Key murine astrocytes stimulated with 1 ug ml LPS were co treated with compounds HAK 2 and HAK five at a concentration of 20 uM each for 24 h. The observed effect of compounds HAK 2 and HAK 5 on LPS stimulation was just like that of OSM induced IL 6 expression in human U343 glioma cells. In comparison to untreated samples LPS induced IL six expression was diminished by 60% in mouse and 50% in rat astrocytes by both HAK compounds. Therefore, suppression of the two, LPS and OSM induced IL six expression in numerous cell forms by structurally relevant compounds is a further indication for robust potency of HAK compounds to target neuroinflammatory processes. Potent inhibition of IL 6 upregulation by compound HAK two in vivo Based on the results obtained from main murine astrocytes, we reasoned that HAK compounds could also suppress elevated IL six expression in vivo.
There fore, bioactivity of your compound HAK two was analyzed selleckchem in the LPS induced mouse septic shock model. In pre paration of this in vivo research, selected HAK compounds have been characterized in detail regarding brain bioavail potential. It was demonstrated by quantitative evaluation of mouse plasma and brain samples working with LC MS MS the compounds are bioavailable and able to pass the blood brain barrier. For more in vivo investigations compound HAK two by using a logBB of 0. 22 was selected. Intraperitoneal injection of one mg kg LPS into C57 B6 mice resulted in an acute elevation of IL 6 concentra tion in plasma, hippocampus and cortex 2 h submit administration. The plasma level of IL six protein was substantially lowered by 55% in mice treated with five mg kg HAK two in parallel as when compared with LPS alone.
Similar results of HAK 2 treatment method were observed for induced IL 6 mRNA in hippocampus and cortex. These data clearly present the solid potency of HAK compounds to modify IL 6 expression in vivo. Effect of HAK compounds on OSM mediated phosphorylation of signal transducer and activator of transcription 3 and extracellular signal regulated kinase one The activation pifithrin �� with the OSM signaling cascade resulting in the stimulation of IL 6 expression is regarded to involve intracellular phosphorylation events. As a result, effects of HAK compounds within the OSM induced phos phorylation of signal transducer and activator of transcrip tion three and extracellular signal regulated kinase 1 were investigated.
Since HAK compounds were proven for being bioactive three to 6 h submit stimulation, U343 cells have been incubated with OSM for six h. In contrast to non sti mulated control, OSM induced phosphorylation of Erk1 as well as STAT3 six h publish stimulation. Interestingly, HAK compounds suppressed STAT3 phosphorylation at serine 727, but neither phosphorylation of pSTAT3Y705 nor pErk1 2T202 Y204. Compound HAK eight showed a substantially reduce result on pSTAT3S727 phosphorylation.