter with a 0.45 μm microporous filter membrane for later use.

[0054] 4. Methodological investigation: (1) Linear result analysis: According to the chromatographic conditions in 1, the standard solutions of different concentrations were injected in sequence, and the concentration of oxalic acid standard (mg / mL) was used as the horizontal axis and the absorption peak area of ​​the standard was used as the vertical axis to draw the standard curve of oxalic acid standard. The results are as follows: Figure 2 (2) Precision experiment: Select an appropriate amount of standard solution, follow the chromatographic conditions 1, inject the sample 6 times continuously, record the peak area, calculate the RSD, and see the results in Table 1.

[0055] Table 1 Precision test results

[0056]

[0057] (3) Stability test: Prepare the solution again according to 3. Inject the sample at 0, 2, 4, 6, 8, 10, and 12 h according to the chromatographic conditions in 2.2.2.1. Integrate the chromatographic peaks, record the peak areas, and calculate the RSD.

[0058] Table 2 Stability test results

[0059]

[0060] (4) Repeatability test: Weigh 5 portions of Pinellia ternata needle crystals of the same weight, prepare the solution according to 2.2.2.3, inject the sample according to the chromatographic conditions in 2.2.2.1, integrate the chromatographic peaks, record the peak areas, and calculate the RSD.

[0061] Table 3 Repeatability test results

[0062]

[0063] (5) Sample recovery experiment: Weigh 5 portions of Pinellia ternata needle crystals, each about 0.2 g, add 0.029 mg of accurately weighed oxalic acid reference substance, prepare the solution according to 3, inject the sample according to the chromatographic conditions of 1, integrate the chromatographic peaks, record the peak area, and calculate the RSD.

[0064] Table 4 Sample recovery test results

[0065]

[0066] 3. Extraction, purification and verification of PTL

[0067] 1. Extraction of PTL crude extract: Weigh an appropriate amount of fresh Pinellia ternata, peel and mince, add 200 mL of deionized water, and grind to a homogenous slurry; centrifuge the Pinellia ternata homogenate at 4000 rpm for 30 minutes, take the supernatant, then slowly add saturated ammonium sulfate, stirring occasionally, until the ammonium sulfate saturation reaches 45%, stand at 4°C overnight, and then centrifuge at 4000 rpm for 15 minutes, take the precipitate, and obtain the Pinellia ternata lectin protein crude extract; after the protein crude extract is completely dissolved in 0.6 mol / L ammonium sulfate solution, centrifuge at 13000 rpm for 10 minutes, take the supernatant, and obtain the sample to be purified. 2. Purification of PTL crude extract: (1) Fill the pipes of the A1 and B1 system pumps to be used with deionized water. Remove the stopper on the chromatography column and connect the upper end of the pre-packed column to the chromatography system. Then break off the lower end, connect the pre-packed column to the AKTA system, and tighten the lower interface (Note: "liquid-liquid connection" is required when replacing the chromatography column); (2) Take 2 mL of the sample to be purified, remove the bubbles in the syringe, inject it into the sample loop, use 1.0 M ammonium sulfate buffer to equilibrate the pre-packed column, and directly elute the pre-packed column with deionized water after the baseline is stable; collect the peak liquid; use a 7000D dialysis bag to dialyze in ultrapure water for 24 hours at 4°C, and then use a freeze dryer to freeze the sample for 24 hours to obtain the purified Pinellia ternata lectin. The purity of the lectin protein was determined to be 82% using Nanodrop.

[0068] 3. Purity verification of PTL: (1) Preparation of sample protein: Take 20 μL of protein and add 5 μL (5×) protein loading buffer, place in a 100°C metal bath for 5 minutes to denature the protein, and store in a -80°C refrigerator for later use; (2) Grouping: Blank group: unpurified lectin protein; Experimental group: purified lectin protein; (3) Prepare 10× Running buffer and make gel, and then calculate the sample volume based on approximately 30 μg of protein per well. When adding the sample, insert the tip of the gun into the channels to avoid sample overflow, and add 5μL of color pre-stained marker to the leftmost channel; (4) Voltage 80V, 30min, after the sample strip enters the separation gel, voltage 120V, 90min; (5) Decolorization solution preparation: 420mL methanol plus 80mL acetic acid, add ultrapure water to 1L; (6) Staining: Place the gel into the prepared Coomassie Brilliant Blue solution, place on a shaker and shake gently for 20min; (7) Decolorization: Recover the Coomassie Brilliant Blue solution, pour in an appropriate amount of decolorization solution, shake quickly for 30min, repeat this step twice, and finally add excess decolorization solution and let it stand at room temperature overnight (to prevent the gel from drying out); (8) Take pictures and observe, the results are as follows Figure 3 shown. Figure 3 It shows that the crude protein extracted from Pinellia ternata using ammonium sulfate precipitation method can remove a large amount of impurities after hydrophobic chromatography and dialysis. The SDS-PAGE gel electrophoresis results show that compared with the unpurified protein, the purified PTL has only a single band, indicating that the purified protein has reached the electrophoretic purity level.

[0069] IV. Sterility verification of Pinellia ternata raphide: (1) Blank group: blank culture medium; Control group: add 200 μL of untreated tap water; Experimental group 1: add 200 μL of Pinellia ternata raphide that has not been sterilized by UV; Experimental group 2: add 200 μL of Pinellia ternata raphide that has been sterilized by UV for 30 minutes; (2) Weigh 9.25 mg of brain heart infusion culture medium powder and 0.925 mg of agar powder and dissolve them in 250 mL of ultrapure water; (3) Autoclave at 121°C for 15 minutes, wait for the culture medium to cool to 50-60°C and pour it into a plate; (4) After the plate cools and solidifies, add the sample and spread it evenly, and place it in a 37°C environment for incubation; (5) Take photos and observe every day. The results are as follows Figure 4 shown. Figure 4After seven days of observation and photography, it was revealed that no bacterial colonies grew on the culture medium of the UV-sterilized Pinellia raphide group, while a large number of bacterial colonies grew on the tap water control group. A small number of bacterial colonies grew on the culture medium of the unsterilized Pinellia raphide group. These results indicate that the purified Pinellia raphide crystals can be used in subsequent cell experiments after UV irradiation without contaminating the cells. V. Bioactivity detection of PTL: (1) Take fresh blood from mice / rabbits, wash it with physiological saline, centrifuge it at 3000rpm for 5min until the supernatant becomes transparent, and the precipitate obtained is mouse / rabbit red blood cells. Resuspend RBCs with physiological saline to a final concentration of 2% (v / v); (2) Add 25μL of physiological saline to each well of a 96-well reaction plate; (3) Add 25μL of physiological saline to the blank group and 25μL of 3mg / mL Pinellia ternata lectin solution to the experimental group; (4) Add 25μL of 2% mouse / rabbit red blood cells to each well and mix gently with a pipette; (5) After standing at room temperature for 1h, take pictures and observe under a microscope. The results are as follows: Figure 5 shown. Figure 5The results showed that after adding PTL for 1 hour, the red blood cells of mice and rabbits began to agglutinate, indicating that the extracted and purified PTL still has biological activity and can be used for subsequent experiments. VI: Cell culture: (1) Take out the RAW 264.7 cell line frozen in the liquid nitrogen tank, quickly place it in a 37℃ water bath and shake the cryotube to thaw. After spraying with alcohol for disinfection, place it in a biosafety cabinet, use a pipette to transfer the cell suspension in the cryotube to the prepared 15mL centrifuge tube, place the 15mL centrifuge tube in a centrifuge, centrifuge at 1000rpm at room temperature for 5 minutes; after disinfection, transfer the 15mL centrifuge tube smoothly to the biosafety cabinet, discard the supernatant, add 2mL complete culture medium to resuspend the cells; transfer the mixed cell suspension to a preheated 10cm cell culture dish, shake it evenly, mark the cell name, date and other information on the culture dish, and place it in a cell culture incubator (37℃, 5% CO2) for culture. (2) Cell passaging: ① Observe the cell status under a microscope. When the density of adherent cells reaches more than 80%, it indicates that the cells can be passaged. ② Spray the cells to be passaged with 75% alcohol and place them in a biosafety cabinet. ③ Discard the original culture medium and add preheated PBS to rinse thoroughly three times. ④ Add 3 mL of prepared complete culture medium and use a pipette to gently blow off the cells on the cell dish. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm at room temperature for 5 minutes. ⑤ Discard the supernatant and resuspend the cells in 1-2 mL of complete culture medium. According to the required passage ratio, take the corresponding cell suspension to the cell culture dish, add an appropriate amount of complete culture medium and shake it gently to evenly mix. Place the culture dish in the incubator for culture. (3) Cell cryopreservation: ① According to steps ①-③ of cell passage, collect cells into 15 mL centrifuge tubes and centrifuge at 1000 rpm for 5 min at room temperature; ② During centrifugation, prepare cell cryopreservation solution (RAW 264.7 cell cryopreservation solution is 8% DMSO + 92% fetal bovine serum), and after centrifugation, discard the supernatant; ③ Add 1 ml of cell cryopreservation solution, resuspend and mix the cells, and mark the cell type and cryopreservation date on the cell cryopreservation tube; ④ Place the cryopreservation tube in a gradient cooling box and quickly store it in a -80°C refrigerator. After 24 h, transfer it from the -80°C refrigerator to a liquid nitrogen tank for long-term storage.

[0070] (4) Cell counting and plating: ① Collect cells into 15 mL centrifuge tubes according to the cell passage procedure; ② Centrifuge at 1000 rpm for 5 min at room temperature and discard the supernatant; ③ Add complete culture medium to resuspend the cells and dilute them to the appropriate density. Use a pipette to gently blow and mix the cells; ④ Quickly aspirate 10 μL of cell suspension and inject it into one side of the cell counting plate to disperse the cells between the glass slides to form a single cell layer; ⑤ Insert the side injected with cells into the fully automatic cell counter, which will display the density of the cell suspension. Subsequently, calculate the volume of cells to be aspirated according to the experimental requirements for plating, add an appropriate amount of complete culture medium and shake to mix, and place in a cell culture incubator for culture.

[0071] VII. Scanning electron microscopy observation of the effect of Pinellia ternata raphide on macrophages: (1) RAW 264.7 cells in good condition were seeded into 6 cm dishes, with each dish containing approximately 1×10 cells. 6 (2) After the cells were observed to adhere to the wall, the cells were divided into 4 groups: blank group: 3 mL PBS was added; the other three groups were added with 3 mL complete culture medium containing 40 μg / mL Pinellia ternata needle crystals; (3) The cells were placed at 37°C and 5% CO2 and incubated for 1 h, 6 h, and 12 h respectively; (4) The culture medium was discarded, and 3 mL PBS buffer was added for washing 3 times, and then fixed with 2.5% glutaraldehyde and placed at 4°C overnight; (5) The cells were dehydrated step by step with 30%, 50%, 70%, 90%, and 100% ethanol by mass fraction, and then gold-sprayed. The morphological changes of the macrophages were then observed using a scanning electron microscope. The results are as follows. Figure 6 shown.

[0072] Figure 6 The results showed that after 1 hour of adding Pinellia raphide crystals, a small number of them began to bind to macrophages, while the majority of macrophages remained structurally intact with no noticeable morphological changes. After 3 hours of addition, the majority of raphide crystals began to bind to cells and pierce the macrophage membranes, entering the cells. Some even penetrated the macrophages, causing swelling and morphological changes. After 6 and 12 hours of addition, the majority of raphide crystals had bound to macrophages, with multiple raphide crystals piercing a single cell. Most macrophages were deformed and their cell membranes were severely damaged. These results indicate that Pinellia raphide crystals can alter macrophage morphology and damage the cell membrane surface. Prolonged incubation significantly affected the structural changes in macrophages. The fact that Pinellia raphide crystals can induce significant changes in macrophage structure within 12 hours further confirms their acute toxicity.

[0073] 8. CCK-8 assay for the effect of ISL on cell viability: (1) ISL was irradiated with UV light and filtered through a 0.45 μm filter membrane for later use; (2) RAW264.7 macrophages were seeded into 96-well plates at a density of 50,000 per well and incubated at 37°C for 24 hours to allow the cells to adhere to the wall; (3) The cells were divided into 8 groups: blank group: only 100 μL PBS was added; experimental group: each group was added with 100 μL of 5 μM, 10 μM, 20 μM, 40 μM, and 10 μM PBS, respectively. M, 60 μM, 80 μM, and 100 μM complete culture medium of M SLS; 3 replicates were set up for each group; (4) The complete culture medium containing the drug was added to the 96-well plate by replacing the liquid, and the cells were incubated for 3 h, 6 h, 12 h, and 24 h, respectively, and the supernatant was discarded; (5) 100 μL of complete culture medium containing 10% CCK-8 was added to each well, and the cells were placed in a 37°C incubator for incubation for 0.5-4 h. The absorbance at 450 nm was measured using an enzyme-labeled instrument. The results are shown in the figure. Figure 7 shown.

[0074] Figure 7 Compared with the blank control, ISL concentrations up to 20 μM had no significant effect on macrophage survival within 3 hours. After 24 hours, when the ISL concentration exceeded 20 μM, macrophage survival began to decline, and cell survival decreased with increasing concentration. This suggests that isoliquiritigenin concentrations up to 20 μM had no significant effect on macrophage activity within 3 hours. Therefore, in subsequent experiments, 10 and 20 μM were selected as the low and high concentration groups.

[0075] IX. Real-time fluorescence quantitative PCR detection of cell-related inflammatory gene expression:

[0076] 1. Effect of Pinellia ternata raphide on the expression of macrophage-related inflammatory genes: (1) Macrophage treatment and grouping: ① Pinellia ternata raphide suspension was irradiated with UV light and then used. RAW264.7 macrophages were plated at 2 million per dish into 6 cm dishes and incubated at 37°C for 24 hours to allow the cells to adhere to the wall. ② The cells were divided into three groups: the blank group was added with 3 mL of PBS solution; experimental group 1: 3 mL of complete culture medium containing 5 μg / mL Pinellia ternata raphide suspension was added; experimental group 2: 3 mL of complete culture medium containing 60 μg / mL Pinellia ternata raphide suspension was added and incubated at 37°C, 5% CO2 for 3 hours.

[0077] (2) RNA extraction: RNA was extracted according to the AxyPrep total RNA miniprep kit, and the centrifugation method was used to obtain RNA. The RNA concentration was determined using Nanodrop. (3) RNA reverse transcription to cDNA: RNA of the same concentration was added to a 0.2 μL centrifuge tube. Different volumes of RNase-free ddH2O and 4 μL 4×gDNAwiperMix were added, vortexed to mix, and heated at 42°C for 2 minutes. 4 μL 5×HiScript III qRT SuperMix was added to the above solution, gently mixed with a pipette, and heated at 37°C for 15 minutes and 85°C for 5 seconds to obtain cDNA. The cDNA was stored at -20°C until use. (4) Real-time quantitative fluorescence PCR detection: The cDNA obtained by reverse transcription was diluted 3 times and operated according to the instructions of the Takara SYBR-Green PCR Master Mix kit. A 10μL qRT-PCR reaction system was used. The reaction system was as follows: TB Green Premix Ex TaqⅡ (Tli RNaseH Plis) (2X) 5μL, PCR Forward Primer (10μM) 0.4μL, PCR Reverse Primer (10μM) 0.4μL, Template cDNA 1μL, ROX Reference Dye 0.2μL, ddH2O 3μL. According to the above system, after adding the reagents to the PCR plate, centrifuge quickly and place it in the PCR amplifier. Set the conditions as shown in the table. Figure 8 Perform PCR reaction. After the reaction is completed, check the amplification curve and melting curve to determine the amplification efficiency and specificity. The results are as follows Figure 9 Data Analysis: Based on the Ct value, calculate the average relative content (2) - ΔΔCt (ΔCt experimental group = Ct experimental group target gene + Ct experimental group internal reference gene, ΔCt control group = Ct control group target gene + Ct control group internal reference gene, ΔΔCt = ΔCt experimental group - ΔCt control group). The Ct value represents the number of amplification cycles required for the fluorescence intensity to reach the threshold. Primer Design: Search for relevant gene sequences and commission Qingke Biotechnology Company for synthesis. Sequences are shown in the sequence listing.

[0078] Figure 9 The results showed that when the cells were incubated with Pinellia ternata raphide crystals for 3 h, compared with the blank group, the 60 μg / mL Pinellia ternata raphide crystals group could significantly upregulate the expression of TNF-α mRNA (****p<0.0001), while the 5 μg / mL Pinellia ternata raphide crystals group had no significant effect on TNF-α mRNA ( Figure 9 a); 5μg / mL poison needle crystal group can slightly upregulate the expression of IL-1βmRNA (*p<0.05), while 60μg / mL poison needle crystal group can significantly upregulate the expression of IL-1βmRNA (****p<0.0001) ( Figure 9 b); Both the 5μg / mL poison needle crystal group and the 60μg / mL poison needle crystal group were able to significantly upregulate the expression of IL-6 mRNA (***p<0.001, ****p<0.0001) ( Figure 9 c) The above results indicate that Pinellia ternata raphide crystals can promote the upregulation of mRNA expression of related inflammatory factors, thereby promoting the secretion of pro-inflammatory factors TNF-α, IL-1β, and IL-6 by macrophages.

[0079] 2. Effect of PTL on the expression of macrophage-related inflammatory genes: (1) PTL was sterilized by ultraviolet irradiation and then filtered through a 0.45μm filter membrane. RAW 264.7 macrophages were plated into 6cm dishes at 2 million / dish and incubated at 37℃ for 24 hours to allow the cells to adhere to the wall; (2) The cells were divided into 4 groups, one of which was added with only 3mL PBS buffer, and the other three groups were added with 3mL complete culture medium containing 25μg / mL, 50μg / mL, and 100μg / mL PTL respectively; (3) The cells were incubated at 37℃ and 5% CO2 for 3h. The mRNA expression level was determined according to step 1 in step 9. The results are as follows: Figure 10 shown.

[0080] Figure 10 After incubating cells with PTL for 3 hours, the addition of different concentrations of PTL significantly upregulated TNF-α and IL-1β mRNA expression compared to the blank control group (**p<0.01, ***p<0.001, ****p<0.0001). Furthermore, TNF-α and IL-1β mRNA expression continued to rise with increasing PTL concentration. These results indicate that PTL can dose-dependently upregulate the mRNA expression of related inflammatory factors, thereby promoting the secretion of pro-inflammatory factors TNF-α and IL-1β by macrophages.

[0081] 3. Effect of ISL on the expression of related inflammatory genes in the inflammatory model induced by Pinellia ternata raphide: (1) Pinellia ternata raphide suspension was irradiated with UV light and then used; (2) RAW 264.7 macrophages were plated at 2 million / dish into 6 cm dishes and incubated at 37°C for 24 hours to allow the cells to adhere to the wall; (3) The cells were divided into 4 groups: blank group: 3 mL PBS buffer was added; modeling group: 3 mL complete medium containing 40 μg / mL Pinellia ternata raphide was added; drug group 1: 3 mL complete medium containing 40 μg / mL Pinellia ternata raphide and 10 μM ISL was added; drug group 2: 3 mL complete medium containing 40 μg / mL Pinellia ternata raphide and 20 μM ISL was added; (4) After adding the drug, the cells were incubated at 37°C and 5% CO2 for 3 hours. The mRNA expression level was determined according to step 2.2.9.1. The results are as follows: Figure 11 shown.

[0082] Figure 11The results showed that after 3 hours of co-incubation of cells with ISL and P. ternata raphide crystals, TNF-α and IL-1β mRNA levels were significantly upregulated in the model group treated with only P. ternata raphide crystals compared to the blank group (****p<0.0001). Compared to the model group, the addition of 10μM and 20μM ISL downregulated TNF-α mRNA expression (*p<0.05, ***p<0.001), with IL-1β mRNA downregulation being particularly significant (****p<0.0001). Furthermore, the downregulation of TNF-α and IL-1β mRNA expression became more pronounced with increasing ISL concentration. These results suggest that ISL can downregulate TNF-α and IL-1β mRNA expression in a dose-dependent manner. ISL inhibits the secretion of pro-inflammatory cytokines TNF-α and IL-1β by macrophages by suppressing the increase in mRNA expression of inflammatory factors induced by P. ternata raphide crystals.

[0083] 4. Effect of ISL on the expression of related inflammatory genes in the PTL-induced inflammatory model: (1) PTL was sterilized by passing through a 0.45μm filter membrane after ultraviolet irradiation; (2) RAW 264.7 macrophages were plated into 6 cm dishes at 2 million / dish and incubated at 37°C for 24 hours to allow the cells to adhere to the wall; (3) The cells were divided into 4 groups: blank group: 3 mL of PBS buffer was added; model group: 3 mL of complete culture medium containing 40μg / mL PTL was added; drug group 1: 3 mL of complete culture medium containing 50μg / mL PTL and 10μM ISL was added; drug group 2: 3 mL of complete culture medium containing 50μg / mL PTL and 20μM ISL was added; (4) After adding the drug, the cells were incubated at 37°C and 5% CO2 for 3 hours. The mRNA expression level was determined according to step 9.1. The results are as follows: Figure 12 shown.

[0084] Figure 12 The results showed that when cells were co-incubated with ISL and PTL for 3 hours, the model group with only PTL added significantly upregulated TNF-α and IL-1β mRNA compared with the blank group (****p<0.0001). Compared with the model group, the addition of 10μM and 20μM ISL downregulated the expression of TNF-α and IL-1β mRNA (***p<0.001, ****p<0.0001). The decrease in TNF-α and IL-1β mRNA expression became more significant with increasing ISL concentration. This indicates that ISL can downregulate TNF-α and IL-1β mRNA expression in a dose-dependent manner and inhibit the increase in mRNA expression of inflammatory factors caused by PTL, thereby inhibiting the secretion of pro-inflammatory factors TNF-α and IL-1β by macrophages.

[0085] 10. Effect of Co-incubation of Pinellia ternata Raphium Crystals and ISL on ROS in Macrophages

[0086] 1. Flow cytometry was used to detect the effect of Pinellia ternata raphe crystals and ISL co-incubation on ROS in macrophages at 488 / 525 nm. The results are as follows: Figure 13 shown.

[0087] Figure 13 Flow cytometry results showed that compared with the blank group, the ROS content of macrophages increased significantly after adding Pinellia ternata raphide crystals. Compared with the raphide crystal group, the addition of 20 μM ISL could inhibit the increase of ROS content ( Figure 14 a and b). The same experimental results can be obtained by fluorescence inverted microscopy. Compared with the blank group, the ROS fluorescence of macrophages was significantly enhanced after the addition of Pinellia ternata raphide crystals. Compared with the raphide crystal group, the addition of 10 and 20 μM ISL can weaken the ROS fluorescence ( Figure 14 c) The above experimental results show that Pinellia ternata raphide crystals can promote the massive production of ROS in macrophages, stimulating the oxidative stress response of macrophages; and the addition of ISL can reduce the increase of intracellular ROS caused by Pinellia ternata raphide crystals, thereby inhibiting the oxidative stress response of macrophages.

[0088] 2. The effect of co-incubation of Pinellia ternata raphide and ISL on ROS in macrophages was detected by microplate reader: (1) RAW 264.7 cells were seeded in 6 cm dishes at 2 million / dish and cultured overnight in a cell culture incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall; (2) According to the experimental design, the cells were divided into 3 groups: blank group: 3 mL PBS buffer was added; modeling group: 3 mL complete culture medium containing 40 μg / mL Pinellia ternata raphide was added; drug group: 3 mL complete culture medium containing 40 μg / mL Pinellia ternata raphide and 20 μM ISL was added; (3) The cells were incubated at 37°C and 5% CO2 for 3 h, 6 h, 8 h, and 12 h, respectively. Discard the culture medium and rinse three times with PBS; (4) Add 3 mL of complete culture medium containing 10 μM DCFH-DA fluorescent probe, incubate at 37°C in the dark for 30 min, discard the culture medium, and rinse three times with PBS; (5) Add 3 mL of PBS and gently blow down the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 500 μL of PBS to resuspend the cells, use a pipette to take 100 μL and add it to a black 96-well plate, and use a fluorescence microplate reader to measure the fluorescence value at 488 / 525 nm. The results are as follows: Figure 14 shown. Figure 14The results showed that the addition of Pinellia ternata raphide crystals for 3, 6, 8, and 12 hours of stimulation increased the fluorescence value of ROS in macrophages (****p<0.0001). However, the addition of 20μM ISL for 3, 6, 8, and 12 hours significantly reduced the relative fluorescence intensity (**p<0.01, ***p<0.001, ****p<0.0001). This indicates that Pinellia ternata raphide crystals can stimulate macrophages for a long time (12 hours), maintaining cellular ROS levels at a high level, causing an imbalance between oxidation and antioxidant effects and leading to oxidative stress. The addition of 20μM ISL can inhibit the continued increase in ROS, reduce intracellular ROS levels, and suppress the oxidative stress response.

[0089] 11. Determination of MDA, an indicator of oxidative stress

[0090] (1) Divide the cells into 4 groups, group them as in step 9.4, discard the supernatant, and wash them 3 times with pre-cooled PBS. Take 300 μL PBS and gently blow off the cells. Place the cell suspension on ice and homogenize it with a homogenizer for 5-10 minutes. Centrifuge at 12000g for 10 minutes and take the supernatant; (2) Determine the protein concentration of the supernatant using a BCA protein concentration assay kit to facilitate the subsequent calculation of the MDA content in the cells; (3) Prepare TBA storage solution: Take 25 mg of TBA and add 6.76 mL of TBA preparation solution to dissolve it, and you can get a TBA storage solution with a concentration of 0.37%. Both TBA preparation solution and TBA storage solution are difficult to dissolve, and can be heated to 70°C to promote dissolution (TBA storage solution needs to be stored at room temperature in the dark); (4) Prepare MDA detection working solution: Take a clean 15 mL centrifuge tube, add 5.25 mL of TBA diluent, 1.75 mL of TBA storage solution and 105 μL of antioxidant and mix well. The MDA working solution is difficult to dissolve and can be heated to 70°C or sonicated to promote dissolution (MDA working solution must be prepared and used immediately). (5) Preparation of MDA standard: Dilute the standard (1 mM) with ultrapure water to 1, 2, 5, 10, 20, and 50 μM for subsequent preparation of the standard curve. (6) Add 0.1 mL of PBS solution to each 1.5 mL centrifuge tube as a blank control, 0.1 mL of the above-mentioned standard solution of different concentrations for preparation of the standard curve, 0.1 mL of sample, and finally 0.2 mL of MDA working solution. (7) After mixing, heat in a metal bath at 100°C for 15 minutes. To prevent boiling and splashing of the liquid during heating, seal the centrifuge tube with sealing film and pierce a small hole with a needle. (8) Cool in a water bath to room temperature and centrifuge at 1000 g for 10 minutes. Take 200 μL of supernatant and add it to a 96-well plate. Use a microplate reader to measure the absorbance at 532 nm. (9) Calculation of MDA content: For cell samples, after calculating the MDA content in the sample, the MDA content in the initial sample can be expressed by the protein content per unit weight, such as mol / mg protein. The result is as follows: Figure 15 shown. Figure 15 The results showed that compared with the blank group, the model group increased the MDA level in macrophages (**p<0.01). Compared with the model group, the addition of different concentrations of ISL reduced the intracellular MDA level. As the ISL concentration increased, the intracellular MDA content decreased. 20μM ISL was able to restore the MDA level of the cells to normal levels (*p<0.05, ***p<0.001). This indicates that the addition of ISL can inhibit lipid oxidation caused by PTL.

[0091] 12. Determination of SOD, an indicator of oxidative stress: (1) Preparation of cell samples: Divide cells into 4 groups, discard the supernatant, and wash 3 times with pre-cooled PBS. Take 300μL PBS to blow off the cells, place the cell suspension on ice, and homogenize with a homogenizer for 5-10 minutes. Centrifuge at 12000g for 10 minutes and take the supernatant as the test sample; (2) Determine the protein concentration using the BCA protein concentration assay kit; (3) Preparation of NBT / enzyme working solution: According to the requirements of each reaction, 158μL SOD detection buffer, 1μL NBT and 1μL enzyme solution are required, and they are mixed to make 160μL NBT / enzyme working solution. The prepared NBT / enzyme working solution should be stored at 4℃ or in an ice bath, and should be prepared and used as soon as possible. (4) Preparation of reaction starting working solution: Dissolve the reaction starting solution (40×) in the kit and mix it evenly. Dilute it by adding 39μL SOD detection buffer to every 1μL reaction starting solution (40×). Mix well to obtain the reaction starting working solution. (5) Refer to Table 5 and use a 96-well plate to set up sample wells and various blank control wells. Add the sample to be tested and other solutions in sequence according to the table. Mix well after adding the reaction starting working solution. Note: The reaction can start after adding the reaction starting working solution. The operation can be carried out at low temperature or with a gun to reduce the error caused by the difference in the time of adding the reaction starting working solution between the wells.

[0092] Table 5 Sample determination reference table

[0093]

[0094] (6) Incubate at 37°C for 30 minutes; (7) Measure the absorbance at 560 nm. The results are as follows: Figure 17 (8) Calculation of total SOD activity in the sample: Inhibition percentage = [(A 空白对照1 -A 空白对照2 )-(A 样品 -A 空白对照3 )] / (A 空白对照1 -A 空白对照2) × 100%, SOD activity units in the test sample = SOD activity units in the detection system = inhibition rate / (1-inhibition percentage) units. SOD activity units can be converted to U / mg protein based on the sample protein concentration and protein dilution factor.

[0095] Figure 16 The results showed that compared with the blank group, the SOD content in the model group was significantly reduced (**p<0.01). Compared with the model group, the addition of 20μM ISL increased the SOD content (**p<0.01). This shows that PTL can generate a large number of free radicals in cells, exceeding the scavenging capacity of the superoxide system, inhibiting the antioxidant system and reducing the SOD content. The addition of ISL can reduce the generation of free radicals ROS, increase the SOD content, and activate the antioxidant capacity in cells.

[0096] 13. Effect of ISL on Macrophage Apoptosis

[0097] (1) Collect 1×10 6 The cells were resuspended in 3 mL of pre-cooled PBS, transferred to a 15 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, washed three times, and the supernatant was discarded; (2) 500 μL of Poptosis Positive Control Solution was added to resuspend the cells, incubated on ice for 30 min, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, PBS was added to resuspend the cells, and the supernatant was discarded; (3) 5× Binding Buffer was diluted with ultrapure water to 1× working solution, and an appropriate amount of pre-cooled 1× Binding Buffer was added to resuspend the cells, and 1×10 6Untreated live cells were mixed. 1× ​​Binding Buffer was added to 1.5 mL and the mixture was divided into three tubes, one of which was a blank control tube and two were single-stained tubes. (4) 5 μL Annexin V-FITC and 10 μL PI were added to the single-stained tubes respectively and incubated at room temperature in the dark for 5 minutes. (5) On the flow cytometer, the voltage of FSC, SSC and fluorescence channels was adjusted using the blank tube, and the compensation of the fluorescence channel was adjusted using the single-stained tube under this voltage condition. (6) Preparation of cell samples: The cells were divided into 4 groups, and the grouping was the same as in 2.2.9.4. 3 mL of pre-cooled PBS was added to gently blow off the cells in the culture dish and transfer them to a 15 mL centrifuge tube. Centrifuged at 1000 rpm for 5 minutes, washed with PBS 3 times, and the supernatant was discarded. Resuspend the cells in 500 μL 1× Binding Buffer; (7) Add 5 μL Annexin V-FITC and 10 μL PI to each tube; (8) Gently vortex to mix and incubate in the dark for 5 minutes; (9) On a flow cytometer, detect Annexin V-FITC (Ex = 488 nm; Em = 530 nm) through the FITC detection channel and detect PI through the PE detection channel (Ex = 561 nm; Em = 585 nm). The results are as follows: Figure 7 shown.

[0098] Figure 17 Compared with the blank group, the total number of apoptotic cells in the model group increased by 17%, of which the number of early apoptotic cells increased by 10.41% and the number of late apoptotic or necrotic cells increased by 6.59%. Compared with the model group, the total apoptotic rate decreased by 8.15% and 9.41% after adding 10μM ISL and 20μM ISL, respectively, of which the number of early apoptotic cells decreased by 5.97% and 6.78%, respectively. This shows that the addition of PTL incubation mainly affects the early stage of cell apoptosis, while the addition of ISL incubation mainly inhibits the early stage of macrophage apoptosis.

[0099] 14. Detection of the binding of PTL to the macrophage membrane surface: (1) FITC-labeled PTL: Slowly add 1 mg / mL FITC to 2 mg / mL PTL solution at a ratio of PTL to FITC = 1 mg: 50 μg, stir and mix, and react at 4°C in the dark for 8 hours; after the reaction is completed, add 5M NH4CI to a final concentration of 50 mM, and terminate the reaction at 4°C in the dark for 2 hours; dialyze the labeled PTL solution in PBS buffer at 4°C to remove FITC that is not bound to PTL; store the labeled PTL solution at 4°C for future use. (2) Flow cytometry detection of the binding of PTL to the macrophage membrane surface: ① RAW 264.7 cells were plated at 2×10 6The cells were seeded at a density of 100 μg / mL in a 6 cm culture dish, and the experiment was performed after the cells attached to the wall; ② Experiment 1 was divided into 9 groups, of which the blank control was divided into three groups: 3 mL of PBS buffer was added, an equal volume of FITC was added, and 3 mL of unlabeled PTL (final concentration of 50 μg / mL) was added; 3 mL of FITC-labeled BSA (final concentration of 50 μg / mL) was added as a negative control; 3 mL of complete culture medium containing 6.25, 12.5, 25, and 50 μg / mL of FITC-labeled PTL was added to the experimental groups, respectively, and after shaking to mix, the cells were placed in a cell culture incubator (37°C, 5% CO2) in the dark and incubated for 3 h; ③ Experiment 2 was divided into 5 groups, 3 mL of FITC-labeled BSA was added, and the blank control was divided into three groups: PBS buffer was used as a blank control, and 3 mL of complete culture medium containing 50 μg / mL FITC-labeled PTL was added and incubated for 0.5 h, 1 h, 3 h, and 6 h, respectively; ④ The culture medium was discarded, 3 mL of PBS buffer was added, and the cells were gently blown off with a pipette and transferred to a 15 mL centrifuge tube. The cells were centrifuged at 1000 rpm for 5 min, and the washing was repeated 3 times. The cells were resuspended in 300 μL PBS and detected by flow cytometry; ⑤ 10,000 cells were collected from each sample group for detection and analysis, and the FITC green fluorescence intensity was detected. The results are shown in the figure. Figure 18 、 19 shown.

[0100] Figure 18 First, PTL was labeled with FITC. Flow cytometry results showed that, compared with the blank control group, the FITC-only group, the unlabeled PTL group, and the FITC-labeled BSA group, stimulation of macrophages with FITC-labeled PTL (6.25, 12.5, 25, 50, and 100 μg / mL) for 3 hours significantly enhanced cell fluorescence in a dose-dependent manner. This indicates that PTL can bind to macrophages, and the binding rate increases significantly with increasing PTL dosage. Figure 19 Flow cytometry was used to examine the effects of PTL (50 μg / mL) on macrophages at different administration times (0.5 h, 1 h, 3 h, and 6 h). The experimental results showed that PTL bound more tightly to proteins on the cell membrane as the administration time increased. These experimental results all indicate that PTL can bind to certain proteins on the macrophage membrane, and that the binding between PTL and macrophages increases with increasing time and dosage.

[0101] (3) Laser confocal microscopy was used to detect the binding of PTL to the macrophage cell membrane: ① RAW 264.7 cells were plated at 1×10 5① The cells were seeded at a density of 100 μg / mL in a glass-bottomed culture dish, and the experiment was carried out after the cells attached to the wall. The cells were divided into 5 groups, and 1 mL of PBS buffer was added as a blank control. 1 mL of complete culture medium containing 50 μg / mL FITC-labeled PTL was added and incubated for 0.5 h, 1 h, 3 h, and 6 h, respectively; ② The culture medium was discarded, and 1 mL of pre-cooled PBS buffer was added to rinse 3 times, 5 min each time; ③ 1 mL of 4% paraformaldehyde was added to fix for 20 min. 1 mL of pre-cooled PBS buffer was added to rinse 3 times, 5 min each time; ④ 1 mL of complete culture medium containing 10 μg / mL H33258 was added and incubated at room temperature for 20 min to stain the cell nucleus. The culture medium was discarded, and 1 mL of pre-cooled PBS buffer was added to rinse 3 times, 5 min each time; ⑤ 1 mL of PBS was added (to prevent the sample from drying out too much), and the state of macrophages was observed and photographed using a laser confocal microscope. The results are shown as follows: Figure 20 shown. Figure 20 Compared with the PBS group, the longer the PTL incubation time, the stronger the green fluorescence of the macrophage cell membrane (green fluorescence represents PTL, and blue fluorescence represents the macrophage nucleus). The results show that with the extension of time, PTL binds more tightly to the cell membrane and gradually enters the cytoplasm. This is consistent with the results of flow cytometry.

[0102] 15. Inhibitory effect of ISL on the binding of PTL to the macrophage membrane surface

[0103] (1) Conventional flow cytometry was used to detect the inhibitory effect of ISL on the binding of PTL to the macrophage cell membrane surface. 10,000 cells were collected from each group of samples for detection and analysis. The FITC green fluorescence intensity was detected. The results are shown in Figure 2. Figure 21 shown.

[0104] Figure 21 Compared with the model group treated with only PTL, the addition of different concentrations of ISL weakened the fluorescence of the cells, and as the ISL concentration increased, the cell fluorescence value decreased (****p<0.0001). This indicates that ISL can inhibit the binding of PTL to related proteins on the macrophage cell membrane, thereby inhibiting the related inflammatory pathways of the cells.

[0105] (2) Conventional laser confocal microscopy was used to detect the inhibitory effect of ISL on the binding of PTL to the macrophage cell membrane surface. The results were as follows: Figure 22 shown.

[0106] Figure 22Laser confocal microscopy was used to observe the fluorescence on the surface of the macrophage cell membrane when cells were co-incubated with different concentrations of ISL and PTL. The confocal microscopy results were consistent with the flow cytometry results. The experimental results showed that compared with the blank group, the FITC fluorescence on the cell membrane surface of the model group was stronger. Compared with the model group with only PTL added, the addition of different concentrations of ISL can weaken the FITC fluorescence on the cell membrane surface. As the ISL concentration increases, the FITC fluorescence on the cell membrane surface becomes weaker. This shows that ISL can inhibit the binding of PTL to related proteins on the macrophage cell membrane.

[0107] 16. Real-time fluorescence quantitative PCR detection of the effect of ISL on the expression of related pathway genes in macrophages

[0108] (1) PTL were sterilized by UV irradiation and then filtered through a 0.45 μm filter. (2) RAW 264.7 macrophages were plated at 2 million cells / dish into 6 cm dishes and incubated at 37°C for 24 hours to allow the cells to adhere. (3) The cells were divided into 4 groups, as described in 2.2.9.4.

[0109] (4) Incubate at 37°C and 5% CO2 for 3 h; (5) Determine the mRNA expression of P38, ERK, JNK, STAT3, and JAK2 according to the above steps. The results are as follows. Figure 23 shown. Figure 23 The results showed that compared with the blank group, the expression of STAT3 and JAK2 mRNA in cells was significantly increased after incubation with PTL (***p<0.001), and the expression of STAT3 and JAK2 mRNA in cells was significantly decreased after incubation with ISL (**p<0.01, ***p<0.001) ( Figure 23 a and b). The above results indicate that ISL has a significant regulatory effect on the expression of inflammatory genes related to the JAK-STAT signaling pathway.

[0110] 17. Detection of the expression of cellular inflammation-related proteins by immunoblotting

[0111] 1. Cell Culture and Grouping: Plate RAW 264.7 macrophages at 2 million cells / dish into 6 cm dishes and incubate at 37°C for 24 hours to allow the cells to adhere. Group the cells as in step 9, step 4.

[0112] 2. Cell protein extraction: Add 200-300 μL of RIPA lysis buffer containing 10% PMSF and 10% protease inhibitors to each culture dish, place on ice and lyse for 15-20 minutes. During the lysis process, shake the lysis buffer in the culture dish from time to time to ensure full contact with the cells. Then use a cell scraper to gently scrape the cells and transfer them to a 1.5 mL centrifuge tube; centrifuge at 4°C, 12000 rpm for 10-20 minutes, remove the centrifuge tube, and aspirate the supernatant into a new 1.5 mL centrifuge tube.

[0113] 3. Determination of cell protein concentration by BCA method: Perform the experiment according to the instructions of the BCA protein concentration determination kit (Biyuntian), and calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0114] 4. Detection of related proteins by immunoblotting: (1) Electrophoresis is the same as step 3 (1) to (7) in step 3; (2) Conventional methods are used for transfer, blocking, incubation with primary antibody, incubation with secondary antibody, and development with ECL kit. The results are as follows: Figure 24 、 25 shown.

[0115] Figure 24 、 25 The results showed that compared with the blank group, in the model group, after 3 hours of PTL stimulation of macrophages, the expression levels of phosphorylated proteins P-P38, P-ERK, P-JNK, P-STAT3, and P-JAK2 in the cells increased (*p<0.05, **p<0.01, ****p<0.0001). This indicates that PTL has the effect of activating the inflammatory signaling pathways MAPK and JAK-STAT. After co-incubation with ISL, the levels of phosphorylated proteins P-P38, P-ERK, P-JNK, P-STAT3, and P-JAK2 in macrophages were significantly decreased (#p<0.05, *p<0.05, **p<0.01, ***p<0.001), indicating that ISL has the effect of inhibiting the activation of inflammatory signaling pathways MAPK and JAK-STAT.

[0116] 18. Effects of ISL on the conjunctiva of rabbits: (1) Animal preparation: Twelve standard rabbits were purchased from Shanghai Slake Laboratory Animal Co., Ltd. and housed at the Hangzhou Normal University Animal Center. All animal experiments were approved by the Hangzhou Normal University Laboratory Animal Ethics Committee, approval number: HSD-20240118-01. (2) The day before the experiment, the rabbits' eyes were checked for normality. The 12 rabbits were randomly divided into four groups and compared using the same-body left-right comparison method. ① Instill 100μg of Pinellia ternata lanceolate into the left eye and an equal amount of normal saline into the right eye; ② Instill 100μg of Pinellia ternata lanceolate + 250μg of PTL into the left eye and an equal amount of normal saline into the right eye; ③ Instill 100μg of Pinellia ternata lanceolate + 250μg of PTL + 100μΜ ISL into the left eye and an equal amount of normal saline into the right eye; ④ Instill 100μg of Pinellia ternata lanceolate + 250μg of PTL + 200μΜ ISL into the left eye and an equal amount of normal saline into the right eye. (3) Fix the rabbit in a rabbit cage, open the upper and lower eyelids, drop the drug solution into the rabbit's eyes, gently close the eyelids for 10 seconds to allow the drug to fully contact the eyes, wait for 2 minutes, rinse with normal saline until there is no foreign matter in the eyes, and observe the rabbit's eye condition after 1.5-2 hours. Take pictures to record the irritation of the rabbit's eyes. After taking pictures, sacrifice the animals and perform pathological examinations. Use normal saline as a blank control. The results are as follows Figure 26 shown.

[0117] Figure 26 Compared with the blank control group, both the nebula crystal group and the nebula crystal plus PTL group caused conjunctival edema and increased ocular secretions in rabbits. Furthermore, compared with the nebula crystal alone group, the nebula crystal plus PTL group showed increased conjunctival edema and increased ocular secretions. Compared with the nebula crystal plus PTL group, the addition of ISL reduced conjunctival edema and ocular secretions in rabbits. This suggests that both nebula crystal and PTL have proinflammatory effects and can cause conjunctival inflammation in rabbits, while ISL can attenuate the inflammation caused by the synergistic effect of nebula crystal and PTL.

[0118] 19. Effects of ISL on peritoneal inflammation in mice: (1) Animal preparation: Five SPF-grade BALB / C female mice, approximately 5 weeks old, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and housed at the Hangzhou Normal University Animal Center. All animal experiments were approved by the Hangzhou Normal University Laboratory Animal Ethics Committee, approval number: HSD-20240118-01. (2) Five mice were randomly divided into five groups: ① 400 μL normal saline (ip); ② 200 μg Pinellia raphide crystals (ip); ③ 200 μg Pinellia raphide crystals + 500 μg PTL (ip); ④ 200 μg Pinellia raphide crystals + 500 μg PTL + 10 mg / kg ISL (ip); ⑤ 200 μg Pinellia raphide crystals + 500 μg PTL + 20 mg / kg ISL (ip).

[0119] (3) Intraperitoneal injection of drugs, 3 hours later, blood was collected from the eye sockets and placed in 1.5 mL anticoagulant tubes. Blood was analyzed using a blood cell analyzer. Mice were killed by cervical dislocation, and peritoneal tissue was taken for pathological examination. The results were as follows: Figure 27 , as shown in Table 6.

[0120] Table 6 Routine blood test results of mice

[0121]

[0122] Figure 27 Table 6 shows that compared with the blank group, the neutrophil count in the nephelium spp. group and the nephelium spp. plus PTL synergistic group increased to 92.7%, while the lymphocyte count decreased to 6.1%. Compared with the nephelium spp. plus PTL synergistic group, 20 mg / kg ISL was able to inhibit the increase in neutrophil count and the decrease in lymphocyte count to a certain extent. This indicates that under the stimulation of nephelium spp. and PTL, mice developed an acute bacterial infection. Both nephelium spp. and PTL are inflammatory in mice, causing severe inflammatory reactions. ISL was able to inhibit the occurrence of inflammation to a certain extent and alleviate the inflammatory response in mice. This is consistent with the experimental results of the rabbit conjunctival experiment.

[0123] 20. Pathological Examination of Rabbit Conjunctival Tissue and Mouse Peritoneal Membrane Tissue

[0124] Paraffin sections of rabbit conjunctival tissue and mouse peritoneal membrane tissue were prepared using conventional methods, and HE staining of tissue sections was performed using conventional methods. Images were collected and analyzed using a fully automatic section scanning imaging system. The results are shown in the figure. Figure 28 、 29 shown. Figure 28HE staining of rabbit conjunctival tissue revealed that the cells in the blank group's conjunctival tissue sections were tightly packed and morphologically intact, with large, darkly stained nuclei, clear borders, and minimal red intercellular matrix. Sections in model groups 1 and 2 showed increased tissue structure, looser cell arrangement, and significantly loosened and edematous intercellular matrix. Numerous proliferating fibroblasts with diverse morphologies, mostly spindle-shaped, had abundant, light-red cytoplasm and were infiltrated with numerous inflammatory cells. Nuclear fragmentation and nuclear condensation occurred to varying degrees, and red intercellular matrix increased. In the presence of ISL, sections in treatment groups 1 and 2 showed increased numbers of tightly packed, morphologically intact cells, large, darkly stained nuclei, clear borders, and reduced inflammatory cell infiltration. The loosened and edematous intercellular matrix was significantly reduced, and the number of proliferating fibroblasts decreased. These results suggest that ISL has a potent anti-inflammatory effect, effectively inhibiting inflammation caused by Pinellia ternata raphe and PTL, and that the anti-inflammatory effect gradually increased with increasing ISL concentration.

[0125] Figure 29 HE staining of mouse peritoneal tissue showed that model group 1 showed no significant vascular dilation and congestion, and had fewer interstitial inflammatory cells. Compared with model group 1, model group 2 showed significant vascular dilation and congestion, more interstitial inflammatory cells, and more pronounced inflammation. The tissue structure of drug group 1 showed fewer interstitial inflammatory cells, significant vascular congestion, and white blood cell aggregation, indicating that drug group 1 was in an early stage of inflammation. Drug group 2 had more interstitial inflammatory cells and less vascular dilation and congestion, indicating that drug group 2 was in the early and late stages of inflammation, and the inflammation was milder than that of drug group 1. These results indicate that high concentrations of ISL have a good anti-inflammatory effect in mice and can improve the inflammation caused by Pinellia ternata raphe and PTL.

[0126] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of isoliquiritigenin in relieving the toxicity of Pinellia ternata.

2. The use of isoliquiritigenin in relieving the toxicity of Pinellia tuber according to claim 1, characterized in that: The isoliquiritigenin can be used to inhibit inflammation caused by Pinellia ternata raphide crystals and Pinellia ternata lectin protein.

3. The use of isoliquiritigenin in relieving the toxicity of Pinellia tuber according to claim 2, characterized in that: The isoliquiritigenin can weaken the inflammatory response caused by Pinellia ternata raphide crystals and Pinellia ternata lectin protein, reduce the secretion of inflammatory factors TNF-α, IL-1β, and IL-6, and reduce the increase of ROS in macrophages caused by Pinellia ternata raphide crystals; inhibit the decrease of oxidative stress indicators SOD and the increase of MDA caused by PTL; inhibit the early apoptosis of macrophages; block the MAPK inflammatory pathway, and inhibit the activation of the JAK-STAT signaling pathway.

4. A Pinellia ternata detoxification drug, characterized in that The pinellia ternata detoxifying drug includes the isoliquiritigenin according to any one of claims 1 to 3.

5. Use of the Pinellia ternata detoxifying drug according to claim 4 in relieving the toxicity of Pinellia ternata.

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