During Gram-negative bacterial infections, the pathological condition of inflammation is initiated bacterial outer cell wall components, especially endotoxins such as LPS [33
]. LPS can directly activate macrophages by triggering the production of pro-inflammatory cytokines such as TNF [34
], IL-1, IL-6, IFN, chemokines such as MCP-1, MIP1 [35
], leukotrienes [36
] and others such as nitric oxide and prostaglandins [37
]. Overproduction of these inflammatory markers amplifies the host immune response and leads to inflammatory complications such as sepsis [38
]. Therefore, the pharmacological interventions of LPS-induced production of inflammatory mediators by macrophages are essential events to control a variety of inflammatory related disorders. In this study, we have for the first time shown that benfotiamine, a fat-soluble analogue of vitamin B1, modulates the production of AA pathway generated inflammatory lipid mediators.
Several studies indicate that the use of natural anti-inflammatory products provides an attractive and safe alternative to modulate various inflammatory disorders [39
]. Due to its therapeutic action in some frequently observed clinical syndromes, thiamine hydrochloride has been advised and used over a long period of time [42
]. There are no reports of adverse effects of oral thiamine, even at dosages of several hundred milligrams a day [44
]. Benfotiamine, a unique derivative of thiamine is the most potent of the allithiamines, a group of lipid-soluble forms of thiamine [46
]. Compared to water-soluble thiamine salts, benfotiamine is absorbed better in the intestine reaching maximum plasma levels of thiamine about 5 times higher and increases active metabolite thiamine diphosphate (ThDP) to 120 folds. The bioavailability is at maximum about 4 times as high as that of thiamine hydrochloride and other lipophilic thiamine derivates [47
]. In fact, a recent randomized, double blind, placebo-controlled clinical study in Germany indicated that benfotiamine at a dose of 600 mg/day and higher had no side effects [48
]. A number of studies have revealed its potential to alleviate diabetic microangiopathy, neuropathy and other oxidative stress-induced pathological conditions in various experimental animal models [49
]. However, no reports are available that show benfotiamine supplementation prevents sepsis complications in animal or human studies. We have recently reported that the anti-oxidative and anti-inflammatory properties of benfotiamine in preventing the LPS-induced cytotoxicity in macrophages as well as preventing the ocular inflammation in rats [24
]. The present study investigates the potential efficacy of benfotiamine in preventing AA pathway dependent inflammatory signals in macrophages. This is an important advance for the use of benfotiamine as an anti-inflammatory food supplement for treating inflammatory disease conditions.
COX-2 and LOX-5 are the critical enzymes of AA metabolism which catalyzes AA to PGs and LKBs, major lipid mediators involved in various inflammatory diseases [32
]. In the present study, we have investigated the effect of benfotiamine on LPS-induced AA pathway generated inflammatory lipid mediators. Our results demonstrate that benfotiamine significantly prevented the AA pathway generated inflammatory lipid mediators as well as the expression of enzymes such as COX-2 and LOX-5, which catalyze the formation of lipid mediators. Thus, inhibition of COX-2 and LOX-5 appears to be responsible for the decreased biosynthesis of pro-inflammatory PGs and LTB4. Septic patients and animals treated with LPS have increased expression of pro-inflammatory cytokines and PGI2 synthase [54
]. Since, PGI2 once formed rapidly gets converted into 6-ketoPGF1, and is involved in vasodilation resulting in reduced blood pressure, and can ultimately lead to sepsis [55
]. In this study, benfotiamine significantly prevented 6-ketoPGF1 levels and PGI2 expression suggesting that benfotiamine could efficiently prevent PGI2 -induced vasculopathy upon bacterial infections.
Lipoxygenases catalyze the oxygenation of AA [56
]. It is known that leukotrienes are important regulators of cancer cell proliferation and cause apoptosis in non-cancer cells [57
]. Therefore, we examined whether benfotiamine could regulate the expression and synthesis of LOX-5. Our results demonstrate that LPS-induced expression and synthesis of LOX-5 in RAW264.7 cells were significantly prevented by benfotiamine ( and ). Our results thus indicate that benfotiamine prevents the expression of two key regulatory enzymes of AA metabolism i.e. COX-2 and LOX-5. Indeed, we observed that as compared to specific COX and LOX inhibitors, benfotiamine is more potent in preventing the LPS-induced macrophage death as well as monocytes adhesion to endothelial cells. During bacterial infections and systemic inflammatory pathologies such as severe sepsis, increased production of ROS causes oxidative stress leading to alterations in protein functions and increased susceptibility to protein degradation [58
]. In addition to proteins, polyunsaturated fatty acids such as AA, present in membrane lipids are highly susceptible to ROS-mediated damage [59
]. Peroxidation of membrane lipids results in the fragmentation of polyunsaturated fatty acids resulting in the production of various cytotoxic and highly reactive aldehydes, such as malonaldehyde and 4-hydroxy-2-nonenal (HNE) [60
]. Since benfotiamine prevents the formation of AA acid metabolites by preventing the activation of cPLA2, we next examined if benfotiamine prevents AA induced generation of HNE. Our results indicate that benfotiamine prevents lipid peroxidation as well as subsequent protein-HNE adducts formation. These results suggest that anti-oxidative properties of benfotiamine could prevent LPS-induced lipid peroxidation and HNE formation.
LPS-induced inflammatory signals are known to mediate through ROS, resulting in the activation of protein kinase C (PKC) and mitogen activated protein kinase (MAPK) [60
] which can cause inflammation by activating transcription factor such as NF-kB. It has been shown earlier that various antioxidants such as vitamin C and vitamin E inhibit ROS-induced inflammatory responses by preventing the activation of NF-kB [63
]. Our current studies indicate that benfotiamine prevents activation of NF-kB as well as Egr-1 and phosphorylation of ERK1/2. Thus prevention of LPS-induced AA metabolizing enzymes and formation of inflammatory lipid mediators by benfotiamine could be mediated regulating redox-sensitive transcription factors.
In summary, this study shows that AA pathway generated inflammatory lipid mediators in response to endotoxin exposure could be prevented by a dietary supplement, benfotiamine. Further, benfotiamine inhibited LPS-induced NF-kB and Egr-1 and expression of key regulatory enzymes of AA metabolism such as cPLA2, COX-2 and LOX-5 in murine macrophages. Our studies indicate that benfotiamine is a more potent anti-inflammatory compound as compared to specific LOX and COX inhibitor. Thus, dual regulation of COX and LOX pathways by benfotiamine could be a novel mechanism by which benfotiamine exhibits its anti-inflammatory response and benfotiamine supplementation could be a potential therapeutic approach to prevent inflammatory complications such as sepsis.