- Open Access
Inhibitory effects of Syzygium jambos extract on biomarkers of endothelial cell activation
BMC Complementary Medicine and Therapies volume 22, Article number: 101 (2022)
Disordered endothelial cell activation plays an important role in the pathophysiology of atherosclerosis, cancer, sepsis, viral infections, and inflammatory responses. There is interest in developing novel therapeutics to regulate endothelial cell function in atherothrombotic, metabolic, vascular, and hematological diseases. Extracts from leaves of the Syzygium jambos (L.) Alston (S. jambos) trees have been proposed to treat cardiovascular diseases and diabetes through unclear mechanisms. We investigated the effects of the S. jambos extract on biomarkers of endothelial dysfunction and immune responses in the human endothelial cell line, EA.hy926.
Leaves of S. jambos were collected, concocted and lyophilized. To study the effects of S. jambos on endothelial cell activation, we used the human endothelial cell line. IL-6 levels were measured using qPCR and ELISA. PDI activity was measured using Insulin Turbidity and Di-E-GSSG assays. CM-H2DCFDA was used to study ROS levels. Migration assay was used to study S. jambos effect on ex vivo human polymorphonuclear and human mononuclear cells.
Our results show that incubation of EA.hy926 cells with ET-1 led to a 6.5 ± 1.6 fold increase in IL-6 expression by qPCR, an event that was blocked by S. jambos. Also, we observed that ET-1 increased extracellular protein disulfide isomerase (PDI) activity that was likewise dose-dependently blocked by S. jambos (IC50 = 14 μg/mL). Consistent with these observations, ET-1 stimulated ex vivo human polymorphonuclear and mononuclear cell migration that also was dose-dependently blocked by S. jambos. In addition, ET-1 stimulation led to significant increases in ROS production that were sensitive to S. jambos.
Our results suggest that the S. jambos extract represents a novel cardiovascular protective pharmacological approach to regulate endothelial cell activation, IL-6 expression, and immune-cell responses.
Endothelial cells play an important role in maintaining vascular tone, platelet aggregation, cell adhesiveness, leukocyte migration, coagulation, and inflammation. Disordered endothelial cell activation contributes to the pathophysiology of diabetes , atherosclerosis, cancer, chronic inflammation, sepsis, and viral infections [2,3,4,5], such as observed in patients with the recently described Coronavirus Disease 2019 (COVID-19) [6, 7]. It is characterized, in part, by increased reactive oxygen species (ROS) and cytokine production, including endothelin-1 (ET-1) and interleukin-6 (IL-6), leading to apoptosis, increased leukocyte migration, accelerated endothelial cell senescence. These cytokines are important contributors to the cytokine release syndrome, also known as cytokine storm that is observed following viral infection, sepsis, and immunotherapy in cancer [8,9,10,11,12].
ET-1 is a 21-amino acid peptide and potent vasoconstrictor that acts via paracrine and autocrine mechanisms to increase vascular permeability leading to rapid and robust leukocyte recruitment . ET-1 plays an important role in sepsis, cytokine release syndrome , and the cardiovascular complications of diabetes . ET-1 induces oxidative stress in part by increasing vascular inflammatory responses, including increased IL-6 production [15, 16] and ROS generation resulting in vascular wall modifications [17, 18]. We reported that ET-1 also increases extracellular protein disulfide isomerase (PDI) activity and is secreted by endothelial cells . PDI is an oxidoreductase enzyme, mostly known for its effects on platelet activation, including integrin-mediated platelet and leukocyte function in thrombogenesis [20, 21], cellular adhesion , nitric oxide (NO) delivery , leukocyte adherence  and oxidative stress sensor in endothelial cells . Therefore, modulation of ET-1 levels and its effects are of great concern in cardiovascular diseases.
The pharmacological applicability of extracts from leaves of the Syzygium jambos (S. jambos) (L.) Alston tree, also known as Eugenia jambos, as a potential source of polyphenols for the treatment of cardiovascular disease, including hypertension, has been previously proposed . S. jambos has been traditionally used to treat diabete . S. jambos has anti-inflammatory activity [28, 29], antioxidant  and is reported to have antimicrobial properties as well [28, 31]. There are reports showing that various polyphenols have anti-inflammatory  and antioxidant properties [33, 34] and can prevent ET-1 effects . However, in humans, the use of plant extracts is limited because of the absence of randomized controlled clinical trials due, in part, to the scarcity of information on the mechanisms by which polyphenols such as S. jambos mediate their effects. Our present study was designed to evaluate the effects of S. jambos on endothelial dysfunction. We hypothesized that the aqueous leave extracts from S. jambos would regulate ET-1-stimulated human endothelial cell function. We show that aqueous extracts from S. jambos inhibits PDI activity and decrease IL-6 expression, ROS production, and cell migration. This work describes novel and important effects of Syzygium jambos extract on endothelin-1 signaling and endothelial cell dysfunction.
Materials and methods
Folin–Ciocalteu reagent (Sigma-Aldrich, USA), gallic acid was purchased from ACROS Organics, EA.hy926 (ATCC, CRL-2922) were obtained from Manassas, VA. IL-6 ELISA kit from R&D Systems (Minneapolis, MN). QCM Chemotaxis 3-μm cell migration assay system was purchased from Millipore (Bedford, MA) and TRIzol reagent from Invitrogen (Thermo Fisher Scientific, USA). The Phusion reverse transcriptase PCR kit was obtained from New England Biolabs (Ipswich, MA). 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) dye from Life Technologies, Trolox, Protein disulfide Isomerase from bovine liver, Rutin, bacitracin, Dithiothreitol (DTT), and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Plant collection, identification, and preparation
Leaves of S. jambos were collected in Puerto Rico in the town of Naguabo (18°14′29.2“ N and 65°45’30.8” W). Vouchers of S. jambos (019663) were numbered and deposited at the George Proctor Herbarium (SJ) in Puerto Rico. Dr. Robert Ross, Botanist from the University of Puerto Rico in Cayey, identified and classified the botanical species. Concoctions were prepared by boiling 30 g of plant material in 100 ml of distilled water. After concentration to 15 ml, the decoctions were filtered through a cheesecloth. Aliquots (5 ml) of the extracts were freeze-dried using a lyophilizer to determine the initial concentration of the rich-polyphenol extracts (μg/ml).
Quality control of extract
The HPLC fingerprinting profile of plant extracts was carried out using a Shimadzu HPLC system with a Reverse Phase C18 column. The HPLC separations were conducted using an acetonitrile gradient with 0.5% acetic acid in water for 75 min with a 0.7 mL/min flow rate. The UV–VIS spectra (700–200 nm) were recorded for each extract using a UV-VIS spectrometer. The maxima absorption wavelengths were identified. The extraction factor (EF) of bioactive molecules from each extract were calculated to compare the yields of extraction in different batches, considering the absorption values (A) recorded for each λmax, multiplied with the dilution factor (d) and applying the following relation: EF = A (λmax)xd. This ensured that a similar amount of bioactive molecules were present in each experiment.
Quantification of phenolic content
The total phenolic content was determined using a spectrophotometry method . The S. jambos extract was prepared as a 1.0 mg/ml aqueous solution. Extract (0.5 ml) was mixed with 1 ml of Folin–Ciocalteu reagent (2 N) and was left to stand at room temperature for 1 min. The sodium carbonate (700 mM Na2CO3) solution (3 ml) was subsequently added, and the solution was allowed to stand at room temperature for 2 h. Supernatant absorbance was measured at 760 nm using a UV-VIS spectrometer. The results were compared with the standards prepared similarly (with known gallic acid concentrations). All samples were analyzed three times in separate experiments and were expressed as gallic acid equivalents in mg/g of dry extract.
Quantification of flavonoid content
The total flavonoid content was determined using a colorimetric method . The S. jambos extract was prepared as a 1.0 mg/ml aqueous solution. The extract (1 ml) was mixed with 0.4 ml of a 5% NaNO2 solution. The mixture was allowed to stay at room temperature for 6 min; 0.4 ml of a 10% AlCl3*9H2O solution was added for 6 min, followed by the addition of a 4 ml 4% NaOH solution. The ethanol solution (70%) was added to reach a final volume of 10 ml. The solution was mixed and kept at room temperature for 15 min. Absorbance was measured at 510 nm using a UV-VIS spectrometer. Comparisons were made against standards prepared similarly (with known rutin concentrations). Results were expressed as rutin mg/g of dry extract. All samples were analyzed three times in separate experiments.
Human endothelial cells EA.hy926 (ATCC, CRL 2922) were maintained in DMEM (containing 4.5 g/L glucose) with 10% FBS as previously described by us . Briefly, twelve hours before treatment, cells were serum-starved in DMEM with 0.2% FBS. At the treatment time, cells were washed with PBS and incubated with vehicle, ET-1, or S. jambos extract in DMEM with 0.2% FBS. The cells were harvested for analyses after 24-h or 48-h incubation.
PDI. activity assays
Insulin turbidity assay
This assay is based on the measurement of the catalytic reduction of insulin as described by Lundstrom and Holmgren . PDI facilitates the reduction of insulin in the presence of Dithiothreitol (DTT). The reduced insulin chains aggregate and the turbidity is monitored spectrophotometrically at 650 nm. The assay was performed in a 96-well plate format and a volume of 100 μl in the presence of 1 mM DTT, 1 μg PDI (Sigma), 0.15 mM bovine pancreas insulin (Sigma), and 0.2 mM EDTA in 100 mM potassium phosphate, pH 7.0. The progress of the reaction was monitored on a 96-well plate reader (Multiscan FC Microplate Reader, Fisher Scientific) at 650 nm for 60 min at 25 °C. The non-enzymatic reduction of insulin by DTT was recorded in control well without PDI.
Di-E-GSSG fluorescence assay
A soluble PDI activity assay was performed following previously described protocols and optimized in our laboratories . Briefly, [E-GSH] formation is recorded for 30 min (ex: 525 nm; em: 545 nm) at room temperature, and fluorescence intensity is recorded and plotted as a function of time to calculate the conjugate’s reduction rate. Measurements were performed in the presence or absence of 16.5 ug/ml Phenylarsine oxide (PAO), a well-known vicinal thiol blocker. PBS was used as vehicle. Supernatant PDI activity was determined by fluorescence recording of [E-GSH] formation.
Quantitative real-time PCR
Total RNA was prepared with 1 mL of TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The Phusion reverse transcriptase PCR kit (New England Biolabs) was used to make 20 μL of cDNA from 1 μg of R.N.A. Gene expression was analyzed using real-time PCR using TaqMan gene expression assay for IL-6 and GAPDH (Applied Biosystems) in a StepOne Plus from ABI. The ΔΔ cycle threshold method was used to determine mRNA levels. IL-6 gene expression was normalized to GAPDH levels.
To determine the concentration of IL-6 in cell culture media of control or treated endothelial cells, an ELISA kit from R&D Systems was used following the manufacturer’s instructions.
Cell migration assay
Cell migration was carried out using the QCM Chemotaxis 3-μm cell migration assay system (Millipore, Bedford, MA). Ex vivo human polymorphonuclear leukocytes (PMN) and mononuclear cells (MNC) from healthy human subjects were isolated using Polymorphprep following approval by the Brigham and Women’s Hospital– Institutional Review Board (Approval num. BWH IRB# 2009P000491) and written informed consent was provided to volunteers who participated in this study. Cells were seeded into the migration chamber as per the manufacturer’s protocol. The supernatant of endothelial cells was incubated for 2 h with the vehicle (PBS), ET-1, or S. jambos extract and placed in the lower chamber. After allowing cell migration for 2 h, PMN and MNC cells that migrated through the membrane were stained with Cyquant and quantified using Spectra Max Gemini EM (Molecular Devices) at 480/520 nm.
Measurements of Reactive Oxygen Species (ROS)
The 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) dye from Life Technologies was used as a probe to measure ROS. Endothelial cells were cultured in 6-well plates and were serum-starved for 12 h in DMEM with 0.2% FBS, washed with fresh DMEM, and incubated with DMEM premixed with 10 μM CM-H2DCFDA for 1 h at 37 °C. Cells were washed twice with PBS to remove the loading buffer, and cells were allowed to recover for 10 min at 37 °C for cellular esterases to deacetylate H2DCFDA. Cells were then stimulated with ET-1 with or without S. jambos extracts and Trolox (200 μM) for 1 h, releasing a variety of ROS species, which oxidize H2DCF into DCF Cells were washed once with Hank’s Balanced Salt Solution and incubated in the same buffer containing 5 μg of CM-H2DCFDA /ml at 37 °C for 30 min. Intracellular fluorescence was detected with excitation at 495 nm and emission at 520 nm using Spectra Max Gemini EM.
Annexin-V apoptosis assay
EA.hy926 cells were cultured in 96-well plates and were serum-starved for 12 h in DMEM with 0.2% FBS before the vehicle, ET-1, or plant extract was added for 24 h. The Annexin V FITC Assay Kit (Cayman Chemical) was used and analyzed using Spectra Max Gemini EM at 485/535 nm per manufacturer protocols to determine the percentage of apoptotic cells.
Data were analyzed by one-way or two-way ANOVA with Bonferroni post-test or Student’s t-test when appropriate. The p-value was set to be <0.05. Data are expressed as mean ± SE of (n) independent experiments unless otherwise stated.
S. jambos inhibits ET-1 induced PDI activity
The concentration of polyphenols and flavonoids in S. jambos aqueous leaf extract was determined using spectrophotometric methods as described in methods. We determined that S. jambos aqueous leaf extract contains polyphenols 135 ± 7 Gallic acid mg/g and flavonoid 99 ± 9 rutin mg/g. Chagas et al. described the extract composition of Syzygium by HPLC-UV/Vis and LC-MS/MS using purified standards as calibrators at different wavelengths to validate its composition as myricetin as the most abundant flavonoid with gallic acid and quercentin  with high antioxidant potency. Eight peaks were identified in the S. jambos aqueous leaf extract HPLC fingerprinting used for quality control (Suppl Fig. 1).
One of the effects of S. jambos that has a lot of interest was its effects on protein disulfide isomerase (PDI). It has been shown that myricetin  and quercentin  inhibit the reductase activity of PDI. As the major components of the S. jambos, we evaluated the effect of extract preparation on PDI activity. We measured in vitro PDI activity in the presence or absence of the S. jambos aqueous leaf extract using the insulin turbidity assay. It shows a significant reduction of 5 μg of purified PDI activity with 5 μg/mL and up to 75% inhibition at 100 μg/mL of S. jambos extract (p = 0.004, n = 3) (Fig. 1A). We also observed that S. jambos extract induced a dose-dependent inhibitory effect on purified PDI with an IC50 of 14 μg/mL (Fig. 1B).
The effectiveness of S. jambos on PDI reductive capacity inhibition compared to other well-known inhibitors was examined in vitro [42,43,44]. These agents’ inhibitory mechanisms are not well known, but it involves covalent binding of an open thiol site to free cysteines in the substrate-binding domain of PDI. In vitro assayed of the extract against phenylarsine oxide, PAO (16.8 ug/mL), bacitracin (1.4 mg/mL) and Rutin (quercetin-3-O-rutinoside, 0.6 ug/mL) is represented in Fig. 2. It shows that 50 μg/mL of S. jambos inhibits purified PDI reductive capacity by 67% but is less effective than Rutin (citrus flavonoid) or bacitracin (antibiotic) at maximal inhibitory concentrations. Previous extractions of S. jambos were not tested on purified PDI activity to assess its crude effective capacity, but in vivo platelets activation, the extract was able to block approximately 60% of ADP-activated platelets .
We previously demonstrated that ET-1 induced PDI activity in endothelial cells  as well as in erythrocytes . To test whether the extract S. jambos can inhibit endothelin-1 (ET-1)-stimulated PDI activity in human endothelial cells, we measured the effect of 50 μg/mL S. jambos on cell-secreted PDI activity using a sensitive fluorescence assay as described in methods (Fig. 3). It showed that ET-1 induced a significant increase in cell-secreted PDI (p = 0.001, n = 6) that was completely blunted by the presence of S. jambos (p < 0.005, n = 6).
S. jambos inhibits IL-6 gene expression and secretion
Mounts of evidence demonstrated that ET-1 effects on the endothelium are part of the inflammatory process. To evaluate the effects of S. jambos on the inflammatory cascade, EA.hy926 cells were treated with 100 nM of ET-1 in the presence or absence of 50 μg/mL of S. jambos for 24 and 48 h (Fig. 4A-B). We observed that ET-1 significantly upregulates IL-6 mRNA expression by 6.5 ± 1.6 folds (p = 0.0002, n = 6), and S. jambos significantly inhibited this upregulation (0.82 ± 0.73 folds, p < 0.001, n = 3) (Fig. 4A). We also observed that IL-6 mRNA expression upregulation by ET-1 is optimal 24 h after stimulation (Fig. 4B). We also observed that S. jambos extract alone had no significant effect on the expression of IL-6 (Fig. 4 A-B), suggesting that the extract bioactive agents interfere with the ET-1 signaling pathway and not the direct IL-6 synthesis.
IL-6 protein secretion was also measured by ELISA to assess whether S. jambos extract directly inhibits its release. EA.hy926 cells were treated with 100 nM ET-1 with and without 50 μg/mL S. jambos for 24 or 48 h as described in Fig. 4A-B. We found that S. jambos inhibited ET-1-induced secretion of IL-6 (p < 0.01, n = 3) (Fig. 4C). In addition, we observed S. jambos extract was able to significantly inhibit ET-1 induced IL-6 expression even though the ET-1 effects were declining 48 h later (p < 0.001, n = 3) (Fig. 4B). This effect was reflected at the synthesis level which we observed a strong decrease in IL-6 secretion after 48 h of incubation with the extract (Fig. 4D).
S. jambos blocks leukocytes migration
The S. jambos leaf oil extract has shown an inhibitory effect on lipopolysaccharide (LPS)-induced migration of eosinophils and neutrophils . To study the effect of S. jambos on endothelin-induced leukocyte migration, we characterized the ex vivo effects of ET-1-stimulated cells human polymorphonuclear (PMN) cells and mononuclear (MNC). Freshly isolated leukocytes were incubated in the supernatant of activated endothelial cells with ET-1 for 1 h in the presence or absence of S. jambos. ET-1 significantly induced the migration of PMN (n = 3, p < 0.001; Fig. 5A) and MNC (n = 3, p < 0.001; Fig. 5B). The chemotactic agent, N-Formyl-Met-Leu-Phe (fMLP), was used as a positive control for these experiments. We observed PMNC and MNC migration was significantly inhibited by the presence of S. jambos in a dose-dependent manner (Fig. 5 A-B). The extract completely blunted the effect of ET-1 cellular migration at 100 μg/mL in both cell types.
S. jambos inhibits ROS production in endothelial cells
Previous studies indicated that S. jambos extract plays a role in ROS regulation; however, contradictory evidence shows an increase in ROS production, leading to apoptosis  or a decrease in ROS, reducing glucose-induced stress . Figure 6 shows ROS levels in EA.hy926 cells using fluorescent dye CM-H2DCFDA as described in Methods to evaluate if S. jambos extract can regulate ET-1 induced ROS production. We observed that pre-incubation of endothelial cells with various concentrations of S. jambos significantly blunted the stimulation of ET-1-induced R.O.S. production by 2-folds compared to vehicle (Fig. 6A, n = 4). We also observed that S. jambos extract blocked the ET-1 stimulated ROS production as low as 25 μg/mL (p < 0.01, n = 4) (Fig. 6A). To determine the antioxidant potential of S. jambos extract, we compared the effect of 50 μg/mL S. jambos with 200 μM Trolox (500 μg/mL), a known powerful scavenger of intracellular ROS. As expected, Trolox completely inhibited ET-1 induced ROS activity (Fig. 6B). We also observed that S. jambos shared a similar function as Trolox, suggesting its strong capacity as an antioxidant with a Trolox equivalent antioxidant capacity of 54% (n = 3) (Fig. 6C).
S. jambos reduces the apoptotic effects of ET-1 stimulated endothelial cells
ET-1 has been shown to induce apoptotic activity in endothelial cells . To evaluate S. jambos extract on ET-1 induced apoptotic effect in endothelial cells, we used Annexin-V Apoptosis Assay (Cayman Chemical) as a measuring tool (Fig. 7). As observed, we found that treatment of endothelial cells with ET-1 for 24 h significantly increased endothelial cells apoptosis (Fig. 7). This effect was significantly blocked by the presence of S. jambos extract by 55%.
Disordered levels of ET-1 and endothelial cell activation play essential roles in endothelial dysfunction and the worsening of vascular complications in diabetes, cardiovascular diseases, and sepsis. As such, it remains an important area of research. This study investigated the effects of S. jambos aqueous leaves extract on ET-1-mediated endothelial cell activation regulation. We showed that the S. jambos extract significantly inhibits purified PDI activity in vitro with a potency comparable to Rutin, a well-known specific inhibitor of PDI activity . We also observed that the S. jambos extract had anti-inflammatory and anti-oxidative properties as it reduced IL-6 secretion/expression, leukocyte migration, and ROS production from ET-1 stimulated endothelial cells. These results suggest that the S. jambos extract represents a novel modulator of ET-1 regulated endothelial cell activation and inflammatory responses. As a regulator of ET-1 mediated endothelial cell and leukocyte function, our data support the contention that extracts from the S. jambos leaves may have medicinal benefits that could be studied in rodent models of cardiometabolic disease.
S. jambos extracts showed a potent effect on ET-1 mediated leukocyte migration. These results are important as ET-1-stimulated IL-6 gene expression has been implicated as an important and independent regulator of endothelial cell activation  as it likewise increased leukocyte recruitment to the endothelium . In addition, IL-6 has been reported to play an important role in response to infection, hematopoiesis, and immune activation . In contrast to what was reported with quercetin effect on IL-6 synthesis , we found no effect of S. jambos (high content of quercetin) on IL-6 expression or secretion, suggesting that ET-1 stimulated IL-6 was mediated by blockade of the ET-1 signaling pathway.
Excess IL-6 levels also stimulate angiogenesis and increased vascular permeability via increased VEGF levels, as observed in rheumatoid arthritis . Our data suggest that S. jambos may serve as an alternative or adjuvant therapeutic approach to control cytokine-induced IL-6 secretion and endothelial cell activation. Our report expands and extends the growing amount of data showing that phytochemicals, such as phenolic compounds in medicinal plants, are associated with increased anti-inflammatory and antioxidant capacity [55, 56]. Our data support the contention that extracts from the S. jambos leaves may have medicinal benefits due to their anti-inflammatory [28, 29], antioxidant properties , and as a regulator of ET-1 mediated endothelial cell function.
ET-1 stimulates ROS production, primarily superoxide anions (O2−), leading to oxidative stress. We observed that S. jambos extract abrogated ET-1 stimulated ROS production in endothelial cells demonstrating that this extract has the capacity of reducing harmful oxidant radicals generated following endothelial cell activation. ET-1 mediates its effects through ETA and ETB receptors. Both receptors have been shown to regulate ROS production [49, 57]. ET-1 activates these receptors to mediate potent vascular contraction, cellular proliferation, and a pro-inflammatory effect. At high intravenous levels, ET-1 causes vasodilation and chronic contractions resulting in organ ischemia and endothelial dysfunction . Under these conditions, ROS generation is elevated [35, 59] and has been proposed to induce lipid peroxidation and reduction of glutathione and SH groups . ET-1 receptor blockade has been shown to restore total glutathione and superoxide dismutase activity [61, 62]. Interaction of ROS with glutathione (GSH) and thiols proteins determines cellular redox homeostasis and maintenance . The potent effects on ROS production that we observed with the extract would suggest that the extract may also block the effects of ET-1 by increasing glutathione (GSH) availability and reducing ROS in addition to its anti-inflammatory actions. GSH plays an important role in the redox regulation of transcription factors and enzymes for signal transduction as an endogenous antioxidant. Indeed, recently it has been shown that flavonoids modulate the expression of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the synthesis of GSH . The authors also demonstrated that flavonoids increase the expression of gamma-glutamylcysteine synthetase and, with it, an increase in intracellular GSH. Thus, we posit that in addition to the potential radical scavenging abilities of S. jambos, this extract also may be important in regulating the procoagulant and pro-inflammatory responses at sites of vascular injury.
In this study, we show that S. jambos regulates PDI activity. PDI is a multifunctional protein that provides essential isomerase and chaperone activities in the endoplasmic reticulum and the plasma membrane . This ubiquitous protein introduces disulfides into proteins and catalyzes the rearrangements of correct disulfides. There is mounting evidence that PDI is involved in many physiological processes that play a major role in inflammatory processes and oxidation-reduction reactions essential for survival. We previously demonstrated that selective blockade of ET-1 receptors decreases PDI secretion in a mouse model of sickle cell disease  while improving the inflammatory and erythrocyte dehydration status, suggesting PDI as a therapeutic target in hematological and vascular diseases. Consequently, there is growing interest in developing novel therapeutics that can regulate ET-1 signaling pathways and PDI activity in various atherothrombotic, vascular and hematological diseases [66, 67]. Currently available selective and nonselective PDI inhibitors that have been used in characterizing PDI function include: Anti-PDI antibodies [RL90 and RL77] [68, 69] (ab5484 & ab2792 from Abcam); Phenyl arsine oxide (PAO); bacitracin ; N-Oxalylglycine; 16F16A; and the more recently described flavonoid, Rutin .
In conclusion, the present in vitro study results show that the aqueous extract of S. jambos leaves blocks secreted PDI activity and IL-6 expression, which then regulates the antioxidant and anti-inflammatory of activated human endothelial and immune cells. These studies suggest that S. jambos polyphenols act as novel regulators of ET-1 activation pathways and strengthen the confirmation of the pharmacological benefits of the phytochemicals present in S. jambos to applications in the management of inflammatory disorders and endothelium dysfunction disorders.
Availability of data and materials
The datasets generated and analysed during the current study are available in Mendeley Data, https://doi.org/10.17632/92ykngb24t.1.
Reactive oxygen species
Protein disulfide isomerase
- S. jambos :
Syzygium jambos (L.) Alston
Tabit CE, Chung WB, Hamburg NM, Vita JA. Endothelial dysfunction in diabetes mellitus: molecular mechanisms and clinical implications. Rev Endocr Metab Disord. 2010;11(1):61–74.
Deanfield JE, Halcox JP, Rabelink TJ. Endothelial function and dysfunction: testing and clinical relevance. Circulation. 2007;115(10):1285–95.
Ince C, Mayeux PR, Nguyen T, Gomez H, Kellum JA, Ospina-Tascón GA, et al. The endothelium in sepsis. Shock. 2016;45(3):259–70.
Teijaro JR, Walsh KB, Cahalan S, Fremgen DM, Roberts E, Scott F, et al. Endothelial cells are central orchestrators of cytokine amplification during influenza virus infection. Cell. 2011;146(6):980–91. https://doi.org/10.1016/j.cell.2011.08.015.
Shi Y, Vanhoutte PM. Macro- and microvascular endothelial dysfunction in diabetes. J Diabetes. 2017;9(5):434–49.
Varga Z, Flammer AJ, Steiger P, Haberecker M, Andermatt R, Zinkernagel AS, et al. Varga, Zsuzsanna et al. “Endothelial cell infection and endotheliitis in COVID-19.” Lancet (London, England). 2020;395(10234):1417-8. https://doi.org/10.1016/S0140-6736(20)30937-5.
Mehra MR, et al. “Cardiovascular Disease, Drug Therapy, and Mortality in Covid-19.” N Engl J Med. 2020;382(25):e102. https://doi.org/10.1056/NEJMoa2007621.
Diehl KJ, Weil BR, Westby CM, MacEneaney OJ, Kushner EJ, Greiner JJ, et al. Effects of endothelin-1 on endothelial progenitor cell function. Clin Chem Lab Med. 2012;50(6):1121–4.
Chen D-D, Dong Y-G, Yuan H, Chen AF. Endothelin 1 activation of endothelin A receptor/NADPH oxidase pathway and diminished antioxidants critically contribute to endothilial progenitor cell reduction and dysfunction in salt-sensitive hypertension. Hypertension. 2012;59:1037–43.
Li J-J, Chen J-L. Inflammation may be a bridge connecting hypertension and atherosclerosis. Med Hypotheses. 2005;64(5):925–9.
Imai M, Okada A, Okada H, Handharyani E, Tsukamoto Y, Adachi K, et al. Rescue with an anti-inflammatory peptide of chickens infected H5N1 avian flu. Nat Preced. 2009. https://doi.org/10.1038/npre.2009.3425.1.
Goto T, Hussein MH, Kato S, Daoud GAH, Kato T, Sugiura T, et al. Endothelin receptor antagonist attenuates oxidative stress in a neonatal sepsis piglet model. Pediatr Res. 2012;72(6):600–5.
Rao RM, Yang L, Garcia-Cardena G, Luscinskas FW. Endothelial-dependent mechanisms of leukocyte recruitment to the vascular wall. Circ Res. 2007;101(3):234–47 Available from: http://circres.ahajournals.org/cgi/doi/10.1161/CIRCRESAHA.107.151860b.
Pernow J, Shemyakin A, Böhm F. New perspectives on endothelin-1 in atherosclerosis and diabetes mellitus. Life Sci. 2012;91(13–14):507-16. https://doi.org/10.1016/j.lfs.2012.03.029.
Mullol J, Baraniuk JN, Logun C, Benfield T, Picado C, Shelhamer JH. Endothelin-1 induces GM-CSF, IL-6 and IL-8 but not G-CSF release from a human bronchial epithelial cell line (BEAS-2B). Neuropeptides. 1996;30(6):551–6.
Stankova J, D’Orleans-Juste P, Rola-Pleszczynski M. ET-1 induces IL-6 gene expression in human umbilical vein endothelial cells: synergistic effect of IL-1. Am J Physiol. 1996;271(4 Pt 1):C1073–8.
Wedgwood S, Black SM. Endothelin-1 decreases endothelial NOS expression and activity through ETA receptor-mediated generation of hydrogen peroxide. Am J Physiol Lung Cell Mol Physiol. 2005;288(3):L480–7 Available from: http://www.ncbi.nlm.nih.gov/pubmed/15531748.
Montezano AC, Touyz RM. Reactive oxygen species and endothelial function - Role of nitric oxide synthase uncoupling and nox family nicotinamide adenine dinucleotide phosphate oxidases. Basic Clin Pharmacol Toxicol. 2012;110(1):87-94. https://doi.org/10.1111/j.1742-7843.2011.00785.x.
Prado GN, Romero JR, Rivera A. Endothelin-1 receptor antagonists regulate cell surface-associated protein disulfide isomerase in sickle cell disease. FASEB J. 2013;27(11):4619–29 Available from: http://www.ncbi.nlm.nih.gov/pubmed/23913858.
Jasuja R, Passam FH, Kennedy DR, Kim SH, van Hessem L, Lin L, et al. Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents. J Clin Investig. 2012;122(6):2104–13.
Cho J. Protein disulfide isomerase in thrombosis and vascular inflammation. J Thromb Haemost. 2013;11(12):2084-91. https://doi.org/10.1111/jth.12413.
Huang EM, Detwiler TC, Milev Y, Essex DW. Thiol-disulfide isomerization in thrombospondin: effects of conformation and protein disulfide isomerase. Blood. 1997;89(9):3205–12.
Bell SE, Shah CM, Gordge MP. Protein disulfide-isomerase mediates delivery of nitric oxide redox derivatives into platelets. Biochem J. 2007;403:283–8.
Hahm E, Li J, Kim K, Huh S, Rogelj S, Cho J. Extracellular protein disulfide isomerase regulates ligand-binding activity of αMβ2 integrin and neutrophil recruitment during vascular inflammation. Blood. 2013;121(19):3789-800, S1-15. https://doi.org/10.1182/blood-2012-11-467985.
Graven KK, Molvar C, Roncarati JS, Klahn BD, Lowrey S, Farber HW. Identification of protein disulfide isomerase as an endothelial hypoxic stress protein. Am J Physiol Lung Cell Mol Physiol. 2002;282(5):L996–1003.
Romero JM. Accion cardiovascular de extractos acuosos de hojas de Syzygium jambos (L.) Alston. Revista Costarricense de Ciencias Medicas. 1995;16(3):17–25.
Gavillán-Suárez J, Aguilar-Perez A, Rivera-Ortiz N, Rodríguez-Tirado K, Figueroa-Cuilan W, Morales-Santiago L, et al. Chemical profile and in vivo hypoglycemic effects of Syzygium jambos, Costus speciosus and Tapeinochilos ananassae plant extracts used as diabetes adjuvants in Puerto Rico. BMC Complement Altern Med. 2015;15(1):244. https://doi.org/10.1186/s12906-015-0772-7.
Sharma R, Kishore N, Hussein A, Lall N. Antibacterial and anti-inflammatory effects of Syzygium jambos L. (Alston) and isolated compounds on acne vulgaris. BMC Complement Altern Med. 2013;13:292. https://doi.org/10.1186/1472-6882-13-292.
Mahmoud MF, Abdelaal S, Mohammed HO, El-Shazly AM, Daoud R, el Raey MA, et al. Syzygium jambos extract mitigates pancreatic oxidative stress, inflammation and apoptosis and modulates hepatic IRS-2/AKT/GLUT4 signaling pathway in streptozotocin-induced diabetic rats. Biomed Pharmacother. 2021;142:112085. https://doi.org/10.1016/j.biopha.2021.112085.
Sobeh M, Esmat A, Petruk G, Abdelfattah MAO, Dmirieh M, Monti DM, et al. Phenolic compounds from Syzygium jambos (Myrtaceae) exhibit distinct antioxidant and hepatoprotective activities in vivo. J Funct Foods. 2018;41:223–31.
Djipa CD, Delmée M, Quetin-Leclercq J. Antimicrobial activity of bark extracts of Syzygium jambos (L.) Alston (Myrtaceae). J Ethnopharmacol. 2000;71(1–2):307-13. https://doi.org/10.1016/s0378-8741(99)00186-5.
Shukla M, Gupta K, Rasheed Z, Khan KA, Haqqi TM. Consumption of hydrolyzable tannins-rich pomegranate extract suppresses inflammation and joint damage in rheumatoid arthritis. Nutrition. 2008;24(7–8):733–43 Cited 2013 May 24. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2577876&tool=pmcentrez&rendertype=abstract.
Ravi K, Ramachandran B, Subramanian S. Protective effect of Eugenia jambolana seed kernel on tissue antioxidants in streptozotocin-induced diabetic rats. Biol Pharm Bull. 2004;27(8):1212–7 Available from: http://www.ncbi.nlm.nih.gov/pubmed/15305024.
Stanely Mainzen Prince P, Kamalakkannan N, Menon VP. Syzigium cumini seed extracts reduce tissue damage in diabetic rat brain. J Ethnopharmacol. 2003;84(2–3):205–9 Available from: http://www.ncbi.nlm.nih.gov/pubmed/12648817.
López-Sepúlveda R, Gómez-Guzmán M, Zarzuelo MJ, Romero M, Sánchez M, Quintela AM, et al. Red wine polyphenols prevent endothelial dysfunction induced by endothelin-1 in rat aorta: role of NADPH oxidase. Clin Sci. 2011;120(8):321–33 Cited 2013 Mar 25. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20977430.
Singleton VL, Rossi JA Jr. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic. 1965;16(3):144–58 Available from: http://www.ajevonline.org/cgi/content/abstract/16/3/144.
Kähkönen MP, Hopia AI, Vuorela HJ, Rauha JP, Pihlaja K, Kujala TS, et al. Antioxidant activity of plant extracts containing phenolic compounds. J Agric Food Chem. 1999;47(10):3954–62.
Coutinho P, Vega C, Pojoga LH, Rivera A, Prado GN, Yao TM, et al. Aldosterone’s rapid, nongenomic effects are mediated by striatin: a modulator of Aldosterone’s effect on estrogen action. Endocrinology. 2014;155(6):2233–43.
Lundström J, Holmgren a. Determination of the reduction-oxidation potential of the thioredoxin-like domains of protein disulfide-isomerase from the equilibrium with glutathione and thioredoxin. Biochemistry. 1991;1993(32):6649–55.
Chagas VT, de Sousa Coelho RMR, Gaspar RS, da Silva SA, Mastrogiovanni M, de Jesus MC, et al. Protective effects of a polyphenol-rich extract from Syzygium cumini (L.) Skeels leaf on oxidative stress-induced diabetic rats. Oxidative Med Cell Longev. 2018;2018:5386079. https://doi.org/10.1155/2018/5386079.
Gaspar RS, da Silva SA, Stapleton J, de Lima Fontelles JL, Sousa HR, Chagas VT, et al. Myricetin, the main flavonoid in Syzygium cumini leaf, is a novel inhibitor of platelet thiol isomerases PDI and ERp5. Front Pharmacol. 2020;10:1678. https://doi.org/10.3389/fphar.2019.01678.
Dickerhof N, Kleffmann T, Jack R, McCormick S. Bacitracin inhibits the reductive activity of protein disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain. FEBS J. 2011;278(12):2034–43.
Zai A, Rudd MA, Scribner AW, Loscalzo J. Cell-surface protein disulfide isomerase catalyzes transnitrosation and regulates intracellular transfer of nitric oxide. J Clin Investig. 1999;103(3):393-9. https://doi.org/10.1172/JCI4890.
Stopa JD, Neuberg D, Puligandla M, Furie B, Flaumenhaft R, Zwicker JI. Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation. JCI Insight. 2017;2(1):e89373. https://doi.org/10.1172/jci.insight.89373.
Rivera A. Reduced sickle erythrocyte dehydration in vivo by endothelin-1 receptor antagonists. Am J Physiol Cell Physiol. 2007;293(3):960–6.
Siani AC, Souza MC, Henriques MGMO, Ramos MFS. Anti-inflammatory activity of essential oils from Syzygium cumini and Psidium guajava. Pharm Biol. 2013;51(7):881-7. https://doi.org/10.3109/13880209.2013.768675.
Ezhilarasan D, Apoorva VS, Ashok VN. Syzygium cumini extract induced reactive oxygen species-mediated apoptosis in human oral squamous carcinoma cells. J Oral Pathol Med. 2019;48(2):115-21. https://doi.org/10.1111/jop.12806.
Atale N, Chakraborty M, Mohanty S, Bhattacharya S, Nigam D, Sharma M, et al. Cardioprotective role of Syzygium cumini against glucose-induced oxidative stress in H9C2 cardiac myocytes. Cardiovasc Toxicol. 2013;13(3):278-89. https://doi.org/10.1007/s12012-013-9207-1.
Dong F, Zhang X, Wold LE, Ren Q, Zhang Z, Ren J. Endothelin-1 enhances oxidative stress, cell proliferation and reduces apoptosis in human umbilical vein endothelial cells: Role of ET B receptor, NADPH oxidase and caveolin-1. Br J Pharmacol. 2005;145(3):323-33. https://doi.org/10.1038/sj.bjp.0706193.
Mantovani A, Sozzani S, Introna M. Endothelial activation by cytokines. Ann N Y Acad Sci. 1997;832:93–116.
Watson C, Whittaker S, Smith N, Vora AJ, Dumonde DC, Brown KA. IL-6 acts on endothelial cells to preferentially increase their adherence for lymphocytes. Clin Exp Immunol. 1996;105(1):112–9.
Tanaka T, Narazaki M, Kishimoto T. Il-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol. 2014;6(10):a016295. https://doi.org/10.1101/cshperspect.a016295.
Liu J, Li X, Yue Y, Li J, He T, He Y. The inhibitory effect of quercetin on IL-6 production by LPS-stimulated neutrophils. Cell Mol Immunol. 2005;2(6):455-60.
Nakahara H, Song J, Sugimoto M, Hagihara K, Kishimoto T, Yoshizaki K, et al. Anti-interleukin-6 receptor antibody therapy reduces vascular endothelial growth factor production in rheumatoid arthritis. Arthritis Rheum. 2003;48(6):1521-9. https://doi.org/10.1002/art.11143.
Zhang Y-J, Gan R-Y, Li S, Zhou Y, Li A-N, Xu D-P, et al. Antioxidant phytochemicals for the prevention and treatment of chronic diseases. Molecules. 2015;20(12):21138–56 Available from: http://www.mdpi.com/1420-3049/20/12/19753.
Gormaz JG, Valls N, Sotomayor C, Turner T, Rodrigo R. Potential role of polyphenols in the prevention of cardiovascular diseases: molecular bases. Curr Med Chem. 2016;23(2):115–28 Available from: http://www.ncbi.nlm.nih.gov/pubmed/26630919.
Elmarakby AA, Loomis ED, Pollock JS, Pollock DM. NADPH oxidase inhibition attenuates oxidative stress but not hypertension produced by chronic ET-1. Hypertension. 2005;45(2):283-7. https://doi.org/10.1161/01.HYP.0000153051.56460.6a.
Gorąca A, Kleniewska P, Skibska B. ET-1 mediates the release of reactive oxygen species and TNF-α in lung tissue by protein kinase C α and β1. Pharmacol Rep. 2016;68(1):121-6. https://doi.org/10.1016/j.pharep.2015.07.007.
Thakali K, Demel SL, Fink GD, Watts SW. Endothelin-1-induced contraction in veins is independent of hydrogen peroxide. Am J Physiol Heart Circ Physiol. 2005;289(3):58–63.
Viswanatha Swamy AHM, Wangikar U, Koti BC, Thippeswamy AHM, Ronad PM, Manjula D, et al. Cardioprotective effect of ascorbic acid on doxorubicin-induced myocardial toxicity in rats. Indian J Pharmacol. 2011;43(5):507-11. https://doi.org/10.4103/0253-7613.84952.
Briyal S, Philip T, Gulati A. Endothelin-a receptor antagonists prevent amyloid-β-induced increase in ETA receptor expression, oxidative stress, and cognitive impairment. J Alzheimers Dis. 2011;23(3):491-503. https://doi.org/10.3233/JAD-2010-101245.
Ozdemir R, Parlakpinar H, Polat A, Colak C, Ermis N, Acet A. Selective endothelin a (ETA) receptor antagonist (BQ-123) reduces both myocardial infarct size and oxidant injury. Toxicology. 2006;219(1–3):142-9. https://doi.org/10.1016/j.tox.2005.11.022.
Chatzi A, Tokatlidis K. The mitochondrial intermembrane space: a hub for oxidative folding linked to protein biogenesis. Antioxid Redox Signal. 2013;19(1):54-62. https://doi.org/10.1089/ars.2012.4855.
Moskaug JO, Carlsen H, Myhrstad MCW, Blomhoff R. Polyphenols and glutathione synthesis regulation. Am J Clin Nutr. 2005;81(1):277S-83S. https://doi.org/10.1093/ajcn/81.1.277S.
Wilkinson KD. The discovery of ubiquitin-dependent proteolysis. Proc Natl Acad Sci U S A. 2005;102(43):15280–2.
Chiu J, Passam F, Butera D, Hogg P. Protein disulfide isomerase in thrombosis. Semin ThrombHemost. 2015;41(07):765–73 Available from: http://www.thieme-connect.de/DOI/DOI?10.1055/s-0035-1564047.
Flaumenhaft R, Furie B, Zwicker JI. Therapeutic implications of protein disulfide isomerase inhibition in thrombotic disease. Arterioscler Thromb Vasc Biol. 2015;35(1):16-23. https://doi.org/10.1161/ATVBAHA.114.303410.
Gallina A, Hanley TM, Mandel R, Trahey M, Broder CC, Viglianti GA, et al. Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry. J Biol Chem. 2002;277(52):50579-88. https://doi.org/10.1074/jbc.M204547200.
Mandel R, Ryser HJP, Ghani F, Wu M, Peak D. Inhibition of a reductive function of the plasma membrane by bacitracin and antibodies against protein disulfide-isomerase. Proc Natl Acad Sci USA. 1993;90(9):4112-6. https://doi.org/10.1073/pnas.90.9.4112.
Lin L, Gopal S, Sharda A, Passam F, Bowley SR, Stopa J, et al. Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b′x Domain. J Biol Chem. 2015;290(39):23543–52.
We thank our laboratory staff for their time, effort, and input during this project and preparing this manuscript. In particular, we thank Jonathan Cosme, Yaritza Santiago, and Pablo Rivera. We especially thank Dr. Estela Estapé for her editorial assistance.
This work was supported by the San Juan Bautista School of Medicine Pilot Projects Program to YIN and NIH NHLBI R01HL096518 to JRR.
Ethics approval and consent to participate
The Brigham and Women’s Hospital- Institutional Review Board (Approval num. BWH IRB# 2009P000491) reviewed and approved the extraction of human polymorphonuclear leukocytes (PMN) and mononuclear cells (MNC) from healthy human subjects protocol. All participants were 18 years-old or older and provided written informed consent for participate in the study. All methods were performed in accordance with the relevant guidelines and regulations.
Permission to collect leaves of S. jambos used in this study was obtained from Municipality of Naguabo. Leaves of S. jambos were collected in Puerto Rico in the town of Naguabo (18°14′29.2″ N and 65°45′30.8″ W). Vouchers of S. jambos (019663) were numbered and deposited at the George Proctor Herbarium (SJ) in Puerto Rico. Dr. Robert Ross, Botanist from the University of Puerto Rico in Cayey, identified and classified the botanical species. Appropriate institutional, national and international guidelines were followed during research.
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Typical fingerprinting chromatogram of S. jambos extract. Eight peaks were selected for the quality control of the S. jambos extract. Gallic acid and Rutin were identified using retention times of standards.
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Inostroza-Nieves, Y., Valentin-Berrios, S., Vega, C. et al. Inhibitory effects of Syzygium jambos extract on biomarkers of endothelial cell activation. BMC Complement Med Ther 22, 101 (2022). https://doi.org/10.1186/s12906-022-03572-7
- Protein disulfide isomerase
- Syzygium jambos
- Endothelial cells
- Reactive oxygen species