Studie från Taiwan

En kommande forskningsrapport från det taiwanesiska universitet Chang Gung University har släppts som en preprint.Dvs studien väntar på granskning innan den godkänns officiellt.

Universitet har jag skrivit om tidigare, här och här.

Den aktuella studien handlar om kroppens försvarsmekanismer vid en bakteriologisk infektion.

Forskarna har undersökt hur ett extract (larixol) ur medicinalväxten euphorbia formosana påverkar läkningsförmågan såväl som rep av vävnadsskada.

 

Såhär beskriver forskarna växten : Euphorbia formosana is one of Taiwanese native medicinal plants.
It has been used in folk medicine for the treatment of snakebite as well as many other dermatoses in Taiwan.
Euphorbia formosana was found to exhibit cytotoxic, antioxidant activities, and antimicrobial, spasmolytic in previous studies.
The ability of euphorbia formosana to mediate pro-apopotic activity intrigued studies to explore its possible application as complementary and alternative medicines.
Euphorbia formosana are rich in diterpenoids, favonoids, fatty acids, amino acids, and steroids in the stem and root bark.
Several studies described the isolation and identification of series of compounds from euphorbia formosana.
Larixol is purified from the root of euphorbia formosana.
Larixol had been demonstrated a selective TRPC6 inhibitor.
Larixol showed anti-inflammatory activities in our studies.
The high concentration of larixol did not inhibit cell viability by MTT or LDH assay. 

Studien är betitlad :

Larixol Inhibits Fmlp-Induced Superoxide Anion Production and Chemotaxis by Targeting the Βγ Subunit of Gi-Protein of Fmlp Receptor in Human Neutrophils

Dess abstract lyder enligt följande :

Abstract

The over-activated neutrophils through G-protein-coupled chemokines receptors (GPCRs) caused inflammation or tissue damage. Therefore, GPCRs or their downstream molecules are major targets for inhibition of uncontrol neutrophil activation. Our studies investigate the action and underlying mechanism of larixol, a diterpene extract from the root of euphorbia formosana, on fMLP-induced neutrophils respiratory burst, chemotaxis, and granular release. The intracellular signaling pathways regulated by larixol were interrupting the interaction of the βγ subunit of Gi-protein of fMLP receptor with downstream molecules induced by the fMLP. Briefly, larixol inhibited fMLP (0.1 μM)-induced superoxide anion production (IC₅₀ :1.98±0.14 μM), the release of cathepsin G (IC₅₀ :2.76±0.15 μM) and chemotaxis in a concentration-dependent manner; however, larixol did not inhibit these induced by PMA (100 nM). Larixol inhibited fMLP-induced Src kinase phosphorylation. Therefore, larixol attenuated the downstream signaling of Src kinases, such as ERK1/2, p38 and AKT phosphorylation. Moreover, larixol inhibited fMLP-induced intracellular calcium mobilization and PKC phosphorylation, the subsequently P47^phox translocation from the cytosol to the plasma membrane. Larixol was found to inhibit the interaction of the βγ subunit of Gi-protein of fMLP receptor with Src kinase or with PLCβ by the immunoprecipitation and duolink assay. Furthermore, larixol did not antagonize the formyl peptide receptors. Larixol did not increase cyclic nucleotide levels in neutrophils. These results suggest that larixol modulated fMLP-induced neutrophils superoxide anion production, chemotaxis, and granular releases by interrupting the interaction of the βγ subunit of Gi-protein with downstream signaling of fMLP receptor.

Materials and methods

2.4
Analyzing cell chemotaxis using live-cell holographic imaging.
To evaluate motility during chemotaxis, neutrophils migrated in an μ-slide chemotaxis (ibidi GmbH, Martinsried, Germany) along an fMLP gradient (0–0.1 μM).
Images of chemotaxis cells were acquired at 60-s intervals over 4 hr. In these experiments, neutrophils were treated various concentrations of larixol (1, 2, 5 or 10 μM) and placed in an μ-slide chemotaxis in a CO2 incubator (37°C).
The cells migration was analyzed with holographic microscope (HoloMonitor M4, Lund, Sweden) equipped with 20X objective. 

Images were imported to Hstudio software and analyzed with the manual tracking and the chemotaxis and migration tools.
The definition of migration distance was the shortest distance from the starting point to the endpoint of the cell.
Motility distance was defined as the accumulated movement of the cell from the starting point to the endpoint of the cell path.
Forward migration distance (FMD) to fMLP was defined as directionally of cells toward chemoattractant and was calculated according to the formula 

FMD= yi=cell end distance of y axis (μm) 1𝑛∑𝑛𝑖 = 1𝑦𝑖

Rose plot shows cell migration and cell mobility. Tracking analysis of a single neutrophil was performed with the integrated software from the phase holographic images acquired in the Method section.
(A) Single neutrophil two dimensions movement trajectories from 20 cells per field of view were displayed as a rose plot.
Incubation of neutrophils with DMSO (0.5 %, resting and control), cyclosporin H (CsH, 1 μM) or larixol (1, 2, 5, 10 μM) before loading on μ-slide.
Cell migration (B), cell motility (C), and forward migration distances (D) were quantified from 20 single-cell tracking data.
Each data point represents the quantitative data from tacking a single cell through 4 h of imaging. 

The integrated software was used to track and analyze 20 cells from one randomly chosen field of view from two wells per condition.

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