CN116098895A - Application of Compound Apostatin-1 in the Preparation of Drugs for Treating Cerebrovascular Diseases - Google Patents

Abstract

The invention relates to an application of a compound Apostatin-1 in preparing a medicament for treating cerebrovascular diseases, after cerebral ischemia, nerve cells in brain tissues undergo programmed necrosis and apoptosis, and autophagy flow is blocked. Whereas Apostatin-1 is capable of inhibiting apoptosis and programmed necrosis by inhibiting tumor necrosis factor receptor-related death domain proteins and restoring autophagy flow. The invention discovers that the compound can obviously reduce the volume of cerebral infarction in the acute stage of focal cerebral ischemia of mice and obviously improve the neurological symptoms thereof, can reduce the damage of neurons, astrocytes and microglial cells induced by oxygen glucose deprivation, has the function of cerebral neuroprotection and has the therapeutic effect on cerebrovascular diseases.

Description

Application of Compound Apostatin-1 in the Preparation of Drugs for Treating Cerebrovascular Diseases

technical field

The invention relates to the technical field of biopharmaceuticals, in particular to the application of a compound Apostatin-1 in the preparation of drugs for treating cerebrovascular diseases.

Background technique

Stroke is a group of diseases whose main clinical manifestations are cerebral tissue ischemia and hemorrhagic injury symptoms, also known as cerebral apoplexy or cerebrovascular accident. The disease has an acute onset and high morbidity and mortality rates. It is the second leading cause of death in the world today, and it is becoming more and more serious with the aging of the population. Acute ischemic stroke (acute cerebral infarction) is the most common type of stroke, accounting for 69.6% to 70.8% of strokes in my country, and the mortality rate of hospitalized patients with acute ischemic stroke in my country is about 2.3% within one month after onset ～3.2%, 9%～9.6% fatality rate at 3 months, 34.5%～37.1% fatality/disability rate, 14.4%～15.4% fatality rate 1 year, 33.4%～33.8% fatality/disability rate, has become The #1 cause of death/disability in our country.

The pathogenesis of ischemic stroke is complex, involving multiple mechanisms such as peroxidation, energy metabolism disorder, calcium overload, and excitatory amino acid toxicity. Tissue plasminogen activator (tissue plasminogen activator, t-PA, intravenous injection) is a drug currently used clinically in the treatment of ischemic stroke. It can dissolve thrombus, recanalize blood vessels, and restore blood flow. However, due to the narrow therapeutic window of tissue plasminogen activator (t-PA) (it is effective only 4.5 hours after stroke) and easy to cause bleeding, only a small number of patients receive thrombolytic therapy. Therefore, finding new therapeutic drugs for ischemic stroke is an urgent problem to be solved. Reducing central nervous cell death after ischemic stroke is a currently recognized therapeutic strategy for ischemic stroke. After the occurrence of ischemic stroke, various forms of cell death such as apoptosis, programmed necrosis, and ferroptosis occurred in the central area of cerebral infarction and the penumbra. Although, it has been found that necroptosis inhibitor Nec-1 and apoptosis inhibitors Z-VEID and Z-IETD can reduce cerebral infarct volume induced by cerebral ischemia. However, single regulation of a certain death pathway may not completely reverse the central nervous system damage induced by ischemic stroke. And, if cell death is guarded, but cell homeostasis is not restored, including the removal of damaged organelles and misfolded proteins in neurons, the cells are left in a persistently disabled and pathological state. Therefore, finding a new target for the treatment of ischemic stroke, a drug with a new mechanism of action and less adverse reactions is an urgent problem to be solved.

Contents of the invention

In order to solve the above technical problems, the present invention provides a new application of Apostatin-1. Apostatin-1 is a targeted inhibitor of Tradd protein and has the functions of reducing apoptosis, programmed necrosis and activating autophagy. In the prior art, only its effect on the treatment of Alzheimer's disease has been studied, and there is no report on the anti-ischemic stroke effect of the drug. The present invention finds for the first time that Apostatin-1 is effective in ischemic stroke and other cerebrovascular diseases. significant protective effect against disease.

The first object of the present invention is to provide the application of compound Apostatin-1 in the preparation of medicaments for treating cerebrovascular diseases.

Further, the cerebrovascular disease is cerebral ischemic disease.

Further, the cerebral ischemic disease is ischemic stroke.

Further, the ischemic stroke is ischemia-reperfusion stroke.

Further, the dosage of the drug is at least 3 nmol.

Further, the drug is administered by intracerebroventricular injection.

The second object of the present invention is to provide a medicine for treating cerebrovascular diseases, said medicine comprising Apostatin-1.

Further, the medicine is composed of 0.1-100% of Apostatin-1 and 99.9-0% of pharmaceutical excipients.

By means of the above solution, the present invention has at least the following advantages:

The present invention finds that Apostatin-1 can treat cerebrovascular diseases including ischemic brain injury, and discovers a new application of the drug. The compound can significantly reduce the volume of cerebral infarction in the acute stage of focal cerebral ischemia in mice and significantly improve its neurological symptoms, has protective effects on brain neurons, and has a good effect especially on cerebral ischemic diseases.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention are described below with detailed drawings.

Description of drawings

In order to make the content of the present invention more clearly understood, the present invention will be further described in detail below according to the specific embodiments of the present invention and in conjunction with the accompanying drawings.

Figure 1 is a schematic diagram of the effect of the compound Apostatin-1 (Apt-1) in Example 1 of the present invention on reducing the volume of cerebral infarction in mice with cerebral ischemia in the acute phase; Mean±SD, n=10;

Figure 2 is a schematic diagram of the effect of the compound Apostatin-1 (Apt-1) in Example 1 of the present invention on improving the neurological symptoms of mice with cerebral ischemia; Mean ± SD, n = 10;

3 is a schematic diagram of the effect of compound Apostatin-1 (Apt-1) in Example 1 of the present invention on improving the right limb usage rate of mice with cerebral ischemia; Mean±SD, n=10; wherein, compared with the model group, * p<0.05, **p<0.01;

4 is a schematic diagram of the effect of the compound Apostatin-1 (Apt-1) and the control drug Edaravone (Edaravone) on improving oxygen-glucose deprivation-induced human neuron cell damage in Example 2 of the present invention; mean±SD, n= 3;

Figure 5 is a schematic diagram of the effect of the compound Apostatin-1 (Apt-1) and the control drug Edaravone (Edaravone) on improving oxygen-glucose deprivation-induced human astrocyte damage in Example 2 of the present invention; mean ± SD, n=3;

6 is a schematic diagram of the effect of the compound Apostatin-1 (Apt-1) and the control drug Edaravone (Edaravone) on improving oxygen-glucose deprivation-induced human microglial damage in Example 2 of the present invention; mean ± SD, n =3; Among them, compared with the normal group (non-OGD), ^## p<0.01, compared with the model group (OGD), **p<0.01.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention.

The applicant's research found that after cerebral ischemia, brain tissue and ischemic astrocytes undergo programmed necrosis and apoptosis, and autophagy flow is blocked. Apostatin-1 inhibits programmed necrosis and apoptosis and activates autophagy by inhibiting tumor necrosis factor receptor-associated death domain protein (TRADD). Therefore, it was further found that Apostatin-1 can treat ischemic stroke.

At the same time, Apt-1 can activate autophagy. According to many literature reports, autophagy flow is blocked during cerebral infarction, and at this time, simply activating autophagy will increase the volume of cerebral infarction. However, we found that Apt-1 can not only activate autophagy, but also protect lysosomes by indirectly inhibiting RIPK1, and promote the restoration of autophagic flux. Therefore, Apt-1 can not only reduce central nervous cell death, but also restore autophagy flow, help cells remove damaged organelles and aging proteins, restore the intracellular environment, and further protect cells. In Example 1, we administered Apt-1 after 6 hours of ischemia-reperfusion, and found that it could still reduce the volume of cerebral infarction induced by cerebral ischemia, indicating that Apt-1 has a wider therapeutic window. In Example 2, the effect of Apt-1 on the in vitro oxygen-glucose deprivation model (this model is applicable to ischemic stroke, traumatic brain injury, neonatal ischemic-hypoxic encephalopathy and other diseases) of different nerve cells was detected. Protective effect, Apt-1 was found to have a protective effect on neurons, astrocytes and microglia induced by oxygen-glucose deprivation.

Example 1

Male C57 mice were randomly divided into a control group (sham operation group), a model group, and an Apostatin-1 (Apt-1) administration group (3 nmol), 10 in each group. The mouse model of transient middle cerebral artery occlusion (tMCAO) was established by suture method. After 60 minutes of ischemia and reperfusion, Apostatin-1 (Apt-1) was injected into the lateral ventricle at 6 hours after reperfusion. The mice were decapitated and the brains were taken out and sliced to observe the effect of Apostatin-1 (Apt-1) on the volume of cerebral infarction in the acute stage of cerebral ischemia. Before the mice were sacrificed, the Longa method was used to score the neurological symptoms of the mice after reperfusion (I/R) in the acute stage of cerebral ischemia. The higher the score, the more obvious the neurological deficit. The use rate of the right limb of the mice was observed through the barrel test to evaluate the improvement of the neurological symptoms of the mice.

Drum test: mice are placed in a drum, and the use of their forelimbs to touch the wall of the drum is observed. After the mouse's forelimbs were lowered from the barrel wall, the situation of the left and right limbs sticking to the barrel wall was recorded. According to this method, each mouse was tested 10 times. The final statistical formula was (the number of movements of the non-impaired forelimb - the number of movements of the damaged forelimb)/(the number of movements of the non-impaired forelimb + the number of movements of the damaged forelimb + the number of common movements of the forelimb). The scoring criteria for neurological symptoms are shown in Table 1.

Table 1

score nervous symptoms 0 Normal, no neurological damage 1 Incomplete extension of the left forelimb, slight neurological damage 2 When the mice walk in circles towards the paralyzed side, the neurological function is moderately damaged 3 The mouse fell to the paralyzed side when walking, and the neurological function was severely damaged 4 Mice can't walk at all, and their consciousness is impaired

The results are shown in Figure 1-3. It can be seen from FIG. 1 that the compound Apostatin-1 (Apt-1) can reduce the volume of cerebral infarction in mice with cerebral ischemia in the acute phase. It can be seen from Fig. 2 that the compound Apostatin-1 (Apt-1) can reduce the neurological symptom score of mice with cerebral ischemia. It can be seen from FIG. 3 that the compound Apostatin-1 (Apt-1) can improve the right limb usage rate of cerebral ischemia mice.

The above results show that: Apostatin-1 (Apt-1) has a certain protective effect on ischemic brain injury, and can be used for the prevention and treatment of this type of disease.

Example 2

Mix human neuron cells (SY5Y), human astrocytes (HA), and human microglial cells (HMC3) with complete medium and inoculate them in sterile 96-well plates (about 1500-2000 per well Cells), placed in a cell incubator for culture after inoculation, using serum-free DMEM:CCK-8 solution = 9:1 ratio to prepare a CCK-8 mixture. After aspirating and discarding the original medium in the 96-well plate, add 100 μl of CCK-8 mixture to each well, and after incubation for 2 hours, measure the absorbance at 450 nm with a microplate reader. Taking Edaravone, a neuroprotective drug currently used clinically, as a positive control drug, the effect of the drug Apostatin-1 (Apt-1) on the cellular oxygen-glucose deprivation reoxygenation model (ie OGD/Re, after 3 hours of ischemia) was tested. The protective effect of reperfusion 24h). Figures 4, 5, and 6 show the CCK-8 detection results of the three types of cells. The results showed that Apostatin-1 had a protective effect on neurons, astrocytes and microglia induced by OGD, and the effect of 10mM dose of Apostatin-1 was equivalent to that of 100mM Edaravone.

Apparently, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation. For those of ordinary skill in the art, on the basis of the above description, other changes or changes in various forms can also be made. It is not necessary and impossible to exhaustively list all the implementation manners here. However, the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

Claims (8)

1. Application of compound Apostatin-1 in the preparation of medicines for treating cerebrovascular diseases. 2. The application according to claim 1, characterized in that: the cerebrovascular disease is cerebral ischemic disease. 3. The application according to claim 2, characterized in that: the cerebral ischemic disease is ischemic stroke. 4. The application according to claim 3, characterized in that: said ischemic stroke is ischemia-reperfusion stroke. 5. The application according to claim 1, characterized in that: the dosage of the drug is at least 3 nmol. 6. The application according to claim 1, characterized in that: the drug is administered by intracerebroventricular injection. 7. A medicament for treating cerebrovascular diseases, characterized in that the medicament includes Apostatin-1. 8. The medicine according to claim 7, characterized in that: the medicine is composed of 0.1-100% Apostatin-1 and 99.9-0% pharmaceutical excipients.

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