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Pediococcus pentosaceus LAB6- and Lactiplantibacillus plantarum LAB12-Derived Cell Free Supernatant Inhibited RhoA Activation and Reduced Amyloid-Β In Vitro

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Abstract

Alzheimer’s disease (AD) is characterized by aggregation of amyloid beta (Aβ) plaque. RhoA may serve as a potential target for prevention against AD given its role in the amyloidogenic pathway. The recent emergence of the gut-brain axis has linked lactic acid bacteria (LAB) to neuroprotection against AD. This study assessed the importance of RhoA inhibition in mediating the neuroprotective potential of LAB. To this end, de Man, Rogosa and Sharpe (MRS) broth fermented by lactobacilli or pediococci were tested against SK-N-SH (a human neuroblastoma cell line) in the presence of RhoA activator II for 24 h after which the RhoA activity was measured using the G-LISA Kit. Fluorescence staining of f-actin stress fibres was performed to validate RhoA inhibition. SK-N-SH was transfected with plasmid expressing amyloid precursor protein (APP) gene. The Aβ concentration in transfected cells exposed to LAB-derived cell free supernatant (CFS) in the presence of RhoA activator II was measured using the ELISA kit. Furthermore, this study measured organic acids in LAB-derived CFS using the gas chromatography. It was found that LAB-derived CFS yielded strain-dependent inhibition of RhoA, with LAB6- and LAB12-derived CFS being the most potent Pediococcal- and Lactiplantibacillus-based RhoA inhibitor, respectively. Lesser stress fibres were formed under treatment with LAB-derived CFS. The LAB-derived CFS also significantly inhibited Aβ in SK-N-SH transfected with APP gene in the presence of RhoA activator II. The LAB-derived CFS was presented with increased lactic acid, acetic acid, butyric acid and propionic acid. The present findings warrant in-depth study using animal models.

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Data Availability

The data that supports the findings of this study is available upon request from the corresponding author.

Abbreviations

AChE:

Acetylcholinesterase

ATP:

Adenosine triphosphate

AD:

Alzheimer’s disease

Aβ:

Amyloid beta

APP:

Amyloid precursor protein

ANOVA:

One-way analysis of variance

ATCC:

American Type Culture Collection

BSA:

Bovine serum albumin

CFS:

Cell free supernatant

CNS:

Central nervous system

DAPI:

4′,6-Diamidino-2-phenylindole

db-cAMP:

Di-butyric cAMP

DPPH:

1,1-Diphenyl-2-picrylhydrazyl

FBS:

Foetal bovine serum

FDA:

Food and Drug Administration

FID:

Flame ionization detector

GC:

Gas chromatography

HRP:

Horseradish peroxidase conjugate

HSTC:

Highest sub-toxic concentration

IC:

Inhibitory concentration

LAB:

Lactic acid bacteria

LPS:

Lipopolysaccharide

MEM:

Eagle’s Minimum Essential Medium

MRS:

De Man, Rogosa and Sharpe

NMDA:

N-methyl-D-aspartate

NSAID:

Non-steroidal anti-inflammatory drugs

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

ROCK:

Rho-associated protein kinase

SCFA:

Short-chain fatty acids

SRB:

Sulforhodamine B

TBE:

Tris-borate-EDTA

TCA:

Tricarboxylic acid

TRITC:

Tetramethylrhodamine

VFA:

Volatile fatty acid

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Funding

The present study acknowledges receipt of financial support from the Ministry of Higher Education Malaysia under the Long-Term Research Grant Scheme [600-RMC/LRGS 5/3 (002/2019)].

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Authors and Affiliations

  1. Collaborative Drug Discovery Research (CDDR) Group, Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Cawangan Selangor, Kampus Puncak Alam, 42300, Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia

    Ramli Muhammad Zaki, Kalavathy Ramasamy, Nor Amalina Ahmad Alwi, Rosmadi Mohd Yusoff & Siong Meng Lim

  2. Faculty of Pharmacy and Health Sciences, Royal College of Medicine Perak, Universiti Kuala Lumpur , Ipoh, 30450, Perak, Malaysia

    Ramli Muhammad Zaki

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  1. Ramli Muhammad Zaki
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  2. Kalavathy Ramasamy
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  3. Nor Amalina Ahmad Alwi
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  4. Rosmadi Mohd Yusoff
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Contributions

Kalavathy Ramasamy and Siong Meng Lim were involved in conceptualisation, project administration and supervision. Ramli Muhammad Zaki performed the majority of the experiments, analysed the data and wrote the original draft. Nor Amalina Ahmad Alwi and Rosmadi Mohd Yusoff constructed the pcDNA 3.1-APP plasmid and validated the APP gene. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Siong Meng Lim.

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Zaki, R.M., Ramasamy, K., Ahmad Alwi, N. et al. Pediococcus pentosaceus LAB6- and Lactiplantibacillus plantarum LAB12-Derived Cell Free Supernatant Inhibited RhoA Activation and Reduced Amyloid-Β In Vitro. Probiotics & Antimicro. Prot. 16, 62–75 (2024). https://doi.org/10.1007/s12602-022-10009-7

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