TW202243679A - 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 medicament for treating acute respiratory distress

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

Acute respiratory distress syndrome (ARDS) is a complex disease that can be caused by multiple factors. According to the Berlin definition, patients are exposed to risk factors within seven days, resulting in acute diffuse pulmonary inflammation, hypoxemia, and respiratory failure (Mattay et al., 2012). is a symptom of acute lung injury, a life-threatening condition. 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. In current clinical medicine, 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.

In 2020, due to the COVID-19 pandemic, the number of patients with ARDS will increase significantly. According to statistics, up to 31% - 42% of hospitalized patients with new coronary pneumonia 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 new coronavirus infection have severe lung inflammation in the acute phase, and varying degrees of pulmonary fibrosis in the recovery phase, resulting in permanent degradation of lung function and even lifelong damage to other organs. Therefore, 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 relieve intensive care in hospitals. Stress on the ward.

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

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

In another aspect, the present invention provides a medicament or a pharmaceutical composition for treating acute respiratory distress, which comprises a therapeutically effective amount of hyaluronic acid.

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

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

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

According to the embodiment of the present invention, administering hyaluronic acid to ARDS model rats 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 anti-inflammatory M2 cell production, increase MMP (matrix-metallopeptidase) activity to promote anti-inflammatory response and decompose fibrosis, and can stimulate the regeneration of alveolar epithelial cells.

According to an embodiment of the present invention, hyaluronic acid is effective in improving decreased blood oxygen saturation levels, alleviating increased respiration rate and/or restoring 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 present invention will be apparent from the following detailed description of several embodiments and from the appended claims.

Unless defined otherwise, 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) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

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

As used herein, the term "acute respiratory distress syndrome (ARDS)" refers to the patient's lung changes resulting from extensive alveolar microvascular damage, which increases the permeability between endothelial cells and causes alveolar Phenomena such as hemorrhage and edema eventually lead to the increase of dead space and shunt in the lung, and the deterioration of lung compliance and oxygenation, resulting in clinical respiratory distress.

As used herein, "hyaluronan or hyaluronic acid, HA for short", also known as hyaluronic acid or uronic acid, exists in the connective tissue and dermis of the human body, and is a transparent jelly-like substance. 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 may include the above hyaluronic acid or its salts with different molecular weights, and their mixtures, and the molecular weight ranges from 10 kDa to 2 MDa.

As used herein, 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. As used herein, the term "normal level" or "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. For example, 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 reduced level means 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.

The term "subject 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 method of treatment of the present invention is diagnosed as suffering from ARDS with the following symptoms: (1) acute onset; (2) decreased oxygenation index (PaO2/FIO2); (3) chest X-ray presentation 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.

The term "individual" or "subject" as used herein 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 experimental animals (such as large rats, mice, guinea pigs, etc.).

As used herein, the term "treating" 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 purpose of curing, curing, relieving , to 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.

As used herein, the term "therapeutically effective amount" refers to the amount of an active ingredient which provides the desired therapeutic or biological effect in the subject being 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 can be by various procedures known in the art, and can be administered systemically, for example, intravenously, intraarterially, or subcutaneously via injection, or by nasal inhalation, or by nasal or oral elongation. Administration into the trachea or tracheostomy.

In order to implement the method of the present invention, the hyaluronic acid containing hyaluronic acid can be sent to the lungs by the usual method in the field of the present invention. One 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. Depending on the mode 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, wherein the weight percentage is calculated based on the weight of the entire composition. A "pharmaceutically acceptable carrier" is nontoxic to the subject at the dosages and concentrations employed and is compatible with hyaluronic acid and any other ingredients of any formulation comprising hyaluronic acid.

In some embodiments, it can be prepared in 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.

The invention is further illustrated by the following examples, which are intended to be illustrative and not limiting. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which 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 severe acute respiratory distress disease animal model

Male rats (SD rats, 230-250 g) were injected intraperitoneally with Zoletil 50 and Xylazine hydrochlotide (Sigma 23076359). After the rats were deeply anesthetized, 5 mg Bo Bleomycin (BLM for short) was dissolved in 200 μl of sterile saline (1 unit activity/1 mg BLM, Nippon Kayaku Co., Ltd.), injected into the bronchi of rats with a 30G needle, and then, Rats lie on the left side at 60 degrees for 90 minutes, resulting in severe, reproducible, left lung injury animal model (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 example uses a mixture of different molecular weight hyaluronic acid, high molecular weight hyaluronic acid, and low molecular weight hyaluronic acid. On the 7th day after the BLM injection, 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 of administration of hyaluronic acid (Figure 1A). All were injected into the mice under anesthesia through the trachea.

1.3 Experimental grouping

The experimental animals were divided into five groups:

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

The second group is the BLM group, that is, on day 0, the rats received intratracheal injection of 5 mg BLM. Starting on day 7 after BLM injection, only 200 µl 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 on day 7 after BLM injury, low molecular weight hyaluronic acid (MW 10 kDa - 100 kDa) was intratracheally administered seven times (Fig. 1).

The fourth group is the BLM+HHA group, that is, on day 0, the rats received 5 mg of BLM intratracheally. Beginning on the 7th day after the 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 5 mg of BLM intratracheally. Beginning on day 7 after BLM injury, mixed molecular weight hyaluronic acid (MW 10 kDa - 2 MDa) was intratracheally administered seven times (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.

1.4 Pulmonary function test of experimental animals-determination of arterial oxygen saturation

After the mice were anesthetized with Isoflurane (Baxter228-194), the hind paws were clamped with a pulse oximeter (Pulseoximeter, NONIN LS1-10R) to detect the oxygen saturation in the arterial blood.

1.5 Testing of Pulmonary Function of Experimental Animals - Determination of Pulmonary Respiratory Frequency

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 4.0 MP45 software, and the rats were kept still in the chamber respiratory rate at time.

1.6 Perfusion fixation of experimental animals and paraffin embedding of tissues

Excess 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.

After fixation, the left and right lungs were sequentially put into alcohols with increasing concentration (70%, 80%, 95%, 100%) to dehydrate each time for 20 minutes. After tissue dehydration, soak in xylene, xylene: paraffin = 1:1 mixture, xylene: paraffin = 1:3 mixture, pure wax. Finally, the tissue is embedded in pure wax.

1.7 Tissue sectioning and taking methods

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 µm 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.

Carry out sagittal section from the outermost side of the lung, make serial slices, and take slices.

1.8 Staining Hematoxylin & Eosin Stain (Hematoxylin & Eosin Stain, referred to as HE Stain)

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. Lung tissue sections were soaked in alcohols of increasing concentrations for dehydration (50%, 70%, 80%, 90%, 95%, 100% in sequence), and then soaked in xylene twice, 5 minutes each time. Finally, the slides were sealed with mounting glue (Permount, Fisher Scientific SP15-500) for optical microscope observation and photographing.

1.9 Cell count of bronchoalveolar lavage (BAL)

外徑1.22 mm)，將插管由左支氣管伸入左肺，以0.5 ml無菌PBS進行沖洗後吸出。接著，進行第二次沖洗，以新的0.5ml無菌PBS沖吸一次；由此得到1 ml肺泡沖洗液。將取得的肺泡沖洗液以1500 rpm離心5分鐘，下層細胞則以1 ml食鹽水回溶，進行細胞計數。 After the rats were perfused with PBS, the whole lung was taken out from the trachea, and a 20G needle was used with a PE cannula (PE60, inner diameter 0.76

1.22 mm in outer diameter), the cannula was inserted from the left bronchus into the left lung, rinsed with 0.5 ml sterile PBS and then sucked out. Then, wash for the second time, flush once with new 0.5ml sterile PBS; thus obtain 1 ml 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 in 1 ml of saline for cell counting.

1.10 Protein Extraction

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

1.11 Immunostaining and Western blotting

Lung tissue slices, or NC paper (NC paper) after electrophoresis of left lung protein, were added 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 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 (secondary antibodies) at room temperature for 60 minutes, and then react with ABC kit (Avidin-biotinylated-horseradish peroxidase complex (ABC) kit, Vector Laboratories) at room temperature for 60 minutes, then react with 0.01 M PBS was washed 3 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). The comparison between the mean values was performed by one-way analysis of variance (One-Way ANOVA) or two-way analysis of variance (Two-Way ANOVA), and then by Turkey’s test for multiple comparisons. For the experimental data, p < 0.05 was regarded as the minimum starting standard for significant differences.

2. Results

2.1 Establishment of severe acute respiratory distress disease animal model

Within seven days of BLM injury, the changes in the lung function of the rats were detected. First, 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. Prior to injury (Day 0), arterial oxygen saturation was typically maintained at 97 %. This decreased to 87.7±1.2 % on the first day after injury, 84.7±1.7 % on the second day, and only 83.7±0.9 % remained on day 7 after injury (Fig. 2A and 2B). 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. Before the injury (Day 0), the partial pressure of oxygen (PO2) in 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 (Fig. 2C). Before the injury (Day 0), the partial pressure of carbon dioxide (PCO2) in the arterial blood was about 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 even increased to 53.8±2.0 mmHg ( Figure 2D). The third detection is the respiratory rate per minute. Before the injury (Day 0), the respiratory rate was about 132.3±11.8 cycles/min; on the first day after the injury, it rose to 188.0±32.6 cycles/min; after that, the breathing rate of the mice gradually accelerated and increased on the seventh day after the injury to 330.3±17.3 cycles/min (Figure 3A and 3B). The results showed that BLM injury caused a decline in lung function, and the arterial blood was hypoxic, resulting in shortness of breath.

From the macroscopic view of the lungs, both the left and right lungs of normal rats are smooth and complete, and the white is the position of the normal alveoli. On the first day after the injury, the left lung had already developed a bumpy surface. On the second day after the injury, in the central area of the left lung, the white alveoli had been partially lost. On the fourth day after the injury, the white alveoli in the central area of the left lung disappeared completely, leaving only the alveoli in the surrounding area. By the 7th day after the injury, the left lung shrank significantly, and only the alveoli remained in the surrounding area (Fig. 4). Next, HE staining was performed on the left lung tissue sections to observe changes in fine patterns (Fig. 5A). Enlarged respectively, the central area and surrounding areas of the left lung (Figure 5B and 5C), the left lung of the rats in the Normal group, whether in the central area or in the surrounding area, is normal alveolar tissue, and connective tissue mostly exists around the bronchi. There is very little connective tissue between the alveoli. On the first day after injury, cellular infiltration between the alveoli began to appear in both the central and peripheral regions. From the second day 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 remained (Fig. 5A-5C). Furthermore, we summed up all the lung tissue sections and calculated the total volume of the left lung. The volume of the left lung in the normal group was about 317.6±19.4 mm ³ cubic millimeters. On the 7th day after BLM injury, it was significantly reduced to about 217.2±17.0 mm ³ , and there was a significant atrophy compared with the normal group (Fig. 5D). In the shrunken left lung at this time, the volume of the alveolar structure also decreased to 59.7±8.4 cm ³ ; the solidified tissue infiltrated by a large number of cells accounted for about 63.8±2.5% of the left lung volume (Fig. 5E and 5F). After BLM injury, the minute respiratory rate of rats increased sharply within seven days, the arterial oxygen index decreased significantly, the alveoli in the left lung decreased, and a large number of cells infiltrated between tissues, all of which were consistent with clinical symptoms of acute respiratory distress. The characteristics are the same. Thus, we successfully established a severe, reproducible, and consistent, animal model of acute respiratory distress. We therefore choose the acute phase, the most severe seventh day, to start treatment.

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

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).

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

，與BLM傷害組相較，並沒有統計上差異。BLM+HHA組大白鼠的動脈血氧飽和度，從第14天到第28天，都比BLM+MIX HA組來的少。從第14天到第28天，BLM+LHA組、與BLM+MIX HA組大白鼠的動脈血氧飽和度，相較BLM組都有明顯的增加。然而，BLM+MIX HA組大白鼠的動脈血氧飽和度在第21天與第28天又較BLM+LHA組有更明顯的改善 (圖7A及7B)。 Arterial oxygen saturation (SpO2) was analyzed with a pulse oximeter to assess 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% (FIGS. 7A and 7B). On the 7th day after the BLM injury, the blood oxygen saturation of the rats in each group decreased significantly, about 84.3±0.7%, until the 28th day, the arterial blood oxygen saturation of the rats in the BLM group did not Improvement, compared with the normal group of rats, showed a significant reduction (Figure 7A and 7B). The arterial blood oxygen saturation of rats in the BLM+HHA group was about 86 on the 28th day.

, compared with the BLM injury group, there was no statistical difference. The arterial blood oxygen saturation of rats in the BLM+HHA group was lower 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. However, 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 (Figure 7A and 7B).

In addition, before BLM injury (Day 0), the partial pressure of oxygen in the arterial blood of rats was about 86.4 - 89.7 mmHg (Fig. 7C). On the 7th day after the BLM injury, the partial pressure of oxygen in the arterial blood of the rats in each group decreased significantly. The partial pressure of oxygen in the arterial blood of the rats in the BLM group did not change much until the 28th day, which was larger than that in the normal group. Compared with white mice, all showed obvious reduction (Fig. 7C). Although the partial pressure of oxygen in arterial blood of rats in the BLM+HHA group tended to rise slightly on the 28th day, there was no statistical difference compared with the BLM group. Moreover, 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. Compared with the BLM group, the partial pressure of oxygen in arterial blood of the rats in the BLM+LHA group increased significantly on the 28th day. The arterial 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 about 46.6 - 47.9 mmHg (Fig. 7D). On the 7th day after the BLM injury, the partial pressure of carbon dioxide in the arterial blood of the rats in each group was significantly higher than that in the normal group, rising to 51.6 – 54.7 mmHg. By the 14th day, 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 the rats in the other groups (BLM group, BLM+LHA group, and BLM+HHA group) had no statistical difference from the normal group rats on day 21 (Fig. 7D).

2.4 Administration of hyaluronic acid can relieve the respiratory rate of mice 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 breathing rate from the 0th day to the 28th day, and the number of breaths within two seconds was maintained at about 4-5 times (Figure 8A and 8F ). On day 7 after BLM injury, the respiratory rate increased significantly in all groups. In the BLM group, from the 7th day to the 28th day, the respiratory rate was significantly faster than that of the normal group (Figure 8B and 8F). The respiration rate of the rats in the BLM+HHA group from the 7th day to the 28th day was similar to that of the BLM group. Moreover, 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 (Fig. 8D and 8F). Compared with the BLM group, the respiratory rate of the rats in the BLM+LHA group decreased significantly on the 14th day, but the respiratory rate was still higher than that of the normal group (Fig. 8C and 8F). Compared with the BLM group, 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 (Fig. 8E and 8F).

2.5 According to macroscopic appearance and HE staining, the administration of hyaluronic acid can improve the atrophy of the left lung of severe acute respiratory distress rats

On the 28th day, 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. In the BLM group, 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. Among them, the white alveolar area of the left lung of the rats in the BLM+MIX HA group, as well as the overall volume of the left lung, were significantly larger than those in the BLM injury group (Figure 9).

Continuous tissue sections of the left lung of rats in each group were stained with HE, and a low-magnification picture was taken (Fig. 10A), and then the central area of the left lung (Fig. 10B) and the peripheral area (Fig. 10C) were enlarged respectively. 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. In the left lung of the rats in the BLM group, 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. In contrast to the rats in the BLM+MIX HA group, although there were a large number of cell infiltrations in the central area of the left lung, there were also many alveolar spaces (white spaces) (Fig. 10B and 10C). All the HE-stained left lung tissue sections were summed up, and the results of statistical quantification showed that the total volume of the left lung in the BLM group was significantly shrunk, and the alveolar volume in the shrunken left lung was significantly reduced (Fig. 10D-10E). Among them, the volume of the left lung of the rats in the BLM+MIX HA group was significantly increased, and the volume of the alveoli in the left lung was larger, which was equivalent to the structure ratio 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 (Fig. 10D-10E).

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

Calculate the number of cells in the flushing fluid of the bronchi and alveoli, the more the number of cells, the more intense the inflammatory response.

On the 28th day, 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. Compared with the BLM group, 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. Compared with the BLM injury 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 from the Normal group. It is speculated that the administration of hyaluronic acid can reduce the lung inflammation of severe acute respiratory distress mice, so the number of cells in the left lung is reduced. In particular, administration of mixed hyaluronic acid of various molecular weights is similar to that of the normal group (Fig. 11A).

After BLM injury, 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 point, the Alveolar Type II cell transforms into myofibroblasts, resulting in reduced alveoli and tissue scarring.

The results of western blotting with anti-N-cadherin antibody showed that only a small amount of N-cadherin existed in the left lung of the rats in the normal group. There will be a large amount of N-cadherin in the left lung of the rats in the BLM group, which represents the massive proliferation of alveolar epithelial cells and the epithelial cell deformation reaction. However, the production of N-cadherin in the left lung of rats in the BLM+MIX HA group was lower than that in the BLM injury group. The production of N-cadherin in the left lung of rats in the BLM+MIX HA group was similar to that of the normal group (Fig. 11B).

2.7 The administration of 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. In the left lung tissue of the rats in the BLM group, a large number of macrophages with small particles appeared. In the left lung tissue of the rats in the BLM+MIX HA group, there were still many small macrophages, but the larger phagocytes were distributed in the connective tissue and in the alveolar space (Fig. 12A). These larger macrophages are the second type of macrophages with anti-inflammatory effects.

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 (Fig. 12B). 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.

Furthermore, 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 (Fig. 12C). 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.

2.8 Administration of hyaluronic acid can stimulate the production of MMP in the left lung of severe acute respiratory distress rats and reduce the inflammatory response

The content of matrix metalloproteinase-9 (Matrix metallopeptidase 9, referred to as MMP-9) protein in the left lung of the rats in each group was quantified. 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 (Fig. 13A).

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 change. MMP-2 in the left lung of rats in the BLM+MIX HA group was significantly increased, 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 lung of severe acute respiratory distress rats, thus reducing the inflammatory response.

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

Type II alveolar epithelial cells express a large amount of Toll-like receptor 4 (TLR-4), which stimulates 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. The expression of TLR-4 in the left lung of the rats in the BLM+MIX HA group 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.

3. Conclusion

After BLM injury, the inflammatory reaction increases, the infiltration of inflammatory cells, the disappearance of type I alveolar epithelial cells, and the massive proliferation of type II alveolar epithelial cells (Alveolar Type II cells) undergo a deformation reaction, deforming into activated myofibroblasts (myofibroblasts). ), synthesize and release collagen, leading to extracellular matrix deposition. Administration of mixed hyaluronic acid with different 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. Therefore, giving hyaluronic acid is believed to also slow down 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 organ isms. 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.; Galesso, 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, as well as the following detailed description of the invention, are better understood when read in conjunction with the accompanying drawings. In order to illustrate the invention, a presently preferred embodiment is shown in the drawings. It should be understood, however, that the 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 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. Figure 2A shows the photographs of arterial blood oxygen saturation of the hind limbs of rats detected every day for 7 days after BLM injury. Fig. 2B is a quantitative graph of arterial saturation in rats every day within 7 days after BLM injury, which shows that arterial blood oxygen saturation drops to the lowest on the seventh day after BLM injury. Figure 2C is a quantitative graph 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. Figure 2D is a quantitative graph of the partial pressure of carbon dioxide in the arterial blood of rats within 7 days after BLM injury, which shows that the partial pressure of carbon dioxide in arterial blood rose to the highest on the seventh day after 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. ß: 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. Figure 3A shows the recordings of the respiration rate of rats detected every day within 7 days after BLM injury, and the respiration rate graph was extracted for 2 seconds. Fig. 3B is a quantitative diagram of the daily respiration rate per minute of rats within 7 days after BLM injury, which shows that the respiration rate reached the highest on the seventh day after 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. ß: 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 of BLM injury, the alveoli in the central region of the left lung had partially disappeared. On the seventh day of the BLM injury, the left lung was shrunk and the alveoli in the central area were absent. 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. Figure 5A is a low magnification image of left lung tissue sections of rats stained with HE at different days, in which the white space is alveolar tissue. Figure 5B is a high-magnification image of HE stained tissue sections in the central region of the left lung (the red frame area in Figure 5A ) of rats at different days. It shows that after BLM injury, cells infiltrate and alveoli gradually decrease. Figure 5C is a high-magnification image of HE-stained tissue sections of the outer peripheral area of the left lung (blue frame area in Figure 5A) of rats at different days, which shows that after BLM injury, there are still some alveoli (white space), but with The number of days of injury increases and the alveoli gradually decrease. Figure 5D is quantification of the total volume of the left lung at different days after injury after HE staining; Figure 5E is the volume of alveoli in the left lung; Figure 5F is the proportion of cell infiltration in the left lung. Figures 5D-5F show that the volume of the left lung after BLM injury gradually decreased, 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. ß: 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. 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. Fig. 8A is a photo of rats in each group undergoing pulse arterial oximeter detection on the 28th day, where the arrow points to the value of arterial blood oxygen saturation. Fig. 7B is the quantitative value of arterial blood oxygen saturation in 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 the administration of hyaluronic acid can slow down the shortness of breath in ARDS sick mice. Figures 8A-8E are 2-second captured recordings of the respiratory rate of rats in each group at different times. Figure 8F is the quantification of the breathing rate per minute of rats in each group 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 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. Fig. 10A is a low magnification picture of the left lung tissue slices of rats in each group on day 28 after HE staining. Figure 10B is a high-magnification magnified 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 magnified photo of the left lung tissue slices of each group on day 28 after HE staining. Figure 10D sums all left lung tissue sections to quantify left lung volume. Figure 10E 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. FIG. 11A 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. FIG. 11B is the left lung of each group of rats on day 28. The concentration of N-cadherin in the left lung was quantified by western blotting to represent the deformation of alveolar epithelial cells. ß: Compared with the rats in the normal group, there is a statistically significant difference, p<0.05. #: Compared with the rats in the BLM group, 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.

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. Fig. 12A is the left lung of rats in each group on day 28, tissue immunostaining was performed with Anti-ED1 antibody, and macrophages were marked. 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. Figure 12B shows the left lung tissues of rats in each group on day 28, which were stained with Anti-CD86 antibody by western blot method to quantify the changes of M1 macrophages. Figure 12C shows the left lung tissues of rats in each group on day 28, stained with Anti-CD206 antibody by western blot method to quantify the changes of 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, 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. FIG. 13A shows the left lung tissues of rats in each group on day 28, which were stained with Anti-MMP9 antibody by western blot method to quantify the protein content of MMP9. Fig. 13B 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. 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 statistically significant 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. 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 for preparing a medicament for treating acute respiratory distress. The use of claim 1, wherein the acute respiratory distress is severe acute respiratory distress. Such as the use of claim 1, wherein the molecular weight of the hyaluronic acid is 10 kDa-2MDa. Such as the use of claim 1, wherein the hyaluronic acid effectively improves the symptoms of ARDS, slows down the respiratory rate that is significantly increased due to ARDS, significantly increases the blood oxygen concentration, improves lung volume and alveolar volume, reduces alveolar epithelial cell deformation, reduces collagen accumulation, Anti-inflammatory and can stimulate the regeneration of alveolar epithelial cells. The use as claimed in claim 1, wherein the hyaluronic acid is effective in improving decreased blood oxygen saturation levels, alleviating increased respiratory rate and restoring alveolar function. The use according to claim 1, wherein the hyaluronic acid is administered through blood injection, intramuscular injection, or subcutaneous injection. The use according to claim 1, wherein the hyaluronic acid is directly delivered from the respiratory tract to the trachea or lung. The use as claimed in Item 7, wherein the hyaluronic acid is directly inhaled through the nasal cavity. The use according to claim 7, wherein the hyaluronic acid is administered through the nasal cavity or the oral cavity to the bronchoscope of the trachea. The use as claimed in item 7, wherein the hyaluronic acid is administered through tracheectomy.

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