Department of Surgery, Section of Pediatric Surgery, Indianapolis, Indiana.
The Indiana University School of Medicine, Indianapolis, Indian.
Shock. 2020 Jun;53(6):737-743. doi: 10.1097/SHK.0000000000001420.
Hydrogen sulfide (H2S) has many beneficial biological properties, including the ability to promote vasodilation. It has been shown to be released from stem cells and increased by hypoxia. Therefore, H2S may be an important paracrine factor in stem cell-mediated intestinal protection. We hypothesized that H2S created through conventional pathways would be a critical component of stem cell-mediated intestinal protection after ischemic injury.
Human bone marrow-derived mesenchymal stem cells (BMSCs) were transfected with negative control siRNA (Scramble), or with siRNA to CBS, MPST, or CTH. Knockdown was confirmed with PCR and H2S gas assessed with AzMC fluorophore. Eight-week-old male mice then underwent intestinal ischemia for 60 min, after which time, perfusion was restored. BMSCs from each of the above groups were then placed into the mouse abdominal cavity before final closure. After 24 h, mice were reanesthetized and mesenteric perfusion was assessed by Laser Doppler Imaging (LDI). Animals were then sacrificed and intestines excised, placed in formalin, paraffin embedded, and stained with H & E. Intestines were then scored with a common mucosal injury grading scale.
PCR confirmed knockdown of conventional H2S-producing enzymes (CBS, MPST, CTH). H2S gas was decreased in MPST and CTH-transfected cells in normoxic conditions, but was not decreased compared with Scramble in any of the transfected groups in hypoxic conditions. BMSCs promoted increased mesenteric perfusion at 24 h postischemia compared with vehicle. Transfected stem cells provided equivalent protection. Histologic injury was improved with BMSCs compared with vehicle. CBS, MPST, and CTH knockdown cell lines did not have any worse histological injury compared with Scramble.
Knocking down conventional H2S-producing enzymes only impacted gas production in normoxic conditions. When cells were transfected in hypoxic conditions, as would be expected in the ischemic intestines, H2S gas was not depressed. These data, along with unchanged perfusion and histological injury parameters with conventional enzyme knockdown, would indicate that alternative H2S production pathways may be initiated during hypoxic and/or ischemic events.
硫化氢（H2S）具有许多有益的生物学特性，包括促进血管扩张的能力。已经表明它可以从干细胞中释放出来，并通过缺氧增加。因此，H2S 可能是干细胞介导的肠道保护中的重要旁分泌因子。我们假设通过常规途径产生的 H2S 将是缺血损伤后干细胞介导的肠道保护的关键组成部分。
用人骨髓间充质干细胞（BMSC）转染阴性对照 siRNA（Scramble）或 CBS、MPST 或 CTH 的 siRNA。通过 PCR 确认敲低，并用 AzMC 荧光探针评估 H2S 气体。然后，8 周龄雄性小鼠接受 60 分钟的肠道缺血，之后恢复灌注。将来自上述各组的 BMSC 放入小鼠腹腔中，然后再关闭。24 小时后，重新麻醉小鼠，通过激光多普勒成像（LDI）评估肠系膜灌注。然后处死动物，切除肠道，置于福尔马林中，石蜡包埋，并用 H&E 染色。用常见的粘膜损伤分级量表对肠道进行评分。
PCR 证实了常规 H2S 产生酶（CBS、MPST、CTH）的敲低。在常氧条件下，MPST 和 CTH 转染细胞中的 H2S 气体减少，但在任何转染组的缺氧条件下，与 Scramble 相比，H2S 气体均未减少。BMSC 在缺血后 24 小时促进肠系膜灌注增加，与载体相比。转染的干细胞提供了等效的保护。与载体相比，BMSC 可改善组织学损伤。与 Scramble 相比，CBS、MPST 和 CTH 敲低细胞系的组织学损伤没有任何恶化。
敲低常规 H2S 产生酶仅在常氧条件下影响气体产生。当细胞在缺氧条件下转染时，正如在缺血肠道中预期的那样，H2S 气体不会被抑制。这些数据以及常规酶敲低时灌流和组织学损伤参数不变，表明在缺氧和/或缺血事件期间可能会启动替代的 H2S 产生途径。
