Dimethyl phthalate exposure induces cognitive impairment via COX2-mediated neuroinflammation.

AIM: The present work explored the mechanism of dimethyl phthalate (DMP, the environmental contaminant) exposure in inducing cognitive impairment. METHODS: Targets and regulatory networks related to DMP-brain injury-cognitive impairment were analyzed through network pharmacology. DMP exposure was carried out to simulate DMP environmental uptake, whereas Morris water maze was performed for examining cognitive impairment. Additionally, inflammatory cytokine levels within tissues were measured. hematoxylin-eosin staining(H&E) and Nissl staining was conducted to examine brain tissue injury, while Western blot was carried out for identifying protein levels. After applying.Small interfering RNA(siRNA-COX2) and celecoxib-COX2 inhibitors separately, we analyzed impacts of DMP. Besides, in vitro experiments were performed to analyze impacts of DMP on microglial activation. RESULTS: As suggested by network pharmacology,Cyclooxygenase-2-PTGS2 (COX2) showed significant relation to DMP, and it exerted its effect via COX2. Following DMP exposure, mice experienced obvious cognitive impairment and brain damage, besides, microglial cells were activated, and inflammatory cytokines were up-regulated. Applying siRNA-COX2 and celecoxib-COX2 suppressed DMP's impact and mitigated mouse cognitive impairment. Based on in vitro analysis, DMP led to microglial activation and neuroinflammation. CONCLUSION: DMP exposure causes neuroinflammation via the COX2-regulated microglial activation, thus leading to cognitive impairment. COX2 may serve as the key action target of DMP.

NADPH oxidase 4 regulate the glycolytic metabolic reprogramming of microglial cells to promote M1 polarization.

This work aimed to investigate the role and mechanism of NADPH oxidase 4 (NOX4) in the polarization of microglial cells. Microglial cells were transfected with the NOX4 overexpression plasmid (pGL3-NOX4), and later treated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) to induce its M1 polarization. Later, the F4/80 + CD86 + cell proportion was detected by flow cytometry (FCM), the inflammatory factor expression levels were analyzed through enzyme-linked immunosorbent assay (ELISA), while ionized calcium binding adapter molecule 1 (IBA-1) and PKM2 expression were measured by immunofluorescence (IF) staining. In addition, dichlorodihydrofluorescein diacetate probe was utilized to detect the reactive oxygen species (ROS) levels, glucose uptake, and glycolysis, as well as lactic acid level. The expression of glycolytic enzymes PKM2, HK2, and citrate (Si)-synthas (CS) was detected by Western-blot (WB) assay. Moreover, the polarization level of microglial cells was detected after ROS expression was suppressed by the ROS inhibitor N-acetylcysteine (NAC). In mouse experiments, LPS was applied in inducing central neuroinflammation in NOX4 knockdown mouse model (KO) and wild-type mice (WT). Thereafter, the inflammatory factor levels and lactic acid level in mouse tissues were detected; IBA-1 and CD86 expression in mice was measured by IF staining; and the expression of glycolytic enzymes PKM2, HK2, and CS in the central nervous system (CNS) was also detected. After NOX4 overexpression in microglial cells, the M1 polarization level was upregulated, the F4/80 + CD86 + cell proportion increased, and inflammatory factors were upregulated. At the same time, the expression of glycolytic enzymes PKM2, HK2, and CS was upregulated. NAC pretreatment suppressed the effects of NOX4, reduced the F4/80 + CD86 + cell proportion, and suppressed the expression of PKM2, HK2, and CS. In the mouse model, the expression levels of CD86 in KO group decreased, and the inflammatory factors were also downregulated. NOX4 promotes glycolysis of microglial cells via ROS, thus accelerating M1 polarization and inflammatory factor expression. In this regard, NOX4 is promising as a new target for the treatment of neuroinflammation.

Paeoniflorin suppresses neuronal ferroptosis to improve the cognitive behaviors in Alzheimer's disease mice.

Aim of this research was to examine the impact of paeoniflorin (Pae) in suppressing the occurrence of ferroptosis in individuals with Alzheimer's disease (AD). The study utilized APP/PS1 mice with AD as the experimental subjects. Following the administration of Pae, the cognitive behaviors of mice were evaluated and the key indexes of ferroptosis were measured, as well as levels of oxidative stress (OS). For in-vitro experiments, Erastin was adopted for inducing the ferroptosis of PC12 cells, and the level of cell ferroptosis was detected after Pae treatment. Pae improved the cognitive ability of AD mice, reduced the level of ferroptosis, decreased the iron ion and MAD levels in brain tissues, and increased SOD expression. In PC12 cells, Pae suppressed the Erastin-induced ferroptosis, mitigated oxidative damage, and reduced the level of ROS. Based on the findings from our research, it was observed that Pae exhibited a specific binding affinity to P53, leading to the suppression of ferroptosis. This mechanism ultimately resulted in the improvement of nerve injury in mice with AD.

Aurantiamide promotes M2 polarization of microglial cells to improve the cognitive ability of mice with Alzheimer's disease.

This work aimed to investigate the effect of aurantiamide (Aur) in promoting the M2 polarization of microglial cells to improve the cognitive ability of mice with Alzheimer's disease (AD). The M2 polarization of BV2 cells was induced by interleukin-4 (IL-4) treatment.Aur promoted the M2 polarization of BV2 cells, and up-regulated the expression of CD206 and SOCS3. In the meantime, it increased TGF-ß1, Arg-1 and IL-10 levels, and promoted the polarization of JAK1-STAT6. Treatment with STAT6 inhibitor antagonized the effect of Aur. Besides, the cognitive ability of AD mice was improved after Aur treatment, meanwhile, the expression of CD206 was up-regulated, while that of IBA-1 was down-regulated. Aur promotes the M2 polarization of microglial cells to improve the cognitive ability of AD mice, and such effect is related to the STAT6 signal.

ADMSC Exo-MicroRNA-22 improve neurological function and neuroinflammation in mice with Alzheimer's disease.

The previous study by our group has found that miRNA-22 can inhibit pyroptosis by targeting GSDMD and improve the memory and motor ability of mice with Alzheimer's disease (AD) mice by inhibiting inflammatory response. In recent years, stem cells and their exosomes have been reported to have good therapeutic effects on AD; therefore, we hypothesize that miRNA-22 is likely to play a synergistic therapeutic effect. In this study, adipose-derived mesenchymal stem cells (ADMSCs) were transfected into miRNA-22 mimic to obtain miRNA-22 loaded exosomes (Exo-miRNA-22), which was further used for the treatment and nerve repair of AD. In brief, 4-month-old APP/PS1 mice were assigned into the control group, Exo and Exo-miRNA-22 groups. After exosome transplantation, we observed changes in the motor and memory ability of mice. In addition, ELISA was used to detect the expression of inflammatory factors in cerebrospinal fluid and peripheral blood, Nissl staining was used to assess the survival of mouse nerve cells, immunofluorescence staining was used to determine the activation of microglia, and Western blot was utilized to detect the expression of pyroptosis-related proteins. As a result, the nerve function and motor ability were significantly higher in mice in the Exo-miRNA-22 group than those in the control group and Exo group. Meanwhile, the survival level of nerve cells in mice was higher in the Exo-miRNA-22 group, and the expression of inflammatory factors was lower than that of the Exo group, indicating Exo-miRNA-22 could significantly suppress neuroinflammation. In vitro culture of PC12 cells, Aß25-35 -induced cell damage, detection of PC12 apoptotic level, the release of inflammatory factors and the expression of pyroptosis-related proteins showed that Exo-miRNA-22 could inhibit PC12 apoptosis and significantly decrease the release of inflammatory factors. In this study, we found that miRNA-22-loaded ADMSC-derived exosomes could decrease the release of inflammatory factors by inhibiting pyroptosis, thereby playing a synergetic therapeutic role with exosomes on AD, which is of great significance in AD research.

Aureusidin derivative CNQX inhibits chronic colitis inflammation and mucosal barrier damage by targeting myeloid differentiation 2 protein.

Our previous study has found that aureusidin can inhibit inflammation by targeting myeloid differentiation 2 (MD2) protein. Structural optimization of aureusidin gave rise to a derivative named CNQX. LPS was used to induce inflammation in intestinal macrophages; flow cytometry, PI staining and Hoechst 33342 staining were used to detect the apoptotic level of macrophages; enzyme-linked immunosorbent assay (ELISA) was utilized to detect the expression level of inflammatory factors (including IL-1ß, IL-18 and TNF-α); immunofluorescence staining was used to investigate the expression of MD2; Western blot was employed to measure the protein level of TLR4, MD2, MyD88 and p-P65. As a result, CNQX with IC50 of 2.5 µM can significantly inhibit the inflammatory damage of macrophages, decrease apoptotic level, reduce the expression level of inflammatory factors and simultaneously decrease the expression level of TLR4, MD2, MyD88 as well as p-P65. Caco-2 cell line was used to simulate the intestinal mucosal barrier in vitro, LPS was employed to induce cell injury in Caco-2 (to up-regulate barrier permeability), and CNQX with IC50 of 2.5 µl was used for intervention. Flow cytometry was used to detect the apoptotic level of Caco-2 cells, trans-epithelial electric resistance (TEER) was measured, FITC-D was used to detect the permeability of the intestinal mucosa, and Western blot was used to detect the expression levels of tight junction proteins (including occludin, claudin-1, MyD88, TLR4 and MD2). As a result, CNQX decreased the apoptotic level of Caco-2 cells, increased TEER value, decreased the expression levels of MyD88, TLR4 and MD2, and increased the protein levels of tight junction proteins (including occludin and claudin-1). C57BL/6 wild-type mice were treated with drinking water containing Dextran sulphate sodium (DSS) to establish murine chronic colitis model. After CQNX intervention, we detected the bodyweight, DAI score and H&E tissue staining to evaluate the life status and pathological changes. Immunohistochemistry (IHC) staining was used to detect the expression of MD2 protein, tight junction protein (including occludin and claudin-1). Transmission electron microscopy and FITC-D were used to detect intestinal mucosal permeability. Western blot was used to detect the expression levels of tight junction proteins (including occludin, claudin-1, MyD88, TLR4 and MD2) in the intestinal mucosa tissue. Consequently, CNQX can inhibit the intestinal inflammatory response in mice with colitis, inhibit the mucosal barrier injury, increase the expression of tight junction proteins (including occludin and claudin-1) and decrease the expression levels of MyD88, TLR4 and MD2. Mechanistically, pull-down and immunoprecipitation assays showed that CNQX can inhibit the activation of TLR4/MD2-NF-κB by binding to MD2 protein. Collectively, in this study, we found that CNQX can suppress the activation of TLR4 signals by targeting MD2 protein, thereby inhibiting inflammation and mucosal barrier damage of chronic colitis.

Performance of Mattis dementia rating scale-Chinese version in patients with mild cognitive impairment and Alzheimer's disease.

BACKGROUND: To identify the applicability of the Chinese Version of Mattis Dementia Rating Scale (DRS-CV). METHODS: The DRS-CV was administered to 483 participants, including 136 normal controls, 167 patients with mild cognition impairment (MCI), and 180 patients with Alzheimer's disease (AD). Receiver Operating Characteristic (ROC) curve was used to evaluate the sensitivity and specificity of the scale. RESULTS: The scores of DRS-CV were ranked in the order of NC > MCI > mild AD > moderate AD group. Memory was the sensitive function affected at a relatively earlier stage of AD. ROC curve analysis indicated the DRS-CV total score and memory subscale showed excellent sensitivity and specificity in the discrimination between MCI from mild AD and mild AD from moderate AD, but poor sensitivity and specificity in the discrimination between MCI and NC. CONCLUSION: The DRS-CV is useful to the early diagnosis and severity of AD, not to the early identification of MCI.

The potential predictive value of salivary cortisol on the occurrence of secondary cognitive impairment after ischemic stroke.

In this study, we aimed to investigate the relationship between salivary cortisol content and secondary mild cognitive impairment (MCI), thereby supporting the prediction of MCI in clinical practice. In this study, the salivary cortisol levels were examined in 120 patients with MCI after cerebral ischemic stroke (CIS) (CIS-MIC) and 80 CIS patients without MIC (CIS). The clinical data were compared among these patients with different cortisol levels. The salivary level of cortisol was significantly higher in patients with CIS-MIC (0.85-3.65 nmol/L) than that in those with CIS (0.52-1.21 nmol/L). The categorized analysis by CIS-MIC quartile showed that patient age, hyperlipidemia, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), etc. were significantly increased with increasing salivary cortisol levels. Moreover, univariate and multivariate logistic regression analyses revealed that the MCI risk of patients in the first quartile was 0.35 and 0.41 times, respectively, of the fourth quartile. Multiple linear regression showed that patient age, the time of rescue, and the salivary cortisol level were independent factors in the Mini-Mental State Exam (MMSE) score of MCI patients. Meanwhile, the receiver operating characteristic (ROC) curve showed that the area under the curve of salivary cortisol as a diagnostic marker for MCI after CIS was 0.982, with sensitivity of 0.973 and specificity of 0.980. In this study, we found that salivary cortisol level was an independent risk factor of MCI after CIS. A higher salivary cortisol level indicated a higher probability of MCI occurrence, and salivary cortisol level can be used as a predictive marker for MCI occurrence.

New mechanism of nerve injury in Alzheimer's disease: ß-amyloid-induced neuronal pyroptosis.

The present study was designed to investigate the role of ß-amyloid (Aß1-42 ) in inducing neuronal pyroptosis and its mechanism. Mice cortical neurons (MCNs) were used in this study, LPS + Nigericin was used to induce pyroptosis in MCNs (positive control group), and Aß1-42 was used to interfere with MCNs. In addition, propidium iodide (PI) staining was used to examine cell permeability, lactate dehydrogenase (LDH) release assay was employed to detect cytotoxicity, immunofluorescence (IF) staining was used to investigate the expression level of the key protein GSDMD, Western blot was performed to detect the expression levels of key proteins, and enzyme-linked immunosorbent assay (ELISA) was utilized to determine the expression levels of inflammatory factors in culture medium, including IL-1ß, IL-18 and TNF-α. Small interfering RNA (siRNA) was used to silence the mRNA expression of caspase-1 and GSDMD, and Aß1-42 was used to induce pyroptosis, followed by investigation of the role of caspase-1-mediated GSDMD cleavage in pyroptosis. In addition, necrosulfonamide (NSA), an inhibitor of GSDMD oligomerization, was used for pre-treatment, and Aß1-42 was subsequently used to observe the pyroptosis in MCNs. Finally, AAV9-siRNA-caspase-1 was injected into the tail vein of APP/PS1 double transgenic mice (Alzheimer's disease mice) for caspase-1 mRNA inhibition, followed by observation of behavioural changes in mice and measurement of the expression of inflammatory factors and pyroptosis-related protein. As results, Aß1-42 could induce pyroptosis in MCNs, increase cell permeability and enhance LDH release, which were similar to the LPS + Nigericin-induced pyroptosis. Meanwhile, the expression levels of cellular GSDMD and p30-GSDMD were up-regulated, the levels of NLRP3 inflammasome and GSDMD-cleaved protein caspase-1 were up-regulated, and the levels of inflammatory factors in the medium were also up-regulated. siRNA intervention in caspase-1 or GSDMD inhibited Aß1-42 -induced pyroptosis, and NSA pre-treatment also caused the similar inhibitory effects. The behavioural ability of Alzheimer's disease (AD) mice was relieved after the injection of AAV9-siRNA-caspase-1, and the expression of pyroptosis-related protein in the cortex and hippocampus was down-regulated. In conclusion, Aß1-42 could induce pyroptosis by GSDMD protein, and NLRP3-caspase-1 signalling was an important signal to mediate GSDMD cleavage, which plays an important role in Aß1-42 -induced pyroptosis in neurons. Therefore, GSDMD is expected to be a novel therapeutic target for AD.

The effects and mechanisms of myeloid differentiation protein 2 on intestinal mucosal permeability in mice with chronic colitis.

The present study was designed to investigate the mechanism of myeloid differentiation protein 2 (MD2) on intestinal mucosa destruction in mice with chronic colitis. Briefly, a chronic colitis mouse model was established by the administration of dextran sulfate sodium (DSS) in transgenic mice of MD2 overexpression (Transgenic, MD2-Tg) and C57BL/6 wild-type mice (MD2-WT). In addition, Caco-2 cells were cultured to form a monolayer cell model in vitro. The small interfering RNA was utilized to silence the MD2 gene in Caco-2 cells, and tumor necrosis factor-α (TNF-α) was used to establish the model of intestinal mucosal inflammation. After DSS induction, the intestinal mucosal tissue inflammation was more severe in MD2-Tg mice than MD2-WT. In addition, the intestinal mucosa was severely damaged, the intestinal mucosal permeability was increased, bacterial translocation was obvious, and the expression levels of MD2, MyD88, Toll-like receptor 4 (TLR4), and HMGB1 in mucosal tissues were significantly increased, while the expression levels of tight junction proteins, occludin, and claudin-1 were significantly lower in MD2-Tg mice compared with those in MD2-WT mice. TNF-α could induce inflammatory apoptosis in Caco-2 cell models. After MD2 silencing, the apoptotic level was decreased, the value of transepithelial electrical resistance was increased, the permeability of intestinal mucosa was decreased, the cellular expression levels of MD2, MyD88, TLR4, and HMGB1 were decreased, while the expression levels of tight junction proteins, occludin and claudin-1 were increased. MD2 could aggravate the destruction of intestinal mucosa in chronic colitis through the HMGB1-TLR4-MyD88 pathway.

The predictive value of serum p-CREB level on secondary cognitive impairment in patients with mild-to-moderate craniocerebral trauma.

The study was designed to investigate the predictive value of phosphorylated CAMP response element binding protein (p-CREB) level in peripheral blood on secondary cognitive impairment in patients with mild-to-moderate craniocerebral trauma. A total of 107 patients with mild-to-moderate craniocerebral trauma were selected, who were admitted to the Second Affiliated Hospital of College of Jiaxing from January 2016 to January 2017. Of them, 30 patients were diagnosed with secondary mild cognitive impairment (MCI) during follow-up, who were assigned to the experimental group. The remaining 77 subjects were assigned to the control group, without significant cognitive impairment. The clinical data of patients were compared between two groups, and the clinical data of patients with different p-CREB levels were compared. Logistic regression analysis was used to investigate the risks of MCI in patients with different p-CREB levels. Moreover, multiple linear regression analysis was employed to assess the influencing factors of scores of Mini-Mental State Examination (MMSE) on patients with secondary MCI. The following pathophysiologic factors, including age, rescuing time, the proportion of hypertension, trauma severity score (AIS-ISS), and serum total cholesterol (TC) were significantly higher in patients in the experimental group compared to those in the control group (all P < 0.05). The serum level of p-CREB ranged from 0.127 to 1.852 ng/ml. Afterwards, the serum levels of p-CREB of patients were divided into four quartiles. The first, second, third, and fourth quartile groups were 0.127-0.548 ng/ml, 0.549-0.982 ng/ml, 0.983-1.412 ng/ml, and 1.413-1.852 ng/ml, respectively. As the level of p-CREB increased, age, rescuing time, the proportion of hypertension, and AIS-ISS gradually decreased, with statistical significance (all P < 0.05). Univariate and multivariate logistic regression analyses demonstrated that the risk of secondary MCI of patients in the first quartile was 1.21 and 1.58 times of the fourth quarter, respectively. Multivariate linear regression analysis showed that age, rescuing time, AIS-ISS, and serum p-CREB level were independent influencing factors of MMSE score in secondary MCI patients. For each increase of 0.1 ng/ml in serum p-CREB level, the MMSE score increased by 0.382 in MCI patients. Serum p-CREB level was an independent risk factor of secondary MCI in patients with mild-to-moderate craniocerebral trauma, whose level was significantly correlated with the injured degree of cognitive impairment. The level of p-CREB is also age-related, and younger patients have a higher level.

Study on antrodia camphorata polysaccharide in alleviating the neuroethology of PD mice by decreasing the expression of NLRP3 inflammasome.

Parkinson's disease (PD) is a neurodegenerative disease, and the role of neuroinflammation in the pathogenesis and progression of PD has been confirmed. The polysaccharides and triterpenoids of antrodia camphorata (a polyporous fungus) harbor diverse and powerful pharmacological effects. In this study, 6-hydroxydopamine was used to construct a PD mouse model. After antrodia camphorata polysaccharide (ACP) intervention, neurobehavioral changes were detected, neurotransmitter changes in striatum were determined by high-performance liquid chromatography, the alterations of striatal NOD-like receptor pyrin domain containing three (NLRP3) were examined by immunohistochemistry, and the expression of NLRP3, IL-1ß, Caspase-1, and proCaspase-1 were detected by western blot. To be specific, the items of neurobehavioral test included open field activity, rotary test, pole test, gait analysis, and swimming test. As a result, 6-hydroxydopamine could lead to PD-like lesions, including tremor, stiffness, attenuated spontaneous activity, and bradykinesia in mice, and the expression of tyrosine hydroxylase in the striatum was decreased. After ACP intervention, the neuroethology of mice was significantly improved, as demonstrated by the elevated levels of dopamine in the striatum and the decreased expression of dopamine in the striatum in NLRP3 inflammasome. NLRP3 inflammasome played an important role in neuroinflammation in PD mice. ACP could reduce the activation of NLRP3 and expression of related inflammatory factors.

[Protective effect of polysaccharide pretreatment on myocardial ischemia reperfusion injury in rats].

OBJECTIVE: To study the protective effects of polysaccharides on myocardial ischemia reperfusion injury in rats after preconditioning. METHODS: The myocardial ischemia reperfusion model was established by reversible left anterior descending coronary artery ligation: 0. 5 h was myocardial ischemia and 2 h was re perfused. A total of 50 healthy SD rats were randomly divided into 5 groups: sham operation group, model group, polysaccharide high dose group, polysaccharide middle dose group and polysaccharide low dose group, 10 rats in each group. The polysaccharide group was given 100, 50, 10 mg/kg of camphora polysaccharides at 30 min before operation, while the model group and the sham operation group were treated with equal dose of saline. The expression of malondialdehyde( MDA), catalase( CAT), superoxide dismutase( SOD), creatine kinase( CK) and creatine kinase isoenzyme( CK-MB) in serum of rats and tumor necrosis factor alpha( TNF-α), interleukin-1ß( IL-1ß) 、interleukin-6( IL-6) in myocardial tissue were detected by Elisa method. The expression of Bcl-2, Bax and Caspase-3 in myocardium was detected by Western-Blot. The infarct size and myocardial tissue HE staining were measured. RESULTS: Compared with the model group, the infarct size of the rats pretreated with polysaccharides decreased significantly, The levels of MDA, CK, CK-MB in serum and TNF-α, IL-1, IL-6 in myocardium were significantly decreased. The expression of SOD and CAT in serum increased significantly. The expression of Bax and caspase-3 was decreased and the expression of Bcl-2 was elevated in myocardium. HE staining showed that the injury degree of myocardial tissue in pretreated rats treated with polysaccharide was significantly lower than that in model group. CONCLUSION: Polysaccharide can reduce the apoptosis of myocardial cells by resisting oxidative stress and inflammatory damage, play a protective role in rats of Myocardial ischemia reperfusion injury.

LncRNA-AC020978 Promotes Metabolic Reprogramming in M1 Microglial Cells in Postoperative Cognitive Disorder via PKM2.

The present work aimed to explore the role of long non-coding RNA (lncRNA)-AC020978 in postoperative cognitive disorder (POCD) and the underlying mechanism. The POCD mouse model was constructed through isoflurane anesthesia + abbreviated laparotomy. The AC020978 expression in brain tissue was silenced after lentivirus injection, then Morris water maze test was conducted to detect the cognitive disorder level, flow cytometry was performed to analyze M1 macrophage level, ELISA was carried out to measure inflammatory factor levels, H&E, Nissl and immunohistochemical staining was performed to detect the pathological changes in brain tissue, and Western blotting assay was adopted to detect protein expression. In addition, microglial cells were cultured in vitro, after lentivirus infection, the effect of AC020978 on the M1 polarization of microglial cells and glycolysis was observed. AC020978 overexpression promoted POCD progression and aggravated cognitive disorder in mice; in addition, the proportion of peripheral and central M1 cells increased, the inflammatory factor levels were upregulated, and microglial cells were activated. By contrast, AC020978 silencing led to cognitive disorder in mice and suppressed microglial cell activation and M1 polarization. In vitro experimental results indicated that AC020978 promoted the expression and phosphorylation of PKM2, which promoted inflammatory response through enhancing microglial cell glycolysis and M1 polarization. AC020978 interacts with PKM2 to promote the glycolysis and M1 polarization of microglial cells, thus regulating cognitive disorder and central inflammation in POCD.

Aurantiamide suppresses the activation of NLRP3 inflammasome to improve the cognitive function and central inflammation in mice with Alzheimer's disease.

AIM: This study was aimed at exploring the mechanism by which aurantiamide (Aur) targeted NLRP3 to suppress microglial cell polarization. METHODS: The 7-month-old APP/PS1 mice and C57BL/6 mice were applied to be the study objects, and Aur was administered intragastrically to APP/PS1 mice at 10 mg/kg and 20 mg/kg. The changes in the neurocognitive function of mice were measured by Morris Water Maze (MWM) test. In the in vitro experiments, the mouse BV2 cells were employed as the study objects, which were subject to treatment with 10 µM and 20 µM Aur and induced with LPS and IFN-γ in order to activate BV2 cells and induce their M1 polarization. RESULTS: Aur was found to suppress the M1 polarization of mouse microglia, reduce central neuroinflammation, and improve the cognitive function in mice. Meanwhile, Aur suppressed the activation and the expression of NLRP3 inflammasome. The results of experiments in vitro demonstrated that Aur inhibited the activation and M1 polarization of BV2 cells. CONCLUSION: Aur targets NLRP3 and suppresses the activation of NLRP3 inflammasome.

Advanced Glycation End-Products (AGEs) Promote Endothelial Cell Pyroptosis Under Cerebral Ischemia and Hypoxia via HIF-1α-RAGE-NLRP3.

This work mainly aimed to explore the role and mechanism of advanced glycation end-products (AGEs) in inducing cerebrovascular endothelial cell pyroptosis under oxygen glucose deprivation (OGD) condition. The mouse cerebral microvascular endothelial cells (BMECs and bEnd.3) were used as the objects to construct the OGD model in vitro. Then, cells were pretreated with AGE-modified human serum albumin (AGE-HSA). Thereafter, CCK-8 assay was conducted to detect cell viability, and flow cytometry (FCM) was performed to measure cell pyroptosis level. Meanwhile, the expression of inflammatory factors was detected by enzyme-linked immunosorbent assay (ELISA). The expression of HIF-α, NLRP3, and RAGE was detected by fluorescence staining. The opening status of cell membrane pore was observed under the electron microscope, and the expression levels of FL-GSDMD, NT-GSDMD, and caspase-1 were measured through Western Blot (WB) assay. Moreover, bEnd.3 cells were treated with siRAN-silenced NLRP3 and HIF-α inhibitor, so as to observe the effect of AGEs on cell pyroptosis level. In the mouse model, the middle cerebral artery occlusion (MCAO) model was constructed by the suture-occluded method. After intraperitoneal injection of AGEs, the pathological changes in mouse brain tissues were detected; the expression levels of NLRP3, ZO-1, and CD31 were determined by histochemical staining, and the levels of inflammatory factors and pyroptosis-related proteins were also detected. Under OGD condition, AGEs induced the pyroptosis of bEnd.3 cells, and the cell pyroptosis rate increased, higher than that of the OGD group. Meanwhile, the levels of inflammatory factors were up-regulated; the expression of HIF-α, NLRP3, and RAGE in cells increased; and the levels of NT-GSDMD and caspase-1 were markedly higher than those of the control and OGD groups. siRNA-NLRP3 or HIF-α inhibitor treatment suppressed pyroptosis and reduced the inflammatory factor levels. In mouse experiments, AGE injection aggravated brain injury in the MCAO mouse model, decreased the expression of ZO-1 and CD31, and elevated the levels of NLRP3 and inflammatory factors. Under cerebral ischemia condition, AGEs can induce endothelial cell pyroptosis via HIF-α-RAGE-NLRP3, thereby further aggravating brain injury.

Mechanism of neocryptotanshinone in protecting against cerebral ischemic injury: By suppressing M1 polarization of microglial cells and promoting cerebral angiogenesis.

AIM: This study explored the protective function and mechanism of neocryptotanshinone (NEO) on cerebral ischemia. METHODS: Lipopolysaccharide/γ-interferon(LPS/IFN-γ)was employed to mimic the polarization of mouse microglial cells BV2. After NEO treatment, the M1 polarization level of BV2 cells was identified using flow cytometry (FCM), fluorescent cell staining and enzyme linked immunosorbent assay(ELISA). Moreover, the mouse endothelial cells bEnd.3 were applied to be the study objects, which were intervened with NEO under the hypoxic condition. Thereafter, based on in-vitro tubule formation assay and fluorescence staining, the in-vitro tubule formation ability of bEnd.3 cells was detected. By adopting middle cerebral artery occlusion(MCAO) method, we constructed the mouse model of cerebral ischemia. After NEO intervention, the pathological changes of brain tissues were identified, while CD34 expression was measured by immunohistochemical (IHC) staining, nerve injury was detected by Nissl staining, and the changes in neurological behaviors of mice were also detected. RESULTS: Our results showed that NEO suppressed M1 polarization of BV2 cells, which exerted its effect through suppressing NF-κB and STAT3 signals, thereby decreasing the levels of iNOS, CD11b and inflammatory factors. NEO stimulated tubule formation in bEnd.3 cells based on the hypoxic situation, which exerted its effect through activating the Vascularendothelial growth factor-Vascular Endothelial Growth Factor Receptor 2-Notch homolog 1(VFGF-VEGFR2-Notch1) signal. Furthermore, NEO suppressed cerebral ischemia in mice and lowered the ischemic penumbra. NEO also improved the neurological behaviors of mice, increased the CD34 levels and decreased the expression of inflammatory factors. CONCLUSION: NEO has well protective effect against cerebral ischemia, and its mechanisms are related to suppressing M1 polarization of microglial cells and promoting cerebral angiogenesis, which are the mechanisms of NEO in treating ischemic encephalopathy.

TL1A promotes the postoperative cognitive dysfunction in mice through NLRP3-mediated A1 differentiation of astrocytes.

AIM: We investigated the mechanism, whereby tumor necrosis factor-like ligand 1A (TL1A) mediates the A1 differentiation of astrocytes in postoperative cognitive dysfunction (POCD). METHODS: The cognitive and behavioral abilities of mice were assessed by Morris water maze and open field tests, while the levels of key A1 and A2 astrocyte factors were detected by RT-qPCR. Immunohistochemical (IHC) staining was used to examine the expression of GFAP, western blot was used to assay the levels of related proteins, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory cytokines. RESULTS: The results showed that TL1A could promote the progression of cognitive dysfunction in mice. Astrocytes differentiated into A1 phenotype, while unobvious changes were noted in astrocyte A2 biomarkers. Knockout of NLRP3 or intervention with NLRP3 inhibitor could inhibit the effect of TL1A, improving the cognitive dysfunction and suppressing the A1 differentiation. CONCLUSION: Our results demonstrate that TL1A plays an important role in POCD in mice, which promotes the A1 differentiation of astrocytes through NLRP3, thereby exacerbating the progression of cognitive dysfunction.

Icariin improves cognitive impairment by inhibiting ferroptosis of nerve cells.

AIM: We investigated the effect and mechanism of Icariin (ICA) on improving neurobehavioral ability of mice with Alzheimer's disease (AD). METHODS: We selected 10-month-old APP/PS1 mice (AD) and wild-type C57BL/6J mice (Normal). After intragastric administration of ICA, Morris water maze was employed to detect neurobehavioral improvements, and to assay key ferroptosis indicators and oxidative stress levels. The common target of ICA for resisting ferroptosis and AD was predicted by network pharmacology. RESULTS: ICA could improve the neurobehavioral, memory and motor abilities of AD mice. It could lower the ferroptosis level and enhance the resistance to oxidative stress. After inhibition of MDM2, ICA could no longer improve the cognitive ability of AD mice, nor could it further inhibit ferroptosis. Network pharmacological analysis revealed that MDM2 might be the target of ICA action. CONCLUSIONS: We found that ICA can inhibit ferroptosis of nerve cells, thereby ameliorating neural damage in mice with AD.