Construction method and application of mitochondrial micro-autophagy animal model

Abstract

The invention relates to a construction method and application of a mitochondrial micro-autophagy animal model, and belongs to the technical field of radiation biology and cell biology. The model can stably induce repeatable mitochondrial micro-autophagy reaction in a plurality of high-sensitivity tissues such as parotid gland, mandibular gland, heart, thymus gland and the like of an experimental animal or a cell line through microwave radiation exposure with set specific parameters. A transmission electron microscope observation shows that after radiation, a plurality of lysosome-like small bodies appear in a mitochondrial membrane in the tissue, a typical inclusion structure is formed, and an ultrastructure characteristic of mitochondrial micro-autophagy is achieved; molecular level detection results show that the expression of a micro-autophagy characteristic signal molecule MIEAP is significantly up-regulated, and activation of a micro-autophagy signal channel and occurrence of micro-autophagy are further verified.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for constructing and applying a mitochondrial microautophagy animal model. Background Technology

[0002] Mitophagy is an important mechanism for quality control through mitochondrial-lysosomal interactions. Mitochondria respond to external stresses by selectively removing excess or damaged mitochondria, thereby ensuring mitochondrial quality and maintaining their function. Mitochondrial microautophagy is one such mechanism.

[0003] Mitochondrial microautophagy is a mechanism by which lysosomes directly engulf and degrade mitochondrial contents. This mode is considered the least known form of autophagy. Mitochondrial microautophagy manifests as the accumulation of lysosome-like organelles within mitochondria without damaging the mitochondrial membrane structure. This phenomenon is called MIEAP-induced accumulation of lysosome-like organelles within mitochondria (MALM). When MALM is inhibited, it leads to high levels of reactive oxygen species (ROS), resulting in the formation of MIEAP-induced vacuoles (MIVs). These vacuoles directly engulf and degrade damaged mitochondria, forming membrane-bound vacuoles ranging in size from nanometers to micrometers. MALM and MIVs are considered important new factors in mitochondrial degradation and crucial mechanisms for maintaining mitochondrial homeostasis. Therefore, MIEAP effectively maintains mitochondrial quality by repairing or clearing unhealthy mitochondria through MALM and MIVs, making them characteristic proteins of mitochondrial microautophagy.

[0004] As a unique mitochondrial quality control mechanism, microautophagy has attracted widespread attention from researchers in recent years. It differs from typical mitophagy (also known as macroautophagy), which involves the engulfment of the entire mitochondria by double-membrane autophagosomes. Morphologically, microautophagy manifests as lysosome-like organelles forming one or more fully encapsulated lysosome-like bodies within the mitochondria without disrupting their structure, and then autonomously degrading them.

[0005] Although studies have reported related phenomena in tumors and neurodegenerative diseases, current research mainly focuses on microautophagy at the cellular level or using gene editing techniques. There is a lack of an in vivo microautophagy experimental model that is reproducible, specific, and does not require gene manipulation. Microautophagy plays a potentially important role in maintaining intracellular homeostasis and stress response, but its research remains relatively lagging, lacking standardized experimental models and specific evaluation methods, which severely restricts its functional exploration and translational applications. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a method for preparing an animal model of mitochondrial microautophagy. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0008] This invention provides the application of microwaves in the preparation of animal models of mitochondrial microautophagy, wherein mitochondrial microautophagy exists in the animal models of mitochondrial microautophagy.

[0009] In the above application, the animal is a mouse.

[0010] In one specific embodiment of the present invention, the mouse is a rat.

[0011] In the above application, the microwave frequency is 2.856 GHz, the microwave power density is 30-50 mW / cm², and the radiation time is 15 minutes.

[0012] In the above application, the microwave is used for radiation once.

[0013] The present invention also provides a method for preparing a mitochondrial microautophagy animal model, comprising the following steps: irradiating a recipient animal with microwaves to obtain a mitochondrial microautophagy animal model, wherein the frequency of the microwaves is 2.856 GHz, the power density of the microwaves is 30-50 mW / cm², and the irradiation time is 15 minutes.

[0014] In the above method, the recipient animal is a mouse.

[0015] In one specific embodiment of the present invention, the mouse is a rat.

[0016] In the above method, the microwave is used for radiation once.

[0017] In the above method, mitochondrial microautophagy exists in the animal model of mitochondrial microautophagy.

[0018] The mitochondrial microautophagy described in this article refers to the abnormal morphology of mitochondria in tissues, including cristae breakage and cavitation, and the formation of one or more intact lysosome-like bodies within the mitochondria without disrupting the mitochondrial membrane structure; and the upregulation of the expression of the characteristic protein MIEAP in tissues as detected by immunohistochemistry and immunoblotting.

[0019] The upregulation of the microautophagy characteristic protein MIEAP refers to the fact that the expression level of the microautophagy characteristic protein MIEAP in the mitochondrial microautophagy animal model is higher than that in the recipient animal.

[0020] In the above method, the expression level of MIEAP protein in the mitochondrial microautophagy animal model is higher than that in the recipient animal.

[0021] The tissues in the aforementioned animal models of mitochondrial microautophagy exhibiting mitochondrial autophagy include the parotid gland, mandibular gland, heart, and thymus.

[0022] The present invention also provides any of the following applications of the mitochondrial microautophagy animal model prepared by the aforementioned method:

[0023] A1) Prepare products for studying mitochondrial microautophagy;

[0024] A2) Prepare products for screening biomarkers of mitochondrial microautophagy;

[0025] A3) Prepare products for evaluating the efficacy of interventional drugs;

[0026] A4) Prepare products for assessing the safety of electromagnetic wave exposure and tissue damage;

[0027] A5) Prepare products for tumor mechanism research and treatment.

[0028] In one specific embodiment of the present invention, the product is an animal.

[0029] In one specific embodiment of the present invention, the animal is a mouse. Specifically, the mouse may be a rat.

[0030] This invention addresses the current lack of stable, reproducible, and non-genetically induced in vivo microautophagy models by employing a microwave-induced microautophagy experimental model. This model not only realistically simulates mitochondrial microautophagy under injury-related physiological and pathological conditions but also possesses excellent biological responsiveness and structural visualization capabilities, making it suitable for in-depth research into the regulatory mechanisms of mitochondrial microautophagy. Furthermore, this model can serve as a platform tool for functional screening and intervention validation, providing a reliable experimental basis for the development of targeted drugs for radiation protection and mitochondrial quality control.

[0031] The model constructed in this invention has the following advantages:

[0032] 1) The induction method is natural, without chemical or genetic intervention, and is more consistent with the actual condition of the body;

[0033] 2) It has a wide range of tissue responses and is applicable to multiple sensitive organs;

[0034] 3) It has strong repeatability, stable model, and is suitable for large-scale screening and evaluation;

[0035] 4) It provides an ideal platform for studying mitochondrial microautophagy, which helps to expand its application in radiobiology, toxicology, pharmacology, tumor mechanism research and treatment.

[0036] The model established in this invention has advantages such as natural induction method, controllable process, and strong tissue specificity. It can be widely used in the study of mitochondrial quality control mechanism, exploration of microautophagy signaling pathway regulation mechanism, evaluation of the biological effects of radiation exposure, and functional screening and validation of targeted intervention drugs, possessing good scientific research value and translational application prospects. Compared with existing models that rely on gene intervention, drug treatment, or stress factors such as hypoxia, this model is closer to real physiological damage conditions, filling the current gap in in vivo experimental models for microautophagy research. Attached Figure Description

[0037] Figure 1 These are the microwave radiation source (a) and the plexiglass radiation box (b) in this embodiment of the invention.

[0038] Figure 2 These are light microscopic images of parotid gland tissue structure in the control group and microwave radiation group in this embodiment of the invention. The scale bar is 50 μm.

[0039] Figure 3 These are light microscopic images of the mandibular gland tissue structure in the control group and microwave radiation group in this embodiment of the invention. The scale bar is 50 μm.

[0040] Figure 4 These are light microscopic images of cardiac tissue structure in the control group and microwave radiation group in this embodiment of the invention. The scale bar is 25 μm.

[0041] Figure 5 These are transmission electron microscopy images of mitochondrial microautophagy in the control group and microwave radiation group of the present invention. The scale bar is 500 nm or 2 μm.

[0042] Figure 6 These are transmission electron microscopy images of mitochondrial microautophagy in the mandibular gland tissue of the control group and microwave radiation group in this embodiment of the invention. The scale bar is 1 μm.

[0043] Figure 7 These are transmission electron microscopy images of mitochondrial microautophagy in heart tissue from the control group and microwave radiation group in this embodiment of the invention. The scale bar is 1 μm or 2 μm.

[0044] Figure 8 These are transmission electron microscopy images of mitochondrial microautophagy in the thymus tissue of the control group and the microwave radiation group in this embodiment of the invention. The scale bar is 2 μm.

[0045] Figure 9This figure shows the in situ MIEAP protein expression in parotid gland tissue of the control group and the microwave radiation group in this embodiment of the invention. The scale bar is 50 μm.

[0046] Figure 10 This figure shows the in situ MIEAP protein expression in the mandibular gland tissue of the control group and the microwave radiation group in this embodiment of the invention. The scale bar is 50 μm.

[0047] Figure 11 This figure shows the in situ MIEAP protein expression in cardiac tissue of the control group and the microwave radiation group in this embodiment of the invention. The scale bar is 50 μm.

[0048] Figure 12 This figure shows the in situ MIEAP protein expression in thymus tissue of the control group and the microwave radiation group in this invention embodiment. The scale bar is 50 μm.

[0049] Figure 13 This describes the immunoblotting results and quantitative analysis of MIEAP protein in the parotid gland control group and the microwave radiation group according to embodiments of the present invention. "-" indicates no radiation, and "+" indicates radiation.

[0050] Figure 14 This describes the immunoblotting results and quantitative analysis of MIEAP protein in the mandibular gland tissue control group and the microwave radiation group according to embodiments of the present invention. "-" indicates no radiation, and "+" indicates radiation.

[0051] Figure 15 This describes the immunoblotting results and quantitative analysis of MIEAP protein in the cardiac tissue control group and the microwave radiation group according to embodiments of the present invention. "-" indicates no radiation, and "+" indicates radiation.

[0052] Figure 16 This describes the immunoblotting results and quantitative analysis of MIEAP protein in the thymus tissue control group and the microwave radiation group according to embodiments of the present invention. "-" indicates no radiation, and "+" indicates radiation. Detailed Implementation

[0053] Example 1: Microwave irradiation of rats

[0054] Wistar rats: purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., animal qualification certificate number 110011241100392723.

[0055] Ten male Wistar rats aged 6–8 weeks were randomly divided into a control group and a radiation group, with five rats in each group. Rats in the microwave radiation group underwent uniform whole-body irradiation using a microwave radiation source with an average power density of 30–50 mW / cm² (a representative image shows 30 mW / cm²). The irradiation was performed once, for a total of 15 minutes. The radiation source was approximately 1.4 m away from the rats, and the microwave frequency was 2.856 GHz. The control group was placed in a radiation box and did not receive irradiation. Results were observed in the rats on day 7 post-irradiation.

[0056] Example 2: Observation of the structure of the parotid gland, mandibular gland and heart in rats

[0057] Seven days after microwave radiation (or sham radiation), rats in the control group and the control group were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (30 mg / kg). Parotid gland, mandibular gland, heart and thymus tissues were harvested, fixed in 10% neutral buffered formalin, routinely dehydrated, cleared, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope.

[0058] Figure 2-3 The results showed that in the control group, the parotid and submandibular glands had clear and full acini, which were round or oval and arranged in small clusters. The cell nuclei had uniform chromatin, and the ducts, blood vessels, and stroma all showed normal structure. Figure 6 (Control). In the radiation group, the parotid gland tissue of rats showed edema and congestion, indicating an inflammatory response. Cavitation of the acini and chromatin condensation and marginalization of some cell nuclei indicated that microwave radiation caused damage to the parotid and submandibular glands of rats.

[0059] Figure 4 The results showed that the control group had normal heart tissue structure with neatly arranged, long spindle-shaped muscle fibers, while the radiation group had disordered, wavy myocardial fibers and condensed chromatin in some cell nuclei, indicating that microwave radiation caused heart damage in rats.

[0060] Example 3: Observation of the ultrastructure of mitochondria in rat parotid gland, mandibular gland, heart and thymus tissues

[0061] Seven days after microwave radiation (or sham radiation), rats in the control group and the microwave radiation group were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (30 mg / kg). Parotid gland, mandibular gland, heart and thymus tissues were harvested. One cubic millimeter of heart tissue was taken, quickly fixed in 2.5% glutaraldehyde for 2 hours, then post-fixed in 1% osmium tetroxide for 2 hours, dehydrated in a gradient of ethanol and acetone, embedded in resin, and after semi-thin sectioning and positioning, ultrathin sections (70 nm thick) were prepared. The sections were double-stained with uranium acetate and lead citrate, and then observed and photographed using a CM120 (Philips, Germany) transmission electron microscope.

[0062] Depend on Figures 5-8The results showed that in the control group, the mitochondria in the parotid gland, mandibular gland, heart, and thymus tissues exhibited normal ultrastructure, with intact mitochondria, normal morphology, and an oval shape. In the microwave-irradiated group, the mitochondria in the parotid gland, mandibular gland, heart, and thymus tissues were swollen, structurally abnormal, with broken and cavitated mitochondrial cristae. One or more intact lysosome-like bodies (marked by red arrows in the figure) were observed within some mitochondria, engulfing damaged mitochondria. These results indicate that microwave radiation induces mitochondrial microautophagy in the parotid gland, mandibular gland, heart, and thymus tissues of rats.

[0063] Example 4: In situ changes in MIEAP expression in rat parotid gland, mandibular gland, heart, and thymus tissues (immunohistochemistry)

[0064] On day 7 after radiation (or sham radiation), rats in the control and microwave radiation groups were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (30 mg / kg body weight). Parotid gland, mandibular gland, heart, and thymus tissues were harvested and prepared into paraffin sections. Immunohistochemical staining was performed using a standard immunohistochemical staining kit. The simplified procedure is as follows: Paraffin-embedded sections were dewaxed and hydrated using standard techniques, treated with H2O2 in 3% methanol to block endogenous peroxidase, and then incubated overnight at 4°C with primary antibody. After washing with PBS, the sections were incubated with peroxidase-labeled secondary antibody at room temperature for 1 hour, followed by staining with diaminobenzidine (ZSGB-Bio, China). Sections incubated with PBS in the control group served as a blank control. Images were taken using a DM3000 (Leica, Germany) microscope.

[0065] The aforementioned immunohistochemical detection kit was purchased from ZSBIO Corporation in China, catalog number PV-9001. The aforementioned secondary antibody was obtained from this kit. The aforementioned primary antibody, MIEAP antibody, was purchased from Bioss Corporation in China, catalog number bs-17642R.

[0066] Figure 9-12 The results showed that the brown areas in the parotid gland, mandibular gland, heart, and thymus tissues were significantly deepened after microwave irradiation, indicating that the expression of the mitochondrial microautophagy characteristic protein MIEAP was significantly upregulated in the tissues. The results indicate that microwave irradiation can induce mitochondrial microautophagy in the parotid gland, mandibular gland, heart, and thymus tissues of rats, which is molecular evidence supporting the activation of the microautophagy pathway.

[0067] Example 5: Changes in MIEAP expression in rat parotid gland, mandibular gland, heart, and thymus tissues (protein immunoblotting)

[0068] On day 7 after radiation (or sham radiation), rats in the control and microwave radiation groups were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (30 mg / kg body weight). Parotid gland, mandibular gland, heart, and thymus tissues were harvested, quickly minced, and lysed with RIPA lysis buffer containing protease inhibitors. The lysis was performed on ice for 30 minutes, followed by centrifugation at 12,000 ×g for 15 minutes. The supernatant was used to determine protein concentration. Equal volumes of protein samples were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder for 1 hour, incubated with the corresponding primary antibody (4°C, overnight) and HRP-labeled secondary antibody (room temperature, 1 hour), and developed using ECL chemiluminescence reagent. Images were analyzed for grayscale using ImageJ software, and protein expression levels were normalized to an internal control. The primary antibody, MIEAP antibody, was purchased from Bioss Pharmaceuticals (China), catalog number bs-17642R, and the internal control GAPDH protein was purchased from Abcam Pharmaceuticals (USA), catalog number ab37168. The aforementioned HRP-labeled secondary antibody was purchased from Abcam, Inc. in the United States, catalog number ab7621.

[0069] Depend on Figures 13-16 The results showed that microwave irradiation significantly upregulated the expression of MIEAP, a characteristic protein of mitochondrial microautophagy, in the parotid gland, mandibular gland, heart, and thymus tissues. These results indicate that microwave irradiation can induce mitochondrial microautophagy in the parotid gland, mandibular gland, heart, and thymus tissues of rats. This provides molecular evidence supporting the activation of the microautophagy pathway.

[0070] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of microwaves in the preparation of mitochondrial microautophagy animal models, wherein mitochondrial microautophagy exists in the mitochondrial microautophagy animal models.

2. The application according to claim 1, characterized in that, The animal in question is a rat.

3. The application according to claim 2, characterized in that, The microwave has a frequency of 2.856 GHz, a power density of 30-50 mW / cm², and a radiation time of 15 minutes.

4. The application according to claim 3, characterized in that, The microwave radiation is used once.

5. A method for preparing a mitochondrial microautophagy animal model, comprising the following steps: irradiating a recipient animal with microwaves to obtain a mitochondrial microautophagy animal model, wherein the frequency of the microwaves is 2.856 GHz, the power density of the microwaves is 30-50 mW / cm², and the irradiation time is 15 minutes.

6. The method according to claim 5, characterized in that, The recipient animal is a mouse.

7. The method according to claim 6, characterized in that, The microwave radiation is used once.

8. The method according to claim 7, characterized in that, Mitochondrial microautophagy exists in the animal model of mitochondrial microautophagy.

9. The method according to claim 8, characterized in that, The expression level of MIEAP protein in the mitochondrial microautophagy animal model was higher than that in the recipient animal.

10. Any of the following applications of the mitochondrial microautophagy animal model prepared by the method of any one of claims 5-9: A1) Prepare products for studying mitochondrial microautophagy; A2) Prepare products for screening biomarkers of mitochondrial microautophagy; A3) Prepare products for evaluating the efficacy of interventional drugs; A4) Prepare products for assessing the safety of electromagnetic wave exposure and tissue damage; A5) Prepare products for tumor mechanism research and treatment.

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