1、The Roles Of H₂S In Arterial Oxygen Saturation And Hemoglobin-Oxygen Binding Affinity In Erythrocytes 2、The Function Of CBS In Intrauterine Growth Retardation

The article contains two parts.One is exploring the roles of H₂S in regulating SaO₂and hemoglobin-O2 affinity in erythrocyte.The other one is focused on the effect of H₂S-producing enzyme cystathionine beta-synthase（CBS）in placenta on intrauterine growth restriction（IUGR）.First partH₂S is one of the three endogenous gaseous transmitters,and involved in multiple physiological and pathological processes.Endogenous H₂S plays an important role during hypoxia.Very recent studies have demonstrated that H₂S mediates O2 sensing in the carotid body response to hypoxia and protectes apoptosis and tissue damage induced by hypoxia.The H₂S-induced posttranslational S-sulfhydration modification has been confirmed to regulate the functions of many proteins and mediates the roles of H₂S in physiological and pathological processes.Recent studies have demonstrated that the plasma level of H₂S was decreased during hypoxia.In this study,we also found the circulatory H₂S was reduced by hypoxia,and GYY4137 could rescue tissue hypoxia and decreased SaO₂（arterial oxygen saturation）induced by 10%O₂.Interestingly,tissue hypoxia and reduced SaO₂ are shown in Cse^-/-mice and rescued by GYY4137.Furtherly,we demonstrate that H₂S could maintain the gas exchange in the lungs and protected lung damage induced by hypoxia.In the previous study,we demonstrated that 2,3-BPG level in erythrocytes of Cse^-/-mice was elevated,meanwhile GYY4137 treatment significantly reduced 2,3-BPG.In addition,treatment of isolated mouse and human erythrocytes with H₂S donor GYY4137significantly decreased 2,3-BPG concentration in the cells.In this study we found H₂S could reduce the production of 2,3-BPG induced by hypoxia in WT mice and insolated human erythrocytes,which was associated with the results in normoxia.Therefore,we furtherly explored the underlying mechanism of H₂S regulating 2,3-BPG in the erythrocytes.We revealed that endogenous H₂S mediated S-sulfhydration of Hb and suppressed the translocalization of Hb to membrane and thus maintained the BPGM largely anchoring to membrane.Hypoxia-induced the reduction of circulatory H₂S contributes decreased S-sulfhydration of Hb.Meanwhile,BPGM was released from membrane to cytosol,and thus more 2,3 BPG was produced.Result:1.H₂S rescued the tissue hypoxia（1）Serum H₂S were significantly decreased in mice suffering from hypoxia.（2）H₂S rescued the hypoxia in WT mice induced by 10%O₂ conditionHypoxia-induced elevation of Hypoxyprobe signals in tissues and decreased SaO₂were reversed by GYY4137.2.H₂S rescued hypoxia induced by Cse knockoutCse-deficiency induced elevation of Hypoxyprobe signals and reduced SaO₂,which were reversed by GYY4137.3.H₂S reduction induced the damage in the lung and decreased SaO2（1）No correlation between decreased SaO₂ in Cse^-/-mouse and blood perfusion in lung（2）GYY4137 could not rescued the SaO₂ through increased pulmonary blood perfusionPinacidil treatment increased the perfusion in lung but had no effect on the SaO₂ and tissue hypoxia.（3）No correlation between decreased SaO₂ in Cse^-/-mouse and pulmonary ventilation（4）H₂S rescued the damage and inflammation in lung of Cse^-/-mice（5）The damage and inflammation in lung induced by hypoxia was reversed by GYY4137 treatment.4.H₂S regulated the levels of 2,3-BPG in RBC（1）H₂S regulated the levels of 2,3-BPG and P50（A）The hypoxia-induced elevation of erythrocyte 2,3-BPG and P50 were reversed by GYY4137 treatment.（B）Using cultured human erythrocytes,we found that GYY4137treatment reduced 2,3-BPG induced by hypoxia.（2）H₂S from peripheral tissues but not erythrocyte regulated the 2,3-BPG production in RBC.5.H₂S suppresses BPGM release from membrane to cytosol（1）H₂S regulates the activity of BPGM（A）Cse^-/-mice showed an increase in cytosolic BPGM activity in erythrocytes compared with WT mice;GYY4137 treatment decreased cytosolic BPGM activity in Cse^-/-mice.Similar effect of GYY4137 on erythrocyte BPGM activity occurred in WT mice.（B）Using cultured mouse and human erythrocytes,we found that GYY4137 treatment inhibited cytosolic BPGM activity.（2）H₂S suppresses BPGM release from membrane to cytosol（A）Western blotting analysis revealed that membrane BPGM levels were significantly decreased while cytosolic BPGM levels were significantly increased in Cse^-/-mice compared with those in WT mice.GYY4137 treatment reduced cytosolic whereas increased membrane BPGM levels.（B）Confocal microscopy showed that BPGM was mainly located on serum membrane in erythrocytes of WT mice.In Cse^-/-mice,there was more BPGM in cytosol of erythrocytes.GYY4137 reduced cytosolic BPGM in erythrocytes of Cse^-/-mice.（C）Western blotting analysis revealed that GYY4137 treatment increased membrane BPGM level whilst decreased cytosolic BPGM level in cultured human and mouse erythrocytes.（D）Confocal microscopy showed that GYY4137 reduced cytosolic BPGM in cultured human and mouse erythrocytes.6.BPGM interacts with band 4.2We performed mass spectrometry combined with Co-IP to identify the interaction of erythrocyte membrane-anchored protein band 4.2 with BPGM.7.H₂S decreases Hb anchoring to the membrane thereby reducing BPGM release from membrane（1）H₂S decreases Hb anchoring to the membrane（A）Membrane heme content was significantly increased in the erythrocytes of Cse^-/-mice compared with WT erythrocytes.In contrast,GYY4137 decreased membrane heme content in erythrocytes of Cse^-/-and WT mice.（B）Accordingly,in cultured mouse and human erythrocytes,GYY4137 treatment significantly decreased membrane heme content.（2）band 4.2 mediated the association of Hb and BPGMCo-IP analysis showed that the association of band 4.2 with Hb was increased whereas the association of band 4.2 with BPGM was decreased in erythrocytes of Cse^-/-mice compared with WT erythrocytes.GYY4137 treatment decreased the association of band 4.2 with Hb whilst increased the association of band 4.2 with BPGM in erythrocytes of Cse^-/-mice.（3）H₂S regulated the association of Hb and BPGM in erythrocyte ghost membraneWe prepared human and mouse erythrocyte ghost membranes and inversely coated on the silicon beads to expose inside out.Then,we incubated silicon beads coated with inverted erythrocyte ghost membranes with Hb in the absence or presence of GYY4137.We found that GYY4137 reduced heme anchored to membrane and BPGM activity in supernatant.8.H₂S decreased Hb anchoring to the membrane via S-sulfhydration of Hb,thereby reducing BPGM release from membrane（1）Idoacetamine reversed GYY4137-induced changes in erythrocyte ghost membraneIdoacetamine reversed GYY4137-induced changes in membrane anchored heme and supernatant BPGM activity in erythrocyte ghost membrane.（2）Mass spectrometric analysiswe intended to identify the proteins which were sulfhydrated by H₂S in erythrocytes by using maleimide assay followed by mass spectrometric analysis.We identified that Hb was the most abundant protein among S-sulfhydrated proteins in mouse RBCs.（3）H₂S regulated the S-sulfhydration of HbUsing biotin-switch technique,（A）we confirmed that profound S-sulfhydration of Hb was exhibited in erythrocytes of WT mice,whereas less S-sulfhydration of Hb occurred in CSE^-/-mice.GYY4137 increased S-sulfhydration of Hb in WT and CSE^-/-mice.（B）No obvious S-sulfhydration of BPGM was found in RBCs of WT and Cse^-/-mice with GYY4137 treatment.（4）The site of S-sulfhydration in Hb was identified9.Downregulation of H₂S contributed to decreased S-sulfhydration of Hb during hypoxia（1）H₂S rescued the translocation of Hb and BPGM induced by hypoxia（A）Membrane heme content was significantly increased in the erythrocytes of mice exposed to hypoxia compared with control erythrocytes.GYY4137 decreased membrane heme content in RBCs obtained from mice exposed to hypoxia.（B）cytosolic BPGM activity in erythrocytes was significantly increased upon hypoxia insult,which was reversed by GYY4137 treatment.（C）Membrane BPGM levels were significantly decreased whilst cytosolic BPGM levels were significantly increased in mice exposed to hypoxia compared with control mice.GYY4137 treatment reduced cytosolic whereas increased membrane BPGM levels.（2）H₂S rescued the association of Hb and BPGM induced by hypoxia in erythrocyte ghost membrane（A）Membrane heme content and cytosolic BPGM activity was significantly increased induced by hypoxia in erythrocyte ghost membrane.（B）GYY4137 treatment reduced membrane heme content and cytosolic BPGM activity in erythrocyte ghost membrane.（3）Hb sulfhydration was decreased upon hypoxiaThe level of Hb sulfhydration was decreased upon hypoxia insult,which was reversed by GYY4137 treatmentConclusionIn normoxia,endogenous H₂S maintain the lung gas exchange and the levels of2,3-BPG in erythrocyte.In hypoxia reduced endogenous H₂S induces the damage in lung gas exchange and decreases SaO₂.Whilst decreased H₂S increases the anchoring of Hb in serum membrane and release of BPGM into cytoplasm in RBCs and promotes the production of 2,3-BPG and O₂ release in peripheral tissues.Second partIUGR is a common pregnancy complication,affecting around 3–8%of pregnancies worldwide.IUGR is a failure of a fetus attaining the full genetic growth potential during pregnancy,causing the insufficient growth and developmental anomaly of fetus.Based on currently available information,dysfunction of placenta is the major cause of IUGR.The placenta is the only extra-embryonic organ,which provides sufficient amounts of oxygen and nutrients for fetus,produces kinds of hormones for pregnancy and induces an immune-privileged environment.The dysfunction of placenta is involved in developmental anomaly of placental vessels,abnormal placental perfusion,reduced placental exchange surface and exchange of nutrients.H₂S in the third gaseous transmitters following nitric oxide（NO）,carbon monoxide（CO）.It has been demonstrated that H₂S plays an important role in regulating physiological and pathological processes such as vasodilatation,inflammation,angiogenesis,apoptosis and oxidative stress.The levels of H₂S in pregnancy is essential for normal fetal growth and development during pregnancy.In our previous study,we demonstrated that H₂S producing enzyme CSE,CBS but not 3-MST is reduced in placenta of women with preeclampsia.To explore the role of H₂S and H₂S producing enzyme in the structure and function of placenta during pregnancy,a genetically engineered pregnant mice with decreased CBS expression in placentas was used.We found that the pregnant mice with decreased CBS showed no preeclampsia complications but IUGR which was rescued by H₂S donor GYY4137.These results demonstrate that reduced H₂S in maternal circulation induced by CBS decrease in placenta is essential for placental structure and function and fetal growth during pregnancy.Results:1.Placental CBS knockdown induced IUGR（1）The pregnant outcome of mice with placenta CBS knockdownA genetically engineered pregnant mouse with decreased CBS expression in placenta was generated and the pregnant outcome was observed.（A）Placenta CBS knockdown had no effect on the gestation period.（B）Compared with negative group(Cbs^f/f♂×Cbs^f/f♀),placenta CBS knockdown significantly decreased fetal and palcental weight measured on term.（C）Placenta CBS knockdown significantly increases the mortality at weaning（3weeks age）.（2）None PE complications was showed in pregnant mice with decreased placental CBSCompared with negative group(Cbs^f/f♂×Cbs^f/f♀),No changes were observed in the ratio of protein/creatinine in urine of pregnancy mice,mean arterial blood pressure（MAP）,morphology of glomeruli and the circulatory levels of sFlt1,sEng,VEGF and PIGF.（3）Placenta CBS knockdown induced IUGRCompared with negative group(Cbs^f/f♂×Cbs^f/f♀),placenta CBS knockdown significantly decreased fetal weight,placental weight and the ratio of fetus/placenta on GD18.5.（4）Placenta CBS knockdown induced IUGR was rescued by GYY4137Compared with negative group(Cbs^f/f♂×Cbs^f/f♀),the level of H₂S was reduced in placenta CBS knockdown mating group.Decreased fetal weight,placental weight and the ratio of fetus/placenta was rescued by GYY4137.2.CBS knockdown damaged the structure of placenta（1）CBS knockdown damaged the structure of placentaCompared with negative placenta(Cbs^f/f/f from Cbs^f/f♂×Cbs^f/f♀),the labyrinth was decreased and the ratio of labyrinth/junctional zone was decreased in the placenta with CBS knockdown.Histological analysis of placentas with H&E and laminin staining revealed that CBS knockdown placentas showed disorganized and impaired vasculature in the labyrinthine zone compared with Cbs^f/f placentas.PAS staining showed the GlyTCs was increased in junctional zone and decidua of CBS decreased placenta.（2）CBS knockdown induced placental damage was rescued by GYY41373.CBS knockdown damaged the hemodynamic of uterine placenta（1）CBS knockdown damaged the uteroplacental hemodynamicDoppler waveform recordings were used to compare the uteroplacental hemodynamic and waveform parameters.Compared with the negative group(Cbs^f/f♂×Cbs^f/f♀),significant changes in umbilical artery,canal and spiral artery blood flow velocity and uterinearterywaveformparametersweremeasuredin Cbs^f/f;Tpbpa/Ada-Cre♂×Cbs^f/f;Tpbpa/Ada-Cre♀mating group.（2）The damaged uteroplacental hemodynamic was rescued by GYY41374.CBS knockdown damaged the remodeling of spiral arteryIHC for cytokeratin（to identify trophoblasts）anda-SMA（to identify smooth muscle）was used to evaluate trophoblast infiltration in the spiral arteries.Compared with the negative placenta,Cbs^f/f;Tpbpa/Ada-Cre placenta showed much less trophoblasts invading whilst morea-SMA staining cells in spiral artery,which was rescued by GYY4137.5.CBS knockdown damaged the mitochondria in placentaCBS knockdown induced mitochondrial superoxide production and resulted in a decrease in mitochondrial membrane potential and ATP production in placenta,which was rescued by GYY4137.ConclusionReduced circulatory H₂S induced by placental CBS knockdown mediated mitochondrial dysfunction in trophoblast,which damaged the placental structure and function,and induced IUGR.