Application of YAP related protein in intestinal ischemia-reperfusion lung injury

Abstract

The invention relates to the technical field of biology, in particular to application of YAP related protein in intestinal ischemia-reperfusion lung injury. The application comprises the application of the overexpressed YAP related protein 1 in treating or inhibiting the intestinal ischemia-reperfusion lung injury; based on the regulation and control effect of a transcriptional regulatory factor of the YAP-associated protein 1 in a Hippo-YAP signal channel, accumulation of overexpressed YAP-associated protein 1 in a cell nucleus is used, so that a large amount of YAP-associated protein 1 is overexpressed in the cell nucleus, expression of downstream Nrf2 genes in the cell nucleus and transfer of the YAP-associated protein 1 in the cell nucleus can be regulated and controlled, and the YAP-associated protein 1 can be expressed in the cell nucleus. According to the present invention, the overexpressed YAP-associated protein 1 can be used to inhibit ferroptosis of intestinal ischemia-reperfusion lung injury tissues, and the overexpressed YAP-associated protein 1 can be used to improve lung injury, such that the overexpressed YAP-associated protein 1 can be used to protect lung epithelium injury caused by intestinal ischemia-reperfusion.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to an application of a YAP-related protein in intestinal ischemia-reperfusion lung injury. Background Art

[0002] Intestinal ischemia-reperfusion (II / R) is a common clinical critical illness. Its occurrence is mainly due to a sharp decrease in intestinal blood supply, and is often related to a series of clinical conditions such as shock, mesenteric artery thrombosis, and intestinal obstruction. II / R not only causes local intestinal damage, but also leads to systemic inflammation and distal organ dysfunction due to impaired intestinal barrier function and intestinal flora displacement, which makes II / R have a high morbidity and mortality. Since the lung is the most sensitive distal organ to II / R, and acute lung injury (ALI) has been proven to be the main cause of death during II / R injury, the mechanism of II / R-induced ALI has not yet been fully elucidated. In addition, ferroptosis is a newly discovered form of cell death in recent years, and its typical characteristics are the accumulation of a large amount of iron, lipid peroxidation, and reactive oxygen species (ROS) during cell death. Ferroptosis is different from cell necrosis, apoptosis, and autophagy, and is a new type of cell death. Ferroptosis is mainly manifested by the integrity of the cell membrane, normal nuclear morphology, mitochondrial atrophy, increased density of mitochondrial membrane, and reduced or disappeared density of mitochondrial cristae. In terms of biochemistry, ferroptosis is mainly caused by the imbalance of lipid peroxidation caused by the decrease of intracellular glutathione peroxidase 4 (GPX4) activity and the consumption of glutathione (GSH), which leads to the accumulation of a large amount of lipid reactive oxygen. Nuclear factor E2-related factor 2 (Nrf2) is an antioxidant molecule and a key gene involved in ferroptosis and lipid metabolism. It can promote target gene transcription by binding to the antioxidant response element (ARE) in the cell nucleus. In addition, Nrf2 can also act as a regulator of the antioxidant system to upregulate the expression of HO-1 to reduce oxidative stress. Current studies have shown that ferroptosis is involved in the occurrence of ALI induced by intestinal II / R, and inhibiting ferroptosis of lung epithelial cells can significantly alleviate lung injury induced by intestinal II / R. Therefore, blocking ferroptosis of lung epithelial cells may be a potential solution for the treatment of ALI induced by intestinal II / R.

[0003] YAP-associated protein 1 (YAP1, also known as YAP) is a downstream transcriptional regulator of the Hippo signaling pathway and plays a core role in controlling organ development and cell proliferation. The Hippo-YAP1 pathway is a kinase cascade reaction. When the Hippo pathway is activated, YAP can be directly phosphorylated and inactivated by large tumor suppressor 1 / 2 (LATS1 / 2), resulting in YAP retention in the cytoplasm. When the Hippo pathway is closed, unphosphorylated YAP enters the nucleus and binds to TEAD transcription factors to promote the transcription of downstream genes. The Hippo pathway has been shown to be a tumor suppressor pathway, which can be demonstrated by inhibiting its downstream YAP molecules. In recent years, the Hippo pathway has played an important role in cerebral ischemia-reperfusion and myocardial ischemia-reperfusion. And there is increasing evidence that the Hippo signaling pathway is related to respiratory diseases. YAP mediates the differentiation of type II alveolar epithelial cells (AECII) to type I alveolar epithelial cells (AECI), while accelerating the resolution of inflammation in LPS-induced ARDS mice. YAP activity is increased in airway epithelial cells of patients with idiopathic pulmonary fibrosis (IPF). However, whether and how YAP is involved in regulating acute lung injury induced by intestinal ischemia-reperfusion remains to be explored. Summary of the invention

[0004] The present application provides an application of a YAP-related protein in intestinal ischemia-reperfusion lung injury to solve the following technical problem: how to fill the gap in YAP in regulating intestinal ischemia-reperfusion-induced acute lung injury.

[0005] In a first aspect, the present application provides an application of a YAP-related protein as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury, and the application includes: using overexpressed YAP-related protein 1 as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury.

[0006] Optionally, the expression level of the overexpressed YAP-associated protein 1 is 1.5 times or more of the expression level of the normal YAP-associated protein 1.

[0007] Optionally, the overexpressed YAP-associated protein 1 is used as a reagent for treating or inhibiting ferroptosis associated with intestinal ischemia-reperfusion lung injury.

[0008] Optionally, the overexpressed YAP-associated protein 1 is used as a reagent for regulating the expression of Nrf2 gene in tissue cells and / or its nuclear translocation in tissue cells in intestinal ischemia-reperfusion lung injury.

[0009] In a second aspect, the present application provides an inhibitory agent for ferroptosis associated with intestinal ischemia-reperfusion lung injury, wherein the inhibitory agent comprises overexpressed YAP-associated protein 1.

[0010] Optionally, the protective agent includes an adeno-associated virus vector carrying an overexpressed YAP-associated protein 1 gene

[0011] In a third aspect, the present application provides a protective agent for intestinal ischemia-reperfusion lung injury, wherein the protective agent comprises overexpressed YAP-associated protein 1.

[0012] Optionally, the protective agent includes an adeno-associated virus vector carrying an over-expressed YAP-associated protein 1 gene.

[0013] In a fourth aspect, the present application provides a therapeutic or inhibitory agent for intestinal ischemia-reperfusion lung injury, wherein the agent comprises overexpressed YAP-associated protein 1.

[0014] Optionally, the agent includes an adeno-associated virus vector carrying an overexpressed YAP-associated protein 1 gene.

[0015] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0016] The present application provides a YAP-related protein as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury, and the application includes: using overexpressed YAP-related protein 1 as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury. Based on the regulatory effect of the transcriptional regulator of YAP-related protein 1 in the Hippo-YAP signaling pathway, overexpression of YAP-related protein 1 is used to accumulate in the cell nucleus, so that a large amount of YAP-related protein 1 is overexpressed in the cell nucleus, and these large amounts of overexpressed YAP-related protein 1 can regulate the expression of the downstream Nrf2 gene in the cell nucleus and the transfer of YAP-related protein 1 in the cell nucleus. Overexpressed YAP-related protein 1 can be used to inhibit the iron death of intestinal ischemia-reperfusion lung injury tissue and overexpressed YAP-related protein 1 can be used to improve lung injury, so that overexpressed YAP-related protein 1 can be used to protect lung epithelial injury caused by intestinal ischemia-reperfusion, thereby filling the gap of YAP-related protein 1 in regulating acute lung injury induced by intestinal ischemia-reperfusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0019] Figure 1 This is a graph showing the effect of overexpression of YAP on II / R-induced lung injury in mice provided in the examples of the present application, wherein: Figure 1 A is a representative HE staining image of the lung tissue of each group of mice under ×200 and ×400 conditions, with the scale bar being 50 μM and 20 μM. Figure 1 B is the protein concentration of bronchoalveolar lavage fluid in each group. Figure 1 C is the ratio of wet weight to dry weight of lung tissue of mice in each group, n=6; in the figure, Sham group is the sham operation group, II / R is the intestinal ischemia-reperfusion group, II / R+NC-AAV is the intestinal ischemia-reperfusion group + control adeno-associated virus group, II / R+YAP-AAV is the intestinal ischemia-reperfusion group + YAP overexpression adeno-associated virus group;

[0020] ****P<0.0001;

[0021] Figure 2 The results of the overexpression of YAP on the local and systemic inflammation of mice with II / R-induced lung injury provided in the examples of the present application are shown in FIG. Figure 2 A is the expression level of IL-1β in serum. Figure 2 B is the expression level of IL-6 in serum. Figure 2 C is the expression level of TNF-α in serum. Figure 2 D is the expression level of IL-1β in bronchoalveolar lavage fluid. Figure 2 E is the expression level of IL-6 in bronchoalveolar lavage fluid. Figure 2 F is the expression level of TNF-α in bronchoalveolar lavage fluid, n=6; **P<0.01, ***P<0.001, ****P<0.0001;

[0022] Figure 3 The transmission electron micrograph of the ultrastructure of lung tissue in mice with II / R-induced lung injury induced by overexpression of YAP provided in the examples of the present application, in which the magnification is ×25000 and the scale bar is 500 nM;

[0023] Figure 4 This is a diagram showing changes in ferroptosis-related proteins in mice with II / R-induced lung injury after overexpression of YAP, provided in the examples of the present application. Figure 4 A is the protein blot band of ferroptosis-related proteins SLC7A11, Nrf2 and GPX4. Figure 4 B is the relative expression analysis result of ferroptosis-related protein SLC7A11. Figure 4 C is the relative expression analysis result of ferroptosis-related protein Nrf2. Figure 4 D is the relative expression analysis result of ferroptosis-related protein GPX4, n=6, Figure 4E is the relative expression analysis result of YAP protein; **P<0.01, ***P<0.001, ****P<0.0001;

[0025] Figure 5 This is a graph showing the expression of ferroptosis-related markers in mice with II / R-induced lung injury by overexpression of YAP provided in the examples of the present application. Figure 5 A is the expression result of MDA, a marker related to ferroptosis. Figure 5 B is ferrous ion Fe, a marker of ferroptosis 2+ The expression results of Figure 5 C is the distribution diagram of the ratio of reduced glutathione GSH / oxidized glutathione GSSG, a marker related to ferroptosis, n=6; ***P<0.001, ****P<0.0001, ns indicates no statistically significant difference;

[0026] Figure 6 The immunofluorescence staining diagram of 4-HNE and GPX4 in lung tissue of mice with II / R-induced lung injury by overexpression of YAP provided in the examples of the present application is as follows: Figure 6 A is the immunofluorescence staining of 4-HNE. Figure 6 B is the immunofluorescence staining image of GPX4, in which the magnification is ×200 and the scale bar is 50 μM;

[0027] Figure 7 The immunofluorescence staining statistical data of 4-HNE and GPX4 in lung tissue of mice with II / R-induced lung injury induced by overexpression of YAP provided in the examples of the present application are shown in FIG. Figure 7 A is the statistical data of immunofluorescence staining of 4-HNE. Figure 7 B is the statistical data of immunofluorescence staining of GPX4, n=3; **P<0.01, ***P<0.001.

[0028] Figure 8 A diagram of the construction process of the intestinal ischemia-reperfusion lung injury model provided in the examples of the present application. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0030] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0031] In this article, the terms including "including" and "including" mean "including but not limited to". "And / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone; where A and B can be singular or plural. Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0032] An embodiment of the present application provides an application of a YAP-related protein as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury, and the application includes: using overexpressed YAP-related protein 1 as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury.

[0033] In some optional embodiments, the expression level of the overexpressed YAP-related protein 1 is 1.5 or more of the expression level of the normal YAP-related protein 1;

[0034] In these embodiments, the expression level of overexpressed YAP-associated protein 1 can be 1.5 or more of the expression level of normal YAP-associated protein 1, and the specific expression level of overexpressed YAP-associated protein 1 can be clarified, thereby clarifying that overexpressed YAP-associated protein 1 can be used as a new target for treating or inhibiting intestinal ischemia-reperfusion lung injury.

[0035] In some optional embodiments, the overexpressed YAP-associated protein 1 is used as a reagent for treating or inhibiting ferroptosis associated with intestinal ischemia-reperfusion lung injury;

[0036] In some optional embodiments, the overexpressed YAP-associated protein 1 is used as a reagent for regulating the expression of Nrf2 gene in tissue cells and / or the nuclear transfer of tissue cells in intestinal ischemia-reperfusion lung injury;

[0037] In these embodiments, since the overexpressed large amount of YAP-associated protein 1 can regulate the expression of the downstream Nrf2 gene in the cell nucleus, and the expression of the downstream Nrf2 gene in the cell nucleus is a key gene involved in ferroptosis and lipid metabolism, the overexpressed YAP-associated protein 1 can be used as a reagent for treating or inhibiting ferroptosis associated with intestinal ischemia-reperfusion lung injury.

[0038] Based on a general inventive concept, the present embodiment provides an inhibitory agent for ferroptosis associated with intestinal ischemia-reperfusion lung injury, wherein the inhibitory agent comprises overexpressed YAP-associated protein 1;

[0039] The inhibitory agent is realized based on the application of the above-mentioned YAP-related protein as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury. The specific application of the YAP-related protein as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury can be referred to the above-mentioned embodiments. Since the inhibitory agent adopts part or all of the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be repeated here one by one.

[0040] In some optional embodiments, the protective agent includes an adeno-associated virus vector carrying an overexpressed YAP-associated protein 1 gene;

[0041] In these embodiments, the protective agent may include an adeno-associated virus vector carrying an overexpressed YAP-related protein 1 gene. Compared with the traditional catheter administration method, the use of an adeno-associated virus vector can specifically achieve overexpression of YAP-related protein 1 in vivo to produce a large amount of YAP-related protein, thereby improving the inhibitory effect of YAP-related protein 1 on ferroptosis associated with intestinal ischemia-reperfusion lung injury.

[0042] Based on a general inventive concept, an embodiment of the present application provides a protective agent for intestinal ischemia-reperfusion lung injury, wherein the protective agent includes overexpressed YAP-associated protein 1.

[0043] The protective agent is realized based on the application of the above-mentioned YAP-related protein as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury. The specific application of the YAP-related protein as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury can refer to the above-mentioned embodiments. Since the protective agent adopts part or all of the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be repeated here one by one.

[0044] In some optional embodiments, the protective agent includes an adeno-associated virus vector carrying an overexpressed YAP-associated protein 1 gene;

[0045] In these embodiments, the protective agent may include an adeno-associated virus vector carrying an overexpressed YAP-associated protein 1 gene. Compared with the traditional catheter administration method, the use of an adeno-associated virus vector can specifically achieve overexpression of YAP-associated protein 1 in vivo to produce a large amount of YAP-associated protein, thereby enhancing the protective effect of YAP-associated protein 1 against intestinal ischemia-reperfusion lung injury.

[0046] Based on a general inventive concept, an embodiment of the present application provides a therapeutic or inhibitory agent for intestinal ischemia-reperfusion lung injury, wherein the agent comprises overexpressed YAP-associated protein 1.

[0047] The inhibitory agent is realized based on the application of the above-mentioned YAP-related protein as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury. The specific application of the YAP-related protein as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury can be referred to the above-mentioned embodiments. Since the inhibitor adopts part or all of the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be repeated here one by one.

[0048] In some optional embodiments, the agent includes an adeno-associated virus vector carrying an overexpressed YAP-associated protein 1 gene;

[0049] In these embodiments, the agent may include an adeno-associated virus vector carrying an overexpressed YAP-associated protein 1 gene. Compared with the traditional catheter administration method, the use of an adeno-associated virus vector can specifically achieve overexpression of YAP-associated protein 1 in vivo to produce a large amount of YAP-associated protein, thereby improving the therapeutic effect or inhibitory effect of YAP-associated protein 1 on intestinal ischemia-reperfusion lung injury.

[0050] The present application is further described below in conjunction with specific examples. The experimental methods in the following examples that do not specify specific conditions are usually measured according to industry standards; if there are no corresponding industry standards, they are carried out according to common international standards, conventional conditions, or conditions recommended by the manufacturer.

[0051] Example 1

[0052] 1. Construction of intestinal ischemia-reperfusion lung injury model (II / R):

[0053] All mice were adaptively reared for one week and fasted for 12 hours before modeling, but were allowed to drink water freely. A dose of 50 mg / kg of sodium pentobarbital was intraperitoneally injected into the mice at a mass concentration of 1%. After the mice were anesthetized, the abdominal hair was removed with a razor and the mice were fixed in a supine position on a warming pad. After laparotomy along the midline of the abdomen, the superior mesenteric artery (SMA) was only separated without clamping; the other two groups were separated and the SMA was clamped after laparotomy (such as Figure 8A), and then release the non-invasive arterial clamp after 1 hour (as shown in Figure 8 B), and reperfusion was performed for 4 h. Group II / R+fer-1 was treated with ferroptosis inhibitor fer-1 24 h before ischemia.

[0054] The mice were pretreated by tail vein injection (0.8 mg / kg). All mice were kept at a constant body temperature during surgery, and the wounds were infiltrated with 0.25% ropivacaine injection for analgesia after surgery.

[0055] Adeno-associated virus was injected through tracheal intubation to establish the YAP overexpression model and control virus model in mouse lung tissue, and the intestinal ischemia-reperfusion model was established on the 28th day after injection.

[0056] 2. Construction of II / R acute lung injury (ALI) model:

[0057] The abdominal midline of the II / R mouse model was incised, and the superior mesenteric artery was occluded for 1 h using a non-invasive vascular clip and reperfused for 4 h to generate the ALI model of II / R. The specific steps were the same as those described above.

[0058] 3. Detection of relevant data:

[0059] 1. Histopathological examination of lung tissue damage:

[0060] After the mice were killed, the right lower lobe lung tissue was immediately taken and placed in 4% paraformaldehyde solution for 48 hours. The lung tissue immersed in the fixative was embedded, sliced ​​and HE stained, and the pathological damage of the lung tissue was observed under an electron optical microscope.

[0061] 2. Elisa detects the expression levels of inflammatory factors in bronchoalveolar lavage fluid and serum:

[0062] After the mice were killed, the chest cavity was opened, the neck of the mice was separated and the trachea was freed. An arterial indwelling needle was carefully inserted into the trachea and fixed. 1 mL of PBS solution was injected through the arterial indwelling needle with a syringe. The lavage was repeated 3 times. Finally, the alveolar lavage fluid was extracted and centrifuged at 1000g at 4°C. After centrifugation for 10 minutes, the supernatant was taken as BALF. The total protein content of BALF was calculated using the BCA method. The standard reference was diluted 2.5 times, 5 times, 10 times, 20 times, and 40 times and added to each well with 50 μL. The sample to be tested was set up in 2 duplicate wells, and 10 μL of the sample to be tested and 40 μL of diluent were added to each well. Except for the blank well, 10 μL of the reaction enzyme was added to each well. After sealing the plate, incubate at 37°C for 60 minutes. Dilute 20 times with distilled water and the concentrated washing solution for use, and wash the plate 5 times with the diluted washing solution for 30 seconds each time. Then add 50 μL of the A and B color display solutions in the kit, react at 37°C in the dark for 15 minutes, then add 50 μL of the stop solution. Finally, measure the OD value at a wavelength of 450 nm and calculate the concentration of the sample to be tested according to the standard curve.

[0063] 3. Kit to detect the expression levels of ferrous ions, GSH / GSSG, and MDA:

[0064] (1) Determination of ferrous ions:

[0065] For specific steps, refer to the instructions of the Geruisi Bio Ferrous Content Detection Kit (Ferrozine Colorimetric Method) (for specific contents of the instructions, refer to: http: / / www.geruisi-bio.com / pro / proshow / id / 810#) Weigh 0.1g of lung tissue, add 1mL of extract, and homogenize in an ice bath. Centrifuge at 12000rpm at 4℃, centrifuge for 5min, take the supernatant and place it on ice for testing. Add 1.2mL of distilled water to reagent 2 to fully dissolve and set aside; take 10μL of standard in an EP tube, and then add 490μL of reagent 3 (prepared according to the instructions) to prepare a 2μL / mL ferrous standard. Mix the ferrous standard thoroughly, place at room temperature for 15min, take 200μL of the mixture into a 96-well plate, and then use an enzyme reader to measure the absorbance at 562nm. The calculation formula for the value after measurement is as follows:

[0066]

[0067] In formula (1), X is Fe in the sample to be measured. 2+ content (nmol / g); A-absorbance value of the test tube; B-absorbance value of the blank tube; S-absorbance value of the standard tube, m-weight of the test sample (g).

[0068] (2) MDA expression level:

[0069] Take out the tissue from the refrigerator and homogenize and lyse it with RIPA lysis buffer (tissue: RIPA = 1:9), centrifuge at 4℃ 12000g, centrifuge for 10min, take the supernatant, and calculate the protein concentration of the supernatant by BCA method. Preparation of TBA storage solution: 25mgTBA is added to 6.76mL TBA preparation solution, so that it is fully dissolved into 0.37% TBA storage solution and then stored in a dark place. Preparation of MDA detection working solution: MDA detection working solution is prepared and used immediately, and is prepared according to the number of samples required for detection. Each sample requires 150μL of TBA diluent, 50μL of TBA storage solution, and 3μL of antioxidant. Dilution of standard: The standard is diluted with distilled water to 1μM, 2μM, 5μM, 10μM, 20μM, and 50μM for subsequent preparation of standard curves. Add samples according to the ratio, mix well, heat in a 100℃ metal bath for 15min, cool to room temperature in a water bath, and centrifuge at room temperature at 1000g for 10min. 200 μL of supernatant was added to a 96-well plate, and then the absorbance was measured at 532 nm using an ELISA reader. The molar concentration of MDA was calculated based on the standard curve, and the MDA content in the initial sample was expressed by the protein content per unit weight.

[0070] (3) Calculation of GSH / GSSG:

[0071] 1. Preparation of the kit:

[0072] (1) Preparation of GSSG stock solution: Add 816 μL of ultrapure water to 5 mg of GSSG provided in this kit, dissolve and mix to obtain the GSSG stock solution with a mass concentration of 10 mM. Except for the portion to be used immediately, the remaining GSSG stock solution should be appropriately divided and stored at -20°C.

[0073] (2) Preparation of DTNB stock solution: Add 1.5 mL of DMSO provided in this kit to 4.5 mg of DTNB provided in this kit, dissolve and mix to obtain the DTNB stock solution. Except for the portion to be used immediately, the remaining DTNB stock solution should be appropriately divided and stored at -20°C.

[0074] (3) Preparation of Protein Removal Reagent M Solution Weigh 0.2 g of protein removal reagent M, add 4 mL of total glutathione detection buffer, and prepare 4 mL of 5% aqueous solution. In addition, the protein removal reagent M solution must be freshly prepared and used only on the same day.

[0075] (4) Preparation of NADPH stock solution (40 mg / mL): Add 100 μL of ultrapure water to the 4 mg of NADPH provided in this kit, dissolve and mix to obtain the NADPH stock solution. Except for the portion used immediately, the remaining NADPH stock solution should be appropriately divided and stored at -70°C.

[0076] (5) Preparation of 5-fold diluted glutathione reductase: Take 50 μL of glutathione reductase, add 200 μL of total glutathione detection buffer, mix well, and obtain 5-fold diluted glutathione reductase.

[0077] (6) Preparation of total glutathione detection working solution: According to the number of samples to be detected, refer to the contents of Table 1 to prepare an appropriate amount of total glutathione detection working solution. The total glutathione detection working solution is obtained by mixing the three reagents in the table in proportion.

[0078] Table 1 Preparation of total glutathione detection working solution

[0079] 1 sample 10 samples 20 samples 5-fold dilution of glutathione reductase 6.6μ L 66μL 132μ L DTNB stock solution 6.6μ L 66μL 132μ L Total Glutathione Assay Buffer 150μL 1.5mL 3mL

[0080] (7) Preparation of 0.5 mg / mL NADPH: Take 10 μL of NADPH stock solution, add 790 μL of total glutathione detection buffer, and mix well to obtain 0.5 mg / mL NADPH. 50 μL of 0.5 mg / mL NADPH is required for each sample tested.

[0081] (8) Preparation of diluted GSH scavenging auxiliary solution: Add 53 μL of GSH scavenging auxiliary solution to 47 μL of ultrapure water and mix immediately. Since the diluted GSH scavenging auxiliary solution is not very stable, it must be freshly prepared each time it is used and can only be used on the same day.

[0082] (9) Preparation of GSH scavenging reagent working solution: Add 89.2 μL of anhydrous ethanol to 10.8 μL of GSH scavenging reagent and mix immediately. The GSH scavenging reagent working solution must be prepared fresh each time.

[0083] 2. Preparation of Standards

[0084] (1) Dilute the 10mM GSSG stock solution with Protein Removal Reagent M solution to a 15μM GSSG solution. Then dilute it to 10μM, 5μM, 2μM, 1μM, and 0.5μM GSSG solutions in sequence. Take six points of 15μM, 10μM, 5μM, 2μM, 1μM, and 0.5μM GSSG solutions to make a standard curve. Note: Since GSSG is not very stable in Protein Removal Reagent M solution, the GSSG solution prepared with Protein Removal Reagent M solution must be freshly prepared before use and cannot be frozen and then used.

[0085] (2) If the GSSG content in the sample needs to be determined, add 20 μL of diluted GSH scavenging auxiliary solution to every 100 μL of standard, and immediately vortex mix. Then add 4 μL of GSH scavenging reagent working solution to every 100 μL, mix immediately, and react at 25°C for 60 minutes. It can be used for subsequent GSSG content detection.

[0086] 3. Preparation of samples and standards for total glutathione content

[0087] Take the tissue and freeze it with liquid nitrogen, then grind it into powder. For every 10 mg of ground tissue powder, add 30 μL of protein removal reagent. Then add 70 μL of protein removal reagent M solution and homogenize thoroughly with a glass homogenizer (for tissues that are easier to homogenize, liquid nitrogen quick freezing and other treatments can be omitted, and an appropriate amount of protein removal reagent M solution can be directly added for homogenization). After placing at 4°C for 10 minutes, centrifuge at 10000g for 10 minutes at 4°C, and take the supernatant for the determination of total glutathione. The sample needs to be temporarily stored at 4°C. Samples that are not measured immediately can be stored at -70°C, but should not exceed 10 days. For the processed tissue samples, it is usually necessary to dilute them appropriately with protein removal reagent M solution before determination, and the dilution multiple is usually 5-20 times.

[0088] 4. Preparation of samples for GSSG content testing

[0089] Take some of the samples prepared above to measure the total glutathione content, add 20 μL of diluted GSH removal auxiliary solution to every 100 μL of sample, and vortex mix immediately. Then add GSH removal working solution to every 100 μL of sample, add 4 μL of GSH removal reagent working solution, and vortex mix immediately, and react at 25°C for 60 minutes. The above reaction can remove up to 50 μM GSH. If the GSH content in the sample is too high, it needs to be appropriately diluted before removing GSH. After the above treatment, it can be used for subsequent determinations.

[0090] 5. Determination of samples and standards

[0091] (1) Refer to Table 2, use a 96-well plate, add samples or standards in sequence, mix well. Add 150 μL of total glutathione detection working solution, mix well, and incubate at 25°C for 5 minutes.

[0092] Table 2 Determination of samples and standards

[0093] Blank Standard curve Sample Sample or standard 0μL 10 μL x μL Protein Removal Reagent M Solution 10 μL 0μL 10-xμL Total glutathione assay working solution 150μL 150μL 150μL Incubate at 25°C or room temperature 5min <![CDATA[5m i n]]> 5min 0.5mg / mL NADPH 50μL 50μL 50μL

[0094] (2) Add 50 μL of 0.5 mg / mL NADPH solution and mix well.

[0095] (3) Immediately measure A412 using an ELISA reader, once every 5 minutes or in real time, for a total of 25 minutes, and obtain 5 data points.

[0096] 6. Calculation of total glutathione content in samples

[0097] After 25 minutes (or 30 minutes to 60 minutes) of reaction, the absorbance is measured only once. A standard curve is made based on the different absorbances measured by standards of different concentrations. The total glutathione (GSSG concentration calculated by the standard curve multiplied by 2) or the GSSG content can be calculated by comparing the sample to the standard curve. The actual calculated total glutathione content is equivalent to multiplying the oxidized glutathione content by 2 and adding the reduced glutathione content. The GSH content can be calculated based on the measured total glutathione content and GSSG content. The calculation formula is: GSH = Total Glutathione-GSSG × 2 (Note: Total Glutathione is the GSSG concentration calculated by the standard curve multiplied by 2, and the GSSG obtained after removing GSH should also be multiplied by 2, because 1 GSSG molecule can be reduced to 2 GSH molecules after the reaction). For example, the concentration of total glutathione measured by this kit is 15 μM (i.e., the GSSG concentration calculated by the standard curve when measuring total glutathione is 7.5 μM, and multiplying by 2 is the total glutathione concentration), and the measured GSSG concentration is 1.2 μM (i.e., the GSSG concentration calculated by the standard curve when measuring the GSSG content alone is 1.2 μM), then the concentration of GSH in the sample is 15-1.2×2=12.6 μM.

[0098] 4. Immunofluorescence detection of GPX4 and 4-HNE expression levels:

[0099] Paraffin-embedded sections were dewaxed, rehydrated, and blocked with 5% BSA for 1 h. Subsequently, the sections were incubated with the following primary antibodies at 4°C overnight: GPX (1:2000; CST, USA), 4-HNE (1:500; CST, USA), and incubated at 4°C overnight. After washing, the corresponding Cy3-labeled secondary antibodies were added dropwise in the dark and incubated at 37°C for 30 min. 4'6-diamidino-2-phenylindole (DAPI) was added in the dark and incubated at 37°C for 5 min to locate the cell nucleus. After washing away the excess DAPI, the liquid on the sections was blotted with absorbent paper and the sections were sealed with a sealing solution containing an anti-fluorescence quencher. The fluorescence intensity was then measured using a microscope.

[0100] 4. Conclusion Analysis:

[0101] By comparing the YAP overexpression group and the lung injury tissues of intestinal ischemia-reperfusion II / R, it can be concluded that YAP has lung injury protection and anti-ferroptosis effects:

[0102] like Figure 1 As shown in the figure, the lung tissues of II / R mice in different groups were observed, and the results showed that overexpression of YAP could significantly alleviate the pathological damage of lung tissue caused by II / R, as manifested by the significant reduction of pathological damage such as pulmonary hemorrhage and alveolar wall thickening. In addition, overexpression of YAP could reduce the increased lung permeability and lung tissue edema caused by II / R.

[0103] like Figure 2 As shown in the results, overexpression of YAP significantly reduced the local and systemic inflammatory responses caused by II / R, especially the inflammatory factors IL-1β, IL-6 and TNF-α. Overexpression of YAP can significantly reduce the expression levels of these inflammatory factors.

[0104] like Figure 3 As shown, overexpression of YAP improved the typical changes of mitochondrial ferroptosis in alveolar type II epithelial cells caused by ALI in II / R mice.

[0105] like Figure 4 As shown, overexpression of YAP can inhibit the expression of ferroptosis-related proteins SLC7A11, Nrf2, and GPX4 in II / R mice.

[0106] like Figure 5 As shown in Figure 2, overexpression of YAP can inhibit the expression of ferroptosis-related biomarkers malondialdehyde (MDA), ferrous ion (Fe 2+ ) expression, and overexpression of YAO can reduce the ratio of reduced glutathione / oxidized glutathione (GSH / GSSG).

[0107] Depend on Figures 2 to 5 It can be seen that overexpression of YAP can inhibit lung tissue-related ferroptosis in II / R mice.

[0108] Since 4-HNE can promote myocardial cell ferroptosis and downregulate the key ferroptosis molecule GPX4 at the protein level, existing studies have shown that 4-HNE can promote the K48 ubiquitination degradation of GPX4. Figure 6 and Figure 7 It can be clearly seen that overexpression of YAP can inhibit lung tissue-associated ferroptosis in II / R mice.

[0109] In summary, the embodiments of the present application provide an application of a YAP-related protein as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury. By examining the effect of YAP on lung tissue in II / R mice, the results showed that overexpression of YAP can play a protective role in lung epithelial damage caused by intestinal ischemia-reperfusion.

[0110] The above is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features applied for by the present application.

Claims

1. A use of a YAP-related protein as a drug for treating or inhibiting intestinal ischemia-reperfusion lung injury, characterized in that: The application includes: using the overexpressed YAP-associated protein 1 to treat or inhibit intestinal ischemia-reperfusion lung injury.

2. The use according to claim 1, characterized in that: The expression level of the overexpressed YAP-associated protein 1 is 1.5 times or more of the expression level of the normal YAP-associated protein 1.

3. The use according to claim 1, characterized in that: The overexpressed YAP-associated protein 1 is used as a reagent for treating or inhibiting ferroptosis associated with intestinal ischemia-reperfusion lung injury.

4. The use according to claim 2, characterized in that: The overexpressed YAP-associated protein 1 is used as a reagent for regulating the expression of Nrf2 gene in tissue cells and / or the nuclear transfer of tissue cells in intestinal ischemia-reperfusion lung injury.

5. An inhibitory agent for ferroptosis associated with intestinal ischemia-reperfusion lung injury, characterized in that: The inhibitory agent includes overexpressed YAP-associated protein 1.

6. The inhibitory agent according to claim 5, characterized in that The protective agent includes an adeno-associated virus vector carrying an overexpressed YAP-associated protein 1 gene 7. A protective agent for intestinal ischemia-reperfusion lung injury, characterized in that: The protective agent includes overexpressed YAP-associated protein 1.

8. The protective agent according to claim 7, characterized in that The protective agent includes an adeno-associated virus vector carrying an over-expressed YAP-associated protein 1 gene.

9. A therapeutic or inhibitory agent for intestinal ischemia-reperfusion lung injury, characterized in that: The agent includes overexpressed YAP-associated protein 1.

10. The drug according to claim 7, characterized in that The agent includes an adeno-associated virus vector carrying an over-expressed YAP-associated protein 1 gene.

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