Semisynthetic quercetin-quinone mitigates BV-2 microglia activation through modulation of Nrf2 pathway.

During brain ageing, microglia, the resident immune cells of the CNS, are immunologically activated and contribute to neuroinflammation, a vicious cycle that supports development of neurological disorders. Therapeutic approaches focus mainly on downregulation of their pro-inflammatory activated state that is associated with health benefits. Electrophilic compounds, such as natural quinones and their reduced pro-electrophilic precursors, flavonoids, represent a wide group of diverse substances with important biological effects. They can cause considerable cytotoxicity when used at higher dosages, but on the other hand, they have versatile health benefits at lower dosages. In this study, we investigated the cytotoxicity and prooxidant profile of synthetic conjugate of two electrophilic compounds, quercetin and 1,4-naphthoquinone, 4'-O-(2-chloro-1,4-naphthoquinone-3-yloxy) quercetin (CHNQ), and its attenuation of inflammatory responses and modulation of Nrf2 pathway in BV-2 microglial cells. CHNQ showed higher cytotoxicity than its precursors, accompanied by promotion of production of reactive oxygen species along with G2/M cell cycle arrest at higher concentrations tested. Nevertheless, at a lower non-toxic concentration, CHNQ, more significantly than did its precursors, downregulated LPS-stimulated microglia cells as documented by decreased iNOS, COX-2 and TNFα protein levels. Moreover, CHNQ most effectively upregulated expression of phase II antioxidant enzyme HO-1 and ß5 subunit of constitutive proteasome. The enhanced anti-inflammatory effect of CHNQ was accompanied by prominent increase in cytosolic expression of Nrf2 and c-Jun, however, induction effect on nuclear Nrf2 translocation was comparable to QUER. Moreover, a conditioned medium from activated BV-2 cells co-treated with quercetin and CHNQ maintained viability of neuron-like PC12 cells. The compounds tested did not show any disturbance of phagocytosis of live or dead PC12 cells. The present experimental data predict a preventive and therapeutic potential of semisynthetic derivative CHNQ in ageing and related pathologies, mediated by activation of proteins of the antioxidant response.

Protective Effect of Semisynthetic and Natural Flavonoid on Aged Rat Microglia-enriched Cultures.

The ROS-mediated lysosomal dysfunction and coinciding deterioration of mitochondrial function are thought to be the prominent mechanisms responsible for aging. Microglia, the resident macrophages in the central nervous system, were postulated to belong to the major targets vulnerable to these detrimental processes, acting as principal drivers in brain aging. The present study investigated the potential protective effect of the semisynthetic flavonoid 3'-O-(3-chloropivaloyl) quercetin (CPQ) and quercetin (Q) on microglia-enriched mixed brain cultures (MBCs) established from aged Wistar rats. Both flavonoids tested suppressed the development of lipofuscin-related autofluorescence in aged cells. Further ensuing protective effects included reduction of protein oxidation markers in aged cells. Moreover, unlike Q, CPQ significantly suppressed sensitivity of aged cells to stimulation of superoxide burst. Other activation markers, cellular hypertrophy and isolectin B4 binding, were also downregulated by treatment with both CPQ and Q. In conclusion, results of our study suggest that both flavonoids tested may protect microglia with a quite comparable efficacy against aging-related accumulated alterations. The protective mechanism can include interference with the ROS-mediated vicious cycles involving lysosomal dysfunction. Nevertheless, the lipophilized quercetin, CPQ, a compound with proposed enhanced biological availability compared to parent molecule, can represent an agent potentially useful for new effective pharmaceutical intervention against brain aging, overcoming the limitations of clinical applicability of quercetin.

Pyridoindole SMe1EC2 as cognition enhancer in ageing-related cognitive decline.

Synthetic pyridoindole-type substances derived from the lead compound stobadine represent promising agents in treatment of a range of pathologies including neurological disorders. The beneficial biological effects were suggested to be likely associated with their capacity to ameliorate oxidative damage. In our study, the effect of supplementation with the derivative of stobadine, SMe1EC2, on ageing-related cognitive decline in rats was investigated. The 20-months-old male Wistar rats were administered SMe1EC2 at a low dose, 0.5 mg/kg, daily during eight weeks. Morris water maze test was performed to assess the spatial memory performances. The cell-based assays of capacity of SMe1EC2 to modulate proinflammatory generation of oxidants by microglia were also performed. The rats treated with SMe1EC2 showed significantly increased path efficiency, significantly shorter time interval of successful trials and exerted also notably lower frequencies of clockwise rotations in the pool compared to non-supplemented aged animals. Mildly improved parameters included test durations, distances to reach the platform, time in periphery of the pool and overall rotations in the water maze. However, the pyridoindole SMe1EC2 did not show profound inhibitory effect on production of nitric oxide and superoxide by activated microglial cells. In conclusion, our study suggests that pyridoindole SMe1EC2, at low doses administered chronically, can act as cognition enhancing agent in aged rats. The protective mechanism less likely involves direct modulation of proinflammatory and prooxidant state of microglia, the prominent mediators of neurotoxicity in brain ageing and neurodegeneration.

Pro-apoptotic effect of new quinolone 7- ethyl 9-ethyl-6-oxo-6,9-dihydro[1,2,5]selenadiazolo [3,4-h]quinoline-7-carboxylate on cervical cancer cell line HeLa alone/with UVA irradiation.

7- ethyl 9-ethyl-6-oxo-6,9-dihydro[1,2,5]selenadiazolo [3,4-h]quinoline-7-carboxylate (E2h) is a new synthetically prepared quinolone derivative, which in our primary study showed cytotoxic effects towards tumor cells. The aim of the present study was to examine the antiproliferative and apoptosis inducing activities of E2h towards human cervical cancer cell line HeLa with/without the presence of UVA irradiation. Further, the molecular mechanism involved in E2h-induced apoptosis in HeLa cells was investigated. Our results showed that both non-photoactivated and photoactivated E2h caused morphological changes and inhibited the cell growth of HeLa cells in a time- and dose-dependent manner. Irradiation increased the sensitivity of HeLa cells to E2h. Quinolone induced S and G2/M arrest and apoptosis in HeLa cells, as characterized by DNA fragmentation and flow cytometry. In addition, E2h elevated the level of reactive oxygen species and activated caspases 3. In conclusions, E2h alone/in combination with UVA irradiation induced apoptosis in HeLa cells through the ROS-mitochondrial/caspase 3-dependent pathway.

Modulation of BV-2 microglia functions by novel quercetin pivaloyl ester.

Chronic inflammation in brain plays a critical role in major neurodegenerative diseases such as Alzheimer's, Parkinson's disease, stroke or multiple sclerosis. Microglia, resident macrophages and intristinc components of CNS, appear to be main effectors in this pathological process. Quercetin, a naturally occurring flavonoid, was proven to downregulate inflammatory genes in microglia. Synthetically modified quercetin, 3'-O-(3-chloropivaloyl) quercetin (CPQ), is assumed to possess better biological availability and enhanced antioxidant properties. In the present study, antineuroinflammatory capability of the novel compound CPQ was assessed in BV-2 microglial cells. Our data show that treatment with CPQ attenuated the production of the inflammatory mediators, nitric oxide (NO) and tumour necrosis factor-α (TNF-α), in LPS-stimulated microglia somewhat more efficiently than did quercetin (p > 0.05 for CPQ vs. quercetin-treated group). Also, protein level of inducible NO synthase (iNOS) in LPS-activated BV-2 microglia was to some extent more effectively supressed by CPQ than by unmodified flavonoid. In consistence with the extent of their effects on pro-inflammatory markers, CPQ and quercetin showed down-regulation of NFκB activation. This quercetin analogue caused also a decline in BV-2 microglia proliferation with interfering with cell cycle progression (p < 0.001 for CPQ vs. quercetin-treated group). However, CPQ did not remarkably affect cell viability. In addition, CPQ showed a minor better suppression of PMA-induced generation of superoxide than did quercetin. Neither CPQ nor quercetin influenced phagocytosis of BV-2 cells. These results point to the therapeutic potential of 3'-O-(3-chloropivaloyl)quercetin (CPQ) as a novel antiinflammatory drug in neurodegenerative diseases, mediating favourable modulation of pro-inflammatory functions of microglia.

Protection or cytotoxicity mediated by a novel quinonoid-polyphenol compound?

Many natural and synthetic quinones and naphthoquinones possess a variety of beneficial pharmacological properties. In plants, the cytotoxic properties of quinones serve in their defensive roles against invading bacteria, fungi and parasites. In this regard many quinones as well as polyphenols, exerting generally toxicity at high dosages, are able to induce favorable hormetic responses at a low dosage. The novel chloronaphthoquinone derivative of quercetin (CHNQ) showed a profound cytotoxicity followed by enhancement of intracellular generation of oxidants in human neonatal B-HNF-3 fibroblasts. Its synthetic precursors, quercetin and 2-chloro-3-hydroxy-[1,4]naphthoquinone, failed to induce these effects, and paradoxically, only CHNQ at a low concentration provided partial protection of the cells against oxidative challenge. Thus, the novel quinonoid-polyphenol CHNQ might have a merit in the search for new prospective agents in prevention and management of ageing and ageing-related pathologies.

Can semi-synthetic flavonoids return old microglia to their youthful state?

A number of studies have indicated that brain inflammation may deteriorate during normal aging and that neuroinflammation is amplified in age-related neurodegenerative diseases. A pivotal role in age-related neuroinflammatory pathologies is attributed to amplified and prolonged activation of microglia. In addition, microglia from the aged brain were reported as senescent displaying many functional impairments. Flavonoids were shown to be promising molecules in modulation of neuroinflammation. Quercetin, a naturally occurring flavonoid, was proven to downregulate inflammatory genes in microglia. Synthetically modified quercetin, 3´-O-(3-chloropivaloyl)quercetin (CPQ), is assumed to posses better biological availability and enhanced antioxidant properties. In the present study, the antineuroinflammatory capacity of CPQ was assessed in BV-2 microglial cells and rat primary microglia. CPQ suppressed more efficiently than its precursor quercetin LPS-induced NO production and iNOS protein expression. However, neither of the compounds tested influenced significantly phagocytosis of BV-2 cells. In addition, CPQ showed a somewhat better suppression of PMA-induced generation of superoxide than did quercetin. Unlike quercetin, CPQ caused a decline in BV-2 microglia proliferation (without any impact on cell viability) along with interference with cell cycle progression. Both compounds tested at 10uM concentration notably enhanced viability of microglia-enriched cultures prepared from 22-month-old rat brains. This was followed by suppression of lipofuscin-like autofluorescence, improvement of lysosomal function and protection of mitochondria in the old microglia. These results can highlight the therapeutic potential of CPQ as a novel antiinflammatory drug in neurodegenerative diseases. In addition, our data suggest that both natural and semisynthetic flavonoids might protect functions of old microglia [VEGA2/0031/12,1/0076/13;APVV-0052-10;ITMS26240220040].