Establishment Of GC-MS Method For Determination Of H₂S In Brain Tissue And Study On The Protective Role And Mechanism Of Endothelial-derived H₂S In Rat Hippocampal Neurons Against Hypoxia/reoxygenation Injury

Ischemic brain injury is a common clinical disease with the highest disability rate and mortality rate in the world.Its pathogenesis is complex,and the clinical treatment effect is not ideal,which seriously affects people’s life and work.Hydrogen sulfide（H₂S）is considered to be the third endogenous gaseous signaling molecule discovered after carbon monoxide（CO）and nitric oxide（NO）.Scientific studies had revealed the presence of physiological concentrations of H₂S in brain tissue.Endogenous H₂S could be produced via l-cystathionine-γ-lyase（CSE）and l-cystathionine-β-synthetase（CBS）catalyzing the l-cysteine（L-Cys）.In addition,3-mercaptopyruvate sulfurtransferase（3-MST）synergizes with cysteine aminotransferase（CAT）to also produce H₂S.Like NO,H₂S is also a vascular endothelial-derived relaxing factor that acts as a signaling molecule in processes such as brain neuromodulation and vasodilation.Previous studies by the group have shown that H₂S released from cerebrovascular endothelium induced hyperpolarization and vasodilatory responses in cerebrovascular smooth muscle cells,promoted the opening of calcium-activated potassium channels in smooth muscle cells,and exerted protective effects against ischemic brain injury.However,it is unclear whether the endothelial-derived H₂S produced by cerebrovascular can penetrate the blood-brain barrier（BBB）and play a protective role on neurons damaged by hypoxia/reoxygenation（H/R）.It is of great significance to the pathological mechanism of ischemic brain injury.In the study of the neurobiological effects of H₂S,the detection of H₂S in brain tissue is very important.Gas chromatography-mass spectrometry（GC-MS）has high sensitivity and low detection limit,and is suitable for the determination of H₂S in biological samples.Therefore,this study intended to establish a GC-MS method to detect the concentration of H₂S in brain tissue,to analyze whether H₂S could pass through the BBB,and to explore the protective effect and mechanism of endothelial-derived H₂S on H/R injury of rat hippocampal neurons.Aims:1.To establish and optimize a method for the determination of H₂S in rat brain tissue by GC-MS,and to investigate whether H₂S can pass through the BBB on whole animals.2.In vitro BBB model to explore whether H₂S can pass through BBB.3.To investigate the protective effect and mechanism of endothelial-derived H₂S on H/R injury in rat hippocampal neurons.Methods:1.The agilent 7890B-7000D gas chromatograph triple quadrupole mass spectrometer with multiple reaction monitoring（MRM）were used to determine the parent ions（m/z394→181,m/z 181→161）of H₂S derivative bispentafluorobenzyl sulfide（Bis）for quantitative and qualitative analysis.The rats were injected with three doses of Na HS in the tail vein,and the brain tissues were taken for derivatization reaction after 0.5 h.The H₂S concentration was detected by GC-MS method.2.Mouse brain microvascular endothelial cells（b End.3 cells）and rat glioma cells（C6cells）were inoculated on both sides of the Transwell chamber to simulate BBB model in vitro.The function of the BBB model was examined by the leakage experiment and the permeability coefficient of sodium fluorescein.The permeability of H₂S on BBB model in vitro was measured by GC-MS.3.To establish an in vitro BBB model,rat hippocampal neurons were inoculated in a12-well plate matched with the Transwell system and grouped as:（1）Control;（2）H/R;（3）Na HS(200μmol·L^-1);（4）L-Cys(50μmol·L^-1);（5）L-Cys(100μmol·L^-1);（6）L-Cys(200μmol·L^-1);（7）CSE inhibitor PPG(100μmol·L^-1);（8）L-Cys(200μmol·L^-1)+PPG(100μmol·L^-1);（9）CBS inhibitor AOAA(1000μmol·L^-1);（10）L-Cys(200μmol·L^-1)+AOAA(1000μmol·L^-1);（11）L-Cys(200μmol·L^-1)+PPG(100μmol·L^-1)+AOAA(1000μmol·L^-1).Groups 3 to 11 all underwent H/R injury and the appropriate drugs were added during the injury.Lactate dehydrogenase（LDH）,neuron specific enolase（NSE）and cell counting kit-8（CCK-8）were used to detect cell injury and cell viability;the concentration of H₂S was detected by GC-MS;the concentration of free Ca²⁺in hippocampal neurons was detected by calcium fluorescence imaging.Results:1.The sodium sulfide（Na HS）in brain tissue samples had a good linear relationship in the range of 0.25～256μmol·L^-1,and the detection limit was 0.1μmol·L^-1.The intra-day and inter-day precision were both less than 15%.There was no obvious matrix effect and the recovery rate was more than 90%.The basal H₂S concentration in the rat cerebral cortex was measured to be（11.84±0.38）μmol·L^-1.After intravenous injection of Na HS,the H₂S concentration in the brain tissue increased significantly and in a dose-dependent manner.2.The leakage experiment showed that the liquid level difference of the blank group became 0 cm after 4 hours of incubation,while the BBB model group could maintain a liquid level difference of≥0.5 cm（liquid level difference float≤0.1cm）after 4 hours of incubation.The permeability coefficient of sodium fluorescein in the in vitro BBB model was significantly lower than that of the blank group,which was（7.89±1.15）×10^-6cm·s^-1.The result were consistent with that in the range of 2-19×10^-6 cm·s^-1 reported in the literature,indicating that the BBB model was successfully established in vitro.3.In the in vitro BBB model,H₂S concentration in the outer chamber（nerve cell side）medium increased significantly and dose-dependently with the increase of Na HS concentration in the inner chamber（endothelial cell side）,indicating that H₂S could penetrate from the endothelial cell side to the nerve cell side through BBB in vitro.In addition,H₂S concentration in the inner chamber（endothelial cell side）medium increased significantly and dose-dependently with the increase of Na HS concentration added to the outer chamber（nerve cell side）,suggesting that H₂S could penetrate from the nerve cell side to the endothelial cell side through BBB in vitro.4.After H/R injury,the leakage of LDH and NSE in the cell supernatant was significantly increased,and the nerve cell viability was significantly decreased（P<0.01vs Control）.Na HS(200μmol·L^-1),L-Cys medium and high doses(100,200μmol·L^-1)pretreatment could significantly reduce the the leakage of LDH and NSE,improved cell viability（P<0.05 or 0.01 vs H/R）.The CSE inhibitor PPG(100μmol·L^-1)and CBS inhibitor AOAA(1000μmol·L^-1)alone had no significant effect on the leakage of LDH and NSE,and the cell viability.However,L-Cys(200μmol·L^-1)reduced the leakage of LDH and NSE and improveed the viability of cell could be weakened by PPG and AOAA（P<0.05 or 0.01 vs L-Cys）.And the weakening effect of the combination of the two was significantly enhanced（P<0.05 or 0.01 vs L-Cys+PPG+AOAA）.5.After H/R injury,the H₂S concentration in the medium supernatant was significantly decreased,and the Ca²⁺fluorescence intensity in rat hippocampal neurons was significantly enhanced（P<0.01 vs Control）.Na HS(200μmol·L^-1)and L-Cys(200μmol·L^-1)pretreatment could significantly increase the H₂S concentration in the medium supernatant,and attenuate the Ca²⁺fluorescence intensity in rat hippocampal neurons（P<0.01 vs H/R）.The CSE inhibitor PPG(100μmol·L^-1)and the CBS inhibitor AOAA(1000μmol·L^-1)alone had no significant effects on H₂S concentration and neuronal Ca²⁺fluorescence intensity.But L-Cys(200μmol·L^-1)pretreatment increased H₂S concentration,the weakened Ca²⁺fluorescence intensity could be attenuated by PPG and AOAA（P<0.05 or 0.01 vs L-Cys）,and the attenuating effect of L-Cys was significantly aggravated by the combination of PPG and AOAA（P<0.05 vs L-Cys+PPG+AOAA）.Conclusions:1.The GC-MS method established in this paper is a reliable and effective method for the detection of H₂S in brain tissue.2.H₂S could enter the brain tissue through the BBB.3.CBS in hippocampal neurons could produce H₂S to protect against self-H/R injury,and H₂S produced by CSE in endothelial cells could also protect against hippocampal neuronal H/R injury through BBB,and the mechanism may be related to the inhibition of Ca²⁺in hippocampal neurons by H₂S.