Application of hesperidin in preparation of medicine for preventing or treating pulmonary arterial hypertension

Abstract

The invention relates to the technical field of medicines, in particular to application of hesperidin in preparation of medicines for preventing or treating pulmonary arterial hypertension. The hesperidin shows a good anti-HAPH effect in in-vivo and in-vitro experiments, and the hesperidin as an EOGT inhibitor has the potential possibility of being researched and developed into an HAPH treatment drug.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to application of hesperidin in preparing a medicine for preventing or treating pulmonary hypertension. Background Art

[0002] High-altitude pulmonary hypertension (HAPH) is clinically classified as the third type of pulmonary hypertension, i.e., pulmonary hypertension caused by lung disease and / or hypoxia. It is a progressive disease caused by chronic hypoxia and the subsequent pulmonary vasoconstriction and remodeling in people living at an altitude of more than 2500m. There is no clear recommendation for the treatment of HAPH patients in the current domestic and international guidelines for the diagnosis and treatment of pulmonary hypertension. The current HAPH treatment plans are mostly based on the first type of pulmonary hypertension, i.e., arterial pulmonary hypertension. Drug treatments are often used, such as endothelin receptor blockers (bosentan, ambrisentan, etc.), carbonic anhydrase inhibitors (acetazolamide, etc.), phosphodiesterase-5 inhibitors (sildenafil, tadalafil, etc.), and angiotensin system inhibitors. Other treatments include intermittent oxygen inhalation or lowering the altitude to increase the patient's inhaled oxygen content. Although the current HAPH treatment methods can reduce the pulmonary artery pressure of HAPH patients to a certain extent, they are not enough to inhibit and delay pulmonary vascular remodeling and prevent right heart failure, thereby achieving the ultimate goal of reducing patient mortality. Therefore, it is of great significance to clarify the molecular mechanism of HAPH disease and develop drugs that can directly reduce pulmonary artery pressure and inhibit and delay pulmonary vascular remodeling for the treatment and prognosis of HAPH patients. Summary of the invention

[0003] The purpose of the present invention is to provide the use of hesperidin in the preparation of drugs for preventing or treating pulmonary hypertension. In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide the use of hesperidin in drugs for preventing or treating pulmonary hypertension, so as to solve the problem that there is no drug with definite efficacy and directly reducing pulmonary artery pressure in the prior art. The present invention establishes a hypoxia-induced HAPH rat model and a PASMCs proliferation model, studies the anti-HAPH pharmacodynamic effects of hesperidin in vivo and in vivo, and evaluates them. On the basis of clarifying its pharmacodynamic effects, it further clarifies the relationship between the pharmacodynamic mechanism of hesperidin and EOGT, providing a basis for the research of HAPH disease treatment drugs and further research on hesperidin anti-HAPH.

[0004] The present invention solves the technical problem by adopting the following technical solutions:

[0005] In a first aspect, the present invention provides use of hesperidin in preparing a medicament for preventing or treating pulmonary hypertension.

[0006] In some embodiments of the present invention, the hesperidin is used to prepare a medicament for preventing or treating high-altitude pulmonary hypertension.

[0007] In some embodiments of the present invention, the hesperidin is used to prepare a medicament for treating high-altitude pulmonary hypertension.

[0008] In some embodiments of the present invention, the hesperidin treats high-altitude pulmonary hypertension by inhibiting pulmonary vascular remodeling.

[0009] In some embodiments of the present invention, the hesperidin treats high-altitude pulmonary hypertension by inhibiting the proliferation of pulmonary arterial smooth muscle cells.

[0010] In some embodiments of the present invention, the hesperidin treats high-altitude pulmonary hypertension by inhibiting the proliferation of pulmonary arterial smooth muscle cells.

[0011] In some embodiments of the present invention, the hesperidin treats high-altitude pulmonary hypertension by inhibiting the expression of EOGT protein in pulmonary artery smooth muscle cells.

[0012] In some embodiments of the present invention, the hesperidin inhibits the expression of EOGT protein in pulmonary artery smooth muscle cells and then inhibits the activation of Notch3 / Bcl-2 signaling pathway to treat high-altitude pulmonary hypertension

[0013] In some embodiments of the present invention, the hesperidin is used as an EOGT inhibitor for preparing a drug for treating high-altitude pulmonary hypertension.

[0014] The present invention includes the following beneficial effects: Hesperidin exhibits good anti-HAPH effects in both in vivo and in vitro experiments, and its mechanism of action is related to its inhibitory effect on EOGT. Hesperidin can inhibit the activation of Notch3 receptors by binding to EOGT, thereby inhibiting the expression of EOGT and blocking the activation of the Notch3 / Bcl-2 signaling pathway in HAPH, thereby inhibiting the proliferation of PASMCs, delaying the pulmonary vascular remodeling of HAPH rats, and ultimately achieving a therapeutic effect on HAPH rats. That is, inhibiting the expression of EOGT is a feasible way to treat HAPH, and hesperidin, as an EOGT inhibitor, has the potential to be studied and developed into a HAPH therapeutic drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 1 is the pulmonary artery pressure waveform of each group of rats in Example 1.

[0016] Figure 2 is the mean pulmonary artery pressure of rats in each group in Example 1.

[0017] Figure 3 This is the HE staining image of the pulmonary artery of each group of rats in Example 1.

[0018] Figure 4 is the percentage of pulmonary vascular wall thickness of each group of rats in Example 1.

[0019] Figure 5 It is the percentage of pulmonary vascular wall area of ​​each group of rats in Example 1.

[0020] Figure 6 Figure 1 shows immunohistochemical staining of EOGT in the pulmonary vessels of rats in each group.

[0021] Figure 7 The figure is a statistical graph of the average optical density of EOGT immunohistochemical staining of the pulmonary vessels of each group of rats in Example 1.

[0022] Figure 8 This is the protein band diagram of the lung tissue of each group of rats in Example 1.

[0023] Fig. 9 Statistical graph of α-SMA protein bands in lung tissue of rats in each group in Example 1.

[0024] Fig.10 Statistical graph of Bcl-2 protein bands in lung tissue of rats in each group in Example 1.

[0025] Fig.11 This is a statistical diagram of EOGT protein bands in the lung tissues of rats in each group in Example 1.

[0026] Fig.12 Statistical graph of Notch3 protein bands in lung tissue of rats in each group in Example 1.

[0027] Fig.13 This is a statistical chart of the CCK8 results of each group in Example 2.

[0028] Fig.14 Graph showing the flow cytometry results for each group in Example 2.

[0029] Fig.15 Statistical graph of cell apoptosis rate in each group in Example 2.

[0030] Fig.16 This is an immunofluorescence staining image of EOGT in each group of PASMCs in Example 2.

[0031] Fig.17 This is the protein band diagram of PASMCs in each group in Example 2.

[0032] Fig.18 This is a statistical diagram of EOGT protein bands in each group of PASMCs in Example 2.

[0033] Fig.19 This is a statistical diagram of the Bcl-2 protein bands in each group of PASMCs in Example 2.

[0034] Fig. 20 This is a statistical diagram of Notch3 protein bands in each group of PASMCs in Example 2.

[0035] Fig.21 This is the molecular docking result of hesperidin and EOGT.

[0036] Fig. 22 This is the binding and dissociation curve of hesperidin and EOGT.

[0037] Fig.23 This is the affinity fitting curve between hesperidin and EOGT.

[0038] Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

[0039] As used herein, the term "prevention" refers to preventing the occurrence of a disease and / or preventing the recurrence of a disease. DETAILED DESCRIPTION

[0040] The present invention specification describes the specific implementation scheme in detail. Those skilled in the art should recognize that the following implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the protection scope of the claims of the present invention. The following examples specifically illustrate the beneficial effects of the present invention.

[0041] Example 1

[0042] The therapeutic effect of hesperidin on HAPH rats

[0043] 1 Materials

[0044] 1.1 Experimental animals

[0045] Male SD rats were provided by the Animal Experiment Center of Xinjiang Medical University (Animal Production License No.: SYXK (New)

[0046] 2018-0003). All animal experiments in this experiment were conducted in accordance with the standards of the Ethics Committee of Xinjiang Medical University (Ethics Standard Number: IACUC-20230308-3).

[0047] 1.2 Reagents

[0048] Hesperidin (Shanghai Yuanye Company, purity >95%); enhanced RIPA lysis buffer, broad-spectrum phosphatase inhibitor, broad-spectrum protease inhibitor (Wuhan Boster Company); BCA protein quantification kit (Beijing Polymer Company); anti-EOGT antibody (abcam, USA); anti-Notch3 antibody (abcam, USA); anti-Bcl-2 antibody (abcam, USA).

[0049] 1.3 Instruments

[0050] Small animal ventilator, Beijing Kosjia Technology Co., Ltd., ks606731; electrophysiological recorder, Chengdu Taimeng Electronics Co., Ltd., BL-420S; microplate reader, Finnish Labsystems Multi-skan MS, 352; small vertical electrophoresis transfer system, American Bio-Rad, Mini-PROTEAN Tetra Cell; chemiluminescent gel imager, American Protein Simple, FluorChem E; low-temperature high-speed centrifuge, American Beckman Coulter Allegra-64R; Northwest special environment artificial experimental cabin, Guizhou Fenglei Aviation Ordnance Co., Ltd., DY-2.

[0051] 2 Methods

[0052] 2.1 Establishment of HAPH animal model

[0053] Normal group; model group; sildenafil group (SDF, 30 mg / kg / d); high-dose hesperidin group (100 mg / kg / d); medium-dose hesperidin group (50 mg / kg / d); low-dose hesperidin group (25 mg / kg / d).

[0054] The rats in the normal group were placed in a plain environment (altitude: 845m, atmospheric pressure 91.7kPa) and fed for 30 days. Except for the normal group, the rats in other groups were placed in a plateau environment (simulated plateau environment altitude: 6000m, atmospheric pressure 47.0kPa) after one week of adaptive feeding, and placed for 22 hours a day for 30 consecutive days to establish the HAPH rat model. During this period, after being placed in the plateau environment for 15 days, hesperidin and sildenafil were given by gavage for 15 days, and normal group and model group rats were given saline by gavage for 15 days (1mL / 100g / d).

[0055] 2.2 Measurement of rat pulmonary artery pressure

[0056] Rats were anesthetized and fixed on the operating table with intraperitoneal injection of sodium pentobarbital (2%, 30 mg / kg), the neck skin was cut, and the trachea was peeled off. The trachea was cut and connected to a small animal ventilator to establish a stable respiratory cycle. The chest skin was cut, and the chest cavity was opened along the midline of the sternum to expose the lungs and heart. The rat heart was opened to expose the pulmonary artery, and a homemade cannula was used and connected to a transducer. The cannula needle was inserted into the pulmonary artery toward the centripetal end, and the pressure waveform and parameters were observed on the BL-420 biological signal acquisition and analysis system, and the stabilized pulmonary artery pressure waveform and the mean pulmonary artery pressure of the rat were recorded.

[0057] 2.3 Pathological examination of rat lungs

[0058] Lung tissues of rats in each group were taken and fixed in 10% formaldehyde solution. The specimens were placed in 80% ethanol, 95% ethanol, and anhydrous ethanol for dehydration, and then placed in xylene for transparency. After transparency, they were embedded. Paraffin-embedded tissue blocks were cut into 5 μm slices, dewaxed with xylene, dehydrated with gradient ethanol, stained with hematoxylin, differentiated with 1% hydrochloric acid ethanol, counter-blued with 1% dilute ammonia, stained with eosin, dehydrated with gradient, and sealed with neutral gum. Each stained slice was observed under an optical microscope, and 25-30 small and medium pulmonary arteries (diameter 300-500 μm) were randomly selected for detection. The percentage of vascular wall thickness (WT%) and the percentage of vascular wall area (WA%) were used to indicate the degree of reconstruction and proliferation of pulmonary tissue arterioles.

[0059] WT% = (outer diameter of pulmonary arteriole - inner diameter of pulmonary arteriole) / (outer diameter of pulmonary arteriole) × 100%

[0060] WA% = (cross-sectional area of ​​pulmonary arteriole wall) / (total cross-sectional area of ​​pulmonary arteriole) × 100%

[0061] 2.4 Immunohistochemical staining of paraffin sections of rat lung tissue

[0062] The paraffin-embedded blocks of rat lung tissue were cut into 5 μm slices. After being placed in a 60°C oven for 4 hours, the slices were immersed in xylene I, II, and III for 15 minutes to dewax. After dewaxing, the slices were immersed in 100% ethanol I and II for 5 minutes, 95%, 90%, 80%, and 70% ethanol for 2 minutes, and then rinsed with PBS buffer 3 times, 3 minutes each time. After completion, antigen repair was performed using the high temperature and high pressure antigen repair method with citric acid buffer. After antigen repair was completed, 3% H 2 O 2React at room temperature for 20 minutes, then rinse with PBS buffer 3 times, 5 minutes each time. After washing, add 5% BSA and block at room temperature for 30 minutes. After blocking, carefully remove the BSA solution on the slice and add the pre-prepared primary antibody working solution, then incubate in a 4°C refrigerator overnight. The next day, rinse with 1×PBST buffer 3 times, 5 minutes each time after the primary antibody incubation. After washing, add the secondary antibody corresponding to the primary antibody and incubate at 37°C for 30 minutes. Rinse 3 times with 1×PBST buffer, 5 minutes each time. Then add DAB colorimetric solution, and immerse in distilled water after staining. Use hematoxylin to stain for 3 minutes, then differentiate in hydrochloric acid alcohol for 5 seconds, and quickly rinse the slice under running water. Then the slices are in 80% ethanol, 95% ethanol for 2 minutes, ethanol I and 100% ethanol II for 5 minutes each, xylene I and II for 5 minutes each, and finally sealed with neutral resin. Observe and take pictures under a microscope to describe the degree of immune response of tissue cells.

[0063] 2.5 Protein immunoblotting of rat lung tissue

[0064] 2.5.1 Total protein extraction and sample preparation from rat lung tissue

[0065] Take out the rat lung tissue frozen at -80℃, quickly cut about 100mg of tissue on ice and place it in a pre-cooled 1.5mL grinding tube. After adding grinding steel balls to the grinding tube, (g) :Lysate (mL) =1:10, add a certain volume of pre-cooled RIPA lysis buffer (RIPA: broad-spectrum phosphatase inhibitor: broad-spectrum protease inhibitor = 100:1:1), place the grinding tube in the instrument adapter, start the instrument and homogenize at 60Hz for 2min. After the homogenization is completed, take out the tissue homogenate, centrifuge it at 4℃12000r / min for 10min, and take the supernatant as the lung tissue protein sample. The supernatant is divided and stored in a -80℃ refrigerator for later use. After the protein of the sample is quantified by the BCA method, the sample is diluted with distilled water according to the quantitative results, and 5×Loading Buffer is added at a volume ratio of protein sample: 5×Loading Buffer = 4:1 and mixed. The final protein content is 5μg / μL. After the sample is mixed, the sample tube is heated in a metal bath at 100℃ for 10min to denature the protein. After denaturation, the sample is cooled to room temperature, divided and stored in a -80℃ refrigerator for later use.

[0066] 2.5.2 Western Blotting

[0067] 2.5.2.1 Gel preparation

[0068] Use a 1.5 mm, 15-well mini glass plate to assemble the gel-making mold. Prepare the required volume of separation gel and concentrated gel according to the instructions of the one-step gel preparation kit. Add a certain volume of separation gel and concentrated gel to the gel-making glass plate, insert a comb, and let it stand for 30 minutes to solidify.

[0069] 2.5.2.2 Protein loading

[0070] Fix the glass plate in the electrophoresis tank, fill the inner tank with electrophoresis buffer (25mM Tris, 192mM Glycine, 0.1% SDS), and add a specified volume of electrophoresis solution to the electrophoresis box. Slowly pull out the comb to expose the sample wells, load the protein sample at a volume of 5μL / well, and finally add the prestained protein marker to the blank well.

[0071] 2.5.2.3 Electrophoresis

[0072] After the sample is loaded, gradient electrophoresis is started. The electrophoresis conditions are: first, electrophoresis at 80 V for 30 min, then at 120 V for 1 h, that is, the electrophoresis is terminated when bromophenol blue migrates close to the bottom of the gel.

[0073] 2.5.2.4 Transfer

[0074] First, prepare the items needed for membrane transfer, place the electrotransfer clip flat in the operation tray, and add a certain amount of electrotransfer buffer (25mM Tris, 190mM Glycine, 20% Methanol) to the tray. Then place the sponge and filter paper on the black and white sides of the electrotransfer clip, with the sponge at the bottom and the filter paper at the top. Take out the glass plate after electrophoresis, open the glass plate, take out the gel and place it on the filter paper on the black side of the electrotransfer clip, and carefully remove the bubbles between the gel and the filter paper so that the two fit tightly. Then, cut the PVDF membrane of appropriate size according to the size of the gel and soak it in methanol for 2 minutes to activate it. Place the activated PVDF membrane on the gel and remove the bubbles between the two so that they fit tightly. Finally, close the electrotransfer clip and put it in the electrotransfer tank in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge" from bottom to top, put the electrotransfer tank into the vertical electrophoresis tank, and electrotransfer for 2 hours under 100V constant voltage conditions.

[0075] 2.5.2.5 Closure

[0076] After the electrotransfer was completed, the PVDF membrane was taken out and blocked in 5% skimmed milk powder prepared in 1×TBST buffer on a slowly shaking shaker at room temperature for 2 h.

[0077] 2.5.2.6 Antibody incubation

[0078] After blocking, the PVDF membrane was washed 3 times with 1×TBST buffer on a high-speed shaking platform for 10 minutes each time. After washing, the PVDF membrane was placed in an antibody incubation box and the primary antibody dilution (1×TBST) was added, and incubated on a slow shaking platform at 4°C for 18 hours. The next day, the primary antibody dilution was recovered, and the PVDF membrane was washed 3 times with 1×TBST buffer on a high-speed shaking platform for 10 minutes each time. Subsequently, the PVDF membrane was placed in an antibody incubation box, the secondary antibody dilution corresponding to the primary antibody was added, and incubated on a slow shaking platform at room temperature for 1 hour.

[0079] 2.5.2.7 Image analysis

[0080] After the secondary antibody incubation, the PVDF membrane was washed three times with 1×TBST buffer on a high-speed shaking shaker for 10 min each time. After washing, the PVDF membrane was imaged in an imager using ECL luminescent liquid, and the band images were collected and analyzed. The protein expression level was expressed as relative expression.

[0081] 3 Results

[0082] 3.1 Effect of hesperidin on pulmonary artery pressure in HAPH model rats

[0083] Figure 1 is the pulmonary artery pressure waveform of each group of rats in Example 1, Figure 2 is the mean pulmonary artery pressure of rats in each group in Example 1.

[0084] like Figure 1 , Figure 2 As shown. After 30 days of hypoxia induction, the mPAP of the model group rats increased significantly compared with the normal group, reaching more than 30 mmHg. The mPAP of the rats in the hesperidin intervention group decreased with the increase of hesperidin intervention concentration, but the lowest was about 20 mmHg, and it could not be completely restored to the level of the normal group. Sildenafil also showed a certain antihypertensive effect, and from Figure 2 It can be seen that the mean pulmonary artery hypertension in the hesperidin 50mg / kg and 100mg / kg groups was lower than that in the sildenafil 30mg / kg group. This shows that hesperidin at a dose of 50mg / kg and 100mg / kg is more effective than sildenafil 30mg / kg in reducing the mean pulmonary artery pressure in HAPH rats.

[0085] 3.2 Effect of hesperidin on pulmonary vascular remodeling in HAPH model rats

[0086] Figure 3 HE staining of pulmonary arteries of rats in each group in Example 1. Figure 4 is the percentage of pulmonary vascular wall thickness of each group of rats in Example 1, Figure 5 It is the percentage of pulmonary vascular wall area of ​​each group of rats in Example 1.

[0087] like Figure 3 As shown in the figure, compared with the normal group, the pulmonary vessels in the model group rats were significantly thickened, and vascular remodeling was inhibited after hesperidin intervention. From the two parameters of WT and WB reflecting the degree of pulmonary vascular remodeling, it can be seen that the two parameters in the model group increased significantly compared with the normal group. The values ​​of these two parameters in the hesperidin intervention group decreased with the intervention concentration, indicating that hesperidin intervention can inhibit pulmonary vascular remodeling in HAPH rats. Figure 4 , Figure 5 It can be seen that the WT and WB parameters of the hesperidin 50mg / kg and 100mg / kg groups were lower than those of the sildenafil 30mg / kg group, indicating that the inhibitory effect of hesperidin at doses of 50mg / kg and 100mg / kg on pulmonary vascular remodeling in HAPH rats is better than that of sildenafil 30mg / kg.

[0088] 3.3 Effect of hesperidin on EOGT expression in pulmonary vessels of HAPH rats

[0089] Figure 6 The EOGT immunohistochemical staining of the pulmonary blood vessels of each group of rats in Example 1 is shown in FIG. Figure 7 The figure is a statistical graph of the average optical density of EOGT immunohistochemical staining of the pulmonary vessels of each group of rats in Example 1.

[0090] like Figure 6 , Figure 7 As shown in the data, after 30 days of hypoxia induction, the expression of EOGT in the lung tissue vessels of the model group rats was significantly upregulated, and the expression of EOGT in the lung tissue of the rats was downregulated after hesperidin intervention, indicating that hesperidin intervention can reduce the expression of EOGT protein in the pulmonary vessels of HAPH rats, that is, the inhibition of pulmonary vascular remodeling by hesperidin on HAPH rats may be related to the downregulation of the expression of glycosylation-modified protein EOGT.

[0091] 3.4 Effect of hesperidin on the expression of α-SMA, EOGT, Notch3, and Bcl-2 in lung tissue of HAPH rats

[0092] Figure 8 This is the protein band diagram of the lung tissue of each group of rats in Example 1, Fig. 9 This is a statistical diagram of α-SMA protein bands in the lung tissues of rats in each group in Example 1. Fig.10 This is a statistical diagram of Bcl-2 protein bands in lung tissue of rats in each group in Example 1. Fig.11 This is a statistical diagram of EOGT protein bands in the lung tissues of rats in each group in Example 1. Fig.12 Statistical graph of Notch3 protein bands in lung tissue of rats in each group in Example 1.

[0093] like Figure 8 , Fig. 9 , Fig.10 , Fig.11 , Fig.12 As shown in the figure, after 30 days of hypoxia induction, the expression of α-SMA, EOGT, Notch3, and Bcl-2 in the lung tissue of the model rats was significantly upregulated, and the expression of α-SMA, EOGT, Notch3, and Bcl-2 in the lung tissue of the rats was downregulated after hesperidin intervention. The downregulation of α-SMA protein expression in lung tissue indicated that pulmonary vascular remodeling was inhibited, and the downregulation of EOGT, Notch3, and Bcl-2 protein expression indicated that the mechanism of hesperidin in alleviating HAPH may be related to inhibiting the activation of the Notch3 / Bcl-2 signaling pathway by downregulating the expression of the glycosylation-modified protein EOGT.

[0094] Example 2

[0095] Inhibitory effect of hesperidin on the proliferation of pulmonary artery smooth muscle cells induced by hypoxia

[0096] 1 Materials

[0097] 1.1 Experimental animals

[0098] Primary cells of SD rat pulmonary artery smooth muscle cells (PASMCs) were produced by our laboratory.

[0099] 1.2 Reagents

[0100] Hesperidin (Shanghai Yuanye Company, purity> 95%); enhanced RIPA lysis buffer, broad-spectrum phosphatase inhibitor, broad-spectrum protease inhibitor (Wuhan Boster Company); BCA protein quantification kit (Beijing Polymer Company); anti-EOGT antibody (Abcam, USA); anti-Notch3 antibody (Abcam, USA); anti-Bcl-2 antibody (Abcam, USA). DMEM culture medium (Gbico, USA); DMSO (BioFroxx, Germany); fetal bovine serum (Gbico, USA); CCK8 (Wuhan Eliruite Company); DAPI (BioFroxx, Germany); BCA protein quantification kit (Beijing Solebold Company); 5× protein loading buffer (Beijing Solebold Company); RIPA lysis buffer (Wuhan Boster Company); SDS-PAGE gel kit (Beijing Botais Company); ECL chemiluminescent liquid (Hefei White Shark Company)

[0101] 1.3 Instruments

[0102] Microplate reader, Labsystems Multi-skan MS, Finland; Mini-PROTEAN Tetra Cell, Bio-Rad, USA; Chemiluminescent gel imager, Protein Simple, USA, FluorChem E; Low-temperature high-speed centrifuge, Allegra-64R, Beckman Coulter, USA

[0103] 2 Methods

[0104] 2.1 CCK8 detection of cell proliferation

[0105] PASMCs in good condition and logarithmic growth phase were taken and inoculated into 96-well cell culture plates at 2000 cells / well, and cultured overnight in a normal environment cell culture incubator (37°C, 5% CO2) for 24 hours. After 24 hours, the original culture medium was discarded, and the corresponding volume of fresh DMEM complete culture medium or different concentrations of hesperidin-containing culture medium was added, and the cells of each group were placed in a conventional incubator (37°C, 5% CO2) or a three-gas incubator (37°C, 5% O2, 90% N2, 5% CO2) for 48 hours of intervention treatment. After 48 hours of culture, the culture plates of the normoxic treatment group and the hypoxic treatment group were taken out and the culture medium was discarded. CCK8 was diluted with basal DMEM culture medium (CCK8:DMEM=1:100) and added to the culture wells of the 96-well plate at 100 μL / well. The cells in the Control group and the Hypoxia group were incubated in a normal environment cell culture incubator for 2 h, and then the absorbance (optical density, OD) was measured at 450 nm using a microplate reader.

[0106] 2.2 Flow cytometry to detect cell apoptosis

[0107] PASMCs in good condition and logarithmic growth phase were selected, and 80,000 cells / dish were inoculated in 60 mm cell culture dishes. After inoculation, they were placed in a normal environment cell culture incubator (37°C, 5% CO2) and cultured overnight for 24 hours. After 24 hours, the original culture medium was discarded, and 4 mL / dish of fresh DMEM complete culture medium or different concentrations of hesperidin-containing culture medium were added again, and each group of cells was placed in a conventional incubator (37°C, 5% CO2) or a three-gas incubator (37°C, 5% O2, 90% N2, 5% CO2) for 48 hours of intervention treatment.

[0108] After 48 hours, the original culture medium in each group of culture dishes was collected, and each group of cells was washed with 2 mL / dish of 4°C pre-cooled PBS buffer and the PBS was combined with the original culture medium. After washing, 0.25% trypsin was added at a volume of 1 mL / dish to digest the cells until the cells fell off, and the original culture medium in each group of culture dishes collected in the previous step was used to terminate the trypsin digestion and collect all the cells in the dish. After collection, the cells were centrifuged at 1000 r / min for 5 minutes. After centrifugation, the supernatant was discarded and the cells were resuspended and washed with 2 mL of pre-cooled PBS buffer, and the cells were continued to be centrifuged at 1000 r / min for 5 minutes. The cell washing was repeated 3 times. After the last wash, the supernatant was discarded, and the cells were stained according to the operating requirements of the kit. After staining, the cells were filtered through a 200-mesh nylon membrane and detected by flow cytometry.

[0109] 2.3 Cell immunofluorescence experiment

[0110] PASMCs in good condition and logarithmic growth phase were selected and inoculated in four-grid glass-bottomed cell culture dishes at 5000 cells / grid. After inoculation, they were placed in a normal environment cell culture incubator (37°C, 5% CO2) and cultured overnight for 24 hours. After 24 hours, the original culture medium was discarded, and the corresponding volume of fresh DMEM complete culture medium or different concentrations of hesperidin-containing culture medium was added again. After that, the cells of each group were placed in a conventional incubator (37°C, 5% CO2) or a three-gas incubator (37°C, 5% O2, 90% N2, 5% CO2) for 48 hours of intervention treatment.

[0111] After 48 hours, the original culture medium was discarded and the cells were gently washed three times with 1 mL / well of pre-cooled PBS buffer. After washing, 500 μL of 4% paraformaldehyde was added to each well and the cells were fixed at room temperature for 10 minutes. The fixative was discarded and the cells were washed three times with 1 mL / well of pre-cooled PBS buffer, each time for 1 minute. After washing, the membrane was permeabilized with 0.25% TritonX-100 at room temperature for 15 minutes. After permeabilization, the permeabilization solution was discarded and the cells were washed three times with 1 mL / well of pre-cooled PBS buffer, each time for 1 minute. After washing, 500 μL of 5% BSA blocking solution prepared with distilled water was added to each well and blocked for 20 minutes at room temperature. After blocking, the blocking solution in the dish was discarded, and 100 μL of the corresponding primary antibody dilution solution (EOGT=1:200:α-SMA=1:300) was added to each well and incubated overnight in a 4°C refrigerator. The primary antibody dilution solution was recovered the next day and washed three times with 1×PBST, each time for 5 minutes. After washing, add the corresponding fluorescent secondary antibody (1:500) prepared with 1×PBST and incubate at 37℃ in the dark for 1h. After the secondary antibody incubation, wash 3 times with 1×PBST, 5min each time. After washing, use DAPI staining working solution (10μg / mL) prepared with 1×PBST at room temperature in the dark for 5min, and wash 3 times with 1×PBST, 5min each time. After washing, observe and take pictures under a laser confocal microscope.

[0112] 2.4 Cell protein immunoblotting

[0113] PASMCs in good condition and logarithmic growth phase were inoculated in 60 mm cell culture dishes at 80,000 cells / dish. After inoculation, they were placed in a normal environment cell culture incubator (37°C, 5% CO2) and cultured overnight for 24 hours. After 24 hours, the original culture medium was discarded, and fresh DMEM culture medium or different concentrations of hesperidin-containing culture medium were added at a volume of 4 mL / dish. The cells in each group were placed in a conventional incubator (37°C, 5% CO2) or a three-gas incubator (37°C, 5% O2, 90% N2, 5% CO2) for 48 hours.

[0114] After 48 hours, cell protein extraction, total cell protein extraction and sample preparation steps were performed, followed by gel preparation, protein loading, electrophoresis, membrane transfer, blocking, antibody incubation and image analysis.

[0115] 3 Results

[0116] 3.1 Effect of hesperidin on hypoxia-induced PASMCs proliferation

[0117] The results of CCK8 experiments are as follows Fig.13 As shown, Fig.13 This is a statistical chart of the CCK8 results of each group in Example 2.

[0118] Hesperidin intervention can significantly inhibit the proliferation of PASMCs induced by hypoxia, and it shows a significant concentration dependence. This shows that hesperidin has an inhibitory effect on the proliferation of PASMCs induced by hypoxia in HAPH.

[0119] Abnormal proliferation of pulmonary artery smooth muscle cells is the main cause of vascular remodeling in HAPH. Inhibiting the proliferation of these cells can inhibit vascular remodeling in HAPH, thereby alleviating HAPH. From the above experimental results, it can be seen that hesperidin inhibits abnormal proliferation of pulmonary artery smooth muscle cells, thereby verifying its effect of inhibiting vascular remodeling in HAPH.

[0120] 3.2 Effect of hesperidin on hypoxia-induced apoptosis of PASMCs

[0121] Flow cytometry was used to detect the apoptosis rate of PASMCs after hesperidin intervened in hypoxia. The experimental results are as follows Fig.14 , Fig.15 As shown, Fig.14 Graph showing the flow cytometry results of each group in Example 2. Fig.15 Statistical graph of cell apoptosis rate in each group in Example 2.

[0122] Hesperidin intervention can significantly increase the apoptosis rate (Q2+Q3) in a concentration-dependent manner. In the HAPH disease state, hypoxia stimulation leads to decreased apoptosis and increased proliferation of PASMCs. Hesperidin can inhibit the abnormal proliferation of PASMCs induced by hypoxia by promoting apoptosis.

[0123] 3.3 Effect of hesperidin on the expression of EOGT, Notch3, and Bcl-2 proteins in PASMCs

[0124] Fig.16 This is the immunofluorescence staining diagram of EOGT in each group of PASMCs in Example 2. Fig.17 This is the protein band diagram of each group of PASMCs in Example 2. Fig.18 This is a statistical diagram of EOGT protein bands in each group of PASMCs in Example 2. Fig.19 This is a statistical diagram of Bcl-2 protein bands in each group of PASMCs in Example 2. Fig. 20 This is a statistical diagram of Notch3 protein bands in each group of PASMCs in Example 2.

[0125] like Fig.16 As shown, immunofluorescence staining was used to detect the expression level of EOGT protein in cells, and green fluorescence indicated EOGT in cells. After hesperidin intervention, the number of green fluorescent particles in the Hypoxia group cells decreased, and the fluorescence intensity was significantly reduced, indicating that hesperidin intervention could inhibit the expression of EOGT in PASMCs.

[0126] Western Blot was used to detect the expression of EOGT, Notch3, and Bcl-2 proteins in PASMCs after hesperidin intervention. Fig.17 , Fig.18 , Fig.19 , Fig. 20 As shown in the figure, the expression of EOGT, Notch3, and Bcl-2 proteins in hypoxia-induced PASMCs was significantly downregulated after hesperidin intervention. In the HAPH disease state, hypoxic stimulation induces the activation of the Notch3 / Bcl-2 signaling pathway in PASMCs, resulting in the inhibition of PASMCs apoptosis and abnormal cell proliferation. Hesperidin intervention can inhibit the activation of the Notch3 / Bcl-2 signaling pathway by downregulating EOGT expression, thereby inhibiting cell proliferation induced by hypoxia.

[0127] Example 3

[0128] Study on the binding of hesperidin to target protein EOGT

[0129] 1 Materials

[0130] 1.1 Reagents

[0131] Hesperidin (Shanghai Yuanye Company, purity> 95%); Biotinylation Kit (Jiangsu Genemore)

[0132] 1.2 Instruments

[0133] ForteBio Octet RED 96e (Sartorius, Germany); Octeto Streptavidin (SA) Biosensor (Sartorius, Germany)

[0134] 2 Methods

[0135] 2.1 Molecular docking of hesperidin and EOGT

[0136] The core target EOGT was used as the receptor and hesperidin as the ligand for molecular docking. First, the 2D structure file of the hesperidin ligand was obtained through the PubChem database, and the result was converted using Chem3D. The PDBID of the receptor EOGT was then obtained through UniProt, and the protein structure of the receptor was obtained from the PDB protein database (RCSB PDB, https: / / www.rcsb.org / ). Finally, the molecular docking between the ligand and the receptor was achieved using Autodock Tools software and Autodock Vina software to evaluate the mutual binding force.

[0137] 2.2 Detection of affinity between hesperidin and EOGT by biofilm interferometry

[0138] First, prepare Assay Buffer PBST, then perform protein EOGT biotinylation and finally perform EOGT solidification. After the above is completed, start loading, add 200μL Assay Buffer to each experimental well, then add 200μL of small molecules configured in the gradient, and add 200μL Assay Buffer as a 0 concentration reference. After the addition is completed, put the experimental plate back into the sample chamber. Start the experimental program setting; 90s contact time, 180s dissociation time, 60s baseline; after confirming the probe position, click Go to start the experiment.

[0139] 3 Results

[0140] 3.1 Molecular docking results of hesperidin and EOGT

[0141] The molecular docking results are as follows Fig.21 As shown, Fig.21 The molecular docking result of hesperidin and EOGT. Hesperidin as a ligand has a relatively stable binding to the receptor EOGT, and the binding energy between the two is S = -7.952 kCal·mol -1 , indicating that the docking results are good and the two have a stable combination.

[0142] 3.2 Results of affinity test between hesperidin and EOGT protein

[0143] Fig. 22 is the binding and dissociation curve of hesperidin and EOGT, Fig.23 This is the affinity fitting curve between hesperidin and EOGT.

[0144] like Fig. 22 , Fig.23 As shown, the binding of hesperidin to EOGT protein is a steady-state model with fast up and fast down, and the KD (M) is 4.40E-05±1.2E-06 using Affinity fitting results, indicating that hesperidin and EOGT are stably bound.

[0145] discuss

[0146] The main pathological changes in HAPH patients are pathological remodeling of pulmonary vessels caused by hypoxia stimulation in high-altitude environments. It is currently believed that excessive proliferation and reduced apoptosis of PASMCs play an extremely important role in hypoxia-induced pulmonary vascular remodeling. The changes in multiple intrinsic cellular mechanisms in PASMCs caused by hypoxia promote the phenotypic transformation of cell proliferation and migration, leading to abnormal proliferation of PASMCs, which in turn promotes changes such as vascular wall thickening, terminal small vessel muscularization and vascular occlusion in pulmonary arterioles, ultimately leading to pulmonary vascular remodeling. Therefore, inhibiting the abnormal proliferation and apoptosis resistance of PASMCs caused by hypoxia stimulation during the occurrence of HAPH may be the key to inhibiting and delaying pulmonary vascular remodeling.

[0147] The results of the study showed that hesperidin has an inhibitory effect on hypoxia-induced PASMCs proliferation and increases PASMCs apoptosis. Hesperidin can reduce mPAP in HAPH rats, delay pulmonary vascular remodeling, and improve the disease state of HAPH rats. And the therapeutic effect of hesperidin on HAPH rats is better than that of sildenafil at a dose of 30 mg / kg / d. Therefore, the results of the study on the anti-HAPH efficacy of hesperidin show that hesperidin has certain anti-HAPH efficacy.

[0148] In the vascular system, the phenotype of vascular smooth muscle cells is strictly regulated by Notch3. The activation of Notch3 is crucial for the proliferation and apoptosis of vascular smooth muscle cells. The activation of Notch3 can inhibit the apoptosis of PASMCs by increasing the expression of Bcl-2, thereby helping the cells survive. In addition, EOGT, as an O-GlcNAc transferase, is responsible for the O-GlcNAc glycosylation modification of proteins. The O-GlcNAc glycosylation modification process of Notch3 receptors, which is responsible for it, plays a decisive role in the activation of Notch3 receptor signals. Hesperidin can significantly reduce the expression levels of EOGT, Notch3, and Bcl-2 proteins in PASMCs and lung tissues of HAPH rats. It can be inferred that the anti-HAPH mechanism of hesperidin may be through inhibiting the expression of EOGT and then inhibiting the activation of Notch3 / Bcl-2 signal transduction, thereby inhibiting the proliferation of PASMCs induced by hypoxia, and finally achieving the anti-HAPH effect.

[0149] In addition, the results of molecular docking and biomembrane interferometry technology showed that hesperidin had a stable binding with EOGT, which further proved that hesperidin inhibited the expression of EOGT by binding to EOGT and then inhibited the activation of Notch3 / Bcl-2 signal transduction, thereby inhibiting hypoxia-induced PASMCs proliferation and ultimately achieving the conclusion of anti-HAPH effect.

[0150] in conclusion

[0151] In summary, hesperidin showed good anti-HAPH effects in both in vitro and in vivo experiments, and its mechanism of action was related to its inhibitory effect on EOGT. Hesperidin can inhibit the Notch3 receptor activation by binding to EOGT and inhibiting the expression of EOGT, thereby blocking the activation of the Notch3 / Bcl-2 signaling pathway in HAPH, thereby inhibiting the proliferation of PASMCs, delaying the pulmonary vascular remodeling of HAPH rats, and ultimately achieving a therapeutic effect on HAPH rats. In other words, inhibiting the expression of EOGT is a feasible way to treat HAPH, and hesperidin, as an EOGT inhibitor, has the potential to be studied and developed into a HAPH therapeutic drug.

[0152] The specification of the present invention describes the specific implementation scheme in detail. Those skilled in the art should recognize that the above implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of hesperidin in the preparation of drugs for preventing or treating pulmonary hypertension.

2. The use according to claim 1, characterized in that: The hesperidin is used for preparing medicine for preventing or treating high-altitude pulmonary hypertension.

3. The use according to claim 2, characterized in that: The hesperidin is used for preparing medicine for treating high-altitude pulmonary hypertension.

4. The use according to claim 2 or 3, characterized in that: The hesperidin treats high-altitude pulmonary hypertension by inhibiting pulmonary vascular remodeling.

5. The use according to claim 2 or 3, characterized in that: The hesperidin treats high-altitude pulmonary hypertension by inhibiting the proliferation of pulmonary artery smooth muscle cells.

6. The use according to claim 2 or 3, characterized in that: The hesperidin treats high-altitude pulmonary hypertension by inhibiting the expression of EOGT protein in pulmonary artery smooth muscle cells.

7. The use according to claim 2 or 3, characterized in that: The hesperidin treats high-altitude pulmonary hypertension by inhibiting the expression of EOGT protein in pulmonary artery smooth muscle cells and thereby inhibiting the activation of the Notch3 / Bcl-2 signaling pathway.

8. The use according to claim 6 or 7, characterized in that: The hesperidin is used as an EOGT inhibitor for preparing a medicine for treating high-altitude pulmonary hypertension.

Images