硫化氢对严重烧伤大鼠重要脏器的影响

目的 观察硫化氢及其所生成的胱硫醚-γ-裂合酶(CSE)在大鼠严重烧伤后的变化规律,分析其对严重烧伤大鼠重要脏器的影响.方法 健康雄性SD大鼠104只,按照随机数字表法分为正常对照组8只、烧伤组48只、硫化氢干预组48只.硫化氢干预组大鼠于伤前5 d起腹腔注射硫氢化钠(56 μmol/kg,每日定时1次),注射5 d后与烧伤组大鼠均造成30%TBSAⅢ度烧伤.于伤后2、6、12、24、48、96 h(每时相点8只)检测烧伤组大鼠血清硫化氢含量,采集心、肝、肾、肺、胃组织标本检测CSE转化率;检测烧伤组和硫化氢干预组大鼠上述各时相点血清ALT、AST、心肌型肌酸激酶同工酶(CK-MB)、尿素氮、肌酐含量,观察上述脏器组织形态学变化.正常对照组检测指标同上.对数据行SNK-q检验、t检验、Pearson相关分析(血清硫化氢含量与CSE转化率、血生化指标).结果 烧伤组大鼠伤后各时相点血清硫化氢含量和各脏器组织CSE转化率普遍低于正常对照组,伤后6或12 h达到最低点.与正常对照组相比,烧伤组伤后2 h血清ALT、AST、CK-MB、尿素氮、肌酐含量明显升高,伤后6 h尿素氮[(32.5±9.8)mmol/L]和肌酐[(65±9)μmol/L]达峰值,伤后12 h ALT[(423±59)U/L]、AST[(993±60)U/L]及CK-MB[(49 261±6637)U/L]达峰值,伤后48 h上述指标仍高于正常对照组(t=3.157～8.386,P值均小于0.05).硫化氢干预组大鼠伤后各时相点血清ALT、AST、CK-MB、尿素氮、肌酐含量变化趋势与烧伤组大致相同,普遍低于烧伤组.组织形态学观察显示,伤后24 h烧伤组大鼠心、肝、肺、肾和胃组织损害严重,硫化氢干预组损伤较之明显减轻.烧伤组血清硫化氢含量与各脏器组织CSE转化率、各生化指标呈显著正相关(r值分别为0.639～0.894、0.301～0.585,P＜0.005或P＜0.001).结论 硫化氢/CSE体系可能参与了大鼠烧伤后的病理生理过程.补充外源性硫化氢对严重烧伤大鼠的重要脏器可产生保护作用.

Abstract：

Objective To investigate the changes in hydrogen sulfide (H2S) and cystathionine gamma-lyase (CSE) in rats with severe burn, and to analyze the effects on important organs. Methods One hundred and four healthy male SD rats were divided into normal control group(NC, n = 8), burn group (B, n =48), and H2S intervention group (HI, n =48) according to the random number table. SD rats in HI group were intraperitoneally injected with NaHS (56 μmol/kg) once a day for 5 days. Then rats in HI and B groups were subjected to 30% TBSA full-thickness burn. Blood sample as well as heart, liver, lung,kidney, and stomach tissue samples were harvested from rats in B group at post burn hour(PBH) 2, 6, 12,24, 48, and 96 respectively for determination of serum content of H2S and CSE activity. Serum content of alanine transaminase (ALT) , aspartate aminotransferase(AST), MB isoenzyme of creatine kinase(CK-MB),urea nitrogen(BUN), and creatinine (Cr) in HI and B groups were examined at each time point. Samples were harvested from above organs in each group for histomorphological observation. Above-mentioned indexes were also determined in NC group as control. Data were processed with SNK-q test, t test,correlation analysis (between serum content of H2S and CSE activities, biochemical indexes). Results Serum content of H2S and CSE activities of above organs (except for lung tissue at PBH 48, 96) in B group within PBH 96 were lower than those in NC group, reaching minimum values at PBH 6 or 12. Compared with those in NC group, serum contents of all biochemical indexes in B group were obviously increased within PBH 48, in which serum contents of BUN [( 32.5 ± 9.8) mmoL/L] and Cr [( 65±9) μmol/L] reached peak at PBH 6, and serum contents of ALT [(423 ±59) U/L], AST [(993 ±60) U/L], and CK-MB [(49 261 ±6637) U/L] peaked at PBH 12. Serum contents of all biochemical indexes in HI group at each time point were significantly decreased as compared with those in B group, but the same change tendencies were showed in both groups. Histomorphological observation showed that all the organs were severely injured in B group at PBH 24, whereas those in HI group were markedly ameliorated. Serum content of H2S in B group was respectively correlated with CSE activities of all organs(with r value from 0. 639 to 0. 894, P values all below 0. 005) and serum contents of biochemical indexes(with r value from 0. 301 to 0. 585, P values all below 0.001). Conclusions H2S/CSE system may take part in pathophysiological process in rats with severe burn. Exogenous H2S replacement therapy can protect important organs of rats with severe burn.     

Ancient origin of the CTH alelle carrying the c.200C>T (p.T67I) variant in patients with cystathioninuria

Espinós C, García‐Cazorla A, Martínez‐Rubio D, Martínez‐Martínez E, Vilaseca MA, Pérez‐Dueñas B, Ko?ich V, Palau F, Artuch R. Ancient origin of the CTH alelle carrying the c.200C>T (p.T67I) variant in patients with cystathioninuria. Hereditary cystathioninuria is due to mutations in the CTH gene that encodes for cystathionase, a pyridoxal‐5′‐phosphate (PLP) dependent enzyme. To date, mutations in this gene have been described in 10 unrelated cystathioninuric patients. Enzyme assays have showed that mutated cystathionase exhibits lower activity than controls. As cystathioninuria is usually accompanied by a wide variety of symptoms, it has been questioned whether it is a disease or just a biochemical finding not associated with the clinical picture of these patients. This is the first report of Spanish patients with cystathioninuria and mild to severe neurological symptoms in childhood. After oral pyridoxine therapy biochemical parameters have normalized but clinical amelioration was not evident. All patients were homozygotes for the c.200C>T (p.T67I) variant which is the most prevalent inactivating mutation in the CTH gene. To further investigate the history of the alleles carrying the c.200C>T transition in Europe, we also constructed the haplotypes on the CTH locus in our Spanish patients as well as in a clinical series of cystathioninuric patients from the Czech Republic harboring the same nucleotide change. We suggest that the CTH p.T67I substitution could have an ancient common origin, which probably occurred in the Neolithic Era and spread throughout Europe.     

A theoretical examination of hydrogen sulfide metabolism and its potential in autocrine/paracrine oxygen sensing

Hydrogen sulfide (H₂S) metabolism has been proposed as the oxygen (O₂) sensing mechanism coupling hypoxia to effector responses in a variety of tissues including vascular and chemoreceptor cells. Implicit in this sensing system is a mechanism for regulating intracellular H₂S concentration, presumably through oxidation. However, verification of this mechanism, or any other pathway of H₂S signaling has been hampered by the lack of suitable methods for measuring intracellular concentration and distribution profiles. Here, intracellular H₂S concentration profiles are modeled using simple monoexponential diffusion equations and current knowledge of H₂S biosynthetic and metabolic pathways. The models predict that; (1) while both mitochondrial oxidation and simple diffusion out of the cell can reduce H₂S concentration, the former is considerably more effective as an effector of intracellular H₂S and (2) exogenously applied H₂S may have unanticipated effects on endogenous signaling processes. In addition, these models provide additional support for mitochondrial H₂S oxidation as the key couple in H₂S-mediated O₂ sensing.     

Hydrogen sulfide mediates the anti-survival effect of sulforaphane on human prostate cancer cells

Hydrogen sulfide (H₂S) is a novel gasotransmitter that regulates cell proliferation and other cellular functions. Sulforaphane (SFN) is a sulfur-containing compound that exhibits anticancer properties, and young sprouts of broccoli are particularly rich in SFN. There is consistent epidemiological evidence that the consumption of sulfur-containing vegetables, such as garlic and cruciferous vegetables, may help reduce the occurrence of prostate cancer. Here we found that a large amount of H₂S is released when SFN is added into cell culture medium or mixed with mouse liver homogenates, respectively. Both SFN and NaHS (a H₂S donor) decreased the viability of PC-3 cells (a human prostate cancer cell line) in a dose-dependent manner, and supplement of methemoglobin or oxidized glutathione (two H₂S scavengers) reversed SFN-reduced cell viability. We further found both cystathionine gamma-lyase (CSE) and cystathionine beta-synthase are expressed in PC-3 cells and mouse prostate tissues. H₂S production in prostate tissues from CSE knockout mice was only 20% of that from wild-type mice, suggesting CSE is a major H₂S-producing enzyme in prostate. CSE overexpression enhanced H₂S production and inhibited cell viability in PC-3 cells. In addition, both SFN and NaHS activated p38 mitogen-activated protein kinases (MAPK) and c-Jun N-terminal kinase (JNK). Pre-treatment of PC-3 cells with methemoglobin decreased SFN-stimulated MAPK activities. Suppression of both p38 MAPK and JNK reversed H₂S- or SFN-reduced viability of PC-3 cells. Our results demonstrated that H₂S mediates the inhibitory effect of SFN on the proliferation of PC-3 cells, which suggests that H₂S-releasing diet or drug might be beneficial in the treatment of prostate cancer.     

同型半胱氨酸相关酶基因多态性与高血压伴周围动脉闭塞性疾病的关系

目的探讨亚甲基四氢叶酸还原酶(MTHFR)基因C677T、胱硫醚B合成酶(CBS)基因844ins68和甲硫氨基合成酶(MS)基因A2756G3种同型半胱氨酸(Hcy)代谢相关酶基因多态性与北京社区汉族老年人群中原发性高血压(EH)、EH伴周围动脉闭塞性疾病(PAOD)易感性的关系。方法PCR扩增老年EH(EH组)、老年EH伴PAOD(EH—PAOD组)患者和老年健康对照组的MTHFR C677T、CBS 844ins68、MS A2756G基因突变点，直接或经限制性内切酶消化后行凝胶电泳，确定基因型并统计基因突变频率。结果EH组100例MTHFR基因3种基因型频率为：C／C29．0％，C／T45．0％，T／T26．0％；EH—PAOD组59例为：C／C15．9％，C／T35．5％，T／T48．6％；对照组100例为：C／C31．0％，C／T50．0％，T／T19．0％。3组MTHFR基因的C677T单核苷酸突变中T突变位点的频率分别为48．5％、64．4％、44．0％。EH—PAOD组与对照组和EH组比较，MTHFR T／T基因型频率和T等位基因频率差异均有统计学意义。而CBS 844ins68、MS A2756G各种基因型频率和等位基因频率在EH组、EH—PAOD组和对照组之间差异无统计学意义。结论MTHFR基因C677T单核苷酸突变可能是北京社区汉族老年人PAOD的遗传性危险因素之一，可能与EH无关。且CBS基因844ins68、MS基因A2756G的突变可能都不足以成为EH和PAOD的遗传危险因子。     

硫化氢对糖尿病大鼠ED的影响和机制研究

 目的：观察硫化氢（H 2S）对糖尿病（DM）大鼠勃起功能障碍（ED）的影响。方法：将70只SD雄性大鼠分为2组，60只腹腔注射链脲佐菌素（STZ）建立DM大鼠模型，10只注射生理盐水为正常对照。阿朴吗啡诱导勃起后检测DM造模成功大鼠的勃起功能情况，将存在ED的36只大鼠随机分成3组：糖尿病对照组（DM组）、糖尿病+硫氢化钠组（DM+NaHS组）和糖尿病+西地那非组（DM+sildenafil组）。DM+NaHS组每天腹腔注射NaHS （50 μmol/kg），DM+sildenafil组每天给予西地那非（5 mg/kg）灌胃，DM组每日腹腔注射生理盐水，注射体积为0.25 mL。持续饲养12周后，观察各组大鼠勃起功能情况，采集各组大鼠动脉血，测定血液中H 2S含量及各组阴茎海绵体组织胱硫醚γ-裂解酶（cystathionine γ-lyase, CSE）活性及CSE表达水平。结果：STZ诱发的DM造模成功率为81.7%（49/60），DM大鼠发生ED的比例为73.5%（36/49），与DM组比，DM+NaHS组和DM+sidenafil组大鼠勃起功能良好，3个实验组大鼠勃起率分别为(33.3±5.5)%、（54.5±5.3）%和(63.6±9.1)%。与正常大鼠组比，各组DM大鼠阴茎海绵体中CSE活性与表达水平均有下降，其中DM+NaHS组大鼠的CSE活性及表达水平显著低于DM组（P<0.05）和DM+sildenafil组（P<0.05）。结论：STZ诱导的糖尿病大鼠中ED发生率较高，H 2S对糖尿病合并ED大鼠勃起功能有一定的促进作用。外源性给予H 2S可以反馈性抑制大鼠阴茎组织中CSE的活性和表达。     

Effects of valproate,an HDAC inhibitor,on the expression of folate carriers and folate metabolism-related genes in the placenta of rats

Valproate (VPA), an antiepileptic drug, is known to inhibit histone deacetylases (HDACs). Exposure to VPA during pregnancy increases several fetal risks. The maintenance of folate level during pregnancy is essential for adequate fetal development, and the placenta plays a critical role in supplying nutrients to the fetus. The aim of this study was to elucidate the effects of VPA on the gene expression of folate carriers and metabolizing enzymes in the rat placenta at both mid and late gestation periods. Pregnant rats were orally administered VPA on a single day or 4 days (repeated administration). Gene expression of folate carriers (Folr1, Slc19a1, Slc46a1) and metabolizing enzymes (Cth, Mtr, Mtrr, Mthfr, Dhfr) was assessed in the placenta on gestational day (GD) 13 or GD20. In the control rats, the expression of Folr1, Slc46a1, Cth, and Mthfr tended to be upregulated, whereas that of Mtrr and Dhfr was downregulated during gestation; the expression of Slc19a1 and Mtr did not change. Repeated VPA administration reduced the placental expression of Folr1and Mtr on GD20 and increased the expression of Dhfr on GD13 compared with the control. These findings indicate that administration of VPA alters the placental gene expression of folate carriers and metabolism-related enzymes.     

胱硫醚β-合酶/胱硫醚γ-裂解酶-硫化氢体系小干扰RNA对大鼠正常肝细胞系BRL凋亡的影响

目的 探讨内源性胱硫醚 β-合酶（CBS）/胱硫醚 γ-裂解酶（CSE）-硫化氢（H2S）体系在生理状态肝细胞凋亡中的影响。方法 培养大鼠正常肝细胞株BRL。小干扰RNA（siRNA）序列筛选：随机分组为阴性siRNA序列转染组、CBS siRNA序列转染组和CSE siRNA序列转染组,转染时间为48h或24h;siRNA转染后细胞凋亡检测：随机分组为siRNA阴性对照组、溶剂对照组、CBS siRNA组、CSE siRNA组、（CBS+ CSE）siRNA组,作用时间为转染后0、4、8、12、24 h;凋亡机制探讨：随机分组为siRNA阴性对照组、CBS siRNA组、CSE siRNA组、（CBS+ CSE）siRNA组,作用时间为转染后4、8、12、24 h;每组均为4个平行样。RT-PCR、Western blot检测BRL细胞中CBS、CSE的表达,siRNA基因静默CBS、CSE,去蛋白法检测H2S生成,Hoechst荧光染色、流式细胞术检测BRL细胞凋亡,荧光染色检测线粒体膜电位（MMP）变化,Western blot检测胞浆内细胞色素C（Cyt C）和激活型caspase 3蛋白表达变化。结果 BRL细胞株存在CBS、CSE表达。CBS/CSE siRNA转染后,细胞内源性H2S生成降低,细胞凋亡且凋亡率随时间延长而逐渐增加。结论 抑制内源性H2S体系可引起生理状态肝细胞凋亡,机制可能部分涉及凋亡线粒体途径。     

Role of hydrogen sulfide in portal hypertension and esophagogastric junction vascular disease

AIM:To investigate the association between endogenous hydrogen sulfide(H2S)and portal hypertension as well as its effect on vascular smooth muscle cells.METHODS:Portal hypertension patients were categorized by Child-Pugh score based on bilirubin and albumin levels,prothrombin time,ascites and hepatic encephalopathy.Plasma H2S concentrations and portal vein diameters(PVDs)were compared between portal hypertension patients and control participants,as well as between portal hypertension patients with varying degrees of severity.In addition,we established a rabbit hepatic schistosomiasis portal hypertension(SPH)model and analyzed liver morphology,fibrosis grade,plasma and liver tissue H2S concentrations,as well as cystathionineγ-lyase(CSE)activity and phosphorylated extracellular signal-regulated kinase(pERK)1/2,B cell lymphoma(Bcl)-2 and Bcl-XL expression in portal vein smooth muscle cells,in addition to their H2S-induced apoptosis rates.RESULTS:In portal hypertension patients,endogenous H2S levels were significantly lower than those in healthy controls.The more severe the disease was,the lower were the H2S plasma levels,which were inversely correlated with PVD and Child-Pugh score.Liver tissue H2S concentrations and CSE expression were significantly lower in the SPH rabbit livers compared with the control animals,starting at 3 wk,whereas pERK 1/2expressions gradually increased 12-20 wk after SPH model establishment.In portal vein smooth muscle cells,increasing H2S levels led to increased apoptosis,while Bcl-2 and Bcl-XL expression decreased.CONCLUSION:H2S prevents vascular restructuring caused by excessive proliferation of smooth muscle cells via apoptosis induction,which helps to maintain normal vascular structures.