CN115770242B - Application of sorafenib in preparing medicament for treating growth hormone dyssecretion after cerebral hemorrhage - Google Patents

Abstract

The invention discloses an application of sorafenib in preparing a medicament for treating growth hormone secretion disorder after cerebral hemorrhage. The invention belongs to the technical field of medicines. The invention realizes the effect of improving the growth hormone secretion disorder occurring in the middle and later stages after the bleeding of the subarachnoid space of the aneurysm by preparing the medicine containing sorafenib. At present, a corresponding clinical treatment means is lack for growth hormone secretion disorder occurring in the middle and later stages after the subarachnoid hemorrhage of the aneurysm, while sorafenib is applied to the preparation of medicines for resisting the growth hormone deficiency after the cerebral hemorrhage, can improve the symptom of the growth hormone deficiency occurring in the middle and later stages after the subarachnoid hemorrhage of the aneurysm, provides a new scheme for the treatment of patients with the growth hormone secretion disorder after the subarachnoid hemorrhage of the aneurysm, is beneficial to improving prognosis of the patients, and has important social benefit.

Description

Application of sorafenib in preparing medicament for treating growth hormone dyssecretion after cerebral hemorrhage

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of sorafenib in preparing a medicine for treating growth hormone secretion disorder after cerebral hemorrhage.

Background

The annual incidence of aneurysmal subarachnoid hemorrhage (Aneurysm subarachnoid hemorrhage, aSAH) is about 10/10 ten thousand, and the total number of aSAH occurring per year can reach 14 ten thousand, calculated as 14 hundred million people. The mortality rate of the aSAH patients is up to 50% and the mortality rate before admission is 10-25%. Even if the treatment is successful, 67% of survivors remain severely disabled, and a heavy burden is imposed on families and society because the age of onset is mainly middle-aged and young. Hypothalamic injury and hyposomatotropin are one of the main causes of the quality of life of patients affected by aSAH and even fatal disability in patients, but the mechanism of occurrence is not yet clear. Neuronal death is the major pathological manifestation of hypothalamic injury following aSAH, but the manner of death and the molecular mechanisms involved are numerous and complex. Clinical trials that have attempted in the past to achieve brain protection by preventing neuronal death have also mostly ended with failure. The aSAH is well developed in the Willis loop. Since the Willis loop is proximal to the hypothalamic arciform nucleus, the patient has hypothalamic-related neuroendocrine dysfunction even after aSAH has stopped its progression, with growth hormone secretion defects being most common. Growth hormone deficiency causes energy synthesis and consumption disorders, leading to cognitive deterioration, growth and development disorders, increased mortality from cardiovascular disease, etc., and is therefore an important factor in poor prognosis for aSAH patients. Growth Hormone Releasing Hormone (GHRH) neurons are located in the hypothalamic arciform nucleus, the highest central point in regulating cognition and growth development. Therefore, the mechanism of occurrence of growth hormone depression after aSAH is deeply studied, and the neuroendocrine protection strategy targeting the mechanism has important practical benefit.

Sorafenib was able to inhibit CRAF, VEGFR-2, VEGFR-3, PDGFR- β at target sites CRAF, BRAF, V EBRAF, c-Kit, FLT-3 and tumor vascular target sites of tumor cells. RAF kinase is serine/threonine kinase, and c-Kit, FLT-3, VEGFR-2, VEGFR-3, PDGFR-beta are tyrosine kinases that act on tumor cell signaling pathways, angiogenesis and apoptosis. Sorafenib is a novel multi-targeting oral drug for treating tumors, and is mainly used for treating hepatocellular carcinoma which cannot be operated or metastasized remotely. In 2013, sorafenib was approved by the FDA for the treatment of refractory local recurrence or metastasis of radioiodinated differentiated thyroid cancer, and phase III clinical trials showed significant improvement in progression free survival, but no significant improvement in overall survival.

There have been reports on early brain injury after sorafenib treatment of aSAH, which refers to brain injury within 72 hours after aSAH. While hypothalamic damage-induced growth hormone decline usually occurs in the late middle-to-late aSAH, some patients remain suffering from persistent neuroendocrine dysfunction, especially growth hormone dysfunction, after discharge from hospital. The incidence of growth hormone deficiency after aSAH was found to be as high as 25.4%. At present, a corresponding clinical treatment means is lack for the growth hormone secretion disorder after aSAH in clinic, and a medicine capable of effectively relieving the growth hormone deficiency after aSAH is needed in the present stage, so that the medicine can be widely applied to clinic to meet the requirement of clinical application.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides application of sorafenib in resisting growth hormone depression after aneurysmal subarachnoid hemorrhage, and the inventor finds that sorafenib can block MAD2L2-JNK-Caspase 1 signal paths and inhibit GHRH neuron cell apoptosis programs, so that a novel treatment scheme for patients with aSAH postgrowth hormone secretion disorder is provided.

In a first aspect of the invention, there is provided the use of sorafenib in the manufacture of a medicament for the treatment of post-cerebral-hemorrhage growth hormone dyssecretion.

Sorafenib (sorafenib) is a novel multi-kinase inhibitor antitumor drug developed by the germany bayer pharmaceutical company and can act on tumor cells and tumor blood vessels simultaneously. Sorafenib formula: c (C) ₂₁ H ₁₆ ClF ₃ N ₄ O ₃ Molecular weight: 464.825, cas number: 284461-73-0, sorafenib is a protein kinase inhibitor with activity in inhibiting a variety of protein kinases, and the results are shown in formula I.

In some embodiments of the invention, the cerebral hemorrhage is an aneurysmal subarachnoid hemorrhage.

In some embodiments of the invention, the post-cerebral hemorrhage growth hormone dysfunction occurs in the middle and later stages of the occurrence of an aneurysmal subarachnoid hemorrhage.

In the present invention, the middle-late stage after occurrence of the aneurysmal subarachnoid hemorrhage refers to the 3 rd month and later time after occurrence of the aneurysmal subarachnoid hemorrhage.

In some embodiments of the invention, the growth hormone secretion disorder is a secretion disorder resulting from the occurrence of focal death in growth hormone releasing hormone neuronal cells.

In some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable adjuvant.

In some embodiments of the invention, the pharmaceutically acceptable adjuvant includes diluents, absorbents, wetting agents, binders, disintegrants, lubricants, colorants, coating materials, solvents, pH adjusters, antibacterial agents, isotonicity adjusting agents, chelating agents.

In some embodiments of the invention, the medicament comprises an oral formulation or an injectable formulation.

In some embodiments of the invention, the oral formulation comprises an oral liquid, a capsule, an oral suspension, a solid dispersion, a capsule, a sustained release agent, a granule, a tablet; the injection preparation comprises intradermal injection, subcutaneous injection, intramuscular injection and intravenous injection.

In some embodiments of the invention, the drug is administered in a dose of 20 to 40mg/kg of mice.

In some embodiments of the invention, the drug is administered at a dose of 30mg/kg of mice.

In some embodiments of the invention, the drug is administered at a frequency of: it is used 1 time every 1-2 days.

In some embodiments of the invention, the drug is administered at a frequency of: it is used 1 time every 1 day.

The beneficial effects of the invention are as follows:

1. the present invention has found that sorafenib can be used to ameliorate growth hormone dyssecretion that occurs late in the middle after bleeding from the subarachnoid space of an aneurysm.

2. According to the invention, through experiments of sorafenib on an aneurysmal subarachnoid hemorrhage animal model, sorafenib can block a MAD2L2-JNK-Caspase 1 signal path and inhibit GHRH neuron cell apoptosis program, so that growth hormone low symptoms after aneurysmal subarachnoid hemorrhage are improved, a new scheme is provided for treating patients with growth hormone secretion disorder after aneurysmal subarachnoid hemorrhage, the prognosis of the patients is improved, and important social benefits are achieved.

Drawings

The invention is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 shows the expression of GSDMD in hypothalamic arciform nucleus GHRH neurons of aSAH mice in comparison of experimental and control groups in the examples of the present invention; wherein panel a is an immunofluorescent staining pattern of GSDMD expression in hypothalamic arciform nucleus GHRH neurons in the aSAH mice of the experimental and control groups; panel B is a graph comparing GSDMD expression level in hypothalamic arciform nucleus GHRH neurons of the aSAH mice; scale bar in the figure = 100 μm; n=3; arc represents an arcuate nucleus;

FIG. 2 is a graph showing Western blot detection results of levels of JNK and P-JNK in hypothalamic arciform nuclear tissue of aSAH mice, wherein A is a graph showing Western blot detection results of levels of JNK and P-JNK in hypothalamic arciform nuclear tissue of aSAH mice, and GAPDH is used as an internal reference protein; panel B is a comparison chart of the phosphorylation expression quantity of P-JNK in hypothalamic arciform nuclear tissues; panel C is a comparative graph of the phosphorylation expression level of T-JNK in hypothalamic arciform nuclear tissue; in the figure, "+" indicates that the corresponding process is performed, and "-" indicates that the corresponding process is not performed;

FIG. 3 shows the detection of caspase-1 protein level in hypothalamic arciform nuclear tissue of aSAH mice by Western blot, comparing the experimental group with the control group in the example of the present invention; wherein, A is a Western blot detection result diagram of caspase-1 protein level in hypothalamic arciform nuclear tissue of aSAH mouse, and GAPDH is used as an internal reference protein; panel B is a comparison chart of the expression level of caspase-1 in the hypothalamic arciform nuclear tissue; in the figure, "+" indicates that the corresponding process is performed and "-" indicates that the corresponding process is not performed;

FIG. 4 is a graph showing the decrease in MAD2L2 expression in hypothalamic arciform nucleus GHRH neurons of aSAH mice in comparison with the control group in the examples of the present invention, wherein graph A is an immunofluorescence staining graph of MAD2L2 expression in hypothalamic arciform nucleus GHRH neurons of aSAH mice in the experimental group and the control group; panel B is a comparison chart of MAD2L2 expression level in hypothalamic arciform nucleus GHRH neurons of an aSAH mouse; scale bar in the figure = 100 μm; n=3; arc represents an arcuate nucleus;

FIG. 5 is a graph showing comparison of GH expression levels of aSAH mice in ELISA test, comparing an experimental group with a control group in the example of the present invention;

FIG. 6 is a graph showing the cognitive ability of aSAH mice according to the comparison between the experimental group and the control group, wherein the graph A is a graph showing the escape latency time of the aSAH mice in the experimental group and the control group from day 1 to day 5; panel B is a comparison graph of the number of times the aSAH mice of the experimental group and the control group pass through the platform on the 6 th day; panel C is a graph comparing the residence time in the target quadrant of the aSAH mice of the experimental and control groups on day 6.

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 mice used in the examples were male C57BL/6 mice, supplied by the university of south medical science laboratory animal center (Guangzhou, china). The mice used in the experiment can obtain standard food and water, and are placed under standard laboratory conditions and subjected to 12-hour bright-dark circulation at room temperature of 22-26 ℃; the feed used was supplied by the university of south medical science laboratory animal center (guangzhou, china).

Sorafenib used in the examples was purchased from seleck, usa; GSDMD is available from proteontech, usa; MAD2L2 was purchased from Santa Cruz, inc., USA; JNK and P-JNK were purchased from CST corporation in the united states; caspase-1 is available from Thermo company, U.S.A.; GH ELISA kit was purchased from Millipore company, USA.

The experimental materials and reagents used in the examples were, unless otherwise specified, consumable materials and reagents conventionally available commercially.

Animal experiments of Sorafenib on growth hormone lowering after cerebral hemorrhage

100 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, 60 mice in the mice are randomly divided into 2 groups (experimental group and control group), 30 mice in each group are treated, and the total weight of the mice in each group is approximately equal by proper adjustment;

each group was treated as follows:

experimental group: constructing an aSAH mouse model while feeding a feed mixed with sorafenib (fed at a dose of 30mg sorafenib/kg mouse) for 7 days;

control group: an aSAH mouse model was constructed and fed with the same amount of placebo mixed feed for 7 days.

1. A mouse model of the subarachnoid hemorrhage of the aneurysm was constructed by an internal carotid artery puncture method.

The experimental mice were anesthetized with 1% pentobarbital (50 mg/1kg mouse body weight) and the anesthetized mice were fixed on a table. Opening a mouse operation body temperature control instrument and a Leica operation microscope, setting the temperature to be 38 ℃, inserting a temperature probe into the anus of a mouse, soaking the neck fur of the mouse with normal saline, preventing the hair from flying randomly during shaving, determining the size range of an operation area to be about 2X 1cm, shaving the fur of the operation area, disinfecting the operation skin area by using skin disinfectant for three times, making an oblique incision with the length of about 1cm from the right side of the median line of the neck, facilitating the separation of blood vessels on the right side, cutting the distal end of the incision to the chin of the mouse (the skin mark is a light red skin dome with the size of rice grains), and cutting the skin and subcutaneous tissue by ophthalmology. The eye forceps blunt separate the platykurtic gland, lymph node and submaxillary gland, the anterior and posterior abdominal junction of the two Abdominal muscles (white aponeurosis) is used as a starting point, the deep fascia is blunt separated from the anterior and posterior abdominal of the two Abdominal muscles towards the neck, the sternocleidomastoid muscle, the sternohyoid muscle, the collarbone hyoid muscle and the sternohyoid muscle are visible, the natural gullet-line-shaped and elongated more than sign (>) sample is formed between the above muscles (the sternocleidomastoid muscle, the posterior abdomen of the two Abdominal muscles, the sternohyoid muscle and the like), the above muscles are blunt separated from the anterior and posterior aspect of the anterior and posterior abdominal muscles, the common carotid artery sheath is visible, the external sheath is full of the blood vessel and the small return vein is covered, the common carotid artery and the vagus nerve in the sheath are clear and variable, the vagus nerve is tightly attached to the dorsal side of the common carotid artery, the same side sternocleiomy muscle is clamped by small curved forceps, the surgical field is fully exposed, the common carotid artery sheath is carefully separated from the common carotid artery with the eye forceps, the vagus nerve is carefully separated from the common carotid artery, the diameter is about 1 μm, and the common carotid artery is separated from the common carotid artery, and the common artery is visible at the position of the left after the common carotid artery and the left and the common carotid artery is separated from the left and 3. The internal carotid artery and the external carotid artery are subjected to blunt separation, the distal end of the external carotid artery and the carotid nerve are subjected to blunt separation, the external carotid artery branches into an upper thyroid artery is exposed, and a double wire (No. 5-0 wire) is threaded at the root of the connection between the external carotid artery and the upper thyroid artery. One of the external carotid arteries is used for ligating, the cervical plexus nerve is prevented from being ligatured by stripping, a certain length of wire is reserved for the second junction of the external carotid artery, the wire is used as a traction wire, simultaneously, the upper carotid artery is ligatured, the upper carotid artery is cut off, the second wire moves upwards along the external carotid artery along the free direction of the distal end, the external carotid artery is ligatured again about 3mm away from the second junction, the external carotid artery is a third junction of the external carotid artery, the external carotid artery is cut off by using neurosurgery micro-shears at a position about 2mm away from the second junction, the traction wire is not reserved for the third junction, the left hand lifts the proximal end stump of the external carotid artery through the traction wire of the second junction, the first branch occipital artery of the external carotid artery is carefully separated by using neurosurgery micro-forceps, the occipital artery is cut off by using neurosurgery micro-forceps, the fibrous shear is fully separated from the residual end of the external carotid artery, the internal carotid artery and the fibrous adventitia around a blood vessel at the bifurcation position of the common carotid artery, the sinus and the carotid artery are closed by using the left-side artery, and the carotid artery are closed by using the left-side of the left coronary artery. Slightly lifting the external carotid artery, shearing a small opening at the near bifurcation of the external carotid artery by using a neurosurgery micro-shear, leading in a capillary tube at the opposite side of the occipital artery stump, pulling the external carotid artery to be approximately in a straight line with the internal carotid artery (the tail of the capillary tube forms an angle of 30-45 degrees with the normal midline of the neck and forms an angle of 30-45 degrees with the horizontal plane, and the head of the capillary tube is inward), inserting the external carotid artery from the external carotid opening to an arterial clamp, rapidly inserting the capillary tube by an assistant, and then flowing out more internal carotid blood, inserting about 12mm, wherein the sense of smoothness is achieved, the capillary tube is rapidly inserted again for 1-2mm, and when the sense of breakthrough is achieved, the front traffic artery is indicated to be already broken (the sense of pricking bean curd residues at the moment). Rapidly extracting the capillary, briefly compressing to stop bleeding by using a small gauze block, lifting the external carotid artery, ligating the external carotid artery at the proximal end of the external carotid artery incision, and opening the common carotid artery clamp for the first knot of the external carotid artery. After the blood flow is unobstructed, the skin suturing operation is finished.

2. Brain tissue extraction and specimen processing

And after the operation molding is finished, 1 to 2 brain tissues are extracted from the experimental group and the control group respectively at the 3d, 7d and 14d, and the brain tissues are extracted and the samples are processed.

The specific operation is as follows:

the randomly extracted mice were intraperitoneally injected with an excess of pentobarbital sodium, anesthetized, and then chest opened, and fixed on a table. Frozen saline was infused from the left ventricle of the mouse until the outflow from the right atrium was observed to have become colorless and transparent, indicating that the circulatory blood flow was clear. And then, continuously pouring 4% paraformaldehyde for fixation, and then, cutting off the head and taking out the brain. The extracted brain was immersed in the tissue fixative for 7d. And cutting a knife at the front end and the rear end of the hypothalamus along the coronal line, reserving the brain tissue at the middle part in a tissue embedding box, and dehydrating and embedding paraffin to obtain brain tissue slices.

3. Immunofluorescence detects the expression of the focal death proteins GSDMD and MAD2L2 on GHRH neurons.

Setting the oven temperature to be 60 ℃ constant, and baking the brain tissue slices overnight for 12-18 h; the slice is quickly put into the xylene in the first cylinder for dewaxing and transparency treatment for 30min before being taken out in the second day and when the slice has residual temperature, wherein the slice is rocked every 10min to help full dewaxing; then changing the slices into a second cylinder of xylene for dewaxing and transparency treatment again for 30 minutes, and shaking the slices every 10 minutes to help full dewaxing; the slices were then immersed in each jar for 5min with a concentration gradient of 100%, 95%, 90%, 80%, 70% alcohol. Placing the soaked slices into a jar filled with double distilled water, and shaking and soaking for three times, each time for 5min; the double distilled water is changed into sodium citrate buffer solution with the same amount and pH value of 8.0, the slices are immersed into a microwave oven, and high fire is set for 10min and medium fire is set for 5min, so that antigen retrieval is carried out; after the mixture is naturally cooled to room temperature; pouring out the sodium citrate buffer solution in the jar, adding the PBS buffer solution, and putting the slices on a shaking table to be slightly immersed for three times, each time for 5min; then soaking in 3% hydrogen peroxide for 20min; transferring to PBS buffer solution, and soaking in shaking table for 5min for three times; blocking with 5% Bovine Serum Albumin (BSA) for 1h at room temperature; discarding BSA, dripping 1 drop of primary antibody (GSDMD and MAD2L 2) prepared according to a specific concentration, placing the primary antibody into a wet box, and incubating the wet box in a refrigerator at 4 ℃ for 12-18 h; discarding the primary antibody, transferring to PBS buffer solution, and soaking in shaking table for three times each for 10min; after the PBS buffer solution is carefully wiped by filter paper, 1 drop of fluorescent secondary antibody diluent solution is dripped, and the solution is incubated for 1h at room temperature in a dark place; sucking out the fluorescent secondary antibody, transferring to PBS buffer solution, and putting on a shaking table to be slightly immersed for three times, wherein each time is 10min; adding 4', 6-diamidino-2-phenylindole (DAPI) under dark condition, incubating for 5min, adding 20mL of double distilled water, and soaking in shaking table for three times each for 10min; 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 in the arciform nucleus region to carry out cell number statistics, the cell number of each group of positive indexes is respectively counted, and the average value is obtained.

4. Western blot detects expression of JNK, P-JNK and caspase 1 in hypothalamic arciform nuclear tissues of two groups of mice.

Fresh arcuate nuclear tissue of the mice of the experimental group and the control group was collected and protein was extracted with cell lysate RIPA. Preparing 12% SDS-PAGE separating gel and 5% SDS-PAGE laminating gel, preparing a mixed solution from the extracted protein and 5×loading Buffer according to the volume ratio of 4:1, then placing the mixed solution in a metal bath, heating the mixed solution at a constant temperature of 100 ℃ for 5min, then placing the mixed solution at a temperature of-4 ℃ for cooling slightly, and then calculating the concentration of the mixed protein according to the volume ratio, wherein the specific operation is as follows:

1) 25 mu L of protein sample and 5 mu L of protein Marker are added to each well;

2) After the sample is added, the electrodes are connected, the voltage is set to be 80V, the glue is applied for 30 minutes, the voltage is changed to 120V, the glue is applied after the glue is applied, and the power supply is turned off after the Marker is completely applied to prepare for film transfer;

3) Loading the glue and polyvinylidene fluoride (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 pre-prepared triethanolamine buffer saline solution (TBS), shaking, washing for three times, and 5 minutes/time;

5) Discarding TBS, adding pre-prepared TBST buffer solution containing 5% skimmed milk (namely TBS buffer solution containing Tween-20), continuously shaking for 1 hour to achieve the effect of sealing the PVDF membrane, discarding TBST buffer solution containing 5% skimmed milk, adding TBST buffer solution without skimmed milk, shaking and cleaning for three times, and 5 minutes/time;

6) Diluting the primary antibody with a TBS solution containing 5% BSA at a dilution ratio of 1:1000;

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 buffer solution to wash the membrane three times for 10 minutes/time;

9) Secondary antibody was mixed with TBST buffer according to 1: diluting with 5000, preparing 5mL of 5% TBST milk prepared in the step (5), and shaking and incubating the PVDF membrane for 1h at room temperature;

10 Three times, 10 minutes/time, with TBST buffer;

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 using the luminescent instrument.

5. ELISA the growth hormone expression in venous blood of two groups of mice was examined.

Growth Hormone (GH) ELISA kit is used for detecting growth hormone expression in mouse venous blood. Two groups of mouse venous blood were collected, left to stand at room temperature for 20min, centrifuged at 12000rpm for 15min at 4℃and the supernatant was transferred to a new EP tube and manipulated according to the kit instructions, wherein the antibody used was GH. OD values were measured with ELISA detector.

6. The water maze detects the cognitive abilities of the mice in the experimental group and the control group.

The mice of the experimental group and the control group were extracted 4 after the completion of the operation molding, and subjected to the water maze test. The randomly extracted mice of each group are placed in a water maze experiment room and are fed for 3 days according to the corresponding setting conditions so as to be familiar with the environment. And then trained 4 times per day for a fixed period of time. At the beginning of training, the platform was placed in the first quadrant and the mice were placed into the pool facing the pool wall from any of the four starting points of the pool wall. The free video recording system recorded the time the mice found the platform and the swimming path, and in 4 exercises, the mice were placed in the water from four different starting points (different quadrants), respectively. After the mice find the platform or the platform cannot be found within 120s (latency is recorded as 120 s), the mice are guided to the platform by the experimenter, rest on the platform for 10s, and then perform the next test.

The water maze test is carried out on two groups of mice according to the test steps for 5 days, the average value of four training latencies of the mice is used as the daily learning score of the mice, and the escape latency duration of the two groups of mice in 5 days is recorded. The station was removed on day 6. Then, the mice are placed into water at the same water inlet point, the swimming path of the mice within 120s is recorded, the times of crossing the target quadrant and the residence time of the mice in the target quadrant are recorded, the spatial positioning capability of the tested mice is observed, and the change rule in the spatial exploration process is observed. The original position of the platform is marked by a circular ring on the computer screen, so that the number of times of crossing the original position of the platform can be recorded and used as statistical data.

Analysis of experimental results

The test results of the experimental group mice and the control group mice in the above examples were analyzed:

1. detection result of GHRH neuron cell apoptosis marker

As shown in fig. 1, immunofluorescence results showed that the expression of GSDMD by the GHRH neurons was reduced in mice of the experimental group compared to the aSAH mice of the control group. Indicating that the GHRH neuron cell apoptosis of the mice in the experimental group is weakened.

2. Detection results of GHRH neuron signal pathway

As shown in fig. 2, western blot results suggest that JNK phosphorylation expression was reduced in hypothalamic arciform nuclear tissues in mice of the experimental group compared to aSAH mice of the control group fed with sorafenib. Demonstrating that sorafenib inhibits the cell pyrosis program by blocking the phosphorylation activation of JNK pathway.

3. Caspase-1 protein expression in GHRH neurons

As shown in FIG. 3, western blot results suggest that caspase-1 expression is down-regulated in hypothalamic arciform nuclear tissues of mice in the experimental group compared to control group of aSAH mice fed sorafenib. caspase-1 is a key protein in the cell scorch program. Again, sorafenib inhibited the apoptosis program of GHRH neurons.

4. MAD2L2 protein expression in GHRH neurons

As shown in fig. 4, immunofluorescence results showed that the mice in the experimental group had reduced expression of MAD2L2 from the GHRH neurons in the sorafenib-fed aah mice compared to the control group, indicating that sorafenib acts on the GHRH neurons by inhibiting the expression of MAD2L2 receptors.

5. Results of detection of mouse GH level

As shown in fig. 5, the ELISA results suggested that the GH levels of sorafenib-fed aSAH mice were significantly recovered in the experimental group compared to the control group of aSAH mice. Indicating that sorafenib is effective in controlling the decline in GH levels in aSAH mice.

6. Results of detection of cognitive level in mice

As shown in fig. 6, the water maze results suggest that the cognitive ability of sorafenib-fed aSAH mice was significantly recovered in the mice of the experimental group compared to the control group. Demonstrating that sorafenib is effective in controlling cognitive impairment in aSAH mice.

In conclusion, sorafenib can inhibit apoptosis of GHRH neuron cells by blocking MAD2L2-JNK-Caspase 1 signaling pathway, thereby improving symptoms of low growth hormone after aSAH.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill 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 (8)

1. The use of sorafenib in the manufacture of a medicament for the treatment of post-cerebral-hemorrhage growth hormone dyssecretion, characterized in that said post-cerebral-hemorrhage growth hormone dyssecretion occurs in the middle and late stages after occurrence of an aneurysmal subarachnoid hemorrhage.

2. The use according to claim 1, wherein the growth hormone secretion disorder is a secretion disorder caused by the occurrence of focal death in growth hormone releasing hormone neuronal cells.

3. The use according to claim 1, wherein the medicament further comprises a pharmaceutically acceptable adjuvant.

4. Use according to claim 3, wherein the pharmaceutically acceptable auxiliary agents comprise diluents, absorbents, wetting agents, binders, disintegrants, lubricants, colorants, coating materials, solvents, pH adjusters, antibacterial agents, isotonicity adjusting agents, chelating agents.

5. The use according to claim 1, wherein the medicament comprises an oral formulation or an injectable formulation.

6. The use according to claim 5, wherein the oral formulation comprises an oral liquid, a capsule, a solid dispersion, a capsule, a sustained release agent, a granule, a tablet; the injection preparation comprises intradermal injection, subcutaneous injection, intramuscular injection and intravenous injection.

7. The use according to claim 1, wherein the medicament is administered in a dose of 20-40 mg/kg of mice.

8. The use according to claim 1, wherein the medicament is administered at a frequency of: it is administered 1 time every 1-2 days.