Hydrogen Sulfide Protects Nerve Cells By Inhibiting ATP-induced Microglial Activation

BackgroundATP（Adenosine triphosphate,ATP）is not only the energy material for storaging in vivo,and also a bio-active substance which is a neurotransmitters between cells for transmission information.A large amounts of ATP was released from damaged cells when ischemia and hypoxia,inflammation injury,reperfusion injury,and so on.Extracellular ATP at high concentrations（mmol / L level）may selectively act on purine receptors,in particular P2X₇ receptor,and induce immune cells activation by regulating the signaling molecular such p38,ERK,JNK,PKC,NF-кB etc.Immune cells which were activated release of some inflammatory mediators.Those inflammatory mediators in turn activate more immune cells,and then secondary death of surrounding neurons are eventually happened.A large number of in vitro and in vivo experiments confirmed that a certain range of concentrations of H₂S has a protective effect on cells with hypoxic-ischemic and chemical injury.This viewpoint have been proved in the nervous system and cardiovascular system.The role of ATP-P2 X purine signaling pathway in the neuroprotection of H₂S is still rarely reported.The intracellular signaling mechanism post P2 X receptor is still unknown.In this study,our experiment was to study the effect of H₂S on microglial activation by ATP and to explore the role of intracellular MAPK signaling pathway,then try to find key targets of neuroprotective effect of H₂S.ObjectivesTo observe the effect of H₂S on the cell vialility of microglia by ATP-induced,release of inflammatory cytokines,MAPK protein expression,conditioned medium forSH-SY5 Ycell,and then to explore the neuroprotective effect and mechanism of H₂S on rat microglia cell by ATP-induced with high concentration。Methods1 The effect of NaHS on the rat microglia vitality induced by ATP1.1 The effect of ATP on the rat microglia vitality The Rat microglia were cultured in high glucose DMEM medium containing 10% fetal bovine serum.Well-differentiated cells with a density of 1x105/ L in the logarithmic phase were inoculated in 96-well culture plates,then were cultured in 37 ℃,5% CO2 culture box for 24 hours.They were randomly divided into six groups:（1）control group: the cells were cultured routinely,not any drug treatment.（2）ATP group:the cells treated with 0.3mmol / L,1 mmol / L,3 mmol / L,5 mmol / L,7 mmol / L ATP respectively.Different concentrations of ATP induced microglia for 6 hours.The cell viability was assayed by MTT assay.We considered the cell viability of control group as 100%.1.2 The effect of NaHS on the rat microglia vitality Cells processing method was as same as 1.1,then six groups were randomly divided into control group,ATP group,NaHS（50,100,200,800μmol / L）+ ATP group.Cells were preincubated with different concentrations of NaHS for 30 minutes,followed by treatment with 3mmol / L ATP for 6 hours.NaHS was always in the reaction system.The cell viability was assayed by MTT assay.We considered the cell viability of control group as100%.1.3 The effect of NaHS and KN-62 on the rat microglia vitalityCells processing method was as same as 1.1.Then they were randomly divided into control group,ATP group,KN-62（P2X₇ receptor antagonists）+ ATP group,NaHS + ATP group.Among this experiment,ATP was 3mmol / L,KN-62 was 500 nmmol / L,NaHS was200μmol / L.KN-62 or NaHS pretreated for 30 minutes,then added ATP for 6 hours,and NaHS and KN-62 was always in the reaction system.The cell viability was assayed by MTT assay.We considered the cell viability of control group as 100%.2 The effect of NaHS on TNF-α,IL-6 release from activated rat microglia induced by ATPRat microglia cells in the logarithmic phase and well-differentiated inoculating in35 mm petri dishes were randomly divided into four groups: control group,ATP group,KN-62 + ATP group,NaHS + ATP group.ATP was 3mmol / L,KN-62 was 500 nmmol / L and NaHS was 200μmol / L.Cells culture supernatants were collected and centrifuged when rat miacroglia cells were stimulated with ATP for 12 hours after pretreated with NaHS for 30 minutes.The change of TNF-α,IL-6 release were measured using a commercially available ELISA kits according to the manufacturer’s instruction.3 The effect of conditioned medium（CM）which treated with ATP or NaHS on neuronal-like SH-SY5 Y cellsMicroglia cells were pre-incubated with KN-62 or NaHS and then treated with ATP.After 12 hours,supernatant fractions were collected,referred to as conditioned medium（CM）.To assess neuronal death by factors released by rat microglial cells following ATP insult,SH-SY5 Y cells were seeded in 35 mm dishes or 96-well culture plates.Cells of dishes were randomly divided into four groups:（1）control group:after rat microglial cells confluented for 12 hours,supernatant fractions were collected,filtered and added onto SH-SY5 Y cells,then cultured.（2）ATP Group: microglia cells treated with ATP for 12 hours,then ATP conditioned medium continued to culture SH-SY5 Y cells.（3）KN-62 + ATP group: conditioned medium pre-incubated with KN-62 for 30 minutes,more than with ATP group,and KN-62 was always present in the reaction system.（4）NaHS + ATP group:conditioned medium pre-incubated with KN-62 for 30 minutes,more than with KN-62 +ATP group.ATP was 5mmol / L,KN-62 was 500 nmmol / L and NaHS was 200μmol /L.Invert microscope observed the change of morphology and adherent cells at different time gradient.SH-SY5 Y cells of 96-well plates were randomly divided into four groups,control group,ATP group,KN-62 + ATP group,NaHS + ATP group.The processing of different drugs treatment was the same as 3.1.To detect the cell viability by MTT,and regarded the control group cell viability as 100%.4 The effect of NaHS on MAPK protein espression in ATP-induced rat microgliaRat microglia cells in the logarithmic phase inoculating in 35 mm petri dished were randomly divided into four groups: control group,ATP（3mmol / L）group,KN-62+ATP group,NaHS+ATP group.After all drugs treated,the cells were collected.Intracellular ERK,p-ERK,P38,p-P38,JNK,and p-JNK protein were analyzed by Western blotting.Results1 The effect of NaHS on the rat microglia vitality induced by ATP1.1 The effect of ATP with different concentration on the vitality of rat microgliaConsidering the cell vitality of control group as 100%,the vitality of 0.3mmol / L ATP were 97.06±1.96%.There were no significant difference in vitality between control group and 0.3mmol / L ATP（P> 0.05）.The vitality of 1mmol / L,3mmol / L,5mmol / L,7mmol /L ATP group were respectively 87.32±2.96%,82.66±3.01%,77.95±4.99% and 73.27±2.32%.They were lower than the control group and had significant difference（P<0.01）.These results showed that ATP presented concentration dependence effect on cell damage.Thenwhile,1mmol / L ATP already showed the feature.1.2 The effect of NaHS with different concentration on the vitality of rat microglia Cells were treated with 3mmol / L ATP for 6 hours after pretreated with 50μmol / L,100μmol / L,200μmol / L,800μmol / L NaHS for 30 minutes.The vitality of 3mmol / L ATP group was 82.66 ± 3.01% considering the cell vitality of control group as 100%.While the vitality of 50μmol / L,100μmol / L,200μmol / L,800μmol / L NaHS were respectively 85.71 ± 2.97%,91.49 ± 4.97%,94.54 ± 5.12%,69.64 ± 6.02%,of which200μmol / L NaHS vitality significantly higher than ATP group（P <0.01）.It indicated that the protection of NaHS on the rat microglia damaged by ATP was in a dose-dependant manner.The optimal concentration of NaHS was 200μmol / L（P <0.01）.At the same time,we could find that high concentration（800μmol / L）of H₂S had toxic effects on cells（P <0.01）.1.3 The effect of cell vitality with NaHS or KN-62 on rat microgliaRat microglia cell divided four groups.One was given 200μmol / L NaHS,another was given 500 nmol / L KN-62,meanwhile pre-incubated for 30 minutes.Added 3mmol / L ATP for 6 hours.The cell viability of control group was 100%.ATP alone group was 82.66±3.01% and lower than the control group（P <0.01）.On the contrary,NaHS + ATP,KN-62 +ATP,respectively were 94.54 ± 3.96%,93.68 ± 2.98%,significantly higher than the ATP group（P <0.05）.These results suggest that NaHS had a similar effect with KN-62.They both could decrese cell death by ATP and improve the cell vitality of rat microglia.It indicated that P2X₇ receptor may be a common site of action.2 The effect of NaHS on TNF-α and IL-6 release from activated rat microglia induced by ATPWe used ELISA assay to detect levels of supernatant TNF-α,IL-6.They reflected the activation of rat microglia.The results showed,after ATP-induced for 24 hours extracellular the release of TNF-α,IL-6 respectively increased 114.68±2.00%（P <0.01）and 76.11 ± 1.97%（P <0.01）compared with control group.Conversely,P2X₇ R specific inhibitor KN-62 pre-incubated for 30 min,then treated with ATP,the release of flammatory factors decreased 34.32±3.01%（P <0.05）and 21.44±2.95%（P <0.05）compared with ATP group.We obtained the same result,NaHS pretreatment may also reduce the effect of activated rat microglia,and manifested that release of TNF-α and IL-6 reduced by 36.00±4.03%（P <0.05）and 17.89±3.99%（P <0.05）.It showed that KN-62 blocked the release of pro-inflammatory cytokines induced by ATP,which suggest that P2X₇ R played an important role in the release of proinflammatory mediators.However,NaHS also could reduce them induced by ATP.This reconfirmed that P2X₇ R could be the target of NaHS in the neuroprotection.3 The effect of conditioned medium（CM）on the SH-SY5 Y cells3.1 The effect of CM on the morphology of SH-SY5 Y cellsRat microglia treated with ATP 、 NaHS or KN-62 mustbe release some pro-inflammatory cytokines,collect broth contain pro-inflammatory cytokines to treat SH-SY5 Y cells.Under an inverted microscope,we observed that normal SH-SY5 Y cells are fusiform,triangular,polygon and three-dimension.There are more particles in the cytoplasm.Cells have apophysis and adhere well.After ATP（5mmol / L）CM treated and with time going,apophysis began to become shorter,cell body became round,volume became shrinkage,and the number of wall off increased.It suggest that the cytotoxious effect of ATP and microglia maybe release cytotoxic factors.But NaHS（200μmol / L）CM could reduce the damage and decrease the number of cells detached.The results suggest that NaHS reduced the release of cytotoxic factors and microglia activation was the key link on neuroprotective effect of NaHS.3.2 The effect of cell vitality on SH-SY5 Y cell induced by CMThe results of SH-SY5 Y cell vitality showed that pure ATP group was 50.41±2.31%and significantly lower（P <0.01）compared with control group which considered as100%.But cell vitality of KN-62+ATP and NaHS+ATP group respectively were 63.01±3.02%,65.48 ± 4.06%.Both of them were higher than ATP group,with statistical significance（P <0.05）.These results suggest that NaHS and KN-62 had similar neuron protective effect,both of them could reduce the SH-SY5 Y cell damage induced by ATP conditioned media.4 The effect of NaHS on MAPK protein expression in ATP-induced rat microgliaThe results of western blotting showed that the expression of p-P38 and p-JNK were significantly increased after 3mmol / L ATP for 1 hours.Compared with the control group increased by 168.05±3.00%（P <0.01）,148.40±2.03%（P <0.01）,but p-ERK1 / 2 did not change significantly（P> 0.05）.With ATP treated for 1 hours after KN-62 incubated for30 min,p-P38,p-JNK protein expression were decreased 54.04±4.87%（P <0.01）,24.69±2.89%（P <0.05）compared with ATP group.Similarly with KN-62,p-P38,p-JNK protein of NaHS+ATP group decreased by 57.46±1.95%（P <0.01）,31.84±2.92%（P <0.05）.In accordance with ATP conditioned medium,they also did not cause the change of p-ERK1 /2 protein（P> 0.05）.These results further demonstrated both NaHS and KN-62 could reduced phosphorylated proteins expression on rat microglia induced by ATP,eapecially p-P38 and p-JNK.It might be to act P2X₇ R and then active MAPK signaling pathway.Mainly the processing associated intracellular p38 and JNK MAPK signaling pathway,whereas little to do with ERK1 / 2.Conclusion（1）High concentrations of extracellular ATP acting on P2X₇ R and activating P38 and JNK MAPK signaling pathway induce microglia activation.（2）After the rat microglia activation,the release of TNF-α,IL-6 increasing,decrease SH-SY5 Y neuronal-like cell viatity.（3）H2S plays a neuroprotective effect via regulating ATP-P2X₇R-MAPK pathway and inhibiting of microglial activation.