Retracted Article: Structural characterization of Mesobuthus martensii Karsch peptides and anti-inflammatory potency evaluation in human vascular endothelial cells

Abstract

Studies have reported that scorpion toxins have excellent anti-cancer effects; however, the anti-inflammatory activity of scorpion peptides has rarely been studied. Here, a series of Mesobuthus martensii Karsch peptides (MMKPs) were isolated and the amino acid sequence was identified. The MMKPs mitigated TNF-α-mediated inflammation in human umbilical vein endothelial cells (HUVECs). The results showed that MMKP-1 (His-Glu-Gly-His) treatment (43.0 μM) significantly attenuated the reactive oxygen species (ROS) generation and mitochondrial membrane potential collapse in HUVECs. Moreover, MMKP-1 down-regulated the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions and blocked the NF-κB pathway to alleviate the damage caused by TNF-α. Of note, our study provides a good reference for the anti-inflammation research on scorpion oligopeptides.

---

1 Introduction

Atherosclerosis, a disease of the large arteries, is the primary cause of heart disease and stroke. In westernized societies, it is the underlying cause of about 50% of all deaths. Endothelial cell damage is a fatal factor in serious cardiovascular diseases, including atherosclerosis, which can cause serious clinical consequences such as myocardial infarction, heart failure and stroke.^1–3 Studies have reported that atherosclerosis is closely related to the inflammatory and proliferative responses of endothelial cells after damage.⁴ During the early phase of atherosclerosis, adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), are secreted by the activated endothelial cells in atherosclerotic lesions, stimulating immune cell and monocyte recruitment and migration into the intimal area of the vascular wall.⁵ Tumor necrosis factor (TNF)-α is one of the most potent pro-inflammatory cytokines and its function is closely connected to the apoptosis of endothelial cells and the development of atherosclerotic lesions.⁶

Nuclear factor-κB (NF-κB) plays a significant role in the transcriptional regulation of inflammatory proteins such as cyclooxygenase-2 (COX-2), ICAM-1, VCAM-1, and E-selectin.^7–9 NF-κB exists in the cytoplasm of unstimulated cells and is bound to its inhibitory protein IκBα. IκBα phosphorylation leads to its degradation and the subsequent translocation of NF-κB to the nucleus, where it activates target gene transcription.¹⁰ During atherosclerosis, NF-κB performs as a regulator of pro-inflammatory and anti-inflammatory gene transcription and also as a regulator of cell survival and proliferation.

Arthropods, such as centipedes and scorpions, are widely used in traditional Chinese medicine. The scorpion has been used in traditional Chinese medicine for many centuries to treat various neuronal problems such as chronic pain, paralysis, apoplexy and epilepsy. Meanwhile, Mesobuthus martensii Karsch is a significant traditional Chinese medicine that has been widely used for anti-thrombosis treatment.¹¹ Mesobuthus martensii Karsch peptides (MMKPs) are a class of molecules that show strong anticoagulant, antithrombotic, and fibrinolysis activities.^12–14 Consequently, identifying the bioactive peptide in Mesobuthus martensii Karsch and exploring the anti-inflammation effect will be of great value and significance. To solve this issue, we isolated and identified MMKPs; we explored their anti-inflammatory potency in human umbilical vein endothelial cells (HUVECs) and illustrated the underlying mechanism.

2 Materials and methods

2.1 Isolation and amino acid sequence analysis of the MMKPs

2.1.1 Preparation of crude protein. All extraction and separation procedures were carried out at 4 °C. Mesobuthus martensii Karsch was minced to a homogenate and defatted. The homogenate and iso-propanol were mixed in a ratio of 1 : 7.5 (w/v) and stirred without interruption for 4 h. Iso-propanol was replaced every 1 h. After that, the supernatant was removed, and the sediment was freeze-dried and stored at −20 °C as the total protein.

The total protein (40 g) was dissolved (5%, w/v) in a 0.20 M phosphate buffer solution (PBS, pH 7.5); then, an ultrasonic cleaner (Shanghai, China) with a straight probe and continuous pulse was used for ultrasonication for 4 h. After centrifugation (8000 × g, 40 min), the supernatant was collected and then fractionated by salting-out with increasing concentrations of (NH₄)₂SO₄ (0, 0.70 and 1.40 mM); the fraction in the 1.40 mM (NH₄)₂SO₄ resulting supernatant was freeze-dried and stored at −20 °C for further analysis.

2.1.2 Ultrafiltration and hydrophobic chromatography. The freeze-dried supernatant was fractionated using ultrafiltration chromatography with 1 kDa molecular weight (MW) cut off membranes (Millipore, Hangzhou, China). Two fractions, namely, MMKP-A (MW < 1 kDa) and MMKP-B (MW > 1 kDa) were collected and freeze-dried.
Hydrophobic chromatography. MMKP-A was dissolved in 1.50 M (NH₄)₂SO₄ prepared with 30 mM PBS (pH 7.5) and loaded onto a Phenyl Sepharose CL-4B hydrophobic chromatography column (2.5 cm × 150 cm), which had previously been equilibrated with the above buffer. A stepwise elution was carried out with decreasing concentrations of (NH₄)₂SO₄ (1.50, 0.75 and 0 M) dissolved in 30 mM PBS (pH 7.5) at a flow rate of 3.0 mL min⁻¹. Each fraction was collected at a volume of 80 mL and was monitored at 280 nm. Fractions were then freeze-dried and the anti-inflammatory activity was detected. The fraction having the strongest anti-inflammatory activity was collected and prepared for anion-exchange chromatography.

2.1.3 Anion-exchange chromatography. The MMKP-A-4 solution (10 mL, 121 mg mL⁻¹) was injected into a DEAE-52 cellulose (Shanghai, China) anion-exchange column (2.0 × 115 cm) pre-equilibrated with deionized water and was stepwise eluted with 1000 mL distilled water, 0.40, 0.80, and 1.60 M (NH₄)₂SO₄ solutions at a flow rate of 2.50 mL min⁻¹. Ten fractions (MMKP-A-4-1 to MMKP-A-4-10) were freeze-dried and the anti-inflammatory activity was detected. The fraction having the strongest anti-inflammatory activity was collected and prepared for Gel filtration chromatography.

2.1.4 Gel filtration chromatography. The MMKP-A-4-4 solution (2 mL, 11.5 mg mL⁻¹) was fractionated on a Sephadex G-25 (Sigma-Aldrich, Shanghai, China) column (2.5 × 120 cm) at a flow rate of 2.5 mL min⁻¹. Each eluate (50 mL) was collected and monitored at 280 nm, and five fractions (MMKP-A-4-4-1 to MMKP-A-4-4-5) were freeze-dried and their anti-inflammatory activity was detected. The fraction having the strongest anti-inflammatory activity was collected and prepared for Reversed Phase-High Performance Liquid Chromatography (RP-HPLC).

2.1.5 MMKP isolation by RP-HPLC. MMKP-A-4-4-3 was finally separated by RP-HPLC (Agilent 10.43 HPLC) on a Zorbax, SB C-18 column (4.6 × 250 mm, 5 μm). The elution solvent system was composed of water–trifluoroacetic acid (solvent A; 100 : 0.1, v/v) and acetonitrile–trifluoroacetic acid (solvent B; 100 : 0.1, v/v). The peptide was separated using a gradient elution from 15% to 65% of solvent B for 40 min at a flow rate of 1.0 mL min⁻¹. The detection wavelength was set at 280 nm and column temperature was 20 °C.

2.1.6 Molecular mass determination and amino acid sequence analysis by HPLC-ESI-MS. Prior to HPLC-ESI-MS analysis, the freeze-dried peptide was rehydrated with 1.0 mL of Milli-Q water. HPLC-ESI-MS was carried out on a SCIEX X500R Q-TOF mass spectrometer (Framingham, U.S.A.). The MS conditions were as follows: ESI-MS analysis was performed using a SCIEX X500R Q-TOF mass spectrometer equipped with an ESI source. The mass range was set at m/z 100–1500. The Q-TOF MS data were acquired in the positive mode and the conditions of MS analysis were as follows: CAD gas flow-rate, 7 L min⁻¹; drying gas temperature, 550 °C; ion spray voltage, 5500 V; declustering potential, 80 V. Software generated data file: SCIEX OS 1.0.

2.2 Reagents and cell culture

The Mesobuthus martensii Karsch was acquired from the Ertiantang pharmacy (Guangzhou, China) and identified by Professor Zhou (Jinan University, Guangzhou, China). All the antibodies were purchased from Cell Signaling Technology (CST, USA). TNF-α was obtained from Sigma-Aldrich (USA); the other chemicals used in the current experiment were purchased from Aldrich or Admas and used without any further purification.

HUVECs were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were grown in a culture medium containing 20% fetal bovine serum, 2 mmol L⁻¹ glutamine, antibiomycins (10 μmol L⁻¹ penicillin G and 10 μmol L⁻¹ streptomycin) (all from Sigma-Aldrich, U.S.A.) in a humidified incubator containing 5% CO₂ at 37 °C.¹⁵

2.3 Cell viability and MMKP protective effect assays

HUVECs were plated in Costa 96-well plates at densities of 1 × 10⁵ cells per milliliter. Cells were treated with 10 μL of fresh FBS-free DMEM (control group) or FBS-free DMEM containing different concentrations of MMKPs for 24 h in an incubator. Then, the plates were washed twice with PBS and treated with 10 μL of CCK-8 solution for 4 h.¹⁶ After that, the absorbance was recorded at 450 nm (BioTek, America).

We detected the protective effects of the MMKPs under a non-toxic concentration, and the HUVECs were treated as follows: control group: DMEM only; proliferative control group: TNF-α at 20 ng mL⁻¹ for 6 h; MMKPs group: TNF-α at 20 ng mL⁻¹ for 6 h and then MMKPs at 40 μM for 24 h. After that, the CCK8 method was used to detect the cell viability.

2.4 Detection of reactive oxygen species (ROS) and mitochondrial membrane potential (Δψ_m)

Free radicals, such as reactive oxygen species (ROS), overproduction or antioxidant capacity decline can result in balance disorders and cause ROS accumulation and oxidative stress, which can lead to fatal damage to DNA and proteins within the cell to subsequently induce apoptosis.¹⁷ DCFH can be oxidized into dichlorofluorescein (DCF), which fluoresces, by intracellular oxidants. Therefore, the intracellular ROS level of HUVECs was measured with a Reactive Oxygen Species assay kit.

The experimental groups were set up as follows: control group: DMEM only; TNF-α group: TNF-α at 20 ng mL⁻¹ for 6 h; MMKP-1-L group: TNF-α at 20 ng mL⁻¹ for 6 h and then MMKP-1 at 43.0 μM for 24 h; MMKP-1-H group: TNF-α at 20 ng mL⁻¹ for 6 h and then MMKP-1 at 215 μM for 24 h. The cells were treated as described above, afterwards, the cells were collected and washed thrice with serum-free medium.¹⁵ HUVECs were incubated in 200 μL of a serum-free medium containing DCFH-DA (25 μM) for 30 min at room temperature and the serum-free medium washed thrice. The cells were collected and assayed by flow cytometric analysis (BD FACS Calibur, Franklin Lakes, CA, USA).

The change in Δψ_m in the HUVECs under TNF-α stimulation was detected by JC-1 staining according to the manufacturer's protocol (Beyotime, China). Briefly, 2 mL of 1 × 10⁶/mL cells were treated as described above in 6-well plates. The cells were washed three times with cold PBS and incubated with 1 μg mL⁻¹ of JC-1 at 37 °C for 30 min without light. The supernatant was removed and washed three times with cold PBS and then assayed by flow cytometric analysis (FACScan, CA). Each treatment was carried out in triplicate and the final results were compared with that of the control group or the TNF-α group.

2.5 Apoptosis and cell cycle flow cytometry analysis

The HUVECs were plated on a 6-well plate at a cell density of 2 × 10⁵ cells per well and treated as the experimental design. After that, the HUVECs were harvested and collected. The apoptosis was detected by Annexin V and PI staining using flow cytometry (FACSCalibur, Franklin Lakes, USA).

For cell cycle analysis, the HUVECs were treated as the experimental design. Then, HUVECs were harvested, fixed in 70% ethanol and stored at −20 °C overnight. After that, the cells were washed with PBS and the cell cycle distribution was detected with PI (propidium iodide) staining using flow cytometry (FACSCalibur, Franklin Lakes, USA).

All the tests were repeated at least 3 times.

2.6 Western blotting analysis

HUVECs were plated in 6-well dishes and treated as the experimental design. After that, HUVECs were washed with cold PBS, scraped, pelleted and lysed in a radioimmunoprecipitation assay (RIPA) buffer. After incubation for 1 h on ice, the cell lysate was centrifuged at 3000 g for 0.5 h at 4 °C.

Lysate protein concentrations were determined by a BCA protein assay kit (Thermo Scientific, USA) and the lysate was adjusted with a lysis buffer.¹⁸ The protein was resolved on a 15% SDS-PAGE and transferred to immobilon polyvinyl difluoride (PVDF) membranes. The blots were blocked with a blocking buffer for 10 min at room temperature and then probed with anti-human antibodies for 1 h at room temperature. Then, the blots were incubated with a peroxidase-conjugated anti-rabbit secondary antibody (1 : 3000 dilution) for 1 h at room temperature. The resulting images were scanned using a scanner (Epson V330 Photo, Japan).

2.7 Statistical analysis

Experiments were repeated at least three times and results are expressed as mean ± SD. Data were analyzed by Student t-test and an analysis of variance (ANOVA) test, followed by a Tukey post test to determine the significant differences between groups. Here, p < 0.05 was considered to be significant.

3 Results

3.1 MMKP amino acid sequence identification and protective potency evaluation

Here, twenty-seven MMKPs were isolated, and the amino acid sequences were identified by HPLC-ESI-MS. The peptide is usually protonated under ESI-MS/MS conditions, and fragmentations mostly occur at the amide bonds because it is difficult to break the chemical bonds of the side chains at such a low energy.¹⁹ Therefore, the b and y ions are the main fragment ions when the collision energy is <200 eV.

The molecular mass determination and peptide characterization of MMKP-1 were analyzed by HPLC-ESI-MS. According to the result, the molecular mass of MMKP-1 was determined to be 479.2014 Da. The ion fragment m/z 156.0762 was regarded as the y1 ion and proved to be [His + H]⁺, while m/z 213.0959 was regarded as the b2 ion and represented the [M − His-Glu + H]⁺ ion. The y1 ion (m/z 110.0709) was the typical fragment [His-COOH]⁺ and m/z 324.1292 was the b3 ion (Fig. 1). On the basis of this, we concluded that the sequence of the peptide was HEGH. The amino acid sequences of MMKPs were identified with the method as MMKP-1; this was further confirmed with the BioTools database and the results are listed in Table 1.

Fig. 1 Structure and MS/MS spectra of MMKP-1.

Table 1 Amino acid sequences and protective potency of MMPKs

Compounds	Amino acid sequences	Cell survival rate (%)
MMPK-1	HEGH	95.9 ± 4.88
MMPK-2	DHRFLH	85.2 ± 8.21
MMPK-3	HDRFLH	88.7 ± 8.09
MMPK-4	YAHRGWS	82.9 ± 8.05
MMPK-5	YAHGSWA	81.7 ± 6.99
MMPK-6	HAGYSWA	83.3 ± 8.22
MMPK-7	HASWEH	90.1 ± 7.73
MMPK-8	WESHAS	86.2 ± 8.80
MMPK-9	SHAYSH	85.0 ± 7.83
MMPK-10	HKYRHD	86.9 ± 7.72
MMPK-11	WGHE	89.8 ± 8.20
MMPK-12	HKFW	87.2 ± 7.08
MMPK-13	FWEH	90.6 ± 5.89
MMPK-14	GAEG	88.2 ± 6.38
MMPK-15	WHGE	82.6 ± 6.08
MMPK-16	GEYHSHE	83.7 ± 8.67
MMPK-17	TKFSYE	84.6 ± 7.50
MMPK-18	YKHEWR	91.5 ± 6.60
MMPK-19	KHGEL	85.0 ± 8.23
MMPK-20	EGHGF	79.9 ± 8.02
MMPK-21	HGEY	86.5 ± 6.34
MMPK-22	YEEGAH	85.6 ± 7.70
MMPK-23	AHEFEL	82.5 ± 8.39
MMPK-24	DSHTS	79.8 ± 7.88
MMPK-25	EAHGHSF	82.4 ± 8.03
MMPK-26	EHGEYF	79.8 ± 7.08
MMPK-27	EGFHL	85.4 ± 6.51
Control group	—	100 ± 9.24
Proliferative control group	—	76.3 ± 6.60

---

Studies have reported that natural products can protect HUVECs against oxidative stress-induced apoptosis and reduced amyloid-β peptide-induced oxidative stress. TNF-α plays a central role in the intestinal inflammation of various inflammatory disorders.²⁰ In the current study, cell viability decreased significantly. TNF-α attenuated the cell survival rate of the control group by 23.7%, which indicated that HUVECs suffered severe damage induced by TNF-α. Thus, experiments were conducted to investigate the protective effects of MMKPs on HUVECs under TNF-α stimulation.

First, the cytotoxic effects of MMKPs were detected in HUVECs and the results showed that peptides exhibited no harmful effects on HUVECs at 0.8 mM. We then detected the protective effects of MMKPs under a non-toxic concentration, and the results showed that MMKP-1 has the best protective effects. Of interest, most MMKPs showed protective potency. After treatments with MMKP-1, 7, 11, 13 and 18 and compared to the result for the proliferative control group, the cell survival rates were up-regulated by 19.6%, 13.8%, 13.5%, 14.3% and 15.2%, respectively. Further studies showed that MMKP-1 significantly attenuated the damage induced by TNF-α, and the survival rates were effectively elevated by 71.1% and 93.2% with 43.0 and 215 μM MMKP-1 treatments (Fig. 2).

Fig. 2 MMKP-1 attenuates ROS production induced by TNF-α. Control group: DMEM only; TNF-α group: TNF-α at 20 ng mL⁻¹ for 6 h; MMKP-1-L group: TNF-α at 20 ng mL⁻¹ for 6 h then MMKP-1 at 43.0 μM for 24 h; MMKP-1-H group: TNF-α at 20 ng mL⁻¹ for 6 h then MMKP-1 at 215 μM for 24 h. The HUVEC viability was detected by CCK-8 method; MMKP-1 inhibited the ROS production under the TNF-α-induced oxidative damages in HUVECs. The values are expressed as means ± SD of triplicate tests. **p < 0.01, *p < 0.05, vs. V, ^##p < 0.01, ^#p < 0.05, vs. control, indicate statistically significant difference.

3.2 MMKP-1 down-regulated the ROS level in HUVECs

ROS plays a fatal role in the oxidative process as an oxidization molecule. The interaction of the cellular immune system with endogenous or exogenous inflammatory stimuli determines the generation of ROS. As reported before, TNF-α is believed to play a fatal role in ROS production and oxidative stress in HUVECs.⁵ Studies have also revealed that the hyperactivation of the inflammatory responses can induce oxidative stress in cells and tissues. Therefore, the intracellular ROS level can be an indicator of oxidative stress.²¹

To investigate the ROS eliminating ability of MMKP-1, the ROS level was detected in HUVECs. We set the ROS content in the control group as 1. As shown in Fig. 2, compared with the ROS level of the control group, the ROS level increases by a factor of 7.81 with TNF-α stimulation. Interestingly, compared with the ROS level of the TNF-α group, the ROS levels decreased by 54.8%, and 75.4%, respectively, after 43.0 and 215 μM MMKP-1 treatments (Fig. 2).

Remarkably, the TNF-α-induced ROS generation was significantly attenuated by the MMKP-1 treatments. Of note, the result confirmed that MMKP-1 could reduce the intracellular oxidative stress and protect HUVECs from oxidative stress damage by scavenging ROS.

3.3 Flow cytometry analysis of cell apoptosis

Cell apoptosis was detected with an Annexin V-FITC/PI assay using flow cytometry, and the results are shown in Fig. 3. Compared with the observation for the control group, the total apoptosis and necrosis rate decreased by about 19.4% with TNF-α stimulation. It is gratifying that the MMKP-1 treatments decreased the total apoptosis and necrosis rates by 11.0% and 14.5% and showed great protective effects. Cell apoptosis provided visual evidence for the intracellular protective potency of MMKP-1 in HUVECs. On the basis of this, the protective effects and the underlying mechanism were further investigated.

Fig. 3 MMKP-1 anti-apoptosis in HUVECs induced by TNF-α. Representative scatter diagrams. Control group: DMEM only; TNF-α group: TNF-α at 20 ng mL⁻¹ for 6 h; MMKP-1-L group: TNF-α at 20 ng mL⁻¹ for 6 h then MMKP-1 at 43.0 μM for 24 h; MMKP-1-H group: TNF-α at 20 ng mL⁻¹ for 6 h then MMKP-1 at 215 μM for 24 h. Cells were stained with Annexin-V and PI. The apoptosis of HUVECs was detected by flow cytometry. The evaluation of apoptosis is via Annexin V: FITC Apoptosis Detection Kit as per manufacture's protocol. The quantitative results are shown at the same time. In each scatter diagram, the abscissa represents the fluorescence intensity of the cells dyed by Annexin V and the ordinate represents the fluorescence intensity of the cells dyed by PI. The lower left quadrant shows the viable cells, the upper left quadrant shows necrotic cells, the lower right quadrant shows the early apoptotic cells, while the upper right quadrant shows late apoptotic cells. The values are expressed as means ± SD of triplicate tests. **p < 0.01, *p < 0.05, vs. V, ^##p < 0.01, ^#p < 0.05, vs. control, indicate statistically significant difference.

3.4 Flow cytometry analysis of cell cycle arresting

Notably, according to the content of DNA in cells, which regulates cell growth and replication, the cell cycle can be divided into three phases: G0/G1, S and G2/M. Excess ROS might cause irreversible damage to biological macromolecules, including DNA, nucleic acids, lipids, and proteins.²²

To further investigate the protective potency of MMKP-1 in the cell cycle phase distributions under TNF-α stimuli, the cell cycle was detected and the profiles are listed in Fig. 4. An increase of 31.4% in the G0/G1 population was observed in HUVECs after TNF-α stimulation. It is suggested that TNF-α blocked the cells in the G0/G1 phase, due to which the cells could not enter the S stage to synthesize DNA; eventually, the proliferation of HUVECs was inhibited. Interestingly, after the MMKP-1 treatments and compared to the result for the TNF-α group, the population of G0/G1 decreased by 13.1% and 27.5%, respectively. It can be concluded that cell cycle arresting also contributed to the protective potency of MMKP-1.

Fig. 4 Cell cycle analysis of HUVECs exposed to MMKP-1. Control group: DMEM only; TNF-α group: TNF-α at 20 ng mL⁻¹ for 6 h; MMKP-1-L group: TNF-α at 20 ng mL⁻¹ for 6 h then MMKP-1 at 43.0 μM for 24 h; MMKP-1-H group: TNF-α at 20 ng mL⁻¹ for 6 h then MMKP-1 at 215 μM for 24 h. Cells were collected, fixed in 70% ethanol, and stained with propidium iodide solution. G0/G1: quiescent state/growth phase; S: initiation of DNA replication; G2/M: biosynthesis/mitosis phases. The values are expressed as means ± SD of triplicate tests. **p < 0.01, *p < 0.05, vs. V, ^##p < 0.01, ^#p < 0.05, vs. control, indicate statistically significant difference.

3.5 MMKP-1 attenuated ICAM-1 and VCAM-1 expressions and blocked NF-κB activation

The activation of the endothelial cells by TNF-α has been known to enhance the expressions of adhesion molecules such as ICAM-1 and VCAM-1.⁵ As shown in Fig. 5, TNF-α up-regulates the expressions of both ICAM-1 and VCAM-1. Interestingly, the up-regulation expressions of ICAM-1 and VCAM-1 induced by TNF-α were clearly attenuated by the MMKP-1 treatments. (Fig. 5).

Fig. 5 MMKP-1 down regulates the expression of ICAM-1 and VCAM-1 and inhibits the activation of NF-κB and IκBα. Control group: DMEM only; TNF-α group: TNF-α at 20 ng mL⁻¹ for 6 h; MMKP-1-L group: TNF-α at 20 ng mL⁻¹ for 6 h then MMKP-1 at 43.0 μM for 24 h; MMKP-1-H group: TNF-α at 20 ng mL⁻¹ for 6 h then MMKP-1 at 215 μM for 24 h. Western blotting analysis of ICAM-1, VCAM-1, pIκBα, IκBα, p-NF-κB, NF-κB abundance in HUVECs. The values are expressed as means ± SD of triplicate tests. **p < 0.01, *p < 0.05, vs. V, ^##p < 0.01, ^#p < 0.05, vs. control, indicate statistically significant difference. GAPDH was used as an internal standard of process control and blot band densitometry was analyzed with Image J software.

NF-κB plays a significant role in regulating the expressions of inflammatory proteins such as ICAM-1 and VCAM-1 in many types of cells.²³ IκBα is the main regulator of NF-κB activation. Inactive NF-κB, bound to its inhibitor IκBα as a complex, is restricted to the cytoplasm. IκBα phosphorylation results in its ubiquitination and degradation; then, NF-κB is released from the NF-κB-IκBα complex and translocated to the nucleus.²⁴ As studied above, MMKP-1 attenuated the ICAM-1 and VCAM-1 expressions; hence, we speculated that MMKP-1 might also affect NF-κB activation. Therefore, the activation of NF-κB and IκBα was further explored.

As shown in Fig. 5, ICAM-1 and VCAM-1 were up-regulated after being induced by TNF-α, which was also accompanied with NF-κB and IκBα activation. However, MMKP-1 attenuated the up-regulation of ICAM-1 and VCAM-1 and decreased the levels of phosphorylated NF-κB and IκBα in TNF-α-induced HUVECs. The results revealed that MMKP-1 inhibited the activation of NF-κB and IκBα, leading to the down-regulation of the adhesion molecule expression.

3.6 MMKP-1 attenuated the collapse of Δψ_m and down-regulated the Bax/Bcl-2 ratio

The Δψ_m balance and mitochondrial integrity are significant for the physiological function of cells. Studies have reported that the collapse of Δψ_m is correlated to the events of the apoptotic process. When the concentration of ROS and oxygen stress reach a certain level, Δψ_m can decline, resulting in the release of apoptosis factors. Once the mitochondrial membrane barrier function is lost, several factors including the loss of redox homeostasis, the metabolic consequences at the bio-energetic level, and the perturbation of ion homeostasis lead to cell death.

Here, a JC-1 fluorescent probe was used to detect Δψ_m. As shown in Fig. 2, with the TNF-α stimuli, cells exhibiting Δψ_m decline increase from 10.0% to 36.3%, indicating an increase by 26.3%. Of interest, with MMKP-1 treatments and compared with the result of the TNF-α group, the cells with Δψ_m decline decreased by 9.20% and 21.9%, respectively. Therefore, we concluded that MMKP-1 may show a protective effect by attenuating the mitochondria damage induced by TNF-α.

The mitochondrial pathway plays a significant role in apoptotic modulation. The mitochondrial-mediated apoptotic pathway can be triggered by several factors including the expressions of B-cell lymphoma 2 (Bcl-2) family members such as Bcl-2 associated protein X (Bax) and B-cell lymphoma-extra large (Bcl-xL). This protein family can affect the permeability of the mitochondrial membrane and trigger the opening of the mitochondrial permeability transition pores in the inner mitochondrial membrane, resulting in the release of cytochrome c and mitochondrial dysfunction.

As shown in Fig. 6, up-regulations of the anti-apoptotic proteins Bcl-2 and Bcl-xL can be observed after MMKP-1 treatments. Usually, the greater the Bax/Bcl-2 ratio, the more percentage of cell apoptosis involved. Here, the TNF-α stimuli induced clear Bax/Bcl-2 ratio up-regulation. However, MMKP-1 attenuated TNF-α-induced Bax and Bid and enhanced Bcl-2 and Bcl-xL expressions. In addition, MMKP-1 attenuated the Bax/Bcl-2 ratio.

Fig. 6 MMKP-1 reversed the Δψ_m collapse and down-regulated the Bax/Bcl-2 ratio. Control group: DMEM only; TNF-α group: TNF-α at 20 ng mL⁻¹ for 6 h; MMKP-1-L group: TNF-α at 20 ng mL⁻¹ for 6 h then MMKP-1 at 43.0 μM for 24 h; MMKP-1-H group: TNF-α at 20 ng mL⁻¹ for 6 h then MMKP-1 at 215 μM for 24 h. (A) MMKP-1 displays a protective effect by attenuating the potential mitochondrial membrane damage induced by TNF-α; (B) MMKP-1 attenuated TNF-α-induced Bax and Bid expressions and down-regulated the Bax/Bcl-2 ratio. Western blotting analyses of Bax, Bid, Bcl-xL and Bcl-2 abundance in HUVECs were explored. The values are expressed as means ± SD of triplicate tests. **p < 0.01, *p < 0.05, vs. V, ^##p < 0.01, ^#p < 0.05, vs. control, indicate statistically significant difference. GAPDH was used as an internal standard of process control and blot band densitometry was analyzed with Image J software.

4 Discussion and conclusion

Inflammation is involved in the initiation, rupture, and thrombosis of atherosclerotic plaques.²⁵ During the early stages of atherosclerosis, inflammatory cell recruitment plays a central role.²⁷ TNF-α is a key cytokine and is involved in nearly every step of inflammation. Both ICAM-1 and VCAM-1 play significant roles in the initiation of early atherosclerosis, preferentially contributing to monocyte adhesion.^5,26 Inhibition of the inflammatory response is widely known to be beneficial in the early stages of atherosclerosis.²⁸ Scorpions have been used in antithrombosis treatments for years; the results showed that the expressions of ICAM-1 and VCAM-1 were up-regulated obviously in TNF-α-induced HUVECs, while this up-regulation was significantly suppressed by the MMKP-1 treatment.

Research demonstrates that oxidative stress can result in adhesion function damage, cell survival and apoptosis, which is mediated through apoptosis-associated proteins such as Bax and Bcl-2.²⁹ In the current study, TNF-α increased intracellular ROS production and caused serious apoptosis in HUVECs. However, MMKP-1 significantly attenuated TNF-α-induced oxidative damage in HUVECs by inhibiting NF-κB activation and regulating Bax and Bcl-2 expressions. Studies have reported that TNF-α can increase the binding of NF-κB to its recognition site in the VCAM-1 promoter, subsequently exhibiting monocyte adhesion to vascular endothelial cells. Here, we observed that the TNF-α-induced increase in IκBα degradation was attenuated by MMKP-1 treatment.

In summary, the results of this study demonstrate that the endothelial dysfunction induced by TNF-α, which is associated with the up-regulation of adhesion molecules, reduced viability and apoptosis in HUVECs, can be reversed by the MMKP-1 treatment. The results revealed that MMKP-1 showed a preventive effect against endothelial dysfunction, which may be via inhibiting oxidative stress, improving endothelial survival and preventing the adhesion function damage. Therefore, we inferred that some MMKPs might be used as potential agents in the prevention and treatment of endothelial cell injury-related diseases.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

We thank Professor Chen (Jinan University, Guangzhou, China) for technical assistance as well as critical editing of the manuscript.

References

1. Y. Shan, R. Zhao, W. Geng, N. Lin, X. Wang, X. Du and S. Wang, Protective effect of sulforaphane on human vascular endothelial cells against lipopolysaccharide-induced inflammatory damage, Cardiovasc. Toxicol., 2010, 10, 139–145.
2. C. Wang, H. Li, P. Fu, S. Zhang and R. Xiu, Serum C-reactive protein and circulating endothelial cells in patients with acute myocardial infarction, Clin. Hemorheol. Microcirc., 2005, 32, 287.
3. S. K. Nadar, G. Y. Lip, K. W. Lee and A. D. Blann, Circulating endothelial cells in acute ischaemic stroke, Thromb. Haemostasis, 2005, 94, 707–712.
4. Y. H. Zhang, Y. H. Zhang, X. F. Dong, Q. Q. Hao, X. M. Zhou, Q. T. Yu, S. Y. Li, X. Chen, A. F. Tengbeh and B. Dong, ACE2 and Ang-(1-7) protect endothelial cell function and prevent early atherosclerosis by inhibiting inflammatory response, Inflammation Res., 2015, 64, 253–260.
5. Y. P. Zhu, T. Shen, Y. J. Lin, B. D. Chen, Y. Ruan, Y. Cao, Y. Qiao, Y. Man, S. Wang and J. Li, Astragalus polysaccharides suppress ICAM-1 and VCAM-1 expression in TNF-α-treated human vascular endothelial cells by blocking NF-κB activation, Acta Pharmacol. Sin., 2013, 34, 1036–1042.
6. N. Vrachnis and F. M. Malamas, Immune aspects and myometrial actions of progesterone and CRH in labor, Clin. Dev. Immunol., 2011, 2012, 937618.
7. Y. S. Li, L. P. Wu, K. H. Li, Y. P. Liu, R. Xiang, S. B. Zhang, L. Y. Zhu and L. Y. Zhang, Involvement of nuclear factor κB (NF-κB) in the downregulation of cyclooxygenase-2 (COX-2) by genistein in gastric cancer cells, J. Int. Med. Res., 2011, 39, 2141.
8. C. Lawson, M. Ainsworth, M. Yacoub and M. Rose, Ligation of ICAM-1 on Endothelial Cells Leads to Expression of VCAM-1 via a Nuclear Factor-κB-Independent Mechanism, in International Plant and Animal Genome Conference Xx, 1999, 1999, pp. 2990–2996.
9. H. Liu, Z. C. Wang, Y. G. Bai, Y. Cai, J. W. Yu, H. J. Zhang, J. X. Bao, X. L. Ren, M. J. Xie and J. Ma, Simulated microgravity promotes monocyte adhesion to rat aortic endothelium via nuclear factor-κB activation, Clin. Exp. Pharmacol. Physiol., 2015, 42, 510–519.
10. S. Shishodia, S. Majumdar, S. Banerjee and B. B. Aggarwal, Ursolic Acid Inhibits Nuclear Factor-κB Activation Induced by Carcinogenic Agents through Suppression of IκBα Kinase and p65 Phosphorylation Correlation with Down-Regulation of Cyclooxygenase 2, Matrix Metalloproteinase 9, and Cyclin D1, Cancer Res., 2003, 63, 4375–4383.
11. G. J. Müller, Scorpionism in South Africa. A report of 42 serious scorpion envenomations, S. Afr. Med. J., 1993, 83, 405.
12. Y. Ren, H. Wu, F. Lai, M. Yang, X. Li and Y. Tang, Isolation and identification of a novel anticoagulant peptide from enzymatic hydrolysates of scorpion (Buthus martensii Karsch) protein, Food Res. Int., 2014, 64, 931–938.
13. Y. M. Song, X. X. Tang, X. G. Chen, B. B. Gao, E. Gao, L. Bai and X. R. Lv, Effects of scorpion venom bioactive polypolypeptides on platelet aggregation and thrombosis and plasma 6-keto-PG F1alpha and TXB2 in rabbits and rats, Toxicon, 2005, 46, 230–235.
14. Y. G. Peng, X. U. Ai-Liang, Y. Huang, Y. Yang, D. Shi, J. Y. Zhao and Y. I. Xiao-Min, Effects of Scorpion Purified Liquid on Fibrinolysis System and Coagulation System in Rats with Thrombosis, Chin. J. Inf. Tradit. Chin. Med., 2011, 18, 47–48.
15. R. L. Adams, I. P. Adams, S. W. Lindow, W. Zhong and S. L. Atkin, Somatostatin receptors 2 and 5 are preferentially expressed in proliferating endothelium, Br. J. Cancer, 2005, 92, 1493.
16. Z. Yin, X. Chen, J. L. Chen, W. L. Shen, T. M. H. Nguyen, L. Gao and H. W. Ouyang, The regulation of tendon stem cell differentiation by the alignment of nanofibers, Biomaterials, 2010, 31, 2163.
17. W. Sangkitikomol, A. Rocejanasaroj and T. Tencomnao, Effect of Moringa oleifera on advanced glycation end-product formation and lipid metabolism gene expression in HepG2 cells, Genet. Mol. Res., 2014, 13, 723–735.
18. J. Dalmau, H. M. Furneaux, R. J. Gralla, M. G. Kris and J. B. Posner, Detection of the anti-Hu antibody in the serum of patients with small cell lung cancer--a quantitative western blot analysis, Ann. Neurol., 1990, 27, 544–552.
19. M. A. Raji, P. Frycak, M. Beall, M. Sakrout, J. M. Ahn, Y. Bao, D. W. Armstrong and K. A. Schug, Development of an ESI-MS screening method for evaluating binding affinity between integrin fragments and RGD-based peptides, Int. J. Mass Spectrom., 2007, 262, 232–240.
20. A. M. Westbrook, B. Wei and K. Hacke, et al., The role of tumour necrosis factor-α and tumour necrosis factor receptor signalling in inflammation-associated systemic genotoxicity, Mutagenesis, 2012, 27(1), 77–86.
21. M. Manczak, T. S. Anekonda, E. Henson, B. S. Park, J. Quinn and P. H. Reddy, Mitochondria are a direct site of A beta accumulation in Alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression, Hum. Mol. Genet., 2006, 15, 1437.
22. T. Takami and I. Sakaida, Iron regulation by hepatocytes and free radicals, J. Clin. Biochem. Nutr., 2011, 48, 103.
23. S. J. Cullen, S. Ponnappan and U. Ponnappan, Catalytic activity of the proteasome fine-tunes Brg1-mediated chromatin remodeling to regulate the expression of inflammatory genes, Mol. Immunol., 2009, 47, 600.
24. W. C. Huang and M. C. Hung, Beyond NF-κB activation: nuclear functions of IκB kinase α, J. Biomed. Sci., 2013, 20, 1–13.
25. K. R. K. Murthy, D. As, M. Z. Abbas and L. Haghnazari, Investigations on the role of insulin and scorpion antivenom in scorpion envenoming syndrome, J. Venomous Anim. Toxins Incl. Trop. Dis., 2003, 9, 202–238.
26. R. S. Wright, J. G. Murphy, K. A. Bybee, S. L. Kopecky and J. M. Lablanche, Statin lipid-lowering therapy for acute myocardial infarction and unstable angina: efficacy and mechanism of benefit, Mayo Clin. Proc., 2002, 77, 1085–1092.
27. C. Viedt, W. Shen, J. Fei, M. Kamimura, G. M. Hänsch, H. A. Katus and J. Kreuzer, HMG-CoA reductase inhibition reduces the proinflammatory activation of human vascular smooth muscle cells by the terminal complement factor C5b-9, Basic Res. Cardiol., 2003, 98, 353–361.
28. Y. J. Li, Y. Chen, Y. You, X. G. Weng, Q. Yang, C. X. Ruan and X. X. Zhu, Effects of shenlian extracts on atherosclerosis by inhibition of the inflammatory response, J. Tradit. Chin. Med., 2011, 31, 344–348.
29. R. Ghosh, A. Alajbegovic and A. V. Gomes, NSAIDs and Cardiovascular Diseases: Role of Reactive Oxygen Species, Oxid. Med. Cell. Longevity, 2015, 9–20.

---