CN108434439B

CN108434439B - Medical application of calreticulin

Info

Publication number: CN108434439B
Application number: CN201810062251.0A
Authority: CN
Inventors: 刘秀华; 王晓礽; 徐菲菲; 宋丹丹; 陶天琪
Original assignee: Chinese PLA General Hospital
Current assignee: Chinese PLA General Hospital
Priority date: 2018-01-23
Filing date: 2018-01-23
Publication date: 2020-02-07
Anticipated expiration: 2038-01-23
Also published as: CN108434439A

Abstract

The invention relates to the field of biomedicine, in particular to medical application of calreticulin. More specifically, the present invention relates to the use of calreticulin for the prevention and/or treatment of acute altitude sickness.

Description

Medical application of calreticulin

Technical Field

Background

Acute altitude disease (AMS) refers to acute hypoxia response or disease that occurs when a person first enters a plateau and is classified into mild and severe AMS depending on its severity. Mild AMS refers to acute altitude reaction; heavy AMS is further classified into: high altitude cerebral edema, high altitude pulmonary edema and mixed type (combined high altitude cerebral edema and high altitude pulmonary edema).

At present, most of the measures for preventing and treating acute altitude diseases are ladder habituation in the altitude entering process and symptomatic treatment aiming at acute hypoxia symptoms such as acute pulmonary edema, cerebral edema and the like of patients. The method has the problems of high stage cost, delayed conventional treatment implementation time, influence on the curative effect and the like. Therefore, there is an urgent need in the art for new, simple, effective drugs for the prevention and treatment of acute altitude diseases.

Summary of The Invention

In a first aspect, the present invention provides the use of calreticulin the manufacture of a medicament for the prevention and/or treatment of acute altitude sickness. In some embodiments, the calreticulin protein is a human calreticulin protein, preferably a recombinant human calreticulin protein. In some embodiments, the calreticulin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1-5. In some embodiments, the acute altitude disease is selected from acute altitude reaction, high altitude pulmonary edema, high altitude cerebral edema, or a combination thereof.

In a second aspect, the present invention provides a pharmaceutical composition for preventing and/or treating acute altitude disease, comprising a therapeutically effective amount of calreticulin, and a pharmaceutically acceptable carrier. In some embodiments, the calreticulin protein is a human calreticulin protein, preferably a recombinant human calreticulin protein. In some embodiments, the calreticulin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1-5. In some embodiments, the acute altitude disease is selected from acute altitude reaction, high altitude pulmonary edema, high altitude cerebral edema, or a combination thereof.

In a third aspect, the present invention provides a method for the prevention and/or treatment of acute altitude sickness comprising administering to a subject in need thereof a therapeutically effective amount of calreticulin. In some embodiments, the calreticulin protein is a human calreticulin protein, preferably a recombinant human calreticulin protein. In some embodiments, the calreticulin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1-5. In some embodiments, the acute altitude disease is selected from acute altitude reaction, high altitude pulmonary edema, high altitude cerebral edema, or a combination thereof.

Drawings

FIG. 1. groups of rats lung tissue wet and dry weights.

FIG. 2. Wet-to-dry weight ratio of lungs of rats in each group.

FIG. 3. Wet and Dry weight of brain tissue in various groups of rats. P <0.05 compared to normal control group and P <0.05 compared to model group.

FIG. 4 shows the wet-to-dry weight ratio of the brain tissue of each group of rats. P <0.05, compared to normal control group, # P <0.05, compared to model group.

FIG. 5. arterial blood pH values of rats in each group.

Figure 6 groups of rats arterial blood PCO 2.

FIG. 7. rat arterial blood PO2 for each group.

FIG. 8. rat arterial blood Beecf from each group.

Fig. 9. rat arterial blood HCO3 for each group.

FIG. 10. Each group of rats arterial blood TCO 2.

FIG. 11. rat arterial blood sO 2% for each group.

FIG. 12 rat arterial blood Na levels for each group.

FIG. 13 arterial blood potassium levels in rats of each group.

FIG. 14. groups of rats arterial blood iCa levels.

Figure 15. rat arterial blood glucose levels for each group.

FIG. 16 shows the arterial hematocrit of rats in each group.

Figure 17 rat arterial blood hemoglobin levels for each group.

FIG. 18 is a graph showing HE staining of rat lung tissue (100X).

FIG. 19. calreticulin cloning strategy.

Detailed Description

In a first aspect, the present invention provides the use of calreticulin the manufacture of a medicament for the prevention and/or treatment of acute altitude sickness.

Acute altitude sickness (AMS) refers to acute hypoxia response or disease that occurs when a person rapidly enters a plateau (e.g., above 3000 meters above sea level), and is classified as mild and severe AMS depending on its severity. Mild AMS refers to acute altitude reaction; heavy AMS is further classified into: high altitude cerebral edema, high altitude pulmonary edema and mixed type (combined high altitude cerebral edema and high altitude pulmonary edema).

When a patient with acute altitude reaction enters the altitude, the symptoms are most obvious on the 1 st to2 th days and gradually relieved later, most of the symptoms disappear basically in 6 to 7 days, and a few symptoms can exist continuously. It is mainly manifested as headache, hypomnesis, insomnia, dreaminess, increased respiratory strength and frequency, tachycardia, etc. Some patients have cyanosis, elevated blood pressure, etc. The acute altitude reaction should be diagnostically identified with viral diseases such as influenza.

The plateau pulmonary edema is generally developed 1 to 3 days after people in plain or low altitude areas rapidly enter the plateau, and is also developed 7 to 14 days later, and the manifestation of the plateau pulmonary edema is the same as that of the general pulmonary edema. The diagnosis of high altitude pulmonary edema should be identified with pneumonia, pulmonary embolism, pneumothorax, cardiogenic pulmonary edema or neurogenic pulmonary edema.

The patients with high altitude cerebral edema mostly have the symptoms of acute altitude sickness, and then have obvious psychoneurosis symptoms such as severe headache, mental abnormality, absentmindedness, obstinate nausea, vomiting and coma. Cerebrospinal fluid examination has increased pressure. High altitude cerebral edema should be diagnostically identified with metabolic or toxic encephalopathy, cerebrovascular accident and craniocerebral trauma.

In some embodiments of the invention, the acute altitude disease is selected from any one or more of acute altitude reaction, high altitude pulmonary edema or high altitude cerebral edema.

Calreticulin (CRT), originally discovered as a calcium binding protein and chaperone in the endoplasmic reticulum of cells, has been shown to regulate protein folding, calcium homeostasis and apoptosis. The inventor surprisingly found that calreticulin can effectively relieve symptoms related to high altitude hypobaria and hypoxia, in particular relieve high altitude pulmonary edema and high altitude cerebral edema, and thus can be used for preventing and/or treating acute altitude diseases. In some embodiments of the invention, the calreticulin is human calreticulin.

The calreticulin proteins described herein encompass functional variants of calreticulin in addition to wild-type calreticulin. In some embodiments, a functional variant of calreticulin comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to wild-type calreticulin and has the biological activity of wild-type calreticulin. In some embodiments, the functional variant of calreticulin has an amino acid sequence in which one or more amino acid residues are substituted, deleted, or added relative to a wild-type calreticulin protein, and has the biological activity of a wild-type calreticulin protein. For example, a functional variant of calreticulin comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions, deletions, or additions relative to wild-type calreticulin. In some embodiments, the amino acid substitution is a conservative substitution. In some embodiments, the wild-type calreticulin protein comprises the amino acid sequence set forth in SEQ ID No.1 or 2.

"polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" may also include modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

Sequence "identity" has a art-recognized meaning and can be calculated using the disclosed techniques as the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along regions of the molecule. (see, e.g., comparative Molecular Biology, desk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: information and genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds, HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heanjad, G.Acem Press, 1987; and Sequence Analysis, Privibiton, M.and device J., Biology, sample J.1991). Although there are many methods for measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to the skilled person (Carrillo, H. & Lipman, D., SIAM tagged Math 48:1073 (1988)).

Suitable conservative amino acid substitutions in peptides or proteins are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Generally, one skilled in The art recognizes that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, e.g., Watson et al, Molecular biology of The Gene,4th Edition,1987, The Benjamin/Cummings pub.co., p.224).

Herein, functional variants of calreticulin also include variants obtained by removal of the native signal peptide of wild-type calreticulin, removal of the endoplasmic reticulum retention sequence, addition of a purification tag (e.g., His tag), addition of a signal peptide that increases protein expression or alters the pattern of protein expression (e.g., a secretory signal peptide), and the like. Such variants do not alter the biological activity of calreticulin. In some embodiments, the functional variant of calreticulin comprises an amino acid sequence set forth in one of SEQ ID NOs 3-5.

In some embodiments, the calreticulin protein may be isolated from its natural source. In some embodiments, the calreticulin protein may be chemically synthesized. In some preferred embodiments, the calreticulin protein is produced by recombinant expression. In some preferred embodiments, the calreticulin protein is a recombinant human calreticulin protein.

Methods for producing proteins by recombinant expression are known in the art. For example, a nucleic acid encoding calreticulin may be cloned into a suitable expression vector, which is then introduced into a suitable host cell, which is cultured under suitable conditions to produce recombinantly expressed calreticulin.

Suitable expression vectors include plasmid vectors, yeast shuttle vectors, baculoviruses, replication-defective adenoviruses, and the like. A large number of available expression vectors suitable for expressing calreticulin of the present invention are known in the art.

Suitable host cells for expressing calreticulin of the invention include prokaryotes, yeast and higher eukaryotic cells. Exemplary prokaryotic host cells includeEscherichia coli cells, Bacillus subtilis cells, and the like. Exemplary yeast host cells are, for example, Saccharomyces cerevisiae, Pichia pastoris, Hansenula cells, and the like. Exemplary higher eukaryotic cells include HEK293 cells, CHO cells (e.g., CHO3E7), and the like. In a preferred embodiment, the calreticulin protein of the invention is produced by ExpicHO-S^TMAnd (4) expressing the cells.

In a second aspect, the present invention provides a pharmaceutical composition for preventing and/or treating acute altitude disease, comprising an effective amount of calreticulin and a pharmaceutically acceptable carrier. The calreticulin and the acute altitude disease are each as defined above.

In a third aspect, the present invention provides a method for the prevention and/or treatment of acute altitude sickness comprising administering to a subject in need thereof a therapeutically effective amount of calreticulin. The calreticulin and the acute altitude disease are each as defined above. Preferably, the subject is a mammal, such as a human.

As used herein, "treating" an individual having a disease or condition means that the individual's symptoms are partially or fully alleviated, or remain unchanged after treatment. "prevention" refers to the prevention of an underlying disease and/or the prevention of worsening of symptoms or progression of a disease.

As used herein, "effective amount" includes "therapeutically effective amount" or "prophylactically effective amount". A "therapeutically effective amount" refers to an amount of a compound (e.g., calreticulin of the present invention) or composition comprising a compound (e.g., calreticulin of the present invention) that is at least sufficient to produce a therapeutic effect upon administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, block, or partially block the symptoms of the disease or disorder. As used herein, a "prophylactically effective amount" refers to an amount of a compound (e.g., a calreticulin of the present invention) or composition comprising a compound (e.g., a calreticulin of the present invention) that will have a desired prophylactic effect when administered to a subject, e.g., to prevent or delay the onset or recurrence of a disease or condition, to reduce the likelihood of the onset or recurrence of a disease or condition. A complete prophylactically effective dose need not occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

The effective amount of calreticulin to be administered depends on the therapeutic objective, the route of administration and the disease condition of the patient. In addition, the attending physician takes into account various factors known to alter the action of drugs, including the severity and type of the disease, the patient's health, weight, sex, diet, time and route of administration, other drugs, and other relevant clinical factors. Therefore, the therapist must titrate the dose of calreticulin and modify the route of administration as needed to obtain the best therapeutic effect. Typically, the clinician will administer the calreticulin until a dose is reached that achieves the desired effect. The progress of such treatment is readily monitored by routine assays.

Generally, the dosage range for administering calreticulin provided herein is one that is large enough to produce a desired effect (e.g., symptomatic improvement of acute altitude sickness). The dosage is not so large as to cause adverse side effects, and the dosage will vary with age, disease state, sex, and extent of disease in the patient, and can be determined by one skilled in the art. The dosage can be adjusted by the individual physician in the event of any adverse side effects. Exemplary doses for preventing or treating acute altitude disease include, but are not limited to, about 0.01mg/kg to about 300mg/kg, e.g., about 0.01mg/kg, about 0.1mg/kg, about 0.5mg/kg, about 1mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg, about 50mg/kg, about 100mg/kg, about 150mg/kg, about 200mg/kg, about 250mg/kg, or about 300 mg/kg. In some preferred embodiments, the calreticulin protein of the invention is administered at a dose of at least 5mg/kg body weight.

For the treatment of acute altitude sickness, the dose of calreticulin may vary depending on the type and severity of the disease. The calreticulin may be administered in a single dose, multiple separate administrations, or by continuous infusion. For repeated administration over several days or longer depending on the disease condition, the treatment can be repeated until the desired suppression of disease symptoms occurs or the desired improvement in the patient's disease condition is achieved. Repeated administration may include increasing or decreasing the amount of calreticulin, depending on the progress of the treatment. Other dosage regimens are also contemplated.

Calreticulin described herein can be administered to a subject by any method known in the art for administering polypeptides, including, for example, systemic or local administration. The calreticulin may be administered by a route such as parenteral (e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intracavity), topical, epidural, or mucosal (e.g., intranasal or oral). The calreticulin-containing compositions may be administered by any convenient route, for example by infusion or bolus injection, absorption through epithelial or cutaneous mucosa (e.g. oral, rectal and intestinal mucosa).

The pharmaceutical compositions provided herein can be in various forms, for example, solid, semi-solid, liquid, powder, aqueous solution, or lyophilized form. Examples of suitable pharmaceutically acceptable carriers are known in the art and include, but are not limited to, water, buffers, saline solutions, phosphate buffered saline solutions, various types of wetting agents, sterile solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, gelatin, glycerol, carbohydrates (such as lactose, sucrose, amylose, or starch), magnesium stearate, talc, silicic acid, viscous paraffin, perfume oils, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, powders, and the like. The pharmaceutical compositions provided herein may contain other additives including, for example, antioxidants, preservatives, antimicrobials, analgesics, binders, disintegrants, colorants, diluents, excipients, extenders, glidants, solubilizers, stabilizers, tonicity agents, vehicles, thickeners, flavoring agents, emulsions (e.g., oil/water emulsions), emulsifying and suspending agents (e.g., acacia, agar, alginic acid, sodium alginate, bentonite, carbomer, carrageenan, carboxymethyl cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, octyl phenol polyether-9, oleyl alcohol, polyvinylpyrrolidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol, tragacanth, xanthan gum and derivatives thereof), solvents, and various ingredients, such as crystalline cellulose, microcrystalline cellulose, citric acid, dextrin, glucose, liquid glucose, lactic acid, lactose, magnesium chloride, potassium metaphosphate, starch, and the like. Such carriers and/or additives may be formulated by conventional methods and may be administered to a subject in a suitable dosage. Stabilizers such as lipids, nuclease inhibitors, polymers and chelating agents can prevent degradation of the composition in vivo.

In the use, pharmaceutical composition and method of the present invention, the calreticulin may also be used in combination with other drugs for treating acute altitude diseases. For example, the pharmaceutical composition may also comprise other drugs for treating acute altitude sickness. Alternatively, the calreticulin may be administered simultaneously with other separately formulated drugs for the treatment of acute altitude sickness. Alternatively, the calreticulin may be administered separately, e.g. sequentially, from other drugs used to treat acute altitude sickness.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art.

The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

Examples

Example 1 recombinant calreticulin preparation

The present invention uses a Chinese Hamster Ovary (CHO) cell expression system to express recombinant calreticulin.

1.1 cloning of the Gene

The human CRT protein is 417 amino acids in length (Genbank accession No.: NM-004343). The inventors deleted the endoplasmic reticulum retention signal of 4 amino acids (KDEL) from the C-terminus of human CRT protein, so that the recombinant CRT protein was mainly expressed in the cytoplasm. Then, the original signal peptide of 17 amino acids from the N-terminus is replaced with an artificial signal peptide that increases expression (MGWSCIILFLVATATGVHS). For purification, a 6 × His purification tag was added to the C-terminus.

According to the recombinant protein sequence designed above, codon optimization of the coding nucleic acid sequence was performed for CHO cells, a Kozak sequence was added to the 5' end, and HindIII and EcoRI sites were added to both ends. The encoding nucleic acid fragment was artificially synthesized and cloned into pUC57 eukaryotic expression vector. The cloning strategy is shown in FIG. 19.

1.2 cell culture and transient transfection

CHO-3E7 cells were maintained in serum-free FreeStyle^TMCHO expression Medium (Life Technologies). Cells were maintained in Erlenmeyer flasks on an orbital shaker at 37 ℃ with 5% CO₂. The day before transfection, cells were seeded at the appropriate density in Erlenmeyer flasks. On the day of transfection, the DNA and transfection reagent are mixed in optimal proportions and then added to the cells to be transfected. Recombinant plasmids encoding calreticulin were transiently transfected into 100ml suspension CHO-3E7 cell cultures. Cell culture supernatants were collected for purification on day 6.

1.3 protein purification and analysis

Cell culture medium was centrifuged and loaded at 1.0 ml/min to 5ml HisTrap^TMFF Crude (GE, Cat. No. 17-5286-01). After washing and elution with the appropriate buffer, the eluted fractions were pooled and the buffer exchanged to PBS ph 7.2. Protein purified by SEC-HPLC, SDS-PAGE and Western blot analysis molecular weight, yield and purity measurements were made using standard protocols. Western blotting was performed using mouse anti-his mAb (GenScript, Cat. No. A00186).

Example 2 pharmacodynamic study of recombinant calreticulin to alleviate altitude hypoxia response

(1) Experimental animals: male SPF-grade SD rats (60, body weight 200-. Quarantine observation is carried out for 3 days. During the period, the animals are observed for physical signs, behavior and activity, fecal characteristics, weight, diet and other indexes, and the animals with diseases are eliminated. Controlling the environmental condition of the animal house at the room temperature of 18-26 ℃; relative humidity is 40% -70%; the light and the shade alternate for 12 hours. The cage, the hamper and the drinking bottle are washed once a day, the ground and the wall of the animal house are disinfected once a week, 0.1 percent of benzalkonium bromide or 0.5 percent of 84 disinfectant is used for wiping and disinfecting, and the two disinfectants are used alternately. The common feed is rat maintenance material purchased from Beijing Huafukang Biotech GmbH. Padding: sterilized granular padding purchased from Beijing Huafukang Biotech GmbH. Drinking water: purified water is drunk and can be freely drunk after acidification.

(2) The main apparatus is as follows: laboratory animal low pressure simulation storehouse (HCP-III Series), Abbotti-start blood gas analyzer, Powerlab physiological recorder, pressure sensor.

(3) Main reagents and preparation thereof: the recombinant calreticulin was prepared and purified as described in example 1, with physiological saline from bexarotene biotechnology limited, beijing, and dexamethasone sodium phosphate injection from soxhlet pharmaceutical limited, drug administration.

(4) Grouping experiments:

① normoxic control group, i.e. intraperitoneal injection of 0.5ml of normal saline

② hypoxia model group, i.e. intraperitoneal injection of 0.5ml of normal saline

③ hypoxia and dexamethasone 5mg/kg group, dexamethasone 5mg/kg is injected into abdominal cavity

④ hypoxia + CRT 5mg/kg group, i.e. intraperitoneal injection of CRT 5mg/kg

⑤ hypoxia + CRT 1mg/kg group, injecting CRT 1mg/kg intraperitoneally

⑥ hypoxia + CRT 0.2mg/kg group, intraperitoneal injection of CRT 0.2mg/kg

(5) Replication of hypoxic model under low pressure:

after 1 hour of intraperitoneal injection, ② - ⑥ groups of rats were placed in a simulated plateau environment low-pressure oxygen chamber at a rate of 10 mS^-1The speed of the temperature-controlled air conditioner is increased to the level of simulated altitude of 6500m at a constant speed, the temperature is set to be 0 ℃ in the daytime (8: 00-18: 00) and 4 ℃ in the nighttime (18: 00-08: 00), and the temperature is continuously increased for 72 hours. After the completion, the air pressure in the cabin is stably increased back to the normal pressure within 45 minutes, and the cabin is taken out. The diet was changed every 24 hours.

When the experiment is finished, 2% sodium pentobarbital is injected into the abdominal cavity with 0.5ml/kg for anesthesia, 2 ml of arterial blood is rapidly taken from the left carotid artery, the arterial blood is sealed and placed in an ice pot, and the blood gas is measured by an Abbotti-start blood gas analyzer; and quickly killing the animal, taking the right lung tissue and the right brain hemisphere for the wet-dry weight ratio of the tissues, taking the middle and lower lobes of the right lung, observing the general lung, taking about 100mg of lung tissue and immediately placing the lung tissue inFixing the mixture in 10 times of 4% paraformaldehyde at normal temperature for at least 48 hours for preparing and observing a light microscope specimen. Another 1 mm³2-3 pieces of large and small lungs were immediately fixed in freshly prepared 3% glutaraldehyde at 4 ℃ for at least 48 hours for preparation and observation of transmission electron microscope specimens. The rest tissues are divided into 3 and 4 parts and immediately put into liquid nitrogen for freezing and storage for later use.

(6) Lung (brain) tissue wet-dry weight ratio: sucking dry blood stain on the right lung tissue by using filter paper, quickly weighing, baking for 48h in a constant-temperature oven at 80 ℃ until the weight is constant, weighing the dry weight, and calculating the wet/dry weight ratio (W/D) of the right lung, namely the wet/dry lung ratio. Brain tissue wet/dry lung weight was calculated as above.

(7) Observation of lung histology: the lung tissues are fixed by 4% neutral formaldehyde, embedded in paraffin, sliced, and subjected to HE staining, 3 lung tissue slices are randomly selected from each rat, three visual fields are randomly selected from each slice, and observation is carried out under an optical lens.

As a result:

(1) wet to dry weight ratio of lung tissue

The results of the wet-to-dry weight ratio of lung tissue are shown in Table 1 and FIGS. 1 and 2. The result of the lung wet weight shows that the lung tissue wet weight of the model group simulating the altitude anoxia is obviously increased (P is less than 0.05) compared with the normal control group; compared with the model group, the dexamethasone group and each group of CRT low, medium and high doses have a decreasing trend, but the difference has no statistical significance (P is more than 0.05); and compared with a positive drug dexamethasone group, the lung wet weight difference of each group of CRT has no significance (P > 0.05).

The analysis result of the lung wet/dry weight ratio shows that compared with a normal control group, the lung tissue wet/dry weight of a model group simulating altitude anoxia is obviously increased (P is less than 0.05); compared with the model group, the wet/dry weight of lung tissues of the dexamethasone group and the CRT low, medium and high doses of each group is obviously reduced (P is less than 0.05); and compared with the positive drug dexamethasone group, the CRT 1ng group is obviously reduced (P <0.05), but the CRT 0.2ng group and the CRT 5ng group have no difference (P > 0.05).

TABLE 1 Wet-to-dry weight ratio of lung tissue in rats of each group

Group of	n	Lung dampness weight (g)	Dry weight of lung (g)	Dry-wet ratio of lung
					Control group
	20	0.47±0.04	0.12±0.02	4.12±0.57
					Model set	16	0.64±0.06^*	0.10±0.01	6.43±0.86^*
Dexamethasone group	16	0.60±0.07^*	0.13±0.01	4.73±0.20^*#
					CRT 0.2ng group	13	0.60±0.04^*	0.12±0.01	4.98±0.16^*#
CRT 1ng group	13	0.61±0.06^*	0.12±0.01	4.87±0.10^*#△
					CRT 5ng group	17	0.62±0.06^*	0.13±0.01	4.93±0.22^*#

Note:^*P<0.05, compared with a normal control group;^#P<0.05, compared to a model set;^△P<0.05 compared to the dexamethasone group. Comparing with normal control group^*P<0.05, comparing with the model group,^#P<0.05。

(2) brain tissue wet/dry weight ratio

The results of the wet-to-dry weight ratios of brain tissues are shown in Table 2 and FIGS. 3 and 4. The result of the wet weight of the brain tissue shows that the wet weight of the lung tissue of the model group simulating the altitude anoxia is obviously increased (P is less than 0.05) compared with the normal control group; compared with the model group, the dexamethasone group and the CRT low, medium and high dose groups have obvious reduction (P is less than 0.05); compared with the positive drug dexamethasone group, the difference of the lung wet weight of each CRT group is not significant (P > 0.05). Brain dry weight: there were no statistical differences between groups.

The analysis result of the brain wet/dry weight ratio shows that compared with a normal control group, the lung tissue wet/dry weight of a model group simulating the altitude anoxia is obviously increased (P is less than 0.05); compared with the model group, the moisture/dry weight of brain tissues of the dexamethasone group and the CRT low, medium and high doses of each group is obviously reduced (P is less than 0.05); CRT was not statistically different (P <0.05) from the positive drug dexamethasone group.

TABLE 2 Wet-to-dry weight ratio of brain tissue of rats in each group

Group of	n	Weight of brain dampness (g)	Brain dry weight (g)	Dry-dry weight ratio of brain
					Control group
	20	1.70±0.12	0.39±0.02	4.30±0.28
					Model set	16	1.89±0.05^*	0.37±0.02	5.06±0.31^*
Dexamethasone group	16	1.71±0.12^*#	0.37±0.04	4.69±0.20^*#
					CRT 0.2ng group	13	1.71±0.08^*#	0.36±0.03	4.74±0.33^*#
CRT 1ng group	13	1.79±0.06^*#	0.36±0.03	4.55±0.22^*#
					CRT 5ng group	17	1.67±0.11^*#	0.37±0.03	4.59±0.22^*#

Note:^*P<0.05, compared with a normal control group;^#P<0.05, compared to the model set.

(3) Blood gas analysis results (Table 3)

As shown in fig. 5, the pH results showed no significant change in pH of the model group simulating altitude hypoxia (P >0.05) compared to the normal control group; but the CRT high dose group was significantly elevated (P <0.05) compared to the model group and the positive drug dexamethasone group, respectively.

As shown in fig. 6, the results of PCO2 showed a significant decrease (P <0.05) in PCO2 in the model group mimicking high altitude hypoxia compared to the normal control group; compared with the model group, the CRT low, medium and high dose groups have obvious increase (P < 0.05); compared with the positive drug dexamethasone group, the CRT low and high dose groups are obviously increased (P is less than 0.05).

As shown in fig. 7, the results of PO2 showed a significant decrease in model group PO2 (P <0.05) that mimics plateau hypoxia, compared to the normal control group; compared with the model group, the CRT high dose is obviously increased (P < 0.05).

As shown in fig. 8, the results of Beecf showed a significant increase in Beecf (P <0.05) in the model group mimicking high altitude hypoxia compared to the normal control group; compared with the model group and the positive drug dexamethasone group, the CRT low, medium and high dose groups are obviously reduced (P is less than 0.05).

As shown in fig. 9, the results of HCO3 showed a significant decrease (P <0.05) in HCO3, a model group mimicking high altitude hypoxia, compared to the normal control group; compared with the model group, the positive drug dexamethasone group, the CRT low, medium and high dose groups are obviously increased (P is less than 0.05); compared with the positive drug dexamethasone group, the CRT low, medium and high dose groups have obvious increase (P <0.05)

As shown in fig. 10, the results of TO2 showed a significant increase in model group TO2 (P <0.05) that simulates high altitude hypoxia, compared TO the normal control group; compared with the model group, the positive drug dexamethasone group, the CRT low, medium and high dose groups are obviously reduced (P is less than 0.05); compared with the positive drug dexamethasone group, the CRT low, medium and high dose groups have obvious reduction (P < 0.05).

As shown in fig. 11, the results of SO 2% showed no significant difference in model group SO 2% that simulated altitude hypoxia (P >0.05) compared to the normal control group; compared with the model group, the positive drug dexamethasone, the CRT low, medium and high dose have no significance (P is more than 0.05).

As shown in fig. 12, the Na results show that the model group simulating altitude hypoxia has no significant difference in Na compared with the normal control group (P > 0.05); CRT low, medium, and high dose groups were decreased compared to the model group (P < 0.05); CRT high dose group was reduced compared to the positive drug dexamethasone group (P < 0.05).

As shown in fig. 13, the results for K show that the model group K that mimics altitude hypoxia is not significantly different (P >0.05) compared to the normal control group; compared with the model group, the positive drug dexamethasone group and the CRT low, medium and high dose groups have no significant difference (P is more than 0.05); CRT high dose group was elevated compared to the positive drug dexamethasone group (P < 0.05).

As shown in fig. 14-15, the results for iCa and Glu show that the model group simulating altitude hypoxia is not significantly different between iCa and Glu (P >0.05) compared to the normal control group; compared with the model group, the positive drug dexamethasone, the CRT low, medium and high dose have no significance (P is more than 0.05).

As shown in fig. 16-17, the results for Hct and Hb showed no significant difference in Hct and Hb in the model group simulating altitude hypoxia (P >0.05) compared to the normal control group; compared with the model group and the positive drug dexamethasone group, the CRT high dose is obviously reduced (P is less than 0.05).

TABLE 3 blood gas test results of each group of experiments

(4) Pathological and pathological changes of lung tissue

Representative pictures of pathological observations of rat lung tissue are shown in FIG. 18. Under the lung tissue of a normal rat, a bronchus mucosa lining single-layer ciliated columnar epithelial cells and mucus cells can be seen under the microscope, and the lining is accompanied with small arteries and veins of the lung; most alveoli are uniformly arranged, the alveolar wall is not obviously thickened, and a small amount of neutrophil infiltration is observed in the interstitium.

The lower trachea of the hypoxemia treatment group rat lung histoscope is highly expanded to be saccular, except for lining single-layer cilium columnar epithelial cells and mucus cells, papillary hyperplasia areas are also seen, no obvious fiber axis is seen, the cell nucleus is slightly enlarged, the dyeing is deep, and the chromatin is uneven; along with the expansion of pulmonary artery and vein, there are more red blood cells in individual vein; approximately 1/2 alveolar walls are slightly thickened, and focal lymphocyte accumulation, more neutrophil infiltration and formation of inflammatory granulation tissue are observed in the stroma, and pulmonary cysts are formed.

Compared with the hypoxia treatment group, the bronchiectasis of the lung tissue of rats in the hypoxia + dexamethasone 5mg/kg group is reduced, most lining cells still can be subjected to papillary hyperplasia, and the dilatation of the artery and vein of the lung is reduced; approximately 1/3 alveolar walls are slightly thickened, the interstitium is infiltrated with more neutrophils, and the area of inflammatory granulation tissue is reduced.

Compared with the hypoxia treatment group, the bronchiectasis of the lung tissue of rats in the hypoxia + CRT 5mg/kg group is reduced, the lining cells have no papillary hyperplasia, and the pulmonary artery and vein have no expansion; approximately 1/5 alveolar walls were slightly thickened, interstitial with moderate neutrophil infiltration, and areas of inflammatory granulation tissue were reduced.

The lung tissue of the rat in the hypoxia + CRT 1mg/kg group is relieved than the bronchiectasis of the hypoxia treatment group, and most lining cells still have high papillary hyperplasia accompanied with the thickening of the wall of the pulmonary artery and vein and the dilatation of the pulmonary vein; the alveolar walls are thickened and fused, more neutrophil infiltration and inflammatory granulation tissue formation are still seen in the interstitium, and individual alveolar edema is formed.

The lung tissue of the rat in the hypoxia + CRT 0.2mg/kg group is relieved of bronchiectasis compared with the hypoxia treatment group, and lining cells have no papillary hyperplasia; the pulmonary artery and vein have no expansion along with the thickening of the pulmonary artery wall; about 1/4 the alveolar wall thickens slightly, the interstitium shows the hyperplasia, expansion and congestion of local small vessels, and more neutrophilic granulocytes infiltrate into the interstitium, and the rest alveoli return to normal obviously.

Compared with the hypoxia + dexamethasone 5mg/kg group, although the protective effect of 1mg/kg CRT treatment on the lung tissue of the hypoxic rat is not obviously different, the protective effect of 0.2mg/kg CRT treatment and 5mg/kg CRT treatment on the lung tissue of the hypoxic rat is obvious compared with that of dexamethasone, and the protective effect is shown to relieve the changes of bronchial lining cell papillary hyperplasia, pulmonary arteriovenous dilatation and alveolar wall thickening. 5mg/kg CRT was significantly better than the 5mg/kg dexamethasone group.

And (4) conclusion:

the human source recombinant CRT obviously reduces pulmonary edema and cerebral edema of a rat model simulating the altitude hypoxia by low, medium and high doses, improves the acid-base balance disorder caused by tissue hypoxia and hypoxia, and has similar treatment effect to dexamethasone; suggesting that CRT is effective in treating acute altitude diseases.

A sequence table:

1 human calreticulin precursor of SEQ ID NO

MLLSVPLLLGLLGLAVAEPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFY GDEEKDKGLQTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFP NSLDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHL YTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASKPEDWDERAKIDDPT DSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQIDNPDY KGTWIHPEIDNPEYSPDPSIYAYDNFGVLGLDLWQVKSGTIFDNFLITNDEAYAEEFGN ETWGVTKAAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEE DKEEDEEEDVPGQAKDEL

2 mature human calreticulin without signal peptide of SEQ ID NO

EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGLQTSQDARF YALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNSLDQTDMHGDSEYNIM FGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHLYTLIVRPDNTYEVKIDNSQ VESGSLEDDWDFLPPKKIKDPDASKPEDWDERAKIDDPTDSKPEDWDKPEHIPDPDA KKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPS IYAYDNFGVLGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDK QDEEQRLKEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEEDKEEDEEEDVPGQAKDE L

3 recombinant human calreticulin without endoplasmic reticulum resident sequence

EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGLQTSQDARF YALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNSLDQTDMHGDSEYNIM FGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHLYTLIVRPDNTYEVKIDNSQ VESGSLEDDWDFLPPKKIKDPDASKPEDWDERAKIDDPTDSKPEDWDKPEHIPDPDA KKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPS IYAYDNFGVLGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDK QDEEQRLKEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEEDKEEDEEEDVPGQA

4 No signal peptide, No endoplasmic reticulum-resident sequence, His-tagged recombinant human calreticulin sequence EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGLQTSQDARF YALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNSLDQTDMHGDSEYNIM FGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHLYTLIVRPDNTYEVKIDNSQ VESGSLEDDWDFLPPKKIKDPDASKPEDWDERAKIDDPTDSKPEDWDKPEHIPDPDA KKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPS IYAYDNFGVLGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDK QDEEQRLKEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEEDKEEDEEEDVPGQAHHH HHH

SEQ ID NO 5 endoplasmic reticulum-free retention sequence, recombinant human calreticulin sequence with artificial signal peptide and His tag

MGWSCIILFLVATATGVHSEPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKF YGDEEKDKGLQTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLF PNSLDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHL YTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASKPEDWDERAKIDDPT DSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQIDNPDY KGTWIHPEIDNPEYSPDPSIYAYDNFGVLGLDLWQVKSGTIFDNFLITNDEAYAEEFGN ETWGVTKAAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEE DKEEDEEEDVPGQAHHHHHH

Artificial signal peptide of SEQ ID NO 6

MGWSCIILFLVATATGVHS。

Claims

1. Use of human calreticulin as the sole active ingredient in the manufacture of a medicament for the treatment of acute altitude sickness, wherein the medicament further comprises a pharmaceutically acceptable carrier.

2. The use of claim 1, wherein the calreticulin is recombinant human calreticulin.

3. The use of claim 1, wherein the calreticulin is selected from the amino acid sequences of SEQ ID NO 1-5.

4. The use of any one of claims 1-3, wherein the acute altitude disease is selected from acute altitude reaction, high altitude pulmonary edema, high altitude cerebral edema, or a combination thereof.