TGR5 overexpression mediated by the inhibition of transcription factor SOX9 protects against hypoxia-/reoxygenation-induced injury in hippocampal neurons by activating Nrf2/ HO-1 signaling

Cerebral ischemia/reperfusion (CI/R) injury is a destructive cerebrovascular disease associated with long-term disability and high mortality rates. TGR5 has been discovered in multiple human and animal tissues and to modulate a variety of physiological processes. The current study sought to reveal the function of TGR5 in CI/R injury and uncover the latent regulatory mechanism.

TGR5 was transcriptionally inhibited by SOX9, and the overexpression of TGR5 played a protective role in CI/R injury.

A hypoxia/reoxygenation (H/R) model was established in mouse hippocampal HT22 cells. The TGR5 expression in the H/R-treated HT22 cells was tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blots. After TGR5 was overexpressed, Cell Counting Kit-8 assays were used to estimate cell viability, and lactate dehydrogenase (LDH) release was assessed by a LDH assay kit. Cell apoptosis was measured by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assays. Cytochrome c release was detected by immunofluorescence assays and western blots were used to analyze the protein levels of apoptosis-related factors. The oxidative stress levels were assessed by corresponding kits. Next, SOX9 expression in the H/R-treated HT22 cells was tested by RT-qPCR and western blots. The interaction between the TGR5 promoter and SOX9 was verified by luciferase reporter and chromatin immunoprecipitation assays. Subsequently, after the H/R-treated HT22 cells had been co-transfected with TGR5 overexpression and SOX9 overexpression plasmids, TGR5 expression was tested by RT-qPCR and western blots, and the above-mentioned functional experiments were repeated. Finally, the expression of Nrf2/HO-1 signaling-related proteins was examined by western blots.

TGR5 expression was significantly decreased in the H/R-exposed HT22 cells. The elevation of TGR5 enhanced the viability, hindered the apoptosis, and alleviated the oxidative stress of the HT22 cells under H/R conditions. Additionally, SOX9 had a strong affinity with TGR5 promoter, and TGR5 was transcriptionally inhibited by SOX9. Further, SOX9 overexpression restored the protective role of TGR5 upregulation in H/R-induced HT22 cell injury. Additionally, TGR5 overexpression mediated by SOX9 inhibition activated Nrf2/HO-1 signaling. Conclusions: TGR5 was transcriptionally inhibited by SOX9, and the overexpression of TGR5 played a protective role in CI/R injury.