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

Abstract

Background: 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.Methods: 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 cotransfected 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.Results: 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.

Taurocholic acid ameliorates hypertension through the activation of TGR5 in the hypothalamic paraventricular nucleus

“…36 Another study showed that TGR5 overexpression in hippocampal neurons can protect against hypoxia-/reoxygenation-induced injury by activating Nrf2 signaling. 37 Here, we also found that after TCA infusion, the TGR5s located on the surface of the neurons in the PVN were activated as their expression increased (Fig. 6).…”