Application of PTX3 inhibitor in preparation of products for treating cardiovascular diseases

Abstract

The invention provides application of a PTX3 inhibitor in preparation of products for treating cardiovascular diseases, and belongs to the technical field of biological medicines. The research finds that the protein level of PTX3 in calcified smooth muscle cells constructed by treating vascular smooth muscle cells with beta-phosphoglycerol is up-regulated, and meanwhile, the content of PTX3 in calcified vascular tissues and plasma induced by vitamin D3 is increased, so that whether a patient suffers from vascular calcification or not can be diagnosed or evaluated by detecting the expression level of PTX3. According to the invention, after the PTX3 is knocked down by using siRNA1 and siRNA2 as shown in SEQ ID NO.1-2, the cell calcification weakening of the PTX3 is knocked down under the induction of beta-phosphoglycerol. Researches find that PTX3 promotes inflammation and aggravates vascular calcification by regulating p65. The invention provides a method for detecting and treating vascular calcification by taking PTX3 as a target spot.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of a PTX3 inhibitor in the preparation of a product for treating cardiovascular diseases. Background Art

[0002] Cardiovascular disease is a circulatory system disease induced by multiple factors. It is the result of the long-term combined effects of obesity, hypertension, hyperlipidemia, hyperuricemia, diabetes and other factors. Due to its complex pathogenesis, limited treatment and prognosis, the morbidity and mortality of cardiovascular disease have remained high, and it has become the primary threat to human health. Vascular calcification (VC) refers to the pathological process of excessive calcium deposition on the blood vessel wall. It is a common pathological manifestation of many cardiovascular diseases, such as atherosclerosis, hypertension, diabetic vascular disease, vascular damage, chronic kidney disease and other diseases. The main risk factors for vascular calcification are hyperlipidemia, diabetes, hyperuricemia, renal failure, hypertension, hyperhomocysteinemia, age, obesity and smoking. Vascular calcification is mainly manifested by increased stiffness of the vascular wall, increased vascular fragility and stiffness, and decreased compliance, which can easily lead to myocardial ischemia, left ventricular hypertrophy and heart failure, and increase the risk of arterial plaques, vascular rupture and aneurysm formation. It is one of the important factors for the high incidence and mortality of cardiovascular and cerebrovascular diseases, and is also an important marker molecule for the occurrence of atherosclerotic cardiovascular events, stroke and peripheral vascular disease. Traditional concepts unanimously believe that vascular calcification is a passive, degenerative, and inevitable terminal process. However, recent clinical and basic research results show that vascular calcification is similar to the process of bone development and cartilage formation, and is an active, adjustable, treatable and preventable process.

[0003] Currently, there is a lack of effective drug targets for early warning, treatment and prognosis assessment of aortic calcification in clinical practice. Therefore, it is necessary to conduct research at different molecular levels and multiple dimensions such as proteomics and metabolomics to develop effective and more specific biomarkers and drug targets for the development of intervention targets for clinical vascular calcification diseases. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an application of PTX3 as a target in the preparation of a product for treating or diagnosing cardiovascular diseases.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides an application of a PTX3 inhibitor in preparing a product for treating cardiovascular diseases.

[0006] The present invention provides an application of a reagent for detecting PTX3 expression in the preparation of a product for diagnosing cardiovascular diseases.

[0007] Preferably, the PTX3 inhibitor comprises one or both of an agent that reduces PTX3 expression and a regulator that reduces PTX3 production.

[0008] Preferably, the regulator that reduces PTX3 production comprises a PTX3 antibody or a protease that degrades PTX3 production.

[0009] Preferably, the agent for reducing the expression of PTX3 includes an agent for knocking down or silencing PTX3.

[0010] Preferably, the agent for knocking down or silencing PTX3 comprises siRNA, shRNA or miRNA.

[0011] Preferably, the sequence of the siRNA is shown as SEQ ID NO.1 and SEQ ID NO.2.

[0012] Preferably, the cardiovascular disease comprises vascular calcification.

[0013] The present invention provides a drug for treating vascular calcification, comprising siRNA1 as shown in SEQ ID NO.1 and siRNA2 as shown in SEQ ID NO.2.

[0014] Preferably, the molar concentration ratio of siRNA1 to siRNA2 is 1:1.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an application of a PTX3 inhibitor in the preparation of a product for treating cardiovascular diseases. The present invention has found that the protein level of PTX3 in calcified smooth muscle cells constructed by treating vascular smooth muscle cells with β-phosphoglycerol is upregulated, and the PTX3 content in calcified vascular tissue and plasma induced by vitamin D3 increases. Therefore, by detecting the expression level of PTX3, it can be used to diagnose or evaluate whether vascular calcification is present. After knocking down PTX3 using siRNA1 and siRNA2 shown in SEQ ID NO.1~2, the calcification of cells knocking down PTX3 is weakened under the induction of β-phosphoglycerol. And the study found that PTX3 promotes inflammation and aggravates the occurrence of vascular calcification by regulating p65. The present invention provides a method for detecting and treating vascular calcification using PTX3 as a new target. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The results of increased expression of PTX3 during in vitro smooth muscle cell calcification, A is a schematic diagram of in vitro induction of smooth muscle cell calcification; B is the calcification of smooth muscle cells added with β-glycerol compared with the control group, and the red color of Alizarin red staining indicates that the cells have calcified; C is the result of increased mRNA level of PTX3 in calcified smooth muscle cells; D is the expression of PTX3 in cells in the control group and smooth muscle cell calcification model group detected by Western Blot; E is the quantitative result of grayscale analysis of protein bands of PTX3 in different groups; Figure 2 The results of increased expression of PTX3 in vascular tissue in the in vivo vascular calcification model, A is a schematic diagram of the in vivo induced vascular calcification model; B is the result of the black deposits of VON-KOSSA staining of the mouse blood vessels in the vitamin D3-treated group, showing the calcified areas of the blood vessels; C is the result of increased calcium ion content in the blood vessels of the mice in the vitamin D3-treated group compared with the control group; D is the result of increased calcium ion content in the serum of the mice in the vitamin D3-treated group compared with the control group; E is the result of increased ALP activity in the serum of the mice in the vitamin D3-treated group compared with the control group; F is the result of increased expression of calcification marker genes in the blood vessels of the mice in the vitamin D3-treated group compared with the control group; G is the result of decreased smooth muscle cell marker proteins in the blood vessels of the mice in the vitamin D3-treated group compared with the control group; H is the result of increased expression of PTX3 in the calcified vascular tissue of the mice in the vitamin D3-treated group compared with the control group; I is the immunofluorescence quantification of PTX3 in the calcified vascular tissue of the mice in the vitamin D3-treated group compared with the control group; Figure 3 This is the result of increased plasma PTX3 levels in the in vivo vascular calcification model; Figure 4 The results of knocking down PTX3 to attenuate cell calcification are shown in Figure 1. A is a schematic diagram of knocking down PTX3 in smooth muscle cells, inducing smooth muscle cell calcification, and detecting the effect of PTX3 on cell calcification; B is the protein changes of PTX3 in the cells of the blank control group, the control group, and the PTX3 knockdown treatment group; C is the quantitative results of grayscale analysis of protein bands of PTX3 in the cells of the blank control group, the control group, and the PTX3 knockdown treatment group; D is the results of Alizarin red staining of smooth muscle cells after PTX3 was knocked down and the smooth muscle cells were treated with calcification medium for 14 days; E is the quantitative results of Alizarin red staining of smooth muscle cells after PTX3 was knocked down and the smooth muscle cells were treated with calcification medium for 14 days; F is the protein expression level of calcification marker protein RUNX2 in smooth muscle cells after PTX3 was knocked down and the smooth muscle cells were treated with calcification medium for 7 days; G is the quantitative results of grayscale analysis of protein bands of calcification marker protein RUNX2 in smooth muscle cells after PTX3 was knocked down and the smooth muscle cells were treated with calcification medium for 7 days; Figure 5The results of PTX3 promoting smooth muscle cell calcification, A is a schematic diagram for detecting the effect of recombinant protein PTX3 on smooth muscle cell calcification; B is the protein expression level of calcification marker protein RUNX2 after BSA or recombinant protein PTX3 was added to the culture medium and smooth muscle cells were treated with calcification medium for 7 days; C is the quantitative result of protein band grayscale analysis of calcification marker protein RUNX2 after recombinant protein PTX3 was added to the culture medium and smooth muscle cells were treated with calcification medium for 7 days; Figure 6 The results of PTX3 promoting vascular calcification by regulating p65, A is the protein levels of phosphorylated p65 and total p65 protein in smooth muscle cells after BSA or recombinant protein PTX3 was added to the culture medium and the smooth muscle cells were treated with calcification medium for 7 days; B is the quantitative result of grayscale analysis of phosphorylated p65 protein band; C is the quantitative result of grayscale analysis of total p65 protein band; D is the protein levels of phosphorylated p65 and total p65 protein in smooth muscle cells after PTX3 was knocked down and the smooth muscle cells were treated with calcification medium for 7 days; E is the quantitative result of grayscale analysis of phosphorylated p65 protein band after PTX3 was knocked down; F is the quantitative result of grayscale analysis of total p65 protein band after PTX3 was knocked down. DETAILED DESCRIPTION

[0017] The present invention provides an application of a PTX3 inhibitor in preparing a product for treating cardiovascular diseases.

[0018] In the present invention, the inhibitor of PTX3 includes one or both of an agent that reduces PTX3 expression and a regulator that reduces PTX3 product. The regulator that reduces PTX3 product includes a PTX3 antibody or a protease that degrades PTX3 product. The agent that reduces PTX3 expression includes an agent that knocks down or silences PTX3. The agent that knocks down or silences PTX3 includes siRNA, shRNA or miRNA. The sequence of the siRNA is shown in SEQ ID NO.1 and SEQ ID NO.2. The present invention uses siRNA1 as shown in SEQ ID NO.1 and siRNA2 as shown in SEQ ID NO.2 to jointly reduce and inhibit the level of PTX3 in smooth muscle cells, and then studies its effect on cell calcification. The study found that inhibiting PTX3 can reduce vascular calcification, which means that siRNA1 and siRNA2 can jointly reduce vascular calcification.

[0019] The present invention provides an application of a reagent for detecting PTX3 expression in the preparation of a product for diagnosing cardiovascular diseases.

[0020] In the present invention, the PTX3 is pentraxin 3. The present invention uses the β-glycerophosphate induced calcified smooth muscle cells and vitamin D3 induced vascular calcification mouse models known in the art to study the effect of PTX3 on vascular calcification. The study found that the expression level of PTX3 in calcified smooth muscle cells and vascular calcification mice was upregulated. Therefore, the present invention first discovered that PTX3 can be used as a detection target for diagnosing cardiovascular diseases or assessing the risk of cardiovascular diseases. The reagent for detecting PTX3 expression includes a PTX3 amplification primer or a PTX3 immunofluorescence reagent.

[0021] In the present invention, the cardiovascular disease is vascular calcification, and the product includes a reagent or a drug.

[0022] The present invention provides a drug for treating vascular calcification, comprising siRNA1 as shown in SEQ ID NO.1 and siRNA2 as shown in SEQ ID NO.2.

[0023] In the above-mentioned drug for treating vascular calcification, the molar concentration ratio of siRNA1 and siRNA2 is 1:1. In the above-mentioned drug, the drug is composed of siRNA1 and siRNA2 as active ingredients, and the drug may also include pharmaceutically acceptable excipients. The present invention has found that the drug composed of siRNA1 and siRNA2 as the only active ingredients can be used to treat vascular calcification.

[0024] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0025] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] In the following examples, the smooth muscle cells were purchased from ScienCell.

[0027] Example 1 (1) PTX3 levels increase during smooth muscle cell calcification in vitro The expression of PTX3 in vascular calcification was detected in an in vitro cell calcification model. At the cellular level, β-glycerophosphate (β-GP) was used to treat vascular smooth muscle cells to cause osteogenic phenotype conversion in smooth muscle cells, thereby constructing a smooth muscle cell calcification model.

[0028] 80% smooth muscle cells were inoculated in a cell culture dish. After the cells adhered to the wall on the second day, β-glycerophosphate with a final concentration of 10 mM was added to the smooth muscle cell culture medium (purchased from ScienCell). The cells were collected after 0 days of stimulation (control group, recorded as Vehicle) and 7 days of stimulation (smooth muscle cell calcification model group, recorded as β-GP). RNA and protein were extracted from the cells in each group. The mRNA level of PTX3 was detected by QPCR, and the protein expression of PTX3 was detected by Western Blot ( Figure 1 A) Alizarin red staining was used to detect β-glycerophosphate-induced smooth muscle cell calcification. Figure 1 The results in B show that compared with the control group, the smooth muscle cells to which β-glycerol phosphate was added underwent calcification, indicating that calcified smooth muscle cells were successfully constructed. Figure 1 The results in C and D show that compared with the control group, the mRNA level of PTX3 in calcified smooth muscle cells was upregulated during the calcification of mouse smooth muscle cells. The protein expression of PTX3 in the cells of the control group and the smooth muscle cell calcification model group during the calcification of human smooth muscle cells was further detected. Figure 1 The results in Figure E showed that the protein level of PTX3 was also upregulated in calcified smooth muscle cells compared with the control group.

[0029] (2) PTX3 expression levels increase in vascular tissue during vascular calcification in vivo At the in vivo level, a mouse model of vascular calcification was constructed using subcutaneous injection of vitamin D3. Ten-week-old C57BL6 / J mice were selected and randomly divided into a control group (Saline group) and a vitamin D3-treated group (VD3 group), with 10 mice in each group. The vitamin D3-treated group received a subcutaneous injection of vitamin D3 at a dose of 400,000 units per kilogram of mouse every day for 3 consecutive days, once a day; the control group received an equal amount of normal saline injected subcutaneously every day for 3 consecutive days. Vascular tissues were then collected from mice in the control group and the vitamin D3-treated group 7 days later. The schematic diagram of the in vivo induced vascular calcification model is shown in Figure 2 A in.

[0030] The blood vessels of mice in the vitamin D3 treatment group were stained with VON-KOSSA. The calcium ion content in the blood vessels and serum of mice in the control group and the vitamin D3 treatment group was detected by the calcium ion content detection kit (MAK022) purchased from Sigma. The ALP activity detection kit (P0321S) purchased from Biyuntian was used to detect the ALP activity in the serum of mice in the control group and the vitamin D3 treatment group. The calcification marker genes (Runx2, Bmp2, Bmp4, Ocn, Osterix and Opn) in the blood vessels of mice in the control group and the vitamin D3 treatment group were detected by real-time fluorescence quantitative PCR. The smooth muscle cell marker proteins (α-SMA and SM22α) in the blood vessels of mice in the control group and the vitamin D3 treatment group were detected by real-time fluorescence quantitative PCR. The protein levels of α-SMA and PTX3 in the blood vessels of mice in the control group and the vitamin D3 treatment group were detected by immunofluorescence, and fluorescent images were formed, and the immunofluorescence of PTX3 in each group was quantified.

[0031] Figure 2 The results showed that the mice in the vitamin D3 treatment group had vascular calcification (see Figure 2 B); Compared with the control group, the calcium ion content in the blood vessels and serum of the mice treated with vitamin D3 increased (see Figure 2 C, D), the ALP activity in the serum of mice treated with vitamin D3 increased (see Figure 2 E in Figure 3), the expression of calcification marker genes in the blood vessels of mice treated with vitamin D3 increased (see Figure 2 F), the smooth muscle cell marker protein in the blood vessels of mice treated with vitamin D3 decreased (see Figure 2 G in the figure), indicating that subcutaneous injection of vitamin D3 successfully established a vascular calcification mouse model.

[0032] Figure 2 The results in H and I show that the fluorescence intensity of PTX3 increases in the vascular tissue of mice with vascular calcification, which further indicates that PTX3 is involved in the occurrence and development of vascular calcification.

[0033] ELISA was used to detect the content of PTX3 in the plasma of mice in the control group and the vitamin D3-treated group.

[0034] Figure 3 The results showed that compared with the control group, the PTX3 content in the plasma of mice with vascular calcification increased, which further indicated that PTX3 was involved in the occurrence and development of vascular calcification and could be used as one of the indicators of vascular calcification.

[0035] Example 2 Low expression of PTX3 at the cellular level attenuates cell calcification At the cellular level, siRNA was used to knock down PTX3 expression and inhibit the PTX3 level in smooth muscle cells to study its effect on cell calcification.

[0036] The experiment was divided into a blank control group (Scrambled), a control group (Scrambled+β-GP) and a PTX3 knockdown treatment group (PTX3-si+β-GP). The treatment method of the PTX3 knockdown treatment group was as follows: smooth muscle cell growth factor, 2% FBS and 1% double antibody were added to the smooth muscle cell culture medium, and smooth muscle cells were inoculated at a density of 80% in the culture dish for culture. On the second day of inoculation, 5μM siRNA1 and 5μM siRNA2 were added at the same time for transfection, and β-glycerophosphate with a final concentration of 10mM was added for stimulation one day after transfection.

[0037] The sequence of siRNA1 is: GGCACAAAGAGGAAUCCAU (SEQ ID NO. 1); The sequence of siRNA2 is: GGGUGGUGGCUUUGAUGAA (SEQ ID NO. 2).

[0038] Scrambled+β-GP is prepared by adding smooth muscle cell growth factor, 2% FBS and 1% double antibody to the smooth muscle cell culture medium, and inoculating smooth muscle cells at a density of 80% in the culture dish for culture. On the second day of inoculation, a random control sequence (siRNA3) with 10μM siRNA is added for transfection. One day after transfection, β-glycerophosphate with a final concentration of 10mM is added for stimulation.

[0039] The sequence of siRNA3 is: UUCUUCGAACGUGUCACGU (SEQ ID NO. 3).

[0040] Scrambled refers to smooth muscle cells transfected with random control sequences without treatment with β-glycerophosphate at a final concentration of 10 mM.

[0041] After stimulation for different time periods (0 days, 7 days, and 14 days), RNA and protein were extracted from smooth muscle cells of each group, or Alizarin red staining was performed (see the experimental procedure). Figure 4 Western Blot was used to detect the expression of PTX3 in each group of cells, and the relative expression levels of PTX3 / β-tubulin were calculated.

[0042] Figure 4The results of B and C showed that compared with Scrambled+β-GP, the expression of PTX3 in cells was low after treatment with PTX3-si+β-GP, indicating that siRNA1 and siRNA2 reduced the expression of PTX3 and successfully constructed smooth muscle cells with low expression of PTX3.

[0043] The cells in the Scrambled+β-GP and PTX3-si+β-GP groups were stimulated with calcification medium containing 10 mM β-glycerophosphate for 14 days, and then fixed with paraformaldehyde (PFA) and stained with Alizarin red, and photographed for analysis.

[0044] Figure 4 The results in D and E showed that low expression of PTX3 reduced the calcium ion concentration in smooth muscle cells.

[0045] After 7 days of stimulation with calcification medium containing 10 mM β-glycerophosphate, the cells were collected, RNA and protein were extracted, and Western blot was used to detect the calcification marker protein RUNX2 in each group of cells.

[0046] Figure 4 The results in F and G showed that inhibition of PTX3 reduced the level of calcification marker protein RUNX2.

[0047] Figure 4 The results showed that inhibition of PTX3 could reduce vascular calcification.

[0048] Example 3 (1) PTX3 aggravates smooth muscle cell calcification 5 mM human recombinant protein PTX3 (hr-PTX3, purchased from abcam, catalog number ab283455) and 10 mM β-glycerophosphate (referred to as hr-PTX3+β-GP group) were added to the smooth muscle cells cultured in vitro, and 5 mM BSA and 10 mM β-glycerophosphate (control group, referred to as BSA+β-GP group) were added at the same time. The blank control group was smooth muscle cells without any protein treatment and β-GP stimulation. After stimulation for different time periods (0 days, 7 days), cells were collected to extract proteins (see Figure 5 (A) Western blot was used to detect the calcification marker protein RUNX2 in each group of cells.

[0049] Figure 5 The results in B and C showed that the calcification marker protein RUNX2 was highly expressed.

[0050] (2) PTX3 promotes vascular calcification by regulating p65 5mM human recombinant protein PTX3 (hr-PTX3, purchased from abcam, catalog number ab283455) and 10mM β-glycerophosphate (referred to as hr-PTX3+β-GP group) were added to the smooth muscle cells cultured in vitro, and 5mM BSA and 10mM β-glycerophosphate (control group, referred to as BSA+β-GP group) were added at the same time. The blank control group was smooth muscle cells without any protein treatment and β-GP stimulation. After stimulation for different time periods (0 days, 7 days), the cells were collected to extract proteins, and the protein levels of phosphorylated p65 and total p65 protein were detected.

[0051] Figure 6 The results showed that the recombinant protein PTX3 significantly increased the protein levels of phosphorylated p65 and total p65 protein (see Figure 6 A~C in it).

[0052] PTX3 was knocked down using the experimental method of Example 2, and after the smooth muscle cells were treated with calcification medium for 7 days, the protein levels of phosphorylated p65 and total p65 protein in the smooth muscle cells of each group were detected.

[0053] Figure 6 The results in D~F showed that knockdown of PTX3 significantly decreased the protein levels of phosphorylated p65 and total p65 protein.

[0054] The above results indicate that PTX3 affects vascular calcification by regulating p65.

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a PTX3 inhibitor in the preparation of a product for treating cardiovascular diseases.

2. Application of a reagent for detecting PTX3 expression in the preparation of products for diagnosing cardiovascular diseases.

3. The use according to claim 1, characterized in that: The PTX3 inhibitor includes one or both of an agent that reduces PTX3 expression and a regulator that reduces PTX3 production.

4. The use according to claim 3, characterized in that: The regulator that reduces the PTX3 product includes a PTX3 antibody or a protease that degrades the PTX3 product.

5. The use according to claim 3, characterized in that: The agent for reducing the expression of PTX3 includes an agent for knocking down or silencing PTX3.

6. The use according to claim 5, characterized in that: The agent for knocking down or silencing PTX3 includes siRNA, shRNA or miRNA.

7. The use according to claim 4, characterized in that: The sequences of the siRNA are shown in SEQ ID NO.1 and SEQ ID NO.

2.

8. The use according to any one of claims 1 to 7, characterized in that: The cardiovascular disease includes vascular calcification.

9. A drug for treating vascular calcification, characterized in that: It includes siRNA1 shown in SEQ ID NO.1 and siRNA2 shown in SEQ ID NO.

2.

10. The drug according to claim 9, characterized in that The molar concentration ratio of the siRNA1 and siRNA2 is 1:1.

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