As an important part of microvascular vessels, microvascular endothelial cells are involved in the exchange of metabolism substances between blood and tissue to maintain homeostasis of the local environment, and its dysfunction plays a key role in the development of a variety of cardiovascular diseases (CVDs), such as atherosclerosis, acute myocardial infarction, ischemic no-reflow and reperfusion injury, heart arrhythmia, and chronic heart failure.1 Cardiac microvascular endothelial cells (CMECs) are first to be injured during myocardial ischemia reperfusion2 and have an effective treatment of myocarditis caused by a virus.3 Injured CMECs can amplify the damage of the primary injury, resulting in endothelial dysfunction (ED) and creating a vicious cycle. Thus, vascular endothelial inflammation contributes to the pathophysiology of CVDs.4 The nuclear factor-kappa B (NF-κB) pathway plays an important role in inflammation, which involves the phosphorylation of inhibitor of NF-κB (IκBα) in response to proinflammatory stimuli, such as lipopolysaccharide (LPS).5,6 After the IκBα phosphorylation, NF-κB becomes free and then translocates into the nucleus, where it induces the expression of inflammatory cytokines [tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), IL-6, etc], adhesion molecules [intercellular adhesion molecule (ICAM-1)], and chemokines.7,8 Therefore, blocking NF-κB pathway may have some potential therapeutic advantages in the treatment of CVDs.
Shenmai formula, a well-known ancient Chinese prescription, is composed of 2 traditional Chinese herbs, Radix Ginseng Rubra (the root of the Panax ginseng C.A.Mey of family Araliaceae) and Ophiopogon japonicas (the root tuber of Ophiopogon japonicus of family Liliaceae) (in a ratio of 1:1). Its major active components are panaxadiol (PD) and panaxatriol (PT) type saponins from Radix Ginseng Rubra, and ophioponins (OP) from O. japonicas.9 This formula has been used to treat CVDs in China for thousands of years. Nowadays, its new clinical application form, Shenmai injection, has been widely used for the adjuvant treatment of acute myocardial infarction,10 shock,11 coronary heart disease,12 viral myocarditis,13,14 congestive heart failure,15 and others. Previous studies showed that Shenmai injection could ameliorate endothelium function in myocardial ischemia/reperfusion injury,16 and inhibit the activity of TNF-α early and reduce IL-6 and IL-8 release, so as to reduce inflammation and tissue damage.17 The latest research suggested that it could effectively protect rat myocardial ischemia through promoting production of nitric oxide (NO) to remove oxygen free radicals, reducing lipid peroxidation, and inhibiting vascular ED.18 Many experiments had confirmed that its main active ingredients, ginsenosides and Radix ophiopogonis, possessed significant vascular protective actions and maintained the stabilization of vascular endothelium function.19–23 Recent in vivo study found that the optimal combination (in a ratio of 1:2:2) of PD, PT, and OP from Shenmai formula could significantly protect the heart,24,25 but whether this combination may extenuate the inflammation injury in endothelium and its mechanism is unclear. In view of the findings above, we conjectured the combination of active components from Shenmai formula might extenuate myocardial ischemia injury by protecting CMECs from inflammatory injury. In this study, we investigated the anti-inflammatory effects of the combination on LPS-induced inflammatory injury in CMECs. Furthermore, to explore the possible mechanisms, we assessed its effects on the NF-κB p65 expression and IκBα phosphorylation in cells that regulates these cytokines release.
Preparation of Components
The main active components of Shenmai formula (PD, PT, and OP) were provided by Zhejiang University (Zhejiang, China). They were prepared as follows:
1 kg Radix Ginseng Rubra pieces (The Ji'an City Jiju Ginseng Industry Co., Ltd., lot number: 12005, Jilin, China) were extracted three times with 6 L 90% ethanol for 2 h per time. The extract was combined and concentrated under reduced pressure. Extract aqueous solution was subjected to chromatography on a D101macroporous resin column (1000 g), and eluted successively with water, 40% and 70% ethanol by three column volumes. The 40% ethanol eluent was PT (ginsenoside Re, Rg1, Rg2, Rh1, etc.), and the 70% ethanol eluent was PD (ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2, etc.).
1kg Ophiopogon japonicus herbs pieces (Hangzhou Traditional Chinese Medicine Electuary Factory, lot number: 070619, Zhejiang, China) were extracted twice with 6 L 70% ethanol for 2 h per time. The extract was combined and concentrated under reduced pressure. Extract aqueous solution was subjected to chromatography on a D101 macroporous resin column (1000 g), and eluted successively with water and 70% ethanol by four column volumes. The 70% ethanol eluent was OP (ophiopogonin D, F, G, H, I, etc.). PD, PT, and OP were completely dissolved in RPMI-1640 medium as a proportion of 1:2:2 under asepsis condition and then cryopreserved at −20°C.
The experiment was approved by the Ethics Committee of Tianjin University of Traditional Chinese Medicine (Permit number: LAEC2013002). In accordance with Nishida's primary culture method of CMECs as described,26 1–3 day old Wistar rats (provided by the experiment animal center of Tianjin, China) were sterilized with 75% ethanol for 3 minutes. The hearts were rapidly excised and placed in a dish containing calcium and magnesium-free D-Hanks buffer to wash out blood cells. After removal of connective tissue, the atria, right ventricle, and all heart valves, the remaining left ventricular tissue was incised along the anterior free wall and washed again with the buffer. To devitalize epicardial mesothelial cells and endocardial endothelial cells, left ventricles were immersed in 70% ethanol for 30 seconds and then extensively washed with the buffer. The remaining heart tissue was cut into the size of 0.5–1.0 mm3. Collagenase (0.1%) and trypsin (0.06%) (mixed with 1:1 ratio) were used to digest for 3–5 minutes in a shaking water bath 4–5 times at 37°C. After adding 10% fetal bovine serum (FBS), the supernatant (except the first time) was filtered through a 75-μm mesh filter and centrifuged at 1000 rpm for 10 minutes. Cells were resuspended in the complete medium [RPMI-1640 medium (HyClone) supplemented with 10% FBS (FBS; GIBCOL) and antibiotics including 1 × 105 U·L−1 penicillin and 1 × 105 μg L−1 streptomycin, 37°C] and cultured in 75 cm2 culture flask for 1.5 hours. CMECs were adherent growth at the bottom of the flask, then cultured in an incubator with a renewal of the complete medium at 1-day intervals. The endothelial identity of CMECs was confirmed by immunofluorescence staining for Von Willebrand factor, a specific marker of endothelial cells, with more than 98% positive staining. Second-generation CMECs in good condition (under the microscope, the cells adherent growth, fast growth, and close arrangement.) were used for further studies and incubated with 1 μg mL−1 LPS for 24 hours as the inflammatory-injured CMECs model.
1. Sham group (control): cell culture
2. LPS group: cell culture + LPS
3. Experimental groups:
* Cell culture + LPS + combination 25 μg/mL
* Cell culture + LPS + combination 50 μg/mL
* Cell culture + LPS + combination 100 μg/mL
Lactate Dehydrogenase Content in CMECs Supernatant Assay
The second-generation CMECs were seeded at a density of 1.0 × 104 cells per well in 96-well plate. On 80% confluency, cells were cultured with free-FBS medium for 24 hours to make them grow in synchronization in the incubator. Cells were treated with LPS in the presence or absence of the combination (25, 50, and 100 μg mL−1) for 24 hours. Then, supernatants of each group were collected from culture media, and the lactate dehydrogenase (LDH) content was measured with LDH assay kit (Biosino Biotechnology Co, Ltd, Beijing, China) according to the manufacture's instructions.
Quantitative Real-time Polymerase Chain Reaction Analysis
Total RNA from CMECs was extracted with Trizol (Shanghai Sangon Biological Engineering Technology & Service Co, Ltd, Shanghai, China) and was reverse transcribed to cDNA with SYBR Premix Ex Taq Reverse Transcription Reagents (TaKaRa Biotechnology Co, Ltd, Dalian, China). All primers were provided by Sangon Biotech (Shanghai, China). The Rat ACTB Endogenous Reference Genes Primers (Cat. PRN02) could obtain more accurate target gene expression (Sangon Biotech, Shanghai, China); the sense and antisense primers for IL-1, IL-6, TNF-α, and ICAM-1 are described in Table 1. The amplification was accomplished in an ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). The expression of related target genes was determined by the 2−ΔΔCT method.27
Enzyme-linked Immunosorbent Assay
CMECs were treated as described above for 6 hours (for testing the protein expression of NF-κB p65) or 24 hours (for testing the protein expression of IL-1, IL-6, and TNF-α), then the supernatant was collected for measuring the concentration of IL-6, IL-1, TNF-α, ICAM-1, and NF-κB p65, according to the manufacture's guidelines of the enzyme-linked immunosorbent assay kit (R&D Systems).
Western Blotting Analysis
After synchronization, CMECs were preincubated with various concentrations (25, 50, and 100 μg mL−1) of the combination for 2 hours before treated with LPS (1 μg mL−1) for 15 minutes.28 Cells were washed twice with ice-cold PBS and lysed with 50 μL RIPA lysis buffer in the presence of PMSF (1 mM) per well. Protein concentration was measured by the bicinchoninic acid method (bicinchoninic acid protein assay kit was purchased from Beijing Solarbio Science & Technology Co, Ltd, China) and mixed with SDS-PAGE loading buffer. After boiling at 100°C for 5 minutes, proteins were separated on 10% (weight per volume) gradient SDS-polyacrylamide gels (BIO-RAD) and transferred to polyvinylidene fluoride membranes (Millipore, GER). Membranes were incubated with 5% (weight per volume) skimmed nonfat milk in TBST buffer for 2 hours at room temperature, followed by incubation with primary antibodies against NF-κB p65 [1:1000; Cell Signaling Technology (CST)], p-IκB α (1:1000; CST), and β-actin (1:1000; CST) overnight at 4°C, and then washed blots were subsequently incubated with HRP–conjugated secondary antibody (1:2000; CST) for 1.5 hours at room temperature. Antibody-positive bands were detected with a chemiluminescence agent (Millipore, GER). The band densities were quantified by Imaging Systems analysis software (VersaDoc Mp5000; BIO-RAD).
Data were presented as mean ± SE. Statistical comparison between different treatments was performed by 1-way analysis of variance with SPSS 13.0 software (SPSS Inc, Chicago, IL). P values less than 0.05 were considered significant.
The Combination Reduced LPS-induced LDH Release in CMECs
To determine whether LPS may induce inflammatory action to injure CMECs and the combination can protect these cells, we detected the LDH content in the cell culture supernatant. LPS significantly increased LDH release by 179.21% in CMECs compared with control group, whereas 25, 50, and 100 μg mL−1 of combination remarkably decreased LDH release induced by LPS at 66.30%, 62.98%, and 59.67% compared with LPS group, respectively (Fig. 1).
The Combination Downregulated LPS-induced IL-1, IL-6, and ICAM-1 mRNA Expression
To examine whether the combination can inhibit LPS-induced mRNA expression of proinflammatory cytokines, such as IL-1, IL-6, TNF-α, and ICAM-1 in CMECs, reverse transcription–quantitative polymerase chain reaction analysis was conducted. LPS significantly upregulated the mRNA expression of IL-1, IL-6, TNF-α, and ICAM-1 compared with control group, whereas the combination (100 μg mL−1) significantly suppressed IL-1 and IL-6 and ICAM-1 mRNA expression (Figs. 2A, B, D), but had no effect on TNF-α mRNA expression compared with LPS group (Fig. 2C).
The Combination Inhibited LPS-induced IL-1, IL-6, TNF-α, and ICAM-1 in CMECs Supernatants
LPS remarkably increased the concentrations of IL-1, IL-6, TNF-α, and ICAM-1 up to 131.30%, 161.38%, 107.83%, and 133.38%, respectively, in the cell culture supernatants compared with control group, whereas the combination (25, 50, and 100 μg mL−1) inhibited the release of these cytokines compared with LPS treatment group at 24 hours after LPS incubation (Figs. 3A–D).
The Combination Inhibited LPS-induced NF-κB Release in CMECs Supernatants
We next investigated whether the combination could inhibit LPS-induced NF-κB release in CMECs supernatants. The result showed that LPS remarkably promoted the NF-κB p65 content in CMECs supernatants compared with control group, whereas the combination (25, 50, and 100 μg mL−1) could reduce its content at 6 hours after LPS treatment (Fig. 4).
The Combination Inhibited the Protein Expression of NF-κB p65 and the Phosphorylation of IκBα in CMECs
We also investigated whether the combination could affect on the protein expression of NF-κB p65 subunit and the phosphorylation of IκBα in CMECs. Treatment with LPS alone increased the protein expression of NF-κB p65 and the phosphorylation of IκBα in CMECs, whereas this effect was suppressed by the combination (100 μg mL−1) (Fig. 5).
Vascular endothelium plays an important role in the pathogenesis of CVDs; the vascular endothelial inflammation can cause diverse CVDs and then amplify the damage of the primary injury, resulting in ED and creating a vicious cycle.21 Results from this study showed that the combination of active components from Shenmai formula remarkably downregulated the mRNA expression of IL-1, IL-6, and ICAM-1 and inhibited their release out of rat CMECs. Furthermore, our initial finding suggested that the combination played anti-inflammatory effects through blocking NF-κB pathway.
After inflammatory injury, the cell membrane permeability was markedly increased, resulting in LDH more easily released to the outside the cell; meanwhile, the synthesis and release of inflammatory cytokines was increased, which might exacerbate cell injury. In this study, we used LPS, a bacterial oligosaccharide endotoxin, to induce inflammatory injury of CMECs and found that the content of LDH and, inflammatory mediators (IL-1, IL-6, TNF-α, and ICAM-1), was increased in the cell supernatant, whereas the combination could diminish LPS-induced inflammatory injury. Actually, LPS can activate IκB kinase to promote the phosphorylation and degradation of the IκB family members, which causes NF-κB separation from IκB and entrance nucleus to induce the gene expression and release of IL-1, IL-6, TNF-α, ICAM-1, etc.25 These cytokines and adhesion molecules induce the cascade reaction and aggravate cellular damage. Here, we found that the combination significantly downregulated the expression of IL-1, IL-6, and ICAM-1 and suggested that it might have a significant anti-inflammatory effect, which was consistent with the previous study.5 Furthermore, we found that the NF-κB p65 protein expression was significantly increased in the CMECs injured by LPS-induced inflammatory indicated that LPS could promote IκB phosphorylation and NF-κB activation corresponding to the early report.24 The combination might remarkably inhibit NF-κB p65 protein expression and reduce the phosphorylation level of IκB, which suggested that the anti-inflammatory effect of the combination might cause inhibition of NF-κB activation and block the NF-κB pathway.
It is noteworthy that TNF-α can also activate NF-κB–like LPS and cause many kinds of cytokines to express, which induces inflammatory cascade reaction and exacerbate inflammatory injury.29 The data in this study suggested that the combination significantly reduced the content of TNF-α in CMECs supernatants, but had no effect on its mRNA expression. It may be because the combination decreases the TNF-α protein level in inflammatory damaged CMECs through effect on the other links of protein synthesis (such as amino acid activation, origination, extension, termination and releasing of a polypeptide chain, and modification after protein synthesis, etc) to ease the inflammatory reaction. The underlying mechanism should be further investigated.
Taken together, our data provided initial proof that the combination could protect the LPS-induced inflammatory injury in rat CMECs by blocking the NF-κB pathway. However, there are many caveats. First, our study only provided an initial description of these anti-inflammatory effects. Further studies are needed to investigate the molecular mechanisms underlying these endothelial anti-inflammatory responses by choosing NF-κB pathway inhibitor. Second, we used LPS to induce inflammatory injury to mimic the inflammation of endothelium, whether the combination could extenuate other inflammatory cytokine (such as TNF-α or IL-1)-induced injury should be investigated. Finally, the other inflammatory mediator, NO synthase (especially inducible NO synthase) and PGE2, should be evaluated to investigate a more systematic and comprehensive study of its anti-inflammatory effects.
Furthermore, some studies suggested that PD, PT, and OP have anti-inflammatory effects. Ginsenoside Rg1 can alleviate inflammation by blocking NF-κB pathway and reducing the release of IL-6.30 PPD- and PPT-type ginsenosides including CK and Rh1 may exhibit strong anti-inflammatory effects by reducing the production of NO, cyclooxygenase-2, and proinflammatory cytokines (such as prostaglandin E2 and TNF-α) and through downregulation of inducible NO synthase, NF-κB, and IκB kinase in LPS-stimulated RAW 264.7 cells.31 Ginsenoside-Re inhibited secretion levels of inflammatory mediators, such as TNF-α, and IL-1β in LPS-stimulated murine macrophage Raw 264.7 cells.32 Ruscogenin extracted from O. japonicas suppresses excessive expression of TNF-α–induced ICAM-1, reduces the migration of NF-κB p65 and DNA binding activity, and blocks NF-κB signaling pathway.33–35 Each extract from O. japonicas exerts anti-inflammatory effects through inhibiting the expression of TNF-α and the secretion of NO induced by LPS in RA W264.7 macrophages.36 Therefore, whether PD, PT, and OP in the combination play a synergistic role in anti-inflammatory is to be further studied.
In summary, our results revealed that the combination remarkably suppressed the synthesis and release of inflammatory mediators including IL-1, IL-6, TNF-α, and ICAM-1 induced by LPS in rat CMECs through blocking the NF-κB pathway. These findings provided a new mechanism underlying the vascular protective activity of the combination of active components from Shenmai formula.
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