Spinosin inhibits activated hepatic stellate cell to attenuate liver fibrosis by targeting Nur77/ASK1/p38 MAPK signaling pathway.

AIM: Liver fibrosis remains a great challenge in the world. Spinosin (SPI), a natural flavonoid-C-glycoside, possesses various pharmacological activities including anti-inflammatory and anti-myocardial fibrosis effects. In this study, we investigate whether SPI can be a potential lead for the treatment of liver fibrosis and explore whether the orphan nuclear receptor Nur77, a negative regulator of liver fibrosis development, plays a critical role in SPI's action. METHODS: A dual luciferase reporter system of α-SMA was established to evaluate the effect of SPI on hepatic stellate cell (HSC) activation in LX2 and HSC-T6 cells. A mouse model of CCl4-induced liver fibrosis was used to test the efficacy of SPI against liver fibrosis. The expression levels of Nur77, inflammatory cytokines and collagen were determined by Western blotting and qPCR. Potential kinase pathways involved were also analyzed. The affinity of Nur77 with SPI was documented by fluorescence titration. RESULTS: SPI can strongly suppress TGF-ß1-mediated activation of both LX2 and HSC-T6 cells in a dose-dependent manner. SPI increases the expression of Nur77 and reduces TGF-ß1-mediated phosphorylation levels of ASK1 and p38 MAPK, which can be reversed by knocking out of Nur77. SPI strongly inhibits collagen deposition (COLA1) and reduces inflammatory cytokines (IL-6 and IL-1ß), which is followed by improved liver function in the CCl4-induced mouse model. SPI can directly bind to R515 and R563 in the Nur77-LBD pocket with a Kd of 2.14 µM. CONCLUSION: Spinosin is the major pharmacological active component of Ziziphus jujuba Mill. var. spinosa which has been frequently prescribed in traditional Chinese medicine. We demonstrate here for the first time that spinosin is a new therapeutic lead for treatment of liver fibrosis by targeting Nur77 and blocking the ASK1/p38 MAPK signaling pathway.

Synthesis, Optimization, and Structure-Activity Relationships of Nicotinamide Phosphoribosyltransferase (NAMPT) Positive Allosteric Modulators (N-PAMs).

Depletion of nicotinamide adenine dinucleotide (NAD+) is associated with aging and disease, spurring the study of dietary supplements to replenish NAD+. The catabolism of NAD+ to nicotinamide (NAM) requires the salvage of NAM to replenish cellular NAD+, which relies on the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT). Pharmacological activation of NAMPT provides an alternative to dietary supplements. Screening for activators of NAMPT identified small molecule NAMPT positive allosteric modulators (N-PAMs). N-PAMs bind to the rear channel of NAMPT increasing enzyme activity and alleviating feedback inhibition by NAM and NAD+. Synthesis of over 70 N-PAMs provided an excellent correlation between rear channel binding affinity and potency for enzyme activation, confirming the mechanism of allosteric activation via binding to the rear channel. The mechanism accounts for higher binding affinity leading to loss of efficacy. Enzyme activation translated directly to elevation of NAD+ measured in cells. Optimization led to an orally bioavailable N-PAM.

Mechanism of Allosteric Modulation of Nicotinamide Phosphoribosyltransferase to Elevate Cellular NAD.

In aging and disease, cellular nicotinamide adenine dinucleotide (NAD+) is depleted by catabolism to nicotinamide (NAM). NAD+ supplementation is being pursued to enhance human healthspan and lifespan. Activation of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD+ biosynthesis, has the potential to increase the salvage of NAM. Novel NAMPT-positive allosteric modulators (N-PAMs) were discovered in addition to the demonstration of NAMPT activation by biogenic phenols. The mechanism of activation was revealed through the synthesis of novel chemical probes, new NAMPT co-crystal structures, and enzyme kinetics. Binding to a rear channel in NAMPT regulates NAM binding and turnover, with biochemical observations being replicated by NAD+ measurements in human cells. The mechanism of action of N-PAMs identifies, for the first time, the role of the rear channel in the regulation of NAMPT turnover coupled to productive and nonproductive NAM binding. The tight regulation of cellular NAMPT via feedback inhibition by NAM, NAD+, and adenosine 5'-triphosphate (ATP) is differentially regulated by N-PAMs and other activators, indicating that different classes of pharmacological activators may be engineered to restore or enhance NAD+ levels in affected tissues.

P2X receptors are expressed on neurons containing luteinizing hormone-releasing hormone in the mouse hypothalamus.

Expression of P2X(1), P2X(2), P2X(3), P2X(4), P2X(5) and P2X(6) receptors, members of a family of ATP-gated cation channels, on neurons containing luteinizing hormone-releasing hormone (LHRH) in the mouse hypothalamus was studied with double-labeling fluorescence immunohistochemistry. This study demonstrated that different combinations of P2X receptor subunits were expressed on the perikarya and axon terminals of LHRH-producing neurons. It was shown for the first time that P2X(2), P2X(4), P2X(5) and P2X(6) receptor subunits were expressed on the perikarya of LHRH-producing neurons and P2X(2) and P2X(6) on their axon terminals. These results suggest that activation of P2X receptors by ATP via different homomeric or heteromeric P2X receptors at both presynaptic and postsynaptic sites could be involved in the regulation of LHRH secretion at the forebrain level.