Tongxinluo Exerts Inhibitory Effects on Pyroptosis and Amyloid-ß Peptide Accumulation after Cerebral Ischemia/Reperfusion in Rats.

Amyloid-ß peptide (Aß) accumulation is a detrimental factor in cerebral ischemia/reperfusion (I/R) injuries accounting for dementia induced by ischemic stroke. In addition to blood brain barrier (BBB), the glymphatic system mediated by aquaporin-4 (AQP-4) on astrocytic endfeet functions as an important pathway for the clearance of Aß in the brain. Cerebral I/R induced astrocytic pyroptosis potentially causes the AQP-4 polarization loss and dysfunctional BBB-glymphatic system exacerbating the accumulation of Aß. Furthermore, Aß toxicity has been identified as a trigger of pyroptosis and BBB damage, suggesting an amplified effect of Aß accumulation after cerebral I/R. Therefore, based on our previous work, this study was designed to explore the intervention effects of Tongxinluo (TXL) on astrocytic pyroptosis and Aß accumulation after cerebral I/R in rats. The results showed that TXL intervention obviously alleviated the degree of pyroptosis by downregulating expression levels of cleaved caspase-11/1, N-terminal gasdermin D, nucleotide-binding oligomerization domain-like receptors pyrin domain containing 3 (NLRP3), interleukin-6 (IL-6), and cleaved IL-1ß and abated astrocytic pyroptosis after cerebral I/R. Moreover, TXL intervention facilitated to restore AQP-4 polarization and accordingly relieve Aß accumulation around astrocytes in ischemic cortex and hippocampus as well as the formation of toxic Aß (Aß 1-42 oligomer). Our study indicated that TXL intervention could exert protective effects on ischemic brain tissues against pyroptotic cell death, inhibit astrocytic pyroptosis, and reduce toxic Aß accumulation around astrocytes in cerebral I/R injuries. Furthermore, our study provides biological evidence for the potential possibility of preventing and treating poststroke dementia with TXL in clinical practice.

Destructive Effects of Pyroptosis on Homeostasis of Neuron Survival Associated with the Dysfunctional BBB-Glymphatic System and Amyloid-Beta Accumulation after Cerebral Ischemia/Reperfusion in Rats.

Neuroinflammation-related amyloid-beta peptide (Aß) accumulation after cerebral ischemia/reperfusion (I/R) accounts for cerebral I/R injuries and poststroke dementia. Recently, pyroptosis, a proinflammatory cell death, has been identified as a crucial pathological link of cerebral I/R injuries. However, whether pyroptosis acts as a trigger of Aß accumulation after cerebral I/R has not yet been demonstrated. Blood-brain barrier (BBB) and glymphatic system mediated by aquaporin-4 (AQP-4) on astrocytic endfeet are important pathways for the clearance of Aß in the brain, and pyroptosis especially occurring in astrocytes after cerebral I/R potentially damages BBB integrity and glymphatic function and thus influences Aß clearance and brain homeostasis. In present study, the method of middle cerebral artery occlusion/reperfusion (MCAO/R) was used for building models of focal cerebral I/R injuries in rats. Then, we used lipopolysaccharide and glycine as the agonist and inhibitor of pyroptosis, respectively, Western blotting for detections of pyroptosis, AQP-4, and Aß 1-42 oligomers, laser confocal microscopy for observations of pyroptosis and Aß locations, and immunohistochemical stainings of SMI 71 (a specific marker for BBB integrity)/AQP-4 and Nissl staining for evaluating, respectively, BBB-glymphatic system and neuronal damage. The results showed that pyroptosis obviously promoted the loss of BBB integrity and AQP-4 polarization, brain edema, Aß accumulation, and the formation of Aß 1-42 oligomers and thus increased neuronal damage after cerebral I/R. However, glycine could inhibit cerebral I/R-induced pyroptosis by alleviating cytomembrane damage and downregulating expression levels of cleaved caspase-11/1, N-terminal gasdermin D, NLRP3 (nucleotide-binding domain, leucine-rich repeat containing protein 3), interleukin-6 (IL-6) and IL-1ß and markedly abate above pathological changes. Our study revealed that pyroptosis is a considerable factor causing toxic Aß accumulation, dysfunctional BBB-glymphatic system, and neurological deficits after cerebral I/R, suggesting that targeting pyroptosis is a potential strategy for the prevention of ischemic stroke sequelae including dementia.

Yi-Zhi-Fang-Dai Formula Exerts Neuroprotective Effects Against Pyroptosis and Blood-Brain Barrier-Glymphatic Dysfunctions to Prevent Amyloid-Beta Acute Accumulation After Cerebral Ischemia and Reperfusion in Rats.

Background: The dysfunctional blood-brain barrier (BBB)-glymphatic system is responsible for triggering intracerebral amyloid-beta peptide (Aß) accumulation and acts as the key link between ischemic stroke and dementia dominated by Alzheimer's disease (AD). Recently, pyroptosis in cerebral ischemia and reperfusion (I/R) injury is demonstrated as a considerable mechanism causing BBB-glymphatic dysfunctions and Aß acute accumulation in the brain. Targeting glial pyroptosis to protect BBB-glymphatic functions after cerebral I/R could offer a new viewpoint to prevent Aß accumulation and poststroke dementia. Yi-Zhi-Fang-Dai formula (YZFDF) is an herbal prescription used to cure dementia with multiple effects of regulating inflammatory responses and protecting the BBB against toxic Aß-induced damage. Hence, YZFDF potentially possesses neuroprotective effects against cerebral I/R injury and the early pathology of poststroke dementia, which evokes our current study. Objectives: The present study was designed to confirm the potential efficacy of YZFDF against cerebral I/R injury and explore the possible mechanism associated with alleviating Aß acute accumulation. Methods: The models of cerebral I/R injury in rats were built by the method of middle cerebral artery occlusion/reperfusion (MCAO/R). First, neurological function assessment and cerebral infarct measurement were used for confirming the efficacy of YZFDF on cerebral I/R injury, and the optimal dosage (YZFDF-H) was selected to conduct the experiments, which included Western blotting detections of pyroptosis, Aß1-42 oligomers, and NeuN, immunofluorescence observations of glial pyroptosis, aquaporin-4 (AQP-4), and Aß locations, brain water content measurement, SMI 71 (a specific marker for BBB)/AQP-4 immunohistochemistry, and Nissl staining to further evaluate BBB-glymphatic functions and neuronal damage. Results: YZFDF obviously alleviated neurological deficits and cerebral infarct after cerebral I/R in rats. Furthermore, YZFDF could inactivate pyroptosis signaling via inhibiting caspase-1/11 activation and gasdermin D cleavage, ameliorate glial pyroptosis and neuroinflammation, protect against BBB collapse and AQP-4 depolarization, prevent Aß acute accumulation and Aß1-42 oligomers formation, and reduce neuronal damage and increase neurons survival after reperfusion. Conclusion: Our study indicated that YZFDF could exert neuroprotective effects on cerebral I/R injury and prevent Aß acute accumulation in the brain after cerebral I/R associated with inhibiting neuroinflammation-related pyroptosis and BBB-glymphatic dysfunctions.

Yi-Zhi-Fang-Dai Formula Exerts a Protective Effect on the Injury of Tight Junction Scaffold Proteins in Vitro and in Vivo by Mediating Autophagy through Regulation of the RAGE/CaMKKß/AMPK/mTOR Pathway.

Alzheimer's disease (AD) is a chronic neurodegeneration disease that is closely related to the abnormal tight junction scaffold proteins (TJ) proteins of the blood-brain barrier (BBB). Recently, Yi-Zhi-Fang-Dai Formula (YZFDF) had exerted a neuronal protective effect against amyloid peptide (Aß) toxicity. Still, the therapeutic mechanism of YZFDF in restoring Aß-induced injury of TJ proteins (ZO-1, Occludin, and Claudin-5) remains unclear. This study aimed to explore the underlying mechanism of YZFDF in alleviating the injury of TJ proteins. We examined the impacts of YZFDF on autophagy-related proteins and the histopathology of Aß in the APP/PS1 double-transgenic male mice. We then performed the free intracellular calcium levels [Ca2+]i analysis and the cognitive behavior test of the AD model. Our results showed that YZFDF ameliorated the injury of TJ proteins by reducing the mRNA transcription and expression of the receptor for advanced glycation end-products (RAGE), the levels of [Ca2+]i, calmodulin-dependent protein kinase ß (CaMKKß), phosphorylated AMP-activated protein kinase (AMPK). Accordingly, YZFDF increased the expression of the phosphorylated mammalian targets of rapamycin (mTOR), leading to inhibition of autophagy (downregulated LC3 and upregulated P62). Moreover, the Aß1-42 oligomers-induced alterations of autophagy in murine mouse brain capillary (bEnd.3) cells were blocked by RAGE small interfering RNA (siRNA). These results suggest that YZFDF restored TJ proteins' injury by suppressing autophagy via RAGE signaling. Furthermore, YZFDF reduced the pathological precipitation of Aß in the hippocampus, and improved cognitive behavior impairment of the AD model suggested that YZFDF might be a potential therapeutic candidate for treating AD through RAGE/CaMKKß/AMPK/mTOR-regulated autophagy pathway.

Muramyl dipeptide promotes Aß1-42 oligomer production via the NOD2/p-p38 MAPK/BACE1 signaling pathway in the SH-SY5Y cells.

The relationship between chronic bacterial colonization in the brain and Alzheimer's disease is attracting extensive attention. Recent studies indicated that the components of bacterial biofilm drive the amyloid-ß production. Muramyl dipeptide, the minimal bioactive peptidoglycan motif common to all bacteria, contributes to the development of many central inflammatory and neurodegenerative disorders. However, the involvement of Muramyl dipeptide in amyloid-ß production is not completely defined. In our present study, wild type mice received an intracerebroventricular injection of normal saline or Muramyl dipeptide. Data showed that the production of Aß1-42 oligomers was significantly increased after Muramyl dipeptide injection in the wild type mice or incubation of the SH-SY5Y cells with Muramyl dipeptide. Moreover, the action of Muramyl dipeptide was dose- and time-dependent. The above results suggested a possibility that the Muramyl dipeptide-induced Aß1-42 oligomer production might be related to the NOD2/p-p38 MAPK/BACE1 pathway. To confirm this, the SH-SY5Y cells were transfected with siRNA NOD2. Data showed that the transfected SH-SY5Y cells exhibited decreased expression of Aß1-42 oligomer, NOD2, p-p38 MAPK, and BACE1 after treatment with Muramyl dipeptide. Finally, SH-SY5Y cells were pretreated with SB203580, an inhibitor of the p-38-MAPK pathway. The results indicated that these pretreated SH-SY5Y cells exhibited decreased expression of Aß1-42 oligomer, p-p38 MAPK, and BACE1 after treatment with Muramyl dipeptide. In conclusion, these results suggested that Muramyl dipeptide was the trigger factor for Aß1-42 oligomer production, which probably acts via the NOD2/p-p38 MAPK/BACE1 signaling pathway.

Aß1-42 oligomer induces alteration of tight junction scaffold proteins via RAGE-mediated autophagy in bEnd.3 cells.

Compelling evidences have shown that amyloid-ß (Aß) peptide is one of the major pathogenic factors resulting in blood-brain barrier (BBB) disruption in Alzheimer's disease (AD). However, the mechanism underlying BBB breakdown remains elusive. In our present study, we employed murine brain capillary endothelial cells (bEnd.3) as an in vitro BBB model to investigate the role of autophagy in Aß1-42 oligo induced BBB disruption. We first identified Aß1-42 oligo cytotoxicity to bEnd.3 cells as observed in the reduced cell viability and downregulation of ZO-1, Occludin and Claudin-5. Based on the observation that both downregulated expression of p-mTOR/m-TOR and upregulated ratio of LC3-II/ß-actin were induced by Aß1-42 oligo, we then applied 3-MA, an inhibitor of autophagy, to test the role of autophagy in Aß1-42 oligo induced Tight junction (TJ) proteins damage. Results have shown that 3-MA partially reversed Aß1-42 oligo induced downregulation of ZO-1, Occludin and Claudin-5, which was further determined by LC3 siRNA. We also used rapamycin to activate autophagy and found that TJ proteins damage induced by Aß1-42 was deteriorated even further. Given that the receptor of advanced glycation end-products (RAGE) is a pivotal receptor that mediates Aß toxicity, RAGE siRNA was utilized to identify the involvement of RAGE in Aß1-42 oligo induced autophagy. The results demonstrated a suppressed autophagy with increased p-mTOR/m-TOR and decreased LC3-II/ß-actin as well as increased ZO-1, Occludin and Claudin-5 in transfected cells after Aß1-42 oligo treatment, as compared to the non-transfected group. In summary, these results suggested that Aß1-42 oligo induced TJ proteins disruption via a RAGE-dependent autophagy pathway.

Aß1-42 induces cell damage via RAGE-dependent endoplasmic reticulum stress in bEnd.3 cells.

Blood-brain barrier (BBB) breakdown has been determined to play a critical role in the pathogenesis of Alzheimer's disease (AD). However, the underlying mechanisms of BBB disruption in AD remain unclear. Our previous study suggested that the receptor for advanced glycation end-products (RAGE) functioned as a signal transduction receptor in Aß1-42-induced damage in endothelial cells. In our present study, we revealed that RAGE-mediated endoplasmic reticulum stress (ERS) is essential for Aß-induced endothelial cell damage. Here, we found that Aß1-42 activated ERS by upregulation of Grp78, xbp-1 and CHOP in endothelial cells and that Aß1-42-resulted lesions, including the upregulations of caspase-12 and caspase-3, the augment of bax/bcl-2 ratio, and the downregulations of ZO-1 and Occludin in bEnd.3 cells, were ameliorated by the pretreatment of salubrinal, an ERS inhibitor. Furthermore, the expressions of Grp78, xbp-1 and CHOP induced by Aß1-42 were blocked by transfection of RAGE small interfering RNA (siRNA), which indicated that Aß1-42 activated ERS in a RAGE-dependent manner. Additionally, bEnd.3 cells transfected with RAGE siRNA showed lower expressions of caspase-12 and caspase-3, decreased bax/bcl-2 ratio, and higher expressions of ZO-1 and Occludin following Aß1-42 treatment, comparing to control cells. In conclusion, our data demonstrated that Aß1-42 induced endothelial cells damage via activation of ERS in a RAGE-dependent manner.

Yi-Zhi-Fang-Dai Formula Protects against Aß1-42 Oligomer Induced Cell Damage via Increasing Hsp70 and Grp78 Expression in SH-SY5Y Cells.

Yi-Zhi-Fang-Dai formula (YZFDF) is an experiential prescription used to cure dementia cases like Alzheimer's disease (AD). In this study, the main effective compounds of YZFDF have been identified from this formula, and the neuroprotective effect against Aß1-42 oligomer of YZFDF has been tested in SH-SY5Y cells. Our results showed that YZFDF could increase cell viability and could attenuate endothelial reticula- (ER-) mediated apoptosis. Evidence indicated that protein folding and endothelial reticula stress (ERS) played an important role in the AD pathological mechanism. We further explored the expression of Hsp70, an important molecular chaperon facilitating the folding of other proteins, and Grp78, the marker protein of ERS in SH-SY5Y cells. Data told us that YZFDF pretreatment could influence the mRNA and protein expression of these two proteins. At last, we also found that YZFDF pretreatment could activate Akt in SH-SY5Y cells. All these above indicate that YZFDF could be a potent therapeutic candidate for AD treatment.

EGb761 improves cognitive function and regulates inflammatory responses in the APP/PS1 mouse.

There is accumulating evidence that the Ginkgo biloba extract EGb761 may help to prevent Alzheimer's disease (AD). However, the underlying mechanism of its action remains to be elaborated. In this study, we examined the effects of EGb761 using the APP/PS1 transgenic mouse model of AD. Two-month-old APP/PS1 mice were supplemented with EGb761 daily for 6months. We found that this chronic treatment with EGb761 improved the cognitive function of these mice and also significantly alleviated amyloid plaque deposition. Although the level of insoluble amyloid beta (Aß) was decreased, the soluble content of Aß was not changed after administration of EGb761. We then determined the changes in central inflammation and observed that the activated microglia around amyloid plaque was increased in these treated mice. We also found that chronic EGb761 treatment downregulated pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS), and upregulated anti-inflammatory cytokines and Arginase-1 (Arg-1), suggesting that EGb761 regulated the phenotype of activated microglia in the APP/PS1 mouse brain. In support of this, pretreatment of the BV2 microglial cell line with EGb761 inhibited the inflammatory reaction to Aß. Furthermore, the addition of conditioned media derived from BV2 cells that were co-treated with Aß and EGb761, protected neurons against treatment of Aß and inhibited apoptotic damage. Taken together, our results demonstrated that EGb761 provided a protective effect in APP/PS1 mouse. This protection was correlated with an inhibition of the pro-inflammatory effects of microglia and an induction of anti-inflammatory effects. These results strongly suggest that EGb761 provides a protective effect in APP/PS1 mouse via regulation of inflammation in the brain.