Immunomodulation of RAW 264.7 Murine Macrophage Functions and Antioxidant Activities of 11 Plant Extracts. (original) (raw)

Abstract

A group of 11 medicinal plants, including Lavandula pubescens, Trigonella foenugricium, Salsola schweinforthi, Calligonum comosum, Silene succulenta, Silene villosa, Bogonvillea glabra, Cakile maritime, Gomphrene celesoids, Mirabilis jalaba, and Silene nocturna growing in Egypt, were extracted and examined for their immunomodulatory and antioxidant activities. RAW 264.7 cells were recruited to investigate the immunomodulatory effect through multiple parameters analysis. First, the proliferation index of macrophages cells was evaluated revealing that Trigonella foenugricium, Silene succulenta and Silene villosa have a significant cytotoxic effect on RAW cells. Interestingly, we observed enhancement of macrophages phagocytic function of by all extracts except Cakile maritime, Gomphrena celosioides and Silene nocturna. Afterwards, macrophages were challenged by incubation with LPS and the effect of various extracts on inflammatory responses was investigated; the generation of NO from activated macrophage was substantially suppressed by 7 extracts namely, Trigonella foenugricium, Calligonum comosum, Silene succulenta, Bougainvillea glabra, Mirabilis jalaba, Gomphrena celosioides and Silene nocturna. TNF-α was decreased by percentage range from 3.8 to 85.8% and Trigonella foenugricium extract showed the highest inhibition of TNF-α release. All extracts except Trigonella foenugricium, Salsola schweinforthi, Silene succulenta and Mirabilis jalaba significantly inhibited COX-2 production from stimulated macrophage. Moreover, evaluating the potential antioxidant activity of these extracts showed that Trigonella foenugricium, Salsola schweinforthi, Calligonum comosum, Bogonvillea glabra and Mirabilis jalaba exhibited some antioxidant activities. Taken together, our results suggest that some of these extracts may have a considerable antinflammatory and antioxidant effects and may be a potential therapeutic choice in the treatment of inflammatory diseases.

Figures (8)

Figure 1. (A) Macrophages cell proliferation assay. Cytotoxicity of plant extracts against RAW 264.7 murine macrophage-like cells expressed as ICs59, Lavandula pubescens, Silene succulenta and Silene villosa exhibited significantly lower IC50 in comparison with the other eight extracts as determined by ANOVA test,(B) Figures demonstrating the effect of the different plants extracts on RAW macrophages as measured by the MIT assay. First, we sought to study the effect of various plants extracts on the growth of RAW 264.7 murine macrophage-like cells. Cells were incubated with gradual concentrations of the extracts for 24h and the cytotoxicity was evaluated using the MTT assay Figure 1A shows the half maximal inhibitory concentration (IC50) of each extract on RAW 264.7 murine macrophage-like cells. Trigonella foenugricium, Silene succulenta and Silene villosa; had a significantly lower

Figure 1. (A) Macrophages cell proliferation assay. Cytotoxicity of plant extracts against RAW 264.7 murine macrophage-like cells expressed as ICs59, Lavandula pubescens, Silene succulenta and Silene villosa exhibited significantly lower IC50 in comparison with the other eight extracts as determined by ANOVA test,(B) Figures demonstrating the effect of the different plants extracts on RAW macrophages as measured by the MIT assay. First, we sought to study the effect of various plants extracts on the growth of RAW 264.7 murine macrophage-like cells. Cells were incubated with gradual concentrations of the extracts for 24h and the cytotoxicity was evaluated using the MTT assay Figure 1A shows the half maximal inhibitory concentration (IC50) of each extract on RAW 264.7 murine macrophage-like cells. Trigonella foenugricium, Silene succulenta and Silene villosa; had a significantly lower

Figure 2. Macrophages phagocytosis assay. (A) Phagocytosis of FITC-zymosan particles (as indicated by white arrows) by confluent macrophage cells as examined by fluorescence microscope (magnification x 400), (B) the overall effect of various plants extracts on phagocytic functions of RAW 264.7 murine macrophage-like cells. Results are expressed as percent of control and presented as mean+tSE. p>0.05 was considered insignificant.

Figure 2. Macrophages phagocytosis assay. (A) Phagocytosis of FITC-zymosan particles (as indicated by white arrows) by confluent macrophage cells as examined by fluorescence microscope (magnification x 400), (B) the overall effect of various plants extracts on phagocytic functions of RAW 264.7 murine macrophage-like cells. Results are expressed as percent of control and presented as mean+tSE. p>0.05 was considered insignificant.

Figure 3. Effect of various plants extracts on NO production by RAW 264.7 murine macrophage-like cells. Cells were incubated with or without LPS (1j1g/mL) for 24h, in the presence or absence of various plants extracts at the indicated concentrations. (A) NO production in the culture medium was determined by Griess reagent. (B) Inhibition of LPS-stimulated NO production from RAW 264.7 murine macrophage-like cells by plants extracts. Data are presented as mean+SE where statistical significance was tested using one way ANOVA with Bonferroni's post hoc test. *p<0.05, **p<0.01 and se" :< 0,001.

Figure 3. Effect of various plants extracts on NO production by RAW 264.7 murine macrophage-like cells. Cells were incubated with or without LPS (1j1g/mL) for 24h, in the presence or absence of various plants extracts at the indicated concentrations. (A) NO production in the culture medium was determined by Griess reagent. (B) Inhibition of LPS-stimulated NO production from RAW 264.7 murine macrophage-like cells by plants extracts. Data are presented as mean+SE where statistical significance was tested using one way ANOVA with Bonferroni's post hoc test. *p<0.05, **p<0.01 and se" :< 0,001.

Figure 4. Effect of various plants extracts on TNF-« production from LPS-stimulated macrophage-like cells. Variable effects were obtained following incubation of various plants extracts as well as LPS with RAW 264.7 murine macrophage-like cells. Results expressed and presented as meantSE and statistical significance was examined using one way ANOVA with Bonferroni's post hoc test.*p <0.05; **p <0.01; ***p <0.001.

Figure 4. Effect of various plants extracts on TNF-« production from LPS-stimulated macrophage-like cells. Variable effects were obtained following incubation of various plants extracts as well as LPS with RAW 264.7 murine macrophage-like cells. Results expressed and presented as meantSE and statistical significance was examined using one way ANOVA with Bonferroni's post hoc test.*p <0.05; **p <0.01; ***p <0.001.

Figure 5. Effect of various plants extracts on COX-2 production by RAW 264.7 murine macrophage-like cells. (A) Inhibition of LPS-mediated COX-2 production from RAW 264.7 murine macrophage-like cells by various plants extracts. Three extracts (Lavandula pubescens, Silene succulenta and Gomphrene celesoids) were found to substantially inhibit LPS-mediated COX-2 production by RAW 264.7 murine macrophage-like cells as compared with the rest of extracts. (B) A representative dotplot demonstrating the effect of various plants extracts on LPS-mediated COX-2 production by RAW 264.7 murine macrophage-like cells.

Figure 5. Effect of various plants extracts on COX-2 production by RAW 264.7 murine macrophage-like cells. (A) Inhibition of LPS-mediated COX-2 production from RAW 264.7 murine macrophage-like cells by various plants extracts. Three extracts (Lavandula pubescens, Silene succulenta and Gomphrene celesoids) were found to substantially inhibit LPS-mediated COX-2 production by RAW 264.7 murine macrophage-like cells as compared with the rest of extracts. (B) A representative dotplot demonstrating the effect of various plants extracts on LPS-mediated COX-2 production by RAW 264.7 murine macrophage-like cells.

Table 1. Summary of all results.

Table 1. Summary of all results.

Figure 6. The half maximal scavenging capacity (SCso) values for various plants extracts., Calligonum comosum, Bogonvillea glabra and Mirabilis jalaba have been shown to have significantly lower SC50 values among all extracts.

Figure 6. The half maximal scavenging capacity (SCso) values for various plants extracts., Calligonum comosum, Bogonvillea glabra and Mirabilis jalaba have been shown to have significantly lower SC50 values among all extracts.

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