Application of reagent for detecting SOCS3 expression level in preparation of product for evaluating treatment effect of mesenchymal stem cells on pulmonary arterial hypertension

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a reagent for detecting the expression level of SOCS3 in preparation of a product for evaluating the treatment effect of mesenchymal stem cells on pulmonary arterial hypertension. By integrating transcriptomics and functional experiments, the invention reveals that MSC activates the expression of SOCS3 through paracrine, and further inhibits the excessive phosphorylation of STAT3, thereby blocking the molecular mechanism of PAAF activation and ECM abnormal deposition. The expression level of the SOCS3 can be used as a marker for judging the inhibition of the mesenchymal stem cells on the activation of the pulmonary adventitia fibroblasts, and also can be used as a marker for judging the treatment effect of the mesenchymal stem cells on the pulmonary arterial hypertension. By up-regulating the expression of SOCS3, the excessive phosphorylation of STAT3 can be inhibited, the activation of pulmonary outer membrane fibroblasts and the expression of Col1 and Col3 can be inhibited, but the proliferative activity is not influenced, so that the PAH can be treated.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of reagents for detecting SOCS3 expression levels in the preparation of products for evaluating the therapeutic effect of mesenchymal stem cells on pulmonary hypertension. Background Technology

[0002] Pulmonary hypertension (PH) is a severe clinical syndrome characterized by progressively increasing pulmonary vascular resistance, ultimately leading to right heart failure and death. PH is systematically classified into five major clinical types: 1) Pulmonary arterial hypertension (PAH); 2) PH associated with left ventricular disease; 3) PH associated with lung disease and / or hypoxia; 4) Chronic thromboembolic pulmonary hypertension (CTEPH); and 5) PH with other mechanisms of unknown origin. Among these, the pathogenesis of PAH is complex and involves multiple pathological changes. Pulmonary artery endothelial cell (PAEC) dysfunction is the initiating factor of PAH, manifested by a decrease in vasodilators (such as nitric oxide and prostacyclin) and an increase in vasoconstrictors (such as endothelin-1), thereby triggering abnormal proliferation of pulmonary arterial smooth muscle cells (PASMCs), leading to thickening of the vessel wall and narrowing of the lumen. Recent studies have found that the chronic inflammatory microenvironment also plays a crucial role in the progression of PAH. Activated macrophages, T lymphocytes, and other inflammatory cells, along with their secreted pro-inflammatory factors such as IL-6 and TNF-α, influence the pathological changes of pulmonary vessels through complex intercellular interactions [3-6]. In addition, pulmonary adventitial fibroblasts (PAAFs) also play an indispensable role in vascular remodeling in PAH. Under the regulation of signaling pathways such as TGF-β / Smad and Wnt / β-catenin, PAAFs transform into myofibroblasts, leading to excessive deposition of extracellular matrix (ECM) in blood vessels, which exacerbates vascular wall fibrosis and stiffening.

[0003] Mesenchymal stem cells (MSCs) have beneficial roles in immune regulation and tissue repair, and have been used in multiple clinical trials to treat various inflammatory diseases, such as graft-versus-host disease, diabetes, pulmonary fibrosis, heart failure, and Crohn's disease. However, the changes in metabolic patterns and pathways in patients with peripheral arterial disease (PAH) after MSC treatment remain unclear. Therefore, it is essential to identify relevant biomarkers and therapeutic targets from a metabolomics perspective to effectively assess the specific regulatory role of MSCs in vascular ECM remodeling in PAH.

[0004] Cytokine signaling inhibitory molecule 3 (SOCS3), a member of the SOCS family, is one of the most potent inhibitory proteins in the SOCS family, negatively regulating the JAK / STAT signaling pathway. Its gene promoter contains a binding site for the JAK2 / STAT3 signaling pathway. As a specific endogenous inhibitor of the JAK / STAT signaling pathway, SOCS3 can inhibit excessive cytokine activation through direct binding to JAK2 sites and phosphorylated tyrosine residues of STAT3, as well as recruitment of ubiquitin-linking enzymes. Currently, the role of SOCS3 in PAH vascular remodeling and pulmonary adventitia fibroblast activation has not been studied. Summary of the Invention

[0005] The purpose of this invention is to provide the application of reagents for detecting SOCS3 expression levels in the preparation of products for evaluating the therapeutic effect of mesenchymal stem cells on pulmonary arterial hypertension (PAH), to elucidate the mechanism of action of MSCs in PAH treatment, and to provide a new potential target for the treatment of PAH.

[0006] This invention provides the application of a reagent for detecting SOCS3 expression levels in the preparation of products for evaluating the therapeutic effect of mesenchymal stem cells on pulmonary hypertension.

[0007] Preferably, the evaluation of the therapeutic effect of mesenchymal stem cells on pulmonary hypertension includes evaluating the therapeutic effect of mesenchymal stem cells on the remodeling of extracellular matrix in pulmonary hypertension vascular cells.

[0008] Preferably, the extracellular matrix remodeling of pulmonary hypertension vessels includes extracellular matrix remodeling of pulmonary hypertension vessels induced by lily alkaloids.

[0009] Preferably, the extracellular matrix remodeling of pulmonary hypertension vessels includes abnormal deposition of collagen in pulmonary hypertension vessels; the collagen includes Collagen 1 and Collagen 3.

[0010] This invention provides the application of a reagent for detecting SOCS3 expression levels in the preparation of products for evaluating the effect of mesenchymal stem cells on inhibiting the activation of pulmonary artery adventitia fibroblasts.

[0011] Preferably, the pulmonary artery adventitia fibroblast activation includes TGF-β1-induced pulmonary artery adventitia fibroblast activation.

[0012] Preferably, the product includes a reagent kit; The reagent used to detect SOCS3 expression levels includes an anti-SOCS3 antibody.

[0013] This invention provides the use of SOCS3 and / or reagents that upregulate SOCS3 expression in the preparation of drugs for treating pulmonary hypertension and / or inhibiting the activation of pulmonary adventitia fibroblasts.

[0014] Preferably, the reagent for upregulating SOCS3 expression includes mesenchymal stem cells.

[0015] Preferably, the SOCS3 and / or the reagent that upregulates SOCS3 expression treats pulmonary hypertension and / or inhibits pulmonary adventitia fibroblast activation by downregulating STAT3 phosphorylation levels.

[0016] Beneficial effects: This invention, through integrated transcriptomics and functional experiments, reveals the molecular mechanism by which MSCs activate SOCS3 expression via paracrine signaling, thereby inhibiting STAT3 hyperphosphorylation and blocking PAAF activation and abnormal ECM deposition. SOCS3 expression levels can serve as a marker for assessing the inhibition of pulmonary artery adventitia fibroblast activation by mesenchymal stem cells (MSCs), and also as a marker for assessing the therapeutic effect of MSCs on pulmonary hypertension (PAH). Upregulating SOCS3 expression can inhibit STAT3 hyperphosphorylation, suppress pulmonary artery adventitia fibroblast activation and Col1 and Col3 expression, without affecting their proliferative activity, thus treating PAH. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1This study aimed to assess the therapeutic effect of MSCs on MCT-induced pulmonary vascular ECM remodeling in PAH. Figure A shows co-localization microscopic images of α-SMA (green) and Col1 (purple) in the distal pulmonary artery, with cell nuclei counterstained with DAPI blue. Scale bar: 10 μm. Figure B shows quantitative analysis of Col1 fluorescence intensity in the distal pulmonary artery. Figure C shows the relative expression level of Col1 mRNA in the distal lung tissue of each group detected by RT-qPCR. Figure D shows co-localization microscopic images of α-SMA (green) and Col3 (red) in the distal pulmonary artery, with cell nuclei counterstained with DAPI blue. Scale bar: 10 μm. Figure E shows quantitative analysis of Col3 fluorescence intensity in the distal pulmonary artery. Figure F shows the relative expression level of Col1 mRNA in the distal lung tissue of each group detected by qRT-PCR. Experimental data are expressed as mean ± standard deviation (n=7-8 per group). Figure 2 To demonstrate the effect of MSCs in inhibiting MCT-induced PAAF activation in PAHs; where A shows representative confocal images of co-localization of α-SMA (green) and vimentin (red) in the distal pulmonary artery of each group, with cell nuclei counterstained with DAPI blue, scale bar: 10 μm; B shows the quantitative analysis of the number of vimentin+ / α-SMA- single-positive cells in the distal pulmonary artery adventitia; C shows the quantitative analysis of the number of vimentin+ / α-SMA+ double-positive cells in the distal pulmonary artery adventitia; experimental data are expressed as mean ± standard deviation (n=7-8 per group). Figure 3 This study aims to identify primary PAAF and establish a TGF-β1-induced PAAF activation model. A shows the cellular morphology of primary PAAF; B shows the immunophenotypic identification of primary PAAF (scale bar: 200 μm); C shows the Western blot results of α-SMA protein expression in PAAF treated with different concentrations of TGF-β1 for 48 h; D shows the immunofluorescence results of α-SMA (green) in PAAF treated with different concentrations of TGF-β1 for 48 h, with cell nuclei stained with DAPI (blue) (scale bar: 200 μm); E shows the Western blot results of α-SMA protein expression in PAAF after different treatment times with 10 ng / mL TGF-β1; F shows the immunofluorescence results of α-SMA (green) in PAAF after different treatment times with 10 ng / mL TGF-β1, with cell nuclei stained with DAPI (blue) (scale bar: 200 μm). Figure 4This study illustrates the inhibitory effect of MSCs on TGF-β1-induced PAAF activation and ECM deposition. A shows a schematic diagram of the transwell non-contact co-culture system of MSCs and PAAFs; B shows Western blot images of α-SMA, Col1, and Col3 protein expression in PAAFs of each group; C shows immunofluorescence staining images of α-SMA (green), Col1 (green), and Col3 (red) expression in PAAFs of each group, with cell nuclei counterstained with DAPI blue, scale bar: 200 μm; D shows quantitative analysis of α-SMA, Col1, and Col3 protein expression; E shows quantitative analysis of the fluorescence intensity of α-SMA, Col1, and Col3; F shows EdU staining images of PAAF proliferation in each group, with cell nuclei counterstained with DAPI blue, scale bar: 400 μm; G shows quantitative analysis of the number of EdU-positive cells; H shows the results of PAAF proliferation activity detected by the CCK-8 assay. Experimental data are expressed as mean ± standard deviation (n=3). Figure 5 This study investigated how MSCs inhibit PAAF activation via the SOCS3 / STAT3 signaling pathway. Specifically, A represents the KEGG pathway enrichment analysis of pulmonary arteries in the MSC-treated group and the MCT-induced PAH group (MCT+MSC vs. MCT+PBS, n=3); B represents the KEGG pathway enrichment analysis of the MSC co-culture treatment group and the TGF-β1-stimulated PAAF group (TGF-β1+MSC vs. TGF-β1+Veh, n=3); C represents Western blot images of SOCS3, p-STAT3, and STAT3 protein expression in PAAF of each group (n=3); D represents the grayscale quantitative analysis of SOCS3 and p-STAT3 / STAT3 protein expression levels (n=3); E represents the relative SOCS3 mRNA expression level in PAAF of each group detected by RT-qPCR (n=3); and F represents representative immunohistochemical images of p-STAT3 expression in the distal pulmonary arteries of each group (n=6-8, scale bar: 10). G represents immunofluorescence confocal microscopy images of SOCS3 expression in the distal pulmonary artery of each group (n=6-8, scale bar: 10 μm); H represents quantitative analysis of p-STAT positive cells in the distal pulmonary artery adventitia (n=6-8); I represents fine quantitative analysis of SOCS3 positivity in the distal pulmonary artery adventitia (n=6-8). Experimental data are expressed as mean ± standard deviation. Figures 1-5 Significance marker: **** P <0.0001; *** P <0.001;** P <0.01;* P <0.05; ns indicates no significant difference. Detailed Implementation

[0019] This invention provides the application of a reagent for detecting SOCS3 expression levels in the preparation of products for evaluating the therapeutic effect of mesenchymal stem cells on pulmonary hypertension.

[0020] In one embodiment, the evaluation of the therapeutic effect of mesenchymal stem cells on pulmonary hypertension (PH) includes evaluating the therapeutic effect of mesenchymal stem cells on extracellular matrix remodeling of PH vascular cells. In another embodiment, the extracellular matrix remodeling of PH vascular cells includes abnormal collagen deposition in PH vascular cells. In yet another embodiment, the collagen includes Collagen 1 and Collagen 3. SOCS3 has the NCBI accession number O14543.

[0021] As one embodiment, the extracellular matrix remodeling of pulmonary hypertension vessels described in this invention includes extracellular matrix remodeling of pulmonary hypertension vessels induced by lily alkaloids.

[0022] This invention provides the application of a reagent for detecting SOCS3 expression levels in the preparation of products for evaluating the effect of mesenchymal stem cells on inhibiting the activation of pulmonary artery adventitia fibroblasts.

[0023] As one embodiment, the pulmonary adventitia fibroblast activation described in this invention includes TGF-β1-induced pulmonary adventitia fibroblast activation.

[0024] As one implementation method, the product of this invention includes a reagent kit.

[0025] As one embodiment, the reagent for detecting SOCS3 expression level according to the present invention includes an anti-SOCS3 antibody.

[0026] This invention, through transcriptome sequencing analysis of pulmonary artery tissue and primary PAAF, revealed that MSC treatment significantly upregulated the cytokine-cytokine receptor interaction pathway, with SOCS3 expression significantly upregulated within this pathway. Upregulation of SOCS3 inhibited JAK2 kinase activity and reduced STAT3 phosphorylation. In vitro and in vivo experiments confirmed that MSC intervention upregulated SOCS3 expression and decreased p-STAT3 expression in PAAF, revealing that MSCs may inhibit PAAF activation and collagen deposition through the STAT3 pathway, providing a theoretical basis for the mechanism by which MSCs regulate vascular ECM remodeling. SOCS3 expression levels can serve as a marker for assessing the inhibition of pulmonary artery adventitia fibroblast activation by mesenchymal stem cells (MSCs) and also as a marker for assessing the therapeutic effect of MSCs on pulmonary hypertension.

[0027] This invention provides the use of SOCS3 and / or reagents that upregulate SOCS3 expression in the preparation of drugs for treating pulmonary hypertension and / or inhibiting the activation of pulmonary adventitia fibroblasts.

[0028] In one embodiment, the reagent for upregulating SOCS3 expression in this invention includes mesenchymal stem cells. In one embodiment, the mesenchymal stem cells in this invention are primary mesenchymal stem cells; in another embodiment, the mesenchymal stem cells in this invention include any one or more generations of primary mesenchymal stem cells from P0 to P3. In one embodiment, the SOCS3 and / or the reagent for upregulating SOCS3 expression in this invention treat pulmonary hypertension and / or inhibit pulmonary adventitial fibroblast activation by downregulating STAT3 phosphorylation levels.

[0029] Aberrant deposition of Col1 and Col3 is the most significant collagen component in PAH vascular remodeling. This invention, through immunofluorescence co-localization, revealed significantly elevated Col1 and Col3 expression in the pulmonary vessels of the PAH model group. MSC treatment significantly inhibited this pathological vascular ECM deposition, resulting in a significant decrease in the expression levels of both collagens. Immunofluorescence co-localization also showed that Col1 and Col3 were primarily enriched in the periphery of α-SMA-positive regions, suggesting that PAAF may be their main source. This invention innovatively combines immunofluorescence double labeling technology and vascular spatial localization methods to confirm that MSCs selectively inhibit the number of activated PAAFs without significantly affecting the number of resting PAAFs. Using a transwell indirect co-culture system, the cellular level was further validated to show that MSCs can significantly inhibit TGF-β1-induced PAAF activation and Col1 and Col3 expression through paracrine effects without affecting their proliferative activity. MSC intervention significantly improved the expression of these collagens.

[0030] To further illustrate the present invention, the application of the reagent for detecting SOCS3 expression level provided by the present invention in the preparation of products for evaluating the therapeutic effect of mesenchymal stem cells on pulmonary hypertension is described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] The reagents and consumables used in the embodiments of this invention are as follows: Recombinant Mouse / Rat TGF-β1 (Suzhou Nearshore), primary antibody α-SMA (Abcam, UK), primary antibody Col1 (Abcam, UK), primary antibody Col3 (Wuhan Saive), primary antibody vWF (Abcam, UK), primary antibody Vimentin (Wuhan Saive), primary antibody SOCS3 (Proteintech, USA), primary antibody p-STAT3 (CST, USA), primary antibody STAT3 (CST, USA), goat anti-mouse IgG H&L (Alexa Fluor® 488) (Abcam, UK), goat anti-rabbit IgG H&L (Alexa Fluor® 594) (Abcam, UK), HRP-conjugated goat anti-mouse secondary antibody (Wuhan Saive), HRP-conjugated goat anti-rabbit secondary antibody (Wuhan Saive), TBS (Wuhan Saive), Tween 20 (Yantai Qingjun), environmentally friendly dewaxing agent (Wuhan Tongsheng), PBS (Gibco, USA), anhydrous ethanol (Beijing Guoyao Group), 75% alcohol disinfectant (Beijing Guoyao Group), 1×Tris-EDTA (pH 9.0) antigen retrieval solution (Wuhan Saiweier), endogenous peroxidase blocking solution (Shanghai Beyotime), DAB chromogenic reagent kit (Wuhan Boster), immunohistochemistry pen (Wuhan Saiweier), autofluorescence quencher (Wuhan Saiweier), BSA (Guangzhou Shunhao), DAPI (Wuhan Saiweier), anti-fluorescence quenching mounting medium (Wuhan Saiweier), microscope slides (Jiangsu Shitai), microscope coverslips (Jiangsu Shitai), TransZol Up high-performance RNA extraction kit (Beijing TransGen, ET111-01-V2), cDNA reverse transcription kit (Shanghai Yisheng), RT-qPCR kit (Shanghai Yisheng), 0.2 mL clear PCR tubes (Corning, USA), DEPC-treated water (0.1%) (Beijing Regen), Transwell chambers of various sizes (Corning, USA), pipettes of various sizes (Jiangsu Naisi), EdU-594 cell proliferation assay kit (Shanghai Beyotime), CCK8 kit (MCE, USA), methanol (Shanghai Maclean), protease and phosphatase inhibitors (Shanghai Beyotime), RIPA Lysis Buffer (ThermoFisher, USA), Micro BCA protein quantification kit (ThermoFisher, USA), ECL chemiluminescence solution (Shanghai Tianneng), PVDF membrane (Merck, Germany), 5× protein loading buffer (Hangzhou Fude), protein marker (ThermoFisher, USA), 10% SDS-PAGE (Shanghai Yamei), SDS-PAGE electrophoresis buffer (Shanghai Beyotime), Western transfer buffer (Shanghai Beyotime), Western primary antibody dilution buffer (Shanghai Beyotime), QuickBlock TMWestern closure solution (Shanghai Beyotime).

[0032] The main instruments used in the embodiments of this invention are: protein electrophoresis apparatus (Bio-Rad, USA), tissue lysis apparatus (Guangzhou Luca Sequencing Instruments), chemiluminescence analyzer (Shanghai Tianneng), spectrophotometer microplate reader (Themo, USA), and microplate reader (Themo, USA).

[0033] Example 1 The therapeutic effect of MSCs on pulmonary vascular ECM remodeling induced by cynomolgus alkaloid (MCT) in PAH 1. Referring to existing technology (Bao Changlei. Therapeutic effects and mechanisms of artemisinin and ROC-325 in experimental animal models of pulmonary arterial hypertension [D]. Northwest A&F University, 2022. DOI:10.27409 / d.cnki.gxbnu.2022.002256.), a single intraperitoneal injection of MCT (50 mg / kg) was administered to 180-220 g SPF-grade male SD rats to construct an MCT-induced PAH animal model.

[0034] 2. Immunofluorescence staining of lung tissue (1) Place lung tissue sections in a 70℃ oven and bake for 2 h; (2) Soak sections in TO clearing solution I and TO clearing solution II for 5 min each for dewaxing; (3) Place sections in a gradient of alcohol (100% I → 100% II → 95% → 80% → 70% → distilled water) for 5 min each; (4) Heat EDTA antigen retrieval solution to boiling (100℃) in advance, place sections in the solution, microwave and maintain the temperature above 95℃ for 10-20 min, then cool to room temperature and wash 3 times with Tris-buffered saline with Tween 20 (TBST) for 5 min each time; (5) Mark tissue with immunohistochemistry pen and block with 5% BSA blocking solution at room temperature for 1.5 h; (6) Add an appropriate amount of primary antibody dilution solution and incubate overnight in a humidified box at 4℃. The antibodies used were: α-smooth muscle actin (α-SMA, ab7817, 1:1000), Collagen 1 (Col1, ab270993, 1:500), Collagen 3 (Col3, GB111629, 1:300), Vimentin (ab92547, 1:300), and SOCS3 (2923S, 1:100); (7) The slides were incubated at room temperature for 30 min in a humidified chamber on the second day, and washed three times with TBST for 5 min each time; (8) The corresponding secondary antibody was added, and the slides were incubated at room temperature in the dark for 1.5 h, and washed three times with TBST for 5 min each time; (9) The slides were stained with DAPI staining solution in the dark for 10 min, and washed three times with TBST for 5 min each time; (10) The slides were mounted with anti-fluorescence quenching mounting medium. After the slides were dried, they were scanned using a panoramic tissue cell quantitative TG scanning system and observed using SlideViewer software.

[0035] 3. RT-qPCR detection of mRNA expression in lung tissue 3.1 RNA was extracted from lung tissue using the TransZol Up high-performance RNA extraction kit.

[0036] 3.2 The lung tissue RNA obtained in step 3.1 was reverse transcribed into cDNA using a cDNA reverse transcription kit. The specific steps are as follows: (1) Residual genomic DNA removal: Prepare a mixture (3 μL of 5×g DNA digester mix, 10 pg~5 μg of total RNA, and RNase-free H2O to a final volume of 15 μL) in an RNase-free centrifuge tube, gently mix with a pipette, and incubate at 42℃ for 2 min to obtain the first step reaction solution. (2) Mix the first step reaction solution with 5 μL of 4×Hifair. ®Mix with SuperMixplus and reverse transcribe according to the program of 25℃ for 5 min, 55℃ for 15 min, and 85℃ for 5 min. The reverse transcription product can be used immediately for qRT-PCR reaction or stored at -20℃ for a short period of time. For long-term storage, it is recommended to aliquot and store at -80℃, avoiding repeated freeze-thaw cycles.

[0037] 3.3 RT-qPCR detection (1) BGI Genomics was commissioned to synthesize detection primers, as shown in Table 1.

[0038] Table 1 Primers for RT-qPCR detection

[0039] (2) Preparation of RT-qPCR detection reaction system: Hieff UNICON® qPCR SYBR Green MasterMix (antibody method, High Rox) 10 μL, upstream primer (10 μM) 0.4 μL, downstream primer (10 μM) 0.4 μL, template DNA 2 μL, sterile ultrapure water to make up to 20 μL, all operations were performed on ice; RT-qPCR detection reaction program: 95℃ pre-denaturation for 30 sec; 95℃ denaturation for 10 sec, 55-60℃ annealing for 20 sec, 72℃ extension for 20 sec, 40 cycles; the instrument default setting for the melting curve stage is 1 cycle.

[0040] 4. Statistical Analysis Statistical analysis was performed using GraphPad Prism software (version 9.5). Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Quantitative analysis was performed on all image data using ImageJ software. P A value <0.05 was considered statistically significant. All quantitative data are expressed as mean ± standard deviation (Mean ± SD).

[0041] 5. Experimental Results Compared with the normal control group (Nor group), the MCT model group had Col1 in the pulmonary vessels ( Figure 1 (A and B) and Col3 ( Figure 1 The expression of D and E was significantly increased (in the middle). P <0.001), while MSC treatment can significantly inhibit this pathological vascular ECM deposition, and significantly reduce the expression levels of the two collagens ( P<0.05). This result was validated at the mRNA level; RT-qPCR results showed that the levels of Col1 and Col3 mRNA in the distal lung tissue of the MSC group were significantly lower than those in the MCT model group. P <0.05, Figure 1 (C and F). Spatial distribution analysis revealed that Col1 and Col3 were mainly enriched in the peripheral portion of the α-SMA-positive region (i.e., the PASMC layer). Figure 1 (A and D in the middle). This characteristic distribution pattern suggests that adventitia PAAF may be the main cellular source of pathological ECM deposition.

[0042] 3. Based on the conclusions of step 2, the regulatory role of MSCs on the activation of pulmonary artery adventitia fibroblasts (PAAFs) was further investigated. Since vascular fibroblasts lack a single specific marker, vimentin can be expressed simultaneously in both vascular fibroblasts and endothelial cells, while α-SMA is expressed not only in PASMCs but also in activated fibroblasts. This step used a combination of double-labeled immunofluorescence co-staining technology (Vimentin / α-SMA) and vascular anatomical localization to identify and analyze PAAFs. Activated PAAFs were defined as vimentin+α-SMA+ double-positive cells located in the peripheral region of α-SMA-positive PASMC layers, while resting PAAFs were vimentin+α-SMA- single-positive cells in the same region. Quantitative analysis showed that compared with the Nor group, the number of both activated and resting PAAFs in the adventitia of the MCT model group was significantly increased. P <0.0001, Figure 2 (AC); After MSC treatment, the number of activated PAAFs was significantly reduced ( P <0.0001, Figure 2 (A and C), but the number of resting PAAFs was not significantly affected ( P >0.05, Figure 2 (A and B in the original text). These results indicate that MSCs primarily improve pathological ECM deposition in blood vessels by inhibiting the conversion of PAAFs from the resting to the activated state, rather than affecting the number of resting cells. This finding provides important experimental evidence for elucidating the specific regulatory targets of MSCs on vascular ECM remodeling during PAH treatment.

[0043] Example 2 1. PAAF extraction, the specific steps are as follows: (1) Take normal male SD rats aged 6-8 weeks and weighing about 200 g, and fix them on the operating table after deep anesthesia with isoflurane. After thoroughly disinfecting the chest and abdomen of the rats with 75% alcohol, use a large autoclaved scissor to cut open the skin of the chest and abdomen along the midline. Then, use another large autoclaved scissor to cut open the diaphragm and ribs to fully expose the lung tissue in the thoracic cavity. At this time, use a small autoclaved scissor to carefully separate and completely cut off the lung tissue. (2) The separated lung tissue was placed in pre-cooled sterile phosphate-buffered saline (PBS) containing 2% penicillin and streptomycin and thoroughly washed to remove blood and impurities. The pulmonary arteries of each lobe were separated under a stereomicroscope, ensuring the integrity of the vessels as much as possible during the separation process. The separated pulmonary arteries were washed three times with pre-cooled sterile PBS containing 2% penicillin and streptomycin. The pulmonary arteries were transferred to a biosafety cabinet and washed several more times with pre-cooled sterile PBS containing 2% penicillin and streptomycin. The pulmonary arteries were transferred to a 6-well plate and cut into small pieces of about 1 mm × 1 mm × 1 mm using autoclaved microscissors. The tissue pieces were then evenly spread on the bottom of the well plate and placed in a 37°C, 5% CO2 incubator for about 10 min to promote tissue adhesion. (3) After tissue attachment, slowly add 2 mL of high-glucose DMEM complete culture medium (containing 20% ​​fetal bovine serum (FBS) and 1% penicillin and streptomycin) to each well of the 6-well plate. When adding the liquid, slowly inject along the well wall with the pipette tip to avoid direct impact on the tissue block. Then place the culture plate in a 37℃, 5% CO2 incubator for further culture; (4) After primary culture for 5-7 days, a large number of spindle-shaped cells can be seen crawling out from the edge of the tissue block under an optical microscope. In a biosafety cabinet, use a sterile pipette tip to gently aspirate the tissue block to avoid damaging the adherent cells. Discard the culture supernatant and gently wash twice with sterile PBS solution, 1 mL each time; (5) Add 1 mL of 0.25% trypsin-EDTA digestion solution to each well, incubate at 37°C for 2-3 min, and add 2 mL of complete culture medium to stop digestion after the cells become round. Pipe the cells to form a single-cell suspension and transfer it to a 15 mL centrifuge tube. If digestion is incomplete, add 1 mL of digestion solution for a second digestion and combine the cell suspensions obtained from the two digestions. (6) After centrifuging the cell suspension at 500 g for 5 min, discard the supernatant and resuspend the cells in 1 mL of complete culture medium. Transfer the cell suspension to a T25 culture flask, gently mix using the "cross method," and incubate at 37℃ in a 5% CO2 incubator for differential adhesion culture. After 60 min, carefully aspirate the culture medium containing unattached cells and gently wash twice with sterile PBS. Continue culturing in high-glucose DMEM complete culture medium (containing 10% FBS and 1% penicillin and streptomycin), changing the medium with fresh medium every 2-3 days. Passage the cells when the confluence reaches approximately 80-90%. All subsequent experiments used primary PAAF cells within the third passage.

[0044] 2. Identification of PAAF Vimentin, a hallmark intermediate fibrous protein of mesodermal mesenchymal cells, is expressed in fibroblasts, endothelial cells, neutrophils, and macrophages, and is an important molecular marker for identifying fibroblasts. α-SMA is mainly expressed in smooth muscle cells and activated fibroblasts, but is negatively expressed in resting fibroblasts. Von willebrand factor (vWF), as an endothelial cell-specific marker, can effectively exclude endothelial cell contamination. Therefore, the immunophenotypic characteristics of vimentin+ / α-SMA- / vWF- can accurately identify fibroblasts. The specific steps of immunofluorescence staining are as follows: (1) Take P1 to replace PAAF, and use 5×10 4 The cells were seeded at a density of 100 cells / well in 24-well plates, and 500 μL of high-glucose DMEM complete medium (containing 10% FBS and 1% penicillin and streptomycin) was added to each well. The plates were then incubated at 37°C and 5% CO2 for 24 h until the cells were fully adhered. (2) After the cells adhere to the wall, carefully aspirate the culture supernatant, add 1 mL of PBS to each well, slowly add it along the wall of the well, gently shake for 5 min and then discard it, wash 3 times, and keep the pipette tip from touching the bottom of the well during the operation to avoid mechanical damage to the adhered cells. (3) Add 1 mL of 4% paraformaldehyde solution to each well, fix at room temperature for 30 min, remove the fixative, and wash 3 times with 1 mL of PBS for 5 min each time; (4) Add 1 mL of 0.1% Triton X-100 solution to each well, permeate at room temperature for 30 min, discard the permeation solution, and wash 3 times with 1 mL of PBS for 5 min each time; (5) Add 1 mL of 5% BSA solution to each well, seal at room temperature for 30 min, remove the sealing solution, no rinsing is required; (6) Add 300 μL of primary antibody working solution [Vimentin (GB11192, 1:300), vWF (ab6994, 1:200), α-SMA (ab7817, 1:500)] to each well, and incubate the 24-well plate at 4℃ overnight (16-18 h). The next day, discard the primary antibody, wash three times with 1 mL PBS for 5 min each time; (7) Add 300 μL of fluorescently labeled secondary antibody working solution (goat anti-mouse IgG H&L (Alexa Fluor® 488) and goat anti-rabbit IgG H&L (Alexa Fluor® 594)) to each well, incubate at room temperature in the dark for 1.5 h, discard the secondary antibody, wash 3 times with 1 mL PBS, 5 min each time; (8) Add 300 μL of DAPI to each well, incubate at room temperature in the dark for 10 min, discard the DAPI staining solution, and wash 3 times with 1 mL of PBS for 5 min each time; (9) To keep the samples moist, add 500 μL of PBS to each well. Immediately acquire images using a Leica DM18 upright fluorescence microscope, or for short-term storage, seal the 24-well plate and store it at 4°C in the dark (image acquisition is recommended within 7 days). The results showed that PAAF cells exhibited typical fibroblast morphological characteristics, with spindle-shaped cell bodies accompanied by slender processes, growing radially or in a whorled pattern, and forming a specific "paving stone" arrangement when the cells merged. Figure 3 (A). Immunofluorescence identification confirmed that the vimentin positivity rate of the isolated cells was >95%, while the expression of smooth muscle cell marker α-SMA and endothelial cell marker vWF was both below 5%, indicating that the obtained PAAF had high purity and met the requirements for subsequent experiments. Figure 3 (B)

[0045] 3. Given that TGF-β1 is a key regulator of pro-fibrotic responses, a systematic experiment was conducted with concentration gradients (0, 5, 10 ng / mL) and time courses (0, 24, 48 h) to confirm that stimulation with 10 ng / mL TGF-β1 for 48 h significantly upregulated α-SMA expression in PAAF. Figure 3 (CF). The establishment of this standardized PAAF activation condition provides a reliable experimental model for subsequent research on the molecular mechanism of MSC-regulated PAAF activation.

[0046] Example 3 Establishment of PAAF / MSC transwell co-culture system (indirect co-culture) 1. MSC resuscitation and culture Human bone marrow-derived MSCs, having completed osteogenic, adipogenic, and chondrogenic differentiation, were passaged to P4 generation and cryopreserved in liquid nitrogen. The water bath was pre-opened and the temperature was set to a constant 37°C. The cryopreserved cells were removed from the liquid nitrogen tank and quickly transferred to the 37°C water bath, gently agitating the cryovials to rapidly thaw the cell suspension. Subsequently, 2 mL of MSC medium (XR MSC Pro complete mesenchymal stem cell medium) was added to dilute the cell suspension, and the cells were centrifuged at 500 g for 5 min, discarding the supernatant. The cell pellet was resuspended in 1 mL of MSC medium, and the cell suspension was seeded into appropriately sized culture flasks and incubated at 37°C in a 5% CO2 incubator. The medium was changed every 2–3 days. When the cells reached 80% confluence, they were digested and passaged using 0.25% trypsin. After continuous passage and large-scale cell expansion, the cells were digested, centrifuged, and the supernatant was discarded. The cells were then resuspended in sterile 0.9% physiological saline, mixed with trypan blue solution at a 1:1 ratio, and counted. Cells were only used when the cell viability was >90%. The cell suspension concentration was adjusted to 3.0 × 10⁻⁶ cells / mL with sterile 0.9% physiological saline. 6 Cells / mL, stored at 4°C, used within 6 hours. All experiments used MSCs within 8 passages.

[0047] 2. Seed 4 × 10⁴ seeds in the upper chamber of a 0.4 μm pore size transwell. 5 One MSC was inoculated at the bottom of another 6-well plate, while 2 × 10⁶ MSCs were simultaneously seeded. 5 Two groups of PAAF cells were cultured separately in their respective complete culture media for 24 h to ensure adequate cell adhesion. Then, the culture medium in the 6-well plates was removed, and the PAAF cells were subjected to 8 h of serum-free, high-glucose DMEM starvation treatment. After treatment, the medium was replaced with 2 mL of serum-free, high-glucose DMEM stimulation medium containing 10 ng / mL TGF-β1. Simultaneously, the transwell chambers containing MSCs were placed on top of the culture system, and 1.5 mL of serum-free, high-glucose DMEM medium was added to the top chamber. Figure 4 (A). After the co-culture system was cultured at 37℃ and 5% CO2 for 48 h, the lower layer PAAF was collected for subsequent functional testing and analysis.

[0048] 3. Western Blot (WB) Detection of Proteins 3.1 Extraction of total cellular protein (1) Take 150 μL of RIPA lysis buffer, add 3 μL of protease inhibitor and 3 μL of phosphatase inhibitor, vortex to mix well and place on ice for later use. (2) Cell washing and lysis: Discard the cell culture medium, wash three times with pre-cooled PBS to completely remove residual culture medium; add 150 μL of pre-cooled RIPA lysis buffer to each well, incubate on ice for 10 min to allow the cells to fully lyse; use a cell scraper to scrape off the cells and repeatedly pipette the lysis buffer to ensure that the cells are completely broken, and collect the lysis buffer into a centrifuge tube. (3) Centrifugation and supernatant collection: Centrifuge at 4℃ and 15000 rpm for 20 min, and carefully aspirate the supernatant to avoid aspirating the precipitate; (4) Protein concentration determination (BCA method): Prepare the standard according to the instructions of the BCA protein quantitative kit, detect the absorbance of the sample, and calculate the protein concentration of each sample; (5) Protein sample preparation and preservation: Mix the protein sample with 5× loading buffer at a ratio of 4:1, boil at 100℃ for 10 min to denature the protein; after aliquoting, store at -20℃ for short-term storage and at -80℃ for long-term storage.

[0049] 3.2 Determination of protein concentration by BCA method (1) Preparation of BCA working solution: Mix reagent A: reagent B = 50:1 before use, store in the dark, and use within 2 hours; (2) Gradient preparation of protein standards: Take 2 μg / μL of protein standard and perform gradient dilution with PBS (2 μg / μL→1 μg / μL→0.5 μg / μL→0.25 μg / μL→0.125 μg / μL→0.0625 μg / μL), and prepare 100 μL for each concentration; (3) Sample loading procedure: Diluted protein standards were added sequentially to 96-well plates according to their concentrations, 20 μL per well, with 3 replicates for each concentration. 19 μL of PBS was added to each well of the sample to be tested, followed by 1 μL of the protein sample to be tested in each well, with 3 replicates for each sample; blank control wells (20 μL PBS) were also included. (4) Reaction system: Add 200 μL of BCA working solution to each well, mix gently, and incubate at 37°C in the dark for 30 min; (5) Detection and Analysis: The absorbance (OD value) at 562 nm was detected using an ELISA reader. Standard Curve Construction: Background values ​​from blank wells were subtracted; a standard curve was fitted using quadratic linear regression with concentration on the x-axis and average OD value on the y-axis. 2 ≥0.99). Sample concentration calculation: measured concentration × dilution factor (20×).

[0050] 3.3 Western Blot Experimental Procedure (1) Prepare the lower layer gel: Select a 10% SDS-PAGE separating gel according to the size of the target molecule (refer to the instructions for preparation). After preparation, mix well and immediately add it between two glass plates (7-8 mL of lower layer gel is usually added for 1.5 mm thick glass plates). Then carefully add 1 mL of isopropanol on the lower layer gel and press it flat. Let it stand at room temperature until it solidifies. (2) Preparing the top gel: After the bottom gel solidifies, discard the isopropanol directly, wash the gel with double-distilled water to remove unpolymerized acrylamide, and blot away any remaining moisture with filter paper. The top gel preparation method follows the instructions for use with the gelling agent. After preparation, mix well and immediately add the gel between two glass plates (2 mL of top gel is typically added to a 1.5 mm thick glass plate). Immediately insert a clean, anhydrous Teflon comb to avoid air bubbles, and allow it to solidify at room temperature. After the top gel solidifies, carefully remove the comb and fix the prepared gel onto the electrophoresis tank. Fill both the inner and outer tanks with Tris-glycine electrophoresis buffer. (3) Sample loading: Take out the protein sample stored at -20℃ and load 20 μL per well (total protein amount per well is 40 μg). Use the pre-stained protein marker as a reference. (4) Electrophoresis: Electrophoresis at 80 V until the sample enters the separating gel, then adjust to 120 V until the bromophenol blue indicator reaches the bottom of the gel; (5) Transfer: The protein was transferred to a 0.22 μm PVDF membrane using the wet transfer method. After gel running, the gel was removed, and a PVDF membrane of similar size was cut. The PVDF membrane was then activated in methanol for 10 s. Then, two layers of filter paper, the gel, the PVDF membrane, and two more layers of filter paper were placed between the plastic clips in the order of "black gel, white membrane," avoiding air bubbles between the gel and the PVDF membrane. The plastic clips were carefully placed into the electrophoresis tank, and transfer buffer (prepared according to the instructions) was added until it covered the top edge of the gel. Transfer conditions: 400 mA constant current transfer for 90-120 minutes. After transfer, the membrane was washed twice with TBST buffer for 5 minutes each time. (6) Blocking: After the transfer is completed, wash the PVDF membrane twice with TBST buffer, then block it with 5% skim milk at room temperature for 2 hours, and wash the membrane twice with TBST buffer for 5 minutes each time; (7) Primary antibody incubation: Add primary antibody dilution buffer and incubate overnight on a shaker at 4°C. The primary antibody concentrations are: α-SMA (ab7817, 1:1000), Col1 (ab34710, 1:1000), Col3 (GB111629, 1:1000), STAT3 (9139S, 1:1000), p-STAT3 (9145S, 1:1000), SOCS3 (2923S, 1:500). The next day, wash the membrane three times with TBST buffer for 10 min each time. (8) Incubation with secondary antibody: Add HRP-labeled secondary antibody (1:5000, Xavier), incubate at room temperature for 1 h, wash the membrane 3 times with TBST buffer, 10 min each time; (9) Add ECL chemiluminescent developing solution and acquire protein band images using a Tanon-5200 multi-functional imaging system. Quantitative analysis of the grayscale values ​​of the analyzed bands is performed using ImageJ software.

[0051] 4. CCK8 assay for cell proliferation Two hours before the end of the PAAF / MSC transwell co-culture system, aspirate the existing culture medium from the wells. Then, add pre-prepared fresh culture medium containing CCK8 reagent (medium:CCK8 = 10:1) to each well, avoiding air bubbles during addition. A blank control group containing only culture medium and CCK8 reagent should be included. Incubate the culture plate at 37°C in a 5% CO2 incubator for 1-4 hours; the incubation time can be adjusted according to the cell condition. After incubation, measure the absorbance (OD value) of each well at 450 nm using a microplate reader. The cell proliferation rate is calculated using the formula: (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0052] 5. EdU assay for cell proliferation Two hours before the end of the PAAF / MSC Transwell co-culture, EdU working solution was added to the culture medium to a final concentration of 10 μM, and the cells were incubated at 37°C for another 2 hours to allow EdU to fully integrate into the DNA synthesis strand of cells in S phase. After incubation, the culture medium was discarded, and cells were fixed with 4% paraformaldehyde solution at room temperature for 30 min, followed by cell membrane permeabilization with 0.3% Triton X-100 for 10 min. Click reaction mixture was prepared according to the kit instructions, added to wells, and incubated in the dark at room temperature for 30 min. After incubation, unbound dye was thoroughly washed with PBS buffer to remove unbound dye, and DAPI was added for nuclear counterstaining for 10 min. Finally, images were observed and acquired under a fluorescence microscope. Five non-overlapping fields of view were randomly selected for image analysis using ImageJ software. During the experiment, care was taken to avoid light exposure.

[0053] Western blot results showed that after stimulation with 10 ng / mL TGF-β1 for 48 h, the expression levels of myofibroblast marker α-SMA and ECM components Col1 and Col3 in PAAF were significantly increased. P <0.05, Figure 4 (B and D). After co-culture with MSCs, the expression of these indicators was significantly reduced ( P <0.05, Figure 4 (B and D). Immunofluorescence staining results further confirmed this finding: the TGF-β1 stimulation group showed obvious α-SMA expression and collagen deposition (Col1 and Col3), while these pathological changes were significantly reduced in the MSC co-culture group. P <0.05, Figure 4 (C and E). EdU proliferation assay and CCK8 assay revealed that although TGF-β1 stimulation significantly increased the proliferation rate of PAAF, no statistically significant difference in proliferation activity was observed between the two groups after MSC co-culture. Figure 4 These results indicate that MSCs significantly inhibit TGF-β1-induced PAAF activation and ECM deposition through paracrine mechanisms, but do not affect PAAF proliferation activity.

[0054] Example 4 Tissue and cell transcriptome sequencing and bioinformatics analysis RNA concentration and purity for sequencing were determined using Nanodrop, ensuring an A260 / A280 ratio > 1.8. RNA integrity was checked using an Agilent 2100 Bioanalyzer, requiring a RIN value > 8.0. Library construction was performed using the mRNA Library Prep Kit, including mRNA purification, fragmentation, cDNA synthesis, end repair, A-tailing, adapter ligation, and PCR amplification. The constructed library was sequenced at 150 bp paired ends. After quality control of the sequencing data, differential expression analysis was performed using DESeq2, selecting genes with |log2FC| > 1 and FDR < 0.05. GO functional annotation and KEGG pathway enrichment analysis were performed on differentially expressed genes to reveal their biological functions and involved metabolic pathways.

[0055] Through pulmonary artery tissue ( Figure 5 (A) and original PAAF ( Figure 5 Transcriptome sequencing analysis of MSCs revealed that MSC treatment significantly upregulated the cytokine-cytokine receptor interaction pathway. Notably, the expression of SOCS3, a key negative regulator in this pathway, was significantly upregulated. At the cellular level, MSC co-culture significantly upregulated SOCS3 expression and downregulated STAT3 phosphorylation levels. P <0.05, Figure 5 (CE). Animal experiments further validated this mechanism: compared with the MCT model group, the number of SOCS3-expressing cells in the pulmonary artery adventitia was significantly increased after MSC treatment (P <0.05, Figure 5 In G and I), the number of cells expressing p-STAT3 was significantly reduced ( P <0.001, Figure 5 (F and H). These results reveal the molecular mechanism by which MSCs activate SOCS3 expression through paracrine action, thereby inhibiting STAT3 hyperphosphorylation and blocking PAAF activation and abnormal ECM deposition. This finding not only further elucidates the mechanism of action of MSCs in treating PAH, but also provides a new potential target for the treatment of PAH.

[0056] As can be seen from the above, SOCS3 can serve as a biomarker for evaluating MSC inhibition of PAAF activation and for the treatment of PAH, providing a new potential target for the treatment of PAH; by activating SOCS3 expression, STAT3 hyperphosphorylation can be inhibited, thereby blocking PAAF activation and abnormal ECM deposition.

[0057] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting SOCS3 expression level in the preparation of a product for evaluating the therapeutic effect of mesenchymal stem cells on pulmonary arterial hypertension.

2. Use according to claim 1, characterized in that, The evaluation of the therapeutic effect of mesenchymal stem cells on pulmonary arterial hypertension comprises evaluation of the therapeutic effect of mesenchymal stem cells on pulmonary arterial hypertension vascular extracellular matrix remodeling.

3. Use according to claim 2, characterized in that, The pulmonary arterial hypertension vascular extracellular matrix remodeling comprises pulmonary arterial hypertension vascular extracellular matrix remodeling induced by monocrotaline.

4. Use according to claim 2, characterized in that, The pulmonary arterial hypertension vascular extracellular matrix remodeling comprises abnormal deposition of collagen in pulmonary arterial hypertension vessels; the collagen comprises Collagen 1 and Collagen 3.

5. Use of a reagent for detecting SOCS3 expression level in the preparation of a product for evaluating the effect of mesenchymal stem cells on inhibiting pulmonary arterial adventitial fibroblast activation.

6. Use according to claim 5, characterized in that, The pulmonary arterial adventitial fibroblast activation comprises TGF-β1-induced pulmonary arterial adventitial fibroblast activation.

7. The use according to any one of claims 1 to 6, characterized in that, The product comprises a kit. The reagent for detecting SOCS3 expression level comprises an anti-SOCS3 antibody.

8. Use of SOCS3 and / or a reagent for up-regulating SOCS3 expression in the preparation of a drug for treating pulmonary arterial hypertension and / or inhibiting pulmonary arterial adventitial fibroblast activation.

9. Use according to claim 7, characterized in that, The reagent for up-regulating SOCS3 expression comprises mesenchymal stem cells.

10. Use according to claim 7, characterized in that, The SOCS3 and / or the reagent for up-regulating SOCS3 expression treat pulmonary arterial hypertension and / or inhibit pulmonary arterial adventitial fibroblast activation by down-regulating STAT3 phosphorylation level.

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