4 Discussion
NO, IL-1β, IL-6, and TNF-α participate in the pathogenesis of inflammatory diseases[13, 14]. We found that Tube inhibited the expression of IL-1β, IL-6, and iNOS and suppressed NO production but did not affect TNF-α expression in LPS-induced RAW264.7 cells. We also measured NF-κB and MAPK signaling pathway-related proteins (e.g., NF-κB p65 and MAKP p38) and found that Tube had no significant effect on the phosphorylation levels[15-20]. These findings suggest that Tube may not inhibit inflammation through the classical NF-κB signaling pathway. Small molecules of bacterial origin can be recognized by cell surface receptors and activate relevant signal transduction, leading to the reprogramming of macrophage energy metabolism from mitochondrial oxidative phosphorylation to a high rate aerobic glycolysis; this reprogramming regulates the synthesis and accumulation of macrophage immune metabolites, which in turn affects the expression of inflammatory factors.
Glucose transporter protein (GLUT1) and lactate dehydrogenase (LDH) play pivotal roles in glycolysis. They convert glucose into pyruvate and lactate, which then enter the tricarboxylic acid (TCA)[21, 22]. Tube increased the mRNA levels of fructose-2,6-bisphosphatase 3, HK2, and GLUT1, the rate-limiting enzyme genes of glycolysis, but decreased mRNA levels of LDH. These findings suggest that Tube causes cellular accumulation of pyruvate; therefore, we measured the pyruvate content in RAW264.7 cells and found thatTube did increase it. Notably, we found little effect of Tube on basal and maximal respiration in RAW264.7 cells; therefore, we tentatively concluded that Tube increases the production of pyruvate, an intermediate product of glycolysis, and does not increase levels of mitochondrial oxidative respiration. Pyruvate is involved in energy metabolism by entering the TCA cycle; therefore,Tube might exert its anti-inflammatory effect by regulating the energy metabolic process.
Macrophage metabolic pathways are altered during inflammatory responses, known as “metabolic reprogramming”[23]. We found that metabolic reprogramming participates in the development of inflammatory responses by regulating the expression of macrophage inflammatory factors[24]. Moreover, we observed an intermediate product, itaconic acid. During LPS stimulation, macrophages upregulate expression of immune response gene 1, a mitochondria-related enzyme that catalyzes the conversion of cis-aconitate into itaconate and inhibits succinate dehydrogenase through competitive binding, creating a metabolic breakpoint in the TCA cycle and blocks the TCA cycle[17, 25] . Itaconic acid inhibits the electron transport chain in mitochondria[25, 26], and it regulates the transcription of IL-1β and IL-6 through the ATF3-IκBζ axis; however, it has no significant effect on the transcription level of TNF-α[8, 10]. Therefore, we measured expression levels of downstream effector molecules of itaconic acid using RT-PCR experiments and found that Tube was associated with the regulatory role of itaconic acid.
IκBζ is a transcriptional regulator of the non-classical NF-κB signaling pathway. It binds to the NF-κB p50 subunit and promotes the transcription of pro-inflammatory factors, including IL-6, IL-1β, and IL-1α[27, 28]. Electrophilic stress induced by itaconic acid inhibited IκBζ by upregulating activating transcription factor 3 (ATF3), reducing the production of the pro-inflammatory factor IL-6[8]. Western blot showed that Tube increased the ATF3 protein level and inhibited IκBζ protein levels. These findings suggest that Tube exerts its anti-inflammatory effect by reprogramming glucose metabolism in macrophages, affecting the metabolism of the small molecule itaconic acid. Finally, to confirm the necessity of the anti-inflammatory effect of itaconic acid on Tube, we used an inhibitor of itaconic acid, citraconate (that competitively inhibits aconitate decarboxylase 1)[29]. Citraconate impaired the protective effect of Tube in the LPS-induced acute inflammation model. Citraconate also attenuated the downregulation of IL-1β and IL-6 by Tube. These results suggest that Tube requires the involvement of itaconic acid to exert its inflammatory inhibitory effects.
In summary, tubeimoside Ⅲ regulates the reprogramming of macrophage glucose metabolism, increasing the content of small metabolic molecule itaconic acid, inhibiting the expression of IL-1β, IL-6, and iNOS, and reducing the inflammatory response.