RSC Advances PAPER View Article Online View Journal | View Issue Structural characterization of Mesobuthus martensii Karsch peptides and anti-inflammatory Cite this: RSC Adv.,2019,9, 19365 potency evaluation in human vascular endothelial cells Man Zheng,a Xiafeng Yan,a Fanli Bu,a Fenglei Zhang,a Zhenhua Li,a Jiali Cui,b Jie liu *c and Minya Dong*a 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-a-mediated inflammation in human umbilical vein endothelial cells (HUVECs). The results showed that MMKP-1 (His-Glu-Gly-His) treatment (43.0 mM) significantly attenuated the reactive oxygen species (ROS) generation and mitochondrial membrane potential collapse in HUVECs. Moreover, Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Received 4th March 2019 MMKP-1 down-regulated the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion Accepted 18th May 2019 molecule-1 (VCAM-1) expressions and blocked the NF-kB pathway to alleviate the damage caused by DOI: 10.1039/c9ra01609k TNF-a. Of note, our study provides a good reference for the anti-inflammation research on scorpion rsc.li/rsc-advances oligopeptides. 1 Introduction Nuclear factor-kB(NF-kB) plays a signicant role in the transcriptional regulation of inammatory proteins such as Atherosclerosis, a disease of the large arteries, is the primary cyclooxygenase-2 (COX-2), ICAM-1, VCAM-1, and E-selec- This article is licensed under a – cause of heart disease and stroke. In westernized societies, it is tin.7 9 NF-kB exists in the cytoplasm of unstimulated cells the underlying cause of about 50% of all deaths. Endothelial cell and is bound to its inhibitory protein IkBa.IkBa phos- damage is a fatal factor in serious cardiovascular diseases, phorylation leads to its degradation and the subsequent Open Access Article. Published on 20 June 2019. Downloaded 9/30/2021 4:07:41 PM. including atherosclerosis, which can cause serious clinical translocation of NF-kB to the nucleus, where it activates consequences such as myocardial infarction, heart failure and target gene transcription.10 During atherosclerosis, NF-kB stroke.1–3 Studies have reported that atherosclerosis is closely performs as a regulator of pro-inammatory and anti- related to the inammatory and proliferative responses of inammatory gene transcription and also as a regulator of endothelial cells aer damage.4 During the early phase of cell survival and proliferation. atherosclerosis, adhesion molecules, such as intercellular Arthropods, such as centipedes and scorpions, are widely adhesion molecule-1 (ICAM-1) and vascular cell adhesion used in traditional Chinese medicine. The scorpion has molecule-1 (VCAM-1), are secreted by the activated endothelial been used in traditional Chinese medicine for many cells in atherosclerotic lesions, stimulating immune cell and centuries to treat various neuronal problems such as monocyte recruitment and migration into the intimal area of chronic pain, paralysis, apoplexy and epilepsy. Meanwhile, the vascular wall.5 Tumor necrosis factor (TNF)-a is one of the Mesobuthus martensii Karsch is a signicant traditional most potent pro-inammatory cytokines and its function is Chinese medicine that has been widely used for anti- closely connected to the apoptosis of endothelial cells and the thrombosis treatment.11 Mesobuthus martensii Karsch development of atherosclerotic lesions.6 peptides (MMKPs) are a class of molecules that show strong anticoagulant, antithrombotic, and brinolysis activi- ties.12–14 Consequently, identifying the bioactive peptide in Mesobuthus martensii Karsch and exploring the anti- a Department of Cardiology, Dongying People's Hospital, Dongying, Shandong, 257091, ff China. E-mail: [email protected]; Fax: +86-755-86671907; Tel: +86-755- in ammation e ectwillbeofgreatvalueandsigni cance. 86671907 To solve this issue, we isolated and identi ed MMKPs; we bPeople's Hospital of Shule County, Kashi, Xinjiang, 844043, China explored their anti-inammatory potency in human umbil- cThe Research Center of Allergy & Immunology, School of Medicine, Shenzhen ical vein endothelial cells (HUVECs) and illustrated the University, Shenzhen, 518060, China. E-mail: [email protected]; Fax: +86- underlying mechanism. 755-86671905; Tel: +86-755-86671905 This journal is © The Royal Society of Chemistry 2019 RSC Adv.,2019,9, 19365–19374 | 19365 View Article Online RSC Advances Paper 2 Materials and methods strongest anti-inammatory activity was collected and prepared for Reversed Phase-High Performance Liquid Chromatography 2.1 Isolation and amino acid sequence analysis of the (RP-HPLC). MMKPs 2.1.5 MMKP isolation by RP-HPLC. MMKP-A-4-4-3 was 2.1.1 Preparation of crude protein. All extraction and nally separated by RP-HPLC (Agilent 10.43 HPLC) on a Zorbax, separation procedures were carried out at 4 C. Mesobuthus SB C-18 column (4.6  250 mm, 5 mm). The elution solvent martensii Karsch was minced to a homogenate and defatted. system was composed of water–triuoroacetic acid (solvent A; The homogenate and iso-propanol were mixed in a ratio of 100 : 0.1, v/v) and acetonitrile–triuoroacetic acid (solvent B; 1 : 7.5 (w/v) and stirred without interruption for 4 h. Iso- 100 : 0.1, v/v). The peptide was separated using a gradient propanol was replaced every 1 h. Aer that, the supernatant elution from 15% to 65% of solvent B for 40 min at a ow rate of À was removed, and the sediment was freeze-dried and stored at 1.0 mL min 1. The detection wavelength was set at 280 nm and À20 C as the total protein. column temperature was 20 C. The total protein (40 g) was dissolved (5%, w/v) in a 0.20 M 2.1.6 Molecular mass determination and amino acid phosphate buffer solution (PBS, pH 7.5); then, an ultrasonic sequence analysis by HPLC-ESI-MS. Prior to HPLC-ESI-MS cleaner (Shanghai, China) with a straight probe and continuous analysis, the freeze-dried peptide was rehydrated with 1.0 mL pulse was used for ultrasonication for 4 h. Aer centrifugation of Milli-Q water. HPLC-ESI-MS was carried out on a SCIEX (8000  g, 40 min), the supernatant was collected and then X500R Q-TOF mass spectrometer (Framingham, U.S.A.). The MS fractionated by salting-out with increasing concentrations of conditions were as follows: ESI-MS analysis was performed (NH4)2SO4 (0, 0.70 and 1.40 mM); the fraction in the 1.40 mM using a SCIEX X500R Q-TOF mass spectrometer equipped with – (NH4)2SO4 resulting supernatant was freeze-dried and stored at an ESI source. The mass range was set at m/z 100 1500. The Q- À20 C for further analysis. TOF MS data were acquired in the positive mode and the 2.1.2 Ultraltration and hydrophobic chromatography. conditions of MS analysis were as follows: CAD gas ow-rate, 7 À1 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. The freeze-dried supernatant was fractionated using ultra l- L min ; drying gas temperature, 550 C; ion spray voltage, tration chromatography with 1 kDa molecular weight (MW) cut 5500 V; declustering potential, 80 V. Soware generated data off membranes (Millipore, Hangzhou, China). Two fractions, le: SCIEX OS 1.0. namely, MMKP-A (MW < 1 kDa) and MMKP-B (MW > 1 kDa) were collected and freeze-dried. 2.2 Reagents and cell culture Hydrophobic chromatography. MMKP-A was dissolved in The Mesobuthus martensii Karsch was acquired from the 1.50 M (NH ) SO prepared with 30 mM PBS (pH 7.5) and 4 2 4 Ertiantang pharmacy (Guangzhou, China) and identied by loaded onto a Phenyl Sepharose CL-4B hydrophobic chroma- Professor Zhou (Jinan University, Guangzhou, China). All the tography column (2.5 cm  150 cm), which had previously been antibodies were purchased from Cell Signaling Technology This article is licensed under a equilibrated with the above buffer. A stepwise elution was (CST, USA). TNF-a was obtained from Sigma-Aldrich (USA); the carried out with decreasing concentrations of (NH ) SO (1.50, 4 2 4 other chemicals used in the current experiment were purchased 0.75 and 0 M) dissolved in 30 mM PBS (pH 7.5) at a ow rate of À1 from Aldrich or Admas and used without any further Open Access Article. Published on 20 June 2019. Downloaded 9/30/2021 4:07:41 PM. 3.0 mL min . Each fraction was collected at a volume of 80 mL purication. and was monitored at 280 nm. Fractions were then freeze-dried HUVECs were purchased from the Cell Bank of the Chinese and the anti-inammatory activity was detected. The fraction Academy of Sciences (Shanghai, China) and were grown in having the strongest anti-inammatory activity was collected a culture medium containing 20% fetal bovine serum, 2 mmol and prepared for anion-exchange chromatography. À À L 1 glutamine, antibiomycins (10 mmol L 1 penicillin G and 10 2.1.3 Anion-exchange chromatography. The MMKP-A-4 À1 À mmol L streptomycin) (all from Sigma-Aldrich, U.S.A.) in solution (10 mL, 121 mg mL 1) was injected into a DEAE-52 a humidied incubator containing 5% CO at 37 C.15 cellulose (Shanghai, China) anion-exchange column (2.0  2 115 cm) pre-equilibrated with deionized water and was stepwise ff eluted with 1000 mL distilled water, 0.40, 0.80, and 1.60 M 2.3 Cell viability and MMKP protective e ect assays À1  (NH4)2SO4 solutions at a ow rate of 2.50 mL min . Ten frac- HUVECs were plated in Costa 96-well plates at densities of 1 tions (MMKP-A-4-1 to MMKP-A-4-10) were freeze-dried and the 105 cells per milliliter.