Research progress of mechanisms for tight junction damage on blood-brain barrier inflammation.

Inflammation in the central nervous system (CNS) contributes to disease pathologies by disrupting the integrity of the blood-brain barrier (BBB). Tight junctions (TJ) are a key component of the BBB. Following hypoxic-ischaemic or mechanical injury to the brain, inflammatory mediators are released such as cytokines, chemokines, and growth factors. Simultaneously, matrix metalloproteinases (MMPs) are released which can degrade TJ proteins. Subsequently, the function and morphology of the BBB are disrupted, which allows immune cells an opportunity to enter into the brain parenchyma. This review summarises the information on the role of TJ protein families in the BBB and provides a comprehensive summary of the mechanisms whereby inflammation breaks down the BBB by increasing degradation of TJ proteins.

PAF Receptor Inhibition Attenuates Neuronal Pyroptosis in Cerebral Ischemia/Reperfusion Injury.

Ischemic stroke is an inflammation-related disease, during which process activation of NLRP3 inflammasome and subsequent pyroptosis play crucial roles. Platelet-activating factor (PAF) is a potent phospholipid regulator of inflammation which exerts its effect via binding specific PAF receptor (PAFR). However, whether PAFR contributes to pyroptosis during ischemia/reperfusion (I/R) injury remains to be elucidated. To explore the underlying effect of PAFR on ischemic stroke from the perspective of pyroptosis, mice were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) injury and primary cultures of mice cerebral cortical neurons were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) injury to mimic I/R in vivo and in vitro, after which indexes associated with pyroptosis were analyzed. Intriguingly, our results indicated that inhibition of PAFR with its inhibitor XQ-1H or PAFR siRNA exerted a neuroprotective effect against I/R injury both in vivo and in vitro. Furthermore, inflammasome activation and pyroptosis after ischemic challenge were attenuated by XQ-1H or PAFR siRNA. Besides, the protection of XQ-1H was abolished by PAF stimulaiton to some extent. Moreover, XQ-1H or PAFR siRNA alleviated the neuronal pyroptosis induced by LPS and nigericin (an NLRP3 activator) in cortical neurons. Taken together, this study firstly demonstrates that PAFR is involved in neuronal pyroptosis after I/R injury, and XQ-1H, a specific PAFR inhibitor, has a promising prospect in attenuating I/R injury from the perspective of anti-pyroptosis.

XQ-1H regulates Wnt/GSK3ß/ß-catenin pathway and ameliorates the integrity of blood brain barrier in mice with acute ischemic stroke.

10-O-(N, N-dimethylaminoethyl) ginkgolide B methanesulfonate (XQ-1H), a novel analog of ginkgolide B, has been preliminarily recognized to show bioactivities against ischemia-induced injury. However, the underlying mechanism still remains to be fully elucidated. The aim of this study was to investigate the effect of XQ-1H against cerebral ischemia/reperfusion injury (CIRI) from the perspective of blood brain barrier (BBB) protection, and explore whether the underlying mechanism is associated with Wnt/GSK3ß/ß-catenin signaling pathway activation. The therapeutic effects of XQ-1H were evaluated in mice subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and in immortalized mouse cerebral endothelial cells (bEnd.3) challenged by oxygen and glucose deprivation/reoxygenation (OGD/R). Results showed that treatment with XQ-1H improved neurological behavior, reduced brain infarction volume, diminished edema, and attenuated the disruption of BBB in vivo. In vitro, XQ-1H increased cell viability and maintained the barrier function of bEnd.3 monolayer after OGD/R. Moreover, the protection of XQ-1H was accompanied with activation of Wnt/GSK3ß/ß-catenin pathway and upregulation of tight junction proteins. Notably, the protection of XQ-1H was abolished by Wnt/GSK3ß/ß-catenin inhibitor XAV939 or ß-catenin siRNA, indicating XQ-1H exerted protection in a Wnt/GSK3ß/ß-catenin dependent profile. In summary, XQ-1H attenuated brain injury and maintained BBB integrity after CIRI, and the possible underlying mechanism may be related to the activation of Wnt/GSK3ß/ß-catenin pathway and upregulation of tight junction proteins.

JLX001 attenuates blood-brain barrier dysfunction in MCAO/R rats via activating the Wnt/ß-catenin signaling pathway.

JLX001, a new dihydrochloride of Cyclovirobuxine D (CVB-D), has bioactivities against ischemia injury. The blood-brain barrier (BBB) disruption is involved in the pathogeneses of ischemic stroke. This study was designed to explore the effect and potential mechanism of JLX001 on the BBB after ischemic stroke. Rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) to mimic cerebral ischemia in vivo. In vitro, rat primary brain microvascular endothelial cells (PBMECs) were cultured and exposed to oxygen-glucose deprivation/reoxygenation (OGD/R). Posttreatment of JLX001 for 15 days after MCAO/R improved the behavior, learning and memory ability. Pretreatment of JLX001 for 3 days significantly attenuated infarct volume, lessened brain edema, mitigated BBB disruption and decreased the neurological deficit score in MCAO/R rats. Moreover, JLX001 increased cell viability and reduced sodium fluorescein leakage after OGD/R injury. In addition, JLX001 increased the expressions of Claudin-5 and Occludin, decreased the expression of MMP-9 both in vivo and in vitro. Moreover, immunofluorescence staining and western immunoblotting results showed that JLX001 increased the expressions of tight junction proteins via activating Wnt/ß-catenin signal pathway in vivo and in vitro, which may be associated with the activation of PI3K/Akt signaling. Besides, XAV939 (an inhibitor of the Wnt/ß-catenin pathway) proved the connection of JLX001 and Wnt/ß-catenin pathway. These results suggest that JLX001 alleviates BBB disruption after MCAO/R and OGD/R possibly by alleviating MMP-9 and activating the Wnt/ß-catenin signaling pathway.

Ma Xing Shi Gan Decoction Protects against PM2.5-Induced Lung Injury through Suppression of Epithelial-to-Mesenchymal Transition (EMT) and Epithelial Barrier Disruption.

This research was designed to explore the effect of Ma Xing Shi Gan decoction (MXD) in alleviating particulate matter less than 2.5 µm in diameter (PM2.5) induced lung injury from the perspective of epithelial barrier protection and inhibition of epithelial-to-mesenchymal transition (EMT). Rats were exposed to PM2.5 to establish a lung injury model in vivo, and a PM2.5-stimulated primary cultured type II alveolar epithelial cell model was introduced in vitro. Our results indicated that MXD alleviated the weight loss and pathologic changes and improved the epithelial barrier dysfunction. MXD also significantly inhibited the TGF-ß/Smad3 pathway, increased the level of ZO-1 and claudin-5, and reversed the EMT process. Notably, the protection of MXD was abolished by TGF-ß in vitro. Our results indicated that MXD has a protection against PM2.5-induced lung injury. The proposed mechanism is reversing PM2.5-induced EMT through inhibiting TGF-ß/Smad3 pathway and then upregulating the expression of tight-junction proteins.

JLX001 improves myocardial ischemia-reperfusion injury by activating Jak2-Stat3 pathway.

AIMS: To investigate the preclinical pharmacodynamics and mechanism of JLX001 against myocardial ischemia reperfusion (MI/R) for clinical application. MATERIALS AND METHODS: In vivo, SD rats were given intragastric administration for 5 days, and the MI/R model was established by ligating/releasing the left anterior descending coronary artery. In vitro, the oxygen-glucose deprivation/reperfusion (OGD/R) model was established after the drug was pre-incubated for 24 h in H9C2 cells. The infract size was determined by TTC staining. Left ventricular function of MI/R rats was detected by echocardiography. The level of histopathological score was determined by hematoxylin-eosin (HE) staining. The level of superoxide dismutase (SOD), malondialdehyde (MDA), creatine kinase (CK), lactic dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) were determined by relevant kits. The level of apoptosis was measured by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Hoechst staining. The expression of p-Jak2, p-Stat3, Bax, Bcl-2, TNF-α, IL-1ß protein were determined by western blot. KEY FINDINGS: JLX001 can significantly improve left ventricular function, reduce myocardial infract size, histopathological score, the level of MDA, CK, LDH, TNF-α, IL-1ß and the expression of Bax protein, significantly increase the activity of SOD, Bcl-2 protein expression, p-Jak2 protein expression, p-Stat3 protein expression in rat heart tissues and H9C2 cells. These effects can be reversed by AG490 which is a specific inhibitor of Jak2-Stat3 pathway. SIGNIFICANCE: JLX001 can alleviate MI/R injury by inhibiting myocardial apoptosis, inflammation, and oxidative stress via Jak2-Stat3 pathway in vivo and in vitro.

Ma xing shi gan decoction eliminates PM2.5-induced lung injury by reducing pulmonary cell apoptosis through Akt/mTOR/p70S6K pathway in rats.

The present study was designed to investigate the anti-apoptosis effect of Ma xing shi gan decoction (MXD) on PM2.5-induced lung injury via protein kinase B (Akt)/mTOR/p70S6K pathway. A UPLC-MS/MS system was introduced for component analysis of MXD. Rats were instilled with PM2.5 solution suspension intratracheally to induce acute lung injury. The rats were then orally administered with MXD (16, 8, and 4 g/kg) once a day for 7 consecutive days. The therapeutic effects of MXD were evaluated by Hematoxylin and Eosin (HE) staining. The apoptotic cell death was analyzed by terminal-deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay. The alterations in cytochrome c (Cytc) and cleaved-caspase-3 (C-caspase-3) were measured by immunohistochemistry (IHC). The expressions of Bax, B-cell lymphoma 2 (Bcl-2), p-Akt, p-mTOR and p-p70S6K were detected by Western blot. In vitro, PM2.5 exposure model was introduced in A549 cell, followed by incubation with MXD-medicated serum. Hoechst staining was used to determine apoptotic rate. The levels of Bax, Bcl-2, p-Akt, p-mTOR and p-p70S6K were detected by Western blot. Our results in vivo indicated that treatment with MXD decreased histopathological changes score, TUNEL-positive cells rate, expressions of Cytc and C-caspase-3. The in vitro results revealed that incubation with MXD-mediated serum decreased apoptotic rate. Both results in vivo and in vitro demonstrated that MXD inhibited pro-apoptotic protein Bax and promoted anti-apoptotic protein Bcl-2 expression. Likewise, MXD activated Akt/mTOR/p70S6K signal pathway, which was also confirmed by Western immunoblotting. In conclusion, MXD attenuates lung injury and the underlying mechanisms may relate to regulating the apoptosis via Akt/mTOR/p70S6K signaling pathway activation.

Ma Xing Shi Gan Decoction Attenuates PM2.5 Induced Lung Injury via Inhibiting HMGB1/TLR4/NFκB Signal Pathway in Rat.

Ma Xing Shi Gan Decoction (MXD), a classical traditional Chinese medicine prescription, is widely used for the treatment of upper respiratory tract infection. However, the effect of MXD against particulate matters with diameter of less than 2.5 µm (PM2.5) induced lung injury remains to be elucidated. In this study, rats were stimulated with PM2.5 to induce lung injury. MXD was given orally once daily for five days. Lung tissues were harvested to assess pathological changes and edema. Myeloperoxidase (MPO) activity and malonaldehyde (MDA) content in lung were determined to evaluate the degree of injury. To assess the barrier disruption, the bronchoalveolar lavage fluid (BALF) was collected to determine the total protein content and count the number of neutrophils and macrophages. For evaluating the activation of macrophage in lung tissue, CD68 was detected using immunohistochemistry (IHC). The levels of inflammatory factors including tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), and interleukin-6 (IL-6) in BALF and serum were measured. In vitro, a PM2.5-activated RAW 264.7 macrophages inflammatory model was introduced. To evaluate the protective effect of MXD-medicated serum, the cell viability and the release of inflammatory factors were measured. The effects of MXD on the High mobility group box-1/Toll-like receptor 4/Nuclear factor-kappa B (HMGB1/TLR4/NFκB) pathway in lung tissue and RAW 264.7 cells were assessed by Western blot. For further confirming the protective effect of MXD was mediated by inhibiting the HMGB1/TLR4/NFκB pathway, RAW 264.7 cells were incubated with MXD-medicated serum alone or MXD-medicated serum plus recombinant HMGB1 (rHMGB1). MXD significantly ameliorated the lung injury in rats, as evidenced by decreases in the pathological score, lung edema, MPO activity, MDA content, CD68 positive macrophages number, disruption of alveolar capillary barrier and the levels of inflammatory factors. In vitro, MXD-medicated serum increased cell viability and inhibited the release of inflammatory cytokines. Furthermore, MXD treatment was found to inhibit HMGB1/TLR4/NFκB signal pathway both in vivo and in vitro. Moreover, the protection of MXD could be reversed by rHMGB1 in RAW 264.7. Taken together, these results suggest MXD protects rats from PM2.5 induced acute lung injury, possibly through the modulation of HMGB1/TLR4/NFκB pathway and inflammatory responses.

Research Progress of Mechanisms and Drug Therapy For Atherosclerosis on Toll-Like Receptor Pathway.

Recent reports have established atherosclerosis (AS) as a major factor in the pathogenetic process of cardiovascular diseases such as ischemic stroke and coronary heart disease. Although the possible pathogenesis of AS remains to be elucidated, a large number of investigations strongly suggest that the inhibition of toll-like receptors (TLRs) alleviates the severity of AS to some extent by suppressing vascular inflammation and the formation of atherosclerotic plaques. As pattern recognition receptors, TLRs occupy a vital position in innate immunity, mediating various signaling pathways in infective and sterile inflammation. This review summarizes the available data on the research progress of AS and the latest antiatherosclerotic drugs associated with TLR pathway.

JLX001 Modulated the Inflammatory Reaction and Oxidative Stress in pMCAO Rats via Inhibiting the TLR2/4-NF-κB Signaling Pathway.

Inflammatory reactions and oxidative stress play critical roles in cerebral ischemic injuries. Microglia are activated after ischemic injury. Activated microglia produce neurotoxic proinflammatory factors and reactive oxygen species (ROS), which have been demonstrated closely related TLR2/4-NF-κB signal pathways. This study was to evaluate the effect of JLX001 against ischemic injury and investigate the mechanisms. The permanent middle cerebral artery occlusion (pMCAO) model was employed in rats. The neurobehavioral score, brain infarction rate, brain water content, pathological changes, immunohistochemical staining, biochemical index (T-AOC, SOD, and MDA), proinflammatory factors (IL-1ß, TNF-α, and NO), expression of TLR2/4 and nuclear translocation of NF-κB p65 were determined. To explore probable underlying mechanism of the neuroprotective effect of JLX001, BV-2 cells were exposed to in oxygen-glucose deprivation (OGD) for 4 h to mimic ischemic injury in vitro. The result showed that JLX001 significantly decreased neurological deficit score, infarct size, and brain edema, attenuated pathological changes, inhibited the activation of microglia, improved the process of oxidative stress, reduced the release of proinflammatory cytokines and downregulated TLR2/4-NF-κB signal pathway. Moreover, OGD reduced BV2 cell viability, induced oxidative damage, increased the release of proinflammatory factors and activated TLR2/4-NF-κB signal pathway, which was significantly reversed by the intervention of JLX001. This study demonstrates that JLX001 is effective in protecting the brain from ischemic injury, which may be mediated by regulating oxidative stress, inflammation and inhibiting TLR2/4-NFκB signal pathway.