Use of scEMC10 in the preparation of a medicament for treating sepsis

Abstract

The application provides a use of scEMC10 in preparation of a medicine for treating sepsis, and relates to the field of biomedical technology. The inventor finds that after LPS-stimulated brain microvascular endothelial cells are treated by scEMC10, a plurality of gene mRNA transcriptions related to EndMT and classic EndMT markers (such as FN1, alpha-SMA, ZEB1, TWIST1 and Snail) induced by LPS can be down-regulated; after a LPS-induced sepsis mouse model is supplemented with scEMC10, the expression of EndMT markers of the brain, liver and kidney can be obviously improved, the bleeding of the brain, lung, liver and kidney of the mouse caused by LPS induction can be obviously inhibited, the survival rate of the mouse is improved, the movement ability of the mouse is improved, and the anxiety level of the mouse is reduced. Therefore, scEMC10 can be used for preparing a medicine for treating sepsis and inhibiting EndMT.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of scEMC10 in the preparation of a medicament for treating sepsis. Background Technology

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection. Its core pathology includes uncontrolled inflammatory response, immune dysfunction, microcirculatory disturbances, and multi-organ damage. Currently, no effective drugs have been successfully developed for the targeted treatment of sepsis. Therefore, the search for new prevention and treatment drugs has significant scientific and clinical value.

[0003] Transforming growth factor β (TGF-β) superfamily signal transduction plays a crucial role in regulating cell growth, differentiation, and development in numerous biological systems. Generally, signal transduction initiates ligand-induced oligomerization of serine / threonine receptor kinases and phosphorylation of cytoplasmic signal transduction molecules. These molecules are Smad2 and Smad3 in the TGF-β / activin pathway, and Smad1 / 5 / 9 in the bone morphogenetic protein (BMP) pathway. Currently, 33 TGF-β family proteins are known to exist in humans, including 3 TGF-β proteins (TGF-β1 / 2 / 3), 10 BMPs (Bone Morphogenetic Proteins), and 11 GDFs (Growth and Differentiation Factors, where GDF2 = BMP9 and GDF8 = myostatin), as well as activin, nodal, inhibin, and AMH / MIS, all of which are dimer, secreted polypeptides. Currently, the effects of this pathway vary greatly and are even opposite in different cells and tissues, but the specific mechanism is still unclear, and there are no reports on its use in treating sepsis.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The primary objective of this invention is to provide the application of scEMC10 in the preparation of medicaments for treating sepsis, thereby providing a new method for the treatment of sepsis.

[0006] A second objective of this invention is to provide the use of scEMC10 in the preparation of medicaments that inhibit endothelial-mesenchymal transition (EndMT).

[0007] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides the use of scEMC10 in the preparation of a medicament for treating sepsis.

[0008] As a further technical solution, the treatment of sepsis includes treating tissue damage or tissue fibrosis caused by sepsis.

[0009] As a further technical solution, the tissue includes one or more of the following: brain microvascular endothelial cells, lungs, liver, or kidneys.

[0010] As a further technical solution, the drug also includes pharmaceutical excipients.

[0011] As a further technical solution, the dosage form of the drug includes an injection.

[0012] Secondly, this invention provides the application of scEMC10 in the preparation of drugs that inhibit endothelial-mesenchymal transition.

[0013] As a further technical solution, the drug also includes pharmaceutical excipients.

[0014] As a further technical solution, the dosage form of the drug includes an injection.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The inventors discovered that LPS-stimulated brain microvascular endothelial cells treated with scEMC10 downregulated LPS-induced EndMT-related gene mRNA transcription and classic EndMT markers (such as Fibronectin, α-SMA, ZEB1, TWIST1, and Snail). In an LPS-induced sepsis mouse model, reintroduction of scEMC10 significantly improved the expression of EndMT markers in the brain, liver, and kidneys, significantly inhibited LPS-induced hemorrhage in the brain, lungs, liver, and kidneys, improved mouse survival, enhanced motor function, and reduced anxiety levels. Therefore, scEMC10 can be used to prepare drugs for treating sepsis and inhibiting EndMT. Attached Figure Description

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0017] Figure 1A. Inhibition of scEMC10 on LPS-induced acute sepsis-induced cerebral hemorrhage. The left figure shows the cerebral hemorrhage results (EB perfusion), and the right figure shows the statistical results of EB leakage. Mouse number = 6. Statistical analysis was performed using an unpaired t-test. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001; B. ELISA detection results of inflammatory factors IL-1β, IL-6, and TNF-α in brain tissue after PBS perfusion. Mouse number = 8. Statistical analysis was performed using an unpaired t-test. ***: p<0.001, ****: p<0.0001; C. ELISA detection results of IL-1β, IL-6, and TNF-α in mouse serum. Mouse number = 8. Statistical analysis was performed using an unpaired t-test. ***: p < 0.001, ****: p < 0.0001; D. Immunofluorescence results of leakage of tight junction protein ZO-1 and EB in different brain regions of brain tissue sections after PBS perfusion. Green: ZO-1, Red: EB, Blue: DAPI (nucleus), CTX: Cortex, MID: Midbrain, HIP: Hippocampus; E. Statistical results of ZO-1 and EB. Mouse number = 6. Statistical analysis was performed using an unpaired t-test. ns: no statistical significance, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001; F. Immunofluorescence results of tight junction protein Occludin and endothelial cell marker CD31 in different brain regions of brain tissue sections after PBS perfusion. Green: Occludin, Yellow: CD31, Blue: DAPI (nucleus), CTX: Cortex, MID: Midbrain, HIP: Hippocampus; G. Occludin and CD31 statistical results. Mouse number = 6. Statistical analysis was performed using unpaired t-tests. ns: no statistical significance, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001; Figure 2A. GO analysis of LPS-treated mouse cerebral endothelial cell line bEnd.3 mRNA-seq sequencing results showed that LPS stimulation significantly altered the cell's response to cytokines and induced changes in the expression of cell migration genes. B. MRNA-seq sequencing results of mouse cerebral endothelial cell line bEnd.3 after LPS or scEMC10 treatment showed that LPS (10 mg / kg) significantly increased the transcription of genes related to EndMT, while scEMC10 inhibited the transcription of these genes. C. Proteomic sequencing results of mouse cerebral endothelial cell line bEnd.3 after LPS or scEMC10 treatment showed that LPS (10 mg / kg) significantly increased the expression of EndMT-related proteins, while scEMC10 inhibited their expression. D. Western blotting analysis of EndMT biomarker protein levels in mice treated with LPS or scEMC10. E. Western blotting analysis of EndMT biomarker protein levels. Statistical analysis of blotting results. Number of mice = 6. Statistical analysis was performed using an unpaired t-test. ns: no statistical significance, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001; F. Immunofluorescence results of endothelial cell marker CD31, mesenchymal cell marker α-SMA, and EB leakage in different brain regions of mouse brain tissue sections after PBS perfusion. Green: CD31, red: α-SMA, purple: EB, blue: DAPI (nucleus). The right figure shows the statistical results of α-SMA fluorescence intensity. Number of mice = 4. Statistical analysis was performed using an unpaired t-test. **: p<0.01, ***: p<0.001; Figure 3 A. Heatmap of EndMT-related genes with different expression levels in HCMECs, comparing the results of the PBS-treated group and the scEMC10-treated group. Downregulated mRNA is shown in blue, and upregulated mRNA is shown in red; B. Western blotting results of EndMT marker protein expression levels in bEnd.3 cells after treatment with scEMC10 (100 ng / ml) and / or TGF-β1 (2 ng / ml) for 48 hours. Mouse count = 3. Statistical analysis was performed using unpaired t-tests. ns: no statistical significance, *: p < 0.05, **: p < 0.01, ***: p < 0.001; Figure 4A. LPS (10 mg / kg)-induced septic pulmonary hemorrhage: the left figure shows the cerebral hemorrhage result (EB perfusion), and the right figure shows the statistical results of EB content. Mouse number = 6. Statistical analysis was performed using an unpaired t-test. ns: no statistical significance; **: p < 0.01; ****: p < 0.0001. B. Immunofluorescence analysis showing the LPS (10 mg / kg)-induced interstitial lung lesions (α-SMA) and hemorrhage (EB) in each group. Scale bar, 10 μm. C. Quantitative analysis of EEA1 positive endosomes and intracellular scEMC10-His density. Mouse number = 6. Statistical analysis was performed using an unpaired t-test. **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. E. LPS (10 mg / kg)-induced septic lung weight-to-body weight ratio showed significant recovery in the scEMC10 treatment group. Mouse number = 6. Statistical analysis was performed using unpaired t-tests. ns: no statistical significance; ***: p < 0.001; ****: p < 0.0001. F. The wet-dry weight ratio of sepsis-induced lung tissue induced by LPS (10 mg / kg) showed significant recovery in the scEMC10 pre-treatment group (number of mice = 6). Statistical analysis was performed using unpaired t-tests. ns: no statistical significance; **: p < 0.01; ***: p < 0.001. G. H&E staining results showed that the scEMC10 pre-treatment group significantly improved the alveolar wall thickening symptoms caused by sepsis. The right figure is a magnified view. Scale bar: 150 μm. H. Statistical graph of alveolar wall thickness in the lungs. Number of mice = 6. Statistical analysis was performed using unpaired t-tests. ns: no statistical significance; *: p < 0.05; **: p < 0.01; ***: p < 0.001. I. Statistical analysis of LPS (10 mg / kg)-induced sepsis and lung injury scores after scEMC10 treatment. Mouse number = 6. Statistical analysis was performed using unpaired t-tests. *: p < 0.05; ***: p < 0.001. Figure 5A. Liver H&E staining and Sirius red staining results after LPS (10 mg / kg)-induced sepsis and scEMC10 treatment showed that liver damage significantly improved after scEMC10 treatment, with earlier injection showing better results. Mouse number = 4. Statistical analysis was performed using unpaired t-tests. ns: no statistical significance, *: p<0.05, **: p<0.01, ***: p<0.001; B. Kidney H&E staining and Sirius red staining results after LPS (10 mg / kg)-induced sepsis and scEMC10 treatment showed that kidney damage significantly improved after scEMC10 treatment, with earlier injection showing better results. Mouse number = 4. Statistical analysis was performed using unpaired t-tests. ns: no statistical significance, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001; C. Immunofluorescence analysis showed the LPS (10 mg / kg)-induced septic sinusoidal interstitial lesions (α-SMA) and vascular density (CD31) in each group. Mouse number = 4. Statistical analysis was performed using unpaired t-tests. ns: no statistical significance, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001; D. Immunofluorescence analysis showed the LPS (10 mg / kg)-induced septic glomerular interstitial lesions (α-SMA) and vascular density (CD31) in each group. Mouse number = 4. Statistical analysis was performed using unpaired t-tests. ns: no statistical significance; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. Figure 6A: The survival rate of the septic mouse model significantly improved after treatment with scEMC10 protein, and the EL group was close to that of the PBS group. Number of mice = 8. Statistical analysis was performed using an unpaired t-test, ns: no statistical significance, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001; B: The body temperature recovery rate of the septic mouse model after treatment with scEMC10 protein was significantly higher than that of the LPS group. C: The food intake recovery rate of the septic mouse model after treatment with scEMC10 protein was significantly higher than that of the LPS group. Number of mice = 8. Statistical analysis was performed using an unpaired t-test, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001; D: The weight recovery rate of the septic mouse model after treatment with scEMC10 protein was significantly higher in the pre-injection group than in the LPS group. Statistical analysis was performed using unpaired t-tests. *: no statistical significance; **: p < 0.01; ****: p < 0.0001. E. Grasping strength test results showed that the gripping strength of the septic mouse model treated with scEMC10 protein was significantly higher than that of the LPS group. Number of mice = 8. Statistical analysis was performed using unpaired t-tests. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. F. Rotamab results showed that the drop time of the septic mouse model at 30 rpm, 35 rpm, and 40 rpm was significantly longer than that of the LPS group after scEMC10 protein treatment. Furthermore, the drop time was longer in the pre-injection group than in the simultaneous injection group. Number of mice = 8. Statistical analysis was performed using unpaired t-tests. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. Open field assays showed that treatment with scEMC10 protein significantly improved anxiety behavior and motor function in the septic mouse model, with more pronounced improvements in the pre-injection group. Mouse count = 8. Statistical analysis was performed using unpaired t-tests. ns: no statistical significance, *: p < 0.05, **: p < 0.01, ****: p < 0.0001. Detailed Implementation

[0018] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0019] In a first aspect, the present invention provides the use of scEMC10 in the preparation of a medicament for treating sepsis.

[0020] The inventors have discovered that scEMC10 has a good therapeutic effect on tissue damage (brain microvascular endothelial cells, lungs, liver or kidneys) and fibrosis caused by sepsis. Therefore, scEMC10 can be used to prepare drugs for the treatment of sepsis.

[0021] In some alternative embodiments, the drug may further include pharmaceutical excipients.

[0022] This invention does not impose specific restrictions on pharmaceutical excipients; they can be selected as needed.

[0023] In some alternative embodiments, the dosage form of the drug includes, but is not limited to, injections, or other dosage forms well known to those skilled in the art.

[0024] Secondly, this invention provides the application of scEMC10 in the preparation of drugs that inhibit endothelial-mesenchymal transition.

[0025] The inventors have discovered that scEMC10 can significantly improve the expression of EndMT markers in the brain, liver, and kidneys. Therefore, scEMC10 has an inhibitory effect on EndMT and can act as an inhibitor of EndMT to improve related diseases caused by EndMT.

[0026] In some alternative embodiments, the drug may further include pharmaceutical excipients.

[0027] This invention does not impose specific restrictions on pharmaceutical excipients; they can be selected as needed.

[0028] In some alternative embodiments, the dosage form of the drug includes, but is not limited to, injections, or other dosage forms well known to those skilled in the art.

[0029] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0030] Example 1 1. Experimental Methods 1.1 Cell Transcriptome Sequencing RNA was extracted from the samples using Trizol, followed by RNA extraction according to the instructions of the EZ-press RNA Purification Kit (Ezbioscience, B0004D). RNA integrity and concentration were assessed using a bioanalyzer (NanoDrop ONE, ThermoFisher). Sequencing was performed by Suzhou Genewiz Biotechnology Co., Ltd. Sequencing data quality was assessed using FastQC software (v0.10.1). Library construction was performed using the ScriptSeq Complete Gold Kit (Epicentre, Illumina), and samples were multi-coded and sequenced at 50 bp paired ends on an Illumina HiSeq2500. Eight samples were used per lane, and each sample was sequenced twice at a depth of 50 million reads.

[0031] 1.2 Immunocytochemistry All solutions were prepared in PBS (GIBCO, Cat#10-010-023). At specified time points, samples were fixed with 4% paraformaldehyde (Thermo Scientific, Cat#28906) for 15 minutes. Subsequently, the free aldehyde group was treated with 0.1 M glycine and washed three times with PBS. After infiltration with 0.3% Triton X-100 for 20 minutes, cells were washed three more times with PBS. Cells were then incubated with blocking solution (10% goat serum (Beyotime, Cat#C0265)) for 30 minutes. Primary antibodies (His-tag 1:100, EEA11:100, CD31 1:100, α-SMA 1:100) were added to 10% blocking solution for 1 hour. After rinsing four times in PBS, cells were incubated with appropriate Alexa Fluor secondary antibodies (anti-mouse 488; anti-rabbit 594, 1:500) for 30 minutes. Cells were then reverse-stained with 40,6-diamino-2-phenylindophenol (DAPI, 0.5 mg / ml, Thermo Scientific, Cat#62248) for 10 minutes. Finally, the cells were washed three times in PBS and then mounted onto microscope slides using Fluoromount (Sigma, Cat#F4680-25). Images were analyzed using ImageJ analysis software (NIH, USA). To quantify fluorescence intensity, the average gray value of the region of interest was measured and the background was subtracted to obtain the fluorescence intensity. High-resolution imaging was performed using an Airyscan super-resolution confocal microscope with a Leica SP8 confocal microscope, and the images were analyzed using ImageJ analysis software.

[0032] 1.3 Cell Transcriptome Sequencing RNA was extracted from the samples using Trizol, followed by RNA extraction according to the instructions of the EZ-press RNA Purification Kit (Ezbioscience, B0004D). RNA integrity and concentration were assessed using a bioanalyzer (NanoDrop ONE, ThermoFisher). Sequencing was performed by Suzhou Genewiz Biotechnology Co., Ltd. Sequencing data quality was assessed using FastQC software (v0.10.1). Library construction was performed using the ScriptSeq Complete Gold Kit (Epicentre, Illumina), and samples were multi-coded and sequenced at 50 bp paired ends on an Illumina HiSeq2500. Eight samples were used per lane, and each sample was sequenced twice at a depth of 50 million reads.

[0033] 1.4 Protein extraction, SDS-PAGE and Western blotting Mouse cerebral cortical tissue was sonicated in RIPA buffer (pH 7.4, with added protease and serine / threonine and tyrosine phosphatase inhibitors). The homogeneous mixture was centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was retained for protein analysis. Sample quantification was performed using the BCA protein assay. The sample (25 μg / lane) was mixed with a reducing sample buffer containing SDS and DTT and heated to 95°C for 5 minutes. Proteins of different molecular weights were separated by SDS-PAGE on an appropriately concentrated polyacrylamide gel and transferred to a PVDF membrane. The membrane was blocked in 5% skim milk for 1 hour and then diluted in blocking buffer with primary antibodies (CD105, TGFBR1, TGFBR2, p-TGFBR1, p-PKA Cα, PKA Cα, p-RhoA, RhoA, E-Cadhern, VE-Cadhern, N-Cadhern, COL1A1, Fibronectin, α-SMA, TWIST1, ZEB1, 1:1000; β-Actin, CST, 1:5000) at 4°C. After thorough washing, the membrane was incubated with peroxidase-conjugated secondary antibody at room temperature for 2 hours, thoroughly washed, and rinsed with a chemiluminescent substrate (Thermo Scientific). Density assays were performed using ImageJ (National Institutes of Health) to quantify the protein expressed in each channel.

[0034] 1.5 Establishment of an LPS-induced sepsis mouse model Eight-week-old C57BL / 6J mice, half male and half female, were intraperitoneally injected with LPS 10 mg / kg, and samples were collected 24 hours later. They were divided into three groups: a PBS control group (PBS), an LPS injection group (LPS), a group receiving scEMC10 (0.5 mg / kg) 12 hours before LPS injection (denoted as EL or scEMC10-LPS), a group receiving scEMC10 (0.5 mg / kg) 2 hours after LPS injection (denoted as LE or LPS-scEMC10), and a group receiving scEMC10 (0.5 mg / kg) 12 hours after LPS injection (denoted as LE-12h or LPS-scEMC10-12h).

[0035] 1.6 H&E staining Paraffin sections were first thoroughly dewaxed with xylene, then hydrated with a gradient of ethanol. The cell nuclei were first stained with hematoxylin for 10 minutes; then briefly differentiated with 1% hydrochloric acid ethanol, followed by thorough blueing with running water or a weakly alkaline solution, and counterstained with eosin for 2 minutes until the cytoplasm turned pink. Finally, the sections were dehydrated with a gradient of ethanol, cleared with xylene, and mounted with neutral resin.

[0036] 2. Experimental Results We induced sepsis in mice using LPS (10 mg / kg) and then analyzed the therapeutic effect of scEMC10 (0.5 mg / kg) on ​​tissue damage. The mice were divided into three groups: a PBS control group, an LPS group, a scEMC10 injection group 12 hours prior to LPS (EL), a scEMC10 injection group 2 hours after LPS (LE), and a scEMC10 injection group 12 hours after LPS (LE-12h). In the LPS-induced sepsis mouse model, treatment with recombinant scEMC10 protein significantly reduced cerebral hemorrhage, with the EL group showing the best recovery in hemorrhage. Figure 1 (A) Compared to the LPS group, the expression of inflammatory factors IL-1β, IL-6, and TNF-α in the mouse brain all decreased after scEMC10 treatment, and the decrease was more significant in the pre-injection group. Figure 1 (B in the text). The results in the serum were basically consistent with those in the brain. Figure 1 (C in the middle).

[0037] To evaluate the functional effects of scEMC10 on tight junctions in brain microvascular endothelial cells, we performed immunofluorescence staining on mouse brain tissue sections for tight junction proteins ZO-1, Occludin, and the endothelial cell marker CD31. The results showed that scEMC10 significantly ameliorated the damage to vascular endothelial cell junctions in various brain regions caused by sepsis. Figure 1 (DG in the middle).

[0038] Numerous studies have confirmed that sepsis can induce multi-organ tissue fibrosis through the TGF-β pathway, specifically by TGF-β inducing endothelial-mesenchymal transition (EndMT) under pathological conditions, leading to tissue fibrosis. LPS vs PBS GO analysis of mRNA-seq results from the mouse brain microvascular endothelial cell line bEnd.3 after 48 hours of treatment with PBS or LPS showed that LPS significantly affected the migration of various cell types. Figure 2 (A) The results of mRNA-seq and heatmap of EndMT-related markers in proteomics after treating the mouse brain microvascular endothelial cell line bEnd.3 with PBS or LPS or LPS and scEMC10 for 48 hours also showed that scEMC10 significantly reduced the expression of interstitial markers (Fibronectin (FN1), Collagen (Col5a3, Col4a3, Col22a1, Col18a1, etc.) induced by LPS, and increased the expression of endothelial markers (CD34, CLDN12, etc.). Figure 2 In mice stimulated with PBS, LPS, or LPS followed by scEMC10, Western blot (WB) results showed that sepsis significantly increased the expression of interstitial brain markers (Fibronectin, Collagen, N-CAD, α-SMA, Snail, TWIST1), while the expression of cadherins highly expressed in endothelial cells (E-cadherin, VE-cadherin) decreased. Treatment with scEMC10 significantly reduced this trend. Figure 2 DE in the middle). Immunofluorescence results showed that the EndMT phenomenon occurred in vascular endothelial cells ( Figure 2 (F in the text).

[0039] In some in vitro cultured endothelial cell lines, TGF-β stimulation alone can induce EndMT. We treated the human brain microvascular endothelial cell line HCMEC with scEMC10 (100 μg / ml) for 48 h and then performed mRNA sequencing. An untreated cell line was used as a control group. RNA sequencing analysis showed that after scEMC10 stimulation of HCMEC, EndMT-related mesenchymal marker genes (MMP15, COL4A1, Fibronectin, ZEB1, etc.) were significantly downregulated, while endothelial markers (CDH5, CLDN5, CD34, etc.) were significantly upregulated. Figure 3In Figure A, PBS_1, PBS_2, and PBS_3 are control groups, and scEMC10_1, scEMC10_2, and scEMC10_3 are experimental groups. After treating bEnd.3 cells with scEMC10 (100 ng / ml) and / or TGF-β1 (2 ng / ml) for 48 hours, Western blotting analysis revealed that TGF-β1-induced EndMT was significantly inhibited by scEMC10. Figure 3 (B in the middle).

[0040] The inhibitory effect of scEMC10 (0.5 mg / kg) on ​​pulmonary fibrosis was analyzed after sepsis was induced by LPS (10 mg / kg). Groups included a PBS control group, an LPS group, a scEMC10 injection group 12 hours prior to LPS (EL), a scEMC10 injection group 2 hours after LPS (LE), and a scEMC10 injection group 12 hours after LPS (LE-12h). EB perfusion results showed that both the EL and LE groups significantly inhibited pulmonary hemorrhage caused by sepsis, while the LE-12h group showed no significant difference compared to the LPS group. Figure 4 (A) Immunofluorescence results showed that the EndMT markers α-SMA and EB intensity (bleeding severity) were significantly reduced in both the EL and LE groups. Figure 4 The results for lung weight and wet / dry ratio also showed that the EL and LE groups had significant improvements compared to the LPS group. Figure 4 EF in the middle. H&E staining results showed that the alveolar septum thickness and damage score in the EL and LE groups were significantly improved compared with the LPS group ( Figure 4 (GI in the text).

[0041] In addition to the improvement in lung damage, we also evaluated the recovery of liver and kidney function using an LPS-induced sepsis mouse model. H&E and Sirius red staining results showed that the EL and LE groups significantly restored the liver and kidney damage caused by the LPS group and inhibited fibrosis. Figure 5 (AB in the text). Immunofluorescence results showed that the EndMT marker α-SMA was significantly reduced in both the liver and kidneys in the EL and LE groups, and vascular damage (CD31) was significantly restored in both groups. Figure 5 (CD in the middle).

[0042] Mouse survival results showed that scEMC10 treatment significantly inhibited LPS-induced sepsis-induced mouse death, and the pre-injection group was close to the PBS group ( Figure 6 (A) In this process, the mice's body temperature, diet, and weight changes were significantly improved (A). Figure 6(BD in the text). Behavioral experiments in mice showed that grip strength, balance, anxiety behavior, and motor ability were significantly improved after scEMC10 treatment, with more pronounced improvements in the pre-injection group. Figure 6 (EG in the middle).

[0043] In summary, we found that scEMC10 treatment selectively inhibited SMAD2 phosphorylation in HCMECs cells, while having no effect on SMAD3 or SMAD1 / 5 / 9. This suggests that the interaction between scEMC10 and CD105 negatively regulates TGFBR1 activity. This finding is consistent with previous studies that CD105 antagonizes TGFBR1 signaling, supporting the model that scEMC10, as a newly discovered ligand in the TGF-β pathway, inhibits the classical TGF-β signaling pathway via CD105. Importantly, this inhibitory effect appears to be compatible with, and may even have a synergistic effect with, scEMC10-induced non-classical downstream signaling. Furthermore, scEMC10 shows some therapeutic efficacy in treating diseases where the TGF-β pathway is activated, such as tissue fibrosis caused by sepsis.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of scEMC10 in the preparation of drugs for treating sepsis.

2. The application according to claim 1, characterized in that, The treatment of sepsis includes treating tissue damage or tissue fibrosis caused by sepsis.

3. The application according to claim 1, characterized in that, The tissues include one or more of the following: brain microvascular endothelial cells, lungs, liver, or kidneys.

4. The application according to claim 1, characterized in that, The drug also includes pharmaceutical excipients.

5. The application according to claim 1, characterized in that, The dosage form of the drug includes injections.

6. Application of scEMC10 in the preparation of drugs that inhibit endothelial-mesenchymal transition.

7. The application according to claim 6, characterized in that, The drug also includes pharmaceutical excipients.

8. The application according to claim 6, characterized in that, The dosage form of the drug includes injections.

Images