Hydrogen Rich Water Attenuates Renal Injury and Fibrosis by Regulation Transforming Growth Factor-β Induced Sirt1

The current research was designed to study the role of hydrogen in renal fibrosis and the renal epithelial to mesenchymal transition (EMT) induced by transforming growth factor-β1 (TGF-β1). Hydrogen rich water (HW) was used to treat animal and cell models. Unilateral ureteral obstruction (UUO) was performed on Balb/c mice to create a model of renal fibrosis. Human kidney proximal tubular epithelial cells (HK-2 cells) were treated with TGF-β1 for 36 h to induce EMT. Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured to test renal function, in addition, kidney histology and immunohistochemical staining of alpha-smooth muscle actin (α-SMA) positive cells was performed to examine the morphological changes. The treatment with UUO induced a robust fibrosis of renal interstitium, shrink of glomerulus and partial fracture of basement membrane. Renal function was also impaired in the experimental group with UUO, with an increase of Scr and BUN in serum. After that, Western-blot was performed to examine the expression of α-SMA, fibronectin, E-cadherin, Smad2 and Sirtuin-1 (Sirt1). The treatment with HW attenuated the development of fibrosis and deterioration of renal function in UUO model. In HK-2 cells, the pretreatment of HW abolished EMT induced by TGF-β1. The down-regulation the expression of Sirt1 induced by TGF-β1 which was dampened by the treatment with HW. Sirtinol, a Sirt1 inhibitor, reversed the effect of HW on EMT induced by TGF-β1. HW can inhibit the development of fibrosis in kidney and prevents HK-2 cells from undergoing EMT which is mediated through Sirt1, a downstream molecule of TGF-β1.

Hydrogen gas alleviates sepsis-induced neuroinflammation and cognitive impairment through regulation of DNMT1 and DNMT3a-mediated BDNF promoter IV methylation in mice

Sepsis-associated encephalopathy (SAE) can cause acute and long-term cognitive impairment and increase the mortality rate in sepsis patients, and we previously reported that 2% hydrogen gas (H2) inhalation has a therapeutic effect on SAE, but the underlying mechanism remains unclear. Dynamic DNA methylation, which catalyzed by DNA methyltransferases (DNMTs), is involved in the formation of synaptic plasticity and cognitive memory in the central nervous system. And brain-derived neurotrophic factor (BDNF), to be a key signaling component in activity-dependent synaptic plasticity, can be induced by neuronal activity accompanied by hypomethylation of its promoter IV. This study was designed to illustrate whether H2 can mediate SAE by alter the BDNF promoter IV methylation mediated by DNMTs. We established an SAE model by cecal ligation and perforation (CLP) in C57BL/6 mice. The Morris water maze test from the 4th to the 10th day after sham or CLP operations were used to evaluate mouse cognitive function. Hippocampal tissues were isolated at the 24 after sham or CLP surgery. Pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6) and High Mobility Group Box 1 (HMGB1) were measured by enzyme-linked immunosorbent assay (ELISA). mRNA or protein levels of DNMTs (DNMT1, DNMT3a and DNMT3b), BDNF promoter IV and total BDNF were detected by RT-PCR and Western blot tests. Immunofluorescence staining were used to determine the expressions of DNMT1 and DNMT3a. The quantitative methylation analysis of the 11 CpG island of the promoter region of BDNF exon IV was determined using theAgena’s MassARRAY EpiTYPER system. We found that 2% H2 inhalation can reduce pro-inflammatory factors, alleviate DNMT1, DNMT3a but not DNMT3b expression, make hypomethylation of BDNF promoter IV at 5 CpG sites, enhance the BDNF levels and then decrease escape latency but increase platform crossing times in septic mice. Our results suggest that 2% H2 inhalation may alleviate SAE through altering the regulation of BDNF promoter IV methylation which mediated by DNMT1 and DNMT3a in the hippocampus of septic mice.