15-Lipoxygenase metabolites of -linolenic acid, [13-(S)-HPOTrE and 13-(S)-HOTrE], mediate anti-inflammatory effects by inactivating NLRP3 inflammasome Naresh Kumar1, Geetika Gupta1, Kotha Anilkumar2, Naireen Fatima1, Roy Karnati1, Gorla Venkateswara Reddy1, Priyanka Voori Giri1 and Pallu Reddanna1*
1School
of Life Sciences, University of Hyderabad, Hyderabad 500046, India Institute of Animal Biotechnology, Hyderabad 500049, India
2National
*Corresponding Author (E-mail:
[email protected])
125
% Growth
100
50 25 0 24
36
Time (h) 125
% Growth
Control 1M 10M 100M 200M
LA
100 75 50 25
Control 1M 10M 100M 200M
ALA
75 50 25 0 24
100
36
Time (h)
125
% Growth
% Growth % Growth
ω-6 PUFAs
AA
75
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Control 1 10M 100M 200M
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ω-3 PUFAs
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Control 1M 10M 100M 200M
DHA
75 50 25
0 24
0
36
Time (h)
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36
Time (h)
13-(S)-HPOTrE Control 1M 10M 100M 200M
B
% Growth
80 60 40 20
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% Growth % Inhibition
80
% Growth
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0
60 40 20
IC50 = 114.34µM
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12
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36 0.1
Time (h)
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10
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1000
13-(S)-HPOTrE (µM)
13-(S)-HOTrE Control 1M 10M 100M 200M
% Growth
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% Growth % Inhibition
100 80
% Growth
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60 40 20
IC50>200µM
0
0 3
6
12
Time (h)
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36
0.1
1
10
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13-(S)-HOTrE (µM)
Supplementary Figure S1| Effects of PUFAs and their 15 LOX metabolites on viability of mouse macrophage cell line, RAW 264.7 cells. In MTT assay, Cells were treated with different concentrations (1, 10, 100 and 200 µM) of A) PUFAs (AA, LA, ALA and DHA) and B) 15 LOX metabolites of ALA [13-(S)-HPOTrE and 13-(S)-HOTrE] for different time points (as mentioned in above graphs). The percent cell growth following treatment was calculated, in comparison with untreated control cells.
B
A
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Count
Count
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010 4
FL1-H
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ROS (Mean Intensity (FL1-H)
Nitrite conc. (µM)
*
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60
Control HOTrE+LPS HPOTrE+LPS ALA+LPS LPS LPS+NAC
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FL1-H
1600 1200 800 400 0
Supplementary Figure S2| Effects of 13-(S)-HPOTrE and 13-(S)-HOTrE on generation of NO and ROS in RAW 264.7 cells. A) Nitrite levels in culture medium of RAW 264.7 cells pre-incubated with ALA (100 µM) for 3 h and further stimulated with or without LPS (100 ng/ml) for the next 24 h. ALA showed significant reduction in NO level as compared to LPS. The values represent mean ±SD of three independent experiments. * indicates significance (p
