| Object:Hydrogen Sulfide (H2S) is a type of gas which is characterized by its smell of "degenerated chicken egg" as well as healthy impairment by long-term contact with it. H2S acts not only toxically, as a abundant articles represented to date, but also plays a role normally in a range of mechanisms in physiologically and pathologically. Moreover, it is recognized as a signal molecule of "third-type gas" following NO (nitric oxide) and CO (carbon monoxide). Normally organisms synthesize their H2S endogenously associated with cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE) activities, which are two key enzymes during its synthesis. CBSs are localized majorly in central nerve system, whereas CSEs are distributed along peripheral nerve system in contrast. Latest researches report that H2S functions as neutral factor (antagonist) against impairments of brain, heart, liver, kidney and the like, from the ischemia-reperfusion injury, through the mechanism of anti-oxidation, restraint of apoptosis, etc. Interestingly, the inflammation plays a key role in evolution of brain ischemia-reperfusion injury. This study is trying to unveil the effects and mechanisms of NaHS as a donor as well as HA (hydroxylamine) and PAG (polyacetylenic glycine) as inhibitors of H2S synthase, with a variety of does, against the impairment of pyramidal neurons residing in CA1 region of hippocampus and the inflammation in brain caused by ischemia-reperfusion (IR) injury. The investigation was exercised on VD model rats by abdominal injecting a range of doses of NaHS and HA and PAG. Meanwhile, the study also attempts to explore the possible mechanism of inflammation response in brain tissue after injury. The experiment was processed by (Ⅰ) HE (hematoxylin & Eosin) staining of sections from rat hippocampal CA1 region for diagnosis of pathological alternation of pyramidal neurons; (Ⅱ) Immunohistochemistry (IHC) and Western Blot (WB) for verification of changes of CBS localization and expression after suffering brain IR injury; (III) ELISA for exploring the roles of inhibitors of H2S synthase mediating the of inflammation of brain caused by IR injury. It is also to justify our hypothesis that H2S plays an important role against the injury of brain from ischemia-reperfusion via the mechanism of anti-inflammatory reaction. Thereafter, this study will provide the evidences towards the function of H2S preventing brain tissue from injury after IR theoretically and experimentally.Methods:The healthy, purebred Wistar rats were selected and body-weights were limited around 310g. After random selection, animals were classified into Normal, Sham, IR and IR+NaHS (H2S therapeutic) groups. Furthermore, IR+NaHS were grouped into 6 subgroups specified by injection of a titer of dose, i.e.,2mg/kg,5mg/kg, 10mg/kg,20mg/kg, as well as IR-NAHS+HA and IR+NaHS+PAG, the latter two act as inhibition groups. Five animals were distributed into each groups or subgroups.The experimental VD rat model was built up by treating rats with 4-VO (4-vessel obstruction) operations, e.g., obstructing both vertebral arteries and both cervical arteries respectively by electric congealment. Of cause sham rats were not applied for the vessel obstruction but they were employed the anesthesia similar to VD animals. The brains tissue of all of rats were dissected, sectioned, and stained by HE, afterward, were applied a microscopy on CA1 regions of hippocampus. Additionally, IHC was used to localizing CBS proteins in hippocampus; ELISA was resorted for quantity detection of both IL-1β(Interleukin-1β) and TNF-a (Tumor Necrosis factor-a) in homogenates of hippocampus; WB was excised lastly for testifying CBS expression.Results:1 Morphology:Pyramidal neurons in CA1 region of hippocampus of normal and sham animals characterize the regular rows, dense perikaryons, integrated shape, full nuclei, and clear nucleoli, as well as demonstrate no injury. In contrast, those in equivalent region of IR group rats show impressive traits of cell injury, including pyramidal neurons rowing sparsely and disorderly, fading away their nuclei. Subsequent comparison of IR with H2S therapeutic groups, 10mg/kg group increases the counts of cell numbers of pyramidal neurons associated with irregular rows of cells; 20mg/kg group manifests equivalent traits to normal and sham with survival neurons. Dissimilarly, the pyramidal neurons in groups of IR+NaHS+HA and IR+NaHS+PAG are apparently and severely destroyed or impaired by IR, compared with subgroup of 20mg/kg of H2S therapeutic rats.2 The expression of protein CBS:IHC, CBS-specific particles in CA1 of normal and sham rats are characterized by light brown-yellow and equal distributed in brain. On the contrary, they are declined apparently in IR rats, even null in certain neurons in some layers and their surrounding areas. The density of CBS-specific particles in low-dose H2S groups, i.e.,20mg/kg shows no big different compared with those in IR. The particles illustrate escalating gradually corresponding to the dose of NaHS uptake. Considerably, CBSs distribute abundantly in CA1 region after treatment with high dose of H2S, i.e.,20mg/kg and a great deal of CBS with dark staining is localized in perikaryon of the pyramidal neurons.On the other hand, CBS particles in the groups of IR+NaHS+HA and IR+NaHS+PAG reduce obviously on the contrary to high-dose H2S group (20mg/kg).3 Westen blot:There is detectable quantity of CBS proteins in CA1 region of hippocampus from normal and sham groups. CBS proteins presenting in IR groups whereas are expressed very low, compared with sham group. Although low-dose H2S therapy does not help animals expression more CBS after IR injury in brain, high-dose H2S therapy provoke animals expressing significantly more CBS that IR rats do. Interestingly, CBS expression has no rising afterwards injection of HA and PAG.4 ELISA:IL-1β and TNF-a are fairly detected by ELISA in CA1 homogenates from normal and sham. The animals, unlike normal and sham, recruit more IL-1β and TNF-a after IR. Nevertheless, the both cytokines are obviously inhibited after H2S uptake therapeutically featured by the peaks of both concentrations falling-off considerable, especially the high-dose H2S group (20mg/kg). IL-1β and TNF-a in IR+NaHS+HA and IR+NaHS+PAG to the contrast to H2S therapeutic group (20mg/kg), soar up.Conclusion:Accepted certain dose of H2S prior to brain ischemia will effectively reduce the extent of injury of pyramidal neurons in CA1 region of hippocampus. More given dose H2S will exercise more effective on protection from injury. Inhibitors of CBS and CSE, i.e., HA and PAG inhibit the protection roles of H2S. It is likely one of the mechanisms of H2S protecting organs from IR injury through the process of HA and PAG inhibiting H2S to promote expression of CBS and to decline expression of inflammatory factors of IL-1β and TNF-a. It is able to testify the function of H2S preventing brain from secondary injury by ischemia-reperfusion (IR). |
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