RhoA/ROCK-2 Pathway Inhibition and Tight Junction Protein Upregulation by Catalpol Suppresses Lipopolysaccaride-Induced Disruption of Blood-Brain Barrier Permeability.

Shan Feng,Huifeng Zhu,Ran He,Hongjin Wang,Li Zou,Ke Liu

doi:10.3390/molecules23092371

Abstract

Lipopolysaccaride (LPS) directly or indirectly injures brain microvascular endothelial cells (BMECs) and damages the intercellular tight junction that gives rise to altered blood-brain barrier (BBB) permeability. Catalpol plays a protective role in LPS-induced injury, but whether catalpol protects against LPS-caused damage of BBB permeability and the underlying mechanism remain to be delineated. Prophylactic protection with catalpol (5 mg/kg, i.v.) consecutively for three days reversed the LPS-induced damage of BBB by decreased Evans Blue (EB) leakage and restored tight junctions in C57 mice. Besides, catalpol co-administrated with LPS increased BMECs survival, decreased their endothelin-1, TNF-Α and IL-6 secretion, improved transmembrane electrical resistance in a time-dependent manner, and in addition increased the fluorescein sodium permeability coefficient of BMECs. Also, transmission electron microscopy showed catalpol protective effects on tight junctions. Fluorescence staining displayed that catalpol reversed the rearrangement of the cytoskeleton protein F-actin and upregulated the tight junction protein of claudin-5 and ZO-1, which have been further demonstrated by the mRNA and protein expression levels of ZO-1, ZO-2, ZO-3, claudin-5, and occludin. Moreover, catalpol concurrently downregulated the mRNA and protein levels of RhoA, and ROCK2, the critical proteins in the RhoA/ROCK2 signaling pathway. This study thus indicated that catalpol, via inhibition of the RhoA/ROCK2 signaling pathway, reverses the disaggregation of cytoskeleton actin in BMECs and prevents down-regulation of junctional proteins, such as claudin-5, occludin, and ZO-1, and decreases endothelin-1 and inflammatory cytokine secretion, eventually alleviating the increase in LPS-induced BBB permeability.

Highlights

The blood-brain barrier (BBB) is a major internal barrier in the human body
The present study found that catalpol exerted a protective role in the damage of LPS-induced BBB permeability by down-regulating the RhoA/ROCK2 signaling pathway as well as up-regulating the brain microvascular endothelial cells (BMECs) tight junction proteins
The damage status of BBB structure can be reflected by the fluorescence intensity of Evans Blue (EB)

Summary

Introduction

The blood-brain barrier (BBB) is a major internal barrier in the human body. It comprises brain microvascular endothelial cells (BMECs), pericytes, astrocytic end-feet, basal laminar cells, neurons, and microglia. The tight junction between BMECs is the fundamental structure of the BBB, the first barrier to maintain homeostasis of the cerebral microenvironment [1]. The tight junction is composed of transmembrane proteins, cytoplasmic attachment proteins, and the cytoskeleton protein F-actin (Figure 1). The transmembrane proteins encompass three types of intact membrane proteins—occludin, claudin, and junction adhesion molecules (JAMs) [2].

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