Application of substance capable of regulating Mfn2-IP3R3 interaction in preparation of medicine for preventing and treating pulmonary arterial hypertension

Abstract

The invention belongs to the technical field of biological medicines, and provides application of a substance capable of regulating Mfn2-IP3R3 interaction in preparation of medicines for preventing and treating pulmonary arterial hypertension aiming at the problems of crosstalk between Mfn2 and IP3R3 and unclear coordination function of the Mfn2 and IP3R3 in the existing pulmonary arterial hypertension occurrence process. According to the substance capable of regulating the Mfn2-IP3R3 interaction, the mitochondrial Ca < 2 + > level is improved by regulating the Mfn2-IP3R3 interaction, so that the Ca < 2 + > steady state is maintained, the mitochondrial integrity is maintained, and the pulmonary artery smooth muscle cell function is recovered. Therefore, cell stress is reduced, and pulmonary arterial hypertension is inhibited. The invention provides a new perspective for the development of medicines for treating pulmonary arterial hypertension, and provides a necessary theoretical basis for the prevention and treatment of pulmonary arterial hypertension by traditional Chinese medicines.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a substance capable of regulating Mfn2-IP3R3 interaction in the preparation of a drug for preventing and treating pulmonary hypertension. Background Art

[0002] Pulmonary arterial hypertension (PAH) is a life-threatening cardiopulmonary disease characterized by abnormal constriction and vascular remodeling of the pulmonary arteries. The incidence of PAH is approximately 7.6 cases per million, and the prevalence is 11-26 cases per million. Studies have shown that irreversible pulmonary vascular remodeling is mainly caused by changes in arterial wall cells, especially excessive proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). Currently, there is no effective treatment that can reverse the progression of the disease. Although existing treatment strategies can relieve symptoms, it is difficult to significantly reduce morbidity and mortality, resulting in poor prognosis for patients. Therefore, there is an urgent need to find new molecules or signaling pathways to target pulmonary artery remodeling and PASMCs proliferation, so as to develop new treatment strategies.

[0003] Mitochondrial fusion protein 2 (Mfn2) is a dynamin-related protein with guanosine triphosphatase (GTP) activity, which is mainly located in the outer mitochondrial membrane and the mitochondrial-associated endoplasmic reticulum membrane. Many studies have shown that MFN2 maintains a close connection between the endoplasmic reticulum (ER) and mitochondria, and regulates the calcium (Ca) 2+ ) transport, lipid metabolism, and glucose metabolism. Furthermore, Mfn2 regulates the balance between mitochondrial fusion and fission. Previously, the applicant's research team discovered that Mfn2 plays a key role in inhibiting the proliferation of PASMCs. They also observed decreased Mfn2 expression in a monocrotaline (MCT)-induced PAH rat model, accompanied by increased mitochondrial autophagy and ER stress. However, the specific mechanism by which Mfn2 influences PAH progression remains unclear and requires further investigation.

[0004] Ca in PASMCs 2+ The concentration of cytosolic free Ca plays a crucial role in regulating pulmonary artery vasoconstriction and vascular remodeling and is closely related to the progression of PAH. 2+ Increased concentrations can cause pulmonary artery vasoconstriction, promote PASMCs proliferation, and lead to vascular wall thickening. The channel proteins mainly involved in calcium transport include inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs). Many studies have reported that IP3R acts as an intracellular Ca 2+ release channels, whose activation leads to intracellular Ca 2+Recent studies have shown that during ER stress, the activation of IP3Rs leads to a large amount of Ca 2+ Mfn2 is released from the ER into the cytosol, thereby inducing mitochondrial fragmentation and cell proliferation. However, the crosstalk between Mfn2 and the type 3 inositol 1,4,5-trisphosphate receptor (IP3R3) and their coordinated functions in the development of PAH remain largely unstudied. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a substance capable of regulating the Mfn2-IP3R3 interaction for use in the preparation of a drug for preventing and treating pulmonary hypertension.

[0006] The present invention aims to explain the binding of Mfn2 to IP3R3 to promote Ca 2+ The molecular mechanism of transport from the ER to mitochondria. Specifically, the present invention manipulates Mfn2 expression in a PAH rat model (in vivo) and a PASMC cell model (in vitro). Furthermore, the present invention will explore the effects of 4-phenylbutyric acid (4-PBA) and Cistanche deserticola phenylethanoid glycosides (CPG), a traditional Xinjiang herbal medicine, on PASMC proliferation. Therefore, the present invention not only helps deepen our understanding of the pathogenesis of PAH but also explores the anti-PAH mechanisms of action of 4-PBA and CPG as novel targeted therapeutics.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A use of a substance capable of regulating Mfn2-IP3R3 interaction in preparing a drug for preventing and treating pulmonary hypertension, wherein the substance regulates the Mfn2-IP3R3 interaction by inhibiting ER stress.

[0009] Preferably, the substance upregulates Mfn2 expression and downregulates IP3R3 expression by inhibiting ER stress, and significantly reduces the expression of ERS-related factors Eif2α and Chop.

[0010] Preferably, the substance downregulates mitochondrial outer membrane Ca by upregulating Mfn2 expression and downregulating IP3R3 expression. 2+ Regulator Vdac1 and upregulate intracellular Ca 2+ Homeostatic regulator SERCA.

[0011] Preferably, the substance downregulates mitochondrial outer membrane Ca 2+ Regulator Vdac1 and upregulate intracellular Ca 2+ The homeostatic regulator SERCA upregulates mitochondrial Ca 2+ concentration, which enhances mitochondrial Ca 2+ uptake capacity.

[0012] Preferably, the substance upregulates mitochondrial Ca 2+ concentration to protect mitochondrial morphology, inhibit mitochondrial autophagy, improve pulmonary hemodynamics, and thereby inhibit pulmonary artery smooth muscle cell proliferation and pulmonary vascular remodeling.

[0013] The present invention also provides a drug for preventing and treating pulmonary hypertension, which includes a substance capable of regulating the interaction between Mfn2 and IP3R3.

[0014] Preferably, the substance capable of regulating the Mfn2-IP3R3 interaction is 4-phenylbutyric acid.

[0015] Preferably, the substance capable of regulating the Mfn2-IP3R3 interaction is Cistanche deserticola phenylethanoid glycoside.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present study found that the regulatory relationship between Mfn2 and IP3R3 plays a crucial role in the pathogenesis of PAH, involving intracellular Ca 2+ The key molecular mechanisms for homeostasis and maintenance of mitochondrial morphology and structure were demonstrated; 4-PBA and CPG regulate Mfn2-IP3R3 interaction to increase mitochondrial Ca 2+ levels, thereby maintaining Ca 2+ This study aims to improve homeostasis, maintain mitochondrial integrity, and restore pulmonary artery smooth muscle cell function, thereby reducing cellular stress and inhibiting PAH. This provides a new perspective for the development of PAH drugs and provides the necessary theoretical basis for the prevention and treatment of pulmonary hypertension with traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figure 2 is the ERS and histopathological changes of the MCT-induced PAH rat model; A is the result of RT-qPCR detection of the mRNA levels of key ER stress-related genes in lung tissue; B is the HE staining result of pulmonary arterioles; C is the TEM image of the mitochondrial structure of PASMC in each group; the green arrows indicate mitochondria;

[0019] Figure 2Figure 3 is the result of the changes in Mfn2 expression and ERS-related gene expression in the TNF-α-induced PAH cell model; A is the change in the gradient concentration of TNF-α stimulation in PASMC over time; B is the immunofluorescence image of the cell substructure; C is the expression result of Mfn2 in the cell substructure; D is the immunofluorescence image of the distribution of Mfn2 in different intervention groups; E is the expression result of Mfn2 in different intervention groups; F is the expression band diagram of silent and overexpressed Mfn2 protein determined by immunoblotting; G is the quantitative expression diagram of silent and overexpressed Mfn2 protein determined by immunoblotting; H is the mRNA level of silent and overexpressed Mfn2; I is the expression of PAH after Mfn2 overexpression. mRNA levels of key ER stress-related genes in cells; J is the mRNA levels of key ER stress-related genes in the PAH cell model after Mfn2 silencing; K is a quantitative graph of the protein levels of key ER stress-related genes in the PAH cell line after Mfn2 overexpression determined by immunoblotting; L is a quantitative graph of the protein levels of key ER stress-related genes in the PAH cell model after Mfn2 silencing determined by immunoblotting; M is a band graph of the protein levels of key ER stress-related genes in the PAH cell line after Mfn2 overexpression determined by immunoblotting; N is a band graph of the protein levels of key ER stress-related genes in the PAH cell model after Mfn2 silencing determined by immunoblotting;

[0020] NC: normal control, TNF-α: TNF-α-induced PAH cell model; OE-Mfn2 NC: Mfn2 overexpression control using an empty vector; OE-Mfn2: Mfn2 overexpression in a PAH cell model; siMfn2NC: Mfn2 silencing control using an empty vector; siMfn2: Mfn2 silencing in a PAH cell model;

[0021] Figure 3 Mfn2 regulates Ca in PAH cells 2+ Homeostasis to maintain mitochondrial integrity; A is immunofluorescence Ca 2+ The probe observed Ca in the endoplasmic reticulum, mitochondria, cytoplasm and different cellular substructures. 2+ Distribution diagram; B is the Ca in the endoplasmic reticulum, mitochondria, and cytoplasm of each group 2+ Concentration statistics; C is immunofluorescence Ca 2+ The Ca2+ probes observed in different groups 2 + Distribution; D: Ca2+ was observed in the endoplasmic reticulum, mitochondria, and cytoplasm of each group. 2+ Concentration statistics; E is a TEM image of mitochondrial ultrastructure, where the green arrows indicate mitochondria;

[0022] Among them, ER:Ca 2+The fluorescent marker in the endoplasmic reticulum is green; Cytoplasm; Ca in the cytoplasm 2+ Labeled with blue fluorescence; Mitochondrion: Ca in mitochondria 2+ Labeled with red fluorescence; NC: normal control; TNF-α: TNF-α-induced PAH cell model; OE-Mfn2 NC: Mfn2 overexpression control using an empty vector; OE-Mfn2: Mfn2 overexpression in a PAH cell model; siMfn2NC: Mfn2 silencing control using an empty vector; siMfn2: Mfn2 silencing in a PAH cell model;

[0023] Figure 4 Figure 3: Effects of IP3R3 inhibition and CPG administration on the Mfn2-IP3R3 signaling pathway; A: Immunoprecipitation analysis of the interaction between Mfn2 and IP3R3; B: Protein level bar graph of key factors in PAH cells determined by immunoblotting; C: Quantitative protein level graph of key factors in PAH cells determined by immunoblotting; D: mRNA levels of key genes in PAH cells;

[0024] NC: normal control; TNF-α: TNF-α-induced PAH cell model; 4-PBA: TNF-α-induced PAH cell model treated with 4-PBA; XC: IP3R3 inhibitor Xestospongin C; CPGs: Cistanche deserticola phenylethanoid glycosides; oe-Mfn2: overexpression of Mfn2 in PAH cell model;

[0025] Figure 5 Mfn2-IP3R3 signaling regulates Ca in PAH cells 2+ Steady-state results; A is the immunofluorescence staining observation of Ca in different groups 2+ Distribution; B is the immunofluorescence staining observation of Ca in different groups 2+ Concentration statistics; C is a TEM image of mitochondrial ultrastructure;

[0026] Figure 6 Figure 2 is the effect of Mfn2-IP3R3 signaling pathway on PAH cells; A is the CCK-8 assay result of cell proliferation in different intervention groups; B is the flow cytometry analysis result of cell apoptosis in different intervention groups; C is the cell apoptosis rate in different intervention groups;

[0027] Among them, NC: normal control; XC: IP3R3 inhibitor Xestospongin C; TNF-α: PAH cell model induced by TNF-α; TNF-α+XC: PAH cell model induced by TNF-α and IP3R3 inhibitor; TNF-α+CPGs group: PAH cell model induced by TNF-α and Cistanche deserticola phenylethanoid glycosides; OE-Mfn2: Mfn2 overexpression in PAH cell model; OE-Mfn2+XC: Mfn2 overexpression in PAH cell model induced by IP3R3 inhibitor; OE-Mfn2 NC: Mfn2 overexpression control using empty vector; OE-Mfn2NC+XC: Mfn2 overexpression control using IP3R3 inhibitor and empty vector;

[0028] Figure 7 Figure 2 is the result of ERS inhibition regulating Mfn2-IP3R3 signaling; A is the result of mRNA level determination of key genes in lung tissue; B is the protein level band diagram of key genes in lung tissue determined by immunoblotting; C is the statistical diagram of protein level of key genes in lung tissue determined by immunoblotting; D is the result of immunofluorescence staining observation of Ca in different groups 2+ Distribution diagram; E is the immunofluorescence staining observation of Ca in different groups 2+ Concentration statistics; F is a TEM image of the mitochondrial ultrastructure in the lung tissue of MCT-induced PAH rats; G is a HE staining result of pulmonary arterioles;

[0029] Among them, ER:Ca 2+ The fluorescent marker in the endoplasmic reticulum is green; Cytoplasm; Ca in the cytoplasm 2+ Labeled with blue fluorescence; Mitochondrion: Ca in mitochondria 2+ Labeled with red fluorescence; NC: normal control; TNF-α: TNF-α-induced PAH cell model; 4-PBA: 4-PBA intervention group. DETAILED DESCRIPTION

[0030] The following is a diagram of the embodiment of the present invention. Figures 1 to 7 The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] 1. Materials and Methods

[0033] 1. Construction and grouping of experimental rats

[0034] Sixty male Wistar rats (180-200 g, 8-10 weeks old) were purchased from the Laboratory Animal Science Research Department, Medical Research Center, First Affiliated Hospital of Xinjiang Medical University (Laboratory Animal Use Permit No. SYXK(Xin)2010-0003). All animal experiments were approved by the Institutional Animal Care and Use Committee of the hospital (Approval No.: IACUC20190416-01). Rats were housed in a standard environment (12-h light / dark cycle, temperature 25-27°C, humidity 50-70%) with free access to food and water. These rats are referred to as normal rats.

[0035] A PAH rat model was established in selected rats using a single intraperitoneal injection of monocrotaline (MCT, 60 mg / kg). Mean pulmonary artery pressure (mPAP) was measured using a BL-420F bioinformatics acquisition system. A PAH rat model was considered successfully established when the measured mPAP was ≥25 mmHg. This PAH rat model is referred to as the PAH rat model.

[0036] The experimental groups are as follows:

[0037] Normal control group (NC group): Normal rats were gavaged with 500 mg / (kg·d) of normal saline for 4 consecutive weeks.

[0038] PAH group: PAH rats were gavaged with 500 mg / (kg·d) normal saline for 4 consecutive weeks.

[0039] Drug treatment group (REV group): PAH rats were first orally administered with 500 mg / (kg·d) normal saline for 2 consecutive weeks, and then administered with 500 mg / (kg·d) 4-phenylbutyric acid (4-PBA) suspension for 2 consecutive weeks.

[0040] Drug prevention group (PRE group): Normal rats were given 4-phenylbutyric acid (4-PBA) suspension 500 mg / (kg·d) for 4 consecutive weeks.

[0041] 2. Hemodynamic testing in animal experiments

[0042] The rats in each group were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (45 mg / kg; E602, Jiangsu Hengrui Pharmaceutical). The right external jugular vein was isolated, and a polystyrene catheter (PE10, Smiths Medical, United Kingdom) was inserted into the right atrium and right ventricle. The mean right ventricular pressure (mRVP) was monitored and recorded in real time using a BL-420F bioinformatics acquisition system (Tech Man, Chengdu, China).

[0043] The subcutaneous fascia and muscle layer of the rat neck were bluntly dissected, and a tracheal ventilator tube was connected through an inverted "T"-shaped incision. Subsequently, the thoracotomy was performed to insert a pulmonary artery catheter, and the mean pulmonary artery pressure (mPAP) was measured and recorded simultaneously with the BL-420F bioinformatics acquisition system (TechMan, Chengdu, China).

[0044] 3. Animal Experimental Determination of Pulmonary Vascular Remodeling Index

[0045] Rats in each group were sacrificed by thoracotomy, and the lungs and hearts were rapidly removed. Organs were washed with pre-chilled saline, and atrial tissue was trimmed. After blotting with filter paper, the free right ventricle (RV) and left ventricle and ventricular septum (LV+S) were weighed, and the right ventricular hypertrophy index (RVHI) was calculated using the formula: RVHI = RV / (LV+S).

[0046] Lung tissue was transversely dissected from the right hilum, fixed with 10% formaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (HE). HE-stained sections were observed under a light microscope (XDS-1A, Mitutoyo, Tokyo, Japan), and images of small pulmonary arteries were acquired at random fields. Images were analyzed using ImageJ software (JEOL, Tokyo, Japan).

[0047] 4. Lung tissue staining and mitochondrial morphology observation of experimental animals

[0048] After euthanasia during thoracotomy, lung tissue (≤1 mm 3 ) and fixed in 2.5% glutaraldehyde (A17876, Alfa Aesar, MA, USA) for 3-4 hours. For electron microscopy sample preparation, the tissue was first rinsed in 0.1 M phosphate buffer (pH 7.4) and then further fixed with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4). Subsequently, the tissue was dehydrated with graded ethanol, embedded in resin, and sectioned into ultrathin sections. After staining with 3% uranyl acetate and lead citrate, the samples were imaged and observed for mitochondrial morphology and ultrastructural changes using an HT7700 transmission electron microscope (HITACHI, Tokyo, Japan).

[0049] 5. In vitro culture and proliferation of pulmonary artery smooth muscle cells

[0050] Commercially purchased pulmonary artery smooth muscle cells (PASMCs) were seeded in Dulbecco's modified Eagle's medium (DMEM) (11330057, Invitrogen, USA) containing 10% fetal bovine serum (FBS) and cultured in a 37°C, 5% CO2 incubator. Cells were passaged using 0.25% trypsin-EDTA (Beyotime, China), and PASMCs from passages 3-6 were used for subsequent experiments. The MTT assay (M5655, Sigma, Germany) was used to detect cell proliferation. After PASMCs were stimulated with tumor necrosis factor-α (TNF-α) (AB9739, Abcam, UK), the optical density (OD) value at a wavelength of 492 nm was measured using a microplate reader at specific time points. PASMCs were stimulated with Xestospongin C (Ab120914, Abcam) and Cistanche deserticola phenylethanoid glycosides (CPG) (Changchun Medicinal Materials Co., Ltd., China), and cell viability was evaluated by OD value to screen the optimal intervention concentration.

[0051] 6. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

[0052] Total RNA was extracted from lung tissue and PASMCs using TRNzol reagent (UN7 E262I8, Invitrogen, CA, USA). Subsequently, mRNA was reverse transcribed into cDNA using PrimeScript RT reagent kit (K1622, Zhenobio, Shanghai, China). Primers for rat genes were designed and synthesized by Sangon Biotech (Shanghai, China). RT-qPCR reactions were performed using PremixExTaq TM Amplification was performed using a real-time PCR system (Shanghai Sangong Biotechnology Co., Ltd.) using PCR products II and ROXplus (P200601, Qiagen, Shanghai, China) for 40 cycles. The specific reaction conditions were: denaturation at 95°C for 10 seconds, annealing at 58°C for 30 seconds, and extension at 60°C for 10 seconds. The relative expression levels of target genes were analyzed using the 2-ΔΔCt method, using GAPDH as an internal reference. The primer sequences used in the present invention are shown in Table 1 below.

[0053] Table 1 Primers used for RT-qPCR reaction

[0054]

[0055]

[0056] 7. Western Blot Analysis

[0057] Proteins were extracted using RIPA lysis buffer, separated on a 10% SDS-PAGE gel, and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The following primary antibodies were used for incubation: anti-Mfn2 (1:1000; AB56889, Abcam, Cambridge, UK), IP3R3 (1:1000; AB125077, Abcam), optic atrophy 1 (Opa1) (1:1000; 612607, BD Biosciences, CA, USA), dynamin-related protein 1 (Drp1) (1:1000; 611112, BD Biosciences), B-cell lymphoma-2 (Bcl-2) (1:1000; 2870, Cell Signaling Technology, MA, USA), protein kinase R-like endoplasmic reticulum kinase (Perk) (1:1000; 3192, Cell Signaling Technology), C / EBP homologous protein (Chop) (1:1000; 2895, Cell Signaling Technology), eukaryotic initiation factor 2α (Eif2α) (1:1000; 9722, Cell Signaling Technology Technology), activating transcription factor 4 (Atf4) (1:1000; 11815, Cell Signaling Technology), voltage-dependent anion channel 1 (Vdac1) (1:1000; sc-390996, Santa Cruz Biotechnology, Inc., TX, USA), sarco / endoplasmic reticulum Ca 2+ ATPase (SERCA) (1:1000; A11136, Thermo Fisher Scientific, MA, USA) and GAPDH (1:1000; 60004-1-1g, Proteintech, IL, USA). Subsequently, the membrane was incubated with secondary antibodies (anti-mouse, Abcam, Cambridge, UK, AB6789, 1:5000 dilution; anti-rabbit, Abcam, Cambridge, UK, AB205718, 1:5000 dilution) at room temperature. Chemiluminescence was detected using ImageLab software (GE6100, Clinx, Shanghai, China), and quantified using ImageJ software.

[0058] 8. Co-immunoprecipitation

[0059] Co-immunoprecipitation (Co-IP) experiments were performed using the Pierce Co-IP kit (Pierce, IL) according to the manufacturer's instructions. 100 μg of purified anti-IP3R3, anti-Mfn2, or anti-Flag antibodies were coupled to the resin. Subsequently, protein samples (1 mg) were incubated with the antibody-coupled resin for 2 hours. After mixing and washing, the protein-antibody complexes were eluted in 50 μL of elution buffer. Finally, the eluted protein samples were subjected to immunoblotting analysis using the corresponding antibodies.

[0060] 9. Construction of cell models overexpressing / silencing Mfn2

[0061] When PASMCs grew to the logarithmic growth phase, 1.5×10 17 Cells were seeded into 96-well plates at a density of 10 cells / well and incubated at 37°C, 5% CO₂ for 24 hours. Viral transfection was performed after the cell density reached 80%-90%. The amount of viral particles required to infect PASMCs with the Mfn2-overexpressing virus and the control virus (GenePharma Co., Ltd., Shanghai, China) was calculated, and the MOI (Multiplicity of Infection) values ​​were set at 12.5, 25, 50, 100, and 200, respectively. For the Mfn2-silencing control virus, the MOI values ​​were set at 2.5, 5, 10, 20, and 40; for the Mfn2-silencing-521 virus, the MOI values ​​were set at 5, 10, 20, 40, and 80; and for the Mfn2-silencing-1176 virus and the Mfn2-silencing-1302 virus, the MOI values ​​were set at 12.5, 25, 50, 100, and 200. Cells were infected for 72 hours and then observed under a fluorescence microscope 96 hours after infection. The optimal MOI value was determined when the fluorescence ratio was ≥80%-90% and the cell morphology change was minimal.

[0062] Before transfection, PASMCs were cultured at a density of 1 × 10 17 Cells were seeded into six-well culture plates at a density of 100 cells / well and cultured to 90% confluence. 24 hours before transfection, a recombinant lentiviral Mfn2 gene expression and silencing plasmid (Shanghai GenePharma Co.) was used at an MOI of 100. The Mfn2 plasmid was synthesized using the LV3 fragment as a cloning vector with BamHI and EcoRI as cloning sites, and its sequence accuracy was verified by the supplier. The lentiviral titer was maintained at 3×10 7 Mfn2 overexpression and silencing plasmids were transfected into PASMCs using a suspension of approximately 20,000 cells / μL.

[0063] 10. Detect Mfn2 expression using immunofluorescence staining

[0064] Immunofluorescence staining was used to detect the expression of Mfn2 in pulmonary artery smooth muscle cells. Paraffin-embedded tissue sections were incubated with an anti-Mfn2 primary antibody (A12771, AB Clone, Shanghai, China) overnight at 4°C. Subsequently, Cy3-conjugated goat anti-rabbit IgG antibody (1:200, SA00009-2, Proteintech, Wuhan, China) or FITC-conjugated goat anti-mouse IgG antibody (1:200, Ab6789, Abcam) was added and incubated at room temperature for 1 hour. Finally, fluorescence images were captured using a confocal microscope.

[0065] 11. Intracellular Ca 2+ Concentration measurement

[0066] The experiment was performed using the GENMED fluorescence detection kit (GMS10267.1v.A, GMS10154v.A, GMS10153.1v.A, Shanghai Jiemei Gene Pharmaceutical Technology Co., Ltd.). The experimental steps are as follows: First, pretreat the cell sample with 500μL of GENMED cleaning solution (reagent A), and then remove the solution. Then, mix 300μL of fresh reagent A with 30μL of staining working solution and add it to the sample. Cytosolic Ca 2+ Detection: Incubate the sample at room temperature in the dark for 15 minutes. After incubation, remove the staining solution and wash the sample with fresh reagent A. Then, use the GENMED fluorescence detection kit for detection. 2+ Detection: Incubate the sample at room temperature in the dark for 60 minutes. After incubation, remove the staining solution and wash the sample with fresh reagent A. Then, detect the sample using the GENMED fluorescence detection kit. The excitation and emission wavelengths of the fluorescence signal are set as follows: Ca in ER 2+ :490 / 525nm, cytoplasmic Ca 2+ :350 / 405-420nm, mitochondrial Ca 2+ :550 / 590nm.

[0067] 12. CCK-8 assay for cell viability

[0068] The cells were plated at 1×10 4 PASMCs were seeded into 96-well plates at a density of 10 cells / well and treated according to experimental grouping. After incubation at 37°C, 5% CO₂ for 24 hours, 20 μL of CCK-8 solution was added to each well and incubated at 37°C for another 4 hours. Subsequently, absorbance was measured at 450 nm using a microplate reader (ThermoMK3, USA) to assess cell viability.

[0069] 13. Detection of cell apoptosis by flow cytometry

[0070] PASMCs were digested with EDTA-free trypsin and then centrifuged at 1500 rpm for 5 minutes at room temperature. Subsequently, the cells were washed with PBS at 4°C and centrifuged again at 1500 rpm for 5 minutes. The cells were resuspended in 300 μL of 1× binding buffer. According to the instructions of the Annexin V-FITC / PI kit (401006, BestBio, Shanghai, China), 5 μL of Annexin V-FITC was added and mixed. The cell suspension was incubated at room temperature in the dark for 15 minutes. Subsequently, 10 μL of PI stain was added, mixed gently, and incubated at room temperature in the dark for 10 minutes. Finally, the detection was performed using a BD-FACS Verse flow cytometer (BD Biosciences, San Jose, USA), and the data were analyzed using FlowJo7.6 software (TreeStar, Oregon, USA).

[0071] 14. Statistical Analysis

[0072] All experiments were performed with at least three biological replicates. To minimize bias, the experimenters were blinded during the experimental procedures and data analysis. Unless otherwise stated, data are expressed as mean ± SD. For continuous variables, the Shapiro-Wilk test was used to assess the normality of the data. If the data were normally distributed, the Student's t test was used to compare the means of two groups, and multiple group comparisons were performed using one-way ANOVA with Tukey's post hoc test. If the data were not normally distributed, the Mann-Whitney U test was used for two-group comparisons, and the Kruskal-Wallis test was used for multiple group comparisons with Dun's post hoc test. Data were analyzed using GraphPad Prism 9.5.1 (Graph Pad Inc., La Jolla, CA, USA), and the significance level was set at P < 0.05.

[0073] During the data analysis phase, the statistical analysts were unaware of the experimental group assignments. Raw data were provided in a coded format, with group labels replaced by anonymous identifiers (e.g., Group A, Group B) assigned by a third-party researcher. This blinding was maintained until all statistical tests were completed to avoid bias in data interpretation.

[0074] 2. Experimental Results

[0075] 1. MCT-induced PAH rat model shows endoplasmic reticulum stress and histological changes

[0076] A PAH rat model was successfully established by intraperitoneal injection of MCT (60 mg / kg). The hemodynamic evaluation results are shown in Tables 2 and 3 below.

[0077] Table 2 Measurement results of hemodynamic parameters in different groups

[0078]

[0079] Table 3 Pulmonary vascular remodeling indices in different groups

[0080]

[0081] Note: WT: ventricular wall thickness; WA: vascular wall area; IA: interstitial area; TA: total vascular area; ED: external diameter (diameter of the external elastic lamina); WT% = 2 × WT / ED × 100%; WA% = (TA-IA) / TA × 100%; IA% = IA / TA × 100%;

[0082] The results in Tables 2 and 3 show that compared with the normal control group, the mean right ventricular pressure (mRVP), mean pulmonary artery pressure (mPAP) and right ventricular hypertrophy index (RVHI) of the PAH rat model group were significantly increased, and the thickness of the pulmonary arterioles increased, including a significant increase in WT%, WA% and IA%.

[0083] RT-qPCR analysis results showed that the expression of key ER stress-related genes was dysregulated, among which Mfn2 expression was significantly decreased, while IP3R3, Perk, Eif2α, Chop and Atf4 expression were significantly upregulated (see results). Figure 1 In addition, HE staining of lung tissue showed that the walls of pulmonary arterioles in the NC group were normal, while the pulmonary vessels of PAH rats underwent significant remodeling, manifested by thickening of the pulmonary artery walls, stenosis of the lumen, disordered tissue structure accompanied by extensive inflammatory cell infiltration and proliferation of collagen fibers in the matrix (see results). Figure 1 Transmission electron microscopy (TEM) further observed that the mitochondrial structure of the NC group was intact, with cristae and intact cell membranes preserved, while the mitochondria in the pulmonary artery smooth muscle cells of PAH rats were swollen and cristae were broken (see the results in Figure 1 These results indicate that MCT-induced PAH rats exhibit significant ER stress activation and histopathological changes, including cell substructure destruction, in the lung tissues.

[0084] 2. Construction of a TNF-α-induced PAH cell model in vitro to evaluate Mfn2 localization and interaction with ERS-related genes

[0085] TNF-α is a key inflammatory cytokine that can promote the proliferation of pulmonary arterial smooth muscle cells (PASMCs) during the development of pulmonary arterial hypertension (PAH). Studies have confirmed that TNF-α can induce the establishment of a PAH cell model. The present invention uses different concentrations of TNF-α (0-160ng / mL) to stimulate primary PASMCs, and detects its effects at 0, 12, 24, 36, 48 and 60 hours to construct a PAH cell model, and then performs an MTT test to evaluate cell viability. The results showed that when the TNF-α concentration was higher than 10ng / mL, the proliferation of PASMCs increased significantly after 12 hours of stimulation, and the effect trends at 24, 36, 48 and 60 hours were similar. Based on this, we determined that 20ng / mL TNF-α stimulation for 12 hours was the optimal condition for constructing a PAH cell model (see results). Figure 2 Immunofluorescence staining further showed that Mfn2 was mainly located near the nucleus and mitochondria (see Figure 2 In addition, compared with the negative control group (NC group), the expression level of Mfn2 in the TNF-α group was significantly decreased, which is consistent with the results of previous studies (see Figure 2 Middle C).

[0086] To further explore the interaction between Mfn2 and endoplasmic reticulum stress (ERS)-related factors, the present invention used RNA interference (RNAi) technology to downregulate Mfn2 expression and observe changes in ERS-related factors. Immunofluorescence staining results showed that in the TNF-α-induced PAH cell model, Mfn2 expression was significantly reduced compared with the control group, while Mfn2 overexpression could restore its expression; conversely, silencing Mfn2 further reduced its expression (see the results). Figure 2 D and E).

[0087] To verify the efficiency of Mfn2 overexpression and silencing, the present invention performed Western blot and RT-qPCR analysis. The results showed that the Mfn2 protein level was significantly increased in the Mfn2 overexpression group, while its expression was significantly decreased in the siMfn2 treatment group (see the results). Figure 2 RT-qPCR analysis further confirmed the corresponding changes in Mfn2 mRNA levels (see Figure 2 The above data indicate that the Mfn2 overexpression and silencing cell model has been successfully constructed, providing a reliable basis for subsequent functional experiments. In addition, RT-qPCR and Western blot results showed that Mfn2 overexpression can reduce the expression of key ERS-related factors (including IP3R3, Perk, Eif2β, Atf4 and Chop) (see results). Figure 2 In contrast, silencing Mfn2 significantly increased the expression of these ERS-related factors (see Figure 2L and N in the middle).

[0088] 3. Mfn2 regulates Ca in PAH cells 2+ Homeostasis to maintain mitochondrial integrity

[0089] The endoplasmic reticulum (ER) is the main intracellular Ca 2+ reservoir, where transient Ca 2+ ions are released into the cytosol and further transferred to the mitochondria, thereby promoting cell signaling and ATP production. However, excess Ca 2+ Release from the ER can lead to mitochondrial Ca 2+ In the state of pulmonary arterial hypertension (PAH), intracellular Ca 2+ Increased concentrations of Ca lead to excessive proliferation of PASMCs. In the PAH cell model, immunofluorescence probes were used to detect Ca 2+ The results showed that compared with the normal control group, the cytoplasmic and ER Ca 2+ The concentration of mitochondrial Ca 2+ The concentration was significantly reduced (see Figure 3 A and B).

[0090] Notably, in addition to the previously identified regulatory interaction between Mfn2 and IP3R3, we also observed that overexpression of Mfn2 in PAH cells significantly increased mitochondrial Ca 2+ concentration, while reducing cytosolic Ca 2+ In contrast, silencing Mfn2 expression resulted in a decrease in cytoplasmic Ca 2+ concentration increased, while mitochondrial Ca 2+ The concentration decreased (see Figure 3 In addition, electron microscopy results showed that Mfn2 overexpression could maintain the complete morphology and structure of mitochondria. In PAH cells with Mfn2 silenced, mitochondria showed obvious swelling and cristae rupture (see Figure 3 These findings suggest that the interaction between Mfn2 and IP3R3 is essential for maintaining mitochondrial structural integrity and Ca2+ regulation in PAH cells. 2+ Homeostasis is crucial.

[0091] 4. Inhibition of IP3R3 and administration of Cistanche deserticola phenylethanol glycoside (CPG) reactivate Mfn2-IP3R3 signaling

[0092] 4-PBA is considered a typical ERS (endoplasmic reticulum stress) inhibitor that helps protein folding, increases protein stability, and reduces persistent UPR (unfolded protein response) signaling. As a low molecular weight fatty acid, 4-PBA prevents misfolded protein aggregation and attenuates ERS-mediated autophagy and apoptosis, and has been used to treat excessive ERS-related diseases. Previous studies have shown that 4-PBA can effectively inhibit key ERS factors in the monocrotaline-induced PAH (pulmonary arterial hypertension) rat model, inhibit pulmonary vascular remodeling and reduce pulmonary artery pressure. In addition, the study showed that under PAH conditions, the expression level of Mfn2 was significantly reduced. Notably, 4-PBA intervention increased the expression of Mfn2, indicating that it may have a potential therapeutic effect in alleviating PAH. However, the specific regulatory mechanism of 4-PBA in the PAH process remains to be further studied.

[0093] Mfn2 has been shown to target and regulate the expression of IP3R3, and regulate ERS and mitochondrial autophagy through the interaction between the two, thereby inhibiting PASMCs (pulmonary artery smooth muscle cells) proliferation and pulmonary vascular remodeling. Based on the previous research results of the present invention, it was preliminarily concluded that there is a significant regulatory correlation between Mfn2 and IP3R3. To further verify this interaction, the present invention performed Co-IP (co-immunoprecipitation) analysis to confirm the interaction between Mfn2 and IP3R3 in the PAH cell model. Under control conditions, a strong interaction was observed between Mfn2 and IP3R3, as shown by the stable signal of the co-immunoprecipitation band. However, TNF-α treatment significantly reduced this interaction, which was further demonstrated by the weakened signal intensity of the co-immunoprecipitation band (see results). Figure 4 (A) This suggests that the TNF-α-induced PAH cell model disrupts the Mfn2-IP3R3 interaction. Interestingly, treatment with the ER stress inhibitor 4-PBA restored the Mfn2-IP3R3 signaling axis to some extent. To validate this finding, the present inventors further applied 4-PBA to a PAH rat model in subsequent animal experiments.

[0094] Cistanche deserticola phenylethanoid glycosides (CPGs) are a traditional plant extract that plays an important role in inflammatory injury by reducing oxidative stress and inhibiting the apoptosis pathway of reperfused cardiomyocytes. Therefore, the present invention hypothesizes that CPGs may have similar functions in regulating the inflammatory injury process of PAH cells. Interestingly, after intervention with the IP3R3 inhibitor Xestospongin C (XC) and CPGs in PAH cell models, Mfn2 and sarcoplasmic reticulum / endoplasmic reticulum Ca2+ expression were significantly increased compared with TNF-α-induced PAH cell models. 2+The expression of β-ATPase (SERCA) was increased. In contrast, the expression of IP3R3, Eif2β, Chop, and Vdac1 was significantly decreased. In addition, inhibition of IP3R3 seemed to enhance the feedback upregulation of Mfn2 expression, and this feedback mechanism was further enhanced under Mfn2 overexpression conditions (see results). Figure 4 It is worth noting that in different intervention groups, the present invention observed that CPGs intervention can restore the expression of Mfn2, but cannot further inhibit the expression of IP3R3, which suggests that CPGs may play a role mainly by directly restoring Mfn2 rather than inhibiting IP3R3. In addition, SERCA is an intracellular Ca 2+ The key genes for homeostasis were significantly restored after CPGs intervention. 2+ The expression of the regulatory factor Vdac1 and the key ERS-related genes Eif2α and Chop were significantly downregulated. These results support that the Mfn2-IP3R3 signaling pathway may maintain Ca2+ in PAH cells by reducing ERS. 2+ Assumption of steady state.

[0095] 5. Mfn2-IP3R3 signaling regulates Ca2+ homeostasis in PAH cells through ER stress, affecting mitochondrial morphology and structure

[0096] Measuring Ca in PAH cells using immunofluorescence probes 2+ The results showed that the mitochondrial Ca concentration of PAH cells in the XC and CPGs intervention groups 2+ The concentration increased significantly, and the cytoplasmic Ca 2+ These changes were more obvious when Mfn2 was overexpressed (see the results in Figure 5 In the TNF-α-induced PAH cell model, transmission electron microscopy revealed mitochondrial membrane damage, cristae fragmentation, and autophagic vesicles containing damaged mitochondrial fragments. However, under the intervention of direct IP3R3 inhibitors, XC and CPG preserved more intact cell membrane and mitochondrial structures, and minimal mitophagy was observed (see results). Figure 5 Middle C).

[0097] 6. Effects of the Mfn2-IP3R3 signaling pathway on the phenotype of pulmonary arterial hypertension cells

[0098] Excessive proliferation of PASMCs is a key factor in the pathogenesis of PAH. Previous studies have shown that activation of Mfn2-IP3R3 signaling upregulates endoplasmic reticulum stress, thereby altering Ca2+ transport between the cytoplasm and mitochondria. 2+ distribution, ultimately compromising the integrity of mitochondrial structure and promoting mitophagy. Figure 6The results of CCK-8 and flow cytometry showed that blocking Mfn2-IP3R3 downstream signaling with the direct inhibitor XC, overexpressing Mfn2, or using CPG significantly reduced the proliferation activity of PAH cells. In addition, CPGs had a more pronounced inhibitory effect on PAH cell proliferation. Compared with the inhibition of IP3R3 alone, overexpression of Mfn2 and inhibition of IP3R3 had a greater inhibitory effect on PAH cell proliferation (see the results). Figure 6 Flow cytometry results also showed that overexpression of XC, CPGs, and Mfn2 all induced significant apoptosis in PAH cells. In addition, CPGs induced more pronounced apoptosis than direct inhibition of IP3R3, while Mfn2 overexpression combined with Mfn2-IP3R3 inhibition led to more pronounced apoptosis in PAH cells (see results). Figure 6 B and C).

[0099] ERS inhibition regulates Mfn2-IP3R3 signaling to balance mitochondrial Ca 2+ Homeostasis and maintenance of mitochondrial integrity.

[0100] The purpose of this part of the study was to determine whether interference with ERS primarily affects Ca2+ regulated by Mfn2-IP3R3 signaling. 2+ Specifically, the results of experiments on PAH cell lines showed that ERS inhibition significantly increased the expression of Mfn2 and simultaneously suppressed the expression of IP3R3. In addition, the expression levels of ERS-related genes (including Perk, Eif2α, Chop, and Atf4) were reduced in the lung tissues of PAH rats treated with 4-PBA (see results). Figure 7 These findings support the present hypothesis that ERS disrupts Ca2+ production by activating aberrant Mfn2-IP3R3 signaling in mitochondria. 2+ homeostasis and induces mitophagy.

[0101] Further cellular substructure analysis confirmed that 4-PBA treatment significantly increased mitochondrial Ca in PAH cells. 2+ concentration, which simultaneously reduced the Ca 2+ Level (results see Figure 7 In addition, electron microscopy showed that after 4-PBA treatment, mitochondrial swelling, cristae destruction, and mitochondrial autophagy in PAH cells were reduced (see Figure 7 In the PAH rat model, 4-PBA treatment significantly reduced mRVP, mPAP, and RVHI levels (results are shown in Tables 4 and 5 below). Histological analysis showed that right ventricular hypertrophy, pulmonary artery wall thickness, and arterial fibrosis were significantly improved in PAH rats (results are shown in Figure 7Most importantly, the present invention demonstrates that 4-PBA can promote and maintain mitochondrial Ca2+ by regulating the Mfn2-IP3R3 pathway through inhibiting ERS in PAH. 2+ transport and homeostasis, protecting mitochondrial structural integrity, and ultimately playing a key role in PAH-associated vascular remodeling.

[0102] Table 4 Measurement results of hemodynamic parameters in different groups

[0103]

[0104] Note: mPAP: maximum mean airway pressure; mRVP: mean right ventricular pressure;

[0105] Table 5 Changes in pulmonary vascular remodeling-related indicators in different groups

[0106]

[0107] Note: WT: wall thickness; WA: vascular wall area; IA: interstitial area; TA: total vascular area; ED: external diameter (diameter of the external elastic lamina); WT% = 2 × WT / ED × 100%; WA% = (TA-IA) / TA × 100%; IA% = IA / TA × 100%; RVHI: right ventricular hypertrophy index;

[0108] In summary, this study elucidates the biological function of Mfn2 and proposes a novel molecular mechanism: ER activation mediates downregulation of Mfn2 expression and concurrent upregulation of IP3R3 expression, while ERS inhibition produces the opposite effect. Furthermore, the present invention discovered that the ER-induced Mfn2-IP3R3 signaling pathway promotes mitophagy, which may be a key mechanism in the development of PAH. Inhibiting ER activation successfully reversed mitophagy in PAH cells, improved pulmonary hemodynamics, and reduced vascular remodeling in PAH rats. Therefore, this study identifies a key signaling pathway, ER activation-induced mitophagy, and further sheds light on the molecular mechanism underlying the development of PAH.

[0109] The present invention confirmed the interaction between Mfn2 and IP3R3 in PAH cells through Co-IP. Their synergistic role in ER-mitochondrial communication is crucial for maintaining cell function and managing PAH-induced cellular stress. In addition, these regulatory factors may act indirectly through different cell signaling pathways or at the ER-mitochondrial interface to maintain the stability of the intracellular environment, including mitochondrial dynamics and Ca. 2+ Signaling regulation. The regulatory relationship between Mfn2 and IP3R3 is crucial for maintaining intracellular Ca 2+ Homeostasis is crucial. 2+As a key signaling molecule, Ca plays an important role in the pathophysiology of PAH. 2+ Disruption of homeostasis may lead to excessive proliferation, inflammation, and vasoconstriction of PASMCs. Dysfunction of Mfn2 and IP3R3 may induce ERS and mitochondrial dysfunction, further disrupting Ca 2+ The present invention uses the ERS inhibitor 4-PBA and a new herbal ingredient CPG, and observes that they can significantly reverse the pathological process of PAH cell models and PAH rats. The present invention shows that 4-PBA can significantly reduce the mPAP of PAH rats induced by MCT and reverse right ventricular remodeling. In addition, 4-PBA can regulate mitochondrial Ca through the Mfn2-IP3R3 signaling axis. 2+ concentration, thereby protecting mitochondrial morphology, reducing mitophagy, and ultimately blocking PAH progression.

[0110] In addition, the present invention confirms that CPG, as an active compound derived from traditional Chinese medicine, exhibits significant efficacy in the treatment of PAH. CPG can restore Mfn2 expression in PAH cell models, inhibit IP3R3, and significantly reduce the expression of ERS-related factors Eif2α and Chop, while downregulating mitochondrial outer membrane Ca 2+ Regulator Vdac1 and restore intracellular Ca 2+ SERCA, a homeostatic regulator. 4-PBA and CPG increase mitochondrial Ca by regulating the Mfn2-IP3R3 interaction. 2+ levels, thereby maintaining Ca 2+ This study aims to restore homeostasis, maintain mitochondrial integrity, and restore pulmonary artery smooth muscle cell function, thereby reducing cellular stress and inhibiting PAH. This provides a new perspective for the development of PAH and provides the necessary theoretical basis for the prevention and treatment of pulmonary hypertension with traditional Chinese medicine.

[0111] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the claims and their equivalents.

Claims

1. Use of a substance capable of regulating Mfn2-IP3R3 interaction in the preparation of a drug for preventing and treating pulmonary hypertension, characterized in that: The substances regulate the Mfn2-IP3R3 interaction by inhibiting ER stress.

2. The use according to claim 1, characterized in that The substance upregulates Mfn2 expression and downregulates IP3R3 expression by inhibiting ER stress.

3. The use according to claim 2, characterized in that The substance downregulates mitochondrial outer membrane Ca by upregulating Mfn2 expression and downregulating IP3R3 expression. 2+ Regulator Vdac1 and upregulate intracellular Ca 2+ Homeostatic regulator SERCA.

4. The use according to claim 3, characterized in that The substance downregulates mitochondrial outer membrane Ca 2+ Regulator Vdac1 and upregulate intracellular Ca 2+ The homeostatic regulator SERCA upregulates mitochondrial Ca 2+ concentration.

5. The use according to claim 4, characterized in that The substance upregulates mitochondrial Ca 2+ concentration to protect mitochondrial morphology and inhibit mitochondrial autophagy, thereby inhibiting pulmonary artery smooth muscle cell proliferation and pulmonary vascular remodeling.

6. A drug for preventing and treating pulmonary hypertension, characterized in that: The drug includes a substance capable of regulating the Mfn2-IP3R3 interaction.

7. The drug for preventing and treating pulmonary hypertension according to claim 6, characterized in that The substance capable of regulating the Mfn2-IP3R3 interaction is 4-phenylbutyric acid.

8. The drug for preventing and treating pulmonary hypertension according to claim 6, characterized in that The substance capable of regulating the Mfn2-IP3R3 interaction is Cistanche deserticola phenylethanoid glycoside.

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