This is the first study investigating the neuroprotection effect of baicalein on VD. Our research has found that (1) baicalein improved cognitive dysfunction in VD rats and significantly improved CCH-induced neuronal damage in the CA1 region of the hippocampus; (2) baicalein treatment reduced glial cell activation and release of pro-inflammatory factors, TLR4/MyD88/NF-κB signaling pathway might play an important role; (3) baicalein modulated the diversity and composition of the intestinal microbiota, and suppressed the relative abundance of inflammation-associated microbiota in VD rats.
CCH plays a key role in the development of cognitive deficits in VD by inducing neuroinflammation, decreasing energy supply, causing neuronal damage and impairing memory. The CCH animal model is considered a good method to study VD. We usually apply the classical animal model of bilateral carotid artery ligation to explore the effects of CCH on cognitive function and its mechanisms [12].
In the MWM test of the study, rats in the model group exhibited spatial learning and memory deficits as escape latency were prolonged, the number of traversing platform positions and the time spent in the target quadrant were reduced. And baicalein treatment significantly improved cognitive dysfunction due to CCH. Due to its high basal metabolism, the hippocampus is more sensitive to hypoxia and ischemia and is one of the earliest brain regions to be pathologically damaged. Neuronal damage in the CA1 region of the hippocampus is the main pathologic change in CCH-induced cognitive impairment [13]. Our study found that baicalein treatment significantly ameliorated CCH-induced neuronal damage in the CA1 region of the hippocampus and improved cognitive impairment.
In the central nervous system, microglia and astrocytes play an important role in the neuroinflammatory response. Glial cell activation and neuroinflammation induced by cerebral hypoperfusion play a crucial role in the pathophysiological mechanisms of VD [14,15]. Activation of immune cells and release of neuroinflammatory factors are associated with synaptic plasticity, neuronal survival, and developmental regulation of nerves that affect learning and memory. Microglia are considered to be macrophages in brain tissue and are important immune cells in the central nervous system. Activated microglia release pro-inflammatory factors such as IL-1β, IL-6, and TNF-α to promote neuronal degeneration in the CCH model [16]. Iba1 is a protein expressed on the surface of microglia. We found an increased number of Iba1 positive cells in the hippocampus of CCH rats, consistent with increased pro-inflammatory factors in the hippocampus. Astrocytes make up 40% of all brain cells, are the most abundant cells in the central nervous system, and mediate inflammatory responses in many diseases [17,18]. GFAP is the most widely used astrocyte marker and can be used to monitor nerve injury and inflammation. Our study showed that GFAP and Iba, as well as TNF-α, IL-6, and IL-1β levels were significantly elevated in the model group, whereas baicalein treatment significantly reduced CCH-induced glial cell activation and pro-inflammatory factor release. It suggests that baicalein can reduce neuroinflammation, exert neuroprotective effects, and attenuate cognitive dysfunction in the CCH model. This is consistent with previous findings that baicalein improves cognitive dysfunction due to other diseases through anti-inflammation [19,20].
In the BCCAO animal model, the TLR4/MyD88 signaling pathway is involved in neuroinflammation after CCH induction and is a major initiator of proinflammatory factors in glial cell activation [21]. TLRs are major regulators of the innate immune system and play an important role in the inflammatory response induced by ischemic brain injury [22]. Among the TLR family members, TLR4 is widely expressed in neurons, microglia, astrocytes, and endothelial cells in the CNS [23]. Microglia play an important role in neuroinflammation as the first line of immune defense in the central nervous system. TLR4 is abundantly expressed in microglia and induces microglia activation [24]. Activated microglia cause an inflammatory cascade response that leads to neurologic dysfunction. A study of ischemia-reperfusion injury suggests that upregulation of TLR4, microglia activation, and elevated levels of inflammatory cytokines induce a neuroinflammatory response and reduce neuronal viability [25]. Our results showed that baicalein treatment reversed the elevation of TLR4 protein in the hippocampus of CCH rats, suggesting that TLR4 may be a potential target for baicalein to regulate VD inflammation.
MyD88 is an important downstream signaling ligand of TLR4 and also an adapter protein of the NF-κB signaling pathway. The NF – κB complex is a homodimer or heterodimer composed of the p50 and p65 subunits. Normally, NF - κB p50 and p65 dimers remain inactivated in the cytoplasm. TLR4 agonists activate the MyD88-dependent intracellular signaling pathway, leading to translocation of the NF-κb p65 subunit into the nucleus and binding to specific DNA sequences, which leads to enhanced transcription of inflammation-related proteins and triggers the production of inflammatory factors [26,27]. Previous studies have found that TLR4 and MyD88 are elevated in surgically induced activated microglia, induce NF-κB p65 phosphorylation and nuclear translocation, elevate inflammatory cytokine levels, promote neuroinflammation, and induce apoptosis in hippocampal neurons [28]. The results of our study show that in CCH rats, TLR4 protein is upregulated, activates its downstream signal MyD88, and contributes to the downstream activation of NF - κB signaling, which is characterized by enhanced phosphorylation of NF-κb p65. While baicalein treatment significantly inhibited the up-regulated expression of TLR4, MyD88, and thus the phosphorylation of NF - κB p65. The above results suggest that the TLR4/MyD88/NF-κB signaling pathway is at least partially involved in CCH, as well as the protective mechanism of baicalein in VD rats.
Changes in the composition of intestinal microbiota are strongly associated with central nervous system disorders. The intestinal microbiota can influence the brain-gut axis and, in turn, cognitive performance. The intestinal flora of healthy people remains in a relatively stable state. Under pathological conditions, certain bacteria in the intestinal microenvironment can overgrow [29]. In our alpha diversity analysis, the model group showed a significant increase in the abundance of intestinal flora compared to the sham group, suggesting that intestinal flora may be involved in the pathogenesis of VD. Baicalein treatment, on the other hand, further increased the alpha diversity of the intestinal microbiota in VD rats.
To further explore which specific species have changed, we conducted a species composition analysis. The gastrointestinal tract is home to 1013-1014 species of microorganisms, most of which are Firmicutes and Bacteroidetes [30]. The same results were obtained in our study, where the species composition of the intestinal flora of rats in all groups was analyzed, with the Firmicutes and Bacteroidetes as the dominant phyla. Among them, rats in the model group showed an increase in the abundance of the Firmicutes and a decrease in the abundance of the Bacteroidetes. Clinical studies have shown a significant reduction in the abundance of Bacteroidetes in the intestinal tract of AD patients, confirming the protective role of the intestinal barrier [31,32]. The fact that Bacteroidetes regulates endothelial cell function and attenuates the inflammatory response is consistent with our finding of an inverse relationship between the bacterium and the presence of dementia [33]. Firmicutes is recognized as a risk factor for the development of Alzheimer's disease [34]. Saji N et al. showed that patients with dementia had a higher ratio of Firmicutes and Bacteroidetes than non-demented patients [35], which is consistent with the results of our study. Treatment with baicalein down-regulated the abundance of Firmicutes and up-regulated the abundance of Bacteroidetes. At the genus level, the abundance of Lactobacillus and Clostridium was up-regulated in the model group of rats. Lactobacillus are known to be common probiotics. Supplementation with probiotics, such as Bifidobacterium and Lactobacillus, significantly improves cognitive performance in AD patients [36]. Intestinal Lactobacillus in AD mice were significantly lower than in the normal group [37]. However, other studies have found increased abundance of Lactobacillus in AD patients [38]. Lactobacillaceae levels are significantly higher in patients with PD combined with cognitive impairment compared to patients with PD without cognitive impairment [39]. Similarly, our study found that Lactobacillus were significantly elevated in CCH rats. Previous studies have found increased relative abundance of Clostridium in the gut of mice with diabetes combined with cognitive impairment [40]. Clostridium were similarly detected in high abundance in the intestinal flora of AD mice [41]. It is suggested that the relative abundance of Clostridium is highly correlated with cognitive impairment. In this study, we found that baicalein down-regulated Clostridium abundance in VD rats.
PCoA analysis showed that the composition of the intestinal microbiota changed significantly between groups. The beta diversity analysis allowed us to determine that the composition of the intestinal flora differed between groups, and in order to find out which flora caused the differences in the communities, we applied LEfSe for the flora difference analysis to look for the biomarker. The methodology combines a statistical analysis of variance and the influence score value of that variance species on the grouping results, while emphasizing statistical significance and biological relevance.
Intestinal microbiota can regulate microglia function by influencing the body's immune function, thereby modulating the secretion of pro-inflammatory factors [42]. Therefore, in our study, the differential species in each group obtained from LDA analysis were subjected to spearman correlation analysis with hippocampal pro-inflammatory factors, thus exploring the link between gut flora and hippocampal inflammation in CCH rats. Consistent with previous studies, Prevotella interacts with the immune system to enhance neuroinflammatory responses, and its abundance is significantly increased in the gut of AD patients and negatively correlates with cognitive performance [43,44,45]. Our study similarly found that the abundance of Prevotella as a marker species for the model group was positively correlated with the levels of pro-inflammatory factors. Our study showed that the abundance of beneficial bacteria with anti-inflammatory effects, such as Blautia and Eubacterium, were significantly increased after the administration of baicalein. Relative abundance of Blautia is low in patients with PD combined with cognitive impairment [46]. The present study likewise found that the abundance of Blautia, a marker species in the high-dose group of baicalein, was positively correlated with cognitive levels and negatively correlated with IL-6 and IL-1β levels, that is to say, the worse the cognition, the lower the abundance of Blautia, and the higher the levels of pro-inflammatory factors.
There are some limitations of this study. First, the TLR4/MyD88/NF-κB signaling pathway involves many upstream and downstream regulators, which we will explore more deeply in future experiments. Second, the interactions between intestinal microbiota and the nervous system are very complex, and the sample size of the intestinal microbiota analyzed in this study was small, more mechanisms of the "brain-gut" axis remain to be discovered. Third, short-chain fatty acids (SCFAs) are the main metabolites produced by intestinal bacteria, and their correlation with neuroinflammation should not be ignored. Whether the neuroprotective effect of baicalein is related to SCFAs still requires further study in the future. Performing intestinal microbiota transplantation may further confirm the potential role of the gut microbes identified in this study.