Fig 1.
Lymphocyte proliferation in borax treated mice stimulated with (a) Ovalbumin and (b) Concanavalin A.
Treatment protocol and other experimental details are same as described in the text. Briefly, the control group of animals (C1) did not receive any treatment. C2, on the other hand, received ovalbumin, but not borax, as per the treatment protocol described in the Materials and Methods section of the text. B1, B2 and B3 indicate different doses of borax administered to mice for 10 consecutive days. Boron increased the lymphocyte proliferative responses of (a) Ovalbumin and (b) Concanavalin A, indicating the B and T cells activation. *p<0.05, **p<0.01 and ***p<0.001, when compared with the control groups; ns = non-significant. The effect of borax was maximum at B3, that is, at a dose level of 4.6 mg/kg b. wt.
Fig 2.
Confocal microscopic images of inducible nitric oxide synthase (iNOS) in peritoneal macrophages isolated from borax treated mice.
Mice were treated with different doses of borax, B1, B2 and B3, as described in Materials and Methods section of the text. The isolated macrophages were incubated with primary iNOS antibody followed by incubation with Alexa Fluor® 488. Images were captured in a LEICA SP5II confocal microscope at 40x (magnification). Arrows indicate increase in fluorescence of iNOS conjugated with Alexa Fluor® 488. DAPI: 4',6-Diamidino-2-phenylindole. In all groups, except the untreated and unstimulated control group, cells were stimulated with lipopolysaccharide (LPS) after isolation from mouse peritoneum.
Fig 3.
Quantification of (a) iNOS and (b) NO in borax treated and control groups of mice.
Mice treated with different doses of borax, as described in the text, were used to isolate the macrophages. Macrophages were cultured and primed with lipopolysaccharide (LPS, 1 ng/ml) for 24 h. The images of iNOS, captured by LEICA SP5II, were quantified using imageJ. NO (nitric oxide) was estimated as nitrite in the supernatant by Griess test. *p<0.05, **p<0.01 and ***p<0.001 indicate significant differences when borax treated groups were compared with LPS-stimulated group. There was a significant (∼) increase in both iNOS and NO by the LPS primed macrophages when compared with unstimulated (control) group. B1, B2 and B3 indicate different doses of borax, as described in the text. The maximum effect was seen in mice treated with 4.6 mg/kg b. wt. borax (B3).
Fig 4.
Effect of borax on the release of (a) TNF-α, (b) IL-1β and (c) IL-6 by the lipopolysaccharide (LPS)-primed macrophages.
Each value in bar diagrams represents Mean ± S.E.M. (n = 6); *p<0.05, **p<0.01 and ***p<0.001, when compared with LPS-stimulated group. Treatment with borax caused a substantial increase in all three cytokines when compared with LPS stimulated group. Maximum activity was shown at a dose level of 4.6 mg/kg b. wt. of borax (B3). B1, B2 and B3 indicate different doses of borax, as described in the text. In all groups, except control, macrophages were challenged with LPS.
Fig 5.
Flow cytometric analysis of B and T cell subsets in borax treated mice.
The splenocytes in suspension were labeled with FITC-conjugated CD8 and PE-conjugated CD19 and CD4 antibodies and analyzed as per the protocol described in methods section. Plots (a) and (e) show the percent population of CD8, CD4 and CD19 cells in untreated control group, which was 8.40 ± 0.65, 9.80 ± 1.65 and 50.40 ± 1.9, respectively. Plots (b), (c) and (d) indicate the percent population of CD8 and CD4 cells in borax treated mice. Plots (f), (g) and (h) show the percent population of CD19 cells. The percent population of CD19 and CD4 cells increased in borax treated mice. Population of CD19 cells increased from 50.40 ± 1.9 to 60.65 ± 1.2, 63.20 ± 2.4 and 66.10 ± 1.2 at different doses of borax, with maximum increase at 4.6 mg/kg b. wt. Percent population of CD4 cells in the suspension was 15.2 ± 1.21, 18.3 ± 0.85 and 20.1 ±1.11 in borax treated groups when compared with the respective control group, Plot (a). CD8 cells did not show a significant change in any of the treatment groups.
Table 1.
Percentage population of CD19, CD4 and CD8 cells in boron treated and untreated groups.