Curcumin (CUR) is well known for its anti-inflammatory and antioxidant effects. However, the endothelial protective effect of CUR in diabetes and the underlying signaling pathway remains unclear. The goal of the current study was to provide evidence regarding the protective mechanism of CUR against the high glucose (HG)-induced damage to human umbilical vein endothelial cells (HUVECs). HG-induced HUVECs injury model was used to evaluate the protective effect and the underlying mechanism of CUR against endothelial injury. The cell viability was determined by the MTT method. The cell reactive oxygen species (ROS) were determined by using flow cytometry. The protein expression levels of Bcl-2, Bax, LC3-II/I, Beclin-1, p62, cleaved caspase-3, IκBα and NF-κB were measured by the western blotting. Results showed that CUR significantly decreased the cell apoptosis, the ROS generation and the inflammatory cytokine NF-κB activity in the HG-induced HUVECs versus the control, P<0.05. In addition, CUR significantly increased the expressions of LC3-II/I, Beclin-1, IκBα and Bax/Bcl-2 in the HG-induced HUVECs versus the control, P<0.05. Furthermore, the addition of autophagy inhibitor 3-MA impaired the autophagy, exacerbated the apoptotic death and increased the ROS and NF-κB levels in HUVECs under the high glucose condition, P<0.05. In brief, autophagy served a protective role in the HG-induced apoptosis in HUVECs and CUR alleviated apoptosis by promoting autophagy and inhibiting the ROS/NF-κB signaling pathway.
Diabetes mellitus (DM) is a chronic metabolic disease, characterized by hyperglycemia. Diabetic angiopathy, one of the most common and serious complications, is also the main cause of mortality in type 2 diabetes mellitus (T2DM) (
The mechanisms by which hyperglycemia influences endothelial function are multiple. The vascular dysfunction in the setting of diabetes is associated with increased vascular oxidative stress and low-grade inflammation (
Autophagy serves a vital role in cellular homeostasis by acting as a housekeeper to eliminate the damaged organelles. The impairment of autophagy, which is observed in the endothelial injury, is implicated especially in the DM-induced endothelial dysfunction. In vascular pathogenesis, autophagy can act as a survival pathway, protecting endothelial cells from oxidative stress (
Curcumin [(CUR); 1,7-bis-(4-hydroxy-3-methoxy-phenyl)-1,6-heptadiene-3,5-dione] is a polyphenol extracted from the
Overall, previous studies have confirmed that HG contents can promote oxidative stress and apoptosis and CUR has a strong antioxidant and antiapoptosis effect, but the exact mechanisms are uncertain. To understand the pharmacological mechanisms of CUR, the current study investigated the antiapoptosis effects of CUR in human umbilical vein endothelial cells (HUVECs). HUVECs were pretreated with various concentrations of CUR before the HG stimulation. Alterations in the expression of autophagy, inflammation and apoptosis-related proteins, cell viability and activation of the ROS were observed. The goal was to provide evidence regarding the protective mechanism of CUR against the HG-induced damage to HUVECs.
The whole experiment was conducted in several parts. To explore the dose-dependent effects of CUR on the viability of HUVECs, the cells were pretreated for 4 h at 37˚C with different concentrations of CUR (5, 10, 20 and 40 µM) and then incubated with HG concentration for 24 h. Exposure of vascular ECs to a glucose level greater than 10 mmol/l (
Immortalized HUVECs (Lonza Group Ltd.) were cultured in Dulbecco's modified Eagle's medium (DMEM; MilliporeSigma), supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified 5% CO2, 37˚C incubator (Thermo Fisher Scientific, Inc.). All parts of the experiment on HUVECs were conducted at the ~3-7 passages. Those HUVECs that were cultured in a medium of 33.3 mmol/l glucose for 24 h served as the HG group (
The colorimetric MTT assay was used to detect the cell viability in the 96-well plates. The MTT substrate was prepared in a physiologically balanced solution, added to HUVECs in culture and incubated for 4 h at 37˚C. Viable cells with active metabolism converted MTT into purple formazan. The optical density of formazan (directly proportional to the number of viable cells) after dissolution in DMSO was measured at 450 nm with a microplate reader (Molecular Devices, LLC.). The mean optical density of 6 wells was used to calculate the cell viability percentage relative to the cell viability of control wells.
The caspase-3 activity was determined with a Caspase-3 Assay kit (Colorimetric) ab39401 (Abcam) according to the manufacturer's protocol. Briefly, the cells were lysed with the cell lysis buffer, incubated on ice for 10 min at 37˚C and centrifuged at 10,000 g for 1 min. The supernatant was collected to measure the protein concentration with the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Inc.). To measure the caspase-3 activity, the sample was mixed with an equal volume of 2X reaction buffer (containing 10 mM DTT) and 200 µM of DEVD-p-NA substrate. The mixture was incubated at 37˚C for 2 h. The optical density was measured at 400 nm with a microplate reader (BioTek Instruments, Inc.).
The cells were seeded in a 6-well plate at a density of 5x104 cells/well. The levels of intracellular ROS were measured with the fluorescent probe dichloro-dihydro-fluorescein diacetate (DCFH-DA; Beijing Baiaosentai Biotechnology). Following treatment with stimuli, the cells were incubated with DCFH-DA (10 µM) at 37˚C for 30 min. The cells and probe were mixed thoroughly for 30 min by inverting the flask once every 3-5 min. The cell suspension was centrifuged at 12,000 x g for 5 min at 4˚C, and the supernatant was discarded. The cells were resuspended and washed 3 times with 1 ml serum-free medium to remove the DCFH-DA that did not enter the cells. The cells were then centrifuged again at 12,000 x g for 5 min at 4˚C, and the supernatant was discarded, followed by the addition of 500 ml phosphate-buffered saline (PBS) to resuspend the cells. After 30 min, the cells were subjected to flow cytometry (using the parameters set for FITC) at an excitation wavelength of 535 nm and an emission wavelength of 610 nm to detect the fluorescence intensity before and after stimulation. The fluorescence images were obtained with fluorescence microscopy (BX41F, Olympus Corporation).
HUVECs were homogenized in RIPA lysis (Wuhan Servicebio Technology Co., Ltd.) buffer to obtain total proteins. According to the previous studies (
Cell apoptosis was determined with an Annexin V-FITC Apoptosis kit (cat. no. C1062s; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. In total, 1x106 cells were collected and the cells from each sample were suspended in 195 µl of 1X Annexin V-FITC binding buffer and 5 µl Annexin V-FITC. The cells were incubated at room temperature for 10 min. Then, each sample was centrifuged at 12,000 x g for 5 min, suspended again in 190 µl of binding buffer, to which was added 10 µl of propidium iodide (PI) working solution. The suspension was mixed and incubated in the dark at room temperature for 15 min. Finally, cell apoptotic rates (early + late) were determined using a flow cytometer (FACSCanto II; BD Biosciences). FlowJo version 7.6.1 software (FlowJo LLC) was used to analyze the data.
Data analysis was conducted with the GraphPad Prism 8 software. All results were presented as mean ± standard deviation (SD). To compare measurements obtained in different test conditions, a one-way analysis of variances (ANOVA) was used to examine the effect of test condition on the dependent variable. The post-hoc analysis was conducted with Tukey's test for pairwise comparisons. P<0.05 was considered to indicate a statistically significant difference.
The first part of the experiment was to measure the cell viability in different cell groups with the purpose to show the protective ability of CUR against the damage induced by HG conditions (
The second part of the experiment was conducted to measure the ROS content for the three groups (Con, HG and HG+CUR) to show the effect of CUR on ROS generation in a HG condition.
The third part of the experiment determined whether CUR could reduce cell apoptosis in the HG-induced injury model.
The fourth part of the experiment was to determine whether the CUR treatment could enhance the cell autophagy of endothelial cells in HG stress. The autophagy-related protein expressions of p62, Beclin1 and LC3-II/I were detected with the western blotting (
The fifth part of the experiment was to investigate the interaction between autophagy and ROS in the HG+CUR group (
To investigate the interaction between cell autophagy and inflammation in the HG+CUR group, the sixth part of the experiment measured the expressions of NF-κB (mediator of inflammatory responses) and IκBα (inhibitor of NF-κB) in different cell groups (
The last part of the experiment investigated the interaction between apoptosis and autophagy by measuring the levels of apoptosis-related cleaved (Cle-) caspase-3, Bcl-2 and Bax in different cell groups (
The current study investigated the effects of CUR on apoptosis and autography in HUVECs under HG conditions. It also explored the molecular mechanism of CUR and the ROS/NF-κB pathway. The current results have shown that CUR can promote autophagy and decrease apoptosis in HUVECs by inhibiting ROS and NF-κB. The present study further supported the putative role of CUR on autophagy induction, revealing the underlying mechanisms that may account for the beneficial effects on endothelial cell apoptosis.
CUR, a hydrophobic polyphenol compound extracted from the spice turmeric, has different pharmacological effects on both
In T2DM, elevated oxidative stress can cause injury to the vascular endothelial cells during the process of diabetic vascular complications (
Autophagy is usually an adaptive mechanism that regulates the cell response to stress and enhances the resistance to apoptosis, and defective autophagy has been linked to increased apoptosis (
Inflammation is probably another key factor for the onset and progression of endothelial dysfunction. Inflammatory processes can enhance vascular ROS generation and endothelial cell apoptosis. The present study found that the CUR treatment inhibited the HG-induced inflammatory response, as evidenced by a decrease in IκBα protein level and an induced NF-κB activity. The enhanced generation of ROS and NF-κB was markedly suppressed by CUR in cells subjected to the HG-induced injury. Furthermore, although the current study investigated ROS and NF-κB separately after autophagy inhibition, the ROS/NF-κB pathway is speculative and generally accepted. Based on these findings, it is hypothesized that ROS could be an important cellular mediator that triggers the NF-κB-dependent pathway after the administration of CUR in HUVECs. It appears that ROS/NF-κB activation is involved in the pathogenesis of the HG-induced cell dysfunction and apoptosis, suggesting that CUR, which can block the ROS/NF-κB signaling, may be effective in protecting the cell activity. A previous study shows that the stimulation of autophagy can reduce oxidative status and rapamycin can protect HUVECs from the damages caused by HG (
Overall, the present study indicated that CUR could promote autography and decrease apoptosis in HUVECs under HG conditions and that CUR treatment markedly restored the HG-reduced ROS protein levels and NF-kB expression. The ROS/NF-κB signaling pathway was, therefore, considered as the potential mechanism involved in the autophagy activation by curcumin. Previous reports have shown that there are a number of different potential intracellular sources of ROS which are capable of influencing or being influenced by NF-κB (
To confirm that the enhanced autophagy due to CUR has a potential role in reducing the generation of NF-κB, caspase-3 and Bax/Bcl-2 in the HG-treated HUVECs, the present study adopted the autophagy inhibitor 3-MA. The results showed that 3-MA induced an additional increase in the Cle-caspase-3, Bax/Bcl-2 and NF-κB levels when compared to the levels in HG treated with CUR. This finding suggested that autophagy served an essential role in reducing caspase-3, Bax/Bcl-2 and NF-κB generation in HG and that CUR could promote autophagy and reduce apoptosis and inflammation. Therefore, the effect of inhibition on autophagy not only exacerbates the HG-induced apoptosis but also diminishes the suppression effects of CUR in NF-κB, providing a scientific basis for further research and clinical application of CUR.
The present study had several limitations. Since it was an
The activation of autophagy is to protect the HG-induced HUVECs. Meanwhile, curcumin protects the HG-induced HUVECs by restoring autophagy, an effect attributed to the inhibition of the ROS/NF-B pathway. Therefore, it was hypothesized that enhancing autophagy by inhibiting ROS/NF-B expression may be a potential therapeutic strategy for treating vascular complications in diabetes. Furthermore, the regulation of endothelial cell autophagy may be another key point of control in regulating vascular function under disease conditions associated with oxidative stress.
The authors would like to thank Dr Chi C Lau, an independent scholar, for his invaluable advice in preparing the manuscript.
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
The study was designed by QHJ, data were collected by HYZ, data analysis and interpretation were performed by XJH and QHJ, and the manuscript was written by QHJ and XJH. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
CUR increases the cell viability in HG. (A) The chemical structure of CUR. (B) The cell viability was measured for cells treated with glucose in different concentrations (5.5-100 mmol/l) and for cells treated with curcumin in different concentrations (5-40 µM) under HG conditions. Each error bar is the standard deviation of the mean (n=6). Significance level between two test conditions: *P<0.05. Con, control group treated with 5.5 mmol/l glucose; HG, high glucose group treated with 33 mmol/l glucose; CUR, curcumin.
CUR reduces the high glucose-induced secretion of ROS. (A) The histogram of counting ROS was measured with the flow cytometry (FITC-A) for the Con, HG and HG+CUR groups. (B) The fluorescence intensity was measured for the three groups. Each error bar is the standard deviation of the mean (n=6). Significance level between two test conditions: **P<0.01. CUR, curcumin; ROS, reactive oxygen species; Con, control group treated with 5.5 mmol/l glucose; HG, high glucose group treated with 33 mmol/l glucose.
CUR reduces the high glucose-induced cell apoptosis. (A) Detection of apoptosis was shown in a two-dimension graph using FITC Annexin V and PI for the Con, HG and HG+CUR groups. (B) The cell apoptosis rate was measured with the TUNEL method for the three groups. Each error bar is the standard deviation of the mean (n=6). Significance level between two test conditions: *P<0.05, **P<0.01. CUR, curcumin; FITC, fluorescein isothiocyanate; PI, propidium iodide; Con, control group treated with 5.5 mmol/l glucose; HG, high glucose group treated with 33 mmol/l glucose.
CUR enhances autophagy for HUVECs incubated in high glucose conditions. (A) The autophagy-related protein expressions were measured with western blotting for the Con, HG and HG+CUR groups. (B) The expressions of LC3II/I, Beclin1 and p62 (after normalization to β-actin) for the three groups. Each error bar is the standard deviation of the mean. Significance level between two test conditions: *P<0.05, **P<0.01. CUR, curcumin; HUVECs, human umbilical vein endothelial cells; Con, control group treated with 5.5 mmol/l glucose; HG, high glucose group treated with 33 mmol/l glucose.
3-MA treatment attenuates the antioxidant effect of CUR. (A) Histogram of ROS was measured with a flow cytometer for the Con, HG and HG+CUR, HG+3MA and HG+CUR+3MA groups. (B) The fluorescence intensity was measured for the five groups. Each error bar is the standard deviation of the mean. Significance level between two test conditions: *P<0.05, **P<0.01. CUR, curcumin; ROS, reactive oxygen species; Con, control group treated with 5.5 mmol/l glucose; HG, high glucose group treated with 33 mmol/l glucose.
3-MA treatment attenuates the anti-inflammatory effect of CUR. (A) Different expressions were measured with western blotting for the Con, HG and HG+CUR, HG+3MA and HG+CUR+3MA groups. (B) The expressions of NF-κB/β-actin and IκBα/β-actin were measured for the five groups. Each error bar is the standard deviation of the mean. Significance level between two test conditions: *P<0.05, **P<0.01. CUR, curcumin; Con, control group treated with 5.5 mmol/l glucose; HG, high glucose group treated with 33 mmol/l glucose.
3-MA treatment attenuates the anti-apoptosis effect of CUR. (A) Different expressions were measured with western blotting for the Con, HG and HG+CUR, HG+3MA and HG+CUR+3MA groups. (B) The levels of Cle-caspase-3/β-actin and Bax/Bcl-2 were measured for the five groups. Each error bar is the standard deviation of the mean. Significance level between two test conditions: *P<0.05, **P<0.01. CUR, curcumin; Con, control group treated with 5.5 mmol/l glucose; HG, high glucose group treated with 33 mmol/l glucose; Cle, cleaved.