阅读说明：本技术 Gyy4137在制备抑制血管内皮细胞焦亡的药物中的应用 (Application of GYY4137 in preparation of medicine for inhibiting scorching of vascular endothelial cells ) 是由 张亮 李玲 何婷婷 张旭琳 于 2021-08-26 设计创作，主要内容包括：本发明提供了GYY4137在制备抑制血管内皮细胞焦亡的药物中的应用,属于生物医药技术领域。H-2S供体GYY4137可通过下调NLRP3炎性小体—Caspase-1信号通路而抑制心血管疾病致病危险因素氧化低密度脂蛋白(oxLDL)和同型半胱氨酸(Hcy)诱导的血管内皮细胞焦亡关键蛋白(NLRP3、Pro-Caspase-1、Caspase-1(P20)、GSDMD、GSDMD-N及IL-18)的表达上调(P<0.05和P<0.01)和血管内皮细胞焦亡关键酶Caspase-1活性,从而抑制血管内皮细胞焦亡。(The invention provides an application of GYY4137 in preparing a medicament for inhibiting scorching of vascular endothelial cells, belonging to the technical field of biological medicines. H 2 The S donor GYY4137 can inhibit the up-regulation of the expression (P) of key proteins (NLRP3, Pro-Caspase-1, Caspase-1(P20), GSDMDM-N and IL-18) of vascular endothelial cell apoptosis induced by oxidized low density lipoprotein (oxLDL) and homocysteine (Hcy) which are pathogenic risk factors of cardiovascular diseases by down-regulating an NLRP3 inflammasome-Caspase-1 signal channel<0.05 and P<0.01) and the key enzyme Caspase-1 activity of the scorch of vascular endothelial cells, thereby inhibitingVascular endothelial cells are burned out.)

The application of GYY4137 in preparing medicines for inhibiting the activity reduction of vascular endothelial cells, inhibiting the expression up-regulation of key protein of scorch of the vascular endothelial cells, inhibiting the activity of Caspase-1 which is a key enzyme of the scorch of the vascular endothelial cells, inhibiting the scorch of the vascular endothelial cells or repairing inflammatory injury of the vascular endothelial cells.

2. The use of claim 1, wherein the decreased activity of vascular endothelial cells, the upregulation of critical protein apoptosis in vascular endothelial cells, the increase in activity of the critical enzyme Caspase-1 in vascular endothelial cell apoptosis, the apoptosis of vascular endothelial cells, or the inflammatory injury of vascular endothelial cells is caused by a causative agent of a cardiovascular disease.

3. The use according to claim 2, wherein the cardiovascular disease causing factors comprise a first factor and/or a second factor; the first factor is oxLDL; the second factor is Hcy and LPS.

4. The use according to claim 1 or 2, wherein the key enzyme of vascular endothelial cell apoptosis is Caspase-1.

5. The use according to claim 1 or 2, wherein the key apoptosis proteins of vascular endothelial cells comprise one or more of NLRP3, Pro-Caspase-1, GSDMD-N and IL-18.

6. The use of claim 1, wherein said vascular endothelial cells comprise human umbilical vein endothelial cells.

7. The use of claim 1, wherein the pharmaceutical dosage form comprises an injectable formulation.

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of GYY4137 in preparation of a medicine for inhibiting scorching of vascular endothelial cells.

Background

Apoptosis is a new programmed, inflammatory mode of cell death discovered following apoptosis and necrosis. Recent studies show that the inflammatory death of vascular endothelial cells, namely cell scorching, plays a key role in the occurrence and development processes of various cardiovascular diseases (hyperlipidemia, hypertension, atherosclerosis, coronary heart disease and heart failure), metabolic diseases (diabetes and complications thereof such as retinopathy) and chronic inflammatory diseases.

The key molecular pathway for cell apoptosis to occur is the signal pathway of cell apoptosis mediated by nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP 3)/cysteine-containing aspartic acid proteolytic enzyme-1 (Caspase-1), and it has been shown that cardiovascular diseases and metabolic disease pathogenic risk factors such as oxidized low density lipoprotein, hyperlipidemia, cholesterol crystal, nicotine, homocysteine, hypersalt, hyperglycemia can cause activation of NLRP3 in vascular endothelial cells, NLRP3 can bind to apoptosis-related spot-like protein (ASC), further recruiting and activating a key protein Caspase-1 generated by cell apoptosis to form a tetramer consisting of P10 subunits and P20 subunits; the activated Caspase-1 can crack and activate downstream endothelin D (Gasderm D, GSDMD) and apoptosis related inflammatory mediators (such as Interleukin-1 beta (Interleukin-1 beta, IL-1 beta) and Interleukin-18 (Interleukin-18, IL-18) precursors), and the N-end domain of the protein of the activated GSDMDM can be assembled on the surface of a cell membrane to form membrane perforation, so that vascular endothelial cell apoptosis is finally triggered.

Research has shown that the key protein in the cell apoptosis signal path can be blocked or knocked out by medicine, which can effectively inhibit the occurrence and development of vascular endothelial cell apoptosis and cardiovascular diseases (atherosclerosis and hypertension). Therefore, the cell apoptosis and the signal path thereof are taken as important intervention targets, which can provide a new idea for protecting the vascular endothelium and treating cardiovascular diseases.

Disclosure of Invention

The invention aims to provide application of GYY4137 in preparing a medicament for inhibiting vascular endothelial cell scorching, H₂The S donor GYY4137 can inhibit the up-regulation of the expression of key protein of apoptosis of vascular endothelial cells by down-regulating NLRP3 inflammasome-Caspase-1 signal path.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of GYY4137 in preparing medicines for inhibiting the activity reduction of vascular endothelial cells, inhibiting the expression up-regulation of key protein for the scorching of the vascular endothelial cells, inhibiting the activity of Caspase-1, inhibiting the scorching of the vascular endothelial cells or repairing inflammatory injury of the vascular endothelial cells.

Preferably, the activity reduction of the vascular endothelial cells, the expression up-regulation of key protein of the scorching of the vascular endothelial cells, the activity increase of Caspase-1 of the key enzyme of the scorching of the vascular endothelial cells, the scorching of the vascular endothelial cells or the inflammatory injury of the vascular endothelial cells are caused by the pathogenic factors of cardiovascular diseases.

Preferably, the cardiovascular disease causing factors comprise a first factor and/or a second factor; the first factor is oxLDL; the second factor is Hcy and LPS.

Preferably, the key enzyme of the vascular endothelial cell apoptosis is Caspase-1.

Preferably, the key protein of the vascular endothelial cell apoptosis comprises one or more of NLRP3, Pro-Caspase-1, GSDMD-N and IL-18.

Preferably, the vascular endothelial cells comprise human umbilical vein endothelial cells.

Preferably, the dosage form of the medicament comprises an injection.

The invention provides GYY4137 for preparing a medicine for inhibiting the activity reduction of vascular endothelial cells, inhibiting the expression up-regulation of key protein for the scorching of the vascular endothelial cells, inhibiting the activity of the key enzyme Caspase-1 for the scorching of the vascular endothelial cells, inhibiting the scorching of the vascular endothelial cells or repairing the inflammatory diseases of the vascular endothelial cellsUse of GYY4137 as H in medicaments for treating lesions₂S donor, significantly increased the activity of HUVECs treated with oxLDL or Hcy + LPS, the causative agent of cardiovascular disease (P)<0.05); oxLDL or Hcy + LPS can remarkably up-regulate the expression of cell apoptosis key marker proteins NLRP3, Caspase-1 (active P20 subunit), GSDMD-N terminal fragment and cytokine IL-18 (P20 subunit) in HUVECs<0.05 or P<0.01); GYY4137 can also inhibit the expression up-regulation (P) of key proteins (NLRP3, Pro-Caspase-1, Caspase-1(P20), GSDMDM-N and IL-18) of oxLDL and Hcy induced apoptosis of vascular endothelial cells by inhibiting the activity of Caspase-1, a key enzyme of apoptosis of vascular endothelial cells and down-regulating the NLRP3 inflammatory corpuscle-Caspase-1 signal pathway<0.05 and P<0.01) to inhibit scorching of vascular endothelial cells.

Drawings

FIG. 1 is a graph showing the effect of varying concentrations of oxLDL and GYY4137 on human umbilical vein endothelial cell activity; wherein A is the effect of different concentrations of oxLDL (25, 50, 100mg/L) on the activity of human umbilical vein endothelial cells, B is the effect of different concentrations of GYY4137(50, 100, 200. mu. mol/L) on the activity of human umbilical vein endothelial cells, and C is the effect of the treatment of specified concentrations of oxLDL (100mg/L) alone and in combination with different concentrations of GYY4137(100, 200. mu. mol/L) on the activity of human umbilical vein endothelial cells; data statistics as mean valuesRepresents, n is 5; compared with the control group, the compound of the formula,^*P<0.05 or^**P<0.01; in comparison with the oxLDL-treated group,^#P<0.05。

FIG. 2 is a graph showing the effect of different concentrations of Hcy and Hcy + LPS on the activity of human umbilical vein endothelial cells; wherein A is the influence of different concentrations of Hcy (50, 100, 250 and 500 mu mol/L) on the activity of human umbilical vein endothelial cells, B is the influence of the combined treatment of different concentrations of Hcy (100, 250 and 500 mu mol/L) and 1 mu g/mL LPS on the activity of human umbilical vein endothelial cells, and C is the influence of the combined treatment of different concentrations of Hcy (100, 250 and 500 mu mol/L) and 10 mu g/mL LPS on the activity of human umbilical vein endothelial cells; data statistics as mean valuesRepresents, n is 5; compared with the control group, the compound of the formula,^*P<0.05 or^**P<0.01。

FIG. 3 is a graph of the effect of varying concentrations of GYY4137 and Hcy + LPS intervention and PAG on human umbilical vein endothelial cell activity; wherein A is the effect of Hcy (500. mu. mol/L) + LPS (10. mu.g/mL) treatment at the indicated concentration and its combined treatment with GYY4137(50, 100, 200. mu. mol/L) at different concentrations on the activity of human umbilical vein endothelial cells, and B is the effect of PAG (1, 5, 10mmol/L) at different concentrations on the activity of human umbilical vein endothelial cells; data statistics as mean valuesRepresents, n is 5; compared with the control group, the compound of the formula,^*P<0.05 or^**P<0.01; compared with the Hcy + LPS-treated group,^#P<0.05。

FIG. 4 is a graph of the effect of different concentrations of oxLDL on human umbilical vein endothelial cell apoptosis; wherein A is oxLDL (25, 50, 100mg/L) with different concentrations to human umbilical vein endothelial cell apoptosis detection index PI⁺Effect of staining, B is PI in human umbilical vein endothelial cells treated with different concentrations of oxLDL (25, 50, 100mg/L)⁺The statistical result of the proportion (%) of the stained cells shows that C is the influence of oxLDL (25, 50 and 100mg/L) with different concentrations on the activity of the human umbilical vein endothelial cell apoptosis detection index LDH; data statistics as mean values Represents, n is 3; compared with the control group, the compound of the formula,^*P<0.05 or^**P<0.01。

FIG. 5 is a graph showing the effect of different concentrations of Hcy on human umbilical vein endothelial cell apoptosis; wherein A is Hcy (100, 250 and 500 mu mol/L) with different concentrations and LPS (10 mu g/mL) combined treatment for detecting human umbilical vein endothelial cell apoptosis index PI⁺Effect of staining, B is P in human umbilical vein endothelial cells treated with different concentrations of Hcy (100, 250, 500. mu. mol/L) in combination with 10. mu.g/mLLPSI⁺The statistical result of the proportion (%) of the stained cells shows that C is the influence of the combined treatment of Hcy (100, 250 and 500 mu mol/L) with different concentrations and 10 mu g/mL LPS on the activity of the human umbilical vein endothelial cell apoptosis detection index LDH; data statistics as mean valuesSD represents, n ═ 3; compared with the control group, the compound of the formula,^**P<0.01。

FIG. 6 is a graph showing the effect of different concentrations of oxLDL on the expression of a pivotal marker protein for apoptosis in human umbilical vein endothelial cells; wherein A is a representative immunoblot diagram of the influence of different concentrations of oxLDL (25, 50, 100mg/L) on the expression of the critical apoptosis marker protein NLRP3 in human umbilical vein endothelial cells treated with different concentrations of oxLDL (25, 50, 100mg/L), C is the analysis result of the relative expression level of the critical apoptosis marker protein Pro-Caspase-1 in human umbilical vein endothelial cells treated with different concentrations of oxLDL (25, 50, 100mg/L), D is the analysis result of the relative expression level of the critical apoptosis marker protein Caspase-1 in human umbilical vein endothelial cells treated with different concentrations of oxLDL (25, 50, 100mg/L), E is the analysis result of the relative expression level of the critical apoptosis marker protein GSDMD in human umbilical vein endothelial cells treated with different concentrations of oxLDL (25, 50, 100mg/L), f is the analysis result of the relative expression level of the pivotal death marker protein GSDMDM-N in the human umbilical vein endothelial cells treated by different concentrations of oxLDL (25, 50 and 100mg/L), and G is the analysis result of the relative expression level of the pivotal death marker protein IL-18 in the human umbilical vein endothelial cells treated by different concentrations of oxLDL (25, 50 and 100 mg/L); data statistics as mean valuesRepresents, n is 3; compared with the control group, the compound of the formula,^*P<0.05 or^**P<0.01。

FIG. 7 is a graph showing the effect of different concentrations of Hcy on the expression of human umbilical vein endothelial cell apoptosis-related protein; wherein A is Hcy (100, 250, 500 mu mol/L) with different concentrations and 10 mu g/mL LPS combined treatment, and the key target of the combined treatment on the scorching in the endothelial cells of the human umbilical veinA representative immunoblot of the effect of expression of proteins, B being the analysis of the relative expression levels of the pivotal marker protein GSDMD in human umbilical vein endothelial cells treated with different concentrations of Hcy (100, 250, 500. mu. mol/L) in combination with 10. mu.g/mL LPS, C being the analysis of the relative expression levels of the pivotal marker protein Caspase-1 in human umbilical vein endothelial cells treated with different concentrations of Hcy (100, 250, 500. mu. mol/L) in combination with 10. mu.g/mL LPS; data statistics as mean valuesRepresents, n is 3; compared with the control group, the compound of the formula,^*P<0.05 or^**P<0.01。

FIG. 8 is a graph showing the H in GYY4137 versus oxLDL and Hcy treated human umbilical vein endothelial cells₂Influence of S content; wherein A is 100mg/L oxLDL or 200. mu. mol/L GYY4137 alone and oxLDL (100mg/L) + GYY4137 (200. mu. mol/L) in combination are used for treating H in human umbilical vein endothelial cells₂Influence of S content, B is H in human umbilical vein endothelial cells treated with Hcy (500. mu. mol/L) + LPS (10. mu.g/mL), 200. mu. mol/L GYY4137, 5mmol/L PAG alone and in combination₂Influence of S content; data statistics as mean valuesRepresents, n is 4; compared with the control group, the compound of the formula,^*P<0.05，^**P<0.01; compared with oxLDL or Hcy + LPS treated groups,^#P<0.05; compared with the Hcy + LPS + PAG treated group,^&P<0.05。

FIG. 9 is a graph of the effect of GYY4137 on oxLDL induced apoptosis of human umbilical vein endothelial cells; wherein A is 100mg/LoxLDL or 200 mu mol/L GYY4137 for single treatment and oxLDL (100mg/L) + GYY4137(200 mu mol/L) combined treatment for human umbilical vein endothelial cell apoptosis detection index PI⁺Effect of staining, PI in human umbilical vein endothelial cells treated with 100mg/LoxLDL or 200. mu. mol/L GYY4137 alone and oxLDL (100mg/L) + GYY4137 (200. mu. mol/L) in combination⁺Percentage (%) of stained cells, C100 mg/LoxLDL or 200. mu. mol/L GYY4137 treatment alone and oxLDL (100mg/L) + GYY4137 (200. mu. mol/L) combination treatment on human umbilical veinEndothelial cell apoptosis detection index LDH activity; data statistics as mean valuesRepresents, n is 3; compared with the control group, the compound of the formula,^*P<0.05; in comparison with the oxLDL-treated group,^#P<0.05。

FIG. 10 is a graph of the effect of GYY4137 on Hcy-induced human umbilical vein endothelial cell apoptosis; wherein A is Hcy (500 mu mol/L) + LPS (10 mu g/mL), 200 mu mol/L GYY4137, 5mmol/L PAG single treatment and combined treatment thereof for detecting human umbilical vein endothelial cell apoptosis index PI⁺Effect of staining, B is PI in human umbilical vein endothelial cells treated with Hcy (500. mu. mol/L) + LPS (10. mu.g/mL), 200. mu. mol/L GYY4137, 5mmol/L PAG alone and in combination⁺The result of statistics on the proportion (%) of the stained cells shows that C is the influence of single treatment and combined treatment of Hcy (500 mu mol/L) + LPS (10 mu g/mL), 200 mu mol/L GYY4137 and 5mmol/L PAG on the activity of human umbilical vein endothelial cell apoptosis detection index LDH; data statistics as mean valuesRepresents, n is 3; compared with the control group, the compound of the formula,^**P<0.01; compared with the Hcy + LPS-treated group,^##P<0.05; compared with the Hcy + LPS + PAG treated group,^&P<0.05,^&&P<0.01。

FIG. 11 is a graph showing the effect of GYY4137 on Caspase-1 activity, a key enzyme for apoptosis, in oxLDL or Hcy treated human umbilical vein endothelial cells; wherein A is the effect of combined treatment of GYY4137(50, 100, 200 mu mol/L) and 100mg/LoxLDL at different concentrations on Caspase-1 enzyme activity in human umbilical vein endothelial cells, and B is the effect of single treatment of Hcy (500 mu mol/L) + LPS (10 mu g/mL), 200 mu mol/L GYY4137, 5mmol/L PAG and combined treatment thereof on Caspase-1 enzyme activity in human umbilical vein endothelial cells; data statistics as mean valuesRepresents, n is 3; compared with the control group, the compound of the formula,^*P<0.05，^**P<0.01，^***P<0.001；compared with oxLDL or Hcy + LPS treated groups,^#P<0.05，^##P<0.01; compared with the Hcy + LPS + PAG treated group,^&&P<0.01。

FIG. 12 is a graph of the effect of GYY4137 on apoptosis-related protein expression in oxLDL treated human umbilical vein endothelial cells; wherein A is a representative immunoblot analysis of the effect of the treatment with 100mg/L oxLDL or 200. mu. mol/L GYY4137 alone and the combined treatment with oxLDL (100mg/L) + GYY4137 (200. mu. mol/L) on the expression of the pivotal apoptosis marker protein in human umbilical vein endothelial cells, B is an analysis of the relative expression level of the pivotal apoptosis marker protein NLRP3 in human umbilical vein endothelial cells treated with 100mg/L oxLDL or 200. mu. mol/L GYY4137 alone and the combined treatment with oxLDL (100mg/L) + GYY4137 (200. mu. mol/L), C is an analysis of the relative expression level of the pivotal apoptosis marker protein Pro-Caspase-1 in human umbilical vein endothelial cells treated with 100mg/L oxLDL or 200. mu. mol/L GYY4137 alone and the combined treatment with oxLDL (100mg/L) + GYY4137 (200. mu. mol/L), and D is an analysis of the relative expression level of the pivotal apoptosis marker protein Pro-Caspase-1 in human umbilical vein endothelial cells treated with 100mg/L oxLDL or 200. mu. mol/L YY4137 alone and the combined treatment with 100mg/L YY4137(100 mg/L Mu mol/L), E is the analysis result of the relative expression level of Caspase-1 in human umbilical vein endothelial cells treated with the combination of 100mg/L of oxLDL or 200 mu mol/L of GYY4137 alone and GSDMD as a key marker of apoptosis in human umbilical vein endothelial cells treated with oxLDL (100mg/L) + GYY4137(200 mu mol/L), F is the analysis result of the relative expression level of GSDMD-N as a key marker of apoptosis in human umbilical vein endothelial cells treated with 100mg/L of oxLDL or 200 mu mol/L of GYY4137 alone and oxLDL (100mg/L) + GYY4137(200 mu mol/L), G is the analysis result of the relative expression level of GSDMD-N as a key marker of apoptosis in human umbilical vein endothelial cells treated with 100mg/L of oxLDL or 200 mu mol/L of GYY4137 alone and oxLDL (100mg/L) + GYY4137(200 mu mol/L) together Analyzing the result; data statistics as mean valuesRepresents, n is 3; compared with the control group, the compound of the formula,^*P<0.05; in comparison with the oxLDL-treated group,^#P<0.05。

FIG. 13 is a table of proteins associated with apoptosis in Hcy-treated human umbilical vein endothelial cells treated with GYY4137The effect of the arrival; wherein A is a representative immunoblot graph of the effect of Hcy (500. mu. mol/L) + LPS (10. mu.g/mL), 200. mu. mol/L GYY4137, 5mmol/L PAG treatment alone and combination thereof on the expression of the key focal death marker protein in human umbilical vein endothelial cells, B is an analysis result of the relative expression level of the key focal death marker protein NLRP3 in human umbilical vein endothelial cells treated alone and combination thereof with Hcy (500. mu. mol/L) + LPS (10. mu.g/mL), 200. mu. mol/L GYY4137, 5mmol/L PAG treatment alone and combination thereof, C is an analysis result of the relative expression level of the key death marker protein Pro-Caspase-1 in human umbilical vein endothelial cells treated alone and combination thereof with Hcy (500. mu. mol/L) + LPS (10. mu.g/mL), 200. mu. mol/L GYY4137, 5mmol/L PAG, d is the relative expression level analysis result of the pyroptosis key marker protein Caspase-1 in the human umbilical vein endothelial cells treated by Hcy (500 mu mol/L) + LPS (10 mu g/mL), 200 mu mol/L GYY4137, 5mmol/L PAG alone and the combination thereof, E is the relative expression level analysis result of the pyroptosis key marker protein GSDMD in the human umbilical vein endothelial cells treated by Hcy (500 mu mol/L) + LPS (10 mu g/mL), 200 mu mol/L GYY4137, 5mmol/L PAG alone and the combination thereof, F is the relative expression level analysis result of the pyroptosis key marker protein GSDMD-N in the human umbilical vein endothelial cells treated by Hcy (500 mu mol/L) + LPS (10 mu g/mL), 200 mu mol/L GYY4137, 5mmol/L PAG alone and the combination thereof, g is the analysis result of the relative expression level of the key focal death marker protein IL-18 in human umbilical vein endothelial cells treated by Hcy (500 mu mol/L) + LPS (10 mu G/mL), GYY4137 at 200 mu mol/L, 5mmol/L PAG alone and the combination thereof; data statistics as mean valuesRepresents, n is 3; compared with the control group, the compound of the formula,^**P<0.01; compared with the Hcy + LPS-treated group,^##P<0.05; compared with the Hcy + LPS + PAG treated group,^&&P<0.01。

Detailed Description

The invention provides application of GYY4137 in preparing medicines for inhibiting the activity reduction of vascular endothelial cells, inhibiting the expression up-regulation of key protein for the scorching of the vascular endothelial cells, inhibiting the activity of Caspase-1, inhibiting the scorching of the vascular endothelial cells or repairing inflammatory injury of the vascular endothelial cells.

In the present invention, said GYY4137 is H having vasodilator and antihypertensive activity₂The small molecular release agent of S is slow release type H₂The hydrogen sulfide released from the S donor, GYY4137, can act directly as an active ingredient; the GYY4137 is commercially available conventionally, and is preferably purchased from Sigma-Aldrich (Cat. No: SML 0100); the reagent adopted when the GYY4137 is used for preparing the solution is preferably dimethyl sulfoxide (DMSO) and water; the solubility of the GYY4137 in water is 1-30 mg/mL.

In the invention, the reduction of the activity of the vascular endothelial cells, the increase of the activity of a key enzyme Caspase-1 of the scorching of the vascular endothelial cells, the up-regulation of the expression of key protein of the scorching of the vascular endothelial cells, the scorching of the vascular endothelial cells or the inflammatory injury of the vascular endothelial cells are preferably caused by cardiovascular disease pathogenic factors.

In the present invention, the cardiovascular disease causing factors preferably include a first factor and/or a second factor; the first factor is oxLDL; the second factor is Hcy and LPS.

In the present invention, the key enzyme for apoptosis of vascular endothelial cells preferably includes Caspase-1.

In the invention, the key protein of the vascular endothelial cell apoptosis preferably comprises one or more of NLRP3, Pro-Caspase-1, GSDMD-N and IL-18.

In the present invention, the vascular endothelial cells preferably include human umbilical vein endothelial cells.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

1 Material

1.1 cells Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from American Type Culture Collection (ATCC) laboratory.

1.2 endothelial cell specific culture medium and fetal bovine serum were purchased from Sciencell, USA; oxidized low density lipoprotein (oxLDL) was purchased from Guangzhou Yiyuan Biotechnology Ltd, homocysteine (Hcy), Lipopolysaccharide (LPS), H₂S donor GYY4137 and endogenous H₂S synthetase inhibitor DL-propargylglycine (D, L-propagylglycine, PAG) was purchased from Sigma-Aldrich, USA; the CCK-8 cell activity detection kit is purchased from Dongzren chemical technology (Shanghai) Co., Ltd; hoechst 33342/Propidium Iodide (PI) double staining kit and Lactate Dehydrogenase (LDH) cytotoxicity detection kit were purchased from Nanjing to establish the bioengineering institute; caspase-1 activity detection kit is purchased from Shanghai Biyuntian biotechnology limited company; the cell protein extraction kit and the skimmed milk powder are purchased from Beijing Solaibao science and technology Limited; NLRP3 rabbit polyclonal antibody, Pro-Caspase-1 rabbit monoclonal antibody, Caspase-1(P20 subunit) rabbit polyclonal antibody, GSDMD rabbit monoclonal antibody, GSDMD-N rabbit monoclonal antibody end fragment antibody are purchased from Abcam company of England, IL-18 rabbit monoclonal antibody is purchased from Wuhan Sanying company, GAPDH mouse monoclonal antibody and horseradish peroxidase labeled secondary antibody (HRP goat anti-rabbit or HRP goat anti-mouse) are purchased from Beijing China fir bridge biotechnology, Inc.; the immunoblotting chemiluminescence substrate kit was purchased from Millipore, USA.

1.3 Main Instrument CO₂Incubator (Thermo Fisher Scientific corporation); high speed cryogenic refrigerated centrifuge (Eppendorf corporation); an automatic microplate reader (Thermo Fisher Scientific Co.); inverted fluorescence microscope (SANYO Co.); electrophoresis apparatus and membrane transfer apparatus (six biotech ltd, beijing).

2 method

2.1 cell culture cryopreserved resuscitated HUVECs at 2X 10⁶cells/L seeded at 25cm²In a cell culture flask, a special endothelial cell culture solution (containing 5% fetal calf serum, 1% double antibody and 1% endothelial growth factor) is added into a cell culture box (37 ℃, 5% CO)₂And saturation humidity), replacing cell culture solution every 2-3 days according to cell growth speed, carrying out passage, taking 2-4 generations of HUVECs in logarithmic growth phase for endothelial cell identification (the positive rate of endothelial cell specificity marker CD31 is more than or equal to 95 percent) and subsequent research, and adjusting the cell density during inoculation to be 1 multiplied by 10 according to different experimental purposes⁶～5×10⁶cells/mL.

2.2 pharmaceutical intervention and grouping

2.2.1 concentration of drug intervention HUVECs in logarithmic growth phase (confluence 80% -90%) were inoculated into a 96-well plate containing 5% fetal bovine serum broth (inoculation density 8X 10) prior to drug intervention³Cells/well) adhered to the wall for 4h, then replaced by serum-free culture solution for starvation culture for 12h, and finally treated by different drugs. Different concentrations of oxLDL (25, 50, 100mg/L), Hcy (50, 100, 250, 500. mu. mol/L) + LPS (1, 10. mu.g/mL), PAG (1, 5, 10mmol/L) and GYY4137(50, 100, 200. mu. mol/L) and a given drug combination (GYY4137(50, 100, 200. mu. mol/L) were used for pretreatment for 4h, 100mg/L of oxLDL or Hcy (500. mu. mol/L) + LPS (10. mu.g/mL) + PAG (5mmol/L) were added for further culture for 24h in complete serum culture medium containing 1% of serum, and then different concentrations of oxLDL, Hcy + LPS, PAG, GYpas 37, the activity of the cells of the HUCs, the activity of cascs, the activity of the pyrogen-signaling proteins of the key pathway proteins and the expression thereof were measured by CCK-8 method (see 2.3 cell activity test), and screening out the optimal interference concentrations of oxLDL, Hcy + LPS, PAG and GYY 4137.

2.2.2 experiments grouping HUVECs in logarithmic growth phase (confluence 80% -90%) were inoculated into 6-well plates containing serum medium (inoculation density 1X 10) prior to drug intervention⁶Cells/well) attached to the wall for 4h, then replaced by serum-free culture solution for starvation culture for 12h, and finally treated with different drugs, and divided into the following 9 groups:

(1) blank control group: culturing HUVECs in complete culture medium containing 1% serum for 24 h;

(2) GYY4137 intervention group: HUVECs were treated with GYY4137 at a final concentration of 200. mu. mol/L for 24 h;

(3) PAG intervention group: treating HUVECs with PAG with final concentration of 5mmol/L for 24 h;

(4) oxLDL treatment group: treating cells with oxLDL at a final concentration of 100mg/L for 24 h;

(5) hcy (500. mu. mol/L) + LPS (10. mu.g/mL): treating the cells with 500. mu. mol/L Hcy + 10. mu.g/mL LPS for 24 h;

(6) hcy + LPS + PAG intervention group: treating the cells with 500. mu. mol/L Hcy + 10. mu.g/mL LPS +5mmol/LPAG for 24 h;

(7) oxLDL + GYY4137 dry run: 50. HUVECs are pretreated for 4h by 100 and 200 mu mol/L GYY4137, and then oxLDL cells with the final concentration of 100mg/L are added for treating for 24 h;

(8) hcy + LPS + GYY4137 dried group: 200 mu mol/L GYY4137 pre-treating HUVECs for 4h, and then adding 500 mu mol/L Hcy +10 mu g/mL LPS to treat cells for 24 h;

(9) hcy + LPS + PAG + GYY4137 dried group: HUVECs were pretreated with 200. mu. mol/L GYY4137 for 4h, and cells were treated with 500. mu. mol/L Hcy + 10. mu.g/mL LPS +5mmol/L PAG for 24 h.

2.3 cell activity detection until the confluency of the HUVECs is about 80-90%, digesting and collecting the HUVECs by using 0.05% trypsin, inoculating the cells into a 96-well plate, culturing for 4 hours in a cell culture box by using a serum-containing culture solution, and replacing the cells after the cells are attached to the wall with a serum-free culture solution for starvation culture for 12 hours. The different drugs mentioned above were then added for 24h intervention. After the intervention, the stock culture was discarded and rinsed with PBS, after which 100. mu.L of culture containing 10% CCK-8 reagent was added to each well, and blank wells (culture containing no cells, only 10% CCK-8) were provided. After incubation for 2h in a cell incubator, the absorbance of each well was measured at a wavelength of 450nm using a microplate reader (absorbance, A). Each set of experiments was set up with 6 replicate wells, replicated 5 times.

Relative cell viability (%) - (sample average a value-blank well average a value)/(blank control average a value-blank well average a value) × 100%.

2.4 Hoechst33342/PI double staining to detect cell apoptosis after the above groups of cells are subjected to different drug intervention treatment for 24h, 10 μ L of Hoechst33342(5mg/L) staining solution is added into a 6-well plate containing cells, the 6-well plate is incubated at 37 ℃ in the dark for 10min, then 5 μ L of PI (2mg/L) is added into the cells, the 6-well plate is incubated at 37 ℃ in the dark for 15min, and PBS solution is added into the 6-well plateRinsing for 2 times, observing and photographing with inverted fluorescence microscope, randomly selecting 3 different fields, and calculating total number of cells and red fluorescence cells in each field, according to literature [ Tang Biao, Tang Wen Jing, Dai Dynasty, etc. ] Panax notoginsenosides inhibits SH-SY5Y cell apoptosis induced by anoxia, sugar deficiency and compound oxygen]J pathophysiology,2020,36 (7):1178-1184.(TANG Biao, TANG Wen-jing, DAI Zi-wei, et al, Panax nonoginseng saponin suspensions recycling oxygen-glucose depletion/reoxygenation-induced shrinkage of SH-SY5Y cells [ J]Chinese Journal of Pathophysiology,2020,36(7):1178-⁺The cell ratio (%) ═ red fluorescent cell number/total cell number × 100%. The above experiment was repeated 3 times.

2.5 detection of Lactate Dehydrogenase (LDH) Release Rate of each group of cells inoculated in a 96-well plate, after 24h of different drug intervention treatment, first of all, each group of cell culture supernatant (25. mu.L) was collected in the 96-well plate, then 25. mu.L of a reaction substrate was added to a serum-free culture medium control and the 96-well plate containing 25. mu.L of the culture supernatant, and incubated at 37 ℃ for 15min, then 25. mu.L of 2, 4-dinitrophenylhydrazine was added to the above reaction sample, and incubated at 37 ℃ for 15min for a color reaction, finally 250. mu.L of a reaction terminator was added to the reaction sample, and incubated at room temperature for 5min for a termination reaction, and finally the A value was measured at 490nm wavelength according to the formula: LDH release rate (%) — (treated sample absorbance-control sample absorbance)/(absorbance for maximum enzyme activity of cells-control sample absorbance) × 100%, LDH content in the supernatant of each group of cell culture solution was calculated. The above experiment was repeated 4 times.

2.6 H₂S content determination after each group of cells are subjected to drug intervention treatment for 24h at different specified concentrations, each group of cells are collected by trypsinization, 500 mu L of precooled phosphate buffer (50mM, pH 6.8) is added into the cells to lyse the homogenate, and a part of cell lysate is taken to determine the total protein concentration in each group of cell lysate; adding phosphate buffer (pH 7.4) containing 2mmol/L pyridoxal phosphate and 10mmol/L L-cysteine into a centrifuge tube containing 430. mu.L of cell lysate, sealing the centrifuge tube with a sealing film, and incubating in a water bath at 37 ℃ for 30 min; then centrifuged through the centrifuge tube cap using a syringe to centrifuge the sealInjecting 250 μ L of 1% zinc acetate into the tube, mixing, and incubating the reaction mixture in a 37 deg.C water bath for 90 min; then adding 250 mu L of trichloroacetic acid (10%, w/v), 133 mu L of LN, N-dimethyl-p-phenylenediamine (20mmol/L) and 133 mu L of ferric trichloride (30mmol/L) into a centrifuge tube in sequence, fully mixing uniformly, and centrifuging for 10min under the conditions of 4 ℃ and 12000 Xg; then, 200 mu L of each group of supernatant and standard reaction solution (NaHS,0-250 mu mol/L) are absorbed and added into a 96-hole enzyme label plate, and the A value of each sample to be detected and the A value of each standard substance are measured by using an enzyme label instrument at the wavelength of 670 nm. Finally, H in each group of cells is calculated according to the protein concentration₂S content (unit: nmol/10)⁶cell). The above experiment was repeated 3 times.

2.7 Caspase-1 enzyme Activity assay

After the HUVECs in each group are subjected to different drug interventions for 24h, cells are collected by digestion with 0.25% pancreatin and low-speed centrifugation at 4 ℃, and 100 mu L of lysate is added into the cells for ice bath lysis for 15 min. The cell lysate was then centrifuged at 16,000 Xg for 15min at 4 ℃ and a small amount of supernatant was aspirated to determine the protein concentration in the cells using the BCA kit. And then, sequentially adding a detection buffer solution, a sample to be detected (containing 50 mu g of protein) and a Caspase-1 reaction substrate Ac-YVAD-pNA (2mM) in the Caspase-1 activity detection kit into the new centrifugal tube, fully mixing uniformly, and incubating for 60-120 min at 37 ℃. And finally, detecting the light absorption value of each sample at the wavelength of 405nm by using a microplate reader, and calculating the Caspase-1 enzyme activity in each group of cells according to a standard curve drawn by a standard substance p-nitroaniline (pNA).

2.8 immunoblotting experiments HUVECs in logarithmic growth phase were inoculated into 6-well plates (1X 10)⁶Cells/well) were treated with different drugs for 24 h. Collecting each group of cells, extracting total protein of the cells according to the kit operation instructions, measuring the protein concentration, and storing at-80 ℃ for later use. An equal amount of 50. mu.g of protein was loaded and separated by electrophoresis on an 8% or 12% SDS-PAGE gel at constant pressure. Transferring the total protein to a PVDF membrane by a wet transfer method, and sealing the PVDF membrane in a 5% skimmed milk powder solution at room temperature for 1-2 h; the blocked PVDF membrane was rinsed with 1 XTSST buffer at room temperature and placed in a solution containing anti-NLRP 3(1:1000), anti-Pro-Caspase-1 (1:1000), anti-CCaspase-1(1:1000), anti-GSDMD-N (1:1000), anti-IL-18 (1:1000) and anti-GAPDH (1:1000) antibody solutions were incubated overnight at 4 ℃; after the redundant primary antibody on the membrane is rinsed by TBST buffer solution, the membrane is put into secondary antibody solution (1:10000) to be incubated for 1-2 h at room temperature. Washing the membrane, developing, exposing and tabletting, observing the result and analyzing. The above experiment was repeated 3 times.

3 statistical treatment

Statistical analysis and histogram plotting were performed using GraphPad Prism 6 software. Statistical results of all experimental data in this study are mean. + -. standard deviationAnd (4) showing. Data comparisons between groups 2 were performed using the t-test and between groups 3 were performed using one-way ANOVA. With P<0.05 and P<A difference of 0.01 is statistically significant.

Results

GYY4137 inhibition of oxLDL or Hcy + LPS-induced reduction of HUVECs Activity

To screen and optimize the optimal concentration of drug used, the CCK-8 method was first used to evaluate oxLDL, Hcy + LPS, PAG or/and H₂Effect of S-delayed donor GYY4137 on HUVECs activity. The experimental results are shown in fig. 1, 2 and 3. Compared with the blank control group, low concentration oxLDL (25mg/L), Hcy (50. mu. mol/L), PAG (1mmol/L) and medium and high concentration GYY4137(50 and 100. mu. mol/L) have no significant effect on HUVECs activity (P>0.05), moderate to high concentrations of oxLDL (50mg/L and 100mg/L), Hcy (100, 250, 500. mu. mol/L) + LPS (1, 10. mu.g/mL) and PAG (5, 10mmol/L) resulted in a significant reduction in HUVECs activity (P. sub.VECs activity)<0.05 and P<0.01), see a in fig. 1, fig. 2 and fig. 3; 200 mu mol/L GYY4137 can significantly increase HUVECs activity (P)<0.05), see B in fig. 1. In addition, GYY4137 significantly increased the activity of oxLDL or Hcy + LPS-treated HUVECs (P) compared to oxLDL-treated and Hcy + LPS-treated groups<0.05), see C in fig. 1 and a in fig. 3.

Establishment of HUVECs cell apoptosis model induced by oxLDL or Hcy + LPS

To further confirm the promotion of oxLDL or Hcy + LPS on the scorching of vascular endothelial cellsFurther, the influence of oxLDL or Hcy + LPS on the cell apoptosis and the expression of apoptosis key proteins of HUVECs is analyzed through LDH activity determination, Hoechst33342/PI staining and immunoblotting. The results showed that low concentrations of oxLDL (25mg/L) resulted in a lower LDH release rate, PI, in HUVECs than the placebo group⁺The proportion of stained cells and the expression of the focal death key protein had no significant effect (P)>0.05), and 50mg/L and 100mg/LoxLDL, Hcy (100, 250, 500. mu. mol/L) + LPS (10. mu.g/mL) make PI of HUVECs⁺The dyeing proportion and the LDH release rate are obviously improved (P)<0.05 and P<0.01), see fig. 4 and 5; in addition, 50mg/L and 100mg/LoxLDL or Hcy (100, 250, 500. mu. mol/L) + LPS (10. mu.g/mL) significantly up-regulated the expression of the key markers of apoptosis NLRP3, Caspase-1 (active P20 subunit), GSDMD, GSDMDM-N-terminal fragment and cytokine IL-18 (P20) in HUVECs as compared to the blank control group<0.05 or P<0.01), but oxLDL had no significant effect on Pro-Caspase-1 expression and Hcy + LPS on GSDMD expression compared with the blank control group (P)>0.05), see fig. 6 and 7.

GYY4137 inhibiting oxLDL or Hcy + LPS induced HUVECs pyro-death, Caspase-1 enzyme activity increase and cell pyro-death key marker protein expression

To clarify the inhibition effect of GYY4137 on oxLDL or Hcy + LPS-induced scorching of HUVECs and possible regulation mechanism thereof, 100mg/LoxLDL, Hcy (500. mu. mol/L) + LPS (10. mu.g/mL), PAG (5mmol/L), Hcy (500. mu. mol/L) + LPS (10. mu.g/mL) + PAG (5mmol/L) and 200. mu. mol/L GYY4137 were selected to intervene HUVECs individually or in combination, and H in each group of cells was detected₂S level, PI⁺The proportion of stained cells, LDH release rate, Caspase-1 activity, and expression of key marker proteins of cellular apoptosis.

The results show that oxLDL, Hcy + LPS, PAG, Hcy + LPS + PAG can remarkably inhibit H in HUVECs after being treated by oxLDL, Hcy + LPS + PAG compared with a blank control group₂Synthesis of S (P)<0.05), and oxLDL, Hcy + LPS + PAG treatment can remarkably up-regulate cellular LDH release rate, PI⁺Proportion of stained cells (P)<0.05 and P<0.01), Caspase-1 enzyme Activity (P)<0.01 and P<0.001) and the expression (P20) of the proteins critical to the apoptosis signal pathway (NLRP3, Pro-Caspase-1, Caspase-1(P20), GSDMD-N and IL-18)<0.05 and P<0.01) (ii) a Compared with the oxLDL group, the Hcy + LPS group and the Hcy + LPS + PAG group, the combined interference of GYY4137 and oxLDL/Hcy + LPS/Hcy + LPS + PAG can obviously improve H in HUVECs₂S content (P)<0.05), and reacting PI⁺The proportion of staining cells, the LDH release rate and the Caspase-1 enzyme activity are obviously reduced (P is less than 0.05 and P is<0.01) and GYY4137 can also obviously inhibit the expression up-regulation (P20), induced by oxLDL or Hcy + LPS, of key proteins of cellular apoptosis (NLRP3, Pro-Caspase-1, Caspase-1(P20), GSDMD-N and IL-18)<0.05 and P<0.01), see fig. 8, 9, 10, 11, 12, 13.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.