Application of MUC1-C segment in the preparation of drugs for the prevention or treatment of pulmonary hypertension

Abstract

The present invention provides the application of the MUC1-C segment in the preparation of drugs for the prevention or treatment of pulmonary hypertension, and belongs to the field of biomedicine technology. The present invention finds that knocking out the MUC1 gene can significantly alleviate the onset of two pulmonary hypertension models MCT / SuHx, and the synthetic MUC1-C inhibitor GO-201-acetate is injected in vivo for MCT-PH modeling. It is found that compared with animals injected with MCT alone, the MCT+GO-201 group of animals has significantly improved right ventricular systolic pressure, pulmonary vascular hypertrophy, vascular fibrosis, and cardiac function. That is, the present invention confirms that knocking out the MUC1 full-segment gene and synthesizing the inhibitor GO-201-acetate can inhibit the function of the MUC1-C segment and reduce the risk of pulmonary hypertension. Therefore, MUC1 and its fragment MUC1-C can become potential therapeutic targets for pulmonary hypertension and can be applied to the research and development of new drugs for the prevention and treatment of pulmonary hypertension.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of the MUC1-C segment in the preparation of drugs for preventing or treating pulmonary hypertension. Background Art

[0002] Pulmonary hypertension (PH) can occur in all age groups and is clinically divided into five categories. The global prevalence is about 1%, and the prevalence and mortality of PH are on the rise. The pathogenesis of pulmonary hypertension is complex. Perivascular inflammatory response is an important part of the pathogenesis of PH and is closely related to pulmonary hemodynamics, vascular remodeling and clinical prognosis.

[0003] At present, the treatments for lowering pulmonary artery pressure include calcium channel blockers, NO / cGMP pathway activators (sildenafil, tadalafil, riociguanide, etc.), prostacyclin pathway activators (epoprostenol, treprostinil, etc.), and endothelin pathway antagonists (bosentan, ambrisentan). Targeted therapy is mainly aimed at dilating pulmonary vasculature, but the pathogenesis of PH is complex, and only less than 10% of PH patients have vasoconstriction as the main pathophysiological feature. Therefore, these drug treatments can alleviate some symptoms and reduce the number of hospitalizations, but they cannot cure the disease, cannot reduce the overall mortality rate, and most of them are very expensive. Therefore, it is very necessary and of great significance to further deepen the understanding of the pathogenesis of pulmonary hypertension and find new targets and treatment strategies.

[0004] MUC1 is a membrane-localized mucin with the most extensive structural and functional research. It is composed of two heterodimers, MUC1-N segment and MUC1-C segment, which are specifically divided into cytoplasmic segment (CT), transmembrane region (TM) and extracellular region (EC). It transmits signals on the cell surface and affects cell function. In the lungs, MUC1 is mainly distributed in the nasal, pharyngeal and laryngeal epithelial cells of the upper airway, and the trachea, bronchus and alveolar epithelial cells of the lower airway. Various hematopoietic cells such as CD 4+ , CD 8+, Th17 lymphocytes, dendritic cells, lymphocytes, macrophages, etc. also express MUC1. The extracellular region of MUC1 (MUC1-EC) is 200 to 500 nm long and can bind to pathogens to prevent pathogens from invading cells. Both sialosylated sugar chain antigen (Krebs von den Lungen-6, KL-6) and CA15-3 are MUC1-EC after shedding, which can be seen in the alveolar lavage fluid or serum of patients with many lung diseases such as asthma, chronic obstructive pulmonary disease, lung cancer, especially interstitial lung disease. More functions of MUC1 occur through its intracellular segment (cytoplasmictail, CT). MUC1-CT has potential phosphorylation sites and can interact with many pathways such as EGFR, MAPK, PI3K / AKT, PDGFRβ, FGFR, Met, ErbB1-4, β-catenin, etc., to produce many functions such as anti-inflammatory, tumor metaplasia promotion, and fibrosis promotion.

[0005] There are few studies on the effect of MUC1 on pulmonary hypertension. One study showed that KL-6 (MUC1-N segment) was elevated in the plasma of patients with type 1 arterial pulmonary hypertension (PAH) and type 3 pulmonary hypertension (pulmonary hypertension associated with lung disease, PPH), and the KL-6 level in PAH patients was correlated with pO2 (r = -0.91), pCO2 (r = 0.80), PAPs (r = 0.82) and CD40L (r = 0.72); another study reported that KL-6 was significantly elevated in patients with connective tissue disease-related PH (CTD-PH), but the specific effect was unclear. Therefore, there is no research on the effect of MUC1 or MUC1-C segment on pulmonary hypertension. Summary of the invention

[0006] In view of the above technical problems, the present invention provides for the first time the use of the MUC1-C segment in the preparation of drugs for the prevention or treatment of pulmonary hypertension. The present invention finds that animals with MUC1 gene knockout can significantly alleviate the onset of two pulmonary hypertension (PH) models MCT / SuHx, including: right ventricular systolic pressure, pulmonary vascular hypertrophy, vascular fibrosis, cardiac function, etc., and by in vivo injection of the synthetic MUC1-C inhibitor GO-201-acetate for MCT-PH modeling, it is found that compared with animals injected with MCT alone, the MCT+GO-201 group of animals has significantly improved right ventricular systolic pressure, pulmonary vascular hypertrophy, vascular fibrosis, cardiac function, etc., and there is no statistical difference with the negative control group. The present invention proves that knocking out the entire MUC1 gene and the synthetic inhibitor GO-201-acetate inhibiting the function of the MUC1-C segment can reduce the risk of pulmonary hypertension. MUC1 and its fragment MUC1-C may become potential therapeutic targets for pulmonary hypertension, and the present invention can be applied to the fields of new drug development for the prevention and treatment of pulmonary hypertension.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] One of the purposes of the present invention is to provide the use of MUC1 in the preparation of drugs for preventing or treating pulmonary hypertension.

[0009] Furthermore, the application is specifically the application of the MUC1-C segment in the preparation of drugs for the prevention or treatment of pulmonary hypertension.

[0010] Furthermore, the application is achieved by inhibiting the function of the MUC1-C segment.

[0011] The second object of the present invention is to provide a drug for preventing or treating pulmonary hypertension, wherein the drug contains a MUC1-C segment function inhibitor.

[0012] Furthermore, the MUC1-C segment function inhibitor is GO-201 acetate.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) The present invention is the first to discover the effect of MUC1 on pulmonary hypertension.

[0015] (2) The present invention discovered the changes in MUC1 expression and cellular localization in rats with pulmonary hypertension: in MCT and SuHx model rats, WB detection of lung tissue protein and immunofluorescence staining of lung tissue sections revealed that the expression of MUC1-N and MUC1-C in the lung tissue of the SuHx model was upregulated, while in the lung tissue sections of the two pulmonary hypertension models, the MUC1-C segment was mainly expressed in AT2 cells.

[0016] (3) The present invention is the first to use GO-201-acetate (rather than TFA salt) for in vivo experiments.

[0017] (4) The present invention is the first to study the effects of MUC1 knockout and the use of MUC1-C inhibitors to protect pulmonary hypertension: in MCT and SuHx model rats, it was found that the pulmonary hypertension phenotype was significantly alleviated after MUC1 knockout, and the use of the MUC1-C inhibitor GO-201-acetate also had a significant improvement effect on the onset of MCT-induced pulmonary hypertension and increased the survival rate. Almost all pulmonary hypertension phenotype evaluation indicators showed no statistical difference from the CTL group, indicating that the improvement effect was extremely significant.

[0018] In summary, the present invention discovered for the first time that MUC1 can be used as a potential therapeutic target for pulmonary arterial hypertension, especially targeting the MUC1-C segment, which is more accurate and effective, providing new ideas for the research and development of new drugs for the prevention and treatment of pulmonary arterial hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The cellular localization and expression changes of MUC1 in the perivascular and alveolar regions in Example 1 of the present invention are shown, wherein A: immunofluorescence staining of MUC1-N, MUC1-C and SPC in lung tissue sections of the MCT / SuHx-PH model; B: WB experiment to detect the expression of MUC1-N and MUC1-C in lung tissue of the SuHx-PH model; C: gray value statistical graph of WB results; D: staining of MUC1-N and MUC1-C in lung vessels of the SuHx-PH model lung tissue; n=4; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by ANOVA;

[0020] Figure 2 MUC1 in Example 1 of the present invention - / - The reduction of pulmonary hypertension in MCT, including: A: Right ventricular pressure (RVP) curve of MCT-PH model; B: Statistical graph of right ventricular systolic pressure (RVSP) of each group; C: HE staining images of lung sections of each group; D: Statistical graph of pulmonary artery wall thickness of each group; E: HE staining image of heart sections of each group; F: Statistical graph of right ventricular hypertrophy index (RVHI); n=6-12; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001by ANOVA;

[0021] Figure 3 MUC1 in Example 1 of the present invention - / - The improvement of SuHx pulmonary hypertension, including: A: right ventricular pressure (RVP) curve of SuHx-PH model; B: right ventricular systolic pressure (RVSP) statistics of each group; C: HE staining of rat heart sections; D: right heart free wall thickness statistics; E: right heart hypertrophy degree statistics; F: HE staining images of lung sections of each group; G: pulmonary artery wall thickness statistics of each group; H: Masson staining of rat lung tissue sections; I: WB results of COL1 and FN expression of rat lung tissue protein; J: B-ultrasound detection of rat PAT and PET waveform; K: PAT / PET ratio statistics; L: right ventricular area change fraction statistics; M: rat tricuspid annular systolic displacement statistics; n=5-8; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001vs Muc1+ / +CTL group, #vs Muc1+ / +SuHx group, $vsMuc1- / -CTL group by ANOVA;

[0022] Figure 4 This is the MS report of the conversion of GO-201 to acetate in Example 1 of the present invention;

[0023] Figure 5 This is the HPLC report of the conversion of GO-201 to acetate in Example 1 of the present invention;

[0024] Figure 6 The COA report of GO-201 converted to acetate in Example 1 of the present invention;

[0025] Figure 7 The figure shows the protection of MCT pulmonary hypertension by MUC1-C inhibitor in Example 1 of the present invention, wherein A: survival curve of rats in each group; B: right ventricular pressure (RVP) curve of SuHx-PH model; C: statistical graph of right ventricular systolic pressure (RVSP) in each group; D: statistical graph of right heart hypertrophy degree; E: PAT and PET waveform of rats detected by B-ultrasound; F: CO waveform of rats detected by B-ultrasound; G: statistical graph of PAT / PET ratio; H: statistical graph of right ventricular free wall diastolic thickness; I: statistical graph of right ventricular free wall systolic thickness; J: statistical graph of cardiac output; K: statistical graph of rat tricuspid annular systolic displacement; n=5-8; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 vs CTL group, # vs MCT group by ANOVA. DETAILED DESCRIPTION

[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention all belong to the scope of the present invention. The reagents and instruments used in the following examples can all be obtained commercially, and the methods used in the examples are consistent with the conventional methods unless otherwise specified.

[0027] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0028] Example 1

[0029] 1 Experimental methods

[0030] 1.1MUC1 - / - Rat construct

[0031] F0 generation Muc1 was obtained from Saiye Biotechnology Co., Ltd. + / - / SD rats, one male and one female, were constructed by microinjection of TALEN into fertilized eggs. The rats were bred and weaned at 3 weeks of age. The pups were separated into cages according to gender at 4 weeks of age. The rat tail tissues were taken to extract DNA. The rat genotypes were identified according to sequencing results after PCR amplification. Muc1 - / - / SD rats, treated with Muc1 - / - / SD male rats and Muc1 - / - / SD female rats were mated in a 1:2 ratio to breed offspring. During the breeding process, the pregnancy cycle, litter size, offspring survival rate, and growth and development status of the rats were observed. A new Muc1 was introduced every 4 generations. + / + / SD rats to ensure their closed group background.

[0032] 1.2 Preparation of GO-201-acetate

[0033] Currently, all products sold on the market are GO-201-TFA. However, due to the toxicity of TFA in vivo, we commissioned Nanjing Peptide Biotechnology Co., Ltd. to synthesize GO-201-acetate lyophilized powder to avoid the cytotoxicity of TFA. It is frozen at -20°C and dissolved in PBS for immediate use.

[0034] 1.3 Construction of MCT pulmonary hypertension model

[0035] The experiment selected 6-8 week old male wild type (MUC1 + / + ) and knockout (MUC1 - / - ) SD rats were treated with monocrotaline (MCT) freeze-dried powder dissolved in PBS to 4 mg / ml, and injected with 50 mg / kg once. The rats were then fed for 28 days and divided into 4 groups: MUC1 + / + CTL group, MUC1 + / + MCT group, MUC1 - / - CTL group, MUC1 - / - MCT group.

[0036] In addition, the MUC1-C segment inhibitor GO-201-acetate (Nanjing Peptide Biotechnology Co., Ltd., NJP25118) was used for in vivo intervention. Wild-type SD rats were intraperitoneally injected with 15 mg / kg / day of GO-201-acetate and a 50 mg / kg dose of MCT-PH model was established. The rats were divided into three groups: CTL group, MCT group, and MCT+GO-201 group.

[0037] After 28 days, the successful establishment of the model was evaluated based on indicators such as the mean pulmonary artery pressure, right ventricular systolic pressure, right ventricular hypertrophy index, and pulmonary vascular wall thickness, as well as the therapeutic effects of the MUC1- / -MCT group and the MCT+GO-201 group.

[0038] 1.4 Construction of SuHx pulmonary hypertension model

[0039] The PH model induced by SU5416 combined with hypoxia (SuHx-PH) was divided into 4 groups: MUC1 + / + CTL group, MUC1 + / + SuHx group, MUC1 - / - CTL group, MUC1 - / - SuHx group, 6-8 week old male wild type (MUC1 + / + ) and knockout (MUC1 - / - ) A single subcutaneous injection of SU5416 (20 mg / kg) was used, and the mice were placed in a hypoxic box with a 10% oxygen concentration for 3 weeks of hypoxia exposure, and then restored to normoxia for 3 weeks. The mean pulmonary artery pressure, right ventricular systolic pressure, right ventricular hypertrophy index, pulmonary vascular wall thickness and other indicators were used to evaluate whether the model was successfully established. And the treatment effect of the MUC1- / -SuHx group was evaluated.

[0040] 1.5 Measurement of right ventricular systolic pressure in rats

[0041] After isoflurane-induced anesthesia of the experimental rats, a Millar catheter was inserted into the jugular vein and then into the right ventricle. The pressure signal was connected to the computer of the small animal physiological instrument to record the right ventricular pressure waveform. The stable waveform was maintained for at least 1 minute. The average right ventricular systolic pressure (RVSP) was obtained by calculating the stable waveform of the right ventricular pressure for at least 30 seconds.

[0042] 1.6 Measurement of cardiac function by color Doppler ultrasound in small animals

[0043] After isoflurane-induced anesthesia, the experimental rats were fixed on the ultrasound workbench, and ultrasound coupling agent was applied to the chest. The color Doppler ultrasound images of the rats' hearts were recorded. Vevo2100 and M250 (13-24MHz) ultrasound probes were used to perform a series of evaluations on the left and right hearts: cardiac output (CO), pulmonary artery blood flow acceleration time to pulmonary artery ejection time ratio (PAT / PET), right ventricular area change fraction (RV FAC%), tricuspid annular systolic displacement (TAPSE), right ventricular end-systolic free wall thickness (RVEDWT) and right ventricular end-diastolic free wall thickness (RVESWT). The influence of breathing should be excluded during the measurement, and at least 3 consecutive cardiac cycles should be recorded and the average value should be taken.

[0044] 1.7 Pathological examination of rat lung tissue

[0045] 10% formalin was perfused through the airway until the lung lobe expanded, and then the lung tissue was dissected and fixed in 10% formalin buffer for 24 hours. The fixed lung tissue was embedded in paraffin blocks and stained with hematoxylin-eosin after sectioning. The morphology of lung tissue, the degree of pulmonary vascular hypertrophy, and the degree of inflammation were observed under a light microscope. Some sections were stained with Masson to observe the collagen deposition of lung tissue and the fibrosis around the pulmonary vessels.

[0046] 1.8 Determination of right heart hypertrophy index

[0047] After the pathological examination of the experimental SD rats, the heart was separated, and the right ventricular wall (RV) was separated from the pulmonary artery outlet. The remaining parts were the left ventricle (LV) + interventricular septum (S); the left ventricle was opened, the blood clot was washed, and the water was sucked out and weighed separately. The right heart hypertrophy index (Fulton index) = RV / (LV+S).

[0048] 1.9 Immunofluorescence staining of lung tissue pathological sections:

[0049] Paraffin sections were sequentially dehydrated with ethanol and dewaxed with xylene; antigen retrieval and blocking with goat serum for 20 minutes at room temperature; primary antibody was added and incubated at 4°C overnight, washed three times with PBS, and fluorescein-labeled secondary antibody was added and incubated at 37°C for 30 minutes; washed three times with PBS, and sealed with DAPI fluorescence quencher.

[0050] 1.10 Immunohistochemical staining of lung tissue pathological sections

[0051] Dewaxing, antigen repair, 3% hydrogen peroxide to block endogenous peroxidase, add goat serum blocking solution, incubate at room temperature for 30 minutes; shake off excess liquid, add primary antibody at 4°C overnight, wash three times with PBS, add biotinylated secondary antibody, incubate at 37°C for 20 minutes; wash three times with PBC, add reagent SABC, incubate at room temperature for 20 minutes; wash three times with PBS; develop color with DAB; counterstain with hematoxylin for 2 minutes, differentiate with hydrochloric acid and alcohol, dry, seal and examine under a microscope.

[0052] 1.11RT-qPCR

[0053] Total RNA from cells or tissue samples was extracted by Trizol method, and RNA concentration was determined by NANO 2000 (Thermo, USA). The miRcute miRNAcDNA First-strand cDNA Synthesis Kit (KR211, TIANGEN, China) was used to prepare miRNAcDNA template. The miRcute miRNA Fluorescence Quantification Detection Kit (FP411, TIANGEN, China) was used for amplification with the following parameters: 94℃ denaturation for 2min, 94℃ amplification for 20s, 60℃ amplification for 30s, and 72℃ amplification for 30s, for a total of 45 cycles, and then signal scanning was performed. The internal reference U6 and microRNAs primers were provided by Genecopoeia (Guangzhou, China). The relative expression of microRNA was calculated according to the 2-△△ct formula.

[0054] 1.12 Western blotting

[0055] Rat lung tissue was obtained and homogenized with RIPA lysis buffer, and the protein concentration was determined by BCA protein concentration determination kit. Total protein was extracted from cells and pulmonary artery smooth muscle tissue, separated by SDS-polyacrylamide gel electrophoresis, and then electrotransferred to nitrocellulose membrane. After blocking the nonspecific binding sites on the membrane with skim milk, affinity-purified corresponding specific antibodies were added in sequence and incubated overnight at 4°C, TBST buffer was shaken quickly 3 times, 5 minutes each time, and peroxidase-coupled anti-rabbit or anti-mouse IgG was added and incubated at room temperature for 60 minutes. After incubation, TBST buffer was quickly washed 3 times, 5 minutes each time, and peroxidase substrate ECL was added after washing. The secondary antibody signal was detected by chemiluminescence imaging, and the grayscale analysis of the target band was performed using Image J software.

[0056] 1.13 Statistical analysis

[0057] All experimental results were expressed as mean x ± SEM, and N represented the number of independent animals or the number of experiments in each experiment. Two independent sample t-tests were used to compare the count data of two groups of samples; one-way analysis of variance (ANOVA) was used to compare the count data of more than two groups of samples. P < 0.05 (two-sided P value) was considered statistically significant.

[0058] 2 Experimental results

[0059] 2.1 Detection of expression changes and cellular localization of MUC1 and its different fragments in PH

[0060] Western blot was used to detect the protein changes of MUC1-N and MUC1-C segments in lung tissues of PH models. The results are as follows Figure 1 shown.

[0061] The results showed that the expression of MUC1-N and MUC1-C was upregulated in SuHx-PH, while MUC1-C was mainly expressed in AT2 by immunofluorescence staining of lung tissue ( Figure 1 ).

[0062] 2.2MUC1- / - protects against the onset of MCT-PH

[0063] The evaluation results of rat survival rate, right ventricular systolic pressure, vascular remodeling, cardiac function, etc. are as follows Figure 2 shown.

[0064] The results showed that the rats in the MUC1- / -MCT group showed a very significant therapeutic effect ( Figure 2 ), so knocking out the MUC1 gene can effectively improve MCT-induced pulmonary hypertension.

[0065] 2.3MUC1- / - protects against the onset of SuHx-PH

[0066] The evaluation results of rat survival rate, right ventricular systolic pressure, vascular remodeling, cardiac function, etc. are as follows Figure 3 shown.

[0067] The results showed that the rats in the MUC1- / -SuHx group also showed a very significant therapeutic effect. + / + CTL group, MUC1 - / - There was no statistical difference in the CTL group ( Figure 3 ), so knocking out the MUC1 gene can significantly improve SuHx-induced pulmonary hypertension.

[0068] 2.4 MUC1-C inhibitor GO-201 acetate conversion

[0069] Currently, GO-201 in the market is basically purified by TFA salt, but TFA has certain biological toxicity. Therefore, the present invention specifically uses GO-201 converted to acetate, and its MS report, HPLC report and COA report are as follows: Figures 4 to 6 shown.

[0070] Figures 4 to 6 It is shown that the MUCl-C inhibitor used in the present invention greatly reduces the TFA content and reduces possible toxic side effects.

[0071] 2.5 MUC1-C inhibitor GO-201 significantly improves MCT-PH

[0072] The comparison results of survival rate, right ventricular systolic pressure, and cardiac function in the MCT+GO-201 group and the MCT group were as follows: Figure 7 shown.

[0073] Depend on Figure 7 It can be seen that the survival rate, right ventricular systolic pressure, and cardiac function of the MCT+GO-201 group improved significantly, and almost all evaluation indicators were not statistically different from those of the CTL group. This shows that inhibiting the function of the MUC1-C segment is very effective in preventing and treating pulmonary hypertension, and has a more precise targeted therapeutic effect than the entire segment ( Figure 7 ).

[0074] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of MUC1 in the preparation of a drug for the prevention or treatment of pulmonary hypertension, characterized in that: The drug is a MUC1 full-segment gene knockout reagent or a MUC1-C segment inhibitor; the MUC1-C segment inhibitor is GO-201-acetate.

2. A drug for preventing or treating pulmonary hypertension, characterized in that: The drug is the MUC1-C segment inhibitor GO-201-acetate used in claim 1.

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