Neonatal bacterial meningitis is a serious life-threatening infectious disease of the central nervous system in pediatrics. The incidence of neonatal bacterial meningitis accounts for approximately 0.2– 6.1‰ of live births [3, 5]. The clinical manifestations of neonatal bacterial meningitis are atypical, and thus early diagnosis can be challenging and can affect timely treatment. The incidence of E. coli meningitis especially among late newborns, increased annually in recent years. Due to atypical clinical symptoms, life-threatening conditions and highly-drug-resistant nature of neonatal E. coli bacterial meningitis [22], understanding the molecular mechanism of the disease and finding a therapeutic direction are necessary.
The BBB is formed by brain microvascular endothelial cells, and the TJ seals the intercellular space and form a physical barrier. TJ is a complex formed by a variety of TJ proteins, which are essential for the integrity and function of the BBB. Damage to the BBB allows pathogenic bacteria to penetrate the brain, which can lead to bacterial meningitis. Different pathogenic bacteria can damage the BBB through various mechanisms; however, the molecular mechanism of the damage of E. coli to the BBB by destroying TJ has not been fully elucidated [23].
In this study, we detected the expression of TJ proteins including, ZO-1, occludin, and claudin-5. These proteins can interact with cytoskeleton scaffold proteins to form atresia bands in cells and strengthen the connection between cells [24]. Studies have shown that a decrease in TJ integrity can lead to the destruction of the BBB. Loss in barrier integrity allows antigens (microorganisms, toxins) in the blood vessels to enter the brain through the intercellular space, consequently destroying the homeostasis of the intracranial environment, leading to inflammatory response [25]. Some pathogens can cause vascular endothelial cell permeability defects by changing the integrity of TJ [26]. LPS is an endotoxin of E. coli, which can destroy the barrier function of microvascular endothelial cells [27]. To clarify the molecular mechanism of E. coli passing through BBB, this study used LPS as a stimulator to understand the expression pattern of TJ proteins in bEND.3 cells, and explore the effect of the PI3K/Akt signaling pathway on cell-cell connections.
bEND.3 cells are vascular endothelial cells commonly constituting the BBB in mice, and is often used in studying BBB in vitro. Lanhui et al. used LPS to stimulate human brain microvascular endothelial cells and found a reduced expression of ZO-1 and occludin, suggesting that TJ proteins play a key role in mediating BBB permeability [28]. In this experiment, we stimulated bEND.3 cells with 10 ng/ml and 1 ug/ml concentrations of LPS for 12 h, and found that the amount of TJ proteins on the cell membrane decreased, especially the expression of ZO-1. The higher the concentration of LPS, the less the amount of ZO-1 on the cell membrane. When 1 ug/ml of LPS was used to stimulate bEND.3 cells, the expression of ZO-1 decreased significantly. However, the expression of occludin and claudin-5 protein did not decrease significantly, which can be attributed to the different research cells selected in this experiment. Dong et al. found that the protein levels of claudin, occludin, and ZO-1 are negatively correlated with the mRNA levels of IL-6, IL-1 β, and TNF- α in mice infected with bacteria [29]. Zhao et al. [30] stimulated human umbilical cord microvascular endothelial cells with different concentrations of LPS in vitro, and found that the content of intracellular reactive oxygen species (ROS) increased significantly. Moreover, the TJ between cells was destroyed, and the amount of TJ protein on the cell membrane decreased. In this experiment, the concentration of ZO-1 protein decreased with the increase of LPS. After LPS stimulation, cells produced many inflammatory factors and ROS; the higher the concentration of LPS, the more inflammatory factors and ROS were produced. Thus, the production of TJ protein was inhibited. We performed cellular immunofluorescence on LPS stimulated and control cells at the cellular level. Compared with the control group, the immunofluorescence of the cell membrane in the LPS stimulation group decreased significantly, indicating that the expression of ZO-1 on the cell membrane also decreased. The results were consistent with the protein level.
LPS-induced neuroinflammation was not only dose- but also time-related [31]. Biesmans et al. [32] have found that neuroinflammation changed over time and certain symptoms in the brains of mice subsided after 24 h of LPS intervention. In this study, decreased TJ protein was most significant when the cells were challenged with 1ug/ml LPS. To further explore the effect of LPS on the TJ of bEND.3 cells, we stimulated the cells with 1ug/ml LPS across different timepoints, and observed changes in the expression pattern of ZO-1, occludin, and claudin-5, which decreased through time. The expression of ZO-1 was reduced especially when stimulated for 24 h, and the difference was statistically significant (P < 0.05). Meanwhile, no significant reduction in the expression of occludin and claudin-5 protein was observed (P > 0.05). Fang et al. [33] stimulated the endothelial cells in vitro with 10 ug/ml LPS and found that LPS can downregulate the expression of ZO-1, claudin-5, and occludin. Moreover, the time with the lowest expression level was consistent with the time with the lowest TEER value.
In this experiment, only the ZO-1 protein in TJ decreased significantly, while occludin and claudin-5 protein did not, which can be due to the cell state and LPS concentration in this study. Most ZO-1 are located at the TJ of cells, and the stimulation of LPS can reduce the expression of TJ protein. Studies have shown that vitamin A can reverse the effect of LPS; however, the specific mechanism remains unclear [34].
PI3K is a family of lipid kinases. Under physiological conditions, PI3K is typically activated by extracellular signals, including growth factors, cytokines, and hormones. The activation and stability of Akt are carefully regulated by phosphorylation of a variety of signal molecules [35]. In this experiment, after stimulating bEND.3 cells with 1ug/ml LPS, we found that the expression of p-PI3K did not change significantly; however, p-Akt began to increase after 2 h and peaked at 4 h. Zheng et al. [36] challenged the human lung microvascular endothelial cells with LPS, and found that p-Akt initially increased and then decreased with the stimulation time, which is consistent with our experiments. Moreover, we speculate that the downregulation of the expression of ZO-1 induced by LPS in bEND.3 cells may be related to the phosphorylation of Akt in the signal pathway. Since the expression of ZO-1 protein began to decrease significantly after 2 h of stimulation and reached the peak 12 h after stimulation, it’s expression decreased significantly earlier than that before the translation. However, there was no significant change in p-PI3K after LPS stimulation. It was considered that PI3K signal molecules were not involved in the regulation of TJ protein. Currently the mechanism of how activated Akt regulates TJ protein has not been elucidated. Studies have shown that LPS can downregulate the phosphorylation of Akt and 4e-bp1 in endothelial cells, subsequently damaging the cell barrier. It is suggested that LPS may reduce the density of TJ protein by inhibiting Akt/mTOR-mediated protein synthesis [18]. Yu et al. [19] reported that TJ fracture can be induced by activating the ROS/AKT/p38MAPK pathway. They proposed that ASK1 and p38 MAPK were triggered by generating a large amount of ROS, which activates Akt, leading to a significant decrease in the expression of ZO-1. Inhibition of production of ROS using N-acetyl-L-cysteine can attenuate the activation of Akt, Ask1, p38-MApk, and the downregulation of ZO-1. In this experiment, the downregulation of TJ protein is considered to be caused by LPS activation of Akt, which in turn activates the downstream signaling molecules.
In conclusion, LPS can downregulate the expression of ZO-1 protein in bEND.3 cells in vitro, through the Akt signal pathway.