Application of metformin in preparation of medicine for treating acute spinal cord injury and secondary lung injury

Abstract

The invention discloses a medicine for treating acute spinal cord injury and secondary lung injury. The medicine contains metformin. The invention also provides an effect of metformin in preparation of a medicine for treating acute spinal cord injury and secondary lung injury. The invention discloses that secondary lung injury exists in an acute spinal cord injury rat model, the autophagy level of lung tissue is reduced compared with that of normal lung tissue, and the apoptosis level is increased. Metformin can reduce cell apoptosis of injured lung tissue of an acute spinal cord injury model rat by promoting cell autophagy, relieve inflammatory response and reduce secondary lung tissue injury caused by acute spinal cord injury. The invention discloses a medicinal composition containing metformin, which lays a foundation for a treatment means for clinically treating acute spinal cord injury and secondary lung injury.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a medicine for treating lung injury secondary to acute spinal cord injury and application thereof. Background Art

[0002] Acute spinal cord injury (ASCI) is an injury to the spinal cord caused by direct or indirect external factors, which causes motor, sensory and sphincter dysfunction in the corresponding segments, as well as changes such as abnormal muscle tone and pathological reflexes. Spinal cord injury is one of the most serious and catastrophic injuries, with high disability and mortality rates, and severely damages motor, sensory and autonomic nervous functions. ASCI not only causes neurological diseases, but also affects organ systems such as respiratory, urinary and reproductive. After ASCI, respiratory diseases are the most common cause of death, and reducing respiratory complications is crucial to preventing patient death. Pneumonia is the most common respiratory complication, accounting for 65.1% of all deaths. Even with antibiotic treatment, lung inflammation cannot be reversed and eventually leads to death, indicating that simply controlling infection cannot prevent lung injury after ASCI. However, some scholars believe that the nerves that control the respiratory muscles are located above the upper thoracic spine area, and respiratory muscle paralysis may be the main cause of lung injury, dyspnea, lung infection and other complications. Although this theory can explain the high level of lung injury caused by ASCI, it cannot explain the occurrence of lung injury after low-level ASCI. Studies have also shown that even in the low-level (T10) ASCI animal model, where the spinal cord injury avoids the upper thoracic region that controls the respiratory muscles, lung damage such as pulmonary hemorrhage, edema, and inflammatory infiltration still occurs, and the damage to the lungs after ASCI lasts until 72 hours after ASCI, and autophagy is inhibited during this process.

[0003] Metformin (chemical formula: C4H11N5) is currently one of the first-choice drugs for the treatment of patients with type 2 diabetes (T2DM), especially for obese diabetic patients. Despite its widespread clinical application, its glucose-lowering mechanism has not been fully elucidated. Studies in recent years have shown that metformin may exert its effects through multiple mechanisms, including anti-inflammation, regulation of cell senescence, and enhancement of autophagy. Specifically, its anti-inflammatory effects may be achieved through reducing oxidative stress, reducing DNA damage, regulating ATM protein kinase activity, and epigenetic effects. On the other hand, metformin can exert its therapeutic effects by enhancing autophagy, which can directly or indirectly inhibit the mTOR signaling pathway, enhance LC3-II-mediated autophagy in dopaminergic neurons, and upregulate the expression levels of autophagy-related proteins. In addition, metformin also activates AMPK by improving mitochondrial function, reducing the concentration of proinflammatory factors, further regulating mTOR signaling and enhancing autophagy in the hippocampus. The above mechanism not only deepens the understanding of the hypoglycemic effect of metformin, but also provides a theoretical basis for its potential application in non-diabetic fields. Future research will further explore its wide application value in the treatment of other diseases. Metformin plays a positive role in acute spinal cord injury, but its role in secondary lung injury is still under further study. Summary of the invention

[0004] The purpose of the present invention is to provide a therapeutic drug and application for acute spinal cord injury secondary lung injury, which is used to solve the problem that the severity of acute spinal cord injury secondary lung injury is high and the treatment scheme needs to be further optimized. In order to achieve the purpose of the present invention, the following technical scheme is adopted: The present invention provides a pharmaceutical composition for treating secondary lung injury caused by acute spinal cord injury. The pharmaceutical composition contains metformin, with a chemical formula of C4H11N5 and a structural formula as shown below:

[0005] The present invention also provides the use of metformin in the preparation of a therapeutic drug for lung injury secondary to acute spinal cord injury. The autophagy level of the lung tissue in the acute spinal cord injury model is lower than that of normal lung tissue, and the apoptosis level is higher. After 3 days of metformin treatment, the autophagy level of the lung tissue is increased, the apoptosis is reduced, the inflammation is reduced, and the lung injury is significantly alleviated.

[0006] Furthermore, acute spinal cord injury includes but is not limited to acute spinal cord injury caused by traumatic factors such as traffic accidents, falls, and violent injuries, and non-traumatic acute spinal cord injury caused by tumors, infections, vascular diseases, degenerative diseases, etc.

[0007] Furthermore, the lung injury includes but is not limited to respiratory muscle weakness, lung infection, release of inflammatory factors, improper fluid management and thromboembolism.

[0008] Furthermore, the dosage of metformin in the rat model of acute spinal cord injury secondary to lung injury was 100 mg / Kg.d, and it was used for 3 consecutive days after lung injury. Beneficial Effects

[0009] 1. The present invention provides a pharmaceutical composition for treating secondary lung injury after acute spinal cord injury. The pharmaceutical composition contains metformin, which can significantly reduce the severity of secondary lung injury after acute spinal cord injury, provides an important basis for reducing secondary lung injury after acute spinal cord injury, and has broad clinical application value.

[0010] 2. The present invention also provides the use of metformin in the preparation of a therapeutic drug for lung injury secondary to acute spinal cord injury. It can significantly reduce lung tissue inflammation and repair tissue damage for lung injuries caused by traumatic and non-traumatic acute spinal cord injuries, such as but not limited to respiratory muscle weakness, lung infection, release of inflammatory factors, improper fluid management and thromboembolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Lung tissue damage in the sham group, control group, and metformin treatment group of acute spinal cord injury at different time points (a. HE staining; b. Pathological score statistics mean±SD, ***p < 0.001, ****p < 0.0001, n= 3, scale bar=100μm) Figure 2 Statistical chart of dry-to-wet weight ratio of lung tissue in the sham operation group, control group, and metformin treatment group in acute spinal cord injury (***p < 0.001, not significant p > 0.05. Data are mean ± SEM, n = 3. SCI: SpinalCord Injury) Figure 3 Immunofluorescence detection of LC3 and RAB7 expression (a. Immunofluorescence labeling method to evaluate the content of LC3 and RAB7 after spinal cord injury, green fluorescence labeled LC3, red fluorescence labeled RAB7. The figure shows the representative results of lung sections at 0, 24, 48 and 72 hours. Magnification ×200; b. Statistical analysis results of LC3 and RAB7 fluorescence intensity, data comparison was performed using one-way analysis of variance and least significant difference test. *p < 0.05, **p < 0.01, ***p < 0.001, no significant p > 0.05. Data are mean ± SEM, n = 3. SCI: Spinal Cord Injury) Figure 4Western blot results (a. Expression results of Beclin-1 and ULK1 in lung homogenate 72 h after acute spinal cord injury. The loading control was GAPDH. And statistical analysis b. Relative quantification of Beclin1 and ULK1 obtained from the blot shown in a (mean ± SD). Data were compared using one-way analysis of variance and least significant difference test. *P < 0.05, **p < 0.01 compared with the control group and the sham operation group, ###p < 0.001 compared with the treatment group and the control group. Data are mean ± SEM, n = 3. SCI: Spinal Cord Injury.) Figure 5 TUNEL staining (a. Fluorescent green dots indicate TUNEL staining of cell nuclei containing DNA strand breaks. The figure shows representative results of lung sections at 0, 24, 48 and 72 h. Magnification × 200. b. Number of TUNEL-positive cells. Data were compared using one-way ANOVA and least significant difference test. ***p < 0.001, ****p < 0.0001, data are mean ± SEM, n = 3. SCI: Spinal Cord Injury.) Figure 6 Results of immunofluorescence staining (a. Immunofluorescence labeling method to evaluate the content of Bax and Bcl-2 after spinal cord injury, green fluorescence labeled Bax, red fluorescence labeled Bcl-2. The figure shows the representative results of lung sections at 0, 24, 48 and 72 hours. Magnification × 200. Statistical analysis results Figure b. Bax and Bcl-2 fluorescence intensity. Data comparison was performed using one-way analysis of variance and least significant difference test. *p < 0.05, **p < 0.01, ***p < 0.001, data are mean ± SEM, n = 3. SCI: Spinal Cord Injury.) Figure 7 ELISA quantitative determination of IL-6 and IL-1β levels in serum and lung tissue of rats 72 hours after acute spinal cord injury. Statistical analysis of the results. Data were compared using one-way analysis of variance and least significant difference test. The control group was compared with the sham operation group ****p < 0.0001, and the treatment group was compared with the control group ##p < 0.01. , ####p < 0.0001. Data are mean ± SEM, n = 3. SCI: Spinal Cord Injury. DETAILED DESCRIPTION

[0012] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0013] Example 1: Detection of Metformin on Gross Assessment of Lung Tissue in an Acute Spinal Cord Injury Secondary Lung Injury Model Construction of rat spinal cord injury model: 144 female wild-type Sprague-Dawley rats were randomly divided into three groups: sham group; control group (vehicle group); metformin treatment group. The sham group only underwent laminectomy, and the other groups were treated with SCI models according to the modified Allen's method under sterile conditions. Briefly, after deep anesthesia with sodium pentobarbital 10 g / L (40 mg / kg, Sigma-Aldrich, St. Louis, MO, USA), laminectomy was performed at the T9-11 level to expose the rat spinal cord. Then, a 10-g metal rod was placed from a height of 2.5 cm through a graduated glass tube into the dorsal surface of the spinal cord. The tail swing or curl of the rat indicated successful injury. The sham group only received laminectomy. Then, the incision was closed in layers, and antibiotics were applied topically. After surgery, the rats in the treatment group were intraperitoneally injected with 100 mg / kg / day of metformin (MCE, USA), and the rats in the sham and control groups were also injected with normal saline. All rats were allowed to recover in an air-conditioned room. Artificial bladder voiding was performed 3 times daily.

[0014] 1. Histological analysis of lung tissue Rats were killed at 24, 48, and 72 hours after surgery or sham surgery, and 40 mL of normal saline and 20 mL of 4% paraformaldehyde were perfused into the lungs. After thoracotomy, lung tissue was fixed with 4% paraformaldehyde solution, dehydrated in ethanol-water solutions of varying concentrations, embedded in paraffin at room temperature, and sectioned. 4 μm thick paraffin sections were collected and stained with hematoxylin and eosin. Figure 1 As shown in a, compared with the sham operation group, the control group showed interstitial widening, inflammatory cell infiltration, and hemorrhage in the lung tissue after ASCI, which gradually worsened over time, and the alveolar structure was damaged 72 hours after ASCI. Inflammatory cell infiltration and hemorrhage appeared in the lung tissue of the treatment group after ASCI, but the degree was less than that of the injury group. The pathological scores at each time point were statistically evaluated ( Figure 1 b).

[0015] 2. Dry-Wet Weight Ratio of Lung Tissue Rats were killed at different time points after surgery or sham surgery, and extrapulmonary tissues were removed and lung samples were collected for evaluation of edema. Samples were weighed (wet weight), then baked at 60°C for 72 hours and weighed again (dry weight). Pulmonary edema was assessed at each time point using the wet weight / dry weight ratio method ( Figure 2 The score of the control group was significantly higher than that of the sham operation group (p < 0.05), and continued to increase over time until 72 hours. Compared with the control group, the score of the treatment group was significantly lower than that of the injury group at 72 hours after injury (P < 0.001).

[0016] Example 2: Detection of the effect of metformin on the autophagy level of lung tissue in the acute spinal cord injury secondary lung injury model Immunofluorescence detection of LC3 and RAB7 expression levels: Rats were killed by an overdose of sodium pentobarbital (80 mg / kg, intraperitoneally) at specific time points after injury, and lung tissues were collected. Paraffin sections of lung tissue (4 μm thick) were dewaxed and hydrated. The sections were microwaved in sodium citrate buffer (pH 6.0) for 15 min and cooled to room temperature. The endogenous peroxidase activity of the sections was then blocked with 0.3% hydrogen peroxide and washed with PBS for 15 min. The samples were then blocked with 5% goat serum for 1 h at room temperature. After discarding the goat serum, the samples were incubated with anti-LC3 antibodies (Abcam) and anti-Rab7 antibodies (Abcam) at 4°C overnight. After the sections returned to room temperature, they were washed with PBS. Some sections were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG H&L (Abcam) and Alexa Fluor 555-conjugated donkey anti-mouse IgG H&L (Abcam), washed with PBS, stained with DAPI, and observed under a laser confocal microscope (Nikon A1R, Tokyo, Japan). Figure 3 a). Figure 3 As shown in (b), the LC3 level in the treatment group was higher than that in the injury group at 72 h after ASCI (P < 0.05), and the RAB7 level in the treatment group was also significantly higher than that in the injury group at 72 h after ASCI (P < 0.001).

[0017] Western Blot detection of ULK-1 and Beclin-1 expression levels: Tissue samples were homogenized in RIPA lysis buffer (P00103J Beyotime, Shanghai, China) containing 1% protease inhibitors. The lysate was centrifuged at 12000g for 15 min at 4°C, and the supernatant containing protein was collected. Then, the total protein concentration was determined using a BCA kit (P0012, Beyotime). The obtained total protein was mixed with 5× SDS-PAGE loading buffer, and tissue samples containing the same amount of protein were loaded on SDS-PAGE at an appropriate concentration for electrophoresis separation according to the molecular weight of the target protein, and then transferred to a PVDF membrane (Millipore, Billerica, MA, USA), which was blocked in 5% skim milk for 1 h at room temperature. Then, the cells were incubated with the following primary antibodies overnight at 4°C: (ULK-1 Rabbit mAb, Abclonal, 1:1000; Beclin-1 Rabbit mAb, Abclonal, 1:1000) and the secondary antibodies (GAPDH: Abclonal, 1:1,0000) for 1 hour at room temperature, and the proteins were prepared for exposure using ECL solution and ChemiDoc Touch Imaging System (Bio-Rad). Figure 4 As shown, the expression levels of ULK-1 (P < 0.05) and Beclin-1 (P < 0.01) were decreased in the control group compared with the sham group, and all these changes were reversed in the treatment group compared with the control group.

[0018] Example 3: Detection of the effect of metformin on apoptosis of lung tissue in the acute spinal cord injury secondary lung injury model TUNEL staining to detect apoptosis levels in lung tissue: TUNEL staining was performed using an in situ cell death detection kit according to the protocol provided by the manufacturer. Briefly, after paraffin sectioning, the TUNEL reaction mixture was applied to the sections, pre-incubated with proteinase-K, and then blocked with a blocking buffer containing a peroxidase-streptavidin coupling solution. Subsequently, the sections were treated with 0.03% diaminobenzidine. Three sections were collected from each rat for subsequent analysis. Fluorescence images were then taken under a fluorescence microscope (Olympus) ( Figure 5 a). Compared with the control group, the metformin treatment group reduced lung tissue apoptosis ( Figure 5 b) (P<0.001).

[0019] Immunofluorescence detection of Bax and Bcl-2 expression levels: Paraffin sections of lung tissue (4 μm thick) were dewaxed and hydrated. The sections were microwaved in sodium citrate buffer (pH 6.0) for 15 min and cooled to room temperature. The endogenous peroxidase activity of the sections was then blocked with 0.3% hydrogen peroxide and washed with PBS for 15 min. The samples were then blocked with 5% goat serum for 1 h at room temperature. After discarding the goat serum, the samples were incubated with anti-Bax antibody (Abcam) and anti-Bcl-2 antibody (Abcam) at 4°C overnight. After the sections returned to room temperature, they were washed with PBS. Alexa Fluor 488-conjugated goat anti-rabbit IgG H&L (Abcam) and Alexa Fluor 555-conjugated donkey anti-mouse IgG H&L (Abcam) were incubated with some sections, washed with PBS, stained with DAPI, and observed under a laser confocal microscope (Nikon A1R, Tokyo, Japan) ( Figure 6 a). Figure 6 b shows that the Bax level in the treatment group was lower than that in the injury group at 72h after ASCI (P< 0.05), and there was no significant difference at 24h and 48h after ASCI. The Bcl-2 level in the treatment group was significantly higher than that in the injury group at 24h, 48h and 72h after ASCI.

[0020] Example 4: Detection of the effect of metformin on the inflammatory level of lung tissue in the acute spinal cord injury secondary lung injury model Lung tissue and serum samples were collected at 72 h, and ELISA kits (Jiangsu ELISA Industry Co., Ltd.) were used to quantitatively determine the levels of IL-6 and IL-1β in rat serum and lung tissue according to the manufacturer's instructions. Figure 7 As shown in the figure, the expression of IL-10 in the treatment group was higher than that in the control group (P< 0.0001), while the expression of IL-6 was lower (P< 0.01).

[0021] The above results show that the use of metformin for 72 hours in a rat model of acute spinal cord injury secondary to lung injury can enhance the autophagy level of lung tissue, reduce apoptosis, significantly reduce lung tissue inflammation, and repair tissue damage.

Claims

1. A pharmaceutical composition for treating secondary lung injury following acute spinal cord injury, characterized in that: Contains metformin.

2. Use of metformin in the preparation of therapeutic drugs for secondary lung injury caused by acute spinal cord injury.

3. The use according to claim 2, characterized in that: The acute spinal cord injury includes, but is not limited to, acute spinal cord injury caused by traumatic factors such as traffic accidents, falls, and violent injuries, and non-traumatic acute spinal cord injury caused by tumors, infections, vascular diseases, degenerative diseases, and the like.

4. The use according to claim 2, characterized in that: The lung damage includes but is not limited to respiratory muscle weakness, lung infection, release of inflammatory factors, improper fluid management and thromboembolism.

5. The use according to claims 2-4, characterized in that: The dosage of metformin in the rat model of acute spinal cord injury secondary to lung injury was 100 mg / Kg.d, and it was used for 3 consecutive days after lung injury.

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