Application of peptidoglycan in preparation of hepatocyte mitochondrial autophagy inducer

Abstract

The invention discloses application of peptidoglycan in preparation of a hepatocyte mitochondrial autophagy inducer, and belongs to the technical field of biological medicine. The invention finds that a bacterial cell wall component peptidoglycan can activate autophagy of damaged mitochondria. Experiments prove that the peptidoglycan serving as the bacterial cell wall component can activate mitochondrial autophagy to remove damaged mitochondrial, further inhibit liver fibrosis and effectively relieve liver injury, so that the peptidoglycan serving as the bacterial cell wall component can be used for preparing the medicine for activating mitochondrial autophagy.

Description

Technical Field

[0001] The invention relates to application of peptidoglycan in preparing a hepatocyte mitochondrial autophagy inducer, and belongs to the technical field of biomedicine. Background Art

[0002] Peptidoglycan, a major component of bacterial cell walls, is composed of linear polysaccharide chains formed by alternating glycosidic bonds between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Current research indicates that the primary function of PGN lies in its immunomodulatory effects, characterized by its ability to activate host immune pathways and cells, thereby influencing the intensity and direction of immune responses.

[0003] As a core organ for metabolism and detoxification in the human body, the liver is highly dependent on the normal function of mitochondria to maintain energy supply and metabolic homeostasis. Mitochondria generate ATP through oxidative phosphorylation, supporting the liver's physiological activities such as biosynthesis, detoxification, and metabolic regulation. Mitochondrial dysfunction is closely related to a variety of liver diseases, including alcoholic liver disease, non-alcoholic fatty liver disease, viral hepatitis, and liver fibrosis. Among them, liver fibrosis, as a key pathological stage in the progression of chronic liver disease, is closely related to mitochondrial function and mitochondrial autophagy.

[0004] Activation of hepatic stellate cells (HSCs) is a key component of liver fibrosis. Mitochondrial dysfunction can significantly impact HSC status: damaged mitochondria produce excessive reactive oxygen species (ROS), which activate HSCs and promote extracellular matrix (ECM) secretion, exacerbating fibrosis. Disturbances in mitochondrial homeostasis (e.g., excessive fission and insufficient fusion) lead to mitochondrial fragmentation, further impairing mitochondrial function and releasing profibrotic signals. Furthermore, disturbances in mitochondrial energy metabolism can alter HSC activation and promote fibrosis progression.

[0005] Mitochondrial autophagy, as a key mechanism for the selective removal of damaged mitochondria, is crucial for the regulation of liver fibrosis. Normal mitophagy can promptly remove dysfunctional mitochondria, reduce ROS production and oxidative stress, inhibit HSC activation and ECM deposition, and thus alleviate liver fibrosis. Conversely, defective mitophagy can lead to the accumulation of damaged mitochondria, aggravate oxidative damage and HSC overactivation, and accelerate the process of fibrosis. Therefore, mitophagy plays a key regulatory role in the occurrence and development of liver fibrosis by maintaining mitochondrial quality and functional homeostasis. Its mechanism research provides potential targets and strategies for the prevention and treatment of liver fibrosis, such as improving mitochondrial function by enhancing mitophagy activity, thereby blocking or reversing liver fibrosis. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides an application of peptidoglycan in preparing a hepatocyte mitochondrial autophagy inducer.

[0007] Preferably, the peptidoglycan is extracted from Escherichia coli.

[0008] The present invention has special requirements for the Escherichia coli substance, as long as it is Escherichia coli.

[0009] In order to verify the role of peptidoglycan, a component of bacterial cell walls, the present invention conducted corresponding cell experiments and animal experiments, which proved that peptidoglycan can indeed activate mitochondrial autophagy in damaged liver cells without affecting the function of normal mitochondria.

[0010] Technical effects of the present invention: (1) The present invention discovered that peptidoglycan, a component of bacterial cell walls, can activate mitochondrial autophagy in damaged liver cells without affecting the function of normal mitochondria, providing a new use for peptidoglycan, a component of bacterial cell walls.

[0011] (2) The present invention has demonstrated through cell experiments and mouse experiments that peptidoglycan, a component of bacterial cell walls, can indeed activate mitochondrial autophagy in damaged liver cells, providing a new idea for the preparation of mitochondrial autophagy activators. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The 3D reconstruction analysis of PGN and mitochondria in Example 2 shows the subcellular localization of PGN and mitochondria in mouse liver cells; Figure A is an immunofluorescence image, Figure B is a 3D reconstruction image, and Figure C is an enlarged image corresponding to the red box.

[0013] Figure 2 These are confocal fluorescence images of AML12 cells treated with PGN or PBS for 48 h in Example 2.

[0014] Figure 3 Flow cytometry histogram of mitochondrial membrane potential in Example 2.

[0015] Figure 4 Figure 3 is a flow cytometry analysis of mitophagy in Huh7 cells based on mtkeima after treatment with PGN for 24 hours in Example 3, wherein Figure A is a representative flow cytometry result; Figure B is a quantitative analysis of the mitophagy level in the cells.

[0016] Figure 5 This is the fluorescence staining and quantification of mtKeima based on 293T cells after PBS or PGN treatment for 24 hours in Example 3.

[0017] Figure 6 This is a protein immunoblot image of the mitochondrial autophagy level in primary hepatocytes after treatment with PGN and 5 mM APAP for 48 h in Example 3.

[0018] Figure 7 For the mitochondrial autophagy flux detection in Example 3, AML12 cells were treated with PGN for 48 h and BAF (400 nmol / L) for 6 h. Protein levels of autophagy-related proteins LC3 and P62 are shown.

[0019] Figure 8 The number of autophagosomes detected by transmission electron microscopy and the statistical graph after AML12 cells were treated with PBS or PGN for 48 hours in Example 2, with arrows indicating autophagosomes.

[0020] Figure 9 Schematic diagram of AAV8-mtkeima-based mitochondrial autophagy detection in the liver of C57B / 6J mice after gavage with PBS or PGN in Example 4.

[0021] Figure 10 These are confocal fluorescence live cell images of the mtkeima-based detection of mitophagy levels in the liver of C57B / 6J mice after gavage with PBS or PGN in Example 4.

[0022] Figure 11 This is the quantitative analysis of mitophagy levels in the liver of C57B / 6J mice after oral gavage with PBS or PGN in Example 4, using confocal fluorescence live cell analysis.

[0023] Figure 12 Schematic diagram of the experimental design for investigating the effect of PGN in the CCL4 liver fibrosis model in Example 5.

[0024] Figure 13 This is the detection of alanine aminotransferase (ALT) activity in the serum of fibrosis mice after gavage with vehicle (corn oil), PBS or PGN in Example 5.

[0025] Figure 14 The figure shows Sirius Red staining of paraffin sections of liver tissue from normal and fibrotic mice following oral gavage with PBS or PGN in Example 5. The figure on the right shows quantitative analysis of the positive fibrosis area in the Sirius Red staining. n is the number of mice in each group.

[0026] Figure 15 Desmin staining of frozen sections of liver tissue from normal and fibrotic mice after oral administration of PBS or PGN. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the contents described above.

[0028] Unless otherwise specified, all reagents used in the embodiments can be purchased through conventional commercial channels.

[0029] The present invention does not protect the experimental methods. The methods involved in the specific embodiments are all routine operations familiar to people in this field and are only for verifying the function of peptidoglycan.

[0030] Preparation of main reagents in the examples: (1) Solution B preparation: 300 μL (5 mg / mL) DNase and 360 μL (5 mg / mL) RNase dissolved in 29.34 mL Tris-HCl (pH 6.8).

[0031] (2) Preparation of 20 mL trypsin solution (50 μg / mL): Dissolve 500 μL (2 mg / mL) trypsin in 19.5 mL of RO water.

[0032] (3) 10 mL of FITC (1 mg / mL): Dissolve 10 mg of FITC in 10 mL of Carbonate Buffer.

[0033] (4) 50 mL Carbonate Buffer (50 mmol / L): Dissolve 29.1 mg of Na2CO3 and 142.81 mg of NaHCO3 in 40 mL of water, adjust to pH 9.2, and then add water to 50 mL.

[0034] Example 1 Preparation method of bacterial cell wall peptidoglycan: (1) Cultivate E. coli culture to OD 600 When the pH value was ≈1.2, centrifuge at 8000×g for 5 min at room temperature, remove the supernatant, add 6 mL of NaCl to resuspend the bacteria, and then distribute them into 2 mL EP tubes, 1 mL per tube, and cook the samples in a metal bath at 100°C for 30 min.

[0035] (2) Wash the cells with sterilized RO water three times, 10 min each time, and then centrifuge at room temperature and 13,000 × g for 5 min to retain the precipitate.

[0036] (3) Resuspend the precipitate in 6 mL of ddH2O and place in a 15 mL tube. Place the sample on ice and sonicate for 1 h (power 25%, on 5 s, off 5 s).

[0037] (4) After the ultrasound is completed, centrifuge at 10,000 × g for 5 min at room temperature and remove the supernatant.

[0038] (5) The precipitate was resuspended in 15 mL of Solution B, incubated on a shaker at 37 °C for 1 h, and centrifuged at 10,000 × g for 5 min at room temperature to collect the precipitate.

[0039] (6) The precipitate was resuspended with 8.3 mL of trypsin solution, mixed at room temperature for 1 h, and then centrifuged at room temperature and 10,000 × g for 5 min. The supernatant was removed and the precipitate was resuspended with 6 mL of sterile RO water, washed with RO water three times, and then centrifuged at room temperature and 13,000 × g for 5 min to collect the precipitate.

[0040] (7) Resuspend the precipitate in 6 mL of sterile RO water, divide into 6 tubes, and boil the sample in a metal bath at 100 °C for 5 min.

[0041] (8) Centrifuge at room temperature and 13,000 × g for 5 min, then resuspend in 1 mL of DMEM in a clean bench to obtain peptidoglycan, which was then stored at -20°C until use.

[0042] Example 2 Peptidoglycan targets and binds to liver cell mitochondria and improves their membrane potential, specifically including the following processes: First, peptidoglycan was labeled with fluorescein isothiocyanate (FITC): 500 μL of PGN (prepared in Example 1) was taken and sonicated in a water bath for 30 min (power 99%, on 5 s, off 5 s); centrifuged at room temperature and 13,000 × g for 5 min, and the pellet was resuspended in 500 μL of FITC (1 mg / mL); wrapped in tin foil to protect from light and incubated at room temperature for 1 h; centrifuged at 17,000 × g for 2 min, discarded the supernatant, and the pellet was resuspended in 1 mL of PBS and washed three times, 5 min each time; finally, the pellet was resuspended in 500 μL of PBS to obtain FITC-PGN and stored at 4°C.

[0043] FITC binds to the lysine residues on the peptidoglycan (PGN) peptide chain, making the peptidoglycan carry green fluorescence. AML12 hepatocytes were treated with 200 μL of FITC-PGN for 48 hours. Subsequently, the mitochondrial outer membrane in the AML12 hepatocyte line was labeled with TOMM20 primary antibody. Next, Alexa Fluor 568-labeled goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody was used to make the mitochondrial outer membrane appear red fluorescent. After photographing under a confocal microscope, three-dimensional (3D) reconstruction was performed. The results are shown below. Figure 1 As shown, it was found that FITC-PGN was indeed attached to the periphery of mitochondria.

[0044] Next, we explored whether peptidoglycan had adverse effects on mitochondrial status. We used transmembrane potential-sensitive MitoTracker Deep Red (MTDR) to label healthy mitochondria and insensitive MitoTracker Green to label all mitochondria. We set up two experiments, PBS and PGN, to detect the status of cell mitochondria. AML12 cells cultured in 35mm confocal dishes were treated with 200μL PBS or 200μL PGN for 48h, and then incubated with 100nM MitoTracker Green in a 37℃ incubator for 15min, washed twice with preheated PBS, and then incubated with 200nM MitoTracker Deep Red in a 37℃ incubator for 15min, washed twice with preheated PBS, and finally added 1ml of DMEM for culture. The cells were observed under a 60× oil lens under a confocal microscope, and the fluorescence images were finally statistically analyzed by ImageJ. The results are shown in the figure. Figure 2 As shown, PGN was found to have no adverse effects on mitochondrial health.

[0045] Mitochondrial membrane potential is a key indicator of mitochondrial status. Under normal circumstances, mitochondria pump protons through the electron transport chain to form a stable membrane potential, supporting efficient ATP synthesis and indicating intact function. During active cellular metabolism, the membrane potential rises to meet energy demand; when metabolism slows, it remains low. However, after apoptosis is initiated, mitochondrial membrane permeability changes, and the membrane potential rapidly depolarizes and decreases, prompting the release of apoptosis-related proteins, mitochondrial dysfunction, and the cell to undergo programmed cell death.

[0046] 293T cells cultured in six-well plates were treated with 200 μL of PGN, 5 mM APAP, and 5 mM PBS for 24 h, then trypsinized and neutralized with DMEM (containing FBS). The cells were centrifuged at 1000 rpm for 3 min, the supernatant discarded, and the cells were resuspended with 200 nM TMRM dye and incubated in a 37°C, 5% CO2 constant temperature cell culture incubator for 30 min. The cells were washed twice with PBS, 3 min each time, and resuspended in PBS (containing 2% FBS). Flow cytometry analysis was performed using the PE channel (excitation light 488 nm, emission light 570 nm). The results showed that the membrane potential of the PGN-treated group was significantly increased compared with the APAP- and PBS-treated groups ( Figure 3 ), this experiment shows that PGN has a certain effect on improving mitochondrial status.

[0047] Example 3 The study on the activation of mitophagy by PGN includes the following steps: The acid-sensitive protein Keima is used. Keima protein exhibits green fluorescence in the neutral environment of the cytoplasm and red fluorescence in the acidic environment of the lysosome.

[0048] The Keima protein was engineered into mito-Keima, a mitochondrial-targeting protein, which causes mitochondria to exhibit green fluorescence. Keima protein characteristics: In acidic environments (such as lysosomes, pH ≈ 4.5-5.5), green fluorescence is quenched, emitting red fluorescence. In neutral environments (such as the cytoplasm or normal mitochondria, pH ≈ 7.0), it can emit both red and green fluorescence. When mitochondria are encapsulated by autophagosomes and fuse with lysosomes to form autolysosomes, mito-Keima enters the acidic environment, significantly enhancing its red fluorescence. The red / green fluorescence intensity ratio can be used to quantitatively reflect mitophagy activity.

[0049] Huh7 cells infected with the mito-Keima lentivirus were treated with 200 μL of DMEM and 200 μL of PGN, respectively, along with 2 μg / mL puromycin. After 24 hours, the cells were trypsinized, washed twice with PBS, and resuspended in PBS containing 2% FBS for flow cytometry analysis. Mito-Keima was dual-excited at 405 nm (neutral pH) and 561 nm (acidic pH), with emission at 620 / 29 nm and 614 / 20 nm, respectively. 10,000 cells were collected for each sample, and data were analyzed using FlowJo. Huh7 cells treated with 50 μM CCCP for 2 hours served as a positive control. Flow cytometry results showed a significant increase in red fluorescence in the PGN-treated group compared to the control group, indicating that PGN activates mitophagy.

[0050] At the same time, 293T cells infected with mito-Keima lentivirus were treated with 200 μL PBS or 200 μL PGN for 24 h, and confocal microscopy was performed. The colocalization of red and green double positive signals was quantitatively analyzed using ImageJ. The results also showed that compared with the control group, the red fluorescence of the PGN-treated group was significantly upregulated, indicating that PGN can activate mitochondrial autophagy ( Figure 4~Figure 5 ).

[0051] LC3 and P62 are autophagy-related proteins. During the autophagy process, P62 acts as an autophagy substrate receptor and promotes the formation of autophagosomes by connecting ubiquitinated mitochondrial membrane proteins with LC3. When autophagy occurs in cells, the protein content of LC3Ⅱ increases, while the protein content of P62 decreases.

[0052] Primary hepatocytes (Huh7 cells) were divided into four groups for experiments. The cells were treated with 200 μL PBS, 200 μL PGN, 5 mM APAP, and 5 mM APAP + 200 μL PGN for 24 hours. The cells were then lysed with RIPA and the supernatant was collected by centrifugation to prepare samples for protein immunoblotting. The results showed that compared with the PBS group, the expression of LC3Ⅱ and P62 in the PGN group was significantly increased ( Figure 6), the protein level of P62 increased significantly after PGN treatment, which may indicate that the mitochondrial autophagy activated by PGN is incomplete. Incomplete autophagy refers to the obstruction of the fusion of autophagosomes and lysosomes, which leads to the accumulation of autophagosomes.

[0053] To verify whether PGN-activated mitochondrial autophagy is complete, an autophagy flux detection experiment was performed using the autophagosome-lysosome fusion inhibitor Bafilomycin A1 (BAF). The cells were divided into four groups: PBS, BAF, PGN, and PGN+BAF. Two groups of AML12 cells were treated with 200 μL PBS for 48 h, and BAF was added to one of the groups 6 h before sampling to a concentration of 400 nmol / L; two groups of AML12 cells were treated with 200 μL PGN for 48 h, and 400 nmol / LBAF was added to one of the groups 6 h before sampling. After sampling, cells in all groups were lysed with RIPA, and the supernatant was collected by centrifugation to prepare samples for protein immunoblotting. The experimental results showed that the levels of autophagy-related proteins were increased, proving that the autophagy flux was complete before BAF treatment ( Figure 7 ).

[0054] In addition, mitochondrial morphology was observed using a transmission electron microscope (TEM). AML12 cells were divided into two groups, PBS and PGN. The two groups of cells were treated with 200 μL of PBS or 200 μL of PGN, respectively, for TEM sample preparation. The sections were stained and photographed. The experimental results showed that compared with the PBS group, the PGN group had more autophagosome-like structures ( Figure 8 ), these results suggest that PGN activates mitophagy.

[0055] Example 4 Study on the ability of PGN to activate mitophagy in mice AAV8-TBG-mtKeima is an adeno-associated virus (AAV)-based tool for detecting mitochondrial autophagy (mitophagy) in cells. It uses the fluorescence differences of mtKeima in different pH environments to visually trace the process of mitochondria from normal state to lysosomal degradation, thereby specifically monitoring the level of mitochondrial autophagy.

[0056] C57B / 6J mice were used as the research subjects and divided into two groups: PBS and PGN. 200µl of PBS or 200µl of PGN were given by gavage three times a week, once every other day, for three weeks. After three weeks, 1.2×10 11 AAV8-TBG-mtkeima virus containing VG (viral genome number) was injected once ( Figure 9).

[0057] Fourteen days after AAV8-TBG-mtkeima virus injection, samples were collected and fresh liver tissue slices approximately 1 mm thick were cut with a blade and placed in a 35 mm confocal microplate containing pre-chilled PBS. Fluorescence staining of mtkeima in the liver cells of the two groups of mice under different pH conditions was analyzed using a 60× oil-immersed confocal microscope. The red fluorescence intensity of the PGN-treated group was significantly higher than that of the PBS-treated group, indicating that mitochondrial lysosomal structures increased in the acidic environment after PGN treatment ( Figure 10-11 ). The results showed that PGN can also activate mitophagy in mice.

[0058] Example 5 PGN alleviates CCL4-induced liver fibrosis In this experiment, carbon tetrachloride ( tetrachloride , CCL4) to establish a liver fibrosis model. In the liver fibrosis model, the fusion of autophagosomes and lysosomes was blocked, resulting in the inability to clear damaged mitochondria in time and their accumulation.

[0059] The experiment was conducted using C57B6 / J male mice aged 8-12 weeks and weighing more than 20g. The mice were randomly divided into three groups. Two groups of mice were intraperitoneally injected with corn oil containing CCL4 (CCL4 concentration of 10% by volume). The first injection volume was 4µl / g, and the second injection volume was 2µl / g starting at 4-5pm every Monday and Thursday. Starting from the third week of corn oil injection, the mice were gavaged with 200µl of PGN or 200µl of PBS. One group received PGN and the other received PBS. Gavage continued three times a week until the sixth week. Blood and liver samples were collected within 15-18 hours after corn oil injection. The remaining group of mice served as a control (Vehicle group) and was injected only with corn oil throughout the entire process. The injection volume and frequency were the same as those of the other two groups. Blood and liver samples were collected at the same treatment time as those of the other two groups.

[0060] The serum alanine aminotransferase (ALT) level statistics showed that the serum ALT of mice increased significantly after CCL4 treatment. The serum ALT (U / L) of PBS and PGN groups was significantly higher than that of the control (Vehicle) group, indicating that the liver fibrosis model was successfully established. The serum ALT level of the PGN treatment group was significantly lower than that of the PBS group ( Figure 13 ).

[0061] The degree of liver fibrosis was assessed by Sirius red staining of liver tissue sections. The results showed that the Vehicle group had lighter staining, while both the PBS and PGN groups showed red staining areas representing fibrosis, and the degree of fibrosis in the PGN group was significantly lower than that in the PBS group ( Figure 14 ).

[0062] Desmin is mainly expressed in activated hepatic stellate cells (HSCs), which are key players in the development of liver fibrosis. Immunohistochemical staining results showed that CCL4 treatment significantly promoted the expression of Desmin, suggesting that CCL4 can activate HSCs, and PGN treatment can effectively inhibit the CCL4-mediated upregulation of Desmin expression, indicating that PGN plays an important regulatory role in the development of liver fibrosis. Figure 15 ). Based on the above experimental results, it is suggested that PGN can effectively alleviate the degree of liver fibrosis in the CCL4-induced liver fibrosis model.

Claims

1. Application of peptidoglycan in the preparation of an inducer of mitochondrial autophagy in hepatocytes.

2. The use according to claim 1, characterized in that: The peptidoglycan is extracted from Escherichia coli.

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