Introduction

Severe burn injury commonly causes a complex traumatic event with various local and systemic effects, causing immeasurable physical and mental damage to the patient1,2. Neovascularization is essential for recovery after burn injury, and an integral link is the coordinated control of endothelial cells at the level of cell migration, proliferation, polarity, differentiation, and intercellular communication, contributing to the formation of functional vascular morphology3. Injury of vascular endothelial cells in the early stage of severe burn not only causes the destruction of vascular endothelial structure, but also seriously weakens the function of endothelial cells, which promotes the occurrence of shock and multiple organ involvement in the early stage of burn injury1.

Endothelial cells maintain the balance of fibrinolysis and coagulation, and play a major regulatory role in angiogenesis4. The proliferation, apoptosis and angiogenesis of vascular endothelial cells are affected by ischemia and hypoxia5,6. Therefore, the regulation of proliferation and apoptosis of endothelial cells is the key target to cope with hypoxic-ischemic state.

Catalpol has been widely reported for its potential protective effects in cardiovascular and cerebrovascular diseases7,8,9.Catalpol attenuates neuronal apoptosis and inhibit cell death in various animal models of ischemia10,11. In addition, Catalpol is also reported to stimulate cell proliferation and differentiation of osteoblasts in alleviating diabetes and its complications12. However, the function and mechanism of Catalpol in angiogenesis after burn injury remain uncharted.

Attributed to the previous reports, it was speculated that Catalpol may maintain endothelial survival by inhibiting apoptosis and promoting proliferation of endothelial cells after glucose oxygen deprivation. Therefore, current study explored whether Catalpol attenuates endothelial loss and attempted to explain the underlying molecular mechanisms.

Materials and methods

Antibodies and reagents

Reagents: Catalpol (HY-N0820, Medchemexpress), Dimethyl sulfoxide (DMSO, HY-Y0320, Medchemexpress), Trizol (R0016, Beyotime) 2 × Rapid Taq Master Mix (P222-01, Vazyme), HiScript III All-in-one RT SuperMix Perfect for qPCR (R333, Vazyme Biotech), One Step TUNEL Apoptosis Assay Kit (C1089, Beyotime), EdU Assay/EdU Staining Proliferation Kit (ab219801, abcam), Immunol Staining Blocking Buffer (P0102, Beyotime), 4% Paraformaldehyde Fix Solution (P0099, Beyotime), DAPI Staining Solution (C1005, Beyotime), Citrate Antigen Retrieval Solution (P0081, Beyotime), Enhanced Endogenous Peroxidase Blocking Buffer (P0100B, Beyotime), Triton X-100 (P0096, Beyotime), Recombinant Human VEGF-A (PH2038, APExBIO), Hif-1α-IN-2 (HY-115903, Medchemexpress).

Antibodies: GFAP Monoclonal antibody (60190-1-Ig, Proteintech), Anti-Bax antibody (50599-2-Ig, Proteintech), Bcl2 Polyclonal antibody (26593-1-AP, Proteintech), Cytochrome c Polyclonal antibody (10993-1-AP, Proteintech), Cleaved Caspase 3 Polyclonal antibody (25128-1-AP, Proteintech), PI3 Kinase p85 alpha Rabbit mAb (R22768, Zenbio), Phospho-PI3 Kinase p85/p55 (Tyr467/Tyr199) Rabbit pAb (341468, Zenbio), AKT Monoclonal antibody (60203-2-Ig, Proteintech), Phospho-AKT (Ser473) Recombinant antibody (80455-1-RR, Proteintech), HIF-1 alpha Polyclonal antibody (20960-1-AP, Proteintech), VEGF-A Polyclonal antibody (19003-1-AP, Proteintech), VEGFR-1 Polyclonal antibody (13687-1-AP, Proteintech), Beta Actin Recombinant antibody (81115-1-RR, Proteintech), GAPDH Monoclonal antibody (60004-1-Ig, Proteintech).

Cell culture and treatment

A cell line of human umbilical vein endothelial cells (HUVECs) was purchased from the Procell Life Technology Co. LTD (CL-0675, Wuhan, China). The HUVECs were cultured with dulbecco’s modified eagle medium (DMEM, PM150210, Procell) in addition with 10% TransSerum® FQ Fetal Bovine Serum (FS301-02, TransGen Biotech, Beijing, China) and 1% Penicillin-Streptomycin (FG101-01, TransGen Biotech). The cells were pre-treated with different dose (1, 10, 100 µM) of Catalpol dissolved in 0.1% DMSO for 24 h, and then replaced the culture media with a deoxygenated and glucose-free DMEM with the cells placing in an anaerobic workstation (Bugbox, Ruskinn Technology) perfused with a gas mixture of 5% CO2 and 95% N2 to induce hypoxia for 6 h at 37 ℃.

Immunofluorescence (IF) staining

4% Paraformaldehyde (PFA) was used to fix the cells for 15 min, then immunoblocking was conducted for 1 h at room temperature (RT). The cells were probed with the primary antibodies of interest overnight at 4 ℃, and further incubated with the fluorescent secondary antibodies at RT for 2 h in dark. After the nucleus counterstained by a diaminidine phenyl indole (DAPI) solution and mounted coverslips using a mounting medium, cell graphs were captured under a fluorescence imaging microscope (Olympus, Tokyo, Japan).

Tunel staining

After gently washing the cells with PBS solution, the cells were fixed with 4% paraformaldehyde for 10 min, dried at room temperature, and then hydrated with PBS solution. Then 50 µl of reagent was added according to the instructions of the TUNEL kit, and the cells were incubated at 37 ° C in the dark for 30 min. Add 30 µl of DAPI reagent (C1005, Beyotime) for 10 min with the cells incubated in the dark for another 5 min. The cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan).

EdU staining

Cells were incubated with medium containing 50 µM EdU for 2 h and then washed three times with PBS. After fixation with 4% paraformaldehyde solution for 30 min, the cells were added to 2 mg/mL glycine for 5 min, and then washed with PBS for 3 times. The cells were permeabilized by adding 0.5% TritonX-100 for 10 min and washed once with PBS. DAPI reagent was added for nuclear staining. Pictures were taken under fluorescence microscope and the EdU positive rate was calculated. EdU positive rate = number of EdU positive cells/total number of cells × 100%.

Western blot

Total protein was extracted using a Total Protein Extraction Kit (KeyGEN) according to the manufacturer’s protocol. After protein concentrations were determined by an Enhanced BCA Protein Quantitation Kit (Beyotime, Shanghai, China), equivalent protein was performed western blot assay. Protein was then probed with the specific primary antibodies at 4 ℃ overnight, afterwards incubated with HRP-labeled secondary antibodies at room temperature for 1 h, then determined the level by an automatic chemiluminescence image analysis system (4600, Tanon, China). Quantification was finally analyzed with the ImageJ software (NIH, NY, USA).

Quantitative RT-PCR

Total RNA was extracted from the cells using a Trizol reagent (YIFEIXUE BioTech, Nanjing, China). After the RNAs were purified by chloroform and isopropanol, reverse transcription was performed using a cDNA Synthesis Kit (R333, Vazyme Biotech, Nanjing, China). The cDNA was quantified by a PCR amplifier (Applied Biosystems, Thermo Fisher, USA). The relative level of the RNAs of interest were calculated using the ΔΔCt method. The primers we employed are listed at Table 1.

Table 1 Primers of interest for qPCR.
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Statistical analysis

Data were presented as mean ± standard deviation, and drawn charts using Prism GraphPad Software (Version 8, San Diego, CA, USA). Differences among multiple groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test. A p Value less than 0.05 was defined as statistically significance.

Results

Catalpol reduces OGD-mediated apoptosis in endothelial cells

Catalpol is known to prevent and improve the function of ischemic heart disease by inhibiting cardiomyocyte apoptosis. To investigate whether Catalpol affects endothelial cell apoptosis, the effect of different concentrations (1 μm, 10 μm, 100 μm) of Catalpol on the expression of apoptosis-related proteins including Bax, Bcl2, Cyto C, C-casp3 in the OGD model was examined (Fig. 1A). The results demonstrated that the Bax/Bcl2 protein expression ratio was highly decreased (Fig. 1A, B) and the expressions of Cyto C and C-casp3 proteins were effectively suppressed in the OGD model with the increase of Catalpol (Fig. 1A, C, D). IF staining showed that the increased expressions of Bax and C-casp3 in the cells under hypoxia and glucose deprivation conditions were reduced by Catalpol, while the result of Bcl2 expression was opposite (Fig. 1E-H). For cell death, the Tunel-positive cells deprived of glucose and oxygen also decreased markedly after high concentration of Catalpol treatment (Fig. 1I-J). It was indicated that Catalpol may have an inhibitory effect on endothelial cell apoptosis under OGD.

Fig. 1
figure 1

Administration of Catalpol reduces OGD-mediated endothelial cell apoptosis. Catalpol (1 μm, 10 μm, 100 μm) was added to the culture medium for 6 h, then the Human umbilical vein endothelial cells were subjected to OGD for 24 h. (A) Western blots were conducted to detect the Bax, Bcl2, Cyto C, C-casp3 expression level. β-Actin was used as the endogenous control. (B-D) Quantitative analysis of Bax/Bcl2 protein ratio and Cyto C, C-casp3 protein expression level. (E) Immunofluorescence staining of Bcl-2, Bax and C-casp3 was used to evaluate the effect of Catalpol on apoptosis signal. Scale bar = 50 μm. (F-H) Quantitative analysis of Bcl-2, Bax, C-casp3 fluorescence level. (I) Representative tunel staining results of control group, OGD group and OGD + Catalpol (1 μm, 10 μm, 100 μm) group. Scale bar = 100 μm. (J) Quantitative analysis of tunel + cell counting from tunel staining. The error bars represent the SD. The data are expressed as mean ± SD from three independent experiments by one-way ANOVA followed by t-test and Tukey’s post hoc analysis (*p < 0.05, **p < 0.01, and ***p < 0.001). OGD, oxygen-glucose deprivation.

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Catalpol promotes endothelial cell proliferation through VEGF/PI3K/AKT pathway

Recent studies have shown that the compound Catalpol possesses the capacity to enhance cell proliferation. Hif-1α is a transcription factor that plays a role in cellular adaptation to hypoxia. It promotes the transcription of a range of genes, including VEGF, which are involved in erythropoiesis, angiogenesis, energy metabolism of nucleosides, cell survival and apoptosis activity and other biological effects. IF results showed that the fluorescence intensity of Hif-1α increased after OGD, which was further enhanced by Catalpol treatment (Fig. 2A). VEGF promotes the proliferation and migration of vascular endothelial cells by activating VEGF receptors (VEGFR) on endothelial cells, thereby promoting neovascularization. A slight increase in VEGF and VEGFR mRNA expression was observed in endothelial cells after OGD treatment, although this was not statistically significant. After treatment with higher concentration (10 μm, 100 μm) of Catalpol, their mRNA expression was remarkably enhanced (Fig. 2B-C). Measurement of VEGF content in the culture medium and the intensity of VEGFR IF staining showed the same results (Fig. 2D-E). Further experiments were conducted to investigate the effect of Catalpol on the PI3K/AKT signaling pathway, which is a key downstream signaling of VEGFR related to cell proliferation. According to the WB results, it was found that compared with the control group, the ratios of p-PI3K/PI3K and p-AKT/AKT expression were decreased under the environment deprived of oxygen and glucose, while these ratios were dramatically increased by the addition of high concentration of Catalpol (Fig. 2F-H). The result of EdU staining indicated that the reduced percentage of EdU-positive cells resulting from OGD was reversed by the high concentration of Catalpol treatment (Fig. 2I-J). Our results demonstrated that Catalpol promotes endothelial cell proliferation by regulating VEGF/PI3K/AKT pathway.

Fig. 2
figure 2

Catalpol promotes endothelial cell proliferation through the VEGF/PI3K/AKT pathway. Catalpol (1 μm, 10 μm, 100 μm) was added to the culture medium for 6 h, then the Human umbilical vein endothelial cells were subjected to OGD for 24 h. (A) Double immunofluorescence labeling of endothelial cells for Hif-1α (green)/DAPI (blue). Scale bar = 50 μm. (B-C) Quantitative analysis of VEGF and VEGFR mRNA level. (D) Determination of VEGF content in the culture medium. (E) Double immunofluorescence labeling of endothelial cells for VEGFR (green)/DAPI (blue). Scale bar = 50 μm. (F) Western blots were conducted to detect the PI3K, p-PI3K, AKT, p-AKT expression level. GAPDH was used as the endogenous control. (G-H) Quantitative analysis of p-PI3K/PI3K and p-AKT/AKT protein ratio. (I) Representative EdU staining results of control group, OGD group and OGD + Catalpol (1 μm, 10 μm, 100 μm) group. Scale bar = 100 μm. (J) Quantitative analysis of EdU + cell counting from EdU staining. The error bars represent the SD. The data are expressed as mean ± SD from three independent experiments by one-way ANOVA followed by t-test and Tukey’s post hoc analysis (*p < 0.05, **p < 0.01, and ***p < 0.001).

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Inhibition of Hif-1α counteracts Catalpol induced endothelial cell proliferation by reducing VEGF expression

Whether inhibition of Hif-1α could counteract Catalpol-induced endothelial cell proliferation after OGD was next investigated. After the effects of higher Catalpol concentrations on endothelial cell proliferation and apoptosis were established, Catalpol at 100 μm concentration was selected for subsequent studies. Subsequent experiments were divided into four groups as shown in Fig. 3A. The expression of Hif-1α and VEGFR measured by IF staining showed that blockage of Hif-1α reduced VEGFR expression, which was rescued by supplement with Recombinant Human VEGF-A (rVEGF) (Fig. 3B-D). Accordingly, the results showed that, along with a remarkable decrease of p-PI3K/PI3K protein ratio, the protein expression of p-AKT compared with AKT also exhibited a marked decline after inhibition of Hif-1α. However, the reduction was reversed by addition of rVEGF (Fig. 3E-G). Meanwhile, EdU-positive cells were reduced with the presence of Hif-1α inhibitor in the OGD model containing Catalpol. Nevertheless, it showed an opposite trend after the addition of rVEGF (Fig. 3H-I). In short, the results demonstrated that suppressing Hif-1α can reduce the expression of VEGF and counteract the proliferation of endothelial cells induced by Catalpol.

Fig. 3
figure 3

Catalpol induced endothelial cell proliferation was counteracted by decreased VEGF expression resulting from inhibition of Hif-1α. (A) Different groups of endothelial cells under different treatments. Catalpol at 100 μm concentration was selected for subsequent experiments. rVEGF = VEGF145 Protein, A recombinant VEGF protein. Hif-1α-IN = Hif-1α-IN-2, A Hif-1α inhibitor. OGD, oxygen-glucose deprivation. (B) Double immunofluorescence labeling of endothelial cells for Hif-1α (green)/DAPI (blue) and VEGFR (green)/DAPI (blue). Scale bar = 50 μm. (C-D) Quantitative analysis of Hif-1α and VEGFR fluorescence level. (E) Western blots were conducted to detect the PI3K, p-PI3K, AKT, p-AKT expression level. GAPDH was used as the endogenous control. (F-G) Quantitative analysis of p-PI3K/PI3K and p-AKT/AKT protein ratio. (H) Representative EdU staining results of different groups. Scale bar = 100 μm. (I) Quantitative analysis of EdU + cell counting from EdU staining. The error bars represent the SD. (*p < 0.05, **p < 0.01, and ***p < 0.001).

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Inhibition of Hif-1α reverses the anti-apoptotic effect of Catalpol through blockage of the VEGF/PI3K/Akt pathway

The role of Hif-1α/VEGF pathway in the anti-apoptotic effect of Catalpol was further investigated. The expression of Cyto C, C-casp3 and Bax/Bcl2 ratio decreased by treatment of Catalpol in the OGD model. The decrease rebounded when Hif-1α is suppressed while supplement with rVEGF overwhelmed the effect of Hif-1α inhibitor (Fig. 4A-D). It was witnessed that Bcl2 fluorescence intensity enhanced by Catalpol was significantly reduced by Hif-1α inhibition, which was rescued by rVEGF supplement (Fig. 4E, F). Nevertheless, the expression of Bax and C-casp3 showed an opposite trend to that of Bcl2 (Fig. 4E, G, H). The percentage of Tunel-positive cells surged after addition of the Hif-1α inhibitor compared with Catalpol treatment alone in the OGD model. And a reduction of Tunel-positive cells was observed by adding rVEGF on the basis of previous treatment (Fig. 4I-J). Taking the previous results together, we suggested that the anti-apoptotic effect of Catalpol may depend on activating the Hif-1α/VEGF/PI3K/Akt pathway in OGD-treated HUVECs.

Fig. 4
figure 4

The anti-apoptotic effect of Catalpol was reversed via inhibition of Hif-1α by blocking the VEGF/PI3K/Akt pathway. Endothelial cells were treated and grouped as in Fig. 3A. (A) Western blots were conducted to detect the Bax, Bcl2, Cyto C, C-casp3 expression level. β-Actin was used as the endogenous control. (B-D) Quantitative analysis of Bax/Bcl2 protein ratio and Cyto C, C-casp3 protein expression level. (E) Immunofluorescence staining of Bcl-2, Bax and C-casp3 was used to evaluate the effect of Hif-1α inhibitor on the anti-apoptotic ability of Catalpol. Scale bar = 50 μm. (F-H) Quantitative analysis of Bcl-2, Bax, C-casp3 fluorescence level. I Representative tunel staining results of different groups. Scale bar = 100 μm. (J) Quantitative analysis of tunel + cell counting from tunel staining. The error bars represent the SD. (*p < 0.05, **p < 0.01, and ***p < 0.001).

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Discussion

In the present study, our data demonstrated that Catalpol is a potent inhibitor of apoptosis and able to facilitate endothelial cell proliferation by promoting the function of VEGF through increasing the expression of Hif-1α, thereby enhancing angiogenesis (Fig. 5).

Fig. 5
figure 5

Catalpol is a potent Hif-1α agonist, which can mediate the proliferation and inhibit the apoptosis of endothelial cells under oxygen and glucose deprivation by activating Hif-1α/VEGF and downstream related pathways.

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The apoptosis, proliferation and migration of endothelial cells play an important role in the dynamic process of vascular network expansion and remodeling in angiogenesis13. Catalpol, the major active component of Rehmanniae radix, has been reported to show potential protective effects in cardiovascular and cerebrovascular diseases7,14. However, its underlying mechanism in angiogenesis remains unclear. Therefore, present study established the OGD model using HUVECs, finding that Catalpol inhibits apoptosis and promotes endothelial cell proliferation after OGD.

The Bax/Bcl-2 ratio is an important determinant of apoptosis15. When Bax is overexpressed in cells, two Bax monosomes form a Bax-Bax homodimer, then the responsiveness of cells to death signals is enhanced to initiate apoptosis. However, when Bcl-2 is highly expressed, Bax-Bax dimer dissociates in large amounts, and a more stable Bcl-2/Bax heterodimer is formed, which antagonizes its apoptosis-inducing effect and prolongs cell survival16. Cyto C is released from mitochondria playing an important role in the progression of apoptosis17. Related studies have shown that Cyto C is also able to activate casepase-318. Caspase-3 is the executor of apoptosis and can be activated by Bax homodimer19. When caspase-3 is activated, procaspase-3 is cleaved into the active fragment cleaved caspase-3 (C-casp3), which acts as a proteolytic enzyme to promote apoptosis. As a representative marker of apoptosis, C-casp3 is frequently present in the cytoplasm during apoptosis20. Our findings showed that Catalpol was able to reduce the expression of Bax, CytoC and C-casp3, along with an increase in Bcl-2 expression, in endothelial cells deprived of oxygen and glucose.

The mechanism by which Catalpol protects endothelial cells from OGD damage was subsequently investigated. VEGF, reported to promote cell proliferation, is secreted by endothelial cells and has been shown to have an important role in angiogenesis and neurogenesis motivation7,21, which is dependent on the PI3K/AKT pathway22. The VEGF/ VEGF-receptor axis consists of a variety of ligands and receptors with overlapping and different ligand-receptor binding specificities, cell type expression, and functions21,23. Activation of the VEGFR pathway triggers a network of signaling processes that promote endothelial cell growth, migration, and survival from preexisting blood vessels22. Previous studies have shown that Catalpol rescues the reduced expression levels of VEGF24and its downstream signal pathways, such as PI3K/AKT pathway25,26. Correspondingly, our study in the OGD model also confirmed the protective effect of Catalpol.

It has been demonstrated that the importance of Hif-1α in promoting angiogenesis during wound healing27,28. When tissues are exposed to hypoxia, various cell types respond by upregulating hypoxia-inducible factor-1α(Hif-1α), which in turn induces the expression of many angiogenic genes. The vascular endothelial growth factor (VEGF) family is one of them29. Hif-1α mediates the downstream pro-angiogenic gene VEGF in response to hyperoxia injury, which can promote endothelial cell proliferation and migration30. Our results showed that the increase of Hif-1α expression was consistent with that of VEGF, and the effect of Catalpol promoting VEGF expression on cell proliferation and anti-apoptosis was blocked after the addition of Hif-1α inhibitor, which is consistent with the reported interaction between Hif-1α and VEGF.

In summary, the present study suggested that Catalpol possesses potent effects on anti-apoptosis and pro-proliferation in OGD-treated HUVECs, which is depended on Hif-1α-induced VEGF production and activation of the VEGFR/PI3K/AKT pathway. Our results may provide a new rationale for the clinical application of Catalpol as a promising burn agent, but its pharmacologic action needs to be explored systematacially in other cells and more in vivo of studies.

Authors’ contributions: Jinrong Ni and Jielin Deng conceived and designed the experiments. Jinrong Ni funded and supervised the research. Jinrong Ni and Qunhu Zhang performed experiments. Luetao Jiang and Haihu Wang performed data analysis. Chengji Zhang assisted the experiments. Jinrong Ni wrote and edited the manuscript.