Application of loganin in preparation of medicine for treating traumatic brain injury diseases

Abstract

The invention provides application of loganin in preparation of drugs for treating traumatic brain injury diseases, and belongs to the technical field of drugs related to brain injury diseases. Loganin can significantly promote proliferation of neural stem cells after brain trauma and induce angiogenesis, and the molecular action mechanism of loganin is closely related to regulation of ephrinB2 and EphB4 protein expression. The discovery provides an important theoretical basis for treating traumatic brain injury by synergistically promoting neurogenesis and angiogenesis through loganin.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology related to brain injury diseases, and particularly relates to the application of loganin in the preparation of drugs for treating traumatic brain injury diseases. Background Technology

[0002] Brain injury is a syndrome of structural and functional disorders of the brain caused by various etiologies such as ischemia, trauma, and inflammation. Traumatic brain injury, a common acute and critical condition in neurosurgery, is often caused by traumatic events such as external impacts or craniocerebral surgery. It can lead to neuronal damage and destruction of vascular structures, subsequently resulting in cerebral edema, increased intracranial pressure, and neurological deficits. Therefore, promoting neurogenesis and angiogenesis in the area surrounding the injury has become one of the key strategies for neurological function repair.

[0003] Existing research has disclosed the effects of loganin on cerebral ischemia-reperfusion injury (The Role and Mechanism of Loganin in Cerebral Ischemia-Reperfusion Injury, Xu Xiaowen et al., *Zhejiang Medical Journal*). Specifically, loganin significantly reduces the cerebral infarction rate and neuronal apoptosis rate in rats with a cerebral ischemia-reperfusion model, reduces cerebral edema, and improves neurological deficit symptoms in the model animals. Its main focus is on inhibiting the inflammatory response and neuronal apoptosis, specifically by regulating the p38 / NF-κB signaling pathway. However, ischemic brain injury and traumatic brain injury have completely different etiologies, initiating pathological processes, and core treatment needs and targets. Existing research has never disclosed that loganin can be used to treat traumatic brain injury. Summary of the Invention

[0004] This invention proposes a new use for loganin, specifically in the preparation of a drug for treating traumatic brain injury.

[0005] This invention proposes the application of loganin in the preparation of drugs for treating traumatic brain injury.

[0006] In a preferred embodiment of the present invention, the traumatic brain injury includes surgical brain injury and traumatic brain injury.

[0007] In this embodiment of the invention, traumatic brain injury is a mechanical injury caused by external impact. The cause is the action of external forces such as impact and penetration. The initial pathology is primary structural damage, including surgical brain injury and traumatic brain injury, such as brain tissue contusion and rupture, blood vessel rupture and hemorrhage, etc., followed by secondary reactions such as ischemia, inflammation, and apoptosis.

[0008] Although traumatic brain injury involves processes such as ischemia, inflammation, and apoptosis, it differs from the secondary response of cerebral ischemia-reperfusion injury (for example, ischemia in traumatic brain injury is mostly caused by ruptured blood vessels rather than vascular occlusion).

[0009] The existing technology does not mention that loganin has the effect of promoting the regeneration of damaged brain tissue, repairing axonal damage, and inhibiting secondary damage around hematoma, which is suitable for traumatic brain injury. Its disclosed mechanism only covers part of the secondary damage link and cannot meet the core treatment needs of traumatic brain injury.

[0010] In a preferred embodiment of the present invention, loganin promotes the proliferation of neural stem cells and induces angiogenesis after brain injury; the target proteins of loganin are ephrin B2 and EphB4. In this embodiment of the present invention, loganin regulates the ephrin B2 / EphB4 signaling pathway by targeting and binding to ephrin B2 and EphB4, thereby promoting the proliferation and differentiation of neural stem cells and accelerating angiogenesis in the brain injury area.

[0011] In a preferred embodiment of the present invention, the effective dose of loganin in the drug is 90 mg / kg to 270 mg / kg. For each kilogram of body weight, the effective daily intake of loganin (e.g., for clinical patients) is 90 mg to 270 mg.

[0012] In a preferred embodiment of the present invention, the dosage form of the drug includes one or more of oral preparations, injections, and topical preparations.

[0013] In a preferred embodiment of the present invention, the oral preparation is a tablet, capsule, granule, oral liquid, or drop; the injectable preparation is an intravenous injection, intramuscular injection, or subcutaneous injection. The topical preparation includes an ointment, gel, patch, or liniment.

[0014] In a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier or excipient, which includes diluents, excipients, binders, disintegrants, lubricants, etc. The diluents and excipients (such as starch and lactose) are used to increase volume for ease of production and administration. The binders (such as cellulose) are used to bind drug powders into granules or tablets. The disintegrants (such as sodium carboxymethyl starch) are used to rapidly disintegrate the drug in the body, releasing the drug. The lubricants (such as magnesium stearate) are used to prevent powder from adhering to machinery, ensuring smooth production.

[0015] This invention has the following advantages:

[0016] This invention proposes the application of loganin in the preparation of drugs for treating traumatic brain injury. Traumatic brain injury directly damages the brain parenchyma and vascular structure, leading to decreased proliferation and differentiation capacity of neural stem cells, inhibited angiogenesis, and accompanied by local inflammation and neuronal apoptosis. The natural compound loganin can significantly promote the proliferation of neural stem cells and induce angiogenesis after traumatic brain injury, and its molecular mechanism of action is closely related to the regulation of ephrinB2 and EphB4 protein expression. This discovery provides an important theoretical basis for the therapeutic effect of loganin on traumatic brain injury by synergistically promoting neurogenesis and angiogenesis. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is the morphological and analytical result of nestin staining in rats with surgically induced brain injury, as shown in Example 1 of this invention. Note: Data are expressed as mean ± standard error; n = 4, ### P <0.001 compared to the sham surgery group; *** P <0.001 compared to the model group, scale bar = 100 micrometers.

[0019] Figure 2 This is the morphological and analytical result of nestin staining in rats with traumatic brain injury using loganin in Example 1 of this invention. Note: Data are expressed as mean ± standard error; n = 4. ### P <0.001 compared to the sham surgery group; *** P <0.001 compared to the model group, scale bar = 100 micrometers.

[0020] Figure 3 The effect of loganin on BrdU in surgically injured rats in Example 1 of this invention. + / Lectin + Staining morphology and analysis results. Note: Data are expressed as mean ± standard error; n = 4. ### P <0.001 compared to the sham surgery group;** P <0.01 compared to the model group, scale bar = 50 micrometers.

[0021] Figure 4 The effect of loganin on BrdU in rats with traumatic brain injury in Example 1 of this invention. + / Lectin+ Dyeing morphology and analysis results. Note: Data are expressed as mean ± SEM; n = 4, ### P <0.001 vs. sham operation group; ** P <0.01 vs. model group, scale bar = 50 μm.

[0022] Figure 5 This is the effect of loganin on the expression levels of ephrinB2 and EphB4 proteins in the brain tissue of rats with surgically induced brain injury 7 days after modeling in Example 1 of the present invention. Note: Data are expressed as mean ± SEM; n = 4, vs. model group, ** P <0.01, *** P <0.001.

[0023] Figure 6 This is the effect of loganin on the expression levels of ephrinB2 and EphB4 proteins in the brain tissue of rats with traumatic brain injury 7 days after modeling in Example 1 of the present invention. Note: Data are expressed as mean ± SEM; n = 4, vs. model group, * P <0.05, ** P <0.01, *** P <0.001. Detailed implementation manners

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Without conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0025] Example 1 Study on traumatic brain injury and surgically induced brain injury rat models using loganin 1.1 Drugs The monomeric compound loganin, with a content greater than 98.5% detected by high performance liquid chromatography. Before use, it was dissolved in distilled water to form a drug solution with the required concentration for experiments. The molecular structure of loganin is shown in Formula I.

[0026] Formula I; 1.2 Experimental animals SPF-grade male Sprague-Dawley rats, 7 - 8 weeks old, weighing 260 - 280 g. Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., certificate number: SCXK (Beijing) 2016 - 0001. Raised conventionally, environmental temperature 24 ± 1°C, humidity 55 ± 5%, fasted for 12 h before surgery without water restriction.

[0027] 1.3 Main experimental instruments Precision craniocerebral injury impactor (Wuhan Yihong Technology Co., Ltd., China); skull drill (Guangdong Aoboer Medical Instrument Co., Ltd., China); cryostat (LEICA, USA); ultrasonic cell disruptor (Ningbo Xinzhi Technology Research Institute, China); benchtop micro-refrigerated centrifuge (Beckman Coulter, USA); full-wavelength microplate reader (Thermo Fisher Scientific, USA); Powerpac Basic electrophoresis apparatus (Bio-Rad, USA); upright fluorescence microscope (Nikon, Japan); chemiluminescent gel imaging system (Alpha, USA).

[0028] 1.4 Main Experimental Reagents Rabbit anti-nestin primary antibody (Sigma-Aldrich, USA); mouse anti-BrdU primary antibody (Roche, USA); mouse anti-Lectin primary antibody (Vector Laboratories, USA); donkey anti-mouse IgG (H+L) secondary antibody AlexaFluor594 (Thermo Fisher Scientific, USA); donkey anti-rabbit IgG (H+L) secondary antibody AlexaFluor 488 (Thermo Fisher Scientific, USA); donkey anti-rabbit IgG (H+L) secondary antibody AlexaFluor 594 (Thermo Fisher Scientific, USA); rabbit anti-ephrinB2 primary antibody (CellSignaling Technology, USA); mouse anti-EphB4 primary antibody (Abcam, USA); horseradish enzyme-labeled goat anti-mouse IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.); horseradish enzyme-labeled goat anti-rabbit IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.); ECL chemiluminescence detection kit (Beijing Botes Biotechnology Co., Ltd.).

[0029] 1.5 Experimental Methods 1.5.1 Establishment of a rat surgical brain injury (SBI) model After weighing, rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50 mg / kg). The hair on the top of their heads was shaved off with a razor blade. After disinfection with povidone-iodine, a 4mm × 4mm window was created 1 mm to the right of the midline, 1 mm anterior to the anterior fontanelle, using a dental titanium drill. A 2mm section was then removed using a sharp instrument dissection method. 2 The right frontal lobe tissue was examined. In the sham surgery group, no brain tissue was removed; all other procedures were the same. On postoperative day 3, patients received pre-prepared, preheated BrdU solution via intraperitoneal injection at a dose of 50 mg / kg twice daily for 4 consecutive days.

[0030] 1.5.2 Establishment of a rat model of traumatic brain injury (TBI) This study established a moderate total intracortical brain injury (TBI) rat model using controlled cortical impaction (CCI). After weighing, rats were anesthetized via intraperitoneal injection of 2% sodium pentobarbital (50 mg / kg) and placed in a stereotactic apparatus. Following preparation and disinfection of the surgical area, the scalp was incised, subcutaneous tissue and periosteum were removed, and a 5.0 mm circular craniectomy was performed in the left frontoparietal cortex (1 mm lateral to the midline and 3 mm posterior to the coronal suture). After craniectomy, a CCI device with a 4.0 mm flat impactor was placed at the center of the craniectomy site. The impact parameters were set to a velocity of 4 m / s, a depth of 2.8 mm, and a dwell time of 150 ms. The dura mater remained intact on the cortex; rats whose dura mater was damaged were excluded. The sham-operated group underwent the same procedures as the TBI model, except for cortical impaction. After suturing the incision, the animals were placed on an experimental animal warming mat until they awoke, after which they were returned to their original cages. On the third day after surgery, patients were given an intraperitoneal injection of pre-prepared and preheated BrdU solution at a dose of 50 mg / kg, twice daily for four consecutive days.

[0031] 1.5.3 Animal grouping and administration Rats were randomly divided into 5 groups: sham-operated group, model group, low-dose loganin group, medium-dose loganin group, and high-dose loganin group. Loganin was dissolved in distilled water, and 3 hours after modeling, loganin was administered by gavage once daily at doses of 30 mg / kg, 90 mg / kg, and 270 mg / kg. The sham-operated group and the model group were given an equal volume of distilled water.

[0032] 1.5.4 Frozen sections of brain tissue Remove the fixed rat brain tissue samples, blot dry with filter paper, trim the blade, protect with isopentane, and flash-freeze in liquid nitrogen for 10 seconds. After freezing with deionized water, slice the tissue. The thickness of the frozen sections was set at 20 μm, and one out of every ten brain samples was taken (anterior fontanelle +1.20 to -0.20 mm). The brain slices were placed in 0.01M PBS solution and stored at 4°C.

[0033] 1.5.5 Nestin and BrdU / Lectin Immunofluorescence Staining Methods Select the mounted tissue sections, circle them with a histochemical pen, and observe the endogenous cell proliferation. BrdU fluorescence staining is required. Pretreatment of the sections is necessary: ​​frozen sections are first rinsed 3 times / 5 min with PBST; then treated with 2 mol / L HCl in a 37℃ constant temperature water bath with shaker for 30 min, followed by rinsing with PBST 3 times / 5 min again. For other staining indicators, no pretreatment is needed; directly wash three times with 3‰ PBST for 5 min each time, ensuring thorough washing on a shaker during the procedure; add 5% serum (of the same species as the secondary antibody) diluted with 3‰ PBST and block at room temperature for 2 h; add 100 μL of primary antibody (prepared according to the antibody instructions) diluted with 5% serum (of the same species as the secondary antibody) per tissue section and incubate at 4℃ for at least 16 h; after 16 h, remove the humidified chamber from the freezer and wash 3 times with 1×PBS solution for 5 min each time; add the corresponding fluorescent secondary antibody (concentration according to the antibody instructions) and incubate at room temperature for 2 h. All operations are performed in the dark; wash 3 times with 1×PBS solution for 5 min each time. min; After the tissue sections have dried, add DAPI mounting medium, cover with a coverslip, gently remove air bubbles, and then take pictures for analysis after drying.

[0034] 1.5.6 Western blot analysis of ephrin B2 and Eph B4 protein levels in brain tissue Fresh rat brain tissue was lysed for BCA protein quantification. The total protein concentration was adjusted to be consistent across groups using lysis buffer. 5X loading buffer was added to the total protein to dilute it to 1× buffer, and the mixture was denatured at 95°C for 10 min. After cooling, it was stored at -80°C and used as needed. 8%-15% separating gel and 5% stacking gel were prepared. Sample loading: Approximately 50 μg of sample was loaded per well. Electrophoresis: Initially, a constant voltage of 60V was set. After the sample transferred from the stacking gel to the separating gel and the marker was completely separated, the voltage was adjusted to a constant voltage of 90V until the sample reached the bottom of the gel. Electrophoresis was then stopped. Transfer: The desired protein bands were cut according to the loading lanes and the marker, and transferred at a constant voltage of 100V for 120 min. Blocking: After transfer, the nitrocellulose (NC) membrane was removed and stained with Ponceau S for 30 seconds. The sample on the gel was then observed. To transfer the protein to the NC membrane, cut out the desired molecular weight protein bands using ophthalmic scissors, place them in 1×TBST washing buffer to remove Ponceau S staining, add 5% skim milk for blocking, and incubate at room temperature with shaking for 2 hours; Primary antibody incubation: Dilute the primary antibody with 5%-10% skim milk, incubate overnight at 4°C, wash 3 times with TBST for 10 minutes each time; Secondary antibody incubation: Dilute the secondary antibody with TBST, incubate at room temperature with shaking for 2 hours, wash 3 times with TBST for 10 minutes each time; Development: Mix equal volumes of ECL chemiluminescence solutions A and B, and add to the NC membrane until completely covered. Place the membrane in a gel imaging system for development and imaging. Analyze the grayscale values ​​of the protein bands using ImageJ software, and calculate the relative expression levels of ephrinB2 and EphB4 proteins using β-actin as an internal reference.

[0035] 1.5.7 Data Analysis Experimental data were analyzed using SPSS 22.0 statistical software, and results are expressed as Mean ± SEM. One-way analysis of variance (ANOVA) was used to compare sample means between groups. P <0.05 indicates statistical significance.

[0036] 2. Experimental Results 2.1 Effect of loganin on the number of nestin-positive cells in the SVZ region of surgically induced brain injury rats Nestin is a specific marker protein for neural stem cells, mainly found in embryonic brain tissue and expressed at the neural progenitor cell stage, serving as a marker for neural progenitor cells. To investigate the effect of loganin on neurogenesis after surgical brain injury (SBI), we used nestin immunostaining to detect cell proliferation in the subventricular zone (SVZ) 7 days after SBI. Results are shown below. Figure 1 .Depend on Figure 1It was found that, compared with the sham surgery group, the proliferation of nestin-positive cells in the SVZ region was significantly increased in the model group 7 days after SBI surgery. P <0.001). Compared with the model group, the medium and high dose groups of loganin showed a further increase in nestin-positive cells, with significant differences. P <0.001).

[0037] 2.2 Effect of loganin on the number of nestin-positive cells in the SVZ region of rats with traumatic brain injury Similarly, to investigate the effects of loganin on neurogenesis in the brain after traumatic brain injury, we used nestin immunostaining to detect cell proliferation in the SVZ region 7 days after TBI modeling. Results are shown below. Figure 2 .Depend on Figure 2 It was found that, compared with the sham surgery group, the proliferation of nestin-positive cells in the SVZ region was significantly increased in the model group 7 days after surgery. P< 0.001). Compared with the model group, the number of nestin-positive cells in the low, medium, and high dose groups of loganin was further increased, showing significant differences. P< 0.001).

[0038] 2.3 Effects of loganin on endothelial cell proliferation in rats with surgically induced brain injury To investigate whether loganin can promote angiogenesis after surgical brain injury (SBI) in rats, we studied its effect on endothelial cell proliferation 7 days after SBI. Lectin can recognize glycosylated structures on the surface of vascular endothelial cells, thereby labeling blood vessels. BrdU was observed around the infarct using immunofluorescence. + / Lectin + Labeled neovascular endothelial cells, results are shown in Figure 3 .Depend on Figure 3 It can be seen that, compared with the sham surgery group, the model group had a lower incidence of peri-infarct BrdU 7 days post-operation. + / Lectin + The proliferation of positive cells increased significantly ( P <0.001). Compared with the model group, the high-dose strychnine group showed significantly lower levels of BrdU. + / Lectin + The number of positive cells increased further, showing a significant difference. P <0.01).

[0039] 2.4 Effects of loganin on endothelial cell proliferation in rats with traumatic brain injury To investigate whether loganin can promote angiogenesis in rats following traumatic brain injury (TBI), we studied its effect on endothelial cell proliferation 7 days after TBI. BrdU was observed around the infarct using immunofluorescence. + / Lectin+ Labeled neovascular endothelial cells, results are shown in Figure 4 .Depend on Figure 4 It can be seen that, compared with the sham surgery group, the model group had a lower incidence of peripheral BrdU infarction 7 days after modeling. + / Lectin + The proliferation of positive cells increased significantly ( P <0.001). Compared with the model group, the BrdU in the medium-dose and high-dose groups of loganin was significantly lower. + / Lectin + The number of positive cells increased further, showing a significant difference. P <0.01).

[0040] 2.5 Regulatory effect of loganin on ephrin B2 and Eph B4 proteins 7 days after surgical brain injury model in rats Western blot analysis of ephrin-B2 and EphB4 protein expression results are shown below. Figure 5 .Depend on Figure 5 It was found that 7 days after SBI modeling, there was no significant difference in the expression levels of ephrin-B2 and EphB4 proteins compared with the sham-operated group. However, after administration of loganin, the expression levels of ephrin-B2 and EphB4 proteins significantly increased. P <0.01, P <0.001).

[0041] 2.6 Regulatory effect of loganin on ephrin B2 and Eph B4 proteins 7 days after traumatic brain injury model in rats Western blot analysis of ephrin-B2 and EphB4 protein expression results are shown below. Figure 6 .Depend on Figure 6 It was found that 7 days after TBI modeling, the expression levels of ephrin-B2 and EphB4 proteins were significantly higher than those in the sham-operated group. P <0.05, after administration of loganin, the expression levels of ephrin-B2 and EphB4 proteins were further significantly increased ( P <0.05, P <0.01, P <0.001).

[0042] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of loganin in the preparation of drugs for treating traumatic brain injury.

2. The application according to claim 1, characterized in that, Traumatic brain injury includes surgical brain injury and traumatic brain injury.

3. The application according to claim 1, characterized in that, The target proteins of the strychnine are ephrinB2 and EphB4.

4. The application according to claim 1, characterized in that, The effective dose of the strychnine is 90 mg / kg to 270 mg / kg.

5. The application according to claim 1, characterized in that, The dosage forms of the drug include oral preparations, injections, or topical preparations.

6. The application according to claim 5, characterized in that, At least one of the following conditions must be met: (1) The oral preparations include tablets, capsules, granules, oral liquids or pills; (2) The injectable includes intravenous injection, intramuscular injection or subcutaneous injection; (3) The topical preparations include ointments, gels, patches or liniments.

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