Research method of influence mechanism of HET0016 on blood-brain barrier injury

Abstract

The invention discloses a method for researching an influence mechanism of HET0016 on blood-brain barrier injury, and relates to the field of medicine. Comprising the following steps: selecting SD rats, and randomly dividing the rats into a false operation group, a TBI group, a TBI + solvent group and a TBI + HET0016 group; the four groups of SD rats are processed, and a TBI model is established; within a certain time after the injury, respectively injecting HET0016 for multiple times; respectively carrying out WB brain tissue sampling and immunofluorescence brain tissue sampling; the expression levels of ZO-1, Occludin and MMP-9 are quantitatively detected through WB, and CD31 and ZO-1 / Occludin are co-localized through immunofluorescence detection; and statistically analyzing and verifying inter-group differences. An immature brain TBI standardized model and an HET0016 administration scheme are established, key technical support is provided for development of pediatric neuroprotective drugs, and the method is suitable for development of novel brain protective agents in pharmaceutical enterprises.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a method for studying the mechanism of influence of HET0016 on blood-brain barrier damage. Background Art

[0002] Traumatic brain injury (TBI) is the leading cause of disability and death among children worldwide. According to the World Health Organization, its annual incidence rate is 200 / 100,000, and about 25% of children have permanent neurological dysfunction. Due to its unique pathophysiological characteristics, the damage and repair mechanism of immature brain tissue is essentially different from that of adults: first, the expression of tight junction proteins (ZO-1, Occludin) between cerebral vascular endothelial cells during development is only 60%-70% of that in adults, and the basement membrane structure is loose, resulting in weak inherent defense capabilities of the blood-brain barrier (BBB); second, the synaptic plasticity of neurons is strong, and abnormal discharge after injury is prone to cause secondary epilepsy; third, the antioxidant enzyme (such as SOD, GSH-Px) system is not fully developed, and the free radical scavenging ability is more than 40% lower than that of adults. Clinical studies have confirmed that the permeability of the BBB after TBI increases sharply by 3-5 times within 24 hours, causing plasma protein leakage, ion imbalance and accumulation of neurotoxic substances in the brain parenchyma, ultimately leading to irreversible neurological damage.

[0003] Current clinical interventions have significant limitations: osmotic dehydrating agents such as mannitol can reduce intracranial pressure in the short term, but they can aggravate blood volume fluctuations and induce renal damage; long-term use of glucocorticoids leads to complications such as immunosuppression and abnormal bone metabolism. In recent years, targeted therapy studies for adult TBI (such as MMP-9 inhibitor BB-94 and antioxidant NAC) have shown attenuated efficacy in juvenile animal models, suggesting the uniqueness of the mechanism of brain injury during development. Studies have shown that 20-hydroxyeicosatetraenoic acid (20-HETE), as a cytochrome P450 metabolite, is abnormally highly expressed after immature brain TBI: on the one hand, by activating the NADPH oxidase-ROS pathway, the tyrosine nitration modification of ZO-1 protein increases by 2.8 times, directly destroying the tight junction structure; on the other hand, it upregulates MMP-9 expression to 4.2 times the physiological state, accelerating the degradation of type IV collagen in the basement membrane. It is worth noting that the density of 20-HETE receptors (GPR75) in juvenile brain vascular endothelial cells is 30% higher than that in adults, and the signal transduction efficiency is improved, which provides a theoretical basis for low-dose targeted intervention. Although HET0016 (20-HETE synthesis inhibitor) can reduce infarct volume by 58% in adult stroke models, its dynamic protective effect and dose-response characteristics on the developing BBB have not yet been clarified, becoming a technical bottleneck that needs to be solved in this field.

[0004] For this purpose, the present invention proposes a research method for the mechanism of HET0016 on blood-brain barrier injury. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies existing in the prior art, and to propose a research method for the mechanism of HET0016 on blood-brain barrier injury.

[0006] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A research method for the mechanism of HET0016 on blood-brain barrier injury, comprising the following steps:

[0008] S1: Select SD rats, and randomly divide the rats into a sham operation group, a TBI group, a TBI + solvent group, and a TBI + HET0016 group;

[0009] S2: Treat the four groups of SD rats to establish a TBI model;

[0010] S3: Inject HET0016 in multiple times at certain time after injury;

[0011] S4: Perform WB brain tissue sampling and immunofluorescence brain tissue sampling respectively;

[0012] S5: Quantitatively detect the expression levels of ZO-1, Occludin and MMP-9 by WB and detect the co-localization of CD31 and ZO-1 / Occludin by immunofluorescence;

[0013] S6: Statistically analyze and verify the differences between groups.

[0014] Preferably: In the step S1, the SD rats are rats 9-10 days after birth.

[0015] Preferably: In the step S2, the method for establishing a TBI model includes the following steps:

[0016] S21: Intraperitoneally inject tribromoethanol for anesthesia. After the anesthesia is completed, use forceps to pull the animal's tongue to the outside of the mouth on one side to prevent suffocation due to posterior displacement of the tongue;

[0017] S22: Then use a new blade to longitudinally cut open the scalp to expose the parietal bone;

[0018] S23: Perform craniotomy using a dental drill and remove the cranial piece;

[0019] S24: Use a flat-bottomed metal probe with a diameter of 3 mm to impact the tissue;

[0020] After the impact is completed, immediately remove the neonatal mice, pull out the tongue with forceps to clear the respiratory tract, stop bleeding the wound, replace the bone piece, and suture the skin tissue;

[0021] S26: Anesthesia recovery is carried out on the warming blanket. After disinfecting the wound with iodophor, the mother rat is placed back beside it.

[0022] Preferably: In the step S21, the mass fraction of tribromoethanol is 2.5%, and the dosage is 0.01 ml / g.

[0023] Preferably: In the step S23, the craniotomy positioning is 2 mm to the left of the sagittal line, between the coronal suture and the lambdoid suture.

[0024] Preferably: In the step S24, the impact parameters are: speed 5.5 m / s, depth 1.5 mm, and duration 50 ms.

[0025] Preferably: In the step S3, HET0016 is intraperitoneally injected three times at 3 h, 27 h, and 51 h after injury, and the injection dose each time is 5 μl / g.

[0026] Preferably: In the step S4, the method for taking WB brain tissue is as follows: Three days after TBI, the cortical tissue at the injured side is taken and placed into a pre-cooled enzyme-free 1.5 ml centrifuge tube marked. Immediately after the tissue is taken, it is stored in a -80 °C freezer.

[0027] Preferably: In the step S4, the method for taking immunofluorescence brain tissue is as follows:

[0028] S41: After deeply anesthetizing each group of neonatal rats, perfusion fixation of the brain is carried out through the heart.

[0029] S42: After anesthesia, the four limbs of the neonatal rats are fixed on the dissection platform. The tissue of the upper abdomen is clamped with forceps and lifted upward. The tissue is cut in a "V" shape with scissors and extended to both sides to fully expose the diaphragm.

[0030] S43: Cut open the diaphragm and the ribs on both sides to fully expose the heart. After inserting the needle of the intravenous infusion set into the left ventricle of the neonatal rat, cut open the right auricle with ophthalmic scissors.

[0031] S44: First, perfusion with normal saline is carried out through the infusion set. After its liver turns white, perfusion with 4% paraformaldehyde is carried out until the leg muscles of the neonatal rat become stiff.

[0032] S45: Finally, the brain tissue is taken out, and the taken brain tissue is immersed and fixed in 4% paraformaldehyde for 24 h.

[0033] The beneficial effects of the present invention are as follows:

[0034] 1. The present invention establishes a standardized model of TBI in the immature brain and a HET0016 administration scheme, providing key technical support for the development of pediatric neuroprotective drugs and being applicable to pharmaceutical enterprises to develop new brain protectants. Description of the Drawings

[0035] Figure 1 Flow chart of the research method for the influence mechanism of HET0016 on blood-brain barrier injury proposed by the present invention;

[0036] Figure 2 Respective sectional images of the research method for the influence mechanism of HET0016 on blood-brain barrier injury proposed by the present invention. Specific implementation manners

[0037] The technical solutions of the present invention will be further described in detail below in conjunction with specific implementation manners.

[0038] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "installation", "connection", "connection", and "setting" should be understood in a broad sense. For example, it can be fixedly connected and set, or detachably connected and set, or integrally connected and set. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0039] Embodiment

[0040] Materials: Male SD rats at 9-10 days after birth were selected as experimental subjects, and a TBI model was established by controlled cortical impact (CCI). The sham operation group only underwent craniotomy. To explore whether HET0016 has a protective effect on the blood-brain barrier of the immature brain.

[0041] Methods: First, in order to study the influence of HET0016 on the blood-brain barrier after TBI in the immature brain, male SD rats at 9-10 days after birth were randomly divided into a sham operation (Sham) group, a TBI group, a TBI + vehicle group, and a TBI + HET0016 group. At 3 h, 27 h, and 51 h after TBI, each group was intraperitoneally injected with HET0016 or solvent at 5 μl / g.

[0042] SD rats were taken out of the animal facility, and their weights were measured and recorded using a balance. The rats were anesthetized by intraperitoneal injection of 2.5% tribromoethanol at a dose of 0.01 ml / g. After anesthesia, the tongue of the animal was pulled to the outside of the mouth with forceps to prevent asphyxiation due to posterior displacement of the tongue. Then, a new blade was used to longitudinally incise the scalp to expose the parietal bone. The dental drill was turned on at a low speed, and a craniotomy was performed 2 mm to the left of the sagittal line, between the coronal suture and the lambdoid suture, and the skull piece was removed for later use. The neonatal rats were fixed on a craniocerebral trauma instrument, and a flat metal probe with a diameter of 3 mm was used to impact the tissue at a speed of 5.5 m / s, a depth of 1.5 mm, and a duration of 50 ms. Immediately after the impact, the neonatal rats were removed, the tongue was pulled out with forceps to keep the airway unobstructed, and after the wound was hemostatic, the bone piece was replaced and the skin tissue was sutured. There was no impact process in the sham operation group, and the remaining steps were the same as those in the experimental group. The rats were anesthetized and resuscitated on a warming blanket. After the wound was disinfected with iodophor, the rats were returned to their mother rats.

[0043] WB brain tissue sampling: Three days after TBI, the rats in each group were directly decapitated at the end of the experiment. Cortical tissue the size of a mung bean around the injured side was taken and placed in a pre-cooled enzyme-free 1.5 ml centrifuge tube marked with the group number. Immediately after the tissue sampling, it was stored in a -80 °C freezer.

[0044] Immunofluorescence brain tissue sampling: After the neonatal rats in each group were deeply anesthetized, the brains were taken by cardiac perfusion. After anesthesia, the four limbs of the neonatal rats were fixed on the dissection platform. The tissue of the upper abdomen was clamped with forceps and lifted upward. A "V"-shaped incision was made with a thread scissors and extended to both sides to fully expose the diaphragm. The diaphragm and the ribs on both sides were cut open to fully expose the heart. After inserting the needle of the intravenous infusion set into the left ventricle of the neonatal rat, the right auricle was cut open with an ophthalmic scissors. First, normal saline was perfused through the infusion set. After the liver turned white, 4% paraformaldehyde was perfused until the leg muscles of the neonatal rat became stiff. Finally, the head was cut off to take the brain, and the taken brain tissue was immersed and fixed in 4% paraformaldehyde for 24 h.

[0045] WB was used to detect the tight junction proteins ZO-1 / Occludin of the blood-brain barrier, and observe whether the protein expression levels increased in the groups injected with HET0016.

[0046] For immunofluorescence staining, CD31 was co-stained with ZO-1 / occludin. CD31 is a marker of endothelial cells, and endothelial cells are the main cell type of the blood-brain barrier. The co-expression of CD31 and ZO-1 / occludin in each group was observed.

[0047] Results: Intraperitoneal injection of HET0016 for three consecutive days after TBI increased the contents of the tight junction proteins ZO-1 and Occludin related to the blood-brain barrier, and decreased the content of the enzyme that hydrolyzes the tight junction proteins. This indicates that HET0016 can effectively repair the blood-brain barrier.

[0048] The present invention establishes a standardized model of immature brain TBI and an HET0016 dosing regimen, providing key technical support for the development of pediatric neuroprotective drugs and being applicable to pharmaceutical companies for the development of new brain protectants.

[0049] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered by the protection scope of the present invention.

Claims

1. A method for studying the mechanism of effect of HET0016 on blood-brain barrier damage, characterized in that: The following steps are involved: S1: SD rats were selected and randomly divided into sham operation group, TBI group, TBI+solvent group, and TBI+HET0016 group; S2: Four groups of SD rats were treated to establish TBI models; S3: HET0016 was injected several times within a certain period of time after injury; S4: brain tissue samples were collected for WB and immunofluorescence respectively; S5: Quantitative detection of ZO-1, Occludin and MMP-9 expression levels by WB and co-localization of CD31 and ZO-1 / Occludin by immunofluorescence; S6: Statistical analysis to verify the differences between groups.

2. The method for studying the mechanism of effect of HET0016 on blood-brain barrier damage according to claim 1, characterized in that: In the S1 step, the SD rats are rats that are 9-10 days old.

3. The method for studying the mechanism of effect of HET0016 on blood-brain barrier damage according to claim 1, characterized in that: In step S2, the method for establishing a TBI model comprises the following steps: S21: Intraperitoneal injection of tribromoethanol for anesthesia. After anesthesia, use forceps to pull the tongue of the animal to one side of the mouth to prevent the tongue from falling back and suffocating. S22: The scalp is then cut longitudinally using a new blade to expose its parietal bones; S23: Perform craniotomy using a dental drill and remove the skull fragment; S24: Use a 3 mm diameter flat-bottomed metal probe to impact the tissue; S25: After the impact, the suckling mouse was removed immediately, the tongue was pulled out with forceps to clear the airway, the bone fragment was put back after the wound stopped bleeding, and the skin tissue was sutured; S26: Perform anesthesia resuscitation on a warming blanket, disinfect the wound with iodine and return the mouse to the mother.

4. The method for studying the mechanism of effect of HET0016 on blood-brain barrier damage according to claim 3, characterized in that: In the step S21, the mass fraction of tribromoethanol is 2.5%, and the dosage is 0.01 ml / g.

5. The method for studying the mechanism of effect of HET0016 on blood-brain barrier damage according to claim 3, characterized in that: In step S23, the craniotomy is positioned 2 mm to the left of the sagittal line, between the coronal suture and the lambdoid suture.

6. The method for studying the mechanism of effect of HET0016 on blood-brain barrier damage according to claim 3, characterized in that: In step S24, the impact parameters are: speed 5.5 m / s, depth 1.5 mm, and duration 50 ms.

7. The method for studying the mechanism of effect of HET0016 on blood-brain barrier damage according to claim 1, characterized in that: In the step S3, HET0016 was intraperitoneally injected three times at 3h, 27h and 51h after injury, with each injection dose being 5 μl / g.

8. The method for studying the mechanism of effect of HET0016 on blood-brain barrier damage according to claim 1, characterized in that: In the step S4, the method for obtaining WB brain tissue is as follows: three days after TBI, the cortical tissue at the injured side is obtained and placed in a marked pre-cooled enzyme-free 1.5 ml centrifuge tube, and the tissue is immediately stored in a -80°C freezer after the tissue is obtained.

9. The method for studying the mechanism of effect of HET0016 on blood-brain barrier damage according to claim 1, characterized in that: In step S4, the method for obtaining brain tissue samples by immunofluorescence is: S41: After the rats in each group were deeply anesthetized, the brains were removed by transcardial perfusion; S42: After anesthesia, fix the limbs of the suckling mouse on the dissection platform, clamp the upper abdominal tissue with forceps and pull it upward, use wire scissors to cut the tissue in a "V" shape and extend it to both sides to fully expose the diaphragm; S43: Cut open the diaphragm and the ribs on both sides to fully expose the heart. After inserting the needle of the intravenous infusion set into the left ventricle of the suckling mouse, use ophthalmic scissors to cut open the right atrial appendage; S44: First, physiological saline was perfused through the infusion tube, and after the liver turned white, 4% paraformaldehyde was perfused until the leg muscles of the suckling mice became stiff; S45: Finally, the brain tissue was removed and fixed by immersion in 4% paraformaldehyde for 24 hours.

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