Method for detecting influence of hypoxia on autophagy of rat pulmonary artery smooth muscle cells

Abstract

The invention discloses a method for detecting the influence of hypoxia on autophagy of rat pulmonary artery smooth muscle cells, and relates to the field of medicine. Comprising the following steps: cell culture: selecting rats in different oxygen states, and separating and culturing pulmonary artery smooth muscle cells through a tissue adherence method; detection: carrying out the following detection on the smooth muscle cells: WB detection of autophagy and ferroptosis related proteins; performing ELISA (enzyme-linked immunosorbent assay) detection on related markers; carrying out qPCR detection on mRNA related to autophagy and ferroptosis; cell autophagy detection; carrying out ROS detection in the cells; and detecting ferrous ions in cells. According to the invention, rat pulmonary artery smooth muscle cells in different oxygen states are respectively subjected to autophagy and ferroptosis related protein detection, related marker detection, autophagy and ferroptosis related mRNA detection, cell autophagy detection, intracellular ROS detection and intracellular ferrous ion detection, so that the influence of autophagy on ferroptosis can be effectively proved; and the blank of medical technology is filled.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a method for detecting the effect of hypoxia on autophagy of rat pulmonary artery smooth muscle cells. Background Art

[0002] Pulmonary hypertension is a pulmonary vascular disease characterized by irreversible pulmonary vascular remodeling (PVR) and pulmonary artery smooth muscle cell (PASMCs) dysfunction, with a progressive increase in pulmonary vascular resistance as its main feature, ultimately leading to right heart failure and premature death.

[0003] Hypoxic pulmonary hypertension (HPH) is an important pathophysiological link in the development of many clinical heart and lung diseases, and is difficult to treat, has a high disability rate, and a high mortality rate. Finding targeted drugs and therapeutic targets is an important need for the prevention and treatment of HPH.

[0004] Autophagy: It is an important mechanism for cells to maintain homeostasis and is directly involved in the regulation of developmental programs, the maintenance of stem cell self-renewal potential, cell differentiation and plasticity. Basal levels of autophagy promote the recycling of organelles and the maintenance of cell homeostasis, while pathogenic factors such as nutrient deficiency, hypoxia, oxidative stress and infection can lead to autophagy disorders and induce diseases. Existing literature has provided valuable findings on the relationship between autophagy and pulmonary hypertension, but there has been no comprehensive and detailed study to explain the complex and contradictory role of autophagy in the process of pulmonary vascular remodeling.

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

[0006] In the existing technology, there is no research method to determine whether autophagy affects the progression of the disease through ferroptosis, resulting in a technical gap.

[0007] To this end, the present invention proposes a method for detecting the effect of hypoxia on autophagy of rat pulmonary artery smooth muscle cells Summary of the invention

[0008] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for detecting the effect of hypoxia on autophagy of rat pulmonary artery smooth muscle cells.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

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

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

[0012] S2: Detection: Perform the following tests on smooth muscle cells:

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

[0014] S22: ELISA detection of relevant markers;

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

[0016] S24: Cell autophagy detection;

[0017] S25: Intracellular ROS detection;

[0018] S26: Detection of intracellular ferrous ions.

[0019] Preferably: in the 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 the step S22, the relevant markers include the autophagy-related marker LC3 and the ferroptosis-related marker GPX4.

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

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

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

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

[0027] The beneficial effects of the present invention are:

[0028] 1. The present invention can effectively prove the effect of autophagy on ferroptosis by detecting autophagy and ferroptosis-related proteins, detecting related markers, detecting autophagy and ferroptosis-related mRNA, detecting cell autophagy, detecting intracellular ROS, and detecting intracellular ferrous ions in rat pulmonary artery smooth muscle cells under different oxygen states, thus filling the gap in medical technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart of a method for detecting the effect of hypoxia on autophagy of rat pulmonary artery smooth muscle cells proposed by the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0031] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", and "set" should be understood in a broad sense, for example, they can be fixedly connected or set, or detachably connected or set, or integrally connected or set. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0032] Embodiment 1:

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

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

[0035] S2: Detection: Perform the following tests on smooth muscle cells:

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

[0037] S22: ELISA detection of relevant markers;

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

[0039] S24: Cell autophagy detection;

[0040] S25: Intracellular ROS detection;

[0041] S26: Detection of intracellular ferrous ions.

[0042] In the step S21, the 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 the step S22, the relevant markers include the autophagy-related marker LC3 and the ferroptosis-related marker GPX4.

[0046] In the 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 the 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. Establishment of a hypoxia model in rat pulmonary artery smooth muscle cells (PASMCs)

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

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

[0054] ①WB detection of autophagy-related proteins and ferroptosis-related proteins LC3, p62, NCOA4, GPX4, Beclin-1, ATG7, etc.;

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

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

[0057] ④ Ferrous ion test kit to detect ferrous ion levels;

[0058] ⑤Reactive oxygen species (ROS) kit was used to detect ROS levels.

[0059] 3. Set up normoxic group (control group), hypoxic group (hypoxia), and intervention group (hypoxia+3-MA group)

[0060] ①WB detection of autophagy-related proteins and ferroptosis-related proteins LC3, p62, NCOA4, GPX4, Beclin-1, ATG7, etc.;

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

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

[0063] ④ Ferrous ion test kit to detect ferrous ion levels;

[0064] ⑤Reactive oxygen species (ROS) kit was used to detect ROS levels.

[0065] Experimental data

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

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

[0068] 2. Prove that hypoxia promotes enhanced autophagy and ferroptosis of PASMC

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

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

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

[0072] (2) ELISA detection of related markers

[0073] ① Elisa detected that the expression level of autophagy-related marker LC3 in the hypoxia group was significantly higher than that in the normoxia group, and the expression level of p63 in the hypoxia group was significantly lower than that in the normoxia group, indicating that hypoxia can promote the enhancement of autophagy in PASMCs;

[0074] ② Elisa detected that the expression level of ferroptosis-related marker GPX4 in the hypoxia group was significantly lower than that in the normoxia group, indicating that hypoxia can promote the enhancement of ferroptosis of PASMCs.

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

[0076] ①qPCR detection of autophagy-related gene Becloin-1: Becloin-1 was significantly expressed at the mRNA level in the hypoxia group compared with the normoxia group, indicating that hypoxia induced the activation of PASMCs autophagy;

[0077] ②qPCR detection of ferroptosis-related gene GPX4: The expression of GPX4 at the mRNA level in the hypoxia group was significantly reduced compared with the normoxia group, indicating that hypoxia induced activation of PASMCs ferroptosis.

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

[0079] The autophagy kit was used to detect cell autophagy: the fluorescence intensity of the hypoxia group was significantly higher than that of the normoxia group, indicating that hypoxia promoted the enhancement of PASMCs autophagy.

[0080] (5) Reactive oxygen species (ROS) kit detection (fluorescence microplate reader)

[0081] Reactive oxygen species (ROS) kit was used to detect intracellular ROS: the fluorescence intensity of the hypoxia group was significantly higher than that of the normoxia group, indicating that hypoxia promotes the generation of ROS in PASMCs. Ferroptosis is a new type of iron-dependent cell death. When iron overload occurs in cells, excessive Fe 2+ The Fenton reaction catalyzes the production of lipid peroxides (LPO) and reactive oxygen species (ROS), resulting in the accumulation of toxic aldehydes in cells, thereby inducing cell death. Therefore, ROS, as a detection indicator of ferroptosis, suggests that hypoxia can promote the enhancement of ferroptosis in PASMCs.

[0082] (6) Ferrous ion test kit (ELISA)

[0083] The ferrous ion kit was used to detect the intracellular ferrous ion content: the ferrous ion content in the hypoxia group was significantly higher than that in the normoxia group, indicating that hypoxia promoted the increase of ferrous ions in PASMCs and activated cell ferroptosis.

[0084] 3. Prove that inhibiting autophagy (using the autophagy inhibitor 3-MA) ​​will alleviate the enhancement of autophagy and ferroptosis caused by hypoxia

[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, and 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 the hypoxia-induced enhanced autophagy of PASMCs;

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

[0088] (2) ELISA detection of related markers

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

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

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

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

[0093] The autophagy kit was used to detect cell autophagy: the fluorescence intensity of the intervention group was significantly lower than that of the hypoxia group, indicating that the autophagy inhibitor 3-MA can alleviate the hypoxia-induced enhanced autophagy of PASMCs.

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

[0095] The reactive oxygen species (ROS) kit was used to detect intracellular ROS: the fluorescence intensity of the intervention group was significantly lower than that of the hypoxia group, indicating that the autophagy inhibitor 3-MA can alleviate the generation of reactive oxygen species in PASMCs induced by hypoxia, thereby alleviating the enhanced ferroptosis caused by hypoxia.

[0096] (6) Ferrous iron test kit detection (ELISA)

[0097] The ferrous ion kit was used to detect the intracellular ferrous ion content: the ferrous ion content in the intervention group was significantly reduced compared with the hypoxia group, indicating that the autophagy inhibitor 3-MA can alleviate the increased ferrous ion production in PASMCs induced by hypoxia, thereby alleviating cell iron death.

[0098] 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 the hypoxia-induced autophagy activation and ferroptosis activation of PASMCs;

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

[0102] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for detecting the effect of hypoxia on autophagy of rat pulmonary artery smooth muscle cells, characterized in that: The following steps are involved: S1: Cell culture: rats in different oxygen states were selected, and pulmonary artery smooth muscle cells were isolated and cultured by tissue adhesion method; S2: Detection: Perform the following tests on smooth muscle cells: S21: WB detection of autophagy and ferroptosis-related proteins; S22: ELISA detection of relevant markers; S23: qPCR detection of autophagy and ferroptosis-related mRNA; S24: Cell autophagy detection; S25: Intracellular ROS detection; S26: Detection of intracellular ferrous ions.

2. The method for detecting the effect of hypoxia on autophagy of rat pulmonary artery smooth muscle cells according to claim 1, characterized in that: In the step S21, the autophagy and ferroptosis related proteins include autophagy related proteins and ferroptosis related proteins.

3. The method for detecting the effect of hypoxia on autophagy of rat pulmonary artery smooth muscle cells according to claim 2, characterized in that: The autophagy-related proteins include LC3, Beclin-1, and ATG7.

4. The method for detecting the effect of hypoxia on autophagy of rat pulmonary artery smooth muscle cells according to claim 2, characterized in that: The ferroptosis-related proteins include LC3, NCOA4, and ATG7.

5. The method for detecting the effect of hypoxia on autophagy of rat pulmonary artery smooth muscle cells according to claim 1, characterized in that: In the step S22, the relevant markers include the autophagy-related marker LC3 and the ferroptosis-related marker GPX4.

6. The method for detecting the effect of hypoxia on autophagy of rat pulmonary artery smooth muscle cells according to claim 1, characterized in that: In the step S23, the related mRNAs include the autophagy-related gene Becloin-1 and the ferroptosis-related gene GPX4.

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

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

9. The method for detecting the effect of hypoxia on autophagy of 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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