Sepsis, which is defined as a severe life-threatening organ dysfunction syndrome caused by a dysregulated host response to infection, initiates a complex inflammatory response cascade process and causes damage to the fate of the individual [22]. Especially during sepsis, subsequent inflammation triggers central nervous system (CNS) dysfunction and affects cognitive function, which induces irreversible consequences of SAE in the host brain [23]. SAE-induced brain damage plays a vital role in the survival and prognosis of septic patients, which is caused not only by an injured organ but also by impaired immunomodulation of the brain and CNS. The impaired activity of the CNS is involved in the acceleration of immune system inflammation, mainly through the blood brain barrier (BBB) and activation of neurons to reach different brain regions [24]. To better simulate and reflect the pathological changes of abdominal infection in different phases of SAE in practice, we chose the classic gold-standard method, namely cecal ligation and puncture (CLP) operation, to establish an experimental SD rat model for sepsis in this study. After CLP operation for 24 h, the vital signs and neurobehavioral test scores and EEG and SEP of the CLP group rats showed worse vital signs (MAP and HR), slower movements, and mental pathological changes with decreased survival percentage compared to the sham group, indicating the successful establishment of the SAE rat model. At present, the treatment and pathological processes of SAE have seen much research published [25]. We demonstrated that TMP has protective effects against SAE injury and improves cognitive function through the activation of autophagy in the hippocampus. Moreover, inhibition of autophagy with chloroquine resulted in lower autophagy levels induced by TMP administration in SAE rats. These results demonstrated that TMP enhances autophagy and is involved in the regulation of SAE.
TMP is an active alkaloid widely applied as a traditional Chinese medicine that exerts anti-inflammatory, antioxidant, neuroprotective, and other excellent properties [26, 27]. Previously we found that TMP ameliorated lipopolysaccharide-induced sepsis in rats by protecting the blood-brain barrier and impairing inflammation and nitrous oxide systems [27]. The experimental results showed that CLP caused infection and inflammation, which passed through the blood-brain barrier (BBB), which is consistent with the current research on the gut-brain axis [28]. In this study, we continued to study autophagy activity from the perspective of TMP administration in SAE rats. We found that TMP significantly increased the escape latency and the time spent in the target quadrant and the frequency of rats crossing the goal platform area in TMP intervention rats, indicating that TMP could ameliorate the spatial learning and memory abilities induced by SAE. In addition, TMP recovered the pathological injury and damaged cellular structure in the CA1 structure of the hippocampus by pathological observation. Therefore, these experimental data suggest that TMP exerts protective effects against SAE-induced cognitive injury.
Toll-like receptors (TLRs) are one of the major pattern recognition receptors (PRRs) expressed by immune and non-immune cells, including neurons, which play a crucial role in generating cytokine storms [29]. TLRs are the first step in the body's defense, and when microbial-associated molecular patterns (MAMPs) and damage or death-associated molecular patterns (DAMPs) are stimulated and enter cells, they recognize and release several pro-inflammatory mediators, which cause an acute inflammatory response meant to control the pathogen and repair the damage [30]. Host-derived endogenous TLR1 plays a significant role in the onset of sepsis-associated cytokine storm and is important for sentry duty in SAE rats. In our study, we found that the expression of TLR1 in the rats of the SAE group was higher than that in the Sham group rats, suggesting that sepsis-induced abdominal infection was encroached into the hippocampus and caused cognitive decline. Subsequently, the pro-inflammatory factors IL-1β, IL-6, and TNF-α in the SAE group were significantly higher than those in the sham group, which demonstrated that SAE induced inflammation in the central nervous system. TMP administration significantly decreased the levels of IL-1β, IL-6, and TNF-α in the TMP group compared with those in the SAE group, indicating that TMP improved the endogenous microenvironment by reducing the levels of inflammatory cytokines. Further studies are needed to investigate the associations between inflammation and autophagy, as well as inflammatory signaling pathways, following TMP administration in SAE rats.
Although numerous studies have shown that autophagy participates in the necessary process of regulation of many diseases [18], but it remains unclear whether autophagy is involved in the pathophysiological basis of SAE disorder and what the mechanism responsible for the TMP-mediated protection, we explore the study and want to find a novel approach for treating the highly lethal illness. The autophagy-related proteins p62, LC3I, LC3II, ATG5, and Beclin1 were used as the characteristic indicators to detect the occurrence of autophagy flux in our study. We found that the expression of p62, ATG5, and Beclin1 was significantly upregulated and the ratio of LC3II/LC3I was also significantly increased compared to the Sham group. In the TMP group, the expression of TLR1 and p62 was significantly downregulated, and the ratio of LC3II/LC3I and the expression of ATG5 and Beclin1 were increased compared to those in the SAE group. To determine the process of autophagy flux in TMP-induced neuroprotection, we established a rapamycin-initiated positive control group (RAPA group) and used it to stimulate autophagy flux in vivo. The same trend was observed for the expression of autophagy-related proteins in the TMP and RAPA groups. Our research findings were consistent with the published results of previous studies [14, 15, 19, 31], accurately illustrating that TMP showed the same experimental results as the mTOR inhibitor rapamycin and demonstrated that TMP and rapamycin had the same effects on the activation of autophagy in the hippocampus under SAE conditions. To further clarify the role of autophagy flux in TMP-improved SAE, we used the autophagy inhibitor CQ to interfere with the process of autophagy in the targeted design groups and to supervise the activity of LC3 and p62 proteins in CQ-treated rats. We found that the administration of CQ significantly upregulated the expression of p62 and downregulated the ratio of LC3II/LC3I in the SAE + CQ and SAE + TMP + CQ groups, indicating that CQ could reverse the level of autophagic flux activated by TMP. The p62 protein participates in the delivery of ubiquitinated capsid proteins to autophagosomes for degradation as one of the autophagy cargo receptors and serves as a recognition signal for autophagy [32]. Our study revealed that the expression of p62 and LC3II proteins was significantly altered in the hippocampus of SAE rats, which could indicate that the activity of autophagy was greatly activated and the degradation of autophagy was unblocked by TMP treatment. However, further investigations are necessary to explore the precise mechanism and role of TMP in the regulation of autophagic flux.