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. 2019 Apr 26;10:289. doi: 10.3389/fphar.2019.00289

FIGURE 3.

FIGURE 3

Comfrey-RE and -OP selectively block COX-2 but not COX-1. (A) Analysis of total COX-enzymatic activity (COX1/2) comparing unstimulated (UT, gray bars) and IL-1β (5 ng/ml, 90 min) stimulated HUVEC (black bars) which were either left untreated (control) or pretreated (30 min) with comfrey-RE (200 μg/ml), comfrey-OP (20 μg/ml), or TAK inhibitor (5 μM). Total COX enzymatic activity is depicted compared to non-stimulated, untreated cells. Error bars represent mean ± SD (n = 2). ∗p < 0.05; ∗∗p < 0.01. Note that both comfrey preparations diminish the IL-1β induced COX-2 activity (black bars), but not the constitutive COX-1 activity (gray bars). (B) Western blot analysis of samples used in A showing that COX-2 expression can only be found in IL-1β induced samples. Treatment of cells with comfrey preparations (RE and OP) reduces COX-2 protein levels. β-actin is shown as loading control. (C) Real-time PCR of IL-1β induced (5 ng/ml; time points of stimulation as indicated) HUVEC either left untreated (CTRL, squares) or pretreated (30 min) with comfrey-RE (200 μg/ml; boxes) and comfrey-OP (20 μg/ml; asterisks). Relative mRNA levels of COX-2 were normalized to GAPDH. Expression levels are depicted as mean fold change compared to non-stimulated cells. Error bars represent mean ± SD (n = 3). (D) Comfrey-OP does not affect COX-1 activity. In vitro analysis of recombinant COX-1 enzymatic activity comparing samples which were either left untreated (control, UT) or treated with the specific COX-1 inhibitor SC-560 (control, black bars) with samples incubated with different concentrations of comfrey-OP (gray bars) or diclofenac (white bars). Total COX enzymatic activity is depicted compared to untreated cells. Error bars represent mean ± SD (n = 3). ∗p < 0.05. (E) Analysis of total COX-enzymatic activity (COX1/2) comparing cells either left untreated (control, UT) or treated with TAK inhibitor (5 μM) (control, black bars) with cells incubated with different concentrations (as indicated) of comfrey-OP, diclofenac, or the COX-1 inhibitor SC-560. Total COX enzymatic activity is depicted compared to untreated cells. Error bars represent mean ± SD (n = 3). ∗p < 0.05; ∗∗p < 0.01.