Pro‑differentiating compounds for human intervertebral disc cells are present in Violina pumpkin leaf extracts

Abstract

Intervertebral disc (IVD) degeneration (IDD) is closely associated with inflammation, oxidative stress and loss of the discogenic phenotype, which current therapies are unable to reverse. In the present study, the effects of acetone extract from Violina pumpkin (Cucurbita moschata) leaves on degenerated IVD cells were investigated. IVD cells were isolated from the degenerated disc tissue of patients undergoing spinal surgery and were exposed to acetone extract and three major thin layer chromatography subfractions. The results revealed that, in particular, the cells benefited from exposure to subfraction Fr7, which consisted almost entirely of p‑Coumaric acid. Western blot and immunocytochemical analysis showed that Fr7 induced a significant increase in discogenic transcription factors (SOX9 and tricho‑rhino‑phalangeal syndrome type I protein, zinc finger protein), extracellular matrix components (aggrecan, collagen type II), cellular homeostasis and stress response regulators, such as FOXO3a, nuclear factor erythroid 2‑related factor 2, superoxide dismutase 2 and sirtuin 1. Two important markers related to the presence and activity of stem cells, migratory capacity and OCT4 expression, were assessed by scratch assay and western blotting, respectively, and were significantly increased in Fr7‑treated cells. Moreover, Fr7 counteracted H2O2‑triggered cell damage, preventing increases in the pro‑inflammatory and anti‑chondrogenic microRNA (miR), miR‑221. These findings strengthen the hypothesis that adequate stimuli can support resident cells to repopulate the degenerated IVD and restart the anabolic machinery. Taken together, these data contribute to the discovery of molecules potentially effective in slowing the progression of IDD, a disease for which there is currently no effective treatment. Moreover, the use of part of a plant, the pumpkin leaves, which is usually considered a waste product in the Western world, indicated that it contains substances with potential beneficial effects on human health.

Keywords: discogenic phenotype; intervertebral disc degeneration; microRNA‑221; pumpkin leaf extracts; p‑Coumaric acid.

Figure 1

Schematic flow chart of the extraction and isolation process of compounds from Violina pumpkin (Cucurbita moschata) leaves (10). GC-MS, gas chromatography-mass spectrometry; NMR, nuclear magnetic resonance.

Figure 2

Effects of Violina pumpkin leaf extracts on cell viability. (A) IVD cells were treated with different concentrations (5, 50 and 250 µg/ml) of Ac or thin layer chromatography subfractions Fr2, Fr6 and Fr7 for 72 h. Cell viability was measured using the MTT assay. The viability of CTR cells was set at 100% (dotted line). Data are presented as the mean ± standard deviation (n=3). ^*P<0.05, ^**P<0.01 vs. CTR; ^#P<0.01 vs. DMSO. (B) IVD cells were treated with 5 µg/ml Ac, Fr2, Fr6 or Fr7 for 72 h. Cell viability was monitored by double staining with calcein AM/PI. Green fluorescence indicates the presence of calcein-labelled live cells, whereas PI-labelled dead cells are revealed by red fluorescence. Merged photomicrographs are shown. Scale bars, 20 µm. (C) IVD cells were treated with 5 µg/ml Ac, Fr2, Fr6 or Fr7 for 72 h and cytoskeletal organization was analysed by Alexa Fluor 488 phalloidin staining and fluorescence microscopy. Representative images of the cells are shown. Nuclei were counterstained with DAPI (blue). Scale bars, 10 µm. Ac, acetone extract; CTR, untreated control; IVD, intervertebral disc; PI, propidium iodide.

Figure 3

Effects of Violina pumpkin leaf extracts on the wound closure of IVD cells. IVD cells were treated with 5 µg/ml Ac, Fr2, Fr6 or Fr7 and subjected to wound scratch assay. Images were captured at 0 and 72 h. Quantitative evaluation and statistical analysis of wound closure percentage were measured using ImageJ software. Data are presented as the mean ± standard deviation (n=3). ^*P<0.01 vs. DMSO; ^#P<0.01 vs. Ac; ^§P<0.01 vs. Fr2; ^{^}P<0.01 vs. Fr6. Representative photomicrographs are shown. Scale bar, 50 µm. Ac, acetone extract; IVD, intervertebral disc.

Figure 4

SOX9, TRPS1 and FOXO3a expression in intervertebral disc cells treated with Violina pumpkin leaf extracts. Protein expression levels were evaluated by immunocytochemistry following treatment with 5 µg/ml Ac, Fr2, Fr6 or Fr7 for 72 h. Representative optical photomicrographs of immunostaining are shown. Scale bars, 20 µm. Protein levels were semi-quantified by densitometric analysis of immunocytochemical staining using ImageJ software and are expressed as fold change relative to DMSO-treated cells. Data are presented as the mean ± SD (n=3). ^*P<0.05 vs. DMSO; ^#P<0.05 vs. Ac; ^§P<0.01 vs. Fr2; ^{^}P<0.05 vs. Fr6. Ac, acetone extract; TRPS1, tricho-rhino-phalangeal syndrome type I protein, zinc finger protein.

Figure 5

ACAN, COL2a1 and MMP13 expression in intervertebral disc cells treated with Violina pumpkin leaf extracts. Protein expression levels were evaluated by immunocytochemistry following treatment with 5 µg/ml Ac, Fr2, Fr6 or Fr7 for 72 h. Representative optical photomicrographs of immunostaining are shown. Scale bars, 20 µm. Protein levels were semi-quantified by densitometric analysis of immunocytochemical staining using ImageJ software and are expressed as fold change relative to DMSO-treated cells. Data are presented as the mean ± SD (n=3). ^*P<0.05 vs. DMSO; ^#P<0.05 vs. Ac; ^§P<0.05 vs. Fr2; ^{^}P<0.05 vs. Fr6. Ac, acetone extract; ACAN, aggrecan; COL2a1, collagen type α1 chain.

Figure 6

Nrf2, SIRT1, SOD2, OCT4 and SOX2 expression in intervertebral disc cells treated with Violina pumpkin leaf extracts. Protein expression levels were evaluated by western blotting after treatment with 5 µg/ml Ac or Fr7 for 72 h. Representative western blots are presented. Relative protein expression levels were calculated by densitometry and the results are presented as fold change relative to DMSO-treated cells. Data are presented as the mean ± SD (n=3). PI3K was used as a loading control. ^*P<0.05, ^**P<0.01 vs. DMSO; ^#P<0.01 vs. Ac. Ac, acetone extract; Nrf2, nuclear factor erythroid 2-related factor 2; SIRT1, sirtuin 1; SOD2, superoxide dismutase 2.

Figure 7

Effects of Ac and Fr7 of Violina pumpkin leaves on damage induced by H₂O₂. (A) IVD cells were pretreated with 5 µg/ml Ac or Fr7 for 6 h and then cultured with different concentrations of H₂O₂ for 16 h. Cell viability was measured using the MTT assay. The viability of DMSO-treated cells was set as 100% (dotted line). Data are presented as the mean ± SD (n=3). ^*P<0.01 vs. DMSO; ^#P<0.05 vs. DMSO/H₂O₂ (0.5 mM). IVD cells were pretreated with 5 µg/ml Ac or Fr7 for 6 h and then cultured with 0.5 mM H₂O₂ for 16 h. (B) Protein expression levels of pro-apoptotic FasR and anti-apoptotic Bcl-2 were evaluated by immunocytochemistry. Representative optical photomicrographs of immunostaining are shown. Scale bars, 20 µm. Protein levels were semi-quantified by densitometric analysis of immunocytochemical staining using ImageJ software and are expressed as fold change relative to DMSO-treated cells. Data are presented as the mean ± SD (n=3). (C) Intracellular ROS production was evaluated. ROS levels are presented as a percentage relative to DMSO-treated cells. Data are presented as the mean ± SD (n=3). (D) Expression levels of miR-221 relative to U6 small nuclear RNA expression were determined by reverse transcription-quantitative PCR. Expression levels are presented as fold change relative to DMSO-treated cells and the data are expressed as the mean ± SD (n=3).^*P<0.05, ^**P<0.01,^***P<0.001 vs. DMSO; ^#P<0.05, ^##P<0.01 vs. DMSO/H₂O₂. Ac, acetone extract; FasR, FAS receptor; H₂O₂, hydrogen peroxide; IVD, intervertebral disc; miR-221, microRNA-221; ROS, reactive oxygen species.

Figure 8

Schematic diagram showing the potential effects of subfraction 7 of Violina pumpkin leaf extract on the recovery of degenerated IVD cells. ACAN, aggrecan; H₂O₂, hydrogen peroxide; IVD, intervertebral disc; miR-221, microRNA-221; ROS, reactive oxygen species; SIRT1, sirtuin 1; SOD2, superoxide dismutase 2.