WO2022236599A1 - 玻尿酸用于制备治疗急性呼吸窘迫症药剂的用途 - Google Patents

玻尿酸用于制备治疗急性呼吸窘迫症药剂的用途 Download PDF

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WO2022236599A1
WO2022236599A1 PCT/CN2021/092837 CN2021092837W WO2022236599A1 WO 2022236599 A1 WO2022236599 A1 WO 2022236599A1 CN 2021092837 W CN2021092837 W CN 2021092837W WO 2022236599 A1 WO2022236599 A1 WO 2022236599A1
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rats
hyaluronic acid
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blm
day
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French (fr)
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傅毓秀
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傅毓秀
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the invention relates to the use of hyaluronic acid for treating acute respiratory distress.
  • Acute respiratory distress syndrome is a complex disease that can be caused by multiple factors.
  • Berlin definition Bact al., 2014
  • patients exposed to risk factors within seven days resulting in acute diffuse inflammation of the lungs, hypoxemia, and respiratory failure (Mattay et al., 2012).
  • Acute respiratory distress disease will destroy the lung structure, and patients will be admitted to the intensive care unit due to respiratory failure and multiple organ failure, resulting in a huge consumption of medical manpower and medical resources.
  • there is no drug to treat acute respiratory distress only supportive therapy, so the fatality rate is extremely high. Therefore, ARDS has already been a rather troublesome disease in the field of intensive care medicine.
  • the development of drugs for acute respiratory distress disease can not only reduce the severity of acute respiratory distress disease, reduce the mortality rate of patients with acute respiratory distress disease, but also avoid subsequent damage to the lungs or other organs, and can also alleviate severe hospital aggravation. Stress in the nursing ward.
  • the present invention aims to provide a new treatment direction for acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • the present invention provides the use of hyaluronic acid for the manufacture of a medicament for treating acute respiratory distress.
  • the present invention provides a medicament or a pharmaceutical composition for treating acute respiratory distress, which comprises a therapeutically effective amount of hyaluronic acid.
  • the present invention provides a method for treating acute respiratory distress, comprising administering to an individual in need thereof a medicament or a pharmaceutical composition comprising a therapeutically effective amount of hyaluronic acid.
  • the method of the invention is particularly suitable for use in severe acute respiratory distress.
  • hyaluronic acid with a molecular weight of 10 kDa-2 MDa can be used in the present invention.
  • administering can effectively improve the symptoms of ARDS, slow down the significantly increased respiratory rate due to ARDS, significantly increase the blood oxygen concentration, improve lung volume and alveolar volume, and reduce alveolar epithelial Cell deformation, reduce inflammation, reduce collagen accumulation, promote the generation of anti-inflammatory M2 cells, increase the activity of MMP (matrix-metallopeptidase) to promote anti-inflammatory response and decompose fibrosis, and can stimulate the regeneration of alveolar epithelial cells.
  • MMP matrix-metallopeptidase
  • hyaluronic acid is effective in improving decreased blood oxygen saturation levels, alleviating increased respiration rate and/or restoring alveolar function.
  • Figure 1 shows the experimental flow chart of inducing severe acute respiratory distress in rats and administering hyaluronic acid. They were divided into 5 groups. Each group injected BLM into the left bronchus of rats on the 0th day, and gave drug treatment on the 7th day. Animals were sacrificed on day 28.
  • Figure 2 shows the change of blood oxygen index in rats within 7 days after BLM injury.
  • Part A of Fig. 2 shows the photographs of arterial blood oxygen saturation of hindlimbs of rats detected every day for 7 days after BLM injury.
  • Part B in Fig. 2 is the quantitative graph of arterial saturation in rats every day within 7 days after BLM injury, which shows that the oxygen saturation in arterial blood drops to the lowest on the seventh day after BLM injury.
  • Part C in Figure 2 is a quantitative map of the partial pressure of oxygen in arterial blood of rats within 7 days after BLM injury, which shows that the partial pressure of oxygen in arterial blood dropped to the lowest on the seventh day after BLM injury.
  • Part D in Figure 2 is a quantitative map of the partial pressure of carbon dioxide in the arterial blood of rats within 7 days after the BLM injury, which shows that the partial pressure of carbon dioxide in the arterial blood rose to the highest on the seventh day after the BLM injury. According to the arterial blood oxygen index, it was confirmed that the animal model of severe acute respiratory distress in rats was successfully established. Compared with Day 0 (Day 0), there is a statistical difference, p ⁇ 0.05. @: Compared with Day 1 (Day 1), there is a statistically significant difference, p ⁇ 0.05. Compared with Day 4, there is a statistical difference, p ⁇ 0.05.
  • Figure 3 shows the changes in the respiration rate of rats within 7 days after BLM injury.
  • Part A in Fig. 3 shows the recordings of the respiration rate of the rats detected every day within 7 days after the BLM injury, and the respiration rate graph of 2 seconds was extracted.
  • Part B in Figure 3 is the quantitative graph of the breathing rate per minute of the rats within 7 days after the BLM injury, which shows that the breathing rate reached the highest on the seventh day of the BLM injury. According to the number of breaths per minute, it was confirmed that the animal model of severe acute respiratory distress in rats was successfully established. Compared with Day 0, there is a statistical difference, p ⁇ 0.05. @: Compared with Day 1, there is a statistically significant difference, p ⁇ 0.05.
  • Figure 4 shows the macroscopic morphology of the lungs of rats after sacrificial perfusion on different days after BLM injury. It can be observed from the figure that on the first day of BLM injury, the surface of the left lung began to appear uneven. On the second day after the BLM injury, the alveoli in the central area of the left lung had partially disappeared. On the seventh day of BLM injury, the left lung shrank and the alveoli in the central area disappeared. According to the macroscopic shape of the left lung, it was determined that the animal model of severe acute respiratory distress in rats was successfully established.
  • Figure 5 shows the microscopic morphology of the left lung of rats at different days after BLM injury.
  • Part A in Figure 5 is a low magnification image of left lung tissue sections of rats stained with HE at different days, and the white space is alveolar tissue.
  • Part B in Figure 5 is a high-magnification view of the tissue sections in the central region of the left lung (the red frame area in Part A in Figure 5) of rats at different days, which shows that after BLM injury, cells infiltrate and alveolar gradually decreases.
  • Part C in Figure 5 is a high-magnification image of tissue sections in the outer peripheral area of the left lung (the blue frame area in Part A in Figure 5) of rats at different days, which shows that there are still some alveoli after BLM injury (white space), only the alveoli progressively decreased with increasing number of days of injury.
  • Part D in Figure 5 is the total volume of the left lung at different days after quantification after HE staining;
  • Part E in Figure 5 is the alveolar volume of the left lung;
  • Part F in Figure 5 is the proportion of left lung cell infiltration.
  • Parts DF in Figure 5 show that the volume of the left lung gradually decreased after BLM injury, the number of alveoli decreased, and the proportion of cell infiltration increased. According to the microscopic morphology of the left lung, it was determined that the animal model of severe acute respiratory distress in rats was successfully established. Compared with the rats on Day 0, there is a statistically significant difference, p ⁇ 0.05.
  • Figure 6 shows that the administration of hyaluronic acid can increase the body weight of ARDS sick mice.
  • the difference was statistically significant, p ⁇ 0.05.
  • p ⁇ 0.05 Compared with the rats in the BLM+MIX HA group on the same day, there was a statistically significant difference, p ⁇ 0.05.
  • ⁇ Compared with the rats in the BLM+LHA group on the same day, there is a statistically significant difference, p ⁇ 0.05.
  • Figure 7 shows that administration of hyaluronic acid can increase the oxygen saturation of arterial blood in ARDS mice.
  • Part A in Fig. 7 is the photos of rats in each group undergoing pulse-type arterial oximeter detection on the 28th day, where the arrow points to the value of arterial blood oxygen saturation.
  • Part B in Fig. 7 is to quantify the values of arterial blood oxygen saturation in rats in each group at different times. Compared with the rats in the normal group on the same day, the difference was statistically significant, p ⁇ 0.05. #: Compared with the rats in the BLM group on the same day, there is a statistically significant difference, p ⁇ 0.05. Compared with the rats in the BLM+MIX HA group on the same day, there was a statistically significant difference, p ⁇ 0.05. ⁇ , Compared with the rats in the BLM+LHA group on the same day, there is a statistically significant difference, p ⁇ 0.05.
  • Figure 8 shows that the administration of hyaluronic acid can slow down the shortness of breath in ARDS sick mice.
  • Parts A and E in Figure 8 are 2-second recordings of the respiratory rate of rats in each group at different times.
  • Part F in Fig. 8 is to quantify the respiration rate per minute of rats in each group at different times. Compared with the rats in the normal group on the same day, there was a statistical difference, p ⁇ 0.05. #: Compared with the rats in the BLM group on the same day, there is a statistical difference, p ⁇ 0.05. Compared with the rats in the BLM+MIX HA group on the same day, there was a statistically significant difference, p ⁇ 0.05.
  • Figure 9 shows that the administration of hyaluronic acid can increase the volume of the left lung of ARDS mice.
  • the picture shows the appearance of the lungs of rats in each group on day 28.
  • the upper row is the frontal photo of the lungs of each group, and the lower row is the back photo of the lungs of each group.
  • Figure 10 shows that the administration of hyaluronic acid can restore the alveolar structure of ARDS mice.
  • Part A in Figure 10 is a low-magnification picture of the left lung tissue slices of rats in each group on the 28th day after HE staining.
  • Part B in Fig. 10 is a high-magnification enlarged central area photograph of the left lung tissue slices of each group on day 28 after HE staining.
  • Part C in Fig. 10 is a high-magnification magnified photo of the left lung tissue slices of each group on day 28 after HE staining.
  • Part D in Figure 10 sums all left lung tissue sections to quantify left lung volume.
  • Part E in Figure 10 quantifies the total alveolar volume of the left lung.
  • Figure 11 shows that the administration of hyaluronic acid can reduce the inflammatory response in the lungs of ARDS mice and the deformation response of alveolar epithelial cells.
  • Part A in Fig. 11 is the quantification of the number of cells in the alveolar flushing fluid of the left lung of rats in each group on day 28 to represent the inflammation situation. The number of cells in the alveolar flushing fluid of the left lung of rats in the BLM group increased significantly.
  • Part B in Fig. 11 is the left lung of rats in each group on day 28, and the concentration of N-cadherin in the left lung was quantified by western blotting to represent the deformation of alveolar epithelial cells.
  • p ⁇ 0.05 Compared with the rats in the normal group, there is a statistically significant difference, p ⁇ 0.05.
  • Figure 12 shows that the administration of hyaluronic acid can promote the second type of macrophages and reduce the first type of macrophages in the left lung of ARDS patients to reduce the inflammatory response.
  • Part A in Figure 12 shows the left lungs of rats in each group on day 28, which were immunostained with Anti-ED1 antibody to mark macrophages. The results showed that most of the phagocytes in the BLM group were smaller. The phagocytes in the BLM+MIX HA group were mostly larger.
  • Part B in Figure 12 is the left lung tissue of rats in each group on day 28, which was stained with Anti-CD86 antibody by western blot method to quantify the changes of M1 macrophages.
  • Part C in Figure 12 is the left lung tissue of rats in each group on day 28, which was stained with Anti-CD206 antibody by western blot method to quantify the changes of M2 macrophages. Compared with the rats in the normal group, there was a statistical difference, p ⁇ 0.05. #: Compared with the rats in the BLM group, there is a statistically significant difference, p ⁇ 0.05.
  • Figure 13 shows that the administration of hyaluronic acid can promote the synthesis of MMP in the left lung of ARDS mice and reduce the inflammatory response.
  • Part A in Figure 13 is the left lung tissue of rats in each group on day 28, which was stained with Anti-MMP9 antibody by western blot method to quantify the protein content of MMP9.
  • Part B in Figure 13 is the left lung tissue of rats in each group on day 28, which was stained with Anti-MMP2 antibody by western blot method to quantify the protein content of MMP2.
  • p ⁇ 0.05 Compared with the rats in the normal group, there was a statistical difference, p ⁇ 0.05.
  • Figure 14 shows that the administration of hyaluronic acid can promote the synthesis of TLR4 in the left lung of ARDS mice to stimulate the regeneration of alveolar epithelial cells.
  • the picture shows the left lung tissues of rats in each group on day 28, stained with Anti-TLR4 antibody by western blot method to quantify the TLR protein content. Compared with the rats in the normal group, there was a statistical difference, p ⁇ 0.05. #: Compared with the rats in the BLM group, there is a statistical difference, p ⁇ 0.05.
  • the articles “a” and “an” refer to one or more than one (ie, at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • ARDS acute respiratory distress syndrome
  • hyaluronic acid hyaluronan or hyaluronic acid
  • HA hyaluronic acid
  • hyaluronic acid can be divided into low molecular weight hyaluronic acid (MW 10kDa-100kDa), medium molecular weight hyaluronic acid (MW 100kDa-1MDa), and high molecular weight hyaluronic acid (>MW 1MDa) (Tavianatou et al., 2019).
  • the hyaluronic acid used in the present invention may include the above hyaluronic acid or its salts with different molecular weights, and mixtures thereof, and the molecular weight ranges from 10kDa to 2MDa.
  • an index related to lung function or a reduced or increased level of a disease symptom as described herein is referenced to its control (or normal) level.
  • control level describes the value that a person of ordinary skill in the art and/or a medical professional would expect a healthy individual or group of people with similar physical characteristics and medical history to have.
  • an elevated level means 5%, 10%, 20%, 30%, 50%, 70%, 90%, 100%, 200%, 300%, 500% or more above a control (or normal) level , compared with the control (or normal) level; and the reduced level refers to 5%, 10%, 20%, 30%, 50%, 70%, 90%, 100% lower than the control (or normal) level, 200%, 300%, 500% or more, compared to control (or normal) levels.
  • subject in need of treatment means a human or non-human animal in need of treatment for ARDS.
  • an individual in need of the methods of treatment of the present invention is diagnosed with ARDS with the following symptoms: (1) acute onset; (2) decreased oxygenation index (PaO2/FIO2); (3) chest X-ray showing Both sides of the lung infiltrate; (4) If the pulmonary artery wedge pressure is measured, it is less than or equal to 18 mm Hg. If there is no data, it means that there is no left atrial hypertension clinically.
  • subject includes human and non-human animals, such as companion animals (such as dogs, cats, etc.), farm animals (such as cows, sheep, pigs, horses, etc.), or laboratory animals (such as large rats, mice, guinea pigs, etc.).
  • companion animals such as dogs, cats, etc.
  • farm animals such as cows, sheep, pigs, horses, etc.
  • laboratory animals such as large rats, mice, guinea pigs, etc.
  • treating refers to administering or administering a composition comprising one or more effective active agents to a subject suffering from a disease, disease condition or disease symptom or disease progression (exacerbation), with the purpose of curing, curing , to alleviate, relieve, alter, ameliorate, ameliorate, enhance, or affect the disease, the condition or symptom of the disease, the disease-induced disorder or the progression of the disorder.
  • terapéuticaally effective amount refers to the amount of an active ingredient which provides a desired therapeutic or biological effect in a subject to be treated. For example, an effective amount for treating ARDS.
  • a therapeutically effective amount can vary depending on various reasons, such as the route and frequency of administration, the body weight and species of the individual receiving the drug, and the purpose of administration. Those skilled in the art can determine the dosage in each case from the disclosure herein, established methods and their own experience.
  • Administration according to the invention may be by various procedures known in the art, and may be administered systemically, for example, intravenously, intraarterially, or subcutaneously via injection, or by nasal inhalation, or nasal or oral administration. Stretching into the trachea for administration, or tracheotomy for administration.
  • the hyaluronic acid containing hyaluronic acid can be sent to the lungs by the usual method in the field of the present invention, and a specific embodiment is to send it to the lungs by injection through the blood, or directly from the respiratory tract, oral cavity (such as nose, mouth, etc.) , or trachea) to the lungs. In another embodiment, delivery to the desired area is by direct injection.
  • Hyaluronic acid as an active ingredient can be formulated into a suitable form of pharmaceutical composition or medical device together with a pharmaceutically acceptable carrier for delivery.
  • the pharmaceutical composition or medical device of the present invention may contain about 0.1% to about 100% by weight of the active ingredient, wherein the weight percentage is calculated based on the weight of the entire composition.
  • a "pharmaceutically acceptable carrier” is nontoxic to the individual at the dosages and concentrations employed and is compatible with hyaluronic acid and any other ingredients of any formulation comprising hyaluronic acid.
  • a suitable isotonic liquid eg, phosphate buffered saline, physiological saline, aqueous dextrose and/or mixtures thereof, and other suitable liquids known to those skilled in the art.
  • the final therapeutic form should be protected from contamination and should be able to inhibit the growth of microorganisms such as bacteria or fungi.
  • One embodiment is to administer a single dose. Alternatively, a slow long-term infusion or multiple short-term daily infusions may be used. Alternating days or dosing once every few days may also be used if desired.
  • hyaluronic acid can be divided into low molecular weight hyaluronic acid (MW 10kDa-100kDa), medium molecular weight hyaluronic acid (MW 100kDa-1MDa), and high molecular weight hyaluronic acid (>MW 1MDa) (Tavianatou et al., 2019).
  • This example uses different A blend of molecular weight hyaluronic acid, high molecular weight hyaluronic acid, and low molecular weight hyaluronic acid.
  • hyaluronic acid was given three times in the second week, and hyaluronic acid was given twice in the third week and the fourth week, that is, on the 7th, 9th, 11th, 14th, 17th, and 21st day after the BLM injury. , 24 days to give hyaluronic acid (Figure 1). All were injected into the mice under anesthesia through the trachea.
  • the experimental animals were divided into five groups:
  • the first group is the normal group, that is, on day 0, the normal rats received intratracheal injection of 200 ⁇ l of normal saline, and on day 7, only 200 ⁇ l of normal saline was injected into the trachea (Figure 1).
  • the second group is the BLM group, that is, on the 0th day, the rats received intratracheal injection of 5 mg BLM. From day 7 after BLM injection, only 200 ⁇ l of normal saline was administered intratracheally without any treatment (Fig. 1).
  • the third group was BLM+LHA group, that is, on day 0, rats received 5 mg BLM intratracheal injection. Starting from day 7 after BLM injury, low molecular weight hyaluronic acid (MW10kDa-100kDa) was intratracheally administered seven times in total (Fig. 1).
  • MW10kDa-100kDa low molecular weight hyaluronic acid
  • the fourth group is the BLM+HHA group, that is, on the 0th day, the rats received 5 mg of BLM intratracheally. Beginning on the 7th day after the BLM injury, high molecular weight hyaluronic acid (>MW 1MDa) was administered intratracheally for a total of seven times (Fig. 1).
  • the fifth group is the BLM+MIX HA group, that is, on day 0, the rats received 5 mg BLM intratracheally.
  • mixed molecular weight hyaluronic acid MW 10kDa-2MDa was intratracheally administered seven times in total (Fig. 1).
  • Rats in each group were measured weekly for their body weight, arterial blood oxygen concentration, and respiratory rate. They were sacrificed on day 28 after BLM administration, and their lung types were observed.
  • mice were anesthetized with Isoflurane (Baxter228-194), the rear paws were clamped with a pulse oximeter (Pulseoximeter, NONIN LS1-10R) to detect the oxygen saturation in the arterial blood.
  • a pulse oximeter Pulseoximeter, NONIN LS1-10R
  • the experimental animals were placed in a closed cylindrical breathing detection chamber (emka Technologies, Whole body plethysmograph), and the changes in the respiratory airflow of the rats within 15 minutes were collected with BIOPAC BSL 4.0MP45 software, and the amount of time the rats were kept in the chamber was quantified. breathing rate during movement.
  • a closed cylindrical breathing detection chamber emka Technologies, Whole body plethysmograph
  • mice Excessive Zoletil 50 and Xylazine hydrochloride (Sigma 23076359) were intraperitoneally injected into the mice, anesthetized to death, and then perfused. After perfusion, the left and right lungs were taken out, placed in an environment at 4°C and fixed for 2 days, and then embedded in paraffin.
  • the tissue wax block was shaved off excess paraffin and trimmed into a trapezoid. Fix the tissue wax blocks on a paraffin microtome. Cut the lung tissue into 5 ⁇ m thick slices. The tissue slices were flattened in warm water at 40°C to 45°C, and then the lung tissue slices were pasted on glass slides and dried on a heating platform at 50°C.
  • the lung tissue sections were first dewaxed, put in xylene, alcohols with decreasing concentration (100%, 95%, 80%, 70% alcohol), placed in hematoxylin solution (Muto Chemical, No. 3008-1) for 5 minutes, then place the lung tissue slices in eosin (Eosin) solution (Muto Chemical, No. 3200-2) for 2.5 minutes, then soak the tissue slices in glacial acetic acid for 3 seconds, and then Rinse with running water.
  • the lung tissue sections were soaked in alcohols with increasing concentrations for dehydration (50%, 70%, 80%, 90%, 95%, 100% in sequence), and then soaked in xylene twice for 5 minutes each time. Finally, the slides were sealed with mounting glue (Permount, Fisher Scientific SP15-500) for optical microscope observation and photographing.
  • the whole lung was taken out from the trachea, a 20G needle was used with a PE cannula (PE60, inner diameter 0.76 ⁇ outer diameter 1.22mm), and the intubation tube was inserted into the left lung from the left bronchus. Rinse with PBS and aspirate. Then, wash for the second time, flush once with new 0.5ml sterile PBS; thus obtain 1ml of alveolar flushing fluid. The obtained alveolar flushing fluid was centrifuged at 1500 rpm for 5 minutes, and the cells in the lower layer were redissolved with 1 ml of saline for cell counting.
  • PE60 PE60, inner diameter 0.76 ⁇ outer diameter 1.22mm
  • NC paper after electrophoresis of left lung protein, were added with primary antibody anti-N cadherin (anti-N cadherin) (Abcam ab18203, 1:1000), anti-ED1 antibody ( Millipore MAB1435, 1:400), anti-CD86 antibody (Proteintech 13395-1-AP, 1:1000), mouse anti-CD206 (Abcam ab646931:1000), anti-MMP9 (Abcam ab76003, 1:1000), anti - MMP2 (Abcam ab92536, 1:1000), anti-TLR4 (Abcam ab30667, 1:1000), react at 4°C for 16 to 18 hours.
  • anti-N cadherin Abcam ab18203, 1:1000
  • anti-ED1 antibody Millipore MAB1435, 1:400
  • anti-CD86 antibody Proteintech 13395-1-AP, 1:1000
  • mouse anti-CD206 Abcam ab646931:1000
  • anti-MMP9 Abcam ab76003, 1:1000
  • anti - MMP2 Anti-TLR4
  • the pulse oximeter detects the oxygen saturation of the arterial blood in the soles of the hind limbs, which can represent the oxygen exchange efficiency of the lungs.
  • Arterial oxygen saturation was typically maintained at 97% prior to injury (Day 0). It would drop to 87.7 ⁇ 1.2% on the first day after injury, then to 84.7 ⁇ 1.7% on the second day, and only 83.7 ⁇ 0.9% remained on the 7th day after injury (parts A and B in Figure 2).
  • the second detection method is to extract blood from the tail artery of mice to detect the partial pressure of oxygen and carbon dioxide in the arterial blood.
  • the oxygen partial pressure (PO2) of the arterial blood was about 88.1 ⁇ 2.7mmHg; on the 4th day after the injury, it would drop significantly to 74.2 ⁇ 4.2mmHg; on the 7th day after the injury, it was only 66.3 ⁇ 3.9mmHg ( Part C in Figure 2).
  • the partial pressure of carbon dioxide (PCO2) in the arterial blood was about 46.4 ⁇ 1.6mmHg; on the 4th day after the injury, it would rise to 49.0 ⁇ 1.3mmHg; on the 7th day after the injury, it would rise to 53.8 ⁇ 2.0mmHg (Part D in Figure 2).
  • the third detection is the respiratory rate per minute.
  • both the left and right lungs of normal rats are smooth and complete, and the white is the position of the normal alveoli.
  • the left lung had already developed a bumpy surface.
  • the white alveoli had been partially lost.
  • the white alveoli in the central area of the left lung disappeared completely, leaving only the alveoli in the surrounding area.
  • the left lung shrank significantly, and only the alveoli remained in the surrounding area (Fig. 4).
  • HE staining was performed on the left lung tissue sections to observe changes in fine patterns (part A in FIG. 5 ).
  • the volume of the left lung in the normal group was about 317.6 ⁇ 19.4 mm3 cubic millimeters. On the 7th day after BLM injury, it was significantly reduced to about 217.2 ⁇ 17.0mm3, which was significantly atrophied compared with the normal group (part D in Figure 5). In the shrunken left lung at this time, the volume of the alveolar structure also decreased to 59.7 ⁇ 8.4cm3; the solidified tissue infiltrated by a large number of cells accounted for about 63.8 ⁇ 2.5% of the left lung volume (parts E and F in Figure 5).
  • the weight of rats in the normal group gradually increased with time. On the 7th day after BLM injury, the body weight of rats in each group stagnated obviously. Afterwards, although the body weight increased slightly over time, the weight of the rats in each group showed a significant decrease compared with the normal group, and the trend of less weight than the normal group continued until the 28th day.
  • the body weight of the rats in the BLM+HHA group was always similar to that of the BLM group.
  • the body weight of the rats in the BLM+HHA group was less than that in the BLM+MIX HA group from the 14th day to the 28th day. Compared with the rats in the BLM group, the body weight of the rats in the BLM+LHA group increased significantly on the 28th day.
  • the body weight of the rats in the BLM+MIX HA group was statistically increased on the 21st day and the 28th day compared with the body weight of the rats in the BLM group ( Figure 6).
  • the arterial blood oxygen saturation of rats in the BLM+HHA group was about 86% on the 28th day, and there was no statistical difference compared with the BLM injury group.
  • the arterial blood oxygen saturation of rats in the BLM+HHA group was less than that in the BLM+MIX HA group from the 14th day to the 28th day. From the 14th day to the 28th day, the arterial blood oxygen saturation of rats in the BLM+LHA group and the BLM+MIX HA group increased significantly compared with the BLM group.
  • the arterial oxygen saturation of the rats in the BLM+MIX HA group was significantly improved on the 21st day and the 28th day compared with the BLM+LHA group (parts A and B in Figure 7).
  • the partial pressure of oxygen in arterial blood of the rats in the BLM+HHA group was still lower than that in the rats in the BLM+MIX HA group on day 28.
  • the partial pressure of oxygen in arterial blood of rats in the BLM+LHA group was significantly higher than that in the BLM group on day 28.
  • the arterial partial pressure of oxygen in rats in the BLM+MIX HA group had significantly increased on the 14th day, and this upward trend continued until the 28th day.
  • the arterial partial pressure of oxygen was significantly improved (Figure 7 Part C of ).
  • the partial pressure of carbon dioxide in the arterial blood of rats was about 46.6-47.9 mmHg (part D in Figure 7).
  • the partial pressure of carbon dioxide in the arterial blood of rats in each group was significantly higher than that in the normal group, rising to 51.6-54.7mmHg.
  • the partial pressure of carbon dioxide in each group was still elevated. Only the partial pressure of carbon dioxide in the arterial blood of rats in the BLM+MIX HA group had significantly decreased on the 14th day, which was lower than that of the BLM group and similar to that of the Normal group.
  • the partial pressure of carbon dioxide in the arterial blood of rats in other groups had no statistical difference from the normal group rats on day 21 (part D in FIG. 7 ).
  • the respiration rate of the rats in the BLM+HHA group was still higher than that of the rats in the BLM+MIX HA group on the 21st day and the 28th day (parts D and F in Figure 8).
  • the respiratory rate of the rats in the BLM+LHA group was significantly reduced on the 14th day, but the respiratory rate was still higher than that of the normal group (parts C and F in FIG. 8 ).
  • the respiratory rate of the rats in the BLM+MIX HA group decreased significantly on the 14th day, and this trend continued until the 28th day; There was no statistical difference between the respiratory rate on day 1 and the 28th day and the normal group (parts E and F in Figure 8).
  • the administration of hyaluronic acid can improve the atrophy of the left lung of severe acute respiratory distress rats
  • the rats in each group were sacrificed, and the left and right lungs were taken to observe the appearance of the lungs.
  • the front view (upper row) and back view (lower row) photos show that white alveolar structures can be seen in the left and right lungs of the rats in the normal group, and the alveoli are complete and smooth.
  • the left lung shrank significantly, a very small amount of alveolar tissue appeared only in the outer periphery of the left lung, and the central area of the left lung had already presented pathological tissue without alveoli.
  • the white alveolar area of the left lung and the overall volume of the left lung of the rats in the BLM+MIX HA group were significantly larger than those in the BLM injury group (Fig. 9).
  • the continuous tissue sections of the left lungs of rats in each group were stained with HE, and the low-magnification pictures (part A in Fig. 10) were taken, and then the central area of the left lung (part B in Fig. 10) and the surrounding area (part B in Fig. Part C of 10).
  • the results showed that the left lung area of the rats in the normal group was larger and there were more alveoli, the connective tissue only appeared around the bronchi, and there was very little connective tissue between alveoli and alveoli.
  • complete alveoli only appeared in the outer periphery of the left lung, and a large number of cells were infiltrated in the central area, and the existence of alveoli was almost invisible.
  • hyaluronic acid can reduce the inflammatory reaction in the left lung of severe acute respiratory distress mice and the EMT reaction with alveolar epithelial cells
  • the alveolar flushing fluid of the left lung of the rats in each group was collected for cell counting.
  • the results showed that the number of cells in the alveolar flushing fluid of the left lung of the rats in the BLM group increased significantly.
  • the number of cells in the alveolar flushing fluid of the rats in the BLM+LHA group and the BLM+HHA group was significantly reduced, but it was still much higher than that in the Normal group.
  • the number of cells in the alveolar flushing fluid of the left lung of the rats in the BLM+MIX HA group was significantly reduced, and there was no statistical difference with the Normal group.
  • hyaluronic acid can reduce the pulmonary inflammatory response of severe acute respiratory distress disease mice, therefore, the number of cells in the left lung is reduced, especially, the administration of hyaluronic acid of various molecular weights is similar to that of the normal group (A in Fig. 11 part).
  • Alveolar Type II cells will proliferate in large quantities and undergo epithelial cell deformation (epithelial mesenchymal transition, referred to as EMT). At this time, N-cadherin (N-cadherin) is expressed in large quantities. At this time, Alveolar Type II cells transform into myofibroblasts, resulting in reduced alveoli and tissue scarring.
  • EMT epithelial mesenchymal transition
  • hyaluronic acid can promote the activation of type II macrophages in the left lung of severe acute respiratory distress rats for anti-inflammation.
  • the left lung tissue sections of rats in each group were immunostained with anti-ED1 antibody to mark macrophages.
  • the results showed that only a small amount of small macrophages existed in the left lung of the rats in the normal group.
  • the left lung tissue of the rats in the BLM group a large number of macrophages with small particles appeared.
  • the larger phagocytes were distributed between the connective tissue and the alveolar space (A in Figure 12 Part), it is speculated that these larger macrophages are the second type of macrophages with anti-inflammatory effect.
  • Type I macrophages were identified by Western blot with anti-CD86 antibody. Type I macrophages have pro-inflammatory effects. The results show. The content of CD86 in the left lung of rats in the BLM group increased. However, the concentration of CD86 in the left lung of rats in the BLM+MIX HA group decreased, which was statistically different from that in the BLM group (Part B in Figure 12). It is speculated that the administration of hyaluronic acid can prevent the differentiation and transformation of type 1 macrophages in the left lung of severe acute respiratory distress mice, so as to reduce the inflammatory response.
  • Anti-CD206 antibody Western blot method was used to mark the second type of macrophages, and the second type of macrophages has anti-inflammatory effect.
  • the concentration of CD206 in the left lung of rats in the BLM+MIX HA group increased statistically compared with the normal (Normal) group and the BLM group (Part C in Figure 12). It is speculated that the administration of hyaluronic acid can promote the differentiation and transformation of type II macrophages in the left lung of severe acute respiratory distress mice to carry out anti-inflammatory reactions.
  • hyaluronic acid can stimulate the production of MMP in the left lung of severe acute respiratory distress rats and reduce the inflammatory response
  • MMP-9 matrix metalloproteinase-9
  • the results showed that the MMP-9 in the left lung of the rats in the BLM group decreased compared with the normal group. situation.
  • the MMP-9 in the left lung of the rats in the BLM+MIX HA group was significantly increased, which was statistically different from that in the BLM group (Part A in Figure 13).
  • MMP-2 matrix metalloproteinase-2
  • the content of matrix metalloproteinase-2 (Matrix metallopeptidase, referred to as MMP-2) protein in the left lung of the rats in each group was quantified.
  • the results showed that the MMP-2 in the left lung of the rats in the BLM group was not significantly higher than that in the normal group.
  • the MMP-2 in the left lung of the rats in the BLM+MIX HA group was significantly increased, which was statistically higher than that of the normal group (Part B in Figure 13). It is speculated that the administration of hyaluronic acid can stimulate the synthesis of MMP in the left lung of severe acute respiratory distress rats, thus reducing the inflammatory response.
  • Type II alveolar epithelial cells express a large number of Toll-like receptor 4 (TLR-4), which can stimulate the regeneration of alveolar epithelial cells (Yang et al., 2012; Liang et al., 2016). Therefore, Western blot method was performed with anti-TLR-4 antibody to observe the content of TLR-4 protein in the left lung of rats in each group. The results showed that there was only a small amount of TLR-4 expression in the left lungs of the rats in the normal group and the BLM injury group. In the BLM+MIX HA group, the expression of TLR-4 in the left lung of the rats was significantly increased, and compared with the normal group and the BLM injury group, there were statistical differences (Figure 14). It is speculated that the administration of hyaluronic acid can increase the production of TLR-4 protein in the left lung of severe acute respiratory distress rats, so as to enhance the regeneration and repair of alveolar epithelial cells.
  • TLR-4 Toll-like receptor 4
  • Hyaluronic acid with different molecular weights has the function of improving ARDS, and hyaluronic acid with mixed molecular weights has the best effect in improving and treating severe acute respiratory distress. Therefore, giving hyaluronic acid is believed to also slow down the threat of the COVID-19 pandemic.

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Abstract

玻尿酸用于制备治疗急性呼吸窘迫症药物的用途。玻尿酸可减少肺组织的病理损伤，促进肺泡再生，增加肺泡功能的功效，对临床急性呼吸窘迫症提供一个新的治疗方向。

Description

玻尿酸用于制备治疗急性呼吸窘迫症药剂的用途 技术领域
本发明涉及玻尿酸用于治疗急性呼吸窘迫症的用途。
背景技术
急性呼吸窘迫症(acute respiratory distress syndrome，简称ARDS)，是一种复杂且多个因素都会导致的疾病。根据柏林定义(Berlin definition)，病患暴露在危险因子七天之内，产生急性肺部弥漫性发炎、低血氧、与呼吸衰竭等现象(Mattay et al.,2012)。是一种急性肺损伤的症状，是具有生命威胁性的疾病。急性呼吸窘迫症会破坏肺部结构，病人会因呼吸衰竭、以及多重器官衰竭，而住进加护病房，造成耗费巨大的医护人力及医疗资源。在目前临床医学上，没有药物可以治疗急性呼吸窘迫症，只有支持性的疗法，因此致死率极高，所以，ARDS在重症医学界原本就已经是一个相当令人头痛的疾病。
2020年，更因为新冠肺炎的大流行，导致ARDS病患大量的增加。根据统计，有高达31％-42％新冠肺炎住院的病人会有ARDS，而其中又有61-81％的病患需要住进加护病房(Wu et al.,2020)(Gibson et al.,2020).这些新冠病毒感染的重症患者，在急性期都有严重的肺部发炎反应，在恢复期也有不同程度的肺脏纤维化现象，造成肺功能永久性的退化、甚至导致其他器官的终生损害。因此，针对急性呼吸窘迫症发展药物，不仅可以减轻急性呼吸窘迫症病情的严重程度、降低急性呼吸窘迫症病人的死亡率、也可以避免后续肺脏或其他器官的损伤情况、更可以缓解医院重症加护病房的压力。
发明内容
本发明旨在提供一种急性呼吸窘迫症(acute respiratory distress syndrome,简称ARDS)新的治疗方向。
一方面，本发明提供玻尿酸用于制造治疗急性呼吸窘迫症药剂的 用途。
另一方面，本发明提供一种用于治疗急性呼吸窘迫症的药剂或医药组合物，其包含治疗有效量的玻尿酸。
再一方面，本发明提供了一种治疗急性呼吸窘迫症的方法，包含对有此需要的个体施用包含治疗有效量的玻尿酸的药剂或医药组合物。
根据本发明，本发明方法特别适用于严重的急性呼吸窘迫症。
根据本发明，本发明可使用分子量10kDa-2MDa的玻尿酸。
根据本发明实施例，对于ARDS模型的大白鼠施打玻尿酸，可有效改善ARDS的症状，可减缓因ARDS明显上升的呼吸频率、使血氧浓度显著回升、改善肺脏体积与肺泡体积、降低肺泡上皮细胞变形、减少发炎反应减少胶原蛋白堆积、促使抗发炎的M2细胞生成、增加MMP(matrix-metallopeptidase)的活性以促进抗发炎反应与分解纤维化，并且可以刺激肺泡上皮细胞的再生。
根据本发明实施例，玻尿酸有效改善降低的血氧饱和度水平，缓解增加的呼吸速率和/或恢复肺泡功能。
在下面的描述中阐述了本发明的一个或多个实施例的细节。从以下几个实施例的详细描述以及从所附的权利要求中，本发明的其它特征或优点将是显而易见的。
附图说明
当结合附图阅读时，将更好地理解前面的概述以及以下对本发明的详细描述。为了说明本发明，在附图中示出了目前较佳的实施例。然而，应该理解的是，本发明不限于所示的精确布置和手段。
在附图中：
图1显示诱发大白鼠产生严重的急性呼吸窘迫症、和施打玻尿酸的实验流程图，共分5组，每组于第0天注射BLM入大白鼠左支气管，第7天给予药物治疗，第28天牺牲动物。
图2显示大白鼠在BLM伤害后7天内，血氧指数的变化。图2中的A部分显示BLM伤害后7天内，每天侦测大白鼠后肢的动脉血氧饱和度的照片。图2中的B部分为BLM伤害后7天内，大白鼠每天动脉饱和度的定量图，其显示BLM伤害第七天，动脉血氧饱和度降到最 低。图2中的C部分为BLM伤害后7天内，大白鼠的动脉血内氧分压指数定量图，其显示BLM伤害后第七天，动脉血氧分压降到最低。图2中的D部分为BLM伤害后7天内，大白鼠的动脉血内二氧化碳分压指数定量图，其显示BLM伤害后第七天，动脉血二氧化碳分压上升到最高。根据动脉血氧指数，确认大白鼠严重的急性呼吸窘迫症动物模式成功建立。
Figure PCTCN2021092837-appb-000001
与第0天(Day 0)相比，有统计差异，p<0.05。@：与第1天(Day 1)相比，具统计差异显著性，p<0.05。
Figure PCTCN2021092837-appb-000002
与Day 4相比，有统计差异，p<0.05。
图3显示大白鼠在BLM伤害后7天内，呼吸速率的变化。图3中的A部分显示BLM伤害后7天内，每天侦测大白鼠的呼吸速率记录图，撷取2秒的呼吸速率图。图3中的B部分为BLM伤害后7天内，大白鼠每天每分钟的呼吸速率定量图，其显示BLM伤害第七天，呼吸频率达到最高。根据每分钟的呼吸次数，确认大白鼠严重的急性呼吸窘迫症动物模式成功建立。
Figure PCTCN2021092837-appb-000003
与Day 0相比，有统计差异，p<0.05。@：与Day 1相比，具统计差异显著性，p<0.05。
图4显示大白鼠在BLM伤害后不同天数，牺牲灌流后，肺部的巨观型态。由图中可观察到，在BLM伤害第一天，左肺表面开始出现不平整的情形。在BLM伤害第二天，左肺中央区域肺泡已经有局部不见。在BLM伤害第七天，左肺萎缩、中央区域肺泡不见。根据左肺巨观型态，确定大白鼠严重的急性呼吸窘迫症动物模式成功建立。
图5显示大白鼠在BLM伤害后不同天数，左肺的微观型态。图5中的A部分为大白鼠在不同天数，左肺组织切片经HE染色的低倍图，其中白色空间为肺泡组织。图5中的B部分为大白鼠在不同天数，左肺中央区域(图5中的A部分中红框区域)组织切片经HE染色的高倍图，其显示BLM损伤之后，都呈现细胞浸润，肺泡逐渐减少。图5中的C部分为大白鼠在不同天数，左肺外周围区域(图5中的A部分中蓝框区域)组织切片经HE染色的高倍图，其显示BLM损伤之后，还有一些肺泡存在(白色空间)，只是随着伤害天数增加，肺泡逐渐减少。图5中的D部分为定量经过HE染色后，伤害后不同天数的左肺总体积；图5中的E部分为左肺肺泡体积；图5中的F部分为左肺细胞浸润占比。图5中的D-F部分显示BLM伤后左肺体积逐渐变小、肺泡变 少、细胞浸润比例增加。根据左肺微观型态，确定大白鼠严重的急性呼吸窘迫症动物模式成功建立。
Figure PCTCN2021092837-appb-000004
与Day 0的大白鼠相比，具统计差异显著性，p<0.05。
图6显示给予玻尿酸，可以增加ARDS病鼠的体重。
Figure PCTCN2021092837-appb-000005
与同日正常组的大白鼠相比，具统计差异显著性，p<0.05。#：与同日BLM伤害组的大白鼠相比，具统计差异显著性，p<0.05。
Figure PCTCN2021092837-appb-000006
与同日BLM+MIX HA组的大白鼠相比，具统计差异显著性，p<0.05。◆，与同日的BLM+LHA组大白鼠相较，有统计差异显著性，p<0.05。
图7显示给予玻尿酸能增加ARDS病鼠动脉血的含氧饱和度。图7中的A部分为第28天，各组大白鼠进行脉冲式动脉血氧仪检测的照片，其中箭头所指为动脉血氧饱和度的数值。图7中的B部分为定量各组大白鼠，在不同时间里，动脉血氧饱和度的数值。
Figure PCTCN2021092837-appb-000007
与同日正常组的大白鼠相比，具统计差异显著性，p<0.05。#：与同日BLM组的大白鼠相比，具统计差异显著性，p<0.05。
Figure PCTCN2021092837-appb-000008
与同日BLM+MIX HA组的大白鼠相比，具统计差异显著性，p<0.05。◆，与同日的BLM+LHA组大白鼠相较，有统计差异显著性，p<0.05。
图8显示给予玻尿酸能减缓ARDS病鼠的呼吸急促。图8中的A-E部分为各组大白鼠在不同时间，呼吸频率的2秒撷取纪录图。图8中的F部分为定量各组大白鼠，在不同时间里，每分钟的呼吸速率。
Figure PCTCN2021092837-appb-000009
与同日正常组的大白鼠相比，有统计差异，p<0.05。#：与同日BLM组的大白鼠相比，有统计差异，p<0.05。
Figure PCTCN2021092837-appb-000010
与同日BLM+MIX HA组的大白鼠相比，具统计差异显著性，p<0.05。
图9显示给予玻尿酸，能提升ARDS病鼠的左肺体积。图为各组大白鼠在第28天的肺脏外观图，上排为各组肺脏正面照，下排为各组肺脏的背面照。
图10显示给予玻尿酸能修复ARDS病鼠的肺泡结构。图10中的A部分为第28天的各组大白鼠左肺组织片，经过HE染色的低倍率图片。图10中的B部分为第28天的各组左肺组织片经过HE染色高倍率放大中央区域的照片。图10中的C部分为第28天的各组左肺组织片经过HE染色高倍率放大左肺外周围的照片。图10中的D部分加总所有左肺组织切片定量左肺体积。图10中的E部分定量左肺的肺泡 总体积。
图11显示给予玻尿酸能降低ARDS病鼠肺脏内的发炎反应、与肺泡上皮细胞的变形反应。图11中的A部分为定量各组大白鼠左肺，在第28天时，肺泡冲洗液内的细胞数量，以代表发炎情形。BLM组大白鼠的左肺的肺泡冲洗液内的细胞数量明显增加。图11中的B部分为第28天的各组大白鼠左肺，以西方墨点法定量左肺N-钙粘蛋白(N-cadherin)的浓度，来代表肺泡上皮细胞的变形。
Figure PCTCN2021092837-appb-000011
与正常组的大白鼠相比，具统计差异显著性，p<0.05。#：与BLM组的大白鼠相比，具统计差异显著性，p<0.05。
Figure PCTCN2021092837-appb-000012
与同日BLM+MIX HA组的大白鼠相比，具统计差异显著性，p<0.05。
图12显示给予玻尿酸能促进ARDS病鼠左肺内第二型巨噬细胞、减少第一型巨噬细胞，以降低发炎反应。图12中的A部分为第28天的各组大白鼠左肺，以Anti-ED1抗体进行组织免疫染色，标定巨噬细胞。显示，BLM组的吞噬细胞多为较小型态。而BLM+MIX HA组的吞噬细胞多为较大型态。图12中的B部分为第28天的各组大白鼠左肺组织，以Anti-CD86抗体进行西方墨点法染色，以定量M1巨噬细胞的变化情形。图12中的C部分为第28天的各组大白鼠左肺组织，以Anti-CD206抗体进行西方墨点法染色，以定量M2巨噬细胞的变化情形。
Figure PCTCN2021092837-appb-000013
与正常组的大白鼠相比，有统计差异，p<0.05。#：与BLM组的大白鼠相比，具统计差异显著性，p<0.05。
图13显示给予玻尿酸能促进ARDS病鼠左肺内MMP的合成，降低发炎反应。图13中的A部分为第28天的各组大白鼠左肺组织，以Anti-MMP9抗体进行西方墨点法染色，以定量MMP9蛋白质含量。图13中的B部分为第28天的各组大白鼠左肺组织，以Anti-MMP2抗体进行西方墨点法染色，以定量MMP2蛋白质含量。
Figure PCTCN2021092837-appb-000014
与正常组的大白鼠相比，有统计差异，p<0.05。#：与BLM组的大白鼠相比，具统计差异显著性，p<0.05。
图14显示给予玻尿酸能促进ARDS病鼠左肺内TLR4的合成，以刺激肺泡上皮细胞的再生。图为第28天的各组大白鼠左肺组织，以Anti-TLR4抗体进行西方墨点法染色，以定量TLR蛋白质含量。
Figure PCTCN2021092837-appb-000015
与正常组的大白鼠相比，有统计差异，p<0.05。#：与BLM组的大白 鼠相比，有统计差异，p<0.05。
具体实施方式
除非另外定义，否则本文使用的所有技术和科学术语具有与本发明所属领域的技术人员通常理解的相同的含义。
如本文所使用的，冠词“一”和“一个”是指冠词的一个或多于一个(即，至少一个)语法对象。举例来说，“一个元素”是指一个元素或多于一个元素。
术语“包括”或“包含”通常以包括/包括允许存在一个或多个特征，成分或组分的意义来使用。术语“包括”或“包含”涵盖术语“由...组成”或“由......组成”。
如本文所使用，“急性呼吸窘迫症(acute respiratory distress syndrome，简称ARDS)”乙辞是病患的肺部变化源于广泛性的肺泡微血管受损，使得内皮细胞间通透性增加，引发肺泡出血及水肿等现象，最后导致肺内死腔及分流增大，肺顺应性与氧合状况变差，而造成临床上的呼吸窘迫病症。
如本文所使用，“玻尿酸(hyaluronan或hyaluronic acid)，简称HA)”，又称透明质酸或醣醛酸，存在于人体的结缔组织及真皮层中，是一种透明的胶状物质。玻尿酸依照分子量，可以区分为低分子量玻尿酸(MW 10kDa-100kDa)、中分子量玻尿酸(MW 100kDa-1MDa)、与高分子量玻尿酸(>MW 1MDa)(Tavianatou et al.,2019)。本发明使用的玻尿酸可包括以上不同分子量的玻尿酸或其盐，及其混合，分子量范围为10kDa-2MDa。
如本文所用，如本文所述的与肺功能有关的指数或与疾病症状的降低或升高的水平是参照其对照(或正常)水平。如本文所使用的，术语“正常水平”或“对照水平”是描述本领域普通技术人员和/或医疗专业人员预期一个健康的个体或具有类似身体特征和病史的人群具有的值。例如，升高的水平表示高于对照(或正常)水平5％，10％，20％，30％，50％，70％，90％，100％，200％，300％，500％或更多，与对照(或正常)水平比较而言；而降低的水平是指低于对照(或正常)水平5％，10％，20％，30％，50％，70％，90％，100％，200％， 300％，500％或更多，与对照(或正常)水平相比而言。
如本文所用的术语“需要治疗方法的个体”是表示需要治疗ARDS的人类或非人类动物。
在一些具体实施态样中，需要本发明治疗方法的个体有以下列症状诊断为患有ARDS：(1)急性发作；(2)氧合指数(PaO2/FIO2)降低；(3)胸部X光呈现两侧肺浸润；(4)肺动脉楔压若有测量，小或等于18毫米汞柱，若无资料，则以临床上无左心房高压现象。
在此使用的术语“个体”或“主体”包括人类和非人类动物，如伴侣动物(如狗，猫等)，农场动物(如牛，绵羊，猪，马等)，或实验动物(如大鼠，小鼠，豚鼠等)。
如本文所用的术语“治疗”是指将包含一种或多种有效活性剂的组合物施用或施用于患有疾病，疾病病症或疾病症状或疾病进展(恶化)的对象，目的是治愈，治愈，缓解，舒解，改变，改善，改良，增进或影响该疾病，该疾病的病症或症状，疾病诱发的障碍或障碍的进展。
本文使用的术语“治疗有效量”是指活性成分在治疗对像中提供所需的治疗或生物作用的量。例如，用于治疗ARDS的有效量。
治疗有效量可以根据各种原因而改变，例如给药途径和频率，接受所述药物的个体的体重和种类，以及给药目的。本领域技术人员可以根据本文的公开内容，确定的方法和他们自己的经验确定每种情况下的剂量。
根据本发明，可以通过本领域已知的各种程序来施用，可以全身给药，例如，经由注射通过静脉内，动脉内给药，或皮下注射给药，或以鼻腔吸入，或鼻腔或口腔伸入气管给药、或气切给药。
为实施本发明的方法，可以将含玻尿酸以本发明领域习用的方法将其送至肺，其中一具体实施例是以注射经血液传送至肺，或可以直接由呼吸道、口腔(例如鼻、口、或气管)递送至肺。另一具体实施例，系通过直接注射递送至期望的区域。作为活性成分的玻尿酸可与药学上可接受的载体一起配制成适当形式的医药组合物或医疗器材用于递送。基于不同的给药方式，本发明的医药组合物或医疗器材可包含约0.1重量％至约100重量％的活性成分，其中基于整个组合物的重量计算重量百分比。“医药上可接受的载体”在所采用的剂量和浓度下 对个体是无毒的，并且与玻尿酸和包含玻尿酸的任何制剂的任何其他成分兼容。
在一些具体实施态样中，可以在合适的等渗液体，例如，磷酸盐缓冲盐水，生理盐水，葡萄糖水溶液和/或其混合物以及本领域技术人员已知的其它合适的液体中制备。应该保护最终的治疗形式免受污染，并应该能够抑制微生物如细菌或真菌的生长。其中一具体实施例是以施用单一的剂量。或者，可以使用缓慢的长期输注或多个短期的每日输注。如果需要，也可以使用每隔几天交替一天或一次给药。
通过以下实施例进一步说明本发明，这些实施例仅用于说明而不是限制。根据本公开内容，本领域技术人员应该理解，可以在所公开的具体实施方式中做出许多改变，并且仍然获得相似或相似的结果，而不偏离本发明的精神和范围。
实施例
1.材料与方法
1.材料与方法
1.1严重的急性呼吸窘迫症动物模式的建立
取雄性大白鼠(SD rats，230-250g)以舒泰50(Zoletil 50)及盐酸二甲苯胺噻嗪(Xylazine hydrochlotide)(Sigma 23076359)腹腔注射，确定大白鼠深层麻醉之后，将5mg博来霉素(bleomycin，简称BLM)溶在200μl的无菌生理食盐水(1unit activity/1mg BLM,Nippon Kayaku Co.,Ltd.)，以30G针头，打入大白鼠的支气管内，接着，大白鼠左侧卧60度、90分钟，造成严重的、可重复性的、左肺损伤的动物模式(Chu et al.,2019)。
1.2玻尿酸的给予
玻尿酸依照分子量，可以区分为低分子量玻尿酸(MW 10kDa-100kDa)、中分子量玻尿酸(MW 100kDa-1MDa)、与高分子量玻尿酸(>MW 1MDa)(Tavianatou et al.,2019)，本实施例使用不同分子量玻尿酸的混合、高分子量玻尿酸、及低分子量玻尿酸。在BLM注射后第7天开始，即第二周给予三次玻尿酸，第三周、和第四周都给予两次玻尿酸，也就是分别在BLM伤害后第7、9、11、14、17、21、24天给予玻尿酸(图1)。均经由气管内打入麻醉中的老鼠体内。
1.3实验分组
本实验动物共分五组：
第一组为正常组，即在第0天，大白鼠气管内接受200μl生理食盐水注射的正常大白鼠，在第7天开始，经气管内只注射200μl生理食盐水(图1)。
第二组为BLM组，即在第0天，大白鼠气管内接受5mg BLM注射的病鼠组。在BLM注射后第7天开始，经气管内只给予200μl生理食盐水，不给予任何治疗(图1)。
第三组为BLM+LHA组，即在第0天，大白鼠气管内接受5mg BLM注射。在BLM伤害后第7天开始，经气管内给予低分子量玻尿酸(MW10kDa-100kDa)，共七次(图1)。
第四组为BLM+HHA组，即在第0天，大白鼠气管内接受5mg BLM注射。在BLM伤害后第7天开始，经气管内给予高分子量玻尿酸(>MW 1MDa)，共七次(图1)。
第五组为BLM+MIX HA组，即在第0天，大白鼠气管内接受5mg BLM注射。在BLM伤害后第7天开始，经气管内给予混合分子量玻尿酸(MW 10kDa-2MDa)，共七次(图1)。
各组大白鼠，每周都测量体重、动脉血氧浓度、与呼吸频率，在BLM给予后第28天牺牲，观察肺脏型态。
1.4实验动物肺功能检测-动脉血氧饱和度的测定
以Isoflurane(Baxter228-194)将老鼠麻醉后，使用脉冲式血氧浓度器(Pulseoximeter,NONIN LS1-10R)夹住后足底，以侦测动脉血中的氧气饱和度。
1.5实验动物肺功能检测-肺部呼吸频率的测定
将实验动物放置于密闭式的圆筒呼吸侦测舱室(emka Technologies,Whole body plethysmograph)，以BIOPAC BSL 4.0MP45软件收集大白鼠15分钟内呼吸气流的变化情形，定量大白鼠在舱室内静置不动时的呼吸频率。
1.6实验动物灌流固定与组织的石蜡包埋
将过量的舒泰50(Zoletil 50)及盐酸二甲苯胺噻嗪(Xylazine hydrochloride)(Sigma 23076359)以腹腔注射方式打入老鼠体内，麻醉 致死后，再进行灌流。灌流后，取出左、右肺脏，置于4℃环境中后固定2天，接着，进行石蜡包埋。
固定后的左、右肺脏，依序放入浓度递增的酒精中(70％、80％、95％、100％)脱水，每次20分钟。组织脱水结束后，放入二甲苯(Xylene)、二甲苯：石蜡＝1：1混合液、二甲苯：石蜡＝1：3混合液、纯蜡中浸泡。最后，组织以纯蜡包埋。
1.7组织切片与取片方式
组织蜡块剃除多余石蜡，并修成梯形。使组织蜡块固定于石蜡切片机上。将肺脏组织切成5μm厚度的切片。组织切片置于40℃至45℃温水中展平，再将肺脏组织切片贴于玻片上，放置50℃加热平台上烘干。
由肺脏最外侧进行矢状切面，进行连续切片、并取片。
1.8染色苏木紫-伊红染色(Hematoxylin & Eosin Stain，简称HE Stain)
肺组织切片先进行脱蜡，依序放入二甲苯、浓度递减的酒精中(100％、95％、80％、70％酒精)、置于苏木精(Hematoxylin)溶液(武藤化学，No.3008-1)中染5分钟，接着，将肺组织片置于伊红(Eosin)溶液(武藤化学，No.3200-2)中染2.5分钟，随即，组织片浸泡于冰醋酸3秒，再以流动的水冲洗。肺组织切片浸泡至浓度递增的酒精内进行脱水(依序为50％、70％、80％、90％、95％、100％)，再浸泡于二甲苯两次，每次5分钟。最后，以封片胶(Permount,Fisher Scientific SP15-500)封片，进行光学显微镜观察及拍照。
1.9支气管肺泡冲洗液(Bronchoalverlar lavage，简称BAL)的细胞计数
大白鼠以PBS灌流完成后，由气管将整个肺取出，以20G针头搭配PE插管(PE60，内径0.76×外径1.22mm)，将插管由左支气管伸入左肺，以0.5ml无菌PBS进行冲洗后吸出。接着，进行第二次冲洗，以新的0.5ml无菌PBS冲吸一次；由此得到1ml肺泡冲洗液。将取得的肺泡冲洗液以1500rpm离心5分钟，下层细胞则以1ml食盐水回溶，进行细胞计数。
1.10蛋白质萃取
取各组大白鼠的肺脏组织，放入钵中加入液态氮，磨碎后加入适量RIPA溶析缓冲液(lysis buffer)(Millipore 20-188)，于4℃作用至隔夜，待肺脏组织均质化后，在4℃以13000rpm离心30分钟，取上清液冻至-20℃冰箱保存。
1.11免疫染色(Immunostaining)与西方墨点法(Western blotting)
将肺组织切片、或左肺蛋白质跑完电泳的NC试纸(NC paper)，分别加入初级抗体抗-N钙粘蛋白(anti-N cadherin)(Abcam ab18203，1:1000)、抗-ED1抗体(Millipore MAB1435，1:400)、抗-CD86抗体(Proteintech 13395-1-AP，1:1000)、小鼠抗-CD206(Abcam ab646931:1000)、抗-MMP9(Abcam ab76003，1:1000)、抗-MMP2(Abcam ab92536，1:1000)、抗-TLR4(Abcam ab30667，1:1000)，于4℃下反应16至18小时。接着，与二级抗体(secondary antibodies)于室温下反应60分钟，再以ABC套组(Avidin-biotinylated-horseradish peroxidase complex(ABC)kit，Vector Laboratories)于室温下反应60分钟后，以0.01M PBS清洗5分钟3次，最后，以DAB[diaminobenzidine，二氨基联苯胺](5mg DAB，30％H2O2 3.5μl in 10ml Tris-HCl pH7.4)进行呈色。
1.12统计分析
所有实验数据以平均值标准误差(Mean±SEM,Standard error of the mean)表示。各平均值间的比较以单因子变异细数分析(One-Way ANOVA)、或双因子变异细数分析(Two-Way ANOVA)，再以Turkey’s test进行多重比较。实验数据皆以p<0.05作为具有显著差异的最低起始标准。
2.结果
2.1严重的急性呼吸窘迫症动物模式的建立
给予BLM伤害七天内，侦测大白鼠的肺功能变化情形。首先，是脉冲式血氧浓度器侦测后肢足底的动脉血含氧饱和度，可代表肺部氧气交换的效能。在伤害之前(Day 0)，动脉血氧饱和度通常维持在97％。伤害后第一天会下降到87.7±1.2％，第二天再下降至84.7±1.7％，在伤害后第7天只剩83.7±0.9％(图2中的A及B部分)。第二种侦测方式是抽取老鼠的尾动脉血液，来侦测动脉血内的含氧分压、与二氧化 碳分压。在伤害之前(Day 0)，动脉血的氧分压(PO2)约88.1±2.7mmHg；伤害后第4天，会显著下降到74.2±4.2mmHg；伤害后第7天只剩66.3±3.9mmHg(图2中的C部分)。在伤害之前(Day 0)，动脉血的二氧化碳分压(PCO2)约46.4±1.6mmHg；伤害后第4天，会上升到49.0±1.3mmHg；伤害后第7天更升高至53.8±2.0mmHg(图2中的D部分)。第三种侦测是每分钟的呼吸频率。在伤害之前(Day 0)，呼吸频率约为132.3±11.8cycles/min；伤害后第1天，会上升到188.0±32.6cycles/min；之后，老鼠的呼吸逐渐加速，在伤害后第7天增加到330.3±17.3cycles/min(图3中的A及B部分)。由此结果显示，BLM伤害造成肺功能下降，动脉血液呈现缺氧的情形，导致呼吸的急促。
由肺的巨观来观察，正常大白鼠不论是左肺、或是右肺，都呈现平滑完整，白色为正常肺泡的位置。在伤害后第一天，左肺已经出现凹凸不平的表面。在伤害后第二天，左肺中央区域，白色肺泡已经有部分丧失。在伤害后第四天，左肺中央区域内的白色肺泡完全不见，仅剩周围区域有肺泡存在。到伤害后第7天，左肺明显萎缩，而且，只剩周围区域有肺泡存在(图4)。接着，左肺组织切片进行HE染色，观察微细型态的变化(图5中的A部分)。分别放大，左肺中央区域、与周围区域(图5中的B及C部分)，Normal组大白鼠的左肺，无论是中央、或周围区域都是正常的肺泡组织，结缔组织多存在于支气管周围，肺泡与肺泡之间的结缔组织极少。在伤害后第一天，不论是中央区域、或周围区域，在肺泡之间开始出现细胞浸润的情形。伤害后第二天到第七天，细胞浸润的情形越来越严重，左肺中央区域的肺泡几乎消失不见。仅在左肺的周边区域，仍然有少许的肺泡存在(图5中的A-C部分)。进一步，我们加总所有的肺组织切片，推算出左肺的总体积，正常组的左肺体积大约317.6±19.4mm3立方毫米。在BLM伤害后第7天，明显降低到217.2±17.0mm3左右，与正常组相比有明显的萎缩(图5中的D部分)。此时萎缩的左肺，肺泡结构的体积也下降到59.7±8.4cm3；大量细胞浸润的实质化组织，则占左肺体积的63.8±2.5％左右(图5中的E及F部分)。在BLM伤害之后，大白鼠每分钟呼吸频率在七天内急遽上升、动脉含氧指数都显著下降、左肺的肺泡减少、组织间大量的细胞浸润，这些，都与临床上的急性呼吸窘迫症 的特性相同。因此，我们成功建立严重的、可重复性的、一致性的、急性呼吸窘迫症的动物模式。我们因此选择急性期、最严重的第七天，开始给予治疗。
2.2给予玻尿酸可以增加严重的急性呼吸窘迫症病鼠的体重
正常组大白鼠随着时间增加，体重也有逐渐上升的情形。在给予BLM伤害后第7天，各组大白鼠体重都有明显停滞的现象。之后，体重随着时间虽然也有些微增长，但各组大白鼠与正常组大白鼠的体重相较，都呈现明显减少的现象，并且，体重较正常组少的趋势一直持续到第28天。BLM+HHA组大白鼠的体重，一直都与BLM组相近。BLM+HHA组大白鼠的体重，从第14天到第28天，都比BLM+MIX HA组来的少。第BLM+LHA组大白鼠的体重，在第28天，与BLM组大白鼠相较，有显著的增加情形。BLM+MIX HA组大白鼠的体重，则在第21天与第28天，都较BLM组大白鼠的体重有统计上的提升(图6)。
2.3给予玻尿酸能够提升严重的急性呼吸窘迫症病鼠的动脉血的氧合指数
以脉冲式血氧浓度仪分析动脉血氧饱和度(SpO2)，由此评估肺部气体交换的功能。结果显示，在实验时间的28天内，正常组大白鼠的血氧饱和度均维持在98.2％左右(图7中的A及B部分)。在给予BLM伤害后第7天，各组大白鼠的血氧饱和度都有明显下降的现象，约为84.3±0.7％，一直到第28天，BLM组大白鼠的动脉血氧饱和度并没有改善，与正常组大白鼠相较，均呈现明显的减少(图7中的A及B部分)。BLM+HHA组大白鼠，在第28天时的动脉血氧饱和度都约为86％左右，与BLM伤害组相较，并没有统计上差异。BLM+HHA组大白鼠的动脉血氧饱和度，从第14天到第28天，都比BLM+MIX HA组来的少。从第14天到第28天，BLM+LHA组、与BLM+MIX HA组大白鼠的动脉血氧饱和度，相较BLM组都有明显的增加。然而，BLM+MIX HA组大白鼠的动脉血氧饱和度在第21天与第28天又较BLM+LHA组有更明显的改善(图7中的A及B部分)。
另外，在BLM伤害之前(Day 0)，大白鼠动脉血内的氧分压约为86.4-89.7mmHg(图7中的C部分)。在给予BLM伤害后第7天，各组大白鼠动脉血氧分压都有明显下降的现象，BLM组大白鼠一直到第 28天，动脉血氧分压并没有太大变化，与正常组大白鼠相较，均呈现明显的减少(图7中的C部分)。BLM+HHA组大白鼠，在第28天时的动脉血氧分压虽然有些微上升的趋势，但与BLM组相较，并没有统计上差异。而且，BLM+HHA组大白鼠的动脉血氧分压，在第28天时，仍然低于BLM+MIX HA组大白鼠。BLM+LHA组大白鼠的动脉血氧分压，在第28天，相较BLM组有明显的增加。BLM+MIX HA组大白鼠的动脉血氧分压在第14天已经显著升高，此上升趋势一直持续到第28天，动脉血氧分压相较BLM组，都有明显的改善(图7中的C部分)。
在BLM伤害之前(Day 0)，大白鼠动脉血内的二氧化碳分压约为46.6-47.9mmHg(图7中的D部分)。在给予BLM伤害后第7天，各组大白鼠动脉血二氧化碳分压都较正常组有明显上升的情形，升高至51.6–54.7mmHg。到第14天，各组动脉血二氧化碳分压仍旧是升高的情形。只有BLM+MIX HA组大白鼠的动脉血二氧化碳分压在第14天已经显著下降，较BLM组减少，与Normal组相近。其余各组大白鼠(BLM组、BLM+LHA组、和BLM+HHA组)动脉血内二氧化碳分压，在第21天，都与正常组大白鼠没有统计差异(图7中的D部分)。
2.4给予玻尿酸能舒缓严重的急性呼吸窘迫症病鼠的呼吸速率
侦测大白鼠的呼吸频率结果显示，正常组大白鼠从第0天到第28天，一直都保持呼吸速率相当稳定的情形，两秒内的呼吸次数约维持4-5次(图8中的A及F部分)。在BLM伤害后第7天，各组的呼吸速率都明显上升。BLM组，在第7天到第28天，呼吸速率都较正常组明显增快(图8中的B及F部分)。BLM+HHA组大白鼠，从第7天到第28天的呼吸速率，都与BLM组相似。而且，BLM+HHA组大白鼠的呼吸速率，在第21天、与第28天时，仍然高于BLM+MIX HA组大白鼠(图8中的D及F部分)。BLM+LHA组大白鼠的呼吸速率在第14天，与BLM组相比，有明显的减少，但是，呼吸频率仍然高于正常组(图8中的C及F部分)。BLM+MIX HA组大白鼠的呼吸速率在第14天，相较BLM组，已有明显的降低，此趋势一直持续到第28天；并且，BLM+MIX HA组大白鼠的呼吸速率在第21天与第28天的呼吸速率与正常组并没有统计差异(图8中的E及F部分)。
2.5由外型巨观及HE染色显示，给予玻尿酸，能改善严重的急性呼吸窘迫症病鼠左肺的萎缩
在第28天，牺牲灌流各组大白鼠，取其左、右肺脏，观察肺部的外观型态。由前面观(上排)、与背面观(下排)照片显示，正常组大白鼠的左、右肺均可以看见白色肺泡结构，肺泡呈现完整、平滑的情形。BLM组的左肺明显萎缩，非常少量的肺泡组织仅出现在左肺的外周围，左肺的中央区域已经呈现没有肺泡的病变组织。其中，BLM+MIX HA组大白鼠，他们左肺的白色肺泡区域、以及整体左肺的体积，与BLM伤害组相比，都有明显的增大(图9)。
各组大白鼠左肺连续的组织切片，经HE染色，取低倍率图片(图10中的A部分)，再分别放大左肺的中央区域(图10中的B部分)、与周边区域(图10中的C部分)。结果显示，正常组大白鼠左肺面积较大、肺泡较多，结缔组织只出现在支气管周围，肺泡与肺泡之间的结缔组织极少。BLM组大白鼠的左肺，完整的肺泡只出现在左肺的外周围，中央区域均为大量细胞浸润、几乎看不到肺泡的存在。而BLM+MIX HA组大白鼠，他们左肺中央区域虽然也有大量的细胞浸润，但也存在不少肺泡空间(白色空间)(图10中的B及C部分)。加总所有HE染色的左肺组织切片，经由统计定量，结果显示，BLM组左肺总体积明显萎缩，萎缩左肺内的肺泡体积显著减少(图10中的D-E部分)。其中，BLM+MIX HA组大白鼠的左肺体积明显提升，左肺肺泡体积较多，与正常组大白鼠左肺的结构比例相当。推测给予混合分子量的玻尿酸，可以提升左肺的整体体积、回复左肺的肺泡空间(图10中的D-E部分)。
2.6给予玻尿酸能降低严重的急性呼吸窘迫症病鼠左肺内发炎反应、与肺泡上皮细胞的EMT反应
计算支气管与肺泡的冲洗液内的细胞数目，细胞数目越多，代表发炎反应越剧烈。
第28天，取各组大白鼠左肺的肺泡冲洗液，进行细胞计数。结果显示，BLM组大白鼠的左肺的肺泡冲洗液内的细胞数量明显增加。BLM+LHA组、和BLM+HHA组大白鼠肺泡冲洗液内的细胞数量，相较于BLM组，都有显著降低的情形，但仍较Normal组来得多。BLM+MIX HA组大白鼠的左肺的肺泡冲洗液的细胞数量，与BLM伤 害组相比，有显著降低，且与Normal组并没有统计差异。推测给予玻尿酸，都能够减少严重的急性呼吸窘迫症病鼠的肺部发炎反应，因此，左肺内的细胞数量减少，尤其，给予混合各分子量的玻尿酸，与正常组相似(图11中的A部分)。
BLM伤害后，Alveolar Type II cell会大量的增生，进行上皮细胞变形反应(epithelial mesenchymal transition，简称EMT)，此时，N-钙粘蛋白(N-cadherin)大量表现。此时，Alveolar Type II cell变型成为myofibroblasts，造成肺泡减少、组织结疤。
以抗-N-钙粘蛋白抗体进行西方墨点法(western blotting)，结果显示，正常组大白鼠的左肺中仅有少量N-钙粘蛋白存在。BLM组大白鼠的左肺中会有大量表现N-钙粘蛋白，代表肺泡上皮细胞大量的增生，进行上皮细胞变形反应。然而，BLM+MIX HA组大白鼠左肺N-钙粘蛋白产量，与BLM伤害组相较，呈现降低情形。BLM+MIX HA组大白鼠左肺N-钙粘蛋白产量，与正常组相近(图11中的B部分)。
2.7给予玻尿酸能促进严重的急性呼吸窘迫症病鼠左肺内第二型巨噬细胞活化，以进行抗发炎作用。
各组大白鼠的左肺组织切片，以抗-ED1抗体进行组织免疫染色，以标定巨噬细胞。结果显示，正常组大白鼠左肺中仅有少量、形态较小的巨噬细胞存在。BLM组大白鼠的左肺组织间，出现大量形态较小颗的巨噬细胞。BLM+MIX HA组大白鼠左肺组织间，仍然有许多形态较小的巨噬细胞存在，但是，出现型态较大的吞噬细胞分布在结缔组织间、及肺泡空间内(图12中的A部分)，推测这些型态较大的巨噬细胞是具有抗发炎作用的第二型巨噬细胞。
以抗-CD86抗体西方墨渍法，标定第一型巨噬细胞。第一型巨噬细胞具有促进发炎的效果。结果显示。BLM组大白鼠的左肺内CD86的含量上升。然而BLM+MIX HA组大白鼠左肺内CD86的浓度下降，与BLM组相比，有统计上差异(图12中的B部分)。推测给予玻尿酸，能避免严重的急性呼吸窘迫症病鼠左肺内第一型巨噬细胞的分化与转型，以降低发炎反应。
进一步，以Anti-CD206抗体西方墨渍法，标定第二型巨噬细胞，第二型巨噬细胞具有抗发炎的作用。BLM+MIX HA组大白鼠左肺内 CD206的浓度升高，与正常(Normal)组、和BLM组相比，都呈现统计上的增加(图12中的C部分)。推测给予玻尿酸，能促进严重的急性呼吸窘迫症病鼠左肺内第二型巨噬细胞的分化与转型，以进行抗发炎反应。
2.8给予玻尿酸能刺激严重的急性呼吸窘迫症病鼠左肺内的MMP生成，降低发炎反应
定量各组大白鼠左肺中基质金属蛋白酶-9(Matrix metallopeptidase 9，简称MMP-9)蛋白质的含量，结果显示，BLM组大白鼠左肺内的MMP-9，与正常组相比，呈现减少的情形。BLM+MIX HA组大白鼠左肺内MMP-9明显增加，较BLM组有统计上的差异(图13中的A部分)。
定量各组大白鼠左肺中基质金属蛋白酶-2(Matrix metallopeptidase，简称MMP-2)蛋白质的含量，结果显示，BLM组大白鼠左肺内的MMP-2，与正常组相比，则没有明显的变化。BLM+MIX HA组大白鼠左肺内MMP-2明显增加，较与正常组有统计上的提升(图13中的B部分)。推测给予玻尿酸，能刺激严重的急性呼吸窘迫症病鼠左肺内合成MMP，因此，降低发炎反应。
2.9给予玻尿酸能改变严重的急性呼吸窘迫症病鼠左肺内TLR-4表现，加速肺泡上皮细胞的再生
第二型肺泡上皮细胞大量表现类铎受体4(Toll-like receptor 4，TLR-4)，会刺激肺泡上皮细胞的再生(Yang et al.,2012；Liang et al.,2016)。因此，以抗-TLR-4抗体进行西方墨渍法，观察各组大白鼠左肺中TLR-4蛋白质的含量。结果显示，正常组与BLM伤害组大白鼠的左肺中仅有少量的TLR-4表现。在BLM+MIX HA组大白鼠的左肺内TLR-4的表现量明显升高，与正常组与BLM伤害组相比，都有统计上的差异(图14)。推测给予玻尿酸，可增进严重的急性呼吸窘迫症病鼠左肺内TLR-4蛋白质的生成，以便增进肺泡上皮细胞的再生与修复。
3.结论
BLM伤害后，发炎反应上升、发炎细胞浸润、第一型肺泡上皮细胞消失、大量增生的第二型肺泡上皮细胞(Alveolar Type II cells)，进行变形反应，变形成为活化态的肌纤维母细胞(myofibroblast)，合成并释 出胶原蛋白，导致胞外基质沉积。给予混合各合分子量的玻尿酸，可以减轻发炎反应、降低肺泡上皮细胞变形、刺激第二型抗发炎的巨噬细胞生成、减少胶原蛋白的沉积、刺激肺泡的再生。不同分子量玻尿酸都有改善ARDS的功能，而混合分子量的玻尿酸，改善、治疗严重的急性呼吸窘迫症的效果最好。因此，给予玻尿酸，相信也可以减缓新冠肺炎(COVID-19)大流行的威胁。
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Claims (10)

  1. 一种玻尿酸制备治疗急性呼吸窘迫症的药剂的用途。
  2. 根据权利要求1所述的用途，其中该急性呼吸窘迫症为严重的急性呼吸窘迫症。
  3. 根据权利要求1所述的用途，其中该玻尿酸的分子量为10kDa-2MDa。
  4. 根据权利要求1所述的用途，其中该玻尿酸有效改善ARDS的症状，减缓因ARDS明显上升的呼吸频率、使血氧浓度显著回升、改善肺脏体积与肺泡体积、降低肺泡上皮细胞变形、减少胶原蛋白堆积、抗发炎、并且可以刺激肺泡上皮细胞的再生。
  5. 根据权利要求1所述的用途，其中该玻尿酸有效改善降低的血氧饱和度水平，缓解增加的呼吸速率和恢复肺泡功能。
  6. 根据权利要求1所述的用途，其中该玻尿酸经由血液注射、肌肉注射、或皮下注射施用。
  7. 根据权利要求1所述的用途，其中该玻尿酸直接由呼吸道递送至气管、或肺。
  8. 根据权利要求7所述的用途，其中该玻尿酸直接由鼻腔吸入。
  9. 根据权利要求7所述的用途，其中该玻尿酸经鼻腔或口腔至气管的支气管镜施予。
  10. 根据权利要求7所述的用途，其中该玻尿酸经气切手术给予。
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CN115317504A (zh) * 2021-05-10 2022-11-11 傅毓秀 玻尿酸用于制备治疗急性呼吸窘迫症药剂的用途

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