A method for detecting the effect of hypoxia on autophagy in rat pulmonary artery smooth muscle cells.

Abstract

This invention discloses a method for detecting the effect of hypoxia on autophagy in rat pulmonary artery smooth muscle cells, relating to the medical field. The method includes the following steps: cell culture, selecting rats under different oxygen states, and isolating and culturing pulmonary artery smooth muscle cells using the tissue adhesion method; detection, performing the following tests on the smooth muscle cells: Western blotting (WB) to detect autophagy and ferroptosis-related proteins; ELISA to detect related biomarkers; qPCR to detect autophagy and ferroptosis-related mRNAs; autophagy detection; intracellular ROS detection; and intracellular ferrous ion detection. This invention effectively demonstrates the effect of autophagy on ferroptosis by detecting autophagy and ferroptosis-related proteins, related biomarkers, autophagy and ferroptosis-related mRNAs, as well as autophagy detection, intracellular ROS detection, and intracellular ferrous ion detection in rat pulmonary artery smooth muscle cells under different oxygen states, filling a gap in medical technology.

Description

Technical Field

[0001] This invention relates to the medical field, and more particularly to a method for detecting the effect of hypoxia on autophagy in rat pulmonary artery smooth muscle cells. Background Technology

[0002] Pulmonary hypertension is a pulmonary vascular disease characterized by irreversible pulmonary vascular remodeling (PVR) and dysfunction of pulmonary artery smooth muscle cells (PASMCs). It is characterized by progressively increasing pulmonary vascular resistance and ultimately leads to right heart failure and premature death.

[0003] Hypoxic pulmonary hypertension (HPH) is a crucial pathophysiological process in the development of many clinical cardiopulmonary diseases, characterized by its difficulty in treatment, high disability rate, and high mortality rate. Finding targeted drugs and therapeutic targets is a vital need for the prevention and treatment of HPH.

[0004] Autophagy is a crucial mechanism for maintaining cellular homeostasis, directly involved in the regulation of developmental processes, the maintenance of stem cell self-renewal potential, cell differentiation, and plasticity. Basal autophagy promotes organelle recycling and cellular homeostasis, while pathogenic factors such as nutrient deficiency, hypoxia, oxidative stress, and infection can lead to autophagy dysregulation and induce disease. Current literature has provided valuable findings regarding the relationship between autophagy and pulmonary hypertension, but a comprehensive and detailed study has yet to explain the complex and contradictory role of autophagy in pulmonary vascular remodeling.

[0005] Ferroptosis is a novel iron-dependent form of cell death that occurs when there is an intracellular iron overload, resulting in the death of cells with excess iron. 2+ The Fenton reaction catalyzes the production of lipid peroxides (LPO) and reactive oxygen species (ROS), leading to the accumulation of toxic aldehydes in cells and inducing cell death. Studies have found that the pathogenesis of pulmonary hypertension (HPH) involves iron homeostasis imbalance. Although the role of ferroptosis in PH remains controversial, most studies have identified ferroptosis as a key factor in pulmonary hypertension.

[0006] In the current technology, there are no research methods to study whether autophagy affects the progression of disease through ferroptosis, thus creating a technological gap.

[0007] Therefore, this invention proposes a method for detecting the effect of hypoxia on autophagy in rat pulmonary artery smooth muscle cells. Summary of the Invention

[0008] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for detecting the effects of hypoxia on autophagy in rat pulmonary artery smooth muscle cells.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A method for detecting the effect of hypoxia on autophagy in rat pulmonary artery smooth muscle cells, comprising the following steps:

[0011] S1: Cell culture: Rat rats with different oxygen states were selected, and pulmonary artery smooth muscle cells were isolated and cultured by tissue adhesion method;

[0012] S2: Detection, the following tests were performed on the smooth muscle cells:

[0013] S21: Western blotting detection of autophagy and ferroptosis-related proteins;

[0014] S22: ELISA detection of relevant biomarkers;

[0015] S23: qPCR detection of autophagy and ferroptosis-related mRNAs;

[0016] S24: Detection of autophagy;

[0017] S25: Intracellular ROS detection;

[0018] S26: Detection of intracellular ferrous ions.

[0019] Preferably, in step S21, the autophagy and ferroptosis-related proteins include autophagy-related proteins and ferroptosis-related proteins.

[0020] Preferably, the autophagy-related proteins include LC3, Beclin-1, and ATG7.

[0021] Preferably, the ferroptosis-related proteins include LC3, NCOA4, and ATG7.

[0022] Preferably, in step S22, the relevant biomarkers include autophagy-related biomarker LC3 and ferroptosis-related biomarker GPX4.

[0023] Preferably, in step S23, the relevant mRNAs include the autophagy-related gene Becloin-1 and the ferroptosis-related gene GPX4.

[0024] Preferably, in step S24, a fluorescent microplate reader is used for detection.

[0025] Preferably, in step S25, a fluorescent microplate reader is used for detection.

[0026] Preferably, in step S26, a fluorescent microplate reader is used for detection.

[0027] The beneficial effects of this invention are as follows:

[0028] 1. This invention effectively demonstrates the effect of autophagy on ferroptosis by detecting autophagy and ferroptosis-related proteins, related markers, autophagy and ferroptosis-related mRNAs, as well as cell autophagy detection, intracellular ROS detection, and intracellular ferrous ion detection in rat pulmonary artery smooth muscle cells under different oxygen states, thus filling a gap in medical technology. Attached Figure Description

[0029] Figure 1 This is a flowchart of a method for detecting the effect of hypoxia on autophagy in rat pulmonary artery smooth muscle cells, as proposed in this invention. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0031] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0032] Example 1:

[0033] A method for detecting the effect of hypoxia on autophagy in rat pulmonary artery smooth muscle cells, comprising the following steps:

[0034] S1: Cell culture: Rat rats with different oxygen states were selected, and pulmonary artery smooth muscle cells were isolated and cultured by tissue adhesion method;

[0035] S2: Detection, the following tests were performed on smooth muscle cells:

[0036] S21: Western blotting detection of autophagy and ferroptosis-related proteins;

[0037] S22: ELISA detection of relevant biomarkers;

[0038] S23: qPCR detection of autophagy and ferroptosis-related mRNAs;

[0039] S24: Detection of autophagy;

[0040] S25: Intracellular ROS detection;

[0041] S26: Detection of intracellular ferrous ions.

[0042] In step S21, autophagy and ferroptosis-related proteins include autophagy-related proteins and ferroptosis-related proteins.

[0043] The autophagy-related proteins include LC3, Beclin-1, and ATG7.

[0044] The ferroptosis-related proteins include LC3, NCOA4, and ATG7.

[0045] In step S22, the relevant biomarkers include autophagy-related biomarker LC3 and ferroptosis-related biomarker GPX4.

[0046] In step S23, the relevant mRNAs include the autophagy-related gene Becloin-1 and the ferroptosis-related gene GPX4.

[0047] In step S24, a fluorescence microplate reader is used for detection.

[0048] In step S25, a fluorescence microplate reader is used for detection.

[0049] In step S26, a fluorescence microplate reader is used for detection.

[0050] Experimental protocol

[0051] 1. Establish a rat pulmonary artery smooth muscle cell (PASMC) hypoxia model.

[0052] ① qPCR was used to detect the level of hypoxia-inducible factor HIF-1α on mRNA.

[0053] 2. Set up a normoxic group (control group) and a hypoxic group (hypoxia group).

[0054] ① Western blotting was used to detect autophagy-related proteins and ferroptosis-related proteins such as LC3, p62, NCOA4, GPX4, Beclin-1, and ATG7.

[0055] ②ELISA detection of autophagy and ferroptosis-related markers such as LC3, p62, and GPX4;

[0056] ③ qPCR detection of the levels of autophagy and ferroptosis-related markers on mRNA;

[0057] ④ Ferrous ion reagent kit for detecting ferrous ion levels;

[0058] ⑤ ROS levels were detected using a reactive oxygen species (ROS) kit.

[0059] 3. Three groups were established: a normoxic group (control group), a hypoxic group (hypoxia group), and an intervention group (hypoxia + 3-MA group).

[0060] ① Western blotting was used to detect autophagy-related proteins and ferroptosis-related proteins such as LC3, p62, NCOA4, GPX4, Beclin-1, and ATG7.

[0061] ②ELISA detection of autophagy and ferroptosis-related markers such as LC3, p62, and GPX4;

[0062] ③ qPCR detection of the levels of autophagy and ferroptosis-related markers on mRNA;

[0063] ④ Ferrous ion reagent kit for detecting ferrous ion levels;

[0064] ⑤ ROS levels were detected using a reactive oxygen species (ROS) kit.

[0065] Experimental data

[0066] 1. Confirm the establishment of the hypoxia model

[0067] qPCR detection of hypoxia-inducible factor (HIF-1α): The hypoxia group showed significantly higher expression of HIF-1α at the mRNA level compared to the normoxic group, indicating that hypoxia induction was successful in the hypoxia group.

[0068] 2. Demonstrates that hypoxia promotes enhanced autophagy and ferroptosis in PASMCs.

[0069] (1) Western blot detection of autophagy and ferroptosis-related proteins

[0070] ① The expression levels of autophagy-related proteins LC3, Beclin-1, and ATG7 were significantly higher in the hypoxia group than in the normoxic group, while the expression level of p62 was significantly lower in the hypoxia group than in the normoxic group, indicating that hypoxia can promote the enhancement of autophagy in PASMCs.

[0071] ② The expression levels of ferroptosis-related proteins LC3, NCOA4, and ATG7 were significantly higher in the hypoxia group than in the normoxic group, while the expression level of GPX4 was significantly lower in the hypoxia group than in the normoxic group, indicating that hypoxia can promote enhanced ferroptosis in PASMCs. Among them, LC3, NCOA4, and ATG7 are ferrophagy-related markers in ferroptosis, suggesting that hypoxia can promote enhanced ferrophagy in PASMCs.

[0072] (2) ELISA detection of related biomarkers

[0073] ①ELISA showed that the expression level of autophagy-related marker LC3 in the hypoxia group was significantly higher than that in the normoxic group, while the expression level of p63 in the hypoxia group was significantly lower than that in the normoxic group, indicating that hypoxia can promote the enhancement of autophagy in PASMCs.

[0074] ②ELISA showed that the expression level of GPX4, a biomarker related to ferroptosis, was significantly lower in the hypoxia group than in the normoxic group, indicating that hypoxia can promote enhanced ferroptosis in PASMCs.

[0075] (3) qPCR detection of autophagy and ferroptosis-related mRNAs

[0076] ①qPCR detection of autophagy-related gene Becloin-1: Compared with the normoxic group, the hypoxia group showed significant expression of Becloin-1 at the mRNA level, suggesting that hypoxia induces and activates autophagy in PASMCs;

[0077] ②qPCR detection of ferroptosis-related gene GPX4: Compared with normoxic group, the expression of GPX4 at the mRNA level was significantly reduced in hypoxia group, suggesting that hypoxia induces ferroptosis in PASMCs.

[0078] (4) Autophagy kit detection (fluorescent microplate reader)

[0079] Autophagy assay kit showed that the fluorescence intensity in the hypoxia group was significantly higher than that in the normoxic group, indicating that hypoxia promotes enhanced autophagy in PASMCs.

[0080] (5) Detection of reactive oxygen species (ROS) using a reagent kit (fluorescent microplate reader)

[0081] Reactive oxygen species (ROS) assay kits were used to detect intracellular ROS levels. The fluorescence intensity was significantly higher in the hypoxia group compared to the normoxic group, suggesting that hypoxia promotes ROS generation in PASMCs. Ferraphobia, as a novel iron-dependent cell death mechanism, occurs when intracellular iron overload leads to the generation of excess Fe. 2+ The Fenton reaction catalyzes the production of lipid peroxides (LPO) and reactive oxygen species (ROS), leading to the accumulation of toxic aldehydes within cells and inducing cell death. Therefore, ROS, as an indicator of ferroptosis, suggests that hypoxia can promote enhanced ferroptosis in PASMCs.

[0082] (6) Ferrous ion detection kit (ELISA reader)

[0083] The ferrous ion assay kit detected intracellular ferrous ion content: the ferrous ion content in the hypoxia group was significantly higher than that in the normoxic group, suggesting that hypoxia promotes the increase of ferrous ions in PASMCs, thereby activating ferroptosis.

[0084] 3. Demonstrating that inhibiting autophagy (using the autophagy inhibitor 3-MA) ​​will alleviate hypoxia-induced increased autophagy and ferroptosis.

[0085] (1) Western blot detection of autophagy and ferroptosis-related proteins

[0086] ① The expression levels of autophagy-related proteins LC3 and ATG7 in the intervention group were significantly lower than those in the hypoxia group, while the expression level of p62 in the intervention group was significantly higher than that in the hypoxia group, indicating that the autophagy inhibitor 3-MA can alleviate hypoxia-induced enhanced autophagy in PASMCs.

[0087] ② The expression levels of ferroptosis-related proteins LC3, NCOA4, and ATG7 were significantly lower in the intervention group than in the hypoxia group, while the expression level of GPX4 was significantly higher in the intervention group than in the hypoxia group. This indicates that the autophagy inhibitor 3-MA can alleviate hypoxia-induced enhanced ferroptosis in PASMCs. Among them, LC3, NCOA4, and ATG7 are ferroptosis-related biomarkers, suggesting that the autophagy inhibitor 3-MA can alleviate hypoxia-induced enhanced ferroptosis in PASMCs.

[0088] (2) ELISA detection of related biomarkers

[0089] Elisa analysis showed that the expression level of GPX4, a biomarker related to ferroptosis, was significantly higher in the intervention group than in the hypoxia group, indicating that the autophagy inhibitor 3-MA can alleviate hypoxia-induced enhanced ferroptosis in PASMCs.

[0090] (3) qPCR detection of autophagy and ferroptosis-related mRNAs

[0091] qPCR detection of autophagy-related gene Becloin-1: The expression level of autophagy-related gene Becloin-1 on mRNA in the intervention group was significantly lower than that in the hypoxia group, indicating that the autophagy inhibitor 3-MA can alleviate hypoxia-induced enhanced autophagy in PASMCs.

[0092] (4) Autophagy kit detection (fluorescent microplate reader)

[0093] Autophagy assay kits were used to detect autophagy in cells: the fluorescence intensity of the intervention group was significantly lower than that of the hypoxia group, suggesting that the autophagy inhibitor 3-MA can alleviate hypoxia-induced enhanced autophagy in PASMCs.

[0094] (5) ROS detection kit (fluorescent microplate reader)

[0095] Intracellular ROS levels were detected using a reactive oxygen species (ROS) assay kit: the fluorescence intensity of the intervention group was significantly lower than that of the hypoxia group, suggesting that the autophagy inhibitor 3-MA can alleviate hypoxia-induced ROS generation in PASMCs, thereby alleviating the enhanced ferroptosis caused by hypoxia.

[0096] (6) Ferrous iron assay kit (ELISA reader)

[0097] The intracellular ferrous ion content was detected by a ferrous ion assay kit: the ferrous ion content in the intervention group was significantly reduced compared to the hypoxia group, suggesting that the autophagy inhibitor 3-MA can alleviate the increased ferrous ion production in PASMCs induced by hypoxia, thereby alleviating ferroptosis.

[0098] Research Conclusions

[0099] ① Hypoxia can induce enhanced autophagy and ferroptosis in rat pulmonary artery smooth muscle cells;

[0100] ② The autophagy inhibitor 3-MA can alleviate hypoxia-induced autophagy activation and ferroptosis activation in PASMCs;

[0101] ③ The autophagy inhibitor 3-MA alleviates hypoxia-induced ferroptosis activation in PSASMCs by inhibiting ferroautophagy.

[0102] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting the effect of hypoxia on autophagy in rat pulmonary artery smooth muscle cells, characterized in that, Includes the following steps: S1: Cell culture: Rat rats with different oxygen states were selected, and pulmonary artery smooth muscle cells were isolated and cultured by tissue adhesion method; S2: Detection, the following tests were performed on the smooth muscle cells: S21: Western blotting was used to detect autophagy and ferroptosis-related proteins. Autophagy-related proteins included LC3, Beclin-1, and ATG7, while ferroptosis-related proteins included LC3, NCOA4, and ATG7. S22: ELISA detects relevant biomarkers, including autophagy-related biomarker LC3 and ferroptosis-related biomarker GPX4; S23: qPCR was used to detect autophagy and ferroptosis-related mRNAs, including autophagy-related gene Beclin-1 and ferroptosis-related gene GPX4. S24: Detection of autophagy; S25: Intracellular ROS detection; S26: Detection of intracellular ferrous ions.

2. The method for detecting the effect of hypoxia on autophagy in rat pulmonary artery smooth muscle cells according to claim 1, characterized in that, In step S24, a fluorescence microplate reader is used for detection.

3. The method for detecting the effect of hypoxia on autophagy in rat pulmonary artery smooth muscle cells according to claim 1, characterized in that, In step S25, a fluorescence microplate reader is used for detection.

4. The method for detecting the effect of hypoxia on autophagy in rat pulmonary artery smooth muscle cells according to claim 1, characterized in that, In step S26, a fluorescence microplate reader is used for detection.

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