CN117122669B - Application of recombinant human growth hormone in treating central diabetes insipidus - Google Patents
Application of recombinant human growth hormone in treating central diabetes insipidus Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/27—Growth hormone [GH], i.e. somatotropin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/12—Antidiuretics, e.g. drugs for diabetes insipidus
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Diabetes (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Endocrinology (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
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Abstract
The invention discloses an application of recombinant human growth hormone in treating central diabetes insipidus. According to the invention, by injecting recombinant human growth hormone into a mouse model with damaged pituitary stems, the recombinant human growth hormone is found to up-regulate a p-STAT5/IGF-1/IGF-1R signal pathway in a posterior pituitary system, promote axon regeneration of large cell neurons in the posterior pituitary system, improve diabetes insipidus, and can be used for preparing diabetes insipidus medicines; the method also provides a new scheme for treating saddle tumor related central urine collapse patients, is beneficial to improving prognosis of patients, and has important social benefits.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of recombinant human growth hormone in treating central diabetes insipidus.
Background
The saddle area is a good development part of intracranial tumor, and because of the relationship between the origin and growth of the tumor and hypothalamus-pituitary shaft, the pituitary shaft is extremely vulnerable to damage and then cause central urinary collapse in the processes of tumor growth and surgical excision, and the current treatment mode only comprises symptomatic support treatment such as posterior pituitary hormone replacement treatment, fluid replacement and the like. Long-term urinary collapse puts great economic stress on the patient. Therefore, the mechanism of the reconstruction of the posterior pituitary system is deeply studied, and the targeted therapeutic mode has great practical benefit.
Recombinant human growth hormone, english name: recombinant Human Growth Hormone (GH), produced by recombinant E.coli secretory expression techniques, is identical in amino acid content, sequence and protein structure to human pituitary growth hormone. In the pediatric field, recombinant human growth hormone is adopted for substitution treatment, so that the growth of the height of children can be obviously promoted, and the growth and development of organs and tissues of the whole body of children can be improved. Meanwhile, the recombinant human growth hormone plays an important role in the reproduction field, the burn field and the anti-aging field. Has been widely used in clinic at present.
Current studies have demonstrated that GH acts directly on the hypothalamus, but its effect is not overly demonstrated. Furthermore, a number of studies have shown that growth hormone can function through the p-STAT5/IGF-1 pathway, which, however, gradually weakens in the central nervous system during development and is responded to by the liver mainly in adults, IGF-1 appears to be secreted more from the liver. In addition to classical effects on growth and metabolism, GH is therapeutically associated with a number of neurological restorations within the central nervous system, including enhancement of neurogenesis, angiogenesis, axon regeneration, and the like.
There is no report on the treatment of central diabetes insipidus of pituitary handle injury by GH, and the invention is mainly aimed at etiology related treatment and can realize functional reconstruction of pituitary handle structure.
Disclosure of Invention
The invention aims to provide the application of GH in treating pituitary stalk injury and/or central diabetes insipidus.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided the use of recombinant human growth hormone in the manufacture of a medicament for the prevention and/or treatment of pituitary stem damage and/or central diabetes insipidus.
Preferably, the central diabetes insipidus comprises central diabetes insipidus caused by pituitary handle injury.
Preferably, the recombinant human growth hormone promotes axonal regeneration of large cell neurons of the posterior pituitary system.
Preferably, the recombinant human growth hormone upregulates the p-STAT5/IGF-1/IGF-1R signaling pathway in the posterior pituitary system.
Preferably, the medicament further comprises at least one other ingredient useful for the prevention and/or treatment of pituitary stem injury and/or central diabetes insipidus.
Preferably, the other drugs useful for preventing and/or treating pituitary stem injury and/or central diabetes insipidus include one or more of pituitary posterior phyllin, desmopressin, chlorpropamide, vasopressin tannate, carbamazepine, hydrochlorothiazide, and the like.
Preferably, the medicament further comprises pharmaceutically acceptable excipients.
Preferably, the pharmaceutically acceptable excipients include: at least one of a diluent, a binder, a solvent, an osmotic pressure regulator, or a buffer.
Preferably, the dosage form of the medicament comprises at least one of an emulsion, a solution, an aerosol, a patch or a drop.
Preferably, the route of administration of the drug comprises at least one of intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration or nasal administration.
Preferably, the single dose of the drug is 0.3-0.5 mg/kg of mice.
Preferably, the drug is administered 1 to 3 times per day.
The beneficial effects of the invention are as follows:
according to the invention, by injecting recombinant human growth hormone into a mouse model with damaged pituitary stems, the recombinant human growth hormone is found to up-regulate a p-STAT5/IGF-1/IGF-1R signal pathway in a posterior pituitary system, promote axon regeneration of large cell neurons in the posterior pituitary system, and improve diabetes insipidus and related functions of the pituitary stems; can be used for preparing medicines for treating pituitary handle injury or diabetes insipidus, also provides a new scheme for treating central uremic collapse patients related to saddle tumor, is beneficial to improving prognosis of patients, and has important social benefit.
Drawings
FIG. 1. Improved central diabetes insipidus in mice with hypophysis separation after treatment with growth hormone; n=6.
Fig. 2. Growth hormone is reactive to two neurons in the posterior pituitary system, manifesting as activation of STAT5, especially in the OXT neurons; scale = 50 μm (left) and 10 μm (right); n=5.
FIG. 3. After the pituitary shaft has been detached for growth hormone treatment, the damaged axon regenerates back across the damaged end to the posterior pituitary, whereas untreated groups are limited to damaged sites; ratio = 200 μm (upper) and 50 μm (lower); n=5.
FIG. 4 shows enhanced IGF-1 and IGF-R expression of AVP and OXT in SON and PVN following growth hormone treatment of pituitary stem lesions; scale = 50 μm (left) and 10 μm (right); n=5.
FIG. 5 serum levels of OXT more closely approach physiological homeostasis after pituitary stem separation for growth hormone treatment; n=5.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
The invention constructs a central urinary collapse model by a mode of damaging the parietal stalk point of the cranium, and researches the effect of growth hormone on improving the central urinary collapse after pituitary stalk separation through the animal experiment. A pituitary-petiole-isolated mouse model was constructed, and growth hormone was dissolved in physiological saline and intraperitoneal injection was performed twice daily for 10 days. Immunofluorescence detects the neuronal loss after pituitary stem disruption, axon degeneration, growth hormone action site, and the double-labeled primary antibodies used here are: p-STAT5+AVP, p-STAT5+OXT, AVP+IGF-1R, OXT +IGF-1 and OXT+IGF-1R. Western blot detects the expression of IGF-1 and IGF-1R in both groups of mouse hypothalamic supraoptic nuclei and paraventricular nuclear tissues. ELISA detects the expression of OXT in venous blood of two groups of mice, and the primary antibody used here is: OXT. Metabolic rate the two groups of mice were tested for improvement in urinary collapse.
Experimental materials:
mice: male C57BL/6 mice were supplied by the university of medical science laboratory animal center in south China. They were able to obtain standard food and water and were placed under standard laboratory conditions with 12 hours of light and dark cycles at room temperature (24.+ -. 2 ℃).
Feed: provided by the university of south medical science laboratory animal center (guangzhou, china).
Recombinant human growth hormone was purchased from chinese gold racing company (sai-zen, national drug standard S10980101, molecular formula: C990H1528N262O300S7, a protein consisting of 191 amino acids); P-STAT5, IGF-1R was purchased from CST corporation of America; IGF-1 was purchased from Abclonal corporation, china; AVP and OXT were purchased from Millipore corporation, usa; secondary antibodies were purchased from Thermo company in the united states; the OXT ELISA kit was purchased from Millipore Inc. of America.
The experimental method comprises the following steps:
grouping:
45 male C57BL/6 mice are fed to a weight of 22+/-4 g according to experimental grouping and treatment, the mice are weighed one by one, the mice are randomly divided into 3 groups of 15 mice, and the total weight of each group of mice is approximately equal by proper adjustment;
experimental group: constructing a pituitary stem damaged mouse model, and simultaneously injecting GH (single administration dosage of 0.35 mg/kg) into the abdominal cavity twice a day for 10 days;
control group: the pituitary stem-destroyed mouse model was constructed while being intraperitoneally injected with physiological saline twice a day for 10 days.
(1) A mouse pituitary stem lesion model (PEL) was constructed using a 3D lesion knife.
Mice were deeply anesthetized with 75mg/kg sodium pentobarbital. The neck coat of the mouse is soaked by physiological saline to prevent the hair from flying randomly during shaving, the size range of an operation area is determined to be about 2x1cm, the coat of the operation area is shaved, skin disinfectant is used for disinfecting the operation skin area three times in sequence, the median skin of the cranium top is cut off for 2cm, and the head of the mouse is mounted on a three-dimensional positioning frame (RWD life science company, chinese Shenzhen). The skull was then adjusted slightly to keep the bregma and lambda points horizontal and a 1 x 2mm bone window was made 1.8mm behind the bregma on the sagittal suture. Next, a 3D printed electric lossy knife (the destroyed knife according to the patent CN209059515U improvement adapted to mice) with a 0.8 mm wide curved head and 0.5 mm thick was lowered at the bone window until it reached the bottom of the skull base, which was more than 6 mm below the skull surface. After 60s compression, a cathodic current of 0.3mA was applied for 30s, the power output was constant (53500; UGO basic, gemonio, italy), whereas the control lesion depth was 5mm, and no current was applied. After the operation, all mice were returned to the original metabolic cage, and their Daily Water Consumption (DWC), daily Urine Volume (DUV) and Urine Specific Gravity (USG) were monitored for 10 days, and the presence or absence of improvement in water electrolyte condition was evaluated based on the monitoring results.
(2) Brain tissue extraction and specimen processing
3d, 7d, 10d mice of the sham operation group are respectively and intraperitoneally injected with excessive sodium pentobarbital for anesthesia and then chest opening is carried out. Frozen saline was infused from the left ventricle of the mouse until the circulating blood flow was clear. And then, continuously pouring a proper amount of 4% paraformaldehyde, and then, cutting off the head and taking out the brain. The extracted brain was immersed in the tissue fixative for 7d. A knife was cut along the coronal line at each of the anterior and posterior ends of the hypothalamus, leaving the brain tissue in the middle portion in the tissue embedding cassette for dehydration and paraffin embedding.
(3) Immunofluorescence is used for detecting the reactivity of large cell neurons of the posterior pituitary system to growth hormone and detecting the regeneration condition of axons after pituitary stalk injury.
The temperature of the oven is set to be 60 ℃ constant, and the slices are put into the oven for baking overnight; taking out the slice in the second day, rapidly putting the slice into the first xylene for dewaxing and transparency for 30min when the slice is still at the residual temperature, wherein the slice is rocked every 10min to help full dewaxing; then, the slices are changed into xylene II to be dewaxed for 30 minutes, and the slices are shaken every 10 minutes to help the full dewaxing; then placing the slices into each jar according to the concentration gradient alcohol for soaking for 5min; placing the slices into a jar filled with double distilled water, and shaking and soaking for three times, each time for 5min; changing double distilled water into EDTA or sodium citrate with pH of 8.0, placing into a microwave oven, setting the flow to high fire for 10min, and repairing antigen with medium fire for 5min; naturally cooling the material for about 2 hours to room temperature; pouring out the sodium citrate buffer solution in the organizing jar, adding the PBS buffer solution, and putting on a shaking table to be slightly immersed for three times, each time for 5min; soaking in 3% hydrogen peroxide for 20min; transferring to PBS buffer solution, and soaking in shaking table for 5min for three times; blocking for 1h at 5% BSA room temperature; discarding BSA, dripping the primary antibody prepared according to the specific concentration, placing in a wet box, and incubating overnight in a refrigerator at 4 ℃; discarding the primary antibody, transferring to PBS buffer solution, and soaking in shaking table for three times each for 10min; carefully wiping the PBS buffer solution with filter paper, then dripping the fluorescent secondary antibody diluent solution, and incubating for 1h at room temperature in a dark place; sucking the secondary antibody, transferring to PBS buffer solution, and soaking in shaking table for three times each for 10min; adding DAPI under dark condition, incubating for 5min, adding double distilled water, and soaking in shaking table for three times (10 min each time); oven drying and slicing at 35deg.C in dark; sealing the film with a quenching inhibitor, airing, and observing and shooting the film under a confocal microscope. Three different visual fields are randomly selected from the supravisual nucleus, the paraventricular nucleus and the median bulge area, and the number of positive index cells of each group is counted and averaged.
(4) Western blot detects the expression of IGF-1 and IGF-1R in both groups of mouse hypothalamic supraoptic nuclei and paraventricular nuclear tissues.
Fresh arciform nuclear tissues of the mice of the experimental group and the control group were collected and proteins were extracted. Preparing 12% SDS-PAGE separating gel and 5% SDS-PAGE laminating gel, preparing a mixed solution from protein and 5×loading Buffer according to the volume ratio of 4:1, heating the mixed solution in a metal bath at the constant temperature of 100 ℃ for 5min, cooling the mixed solution at the temperature of-4 ℃, and calculating the concentration of the mixed protein according to the volume ratio.
1) 25 μl of protein sample and 5 μl of protein Marker were added to each well;
2) After the sample is added, the electrode is connected well, the black port is connected with the negative electrode, the red port is connected with the positive electrode, the power switch is turned on, the set voltage is 80v, the glue is applied for 30 minutes, the voltage is changed into 120v after the glue is applied, the power is turned off after the marker is completely applied, and the film is ready to be transferred;
3) Clamping the glue and the PVDF film by using a sandwich clamp, then loading the clamped glue and PVDF film into a film transferring groove, and setting 300mA constant current ice for film transferring for 90 minutes;
4) Taking out the PVDF film, cutting out corresponding strips according to the relative molecular mass of a marker, putting the strips in a box, adding a pre-prepared TBS, shaking, and cleaning for three times, wherein the time is 5 minutes/time;
5) Discarding TBS, adding pre-prepared TBST containing 5% skimmed milk, shaking for 1 hr to seal PVDF membrane, discarding TBST containing skimmed milk, adding TBST containing no skimmed milk, shaking, and cleaning for three times for 5 min/time;
6) Diluting the primary antibody with a TBS solution containing 5% bsa;
7) Pouring the primary anti-dilution liquid into a box to incubate PVDF membrane, and standing overnight in a refrigerator at 4 ℃;
8) Recovering an anti-dilution liquid, adding TBST to wash the membrane three times for 10 minutes/time;
9) Secondary antibody was prepared as 1: diluting with 5000, preparing 5ml of 5% TBST milk, and incubating the PVDF membrane for 1h at room temperature under shaking;
10 Three times of TBST film washing for 10 minutes/time;
11 Taking out the PVDF film by using tweezers, draining water on filter paper, spreading in a chemiluminescent instrument, uniformly dripping 1ml of pre-prepared luminescent working solution onto the PVDF film, and detecting the result by the luminescent instrument.
(5) The ELISA detects the expression of OXT in venous blood of both groups of mice.
Two groups of mouse venous blood were collected, left to stand at room temperature for 20min, centrifuged at 12000g/15min, and the supernatant was transferred to an additional EP tube:
(1) coating by adding antibody, standing overnight at 4 ℃, washing for three times, and drying;
(2) adding antigen to be detected, washing for three times at 37 ℃ for 30 minutes, and polishing;
(3) adding enzyme-labeled antibody, washing for three times at 37 ℃ for 30 minutes, and polishing;
(4) adding substrate solution to 37 ℃ for 15 minutes, and adding stop solution;
(5) OD values were measured with ELISA detector.
Experimental results:
1. behavioural conditions after growth hormone treatment
As shown in FIG. 1, after the pituitary stalk injury of mice after the growth hormone treatment, the water intake, urine volume and urine specific gravity of the mice are improved to different degrees, which suggests improvement of diabetes insipidus.
2. Results of detecting responsiveness of large cell neurons of posterior pituitary system to growth hormone
Of SON and PVN, the OXT neuron has good reactivity to growth hormone (pSTAT 5 shows reactivity to growth hormone), as shown in fig. 2.
3. Detection result of pituitary stem axon regeneration condition after growth hormone treatment
As shown in fig. 3, AVP and OXT neuronal axons regenerate back to the posterior pituitary in the median bulge following growth hormone treatment, whereas untreated group axons failed to cross the damaged end.
4. Conditions of large cell neuron IGF-1/IGF-1R expression
As shown in FIG. 4, there was a different degree of elevation of IGF-1 and IGF-1R after growth hormone treatment.
5. Elisa results of mouse serum OXT after growth hormone treatment
As shown in fig. 5, serum OXT levels more approached physiological homeostasis following growth hormone treatment.
The present invention has been described in detail in the above embodiments, but the present invention is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (5)
1. The application of recombinant human growth hormone in preparing medicine for treating central diabetes insipidus caused by pituitary injury is provided.
2. The use according to claim 1, wherein the medicament further comprises pharmaceutically acceptable excipients.
3. The use according to claim 2, wherein the pharmaceutically acceptable excipients comprise: at least one of a diluent, a binder, or an osmotic pressure regulator.
4. The use of claim 1, wherein the dosage form of the medicament comprises at least one of an emulsion, a solution, an aerosol, a patch or a drop.
5. The use of claim 1, wherein the route of administration of the medicament comprises at least one of intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, or nasal administration.
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| CN108853043A (en) * | 2018-09-28 | 2018-11-23 | 郑士平 | A kind of drug and application thereof for treating central diabetes insipidus |
| CN113834889A (en) * | 2021-09-29 | 2021-12-24 | 中国医学科学院北京协和医院 | Pituitary stalk blocking syndrome biomarker and determination method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108853043A (en) * | 2018-09-28 | 2018-11-23 | 郑士平 | A kind of drug and application thereof for treating central diabetes insipidus |
| CN113834889A (en) * | 2021-09-29 | 2021-12-24 | 中国医学科学院北京协和医院 | Pituitary stalk blocking syndrome biomarker and determination method and application thereof |
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