Edited by:
Reviewed by:
*Correspondence:
This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Increasing evidence shows that inflammation plays a vital role in the occurrence and development of ischemia/reperfusion (I/R). Suppression of excessive inflammation can ameliorate impaired cardiac function, which shows therapeutic potential for clinical treatment of myocardial ischemia/reperfusion (MI/R) diseases. In this study, we investigated whether Ginkgolide C (GC), a potent anti-inflammatory flavone, extenuated MI/R injury through inhibition of inflammation.
Nowadays, cardiovascular disease becomes one of the leading cause of disability and death worldwide, and one of the most popular and dangerous cardiovascular diseases is myocardial ischemia/reperfusion (MI/R) injury (
CD40, one member of tumor necrosis factor receptor (TNFR) family, serves as a transmembrane type I receptor (
Increasing evidence suggested that NF-κB activation elevated in the MI/R-related infarct area, inflammation was suppressed when NF-κB activation was inhibited, and cardiac preservation was provided (
Ginkgolide C (GC,
Chemical structure of GC.
Thus, taking into account activation of inflammatory response during MI/R injury, we have not only investigated the protective effect of GC both
GC (PubChem CID: 161120), Aspirin (PubChem CID: 2244), 2, 3, 5-Triphenyltetrazolium chloride (TTC), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and chloral hydrate were bought from Sigma (St. Louis, MO, United States). DMEM medium (high glucose) and newborn calf serum were products of Gibco (Grand Island, NY, United States). Trypsin was purchased from Amresco (Solon, OH, United States). TNF-α, IL-1β and IL-6 ELISA kits were bought from Sigma (St. Louis, MO, United States). Anti-ICAM-1, anti-VCAM-1, anti-iNOS, anti-CD40, anti-NF-κB p65, anti-p-IκB-α, anti-IKK-β, anti-β-actin, anti-Histone, goat anti-rabbit and anti-mouse IgG antibodies were purchased from Santa Cruz (Santa Cruz, CA, United States). Nuclear-Cytosol Extraction Kit and ECL plus kit was purchased from Jiancheng (Nanjing Jiancheng Bioengineering Inc., Nanjing, China)
Adult male Sprague-Dawley rats (250 ± 20 g) were provided by Experimental Animal Center of Shandong University. Rats were raised in a controlled environment (temperature 18–22°C, humidity 40–70%) with a 12 h light-dark cycle and allowed to eat and drink freely until the experiment. All the experiments were approved by the ethics committee of the Shandong University (Permission No. 2013-092).
MI/R surgery was precisely implemented according to the procedure in
Rats were randomly divided into 6 groups as follows (
Infarct size was assessed by TTC staining technique in accordance with a previous study report (
The third heart slice was fixed in 3.0% glutaraldehyde buffered fixative (pH 7.2) for 2–3 days. After being rinsed in PBS, the specimens were embedded in Polybed 812. Then, 90 nm-thick specimens were made and observed under electron microscope (JEM-2000EX).
The third heart slice was stained with hematoxylin and eosin (HE) after fixation. The degree of histopathological damage was assessed with pathological scores according to the criteria reported by the previous study (
The expressions of ICAM-1, VCAM-1 and iNOS were examined by immunohistochemistry. In brief, the third frozen heart slice incubated with anti-ICAM-1, anti-VCAM-1, and anti-iNOS antibodies overnight at 4°C after being blocked by 10% normal serum. Then, anti-rabbit IgG was applied to incubate the heart slices combined with primary antibody for 30 min. Consequently, immunohisochemical staining protocol was applied to heart slice in accordance with the instructions. Images were obtained using fluorescence microscope. The optical density of positive staining area was quantified by Image-Pro Plus software (version 6.0, Media Cybernetic, United States) and the results were expressed as mean optical density mean ± SD.
The homogenized tissue samples were sonicated to release the MPO from tissue into the supernatant. Then,
Ventricular myocytes isolated from 1 to 4-day-old Sprague-Dawley rats were treated with H/R injury according to the procedure in
The cells were randomly divided into 6 groups as follows (
When ventricular myocytes reached 80–85% confluence, the cells in control group were transfected with pGPU6/Hygro while other groups’ cells were transfected with pGPU6/Hygro-CD40. The transfection should take 24 h using the GenePharma Transfection Reagent before the pretreatment of GC, Aspirin and H/R procedure.
Cell survival of ventricular myocytes was determined by MTT assay. After H/R procedure, 5 mg/mL MTT was added to the medium to incubate the cells for 4 h at 37°C. MTT was then removed, and cells were lysed with DMSO for 15 min. Absorbance was tested at wavelength of 490 nm by a microplate reader. Results were expressed as the percent of the optical density (OD) of control cells.
Following 120 min reperfusion, all rats were deeply anesthetized, and blood samples were obtained. Cell supernatant was collected from medium after H/R procedure. The concentrations of TNF-α, IL-1β and IL-6 were detected by ELISA kits.
Cytoplasmic and nuclear proteins from cells were extracted by Nuclear-Cytosol Extraction Kit (Applygen Technologies Inc, Beijing, China). Cultured medium from plates was removed and astrocytes were detached with cold PBS and centrifuged at 1000 rpm for 5 min at 4°C. Pellets were resuspended in 200 μL of cytosol extraction buffer A and incubated on ice for 10 min. Then, 10 μL cytosol extraction buffer B was added, incubated on ice for 1 min and centrifuged at 1000 ×
The quantity of total protein was assessed by BCA assay. Fifty microgram of protein was loaded to SDS-PAGE gel and then, transferred to a PVDF membrane. After being blocked with 5% skim milk, the membrane was incubated with primary antibody (1:800) that recognized ICAM-1, VCAM-1, iNOS, CD40, NF-κB p65, p-IκB-α and IKK-β proteins. After incubation for 4 h at 37°C, horseradish peroxidase-conjugated secondary antibody (1:1000) was added to the membrane and the immune complexes was determined using an ECL plus kit. Images were taken using the Gel Imaging System with Quantity One software.
All data values were expressed as the mean ± standard deviation (SD).
As shown in
Effect of GC on infarct size in MI/R injury rat model. Treatment with Aspirin and GC significantly reduced the infarct size in MI/R injury rat model. Data were expressed as mean ± SD (
In the present study, mitochondrial alignment and myofibrillar banding appeared normal in the control group (
Effects of GC on the ultrastructure of myocardial tissue, histopathological changes, myocardial PMNs counting and MPO activity.
Pathological changes were applied to further evaluate the protective effect of GC on MI/R injury. After I/R occurred, the tissue in I/R injured area became necrotic, and the structure of intercalated disk and gap junction was severely impaired. However, treatment with GC and Aspirin could largely improve the histological features caused by I/R injury, characterized by alleviative inflammatory infiltration and recoverable structure of ischemic myocardial tissue (
To quantify neutrophilic infiltration, myocardial MPO activity was subsequently investigated. Very low MPO activity (1.21 ± 0.05 U/g protein) was tested in control group (
The concentration of inflammatory cytokines following I/R procedure in serum were detected by ELISA.
Effects of GC on serum inflammatory cytokines after I/R in rats.
Group | Dose (mg/kg) | TNF-α (pg/mL) | IL-1β (pg/mL) | IL-6 (pg/mL) |
---|---|---|---|---|
Control | 25.50 ± 6.91 | 86.63 ± 12.52 | 29.74 ± 5.07 | |
I/R | 76.10 ± 12.91## | 257.23 ± 18.69## | 95.87 ± 4.80## | |
I/R + Aspirin | 16 | 31.41 ± 6.99∗∗ | 102.92 ± 7.31∗∗ | 40.87 ± 5.29∗∗ |
I/R + GC | 8 | 63.01 ± 6.93∗ | 200.96 ± 8.71∗∗ | 87.55 ± 4.50∗ |
16 | 49.23 ± 7.68∗∗ | 156.96 ± 13.88∗∗ | 67.53 ± 5.34∗∗ | |
32 | 35.79 ± 7.23∗∗ | 129.58 ± 7.05∗∗ | 53.58 ± 6.01∗∗ |
Immunostaining was used to visualize the expressions of ICAM-1, VCAM-1 and iNOS in myocardial tissue. Compared with the control group, I/R procedure significantly increased the expressions of ICAM-1, VCAM-1 and iNOS (
Effect of GC on ICAM-1, VCAM-1 and iNOS expressions in myocardial tissue after I/R procedure. The tissue was observed using a microscope at a magnification × 400.
As shown in
Effects of GC on cell viability after H/R procedure. Data were expressed as mean ± SD (
As shown in
Effects of GC on the supernatant inflammatory cytokines of ventricular myocytes.
Group | Concentration (μM) | TNF-α (pg/mL) | IL-1β (pg/mL) | IL-6 (pg/mL) |
---|---|---|---|---|
Control | 6.45 ± 1.41 | 34.02 ± 8.85 | 48.94 ± 6.82 | |
H/R | 54.61 ± 9.36## | 907.96 ± 32.08## | 612.16 ± 41.17## | |
H/R + Aspirin | 10 | 17.33 ± 3.92∗∗ | 207.46 ± 81.12∗∗ | 159.03 ± 9.08∗∗ |
H/R + GC | 1 | 46.38 ± 4.08∗ | 531.29 ± 54.70∗∗ | 574.98 ± 48.95∗∗ |
10 | 42.35 ± 4.47∗∗ | 427.73 ± 43.31∗∗ | 427.75 ± 42.88∗∗ | |
100 | 15.86 ± 2.91∗∗ | 275.89 ± 78.23∗∗ | 263.16 ± 15.52∗∗ |
As shown in
Effects of GC on the expressions of CD40, ICAM-1, VCAM-1, iNOS, NF-κB p65, p-IκB-α and IKK-β by Western blot after H/R procedure.
As shown in
Activation of NF-κB pathway is thought to be a key signaling event involved in the pathogenesis of MI/R. To determine whether GC inhibited H/R-induced cardiac NF-κB activation, we first examined the translocation of NF-κB p65 from cytoplasm to the nucleus by western blot. As shown in
NF-κB translocation is preceded by the phosphorylation and ubiquitination of IκB-α, we then studied whether IκB-α was also in relation to the effect of GC on NF-κB pathway activation. First, we checked whether the total IκB-α had degradated. The results showed that the total IκB-α in each group was not different (
Substantial evidence unequivocally shows that a wide variety of influential factors regulate the activity of NF-κB, especially IKK-β. Then, we investigated the effect of GC on IKK-β activity. The results revealed a significant inhibitory effect on the H/R-induced IKK-β activation in the presence of GC (
The cell viability in H/R + CD40 silencing group was significantly lower than that in control group (
Effects of GC on cell viability
The ventricular myocytes preincubated with pGPU6/Hygro expressed little CD40 after transfection in control group, and preincubation of pGPU6/Hygro-CD40 successfully and stably inhibited CD40 expression in all groups (
The expressions of TNF-α, IL-1β, IL-6, ICAM-1, VCAM-1 and iNOS in H/R + CD40 silencing group were significantly elevated compared with control group. After the procedure of CD40 gene silencing, GC could not regulate the expressions of TNF-α, IL-1β, IL-6, ICAM-1, VCAM-1 and iNOS (shown in
Effects of GC on the supernatant inflammatory cytokines of ventricular myocytes under the condition of CD40 gene silencing.
Group | Concentration (μM) | TNF-α (pg/mL) | IL-1β (pg/mL) | IL-6 (pg/mL) |
---|---|---|---|---|
Control | 6.64 ± 0.94 | 35.40 ± 7.11 | 48.42 ± 9.09 | |
H/R + CD40- | 70.08 ± 11.39## | 1036.01 ± 105.01## | 603.53 ± 59.57## | |
H/R + GC + CD40- | 1 | 69.27 ± 5.88 | 1041.32 ± 62.15 | 600.48 ± 107.81 |
10 | 68.88 ± 7.47 | 1037.66 ± 52.72 | 601.44 ± 80.31 | |
100 | 68.56 ± 4.19 | 1023.08 ± 72.66 | 598.30 ± 60.98 |
H/R + CD40 silencing group procedure significantly affected the translocation level of NF-κB, activation of IκB-α phosphorylation and up-regulation of IKK-β activity. Nonetheless, all GC groups showed no difference in NF-κB p65 translocation, IκB-α phosphorylation and IKK-β activity compared with H/R + CD40 silencing group (shown in
This is the first investigation studied on I/R rats subjected to GC to examine whether GC exerts significant protective effect against MI/R injury
It has been broadly illuminated that inflammation played a vital role in the entire process of MI/R injury, and its effects refer to myocytes dysfunction, inflammatory cytokines overexpression, neutrophil accumulation and myocardium ischemia (
It is clear that lack of blood to heart results in insufficient blood and oxygen transmitted to the beat of the heart muscle, named ischemia, following by damage or dysfunction of the cardiac tissue (
Importantly, neutrophils “shoot at first sight,” releasing reactive oxygen species and proteases, thereby causing extensive collateral damage to the ischemic myocardium and increasing infarct size (
Moreover,
NF-κB could induce the transcription and expression of multiple cytokines related to immunity and inflammation and other relative gene so as to cause heart damage following pathologic process of I/R injury (
It is now clear that NF-κB signaling is tightly regulated at the level of IκB phosphorylation. The IKK complex, composed of IKK (α, β, and γ), is activated by phosphorylation of IKK-α or IKK-β on serine residues within their activation loops either by upstream kinases or through autophosphorylation (
Indeed, much evidence was given that activation of NF-κB occurred at the very early stages of I/R process and then sequentially modulated the occurrence of downstream inflammatory factors (
CD40 signaling is now widely accepted as a model of the non-canonical NF-κB pathway, which will strongly trigger its downstream signaling events (
According to the fact, we concluded that CD40 played the vital role in regulating inflammation for ventricular myocytes in response to H/R injury. Therefore, we silence the CD40 gene in ventricular myocytes stimulated by H/R to verify our hypothesis. As we expected, the cardioprotective effect of GC disappeared after CD40 gene was silenced. Consequently, it was evident that GC protected heart from MI/R injury through inhibiting NF-κB pathway via CD40. It is also consistent with other investigations reporting that inhibition of CD40 reduces the activation of NF-κB.
Taken together, our results obtained from this work demonstrated that GC protected rat from inflammatory insult by occlusion of LAD coronary artery and inhibited H/R-induced inflammatory damage to ventricular myocytes through blocking CD40 dependent NF-κB signal pathway on the basis of following evidence: (1) GC improved cardiac outcomes of MI/R injury and alleviated PMNs infiltration in rats, (2) GC inhibited overproduction of inflammatory factors (TNF-α, IL-1β and IL-6) and overexpression of inflammatory proteins (ICAM-1, VCAM-1 and iNOS) both
Schematic diagram describing the mechanism in the inhibitory effect of GC on H/R-induced ventricular myocytes inflammatory injury:
GC can exhibit significant cardioprotective effects through reducing infarct size, inhibiting inflammatory response, improving myocardial histological structure and alleviating PMNs infiltration during I/R injury. Inhibition of excessive inflammation via suppressing CD40/NF-κB signal pathway should be the key mechanism of GC in the protective of MI/R injury. Thus, GC will be a prospective and preventive agent in the management of MI/R injury.
Animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures involved in the use of the laboratory animals were approved by the ethics committee of Shandong University (Permission No. 2013-092).
RZ, DH, ZL, YZ, and PL performed the research. JL, GY, SL, BH, and JbL designed the research study. RZ and CS analyzed the data. RZ wrote and edited the paper.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.