In our previous study, we uncovered that ketamine exerted protective effect on hippocampus neurons and cecal epithelial cells via repressing different doses of TNF-induced SIRS. Here, we revealed that ketamine pre-treatment prevented BBB damage and alleviated neuroinflammation by reducing cecum-derived HMGB1 release. Other than that, protective management like Rip3 knockout and ketamine pre-treatment relieved TNF-induced SIRS symptoms and brain damage. From the assay system in vitro, we reconfirmed that ketamine pre-treatment downregulated the level of pro-inflammatory cytokines. Furthermore, we found that protecting the cecum Rip3 knockout, ketamine pre-treatment, or cecectomy could limit circulating HMGB1 generation. We also demonstrated that anti-HMBG1 and cecectomy therapy applications were sufficient to safeguard BBB relative intactness and alleviate microglia over-activation. Thus, our work here provided a validated evidence of the crucial role of the cecum in the integrity of BBB and the neuroinflammation during SIRS. More importantly, our study provided a new theoretical view and therapeutic target for the application of ketamine in SIRS.
A complex pathophysiologic state responding to severe attacks like infection, trauma, and other injuries is the feature of SIRS. The recognition of effective treatments is an urgent issue ahead for all of us to solve. Many studies have reported various anticipated candidates such as “anti-mediators,” receptor blockers and inflammatory regulatory reagents, etc1. The final desired aim of those managements is to prolong patients’ life and improve prognosis as much as possible. SIRS is usually associated with an aggressive amount of pro-inflammatory cytokines and outraged cytokine storm19. Cytokine storm extravagates the immune hyperactivation of early acute period and immunosuppression of terminal stage. From the studies of patients with SIRS, scientists have confirmed that the level of various pro-inflammatory cytokines, including IL-6, IL-18, IL-15, TNF, IL-1β, and IFN-γ has up-regulated importantly in circulation with severe infectious diseases, as recently widespread COVID-19, previous reported MHV, SARS-CoV-220, and non-infectious disease. According to the study by Karki, synergism of TNF and IFN-γ triggered inflammatory cell death, tissue damage, and mortality in SIRS patients. In our mice model, the application of TNF could roughly match the disease states like SIRS. TNF is one of the classical necroptosis triggers of cell death pathways. Necroptosis is usually executed by RIP3-MLKL signaling21, a main cellular biological mechanism of SIRS3, 12. When SIRS is primed, multiple organ failure is irreversible. However, several types of research, including our results, have observed the sensitivity of cecum during the development of SIRS11, and Rip3−/− mice have found that cecum was the only organ attenuated11. In this study, in addition to showing relief of SIRS symptoms, as previous research reported in Rip3−/− mice, the BBB function was also protected. After SIRS occurs extravagantly, end-organ dysfunctions usually occur in each stage with the progress of the disease. Adult respiratory distress syndrome disseminated intravascular coagulation and acute renal failure are three common pathological attack22. However, nervous system damage like brain edema, septic encephalopathy even brain death is usually irreversible. In our research, we confirmed the status of ketamine in the protection of BBB function and reasoned that the cecum might be a vulnerable organ when confronted with inflammation and stress challenges. So protecting cecum with ketamine pre-treatment, Rip3−/− mice, and even pre-cecectomy might work to some extent.
There are accumulating shreds of evidence indicating that SIRS-associated encephalopathy23 features microglia activation, neuronal death, and loss of different regions of the brain, and metabolic changes24. Microglia over-activation evokes pro-inflammatory molecules and cytokines migrating around neurons25, 26, 27. In our study, the brain of the SIRS mice model represents the typical activated microglia morphological change, such as larger soma size and reduced branch complexity in vivo. Also, NF-κB downstream maker genes, including IFN-γ, IL-6, and Cx3cl10 up-regulate in vitro on TNF administration. These cytokines have been proven to promote endothelial cells expressing adhesion molecules, facilitating the invasion of circulating immune cells and subsequently enabling more pronounced neuroinflammation. For example, TNF has been shown to disrupt endothelial integrity. Moreover, the change of adherent junction molecules like VE-cadherin down-regulated and cell adhesion molecules expressing on endothelial cells up-regulated strikingly, promoting the permeability of the BBB28. Endothelial cells have been reported to release CCL5, inducing migrating microglia to express CLDN5 and infiltrate through the neurovascular unit during moderate SIRS. The microglia also play important role in maintaining BBB integrity 29. Haruwaka et al. noted that the over-activated microglia engulfed astrocytic fragments and accelerated BBB leakage. Our results firmly proved that microglia participated in BBB damage and showed the phagocytic phenotype change. Besides the direct role of microglia in the neurovascular unit, the IL-1β produced by microglia could disrupt the BBB function directly30. As our data represented in this study, ketamine pre-treatment reversed BBB damage, alleviated microglia activation, and reduced the amount of IL-1β production in the brain. Ketamine may play a direct role in brain protection. Whether related to other effects is our focus.
HMGB1, a typical alarmin, is actively and passively released from immunocompetent cells and damaged cells31. Under inflammatory stress, HMGB1 dissociates from chromatin and promotes inflammatory reaction further after cell death. The actively non-classical secretion mechanism involving the JAK/STAT1-regulated pathway in assisting HMGB1 in translocating out of the nucleus to the cytoplasm32. HMGB1 promotes lipopolysaccharide (LPS) entering cells and induces pyroptosis in sepsis, exacerbating the ill condition of SIRS33. Besides, HMGB1 is usually selected as an indicator reflecting disease severity in patients34. In this study, we found that the amount of serum HMGB1 in the TNF group was more than that in the Sham group. We used HMGB1 neutralizing antibody to rescue the SIRS mice model, noting that sickness and BBB damage were alleviated. These beneficial effects match previous studies35. For the HMGB1 to BBB function, HMGB1 can bind to TLRs and RAGE on endothelial cells, promoting further TNF and other cytokines release36. As the receptor of HMGB1, RAGE exists the other type of soluble RAGE or endogenous secretory RAGE, serving as a “decoy” to tie up RAGE ligands like HMGB1. Consequently, NF-κB signaling activation is accompanied by the increased level of the IL-1β, decreased tight junction protein like ZO-1, and inducing neuroinflammatory damage at the BBB37, 38. The above analysis suggests that the BBB damage and neuroinflammation caused by TNF-induced SIRS may be related to the increase of HMGB1 in the serum.
Cecum plays a critical role during the SIRS process. Our previous study has detected severe damage and necroptosis that occurred in the cecum of TNF-induced SIRS mice9. More specifically, consolidated evidence has also suggested that cecum damage increased circulating HMGB1 levels and elevated inflammatory cytokines released by over-activated microglia and led to a neurological disorder, thereafter39, 40. In our study, SIRS mice receiving cecectomy surprisingly improved SIRS mice outcomes, including decreasing circulating HMGB1 level, improving BBB permeability, and inhibiting over-activated microglia. We also found that Rip3 knockout decreased HMGB1 release and alleviated BBB damage. It was reported that Rip3 knockout could prevent cecum damage and alleviate SIRS response13. Above all, the increase of HMGB1 in the serum is likely to be closely related to the cecal injury. These results broadened a novel horizon on the role of cecum during SIRS-associated BBB damage and neuroinflammation.
Ketamine, a non-competitive inhibitor to N-methyl-D-aspartate (NMDA) receptor, has the ability of anesthesia and analgesia. Besides, the anti-inflammatory ability of ketamine is the other advantage not to be ignored. Low-dose of ketamine (0.15 ~ 0.25 mg/kg (single i.v. bolus) can decrease pro-inflammatory cytokines after surgery, favoring more optimizing postoperative outcomes41. Ketamine inhibits NF-κB signaling, decreasing TNF, IL-6, C-reactive protein (CRP), and/or inducible nitric oxide synthase42. Additionally, ketamine remarkably limits the diapedesis of the neutrophils to the site of inflammation42 and resolves the inflammatory reaction by promoting immune cell apoptosis and further reducing pro-inflammatory cytokine release43. Moreover, clinical evidence has proved that the severe state of critically ill patients and experimental septic shock could be improved with ketamine intervention44. Overall, SIRS is the terminal disease state. Our previous work has provided valid evidence that ketamine intervention greatly prolonged the survival of mice and reduced SIRS symptoms8, 9, and NF-κB signaling downstream genes expression has been inhibited in our system in vivo and in vitro45. In this study, to our surprise, ketamine pre-treatment also exerted a protective effect on BBB integrity and neuroinflammation. Furthermore, ketamine pre-treatment also reduced cecum damage and decreased circulating HMGB1 levels. This protective effect of ketamine matches well with the previous reports46. Besides the previous HMGB1 inhibiting agents reported47, ketamine might be a novel inhibiting drug that could be applied to clinical practice as well. However, although the application of HMGB1 blocking regents has been reported to obtain a successful effect in reducing mortality in the SIRS mice model, there is no current evidence for the clinician to apply it to humans. And this question entails being solved in the future5.
In conclusion, this study provided a shred of validated evidence that cecum plays a crucial role in the integrity of BBB and neuroinflammation during SIRS. More importantly, our work provided a new theoretical view and therapeutic application prospect of ketamine in alleviating intestinal brain injury in TNF-induced severe SIRS.