Caffeic Acid Ester Fraction from Erigeron Breviscapus Inhibits Microglial Activation and Provides Neuroprotection

objective： To investigate the effects of caffeic acid ester fraction （Caf） from Erigeron breviscapus, mainly composed of dicaffeoylquinic acids （diCQAs）, on microglial activation in vitro and focal cerebral ischemia in vivo. Methods： The production of nitric oxide （NO）, tumor necrosis factor α （TNF-α）, and interleukin-1β （IL-1 β ） induced by lipopolysaccharide （LPS） treatment in rat primary cultured microglia were measured by Griess reaction or enzyme-linked immunosorbent assay. Cell viability of cortical neurons was measured using AlamarBlue reagent. The behavioral tests and the infarct area of brain were used to evaluate the damage to central nervous system in rat middle cerebral artery occlusion （MCAO） model of cerebral ischemia. Real time polymerase chain reaction was used to determine the expression of inducible nitric oxide synthase （iNOS）, TNF-α and IL-1 β mRNA in ischemic cerebral tissues. Results： Caf inhibited the production of NO, TNF-α and IL-1β induced by LPS treatment in primary microglia in a dose-dependent manner. Exposure of cortical neurons to conditioned medium from Caf-treated microglia increased neuronal cell viability （P〈0.01） compared with conditioned medium from LPS-treated alone. In MCAO rat model of cerebral ischemia, Caf could significantly improve neurobehavioural performance and reduce pementage infarct volume compared with the vehicle group （P〈0.05）. Caf could also significantly inhibit the up-regulation of iNOS, TNF-α, and IL-1 β gene expressions in ischemic cerebral tissues. Conclusion： Car could suppress microglial activation, which may be one mechanism of its neuroprotective effect against ischemia.

Inhibition of temperature-sensitive TRPV3 channel by two natural isochlorogenic acid isomers for alleviation of dermatitis and chronic pruritus

Genetic gain-of-function mutations of warm temperature-sensitive transient receptor potential vanilloid 3(TRPV3)channel cause Olmsted syndrome characterized by severe itching and keratoderma,indicating that pharmacological inhibition of TRPV3 may hold promise for therapy of chronic pruritus and skin diseases.However,currently available TRPV3 tool inhibitors are either nonselective or less potent,thus impeding the validation of TRPV3 as therapeutic target.Using whole-cell patch-clamp and single-channel recordings,we report the identification of two natural dicaffeoylquinic acid isomers isochlorogenic acid A(IAA)and isochlorogenic acid B(IAB)that selectively inhibit TRPV3 currents with IC50 values of 2.7±1.3 and 0.9±0.3μmol/L,respectively,and reduce the channel open probability to 3.7±1.2%and 3.2±1.1%from 26.9±5.5%,respectively.In vivo evaluation confirms that both IAA and IAB significantly reverse the ear swelling of dermatitis and chronic pruritus.Furthermore,the isomer IAB is able to rescue the keratinocyte death induced by TRPV3 agonist carvacrol.Molecular docking combined with site-directed mutations reveals two residues T636 and F666 critical for the binding of the two isomers.Taken together,our identification of isochlorogenic acids A and B that act as specific TRPV3 channel inhibitors and gating modifiers not only provides an essential pharmacological tool for further investigation of the channel pharmacology and pathology,but also holds developmental potential for treatment of dermatitis and chronic pruritus.