CN116459272A - Application of Bax inhibitor1 in treating amyotrophic lateral sclerosis - Google Patents
Application of Bax inhibitor1 in treating amyotrophic lateral sclerosis Download PDFInfo
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- CN116459272A CN116459272A CN202310500437.0A CN202310500437A CN116459272A CN 116459272 A CN116459272 A CN 116459272A CN 202310500437 A CN202310500437 A CN 202310500437A CN 116459272 A CN116459272 A CN 116459272A
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Abstract
The invention is applicable to the technical field of medicines, and provides an application of Bax inhibitor1 in treating amyotrophic lateral sclerosis, including an application of BI1 in preparing a medicine for preventing and/or treating amyotrophic lateral sclerosis. The present invention discovers that BI1 inhibits SOD1 G93A Induced apoptosis of NSC-34 cells, cyto-C release, mitochondrial transport of BAX, nuclear reversal of TDP-43 and mitochondrial abnormalities; inhibit TDP-43 degradation and transmission of pathological TDP-43 in exosomes; BI1 can activate autophagy and reduce SOD1 G93A Induced active oxygen levels; BI1 interacts with TDP-43 to inhibit apoptosis; the important role of BI1 in regulating ALS pathogenesis through mediating TDP43 is revealed, a powerful theoretical basis is provided for revealing the ALS pathogenesis, and a novel direction and a novel effect are provided for further developing small molecule therapeutic compoundsA target.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an application of Bax inhibitor1 in treating amyotrophic lateral sclerosis.
Background
Amyotrophic Lateral Sclerosis (ALS) is a fatal, adult-onset motor neuron degenerative disease that is primarily manifested as motor neuron degeneration, and thus how to alleviate motor neuron death is one of the important strategies for treating ALS. Baxinhibitor1 (BI 1), also known as transmembrane Bax Inhibitor Motif Containing (TMBIM 6), exerts its anti-apoptotic activity by interacting with many proteins, such as Bcl2 and Bclxl proteins at the N-terminus. Although BI1 plays an important role in inhibiting Bax activity, it has also been reported that BI1 has a certain neuroprotective effect and also has an important effect on mitochondrial dysfunction and the occurrence of autophagy, but the role of BI1 in the pathogenesis of neurodegenerative diseases is not clear.
More than 95% of patients with Amyotrophic Lateral Sclerosis (ALS) have diseased spinal motor neurons characterized by the mismetabolism of the RNA/DNA binding protein TDP-43 (ALS-TDP), particularly the presence of cytosol. Studies have shown that over 40 disease-related mutations are found in TDP-43 and some documents report that TDP-43 mutations enhance aggregation, leading to ALS. It is worth mentioning that about 95% of the individuals show no mutation in TDP-43, and that abnormal levels and incorrect localization of TDP43 may also lead to the occurrence of ALS. Normally, TDP-43 shuttles between the nucleus and the cytoplasm, whereas under pathological conditions TDP-43 remains mainly in the cytoplasm and forms aggregates, the phenomenon of nuclear consumption being evident. At the same time, TDP-43 is involved in regulating mitochondrial dysfunction and enhancing mitochondrial autophagy. Autophagy is essential for maintaining normal function of the Central Nervous System (CNS), and can avoid misfolding and accumulation of aggregated proteins, and impaired autophagy is associated with the pathogenesis of various neurodegenerative diseases. Autophagy can be induced by ER stress, mitochondrial injury, free radical and genotoxic stress, etc. Stress-induced autophagy and basal autophagy are critical for maintaining cell homeostasis in vitro and in vivo.
Fusion of autophagosomes to lysosomes degrades cargo within the cytosol, and the relationship between the rate of formation of different autophagosomes and the rate of clearance of cargo by lysosomes is referred to as "autophagy flux". The fusion of autophagosomes to lysosomes is impaired, or inhibition of lysosomal acidification prevents degradation of autophagosome contents and promotes accumulation of autophagosomes, thereby inhibiting "autophagy flux". Blocking of autophagy flux can be observed in different human diseases, and neurodegenerative diseases are characterized by reduced autophagy flux, many details of the autophagy lysosomal fusion process remain unknown, possibly involving additional proteins whose mutations/deletions may affect autophagosome turnover. Exosomes perform the task of removing unwanted or toxic intracellular proteins from the cells. They also act as intercellular messengers. TDP-43 can be detected in exosomes secreted by nerve cells, and protein aggregation and autophagy inhibition are major factors promoting secretion of TDP-43 exosomes. For this reason we propose the use of Bax inhibitor 1 in the treatment of amyotrophic lateral sclerosis.
Disclosure of Invention
The invention aims to provide an application of Bax inhibitor 1 in treating amyotrophic lateral sclerosis, and aims to solve the problems in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
use of Bax inhibitor 1 in the treatment of amyotrophic lateral sclerosis, including use of BI1 in the manufacture of a medicament for the prevention and/or treatment of amyotrophic lateral sclerosis.
Further, the BI1 plays a role in transfecting cells.
Further, the preparation steps of the transfected cells are as follows:
step S1, culturing NSC-34 cells in a culture dish of 60 mm, transferring into pcmV6/TMBIM6-Myc-DDK-tagged plasmids by a transient method, respectively diluting 10 times, 100 times and 1000 times after culturing 48 h, transferring into a culture dish of 100mm, continuously culturing, screening by G418, picking out screened single cells, culturing in a 96-well plate, transferring into 24-well plate and 6-well plate in sequence after the cells are full, enlarging and culturing, and collecting cell samples to obtain NSC-34 cells stably over-expressing BI 1;
step S2, expanding culture of SOD1 G93A Adding 50 mug/mL of antibiotics into LB culture solution of 5 mL, shaking culturing at 37 ℃ for 8 hours, pouring into LB culture medium of 300 mL, and culturing overnight; extracting transfection-grade plasmid by using a large extraction kit for removing endotoxin;
and S3, transferring the transfection-grade plasmid into NSC-34 cells and BI1-NSC 34 cells to obtain transfected cells.
Further, in the step S2, in the LB culture solution of 5 mL, the following steps 1: antibiotic was added at a rate of 1000. Mu.g/mL.
Further, the step S3 includes the following steps:
step S31, inoculating BI1-NSC 34 cells and NSC-34 cells on a cell plate of 60 mm one day before transfection, wherein the inoculation density is 80-85%, and incubating the cells in a 5% CO2 cell incubator at 37 ℃ with 10% fetal bovine serum and 1% of G418 DMEM culture medium;
step S32, following transfection reagent (μL): transfection reagent and transfection-grade plasmid (μg) =3:1-2:1 were added to two OPTI-MEM containing tubes, respectively, and incubated at room temperature for 5 min;
step S33, uniformly mixing the two test tubes in the step S32, and incubating for 20 min at normal temperature;
step S34, adding the mixed solution of step S33 into the cells in step S31, and gently mixing the cells to obtain transfected cells.
Compared with the prior art, the invention has the beneficial effects that:
1. BI1 inhibits SOD1 G93A Induced apoptosis of NSC-34 cells, cyto-C release, mitochondrial transport of BAX, nuclear reversal of TDP-43 and mitochondrial abnormalities; BI1 can activate autophagy and reduce SOD1 G93A Induced reactive oxygen levels and enhance autophagy by promoting competitive binding between Bax and BCL-2 and between TDP-43, MFN2 and Beclin1 to disrupt the interaction of Beclin1 and BCL-2; BI1 interaction with TDP-43 indirectly reduces the interaction of TDP-43 with Bax to inhibit apoptosis; BI1 inhibits TDP-43 (43 KD) degradation and transmission of pathological TDP-43 in exosomes.
2. The important role of BI1 in regulating ALS pathogenesis through mediating TDP43 is revealed, a powerful theoretical basis is provided for revealing ALS pathogenesis, and a novel direction and target are provided for further development of small molecule therapeutic compounds.
Drawings
FIG. 1 shows overexpression of SOD1 in NSC-34 cells G93A Is to be identified.
FIG. 2 shows SOD1 G93A Inducing apoptosis of NSC-34 cells.
FIG. 3 is a diagram of transient transfection of BI1 versus SOD1 G93A Induction of the effects of apoptosis in NSC-34 cells.
FIG. 4 is a stable cell line fluorescent staining (FITC).
FIG. 5 is an identification of stable cell lines.
FIG. 6 is a diagram of BI1 vs. SOD1 G93A The effect of inducing a change in cell viability.
FIG. 7 is a diagram of BI1 vs. SOD1 G93A Effect of induced apoptosis.
FIG. 8 shows the detection of SOD1 by transmission electron microscopy G93A Effects on apoptosis of BI1-NSC34 cells.
FIG. 9 TUNEL detection of BI1-NSC34 cells versus SOD1 G93A Effect of induced apoptosis.
FIG. 10 is inhibition of SOD1 after BI1 overexpression G93A Induced protein transfer.
FIG. 11 is a diagram of BI1 vs. SOD1 G93A Influence of induced changes in mitochondrial membrane potential.
FIG. 12 is a graph of BI1 vs. SOD1 G93A Effect of induced mitochondrial damage (scale: 2 μm).
FIG. 13 is a diagram of BI1 vs. SOD1 G93A Inducing the effects of axonal injury in NSC-34 cells.
FIG. 14 is a chart of BI1 vs. SOD1 G93A Effects of MAM change in induced NSC-34 cells.
FIG. 15 is a chart of BI1 vs. SOD1 G93A The effect of inducing autophagosome changes in NSC-34 cells (scale: 2 μm).
FIG. 16 shows SOD1 in time gradient G93A Effects on autophagy of NSC-34 cells.
FIG. 17 shows the result of the reaction of chlorquinone on SOD1 G93A Blocking the effects of autophagy in NSC-34 cells.
FIG. 18 shows the result of the comparison of Rapamycin with SOD1 G93A Blocking the effects of autophagy in NSC-34 cells.
FIG. 19 is a chart of BI1 vs. SOD1 G93A Blocking the effects of autophagy in NSC-34 cells.
FIG. 20 is a chart of BI1 vs. SOD1 G93A Inducing the effects of autophagy-related proteins in NSC-34 cells.
FIG. 21 is a graph of BI1 vs. SOD1 G93A Inducing the effects of ROS levels in NSC-34 cells.
FIG. 22 shows the level of interaction of BI1 with TDP-43 in cells.
FIG. 23 is the interaction of Bax and TDP-43.
FIG. 24 is the interaction of Bax and BCL-2.
FIG. 25 shows the interaction of Beclin1 and BCL-2.
FIG. 26 is an interaction of MFN2 and TDP-43.
FIG. 27 shows the interaction of Beclin1 and TDP-43.
FIG. 28 is a chart of BI1 vs. SOD1 G93A Influence of TDP-43 in induced NSC-34 extracellular body.
FIG. 29 shows the result of the exosome inhibitor (GW 4869) on SOD1 G93A Blocking NSC-34 cell self-assemblyEffects of phagocytosis.
FIG. 30 shows BI1 vs. SOD1 after GW4869 is added G93A Blocking the effects of autophagy.
Description of the embodiments
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Specific implementations of the invention are described in detail below in connection with specific embodiments.
The use of the Bax inhibitor 1 in the preparation of a medicament for preventing and/or treating amyotrophic lateral sclerosis comprises the use of BI1, wherein the BI1 plays a role in transfected cells.
As a preferred embodiment of the present invention, the transfected cells are prepared by the steps of:
step S1, culturing NSC-34 cells in a 60 mm culture dish, and transferring into pcMV6/TMBIM6 by a transient method
After 48-h, respectively diluting 10 times, 100 times and 1000 times, transferring into a 100 mm culture dish for continuous culture, then adopting G418 to select, selecting single cells after the selection to culture in a 96-well plate, transferring into a 24-well plate and a 6-well plate in sequence for expansion culture after the cells are full of the culture, and collecting cell samples to obtain NSC-34 cells (BI 1-NCS 34) for stably over-expressing BI 1;
step S2, expanding culture of SOD1 G93A Plasmids and LacZ plasmid (Kan resistance) were grown in LB medium 5 mL according to 1: adding 50 mug/mL of antibiotic at a ratio of 1000, shaking culture at a constant temperature of 37 ℃ for 8 h, pouring into 300 mL LB medium (Kan final concentration is 50 mug/mL), and culturing overnight; extracting transfection-grade plasmid by using a large extraction kit for removing endotoxin;
And S3, transferring the transfection-grade plasmid into NSC-34 cells and BI1-NSC 34 cells to obtain transfected cells.
As a preferred embodiment of the present invention, the step S3 includes the steps of:
step S31, inoculating BI1-NSC 34 cells and NSC-34 cells on a cell plate of 60 mm one day before transfection, wherein the inoculation density is 80-85%, and incubating the cells in a 5% CO2 cell incubator at 37 ℃ with 10% fetal bovine serum and 1% of G418 DMEM culture medium;
step S32, will be performed with transfection reagent (μl): transfection reagent and transfection-grade plasmid (μg) =3:1-2:1 were added to two OPTI-MEM containing tubes, respectively, and incubated at room temperature for 5 min;
step S33, uniformly mixing the two test tubes in the step S32, and incubating for 20 min at normal temperature;
step S34, adding the mixed solution of step S33 into the cells in step S31, and gently mixing the cells to obtain transfected cells.
The invention is explored as follows:
1.1 overexpression of SOD1 G93A Apoptosis in NSC-34
Respectively using pcMV6/LacZ-Myc, pcMV6/SOD1 G93A Transfection of NSC-34 cells with FLAG-Myc plasmid, wherein pcmV6/LacZ-Myc is the control group, pcmV6/SOD1 G93A FLAG-Myc with FLAG tag, and pcMV6/SOD1 with FALG tag by laser confocal microscope detection G93A The FLAG-Myc plasmid fluoresced green under the microscope. As detected by a laser confocal microscope, as shown in FIG. 1, compared with LacZ group, SOD1 is transferred G93A NSC-34 cells of (C) show green fluorescence under laser, which proves SOD1 G93A Overexpression was successful in NSC-34 cells.
Meanwhile, we perform Western-Blotting experiment to test SOD1 G93A Inducing apoptosis of NSC-34 cells. When cells undergo apoptosis, caspase-3 protease is activated by self-cleavage from 32KD to 17KD and 12KD fragments, and the main cleavage substrate of activated Caspase-3 is PARP (DNA repair enzyme) which can be cleaved into 85KD and 31KD, and the loss of enzyme activity after the cleavage of PARP accelerates the instability of cells and marks the occurrence of apoptosis. Transfection of SOD1 in NSC-34 cells G93A And setting a blank control group, detecting the expression change of PARP and Pro-Caspase-3 by using a Western-Blot after 24 and h, and selecting GAPDH as an internal reference protein. The results are shown in FIG. 2Transfection of SOD1 compared to the control group G93A The expression level of Pro-caspase3 in the group is obviously reduced, which proves that the Pro-caspase3 can be cut and activated, and the expression level of PARP 110KD fragment thereof, namely SOD1, is obviously reduced G93A Activation of caspase3 may be induced in NSC-34 cells and cleavage of the downstream substrate PARP, with apoptosis occurring.
1.2 transient transfection of BI1 versus SOD1 G93A Influence of induction of apoptosis of NSC-34 cells
To clarify BI1 vs. SOD1 G93A Whether induced apoptosis of NSC-34 cells is functional, we first examined whether BI1 overexpression affects SOD1 by transiently transfecting the BI1 plasmid G93A Induced apoptosis of NSC-34 cells. First, transient transfection of BI1 plasmid in NSC-34 cells followed by transfection of SOD1 after 24 h G93A And setting a blank control group, detecting the expression change of PARP through Western-Blot after 48 and h, and selecting GAPDH as an internal reference protein. As a result, FIG. 3 shows SOD1 G93A The expression level of PARP over-expressed (group 2) was significantly reduced, indicating that apoptosis occurred in the cells at this time. And SOD1 is transfected after transient transfection of BI1 plasmid G93A (group 4) the PARP upstream expression level is remarkably increased, and compared with the control group, the PARP expression level is basically consistent, and the SOD1 can be effectively relieved G93A Apoptosis is induced.
1.3 identification of NSC-34 cell lines of stable over-expressed BI1
Verification of SOD1 by transient transfection of BI1 G93A After the induced apoptosis of NSC-34 cells had an inhibitory effect, we constructed a BI 1-overexpressing NSC-34 cell line by stably transfecting the pcmV6/TMBIM6-Myc-DDK-tagged plasmid for further investigation. Stably transfecting pcMV6/TMBIM6-Myc-DDK-tagged plasmid to construct a NSC-34 cell line which over-expresses BI1, detecting Myc tag by immunofluorescence, and displaying green Fluorescence (FITC) under a laser confocal microscope. The results are shown in FIG. 4, which shows successful construction of the NSC-34 cell line stabilizing the over-expressed BI 1. Extracting whole protein of a BI1 stable cell line, setting NSC-34 cell protein as a blank control group, detecting BI1-Myc protein by using Western-Blot, and selecting GAPDH as an internal reference protein. Meanwhile, the Western-Blot identification result is shown in FIG. 5, compared with the control group, the BI1-Myc band can be obviously seen, Indicating successful transfer of BI1, successful construction of a stable over-expressed BI1 NSC-34 cell line, hereinafter referred to as BI1-NCS34.
1.4 BI1 can alleviate SOD1 G93A Induced apoptosis of NSC-34 cells
To investigate the stable over-expression of BI1 to SOD1 in NSC-34 cells G93A Inducing the influence of apoptosis, and performing CCK-8 experiment to detect the change of cell viability. Transfection of SOD1 in NSC-34 and BI1-NSC34 cells G93A The transfected LacZ was set as a blank control group, incubated with CCK-8 kit in a time gradient, and absorbance was detected at the microplate reader 450 nm (P compared to the control group)<0.001). As a result, as shown in FIG. 6, NSC-34 cells were transfected with SOD1 as compared with the control group G93A The activity of the post-cells is significantly reduced, while the cellular activity is significantly increased after BI1 is overexpressed.
Meanwhile, BI1-NSC34 was examined for SOD1 by Western-mapping G93A Inhibition of apoptosis induced. Transfection of SOD1 in NSC-34, BI1-NSC34 cells G93A And setting a blank control group, carrying out transfection on 24 h, extracting cell whole protein, carrying out Western-Blotting test, taking GAPDH as a protein reference, and detecting the expression quantity change of apoptosis marker proteins PARP and casp 3. The results are shown in FIG. 7, which shows SOD1 compared with the control group G93A The expression level of Pro-caspase3 is obviously reduced, and the expression level of caspase3 is obviously increased, which indicates that the NSC-34 cells are transfected with SOD1 G93A Pro-caspase3 can be induced to cleave, producing activated caspase3; and the expression quantity of the full length PARP is obviously reduced compared with the expression quantity of other three groups, and the expression quantity of the PARP cutter is increased. These results indicate that cleavage of PAPR by activated caspase3 results in its loss of DNA repair enzyme activity, thereby inducing apoptosis. After the BI1 is over-expressed, the expression level of Pro-caspase3 is obviously increased, the expression level of activated caspase3 is greatly reduced, the expression level of PARP full length is obviously increased, and the expression level of PARP cutter is also obviously reduced.
In addition, we also used transmission electron microscopy to observe apoptosis. Transfection of SOD1 in NSC-34, BI1-NSC 34 cells G93A And a blank control is arranged, and after 24 h transfection, the cells are slicedAnd (3) preparing slices by embedding and other means, and observing mitochondrial change under a transmission electron microscope, wherein N is cell nucleus. The results are shown in FIG. 8, where NSC-34 was transfected with SOD1 compared to the control group G93A The cell nuclei of the group are deformed, nuclear membrane is shrunken and the cell nuclei are shrunken under an electron microscope, which indicates that the SOD1 is transfected G93A Post-cell apoptosis, while transfection of SOD1 in BI1-NSC 34 cells G93A After that, the cell nucleus is observed to be normal under an electron microscope, which proves that the BI1 can inhibit the SOD1 G93A Inducing apoptosis of NSC-34 cells. In addition, apoptosis was observed under a laser confocal microscope using TUNEL detection kit. In FIG. 9, (A) transfection of SOD1 in NSC-34, BI1-NSC 34 cells G93A A blank control group was set, and after 24 h transfection, immunofluorescence experiments were performed to detect the fluorescence level. (B) Statistical results of the number of TUNEL-stained positive cells (P compared to control group)<0.0001). The results are shown in FIG. 9, which shows SOD1 compared with the control group G93A The green fluorescence of the group was significantly enhanced, whereas in BI1-NSC 34 cells, the green fluorescence was compared to NSC-34 transfected SOD1 G93A And significantly weakens.
2.1 BI1 inhibits Cyto-C release, mitochondrial transport of BAX and nuclear reversal of TDP-43
The redistribution of TDP-43 from the nucleus to the cytoplasm in fALS patients has been considered a common pathological hallmark. Meanwhile, BAX acts as a pro-apoptotic protein, altering mitochondrial membrane permeability, entering mitochondria from the influx of cytoplasm, and Cyto-C is released from mitochondria to cytoplasm, and apoptosis occurs. When SOD1 G93A When the above reaction is induced, BI1 as a gene for inhibiting apoptosis, can inhibit the mislocalization of BAX, TDP-43 in mitochondria, and the release of Cyto-C? For this hypothesis, western-Blotting was performed to examine the expression and transfer of TDP-43, BAX and Cyto-C.
Transfection of SOD1 in NSC-34, BI1-NSC 34 cells G93A Setting blank control, extracting cell nucleus, cytoplasm and mitochondrial proteins after transfection of 24H for Western-Blot test, taking H3 as nuclear protein reference, COX IV as mitochondrial protein reference and action as cytoplasmic reference. As shown in FIG. 10, the results show that the SOD was higher than that of the control group 1G93A In the group, TDP-43 (43 KD) in the nucleus of cellsThe amount of the expression is obviously reduced, the level of the TDP-43 in cytoplasm is obviously increased, and the expression level of the TDP-43 (43 KD) is obviously increased in mitochondrial protein components; in addition, the expression quantity of Cyto-C in mitochondria is obviously reduced, and the expression quantity in cytoplasm is obviously improved; the expression level of BAX in the cytoplasm is significantly reduced and transferred to mitochondria. While LacZ and SOD1 are transfected at BI1-NSC34 G93A After that, compared with the control group, the expression level of TDP-43 in the nucleus is basically consistent, the expression level of TDP-43 in cytoplasm is also obviously reduced, and the expression level of TDP-43 in mitochondria is also obviously reduced. In addition, we found that TDP-43 (100 KD) accumulated in the cytoplasm.
3.1 BI1 can improve SOD1 G93A Mitochondrial abnormalities in primed NSC-34 cells
3.1.1 BI1 inhibits SOD1 G93A Induced changes in mitochondrial membrane potential
We used mitochondrial membrane potential detection experiments to examine BI1 versus SOD1 G93A Effects of induced mitochondrial dysfunction. In FIG. 11, (A) transfection of SOD1 in NSC-34, BI1-NSC34 cells G93A And setting a blank control, carrying out cell incubation according to a JC-1 kit after 24 h transfection, wherein the JC-1 kit is a bifluorescent dye, when the membrane potential is reduced, exciting FITC to be green fluorescent, when the membrane potential is increased, exciting PE to be red fluorescent, and selecting DAPI to dye the cell nucleus to determine the cell. (B) Statistics of number of positive cells for JC-1 membrane potential decrease (P compared to control group) <0.0001). (C) Statistics of number of JC-1 membrane potential elevation positive cells (P compared with control group)<0.0001). As shown in FIG. 11, the results of transfection of SOD1 were compared with the control group G93A In NSC-34 cells, the green fluorescence is obviously enhanced and the red fluorescence intensity is reduced, indicating SOD1 G93A The decrease of mitochondrial membrane potential, which is the key to mitochondrial energy supply, is initiated, neurotransmission is blocked, and cells undergo apoptosis. While transfection of SOD1 in BI1-NSC34 cells G93A After transfection of SOD1 compared with NSC-34 G93A Its green fluorescence is significantly reduced and its red fluorescence is significantly increased.
3.1.2 BI1 inhibits SOD1 G93A Resulting in an increased number of damaged mitochondria
Mitochondria need to maintain their function and reduce the number of functionally impaired mitochondria by constantly fusing and fissuring. SOD1 G93A Not only can induce the imbalance of fusion fission related proteins of mitochondria, but also can induce the abnormal and fragmented increase of mitochondria so as to reduce the number of healthy mitochondria, and a transmission electron microscope is adopted to directly observe BI1 to SOD1 G93A Induced mitochondrial abnormalities affect. Transfection of SOD1 in NSC-34, BI1-NSC34 cells G93A And setting a blank control, tabletting after 24 h by means of slicing, embedding and the like, and observing mitochondrial change under a transmission electron microscope, wherein N is a cell nucleus. As a result, as shown in FIG. 12, SOD1 was overexpressed in NSC-34 cells compared to the control group G93A The healthy mitochondria have a reduced number of mitochondria and a shallower mitochondrial matrix, a shorter cristae, and even a disappearance or transformation of small vacuolated structures. While transfection of SOD1 in BI1-NSC34 cells G93A After transfection of SOD1 compared with NSC-34 G93A The mitochondrial morphology size returns to normal and healthy mitochondria numbers increase. This suggests that BI1 is capable of slowing down SOD1 G93A Induced mitochondrial damage.
3.1.3 BI1 can inhibit SOD1 G93A Promoting axonal integrity of NSC-34 cells
Mitochondria power axon transport between synapses, and thus mitochondria are critical for neuronal function and survival. Because of SOD1 G93A Inducing mitochondrial damage of NSC-34 cells, leading to dysfunction of neuronal cell information transmission and further apoptosis. To investigate whether BI1 can inhibit SOD1 G93A Disruption of neuronal axon integrity since the main component of neuronal axons is beta-Tubulin-based Tubulin, we used immunofluorescence techniques to observe the distribution of beta-Tubulin in cells. In FIG. 13, (A) transfection of SOD1 in NSC-34, BI1-NSC34 cells G93A And setting a blank control group, and performing immunofluorescence experiment to detect fluorescence change of beta-Tubulin after 24-h. (B) Cell axon length statistic plot (P compared to control group) <0.0001). The results are shown in FIG. 13, which shows overexpression of SOD1 in NSC-34 cells compared to control G93A Beta of itTubulin is greatly reduced in cell distribution, exists substantially around the nucleus, and severely disrupts the integrity of axons. While transfection of SOD1 in BI1-NSC34 cells G93A After transfection of SOD1 compared with NSC-34 G93A Its β -Tubulin distribution is prolonged and axons are intact.
3.1.4 BI1 modulation of MAM
BI1 to SOD1 was observed by transmission electron microscopy G93A Influence of induced MAM change. In FIG. 14, (A) transfection of SOD1 in NSC-34, BI1-NSC34 cells G93A And setting a blank control, observing MAM change under a transmission electron microscope after transfection of 24 h, and selecting MAM amplifying regions in a white virtual frame, wherein N is as follows: a cell nucleus; m: mitochondria; ER: endoplasmic reticulum (scale: 2 μm). (B) MAM spacing statistics (P compared to control group)<0.05). As shown in FIG. 14, the results of transfection of SOD1 alone compared with the control group G93A The distance between MAM in the group is obviously increased, and mitochondrial cristae is blurred, etc., and after BI1 is over-expressed, the distance between MAM is obviously reduced, and the contact area between mitochondrial membrane and endoplasmic reticulum membrane is increased.
4.1 BI1 activates autophagy and inhibits SOD1 G93A Induced ROS
Studies have shown that in SOD1 G93A Autophagy disorders, and thus apoptosis, are caused in ALS disease models by mutations. Since BI1 can inhibit the progress of apoptosis, to further investigate whether BI1 can regulate apoptosis via autophagy pathway, we first used transmission electron microscopy to observe changes in autophagosomes within cells. Transfection of SOD1 in NSC-34, BI1-NSC34 cells G93A And setting a blank control, tabletting by means of slicing, embedding and the like after 24 h, observing autophagosome change under a transmission electron microscope, wherein N is a cell nucleus, and an enlarged area is arranged in a white virtual frame. As a result, FIG. 15 shows SOD1 G93A The number of induced autophagosomes was significantly reduced and the number of autophagosomes was significantly increased after BI1 overexpression, indicating that BI1 was able to activate autophagy.
To further verify this result, we detected changes in autophagy marker proteins and related proteins by Western-Blot experiments. First by detecting SOD1 G93A Changes in autophagy-related proteins after various times of action (18 h, 24 h and 48 h). At the position ofTransfection of LacZ and SOD1 in NSC-34 cells at gradient time (18 h, 24 h and 48 h) G93A After different times of action, collecting whole protein extract for Western-Blot inspection, selecting GAPDH as reference protein, and detecting the expression levels of autophagy-related proteins MFN2 and GRP75, apoptosis-related proteins PARP and TDP-43 on MAM, wherein the autophagy-related proteins LC3B, beclin1 and the apoptosis-related proteins PARP are selected from GAPDH. The results are shown in fig. 16, 18 h, and PARP gradually decreases, indicating that PARP cleavage becomes stronger with longer time. Autophagy marker protein LC3B is less and less, indicating that autophagy is weaker with time. While another protein Beclin1 regulating autophagy had slightly higher protein expression levels at 18 h, and decreased protein levels of Beclin1 over time. Protein MFN2 protein levels, which are closely related to mitochondrial function, decrease over time, and GRP75 protein levels vary somewhat similarly to Beclin1, with 18-24 h, GRP75 protein levels increasing, 48 h, and GRP75 protein levels decreasing. Studies have shown that TDP43 leads to mitochondrial dysfunction and enhances autophagy. Western-Blot results showed that TDP43 protein expression levels decreased with time. Unless otherwise indicated, the transfection times we selected for the following experiments were 24 h. The above data illustrate SOD1 G93A Inhibiting autophagy after overexpression, in order to study BI1 on SOD1 G93A Inhibition of induced autophagy we first examined the effects of autophagy inhibitors (chlorquinone) and autophagy activators (Rapamycin) on SOD1 by Western-Blot experiments G93A Effect of induced autophagy inhibition.
NSC-34 cells were inoculated in 35 mm dishes, 0.25. Mu.L of chloroquinone was added, and a blank was placed at a final concentration of 2.5. Mu. Mol/mL with 1 h, and then LacZ, SOD1 was transfected G93A Collecting the whole protein extract after 24-h for Western-Blot experiment examination, selecting GAPDH as an internal reference protein, and detecting autophagy abundance marker protein Beclin1 and autophagy related proteins MFN2 and GRP75 on MAM. As shown in FIG. 17, the addition of chloroquine, an autophagy inhibitor, did not affect SOD1 G93A The induced protein levels were reduced and the Beclin1, GRP75 and MFN2 protein levels remained reduced. NSC-34 was inoculated in 35 mm dishes, 2.5. Mu.L of Rapamycin was added, and a blank was placed at a final concentration of 2.5 nmAfter ol/mL action 1 h, lacZ and SOD1 were transfected again G93A And collecting the whole protein extract after 24. h to perform Western-Blot experiments, selecting GAPDH as an internal reference protein, and detecting the expression levels of autophagy-related proteins MFN2 and GRP75, apoptosis marker proteins Pro-casp3 and TDP-43 on MAM (autophagy-related protein) by using the GAPDH as an internal reference protein. As shown in FIG. 18, the addition of rapamycin, an autophagy activator, first restored LC3B expression, indicating that autophagy was indeed activated, alleviating SOD1 G93A Activation of induced caspase-3 suggests that activation of autophagy does block the occurrence of apoptosis. Second, can obviously relieve SOD1 G93A The induced protein levels of Beclin1 and GRP75 were reduced, but had little effect on MFN 2. Finally, the addition of autophagy activator, while not significantly affecting TDP43 (43 KD), attenuated TDP-43 (35 KD) production.
The above data demonstrate that autophagy inhibitors and activators are at SOD1 G93A Inducing apoptosis in order to reveal the role of BI1 for SOD1 G93A Inducing the effects of autophagy-related proteins during apoptosis we detected changes in related proteins by Western-Blot experiments. Transfection of SOD1 in NSC-34, BI1-NSC 34 cells G93A And setting a blank control group, carrying out Western-Blot detection on a whole protein extract after 24-h transfection, selecting GAPDH as an internal reference protein, and detecting the expression quantity of autophagy-related proteins GRP75, MFN2 and TDP-43 on MAM (autophagy-related protein) by using the GAPDH as an internal reference protein LC3B, beclin 1. As shown in FIG. 19, BI1 overexpression, similar to rapamycin, can alleviate SOD1 G93A Induced decrease in LC3B, beclin1 and GRP75 protein levels, except that BI1 also relieves SOD1 G93A Induced decrease in MFN2 protein levels. Although in BI1 overexpressing cells, SOD1 G93A After overexpression, TDP43 (100 KD) was still detected, but BI1 significantly eases the degradation of TDP43 (43 KD) and the generation of TDP43 (35 KD).
Meanwhile, we detected BI1 for SOD1 using immunofluorescence technique G93A Effects of autophagy-related proteins during induction of apoptosis in FIG. 20, (A) transfection of SOD1 in NSC-34, BI1-NSC 34 cells G93A Setting blank group, transferringAfter 24-h staining, autophagy-related protein changes were detected by immunofluorescence, wherein Beclin1, LC3B autophagy marker proteins were shown with FITC green fluorescence and MFN2 with PE red fluorescence. (B) Beclin1 fluorescence intensity statistic plot (P compared to control group)<0.001). (C) LC3B fluorescence intensity statistic plot (P compared to control group)<0.001). (D) MFN2 fluorescence intensity statistic plot (P compared to control group)<0.001). As a result, FIG. 20 shows that SOD1 was converted in one single pass G93A The fluorescent intensity of Beclin1, LC3B and MFN2 of the group is obviously reduced, the fluorescent level of LC3B is obviously enhanced after BI1 is over-expressed, and the fluorescent level of Beclin1 and MFN2 is also obviously enhanced, which proves that BI1 relieves SOD1 G93A Induced decrease in LC3B, beclin1 and MFN2 protein levels activates autophagy.
Next, we measured BI1 for SOD1 G93A An effect of inducing the generation of oxidative stress (ROS). In FIG. 21, (A) transfection of SOD1 in NSC-34, BI1-NSC 34 cells G93A And setting a blank control group, and after 24 h transfection, incubating by a reactive oxygen species kit, and selecting FITC green fluorescence excitation to detect the ROS level in the cells. (B) ROS positive cell number statistics plot (P compared to control group)<0.0001). As shown in FIG. 21, SOD1 G93A Can obviously strengthen the generation of ROS, and obviously block the generation of ROS after the BI1 is over expressed.
5.1 BI1 can bind to TDP-43 to inhibit apoptosis and regulate binding between autophagy-related proteins to enhance autophagy
To further elucidate the molecular mechanisms by which BI1 acts on ALS pathogenesis, we next explored interactions between related proteins in this process. Transfection of SOD1 in NSC-34, BI1-NSC 34 cells G93A A blank was placed, 24 h followed by immunofluorescence interaction to detect BI1 interaction with TDP-43. Wherein DAPI marks nuclei, BI1 selects FITC to excite green fluorescence, TDP-43 selects PE to excite red fluorescence, and Merge's image is three-channel integration image (as shown in FIG. 22). Transfection of SOD1 in NSC-34, BI1-NSC 34 cells G93A A blank group was set, 24 h, and the interaction between Bax and TDP-43 was detected by immunofluorescence interaction. Wherein DAPI marks the nucleus, bax FITC excitation is selected to emit green fluorescence, TDP-43 is selected to emit PE excitation to emit red fluorescence, and Merge images are three-channel integrated images (as shown in FIG. 23). The fluorescence results are shown in FIGS. 22 and 23, in which the interaction between TDP43 and Bax was found to be SOD1 G93A The overexpression is strongest and the interaction of TDP43 with Bax is reduced when BI1 is overexpressed. SOD1 G93A In the over-expression group, the TDP43 and the BI1 are least bound, and the bound cells are basically cells undergoing apoptosis, and the cell nucleus is obviously degraded by shrinkage, so that the TDP43 and the BI1 are not only bound in cytoplasm, but also form a complex in the cell nucleus. The cell morphology also changes obviously, and the cells of the other three groups all tend to be long fusiform, only SOD1 G93A The group cells are severely deformed, also due to apoptosis. Whereas BI1 overexpression can alleviate cell degeneration, cell nucleus arrest, and TDP43-BI1 interaction is also enhanced.
Although BI1 is an inhibitor of Bax, it has been reported that BI1 does not interact directly with Bax, in order to further clarify the interaction of BI1 with TDP43 at SOD1 G93A The role in the signaling pathway of induced apoptosis, we studied the interactions between several other related proteins. Transfection of SOD1 in NSC-34, BI1-NSC 34 cells G93A A blank group was set, and the interaction between Bax and BCL-2 was detected by immunofluorescence interaction after 24. 24 h. Wherein DAPI marks the cell nucleus, bax selects FITC to excite to be green fluorescence, BCL-2 selects PE to excite to be red fluorescence, and Merge images are three-channel integration images. The results are shown in FIG. 24, in which Bax and BCL-2 interact with SOD1, compared to the control group G93A In (2) and the interaction between Bax and BCL-2 is obviously enhanced after BI1 is over expressed. This suggests that BI1 may inhibit apoptosis by enhancing the interaction of BCL-2 with Bax. Transfection of SOD1 in NSC-34, BI1-NSC 34 cells G93A A blank group was set up, and the interaction between Beclin1 and BCL-2 was detected by immunofluorescence interaction after 24. 24 h. Wherein DAPI marks the cell nucleus, beclin1 selects FITC to excite to be green fluorescent, BCL-2 selects PE to excite to be red fluorescent, and Merge images are three-channel integration images. The results are shown in FIG. 25, which shows SOD1 compared with the control group G93A The fluorescence of Beclin1 and BCL-2 of the group is obviously enhanced, which indicates the protein interaction between the twoWith a significant increase. And after BI1 is over-expressed, both fluorescence intensities are significantly reduced. Transfection of SOD1 in NSC-34, BI1-NSC 34 cells G93A A blank group was set, 24 h, and the interaction of MFN2 and TDP-43 was detected by immunofluorescence interaction. Wherein DAPI marks the cell nucleus, MFN2 selects FITC to excite to be green fluorescence, TDP-43 selects PE to excite to be red fluorescence, and Merge images are three-channel integration images. As shown in FIG. 26, SOD1 was compared with the control group G93A The fluorescence intensity of MFN2 and TDP-43 was reduced in the group, while the MFN2 expression level was up-regulated after BI1 overexpression, and the interaction with TDP-43 was enhanced. Transfection of SOD1 in NSC-34, BI1-NSC 34 cells G93A A blank group was set, 24 h, and then the interaction between Beclin1 and TDP-43 was detected by immunofluorescence interaction. Wherein DAPI marks the cell nucleus, beclin1 selects FITC to excite to be green fluorescence, TDP-43 selects PE to excite to be red fluorescence, and Merge images are three-channel integration images. As a result, FIG. 27 shows that SOD1 was obtained in comparison with the control group G93A The interaction of Beclin1 and TDP-43 of the group was not significantly altered. And after BI1 is over-expressed, the fluorescence intensity of the two is enhanced, namely the interaction of the two is enhanced.
6.1 BI1 inhibits the transmission of pathological TDP-43 in exosomes and enhances autophagy
Under pathological conditions of abnormal TDP-43 aggregation, secretion of exosome TDP-43 will increase, and exosome containing pathological TDP-43 plays a key role in disease transmission and development. To clarify the role of BI1, we examined the changes in TDP43 and BI1 in the exosome component when BI1 is overexpressed. Transfection of SOD1 in NSC-34, BI1-NSC 34 cells G93A And setting a blank control group, extracting cell exosome components through an exosome extraction kit after 24 h, and detecting the expression quantity of pathological TDP-43 and BI1 through Western-Blot, wherein CD81 is a marker protein for detecting exosomes. As shown in FIG. 28, the higher expression of TDP43 (35 KD) and SOD1 was detected in the exosome fraction G93A After overexpression, TDP43 (35 KD) secretion was increased, whereas BI1 after overexpression, TDP43 (35 KD) secretion was decreased. A substantial increase in BI1 was also detected in the BI1 over-expressed component in the exosome component, indicating that BI1 is also secreted by the exosome pathway. Inoculation of NSC-34 Fine in 35 mm Petri dishesCells, 5.8. Mu.L GW4869 was added, and a blank control was set, and after 1h of effect at a final concentration of 10 nmol/mL, lacZ, SOD1 was transfected into NSC-34 cells G93A Collecting the whole protein extract after 24-h for Western-Blot experiment examination, selecting GAPDH as an internal reference protein, and detecting the expression level of autophagy abundance marker proteins Beclin1 and LC3B and MAM autophagy related proteins MFN2 and GRP75 and TDP-43. After the exosome inhibitor (GW 4869) was added to the cell fraction, the result of Western-Blot analysis showed that the expression of LC3B was increased as shown in FIG. 29, indicating that the exosome was most likely SOD1 G93A Compensation pathway for induced autophagy inhibition. But GW4869 is specific to SOD1 G93A The induced reduction of Beclin1 and MFN2 has little influence, and can relieve SOD1 G93A The induced GRP75 protein content is reduced. The degradation of TDP43 (43 KD) was not significantly affected, while the production of TDP43 (35 KD) in the cellular fraction was not substantially affected. Inoculating BI1-NSC34 cells in 35 mm culture dish, adding 5.8 μl GW4869, and setting blank control, and transfecting SOD1 on BI1-NSC34 cells after final concentration of 10 nmol/mL for 1 hr G93A And setting a blank control group, collecting a whole protein extract after 24-h for Western-Blot experiment examination, selecting GAPDH as an internal reference protein, and detecting the expression amounts of autophagy-related proteins and TDP-43 on the films of the Beclin1 and LC3B autophagy abundance marker proteins, and the MFN2 and GRP75 MAM. In BI1 overexpressing cells, the results are shown in FIG. 30, although SOD1 cannot be blocked G93A The induced Beclin1 is reduced, but GW4869 can obviously increase the expression (Lane 3) of Beclin1, can also enhance the protein expression of GRP75 and MFN2, relieve the degradation of TDP43 (43 KD), recover the expression of LC3B and enhance autophagy.
The experiments employed in the above studies were specifically as follows:
1. the immunoblotting (Western-Blot) experiment is specifically:
1) Sampling: adding proper amount of protein lysate according to the cell number, and breaking cell wall in ultrasound (4 deg.C, 20 s);
2) Determining the concentration of the sample protein using BCA method;
3) Adding 4X Sample Loading Buffer according to the volume of the sample, and carrying out boiling water bath on the sample for 7 min;
4) Calculating the sample loading volume by the sample concentration measured by the BCA method, and then starting the experimental operation of SDS-PAGE gel electrophoresis;
5) Transferring: proteins on the PAGE gel were transferred to PVDF membrane (90V voltage generated, 4℃used, 2 h);
6) Blocking with TBST blocking solution containing 5% skimmed milk powder for 45 min (room temperature, slow shaking);
7) Adding corresponding primary antibody, and preparing at 4 ℃ overnight (primary antibody, sealing solution and TBST in a certain proportion);
8) Washing the film: TBST is washed for 5 times, each time for 6 min;
9) Adding a corresponding secondary antibody, and slowly shaking at room temperature of 1 h (the secondary antibody, a sealing liquid and TBST are prepared according to a certain proportion);
10 Film washing: TBST is washed 5 times for 6 min/time;
11 Exposure treatment with ECL chemiluminescent developer, and analytical development.
2. TUNEL staining and immunofluorescence assay are specifically:
apoptosis occurs to activate DNA endonucleases, which cleave genomic DNA between nucleosomes. Upon cleavage of genomic DNA, exposed 3' -OH was added dUTP (fluoroscein-dUTP) under the catalysis of terminal deoxynucleotidyl transferase (Terminal Deoxynucleotidyl Transferase, tdT), and Fluorescein (FITC) -labeled dUTP was green-fluorescent under a fluorescence microscope, so that apoptosis was observed by fluorescent microscope detection.
1) The culture broth was discarded and rinsed 2 times with ice PBS;
2) Fixing 4% paraformaldehyde for 25-30 min;
3) Ice PBS rinse 3 times;
4) 0.3% Triton X100 PBS solution was allowed to pass through for 5 min at room temperature;
5) Ice PBS rinse 3 times;
6) Adding TUNEL staining solution, and incubating in a dark place for 2 h;
7) Rinsing with 0.075% PBST for 4 times, 7-8 min each;
8) DAPI staining for 5 min;
9) Washing with ice PBS for 2 min for 3 times;
10 And (3) tabletting and observing.
3. The CCK-8 experiment is specifically as follows:
use of CCK-8 kit for transfection of SOD1 G93A The NSC-34 cells of LacZ and the BI1-NSC 34 cells open cell activity assay because WST-8 (2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium monosodium salt) can be reduced by intramitochondrially dehydrogenases to yield a highly water-soluble orange-yellow formazan product (formazan) whose shade of color is proportional to cell proliferation, inversely proportional to cytotoxicity, and linearly related to the same cell, shade of color and cell number in the presence of an electron-coupling reagent. Therefore, when the initial number of cells is almost uniform, the absorbance after the gradient time can indirectly reflect the number of living cells. The method can adopt an enzyme-labeled instrument to measure the OD value to judge the cell activity intensity, and comprises the following steps:
1) The day before the experiment, the number of cells is generally about 106;
2) The volume of the addition to the 96-well plate is 10:1 and thoroughly mixing;
3) Culturing in a 37 ℃ incubator;
4) The OD values of the samples were measured with a microplate reader at 450 nm every 30 min.
4. The JC-1 mitochondrial membrane potential detection specifically comprises the following steps:
since the polymer formed by mainly concentrating JC-1 in the mitochondrial matrix can emit red fluorescence when the mitochondrial membrane potential is raised, JC-1 cannot accumulate in the mitochondrial matrix when the mitochondrial membrane potential is lowered, and the polymer is a monomer emitting green fluorescence. Meanwhile, the reduction of the mitochondrial membrane potential is a marked event in the early apoptosis stage, so that the observation of the intensity of mitochondrial rubrene-green fluorescence under a fluorescence microscope can intuitively represent the apoptosis condition of cells. Transfection of LacZ, SOD1 with mitochondrial membrane potential detection kit (JC-1) G93A Apoptosis detection is carried out on BI1-NSC 34 cells and NSC-34 cells, and finally, fluorescence intensity is observed through a laser confocal microscope to judge the apoptosis degree, wherein the method comprises the following specific steps:
1) Preparation of JC-1 dyeing working solution: for example, JC-1 was diluted by adding 8 mL ultrapure water to each 50. Mu.L JC-1 (200X), and 2 mL staining buffer (5X) was added and mixed well;
2) A positive control was set. CCCP contained in the kit was set at 1: adding the mixture into a cell culture medium according to the proportion of 1000, diluting to 10 mu L, and treating for 20 min;
3) Loading JC-1: after PBS washing, JC-1 working solution was added, incubated at 37℃for 20 min, and then washed twice with JC-1 buffer and observed under a fluorescence microscope.
5. The ROS reactive oxygen species detection is specifically:
adopts the Biyundian active oxygen detection kit to transfect LacZ and SOD1 G93A The BI1-NSC34 cells and NSC-34 cells were examined for reactive oxygen species and observed under a fluorescent microscope.
6. The exosome extraction is specifically as follows:
1) Collecting cell supernatant, centrifuging at 4deg.C at 3000 Xg for 30 min, and discarding precipitate;
2) Sample and EPS-C amount 2:1, mixing the materials reversely, and standing the materials at 4 ℃ overnight;
3) Precipitating exosomes by centrifugation at 10000 Xg for 30 min at 4 ℃;
4) Discarding the supernatant, collecting the precipitate, centrifuging at 10000 Xg for 5 min, and carefully sucking the supernatant with 10 μl gun head while avoiding the precipitate;
5) To the precipitate, 30. Mu.L of ERS-C solution was added and blown down uniformly. Namely exosomes.
7. The protein immunofluorescence interaction is specifically as follows:
1) The 24-well plate cells were discarded and washed 2 times with ice PBS;
2) Fixing 4% paraformaldehyde at room temperature for 30 min;
3) Ice PBS 3 times, each time 5 min;
4) A0.3% Triton X100 PBS solution was allowed to pass through for 5 min at room temperature.
5) Ice PBS rinse 3 times;
6) Incubating 5% lamb serum and 0.1% Tween20-PBS solution at room temperature for 45 min;
7) After blocking, the antibody and blocking solution 1: preparing primary antibody according to a proportion of 300, and incubating overnight;
8) PBST is washed four times for 7-8 min each time;
9) Preparing a fluorescent secondary antibody, and incubating for 2 h at room temperature and in a dark place;
10 PBST is washed four times for 7-8 min each time;
11 Sealing tablet, and observing under a fluorescence microscope.
8. The mitochondrial extraction operation is specifically as follows:
1) Collection of 2X 10 7 Cells were washed twice with 1 mL precooled PBS, centrifuged at 3000×g for 3 min, and the supernatant discarded;
2) Adding 800 mu L of MIBI into the cell sediment, vibrating for 5s, and standing on ice for 2 min;
3) Adding 10 mu L of MIBII, and shaking vigorously for 5s;
4) Incubation on ice for 5 min, during which shaking is carried out once per minute;
5) Adding 800 mu L of MIBIII, and mixing the mixture for 5 to 6 times in a reverse way;
6) Centrifuging at 4deg.C and 700 Xg for 10 min;
7) Transferring the supernatant to a new centrifuge tube, and centrifuging at 4 ℃ for 15 min at 12000 Xg;
8) Collecting the supernatant to obtain cytoplasmic protein;
9) Adding 500 mu LMIBIII to the precipitate for resuspension;
10 4 ℃, and centrifuging for 15 min at 12000 Xg;
11 Discarding the supernatant, precipitating to obtain mitochondria, and adding protein lysate to perform subsequent protein electrophoresis analysis.
9. The extraction operation of the nucleoprotein is specifically as follows:
1) Collection of 2X 10 7 Cells were washed with 1 mL pre-chilled PBS and centrifuged at 500 Xg for 5 min;
2) Discarding the supernatant, washing the cells with ice PBS, and centrifuging at 500 Xg for 5 min;
3) Removing the supernatant, drying the precipitate as much as possible, and adding 200 mu LCESA;
4) The maximum rotation speed is vortex 15 s, so that cells are completely suspended, and then the sample is placed on ice for 15 min for incubation, so that the cells are expanded;
5) Adding 10 mu LCESB, swirling at the highest speed of 15 s, placing on ice and incubating for 2 min;
6) Centrifuging at 4 ℃ for 5 min at 16000 Xg;
7) Removing supernatant to obtain cytoplasmic extract;
8) To remove possible residual proteins in the nuclei, steps 6, 7 were repeated and the cell pellet was rinsed with ice PBS;
9) 100. Mu.L of ice NES was added to the pellet to suspend it and placed on ice for 30 min, vortexing 15 s every 10 min;
10 4 ℃, and centrifuging at 16000 Xg for 5 min;
11 The supernatant is removed to obtain the nucleoprotein extract, and the subsequent experimental analysis can be performed.
10. The transmission electron microscope method comprises the following steps:
pretreating cells by using a transmission electron microscope cell sample fixing solution, embedding, slicing, finally tabletting, and observing the cell sample by using a JEM-1400 transmission electron microscope.
11. Statistical analysis
Fluorescence intensities in the figures of the present application were calculated using Image J analysis, data were tested for significance using GraphPad prism8.0 software for analysis of variance, and data were expressed as mean ± standard deviation (x ± s). P <0.05, p <0.01, p <0.001, p <0.0001 are the decisions of the difference.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and improvements can be made by those skilled in the art without departing from the spirit of the present invention, and these should also be considered as the scope of the present invention, which does not affect the effect of the implementation of the present invention and the utility of the patent.
Claims (8)
1. Use of Bax inhibitor 1 for the treatment of amyotrophic lateral sclerosis, characterized by comprising use of BI1 for the preparation of a medicament for the prevention and/or treatment of amyotrophic lateral sclerosis.
2. The use of Bax inhibitor 1 according to claim 1 for the treatment of amyotrophic lateral sclerosis, wherein the BI1 plays a role in transfecting cells.
3. Use of Bax inhibitor 1 according to claim 2 for the treatment of amyotrophic lateral sclerosis, characterized in that the transfected cells are prepared by the steps of:
step S1, culturing NSC-34 cells in a 60 mm culture dish, and transferring into pcMV6/T by a transient method
MBIM6-Myc-DDK-tagged plasmid is respectively diluted 10 times, 100 times and 1000 times after 48-h culture, transferred into a 100mm culture dish for continuous culture, then G418 screening is adopted, screened single cells are selected for culture in a 96-well plate, and transferred into a 24-well plate and a 6-well plate for expansion culture in sequence after the cells are full of growth, and cell samples are collected, so that NSC-34 cells which stably over express BI1 are obtained;
Step S2, expanding culture of SOD1 G93A The plasmid and LacZ plasmid are added with 50 mug/mL of antibiotics in LB culture solution of 5 mL, shake-cultured at a constant temperature of 37 ℃ for 8 h, poured into LB culture medium of 300 mL and cultured overnight; extracting transfection-grade plasmid by using a large extraction kit for removing endotoxin;
and S3, transferring the transfection-grade plasmid into NSC-34 cells and BI1-NSC 34 cells to obtain transfected cells.
4. Use of Bax inhibitor 1 according to claim 3 for the treatment of amyotrophic lateral sclerosis, wherein in step S2, the composition is prepared according to 1 in LB broth of 5 mL: antibiotic was added at a rate of 1000. Mu.g/mL.
5. Use of Bax inhibitor 1 according to claim 3 for the treatment of amyotrophic lateral sclerosis, wherein step S3 comprises the steps of:
step S31, inoculating BI1-NSC 34 cells and NSC-34 cells on a cell plate of 60 mm one day before transfection, and incubating in a cell incubator;
s32, respectively adding a transfection reagent and a transfection-grade plasmid into two OPTI-MEM-containing test tubes, and incubating for 5 min at normal temperature;
step S33, uniformly mixing the two test tubes in the step S32, and incubating for 20 min at normal temperature;
step S34, adding the mixed solution of step S33 into the cells in step S31, and gently mixing the cells to obtain transfected cells.
6. The use of Bax inhibitor 1 according to claim 5 for the treatment of amyotrophic lateral sclerosis, wherein in step S31 BI1-NSC 34 cells, NSC-34 cells are seeded on a cell plate of 60 mm one day before transfection at a seeding density of 80-85%.
7. The use of Bax inhibitor 1 according to claim 6 for the treatment of amyotrophic lateral sclerosis, wherein in step S31, at 37 ℃, 5% co 2 The cell culture chamber was incubated with 10% fetal bovine serum and 1% G418 DMEM medium.
8. The use of Bax inhibitor 1 according to claim 5 for the treatment of amyotrophic lateral sclerosis, wherein in step S32, the following transfection reagents: transfection-grade plasmid = 3:1-2:1 ratio the transfection reagent and transfection-grade plasmid were added to two OPTI-MEM containing tubes, respectively.
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