Curcuma oil reduces endothelial cell mediated inflammation in post myocardial ischemia
/reperfusion in rats
A Manhas, V Khanna, P Prakash, D Goyal, R Malasoni, A Naqvi, A Dwivedi, M Dikshit, K Jagavelu
,Department of Pharmacology and Department of Pharmaceutics, CSIR-Central Drug Research Institute, Lucknow226031, India. Present address: Department of Internal Medicine, University of Iowa Health
care, Iowa City, IA 52242, USA.
Corresponding Author:Kumaravelu Jagavelu, Ph.D Senior Scientist Pharmacology Division,Room PCS-202,Central Drug Research Institute-CSIR,CDRI/B.S.10/1, Sector 10,Jankipuram extension, Sitapur
Road, Lucknow-226031, India. Phone 91-522-2771940, 2771960 ext 4602 E-mail:
Financial Support: This work was supported by the grants from CSIR-CDRI Network project:
"Towards holistic understanding of complex diseases: Unraveling the threads of complex disease (THUNDER, BSC0102), UNDO BSC0103 of Council of Scientific and Industrial Research (CSIR), Government of India and Ramalingaswami fellowship (K.J) of Department of Biotechnology (DBT), Government of India.
Conflict of Interest
The authors have no conflict of interest.
Abstract:
Endothelial cells initiated inflammation persisting in post myocardial infarction needs to be controlled and moderated for avoiding fatal complications. Curcuma oil (C.oil, Herbal Medicament) a standardized hexane soluble fraction of Curcuma longa has possessed neuroprotective effect. However, its effect on MI/RP and endothelial cells remain incompletely defined. Here, using in vivo rat myocardial ischemia/reperfusion (MI/RP) injury model and in vitro cellular approaches using EA.hy926 endothelial cells, ELISA, RT-PCR and myograph, we provide evidence that with effective regimen and preconditioning of rats with C.oil (250 mg/kg, P.O.), before and after MI/RP surgery protects rats from MI/RP induced injury. C.oil treatment reduces left ventricular ischemic area and endothelial cell induced inflammation, specifically in the ischemic region (*p < 0.0001) and improved endothelial function by reducing the expression of pro-inflammatory genes and adhesion factors on endothelial cells both in vitro and in vivo.Furthermore, mechanistic studies have revealed that C.oil reduced the expression of adhesion factors like E-selectin (#p = 0.0016) and ICAM-1 ($p = 0.0069) in initiating endothelial cells induced inflammation. In line, to the RT-PCR expression data, C.oil reduced the adhesion of inflammatory cells to endothelial cells as assessed by the interaction of THP-1 monocytes with the endothelial cells using flow based adhesion and under inflammatory conditions. Conclusion:
These studies provide evidence that salutary effect of C.oil on MI/RP could be achieved with pre
and post treatment of rats, C.oil reduced MI/RP induced injury by reducing the endothelial cell mediated inflammation, specifically in the ischemic zone of MI/RP rat heart.
Key Words: Myocardial infarction, Cardiac rupture, Herbal Medicament, Endothelial cell, Inflammation
Introduction:
Curcumin, extracted from the Curcuma longa rhizomes is an oldest and first hand traditional medicine for many diseases in South Asia (1). It has been extensively used in Ayurvedic medicine to treat common bacterial infections, arthritis and wound healing owing to its antimicrobial, anti-proliferative, antioxidant and anti-inflammatory properties (2).Several fractions of standardized hexane soluble fraction, Herbal Medicament (HM), a patented compound from CDRI, or Curcuma oil, (C.oil) have been isolated with different enriched compounds; fraction A with ar-turmerone and turmerone, fraction B with curcumene and zingiberine and fraction C with curcumerozone, germacrone, zedoarone, sedoarondiol and others (3, 4). C.oil, a volatile and highly lipophilic component of curcuma having a greater efficacy, has been endorsed with antimicrobial, antifungal, anti-inflammatory and antifibrotic properties. The effective nature of its lipophilic activity has reduced cerebral ischemia (5) and stroke (6) but not myocardial infarcted area (7). Inflammation ensuing MI/RP presents a greater risk for the myocardial tissue, with the endothelium initiating and progressing the inflammation leading to increased cardiac damage (8).
Vascular endothelium plays a vital role in the initiation of pathogenesis of numerous thrombotic (9), inflammatory and cardiovascular diseases. Myocardial ischemia/reperfusion (MI/RP) injury induces a profound inflammatory condition that results in exuberant wound healing response to myocardial injury, angiogenesis, tissue destruction and fibrosis (10, 11). The persistent inflammation involves diminished nitric oxide production (12), influx of neutrophils (13, 14), myocardial and endothelial cell injury (15) leading to myocardial scarring. Although, the essential process of inflammation is required, it has to be tightly regulated for healthy healing of myocardial tissue (16, 17).
Previous studies have reported the positive effects of C.oil on platelets, in which C.oil has conferred protection against thromboembolism and increased time to occlusion in arterial thrombosis by inhibiting platelet aggregation, but could not confer protection against MI/RP injury in the rats (7). The endothelial cells lining the arterial wall are the first line of cells exposed to the C.oil, once in the circulation. These cells mediate the adhesion of various pro- inflammatory cells for the initiation of inflammation. However, the effect of C.oil on endothelial cells induced inflammatory responses during post myocardial infarction/reperfusion injury has not been elucidated. Hence we hypothesized that C.oil might reduce the endothelial cell induced inflammatory factors ensuing MI/RP induced injury and further a regimen change in C.oil treatment could confer protection against MI/RP induced injury. In support of this hypothesis, we demonstrated that C.oil treatment, pre and post MI/RP injury has reduced the infarct size in the rat and the endothelial cell induced inflammatory responses. Similarly, C.oil significantly reduced the expression of several adhesion factors as well as interaction of inflammatory cells with endothelium. Thus, suggesting that C.oil could limit the endothelial cells induced inflammatory reaction following MI/RP in rats.
Materials and methods
Animal, cells and Reagents
Male Wistar rats (270-320gm) were obtained from the National Laboratory Animal Centre of CSIR-Central Drug Research Institute (CDRI), Lucknow, India. All the animals had free access to standard chow pellet and water ad libitum and maintained at the optimum temperature at 25 ± 2°C, 12 hours a day/night cycle. All the animal procedures were approved by the Institutional Animal Ethics Committee of the CDRI (Permit No. IAEC/2012/60). EA.hy926 endothelial cells generated by C. J. S. Edgell (was generously provided as a gift from the University of North Carolina, Lineberger Comprehensive Cancer Centre) (18, 19) and human THP-1 cells from Acute Monocytic Leukemia patients were cultured in DMEM and RPMI-1640 (Himedia) respectively supplemented with 10% FBS and 1% penicillin/streptomycin. Curcuma longa rhizomes were procured from the market and their authenticity was established with the specimen from CSIR-CDRI, Botany division specimen voucher Accession No. 24660, Field No.
11796 dated 1985 and its HPLC based standardization (4) has shown to contain ar-turmerone, turmerone and curlone.
Myocardial Ischemia Reperfusion (MI/RP) Injury
Wistar rats were either treated with vehicle (0.25% CMC, Sigma) or C. oil (250 mg/kg), a standardized hexane soluble fraction synthesized by the department of Pharmaceutics, CSIR- CDRI as described in (4) orally for 3 days pre and post MI/RP procedure. MI/RP procedure was performed as described in a previous paper from our laboratory (7). In brief, rats were anesthetized with ketamine/xylazine (80/10 mg/kg, i.p.). Left anterior descending coronary artery was ligated for a period of 30 min by using 5-0 suture (Ethicon, non-absorbable surgical suture U.S.P, Johnson & Johnson) and reperfused for a period of 3 days. After 3 days of MI/RP, the animals were sacrificed for further studies. A treatment schedule was carried out as described in Fig. 1A. To determine the left ventricular infarct size, heart was quickly excised and sliced into five 1.0 mm thick sections perpendicular to the long axis of the heart. Slices were then placed in 1% pre warmed 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) prepared in phosphate-buffered saline (pH 7.4) at 37°C for 30 min followed by rinsing with distilled water to remove traces of TTC. Viable tissue stained pale red and dead tissue remains uncolored. The tissue samples were fixed in 10% formaldehyde and infarct tissue was photographed using a surgical microscope (Leica S6 D, Germany) and quantified by using Leica QWin Plus software (Leica QWin Plus V 3.5.1, Switzerland). Myocardial infarct size was expressed as a percentage of total heart ventricular slice surface area (7).
Biochemical estimation
CK-MB activity was measured in serum collected on the day of surgery, after a period of 3 hours of MI/RP. The assay was performed as per manufacturer’s protocol (Merck Diagnostic). The change in absorbance (∆A) per minute was measured spectrophotometrically (PowerWave HT Microplate Spectrophotometer, BioTek, USA) at the wavelength of 340 nm and enzyme activity was expressed as CK-MB activity (U/L) = ∆A/min x 1650 (7).
Endothelial dysfunction
The thoracic aorta was excised and placed in ice-cold Kreb’s bicarbonate solution (118mM NaCl; 5mM KCl; 2.5mM CaCl2; 1.2mM MgSO4; 1.2mM KH2PO4; 25mM NaHCO3; 11mM Glucose; and 0.03mM EDTA; pH 7.4) and trimmed of adhering connective tissue and fat.
Transverse 4-mm wide aortic rings were cut and mounted in 10 ml organ baths containing Kreb’s solution, maintained at 37°C and continuously oxygenated with 95% O2 and 5% CO2. The rings were equilibrated for 90 min, during which the bathing fluid was changed every 15 min and the rings were kept under a constant tension of 2 g throughout the experiment. Isometric measurements were recorded with force transducers (FSG-01, Budapest, Hungary) using S.P.E.L. solution Pack for Experimental Laboratories ADVANCE ISOSYS data acquisition and analysis software (Experimetria, Budapest, Hungary). Ring viability and maximum contraction were assessed by 80mM KCl (20, 21).
FIGURE 1. C.oil reduced the ischemic zone in MI/RP-injured rats. A, Treatment schedule for rats before and after MI/RP injury. B, CK-MB levels (U/L) in serum obtained after 3 hours of reperfusion injury. *P = 0.0195 versus vehicle MI/RP. C and D, TTC-stained heart sections posttreatment with CMC (0.25%) and C.oil (250 mg/kg) after 3 days of reperfusion injury. E,
Percent infarct size of above TTC-stained heart sections. All the values are expressed in mean 6 SEM. *P , 0.001 versus vehicle MI/RP. N = 3.
Positive stains were quantified by using Leica QWin Plus soft- ware (Leica QWin Plus version 3.5.1, Switzerland).
ELISA
ELISA was performed on plasma samples collected on the last day of the experiment. Blood was withdrawn via cardiac puncture in a plastic syringe supplemented with 1.9 % tri-sodium citrate.
TNF-α, IL-6 and IFN-γ (BD Biosciences) was analyzed as per the manufacturer’s protocol.
Immunofluorescence
Experimental hearts were embedded in OCT medium and 8 micron serial sections were harvested using a cryotome. The slides were stained using antibodies vWF (Sigma) and E- selectin (Abcam). Stained slides were mounted by using anti fade reagent and were micrographed using a fluorescent microscope (Leica DM6000). Positive stains were quantified by using Leica QWin Plus software (Leica QWin Plus V 3.5.1, Switzerland).
Isolation of Rat myocardial endothelial cells
Heart was dissected, washed with ice-cold phosphate buffer saline (pH 7.4) and minced.
Digestion of tissue was done by using Collagenase (2 mg/ml, Sigma) supplemented with BSA (1%, Sigma) at 37°C. CD31 positive cells were then positively selected using sheep anti-Rat IgG Dynabeads (Invitrogen, India) coated with Rat Anti-Mouse CD31 (BD Biosciences) for 30 min.
CD31 positive endothelial cells were separated by using magnetic particle concentrator (Invitrogen, India) and processed further for RT-PCR. (22).
Real-time Polymerase Chain Reaction
Total RNA was extracted from EA.hy926 endothelial cells and myocardial endothelial cells using Trizol (Invitrogen), and complementary DNA (cDNA) was synthesised using RevertAid H Minus First Strand cDNA Synthesis Kit as per the manufacturer’s protocol (Fermentas) with 1µg of total RNA. Real time PCR was performed using Roche light cycler detection systems. Gene specific primers were designed for vWF, PDGF, PDGF-B, Lox-1, Annexin-V, ICAM-1, E- selectin, GAPDH and 18s RNA can be provided upon request. Messenger RNA levels for each gene was normalized to GAPDH or 18s RNA and the data were shown as fold changes.
Flow based Cell-Cell interaction
EA.hy926 endothelial cells were grown on collagen G (PAN biotech, Germany) coated fields and either treated with 20 µg/ml dose of C.oil, vehicle control or TNF-α (10 ng/ml) for 6-8 hours. Calcein-AM (C3100 MP, Invitrogen, India) labeled THP-1 monocytic cells were allowed to interact with EA.hy926 endothelial cells under flow conditions with a flow rate of 1 ml/min or under static conditions for 10 min. Non adhered cells were washed off with phosphate buffered saline and the adhered cells were fixed with neutral buffered formalin (pH 7.4) for a period of 2 min and mounted using an aqueous mounting medium (Fisher Scientific, India). The interaction of EA.hy926 endothelial cells with THP-1 monocytes was micrographed using a fluorescent microscope (Leica DM5000). All the fields were mounted by using glycerol followed by fixation
with interaction was quantified by using Leica Qwin software (Leica QWinPlus V 3.5.1, Switzerland).
Statistics
The variation between data groups were evaluated for significance by the use of the unpaired t- test or one-way analysis of variance (ANOVA), as appropriate and Dunnett's Multiple Comparison post test (Graph Pad prism Software Inc., San Diego, CA). Data are expressed as Mean ± SEM, unless noted otherwise. For all tests, a probability value of ≤ 0.05 was considered as statistical significance.
Results
Pre and post treatment of C.oil reduced the ischemic zone in MI/RP injured rats
As a first step to explore the role of C.oil in post MI/RP inflammation, we fed the rats with C.oil pre and post MI/RP injury (Fig. 1A). To confirm the successful MI/RP surgery, the well accepted marker isoenzyme CK-MB, expressed in damaged myocardium, was measured. We observed a significantly reduced levels of CK-MB in C.oil treated MI/RP rats (253.77 U/L vs. 464.677 U/L,
*p = 0.0195) compared to that of the vehicle MI/RP rats (Fig. 1B). Gross observation of 2,3,5, - triphenyltetrazolium chloride (TTC) stained heart sections harvested after 3 days post MI/RP revealed that the ischemic zone in the left ventricles was significantly reduced in C. oil treated group as compared to the vehicle MI/RP group (Fig. 1C - 1D). Quantification of ischemic ventricular area showed a significantly reduced ischemic zone in C.oil treated MI/RP group (5.3576% vs. 15.5218%, *p < 0.0001) compared to the vehicle MI/RP left ventricular area (Fig.
1E). However, ischemic zone was not observed in sham control of both the groups (Fig. 1C - 1D) C.oil treatment reduced inflammatory cytokine mediators
Inflammatory cytokines, released after MI/RP, play an important role in the progression of the disease leading to a cascade of signaling events that affects the endothelial function. Hence we measured the levels of potent circulating inflammatory cytokine mediators like TNF-α, IL-6 and IFN-γ by ELISA. C.oil treatment pre and post MI/RP injury, significantly reduced the circulating protein levels of TNF-α (38.68 ρg/ml vs. 90.12 ρg/ml, *p < 0.0001), IL-6 (40.57 ρg/ml vs. 73.95 ρg/ml, *p < 0.0001) and IFN-γ (28.18 ρg/ml vs. 79.45 ρg/ml, *p = 0.0179) as compared to that of vehicle MI/RP group. However, non-significant minor reductions in the inflammatory mediators were observed in the sham group upon C.oil treatment compared to vehicle sham (Fig. 2A-2C).
Endothelial function is improved upon C.oil treatment
We then examined the consequences of the decreased expression of inflammatory mediators on the endothelium in the C.oil treated MI/RP rats. To determine the endothelial function in the C.oil treated MI/RP rats, thoracic aortic rings were excised and acetylcholine (Ach) (3 nM to 300 µM) induced concentration dependent relaxation pattern was performed. C.oil treated aortic rings showed a significantly effective relaxation (68.0% ± 7.1 vs. 43.40% ± 6.5, *p < 0.01)) at 300 µM dose and (65.1% ± 7.1 vs. 40.80% ± 6.0, *p < 0.01) at 30 µM acetylcholine dose (Fig. 3A) as compared to that of vehicle MI/RP aortic rings. Further, C.oil treated aortic rings relaxed at par with vehicle treated sham group at 30µM acetylcholine dose (68.0% ± 7.1 vs. 86.80% ± 2.70, @p
< 0.001). Sham control of both the groups failed to show any significant change in relaxation pattern (86.80% ± 2.70, @p < 0.001 and 87.20% ± 2.40 #p < 0.001) at 300 µM and (80.80% ±
2.80, @p < 0.001 and 77.90% ± 2.10, #p < 0.001) at 30 µM acetylcholine dose (Mean ± SEM, Fig. 3B). These results suggest that C. oil treatment improved the endothelial function in MI/RP rats.
FIGURE 2. Reduction in inflammatory cytokine levels after C.oil treatment. A, Plasma TNF-a level. B, Plasma IL-6 level. C, Plasma IFN-g level, in the C.oil (250 mg/kg)-treated MI/RP rats, after 3 days of reperfusion injury. All the values are expressed in mean 6 SEM. *P , 0.0001, #P = 0.0179 versus vehicle MI/RP. N = 3.
FIGURE 3. Endothelial function is improved on C.oil treatment. Concentration-dependent acetylcholine (3 nM–300 mM) induced relaxation in rat thoracic aortic rings precontracted with phenylephrine
hydrochloride (1 mM), after 3 days of MI/RP injury. All the values are expressed in mean 6 SEM. *P , 0.01 C.oil MI/RP versus vehicle MI/RP, @P , 0.001 vehicle sham versus vehicle MI/RP, and #P , 0.001 C.oil sham versus vehicle MI/RP. N = 3.
C.oil treatment controls the inflammatory and apoptotic markers induced by MI/RP on endothelial cells
Next we assessed whether the C.oil administration affects the inflammatory factors specifically on the cardiac endothelial cells. Cardiac endothelial cells were characterized by the presence of its biomarker vWF and CD31 (Supplementary Fig.S1). Real-time PCR expression profile of cardiac endothelial cells isolated specifically from distant and ischemic zone three days after reperfusion revealed that pre and post treatment with C.oil reduced the expression of pro inflammatory factor von Willebrand Factor (vWF) as compared to the distant myocardial endothelial cells (fold change 0.595 vs. 1.65, *p = 0.0010) (Fig. 4A). Similarly the expression of platelet derived growth factor (PDGF) (fold change 1.63 vs. 4.30, *p = 0.0005) (Fig. 4B) and Lox-1 (1.35 vs. 2.44, *p<0.0196) (Fig.4C) were also significantly reduced in the MI/RP ischemic zone as compared to distant myocardial endothelial cells. Furthermore, apoptosis marker annexin V was also considerably reduced in C.oil treated endothelial cells of MI/RP ischemic zone (fold change 2.09 vs. 3.22, *p = 0.0190) (Fig. 4D) as compared to the distant myocardial endothelial cells. These results suggest that inflammatory responses were increased only in the ischemic zone of MI/RP heart and not in distant zone. Thus the increased inflammatory response is attenuated with C.oil treatment in ischemic zone.
C.oil inhibits adhesion of monocytes to endothelial cells both under static and flow based conditions
To further assess the effect of C.oil on the functionality of the adhesion factors expressed in the endothelial cells, in vitro cell-cell interaction assay was carried out either under static or flow based conditions. C.oil (20 µg/ml) treated EA.hy926 endothelial cells significantly reduced calcein-AM labelled THP-1 cell adherence onto endothelial cells under static conditions as compared to the vehicle treated control cells (268.233 vs. 525.352, *p = 0.0267) (Fig. 5A -5B) and per se control (Supplementary Fig. S2). Flow based conditions were known to up-regulate the several adhesion factors on endothelial cells. Hence, we assessed the effect of C.oil (20 µg/ml) on EA.hy926 endothelial cell under flow based conditions, Calcein-AM labelled THP-1 cells were allowed to interact with EA.hy926 endothelial cells under flow based conditions. C.oil (20 µg/ml) treated EA.hy926 endothelial cells had shown significantly reduced interaction (168.991 vs. 554.193, *p = 0.0008) (Fig. 5A - 5B) as compared to vehicle control and cells without any treatment (Supplementary Fig. S2). Next, we investigated whether C.oil could reduce inflammation mediated adhesion of THP-1 monocytes. C.oil treatment significantly reduced the TNF-α induced adhesion of THP-1 monocytes onto EA.hy926 endothelial cells both under static (477.367 vs. 1519.23, *p = 0.0005) (Fig. 5D) and flow based conditions (459.834 vs.
1533, *p = 0.0003) (Fig. 5D). As a control, phase contrast images show equal number of endothelial cells were plated in both the groups (Supplementary Fig. S3A - 3D). These results suggested that C.oil reduced the inflammatory conditions by inhibiting the interaction of THP-1 monocytes with the EA.hy926 endothelial cells.
FIGURE 4. C.oil treatment control inflammatory and apoptotic marker on endothelial cells, induced by MI/RP. A, Expression of von Willebrand factor, *P = 0.0010 versus vehicle MI/RP. B, The expression of PDGF, *P , 0.0005 versus vehicle MI/RP. C, Expression of Lectin-type oxidized LDL receptor-1 (Lox-1), *P = 0.0196 versus vehicle MI/RP. D, Expression of Annexin-V, *P = 0.0190 versus vehicle MI/RP, on endothelial cells isolated from heart after 3 days of reperfusion injury. All the values are expressed in mean 6 SEM. N = 3.
C.oil inhibits the expression of important adhesion factors on endothelial cells
To understand the underlying mechanism behind the reduced adhesion of THP-1 monocytic cells with the EA.hy926 endothelial cells, we performed real-time PCR for adhesion factors expressed on the C.oil treated EA.hy926 cells. C.oil (20 µg/ml) treatment for 6 hours on EA.hy926 cells, significantly reduced the mRNA expression levels of vWF (fold change 0.32, *p = 0.0323), PDGF-B (fold change 0.579, @p = 0.0439), ICAM-1 (fold change 0.398, $p = 0.0069) and E- selectin (fold change 0.248, #p = 0.0016) as compared to control (Fig. 6A). We substantiated these results in MI/RP hearts. C.oil treated MI/RP rat hearts had significantly reduced the endothelial specific expression of adhesion factor E-selectin (5.47% vs. 17.49%, *p = 0.0021) (Fig. 6B - 6C) and pro-inflammatory protein vWF (12.38% vs. 27.03%, #p = 0.0045) (Fig. 6D - 6E) compared to the vehicle MI/RP rat hearts. These results suggest that both in in vivo disease condition and in vitro, C.oil reduced the expression of vWF and E-selectin on endothelial cells.
FIGURE 5. C.oil inhibits adhesion of monocytes to endothelial cells. A, Adhesion of THP-1 monocytes onto C.oil (20 mg/mL)- treated EA.hy926 cells under static- (left panel) and flow-based conditions (right panel). B, Quantified data of THP-1 monocytes adhered onto EA.hy926 cells, *P= 0.0267 versus control and #P = 0.0008 versus control. C, Adhesion of THP-1 monocytes onto C.oil (20 mg/mL)-treated EA.hy926 cells after TNF-a (10 ng/mL) stimulation under static- (left panel) and flow-based conditions (right panel). D, Quantified data of THP-1 monocytes adhered onto EA.hy926 cells, *P = 0.0005 versus control and #P = 0.0003 versus control. All the values are expressed in mean 6 SEM. N = 4.
FIGURE 6. C.oil inhibits the expres- sion of adhesion factors on endothelial cells. A, RT-PCR for expression of adhesion factors on EA.hy926 cells after 6 hours of C.oil (20 mg/mL) treatment. *P = 0.0323, @P = 0.0439, $P = 0.0069, and #P = 0.0016 versus control. B, The expression of E-selection in vehicle (CMC, 0.25%) and C. oil (250 mg/kg)-treated MI/RP heart. C, Percent mean fluorescence inten- sity, *P = 0.0021 versus vehicle MI/RP. D, Expression of vWF in vehicle and C.oil-treated MI/RP heart. E, Percent mean fluorescence intensity, #P = 0.0045 versus vehicle MI/RP. All the values are expressed in mean 6 SEM. N = 3.
Discussion
Uncontrolled inflammatory cell recruitment resulting post myocardial infarction leads to fatal complications such as ventricular rupture. Post myocardial inflammatory cell recruitment needs
to be regulated tightly to allow infarct healing and minimizing the ventricular rupture (23). Our present work made a number of important observations on how C.oil reduces post myocardial inflammation. Specifically, our study provides the following new findings. 1. C.oil treatment reduced the infarct size. 2. C.oil treatment reduced the inflammatory factors specifically on myocardial endothelial cells in the infarct zone without affecting the distant area. 3. C.oil reduced the adhesion of THP-1 monocytic cells with endothelial cells, which is a prerequisite for initiating inflammation.
Previous studies have shown, C.oil has a neuroprotective effect against cerebral stroke in the rat MCAo model, but not in MI/RP injury (6, 7). The previous dose regimen of the C.oil did not elicit the protective effect on MI/RP injury, because the rats were fed with C. oil only for 3 days, this could have led to a partial inhibition of inflammatory processes as evident in the platelet aggregation time (7). However, preconditioning of rats with C.oil before and after MI/RP injury significantly reduced the infarct size as assessed by the TTC staining and also reduced the release of potent pro-inflammatory factors like TNF-α, IL-6 and IFN-γ which are responsible for initiation and progression of several inflammatory processes (24, 25), suggesting that C.oil impedes with the post MI/RP inflammatory processes. The reduction in MI/RP infarct size could be partly attributed to the regimen change of C.oil treatment, as this pretreatment could reduce the inflammatory factors and reactive oxygen species that could gravitate the MI/RP region.
Further, our results obtained from the endothelial cells specifically from myocardial distant and ischemic region clearly shows that inflammatory milieu and apoptotic markers is increased only in the ischemic zone as compared to distant zone, suggesting that the MI/RP stress to the heart has increased the inflammatory signaling mechanism and is activated only in the ischemic zone.
Hence, controlling the inflammation in a regulated fashion is very important in minimizing the eventual thrombosis formation. In line, these results are corroborated with the previous data suggest that a reduction in the inflammatory milieu or improving antioxidants post MI could reduce the chance of post MI/RP inflammatory cascade.
Endothelial dysfunction of vasculature is the causative factor for the initiation of inflammation contributing to congestive heart failure and elevated peripheral resistance (26). Myocardial ischemia increases the oxidative endothelial injury and other inflammatory cytokines increasing the endothelial dysfunction (27, 28). This could be the effect from the parent compound Curcumin which showed beneficial effects on arterial dysfunction by improving acetylcholine induced endothelial-dependent dilation in aged mice (29) and also reduced the angiotensin II mediated AT-1 receptor upregulation (30). Our results indicate that post-MI/RP induced endothelial dysfunctions could be due to the increased cytokine levels, which are significantly reduced with C.oil treatment and brought back to normalcy. This net reduction in circulating inflammatory cytokines might be due to pre and post treatment with C.oil prior to surgery. These results corroborate with the several of the publications reported earlier of having an anti- inflammatory properties with its parent compound, Curcumin (31). However, our results from the C.oil treatment (a fraction of Curcumin), significantly improved endothelial function in C.oil treated MI/RP rat is convincing, suggesting that C.oil could improve endothelial function despite increased inflammatory milieu.
Curcumin has been proved to have an antiplatelet and anti-inflammatory effect in different experimental conditions. In line with the effect of parent compound, C.oil treatment has significantly reduced the platelet aggregation and thrombosis (7). The anti-thrombotic effect could be due to reduced adhesion factors expressed on endothelial cells. Endothelial cells have
been reported with an exclusive capacity to detect the shear stress and respond accordingly.
Further, endothelial cells, when activated or in response to the ischemic stress, increase the expression of adhesion factors for initiating the inflammatory reactions (32). Our results demonstrate that EA.hy926 endothelial cells treated with C.oil (per se) and TNF-α, induced adhesion of THP-1 monocytes have reduced both under static as well as in flow based conditions, suggesting that the C.oil might have reduced the expression of adhesion factors.
Further, in severe MI/RP, inflammatory cell recruitment after myocardial infarction needs to be tightly regulated to permit the infarct healing while avoiding fatal complications such as cardiac rupture (6, 17). The reduction in the adhesion of monocytes to endothelial cells is in part due to the reduced expression of E-selectin, which regulates the adhesion by binding sialylated carbohydrates expressed on the leukocytes (33). C.oil inhibition could be in part by binding to the E-selectin and thereby reducing the adhesion or it might directly reduce the transcriptional regulation of E-selectin as assessed by the decreased expression of E-selectin mRNA and protein content in C.oil treated endothelial cells and MI/RP hearts respectively. Together, our results with C. oil treatment show significantly reduced the adhesion of monocytes on EA.hy926 endothelial cells, suggesting a reduced inflammatory reaction.
Acknowledgements
The authors acknowledge Sheeba Samuel for cryo-sectioning and Varun Pathak for secretarial support.This manuscript bears CDRI communication Number 8665.
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