阅读说明：本技术 女贞苷在制备防治钙化性主动脉瓣疾病药物中的应用 (Application of ligustrin in preparation of medicine for preventing and treating calcified aortic valve diseases ) 是由 王勇军 董念国 韩东 周廷文 于 2021-08-04 设计创作，主要内容包括：本发明涉及女贞苷在制备治疗钙化性主动脉瓣疾病药物中的应用。本发明经研究发现女贞苷可以有效下调瓣膜间质细胞中成骨分化标志基因RUNX2和Osterix的表达,抑制瓣膜间质细胞成骨分化,从而延缓或者抑制瓣膜钙化,并提供了一种通过女贞苷处理后可显著改善瓣膜间质细胞的体外成骨分化的方法,进而为钙化性主动脉瓣疾病的预防和治疗提供了新的药物选择。(The invention relates to application of ligustrazine in preparing a medicament for treating calcified aortic valve diseases. According to the invention, the ligustilide is found to be capable of effectively reducing the expression of osteogenic differentiation marker genes RUNX2 and Osterix in valve interstitial cells and inhibiting osteogenic differentiation of the valve interstitial cells, so that valve calcification is delayed or inhibited, and a method capable of remarkably improving in-vitro osteogenic differentiation of the valve interstitial cells after treatment of the ligustilide is provided, so that a new medicine selection is provided for prevention and treatment of calcified aortic valve diseases.)

1. Application of ligustilide in preparing medicine for preventing and treating calcified aortic valve diseases is provided.

2. The use of ligustilide in the preparation of a medicament for the prevention and treatment of calcified aortic valve disease according to claim 1, wherein the ligustilide is a compound extracted from the leaves of ligustrum lucidum ait of the genus ligustrum of the family meliaceae, CAS number 260413-62-5, molecular formula C₃₃H₄₀O₁₈。

3. The pharmaceutical according to claim 2, wherein the concentration of said ligustrazine is 10 μ M to 50 μ M.

4. The pharmaceutical of claim 3, wherein the concentration of said ligustrazine is 25 μ M.

5. The use of ligustilide in the preparation of a medicament for the prevention and treatment of calcified aortic valve diseases according to claim 1, wherein the ligustilide is used in the preparation of a medicament for the intervention of osteogenic differentiation of valve stromal cells.

6. The use of ligustilide in the preparation of a medicament for the prevention and treatment of calcified aortic valve disorders according to claim 1, wherein the ligustilide is used in the preparation of a medicament for modulating the expression or activity of CasR.

7. A cell culture method for inhibiting osteogenic differentiation of valve interstitial cells is characterized in that human valve interstitial cells are induced and cultured for 7-14 days by using a calcification culture medium containing ligustilide, wherein the composition of the calcification culture medium containing the ligustilide is as follows: 25 mu M of ligustilide, 50mg/mL of vitamin C, 5mmol/L of beta-glycerophosphoric acid, 100nmol/L of dexamethasone, 2% of fetal calf serum and a high-sugar medium (DMEM).

8. The method of claim 7, wherein the induction culture is carried out in a mixed gas containing 5 vol.% carbon dioxide and 95 vol.% oxygen at a constant temperature of 37 ℃.

The technical field is as follows:

the invention relates to the field of biological medicines, and in particular relates to application of ligustrazine in preparation of a medicine for preventing and treating calcified aortic valve diseases.

Background art:

calcified Aortic Valve Disease (CAVD) is a progressive disease of high morbidity and mortality in the elderly, with major pathophysiological changes being fibroproliferative calcification of the aortic valve leaflets leading to valve stiffening and causing hemodynamic changes affecting cardiac function. From the past, CAVD is considered to be an age-related degenerative disease, which is a degenerative and hardening process of valve tissue with age. However, basic research in recent years indicates that CAVD is an active progress process involving complex pathological changes such as endothelial injury, inflammatory cell infiltration, extracellular matrix remodeling, and osteogenic differentiation of valve interstitial cells. Cells in valve tissue mainly include Valve Endothelial Cells (VECs), Valve Interstitial Cells (VICs), valve precursor cells, and the like, and research has proved that calcium salt increase caused by osteogenic differentiation of valve interstitial cells may be an important initiation factor for valve calcification. Currently, CAVD lacks an effective clinically available drug treatment regimen, the primary treatment of which is aortic valve replacement surgery. However, the patient who is subjected to the surgical operation inevitably bears a high medical operation risk and an economic burden. Therefore, exploring the specific pathogenesis of CAVD, the effective prevention and/or treatment of calcified aortic valve disorders by non-surgical methods is currently an urgent need for clinical treatment of CAVD.

Calcium-sensing receptors (casrs), a member of the G-protein coupled receptor family, are involved in regulating the synthesis and secretion of parathyroid hormone and are widely distributed in the parathyroid gland, gastrointestinal tract, nervous system and bone tissues, and are capable of sensing and affecting changes in extracellular calcium ion concentrations. The study found that activation of CasR in valve stromal cells may play a key role in promoting the progression of cav, suggesting the possibility of improving calcified aortic valve disease by drug modulation of CasR expression or activity.

The ligustilide is a special component of traditional Chinese medicine glossy privet fruit, has definite pharmacological activity, and has obvious effects on maintaining calcium metabolism balance and preventing senile osteoporosis. The patent with the patent application number of CN201910418873.7 provides the application of acteoside and/or ligustrazine A in preparing the medicine for preventing and treating the vascular endothelial cell inflammatory injury. Recent studies have also shown that ligustrazine is a Potential CasR antagonist (Feng R, et al. protective Effects of Ligustroflavone, an Active Compound from Ligustrum lucidum, on Diabetes-Induced Osteoporosis in Mice: A Potential cancer as Calcium-Sensing Receptor antagonist. am J Chin Med.2019; 47(2):457 + 476). However, no report has been found on the study between ligustrazine and calcified aortic valve diseases.

The invention content is as follows:

technical problem to be solved

Aiming at the background, the invention discovers that the ligustilide can effectively reduce the expression of osteogenic differentiation marker genes RUNX2 and Osterix in valve interstitial cells through research, inhibit the osteogenic differentiation of the valve interstitial cells, so as to delay or inhibit the calcification of the valve, provides the application of the ligustilide in preparing the medicament for preventing and treating the calcified aortic valve diseases, provides a method for obviously improving the in-vitro osteogenic differentiation of the valve interstitial cells after the treatment of the ligustilide, and further provides a new medicament selection for preventing and treating the calcified aortic valve diseases.

(II) technical scheme

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention discloses the first aspect, provides the application of ligustrazine in preparing the medicament for preventing and treating calcified aortic valve diseases, and in the invention, the ligustrazine refers to the CAS number of 260413-62-5; a substance with a molecular formula of C33H40O18 has a chemical structure shown in FIG. 1A.

Specifically, the ligustrazine is used for preparing a medicament for interfering the osteogenic differentiation of valve interstitial cells.

Further, the ligustrazine is used for preparing a medicine for regulating CasR expression or activity.

Further, the concentration of the ligustrazine is 10-50 mu M.

Further, the concentration of the ligustrazine is 25 μ M.

Ligustrum lucidum is a potential antagonist of CasR, shows that the Ligustrum lucidum can inhibit osteogenic differentiation of valve interstitial cells under an in vitro osteogenic differentiated cell model induced by osteogenesis, and proves that the Ligustrum lucidum can lower the expression of osteogenic differentiation marker genes RUNX2 and Osterix by inhibiting the expression of a calcium sensitive receptor CasR to play a role in resisting calcification.

The invention discloses a second aspect, which provides a cell culture method for inhibiting osteogenic differentiation of valve interstitial cells, wherein human valve interstitial cells are induced and cultured for 7-14 days by using a calcification culture medium containing ligustilide, and the composition of the calcification culture medium containing the ligustilide is as follows: 25 mu M of ligustilide, 50mg/mL of vitamin C, 5mmol/L of beta-glycerophosphoric acid, 100nmol/L of dexamethasone, 2% of fetal calf serum and a high-sugar medium (DMEM).

Specifically, the induction culture is carried out in a mixed gas with 5% by volume of carbon dioxide and 95% by volume of oxygen, and the culture temperature is constant at 37 ℃.

(III) advantageous effects

The invention has the following beneficial effects: the invention provides application of ligustilide in preparation of a medicine for preventing and treating calcified aortic valve diseases, wherein the ligustilide can effectively inhibit CasR expression and can reduce the expression of valve interstitial cell osteogenic differentiation marker genes RUNX2 and Osterix, so that the valve interstitial cell osteogenic differentiation is inhibited, and the aortic valve calcification resisting effect is exerted.

Description of the drawings:

in order to more clearly illustrate the technical solution in the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below.

FIG. 1A shows the chemical structure of Ligustrum lucidum ait;

fig. 1B is a graph of the change in CasR protein levels in vitro calcification-induced culture conditions of human valvular stromal cells improved by treatment with 25 μ M concentration of ligustrin: the control group represents the conventional non-calcification induction medium culture condition, OM represents the calcification induction medium culture condition, and OM + ligustilide represents the ligustilide-treated group.

FIG. 2 is a graph showing that the change of the immunofluorescence level of CasR protein in vitro calcification-induced culture conditions of human valvular interstitial cells improved by the treatment of 25 μ M ligustrin: the control group represents the conventional non-calcification induction medium culture condition, OM represents the calcification induction medium culture condition, and OM + ligustilide represents the ligustilide-treated group.

Fig. 3A is a graph showing that the change of protein level of calcification marker gene of human valve mesenchymal cells under in vitro calcification-inducing culture conditions is improved by treating 25 μ M of ligustilide: the control group represents the conventional non-calcification induction medium culture condition, OM represents the calcification induction medium culture condition, and OM + ligustilide represents the ligustilide-treated group.

Fig. 3B is a graph showing the change in alkaline phosphatase activity of human valvular stromal cells improved by treatment with 25 μ M ligustilide concentration under in vitro calcification-inducing culture conditions: the control group represents the conventional non-calcification induction medium culture condition, OM represents the calcification induction medium culture condition, and OM + ligustilide represents the ligustilide-treated group.

Fig. 4A is a graph of calcium nodule formation of human valve stromal cells improved by treatment with 25 μ M concentration of ligustilide under in vitro calcification-inducing culture conditions: the control group represents the conventional non-calcification induction medium culture condition, OM represents the calcification induction medium culture condition, and OM + ligustilide represents the ligustilide-treated group.

Fig. 4B is a graph of the change in calcium content of human valve stromal cells improved by treatment with 25 μ M concentration of ligustilide under in vitro calcification-inducing culture conditions: the control group represents the conventional non-calcification induction medium culture condition, OM represents the calcification induction medium culture condition, and OM + ligustilide represents the ligustilide-treated group.

Fig. 5A is a graph showing that the treatment of ligustrazine with a concentration of 25 μ M improves the protein level change of calcification marker genes caused by CasR overexpression of human valvular stromal cells under in vitro calcification-inducing culture conditions: OM represents calcification induction culture medium culture conditions, OM + CasR represents a CasR overexpression group, and OM + CasR + ligustrin represents a ligustrin treatment group.

Fig. 5B is a graph of calcium nodule changes caused by CasR overexpression in human valvular stromal cells improved by treatment with 25 μ M concentration of ligustilide under in vitro calcification-inducing culture conditions: OM represents calcification induction culture medium culture conditions, OM + CasR represents a CasR overexpression group, and OM + CasR + ligustrin represents a ligustrin treatment group.

Fig. 5C is a graph of quantitative changes in calcium caused by CasR overexpression in vitro calcification-induced culture conditions for human valvular stromal cells improved by 25 μ M ligustilide treatment: OM represents calcification induction culture medium culture conditions, OM + CasR represents a CasR overexpression group, and OM + CasR + ligustrin represents a ligustrin treatment group.

The specific implementation mode is as follows:

the technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

Step one, constructing an osteogenic differentiated cell model of human valve interstitial cell induced by a calcification induction culture medium.

Taking human valve interstitial cells with the culture density of about 70 percent, starving the human valve interstitial cells overnight by using a DMEM high-sugar culture medium containing 2 percent fetal calf serum, performing osteogenic differentiation induction on the human valve interstitial cells by newly configuring an osteogenic induction culture medium (50mg/mL vitamin C, 5mmol/L beta-glycerophosphate, 100nmol/L dexamethasone and a high-sugar DMEM culture medium containing 2 percent fetal calf serum in a solvent) on the second day, and detecting the expression changes of CasR, calcification marker genes RUNX2 and Osterix proteins of different treatment groups 7 days after induction. The effect of different treatments on the osteogenic differentiation of valve stromal cells was examined after 21 days of induction.

Step two, the ligustrazine inhibits the osteogenic differentiation phenotype of human valve interstitial cell cells.

By adopting the human valve interstitial cell osteogenic differentiation model constructed in the first step, the human valve interstitial cells are divided into 3 different treatment groups, namely a control group (normal culture medium), an OM group (osteogenic induction culture medium) and an OM + ligustilide group (osteogenic induction culture medium + ligustilide), wherein the ligustilide is purchased from MedChemexpress (https:// www.medchemexpress.cn/angustoflex. html), and the product catalog HY-N0546 is the final concentration in the culture medium. Extracting protein after all groups are treated for 7 days at the same time to detect the expression change of the CasR, calcification marker genes RUNX2 and Osterix protein; observing the expression change of the CasR by immunofluorescence staining; after 21 days of treatment, each group was stained with alizarin red, and osteogenic differentiation phenotypic changes were observed.

From FIGS. 1B and 2, it can be seen that the expression level of CasR protein is significantly increased (P < 0.05) compared to the control group 7 days after OM treatment of human valve stromal cells. And when the ligustrazine with the concentration of 25 mu M is added into the OM treatment group, the up-regulation effect of the expression of the CasR protein induced by the OM culture medium is obviously inhibited (P is less than 0.05).

As can be seen from fig. 3A and 3B, the expression levels of RUNX2 and Osterix (osteogenic differentiation marker gene) proteins were significantly increased (P < 0.05) compared to the control group 7 days after OM treatment of human valve stromal cells. And when the ligustrazine with the concentration of 25 mu M is added into the OM treatment group, the up-regulation effect of RUNX2 and Osterix protein expression induced by OM culture medium is obviously inhibited (P is less than 0.05). After 7 days of OM treatment of human valve stromal cells, cellular alkaline phosphatase activity was significantly increased compared to the control group (P < 0.05). When ligustilide is added into OM treatment group at a concentration of 25 μ M, the OM culture medium-induced increase effect of cell alkaline phosphatase activity is significantly inhibited (P < 0.05).

As can be seen from fig. 4A and 4B, alizarin red staining and calcium quantitative analysis were performed on each group of human valve stromal cells 21 days after treatment, and the results showed that the alizarin red positive staining and calcium content of the cells were significantly increased compared to the control group (P < 0.05) 7 days after the OM treatment. And when the Ligustrum lucidum ait with the concentration of 25 mu M is added into the OM treatment group, the red positive staining of the cell alizarin and the calcium content increasing effect (P is less than 0.05) induced by the OM culture medium are obviously inhibited.

The results show that the ligustilide can inhibit the in vitro osteogenic differentiation phenotype transformation of human valve interstitial cells.

Step three, the ligustilide can inhibit the human valve interstitial cell osteogenic differentiation phenotype enhancement effect caused by CasR overexpression.

The over-expression adenovirus is constructed by separating and culturing valve interstitial cells. After transfecting the valve interstitial cells by using the over-expression adenovirus for 72 hours, inducing the valve interstitial cells by using an osteogenic differentiation induction culture medium for 7 days to construct an in vitro osteogenic differentiation model. And (3) evaluating the calcification degree of valve interstitial cells by using alizarin red staining, calcium quantitative analysis and detection of RUNX2 and Osterix (osteogenic differentiation marker gene) protein expression level. Alizarin red staining and calcium quantification showed a significant increase in calcium salt nodules and calcium salt deposition (P < 0.05) in valve mesenchymal cells after CasR overexpression compared to the control group. While ligustrin treatment inhibited the effect of calcium salt nodule and calcium salt deposit increase in human valve stromal cells caused by CasR overexpression (P < 0.05), as shown in FIGS. 5A and 5B.

The RUNX2 and Osterix protein expression level detection shows that the expression levels of RUNX2 and Osterix protein of valve mesenchymal cells after over-expressing CasR are obviously increased (P is less than 0.05) compared with a control group. While the treatment of the ligustrin can inhibit the up-regulation effect of the expression level of human valve interstitial cell RUNX2 and Osterix protein caused by CasR over-expression (P is less than 0.05), as shown in figure 5C.

The results show that the ligustilide can inhibit the osteogenic differentiation phenotype transformation of human valve mesenchymal cells by inhibiting the expression of CasR.

In conclusion, the ligustrazine can effectively reduce the expression of osteogenic differentiation marker genes RUNX2 and Osterix in valve interstitial cells and inhibit the osteogenic differentiation of the valve interstitial cells, so that the valve calcification is delayed or inhibited, and a new medicine selection is provided for the prevention and treatment of calcified aortic valve diseases.

Finally, it should be noted that the above examples are only used for illustrating the present invention and do not limit the protection scope of the present invention. In addition, after reading the technical content of the invention, the skilled person can make various changes, modifications or variations to the invention, and all the equivalents thereof also belong to the protection scope defined by the claims of the present application.