Application of lisinopril in preparation of medicine for treating amyotrophic lateral sclerosis

Abstract

The invention is applicable to the technical field of biological medicines, and provides application of lisinopril in preparation of a medicine for treating amyotrophic lateral sclerosis. The invention has important value for promoting research and development of amyotrophic lateral sclerosis (ALS) treatment medicines and improving prognosis of patients. In-vitro experiments show that lisinopril can prevent mitochondria damage of NSC34 cells induced by SOD1G93A, expression of Beclin 1, LC3 and p62 is regulated by inhibiting a TGF [beta] 1 / PI3K / AKT / mTOR signal channel, cell autophagy is activated, and neuronal apoptosis is inhibited. In-vivo experiments show that lisinopril can activate in-vivo BI1 expression, regulate ALS model mouse lipid content and intervene in a glycerophospholipid metabolic process, and also can inhibit motor neuron death, myelin sheath shedding and neuromuscular junction degeneration and promote mitochondrial biogenesis by influencing autophagy regulated by a TGF [beta] 1 / PI3K / AKT / mTOR pathway.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of lisinopril in preparation of a medicine for treating amyotrophic lateral sclerosis. BACKGROUND

[0002] Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease, mainly attacking upper and lower motor neurons. After the onset of the disease, patients will have symptoms such as muscle atrophy, paralysis, and respiratory failure, and most patients will die within 1-5 years after the onset. Among ALS cases, about 90% are sporadic cases (sALS), and 10% are familial (fALS). The pathogenesis is extremely complex, involving immune abnormalities, oxidative stress, autophagy disorders, and other aspects. Currently, a variety of related biomarkers have been found, such as TDP43, SOD1, etc.

[0003] In existing research, 187 Superoxidedismutase 1 (SOD1) variations have been described in ALS patients, which exist in about 18.9% of fALS and 1.2% of sALS cases, and most of them present autosomal dominant inheritance. Among fALS, 155 mutations have been described, and G93A mutation (glycine at position 93 is changed to alanine) is one of the mutations that has been studied in depth. It is the first mutation studied in transgenic mouse models and belongs to the pseudo-WT mutation, and the SOD1 enzyme activity is not impaired. However, this gain-of-function mutation will cause increased oxidative activity, excessive production of hydrogen peroxide radicals, and increased protein-protein interactions, thereby affecting the normal physiological processes of motor neurons. Motor neurons are sensitive to oxidative stress, and the main reactant ROS increases during oxidative stress, which inhibits axonal transport, causes accumulation of mutant SOD1, damages mitochondria, proteasomes, and protein folding chaperones, and ultimately causes damage to motor neurons. This damage promotes the development of ALS disease through pathways such as affecting mitochondrial function, disrupting intracellular homeostasis, activating programmed cell death mechanisms (i.e., apoptosis), and promoting the formation of toxic inclusion bodies.

[0004] Meanwhile, the relationship between lipid metabolism and ALS has gradually attracted attention. Lipids, as important non-water-soluble organic matter in the body, are divided into fatty acids, sphingolipids, glycerolipids, glycerophospholipids, sterols, prenol lipids and glycolipids. These lipid categories are interrelated and collectively form a complex lipid metabolism network. Lipids mainly play three core functions in the body: as energy reserve substances (such as cholesterol; constitute the basic structure of the cell membrane (phospholipids, sterols and sphingomyelin form lipid bilayers and lipid rafts); as important signal molecules involved in the regulation of inflammatory response, oxidative stress and other physiological processes. In recent years, studies have found that lipid metabolism disorders are closely related to the pathogenesis of ALS. For example, abnormal sphingolipid metabolism caused by SPTLC1 gene mutation and elevated arachidonic acid levels can promote motor neuron death; clinical observations have also found that a decrease in serum triglyceride levels in ALS patients is associated with poor prognosis, and increasing lipid intake can significantly improve the survival rate of model animals, indicating that lipid metabolism regulation is expected to become a new direction for the treatment of ALS.

[0005] In addition, at the molecular mechanism level, the mTOR signaling pathway (especially the mTORC1 complex) plays a key regulatory role in the autophagy process through the PI3K / AKT pathway. After activation of the pathway, downstream targets such as ULK1 are phosphorylated to inhibit autophagy and promote apoptosis. TGFβ1 not only acts through the classic Smad pathway, but also activates mTOR signaling through the non-classical pathway (PI3K / AKT), further inhibiting autophagy and accelerating disease progression. Activating the autophagy pathway is considered a potential treatment strategy for ALS, and anti-apoptotic proteins like BI1 (TMBIM6) are involved in regulating apoptosis, calcium homeostasis and other processes, and are related to neurodegenerative diseases. In ALS, dysfunction of the autophagy-lysosome system affects neuronal stability.

[0006] However, there are currently very limited treatment options for ALS. Despite some research on the pathogenesis and related molecular mechanisms of ALS, there is still a lack of effective treatment drugs and methods. The existing treatment methods are difficult to fundamentally stop the progression of the disease and cannot meet the treatment needs of patients. Therefore, the present application proposes the use of lisinopril in the preparation of a drug for treating amyotrophic lateral sclerosis. As an angiotensin-converting enzyme inhibitor, lisinopril has not only antihypertensive effects but also multiple neuroprotective potentials. It can inhibit the production of Ang II, block the activation of the PI3K / AKT / mTOR pathway, reduce fibrosis, down-regulate TGFβ1 expression, and alleviate inflammation. Animal experiments have confirmed its ability to prolong lifespan and delay aging. In particular, it can act on both lipid metabolism and the mTOR signaling pathway, making it a promising multi-target drug for treating ALS and providing a new approach and direction for the treatment of ALS. SUMMARY

[0007] The application aims to provide an application of lisinopril in preparation of a drug for treating amyotrophic lateral sclerosis (ALS), and aims to solve the problems in the background art.

[0008] The application aims to provide an application of lisinopril in preparation of a drug for treating amyotrophic lateral sclerosis (ALS), and aims to solve the problems in the background art. The application aims to provide an application of lisinopril in preparation of a drug for treating amyotrophic lateral sclerosis (ALS), and aims to solve the problems in the background art. inhibiting SOD1 G93A inducing apoptosis; improving mitochondrial membrane potential and mitochondrial number; regulating the expression of MAP2 and beta-Tubulin, and maintaining the integrity of neuron axons; activating autophagy through the TGFbeta1 / PI3K / AKT / mTOR pathway; promoting the transformation of fast muscle fibers into slow muscle fibers, and increasing ATP content; inhibiting the damage of spinal motor neurons and myelin sheaths; improving the integrity of neuromuscular junctions; inhibiting muscle fibrosis and muscle atrophy; regulating lipid metabolism, reducing the content of sphingomyelin, ceramide, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol, and increasing the content of triglyceride.

[0009] Further, the drug is used for prolonging the life span of ALS model mice, delaying the onset time or improving the motor function.

[0010] Further, the drug reduces muscle fibrosis by inhibiting the TGFbeta1 / Smad2 / 3 signaling pathway.

[0011] Further, the drug improves the ALS symptoms by regulating glycerophospholipid metabolism (the content of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol), sphingolipid metabolism (the content of sphingomyelin and ceramide), glyceride metabolism (the content of triglyceride) or autophagy pathway.

[0012] Compared with the prior art, the application has the following beneficial effects: The application proves that lisinopril plays a key role in ALS treatment, and lays a foundation for drug design targeting Bax inhibitor 1 (BI1). In in vitro experiments, lisinopril can effectively prevent SOD1 G93AThe induced NSC34 cell mitochondrial membrane potential decline caused the damage of mitochondrial structure and function. At the same time, lisinopril regulates the expression of Beclin 1, LC3 and p62 by inhibiting the TGFβ1 / PI3K / AKT / mTOR signaling pathway, activates the autophagy process, and then inhibits the apoptosis of neurons. In vivo experiments show that lisinopril can activate the expression of BI1 in vivo, regulate the content of triglyceride (TG), sphingomyelin (SM), ceramide (Cer), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) and other lipids in ALS model mice, and intervene in the glycerophospholipid metabolism process. In addition, lisinopril also regulates autophagy through the TGFβ1 / PI3K / AKT / mTOR pathway, inhibits the death of motor neurons, demyelination, and neuromuscular junction (NMJ) degeneration, and promotes the biogenesis of mitochondria. These results fully demonstrate that lisinopril plays a major role in the treatment of ALS disease, and the revelation of its mechanism provides an important structural basis for designing new drugs targeting BI1 in the future, and has important application value for promoting the research and development of ALS treatment drugs and improving patient prognosis. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 For Tunel detection of the effect of lisinopril on cell apoptosis.

[0014] Figure 2 For the effect of lisinopril on the mitochondrial membrane potential of NSC34 cells.

[0015] Figure 3 For the effect of lisinopril on SOD1 G93A Induced mitochondrial number of NSC34 cells.

[0016] Figure 4 For the effect of lisinopril on the axon of NSC34 cells; wherein DAPI locates the nucleus, showing blue fluorescence, β3-Tubulin selects FITC to excite green fluorescence, and MAP2 selects R-PE to excite red fluorescence.

[0017] Figure 5 For the effect of lisinopril on SOD1 G93A inhibits NSC34 cell autophagy; wherein A is the effect of lisinopril on the expression of BI1, p-ULK, p-mTOR, p70S6k, Beclin 1, LC3 and p62 proteins; B is the effect of lisinopril on the expression of PI3K, p-AKT and TGFβ1 proteins.

[0018] Figure 6Effects of Lisinopril on the onset time, lifespan and motor ability of ALS mice; A: ALS CON and ALS Lisinopril male mice onset time statistics; B: ALS CON and ALS Lisinopril female mice onset time statistics; C: ALS CON and ALS Lisinopril male mice lifespan statistics; D: ALS CON and ALS Lisinopril female mice lifespan statistics; E: ALS CON and ALS Lisinopril male mice four limbs grip strength statistics; F: ALS CON and ALS Lisinopril female mice four limbs grip strength statistics; G: ALS CON and ALS Lisinopril male mice running time statistics; H: ALS CON and ALS Lisinopril female mice running time statistics.

[0019] Figure 7 Footprints of ALS CON and ALS Lisinopril female and male mice before and after onset.

[0020] Figure 8 Effects of Lisinopril on the fast and slow muscle transformation of ALS mice; A: MYH1 and MYH7 double labeling staining results; B: ATP content detection results; C: HK content detection results; D: PPARG transcription detection results; E: Lipid droplet oil red staining results.

[0021] Figure 9 Effects of Lisinopril on the number of Nissl bodies and myelin structure in the spinal cord of ALS mice; A: Effects of Lisinopril on the number of Nissl bodies in the spinal cord of ALS mice; B: Effects of Lisinopril on the myelin sheath in the spinal cord of ALS mice.

[0022] Figure 10 Gastrocnemius muscle NMJ staining of ALS CON and ALS Lisinopril mice.

[0023] Figure 11 Effects of Lisinopril on muscle fibrosis in ALS mice; A: ALS CON and ALS Lisinopril mice skeletal muscle Sirius red staining; B: ACP5 expression level statistics; C: FN expression level statistics.

[0024] Figure 12 Effects of Lisinopril on muscle atrophy in ALS mice.

[0025] Figure 13 180-day ALS CON and ALS Lisinopril mouse soleus muscle transmission electron microscopy images.

[0026] Figure 14 Lisinopril regulation of the TGFβ signaling pathway; A: Lisinopril inhibits the activation of the TGFβ1 / Smad2 / 3 pathway and the TGFβ1 / PI3K / AKT pathway before and after the onset of the ALS model mice; B: Lisinopril inhibits the activation of the mTOR signaling pathway to activate autophagy.

[0027] Figure 15 PCA analysis of QC and all metabolite samples.

[0028] Figure 16 Score scatter plot and ordering validation plot of PLS-DA; wherein A is the PLS-DA score scatter plot of ALS CON group and ALS Lisinopril group; B is the PLS-DA ordering validation plot of ALS CON group and ALS Lisinopril group.

[0029] Figure 17 Volcano plot of differential lipid compounds between ALS Lisinopril group and ALS CON group; wherein A is the volcano plot under negative ion mode; B is the volcano plot under positive ion mode.

[0030] Figure 18 Correlation analysis of differential lipid compounds between ALS Lisinopril group and ALS CON group.

[0031] Figure 19 Heat map of cluster analysis of differential lipid compounds between ALS Lisinopril group and ALS CON group.

[0032] Figure 20 KEGG enrichment bubble chart.

[0033] Figure 21 KEGG classification chart of differential lipid compounds. DETAILED DESCRIPTION

[0034] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below, but it should not be understood as limiting the scope of the present application.

[0035] The specific implementation of the present application will be described in detail below in combination with specific examples.

[0036] Example 1: Effect of Lisinopril on SOD1 G93A induced NSC34 cells; (1) TUNEL staining to detect cell apoptosis: this method identifies apoptotic cells by detecting 3'-OH ends produced by DNA fragmentation. The experimental procedure is as follows: NSC34 cells were inoculated in 24-well plates containing glass slides, 24 h later, 15 μM lisinopril (7.5 mM stock solution diluted) was added, 1 h later, LacZ and SOD1 G93APlasmid. After 24 h of continuous culture, 4% paraformaldehyde was used for fixation for 30 min, and 0.3% Triton X-100 was used for permeabilization for 10 min. Then, TUNEL detection solution was added for incubation at 37 °C for 1 h in the dark, and DAPI was used for nuclear staining for 5 min. After PBS washing, the sample was mounted, and finally observed and photographed by confocal microscopy. This method can specifically detect apoptotic cells by using TdT enzyme to connect markers to DNA break ends.

[0037] (2) MPTP fluorescence detection of mitochondrial membrane permeability: MPTP detection kit was used to evaluate the change of mitochondrial membrane permeability, and its principle was based on Calcein AM fluorescent probe: Calcein AM in living cells was hydrolyzed into membrane non-permeable Calcein (green fluorescence), and CoCl2 could quench cytoplasmic fluorescence while retaining mitochondrial fluorescence; if MPTP is open, mitochondrial fluorescence is also quenched. The experimental procedure is as follows: after the NSC34 cells were washed with PBS, the fluorescence quenching solution was added for incubation at 37 °C for 45 min in the dark, and then the pre-warmed culture medium was replaced for further incubation for 30 min. After DAPI staining, the sample was mounted and observed by confocal microscopy. This method can specifically reflect the open state of MPTP by changes in fluorescence distribution, indicating mitochondrial dysfunction.

[0038] (3) Western blot experiment to detect the expression of related proteins in NSC34 cells: the cells were divided into LacZ group, SOD1 G93A group and corresponding lisinopril treatment group, and 4 μg of plasmid was transfected for 24 h of culture. After cell lysis, the protein sample was obtained by centrifugation at 4 °C and 12000 rpm for 10 min, and quantified by BCA method. The protein was denatured by boiling at 100 °C for 10 min. 6%-12.5% gradient separation gel was used for SDS-PAGE electrophoresis (50 V for 10 min, 80 V for 20 min, and 120 V for 1 h), and then transferred to PVDF membrane at 200 mA constant current. After blocking with 5% skim milk at room temperature for 1 h, the primary antibody (p-mTOR 1:800, etc.) was incubated at 4 °C overnight, and the secondary antibody (1:2000) was incubated at room temperature for 1 h. The membrane was washed with TBST for 5 times (7 min each time), and finally developed by ECL. This method can sensitively detect the difference in expression level of target protein.

[0039] (4) Immunofluorescence staining of MAP2 and β-Tubulin: NSC34 cells were divided into LacZ group, SOD1 G93AGroups and corresponding Lisinopril (15 μΜ) treatment groups, 1 μg plasmid was transfected and cultured for 24 h. 4% paraformaldehyde was used to fix cells at room temperature for 30 min, 0.3% Triton X-100 was used to permeabilize cells for 7 min, and 5% goat serum was used to block cells at room temperature for 45 min. Primary antibody (MAP2 and β-Tubulin 1:300) was incubated at 4°C overnight, and fluorescent secondary antibody (1:200) was incubated at room temperature for 2 h in the dark. Cells were washed 4 times (7 min each time) with PBST, and then mounted for confocal microscopy observation. This method can clearly show the structural characteristics of neuron dendrites (MAP2) and axons (β-Tubulin).

[0040] 1.1 Lisinopril inhibits SOD1 G93A induced NSC34 cell death; The effect of Lisinopril on SOD1 G93A induced NSC34 cell apoptosis was evaluated by the TUNEL experiment described above. The results are shown in Figure 1 Compared with the LacZ group, the green fluorescence (apoptosis signal) of the SOD1 G93A group was significantly enhanced, while the fluorescence intensity of the Lisinopril treatment group was significantly reduced. This indicates that Lisinopril can effectively inhibit SOD1 G93A induced cell apoptosis.

[0041] 1.2 Lisinopril inhibits SOD1 G93A induced decrease of mitochondrial membrane potential in NSC34 cells; The effect of Lisinopril on SOD1 G93A induced mitochondrial dysfunction was detected by the MPTP experiment described above. The results are shown in Figure 2 Compared with the LacZ group, the green fluorescence of NSC34 cells transfected with SOD1 G93A was significantly reduced, indicating that SOD1 G93A induces a decrease in mitochondrial membrane potential, while the fluorescence intensity of the Lisinopril treatment group was significantly higher than that of the SOD1 G93A group, indicating that Lisinopril can inhibit SOD1 G93A induced decrease of mitochondrial membrane potential.

[0042] 1.3 Lisinopril affects SOD1 G93A induced number of mitochondria in NSC34 cells; To investigate the effect of Lisinopril on SOD1 G93A induced number of mitochondria in NSC34 cells, transmission electron microscopy was used to observe the mitochondria in the cells. The experimental procedure was as follows: NSC34 cells were treated with Lisinopril at a final concentration of 15 μmol, and the control group was treated with an equal amount of PBS buffer. After 1 h, LacZ and SOD1 G93APlasmid, 24 h later, centrifugal collection of cells and the addition of electron microscopy fixation fluid, and then by embedding means of sheet, under the transmission electron microscope to observe the number of mitochondria.

[0043] Results as Figure 3 shown, NSC34 cells overexpressing SOD1 G93A After compared with the control group, the number of mitochondria decreased significantly, and after treatment with lisinopril, the number of mitochondria increased. Lisinopril can slow down SOD1 G93A induced mitochondrial damage and promote mitochondrial biogenesis.

[0044] 1.4 Lisinopril affects the expression of MAP2 and tubulin in SOD1 G93A induced NSC34 cells; MAP2 is a protein involved in microtubule assembly, which is a key step in neurogenesis and a complex process of neuron formation and development. β3-Tubulin is the main component of microtubules in neurons, which plays a key role in axon guidance, maturation and maintenance. There is a large amount of MAP2 in the cell body and dendrites of mature neurons, while the main component of axons is β3-Tubulin-based tubulin. In ALS disease, SOD1 G93A mutation can lead to a decrease in MAP2 content, affecting neurite growth, while also damaging the integrity of neuronal axons, further leading to apoptosis. To explore whether lisinopril can inhibit SOD1 G93A mediated vulnerability of motor neurons, the distribution and expression of MAP2 and β3-Tubulin in NSC34 cells were observed by immunofluorescence staining of MAP2 and β-Tubulin.

[0045] Results as Figure 4 shown, compared with the LacZ group, the distribution of MAP2 and β3-Tubulin in the cells of the SOD1 G93A group decreased significantly, and β3-Tubulin was mainly present near the nucleus, indicating that the integrity of the axon was severely damaged. While the lisinopril group relative to the SOD1 G93A group, the distribution of MAP2 and β3-Tubulin in the cells increased significantly, and the distribution of β3-Tubulin was also extended, and the axon was relatively complete, indicating that lisinopril can inhibit SOD1 G93A mediated vulnerability of motor neurons.

[0046] 1.5 Lisinopril activates autophagy through the TGFβ pathway; Autophagy plays a central role in degrading misfolded proteins such as mutant SOD1, studies have shown that in SOD1 G93AIn ALS disease model, autophagy is inhibited, so it can't remove protein aggregates, leading to cell death and neurodegeneration. BI1 can activate autophagy through TGFβ signaling pathway. In order to further explore whether BI1 activator Lisinopril can activate autophagy through TGFβ1 / PI3K / AKT / mTOR pathway to inhibit apoptosis, Western blot experiment was used to detect the content of BI1 and TGFβ1 / PI3K / AKT / mTOR pathway related proteins and autophagy related proteins Beclin 1, LC3 and p62. The experimental process is as follows: NSC34 cells were treated with Lisinopril at a final concentration of 15 μmol, and the control group was treated with the same amount of PBS buffer. After 1 h, 4 μg of LacZ and SOD1 G93A plasmid was transfected according to different groups. After 24 h, whole protein extract was collected for Western blot experiment. GAPDH was selected as internal reference protein to detect the changes of BI1, TGFβ1, PI3K, p-AKT, p-mTOR, p70S6k, p-ULK and autophagy related proteins Beclin 1, LC3 and p62.

[0047] The results are shown in Figure 5 A and B, compared with SOD1 G93A group, the content of BI1, Beclin 1, LC3 and p-ULK increased, and the content of TGFβ1, PI3K, p-AKT, p-mTOR, p-70S6k and p62 decreased, indicating that after treatment with Lisinopril, the expression of BI1 was activated, autophagy was activated by inhibiting TGFβ1 / PI3K / AKT / mTOR pathway, and autophagy was normally flowed.

[0048] Example 2: Effect of Lisinopril on ALS model mice; (1) Spinal cord Luxol Fast Blue staining: After the paraffin section of spinal cord was deoxidized by dimethylbenzene, it was washed with distilled water, and then immersed in 0.1% Luxol Fast Blue (LFB) solution for 12 h. After washing with distilled water, it was immersed in 95% alcohol. 0.05% lithium carbonate aqueous solution was used for color separation. 70% alcohol was used for further color separation, and the spinal cord gray matter and white matter were observed under a microscope every 1 min during the process. The section was washed with ddH2O, then transparentized with dimethylbenzene, and mounted with neutral resin.

[0049] (2) Sirius red staining; the specific process is as follows: Dewaxing of paraffin sections to water: Place longitudinal paraffin sections of skeletal muscle in xylene (5 min, 3 times), absolute ethanol (1 min), 95% ethanol (1 min), 70% ethanol (1 min), and distilled water rinse (5 min), sequentially.

[0050] Staining with Sirius red staining solution: Drop stain sections with saturated picric acid Sirius red staining solution for 15 min.

[0051] Rinsing: Rinse with absolute ethanol.

[0052] Mounting: Bake sections in a 60 °C oven, clear sections with xylene, and mount with neutral balsam. Polarized light microscopy: Photograph sections using polarized light microscopy.

[0053] (3) Fast and slow muscle immunofluorescence double staining; the specific process is as follows: Dewaxing of paraffin sections: The steps are the same as the first step of Sirius red staining. Fixing: Place at room temperature for 15 min with paraformaldehyde, and wash with PBS for 3 times.

[0054] Permeation: Incubate with 0.1% Triton x 100 at room temperature for 10 min, and wash with PBS for 3 times.

[0055] Blocking: Prepare blocking solution (5% goat serum and 0.1% Tween 20 in PBS), and block at room temperature for 1 h.

[0056] Incubate with MYH7 (1:200) antibody at 4 °C overnight.

[0057] Wash with PBST for 4 times, and incubate with HRP enzyme-labeled secondary antibody at room temperature for 2 h in the dark.

[0058] Wash with PBST for 4 times, each for 5 min.

[0059] Add TSA reagent: Drop tyramide-CY3 on the tissue, and incubate at room temperature for 20 min.

[0060] Wash with PBS for 3 times, each for 5 min.

[0061] Section repair: Place the section in 1x citric acid repair solution, and repair in a microwave oven at high heat for 6 min, and naturally cool to room temperature.

[0062] Blocking: Prepare blocking solution (5% goat serum and 0.1% Tween 20 in PBS), and block at room temperature for 1 h.

[0063] Incubate with MYH1 (1:200) antibody at 4 °C overnight.

[0064] PBST wash 4 times, incubate with HRP enzyme-labeled secondary antibody for 2 h at room temperature in the dark.

[0065] PBST wash 4 times, each for 5 min.

[0066] Drop tyramide-488 on the tissue, incubate for 20 min at room temperature, then wash with PBS for 3 times, each for 5 min.

[0067] DAPI re-stain the nucleus: drop DAPI staining solution on the tissue, incubate for 6 min at room temperature in the dark.

[0068] Wash with PBS for 3 times, each for 7 min, then drop anti-fade mounting medium.

[0069] Microscopy: use confocal laser microscope to observe and take pictures.

[0070] 2.1 Lisinopril affects the onset time, lifespan and motor ability of ALS mice; To explore the effect of lisinopril on ALS mice, the mice were divided into two groups: ALS lisinopril group (ALS Lisinopril) mice were given 30 mg / Kg of lisinopril by gavage daily for 14 consecutive days, and ALS CON group mice were given the same amount of PBS.

[0071] ALS patients often accompany the loss of motor function, and eventually die of respiratory failure. In this study, the whole time node from birth, onset to final death of ALS model mice before and after lisinopril treatment was monitored, and the survival curve was drawn. The results showed that the treatment of lisinopril can prolong the lifespan of female and male ALS mice and delay the onset time of disease (Fig. 1A-D). Figure 6

[0072] To explore the therapeutic effect of lisinopril on the motor function of ALS model mice, the experiment selected running test, grip strength test and footprint test for evaluation. The results showed that compared with ALS CON group, the exercise time and stride of female and male mice on the treadmill from 150 days to 180 days were improved after lisinopril treatment, and the improvement effect of lisinopril on the motor function of female mice was stronger than that of male mice (Fig. 2G and H, Figure 6 Figure 7 ). In addition, lisinopril treatment also enhanced the grip strength of female and male ALS model mice during 150 days to 180 days (Fig. 2E and F). Figure 6

[0073] 2.2 Lisinopril increases the content of ATP in the skeletal muscle of ALS mice; With the development of ALS disease, the number of fast and slow muscle fibers will decrease, but after administration of lisinopril, the experiment observed that the number of fast and slow muscle fibers increased and the fast muscle fibers transformed into slow muscle fibers (Fig. 3A and B).​​​Figure 8 (A). Because slow-twitch muscle fibers are rich in mitochondria and can effectively utilize oxygen to oxidize fatty acids and glucose, producing a large amount of ATP, the ATP content in skeletal muscle was further measured. The results showed that the ATP content increased significantly after administration of lisinopril. Figure 8 (B)

[0074] To clarify whether the increase in ATP was due to fatty acid oxidation or glucose oxidation, the transcription of PPARG, the accumulation of lipid droplets, and the content of HK in the skeletal muscle of ALS model mice before and after lisinopril treatment were examined. The results showed that lisinopril could increase the content of HK (…). Figure 8 (C), inhibiting the transcription of PPARG ( Figure 8 (D), and increases the production of lipid droplets ( Figure 8 (E). In summary, lisinopril can promote the conversion of fast-twitch muscle fibers to slow-twitch muscle fibers in the skeletal muscle of ALS mice and improve ATP production efficiency by promoting glucose oxidation, thereby meeting the energy requirements of ALS mice. Its effects on PPARG and lipid droplets also suggest that lisinopril has a certain role in lipid metabolism.

[0075] 2.3 Lisinopril inhibits damage to spinal motor neurons and myelin sheath in ALS mice; In the early stages of ALS, the number of Nissl bodies may remain relatively stable or show a slight decreasing trend. However, as the disease progresses, the degeneration or even death of motor neurons severely impairs neuronal function, leading to a significant decrease in the number of Nissl bodies. This study examined the number of Nissl bodies in the spinal cord of ALS mice at 150 and 180 days of age. The results showed that the number of Nissl bodies in the spinal cord of ALS mice at 180 days of age was decreased compared to that of 150-day-old ALS mice; however, after treatment with lisinopril, the number of Nissl bodies in the spinal cord of ALS mice at both 150 and 180 days of age significantly increased. Figure 9 (A). This study confirmed that lisinopril promotes the survival of motor neurons.

[0076] LFB staining of mouse spinal cord revealed that, compared with the 150-day ALS CON group, ALS mice treated with lisinopril had smaller areas of myelin cavity at 150 days. As the disease progressed to 180 days, ALS CON mice showed extensive myelin cavity formation and severe demyelination; while lisinopril treatment improved myelin cavity formation and reduced myelin loss in ALS mice at 180 days. Figure 9 (Middle B). This indicates that as the disease progresses, the degree of myelination and demyelination in the spinal cord of ALS mice gradually worsens, and lisinopril treatment can effectively prevent myelin sheath loss, thereby maintaining the transmission of nerve impulses.

[0077] 2.4 Lisinopril affects the integrity and function of the NMJ; The neuromuscular junction (NMJ) is a specialized chemical synaptic structure between the axon terminal of a motor neuron and a skeletal muscle fiber, composed of a presynaptic membrane, a synaptic cleft, and a postsynaptic membrane. To assess whether lisinopril could reduce NMJ degeneration in the gastrocnemius muscle of ALS model mice, 50 μm longitudinal sections of the gastrocnemius muscle were co-stained with α-Bungarotoxin (red) and anti-SV2A / neurofilament H antibody (green), and photographed using a confocal microscope at 200x magnification (scale bar 10 μm). The results are as follows: Figure 10 As shown, compared with the ALS CON group, the lisinopril treatment group improved the denervation of the axonal terminals at the NMJ in ALS model mice. The overlapping red and green areas indicate that the NMJ structure is intact and the denervation of skeletal muscle is improved.

[0078] 2.5 Lisinopril inhibits muscle fibrosis in ALS mice; To investigate the effect of lisinopril on skeletal muscle fibrosis in ALS mice, longitudinal paraffin sections (6–7 μm thick) of skeletal muscle from ALS CON group and ALS lisinopril group mice were stained with Sirius red and photographed using a polarized light microscope at 200x magnification. Red (or yellow) represented type I collagen fibers, and green represented type III collagen fibers. The results showed that the skeletal muscle of ALS mice exhibited typical fibrotic characteristics of decreased type III collagen and increased type I collagen, and lisinopril treatment significantly inhibited this pathological transformation. Figure 11 (A). In addition, qPCR results ( Figure 11 Further studies (B and C) showed that lisinopril can reduce the expression levels of fibrosis marker genes ACP5 and FN, although the expression levels in the 180-day treatment group were higher than those in the 150-day group. Overall, lisinopril primarily improves skeletal muscle function degeneration in ALS by delaying collagen type conversion and the fibrosis process.

[0079] 2.6 Lisinopril inhibits muscle atrophy in ALS mice; Muscle atrophy is a key pathological feature of ALS, manifested as a decrease in the cross-sectional area (CSA) and number of muscle fibers. Skeletal muscle samples were collected from ALS CON and ALS lisinopril groups at 150 and 180 days of age. 10 μm transverse sections were prepared, stained with hematoxylin and eosin (HE), and photographed using a Nikon microscope at 400x magnification. Figure 12 Analysis revealed that in untreated ALS mice, the coronary subtraction angiography (CSA) of myofibrils decreased significantly with disease progression (peak value of 1000-1500 μm² at 150 days → peak value of 500-1000 μm² at 180 days); while the lisinopril treatment group maintained CSA at around 2000 μm² at both 150 and 180 days. Figure 12), which confirmed that the drug could effectively inhibit the progression of ALS-related muscle atrophy.

[0080] 2.7 Lisinopril affects the morphology and quantity of mitochondria in the muscle tissue of ALS mice; The soleus muscle of the ALS CON group and the ALS lisinopril group mice at 180 days after birth was taken for longitudinal section, and after embedding and other processing, the number and structure of mitochondria were observed under a transmission electron microscope at an accelerating voltage of 80 kv. The results showed that (Fig. 4A and B), compared with the ALS CON group, the number of mitochondria in the ALS lisinopril group mice was more; and the mitochondria of the ALS CON group mice appeared obvious swelling and rupture, while the morphology and structure of the mitochondria of the ALS lisinopril group mice were relatively normal. This indicated that lisinopril could inhibit the decrease in the number of mitochondria and the damage to the structure of mitochondria in the ALS model mice. Figure 13

[0081] 2.8 Lisinopril inhibits the activation of the TGFβ signaling pathway in ALS mice; Western blot was used to detect the expression changes of related proteins (TGFβ1, Smad2 / 3, p-Smad2 / 3, PI3K, p-AKT, BI1, p-ULK, p-mTOR, p-70S6k, Beclin 1, LC3, p62 and GAPDH proteins) in the TGFβ pathway. The results showed that (Fig. 5A and B), after administration of lisinopril, the expression of BI1 in the skeletal muscle of ALS mice increased, indicating that lisinopril could activate the expression of BI1 in vivo in ALS mice. At the same time, lisinopril reduced the expression of TGFβ1 in ALS mice and inhibited the activation of Smad2 / 3, indicating that lisinopril could inhibit muscle fibrosis and lipid metabolism through the TGFβ1 / Smad2 / 3 signaling pathway. In addition, lisinopril reduced the content of PI3K, p-AKT, p-mTOR and p62, and increased the content of Beclin 1 and LC3, indicating that lisinopril could inhibit the TGFβ1 / PI3K / AKT / mTOR pathway and activate autophagy in vivo in ALS mice. Figure 14

[0082] Example 3: Effect of lisinopril on lipid metabolism in ALS mice; (1) Sample processing: 60-day-old ALS mice were continuously administered with lisinopril for 14 days (the control group was given PBS), and the hind limb skeletal muscle was taken at 150 days of age (n=8 / group, 150 mg / sample), and then was quickly frozen in liquid nitrogen and sent to the laboratory for detection and analysis by liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology.

[0083] (2) Data analysis: Preprocessing: Lipidsearch software was used to extract and align the detection data to obtain the qualitative and quantitative results of lipids.​​

[0084] Differential screening: By principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), the variable projection importance (VIP) > 1.0, the difference multiple (FC) > 1.5 or <0.667, and p Differential lipids with values <0.05.

[0085] Visualization and pathway analysis: The volcano plot was drawn by ggplot2 software, and the heat map was drawn by Pheatmap software to intuitively display the differential lipids. At the same time, with the help of Metaboanalyst 4.0 platform and combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the metabolic pathways were analyzed.

[0086] (3) Experimental results; 3.1 Principal component analysis; The peaks extracted from all experimental samples and quality control (QC) samples were subjected to PCA analysis. As shown in Figure 15 , the QC samples were distributed together, indicating that the QC samples had small differences, indicating that the entire method was stable and the data quality was high. In addition, the ALS CON group and the ALS Lisinopril group samples had certain distribution differences in the figure, showing a separation trend, indicating that the two groups of samples had characteristic differences in the principal component analysis dimension. And the sample points within the same group were relatively clustered, indicating that the similarity between the samples in the same group was high.

[0087] 3.2 Differential metabolite analysis of ALS Lisinopril group and ALS CON group; PLS-DA model establishment: In order to further analyze the differences between ALS Lisinopril group and ALS CON group, and to mine the significantly different lipid metabolites between the two groups. First, the PLS-DA statistical model was subjected to supervised pattern discriminant analysis, and the model evaluation parameters (R2, Q2) obtained by 7-fold cross-validation (7-cycle cross-validation, when the number of biological replicates n<=3, k-cycle cross-validation, k=2n) were obtained. If R2 and Q2 are closer to 1, the model is more stable and reliable. To determine the quality of the model, the model was also subjected to ranking verification to test whether the model had "overfitting" phenomenon. The specific method is to randomly disturb the grouping label of each sample and then build a model and make a prediction. Each modeling corresponds to a set of R2 and Q2 values. According to the Q2 and R2 values after 200 disturbances and modeling, a regression line is drawn. When the R2 data is greater than the Q2 data and the Q2 regression line and the Y-axis intercept are less than 0, it indicates that the model does not have "overfitting" phenomenon. From Figure 16As can be seen from the middle A and B, R2Y is greater than Q2Y, R2data is greater than Q2data, and the regression line of Q2 and Y axis intercept is less than 0, indicating that the model is well established and there is no overfitting problem, which can better describe the sample and can be used as a prerequisite for model biomarker group searching, and is suitable for later analysis. In addition, it is found that the two groups of samples have a clear separation trend in the analysis graph, which indicates that there is a significant difference in lipid metabolism profile between the ALS captopril group and the ALS CON group.

[0088] Screening of differential metabolites: The present application screens differential lipid compounds based on multi-parameter analysis, and evaluates the contribution of compounds to the grouping by VIP value (variable projection importance) > 1.0, combined with FC value (fold change) > 1.5 (up-regulation) or <0.667 (down-regulation) and P value <0.05 (T test significance) as screening criteria. The analysis results show that 336 significant differential lipids are identified between the ALS captopril group and the ALS CON group, of which 116 are up-regulated and 220 are down-regulated (Table 2) Figure 17 Middle A and B), and these differential lipids may be involved in the therapeutic mechanism of captopril for ALS.

[0089] 3.3 Correlation and clustering analysis of differential lipid metabolites; Correlation analysis of differential lipid compounds: Lipid molecules have strong correlation, and by analyzing the correlation of lipid molecules in different samples, lipid molecules with strong correlation in samples can be found. The correlation analysis results of the significant differential lipid metabolites between the ALS captopril group and the ALS CON group show that most glyceride subtype lipids are negatively correlated with sphingomyelin (d37:1), and most phosphatidylcholine subtype molecules and phosphatidylethanolamine subtype molecules are positively correlated (Table 3) Figure 18 ).

[0090] Cluster analysis heat map: Cluster analysis was used to determine the metabolic patterns of lipid compounds under different experimental conditions. Generally, lipid compounds with similar metabolic patterns may have similar functions, or be involved in the same metabolic process or cellular pathway. Therefore, by clustering lipid compounds with the same or similar metabolic patterns, the functions of certain lipid compounds can be inferred. In this experiment, in order to evaluate the rationality of the candidate lipids, more comprehensively and intuitively show the relationship between the samples and the expression pattern differences of the lipids in different samples, cluster analysis was performed on the 336 differential lipid molecules screened between the ALS Lisinopril group and the ALS CON group. It was found that in the ALS Lisinopril group, the content of triglyceride (TG) was generally high, while the expression of sphingomyelin (SM), ceramides (Cer), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) was generally high in ALS CON Figure 19 ).

[0091] 3.4 Enrichment pathway analysis of differential lipid metabolites; KEGG enrichment results are in KEGG Pathway units, and hypergeometric test is used to calculate the P-value of pathway enrichment, where P-value < 0.05 is the threshold, and the KEGG pathway that meets this condition is defined as significantly enriched KEGG pathway. However, in lipid metabolomics research, the number of metabolites obtained is usually only a few hundred to more than a thousand, and the data set is relatively small, so the KEGG analysis of metabolomics usually does not strictly set the P-value threshold. In this study, the significant difference in lipid metabolites between the ALS lisinopril group and the ALS CON group was analyzed by pathway enrichment, and the results were plotted into a bubble chart. Each bubble in the chart represents a pathway that is enriched, and the redder the bubble color, the closer the P-value is to 0, indicating that the reliability of the pathway test is stronger and more statistically significant. The size of the bubble represents the number of differential lipid compounds in the corresponding pathway, and the larger the bubble, the more differential lipid compounds in the pathway. Among them, the KEGG analysis in this study significantly enriched the glycerophospholipid metabolism, phosphatidylinositol signaling system, glycosylphosphatidylinositol-anchor biosynthesis, and autophagy pathways Figure 20 ). The KEGG classification results also show that these differential lipid compounds are mainly related to lipid metabolism, nervous system, and signal transduction Figure 21 ). Then, the differential metabolites were compared with the differential metabolites in the screened differential metabolic pathways, and a total of 6 lipid metabolites were screened, namely phosphatidic acid, lysophosphatidylcholine, cardiolipin, PI, PS, and PE, and these 6 lipid metabolites belong to glycerophospholipids, which suggests that glycerophospholipids may play an important role in the development of ALS disease.

[0092] In summary, lisinopril plays a key role in the treatment of ALS, laying the foundation for drug design targeting Bax inhibitor 1 (BI1). In in vitro experiments, lisinopril can effectively prevent SOD1 G93AThe induced NSC34 cell mitochondrial membrane potential decline caused the damage of mitochondrial structure and function. At the same time, lisinopril regulates the expression of Beclin 1, LC3 and p62 by inhibiting the TGFβ1 / PI3K / AKT / mTOR signaling pathway, activates the autophagy process, and then inhibits neuronal apoptosis. In vivo experiments show that lisinopril can activate the expression of BI1 in vivo, regulate the content of triglyceride (TG), sphingomyelin (SM), ceramide (Cer), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) and other lipids in ALS model mice, and intervene in the glycerophospholipid metabolism process. In addition, lisinopril also regulates autophagy through the TGFβ1 / PI3K / AKT / mTOR pathway, inhibits the death of motor neurons, demyelination, neuromuscular junction (NMJ) degeneration, and promotes mitochondrial biogenesis. These results fully demonstrate that lisinopril plays an important role in the treatment of ALS disease, and the revelation of its mechanism provides an important structural basis for designing new drugs targeting BI1 in the future, and has important application value for promoting the research and development of ALS treatment drugs and improving patient prognosis.

[0093] The above is only the preferred embodiment of the present application, it should be noted that for those skilled in the art, without departing from the concept of the present application, can make several deformation and improvement, these should also be considered as the protection scope of the present application, these will not affect the effect and practicality of the present application.

Claims

1. The use of lisinopril in the preparation of drugs for treating amyotrophic lateral sclerosis (ALS), characterized in that, Lisinopril works through the following mechanism: Inhibit SOD1 G93A Induced apoptosis; Improve mitochondrial membrane potential and mitochondrial number; Regulates the expression of MAP2 and β-Tubulin to maintain neuronal axonal integrity; Autophagy is activated through the TGFβ1 / PI3K / AKT / mTOR pathway; It promotes the conversion of fast-twitch muscle fibers into slow-twitch muscle fibers and increases ATP content; Inhibits damage to spinal motor neurons and myelin sheath; Improve the integrity of the neuromuscular junction; Inhibits muscle fibrosis and muscle atrophy; It regulates lipid metabolism, reducing the levels of sphingomyelin, ceramide, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol, while increasing the levels of triglycerides.

2. The application according to claim 1, characterized in that, The drug is used to prolong the lifespan of ALS model mice, delay the onset of disease, or improve motor function.

3. The application according to claim 1, characterized in that, The drug reduces muscle fibrosis by inhibiting the TGFβ1 / Smad2 / 3 signaling pathway.

4. The application according to claim 1, characterized in that, The drug improves ALS symptoms by regulating glycerophospholipid metabolism, sphingolipid metabolism, glyceride metabolism, or autophagy pathways.

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