Application of Rab26 protein in preparation of medicine for preventing or treating cardiac hypertrophy

Abstract

The invention relates to the technical field of biological medicine, in particular to application of Rab26 protein in preparation of medicine for preventing or treating cardiac hypertrophy. When the expression level of the Rab26 gene is low (less Rab26 protein is generated), the cardiac muscle cell hypertrophy degree is obviously increased; when the Rab26 gene is over-expressed (more Rab26 protein is generated), the cardiac muscle cell hypertrophy degree is obviously reduced. Therefore, the Rab26 protein is used for preparing the medicine for preventing or treating cardiac hypertrophy.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of Rab26 protein in preparing medicine for preventing or treating myocardial hypertrophy. Background Art

[0002] Myocardial hypertrophy is usually an early stage of heart failure, which will lead to further deterioration of heart function over time and eventually develop into end-stage heart failure.

[0003] Pathological myocardial hypertrophy is a phenomenon of myocardial cell hypertrophy and proliferation caused by long-term high load, ischemia or other pathological stimulation of the heart. This process is not only accompanied by changes in myocardial structure and function, but also involves a series of complex molecular mechanisms, including activation of intracellular signal transduction pathways, inflammatory response, oxidative stress response, etc. Although there are certain treatment methods, the effective means to prevent pathological myocardial hypertrophy from progressing to heart failure are still very limited, and new treatment strategies are urgently needed. Summary of the invention

[0004] Based on this, the present invention provides the use of Rab26 protein in preparing a drug for preventing or treating myocardial hypertrophy, which at least solves one problem in the prior art.

[0005] In a first aspect, the present invention provides use of Rab26 protein in preparing a drug for preventing or treating myocardial hypertrophy.

[0006] Rab26 protein is mainly expressed in cardiomyocytes. When the expression level of Rab26 gene is low (less Rab26 protein is produced), the degree of cardiomyocyte hypertrophy increases significantly; when Rab26 gene is overexpressed (more Rab26 protein is produced), the degree of cardiomyocyte hypertrophy decreases significantly. Therefore, Rab26 protein can be used to prepare drugs for preventing or treating myocardial hypertrophy. Rab26 protein can promote the degradation of eEF1α1 protein, thereby slowing down the occurrence and development of myocardial hypertrophy.

[0007] In some optional embodiments, the myocardial hypertrophy is pathological myocardial hypertrophy.

[0008] In some optional embodiments, Rab26 protein alleviates cardiac hypertrophy by regulating the degradation of eEF1α1.

[0009] In a second aspect, the present invention provides a drug for preventing or treating myocardial hypertrophy, comprising Rab26 protein.

[0010] In some optional embodiments, the myocardial hypertrophy is pathological myocardial hypertrophy.

[0011] In some optional embodiments, the drug for preventing or treating myocardial hypertrophy further comprises a pharmaceutically acceptable excipient.

[0012] In some optional embodiments, the pharmaceutically acceptable excipient is at least one of a solvent and a diluent.

[0013] In some optional embodiments, the pharmaceutically acceptable excipient is water or physiological saline.

[0014] In some optional embodiments, the drug for preventing or treating myocardial hypertrophy is in the form of an injection.

[0015] Due to the adoption of the above technical solution, the embodiments of the present invention have at least the following beneficial effects: (1) By constructing Rab26 gene-specific knockout and overexpression mouse models, the potential of Rab26 protein in improving cardiac function was verified; Rab26 gene overexpression can significantly improve cardiac ejection fraction and single-cell contractile function, and reduce cardiomyocyte hypertrophy, providing a new idea for the clinical treatment of pathological myocardial hypertrophy; (2) It was discovered that Rab26 protein regulates the degradation of eEF1α1 through the autophagy pathway. This discovery provides new insights for a deeper understanding of the molecular mechanism of heart failure, provides new molecular targets for drug development and clinical treatment, and promotes the development of heart failure treatment research. By precisely targeting Rab26 and eEF1α1 regulation, customized treatment options can be provided for patients with different types of pathological myocardial hypertrophy, thereby improving treatment outcomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The Western blot test results and statistical data of each group of mice or rats in Example 1 are shown in FIG.

[0017] Figure 2 These are the results of immunofluorescence staining of the Sham group and TAC group in Example 1.

[0018] Figure 3 These are the results of immunofluorescence co-localization analysis of the Sham group and the TAC group in Example 1.

[0019] Figure 4 These are the immunofluorescence staining results of the control group and the PE group of adult mouse cardiomyocytes and HL-1 cardiomyocytes in Example 1.

[0020] Figure 5 These are the results of immunofluorescence staining analysis of the control group and PE group of adult mouse cardiomyocytes and HL-1 cardiomyocytes in Example 1.

[0021] Figure 6 The Western blot test results and statistical data of each group of mice in Example 2 are shown in FIG.

[0022] Figure 7 These are the cardiac ultrasound test results of each group of mice in Example 2.

[0023] Figure 8 These are the cardiac function test results of each group of mice in Example 2.

[0024] Fig. 9 are the heart weight / body weight ratio and heart weight / tibia length ratio of each group of mice in Example 2.

[0025] Fig.10 These are the atrial natriuretic peptide and brain natriuretic peptide values ​​of each group of mice in Example 2.

[0026] Fig.11 The following are the sizes of pathologically stained cardiomyocytes of each group of mice in Example 2.

[0027] Fig.12 This is a statistical diagram of the cross-sectional area of ​​pathologically stained cardiomyocytes in each group of mice in Example 2.

[0028] Fig.13 These are the results of single cardiomyocyte function testing of each group of mice in Example 3.

[0029] Fig.14 is the oxygen consumption of mitochondria in myocardial cells of each group of mice in Example 3.

[0030] Fig.15 These are the Seahorse test results for each group of mice in Example 3.

[0031] Fig.16 The Western blot test results and statistical data of each group of mice in Example 4 are shown in FIG.

[0032] Fig.17 These are the cardiac ultrasound test results of each group of mice in Example 4.

[0033] Fig.18 These are the cardiac function test results of each group of mice in Example 4.

[0034] Fig.19 are the heart weight / body weight ratio and heart weight / tibia length ratio of each group of mice in Example 4.

[0035] Fig. 20 These are the atrial natriuretic peptide and brain natriuretic peptide values ​​of each group of mice in Example 4.

[0036] Fig.21 The following are the sizes of pathologically stained cardiomyocytes of each group of mice in Example 4.

[0037] Fig. 22 This is a statistical diagram of the cross-sectional area of ​​pathologically stained cardiomyocytes in each group of mice in Example 4.

[0038] Fig.23 These are the results of single cardiomyocyte function testing of each group of mice in Example 5.

[0039] Fig.24 is the oxygen consumption of mitochondria in myocardial cells of each group of mice in Example 5.

[0040] Fig.25 These are the Seahorse test results for each group of mice in Example 5.

[0041] Fig.26 This is the result of co-immunoprecipitation of Rab26 and eEF1A proteins in Example 6.

[0042] Fig. 27 This is the immunofluorescence co-localization result of Rab26 and eEF1A proteins in Example 6.

[0043] Fig.28 This is the result of immunofluorescence co-localization analysis of Rab26 and eEF1A proteins in Example 6.

[0044] Fig.29 These are the Doulink-PLA analysis results of Rab26 and eEF1A proteins in Example 6. DETAILED DESCRIPTION

[0045] The following is a clear and complete description of the concept of the present invention and the technical effects produced, so as to fully explain the purpose, scheme and effect of the present invention.

[0046] As a member of the Rab protein family, Rab26 protein has been found to be involved in a variety of cellular processes, such as endocytosis, cell migration and secretion. However, the function of Rab26 protein in heart disease has not been fully studied, especially its role in myocardial hypertrophy and heart failure remains unclear.

[0047] The inventors found that the expression of Rab26 protein was significantly downregulated in the mouse model of pathological myocardial hypertrophy, especially in spontaneously hypertensive rats, aortic arch stenosis mice and angiotensin II-induced pathological myocardial hypertrophy mouse models. Immunofluorescence staining results showed that Rab26 protein was mainly expressed in cardiomyocytes, suggesting that it may play a key role in myocardial function and pathological changes. In the myocardial Rab26 gene-specific knockout mouse model, the loss of the Rab26 gene aggravated the deterioration of cardiac function, including decreased cardiac ejection fraction, weakened single-cell contractile function and a significant increase in myocardial cell hypertrophy.

[0048] Through further research, the inventors found that Rab26 protein may alleviate the process of pathological myocardial hypertrophy by regulating the degradation of eEF1α1 protein. The eEF1α1 gene is an important factor involved in protein synthesis. Rab26 protein can bind to eEF1α1 protein, and there is a negative correlation between the two, suggesting that Rab26 protein may regulate the degradation of eEF1α1 protein through the autophagy pathway, thereby slowing down the occurrence and development of myocardial hypertrophy.

[0049] Accordingly, the present invention provides the use of Rab26 protein in the preparation of a drug for preventing or treating myocardial hypertrophy, and provides a drug for preventing or treating myocardial hypertrophy, providing a new targeted treatment strategy for treating pathological myocardial hypertrophy. The technical scheme and technical effects of the present invention can be reflected by the following examples.

[0050] Example 1: Determining the changing trend of Rab26 protein in a mouse model of pathological myocardial hypertrophy For animal experiments, the following experimental groups were set up: Spontaneously hypertensive rats (SHR group): Spontaneously hypertensive rats were purchased from Vital River Company and were normally raised for 16 weeks.

[0051] Spontaneously hypertensive rat control group (WKY group): Spontaneously hypertensive rats (Wistar-Kyoto) were purchased from Weitonglihua Company and raised normally for 16 weeks.

[0052] Angiotensin II-induced group (Ang-II group): C57 mice were purchased from Vital River Company and treated with angiotensin II (1000 ng / kg·min) via micropump injection for four weeks.

[0053] Angiotensin II-induced control group (Control group): C57 mice were purchased from Vital River Company and treated with PBS via micropump injection for four weeks.

[0054] Aortic arch stenosis surgery group (TAC group): 8-9 week old C57BL / 6J male mice were selected, the hair on the front of the neck of the mice was fully exposed, and the neck hair of the mice was removed with depilatory cream. The mice with hair removed were then placed in an anesthesia box filled with 1%-2% isoflurane gas for anesthesia. When the mice were in anesthesia, the limbs and teeth of the mice were fixed on the mouse board, and the anesthesia state was maintained with 1% isoflurane. The skin of the neck of the mice was fully disinfected with medical iodine, and then the skin of the mice was cut open along the front edge of the neck. The thyroid tissue and muscle layer in front of the neck were bluntly separated with tweezers, and the thyroid tissue was gently pushed to both sides (try not to affect the blood supply of the thyroid tissue), and the front neck tissue was separated cleanly. Then use ophthalmic scissors to cut the mouse sternum to the second rib, use a retractor to hook the mouse neck muscles and fix them upward, fully expose the trachea below, bluntly separate the muscle tissue in front of the trachea, and fully expose the right common carotid artery; find the branch blood vessels of the aortic arch, use a pre-threaded needle to pass the 6-0 silk thread through the aortic arch between the first and second branches of the aortic arch, pull out the silk thread after bypassing the aortic arch, clamp the thread head with tweezers for surgical knotting, first fix a 27G needle in the knot, and carefully pull out the needle after tightening the knot, so that the ascending aorta can be circumscribed to the same size, and the degree of aortic arch narrowing is 50%-60%; then suture the sternum and skin layer by layer, and disinfect the local area again.

[0055] Sham group: 8-9 week old C57BL / 6J male mice were selected, and the treatment of mice was basically the same as that of TAC group, except that only threading was performed and the aortic arch was not ligated.

[0056] The mice or rats in each group were fed with regular diet, drinking water and lighting for 4 weeks after surgery, and then cardiac function tests, Western blot tests and immunofluorescence staining were performed. Figure 1-Figure 3 As shown in Figure 2, Rab26 protein expression was downregulated in various types of myocardial hypertrophy models. Figure 1 Middle A and Figure 1 As shown in Figure B, compared with the Sham group, the ratio of Rab26 protein content to β-Tubulin protein content (Rab26 / β-Tubulin) in the heart tissue of mice in the TAC group decreased by about 50%; Figure 1 Middle C and Figure 1 As shown in D, compared with the WKY group, the Rab26 / β-Tubulin in the heart tissue of the SHR rats decreased by about 20%; Figure 1 ZhongE and Figure 1 As shown in F, compared with the Control group, the Rab26 / β-Tubulin in the heart tissue of mice in the Ang-II group decreased by about 45%. Figure 2 and Figure 3As shown, compared with the Sham group, the expression of Rab26 protein in cardiomyocytes of mice in the TAC group was downregulated.

[0057] In addition, for cell experiments, set up the following experimental groups: Phenylephrine stimulation group (PE group): HL-1 cardiomyocytes and adult mouse cardiomyocytes were cultured in vitro and stimulated with phenylephrine (50 μmol / L, 48 hours) to construct a cardiomyocyte hypertrophy model.

[0058] Control group: HL-1 cardiomyocytes and adult mouse cardiomyocytes were treated with PBS.

[0059] Western blot detection and immunofluorescence staining were performed 48 hours after modeling. Figure 4 and Figure 5 As shown, the expression of Rab26 protein in HL-1 cardiomyocytes and adult mouse cardiomyocytes in the PE group was significantly downregulated.

[0060] Example 2: Overexpression of Rab26 gene in cardiomyocytes improves pathological myocardial hypertrophy For animal experiments, the following experimental groups were set up: Vector group: 4-6 week old C57 mice were selected and injected with Vector (2×10^11 vg) each, and the model was established 4 weeks later.

[0061] Rab26 group: C57 mice aged 4-6 weeks were selected and each mouse was injected with adeno-associated virus type 2 (2×10^11 vg) to overexpress the Rab26 gene through adeno-associated virus type 2, and the model was established after 4 weeks.

[0062] Vector+TAC group: Based on the Vector group, aortic arch coarctation surgery was performed. The operation of aortic arch coarctation surgery was the same as that in Example 1.

[0063] Vector+Sham group: Based on the Vector group, sham surgery was performed. The operation of the sham surgery was the same as that in Example 1.

[0064] Rab26+TAC group: Based on the treatment of Rab26 group, aortic arch coarctation surgery was performed. The operation of aortic arch coarctation surgery was the same as that of Example 1.

[0065] Rab26+Sham group: Based on the results of the Rab26 group, sham surgery was performed. The operation of the sham surgery was the same as that in Example 1.

[0066] The mice in each group were fed with regular diet, drinking water and lighting for 4 weeks after surgery, and then subjected to cardiac function test, morphological test, plasma ANP and BNP test, Western blot test, and immunofluorescence staining. Figure 6 As shown in Figure 2, the Rab26 gene-specific overexpression model of mouse cardiomyocytes was successfully constructed. Figure 7 and Figure 8 As shown in the figure, compared with other groups of mice, the heart function of mice with overexpression of Rab26 gene was good, indicating that overexpression of Rab26 can significantly improve the heart function of mice with heart failure. Fig. 9 As shown in Figure 2, the HW / BW and HW / TL ratios of mice with overexpression of the Rab26 gene were smaller, indicating that overexpression of the Rab26 gene can improve the heart weight of mice with heart failure. Fig.10 As shown in Figure 2, the ANP and BNP levels of mice with overexpression of the Rab26 gene were lower, indicating that overexpression of the Rab26 gene can significantly improve the levels of heart failure markers. Fig.11 and Fig.12 As shown, H&E and WGA staining results showed that Rab26 gene overexpression improved cardiac hypertrophy.

[0067] Example 3: Overexpression of the cardiomyocyte Rab26 gene improves cardiomyocyte contractile function and mitochondrial metabolism in mice with pressure overload heart failure For animal experiments, the following experimental groups were set up: Vector group: 4-6 week old C57 mice were selected and injected with Vector (2×10^11 vg) each, and the model was established 4 weeks later.

[0068] Rab26 group: C57 mice aged 4-6 weeks were selected and each mouse was injected with adeno-associated virus type 2 (2×10^11 vg) to overexpress the Rab26 gene through adeno-associated virus type 2, and the model was established after 4 weeks.

[0069] Vector+TAC group: Based on the Vector group, aortic arch coarctation surgery was performed. The operation of aortic arch coarctation surgery was the same as that in Example 1.

[0070] Vector+Sham group: Based on the Vector group, sham surgery was performed. The operation of the sham surgery was the same as that in Example 1.

[0071] Rab26+TAC group: Based on the treatment of Rab26 group, aortic arch coarctation surgery was performed. The operation of aortic arch coarctation surgery was the same as that of Example 1.

[0072] Rab26+Sham group: Based on the results of the Rab26 group, sham surgery was performed. The operation of the sham surgery was the same as that in Example 1.

[0073] The mice in each group were fed with regular diet, drinking water and lighting for 4 weeks after surgery, and then single cardiomyocytes were isolated and tested for function, and Seahorse test was performed. Fig.13 As shown in the results, Rab26 gene overexpression can improve the contractile function of cardiomyocytes, and indicators such as PS, ±dL / dt, and TR90 have recovered to a certain extent compared with other groups. Fig.14 and Fig.15 As shown in the figure, after TAC treatment, all indicators of primary cardiomyocytes of mice were reduced, and mitochondrial respiratory indicators such as basal respiratory value, ATP production, and maximum oxygen consumption were improved after overexpression of Rab26 gene. The above results show that overexpression of Rab26 gene can improve the contractile function and mitochondrial capacity metabolism of mouse cardiomyocytes.

[0074] Example 4: Knockout of Rab26 gene in cardiomyocytes aggravates pathological myocardial hypertrophy For animal experiments, the following experimental groups were set up: WT group: Rab26 flox / flox The mice served as the control group were intraperitoneally injected with tamoxifen (40 mg / kg) for three consecutive days.

[0075] CKO group: Cre-Rab26 flox / flox Mice were treated as Rab26 conditional knockout mice and injected intraperitoneally with tamoxifen (40 mg / kg) for three consecutive days.

[0076] WT+TAC group: Based on the WT group, aortic arch coarctation surgery was performed, and the operation of aortic arch coarctation surgery was the same as that of Example 1.

[0077] CKO+TAC group: Based on the CKO group, aortic arch coarctation surgery was performed, and the operation of aortic arch coarctation surgery was the same as that in Example 1.

[0078] The mice in each group were fed with regular diet, drinking water and lighting for 4 weeks after surgery, and their cardiac function and morphology were tested, and H&E and Masson staining were performed. Fig.16 As shown in Figure 2, a cardiomyocyte Rab26 gene-specific knockout mouse model was successfully constructed. Figure 17-Figure 18 As shown in Figure 2, compared with other groups of mice, Rab26 knockout can significantly aggravate the deterioration of cardiac function in heart failure mice. Fig.19 As shown in Figure 2, knocking out the Rab26 gene can significantly increase the heart weight of mice with heart failure. Fig. 20 As shown in Figure 2, knocking out the Rab26 gene can significantly aggravate the levels of heart failure markers. Figure 21-22 As shown, knocking out the Rab26 gene can significantly aggravate cardiomyocyte hypertrophy in heart failure mice.

[0079] Example 5: Cardiomyocyte Rab26 gene knockout aggravates cardiomyocyte contractile function and mitochondrial metabolism in pressure-overload heart failure mice For animal experiments, the following experimental groups were set up: WT group: Rab26 flox / flox The mice served as the control group were intraperitoneally injected with tamoxifen (40 mg / kg) for three consecutive days.

[0080] CKO group: Cre-Rab26 flox / flox Mice were treated as Rab26 conditional knockout mice and injected intraperitoneally with tamoxifen (40 mg / kg) for three consecutive days.

[0081] WT+TAC group: Based on the WT group, aortic arch coarctation surgery was performed, and the operation of aortic arch coarctation surgery was the same as that of Example 1.

[0082] CKO+TAC group: Based on the CKO group, aortic arch coarctation surgery was performed, and the operation of aortic arch coarctation surgery was the same as that in Example 1.

[0083] The mice in each group were fed with regular diet, drinking water and lighting for 4 weeks after surgery, and then single cardiomyocytes were isolated and tested for function and Seahorse test. Fig.23 As shown in Figure 3, cardiomyocyte-specific knockout of Rab26 aggravated cardiomyocyte contractile function. Fig.24 and Fig.25 As shown in the figure, after TAC treatment, all indicators of primary cardiomyocytes of mice were reduced, and myocardial-specific knockout of Rab26 aggravated mitochondrial respiratory indicators such as basal respiration value, ATP production, and maximum oxygen consumption. The above results indicate that specific knockout of the Rab26 gene aggravates the contractile function and mitochondrial energy metabolism of mouse cardiomyocytes.

[0084] Example 6: Interaction between Rab26 protein and eEF1α1 protein The proteins that bind to Rab26 protein were analyzed by immunoprecipitation mass spectrometry. The mass spectrometry results showed that Rab26 protein can interact with eEF1α1 protein, and the binding between the two was further verified by immunoprecipitation, immunofluorescence and PLA. Figure 26-Figure 29 As shown, Rab26 can clearly bind to eEF1α1.

[0085] In the above examples, the method for detecting mice (or rats) is as follows: ① Heart function test: The Visual Sonics Vevo 2100 small animal ultrasound detection system was used to perform M-mode ultrasound on mice and record the heart rate. During the detection, the measurement frequency was 15 MHz, the depth was 3 cm, and the speed was 200 mm / s. In order to obtain a relatively clear parasternal left ventricular long axis image, the direction and position of the ultrasound probe can be appropriately adjusted, and the corresponding M-mode ultrasound image can be measured and recorded at the level of the left ventricular papillary muscle, and the left ventricular ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter at end-systolic (LVID;s), and left ventricular internal diameter at end-systolic (LVID;d) and other indicators were recorded.

[0086] ②Hemodynamic pressure detection: A 1.4F Millar catheter was used to measure hemodynamic indices, including aortic systolic pressure (SP) and diastolic pressure (DP). The detection steps were as follows: fully expose the hair on the front of the mouse neck, use a depilatory cream to remove the neck hair of the mouse, and then place the depilated mouse in an anesthesia box filled with 1%-2% isoflurane gas for anesthesia; when the mouse was in an anesthetized state, fix the mouse's limbs and teeth on a mouse board, and use 1% isoflurane to maintain an anesthesia state; cut the mouse skin along the median front edge of the neck, use forceps to bluntly separate the anterior cervical thyroid tissue and muscle layer, and gently push the thyroid tissue to both sides (try not to affect the blood supply of the thyroid tissue), and after the anterior cervical tissue is separated, use ophthalmic scissors to cut the mouse sternum to the second rib, use a retractor to hook the mouse neck muscle and fix it upward, fully expose the trachea below, and bluntly separate the muscle tissue in front of the trachea. , fully expose the right common carotid artery; strip the tissues around the common carotid artery and the carotid sheath, and carefully separate the accompanying vagus nerve; after separating the tissues around the carotid artery, tie a slipknot with 6-0 silk thread at the proximal end of the artery, tie a dead knot with 6-0 silk thread at the distal end, and put a silk thread under the right common carotid artery in advance for use; use vascular microscissors to cut a "V"-shaped small opening at the distal end of the right common carotid artery (try to face the opening upward), fix the blood vessel with micro forceps and slightly open the "V"-shaped incision, slowly insert the Millar catheter from the opening of the right common carotid artery, tie a knot with the spare silk thread, slowly loosen the slipknot at the proximal end of the right common carotid artery, push the Millar catheter forward to the aorta, stay for 2 minutes, and start recording data after the signal is stable; then continue to push the catheter forward into the left ventricle, stay for 2 minutes, and record the signal again after the signal is stable. Use the instrument software paired with the Millar catheter for data analysis.

[0087] ③Morphological detection: The mice were anesthetized and killed, and their weights were recorded. The hearts and lungs were quickly cut and the weights of the hearts and lungs were recorded. A part of the myocardial tissue was embedded in OCT and frozen, and then stained with WGA and Tunel, etc. The other part of the myocardial tissue was fixed with 4% paraformaldehyde and paraffin-sectioned, and then stained with H&E and Masson, etc. The HW / BW and HW / TL ratios were calculated based on the heart weight (HW), body weight (BW) and tibia length (TL) data of the mice.

[0088] ④ Plasma ANP and BNP detection: The mice were anesthetized and killed, and the mouse plasma was collected. The competitive method was used to detect the content of central natriuretic peptide (ANP) or brain natriuretic peptide (BNP) in the samples using Nanjing Jiancheng ANP (H180-1-2) and BNP (H166-1-2) kits. The samples were added to the enzyme-labeled wells pre-coated with antibodies, and then the recognition antigens labeled with biotin were added. The samples were incubated at 37°C for 30 minutes. The two competed with the solid phase antibody to form an immune complex. The unbound biotin antigen was removed by washing with PBST, and then avidin (HRP) was added. The cells were incubated at 37°C for 30 minutes. Avidin bound to the biotin antigen. After washing, the bound HRP catalyzed tetramethylbenzidine (TMB) to blue, which was then converted to yellow under the action of acid. There was an absorption peak at a wavelength of 450nm, and the absorbance value was negatively correlated with the concentration of the antigen in the sample.

[0089] ⑤ Single cardiomyocyte isolation and functional testing: After the mice were anesthetized and disinfected, the heart was quickly removed and perfused with KHB buffer for 15 min. Collagenase D (0.05 mg / ml) was added and digestion was terminated after 15-20 min. The left ventricle was then cut into pieces and long strips of cardiomyocytes were observed under a microscope. The isolated cardiomyocytes were placed on a transparent slide and covered with contraction buffer. Suprathreshold stimulation was given at a frequency of 0.5 Hz and an interval of 3 ms. The phase contrast microscope was connected to a computer monitor, and the peak shortening (PS), ± maximum shortening / re-lengthening rate (± dL / dt), and shortening and re-lengthening duration (TPS, TR90) values ​​were recorded using IonOptixsoft-edge software.

[0090] ⑥Myocardial cell apoptosis staining: Use an immunohistochemistry pen to draw marks around the OCT-embedded heart tissue slices, and wash the tissue slices with PBS 3 times, 5 minutes each time. After discarding the PBS, fix the tissue slices with 4% paraformaldehyde at room temperature for 15 minutes. Wash the tissue slices with PBS 3 times, 5 minutes each time. Add PBS containing 0.5% Triton X-100 to the surface of the slices, incubate at room temperature for 5 minutes, and then wash the tissue slices with PBS 3 times, 5 minutes each time. Block the tissue slices with goat serum blocking solution at room temperature for 1 hour. Aspirate the goat serum blocking solution, add the pre-configured Tunel detection solution, and incubate at 37℃ in the dark for 60 minutes. After discarding the Tunel detection solution, wash the tissue slices with PBS 3 times, 5 minutes each time. Finally, add a small amount of anti-fluorescence quenching sealing agent to the surface of the heart tissue, seal the slices, and perform laser confocal imaging detection and photography as soon as possible.

[0091] ⑦Western blot detection: Western blot was used to detect the expression of ANP, BNP, Rab26, eEF1α1 and other proteins and their active components in myocardial tissue or cardiomyocytes.

[0092] ⑧Detection of cardiomyocyte hypertrophy: Use an immunohistochemistry pen to draw marks around the OCT-embedded heart tissue slices, and wash the tissue slices with PBS 3 times, 5 minutes each time. After discarding PBS, fix the tissue slices with 4% paraformaldehyde at room temperature for 15 minutes. Wash the tissue slices with PBS 3 times, 5 minutes each time. Block the tissue slices with goat serum blocking solution at room temperature for 1 hour. Aspirate the goat serum blocking solution, add the pre-prepared WGA dye solution, place it in a wet box at 37°C and incubate for 30 minutes, discard the WGA dye solution, and wash the tissue slices with PBS 3 times, 5 minutes each time. Finally, add a little anti-fluorescence quenching sealing agent to the surface of the heart tissue, seal the slice, and perform laser confocal imaging detection and photography as soon as possible.

[0093] ⑨Seahorse detection: The Seahorse energy metabolism analyzer was used to detect the mitochondrial oxygen consumption (OCR) of mouse cardiomyocytes, and to quantify and analyze various indicators of mitochondrial respiratory function, including basal respiration, ATP production, maximum oxygen consumption, and spare respiration capacity.

[0094] The above is only a preferred embodiment of the present invention. The present invention is not limited to the above implementation. As long as the technical effect of the present invention is achieved by the same or equivalent means, it should belong to the protection scope of the present invention. Within the protection scope of the present invention, its technical scheme and / or implementation method can have various modifications and changes.

Claims

1. Application of Rab26 protein in the preparation of drugs for preventing or treating myocardial hypertrophy.

2. The use according to claim 1, characterized in that: Myocardial hypertrophy is pathological myocardial hypertrophy.

3. The use according to claim 1, characterized in that: Rab26 protein alleviates cardiac hypertrophy by regulating the degradation of eEF1α1.

4. A drug for preventing or treating myocardial hypertrophy, characterized in that: Including Rab26 protein.

5. The drug for preventing or treating myocardial hypertrophy according to claim 4, characterized in that: Myocardial hypertrophy is pathological myocardial hypertrophy.

6. The drug for preventing or treating myocardial hypertrophy according to claim 4, characterized in that: The drug for preventing or treating myocardial hypertrophy further comprises a pharmaceutically acceptable excipient.

7. The drug for preventing or treating myocardial hypertrophy according to claim 6, characterized in that: The pharmaceutically acceptable excipient is at least one of a solvent and a diluent.

8. The drug for preventing or treating myocardial hypertrophy according to claim 7, characterized in that: The pharmaceutically acceptable excipient is water or saline.

9. The drug for preventing or treating myocardial hypertrophy according to claim 8, characterized in that: The drug for preventing or treating myocardial hypertrophy is in the form of an injection.

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