Use of anisodamine hydrobromide

Abstract

Use of anisodamine hydrobromide in the preparation of a drug for treating sepsis and / or septic shock. Anisodamine hydrobromide can ameliorate CLP-induced changes in vital signs of rats, improve the morphology of lung tissue, regulate related target proteins, and produce a good therapeutic effect on sepsis and / or septic shock.

Description

Use of anisodamine hydrobromide

[0001] The present application claims priority to the Chinese patent application No. 202411235628.X filed on September 04, 2024, and entitled "Use of anisodamine hydrobromide", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of medicine, in particular to the use of anisodamine hydrobromide. BACKGROUND

[0003] 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 methods in clinical practice.

[0004] 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 clinical treatment of septic shock in China, and it was not until the production of the drug was limited that the clinical application was restricted. In 2016, China began to increase the production of anisodamine hydrobromide, and the clinical application also increased. Tang Hong et al. once 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. The half-life of the drug is 40 minutes, and the drug will not accumulate in the body for a long time.

[0005] However, the effect of anisodamine hydrobromide in the treatment of sepsis shock and its mechanism have not been reported. SUMMARY

[0006] Therefore, the present application aims to provide the use of anisodamine hydrobromide.

[0007] The present application provides the use of anisodamine hydrobromide in the preparation of a drug for regulating proteins in lung tissue.

[0008] The proteins in lung tissue include: lung tissue plasma membrane microvesicle associated protein, lung tissue energy metabolism associated 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.

[0009] In the present application, the lung tissue plasma membrane microvesicle associated protein includes Caveolin-1 and / or Src;

[0010] The lung tissue energy metabolism associated protein includes ATP5A, ATP5B and / or ATP5D;

[0011] The lung tissue matrix metalloproteinase includes MMP-2 and / or MMP-9;

[0012] The lung tissue adherent junction protein includes VE-cadherin and / or α-catenin;

[0013] The lung tissue tight junction protein includes JAM1 and / or Claudin-5;

[0014] The lung tissue basement membrane protein includes Collagen IV and / or Laminin.

[0015] In the present application, the regulation of proteins in lung tissue includes:

[0016] 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.

[0017] The present application also provides the use of anisodamine hydrobromide in the preparation of a drug for protecting lung tissue of patients with sepsis and / or septic shock.

[0018] In the present application, the protection of lung tissue of patients with sepsis includes: inhibiting the increase of lung tissue MPO, CD68 and CD18 positive cell area ratio, 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.

[0019] The present application also provides the use of anisodamine hydrobromide in the preparation of a medicament for improving the vital signs of a patient with sepsis and / or septic shock.

[0020] In the present application, the vital signs include blood gas indicators, arterial pressure, heart rate and / or body temperature.

[0021] In some embodiments, the blood gas indicators include arterial oxygen partial pressure, blood oxygen saturation and / or blood pH.

[0022] In the present application, the use of anisodamine hydrobromide in the preparation of a medicament for improving the survival rate of a patient with sepsis and / or septic shock.

[0023] Preferably, the dose of anisodamine hydrobromide administered in the present application is 0.6 mg / kg to 2.4 mg / kg.

[0024] Preferably, the administration time of anisodamine hydrobromide in the present application is 6 hours after the onset of the disease.

[0025] The present application also provides a method for treating sepsis and / or septic shock, which comprises administering anisodamine hydrobromide.

[0026] The subject of the treatment is a human or a mammal. The mammal is a primate, a canine, a feline or a rodent.

[0027] The present application provides a new use of anisodamine hydrobromide. Research shows that anisodamine hydrobromide improves the vital signs of rats caused by CLP, improves the morphology of lung tissue, and regulates related target proteins, thereby playing a good therapeutic role on sepsis and / or septic shock. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 shows the changes in blood flow of rats at different time points within 24 hours after CLP modeling;

[0029] Figure 2 shows the effect of different doses of anisodamine hydrobromide on the survival rate of rats within 7 days after CLP modeling;

[0030] Figure 3 shows the effect of different doses of anisodamine hydrobromide on the heart rate of rats within 72 hours after CLP modeling;

[0031] Figure 4 shows the effect of different doses of anisodamine hydrobromide on the rectal temperature of rats within 72 hours after CLP modeling;

[0032] Figure 5 shows the effect of different doses of anisodamine hydrobromide on the mean arterial pressure of rats within 72 hours after CLP modeling;

[0033] Figure 6 Effects of anisodamine hydrobromide on blood gas analysis changes in CLP rats at 7 days, wherein: A, arterial partial pressure of oxygen; B, arterial partial pressure of carbon dioxide; C, arterial oxygen saturation; D, arterial pH; E, arterial lactic acid content; Control, normal control group; CLP, CLP model group; CLP+L, CLP modeling + anisodamine low-dose group; CLP+M, CLP modeling + anisodamine medium-dose group; CLP+H, CLP modeling + anisodamine high-dose 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;

[0034] Figure 7 Effects of anisodamine hydrobromide on lung Evans blue exudation changes in CLP rats at 7 days, wherein: A, lung Evans blue exudation images of rats in each group; B, statistical results of lung tissue Evans blue exudation amount of rats in each group; #p<0.05 vs Control; **p<0.01 vs CLP; ##p<0.01 vs Control, N=6;

[0035] Figure 8 Effects of anisodamine hydrobromide on lung tissue wet / dry weight ratio in CLP rats at 7 days;

[0036] Figure 9 Effects of anisodamine hydrobromide on lung microvascular leukocyte adhesion in CLP rats, wherein: A, lung microvascular leukocyte adhesion graph of control rats; B, lung microvascular leukocyte adhesion graph of CLP rats; C, lung microvascular leukocyte adhesion graph of rats in CLP modeling + anisodamine hydrobromide high-dose administration group; D, statistical graph of intravascular leukocyte adhesion number in each group; E, statistical graph of extravascular leukocyte adhesion number in each group; F, statistical graph of rolling leukocyte number in each group; Red arrows point to leukocytes adhering to lung microvessels; N=6;

[0037] Figure 10 Effects of anisodamine on lung microvascular exudation changes in CLP rats, wherein: A, lung microvascular leukocyte exudation graph of control rats; B, lung microvascular leukocyte exudation graph of CLP rats; C, lung microvascular leukocyte exudation graph of rats in CLP modeling + anisodamine high-dose administration group; Iv, intravascular mean fluorescence intensity; N=6;

[0038] Figure 11 HE staining morphological graph of lung microvessels and surrounding tissues of rats in each group;

[0039] Figure 12 HE staining morphological graph of lung terminal bronchial epithelial cells and surrounding tissues of rats in each group;

[0040] Figure 13 Immunohistochemical staining graph of lung tissue MPO, CD68 and CD18 of rats in each group;

[0041] Figure 14 is an immunofluorescence staining diagram of Caveolin-1, a caveolae-related protein in lung tissue of rats in each group;

[0042] Figure 15 is an immunofluorescence staining diagram of Occludin, a tight junction protein of microvascular endothelial cells in lung tissue of rats in each group;

[0043] Figure 16 is an immunofluorescence staining diagram of Claudin-5, a tight junction protein of microvascular endothelial cells in lung tissue of rats in each group;

[0044] Figure 17 is an immunofluorescence staining diagram of VE-Cadherin, an adherens junction protein of microvascular endothelial cells in lung tissue of rats in each group;

[0045] Figure 18 is an immunofluorescence staining diagram of Collagen IV, a basement membrane protein in lung tissue of rats in each group;

[0046] Figure 19 is an immunofluorescence staining diagram of Laminin, a basement membrane protein in lung tissue of rats in each group;

[0047] Figure 20 is the relative expression amount of JAM-1 and Claudin-5 proteins; n = 6;

[0048] Figure 21 is the relative expression amount of VE-Cadherin and α-catenin proteins; *P < 0.05 vs the control group; #P < 0.05 vs the model group; n = 6;

[0049] Figure 22 is the relative expression amount of MMP-2 and MMP-9 proteins; *P < 0.05 vs the control group; n = 6;

[0050] Figure 23 is the relative expression amount of ATP5A, ATP5B and ATP5D proteins; *P < 0.05 vs the control group; n = 6;

[0051] Figure 24 is the relative expression amount of Caveolin-1 and Src proteins; n = 6. DETAILED DESCRIPTION

[0052] The present application provides the use of anisodamine hydrobromide, and those skilled in the art can refer to the content herein to appropriately improve the process parameters for implementation. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are all considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0053] The test materials used in the present application are all ordinary commercially available products and can be purchased in the market. The present application will be further described below in combination with examples:

[0054] Example 1

[0055] The CLP rat model was used to evaluate the improvement effect of anisodamine hydrobromide on the inflammatory response caused by CLP as a starting point; the improvement effect of anisodamine hydrobromide on the pulmonary microcirculation disorder caused by CLP was evaluated as a starting point, and the mechanism of anisodamine hydrobromide in improving pulmonary microcirculation disorder was elucidated.

[0056] 1. Materials and methods

[0057] 1.1 Experimental animals

[0058] SPF male Wistar rats (body weight about 200-220 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animal production license number: SCXK (Jing) 2016-0006; animal use license number: SYXK (Jing) 2017-0033. Wistar rats were bred in an animal laboratory with a temperature of 23±2℃, humidity of 40±5%, and 12 hours of light / dark alternation. In this environment, animals were free to take 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 scheme was approved by the Animal Ethics Committee of Peking University Medical Department (LA2019349).

[0059] Table 1 Experimental reagents

[0060] Table 2 Specific antibody reagents

[0061] 1.2 Experimental instruments and equipment

[0062] Table 3 Experimental instruments and equipment

[0063] 1.3 Experimental methods

[0064] 1.3.1 Pre-experiment (drug dose screening) animal grouping and administration method

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

[0066] normal control group (Control),

[0067] CLP model group (CLP),

[0068] CLP + anisodamine low-dose group (CLP + ADM(L)), the administration dose is 0.6 mg / kg,

[0069] CLP + anisodamine medium-dose group (CLP + ADM(M)), the administration dose is 1.2 mg / kg,

[0070] CLP + anisodamine high-dose group (CLP + ADM(H)), the administration dose is 2.4 mg / kg,

[0071] Each group has 6 rats, and a total of 30 rats. The pre-experiment (drug dose screening) animal grouping is shown in Table 4.

[0072] Table 4 Pre-experiment (drug dose screening) animal grouping Note: The samples required for vital sign detection and histological staining are from the same rat.

[0073] Control group: Under the assistance of isoflurane and gas anesthesia machine, the rat was anesthetized, the abdominal cavity was opened with a longitudinal incision of about 2 cm in the middle of the abdomen, the cecum was found, the mesentery of the distal end of the cecum and the large intestine was carefully separated, then the cecum was pushed back into the abdominal cavity, and the abdominal cavity was closed and sutured. The sample was taken and detected after seven days of operation.

[0074] CLP group: Under the assistance of isoflurane and gas anesthesia machine, the rat was anesthetized, the abdominal cavity was opened with a longitudinal incision of about 2 cm in the middle of the abdomen, the cecum was found, the mesentery of the distal end of the cecum and the large intestine was carefully separated, the distal end of the cecum was tightly ligated with a sterile No. 4 line at 1 / 2, and a 10 ml sterile syringe needle was used to puncture through the center of the distal end of the cecum that had been ligated, then the cecum was pushed back into the abdominal cavity, and the abdominal cavity was closed and sutured. The sample was taken and detected after seven days of operation.

[0075] CLP + ADM(L) group: Under the assistance of isoflurane and gas anesthesia machine, the rat was anesthetized, the abdominal cavity was opened with a longitudinal incision of about 2 cm in the middle of the abdomen, the cecum was found, the mesentery of the distal end of the cecum and the large intestine was carefully separated, the distal end of the cecum was tightly ligated with a sterile No. 4 line at 1 / 2, and a 10 ml sterile syringe needle was used to puncture through the center of the distal end of the cecum that had been ligated, then the cecum was pushed back into the abdominal cavity, and the abdominal cavity was closed and sutured. Low-dose anisodamine hydrobromide treatment was given at 6 hours after the operation. The sample was taken and detected after seven days of operation.

[0076] CLP + ADM(M) group: Under the assistance of isoflurane and gas anesthesia machine, the rat was anesthetized, the abdominal cavity was opened with a longitudinal incision of about 2 cm in the middle of the abdomen, the cecum was found, the mesentery of the distal end of the cecum and the large intestine was carefully separated, the distal end of the cecum was tightly ligated with a sterile No. 4 line at 1 / 2, and a 10 ml sterile syringe needle was used to puncture through the center of the distal end of the cecum that had been ligated, then the cecum was pushed back into the abdominal cavity, and the abdominal cavity was closed and sutured. Medium-dose anisodamine hydrobromide treatment was given at 6 hours after the operation. The sample was taken and detected after seven days of operation.

[0077] CLP+ADM(H) group: Under the assistance of isoflurane and gas anesthesia machine, the abdominal median incision was made to open the abdominal cavity for about 2 cm, the cecum was found, the mesentery of the distal end of the cecum was carefully separated, the distal end of the cecum was tightly ligated with a sterile No. 4 thread, and a 10 ml sterile syringe needle was used to puncture the central part of the distal end of the cecum, then the cecum was pushed back into the abdominal cavity, and the abdominal cavity was closed and sutured. High-dose hyoscyamine hydrobromide treatment was given at 6 hours after the operation. The samples were taken and detected after 7 days of operation.

[0078] 1.3.2 Detection of vital signs of rats (heart rate, mean arterial pressure, anal temperature)

[0079] The heart rate, mean arterial pressure and anal temperature of Wistar rats in each group were measured at baseline (0 hour), 6 hours, 24 hours and 72 hours using a non-invasive physiological recorder (Softron BP-98E; Softron, Shinano, Japan). The body weight of rats at each time point was measured using an electronic balance (Scout SE; Ohaus Instruments Co., Ltd., Jiangsu, China).

[0080] 1.3.3 Blood gas analysis (partial pressure of carbon dioxide, partial pressure of oxygen, oxygen saturation, pH value)

[0081] The rats in each group were taken at seven days, and anesthetized by intramuscular injection of urethane (1.5 g / kg, 2.5 mL / kg). Arterial blood was collected through the abdominal aorta and placed in a heparin anticoagulant (25 mg / mL) EP tube. The fresh blood collected was immediately detected for the partial pressure of carbon dioxide, partial pressure of oxygen, oxygen saturation and pH value of rat arterial blood using a blood gas analyzer (ABL80 FLEX; Radiometer, California, USA).

[0082] 1.3.4 Dynamic visualization of microcirculation in lung

[0083] Acute lung injury animal model was established by intramuscular injection of urethane (1.5 g / kg, 2.5 mL / kg) in rats. The rats were placed in supine position and fixed on the animal observation board. The femoral vein was cannulated. A 1-2 cm incision was made in the middle of the neck, and the trachea was exposed. The trachea was cut into an inverted T-shaped incision and inserted into the animal breathing tube. The small animal respirator (ALC-V8; Shanghai Olimpia Biotech Co., Ltd., Shanghai, China) was turned on to give mechanical assisted ventilation (tidal volume 15 mL / kg, respiratory rate 75 times / min). The left chest contour of the rat was exposed, and the ribs were cut open from the 3rd to 5th intercostal space on the left side to expose the left lung tissue. 37°C normal saline was intermittently added to keep the lung surface moist and the lung tissue viable. Five minutes before observation, the fluorescent markers fluorescein isothiocyanate (FITC) (50 mg / kg, 20 mg / mL) and rhodamine 6G (1.5 mg / kg, 1 mg / mL) were slowly injected through the femoral vein retention cannula. FITC labels dextran in plasma, and rhodamine 6G labels leukocytes in blood vessels. The animal was placed under a stereomicroscope (BX51WI; Olympus, Tokyo, Japan) for dynamic visualization of pulmonary microcirculation observation with the aid of a hypersensitive camera (USS-301; UNIQ, California, USA), a color display (20PF5120; Philips, Eindhoven, Netherland), and other instruments. During the observation period, the respirator was turned off to give oxygen positive pressure ventilation (pressure 12-13 cm H2O) to keep the lung tissue in an expanded state. FITC fluorescence was observed at 530 nm green light band, and rhodamine 6G fluorescence was observed at 590 nm red light band. Lung microvessels with a diameter of 30-50 μm were observed and photographed under a microscope. Image-J software was used to detect the FITC fluorescence intensity in the lung microvessels and the extravascular lung interstitium. The change in plasma exudation was represented by the ratio of the FITC fluorescence intensity in the extravascular lung interstitium to the FITC fluorescence intensity in the lung microvessels. The number of leukocytes adhering to the microvessel wall in the image was counted.

[0084] 1.3.5 Lung Evans blue exudation detection

[0085] Another 24 hours after the acute lung injury model of rats in each group, intramuscular injection of urethane (1.5 g / kg, 2.5 mL / kg) for anesthesia. Isolation of femoral vein blood vessels, and femoral vein intubation, through the femoral vein once slow injection of 2% Evans blue dye (50 mg / kg, 20 mg / mL). Evans blue in vivo circulation for 1 hour, cut the chest, using normal saline for lung circulation perfusion 5 min, flush the residual blood in the lung tissue. Cut the right middle and lower lobe of the lung tissue of rats, placed under the stereoscope to take pictures, weighing the lung tissue, recorded as wet weight. The tissue is cut into small pieces, placed in a 10 ml EP tube, 1 mL of formamide solution was added for every 100 mg of wet lung weight, and then soaked in a 60°C water bath for 18 hours. After that, the centrifuge (Allegra 64R Beckman; Palo Alto, CA, USA) was used at 5000g for 5 min. The upper liquid was aspirated, and the multifunctional microplate reader (Synergy 2; Bio-Tek, Vermont, USA) was used to detect the absorbance values at 740 nm and 610 nm wavelengths. The corrected Evans blue absorbance at 610 nm (OD 610correct ) was calculated, and the standard curve obtained from the measurement of the standard sample was plotted. The standard curve method was used to calculate the lung Evans blue content of each group of rats. The average lung Evans blue content of the rats = total Evans blue content (μg) / wet lung weight (g).

[0086] 1.3.6 Wet / dry weight ratio of lung tissue

[0087] After 7 days of CLP modeling, each group of rats was taken, and intramuscular injection of urethane (1.5 g / kg, 2.5 mL / kg) was used for anesthesia. Arterial blood was collected through the abdominal aorta, and the right upper lobe of the lung, 3-5 cm of intestinal tissue, and intact brain tissue of the rats were weighed and recorded as wet weight. The lung, intestine, and brain tissues were placed in a 60°C electric heating constant temperature drying oven (202-2AB; Test Instrument, Tianjin, China) for drying. After 72 hours, they were taken out and weighed and recorded as dry weight. The wet / dry weight ratio of the lung, intestine, and brain tissues of the rats = wet weight / dry weight.

[0088] 1.3.7 Lung tissue HE staining

[0089] Anesthetize the rats in each group by intramuscular injection of urethane (1.5 g / kg, 2.5 mL / kg). Take the middle lobe of the right lung of the rats, wash the surface residual blood, and place it in a 4% paraformaldehyde solution for fixation for 48 hours. After the tissue is fixed, cut it open at the middle transverse section of the lung tissue with a blade, and sequentially dehydrate in 70% ethanol for 48 hours, 80% ethanol for 24 hours, 95% ethanol for 12 hours, and anhydrous ethanol for 4 hours. Then, immerse in xylene for transparency for 20 min at 60°C for 4 hours. After the tissue is completely immersed in wax, embed it and make paraffin sections. Cut 5 μm paraffin sections using a fully automatic paraffin section machine (Leica 2M2255; Leica, Mannheim, Germany) and place them on glass slides. Place the sections in a drying oven (202-2AB; Tianjin Test Instrument Co., Ltd., Tianjin, China) at 37°C overnight for drying. Store them in a section box at room temperature.

[0090] Hematoxylin-eosin (HE) staining process:

[0091] 1. Paraffin section deparaffinization and hydration

[0092] Deparaffinization: xylene immersion for 2 times, 10 min each time;

[0093] Hydration: anhydrous ethanol for 5 min, 95% ethanol for 2 min, 80% ethanol for 2 min, ddH2O for 1 min;

[0094] 2. Use Mayer hematoxylin dye for nuclear staining for 5 min, and wash the excess dye with ddH2O;

[0095] 3. Use eosin dye for cytoplasmic staining for 10 s, and wash the excess dye with ddH2O;

[0096] 4. 70% ethanol washing for 1 time, 1-3 s; 80% ethanol washing for 3 times, 1-3 s each time;

[0097] 5. 95% ethanol washing for 1 min, and anhydrous ethanol washing for 2 times, 5 min each time;

[0098] 6. Immersion of the sections in xylene for transparency for 2 times, 5 min each time;

[0099] 7. Use neutral gum to seal the sections, and observe the morphological changes of the lung tissue under a microscope.

[0100] 8. Use Image-J software to analyze and statistically process the alveolar septum thickness and lung interstitial area.

[0101] 1.3.8 Lung tissue immunohistochemical staining

[0102] Immunohistochemical (IHC) staining process, referring to rabbit / mouse two-step method detection kit (Zhongshanjingqiao, Beijing, China):

[0103] 1. Paraffin section deparaffinization, hydration

[0104] Deparaffinization: xylene immersion 2 times, 10 min each time;

[0105] Hydration: anhydrous ethanol 5 min, 95% ethanol 2 min, 80% ethanol 2 min, ddH2O 1 min;

[0106] 2. Citrate antigen repair, high heat in microwave oven for 10 min, then naturally cool at room temperature, use phosphate buffered saline (PBS) to wash 3 times, 5 min each time;

[0107] 3. Use 0.3% Triton X-100 to break the membrane, stand at 37°C for 30 min, use PBS to wash 3 times, 5 min each time;

[0108] 4. To eliminate endogenous peroxidase activity, add endogenous peroxidase blocker and incubate at room temperature for 10 min, use PBS to wash 3 times, 3 min each time, use immunohistochemical pen to circle the tissue area;

[0109] 5. Goat serum blocking at room temperature for 30 min, add primary antibody MPO (1:500) or CD68 (1:500), incubate overnight at 4°C; the next day, warm up for 1 h, use PBS to wash 3 times, 5 min each time;

[0110] 6. Add reaction enhancer, incubate at room temperature for 30 min, use PBS to wash 3 times, 5 min each time;

[0111] 7. Add enhanced enzyme-labeled goat anti-rabbit / mouse lgG polymer solution dropwise, incubate at room temperature for 30 min, use PBS to wash 3 times, 5 min each time;

[0112] 8. Add DAB color developing liquid dropwise, 10-15 s, rinse with tap water, wash with ddH2O;

[0113] 9. Use Mayer hematoxylin for nuclear staining for 5 min, wash excess dye with ddH2O;

[0114] 10. 70% ethanol washing 1 time, 1-3 s; 80% ethanol washing 3 times, 1-3 s each time;

[0115] 11. 95% ethanol washing 1 min, anhydrous ethanol washing 2 times, 5 min each time;

[0116] 12. The slices were soaked in xylene for 2 times, 5 min each time;

[0117] 13. The slices were sealed with neutral gum and observed under microscope for the changes of positive brown area in lung tissue morphology.

[0118] 14. The positive brown area was analyzed and counted using Image-J software.

[0119] 1.3.9 Western Blot detection

[0120] The rats in each group were anesthetized by intramuscular injection of urethane (1.5 g / kg, 2.5 mL / kg). The right lower lobe of the rat lung was taken, the surface residual blood was washed, and the EP tube was filled, and stored at -80°C for standby. Add 1 mL of 1x RIPA lysis buffer, protease inhibitor (1:100) and phosphatase inhibitor (1:100) to each 100 mg of lung tissue. Use the tissue grinder (F6 / 10F013200021; ThermoFisher Scientific, Waltham, USA) to grind the tissue thoroughly, and freeze-thaw in liquid nitrogen for 3 times. Centrifuge at 13000 rpm, 4°C for 30 min (5417R; Eppendorf, Hamburg, Germany). Take the supernatant, add 5x protein gel denaturation buffer, and put it into a dry thermostat (DH100-2; Hangzhou Ruicheng Instrument Co., Ltd., Hangzhou, China) to boil for 15 min. After cooling, centrifuge and mix well, and store at -80°C for standby.

[0121] Protein quantification: Take the protein stock solution and use the BCA protein quantification kit (Beijing Puli Lei Gene Technology Co., Ltd., Beijing, China) to quantify the protein. After diluting the standard and the sample to be tested to a certain proportion, add the working solution (mix the working solution A and the working solution B to uniformity at a ratio of 50:1 by volume), incubate at 37°C for 30 min, develop color, and use a multifunctional enzyme marker (Synergy 2; Bio-Rad, Hercules, CA, USA) to measure the absorbance value at 560 nm. Draw a standard curve to calculate the protein concentration of the sample to be tested.

[0122] Electrophoresis: Add an equal amount of protein of each group to the polyacrylamide gel well, and perform constant voltage electrophoresis. Concentrate the gel at 80V (about 30 min), and separate the gel at 120V (about 90 min). When the bromophenol blue runs to the bottom of the separation gel, stop the electrophoresis. The electrophoresis buffer system is shown in Table 3.5.

[0123] Table 5 10x electrophoresis buffer system

[0124] Dilute the above reagents to 1 L of ddH2O.

[0125] Electrotransfer and development: Subsequently, using sandwich method, according to the order of cathode plate-sponge pad-filter paper-protein gel-PVDF membrane-filter paper-sponge pad-anode plate. Using 250 mA, 90 min constant current electrotransfer to transfer protein bands from protein gel to polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA) under high current. After the end of the transfer, the PVDF membrane was placed in 5% skim milk or 5% BSA in 1x TBST solution, room temperature blocking for 1 hour to block non-specific binding sites. Dilute the primary antibody (1:1000) in 5% skim milk or 5% BSA in 1x TBST solution, 4°C overnight. After warming up for 1 h the next day, the PVDF membrane was washed 3 times with TBST solution for 5 minutes each time. HRP-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, USA) was prepared in 5% skim milk or 5% BSA in 1x TBST solution and incubated at room temperature for 1 h. The PVDF membrane was washed 3 times with TBST solution for 5 minutes each time. ECL luminescent liquid (Beijing Proliferation Gene Technology Co., Ltd., Beijing, China) was added to each membrane, and the Bio-Rad chemical imaging exposure system (1708370; Bio-Rad, Texas, USA) was used to develop in the darkroom. The exposure results were analyzed semi-quantitatively using ImageLab (Bio-Rad, Richmond, CA, USA) and Image J software, and the relative expression of each group of target proteins = target protein band exposure gray value / corresponding internal standard exposure gray value. The electrotransfer buffer system is shown in Table 3.6, and the TBST system is shown in Table 3.7.

[0126] Table 6 10x Electrotransfer Buffer System

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

[0128] Table 7 10x TBST System

[0129] The above reagents were made up to 1 L of ddH2O.

[0130] 1.3.10 Statistical Methods

[0131] All data results were expressed as mean ± SEM (Mean ± SEM), and the software of GraphPad Prism 7.0 (GraphPad Software, CA, USA) was used for plotting and data analysis. One-way ANOVA or Two-way ANOVA statistical methods were applied for pairwise comparison and statistics between data, and Bonferroni correction was used for pairwise comparison of values between groups. When p < 0.05, it was considered that there was significant difference, which was statistically significant.

[0132] 2. Experimental results

[0133] 2.1 Blood flow was lowest at 6 hours after CLP modeling, suitable for anisodamine hydrobromide treatment

[0134] The change of blood flow of rats was continuously detected within 24 hours after CLP modeling, with 0 hour as the baseline. The experimental results are shown in Figure 1. The blood flow was lowest at 6 hours after CLP modeling, so anisodamine was given at this time for treatment.

[0135] 2.2 Effect of anisodamine hydrobromide treatment at 6 hours after CLP modeling on the survival rate of rats for 7 days

[0136] The mortality rate of rats in each group was continuously monitored within 7 days after CLP modeling, with 0 hour as the baseline. The experimental results are shown in Figure 2. The survival rate of rats gradually decreased at 1 day, 2 days, 3 days and 7 days after modeling. The medium and high doses of anisodamine hydrobromide could increase the survival rate for 7 days. Among them, the medium and high doses of anisodamine significantly increased the survival rate for 7 days after CLP modeling.

[0137] 2.3 Effect of anisodamine hydrobromide on heart rate of rats after CLP modeling

[0138] The change of heart rate of rats in each group was continuously monitored within 72 hours after CLP modeling, with 0 hour as the baseline. The experimental results are shown in Figure 3. The heart rate of rats significantly increased at 6 hours and 24 hours after CLP modeling. The medium and high doses of anisodamine hydrobromide could significantly inhibit the increase of heart rate of rats at 24 hours after CLP modeling.

[0139] 2.4 Effect of anisodamine hydrobromide on anal temperature of rats after CLP modeling

[0140] The change of anal temperature of rats in each group was continuously monitored within 72 hours after CLP modeling, with 0 hour as the baseline. The experimental results are shown in Figure 4. The anal temperature of rats significantly decreased at 24 hours and 72 hours after CLP modeling. The low, medium and high doses of anisodamine hydrobromide could improve the decrease of anal temperature of rats at 72 hours after CLP modeling.

[0141] 2.5 Effect of anisodamine hydrobromide on mean arterial pressure of rats after CLP modeling

[0142] The mean arterial pressure of each group of rats was continuously monitored within 72 hours after CLP modeling, with 0 hour as the baseline. The experimental results are shown in Figure 5. The mean arterial pressure of rats decreased after 6 hours of CLP modeling, but there was no significant difference. Anisodamine slightly alleviated the mean arterial pressure of rats in each group, but had no significant effect.

[0143] 2.6 Effect of anisodamine hydrobromide on blood gas analysis of rats after 7 days of CLP modeling

[0144] Figure 6 is the result of blood gas analysis of rats in each group. Compared with the control group, the arterial partial pressure of oxygen, oxygen saturation, and pH value of rats in the CLP model group significantly decreased, and the arterial partial pressure of carbon dioxide and lactic acid content had no significant difference. Anisodamine hydrobromide significantly inhibited the decrease in arterial partial pressure of oxygen, the decrease in oxygen saturation, and the decrease in blood pH value of rats caused by CLP modeling.

[0145] 2.7 Effect of anisodamine hydrobromide on Evans blue exudation of rats after 7 days of CLP modeling

[0146] Figure 7 is the result of lung Evans blue exudation of rats in each group, and the blue area in Figure 7A is the exuded Evans blue. The amount of Evans blue exudation in the Control group was low. Compared with the Control group, the amount of Evans blue exudation of rats after CLP modeling significantly increased. Anisodamine hydrobromide significantly inhibited the increase in the amount of Evans blue exudation caused by CLP. The statistical results are shown in Figure 7B.

[0147] 2.8 Effect of anisodamine hydrobromide on the wet / dry weight ratio of lung tissue of rats after 7 days of CLP modeling

[0148] Figure 8 is the result of the wet / dry weight ratio of lung tissue of rats in each group, and there was no significant difference between the Control group, the model group, and the anisodamine low, medium, and high dose administration groups.

[0149] 2.9 Effect of anisodamine hydrobromide on leukocyte adhesion in lung microvessels of CLP rats

[0150] Figures 9A-C are images of leukocytes labeled with rhodamine 6G adhering to lung microvessels observed using a dynamic visualization microcirculation system, which confirmed that anisodamine hydrobromide can inhibit leukocyte adhesion caused by CLP modeling. There was no leukocyte adhesion in the lung microvessels of rats in the Control group. Compared with the Control group, a large number of leukocytes adhered to the lung microvessel wall in the CLP model group. Anisodamine hydrobromide significantly cleared the leukocyte adhesion caused by CLP. Anisodamine hydrobromide can not only inhibit leukocyte adhesion to lung microvessels, but also clear leukocytes that have adhered to lung microvessels. The statistical results of the number of leukocyte adhesion in lung microvessels are shown in Figure 9D.

[0151] 2.10 Effects of anisodamine hydrobromide on lung microvascular permeability in CLP rats

[0152] Fig. 10 A-C are the results of FITC-dextran extravasation observed using the upright dynamic visualization microcirculation system. The lung microvessels of the Control group did not have obvious extravasation. Compared with the Control group, the lung microvessels of the CLP 6h model group had obvious extravasation of FITC-dextran to the outside of the lung microvessels. High-dose anisodamine hydrobromide significantly inhibited the extravasation of FITC-dextran caused by CLP. The ratio of the FITC fluorescence intensity outside to inside the lung microvessels reflects the permeability of the lung microvessels. Statistical analysis of the lung microvascular extravasation of rats in each group is shown in Fig. 10D.

[0153] 2.11 Effects of anisodamine hydrobromide on lung tissue morphology in CLP rats

[0154] Fig. 11 is the HE staining result of the lung microvessels and surrounding tissues of rats in each group. The lung microvessels and surrounding tissues of rats in the Sham group did not have obvious edema and had few inflammatory cells. After CLP modeling, the lung microvessels and surrounding tissues of rats in the model group had obvious edema, the alveolar septum was obviously thickened, and inflammatory cell infiltration was observed in the alveolar cavity, accompanied by diffuse alveolar damage. The high-dose anisodamine hydrobromide group significantly improved the morphological changes of the lung microvessels caused by CLP modeling.

[0155] Fig. 12 is the HE staining result of the terminal bronchial epithelial cells and surrounding tissues of rats in each group. The terminal bronchial epithelial cells of rats in the Sham group were closely arranged, the villus morphology was complete, and no obvious changes occurred. After CLP modeling, the terminal bronchial epithelial cells of rats in the model group were loosely arranged, and the villus morphology was damaged and shed. The high-dose anisodamine hydrobromide group significantly improved the morphological arrangement disorder of the terminal bronchial epithelial cells caused by CLP.

[0156] 2.12 Effects of anisodamine hydrobromide on the proportion of MPO, CD68 and CD18 positive cells in lung tissue of CLP rats

[0157] Fig. 13 is the result of immunohistochemistry of lung tissue of rats in each group, which shows the inflammatory cell infiltration in the lung tissue of rats. 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 rats in the Sham group was relatively low. Compared with the Sham group, the proportion of MPO, CD68 and CD18 positive cells in the lung tissue of rats in the CLP model group was significantly increased. Anisodamine hydrobromide significantly inhibited the increase in the proportion of MPO, CD68 and CD18 positive cells in the lung tissue caused by CLP.

[0158] 2.13 The influence of anisodamine hydrobromide on the expression of Caveolin-1 in lung tissue of CLP rats

[0159] The expression level of Caveolin-1 was detected by immunofluorescence staining, as shown in Figure 14. Compared with the Sham group, the expression level of Caveolin-1 in the CLP model group did not change significantly. Compared with the CLP model group, the anisodamine hydrobromide administration group significantly reduced the expression of Caveolin-1.

[0160] 2.14 The influence of anisodamine hydrobromide on the expression of Occludin, Claudin-5 and VE-Cadherin in lung tissue of CLP rats

[0161] Figures 15-17 are the results of immunofluorescence staining of tight junction proteins Occludin, Claudin-5 and adherent junction protein VE-Cadherin extracted from lung tissue of rats in each group. The expression of Occludin, Claudin-5 and VE-Cadherin in the intercellular space of vascular endothelial cells in the Sham group was complete and continuous. Compared with the Sham group, the expression of tight junction proteins Occludin, Claudin-5 and adherent junction protein VE-Cadherin between vascular endothelial cells in the lung tissue of rats in the CLP model group degraded and presented a discontinuous state. Anisodamine hydrobromide significantly improved the degradation and discontinuous state of Occludin, Claudin-5 and VE-Cadherin caused by CLP.

[0162] 2.15 The influence of anisodamine hydrobromide on the expression of Collagen IV and Laminin in lung tissue of CLP rats

[0163] The morphology of basement membrane proteins Collagen IV and Laminin was observed by immunofluorescence. As shown in Figures 18 and 19, the expression of Collagen IV and Laminin in the Sham group was complete and continuous, while the expression of Collagen IV and Laminin in the CLP model group was relatively reduced and appeared degraded and broken. Compared with the CLP model group, the morphology of Collagen IV and Laminin in the anisodamine hydrobromide administration group was more continuous.

[0164] 2.16 The influence of anisodamine hydrobromide on the expression of JAM1 and Claudin-5 in lung tissue of CLP rats

[0165] The expression levels of junctional adhesion molecule 1 (JAM-1) and Claudin-5 were detected by Western blot. The results are shown in Figure 20. Compared with the control group, the expression of JAM-1 in the model group was significantly down-regulated, and the high-dose anisodamine treatment reversed the down-regulation of JAM-1; the expression level of Claudin-5 showed no significant change.

[0166] 2.17 Effect of anisodamine hydrobromide on the expression of lung tissue adhesion junction proteins VE-cadherin and α-catenin in CLP rats

[0167] The expression levels of adhesion junction proteins VE-cadherin and α-catenin were detected by Western blot. The results are shown in Figure 21. Compared with the control group, the expression of VE-cadherin in the model group was reduced, and the expression of α-catenin showed no significant change, and the high-dose anisodamine treatment improved the down-regulation of VE-cadherin.

[0168] 2.18 Effect of anisodamine hydrobromide on the expression of lung tissue matrix metalloproteinases MMP-2 and MMP-9 in CLP rats

[0169] The expression levels of matrix metalloproteinases MMP-2 and MMP-9 were detected by Western blot. The results are shown in Figure 22. Compared with the control group, the expression of MMP-2 and MMP-9 in the model group was up-regulated; compared with the model group, the expression of MMP-9 in the model + anisodamine high-dose group was down-regulated, and the expression level of MMP2 showed no significant change.

[0170] 2.19 Effect of anisodamine hydrobromide on the expression of lung tissue energy metabolism related proteins ATP5A, ATP5B and ATP 5D in CLP rats

[0171] The expression levels of energy metabolism related proteins ATP5A, ATP5B and ATP 5D were detected by Western blot. The results are shown in Figure 23. Compared with the control group, the expression of ATP5A in the model group was reduced, and the expression levels of ATP5B and ATP 5D showed no significant change. Compared with the model group, the expression levels of ATP5A, ATP5B and ATP 5D in the model + anisodamine high-dose group showed no significant change.

[0172] 2.20 Effect of anisodamine hydrobromide on the expression of lung tissue plasma membrane microvesicle related proteins Caveolin-1 and Src in CLP rats

[0173] The expression levels of caveolin-1 and Src of the plasma membrane microvesicle related protein were detected by Western blot. The results are shown in Figure 24. Compared with the control group, the expression of caveolin-1 and Src in the model group had no significant change.

[0174] 3. Conclusion

[0175] The present application proves that anisodamine hydrobromide improves the changes in vital signs of rats caused by CLP, improves the changes in heart rate and anal temperature of rats, increases mean arterial pressure of rats, inhibits the increase in arterial carbon dioxide partial pressure and lactic acid of rats caused by CLP, and significantly restores oxygen partial pressure, blood oxygen saturation and pH value. Anisodamine hydrobromide improves the morphological damage of lung tissue caused by CLP, inhibits the edema of lung microvessels and surrounding tissues, increases lung parenchyma, thickens lung interstitium, reduces alveolar cavity area, improves the loose arrangement of terminal bronchial epithelial cells, partial shedding of villi and morphological damage. Anisodamine hydrobromide inhibits the adhesion of rhodamine 6G labeled leukocytes caused by CLP, inhibits the inflammatory cell infiltration of lung tissue, increases the proportion of CD68 and MPO positive cell areas by immunohistochemistry, inhibits the FITC-dextran exudation of microvessels and the exudation of lung Evans blue, inhibits the increase in wet / dry weight ratio of lung tissue, and improves lung interstitial edema. The present application further proves that anisodamine hydrobromide inhibits the low expression of endothelial cell tight junction proteins JAM-1, Occludin, Claudin-5 and adherent junction protein VE-Cadherin in lung tissue.

[0176] The above is only the preferred embodiment of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Anisodamine hydrobromide for use in the preparation of a medicament for regulating proteins in lung tissue. The proteins in the lung tissue include: The lung tissue plasma membrane caveolae-associated proteins, lung tissue energy metabolism-associated proteins, lung tissue matrix metalloproteinases, lung tissue adherens junction proteins, lung tissue tight junction proteins, lung tissue basement membrane proteins and / or lung tissue microvascular endothelial cell junction proteins.

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

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

4. Anisodamine hydrobromide for use in the preparation of a medicament 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 increases in the proportion of lung tissue MPO, CD68 and CD18 positive cell area, improving the morphological arrangement of terminal bronchial epithelial cells, improving lung microvascular morphological changes, improving lung microvascular permeability, clearing lung microvascular leukocyte adhesion and / or inhibiting Evans blue exudation.

6. Anisodamine hydrobromide for use in the preparation of a medicament 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 partial pressure of oxygen, blood oxygen saturation and / or blood pH.

9. Anisodamine hydrobromide for use in the preparation of a medicament for improving the survival rate of a patient with sepsis and / or septic shock.

10. 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.

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