IM-133N modulates cytokine secretion by RAW264.7 and THP-1 cells

Figures & data

Figure 1. Mass spectra showing compounds of IM-133N extract. Spectrometric conditions are described in the Methods.

Table 1. List of compounds in IM-133N identified by mass spectrometry.

Compound	Molecular formula	Actual molecular weight	Obtained molecular ion	Molecular structure
Symplososide	C27H26O10	510.50 g/mol	509.01 m/z
Symponoside	C27H26O11	526.50 g/mol	525.07 m/z
Symploveroside	C29H32O10	540.57 g/mol	539.91 m/z
Symplocomoside	C27H26O12	542.50 g/mol	541.13 m/z
Salireposide	C20H22O9	406.38 g/mol	405.36 m/z
Symconoside–A	C26H32O14	568.50 g/mol	567.22 m/z
Oleanolic acid	C30H48O3	456.70 g/mol	454.90 m/z
Betulic acid	C30H48O3	456.70 g/mol	454.90 m/z
Mesquitol	C15H14O6	290.26 g/mol	288.79 m/z

Note: The compounds which are mentioned in the Table were reported in the literature; however, the complete structural elucidation of each compound in IM-133N extract was not confirmed here.

Table 2. Primers used for amplifications.

Gene	Source	Primer sequences	Annealing T°
IL-10	Mouse	Forward 5′-CCAGTTTTACCTGGTAGAAGTGATG-3′ Reverse 5′-TGTCTAGGTCCTGGAGTCCAGCAGACTCAA-3′	60 °C
	Human	Forward 5′-ATGCCCCAAGCTGAGAACCAAGAC-3′ Reverse 5′-TCTCAAGGGGCTGGGTCAGCTATCCCA-3′	60 °C
IL-6	Mouse	Forward 5′-TTCCTCTCTGCAAGAGACT-3′ Reverse 5′-TGTATCTCTCTGAAGGACT-3′	60 °C
	Human	Forward 5′-GTACCCCCAGGAGAAGATTC-3′ Reverse 5′-CAAACTGCATAGCCACTTTC-3′	60 °C
CXCL1 (mouse) IL-8	Mouse	Forward 5′-CACCTCAAGAACATCCAGAGCT-3′ Reverse 5′-CAAGCAGAACTGAACTACCATCG-3′	55 °C
	Human	Forward 5′-GCTTTCTGATGGAAGAGAGC-3′ Reverse 5′-GGCACAGTGGAACAAGGACT-3′	60 °C
IFNγ	Mouse	Forward 5′-TGGCATAGATGTGGAAGAAAAGAG-3′ Reverse 5′-TGCAGGATTTTCATGTCACCAT-3′	60 °C
	Human	Forward 5′-TGACCAGAGCATCCAAAAGA-3′ Reverse 5′-TGTATTGCTTTGCGTTGGAC-3′	60 °C
TNFα	Mouse	Forward 5′-GTTCTATGGCCCAGACCTCACA-3′ Reverse 5′-TACCAGGGTTTGAGCTCAGC-3′	60 °C
	Human	Forward 5′-ATGAGCACTGAAAGCATGATC-3′ Reverse 5′-TCACAGGGCAATGATCCCAAAGTAGACCTGCCC-3′	60 °C
iNOS	Mouse	Forward 5′-TCTTGGTCAAAGCTGTGCTC-3′ Reverse 5′-CATTGCCAAACGTACTGGTC-3′	60 °C
GAPDH	Mouse	Forward 5′-ACCACAGTCCATGCCATCAC-3′ Reverse 5′-TCCACCACCCTGTTGCTGTA-3′	60 °C
	Human	Forward 5′-ATGAAGATCCTGACCGAGCGT-3′ Reverse 5′-AACGCAGCTCAGTAACAGTCCG-3′	58 °C

Figure 2. Cytotoxicity of IM-133N on (a) RAW264.7 and (b) THP-1 cells. Cells were incubated for 24 h with differing concentrations of IM-133N and cell viability was then determined using an MTT assay. Data are expressed as mean (±SE) cytotoxicity (i.e. percentage reduction in viable cell number vs control) from three assays using n = 3 samples/IM-133N concentration.

Table 3. Effect of IM133N on NO and TNFα secretion.

	RAW264.7		THP-1
Treatment	NO^a	TNFα^b	TNFα^b
Control	2.33 ± 0.15	1.03 ± 0.11	0.77 ± 0.21
IM-133N (25 μg/ml)	13.01 ± 5.03*#	8.99 ± 0.42*#	6.12 ± 0.76*#
IM-133N (50 μg/ml)	27.42 ± 3.29*#	11.52 ± 0.82*#	10.09 ± 0.91*#
IM-133N (100 μg/ml)	51.12 ± 4.97*#	20.58 ± 1.05*#	19.31 ± 1.86*#
LPS (1 μg/ml)	67.91 ± 6.56*	25.09 ± 2.33*	23.77 ± 2.91*

RAW264.7 and THP-1 cells were cultured with IM-133N or LPS (1 μg/ml) and supernatants were harvested ^a24 h later and assayed for NO or ^b4 h later and assayed for TNFα.

Values are mean ± SD of three individual experiments, performed in triplicate/dose. Values for NO are in terms of µM; those for TNFα are in pg/ml.

*Value significantly different from control or #from LPS control (p < 0.05).

Figure 3. Densitometric analysis of gene transcripts obtained from RAW264.7 cells. Cells were incubated with the IM-133N for 4 h (for TNFα gene amplification) or for 24 h (for iNOS and the other cytokines investigated) before being harvested. Relative levels of each gene’s expression were normalized to that for GAPDH. Values shown depict arbitrary units. Values are mean ± SD of three individual experiments using n = 3 samples/IM-133N concentration. *Value significantly different from control or # from LPS control (p < 0.05).

Figure 4. Densitometric analysis of gene transcripts obtained from THP-1 cells. Cells were incubated with the IM-133N for 4 h (for TNFα gene amplification) or for 24 h (for the other cytokines investigated) before being harvested. Relative levels of each gene’s expression were normalized to that for GAPDH. Values shown depict arbitrary units. Values are mean ± SD of three individual experiments using n = 3 samples/IM-133N concentration. * Value significantly different from control or # from LPS control (p < 0.05).