New use of Areg

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses a new use of Areg. The application discloses the following: a preparation for promoting expression of Areg or Areg protein is used for preparing a drug for preventing and treating myocardial infarction; the preparation for promoting expression of Areg or Areg protein is used for preparing a drug for preventing and treating heart dysfunction; a preparation for inhibiting expression of Areg is used for preparing a biological model of myocardial infarction or a biological model of adverse ventricular remodeling; a preparation for detecting expression level of Areg is used for preparing a product for screening (diagnosing) myocardial infarction. It is found that expression of Areg protein is increased after myocardial infarction of a mouse, and it is found that knocking out Areg can aggravate adverse ventricular remodeling after myocardial infarction of a mouse. It is verified that by increasing expression level of Areg protein, autophagosome formation is inhibited, myocardial cell apoptosis is reduced, loss of functional myocardial cells is reduced, thereby ventricular remodeling is improved, and heart function is protected.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a new use of Areg. BACKGROUND

[0002] Myocardial infarction (MI) is one of the most common cardiovascular diseases, and is a cardiovascular disease with a high mortality rate. The main cause of death is heart failure. Improving adverse ventricular remodeling after myocardial infarction can significantly improve the prognosis of myocardial infarction. Myocardial infarction causes a significant loss of myocardial cell function, which is the basic pathological process leading to heart failure. Early and late reduction of functional myocardial cell loss and control of ventricular remodeling can effectively improve the clinical efficacy.

[0003] Amphiregulin (Areg) is a member of the epidermal growth factor (EGF) family. It binds to the EGF receptor (EGFR) to activate the downstream signaling pathway in a paracrine, autocrine and paracrine manner.

[0004] Currently, there is no research on the correlation between amphiregulin and myocardial infarction, heart dysfunction, etc., and no researchers have proposed a scheme for using amphiregulin as a target to diagnose and treat myocardial infarction, heart dysfunction, etc. SUMMARY

[0005] To solve the above problems, the present application provides a new use of Areg, mainly to solve the problem that the current detection means for myocardial infarction and its induced ventricular remodeling are not perfect, and the treatment means also needs to be improved.

[0006] To solve the above problems, the present application adopts the following technical scheme:

[0007] The first aspect of the present invention provides The application of the preparation for promoting the expression of Areg or the Areg protein in the preparation of a medicine for preventing and treating myocardial infarction.

[0008] Myocardial infarction (MI) is one of the most common cardiovascular diseases, and is a cardiovascular disease with a high mortality rate. The Areg protein is at least one of a high expression Areg protein, an overexpression Areg protein, and a recombinant Areg protein that can be injected exogenously (including a recombinant Areg protein known before the filing date of the present application). High expression and overexpression are both relative to normal expression. The purpose of the recombinant Areg protein that can be injected exogenously is to increase the total amount of Areg protein, which can also increase the expression of the protein and play a regulatory role.

[0009] Prevention and treatment mainly refer to prevention and treatment. Prevention is to intervene in advance to prevent myocardial infarction; treatment is to treat patients who have already shown pathological manifestations of myocardial infarction. The role of the preparation for promoting Areg expression or Areg protein is mainly to improve cardiac dysfunction after myocardial infarction, avoid further deterioration, and improve autophagosomes in myocardial cells through regulation.

[0010] The preparation for promoting Areg expression is at least one or more combinations of AAV9-Areg and an EP2 receptor agonist, and should also include other preparations that have been proven to promote Areg expression. The EP2 receptor agonist includes prostaglandin E2, and AAV9-Areg is an AAV9 adeno-associated virus that can specifically overexpress Areg in the heart. The role of high expression or overexpression of Areg protein is to activate the EGFR / PI3K / Akt / mTOR signaling pathway.

[0011] The second aspect of the present invention provides The preparation for promoting Areg expression or Areg protein is used in the preparation of a drug for preventing and treating cardiac dysfunction.

[0012] Among them, the Areg protein is at least one of high expression Areg protein, overexpression Areg protein, and exogenous injectable recombinant Areg protein. High expression and overexpression are relative to normal expression. The purpose of exogenous injectable recombinant Areg protein is to increase the total expression level of Areg protein, which also has a regulatory effect. Myocardial infarction leads to a large loss of functional cardiomyocytes and chronic ventricular remodeling, which is the basic pathological process leading to heart failure. Therefore, the use of the preparation for promoting Areg expression or Areg protein can also treat heart failure. The role of the preparation for promoting Areg expression or Areg protein in preventing and treating cardiac dysfunction is to inhibit ventricular remodeling, and by inhibiting adverse ventricular remodeling, further development into heart failure (heart dysfunction with an ef value less than 50 is heart failure according to clinical standards) can be effectively avoided.

[0013] Similarly, the preparation for promoting Areg expression is at least one or more combinations of AAV9-Areg and an EP2 receptor agonist, and should also include other preparations that have been proven to promote Areg expression. The EP2 receptor agonist includes prostaglandin E2, and AAV9-Areg is an AAV9 adeno-associated virus that can specifically overexpress Areg in the heart. The role of high expression or overexpression of Areg protein is to activate the EGFR / PI3K / Akt / mTOR signaling pathway.

[0014] The third aspect of the present invention providesThe preparation for inhibiting Areg expression is used for preparing any one of a myocardial infarction biological model, a ventricular adverse remodeling biological model and a heart dysfunction biological model.

[0015] At present, the myocardial infarction biological model is mainly prepared by permanently ligating a left anterior descending (LAD) coronary artery of a mouse to cause myocardial infarction, but this physical myocardial infarction modeling has a significant influence on the artery of the mouse, which increases the heterogeneity of the model. The present application can directly regulate the protein expression level in the mouse by using a biological preparation, so that the same development process as the myocardial infarction disease is obtained, thereby obtaining a better biological model. The heart dysfunction biological model can be a heart failure biological model when needed, and the severity of the heart dysfunction can be controlled by controlling the degree of inhibition of Areg expression. When the heart dysfunction is more severe, the biological model is a heart failure biological model.

[0016] The preparation for inhibiting Areg expression includes shRNA and anti-Areg, and both of them can interfere with the expression of Areg to achieve knockdown or even knock out the expression of Areg. Thus, the EGFR / PI3K / Akt / mTOR pathway can be inhibited by knocking down the expression of Areg in the animal model, which leads to an increase in myocardial cell apoptosis and further induces heart dysfunction, so that the animal model has pathological manifestations such as myocardial infarction. With the deepening of the degree of inhibition of Areg expression, the animal model can further develop from myocardial infarction to ventricular adverse remodeling and heart failure. Thus, the purpose of artificially preparing an animal research model with a specific disease is achieved. The pathological changes in a healthy animal can be formed by controlling the expression of Areg, thereby providing a suitable model for scientific research. In many cases, the biological model is an animal model.

[0017] The fourth aspect of the present invention providesThe application of the preparation for detecting the expression level of Areg in the preparation of products for screening (screening includes diagnosis and risk screening) myocardial infarction disease or detecting the treatment effect of myocardial infarction. Since the expression level of Areg is positively correlated with myocardial infarction, the increase of the expression level of Areg in vivo without external interference indicates the occurrence of myocardial infarction, and with the improvement of the pathology of myocardial infarction, the expression level of Areg decreases with the decrease of macrophage infiltration after the alleviation of myocardial infarction. Therefore, the risk of pathological changes of myocardial infarction can be screened by detecting the expression level of Areg, and when the expression level of Areg is more than 4 times of the normal expression level of heart, it is clear that myocardial infarction occurs, which provides a new basis for clinical diagnosis. At the same time, after the treatment of myocardial infarction, the treatment effect can also be characterized by detecting the expression level of Areg, and when the expression level of Areg returns to normal, it can be considered that myocardial infarction has been effectively treated, and if the expression level of Areg is still high after targeted treatment, it can be considered that myocardial infarction has not been effectively treated. Of course, when the expression level of Areg is used as a diagnosis of the treatment effect, other factors that can affect the expression of Areg generally need to be excluded, that is, the preparation or Areg protein for promoting the expression of Areg is generally not used to prevent and treat myocardial infarction in the corresponding treatment scheme, unless the interference of the preparation or Areg protein for promoting the expression of Areg can be effectively excluded (for example, when the expression level of the preparation or exogenous Areg protein for promoting the expression of Areg can be quantified).

[0018] In order to better detect and diagnose, the preparation for detecting the expression level of Areg at least includes one of a probe and a primer, and the primer is at least

[0019] Forward primer: 5'-GGTCTTAGGCTCAGGCCATTA-3' Reverse primer: 5'-CGCTTATGGTGGAAACCTCTC-3'.

[0020] The beneficial effects of the application are: it is found that the expression of Areg protein increases after myocardial infarction in mice, and it is also found that the knockout of Areg will aggravate the ventricular adverse remodeling after myocardial infarction in mice. It is verified that by increasing the expression level of Areg protein, autophagosome formation is inhibited, myocardial cell apoptosis is reduced, functional myocardial cell loss is reduced, thereby improving ventricular remodeling and protecting heart function. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1-2 The expression of Areg gene and protein in the heart of mice after myocardial infarction is increased;

[0022] Figure 3 The Areg- / - mouse is successfully constructed;

[0023] Figure 4 The heart function of Areg- / - mouse after myocardial infarction is deteriorated;

[0024] Figure 5Cardiac function in Areg- / - mice under steady-state conditions was no different from that in WT mice;

[0025] Figure 6 In Areg- / - mice, the mortality rate after myocardial infarction was increased, the infarct area was larger, and cardiac fibrosis was increased.

[0026] Figure 7 The results showed that AAV9-Areg mice exhibited improved cardiac function after myocardial infarction, while sh-Areg mice showed deterioration of cardiac function after myocardial infarction.

[0027] Figure 8 The exogenous supplementation of Areg improved cardiac function in mice after myocardial infarction, while the injection of Anti-Areg worsened cardiac function in mice after myocardial infarction.

[0028] Figure 9 Volcano plot of differentially sequenced genes from the cardiac transcriptomes of Areg- / - mice and WT mice;

[0029] Figure 10 Areg deficiency inhibits the cardiac EGFR / PI3K / Akt / mTOR pathway after myocardial infarction (MI).

[0030] Figure 11 Areg deficiency promotes the formation of autophagosomes in cardiomyocytes and inhibits the clearance of autophagosomes;

[0031] Figure 12 Areg deficiency promotes cardiomyocyte apoptosis;

[0032] Figure 13-14 Areg activates the EGFR / PI3K / Akt / mTOR signaling pathway in cardiomyocytes, thereby regulating autophagy and apoptosis in cardiomyocytes;

[0033] Figure 15 Areg cells regulate autophagy and apoptosis in cardiomyocytes by activating the EGFR / PI3K / Akt / mTOR signaling pathway. Detailed Implementation

[0034] The invention will be further described below with reference to examples.

[0035] Experimental content

[0036] 1. Increased Areg expression in the heart of mice after myocardial infarction (MI)

[0037] The inventors, through PCR and Western blot analysis of heart tissues from mice at different time points after myocardial infarction (MI), found that both Areg gene expression and protein expression were elevated in mouse heart tissues. Figure 1 (A and B), and is mainly expressed in the infarct junction area. Figure 1 (C and D). Flow cytometry sorting of cardiac CD45+ and CD45 - cells, and quantified Areg transcription level, CD45 + cells expressed higher level of Areg than CD45 - cells Figure 1 E). Moreover, in CD45 + cells, Areg was highly expressed in cardiac macrophages, but lowly expressed in CD3 + T cells, and almost not expressed in CD19 + B cells + + Figure 1 F). To detect the proportion of Areg secretion from the above cells in heart, the inventors detected Areg secretion in myocardial infarction heart 7 days after myocardial infarction by flow cytometry, and the results showed that 79.71% of Areg was produced by CD45 + cells. In CD45 + cells, 74.72% of Areg was secreted by CD11b + F4 / 80 + macrophages, 12.20% of Areg was secreted by CD45 + CD3 + cells, and CD45 + CD19 + B cells did not secrete Areg Figure 2 G). These data showed that after acute myocardial infarction, Areg was up-regulated in the border zone of myocardial infarction ( Figure 1 A, more than 4 times the expression level in normal heart, indicating the occurrence of myocardial infarction), and macrophages were the main source of Areg.

[0038] 1) PCR reaction:

[0039] The primer sequences are as follows:

[0040]

[0041] 2. Areg deficiency after MI aggravates mouse heart dysfunction

[0042] The inventors used Areg - / - mice to study the role of Areg after myocardial infarction. First, the heart was collected to detect the expression level of Areg under steady state. The Areg protein expression level of Areg - / - mice was significantly lower than that of WT mice Figure 3 A, indicating that Areg - / - mice were successfully constructed. The heart function of Areg - / - mice was analyzed by echocardiography 14 days after myocardial infarction Figure 4 ​​A). Areg deficiency resulted in decreased EF, FS (heart failure when EF value is lower than 50), increased left ventricular dilation index (LVEDD, LVEDV), increased left ventricular systolic index (LVESD, LVESV), increased heart weight / body weight ratio, and increased lung weight / body weight ratio Figure 4 B-I). Meanwhile, in the steady state, Areg - / - There was no difference in FS, EF, LVEDD, LVEDV, LVESD, and LVESV between the mice and WT mice Figure 5 A-G). Areg - / - The survival rate of the mice 14 days after MI was significantly lower than that of the WT group Figure 6 A). In addition, Masson staining showed that Areg - / - The infarct scar area of the mice increased Figure 6 B and C). Similarly, by evaluating the interstitial fibrosis in the area surrounding the infarct through collagen volume fraction (CVF), Areg - / - The interstitial fibrosis of the mice increased Figure 6 D and E). The results showed that Areg protected ventricular remodeling after myocardial infarction.

[0043] The inventors further clarified the role of Areg after myocardial infarction using sh-RNA and AAV9. Areg was locally overexpressed in the heart using AAV9 adeno-associated virus. The expression level of Areg protein in AAV0-Areg mice was significantly higher than that in control mice Figure 7 A), indicating that Areg overexpression was successful. The heart function of the mice 14 days after myocardial infarction was analyzed by echocardiography, and Areg overexpression increased EF and FS Figure 7 B-D). Next, the inventors used sh-RNA to inhibit Areg expression. The expression level of Areg protein in sh-Areg mice was significantly lower than that in control mice Figure 7 E), indicating that Areg knockdown was successful. The heart function of the mice 14 days after myocardial infarction was analyzed by echocardiography, and inhibition of Areg expression decreased EF and FS Figure 7 F-H).

[0044] The inventors further clarified the role of Areg after myocardial infarction using recombinant Areg protein (RD, 989 AR) and Anti-Areg (Amphiregulin Antibody, Novus, AF989) antibody. The heart function of the mice 14 days after myocardial infarction was analyzed by echocardiography, and exogenous supplementation of recombinant Areg expression increased EF and FS, and Anti-Areg treatment decreased EF and FS Figure 8A-C).

[0045] 3. Areg deficiency after MI inhibits EGFR / PI3K / Akt / mTOR pathway

[0046] Areg deficiency after MI - / - RNA sequencing analysis of myocardium of Areg - / - mice and WT mice. The results showed 957 differentially expressed genes (DEGs) compared to Areg Figure 9 mice. KEGG pathway enrichment analysis showed that DEGs were enriched in PI3K-Akt signaling pathway, EGF receptor signaling pathway, mTOR signaling pathway and apoptosis signaling pathway Figure 10 A). EGFR / PI3K / Akt / mTOR signaling pathway is a classic regulatory axis of myocardial infarction. Subsequently, the inventors detected the phosphorylation level of EGFR / PI3K / Akt / mTOR by western blotting. Compared with WT mice, the ratio of p-EGFR / EGFR, p-PI3K / PI3K, p-Akt / Akt and p-mTOR / mTOR was down-regulated in Areg - / - mice Figure 10 B and C). In summary, these data suggest that Areg deficiency inhibits EGFR / PI3K / Akt / mTOR pathway after myocardial infarction.

[0047] 4. Areg deficiency promotes autophagosome formation and inhibits autophagosome clearance after MI

[0048] PI3K / Akt / mTOR pathway as a regulator of autophagy has attracted widespread attention. To investigate whether autophagy is involved in the Areg protective mechanism, the inventors first detected the protein levels of LC3-II (autophagy marker) and p62 (autophagy adaptor protein) in the heart tissue of Areg - / - and WT mice 7 days after MI. Areg deficiency increased the protein levels of LC3-II and p62 Figure 11 A). Since P62 is degraded during autophagosome processing, these results suggest that Areg deficiency leads to impaired autophagosome clearance. Since autophagy is a dynamic process, the inventors used autophagy inhibitor 3-methyladenine (3-MA, an autophagy inhibitor that blocks the upstream step of autophagy) and autophagosome-lysosome fusion inhibitor Bafilomycin-A1 (BFA, an autophagy inhibitor that blocks the downstream step of autophagy) to confirm the autophagy flux of Areg - / - mice. The results showed that 3-MA could not inhibit the accumulation of LC3-II induced by Areg deficiency Figure 11B). On the other hand, BFA did not significantly promote Areg - / - The accumulation of LC3-II in mouse, BFA also did not inhibit the accumulation of LC3-II caused by Areg deficiency Figure 11 C). The results suggest that during myocardial infarction, Areg deficiency promotes autophagosome synthesis, but inhibits autophagosome clearance. At the same time, electron microscopy analysis showed that Areg - / - Autophagic vacuoles in mouse cardiomyocytes were significantly increased Figure 11 D). In summary, these findings suggest that after myocardial infarction, Areg deficiency promotes autophagosome formation and inhibits autophagosome clearance, resulting in the accumulation of a large number of autophagosomes in cardiomyocytes.

[0049] 5. Areg deficiency promotes cardiomyocyte apoptosis after MI

[0050] Areg deletion significantly increased the expression of pro-apoptotic proteins Cleaved caspase-3 and Bax, while reducing the expression of anti-apoptotic protein Bcl-2 Figure 12 A and B). By TUNEL detection of apoptotic cardiomyocytes, the results showed that Areg - / - Mice had more TUNEL+ cardiomyocytes in the infarct border zone of the heart than WT mice Figure 12 C and D). These results suggest that Areg deficiency promotes cardiomyocyte apoptosis after myocardial infarction.

[0051] 6. Areg activates the EGFR / PI3K / Akt / mTOR signaling pathway to regulate autophagy and apoptosis of cardiomyocytes

[0052] First, neonatal mouse cardiomyocytes (NMCMs) were isolated from 3-day-old WT mice and cultured under hypoxic conditions to simulate the environment after myocardial infarction. Next, the inventors evaluated the expression levels of phosphorylated EGFR, PI3K, Akt and mTOR before and after treatment with recombinant Areg (100 pg / mL). In both hypoxic and normoxic conditions, the p-EGFR / EGFR, p-PI3K / PI3K, p-Akt / Akt and p-mTOR / mTOR ratio in the recombinant Areg stimulation group were higher than in the control group Figure 13 A and B).

[0053] Autophagic flux reflects the dynamic process of autophagy and is a reliable indicator of autophagic activity. To further understand the effect of Areg on autophagic flux, the inventors transfected NMCMs with mRFP-GFP-LC3 virus. Consistent with the in vivo research data, hypoxia led to an increase in autophagosome formation in NMCMs Figure 13 C and 14D), but hypoxia did not increase the number of autolysosomes (red dots) in NMCMs Figure 13C and 14E), Areg significantly reduced the number of autophagosomes and increased the number of autolysosomes in NMCMs under hypoxia condition Figure 13 C and 14D-E). Moreover, Areg significantly reduced the number of autophagosomes in NMCMs under hypoxia condition in the presence of BFA, which inhibits lysosomal acidification and prevents autophagosome-lysosome fusion Figure 13 C and 14D). Meanwhile, recombinant Areg significantly reduced the expression of LC3-II under hypoxia condition. However, it had no effect on LC3-II under normoxia condition Figure 14 F and G). These data suggest that Areg promotes the clearance of autophagosomes and inhibits the formation of autophagosomes in NMCMs.

[0054] Recombinant Areg significantly reduced the expression of cleaved caspase-3 and Bax and increased the expression of Bcl-2 under hypoxia condition, but had no effect on the expression of these markers under normoxia condition, which might be related to the fact that NMCMs themselves have less apoptosis under normoxia condition Figure 14 H and I), which suggest that Areg inhibits the apoptosis of NMCMs.

[0055] 7. Areg regulates autophagy and apoptosis of cardiomyocytes by activating EGFR / PI3K / Akt / mTOR signaling pathway

[0056] In NMCMs, treatment with PI3K or Akt / mTOR inhibitors can significantly reverse the effects of Areg on autophagosome formation and autophagosome clearance Figure 15 A-C). Similarly, treatment with PI3K and Akt / mTOR inhibitors (LY294002 and Coenzyme Q0) significantly reversed the decrease in the protein levels of LC3-II, cleaved caspase-3, and Bax induced by Areg, as well as the increase in Bcl-2 induced by Areg Figure 15 D and E). Therefore, PI3K and Akt / mTOR inhibitors counteract the effects of recombinant Areg on autophagy and apoptosis of NMCMs. Taken together, these results suggest that Areg promotes the clearance of autophagosomes, inhibits the formation of autophagosomes and apoptosis of NMCMs through the EGFR / PI3K / Akt / mTOR signaling pathway.

[0057] CONCLUSION

[0058] The inventors found that the expression of Areg protein increased after myocardial infarction in mice, and that knocking out Areg exacerbated ventricular adverse remodeling after myocardial infarction in mice. After the relief of myocardial infarction, the infiltration of macrophages decreased, and the expression level of Areg decreased accordingly. Areg - / -The mouse reduces Areg expression from the gene level, sh-Areg reduces Areg expression from the transcription level, and the anti-Areg antibody inhibits Areg action from the protein level. The specific mechanism is that the heart macrophage-based cells secrete Areg, bind to the surface EGFR of cardiomyocytes, activate the downstream PI3K / Akt / mTOR pathway, inhibit the formation of autophagosomes in cardiomyocytes, promote the clearance of autophagosomes, reduce cardiomyocyte apoptosis, thereby reducing the loss of functional cardiomyocytes, improving ventricular remodeling, and protecting heart function.

[0059] Those skilled in the art can clearly understand that various modifications can be made to the above embodiments without departing from the overall spirit and concept of the present application. All of them fall within the protection scope of the present application. The protection scheme of the present application is subject to the claims attached to the present application.

Claims

1. The application of AAV9-Areg or Areg protein in the preparation of drugs for the prevention and treatment of myocardial infarction, among which, The AAV9-Areg or the Areg protein in the drug exerts its effects by inhibiting ventricular remodeling, reducing cardiomyocyte apoptosis, and improving cardiac function.

2. The application according to claim 1, wherein, The Areg protein is at least one of the following: highly expressed Areg protein, overexpressed Areg protein, or exogenously injectable recombinant Areg protein.

3. The application according to claim 1 or 2, wherein, The Areg protein functions by activating the EGFR / PI3K / Akt / mTOR signaling pathway, inhibiting autophagosome formation and promoting autophagosome clearance, and reducing cardiomyocyte apoptosis.

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