Application of anisodamine hydrobromide

Abstract

The invention relates to the technical field of medicines, in particular to application of anisodamine hydrobromide. The invention provides new application of anisodamine hydrobromide, and researches show that anisodamine hydrobromide can improve rat vital sign changes caused by CLP, improve lung tissue morphology and regulate related target proteins, so that the anisodamine hydrobromide has a good treatment effect on sepsis and / or septic shock.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the use of anisodamine hydrobromide. BACKGROUND

[0002] Sepsis refers to a life-threatening organ dysfunction caused by a dysregulated host response to infection, which is an important clinical problem faced by emergency and critical care disciplines. Sepsis shock, also known as septic shock, is a severe condition caused by sepsis, mainly manifested as insufficient perfusion of patient tissues and a state of persistent hypotension after volume testing. Refractory hypotension and vascular paralysis caused by sepsis shock are the main causes of death in patients with sepsis. The main measures for clinical treatment of sepsis shock include immune regulation, organ function support, fluid resuscitation, and anti-infection. Through clinical practice, even after the above treatment of sepsis shock, the body circulation is in a complex state, and microcirculation disorder still occurs, which interferes with the local cell oxygen supply and causes organ dysfunction. Therefore, the key measure for clinical treatment of sepsis shock should be to improve microcirculation. However, since the mechanism of sepsis shock has not been fully understood, there is a lack of mechanism-based treatment in clinical practice.

[0003] Anisodamine hydrobromide is a drug prepared by extracting natural alkaloids from the roots of Solanaceae plants. As early as 1965, anisodamine hydrobromide was used in the clinical treatment of septic shock in China, and until the production of the drug was limited, the clinical application was limited. In 2016, China began to increase the production of anisodamine hydrobromide, and the clinical application also increased. Tang Hong et al. showed through animal experiments that anisodamine hydrobromide has mechanisms such as promoting fibrinolysis, antagonizing blood coagulation, anti-lipid peroxidation, anti-cholinergic receptor, stabilizing cytoplasmic membrane, and anti-inflammatory, and is used for the treatment of sepsis shock with good effect. The relevant guidelines point out that when anisodamine hydrobromide is used for drug treatment of shock, the dosage should be determined according to the patient's condition, and the dosage should be truly individualized. After drug administration, the patient's condition is improved, and the drug administration interval is gradually prolonged until the drug is stopped. The effect is seen after intravenous injection for 1-2 minutes, and the drug can be quickly excreted through the kidney, and the half-life of the drug is 40 minutes, and the drug will not accumulate in the body for a long time.

[0004] However, the effect and mechanism of anisodamine hydrobromide in the treatment of sepsis shock have not been reported. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to provide the use of anisodamine hydrobromide.

[0006] In the present application, the use of anisodamine hydrobromide in the preparation of a drug for regulating proteins in lung tissue is provided.

[0007] The proteins in the lung tissue include: lung tissue plasma membrane microvesicle related protein, lung tissue energy metabolism related protein, lung tissue matrix metalloproteinase, lung tissue adherent junction protein, lung tissue tight junction protein, lung tissue basement membrane protein and / or lung tissue microvascular endothelial cell junction protein.

[0008] In the present application, the lung tissue plasma membrane microvesicle related protein includes Caveolin-1 and / or Src;

[0009] The lung tissue energy metabolism related protein includes ATP5A, ATP5B and / or ATP5D;

[0010] The lung tissue matrix metalloproteinase includes MMP-2 and / or MMP-9;

[0011] The lung tissue adherent junction protein includes VE-cadherin and / or alpha-catenin;

[0012] The lung tissue tight junction protein includes JAM1 and / or Claudin-5;

[0013] The lung tissue basement membrane protein includes Collagen IV and / or Laminin.

[0014] In the present application, the regulation of the proteins in the lung tissue includes:

[0015] Maintaining the level of ATP5A, inhibiting the level of MMP-9 and / or MMP-2, promoting the expression of VE-cadherin, reversing the down-regulation of JAM-1, maintaining the level of Collagen IV and / or Laminin, improving the degradation of Occludin, Claudin-5 and / or VE-Cadherin.

[0016] The present application also provides the use of anisodamine hydrobromide in the preparation of a drug for protecting the lung tissue of a patient with sepsis and / or septic shock.

[0017] In the present application, the protection of the lung tissue of the patient with sepsis includes: inhibiting the increase of the proportion of lung tissue MPO, CD68 and CD18 positive cell area, improving the morphological arrangement disorder of the terminal bronchial epithelial cells, improving the morphological changes of lung microvessels, improving the permeability of lung microvessels, clearing the leukocyte adhesion of lung microvessels and / or inhibiting the amount of Evans blue exudation.

[0018] The present application also provides the use of anisodamine hydrobromide in the preparation of a drug for improving the vital signs of a patient with sepsis and / or septic shock.

[0019] In the present application, the vital signs include: blood gas index, arterial pressure, heart rate and / or body temperature.

[0020] In some embodiments, the blood gas parameters include: arterial oxygen partial pressure, oxygen saturation, and / or blood pH.

[0021] In this invention, scopolamine hydrobromide is used in the preparation of a drug that improves the survival rate of patients with sepsis and / or septic shock.

[0022] Preferably, the dosage of scopolamine hydrobromide in this invention is 0.6 mg / kg to 2.4 mg / kg.

[0023] Preferably, the administration time of scopolamine hydrobromide in this invention is 6 hours after the onset of the disease.

[0024] The present invention also provides a treatment for sepsis and / or septic shock, comprising administration of anisodamine hydrobromide.

[0025] The treatment is administered to humans or other mammals. The mammals are primates, canines, felines, or rodents.

[0026] This invention provides a new use for scopolamine hydrobromide. Studies have shown that scopolamine hydrobromide improves the changes in vital signs in rats induced by CLP, improves lung tissue morphology, and regulates related target proteins, thereby playing a good therapeutic role in sepsis and / or septic shock. Attached Figure Description

[0027] Figure 1 Changes in rat blood flow at different time points within 24 hours of CLP modeling;

[0028] Figure 2 Effects of different doses of anisodamine hydrobromide on the survival rate of rats within 7 days of CLP modeling;

[0029] Figure 3 Effects of different doses of anisodamine hydrobromide on heart rate in rats within 72 h of CLP modeling;

[0030] Figure 4 Effects of different doses of anisodamine hydrobromide on anal temperature in rats within 72 h after CLP modeling;

[0031] Figure 5 Effects of different doses of anisodamine hydrobromide on mean arterial pressure in rats within 72 h of CLP modeling;

[0032] Figure 6Effects of anisodamine hydrobromide on blood gas analysis changes in CLP rats over 7 days, where A, arterial blood oxygen partial pressure; B, arterial blood carbon dioxide partial pressure; C, arterial blood oxygen saturation; D, arterial blood pH; E, arterial blood lactate content; Control, normal control group; CLP, CLP model group; CLP+L, CLP modeling + low-dose anisodamine group; CLP+M, CLP modeling + medium-dose anisodamine group; CLP+H, CLP modeling + high-dose anisodamine group; *p<0.05 vs Control; #p<0.05 vs CLP; **p<0.01 vs Control; ##p<0.01 vs CLP; ***p<0.001 vs Control; ###p<0.001 vs CLP, N=6;

[0033] Figure 7 Effects of scopolamine hydrobromide on changes in Evans blue exudate in the lungs of CLP rats over 7 days, where: A, images of Evans blue exudate in the lungs of rats in each group; B, statistical results of Evans blue exudate in the lung tissues of rats in each group; #p<0.05 vsControl; **p<0.01 vs CLP; ##p<0.01 vs Control, N=6;

[0034] Figure 8 Effect of scopolamine hydrobromide on the wet-to-dry weight ratio of lung tissue in CLP rats after 7 days;

[0035] Figure 9 The effect of anisodamine hydrobromide on leukocyte adhesion in pulmonary microvessels of CLP rats, including: A, leukocyte adhesion in pulmonary microvessels of control group rats; B, leukocyte adhesion in pulmonary microvessels of CLP group rats; C, leukocyte adhesion in pulmonary microvessels of CLP modeling group + high-dose anisodamine hydrobromide administration group rats; D, statistical graph of intravascular leukocyte adhesion in each group; E, statistical graph of extravascular leukocyte adhesion in each group; F, statistical graph of intravascular rolling leukocyte count in each group; red arrows indicate leukocytes adhered to pulmonary microvessels; N=6;

[0036] Figure 10 Effects of anisodamine on pulmonary microvascular exudation in CLP rats, including: A, pulmonary microvascular leukocyte exudation in control group rats; B, pulmonary microvascular leukocyte exudation in CLP group rats; C, pulmonary microvascular leukocyte exudation in CLP modeling + high-dose anisodamine administration group rats; Iv, mean fluorescence intensity within microvessels; N=6;

[0037] Figure 11 HE staining morphology of lung microvessels and surrounding tissues in rats of each group;

[0038] Figure 12HE staining morphology of terminal bronchial epithelial cells and surrounding tissues in rats of each group.

[0039] Figure 13 Immunohistochemical staining images of MPO, CD68, and CD18 in lung tissues of rats in each group;

[0040] Figure 14 Immunofluorescence staining images of Caveolin-1, a protein associated with microvesicles of the lung membrane in rats from each group;

[0041] Figure 15 Immunofluorescence staining of Occludin, a tight junction protein in microvascular endothelial cells of rat lung tissue in each group;

[0042] Figure 16 Immunofluorescence staining of Claudin-5 tight junction protein in microvascular endothelial cells of rat lung tissue in each group;

[0043] Figure 17 Immunofluorescence staining of VE-Cadherin, a microvascular adhesion junction protein in lung tissue of rats in each group;

[0044] Figure 18 Immunofluorescence staining images of Collagen IV basement membrane protein in lung tissue of rats in each group;

[0045] Figure 19 Immunofluorescence staining images of laminin basement membrane protein in lung tissue of rats in each group;

[0046] Figure 20 Relative expression levels of JAM-1 and Claudin-5 proteins; n=6;

[0047] Figure 21 Relative expression levels of VE-Cadherin and α-catenin proteins; *P<0.05 vs. control group; #P<0.05 vs. model group; n=6;

[0048] Figure 22 Relative expression levels of MMP-2 and MMP-9 proteins; *P<0.05 vs. control group; n=6;

[0049] Figure 23 Relative expression levels of ATP5A, ATP5B, and ATP5D proteins; *P<0.05 vs. control group; n=6;

[0050] Figure 24 Relative expression levels of Caveolin-1 and Src proteins; n=6. Detailed Implementation

[0051] The present invention provides the use of anisodamine hydrobromide. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0052] The test materials used in the present invention are all ordinary commercially available products and can be purchased on the market. The following further elaborates the present invention in combination with examples:

[0053] Example 1

[0054] Using the CLP rat model, taking the inflammatory response as the entry point, evaluating the improvement effect of anisodamine hydrobromide on the inflammatory response caused by CLP; taking the intercellular connection of vascular endothelial cells as the entry point, evaluating the improvement effect of anisodamine hydrobromide on the pulmonary microcirculation disorder caused by CLP, and clarifying the mechanism of action of anisodamine hydrobromide in improving pulmonary microcirculation disorder.

[0055] 1. Materials and Methods

[0056] 1.1 Experimental Animals

[0057] SPF-grade male Wistar rats (weighing about 200 - 220 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animal production license number: SCXK (Beijing) 2016 - 0006; animal use license number: SYXK (Beijing) 2017 - 0033. The Wistar rats were housed in an animal laboratory with a temperature of 23 ± 2 °C, a humidity of 40 ± 5%, and a 12-hour light / dark cycle. Under this environment, the animals had free access to water and food. The experimental operation process was carried out in accordance with the guidelines of the Animal Research Committee of Peking University, and the experimental protocol was approved by the Laboratory Animal Ethics Committee of the Health Science Center of Peking University (LA2019349).

[0058] Table 1 Experimental Reagents

[0059] Table 2 Specific Antibody Reagents

[0060]

[0061] 1.2 Experimental Instruments and Equipment

[0062] Table 3 Experimental Instruments and Equipment

[0063] 1.3 Experimental Methods

[0064] 1.3.1 Preliminary Experiment (Drug Dosage Screening): Animal Grouping and Administration Methods

[0065] Wistar rats were randomly divided into 5 groups:

[0066] Normal control group (Control)

[0067] CLP Model Group (CLP)

[0068] The low-dose group of CLP + anisodamine (CLP + ADM(L)) was administered at a dose of 0.6 mg / kg.

[0069] The CLP + anisodamine medium-dose group (CLP + ADM (M)) was administered at a dose of 1.2 mg / kg.

[0070] The high-dose group of CLP + anisodamine (CLP + ADM (H)) was administered at a dose of 2.4 mg / kg.

[0071] Six rats were grouped, for a total of 30 rats. Preliminary experiment (drug dosage screening): Animal grouping is shown in Table 4.

[0072] Table 4. Animal grouping in the preliminary experiment (drug dosage screening)

[0073]

[0074] Note: All samples required for vital sign detection and histological staining were from the same rat.

[0075] Control group: Rats were anesthetized with isoflurane and gas anesthesia. A midline incision was made in the abdomen to open the abdominal cavity, approximately 2 cm long. The cecum was located, and its distal end was carefully separated from the mesentery of the large intestine. The cecum was then pushed back into the abdominal cavity, which was closed and sutured. Samples were collected and tested seven days after surgery.

[0076] CLP group: Rats were anesthetized with isoflurane and gas anesthesia. A midline abdominal incision of approximately 2 cm was made to open the abdominal cavity. The cecum was located, and its distal mesentery to the large intestine was carefully separated. The distal half of the cecum was ligated with sterile No. 4 suture, and a 10 ml sterile syringe needle was used to puncture the distal center of the ligated cecum. The cecum was then pushed back into the abdominal cavity, which was closed and sutured. Samples were collected and tested seven days after surgery.

[0077] CLP+ADM(L) group: Rats were anesthetized with isoflurane and gas anesthesia. A midline abdominal incision of approximately 2 cm was made to open the abdominal cavity. The cecum was located, and its distal mesentery to the large intestine was carefully separated. The distal half of the cecum was ligated with sterile No. 4 suture, and a 10 ml sterile syringe needle was used to puncture the ligated cecum at its distal center. The cecum was then pushed back into the abdominal cavity, which was closed and sutured. Low-dose anisodamine hydrobromide was administered 6 hours after surgery. Tissue samples were collected and tested seven days after surgery.

[0078] CLP+ADM(M) group: Rats were anesthetized with isoflurane and gas anesthesia. A midline abdominal incision of approximately 2 cm was made to open the abdominal cavity. The cecum was located, and its distal mesentery to the large intestine was carefully separated. The distal half of the cecum was ligated with sterile No. 4 suture, and a 10 ml sterile syringe needle was used to puncture the ligated cecum at its distal center. The cecum was then pushed back into the abdominal cavity, which was closed and sutured. A medium dose of anisodamine hydrobromide was administered 6 hours after surgery. Tissue samples were collected and tested seven days after surgery.

[0079] CLP+ADM(H) group: Rats were anesthetized with isoflurane and gas anesthesia. A midline abdominal incision of approximately 2 cm was made to open the abdominal cavity. The cecum was located, and its distal mesentery to the large intestine was carefully separated. The distal half of the cecum was ligated with sterile No. 4 suture, and a 10 ml sterile syringe needle was used to puncture the ligated cecum at its distal center. The cecum was then pushed back into the abdominal cavity, which was closed and sutured. High-dose anisodamine hydrobromide was administered 6 hours after surgery. Tissue samples were collected and tested seven days after surgery.

[0080] 1.3.2 Detection of vital signs in rats (heart rate, mean arterial pressure, rectal temperature)

[0081] Heart rate, mean arterial pressure, and rectal temperature of Wistar rats in each group were measured at baseline (0 hours), 6 hours, 24 hours, and 72 hours using a non-invasive physiological recorder (Softron BP-98E; Softron, Shinano, Japan). Body weight of the rats was measured at each time point using an electronic balance (Scout SE; Ohaus Instruments Co., Ltd., Jiangsu, China).

[0082] 1.3.3 Blood gas analysis (partial pressure of carbon dioxide, partial pressure of oxygen, blood oxygen saturation, pH value)

[0083] Seven days later, rats in each group were anesthetized by intramuscular injection of urethane (1.5 g / kg, 2.5 mL / kg). Arterial blood was collected via the abdominal aorta and placed in heparin-anticoagulated (25 mg / mL) EP tubes. The fresh blood was immediately analyzed using a blood gas analyzer (ABL80 FLEX; Radiometer, California, USA) to measure the partial pressure of carbon dioxide, partial pressure of oxygen, blood oxygen saturation, and pH value in the rat arterial blood.

[0084] 1.3.4 Dynamic Visualization of Lung Microcirculation Detection

[0085] Rats in each group of the 24-hour acute lung injury animal model were anesthetized by intramuscular injection of urethane (1.5 g / kg, 2.5 mL / kg). The rats were placed in a supine position and fixed on an animal observation board. The femoral vein was isolated and cannulated. A 1-2 cm incision was made in the center of the neck to expose the trachea. The trachea was cut into an inverted T-shape and a breathing tube was inserted. Mechanical ventilation was provided using a small animal ventilator (ALC-V8; Shanghai Aolite Biotechnology Co., Ltd., Shanghai, China) (tidal volume 15 mL / kg, respiratory rate 75 breaths / min). The left thoracic contour of the rat was exposed, and the ribs were incised between the 3rd and 5th intercostal spaces on the left side of the thoracic cavity to expose the left lung tissue. 37°C saline was intermittently dripped to maintain lung surface moisture and lung tissue viability. Five minutes prior to observation, fluorescently labeled fluorescein isothiocyanate (FITC) (50 mg / kg, 20 mg / mL) and rhodamine 6G (1.5 mg / kg, 1 mg / mL) were slowly injected via a femoral vein cannula. FITC labeled dextran in plasma, and rhodamine 6G labeled leukocytes in blood vessels. Animals were placed under an upright microscope (BX51WI; Olympus, Tokyo, Japan) and subjected to dynamic visualization of lung microcirculation using instruments such as a high-sensitivity camera (USS-301; UNIQ, California, USA) and a color monitor (20PF5120; Philips, Eindhoven, Netherland). During observation, the ventilator was turned off, and positive pressure ventilation (12–13 cm H2O) was administered to maintain lung expansion. FITC fluorescence was observed at 530 nm green light and Rhodamine 6G fluorescence at 590 nm red light. Pulmonary microvessels with diameters of 30–50 μm were observed and imaged under a microscope. Image-J software was used to detect the FITC fluorescence intensity within and outside the pulmonary interstitium of the microvessels. Changes in plasma exudation were represented by the ratio of FITC fluorescence intensity in the extravascular interstitium to that in the pulmonary microvessels. The number of white blood cells adhering to the microvessel walls was counted in the images.

[0086] 1.3.5 Detection of Evans Blue Exudates in the Lungs

[0087] Rats from each group with an acute lung injury model (24 hours prior) were anesthetized by intramuscular injection of urethane (1.5 g / kg, 2.5 mL / kg). The femoral vein was isolated and cannulated. A single, slow injection of 2% Evans blue solution (50 mg / kg, 20 mg / mL) was administered via the femoral vein. After 1 hour of Evans blue circulation, the thoracic cavity was opened, and pulmonary perfusion with physiological saline was performed for 5 minutes to flush out residual blood from the lung tissue. The right middle and lower lobes of the lung tissue were harvested, photographed under a stereoscope, and weighed as wet weight. The tissue was minced and placed in 10 ml EP tubes. 1 mL of formamide solution was added for every 100 mg of wet lung weight. The tubes were soaked in a 60 °C water bath for 18 hours and then centrifuged at 5000 g for 5 minutes using an Allegra 64RBeckman (Palo Alto, CA, USA). Aspirate the supernatant and use a multi-sensor microplate reader (Synergy 2; Bio-Tek, Vermont, USA) to read the absorbance values ​​at 740 nm and 610 nm. Calculate the 610 nm corrected Evans blue absorbance (OD). 610 correct A standard curve was plotted using measurements of the standard samples, and the Evans blue content in the lungs of rats in each group was calculated using the standard curve method. The average Evans blue content in lung tissue = total Evans blue content (μg) / wet weight of lung tissue (g).

[0088] 1.3.6 Detection of wet / dry weight ratio of lung tissue

[0089] Seven days after CLP modeling, rats from each group were anesthetized by intramuscular injection of urethane (1.5 g / kg, 2.5 mL / kg). Arterial blood was collected via the abdominal aorta, and the upper lobe of the right lung, 3-5 cm of intestinal tissue, and intact brain tissue were weighed and recorded as wet weight. The lung, intestinal, and brain tissues were placed in a 60 ℃ electric thermostatic drying oven (202-2AB; Test Instruments, Tianjin, China) to dry. After 72 hours, they were removed, weighed, and recorded as dry weight. The wet-to-dry weight ratio of rat lung, intestinal, and brain tissues = wet weight / dry weight.

[0090] 1.3.7 HE staining of lung tissue

[0091] Rats in each group were anesthetized by intramuscular injection of urethane (1.5 g / kg, 2.5 mL / kg). The right middle lobe of the lung was harvested, and any remaining blood was cleaned off. The tissue was then fixed in 4% paraformaldehyde solution for 48 hours. After fixation, the lung tissue was cut in the middle transverse section and dehydrated sequentially in 70% ethanol for 48 hours, 80% ethanol for 24 hours, 95% ethanol for 12 hours, and anhydrous ethanol for 4 hours. The tissue was then cleared in xylene for 20 minutes and immersed in paraffin at 60°C for 4 hours. After complete paraffin infiltration, the tissue was embedded and paraffin sections were prepared. 5 μm thick paraffin sections were cut using an automated paraffin microtome (Leica 2M2255; Leica, Mannheim, Germany) and placed on glass slides. The sections were then dried overnight at 37°C in a drying oven (202-2AB; Tianjin Tester Instruments Co., Ltd., Tianjin, China). The sections were then stored at room temperature in a slide holder.

[0092] Hematoxylin-eosin (HE) staining procedure:

[0093] 1. Dewaxing and hydration of paraffin sections

[0094] Dewaxing: Immerse twice in xylene, 10 minutes each time;

[0095] Hydration: anhydrous ethanol for 5 min, 95% ethanol for 2 min, 80% ethanol for 2 min, ddH2O for 1 min;

[0096] 2. Stain cell nuclei with Mayer hematoxylin dye for 5 min, then wash off excess dye with ddH2O;

[0097] 3. Stain the cytoplasm with eosin dye for 10 seconds, then wash away excess dye with ddH2O;

[0098] 4. Clean once with 70% ethanol for 1-3 seconds; clean three times with 80% ethanol for 1-3 seconds each time.

[0099] 5. Wash with 95% ethanol for 1 min, then wash twice with anhydrous ethanol for 5 min each time;

[0100] 6. Immerse the slices in xylene twice for 5 minutes each time to allow them to clear.

[0101] 7. Mount the slides with neutral resin and observe the morphological changes in lung tissue under a microscope.

[0102] 8. Image-J software was used to analyze and statistically analyze alveolar septal thickness and lung interstitial area.

[0103] 1.3.8 Immunohistochemical staining of lung tissue

[0104] Immunohistochemical (IHC) staining procedure, referring to the rabbit / mouse two-step detection kit (Zhongshan Jinqiao, Beijing, China):

[0105] 1. Dewaxing and hydration of paraffin sections

[0106] Dewaxing: Immerse twice in xylene, 10 minutes each time;

[0107] Hydration: anhydrous ethanol for 5 min, 95% ethanol for 2 min, 80% ethanol for 2 min, ddH2O for 1 min;

[0108] 2. Citrate antigen retrieval: Microwave on high for 10 min, then allow to cool naturally at room temperature, and wash three times with phosphate-buffered saline (PBS) for 5 min each time.

[0109] 3. Disrupt the membrane using 0.3% Triton X-100, incubate at 37°C for 30 min, and wash three times with PBS for 5 min each time;

[0110] 4. To eliminate endogenous peroxidase activity, add endogenous peroxidase blocking agent and incubate at room temperature for 10 min. Wash with PBS 3 times, 3 min each time, and circle the tissue area with an immunohistochemical pen.

[0111] 5. Block sheep serum at room temperature for 30 min, add primary antibody MPO (1:500) or CD68 (1:500), and incubate overnight at 4°C; warm to room temperature for 1 h the next day, and wash 3 times with PBS for 5 min each time;

[0112] 6. Add reaction enhancement solution, incubate at room temperature for 30 min, and wash three times with PBS for 5 min each time;

[0113] 7. Add the enhanced enzyme-labeled goat anti-rabbit / mouse IgG polymer solution, incubate at room temperature for 30 min, and wash three times with PBS for 5 min each time;

[0114] 8. Add DAB colorimetric solution, wait 10-15 seconds, rinse with tap water, and wash with ddH2O;

[0115] 9. Stain cell nuclei with Mayer hematoxylin for 5 min, then wash away excess dye with ddH2O;

[0116] 10. Clean once with 70% ethanol for 1-3 seconds; clean three times with 80% ethanol for 1-3 seconds each time.

[0117] 11. Wash with 95% ethanol for 1 min, then wash twice with anhydrous ethanol for 5 min each time;

[0118] 12. Soak the sections in xylene twice for 5 minutes each time until clear.

[0119] 13. Using neutral resin as a mounting medium, observe the changes in morphologically positive brown areas of lung tissue under a microscope.

[0120] 14. Use Image-J software to analyze and statistically analyze the positive brown areas.

[0121] 1.3.9 Western Blot Detection

[0122] Rats in each group were anesthetized by intramuscular injection of urethane (1.5 g / kg, 2.5 mL / kg). The lower lobe of the right lung of each rat was harvested, cleaned of any residual blood, placed in EP tubes, and stored at -80℃ for later use. 1 mL of 1×RIPA lysis buffer, a protease inhibitor (1:100), and a phosphatase inhibitor (1:100) were added to every 100 mg of lung tissue. The tissue was thoroughly homogenized using a tissue homogenizer (F6 / 10F013200021; ThermoFisherScientific, Waltham, USA), and subjected to three freeze-thaw cycles in liquid nitrogen. The tissue was centrifuged at 13000 rpm, 4℃, for 30 min (5417R; Eppendorf, Hamburg, Germany). The supernatant was collected, and 5× protein gel denaturation buffer was added. The mixture was boiled for 15 min in a dry incubator (DH100-2; Hangzhou Ruicheng Instrument Co., Ltd., Hangzhou, China). After cooling, the mixture was centrifuged to mix thoroughly and stored at -80℃ for later use.

[0123] Protein quantification: Protein stock solution was quantified using a BCA protein quantification kit (Beijing Pulilai Gene Technology Co., Ltd., Beijing, China). Standards and test samples were diluted at a certain ratio and then added to working solution (working solution A and working solution B were mixed at a volume ratio of 50:1 until homogeneous). The mixture was incubated at 37℃ for 30 min for color development. The absorbance at 560 nm was measured using a multi-functional microplate reader (Synergy 2; Bio-Rad, Hercules, CA, USA). A standard curve was plotted to calculate the protein concentration of the test sample.

[0124] Electrophoresis: Add equal amounts of protein from each group to the wells of a polyacrylamide gel and perform electrophoresis at a constant voltage: 80 V for the stacking gel (approximately 30 min) and 120 V for the separating gel (approximately 90 min). Electrophoresis is stopped when the bromophenol blue reaches the bottom of the separating gel. The electrophoresis buffer system is shown in Table 3.5.

[0125] Table 5 10× Electrophoresis Buffer System

[0126]

[0127] The above reagents were diluted to 1 L of ddH2O.

[0128] Electroporation and Development: A sandwich method was then used, placing the membrane in the following order: cathode plate - sponge pad - filter paper - protein gel - PVDF membrane - filter paper - sponge pad - anode plate. Protein bands were transferred from the protein gel to the polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA) under high current using electroporation at 250 mA for 90 min. After transfer, the PVDF membrane was placed in 1×TBST solution of 5% skim milk or 5% BSA and blocked at room temperature for 1 hour to block non-specific binding sites. Primary antibody was prepared by diluting in 1×TBST solution of 5% skim milk or 5% BSA and incubated overnight at 4°C. The membrane was warmed to room temperature for 1 hour the next day and then washed three times with TBST solution for 5 minutes each time. HRP-labeled secondary antibodies were prepared in 1×TBST solution of 5% skim milk or 5% BSA (Cell Signaling Technology, Danvers, MA, USA) and incubated at room temperature in the dark for 1 h. The PVDF membranes were washed three times with TBST solution for 5 minutes each time. ECL chemiluminescence solution (Beijing Pulilai Gene Technology Co., Ltd., Beijing, China) was added to each membrane, and development was performed in a darkroom using a Bio-Rad chemical imaging exposure system (1708370; Bio-Rad, Texas, USA). The results were semi-quantitatively analyzed using ImageLab (Bio-Rad, Richmond, CA, USA) and ImageJ software. The relative expression level of the target protein in each group was calculated as: target protein band exposure gray value / corresponding internal control exposure gray value. The electroporation buffer system is shown in Table 3.6, and the TBST system is shown in Table 3.7.

[0129] Table 6 10× Electroporation Buffer System

[0130]

[0131] The above reagents were diluted to 1 L of ddH2O.

[0132] Table 7 10×TBST System

[0133]

[0134] The above reagents were diluted to 1 L of ddH2O.

[0135] 1.3.10 Statistical Methods

[0136] All data are expressed as mean ± standard error (Mean ± SEM). GraphPad Prism 7.0 (GraphPad Software, CA, USA) software was used for graphing and data analysis. One-way ANOVA or two-way ANOVA was applied for pairwise comparisons and statistical analysis, with Bonferroni correction used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0137] 2. Experimental Results

[0138] 2.1 Blood flow is lowest 6 hours after CLP modeling, making it a suitable time for scopolamine hydrobromide administration.

[0139] Using 0 hours as the baseline, changes in rat blood flow were continuously monitored within 24 hours of CLP modeling. The experimental results are as follows: Figure 1 As shown, blood flow was lowest 6 hours after CLP modeling, so scopolamine was administered at this time.

[0140] 2.2 Effect of scopolamine oxalool treatment 6 hours after CLP modeling on the seven-day survival rate of rats

[0141] Using 0 hours as the baseline, the mortality rate of rats in each group was continuously monitored for 7 days after CLP modeling. The experimental results are as follows: Figure 2 As shown, the survival rate of rats gradually decreased after 1, 2, 3, and 7 days of modeling. Medium and high doses of anisodamine hydrobromide increased the seven-day survival rate. Among them, medium and high doses of anisodamine significantly increased the seven-day survival rate after CLP modeling.

[0142] 2.3 Effects of scopolamine hydrobromide on heart rate in rats after CLP modeling

[0143] Using 0 hours as the baseline, the heart rate of rats in each group was continuously monitored for 72 hours after CLP modeling. The experimental results are as follows: Figure 3 As shown, the heart rate of rats with CLP modeling increased significantly at 6 and 24 hours. Medium and high doses of anisodamine hydrobromide significantly inhibited the increase in heart rate in rats 24 hours after CLP modeling.

[0144] 2.4 Effect of scopolamine hydrobromide on rectal temperature in rats after CLP modeling

[0145] Using 0 hours as the baseline, the changes in rectal temperature of rats in each group were continuously monitored for 72 hours after CLP modeling. The experimental results are as follows: Figure 4As shown, the rectal temperature of rats was significantly reduced at 24 and 72 hours after CLP modeling. Low, medium, and high doses of anisodamine hydrobromide could improve the reduction in rectal temperature in rats 72 hours after CLP modeling.

[0146] 2.5 Effect of scopolamine hydrobromide on mean arterial pressure in rats after CLP modeling

[0147] Using 0 hours as the baseline, changes in mean arterial pressure were continuously monitored in each group of rats within 72 hours after CLP modeling. The experimental results are as follows: Figure 5 As shown, the mean arterial pressure of rats decreased 6 hours after CLP modeling, but the difference was not statistically significant. Scopolamine in each group slightly alleviated the mean arterial pressure in rats, but had no significant effect.

[0148] 2.6 Effect of scopolamine hydrobromide on blood gas analysis in rats 7 days after CLP modeling

[0149] Figure 6 These are the results of blood gas analysis in each group of rats. Compared with the control group, the CLP model group showed significantly decreased arterial blood oxygen partial pressure, blood oxygen saturation, and pH, while there were no significant differences in arterial blood carbon dioxide partial pressure and lactate content. Scopolamine hydrobromide significantly inhibited the decrease in arterial blood oxygen partial pressure, blood oxygen saturation, and blood pH induced by the CLP model in rats.

[0150] 2.7 Effect of scopolamine hydrobromide on Evans blue exudation in rats 7 days after CLP modeling

[0151] Figure 7 These are the results of changes in Evans blue exudate in the lungs of rats in each group. Figure 7 The blue area in section A represents the exudated Evans blue. The control group showed lower Evans blue exudation. Compared to the control group, the Evans blue exudation in rats induced by the CLP model was significantly increased. Scopolamine hydrobromide significantly inhibited the increase in Evans blue exudation induced by CLP. Statistical results are shown below. Figure 7 As shown in B.

[0152] 2.8 Effect of scopolamine hydrobromide on the wet-to-dry weight ratio of rat lung tissue 7 days after CLP modeling

[0153] Figure 8 The results showed that the wet-to-dry weight ratio of lung tissue in each group of rats was significantly different among the control group, the model group, and the low, medium, and high dose groups of anisodamine.

[0154] 2.9 Effects of scopolamine hydrobromide on leukocyte adhesion in pulmonary microvessels of CLP rats

[0155] Figure 9The AC study used a dynamic visualization microcirculation system to observe images of rhodamine 6G-labeled leukocytes adhering to pulmonary microvessels, confirming that anisodamine hydrobromide can inhibit leukocyte adhesion induced by the CLP model. No leukocyte adhesion occurred in the pulmonary microvessels of rats in the control group. Compared with the control group, a large number of leukocytes adhered to the walls of pulmonary microvessels in the CLP model group. Anisodamine hydrobromide significantly cleared CLP-induced leukocyte adhesion. Anisodamine hydrobromide can both inhibit leukocyte adhesion to pulmonary microvessels and clear leukocytes already adhered to pulmonary microvessels. Statistical results of the number of leukocytes adhering to pulmonary microvessels are shown below. Figure 9 As shown in D.

[0156] 2.10 Effects of scopolamine hydrobromide on pulmonary microvascular permeability in CLP rats

[0157] Figure 10 The AC result was obtained by observing and photographing FITC-dextran exudation using an upright dynamic visualization microcirculation system. No significant exudation was observed in the pulmonary microvessels of the Control group. Compared with the Control group, FITC-dextran significantly exuded into the lateral pulmonary microvessels in the CLP 6-hour model group. High-dose anisodamine hydrobromide significantly inhibited CLP-induced FITC-dextran exudation. The ratio of FITC fluorescence intensity on the lateral to medial sides of pulmonary microvessels reflected the permeability of pulmonary microvessels. Statistical analysis of pulmonary microvessel exudation in each group of rats is as follows: Figure 10 As shown in D.

[0158] 2.11 Effects of scopolamine hydrobromide on lung tissue morphology in CLP rats

[0159] Figure 11 HE staining results of pulmonary microvessels and surrounding tissues in rats of each group. In the Sham group, no significant edema was observed in the pulmonary microvessels and surrounding tissues, and inflammatory cells were relatively few. After CLP modeling, the model group rats showed significant edema in the pulmonary microvessels and surrounding tissues, significant thickening of the alveolar septa, and inflammatory cell infiltration within the alveolar spaces, accompanied by diffuse alveolar damage. High-dose anisodamine hydrobromide significantly improved the morphological changes in pulmonary microvessels induced by CLP modeling.

[0160] Figure 12 These are the HE staining results of terminal bronchial epithelial cells and surrounding tissues in rats from each group. In the Sham group, the terminal bronchial epithelial cells of rats were tightly packed, with intact villi and no significant changes. After CLP modeling, the terminal bronchial epithelial cells of rats in the model group were loosely packed, and the villi morphology was damaged and shed. High-dose anisodamine hydrobromide significantly improved the CLP-induced disordered morphology of terminal bronchial epithelial cells.

[0161] 2.12 Effects of scopolamine hydrobromide on the proportion of MPO, CD68, and CD18 positive cell regions in lung tissue of CLP rats

[0162] Figure 13 The immunohistochemical results of lung tissue from each group of rats demonstrate the inflammatory cell infiltration in the lung tissue. MPO is a neutrophil marker, CD68 is a monocyte / macrophage marker, and CD18 is an adhesion molecule. The proportion of MPO, CD68, and CD18 positive cells in the lung tissue of the Sham group rats was relatively low. Compared with the Sham group, the proportion of MPO, CD68, and CD18 positive cells in the lung tissue of the CLP model group rats was significantly increased. Scopolamine hydrobromide significantly inhibited the CLP-induced increase in the proportion of MPO, CD68, and CD18 positive cells in the lung tissue.

[0163] 2.13 Effect of scopolamine hydrobromide on the expression of Caveolin-1, a microvesicle-associated protein in the lung tissue of CLP rats

[0164] The expression level of Caveolin-1, a plasma membrane microvesicle-associated protein, was detected using immunofluorescence staining. Figure 14 Compared with the Sham group, the expression level of Caveolin-1 in the CLP model group showed no significant change. Compared with the CLP model group, the anisodamine hydrobromide administration group significantly reduced Caveolin-1 expression.

[0165] 2.14 Effects of scopolamine hydrobromide on the expression of Occludin, Claudin-5, and VE-Cadherin in lung tissue of CLP rats

[0166] Figures 15-17 The images show the immunofluorescence staining results of the tight junction proteins Occludin, Claudin-5, and VE-Cadherin extracted from the lung tissue of rats in each group. In the Sham group, the expression of Occludin, Claudin-5, and VE-Cadherin in the intercellular spaces of vascular endothelial cells was intact and continuous. Compared with the Sham group, the expression of Occludin, Claudin-5, and VE-Cadherin in the lung tissue of rats in the CLP model group was degraded and discontinuous. Scopolamine hydrobromide significantly improved the degradation and discontinuous state of Occludin, Claudin-5, and VE-Cadherin induced by CLP.

[0167] 2.15 Effects of scopolamine hydrobromide on the expression of Collagen IV and Laminin in the basement membrane of lung tissue from CLP rats

[0168] The morphology of basement membrane proteins Collagen IV and Laminin was observed using immunofluorescence. Figure 18 and 19 It is evident that Collagen IV and Lamin expression was intact and continuous in the Sham group, while Collagen IV and Lamin expression was relatively reduced and showed degradation and fragmentation in the CLP model group. Compared with the CLP model group, the morphology of Collagen IV and Lamin was more continuous in the anisodamine hydrobromide administration group.

[0169] 2.16 Effects of scopolamine hydrobromide on the expression of tight junction proteins JAM1 and Claudin-5 in lung tissue of CLP rats

[0170] The expression levels of the tight junction proteins junctional adhesion molecule 1 (JAM-1) and Claudin-5 were detected by Western blot. The results are as follows: Figure 20 As shown, compared with the control group, JAM-1 was significantly downregulated in the model group, and high-dose anisodamine hydrobromide treatment reversed the downregulation of JAM-1; the expression level of Claudin-5 did not change significantly.

[0171] 2.17 Effects of scopolamine hydrobromide on the expression of adhesion and connexin proteins VE-cadherin and α-catenin in lung tissue of CLP rats

[0172] The expression levels of adhesion junction proteins VE-cadherin and α-catenin were detected by Western blot. The results are as follows: Figure 21 As shown, compared with the control group, the expression of VE-cadherin in the model group was reduced, while the expression of α-catenin was not significantly changed. High-dose anisodamine treatment improved the downregulation of VE-cadherin.

[0173] 2.18 Effects of scopolamine hydrobromide on the expression of matrix metalloproteinases MMP-2 and MMP-9 in lung tissue of CLP rats

[0174] The expression levels of matrix metalloproteinases MMP-2 and MMP-9 were detected by Western blot. The results are as follows: Figure 22 As shown, compared with the control group, the expression of MMP-2 and MMP-9 was upregulated in the model group; compared with the model group, the expression of MMP-9 was downregulated in the model + high dose of scopolamine, while the expression level of MMP2 did not change significantly.

[0175] 2.19 Effects of scopolamine hydrobromide on the expression of energy metabolism-related proteins ATP5A, ATP5B, and ATP5D in lung tissue of CLP rats

[0176] The expression levels of energy metabolism-related proteins ATP5A, ATP5B, and ATP5D were detected using Western blot. The results are as follows: Figure 23 As shown, compared with the control group, the expression of ATP5A in the model group was reduced, while the expression levels of ATP5B and ATP5D showed no significant changes. Compared with the model group, the expression levels of ATP5A, ATP5B, and ATP5D in the model + high-dose anisodamine group showed no significant changes.

[0177] 2.20 Effects of scopolamine hydrobromide on the expression of Caveolin-1 and Src, microvesicle-associated proteins in lung tissue of CLP rats

[0178] The expression levels of the plasma membrane microvesicle-related proteins Caveolin-1 and Src were detected by Western blot. The results are as follows: Figure 24 As shown, compared with the control group, the expression of Caveolin-1 and Src in the model group did not change significantly.

[0179] 3. Conclusion

[0180] This invention confirms that anisodamine hydrobromide improves vital signs changes in rats induced by cyclophosphamide (CLP), improves heart rate and rectal temperature changes, increases mean arterial pressure, inhibits CLP-induced increases in arterial carbon dioxide partial pressure and lactate, and significantly restores oxygen partial pressure, blood oxygen saturation, and pH. Anisodamine hydrobromide improves CLP-induced morphological damage to lung tissue, inhibits pulmonary microvascular and surrounding tissue edema, increases lung parenchyma, thickens pulmonary interstitium, reduces alveolar cavity area, and improves loose arrangement of terminal bronchial epithelial cells, partial villus loss, and morphological destruction. Anisodamine hydrobromide inhibits CLP-induced adhesion of rhodamine 6G-labeled leukocytes; inhibits inflammatory cell infiltration in lung tissue, increases the proportion of CD68 and MPO positive cells in immunohistochemical staining; inhibits microvascular FITC-dextran exudation and pulmonary Evans blue exudation; inhibits the increase in lung tissue wet / dry weight ratio, and improves pulmonary interstitial edema. This study further confirms that scopolamine hydrobromide inhibits the low expression of tight junction proteins JAM-1, Occludin, Claudin-5, and adhesion junction protein VE-Cadherin in lung tissue endothelial cells.

[0181] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of anisodamine hydrobromide in the preparation of a drug for regulating proteins in lung tissue. The proteins in the lung tissue include: The lung tissue plasma membrane caveolae-related protein, lung tissue energy metabolism-related protein, lung tissue matrix metalloproteinase, lung tissue adherin, lung tissue tight junction protein, lung tissue basement membrane protein and / or lung tissue microvascular endothelial cell junction protein.

2. The use according to claim 1, wherein, The lung tissue plasma membrane caveolae-related protein comprises Caveolin-1 and / or Src; The lung tissue energy metabolism-related protein comprises ATP5A, ATP5B and / or ATP5D; The lung tissue matrix metalloproteinase comprises MMP-2 and / or MMP-9; The lung tissue adherin comprises VE-cadherin and / or α-catenin; The lung tissue tight junction protein comprises JAM1 and / or Claudin-5; The lung tissue basement membrane protein comprises Collagen IV and / or Laminin.

3. Use according to claim 1 or 2, characterized in that, The regulation comprises: Maintaining the level of ATP5A, inhibiting the level of MMP-9 and / or MMP-2, promoting the expression of VE-cadherin, reversing the down-regulation of JAM-1, maintaining the level of Collagen IV and / or Laminin, and improving the degradation of Occludin, Claudin-5 and / or VE-Cadherin.

4. Use of anisodamine hydrobromide in the preparation of a drug for protecting lung tissue of a patient with sepsis and / or septic shock.

5. Use according to claim 4, characterized in that, The protection of lung tissue of a patient with sepsis comprises inhibiting the increase of the proportion of lung tissue MPO, CD68 and CD18 positive cells, improving the morphological arrangement disorder of terminal bronchial epithelial cells, improving the morphological changes of lung microvessels, improving lung microvascular permeability, clearing lung microvascular leukocyte adhesion and / or inhibiting the amount of Evans blue exudation.

6. Use of anisodamine hydrobromide in the preparation of a drug for improving vital signs of a patient with sepsis and / or septic shock.

7. Use according to claim 6, characterized in that, The vital signs comprise blood gas indicators, arterial pressure, heart rate and / or body temperature.

8. Use according to claim 7, characterized in that, The blood gas indicators comprise arterial oxygen partial pressure, blood oxygen saturation and / or blood pH value.

9. Use of anisodamine hydrobromide in the preparation of a drug for improving the survival rate of a patient with sepsis and / or septic shock.

10. The use according to any one of claims 1 to 9, characterized in that, The dose of anisodamine hydrobromide is 0.6 mg / kg to 2.4 mg / kg.

Images