Determination of the Pharmacokinetics and Tissue Distribution of Methyl 3,4-Dihydroxybenzoate (MDHB) in Mice Using Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND AND OBJECTIVES: Methyl 3,4-dihydroxybenzoate (MDHB) has the potential to prevent neurodegenerative diseases (NDDs). The present work aims to reveal the pharmacokinetics and tissue distribution characteristics of MDHB. METHODS: The pharmacokinetics and tissue distribution of MDHB were analyzed using LC-MS/MS after a single intragastric administration (50 to 450 mg/kg) in mice, and samples were collected from five animals at specific time points. RESULTS: Pharmacokinetic parameters of MDHB following intragastric administrations were: the time to peak concentration (Tmax) ranged from 0.033 to 0.07 h, the peak concentration (Cmax) ranged from 12,379.158 to 109798.712 µg/l, the elimination half-life (t1/2z) ranged from 0.153 to 1.291 h, the area under the curve (AUC0-∞) ranged from 640.654 to 20,241.081 µg/l × h, the mean residence time (MRT0-∞) ranged from 0.071 to 0.206 h, the apparent volume of distribution (Vz/F) ranged from 17.538 to 45.244 l/kg, and the systemic clearance (Clz/F) ranged from 22.541 to 80.807 l/h/kg. The oral bioavailability of MDHB was 23%. The maximum MDHB content was detected in the stomach, and the minimum content was observed in the testes; the peak content in the brain was 15,666.93 ng/g. CONCLUSIONS: The pharmacokinetic characteristics of MDHB include fast absorption, high systemic clearance, a short half-life and an oral bioavailability of 23%. Additionally, MDHB permeates the blood-brain barrier (BBB) and is rapidly distributed to all organs. The identification of the pharmacokinetics of MDHB following its oral administration will contribute to further preclinical and clinical studies of its effects.

PI3K/Akt signaling pathway is involved in the neurotrophic effect of senegenin.

Neurodegenerative diseases are frequently associated with the loss of synapses and neurons. Senegenin, extracted from the Chinese herb Polygala tenuifolia Willd, was previously found to promote neurite outgrowth and neuronal survival in primary cultured rat cortical neurons. The aim of the present study was to investigate the underlying mechanisms of senegenin-induced neurotrophic effects on rat cortical neurons. Primary cortical rat neurons were treated with various pharmacological antagonists and with or without senegenin, and subjected to MTT and western blot analysis to explore the effects of senegenin on cell survival as well as the activation of signaling pathways. Neurite outgrowth and neuronal survival induced by senegenin were significantly inhibited by A2A receptor antagonist ZM241385 and specific phosphoinositide-3 kinase (PI3K) inhibitor LY294002, but not by tropomyosin receptor kinase A receptor inhibitor K252a, mitogen-activated protein kinase kinase inhibitor PD98059 or protein kinase C inhibitor GÖ6976. Furthermore, senegenin enhanced the phosphorylation of Akt, which was blocked by LY294002. The present study revealed that the PI3K/Akt signaling pathway may be involved in the neurotrophic effects of senegenin.

Methyl 3,4-dihydroxybenzoate extends the lifespan of Caenorhabditis elegans, partly via W06A7.4 gene.

To identify and analyze the compounds that delay aging and extend the lifespan is an important aspect of the gerontology research. A number of compounds, including the ones with the antioxidant properties, have been shown to extend the lifespan of Caenorhabditis elegans. Here, we report that methyl 3,4-dihydroxybenzoate (MDHB), a small antioxidant molecule, prolongs the C. elegans' lifespan under normal as well as stress conditions, delays the age-associated decline in the pharyngeal pumping rate, and obviously enhances the abilities of scavenging intracellular reactive oxygen species (ROS). To further investigate the mechanism underlying the anti-aging action of MDHB, microarray analyses were performed, which demonstrated that 13 genes were differentially expressed in worms treated with MDHB for 48 and 144 h in common. RNA interference of W06A7.4 (NM_001269697.1), the most significantly up-regulated gene, shortened the lifespan of worms by 14%, compared with the L4440 control. Our findings demonstrate that W06A7.4 is a potentially positive determinant of the MDHB induced C. elegans' lifespan extension effect.

Neuroprotective effects of methyl 3,4-dihydroxybenzoate against H2O2-induced apoptosis in RGC-5 cells.

In the present study, we investigated the protective effect of methyl 3,4-dihydroxybenzoate (MDHB) against H2O2-induced apoptosis in RGC-5 cells. The RGC-5 cells were cultured in plates for 24 h, which were then pretreated with dimethyl sulfoxide, different concentrations of MDHB, or probucol for 12 h prior to addition of 300 µM H2O2 for 24 h. The cell viability was detected by MTT assay. The rate of apoptosis, level of lipid peroxidation, and mitochondrial membrane potential (MMP) were detected by flow cytometry. Western blot analysis was also used to measure the expression level of Bcl-2, Bax, caspase 9, and caspase 3 proteins in H2O2-treated RGC-5 cells. Our study showed that the cell viability of RGC-5 cells significantly decreased after treatment with 300 µM H2O2 for 24 h, but MDHB (8, 16, 32 µM) increased RGC-5 cell survival, suppressed the rate of apoptosis, scavenged reactive oxygen species, and restored MMP. MDHB also obstructed H2O2-induced apoptosis by regulating the expression of Bcl-2 and Bax, as well as suppressing the activation of caspase 9 and caspase 3. Our results showed that MDHB is an effective neuroprotective compound that mitigates oxidative stress and inhibits apoptosis in RGC-5 cells.

Methyl 3,4-dihydroxybenzoate protects primary cortical neurons against Aß25₋35-induced neurotoxicity through mitochondria pathway.

Amyloid-ß peptides (Aß), which can aggregate into oligomers or fibrils in neurons, play a critical role in the pathogenesis of Alzheimer's disease (AD). Methyl 3,4-dihydroxybenzoate (MDHB), a phenolic acid compound, has been reported to have antioxidative and neurotrophic effects. The present study investigated the neuroprotective effects of MDHB against Aß-induced apoptosis in rat primary cortical neutons. The primary cortical neurons were pretreated with different concentrations of MDHB for 24 hr, then incubated with 10 µM Aß25-35 for 24 hr. The results showed that Aß25-35 could induce neurotoxicity as evidenced by the decreased cell viability and the increased apoptotic rate. In parallel, Aß25-35 significantly increased the reactive oxygen species accumulation and decreased mitochondrial membrane potential. However, pretreatment of the primary cortical neurons with MDHB could effectively suppress these cellular events caused by Aß25-35 exposure. In addition, MDHB could increase the level of Bcl-2, decrease the level of Bax, and inhibit the activation of caspase-9 and caspase-3 in Aß25-35 -treated primary cortical neurons. All these results were beneficial in their protective effect against Aß-induced neurotoxicity. Our results suggest that MDHB has a neuroprotective effect that provides a pharmacological basis for its clinical use in the treatment of AD.