Research study - OA Publishing

Research study

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F : Almelkar SI, Patwardhan AM. Bacopa monniera extract plays a significant role in lipofuscin scavenging in cultured human vascular endothelial cells. OA Alternative Medicine 2013 Feb 02;1(1):5.

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Bacopa monniera extract plays a signifi cant role in lipofuscin scavenging in cultured human vascular endothelial cells

SI Almelkar1*, AM Patwardhan2

* Corresponding authorEmail: [email protected] Department of Cardiovascular and Thoracic

Surgery, Seth G.S. Medical College and KEM Hospital, Parel, Mumbai 400012, Maharash-tra, India

2 Department of Cardiovascular and Thoracic Surgery, JNMC, Acharya Vinoba Bhave Rural Hospital, Sawangi (Meghe), Wardha 442004, Maharashtra, India

AbstractIntroductionWe investigated the generation of lipofuscin granules in human umbil-ical vein endothelial cells that were exposed to hydrogen peroxide (H2O2, 100 µM) for 1 h. Lipofuscin granule scavenging was studied using the Bacopa monniera (Brahmi) extract (100 µg) for 2 h, 4 h and 6 h expo-sures. We used an endothelial cell culture model to investigate the antioxidant effects of the B. monniera extract on lipofuscin granules scavenging in cultured human umbilical vein endothelial cells.Materials and methodsTo understand the protective role of the B. monniera extract in post-oxidative stressed human umbilical vein endothelial cells, an experiment was set-up with the following exposure regimen: pre-B. monniera extract (100 µg) for 2 h plus post-H2O2 (100 µM) for 1 h. To analyse whether pre-oxidative stressed human umbilical vein endothelial cells could be protected with exposure to post-B. monniera extract and whether the lipofuscin granules could be scavenged by post-B. monniera extract treatment, the following exposure regimen was provided: pre-H2O2 (100 µM) for 1 h plus post-B. monniera extract (100 µg) for 2 h.

Introduction

due to the iron-catalysed oxida-tion/polymerisation of protein and lipid residues3. It is believed that th-ese changes occur not only due to continuous oxidative stress (causing oxidation of mitochondrial constitu-ents and autophagocytosed materi-al) but also because of the inherent inability of cells to completely remo-ve oxidatively damaged structures (biological garbage)4. Accumulation of lipofuscin granules is mostly obs-erved in various neurological disor-ders2. There are no reported studies of B. monniera and its antioxidant activities, which play a significant role in lipofuscin scavenging in cult-ured human umbilical vein endothe-lial cells (HUVECs). Therefore, this study is the first to show that the B. monniera extract (BME) plays a piv-otal role by strongly supporting the scavenging of lipo-fuscin granules in cultured HUVECs.

Material and methodsThis work conforms to the values laid down in the Declaration of Helsinki (1964). The protocol of this study has been approved by the relevant ethical committee related to our institu-tion in which it was performed. All subjects gave full informed consent to participate in this study.

BME preparationThe dried herb was macerated in 95% methanol and the crude extract of B. monniera was prepared and filtered with Whatman no.1 filter paper2. The extract was stored at −30°C5.

Harvest and expansion of HUVECs6

The isolation protocol for HUVECs was adapted from Baudin et al. 20077.The umbilical cord was collected immediately and the cord was rinsed

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ResultsThe presence of lipofuscin granule formation was confirmed by the Zie-hl-Neelson’s staining method and microscopic examinat-ion. Exposur-es to H2O2 (100 µM) showed the ab-undant generation and formation of lipofuscin granules. B. monniera ext-ract exposures of 2 h, 4 h and 6h (100 µg), showed the abundant gen-eration and formation of lipofuscin granules. B. monniera extract expos-ures of 2 h, 4 h and 6h (100 µg), sho-wed a complete absence of lipofusc-in granules. Pre-B. monniera extract (100 µg) exposure for 2 h plus post-H2O2 (100 µM) exposure for 1 h, resulted in extremely reduced lipof-uscin granules. H2O2 exposure for 1 h and pre-H2O2 (100 µM) exposure for 1 h plus post-B. monniera extract (100 µg) exposure for 2 h, resulted in the moderate appearance of lipofuscin granules.ConclusionThis study concluded that the B. mo-nniera extract has potential antioxi-dant properties, which play a pivotal role in the analysis of lipofuscin gra-nules scavenging in cultured human umbilical vein endothelial cells.

Medicinal plants that have been use-ful in history are obvious choices as sources with significant pharmacol-ogical and biological activities1. One such medicinal plant is Bacopa mon-niera, commonly termed as Brahmi2. Studies in the past have focused on B. monniera’s cognitive enhancing effects that specifically include me-mory, learning and concentration. Post-mitotic cells accumulate a non-degradable, intralysosomal substan-ce called lipofuscin which is formed

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and washed with sterile phosphate buffered saline (PBS). One end of the umbilical vein was clamped using artery forceps and an enzyme cock-tail of 0.15% collagenase type IV (Sigma Aldrich, St. Louis, Missouri, USA) and dispase II (Roche, Nutley, New Jersey, USA) was filled into the lumen cavity of the vein for dislodging the endothelial cells (ECs). The ECs were incubated for 20 min at 37°C. After completion of incuba-tion, the vein was flushed with M199 medium (Invitrogen, Carlsbad, Cali-fornia, USA). The cells in suspension were centrifuged at 1500 rpm for 10 min. Cell pellets were refreshed twice by centrifugation and the final pellet was again resuspended in an endothelial cell growth medium (ECGM) (PromoCell GmbH, Germany) containing 20% foetal bovine serum (FBS) (Invitrogen, Carlsbad, Cali-fornia, USA); isolated HUVECs were plated in tissue culture flasks. After 12 h of incubation, the HUVECs were fed with complete ECGM containing 20% FBS, 2 mM L-glutamine, 10 µg/mL heparin and penicillin 5 unit/mL and incubated at 37°C with 5% CO2.

Lipofuscin granules stained by Ziehl-Neelson method for control cells (without exposure), oxidative stress (H2O2) for 1 h8

HUVECs were cultured onto cov-er slips until resembling a cobble stone appearance. HUVECs without

any exposure served as the control. The HUVECs were exposed to oxida-tive stress (H2O2) for 1 h (100 µM) and were fixed with 4% paraform-aldehyde and refreshed with PBS 1 × (5 min × 3 washes). The HUVECs were washed with 70% isopropanol (10 min × 2 washes) and stained with carbolfuscin for 30 min at 60oC. The HUVECs were further rinsed in acid-alcohol (1% HCl in 70% ethanol) until they turned light pink. They were washed in distilled water for 5 min, counter stained (0.5% tolu-idene blue) for 10 min and rinsed again with distilled water for 5 min. Isopropyl alcohol (70%) was used to dehydrate the cells. The cover slips were mounted with dibutyl phthalate xylene (DPX). Stained HUVECs were observed under a bright field micro-scope for lipofuscin granules.

Exposure to BME for 2 h, 4 h and 6 h, pre-BME exposure for 2 h plus post-H2O2 for 1 h and pre-H2O2 exposure for 1 h plus post-BME for 2 h to cultured HUVECs to study the eff ect of BME on lipofuscin granules8

HUVECs were cultured on cover slips until they reached a cobble stone morphology. HUVECs were further processed as follows: BME exposure for 2 h, 4 h and 6 h, pre-BME expo-sure for 2 h plus post-H2O2 for 1 h and pre-H2O2 exposure for 1 h plus post-BME for 2 h.

All exposed HUVECs were fixed with 4% paraformaldehyde and refreshed with PBS 1 × (5 min × 3 washes). The HUVECs were washed with 70% isopropanol (10 min × 2 washes) and stained with carbolfuscin for 30 min at 60oC. The HUVECs were rinsed in acid-alcohol (1% HCl in 70% ethanol) until they turned light pink. The HUVECs were washed with distilled water for 5 min, counter stained (0.5% toluidene blue) for 10 min and rinsed again with distilled water for 5 min. Isopropyl alcohol (70%) was used to dehydrate the cells. The cover slips were mounted with DPX. Stained HUVECs were observed under a bright field micro-scope for lipofuscin granules.

ResultsB. monniera crude extractA 12% yield of sticky, crude extract was obtained from B. monniera and further used for experimental work.

Isolation and culture of HUVECsHUVECs were isolated in clusters of 30. HUVECs were attached to the plate surface immediately after seeding for 2 h. HUVECs started expanding in clusters and reached confluence on the 10th day (Figure 1). On 70% confluence, the HUVECs resembled the perfect cobblestone morphology, and the cell density was 3 × 105 cells/cm2.

Figure 1: HUVECs showed a cobble stone morphology in culture.

Figure 2: HUVECs in control (with-out treatment) showed a very low appearance of lipofuscin granules.

Figure 3: HUVECs treated with H2O2 (100 µM) showed abundant lipofus-cin granules.

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Lipofuscin granules in the control cells and in cells exposed to oxidative stress (H2O2 for 1 h)The current investigation reveals the appearance and occurrence of lipo-fuscin granules in cultured HUVECs. HUVECs without any exposure served as controls (normal) and showed a much lower presence of lipofuscin granules (Figure 2).

Oxidant exposures (H2O2 for 1 h) to cultured HUVECs, led to lipo-fuscin granule generation. Exposure of H2O2 for 1 h at a concentration of 100 µM resulted in maximum damage to the cultured HUVECs; the cells showed distorted cell and nuclear membranes with copious formations of lipofuscin granules in the cytosol region (Figure 3).

Exposure to BME for 2 h, 4 h and 6 h, pre-BME exposure for 2 h plus post-H2O2 for 1 h and pre-H2O2 exposure for 1 h plus post-BME for 2 h to cultured HUVECs to study the eff ect of BME on lipofuscin granules Exposure to BME (100 µg) treatment for 2 h (Figure 4), 4 h (Figure 5) and 6 h (Figure 6), showed the absence of or the lack of lipofuscin granule formation and an intact cell and nuclear membrane. Pre-BME (100 µg) exposure for 2 h plus post-H2O2 (100 µM) for 1 h, showed the pres-ence of very reduced amounts of lipo-fuscin granule formation with intact nuclear membranes and very little injury to the cell membranes (Figure 7). Pre-H2O2 (100 µM) exposure for 1 h plus post-BME (100 µg) for 2 h,

resulted in the moderate appear-ance of lipofuscin granule formation with little nuclear and cell membrane damage (Figure 8).

Discussion Cells are constantly subjected to free-radical attack. Free radicals are scavenged in the cells by the antioxi-dant system present in the cell. The unscavenged free radicals exert their cytotoxic action on cell membrane lipids. These peroxidised membranes are engulfed by lysosomes which may bring about damage to the lyso-somal enzymes. The residual body of lysosomes that are unable to digest cell membranes turns into lipofuscin granules. In advanced aging, the rate of accumulation of lipofuscin gran-ules increases in tissues like the heart and brain2.

B. monniera is a herb, and a variety of studies have been carried out to study its antioxidative properties. Past research investigating BME exposure in neurons, established the lipofuscin scavenging property of B. monniera2. We tried to investigate the role of BME in lipofuscin scav-enging in cultured HUVECs. HUVECs without any exposure served as the controls and showed very little pres-ence of lipofuscin granules. Exposure to 1 h of oxidative stress (H2O2) at a concentration of 100 µM resulted in

Figure 6: HUVECs treated with BME for 6 h (100 µg) showed a complete absence of lipofuscin granules.


Figure 7: HUVECs treated with pre-BME (100 µg) for 2 h plus post-H2O2 (100 µM) for 1 h showed re-duced lipofuscin granules.


Figure 8: HUVECs treated with pre-H2O2 (100 µM) for 1 h plus post-BME (100 µg) for 2 h showed moder-ate lipofuscin granules.

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maximum damage to the cultured HUVECs, such as distorted cell and nuclear membranes and abundant formation of lipofuscin granules in the cytosol region. This firmly explains that dose-dependant H2O2 exposure for 1 h brings about cellular damage and is involved in the forma-tion of lipofuscin granules in cultured HUVECs.

Exposure to BME (100 µg) for 2 h, 4 h and 6 h, showed the absence of lipofuscin granule formation and intact cell and nuclear membranes. Pre-BME (100 µg) exposure for 2 h plus post-H2O2 (6.25 µM to 100 µM) for 1 h, showed negligible/reduced amount of lipofuscin granule forma-tion with intact nuclear membrane and very little injury to the cell membrane. This illustrates that pre-BME exposure for 2 h provides the necessary anti-oxidative protec-tion to cultured HUVECs while the results from the post-H2O2 exposure for 1 h illustrate that there was a lack of or minimal lipofuscin granule formation. Therefore, it is deduced that pre-BME exposure has a protec-tive role against lipofuscin granule formation even after post-H2O2 expo-sures. Pre-H2O2 (100 µM) exposure for 1 h plus post-BME (100 µg) expo-sure for 2 h, resulted in the moderate appearance of lipofuscin granule formation with little nuclear and cell membrane damage. This shows that pre-H2O2 exposures bring about the moderate generation of lipofuscin granules, but after post-BME expo-sures, the generation of lipofuscin granules is halted due to the BME antioxidative properties.

Conclusion This investigation shows that BME is an antioxidant which plays a pivotal role in lipofuscin scavenging in stressed and normal HUVECs. H2O2 (100 µM) treatment is responsible for oxidative impair and brings about the extreme generation or formation of lipofuscin granules. BME expo-sures for 2 h, 4 h and 6h (100 µg), bring about scavenging of lipofuscin granules at different time intervals. Pre-BME (100 µg) exposure for 2 h brings about reduced lipofuscin formation. After post-H2O2 (100 µM) exposure for 1 h, there is no further rise in lipofuscin. Pre-H2O2 (100 µM) exposure for 1 h plus pos-tBME (100 µg) exposure for 2 h, resulted in the moderate appearance of lipo-fuscin granules.

Abbreviations listBME, Bacopa monniera extract; ECs, endothelial cells; ECGM, endothe-lial cell growth medium; FBS, foetal bovine serum; PBS, phosphate buff-ered saline.

AcknowledgementWe gratefully thank the Indian Council of Medical Research (ICMR), File No: 45/47/2010/BMS/TRM IRIS Cell: 2010-06230, Government of India, for providing a senior research fellowship to our first author. We are also thankful to Dr NB Agrawal for providing research funds through Dr PK Sen, Research Society, GSMC and KEM Hospital, Parel, Mumbai. We thank Dr BM Kandalkar (Professor and Head of Department, Depart-ment of Pathology, GSMC and

KEM Hospital, Mumbai, India) for his generosity in helping us and providing his support with the tissue culture laboratory. We especially thank Mrs Seema V Sharma (Senior Scientific Officer, CVTC) Mrs Rajeshri (JSO), Mr Sunil Chaudhary, Mrs Nidhi Taneja, Mr Janardhan and other CVTC staff for their laboratory support.

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