CN111956647A

CN111956647A - Application of hydroxychloroquine in preparation of medicine for treating brain injury

Info

Publication number: CN111956647A
Application number: CN202010374622.6A
Authority: CN
Inventors: 林丽; 胡健; 谭显西
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2020-05-06
Filing date: 2020-05-06
Publication date: 2020-11-20

Abstract

The invention discloses an application of hydroxychloroquine in preparing a medicament for treating brain injury, and experimental results show that hydroxychloroquine can better improve the nerve function and the injured area tissue of a TBI model mouse, can reduce neuroinflammation or be used for stabilizing the integrity of a blood brain barrier, and can be used as a potential medicament for treating TBI.

Description

Application of hydroxychloroquine in preparation of medicine for treating brain injury

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of hydroxychloroquine in preparation of a medicine for treating brain injury.

Background

Traumatic Brain Injury (TBI) is the leading cause of death and long-term disability, with the incidence of Brain trauma increasing year by year as society develops. Worldwide, about 1000 million people are hospitalized or died due to TBI every year, and studies of the world health organization show that brain damage will become a critical disease of disability and death by 2020. At present, the treatment of traumatic brain trauma does not have a standard treatment scheme and does not have a specified medicine application, the clinical treatment mainly comprises surgical debridement and trauma sequelae improvement, and the auxiliary treatment (acupuncture, psychotherapy and the like) of sports rehabilitation and the like is still the main choice for the treatment of the brain trauma.

Neuropathophysiological processes occurring after TBI are intricate and complex, including primary brain injury due to tissue defect caused by mechanical external force and secondary brain injury caused by oxidative stress, apoptosis, neuroinflammation, blood brain barrier destruction, and the like. Acceleration/deceleration of primary injuries, such as collisions or skull collisions, can only be achieved by patient education or improved protective equipment, without predictability. Therefore, research on the treatment of traumatic brain injury has focused mainly on reducing the effects of secondary injury.

Hydroxychloroquine (HCQ) is a safe medicament which has been clinically used for a long time, is used for treating malaria for decades, is also widely used for treating autoimmune diseases such as Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis (RA) and the like, and has generally verified the safety clinically. Clinical findings show that HCQ accumulates in various tissues including the brain to a sufficient drug level after a long-term HCQ treatment, but there is no report of application of HCQ to brain injury.

Disclosure of Invention

The invention provides application of hydroxychloroquine in preparing a medicament for treating brain injury, and thus provides a novel potential medicament for treating brain injury.

An application of hydroxychloroquine in preparing a medicament for treating brain injury, wherein said medicament is used for treating brain injury or a disease associated with brain injury.

Preferably, the brain injury is secondary brain injury.

The experimental results show that the medicament is used for promoting the recovery of nerve functions after brain injury, and can be used for reducing necrotic abscission of tissues in the injured area.

Preferably, the medicament may also be used to reduce neuroinflammation or to stabilize the integrity of the blood brain barrier.

Compared with the prior art, the invention has the beneficial effects that:

through animal experiments, the hydroxychloroquine is found to be capable of promoting the recovery of nerve functions after brain injury, can be used for reducing necrosis and shedding of tissues in an injured area, reducing the release of nerve inflammation factors after brain trauma and relieving the injury of a blood brain barrier, and can be used as a potential medicine for treating brain injury.

Drawings

FIG. 1 is a graph showing the improvement of HCQ in the nerve function impairment in example 2 of the present invention;

FIG. 2 is a graph showing the improvement of HCQ in tissue defects in example 2 of the present invention;

FIG. 3 is a graph showing the effect of HCQ on the release of neuroinflammatory factor following brain trauma in mice in example 3 of the present invention;

FIG. 4 is a graph of the effect of HCQ on Blood Brain Barrier (BBB) disruption in mice after TBI in example 4 of the present invention.

Detailed Description

Example 1 construction of mouse brain Trauma (TBI) model

A brain trauma model is established using controlled cortical percussive injury (CCI). The mice were weighed, anesthetized by intraperitoneal injection of 4% chloral hydrate (100ul/10g)), and the head of the anesthetized mice was shaved off and fixed in a brain stereotaxic apparatus. The fixation standard is to keep the skull horizontal at the left and right positions and the tangent horizontal at the front and back positions. After iodine tincture is applied for disinfection, the skin of the head is cut along the median sagittal direction, and bregma, the herringbone point and the left brain part on the skull are exposed. 0.6mm behind bregma and 1mm lateral to midline, drilling skull with dental drill, grinding into round window with diameter of 4mm, adjusting the striking tube of the striking device to keep perpendicular to brain contact part, and ensuring vertical striking. The striking coefficient is: speed 4m/s, residence time 200ms, diameter 3mm, depth 1 mm. After the suction bleeding is finished, the incision is sutured, and the animal is normally raised after waking. The model is successfully made by 30 mice in the time, and the mice in the Sham operation group (Sham) are not beaten after the bone window is ground, namely normal control.

Example 2 pharmacodynamic test of HCQ on TBI model mice

The experiment is divided into three groups of Sham group, TBI + Vehicle group and TBI + HCQ group, and each group contains 15 animals. Mice in the TBI + HCQ group were administered 30mg/kg i.p. immediately after molding, twice a day, and mice in the other groups were administered a calculated amount of physiological saline for three days.

Behavioral experiments: all mice need to perform three continuous days of behavioral adaptation experiments including roller experiments and grip force experiments before modeling, and the mice with behavioral abnormalities are eliminated. And (3) performing behavioral experiments on all mice 24h and 3 days after molding, and evaluating the nerve functions of the mice in each group. As shown in FIG. 1, the results show that normal mice (Sham group) did not drop on the roller for about 400s (three experiments were averaged), the dropping time was significantly accelerated in TBI group mice compared to normal group, the difference was statistically significant (P <0.005), and the dropping time was prolonged in dosing group compared to TBI group, the difference was statistically significant (A, 24h, P < 0.01; 3d, P < 0.05). In the gripping force experiment, the gripping force of forelimbs of normal mice is about 100g (5-time gripping and averaging), the gripping force of forelimbs of brain trauma mice is obviously reduced, and compared with a normal group, the gripping force of forelimbs of the mice has statistical significance (B, P is less than 0.01), and after HCQ is given, the gripping force of forelimbs of the mice is improved, and compared with a TBI group, the gripping force of forelimbs of the mice has statistical significance (B, P is less than 0.01). The experimental results show that after the brain trauma of the mouse, the nerve function is damaged to a certain extent, and the symptoms are that the motor ability is reduced (the falling time of the roller is shortened) and the forelimb is powerless. However, administration of HCQ improved impaired neurological function in mice and was therapeutic.

Tissue defect evaluation: after 3 days, the mouse is anesthetized by intraperitoneal injection of water and chloral, the chest cavity is opened to fully expose the heart, the right auricle is cut, a perfusion tube is inserted from the apex of the heart of the left ventricle, cold normal saline is firstly instilled quickly, so that blood in the tissues is washed clean until the tissues such as the liver, the lung and the like are completely whitened. Then 4% paraformaldehyde (30ml-50ml) is used for perfusion fixation until the liver of the mouse becomes hard and the limbs become rigid, thus completing the fixation. Cutting off the head, taking the whole brain tissue, placing in 4% paraformaldehyde solution, and fixing at 4 deg.C for 24 hr. Gradient dehydration, transparency and paraffin embedding. Finally, serial coronal sections (5 μm) were performed and hematoxylin-eosin was performed. 5 mice were selected for each group, and 5 sections containing the lesion area were randomly selected for each mouse, observed under a microscope and photographed, and the defect area was counted. As shown in figure 2, after the brain trauma of the mouse, the tissue of the injured area is necrotized and shed to form a concave hole. Compared with the TBI group, the mice in the administration group have less brain tissue defects and the difference has statistical significance (P <0.05), which indicates that HCQ can reduce necrotic abscission of tissues in the damaged area.

Example 3 Effect of HCQ on neuroinflammatory factor Release following brain trauma in mice

After the TBI3 days, the mouse is anesthetized by intraperitoneal injection of chloral hydrate, the chest is opened to fully expose the heart, the right auricle is cut, an infusion tube is inserted from the apex of the left ventricle, and normal saline is quickly instilled to wash the blood in the tissues until the tissues such as the liver, the lung and the like are completely whitened. Cutting off the head, taking the brain tissue around the injury, and extracting RNA by adopting a Trizol method. Q-PCR (real-time fluorescence quantification) for inflammatory factor detection: expression levels of iNOS, IL-1. beta. and TNF-. alpha. As shown in FIG. 3, the inflammatory factor expression level was increased in the injured group (TBI + Vehicle group) and decreased in the HCQ group (TBI + HCQ group) compared to the injured group (sham group), with the difference being statistically significant (. about.P)<0.005 vs sham group;^##P<0.01，^###P<0.005 in comparison with TBI group), indicating that HCQ has an inhibitory effect on the release of neuroinflammatory factors after brain trauma in mice.

Example 4 Effect of HCQ on Blood Brain Barrier (BBB) disruption in mice post TBI

BBB permeability was measured by evans blue method. After 72 hours, each group of mice is injected with Evans blue (4 percent, 3mL/kg) through tail veins, and after 3 hours of injection, the mice are anesthetized by intraperitoneal injection with chloral hydrate, the thoracic cavity is opened to fully expose the heart, meanwhile, the right auricle is cut, a perfusion tube is inserted from the apex of the heart of the left ventricle, and normal saline is quickly instilled, so that blood in the tissues is washed clean until the tissues such as the liver, the lung and the like are completely whitened. The brain is taken by cutting the head and the damaged area is photographed. Then, the brain tissue around the injury is soaked in trichloroacetyl with the same volume overnight, the supernatant is obtained by centrifugation, and the content of evans blue is detected by a microplate reader under the wavelength of 620 nm. As shown in FIG. 4, after brain injury, the blood brain barrier was disrupted, the permeability was increased, and the content of Evans blue was increased in the injured group (TBI + Vehicle group) and decreased in the infiltration of Evans blue in the HCQ-treated group (TBI + HCQ group) compared to the normal group (sham group), and the difference was statistically significant (. about.. about.P)<0.001, compared to the sham group;^##P<0.01, compared to TBI group), indicating that HCQ has a reparative effect on the BBB.

Claims

1. The application of hydroxychloroquine in preparing a medicament for treating brain injury is characterized in that the medicament is used for treating brain injury or diseases related to brain injury.

2. The use of hydroxychloroquine in the manufacture of a medicament for the treatment of a brain injury according to claim 1, wherein said brain injury is a secondary brain injury.

3. The use of hydroxychloroquine in the manufacture of a medicament for the treatment of brain injury according to claim 1, wherein said medicament is for promoting recovery of neurological function following brain injury.

4. The use of hydroxychloroquine in the manufacture of a medicament for treating brain injury as in claim 1, wherein said medicament is for reducing neuroinflammation and reducing the release of neuroinflammatory factors.

5. Use of hydroxychloroquine in the manufacture of a medicament for the treatment of brain injury according to claim 1, wherein said medicament is for reducing necrotic exfoliation of tissue in the injured area.

6. The use of hydroxychloroquine in the manufacture of a medicament for treating brain injury according to claim 1, wherein said medicament is for stabilizing the integrity of the blood-brain barrier and reducing injury to the blood-brain barrier.