Neuroprotection And Cognitive Protection By Dth Through TLR4/NF-?B & MAPK Signaling Pathway Inhibiting Inflammatory Response

Objective The aim of the present study was to study the neuroprotective role and cognitive protection of DHT in neuroinflammatory conditions induced by LPS though inhibiting TLR4/ NF-?B/MAPK signaling pathways,including the functional mechanism.Methods Part?,To test that the effects of different concentrations of DHT and LPS on the activity of BV2 microglial cell lines and primary microglial cells by CCK8.The effect of DHT on the morphological changes of these cells induced by LPS was observed by a fluorescence microscope.Then the expression levels of iNOS and COX-2 proteins were measured by Western Blot;the NO,TNF-?,PGE2,and IL-1? levels in cell supernatants were tested by the ELISA kit;the m RNA levels of TNF-?,IL-1?,IL-6,iNOS,COX-2 were examined by RT-PCR.The concentration of these imflammatory factors were compared between DHT treated or not.In LPS-induced BV2 microglia and primary microglia,the effects of DHT on the activation of TLR4/NF-?B and TLR4/MAPK signaling pathways were detected by Western Blot,respectively.The TLR4 inhibitor CLI-095 was used to study whether DHT can inhibit the phosphorylation of NF-?B and MAPK by inhibiting the expression of TLR4.In SH-SY5 Y neurons,the effects of DHT,LPS and supernatants from LPS-treated microglia on the cell viability were detected by CCK8.Part ?,C57BL/6 mouse was chioced as neuroinflammation model by LPS-intraperitoneal injection.There are five groups in the animal experiment: control,castration,LPS,castration + LPS and castration + DHT + LPS.The morphology and number of microglia and astrocytes in the hippocampus and cortex were detected by immunohistochemistry.Western Blot was used to test hippocampal and cortical Iba-1 and GFAP protein levels.The levels of TNF-?,IL-1?,and IL-6 in LPS-injected mouse serum and brain tissue homogenates were measured by ELISA,and the m RNA levels of anti-inflammatory factors IL-4,IL-10,and IL-13 in brain tissue were detected by RT-PCR.Western Blot was used to detect hippocampal and cortical iNOS and COX-2 protein levels.In the hippocampus and cortex of LPS-injected mice,Western Blot was used to detect the expression and phosphorylation of TLR4-mediated NF-?B(p65)and MAPK(p38,ERK,JNK,AKT)pathways.The expression of A?,apoptosis-related proteins Bcl-2,Bax,Caspase-3 and the synaptophysin-related protein synaptophysin were examined by Western Blot.The morphology and number of neurons in the hippocampus and cortex were detected by Nissl staining.Part?,There are five groups in the this part including control,castration,LPS,castration + LPS and castration + DHT + LPS.Behavioral tests,including the open field test,the polet test and the morris water maze,were used to detect locomotor activity,the motor incoordination and cognitive ability in LPS-injected mice.Results Part?,By CCK8 assay,we found that 10 nM DHT and 100 ng/ml LPS did not affect the cell viability of BV2 microglia and primary microglia,but LPS can significantly induce morphological alteration.DHT significantly inhibited the expression of iNOS and COX-2,and the release of NO,PGE2,inhibited the m RNA levels of TNF-?,and IL-1? by RT-PCR tests.In LPS-treated BV2 microglia and primary microglia,Western Blot showed that DHT significantly inhibited the increase in TLR4,My D88 protein expression and the phosphorylation of NF-?B p65 and MAPK p38.By using the TLR4 inhibitor CLI-095,we found that DHT and CLI-095 have similar inhibitory effects.DHT-pretreated supernatant could significantly increase the cell viability of SH-SY5 Y cell.Part?,Western Blot test further found that the expression of GFAP and Iba-1 were increased in the hippocampus and cortex after LPS injected mouse brains.The concentrations of GFAP and Iba-1 in the castration+DHT+LPS group were significant lower than castration+LPS group.The level of proinflammatory cytokines including TNF-?,IL-1?,IL-6 in brain and serum were significantly increased in the LPS group,the castration+LPS group was significantly increased the level of these factors,but the expression of these inflammatory factors in castration+DHT+LPS group were lower than in castration+LPS group.In the hippocampus and cortex,the expression of TLR4,and the phosphorylation of p65,p38,ERK and JNK in castration+DHT+LPS group were lower than castration+LPS group.The concentrations of A? and caspase 3 in the hippocampus and the cortex in castration+LPS group were higher than in control,but those in castration+DHT+LPS group were lower than in castration+LPS group.Nissl staining further revealed that neuron damage in the hippocampus and the cortext was significantly lighten in castration+DHT+LPS group.Part ?,In open field test,the total distance were more longer in castration+DHT+LPS group than in castration +LPS group.In the pole test,the time to place their four paws on the floor were significantly shortened in castration+DHT+LPS group than in castration+LPS group.In the morris water maze,the time to find the platform,spent in the target quadrant and crossing numbers in castration+DHT+LPS group were significantly prolonger than in castration+LPS group.These results indicate that DHT can improve LPS-induced cognitive impairment.Conclusions According to these results above,we found that in LPS-induced neuroinflammation,DHT exerts the neuroprotective effect,and cognitive protection.The mechanisms of the neuroprotective effects may be inhibit inflammatory responses though the TLR4-mediated NF-?B and MAPK signaling pathways.