TWI816119B - Use of hyaluronan for manufacturing a medicament for treating acute respiratory distress syndrome - Google Patents

Abstract

The present invention relates to a use of hyaluronan for manufacturing a medicament for treating acute respiratory distress syndrome (ARDS). The present invention provides an efficacy in reducing pathological injury in lung tissue and stimulating alveolar regeneration, increasing lung function, and accordingly provides a new clinical approach to curing ARDS.

Description

Use of hyaluronic acid in the preparation of pharmaceuticals for the treatment of acute respiratory distress syndrome

The present invention relates to the use of hyaluronic acid for treating acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a complex disease caused by multiple factors. According to the Berlin definition, patients are exposed to risk factors within seven days, resulting in acute diffuse lung inflammation, hypoxemia, and respiratory failure (Mattay et al., 2012). It is a symptom of acute lung injury and a life-threatening disease. Acute respiratory distress syndrome 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. In current clinical medicine, there are no drugs to treat acute respiratory distress syndrome, only supportive therapy, so the mortality rate is extremely high. Therefore, ARDS is already a very troublesome disease in the critical care medical community.

In 2020, the COVID-19 pandemic has led to a large increase in ARDS patients. According to statistics, as many as 31% - 42% of patients hospitalized with COVID-19 will have ARDS, and 61-81% of them need to be admitted to the intensive care unit (Wu et al., 2020) (Gibson et al., 2020 ). These severe patients with COVID-19 infection have severe pulmonary inflammation in the acute phase and varying degrees of pulmonary fibrosis in the recovery phase, causing permanent deterioration of lung function and even lifelong damage to other organs. Therefore, developing drugs for acute respiratory distress disease can not only reduce the severity of acute respiratory distress disease and reduce the mortality rate of acute respiratory distress disease patients, but also avoid subsequent damage to the lungs or other organs, and can also alleviate critical care in hospitals. Ward pressure.

The present invention aims to provide a new treatment direction for acute respiratory distress syndrome (ARDS).

In one aspect, the present invention provides use of hyaluronic acid for the manufacture of a medicament for treating acute respiratory distress syndrome.

On the other hand, the present invention provides a medicament or pharmaceutical composition for treating acute respiratory distress syndrome, which contains a therapeutically effective amount of hyaluronic acid.

In yet another aspect, the present invention provides a method of treating acute respiratory distress syndrome, comprising administering to an individual in need thereof a medicament or pharmaceutical composition comprising a therapeutically effective amount of hyaluronic acid.

According to the invention, the method of the invention is particularly suitable for use in severe acute respiratory distress syndrome.

According to the present invention, hyaluronic acid with a molecular weight of 10kDa-2MDa can be used.

According to embodiments of the present invention, administration of hyaluronic acid to ARDS model rats can effectively improve the symptoms of ARDS, slow down the respiratory rate significantly increased due to ARDS, significantly increase blood oxygen concentration, improve lung volume and alveolar volume, and reduce alveolar epithelium. Cell deformation, reducing inflammatory responses, promoting the generation of anti-inflammatory M2 cells, increasing MMP (matrix-metallopeptidase) activity to promote anti-inflammatory responses and decomposing fibrosis, and stimulating the regeneration of alveolar epithelial cells.

According to embodiments of the present invention, hyaluronic acid effectively improves reduced blood oxygen saturation levels, alleviates increased respiratory rates and/or restores alveolar function.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the following detailed description of several embodiments and from the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the articles "a" and "an" refer to one or more than one (ie, at least one) grammatical object of the article. For example, "an element" means one element or more than one element.

The term "comprises" or "includes" is generally used in the sense of including/including allowing for the presence of one or more features, ingredients or components. The term "includes" or "includes" encompasses the term "consists of" or "consisting of."

As used in this article, the term "acute respiratory distress syndrome (ARDS)" refers to the patient's lung changes resulting from extensive alveolar capillary damage, which increases inter-endothelial cell permeability and triggers alveolar Phenomenons such as bleeding and edema eventually lead to an increase in dead space and shunts in the lungs, deterioration of lung compliance and oxygenation, and clinical respiratory distress.

As used in this article, "hyaluronan or hyaluronic acid (HA)", also known as hyaluronic acid or uronic acid, is a transparent gel-like substance that exists in the connective tissue and dermis of the human body. According to the molecular weight, hyaluronic acid can be divided into low molecular weight hyaluronic acid (MW 10 kDa - 100 kDa), medium molecular weight hyaluronic acid (MW 100 kDa - 1 MDa), and high molecular weight hyaluronic acid (> MW 1 MDa) (Tavianatou et al., 2019). The hyaluronic acid used in the present invention can include hyaluronic acid or its salts with different molecular weights as above, and mixtures thereof, with the molecular weight ranging from 10 kDa to 2 MDa.

As used herein, an index related to lung function or a reduced or increased level of disease symptoms as described herein is with reference to its control (or normal) level. As used herein, the terms "normal level" or "control level" describe values that one of ordinary skill in the art and/or medical professionals would expect a healthy individual or a population with similar physical characteristics and medical history to have. For example, elevated levels mean 5%, 10%, 20%, 30%, 50%, 70%, 90%, 100%, 200%, 300%, 500% or more above control (or normal) levels , compared with the control (or normal) level; and the reduced level refers to 5%, 10%, 20%, 30%, 50%, 70%, 90%, 100%, below the control (or normal) level. 200%, 300%, 500% or more compared to control (or normal) levels.

The term "individual in need of treatment" as used herein means a human or non-human animal in need of treatment for ARDS.

In some embodiments, an individual in need of the treatment method of the present invention is diagnosed as suffering from ARDS if he or she has the following symptoms: (1) acute attack; (2) decreased oxygenation index (PaO2/FIO2); (3) chest X-ray presentation Pulmonary infiltration on both sides; (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 is assumed that there is no clinical left atrial hypertension.

As used herein, the terms "individual" or "subject" include human and non-human animals, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.), or laboratory animals (e.g., large animals). rats, mice, guinea pigs, etc.).

The term "treatment" as used herein refers to the administration or administration of 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 intent of curing, curing, alleviating , relieve, alter, ameliorate, ameliorate, enhance or influence the disease, the symptoms or symptoms of the disease, the disorder induced by the disease or the progression of the disorder.

As used herein, the term "therapeutically effective amount" refers to an amount of an active ingredient that provides the desired therapeutic or biological effect in a subject to be treated. For example, an effective amount for treating ARDS.

The therapeutically effective amount may vary depending on various reasons, such as the route and frequency of administration, the 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 based on the disclosure herein, established methods, and their own experience.

Administration according to the present invention may be by various procedures known in the art, and may be administered systemically, for example, by injection, intravenously, intraarterially, or subcutaneously, or by nasal inhalation, or by nasal or oral administration. Administration into the trachea or tracheotomy.

In order to implement the method of the present invention, the hyaluronic acid-containing acid can be delivered to the lungs by a method commonly used in the field of the present invention. One specific embodiment is to deliver it to the lungs through the blood via injection, or it can be directly delivered from the respiratory tract or oral cavity (such as the nose, mouth, etc.) , or trachea) delivered to the lungs. In another embodiment, delivery is by direct injection to the desired area. Hyaluronic acid as an active ingredient can be formulated together with a pharmaceutically acceptable carrier into a suitable form of pharmaceutical composition or medical device for delivery. Based on different modes of administration, the pharmaceutical composition or medical device of the present invention may contain about 0.1% to about 100% by weight of the active ingredient, where the weight percentage is calculated based on the weight of the entire composition. A "pharmaceutically acceptable carrier" is non-toxic to the subject at the doses and concentrations employed and is compatible with hyaluronic acid and any other ingredients of any formulation containing hyaluronic acid.

In some embodiments, it may be prepared in a suitable isotonic liquid, such as phosphate buffered saline, physiological saline, aqueous glucose solution and/or mixtures thereof, as well as 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 one dose every few days can also be used if needed.

The present invention is further illustrated by the following examples, which are for illustration only and not for limitation. Those skilled in the art should, in light of this disclosure, appreciate that many changes could be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example

1. Materials and methods

1.1 Establishment of animal model of severe acute respiratory distress syndrome

Male SD rats (230-250 g) were intraperitoneally injected with Zoletil 50 and Xylazine hydrochlotide (Sigma 23076359). After confirming that the rats were deeply anesthetized, 5 mg of Bo Bleomycin (BLM) was dissolved in 200 µl of sterile saline (1 unit activity/1 mg BLM, Nippon Kayaku Co., Ltd.), and injected into the bronchi of the rat with a 30G needle. Then, Rats lying on their left side at 60 degrees for 90 minutes caused an animal model of severe, reproducible, left lung injury (Chu et al., 2019).

1.2 Administration of hyaluronic acid

According to the molecular weight, hyaluronic acid can be divided into low molecular weight hyaluronic acid (MW 10 kDa - 100 kDa), medium molecular weight hyaluronic acid (MW 100 kDa - 1 MDa), and high molecular weight hyaluronic acid (> MW 1 MDa) (Tavianatou et al., 2019). This embodiment uses a mixture of hyaluronic acid with different molecular weights, high molecular weight hyaluronic acid, and low molecular weight hyaluronic acid. Starting from the 7th day after BLM injection, that is, hyaluronic acid will be administered three times in the second week, and hyaluronic acid will be administered twice in the third and fourth weeks, that is, on the 7th, 9th, 11th, 14th, 17th, and 21st days after BLM injury. , hyaluronic acid was given for 24 days (Figure 1A). All were injected into anesthetized mice via the trachea.

1.3 Experimental grouping

The experimental animals are divided into five groups:

The first group is the normal group, that is, the normal rats received an intratracheal injection of 200 µl of normal saline on day 0. Starting from the 7th day, only 200 µl of normal saline was injected into the trachea (Figure 1).

The second group is the BLM group, which is the group of sick mice that received 5 mg BLM injection into the trachea on day 0. Starting on the 7th day after BLM injection, only 200 µl of normal saline was administered intratracheally without any treatment (Figure 1).

The third group is the BLM+LHA group, that is, on day 0, the rats received an intratracheal injection of 5 mg BLM. Starting on day 7 after BLM injury, low molecular weight hyaluronic acid (MW 10 kDa - 100 kDa) was administered intratracheally for a total of seven times (Figure 1).

The fourth group is the BLM+HHA group, that is, on day 0, the rats received 5 mg BLM injection into the trachea. Starting on the 7th day after BLM injury, high molecular weight hyaluronic acid (> MW 1 MDa) 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 an intratracheal injection of 5 mg BLM. Starting on the 7th day after BLM injury, mixed molecular weight hyaluronic acid (MW 10 kDa - 2 MDa) was administered intratracheally for a total of seven times (Figure 1).

The body weight, arterial blood oxygen concentration, and respiratory rate of the rats in each group were measured every week. They were sacrificed on the 28th day after BLM administration and the lung morphology was observed.

1.4 Pulmonary function testing of experimental animals-measurement of arterial blood oxygen saturation

After the mice were anesthetized with Isoflurane (Baxter228-194), a pulse oximeter (Pulseoximeter, NONIN LS1-10R) was used to clamp the soles of the hind feet to detect the oxygen saturation in the arterial blood.

1.5 Pulmonary function testing of experimental animals-measurement of lung respiratory rate

The experimental animals were placed in a closed cylindrical respiratory detection chamber (emka Technologies, Whole body plethysmograph), and BIOPAC BSL 4.0 MP45 software was used to collect the changes in respiratory airflow of the rats within 15 minutes. The rats were quantified while standing still in the chamber. respiratory rate.

1.6 Perfusion fixation of experimental animals and paraffin embedding of tissues

Excessive amounts of Zoletil 50 and Xylazine hydrochloride (Sigma 23076359) were intraperitoneally injected into the mice. After anesthesia, they were killed and then perfused. After perfusion, the left and right lungs were removed, fixed in a 4°C environment for 2 days, and then embedded in paraffin.

After fixation, the left and right lungs were dehydrated by sequentially placing them in alcohol of increasing concentration (70%, 80%, 95%, 100%) for 20 minutes each time. After the tissue is dehydrated, soak it in xylene (Xylene), a mixture of xylene: paraffin = 1:1, a mixture of xylene: paraffin = 1:3, and pure wax. Finally, the tissue was embedded in pure wax.

1.7 Tissue sectioning and slice removal methods

Excess paraffin is shaved off from the tissue wax block and shaped into a trapezoid shape. Fix the tissue wax block on the paraffin microtome. Cut the lung tissue into 5 µm thick sections. Place the tissue slices in warm water at 40°C to 45°C to flatten them, then place the lung tissue slices on glass slides and place them on a 50°C heating platform to dry.

A sagittal section was made from the outermost side of the lung, and serial sections were taken.

1.8 Dyeing Hematoxylin & Eosin Stain (HE Stain for short)

The lung tissue sections were first dewaxed and placed in xylene, alcohol of decreasing concentration (100%, 95%, 80%, 70% alcohol), and hematoxylin solution (Muto Chemical, No. 3008-1) for 5 minutes, then place the lung tissue pieces in Eosin solution (Muto Chemical, No. 3200-2) for 2.5 minutes, then soak the tissue pieces in glacial acetic acid for 3 seconds, and then Rinse with running water. The lung tissue sections were immersed in increasing concentrations of alcohol 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 mounted with mounting gel (Permount, Fisher Scientific SP15-500) for observation and photography under an optical microscope.

1.9 Cell count in bronchoalveolar lavage (BAL)

After the rat was perfused with PBS, the entire lung was removed from the trachea, and a 20G needle was used with a PE cannula (PE60, inner diameter 0.76 (outer diameter 1.22 mm), extend the intubation tube from the left bronchus into the left lung, flush with 0.5 ml sterile PBS and then aspirate. Then, perform a second flush and inhale once with new 0.5 ml of sterile PBS; thus, 1 ml of alveolar flush fluid is obtained. The obtained alveolar lavage 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.

1.10 Protein extraction

Take the lung tissue of rats from each group, put it into a bowl and add liquid nitrogen, grind it and add an appropriate amount of RIPA lysis buffer (Millipore 20-188), and incubate at 4°C overnight until the lung tissue is homogenized. Finally, centrifuge at 13,000 rpm for 30 minutes at 4°C, and freeze the supernatant to a -20°C refrigerator for storage.

1.11 Immunostaining and Western blotting

Take lung tissue sections or NC paper that has run electrophoresis on left lung proteins, and add primary antibodies anti-N cadherin (Abcam ab18203, 1:1000) and anti-ED1 antibodies ( Millipore MAB1435, 1:400), anti-CD86 antibody (Proteintech 13395-1-AP, 1:1000), mouse anti-CD206 (Abcam ab64693 1:1000), 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. Then, react with secondary antibodies for 60 minutes at room temperature, and then react with ABC kit (Avidin-biotinylated-horseradish peroxidase complex (ABC) kit, Vector Laboratories) for 60 minutes at room temperature, and then use 0.01 M PBS was washed three times for 5 minutes, and finally, DAB [diaminobenzidine, diaminobenzidine] (5mg DAB, 30% H ₂ O ₂ 3.5 µl in 10 ml Tris-HCl pH7.4) was used for color development.

1.12 Statistical analysis

All experimental data are expressed as Mean ± SEM (Standard error of the mean). Comparisons between the average values were performed using One-Way ANOVA or Two-Way ANOVA, followed by Turkey’s test for multiple comparisons. Experimental data all use p < 0.05 as the lowest starting standard for significant differences.

2. Results

2.1 Establishment of animal model of severe acute respiratory distress syndrome

Within seven days of BLM injury, the lung function changes of rats were detected. First, the pulse oximeter detects the oxygen saturation of arterial blood in the soles of the hind limbs, which can represent the efficiency of oxygen exchange in the lungs. Before injury (Day 0), arterial oxygen saturation was usually maintained at 97%. It will drop to 87.7±1.2% on the first day after injury, then drop to 84.7±1.7% on the second day, and only 83.7±0.9% remains on the 7th day after injury (Figure 2A and 2B). The second detection method is to extract blood from the tail artery of mice to detect the oxygen partial pressure and carbon dioxide partial pressure in the arterial blood. Before the injury (Day 0), the oxygen partial pressure (PO2) of the arterial blood was about 88.1±2.7 mmHg; on the 4th day after the injury, it dropped significantly to 74.2±4.2 mmHg; on the 7th day after the injury, it was only 66.3±3.9 mmHg (Figure 2C). Before the injury (Day 0), the partial pressure of carbon dioxide (PCO2) in the arterial blood was approximately 46.4±1.6 mmHg; on the 4th day after the injury, it rose to 49.0±1.3 mmHg; on the 7th day after the injury, it increased to 53.8±2.0 mmHg ( Figure 2D). The third type of detection is respiratory rate per minute. Before the injury (Day 0), the respiratory frequency was approximately 132.3±11.8 cycles/min; on the 1st day after the injury, it increased to 188.0±32.6 cycles/min; after that, the mouse's breathing gradually accelerated and increased on the 7th day after the injury. to 330.3±17.3 cycles/min (Figure 3A and 3B). The results show that BLM injury causes a decrease in lung function and a lack of oxygen in the arterial blood, resulting in rapid breathing.

Observed from the macroscopic view of the lungs, both the left and right lungs of normal rats appear smooth and complete, and the white color indicates the position of normal alveoli. On the first day after the injury, the left lung already had a bumpy surface. On the second day after the injury, the white alveoli in the central area of the left lung had been partially lost. On the fourth day after the injury, the white alveoli in the central area of the left lung were completely missing, leaving only alveoli in the surrounding area. On the 7th day after the injury, the left lung shrank significantly, and only the alveoli remained in the surrounding area (Figure 4). Next, left lung tissue sections were stained with HE to observe subtle morphological changes (Figure 5A). Magnified respectively, the central area and the surrounding area of the left lung (Figure 5B and 5C). The left lung of the rats in the Normal group has normal alveolar tissue in both the central and surrounding areas. Connective tissue mostly exists around the bronchi, and the alveoli and There is very little connective tissue between the alveoli. On the first day after injury, cell infiltration begins to appear between the alveoli, whether in the central area or the surrounding area. From the second to the seventh day after the injury, the cellular infiltration became more and more serious, and the alveoli in the central area of the left lung almost disappeared. Only in the peripheral area of the left lung, a few alveoli still exist (Figure 5A-5C). Furthermore, we summed up all the lung tissue sections to calculate the total volume of the left lung. The volume of the left lung in the normal group was approximately 317.6±19.4 ^mm3 . On the 7th day after BLM injury, it was significantly reduced to about 217.2±17.0 ^mm3 , showing significant atrophy compared with the normal group (Fig. 5D). At this time, the volume of the atrophic left lung's alveolar structure also dropped to 59.7±8.4 cm ³ ; the parenchymal tissue with large amounts of cell infiltration accounted for about 63.8±2.5% of the volume of the left lung (Figures 5E and 5F). After BLM injury, the rat's respiratory rate per minute increased sharply within seven days, the arterial oxygen index dropped significantly, the alveoli in the left lung decreased, and a large number of cells infiltrated between tissues. These are all consistent with clinical acute respiratory distress syndrome. The characteristics are the same. Therefore, we successfully established a severe, reproducible, and consistent animal model of acute respiratory distress disease. Therefore, we chose the acute phase, the most severe day on the seventh day, to start treatment.

2.2 Administration of hyaluronic acid can increase the weight of mice with severe acute respiratory distress syndrome

The rats in the normal group gradually increased in weight as time went by. On the 7th day after BLM injury, the weight of rats in each group significantly stagnated. After that, although the body weight increased slightly over time, the weight of the rats in each group showed a significant decrease compared with the rats in the normal group, and the trend of being less in weight than the normal group continued until the 28th day. The weight of the rats in the BLM+HHA group has always been 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 to the 28th day. On the 28th day, the body weight of the rats in the BLM+LHA group increased significantly compared with the rats in the BLM group. The body weight of the rats in the BLM+MIX HA group was statistically higher than that of the rats in the BLM group on days 21 and 28 (Figure 6).

2.3 Administration of hyaluronic acid can increase the oxygenation index of arterial blood in rats with severe acute respiratory distress syndrome

Pulse oximetry is used to analyze arterial oxygen saturation (SpO2) to evaluate the function of pulmonary gas exchange. The results showed that during the 28 days of the experiment, the blood oxygen saturation of the rats in the normal group was maintained at around 98.2% (Figures 7A and 7B). On the 7th day after BLM injury, the blood oxygen saturation of rats in each group dropped significantly, about 84.3±0.7%. Until the 28th day, the arterial oxygen saturation of the rats in the BLM group did not decrease. Improvement, compared with the normal group of rats, showed a significant reduction (Figure 7A and 7B). The arterial oxygen saturation of rats in the BLM+HHA group was approximately 86 on the 28th day. , compared with the BLM injury group, there was no statistical difference. The arterial oxygen saturation of rats in the BLM+HHA group was lower than that in the BLM+MIX HA group from the 14th to the 28th day. From the 14th to the 28th day, the arterial blood oxygen saturation of rats in the BLM+LHA group and BLM+MIX HA group increased significantly compared with the BLM group. However, the arterial oxygen saturation of rats in the BLM+MIX HA group was more significantly improved than that in the BLM+LHA group on days 21 and 28 (Figures 7A and 7B).

In addition, before BLM injury (Day 0), the partial pressure of oxygen in the arterial blood of rats was approximately 86.4 - 89.7 mmHg (Figure 7C). On the 7th day after BLM injury, the partial pressure of arterial blood oxygen of rats in each group decreased significantly. Until the 28th day, the partial pressure of arterial blood oxygen of rats in the BLM group did not change much, which was significantly different from that of the normal group. Compared with white mice, all showed significant decreases (Figure 7C). Although the partial pressure of arterial blood oxygen in the BLM+HHA group of rats showed a slight upward trend on the 28th day, there was no statistical difference compared with the BLM group. Moreover, the arterial blood oxygen partial pressure of rats in the BLM+HHA group was still lower than that of rats in the BLM+ MIX HA group on day 28. The arterial blood oxygen partial pressure of rats in the BLM+LHA group increased significantly compared with the BLM group on the 28th day. The arterial blood oxygen partial pressure of rats in the BLM+ MIX HA group had significantly increased on the 14th day, and this upward trend continued until the 28th day. Compared with the BLM group, the arterial blood oxygen partial pressure was significantly improved (Figure 7C) .

Before BLM injury (Day 0), the partial pressure of carbon dioxide in the arterial blood of rats was approximately 46.6 - 47.9 mmHg (Figure 7D). On the 7th day after BLM injury, the arterial blood carbon dioxide partial pressure of rats in each group increased significantly compared with the normal group, rising to 51.6 – 54.7 mmHg. By the 14th day, the arterial blood carbon dioxide partial pressure in each group was still elevated. Only the arterial blood carbon dioxide partial pressure 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 the Normal group. The partial pressure of carbon dioxide in the arterial blood of the other groups of rats (BLM group, BLM+LHA group, and BLM+HHA group) on day 21 was not statistically different from that of the rats in the normal group (Figure 7D).

2.4 Administration of hyaluronic acid can slow down the breathing rate of rats with severe acute respiratory distress syndrome

The results of detecting the respiratory rate of the rats showed that the rats in the normal group maintained a fairly stable respiratory rate from day 0 to day 28, with the number of breaths within two seconds maintaining about 4-5 times (Figure 8A and 8F ). On the 7th day after BLM injury, the respiratory rate of each group increased significantly. In the BLM group, from the 7th to the 28th day, the respiratory rate was significantly faster than that of the normal group (Figures 8B and 8F). The respiratory rate of rats in the BLM+HHA group from the 7th to the 28th day was similar to that of the BLM group. Moreover, the respiratory rate of rats in the BLM+HHA group was still higher than that of rats in the BLM+ MIX HA group on days 21 and 28 (Figures 8D and 8F). The respiratory rate of rats in the BLM+LHA group was significantly reduced compared with the BLM group on day 14, but the respiratory rate was still higher than that of the normal group (Figures 8C and 8F). The respiratory rate of the rats in the BLM+MIX HA group was significantly lower on the 14th day compared with the BLM group, and this trend continued until the 28th day; and, the respiratory rate of the rats in the BLM+MIX HA group was on the 21st day. There was no statistical difference between the respiratory rate on day 28 and the normal group (Figures 8E and 8F).

2.5 Macroscopic appearance and HE staining show that administration of hyaluronic acid can improve the atrophy of the left lung of mice with severe acute respiratory distress syndrome.

On the 28th day, the rats in each group of perfused rats were sacrificed, and their left and right lungs were removed to observe the appearance of the lungs. The photos from the front view (upper row) and back view (lower row) show that white alveolar structures can be seen in both the left and right lungs of the rats in the normal group, and the alveoli appear complete and smooth. The left lung of the BLM group shrank significantly, a very small amount of alveolar tissue appeared only on the outer periphery of the left lung, and the central area of the left lung already showed diseased tissue without alveoli. Among the rats in the BLM+MIX HA group, the white alveolar area of their left lung and the overall volume of the left lung were significantly increased compared with the BLM injury group (Figure 9).

Continuous tissue sections of the left lungs of rats in each group were stained with HE, and low-magnification pictures were taken (Figure 10A), and then the central area of the left lung (Figure 10B) and the peripheral area (Figure 10C) were enlarged. The results showed that the left lung area of the rats in the normal group was larger and there were more alveoli. Connective tissue only appeared around the bronchi, and there was very little connective tissue between alveoli. In the left lung of the rats in the BLM group, complete alveoli appeared only on the outer periphery of the left lung. The central area was infiltrated with a large number of cells, and almost no alveoli could be seen. As for the rats in the BLM+MIX HA group, although there was a large amount of cell infiltration in the central area of their left lung, there was also a lot of alveolar space (white space) (Figure 10B and 10C). All HE-stained left lung tissue sections were summed up and statistically quantified. The results showed that the total volume of the left lung in the BLM group significantly shrunk, and the alveolar volume in the atrophied left lung was significantly reduced (Figure 10D-10E). Among them, the volume of the left lung of the rats in the BLM+MIX HA group was significantly increased, and the alveolar volume of the left lung was larger, which was similar to the structural proportion of the left lung of the rats in the normal group. It is speculated that administration of mixed molecular weight hyaluronic acid can increase the overall volume of the left lung and restore the alveolar space of the left lung (Figure 10D-10E).

2.6 Administration of hyaluronic acid can reduce the inflammatory reaction in the left lung of severe acute respiratory distress syndrome mice and the EMT reaction with alveolar epithelial cells.

Count the number of cells in the bronchial and alveolar washing fluid. The greater the number of cells, the more severe the inflammatory response.

On the 28th day, the alveolar lavage fluid from the left lungs of rats in each group was taken for cell counting. The results showed that the number of cells in the alveolar lavage fluid of the left lung of rats in the BLM group increased significantly. The number of cells in the alveolar lavage fluid of rats in the BLM+LHA group and BLM+HHA group was significantly lower than that in the BLM group, but it was still much higher than that in the Normal group. The number of cells in the alveolar lavage fluid of the left lung of rats in the BLM+MIX HA group was significantly lower than that in the BLM injury group, and was not statistically different from the Normal group. It is speculated that the administration of hyaluronic acid can reduce the inflammatory response in the lungs of severe acute respiratory distress syndrome mice. Therefore, the number of cells in the left lung is reduced. In particular, the administration of hyaluronic acid mixed with various molecular weights is similar to the normal group (Figure 11A).

After BLM injury, Alveolar Type II cells will proliferate in large quantities and undergo epithelial mesenchymal transition (EMT). At this time, N-cadherin (N-cadherin) will be expressed in large quantities. At this time, Alveolar Type II cells transform into myofibroblasts, causing alveoli reduction and tissue scarring.

Western blotting with anti-N-cadherin antibodies showed that only a small amount of N-cadherin was present in the left lungs of the normal group of rats. A large amount of N-cadherin was expressed in the left lungs of rats in the BLM group, representing a large amount of alveolar epithelial cell proliferation and epithelial cell deformation reaction. However, the N-cadherin production in the left lung of rats in the BLM+MIX HA group was reduced compared with the BLM injury group. The N-cadherin production in the left lung of rats in the BLM+MIX HA group was similar to that in the normal group (Figure 11B).

2.7 Administration of hyaluronic acid can promote the activation of type II macrophages in the left lungs of mice with severe acute respiratory distress syndrome to perform anti-inflammatory effects.

The left lung tissue sections of rats in each group were immunostained with anti-ED1 antibody to identify macrophages. The results showed that only a small number of small macrophages existed in the left lungs of the rats in the normal group. A large number of smaller macrophages appeared in the left lung tissue of rats in the BLM group. In the BLM+MIX HA group, there are still many small macrophages in the left lung tissue of rats, but larger phagocytes appear distributed among the connective tissue and in the alveolar space (Figure 12A). It is speculated that These larger macrophages are type II macrophages with anti-inflammatory effects.

Anti-CD86 antibody Western blot method was used to calibrate type I macrophages. Type I macrophages have a pro-inflammatory effect. The results are displayed. 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, and there was a statistical difference compared with the BLM group (Figure 12B). It is speculated that the administration of hyaluronic acid can prevent the differentiation and transformation of type 1 macrophages in the left lungs of severe acute respiratory distress syndrome mice, thereby reducing the inflammatory response.

Furthermore, Anti-CD206 antibody Western ink blot method was used to calibrate type II macrophages. Type II macrophages have anti-inflammatory effects. The concentration of CD206 in the left lung of rats in the BLM+MIX HA group increased statistically, and compared with the Normal group and the BLM group, it showed a statistical increase (Figure 12C). It is speculated that the administration of hyaluronic acid can promote the differentiation and transformation of type II macrophages in the left lungs of severe acute respiratory distress syndrome mice to carry out anti-inflammatory responses.

2.8 The administration of hyaluronic acid can stimulate the production of MMP in the left lungs of rats with severe acute respiratory distress syndrome and reduce the inflammatory response.

The protein content of Matrix metallopeptidase 9 (MMP-9) in the left lungs of rats in each group was quantified. The results showed that MMP-9 in the left lungs of rats in the BLM group was reduced compared with the normal group. situation. MMP-9 increased significantly in the left lungs of rats in the BLM+MIX HA group, which was statistically different from the BLM group (Figure 13A).

The protein content of Matrix metallopeptidase (MMP-2) in the left lungs of rats in each group was quantified. The results showed that MMP-2 in the left lungs of rats in the BLM group was not significantly lower than that in the normal group. changes. MMP-2 in the left lung of rats in the BLM+MIX HA group increased significantly, which was statistically higher than that in the normal group (Figure 13B). It is speculated that the administration of hyaluronic acid can stimulate the synthesis of MMP in the left lungs of severe acute respiratory distress syndrome mice, thus reducing the inflammatory response.

2.9 Administration of hyaluronic acid can change the expression of TLR-4 in the left lungs of severe acute respiratory distress syndrome mice and accelerate the regeneration of alveolar epithelial cells.

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, anti-TLR-4 antibody was used to perform Western ink blotting to observe the TLR-4 protein content in the left lungs 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. The expression amount of TLR-4 in the left lung of rats in the BLM+MIX HA group was significantly increased, and there were statistical differences compared with the normal group and the BLM injury group (Figure 14). It is speculated that the administration of hyaluronic acid can increase the production of TLR-4 protein in the left lungs of severe acute respiratory distress syndrome mice, thereby promoting the regeneration and repair of alveolar epithelial cells.

3.Conclusion

After BLM injury, the inflammatory response increases, inflammatory cells infiltrate, type I alveolar epithelial cells disappear, and massive proliferation of type II alveolar epithelial cells (Alveolar Type II cells) undergoes a deformation reaction and deforms into activated myofibroblasts. ), synthesize and release collagen, leading to extracellular matrix deposition. Mixing hyaluronic acid of various molecular weights can reduce inflammation, reduce alveolar epithelial cell deformation, stimulate the production of type II anti-inflammatory macrophages, reduce collagen deposition, and stimulate alveolar regeneration. 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 syndrome. Therefore, it is believed that giving hyaluronic acid can also mitigate the threat of the COVID-19 pandemic. References 1. Matthay MA et al., The acute respiratory distress syndrome. J Clin. Invest. 2012. 122(8): 2731- 2740. 2. Gibson PG et al., VOVID-19 ARDS: clinical features and differences from typical pre-COVID-19 ARDS. Med. J. Aust. 2020. 3. Wu C, Chen X, Cai Y, Xia Ja, Zhou X, Xu S, Huang H, Zhang L, Zhou X, Du C, Zhang Y , Song J, Wang S, Chao Y, Yang Z, Xu J, Zhou X, Chen D, Xiong W, Xu L, Zhou F, Jiang J, Bai C, Zheng J, Song Y: Risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in Wuhan, China. JAMA Internal Medicine 2020; 180:934- 943. 4. Zhang E YY, Zhang J, Ding G, Chen S, Peng C, Lavin MF, Yeo AJ, Du Z, Shao H. Efficacy of bone marrow mesenchymal stem cell transplantation in animal models of pulmonary fibrosis after exposure to bleomycin: A meta analysis. Exp Ther Med. 2019; 17: 2247-55. 5. Giji S, Arumugam M. Isolation and characterization of hyaluronic acid from marine organisms. Adv Food Nutr Res. 2014; 72: 61-77. 6. Hadidian Z, Pirie N. The preparation and some properties of hyaluronic acid from human umbilical cord. Biochem J. 1948; 42: 260-65. 7. Chu KA, Wang SY, Yeh CC, Fu TW, Fu YY, Ko TL, Chiu MM, Tsai PJ, Fu YS. Reversal of bleomycin-induced rat pulmonary fibrosis by a xenograft of human umbilical mesenchymal stem cells from Wharton's jelly. 2019 Theranostics. 22(9): 6646 - 6664. 8. Liang, J., Y. Zhang, T. Xie, N. Liu, H. Chen, Y. Geng, A. Kurkciyan, JM Mena, BR Stripp, D. Jiang & PW Noble (2016) Hyaluronan and TLR4 promote surfactant-protein-C-positive alveolar progenitor cell renewal and prevent severe pulmonary fibrosis in mice. Nat Med, 22 , 1285-1293. 9. Tavianatou, AG; Caon, I.; Franchi, M.; Piperigkou, Z.; Galeso, D.; Karamanos, NK Hyaluronan: molecular size-dependent signaling and biological functions in inflammation and cancer. FEBS J. 2019. 286: 2883- 2908. 10 . Yang HZ, Wang JP, Mi S, Liu HZ, Cui B, Yan HM, et al. TLR4 activity is required in the resolution of pulmonary inflammation and fibrosis after acute and chronic lung injury. Am J Pathol. 2012; 180: 275 -292.

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The foregoing summary and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. For purposes of illustrating the invention, there is shown in the drawing a presently preferred embodiment. It should be understood, however, that this invention is not limited to the precise arrangements and instrumentalities shown.

In the attached picture:

Figure 1 shows the experimental flow chart of inducing severe acute respiratory distress syndrome in rats and administering hyaluronic acid. It is divided into 5 groups. Each group injected BLM into the left bronchus of rats on day 0, and was given drug treatment on day 7. Sacrifice animals on day 28.

Figure 2 shows the changes in blood oxygen index of rats within 7 days after BLM injury. Figure 2A shows photos of arterial blood oxygen saturation in the hind limbs of rats measured daily within 7 days after BLM injury. Figure 2B is a quantitative graph of daily arterial saturation in rats within 7 days after BLM injury, which shows that arterial blood oxygen saturation dropped to the lowest on the seventh day after BLM injury. Figure 2C is a quantitative graph of the oxygen partial pressure index in the arterial blood of rats within 7 days after BLM injury, which shows that the arterial blood oxygen partial pressure dropped to the lowest on the seventh day after BLM injury. Figure 2D is a quantitative graph of the partial pressure of carbon dioxide index in the arterial blood of rats within 7 days after BLM injury, which shows that the partial pressure of carbon dioxide in the arterial blood rose to the highest level on the seventh day after BLM injury. Based on the arterial blood oxygen index, it was confirmed that the animal model of severe acute respiratory distress syndrome in rats was successfully established. ß: Compared with Day 0, there is a statistical difference, p<0.05. @: Compared with Day 1, the difference is statistically significant, p<0.05. ♣: Compared with Day 4, there is a statistical difference, p<0.05.

Figure 3 shows the changes in respiratory rate of rats within 7 days after BLM injury. Figure 3A shows that within 7 days after BLM injury, the respiratory rate record of the rat was detected every day, and the respiratory rate chart of 2 seconds was captured. Figure 3B is a quantitative chart of the respiratory rate per minute of the day in rats within 7 days after BLM injury. It shows that the respiratory rate reaches the highest on the seventh day of BLM injury. Based on the number of breaths per minute, it was confirmed that the animal model of severe acute respiratory distress syndrome in rats was successfully established. ß: Compared with Day 0, there is a statistical difference, p<0.05. @: Compared with Day 1, the difference is statistically significant, p<0.05.

Figure 4 shows the macroscopic morphology of the lungs of rats after sacrificial perfusion at 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 become uneven. On the second day after the BLM injury, the alveoli in the central area of the left lung were partially missing. On the seventh day of BLM injury, the left lung shrank and the alveoli in the central area disappeared. Based on the macroscopic morphology of the left lung, the animal model of severe acute respiratory distress syndrome in rats was successfully established.

Figure 5 shows the microscopic morphology of the left lung of rats at different days after BLM injury. Figure 5A is a low-power image of HE-stained left lung tissue sections of rats on different days, in which the white space is the alveolar tissue. Figure 5B is a high-power image of HE-stained tissue sections in the central area of the left lung (red box area in Figure 5A) of rats on different days. It shows that after BLM injury, cell infiltration was present and the alveoli were gradually reduced. Figure 5C is a high-power image of HE-stained tissue sections of the outer peripheral area of the left lung (blue box area in Figure 5A) of rats on different days. It shows that after BLM injury, there are still some alveoli (white spaces), but as the As the number of days of injury increases, the alveoli gradually decrease. Figure 5D shows the total volume of the left lung at different days after injury after quantitative HE staining; Figure 5E shows the alveolar volume of the left lung; Figure 5F shows the proportion of cell infiltration in the left lung. Figures 5D-5F 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. Based on the microscopic morphology of the left lung, it was determined that the animal model of severe acute respiratory distress syndrome in rats was successfully established. ß: Compared with the white rats on Day 0, the difference is statistically significant, p<0.05.

Figure 6 shows that administration of hyaluronic acid can increase the weight of ARDS mice. ß: Compared with the rats in the normal group on the same day, there is a statistically significant difference, p<0.05. #: Compared with the rats in the BLM injury 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 is a statistically significant difference, p<0.05. u, 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. Figure 8A shows the photos of rats in each group undergoing pulse arterial oximeter testing on day 28, in which the arrow points to the value of arterial blood oxygen saturation. Figure 7B quantifies the arterial blood oxygen saturation values of rats in each group at different times. ß: Compared with the rats in the normal group on the same day, there is a statistically significant difference, 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 is a statistically significant difference, p<0.05. u, 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 administration of hyaluronic acid can alleviate the shortness of breath in ARDS mice. Figures 8A-8E are 2-second captured recordings of respiratory rates of rats in each group at different times. Figure 8F quantifies the breathing rate per minute of each group of rats at different times. ß: Compared with the rats in the normal group on the same day, there is 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 is a statistically significant difference, p<0.05.

Figure 9 shows that administration of hyaluronic acid can increase the left lung volume of ARDS mice. The picture shows the appearance of the lungs of rats in each group on the 28th day. The upper row is the front view of the lungs in each group, and the lower row is the back view of the lungs in each group.

Figure 10 shows that administration of hyaluronic acid can repair the alveolar structure of ARDS mice. Figure 10A is a low-magnification picture of left lung tissue slices of rats in each group on day 28 after HE staining. Figure 10B is a high-magnification photo of the central area of the left lung tissue slices of each group on day 28 after HE staining. Figure 10C is a high-magnification photo of the outer periphery of the left lung stained with HE on the left lung tissue pieces of each group on day 28. Figure 10D sums all left lung tissue sections to quantify left lung volume. Figure 1OE quantifies the total alveolar volume of the left lung.

Figure 11 shows that administration of hyaluronic acid can reduce the inflammatory reaction and deformation reaction of alveolar epithelial cells in the lungs of ARDS mice. Figure 11A quantifies the number of cells in the alveolar wash fluid of the left lungs of rats in each group on day 28 to represent inflammation. The number of cells in the alveolar lavage fluid of the left lung of rats in the BLM group increased significantly. Figure 11B shows the left lungs of each group of rats on day 28. The concentration of N-cadherin (N-cadherin) in the left lung was quantified by Western blotting method to represent the deformation of alveolar epithelial cells. ß: Compared with the rats in the normal group, the difference is statistically significant, p<0.05. #: Compared with the rats in the BLM group, the difference is statistically significant, p<0.05. ♠: Compared with the rats in the BLM+MIX HA group on the same day, there is a statistically significant difference, p<0.05.

Figure 12 shows that administration of hyaluronic acid can promote type II macrophages and reduce type I macrophages in the left lungs of ARDS mice, thereby reducing the inflammatory response. Figure 12A shows the left lungs of rats in each group on day 28. Anti-ED1 antibody was used for tissue immunostaining to identify macrophages. It showed that the phagocytes in the BLM group were mostly smaller. The phagocytes in the BLM+MIX HA group were mostly larger in size. Figure 12B shows the left lung tissue of each group of rats on day 28. Western blot staining was performed with Anti-CD86 antibody to quantify the changes in M1 macrophages. Figure 12C shows the left lung tissue of each group of rats on day 28. Western blot staining was performed with Anti-CD206 antibody to quantify the changes in M2 macrophages. ß: Compared with the rats in the normal group, there is a statistical difference, p<0.05. #: Compared with the rats in the BLM group, the difference is statistically significant, p<0.05.

Figure 13 shows that administration of hyaluronic acid can promote the synthesis of MMP in the left lungs of ARDS mice and reduce the inflammatory response. Figure 13A shows the left lung tissue of each group of rats on day 28. Western blot staining was performed with Anti-MMP9 antibody to quantify the MMP9 protein content. Figure 13B shows the left lung tissue of each group of rats on day 28. Western blot staining was performed with Anti-MMP2 antibody to quantify the MMP2 protein content. ß: Compared with the rats in the normal group, there is a statistical difference, p<0.05. #: Compared with the rats in the BLM group, the difference is statistically significant, p<0.05.

Figure 14 shows that administration of hyaluronic acid can promote the synthesis of TLR4 in the left lungs of ARDS mice to stimulate the regeneration of alveolar epithelial cells. The picture shows the left lung tissue of each group of rats on day 28. Western blot staining was performed with Anti-TLR4 antibody to quantify the TLR protein content. ß: Compared with the rats in the normal group, there is a statistical difference, p<0.05. #: Compared with the rats in the BLM group, there is a statistical difference, p<0.05.

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Claims (10)

A use of hyaluronic acid in preparing a medicament for treating acute respiratory distress syndrome (ARDS), wherein the ARDS is severe ARDS, which causes alveoli reduction, and wherein the hyaluronic acid is a hyaluronic acid mixture containing low molecular weight hyaluronic acid, medium molecular weight hyaluronic acid and high molecular weight hyaluronic acid. Such as the use of claim 1, wherein the agent is administered after the occurrence of severe ARDS. Such as the use of claim 1, wherein the molecular weight of the hyaluronic acid is 10kDa-2MDa. Such as the use of claim 1, in which the agent effectively improves the symptoms of ARDS, slows down the respiratory rate significantly increased due to ARDS, significantly increases blood oxygen concentration, improves lung volume and alveolar volume, reduces alveolar epithelial cell deformation, anti-inflammation, and can Stimulates the regeneration of alveolar epithelial cells. The use of claim 1, wherein the agent is effective in improving reduced blood oxygen saturation levels, alleviating increased respiratory rate and restoring alveolar function. The use of claim 1, wherein the pharmaceutical agent is administered via blood injection, intramuscular injection, or subcutaneous injection. Such as the use of claim 1, wherein the pharmaceutical agent is delivered directly from the respiratory tract to the trachea or lungs. Such as the use of claim 7, wherein the pharmaceutical agent is directly inhaled through the nasal cavity. Claim the use of item 7, wherein the agent is administered through a bronchoscope from the nasal cavity or oral cavity to the trachea. Claim the use of item 7, wherein the agent is administered through tracheostomy.