Table 1
Forward and reverse sequences of applied primers
| Gene | Forward | Reverse |
|---|---|---|
| GAPDH | 5′-AACGACCCCTTCATT-GAC-3′ | 5′-TCCACGACATACTCAG-CA-3′ |
| COX-1 | 5′-TCTCAGGCTA-CACCCTAGACCA-3′ | 5′-ATCGGGGTAGTCCGAG-TAACGT-3′ |
| COX-2 | 5′-CAGCACTTCACGCAT-CAGTT-3′ | 5′-CGCAGTTTACGCTGTC-TAGC-3′ |
Table 2
Values of IC50 obtained by MTT test
| Cell line | IC50 [μg/mL] |
|---|---|
| RAW264.37 | not cytotoxic at concentration range tested |
| PC-12 | 193.1 |
Fig. 1
The effect of LevidorTM extract on pro-inflammatory markers (IL-6, TNF-α) induced by LPS in the RAW267.7 cells. All results are given as means ± SD of three independent determinations. Negative control (C−) refers to cells treated with solvent, while positive control (C+) relates to cells treated with inflammatory inducer only. Significantly different values are marked as (*) p ≤ 0.05
Fig. 2
Changes in COX-1 (A) and COX-2 (B) gene expression in the U937 cells after 48 h pre-treatment with PMA (10 ng/mL), followed by 40 h of incubation without treatment, 4 h treatment with 1, 2,5, 5, 10, 25, and 50 μg/mL of LevidorTM as well as LPS addition (1 μg/mL) for 2, 4, 6, and 16 h. Results are presented as fold changes in the expression of investigated genes. Negative control (C−) refers to cells treated with PMA, while positive control (C+) relates to cells treated also with inflammatory inducer. Significantly different values are marked as (*) p ≤ 0.05. The results are calculated based on three independent experiments
Fig. 3
Western blot analysis of the modulation of COX-1 and COX-2 protein abundance in the U937 after 48 h pre-treatment with PMA (10 ng/mL), followed by 40 h of incubation without treatment, 4 h treatment with 1, 2.5, 5, 10, 25, and 50 μg/mL of LevidorTM as well as LPS addition (1 μg/mL) for 24 h. Negative control (C−) refers to cells treated with PMA, while positive control (C+) relates to cells treated also with inflammatory inducer (LPS)
Fig. 4
Visualisation of COX-1 and COX-2 protein in the U937 cells after 24 h pre-treatment with PMA (10 ng/mL), followed by 24 h of incubation without treatment, 4 h treatment with 10, 25, and 50 μg/mL of LevidorTM as well as TNF-α addition (100 ng/mL) for 20 h. Negative control (C−) refers to cells treated with PMA, while positive control (C+) relates to cells treated also with inflammatory inducer (LPS)