In this study, we first demonstrated the role of TGR5 activation in neuroinflammation and long-term cognitive impairment after CLP in rats and that its effects may occur via the TGR5/cAMP/PKA/CREB signaling pathway. We found the following: 1) The expression of TGR5 in the hippocampus increased in a time-dependent manner and peaked at 3 d after CLP. The results of immunofluorescence analysis showed that TGR5 was expressed in microglial cells. 2) Although the intranasal administration of INT-777, a selective inhibitor of TGR5, at 1 hour after CLP did not improve the survival and change in body weight of the rats, it reduced hippocampal microglial activation, neutrophil infiltration, and the expression of IL-1β in the hippocampus; it also reduced the clinical score 6 hours after surgery and improved spatial memory and anxiety-like behavior at 15 days. 3) Intracerebroventricular administration of TGR5 CRISPR and SQ22536 offset the protective effect of INT-777. These observations suggest that TGR activation inhibits neuroinflammation, at least in part, through the cAMP/PKA/CREB signaling pathway, thereby improving long-term cognitive impairment after CLP. INT-777, a selective inhibitor of TGR5, improved cognitive impairment and anxiety-like behavior in CLP rats.
Sepsis-associated encephalopathy (SAE) is the most common cause of encephalopathy in ICUs[4]. After discharge, up to 50% of sepsis survivors develop cognitive impairment that affects memory, attention, verbal fluency and executive function[3, 26]. In addition, sepsis patients exhibit a higher incidence of mental illnesses, such as depression, anxiety and self-harm, than the general population[27]. Therefore, neurological dysfunction severely affects the quality of life of sepsis survivors and imposes a huge economic and social burden. In animal models of sepsis, surviving animals showed the same cognitive impairments and mental disorders shown by sepsis survivors[8, 28, 29]. In our study, we observed the same phenomenon. The Morris water maze test can be altered in numerous ways to investigate working memory, reference memory and task strategy[30]. The sepsis survivors in our study were found to spend significantly more time on the platform, swim longer, and spend less time in the target quadrant after the platform was removed. An elevated-plus maze experiment assessed anxiety or the fear of an open space in the rats; normally, animals consciously avoid the open arms and stay in a closed arm[31]. In our experiment, CLP rats took more time to enter an open arm. Not only the Morris water maze experiment but also the elevated plus-maze experiment shows the function of the hippocampus and prefrontal cortex in animals[32, 33]. Therefore, to further explore the mechanism by which INT-777 improves cognitive impairment in rats after CLP, we selected hippocampal tissue for further study.
The underlying pathologic changes in brain injury after sepsis are multifactorial and highly complex. Inflammatory cytokines, BBB destruction, ischemic processes, neurotransmitter changes, and mitochondrial dysfunction may be involved in this complex process, but its specific mechanisms have not yet been established[34]. However, uncontrolled neuroinflammation and ischemic injury are closely related to brain injury after sepsis[35]. Neuroinflammation is a major component of the pathology and progression of many neurological and neurodegenerative diseases[36]. Uncontrolled inflammation is a major cause of the abnormal function of brain cells (including microglia, neurons, and endothelial cells) and their increased apoptosis [37]. Microglial cells are the main macrophage cells in the brain, representing most of the brain immune system. Overactivation of microglial cells releases various harmful inflammatory mediators and increases the release of reactive oxygen species by destroying the stability of the blood-brain barrier, thus exacerbating brain dysfunction[38]. Indeed, studies have shown that inhibiting microglia helps reduce neuroinflammation in sepsis, which in turn improves long-term cognitive function[39]. Our results showed that the number of microglial cells in the hippocampus of rats at 24 hours after CLP was significantly increased compared to that in rats in the sham group, and MPO staining suggested that neutrophil infiltration was increased, with the inflammatory mediator IL-1β showed the same trend. The results of western blot analysis performed for further quantitative analysis also indicated that the protein expression levels of Iba-1, MPO and IL-1β in the hippocampus after CLP were significantly increased. Combined with the results of other studies, these results show that inhibition of early neuroinflammation after CLP may be an effective strategy to improve later cognitive dysfunction.
TGR5 (also called G-protein-coupled bile acid receptor, Gpbar1) is a member of the GPCR superfamily[9]. Surprisingly, numerous studies have shown that TGR agonists may be agents for the treatment of metabolic, inflammatory and digestive diseases[40]. In neurological diseases, activation of TGR5 by a different agonist also shows neuroprotective effects. McMillin et al. found that activating TGR5 could alleviate neuroinflammation and improve outcomes through neuron and microglia paracrine signaling in a mouse model of hepatic encephalopathy[15]. Lewis et al. also found that clinical scores in experimental autoimmune encephalitis model mice were significantly reduced after treatment with TGR5 agonists, which was associated with the reduced activation of monocytes and microglia cells[41]. 6α-Ethyl-23(S)-methylcholic acid (6-EMCA, INT-777) is a selective, specific agonist for TGR5, and Zuo et al. found that intranasal administration of INT-777 was also beneficial for SAH rats[12]. In our study, after the intranasal administration of INT-777 one hour after CLP, cognitive impairment and anxiety-like behavior in CLP rats significantly improved and the number of activated microglia and neutrophil infiltration were significantly reduced, and the results of WB analysis showed that inflammatory factors were also downregulated. After TGR5 CRISPR-mediated knockout of TGR5 in CLP animals, the neuroprotective effect of INT-777 disappeared. All these results suggest that INT-777 can inhibit microglial activation and inflammation by activating TGR5, thereby improving long-term cognitive impairment.
G protein coupled receptors (GPCRs) are thought to play a role in signal transduction by producing a number of second messengers, including cAMP. When activated by INT-777, TGR5 leads to increased intracellular cAMP levels and activates subsequent signaling pathways[42]. Studies have shown that the TGR5-cAMP-PKA axis is the basis of bile acid-mediated anti-inflammatory effects[19]. PKA promotes phosphorylation of the transcriptional activation sites of CREB, which further promotes the binding of CREB to the transcriptional coactivator CBP and the formation of complexes that block the transcription of inflammatory genes[43]. CREB signaling also reduces the activation of proinflammatory microglial cells, leading to neuroprotective effects[44].
In the current study, we found that INT-777 increased the expression of cAMP/P-PKA/P-CREB after CLP, while CRISPR-mediated TGR5 knockout inhibited the expression of these proteins. To further confirm our hypothesis, SQ22536, an adenylyl cyclase inhibitor, was used to inhibit cAMP, and the results showed that the expression of TGR5 was not changed, but the expression of cAMP/p-PKA/p-CREB was decreased, and expression of the inflammatory cytokines IL-1β, IL-6 and TNF-α was significantly increased, suggesting that the neuroprotective effect of TGR5 had been reversed by SQ22536. These findings support the idea that the improvement in cognitive impairment observed in rats after CLP with INT-777 treatment is achieved through the TGR5/cAMP/PKA/CREB signaling pathway.
This study has some limitations. First, although TGR5 plays a role in neuroprotection through different mechanisms, such as resistance to apoptosis, oxidative stress resistance, and the maintenance of blood-brain barrier integrity, in this study, the role of TGR5 in only neuroinflammation was studied. Second, our study showed that TGR5 is also minimally expressed on astrocytes, and the current study confirms that astrocytes are also strongly associated with neuroinflammation[45], and we intend to further evaluate the effect of INT-777 treatment on astrocytes in neuroinflammation in a follow-up study. Third, INT-777 was administered at 1 hour after CLP, and whether the administration of INT-777 within a time frame of 3 hours, 6 hours or longer after CLP would still have a significant neuroprotective effect was not evaluated, although this information would be more valuable for clinical application.