The effect of tissue necrosis factor-alpha inhibitor and AMT on cell death and nitric oxide production in diabetic murine macrophages
Barr, Jessica L.
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Many dental diseases have an inflammatory component. These diseases share many cellular processes common to all forms of inflammation; inflammatory cell recruitment, cytokine release, and subsequent cellular repair. Systemic conditions, such as diabetes, drastically alter the capacity to mount an adequate host response, thus creating a chronic situation with many side effects. This study evaluated the effect of Type II diabetes mellitus on murine macrophage nitric oxide production, apoptosis and necrosis under various stimulatory conditions. The effect of TNF-[alpha] inhibitor and COX2 inhibitor, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) on these parameters was also evaluated. Peritoneal murine macrophages from diabetic models and controls that had P. gingivalis or vehicle only (PBS) injection were harvested. TNF-[alpha] receptor inhibitor and AMT were added. In addition, various concentrations of LPS stimuli were added in-vitro (1, 10, 100ng/ml). The dose response and time course of NO production, apoptosis, necrosis and the effect of inhibitors on these cellular processes were evaluated. The NO production was evaluated using Griess reagent, the apoptosis was evaluated via ELISA and the necrosis was quantified by lactase dehydrogenase (LDH) assay. The results demonstrated a greater response of the non-diabetic controls to in-vitro LPS stimulation which was demonstrated by a significantly larger amount of NO being produced by the control cells following in-vitro stimulation. The greatest amount of NO produced by both the diabetic and control groups was demonstrated at maximum stimulation (100ng/mL).In addition, following both in-vivo and in-vitro stimulation, the control group again demonstrated a significantly greater amount of NO production. A significant maximun NO production at 5 days was seen for the control groups under all stimulatory conditions and for the diabetic group following in-vivo stimulation. The diabetic macrophages underwent significantly more apoptosis than the control especially under in-vivo bacterial stimulation. With in-vitro and in-vivo bacterial challenge, macrophage apoptosis significantly increased in control group, which suggests macrophage apoptosis is prompted by activation and diabetic macrophages might be pre-activated in-vivo. TNF-[alpha] inhibitors only reduced NO production by macrophages stimulated in-vitro but was not effective on those primed by in-vivo bacterial injection. The inhibitors to TNF-[alpha] or iNOS were most effective for both the diabetic and control group in reducing apoptosis at day 5 with both in-vivo and in-vitro (maximum) stimulation but not with in-vivo stimulation alone. In a similar manner, the inhibitors were most effective in reducing necrosis of macrophages with combined stimulation when the maximum necrosis was seen at 8 days for both groups. Based on the present studies, it is deduced that the diabetic macrophages are less effective in responding to infectious agents when compared to non-diabetic controls. Increased apoptosis in the diabetic model may be attributed to an alteration in the status of initial priming, activation and sequential process of those macrophages. Nitric oxide promotes macrophage apoptosis and TNF directly or indirectly affects macrophage apoptosis at least partially by promoting NO production.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Thesis (M.S.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 2005 (Endodontics).Includes bibliographical references (leaves 87-87).
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