CN117180268A - Cytoprotective preparation containing lipoic acid or lipoamide analogue for alcoholic liver injury and application thereof - Google Patents
Cytoprotective preparation containing lipoic acid or lipoamide analogue for alcoholic liver injury and application thereof Download PDFInfo
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- CN117180268A CN117180268A CN202311231479.5A CN202311231479A CN117180268A CN 117180268 A CN117180268 A CN 117180268A CN 202311231479 A CN202311231479 A CN 202311231479A CN 117180268 A CN117180268 A CN 117180268A
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- ethanol
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- lipoamide
- acetaldehyde
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- AGBQKNBQESQNJD-UHFFFAOYSA-M lipoate Chemical compound [O-]C(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-M 0.000 title claims abstract description 52
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- 206010067125 Liver injury Diseases 0.000 title claims abstract description 21
- 231100000753 hepatic injury Toxicity 0.000 title claims abstract description 21
- FCCDDURTIIUXBY-UHFFFAOYSA-N lipoamide Chemical class NC(=O)CCCCC1CCSS1 FCCDDURTIIUXBY-UHFFFAOYSA-N 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 138
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 claims abstract description 66
- -1 2- (dimethylamino) ethyl Chemical group 0.000 claims abstract description 11
- 206010019837 Hepatocellular injury Diseases 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 2
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Abstract
The invention relates to a cytoprotective preparation for alcoholic liver injury containing lipoic acid or lipoamide analogue and application thereofN- (2- (dimethylamino) ethyl) -5- (1, 2-dithiolan-3-yl) pentanamide (abbreviated DMAE-LA). The invention confirms the effective dosage range of LA and DMAE-LA for inhibiting ethanol and acetaldehyde to induce cell death; adopts PI dye fluorescence imaging to confirm that LA and DMAE-LA improve physiological interference of ethanol to AML12 cell plasma membrane potential; the DHE super-oxygen fluorescent probe is utilized to prove that LA and DMAE-LA effectively remove active oxygen; DMAE-LA is more effective in protecting mitochondrial and lysosomal integrity than LA. The lipoic acid or lipoamide analogues of the invention can protect cells from cytotoxicity induced by ethanol or acetaldehyde, and canIs used as a sulfur-containing medicament with good protecting effect on liver tissue.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a cell protection preparation for alcoholic liver injury of lipoic acid or lipoamide analogues and application thereof.
Background
At present, alcoholic liver disease (alcoholic liver disease; ALD) is the chronic liver disease with the greatest prevalence worldwide, approximately contributing to 9.2% of the burden of all diseases in society in developed countries (calculated as incapacitation of adjusting life years disability adjusted life years [ DALY ]), and the same development situation has also emerged in China. In China, the proportion of frequent drinkers in 2015 adults is increased by 2.5 times compared with 2000, and correspondingly, the incidence of ALD is increased from 2.27% in 2000 to 8.74% in 2015, and the rate of excessive drinking (alcohol use disorder; AUD) is about 5% -7.5%. Long-term alcohol abuse can place a severe burden on other organs such as the heart, leading to a variety of chronic diseases.
In the human body, most of the consumed alcohol (ethanol) is metabolized in liver cells by alcohol dehydrogenase to acetaldehyde, which is then metabolized by acetaldehyde dehydrogenase (acetaldehyde dehydrogenase; ALDH) 2 to acetic acid, which is then decarboxylated to water and carbon dioxide. Acetaldehyde, an intermediate of metabolism, has strong toxicity, mutagenicity and carcinogenicity, and can be directly combined with proteins, lipids or nucleic acids to form a complex in a human body, so that the structure and function of a cell macromolecule are destroyed, resulting in damage of tissues and organs, especially the liver, which is a main metabolic site of alcohol, and thus, acetaldehyde is considered as one of the main factors for promoting the progress of alcoholic liver diseases. Aldehyde group-containing small molecule compounds such as acetaldehyde can form Schiff base with the sulfhydryl group of cysteine residues in cells to generate alkylation injury, and the sulfhydryl group-containing molecules are the most important components of a reactive oxygen species (reactive oxygen species; ROS) elimination system in a body. Acetaldehyde has higher volatility and great difficulty in experimental operation. Moreover, the active aldehyde group of the acetaldehyde molecule can generate wide biological macromolecule injury, and different researches start from different fields and research platforms, so that different conclusions such as DNA injury, autophagy, endoplasmic reticulum stress, plasma membrane injury, cell necrosis, iNOS activation and the like can be found by the acetaldehyde. But there is no more intensive systematic study to clear the causal relationship between these lesions. At present, the specific action mechanism of ROS on toxic injury of alcohol-related acetaldehyde metabolic injury is not completely clear, so that it is difficult to find a general and efficient prevention and treatment scheme for alcohol liver. Studies on cytotoxicity of small molecules with aldehyde groups such as acrolein and gossypol have shown that depletion of small molecules with intracellular thiol groups caused by aldehyde groups is found to be a main driving mechanism of toxicity, and thus ROS accumulate in large amounts, causing Oxidative Stress (OS), mitochondrial dysfunction, DNA damage, endoplasmic reticulum stress, and the like, and finally causing cell death. Therefore, the acetaldehyde induced cell stress is a key cut-in point for solving the toxicity tolerance of the acetaldehyde, and the acetaldehyde induced cell stress deserves further deep analysis and research on the molecular mechanism of the acetaldehyde, so that a control scheme for relieving the alcoholic disease is developed in a targeted system.
Disclosure of Invention
The invention aims to provide a cell protection preparation for alcoholic liver injury of lipoic acid or lipoamide analogues and application of the cell protection preparation as a cell protection agent in alcoholic liver injury, and provides a novel sulfur-containing medicine which is widely suitable for preventing and treating alcoholic diseases.
The present invention provides a cytoprotective preparation for alcoholic liver injury containing lipoic acid or lipoamide analogue.
Further, the lipoamide analogue isN- (2- (dimethylamino) ethyl) -5- (1, 2-dithiolan-3-yl) pentanamide (abbreviated DMAE-LA).
Furthermore, the dosage of the lipoamide analogues is 0.2-25 mu M.
The application of the preparation is used as a medicine with cytoprotective effect on liver cells.
Use of lipoamide analogues for protecting alcoholic liver cell damage.
Use of lipoic acid or lipoamide analogues for the preparation of a cytoprotective preparation for alcoholic liver injury.
Lipoic Acid (LA) is a natural organic sulfur antioxidant, contains disulfide bonds, has strong antioxidation, and can directly neutralize physiological and pathological related ROS such as super oxygen (super oxide), hypochlorous acid (hypochlorous acid; HOCl), hydroxyl radical (hydroxyl radical) and the like in cells. And LA has low toxicity, has the characteristics of fat solubility and water solubility, and can remove free radicals in cells, chelate metals and recover glutathione level in cells. The research of the invention discovers that LA not only can reduce peroxidation of lipid; reducing Reactive Oxygen Species (ROS) accumulation by increasing Glutathione (GSH) levels; LA can also induce apoptosis in liver cancer cells by modulating intracellular ROS production and activation of p53 protein, the efficacy of which is linked to endoplasmic reticulum stress and activation of unfolded proteins; meanwhile, the invention discovers that LA can play a good role in treating alcoholic liver injury in a living body model.
LA is taken up by the cells and reduced to its potent dithiol form, dihydrolipoate (DHLA), most of which is rapidly expelled from the cells. In order to improve the retention in cells, the invention further synthesizes lipoamide analoguesN- (2- (dimethylamino) ethyl) -5- (1, 2-dithiolan-3-yl) pentanamide (abbreviated DMAE-LA). The invention establishes an ethanol and acetaldehyde induced hepatocyte damage model in mature hepatocyte line models including cells such as HepG2 of a human-derived hepatocyte line, AML12 of a mouse-derived non-tumor hepatocyte line and the like. And further, the quantitative effect relationship of ethanol injury is evaluated by utilizing PI cell membrane potential fluorescence imaging and the like, and the spatial position of ethanol induced active oxygen production in DMAE-LA protected cells is studied by combining a probe such as MitoTracker Deep Red and an HE super-oxygen fluorescent probe. Meanwhile, further research finds that the DMAE-LA has better efficacy in protecting the alcoholic cell injury.
Compared with LA, DMAE-LA has a protective effect on ethanol or acetaldehyde-induced AML12 mouse liver cell (or HepG2 liver cell) cell death at a dose which is 20 times smaller (0.2-25 mu M); DMAE-LA more effectively counteracts the inhibitory effect of ethanol on hepatocyte proliferation; the drug effect of the DMAE-LA for relieving ethanol-induced potential injury of liver cell membranes is stronger; DMAE-LA can more effectively improve the intracellular lysosome stress induced by ethanol; DMAE-LA can effectively improve intracellular mitochondrial stress induced by ethanol with smaller dosage; DMAE-LA was effective in improving ethanol-induced intracellular oxidative stress at lower doses. Compared with LA, the DMAE-LA has better drug effect and can be used for a general and efficient prevention and treatment scheme of alcoholic diseases.
Compared with the prior art, the invention verifies the drug effect of LA and LAP for protecting acetaldehyde to induce liver cell death through CCK-8 and other cell death experiments; further characterization of the efficacy of LA and LAP by measuring total GSH (glutathione) levels and MDA (lipid peroxide malondialdehyde) levels in the cell; the mechanism of acetaldehyde induced active oxygen generation and the reasons for mitochondrial injury and lysosome injury are researched through probes such as MitoTracker, related signal paths are deeply dug, the theoretical framework is verified and perfected, and a core initiation mechanism of acetaldehyde toxicity is found. The lipoic acid or lipoamide analogue can be used as a medicament with cytoprotective effect on liver cells and is a general and efficient prevention and treatment scheme of alcohol diseases.
Drawings
FIG. 1 shows that DMAE-LA has better protective effect on ethanol-induced mouse hepatocyte AML12 cell death than LAp<0.05，**p<0.01，***p<0.001 Comparison to DMSO group; # p<0.05， ## p<0.01， ### p<0.001 Comparison to the ethanol (mM) group;
FIG. 2 shows that DMAE-LA has better protective effect on ethanol-induced human hepatocyte HepG2 cell death than LAp<0.05，**p<0.01，***p<0.001 Comparison to DMSO group; # p<0.05， ## p<0.01， ### p<0.001 Comparison to the ethanol (mM) group;
FIG. 3 shows DMAE-LA has better protective effect on acetaldehyde-induced mouse hepatocyte AML12 cell death than LAp<0.05，**p<0.01，***p<0.001 Comparison to DMSO group; # p<0.05， ## p<0.01， ### p<0.001 Comparison to group acetaldehyde (mM);
FIG. 4 shows DMAE-LA has better protective effect on acetaldehyde-induced human hepatocyte HepG2 cell death than LAp<0.05，**p<0.01，***p<0.001 Comparison to DMSO group; # p<0.05， ## p<0.01， ### p<0.001 Comparison to group acetaldehyde (mM);
FIG. 5 is a PI imaging analysis of DMAE-LA protected ethanol induced damage to AML12 mouse hepatocyte cell membrane potential;
FIG. 6 is the protective effect of DMAE-LA on ethanol-induced mitochondrial stress in AML12 mouse hepatocytes;
FIG. 7 is the protective effect of DMAE-LA on ethanol-induced AML12 mouse hepatocyte lysosomal stress;
FIG. 8 is that DMAE-LA reduced the level of ROS associated with ethanol-induced intracellular oxidative stress in HepG2 human liver cells.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.
Example 1
Performance study of LA and lipoamide analogues DMAE-LA to protect against alcoholic cell damage.
1. Cell culture
(1) Cell culture
Human liver cells HepG2 cells were cultured in MEM medium (batch number: PM150411, wohaze life technologies Co., ltd.) +10% serum+1% antibiotics at 37℃with 5% CO 2 Culturing in an incubator. Murine non-tumor hepatocytes AML12 cells were cultured in DMEM/F12 medium (batch number: CM-0602, wohaze life technologies Co., ltd.) +10% serum+1% antibiotics at 37deg.C, 5% CO 2 Culturing in an incubator.
(2) Cell passage
And observing the cells in the cell bottle under a microscope, and passaging when the cells are converged to 80-90%. When cells were digested with trypsin and cell pseudopodia was observed to begin to shrink and round, the digestion was stopped by adding a serum-containing culture medium. After carefully blowing the cells to homogeneity, the cells were sub-bottled for passage at a ratio of 1:3.
(3) Experimental cell plating
The AML12 cells (or HepG2 cells) in logarithmic growth phase are digested to prepare single cell suspension, and the cell concentration is regulated after cell counting. And (3) planting the regulated cell suspension in a pore plate required by experiments, slowly placing the cell suspension in an incubator for culture, and treating the cells according to the experimental requirements after the cells are completely adhered.
TABLE 1 reference concentration of cell suspensions under different experimental conditions
2. Lipooctanoamide analogue DMAE-LA protective alcoholic liver cell injury condition screening
The interfering concentrations of LA and DMAE-LA were screened using the cell viability assay (cell counting kit-8 assay; CCK-8). The liver cell line model (HepG 2 cell of human source and AML12 cell of mouse source) which is common and important is selected, and alcohol liver model is established by using ethanol or acetaldehyde induction. The sulfur-containing drug lipoic acid and its lipoamide analogs (including LA, DMAE-LA) were then validated.
(1) Experimental grouping:
1) Murine non-tumor hepatocyte line ethanol induced hepatocyte injury model (AML 12):
(1) a negative control group; (baseline without drug treatment)
(2) A vehicle group; (DMSO)
(3) A model group; (600 mM ethanol was added to create an alcoholic hepatocyte injury model)
(4) Experiment group 1; (600 mM ethanol. Fwdarw. 25, 100, 400. Mu.M dose of LA, respectively)
(5) Experiment group 2; (600 mM ethanol. Fwdarw. 0.04, 0.2, 1. Mu.M dose of DMAE-LA, respectively)
2) Ethanol-induced hepatocyte injury model of human liver cell line (HepG 2):
(1) a negative control group; (baseline without drug treatment)
(2) A vehicle group; (DMSO)
(3) A model group; (ethanol 400 mM is added to establish an alcoholic liver cell injury model)
(4) Experiment group 1; (ethanol 400 mM is added to establish an alcoholic hepatocyte injury model → 5, 25, 100. Mu.M doses of LA are added respectively)
(5) Experiment group 2; (ethanol 400 mM was added to model alcoholic liver cell injury. The addition of DMAE-LA at 0.2, 1, 5. Mu.M doses, respectively)
3) Murine non-tumor hepatocyte lineage acetaldehyde-induced hepatocyte injury model (AML 12):
(1) a negative control group; (baseline without drug treatment)
(2) A vehicle group; (DMSO)
(3) A model group; (addition of 10 mM acetaldehyde to establish an alcoholic liver cell injury model)
(4) Experiment group 1; (addition of 10 mM acetaldehyde. Fwdarw. Addition of 1, 5, 25. Mu.M dose of LA, respectively)
(5) Experiment group 2; (addition of 10 mM acetaldehyde. Fwdarw. Addition of DMAE-LA in a dose of 0.2, 1, 5. Mu.M, respectively)
4) Model of acetaldehyde-induced hepatocyte injury of human liver cell line (HepG 2):
(1) a negative control group; (baseline without drug treatment)
(2) A vehicle group; (DMSO)
(3) A model group; (addition of 10 mM acetaldehyde to establish an alcoholic liver cell injury model)
(4) Experiment group 1; (addition of 10 mM acetaldehyde to model alcoholic liver cell injury. To 5, 25, 100. Mu.M doses of LA)
(5) Experiment group 2; (addition of 10 mM acetaldehyde to model alcoholic liver cell injury. To 1, 5, 25. Mu.M dose of DMAE-LA, respectively)
(2) The experimental process comprises the following steps:
the cell viability was measured using a CCK-8 assay kit (lot number: C0038, shanghai Biyun biotechnology Co., ltd.). 96-well plates were 1X 10 5 cells were seeded at cell/mL density, 6 duplicate wells were used, 100. Mu.L of cell suspension was added to each well, 100. Mu.L of cell-free complete medium was used as zeroing wells, and drug was added 24 hours after cell seeding. The dishes were placed in 5% CO 2 Incubate at 37℃for 24 hours in incubator. 10. Mu.L of CCK-8 solution was added and incubated in an incubator for 1 hour, and absorbance was measured at 450 nm using a microplate reader.
(3) Experimental results:
LA has a protective effect on ethanol (or acetaldehyde) induced cell death of AML12 mouse hepatocytes (or HepG2 human hepatocytes) as measured by AML12 cells (or HepG2 cells) cell activity (24 h); the DMAE-LA (0.2-25 mu M) with a significantly smaller dosage range has a good protection effect on ethanol (or acetaldehyde) induced cell death of AML12 mouse liver cells (or HepG2 liver cells), further shows that the DMAE-LA can also effectively protect ethanol metabolism poison acetaldehyde induced liver injury, and the DMAE-LA has better drug effect, as shown in figures 1,2, 3 and 4.
3. DMAE-LA improves the effect of ethanol on cell membrane potential
The fluorescent dye PI (propidium iodide) is a nuclear staining reagent capable of staining DNA and is commonly used for apoptosis detection. It is an analogue of ethidium bromide that releases red fluorescence upon intercalation into double stranded DNA. Although PI cannot pass through living cell membranes, it can pass through broken cell membranes to stain nuclei. Red staining indicates that plasma membrane potential of cells that have been or are dying cannot be maintained, so PI can cross cell membranes to stain nucleic acids.
(1) Experimental grouping:
(1) a control group; (baseline without drug treatment)
(2) A model group; (600 mM ethanol was added to create an alcoholic hepatocyte injury model)
(3) Positive control experimental group; (600 mM ethanol. Fwdarw. 100. Mu.M dose of LA was added)
(4) DMAE-LA experimental group; (600 mM ethanol. Fwdarw. 25. Mu.M DMAE-LA was added separately)
(2) The experimental process comprises the following steps:
evaluation of DMAE-LA by PI dye fluorescence imaging can effectively improve the influence level of ethanol on the potential damage of hepatic cell membranes. AML12 cells in the logarithmic growth phase are inoculated into a 12-well plate, the cells are divided into 4 groups, a control group, a model group, a positive control experimental group and a DMAE-LA experimental group are established in experiments, 3 compound holes are formed in each group, and PI with the final concentration of 1.5 mu mol/L is added. Immediately after PI dye input, imaging (red fluorescence) was performed, the photographing exposure time was set to 700 ms, and 20-fold (objective) fluorescence imaging was performed. ZEISS Observer A1 was photographed by observation with an inverted fluorescence microscope. Fluorescence intensities were measured using Image J software to select 3 low power mirror fields, and their average was taken and statistically analyzed by GraphPad Prism 8.
(3) Experimental results:
through PI staining imaging of AML12 cells, LA has a protective effect on ethanol-induced liver cell death of AML12 mice, and DMAE-LA has a protective effect on ethanol-induced liver cell death of AML12 in a smaller dosage range, which shows that the effect of the DMAE-LA on protecting ethanol-induced liver injury is stronger, as shown in figure 5.
4. DMAE-LA improves ethanol-induced mitochondrial stress in hepatocytes
Mitochondrial ROS injury under drugs was assessed by MitoTracker Deep Red live intracellular confocal imaging. MitoTracker Deep Red (lot number: M22426, invitrogen) is a far infrared fluorescent dye (absorption/emission wavelength 644/665 nm) that stains mitochondria in living cells and can be used for mitochondrial localization.
(1) Experimental grouping:
(1) a control group; (baseline without drug treatment)
(2) A technical reference group; ( Adding CCCP group with proper concentration; CCCP, carboyl cyanide 3-chlorophenylhydrozone, is a powerful oxidative phosphorylation uncoupler of mitochondria, leading to loss of membrane potential across the inner mitochondrial membrane )
(3) A model group; (600 mM ethanol was added to create an alcoholic hepatocyte injury model)
(4) Positive control experimental group; (600 mM ethanol. Fwdarw. 100. Mu.M dose of LA was added)
(5) DMAE-LA experimental group; (600 mM ethanol. Fwdarw. 25. Mu.M DMAE-LA was added separately)
(2) The experimental process comprises the following steps:
AML12 cells in the logarithmic growth phase were inoculated into 3.5. 3.5 cm confocal proprietary basal cell dishes, the cells were divided into 5 groups, and after overnight cell attachment. After 1 hour of pretreatment with LA/DMAE-LA, ethanol was added for another 1 hour, and after 3 times of washing with HBSS, 25 nM MitoTracker Deep Red fluorescent probe (probe diluted with DMSO, system incubated with serum-free medium) was added and incubated in a cell incubator at 37℃for 30 minutes. Fluorescence imaging was performed using a Zeiss 880 confocal microscope live cell workstation.
(3) Experimental results:
LA and DMAE-LA were effective in protecting ethanol-induced mitochondrial oxidative damage by fluorescence imaging within MitoTracker Deep Red living cells, demonstrating that LA and DMAE-LA have a modulating effect on ethanol-induced intracellular ROS oxidative stress. While DMAE-LA is effective in improving ethanol-induced intracellular mitochondrial stress at smaller doses (only 1/4 of LA dose) compared to LA, as shown in figure 6.
5. DMAE-LA improves ethanol-induced intracellular lysosomal stress
Lysosomal ROS injury under drug was assessed by LysoTracker Deep Red live intracellular confocal imaging. LysoTracker Deep Red probes consist of a fluorescent group bound to a weak base and are only partially protonated at neutral pH. This property allows the LysoTracker Deep Red probe to freely permeate the cell membrane and label living cells. LysoTracker Deep Red is a dark red fluorescent dye that can be used to label and track acid organelles in living cells.
(1) Experimental grouping:
(1) a control group; (baseline without drug treatment)
(2) A model group; (ethanol 400 mM is added to establish an alcoholic liver cell injury model)
(3) Positive control experimental group; (addition of 400 mM ethanol. Fwdarw. Addition of 100. Mu.M dose of LA, respectively)
(4) DMAE-LA experimental group; (addition of 400 mM ethanol. Fwdarw. Addition of 5. Mu.M dose of DMAE-LA, respectively)
(2) The experimental process comprises the following steps:
AML12 cells in the logarithmic growth phase were inoculated into 3.5. 3.5 cm confocal proprietary basal cell dishes, the cells were divided into 6 groups, and after overnight cell attachment. After 1 hour of pretreatment with LA/DMAE-LA, ethanol was added for another 1 hour, and after 3 times of washing with HBSS, 25 nM LysoTracker Deep Red fluorescent probe (probe diluted with DMSO, system incubated with serum-free medium) was added and incubated in a cell incubator at 37℃for 30 minutes. Fluorescence imaging was performed using a Zeiss 880 confocal microscope live cell workstation.
(3) Experimental results:
LA and DMAE-LA were effective in protecting ethanol-induced mitochondrial oxidative damage by fluorescence imaging within LysoTracker Deep Red living cells, further demonstrating that LA and DMAE-LA have a modulating effect on ethanol-induced intracellular ROS oxidative stress. DMAE-LA also effectively ameliorates ethanol-induced intracellular lysosomal stress at a smaller dose (20-fold smaller than LA) and in a dose-dependent manner compared to LA, as shown in figure 7. In addition, as can be seen from FIG. 7, only DMAE-LA effectively protected the intracellular lysosomes (an organelle important for cell homeostasis, metabolism, immunity and survival) exhibited by Lyso-Tracker staining, whereas LA did not. Abnormal assimilation of redox-active iron exacerbates oxidative tissue damage, while the most important redox-active iron cell pools are present in lysosomes, making these organelles susceptible to oxidative stress. In experiments using airway epithelial cells and macrophages, chelation of iron in the lysosomes effectively prevented hydrogen peroxide-induced lysosomal rupture and subsequent cell death.
6. DMAE-LA inhibits ethanol-induced intracellular ROS formation (reduces oxidative stress)
DMAE-LA was evaluated by fluorescence imaging of DHE (dihydroethidium) to inhibit ethanol-induced intracellular ROS formation (decrease oxidative stress). DHE is the most commonly used fluorescent probe for detecting superoxide anions and can effectively detect active oxygen. The dye can enter cells freely and is dehydrogenated to form ethidium bromide under the action of superoxide anions in the cells. Ethidium bromide can bind to RNA or DNA to produce red fluorescence. When the level of superoxide anions in the cells is higher, more ethidium bromide is generated, the red fluorescence is stronger, and conversely, the red fluorescence is weaker. Thus, the hydrogen ethidium can be used for detecting the level of superoxide anions. The superoxide anion is used as a free radical and is provided with an unpaired electron, and the magnetization characteristics of the superoxide anion are unchanged before and after single electron reduction. In the oxidation reaction in the human body, four electrons are required for complete reduction of a single oxygen molecule. If the oxygen molecules are reduced by only a single electron added, the intermediate product formed is superoxide anion (O 2 •− ) The properties are very active, and the reaction with macromolecules such as proteins, polysaccharides and the like in the body is easy to lose activity. In Reactive Oxygen Species (ROS), the generation of other species of ROS is derived from superoxide anions.
(1) Experimental grouping:
(1) a control group; (baseline without drug treatment)
(2) A technical reference group; (addition of the appropriate concentration of the anti-mycin A group, namely antimycin A, is the first known and most potent mitochondrial respiratory chain inhibitor and can be used as a model for studying mitochondrial respiration and superoxide production mechanisms)
(3) A model group; (ethanol 400 mM is added to establish an alcoholic liver model)
(4) Positive control experimental group; (addition of 400 mM ethanol. Fwdarw. Addition of 250. Mu.M dose of LA, respectively)
(5) DMAE-LA experimental group; (addition of 400 mM ethanol. Fwdarw. Addition of 5. Mu.M dose of DMAE-LA, respectively)
(2) The experimental process comprises the following steps:
DHE fluorescent probes were used to detect ROS levels in ethanol-induced cells-injured hepatocytes following DMAE-LA action. HepG2 cells in the logarithmic growth phase were inoculated into 12-well plates, the cells were divided into 5 groups, 3 duplicate wells were placed in each group, DHE was added at a final concentration of 5. Mu. Mol/L, and incubated in an incubator at 37℃for 30 min. After the incubation, the ZEISS Observer A1 is observed and photographed by a phase contrast inverted fluorescence microscope. Fluorescence intensities were measured using Image J software to select 3 low power mirror fields, and their average was taken and statistically analyzed by GraphPad Prism 8.
(3) Experimental results:
through DHE living cell fluorescence imaging, LA and DMAE-LA can effectively protect ethanol from inducing ROS oxidative damage, and further prove that LA and DMAE-LA have regulating effect on ethanol-induced ROS oxidative stress in liver cells. And compared with LA, the DMAE-LA can effectively improve the intracellular oxidative stress induced by ethanol with smaller dosage (only 1/4 of the dosage of LA is needed).
Experiments show that the protection effect of DMAE-LA on acetaldehyde-induced alcoholic liver injury is similar to that of DMAE-LA, so that experiments and data of the protection effect of DMAE-LA on acetaldehyde-induced alcoholic liver injury are omitted in the embodiment.
Claims (7)
1. A cytoprotective preparation for alcoholic liver injury containing lipoic acid or lipoamide analogue is provided.
2. According to claimThe cytoprotective preparation for alcoholic liver injury of claim 1, wherein the lipoamide analogue isN- (2- (dimethylamino) ethyl) -5- (1, 2-dithiolan-3-yl) pentanamide.
3. The cytoprotective preparation for alcoholic liver injury according to claim 1, wherein the lipoamide analogue is used in a dosage of 0.2-25 μm.
4. The cytoprotective formulation of alcoholic liver injury of claim 1, wherein the alcoholic liver injury is induced by ethanol or acetaldehyde.
5. Use of a cytoprotective agent for alcoholic liver injury according to claim 1 as a cytoprotective agent for alcoholic liver injury.
6. Use of lipoic acid or lipoamide analogues to protect alcoholic hepatocellular injury.
7. Use of lipoic acid or lipoamide analogues for the preparation of a cytoprotective preparation for alcoholic liver injury.
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