Probiotics Improve Cognitive Impairment by Decreasing Bacteria-Related Pattern Recognition Receptor-Mediated Inflammation in the Gut-Brain Axis of Mice.

INTRODUCTION: Some studies have found that probiotics can improve cognitive impairment in Alzheimer's disease, although the specific molecular mechanism by which this occurs has not been reported. Our previous research found that probiotics inhibited bacteria-related Toll-like receptor 4- and retinoic-acid-inducible gene-I-mediated nuclear factor-κB signaling pathways to improve cognitive impairment. However, it is unclear whether probiotics have similar effects on other pattern recognition receptors that respond to bacteria. METHODS: Nine-month-old senescence-accelerated mouse prone 8 (SAMP8) mice received ProBiotic-4 (a mixture of Lactobacillus acidophilus, Bifidobacterium bifidum, Lactobacillus casei, and Bifidobacterium lactis) orally for 12 weeks. The effects on other bacteria-related pattern recognition receptors were then investigated. RESULTS: ProBiotic-4-treated SAMP8 mice showed improvement in memory deficits, synaptic and cerebral neuronal injuries, and microglial activation. ProBiotic-4 also markedly increased the expression of intestinal tight junction proteins (i.e., claudin-1, occludin, and zonula occluden-1), decreased the expression of interleukin-1ß at both the mRNA and protein levels, and reduced the expression of caspase-11, cleaved caspase-1, and α-kinase 1 (ALPK1) in the intestine and brain. CONCLUSIONS: These findings suggest that probiotics may have therapeutic potential for the treatment of inflammation in the gut-brain axis and for cognitive impairment. The mechanism of action of probiotics appears to be related to inhibition of the caspase-11/caspase-1 pathway and reduction of ALPK1 expression.

Phthalide derivative CD21 attenuates tissue plasminogen activator-induced hemorrhagic transformation in ischemic stroke by enhancing macrophage scavenger receptor 1-mediated DAMP (peroxiredoxin 1) clearance.

BACKGROUND: Hemorrhagic transformation (HT) is a critical issue in thrombolytic therapy in acute ischemic stroke. Damage-associated molecular pattern (DAMP)-stimulated sterile neuroinflammation plays a crucial role in the development of thrombolysis-associated HT. Our previous study showed that the phthalide derivative CD21 attenuated neuroinflammation and brain injury in rodent models of ischemic stroke. The present study explored the effects and underlying mechanism of action of CD21 on tissue plasminogen activator (tPA)-induced HT in a mouse model of transient middle cerebral artery occlusion (tMCAO) and cultured primary microglial cells. METHODS: The tMCAO model was induced by 2 h occlusion of the left middle cerebral artery with polylysine-coated sutures in wildtype (WT) mice and macrophage scavenger receptor 1 knockout (MSR1-/-) mice. At the onset of reperfusion, tPA (10 mg/kg) was intravenously administered within 30 min, followed by an intravenous injection of CD21 (13.79 mg/kg/day). Neuropathological changes were detected in mice 3 days after surgery. The effect of CD21 on phagocytosis of the DAMP peroxiredoxin 1 (Prx1) in lysosomes was observed in cultured primary microglial cells from brain tissues of WT and MSR1-/- mice. RESULTS: Seventy-two hours after brain ischemia, CD21 significantly attenuated neurobehavioral dysfunction and infarct volume. The tPA-infused group exhibited more severe brain dysfunction and hemorrhage. Compared with tPA alone, combined treatment with tPA and CD21 significantly attenuated ischemic brain injury and hemorrhage. Combined treatment significantly decreased Evans blue extravasation, matrix metalloproteinase 9 expression and activity, extracellular Prx1 content, proinflammatory cytokine mRNA levels, glial cells, and Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) pathway activation and increased the expression of tight junction proteins (zonula occludens-1 and claudin-5), V-maf musculoaponeurotic fibrosarcoma oncogene homolog B, and MSR1. MSR1 knockout significantly abolished the protective effect of CD21 against tPA-induced HT in tMCAO mice. Moreover, the CD21-induced phagocytosis of Prx1 was MSR1-dependent in cultured primary microglial cells from WT and MSR1-/- mice, respectively. CONCLUSION: The phthalide derivative CD21 attenuated tPA-induced HT in acute ischemic stroke by promoting MSR1-induced DAMP (Prx1) clearance and inhibition of the TLR4/NF-κB pathway and neuroinflammation.

[Effects of Low Concentration Hydrogen Inhalation on Asthma and Sleep Function in Mice].

OBJECTIVE: This study was designed to investigate the effects of low concentration hydrogen inhalation on asthma and sleep function in mice and the potential mechanism. METHODS: In the asthma experiment, BALB/c mice were randomly divided into normal control group, asthma model group and hydrogen treatment group. After establishing ovalbumin (OVA)-induced asthma model, the hydrogen treatment group mice were treated by inhalation of hydrogen (24-26 mL/L per day) for 7 consecutive days, and the normal control group and asthma model group mice received similar treatment by inhalation of air. The levels of interleukin (IL)-4, IL-13, and interferon-γ (IFN-γ) in bronchoalveolar lavage fluid (BALF) were measured by commercially available ELISA kits. The levels of malondialdehyde (MDA) and glutathione (GSH), as well as the activity of superoxide dismutase (SOD) in lung tissue were detected by colorimetric assays. The pathological changes in lung tissue were assessed by HE staining. In the sleep experiment, ICR mice were randomly divided into blank control group and 1 d, 3 d, 5 d hydrogen treatment groups and diazepam group. The effects of inhalation of 24-26 mL/L per day hydrogen on the sleep duration induced by intraperitoneal injection of upper-threshold dose of sodium pentobarbital and the sleep latency in response to subthreshold dose were evaluated. RESULTS: In the asthma experiment, the asthma model group showed higher levels of IL-4 and IL-13 ( P<0.05) and lower levels of IFN-γ ( P<0.001) in BALF, as compared to the normal control group. The content of MDA in lung tissue was also significantly increased ( P<0.01), companied by a decreased GSH concentration ( P <0.05) and a mildly reduced SOD activity ( P>0.05). Compared to the asthma model group, treatment with hydrogen significantly decreased the levels of IL-4 and IL-13 and increased the level of IFN-γ in BALF ( P<0.05). Moreover, without alteration of the MDA production ( P>0.05), hydrogen inhalation greatly increased GSH level and restored the SOD activity ( P<0.05) in lung tissue. Additionally, the HE staining data showed that the hydrogen treatment attenuated the pulmonary histopathological changes. In the sleep experiment, compared with the blank control group, the sleep latency was significantly shorter ( P<0.05) and the sleep duration was longer ( P<0.001) in all the hydrogen treatment groups after receiving an upper-threshold dose of sodium pentobarbital. Meanwhile, in all the hydrogen treatment groups, the sleep latency was significantly longer ( P<0.001) and the sleep duration was shorter ( P<0.001) when compared to the diazepam group. Compared with the blank control group, after intraperitoneal injection of a subthreshold dose of sodium pentobarbital, the sleep latency was significantly increased in both 1 d and 5 d hydrogen treatment groups, and there was no significant difference as compared to the diazepam group. In the 3 d hydrogen treatment group, the sleep latency was only slightly increased ( P>0.05), which was significantly lower than that of the diazepam group ( P<0.05). CONCLUSION: Low concentration hydrogen inhalation could alleviate OVA-induced asthma in mice, and the mechanism might be related to the anti-oxidative and anti-inflammatory effects of hydrogen. Also, low concentration hydrogen inhalation could improve sleep function in mice.

Aging-related renal injury and inflammation are associated with downregulation of Klotho and induction of RIG-I/NF-κB signaling pathway in senescence-accelerated mice.

BACKGROUND AND AIMS: The predominant distribution of the antiaging Klotho protein in both the kidneys and brain may point to its essential role in protecting against dysfunction of the kidney-brain axis during the aging process. Our previous study showed that the downregulation of Klotho was involved in aging-related cognitive impairment in aged senescence-accelerated mouse prone-8 (SAMP8) mice. The present study investigated the potential role of Klotho in aging-associated inflammation and renal injury. METHODS: Age- and gender-matched groups of SAMP8 mice and their corresponding normal control senescence-accelerated mouse resistant-1 (SAMR1) were used to investigate the potential role of Klotho in aging-associated inflammation and renal injury. RESULTS: Compared with aged SAMR1 controls, early-stage chronic kidney disease (CKD), which is associated with an increase in the urinary albumin-to-creatinine ratio, inflammatory cell infiltration, glomerulosclerosis, and tubulointerstitial fibrosis, was observed in aged SAMP8 mice. Furthermore, the aging-related loss of Klotho-induced activation of the retinoic acid-inducible gene 1/nuclear factor-κB (RIG-I/NF-κB) signaling pathway and subsequent production of the proinflammatory mediators tumor necrosis factor α, interleukin-6, and inducible nitric oxide synthase in the kidneys of aged SAMP8 mice compared with SAMR1 controls. CONCLUSIONS: The present results suggest that aging-related inflammation and the development of early-stage CKD are likely associated with the downregulation of Klotho and induction of the RIG-I/NF-κB signaling pathway in 12-month-old SAMP8 mice. Moreover, aged SAMP8 mice with cognitive deficits and renal damage may be a potential mouse model for investigating the kidney-brain axis in the aging process.

[Protective effect of ligustilide against low potassium induced apoptosis in cultured rat cerebellar granule neurons].

OBJECTIVE: To investigate the effect of ligustilide (LIG) on low potassium-induced apoptosis in primary cultured cerebellar granule neurons (CGN). METHODS: Apoptosis was induced by low potassium in cultured neonatal rat CGN in vitro. The CGN was divided into control/model/CGP54626 + LIG and LIG group. The neuronal viability of each group was measured by MTT assay. The protein expression levels of the key insulin-like growth factor 1 (IGF)-1 signaling effectors,including the phosphorylated IGF-1 receptor (IGF-1R), Akt, ERK1/2, CREB and activated caspase 3 were examined by Western blot analysis. RESULTS: LIG ranging from 2.5 to 20 micromol/L could protect against low potassium-induced apoptosis of CGN ini a concentration-dependent manner. 20 micromol/L LIG significantly induced upregulation of the phosphorylated levels of IGF-1, Akt, ERK1/2 and CREB, and downregulation of cleaved-caspase 3 expression, which could be blocked by a selective gamma-aminobutyric acid B (GABAs) receptor antagonist CGP54626. CONCLUSION: LIG concentration-dependently protects against low potassium-induced apoptosis in CGN at least partly through GABAa receptor activation and its downstream IGF-1 signaling pathway.

Therapeutic efficacy of a novel non-peptide αvß3 integrin antagonist for pathological retinal angiogenesis in mice.

αvß3 integrin has been reported as a promising therapeutic target for angiogenesis. In the present study, we tested the antiangiogenic activity of 3-[3-(6-guanidino-1-oxoisoindolin-2-yl) propanamido]-3-(pyridin-3-yl) propanoic acid dihydrochloride (GOPPP), a novel non-peptide αvß3 antagonist. Both human umbilical vein endothelial cells (HUVECs) and a mouse model of oxygen-induced retinopathy (OIR) were investigated separately. HUVEC adhesion, proliferation, migration, ERK1/2 and Akt phosphorylation were assessed. C57BL/6 mice were used for the studies in the OIR model. After exposure to 75% oxygen from postnatal day (PD) 7 to PD12, the mice were returned to room air, and GOPPP was intravitreally administered on PD12. Retinal neovascularization was evaluated on PD17. Hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) protein levels and ERK1/2 phosphorylation were determined by Western blot analysis of retina proteins. GOPPP significantly inhibited the pro-angiogenic effects of vitronectin on HUVECs, including adhesion, proliferation, and migration, and inhibited ERK1/2 and Akt phosphorylation. Retinal neovascularization in the OIR model was significantly suppressed by intravitreal administration of 50 ng GOPPP. The pro-angiogenic factors HIF-1α and VEGF induced by hypoxia were significantly inhibited by GOPPP in OIR mice. GOPPP administration also inhibited ERK1/2 phosphorylation in the OIR model. These results indicate that GOPPP, a novel αvß3 integrin antagonist, may have potential for the treatment of pathological retinal angiogenesis.

QBT improved cognitive dysfunction in rats with vascular dementia by regulating the Nrf2/xCT/GPX4 and NLRP3/Caspase-1/GSDMD pathways to inhibit ferroptosis and pyroptosis of neurons.

BACKGROUND: The novel phthalein component QBT, extracted from Ligusticum chuanxiong, shows promising biological activity against cerebrovascular diseases. This study focused on ferroptosis and pyroptosis to explore the effects of QBT on nerve injury, cognitive dysfunction, and related mechanisms in a rat model of vascular dementia (VaD). METHODS: We established a rat model of VaD and administered QBT as a treatment. Cognitive dysfunction in VaD rats was evaluated using novel object recognition and Morris water maze tests. Neuronal damage and loss in the brain tissues of VaD rats were assessed with Nissl staining and immunofluorescence. Furthermore, we investigated the neuroprotective mechanisms of QBT by modulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/cystine-glutamate antiporter (xCT)/glutathione peroxidase 4 (GPX4) and Nod-like receptor family pyrin domain-containing 3 (NLRP3)/cysteine-requiring aspartate protease-1 (Caspase-1)/Gasdermin D (GSDMD) pathways to inhibit ferroptosis and pyroptosis both in vivo and in vitro. RESULTS: Our findings indicated that QBT significantly ameliorated neuronal damage and cognitive dysfunction in VaD rats. Additionally, QBT reversed abnormal changes associated with ferroptosis and pyroptosis in the brains of VaD rats, concurrently up-regulating the Nrf2/xCT/GPX4 pathway and down-regulating the NLRP3/Caspase-1/GSDMD pathway to inhibit ferroptosis and pyroptosis in neuronal cells, thereby exerting a neuroprotective role. CONCLUSION: In summary, QBT effectively mitigated neuronal damage and cognitive dysfunction in VaD rats, demonstrating a neuroprotective effect by inhibiting ferroptosis and pyroptosis in neuronal cells. This study offers a novel perspective and theoretical foundation for the future development of drugs targeting VaD.

A novel phthalein component ameliorates neuroinflammation and cognitive dysfunction by suppressing the CXCL12/CXCR4 axis in rats with vascular dementia.

ETHNOPHARMACOLOGICAL RELEVANCE: Chuanxiong, a plant of the Umbelliferae family, is a genuine medicinal herb from Sichuan Province. Phthalides are one of its main active components and exhibit good protective effect against cerebrovascular diseases. However, the mechanism by which phthalides exert neuroprotective effects is still largely unclear. AIM OF THE STUDY: In this study, we extracted a phthalein component (named as QBT) from Ligusticum Chuanxiong, and investigated its neuroprotective effects against vascular dementia (VaD) rats and the underlying mechanism, focusing on the chemokine 12 (CXCL12)/chemokine (C-X-C motif) receptor 4 (CXCR4) axis. METHODS: A rat model of VaD was established, and treated with QBT. Cognitive dysfunction in VaD rats was assessed using the Y-maze, new object recognition, and Morris water maze tests. Neuronal damage and inflammatory response in VaD rats were examined through Nissl staining, immunofluorescence, enzyme-linked immunospecific assay, and western blotting analysis. Furthermore, the effects of QBT on CXCL12/CXCR4 axis and its downstream signaling pathways, Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/nuclear factor-κB (NF-κB), were investigated in VaD rats and BV2 microglial cells exposed to oxygen glucose deprivation. RESULTS: QBT significantly alleviated cognitive dysfunction and neuronal damage in VaD rats, along with inhibition of VaD-induced over-activation of microglia and astrocytes and inflammatory response. Moreover, QBT exhibited anti-inflammatory effects by inhibiting the CXCL12/CXCR4 axis and its downstream JAK2/STAT3 and PI3K/AKT/NF-κB pathways, thereby attenuating the neuroinflammatory response both in vivo and in vitro. CONCLUSION: QBT effectively mitigated neuronal damage and cognitive dysfunction in VaD rats, exerting neuroprotective effects by suppressing neuroinflammatory response through inhibition of the CXCL12/CXCR4 axis.

Modulation of Xiongdanjiuxin pills on the gut-liver axis in high-fat diet rats.

AIM: Xiongdanjiuxin pill (XP) is a traditional Chinese medicine formula for the prevention and treatment of hyperlipidemia (HLP) and related complications. In this study, the gut-liver axis was used as the breakthrough point to analyze the therapeutic effect and potential mechanism of XP on HLP model rats and related complications. MAIN METHODS: We used high-fat diet (HFD) to establish the HLP model of rats and treated them with XP. The 16S rRNA sequencing method was used to explore the effect of XP on the gut microbiota of HFD rats, and the effects of XP on ileum pathology, intestinal barrier and circulatory inflammation in HFD rats were also investigated. We further explored the molecular mechanism of XP treating liver inflammation in rats with HFD by regulating toll-like receptor 4 (TLR4) signaling. KEY FINDINGS: We found that XP could regulate the imbalance of gut microbiota in HFD rats, and up-regulate the expression of tight junction protein in intestinal epithelium of HFD rats, thereby improving the intestinal barrier damage and intestinal inflammatory response. In addition, XP could significantly reduce the levels of inflammatory cytokines in HFD rats, and inhibit TLR4 signaling pathway, thereby reducing liver inflammation in HFD rats. SIGNIFICANCE: XP can effectively improve the imbalance of gut-liver axis in hyperlipidemic rats and alleviate the inflammatory damage of liver. Its mechanism may be related to regulating the disorder of gut microbiota and inhibiting TLR4 signal pathway, so as to achieve the therapeutic effect on hyperlipidemic fatty liver in rats.

Phthalide derivative CD21 regulates the platelet- neutrophil extracellular trap-thrombin axis and protects against ischemic brain injury in rodents.

Prothrombotic and proinflammatory properties of neutrophil extracellular traps (NETs) contribute to brain damage after ischemic stroke. CD21 is a novel phthalide neuroprotectant against cerebral ischemia in rodents. This study investigated effects of CD21 on the platelet-NET-thrombin axis and ischemic brain injury and the underlying mechanism. CD21 exerteddose-dependent neuroprotectionin rats that were subjected to2 h middle cerebral artery occlusion,dose-dependentlyinhibited adenosine diphosphate-mediatedplatelet aggregationin rats, and dose-dependentlyexertedanti-thrombotic activityin rodents that received a collagen-epinephrine combination, ferric chloride, or an arteriovenous shunt. Equimolar CD21 doses exerted stronger efficacy than 3-N-butylphthalide (NBP, natural phthalide for the treatment of ischemic stroke). CD21 dose-dependently improved regional cerebral blood flow, neurobehavioral deficits, and infarct volume in mice that were subjected to photothrombotic stroke (PTS). CD21 (13.79 mg/kg, i.v.) significantly decreased NET components (plasma dsDNA concentrations; mRNA levels of elastase, myeloperoxidase, and neutrophil gelatinase-associated lipocalin and protein level of citrullinated histone H3 in ischemic brain tissues), mRNA and protein levels of peptidyl-arginine deiminase 4 (PDA4, NET formation enzyme), and mRNA levels of NET-related inflammatory mediators (interleukin-1ß, interleukin-17A, matrix metalloproteinase 8, and matrix metalloproteinase 9) in ischemic brain tissues, despite no effect on mRNA levels of deoxyribonuclease I (NET elimination enzyme). Pretreatment with compound C (inhibitor of adenosine monophosphate-activated protein kinase [AMPK]) significantly reversed the inhibitory effects of CD21 on NETs, PDA4, and inflammatory mediators in PTS mice. These results suggest that CD21 might regulate the platelet-NET-thrombin axis and protect against ischemic brain injury partly through the induction of AMPK activation.

[Protective effects of Z-ligustilide against cytotoxicity induced by Abeta25-35 in neuron cells].

OBJECTIVE: To investigate the effects of Z-ligustilide (LIG) on Abeta25-35-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. METHODS: Cells were incubated with 0.1, 1.0, 2.5, 5.0 microg/mL LIG and then were exposed to 50 micromol/L Abeta25-35 to induce cytotoxicity in SH-SY5Y cell to establish the AD model in vitro. Cell viability was test by MTT method. Pro- and anti-apoptosis protein levels were investigated by Western blot. RESULTS: After exposure to Abeta25-35, the cell viability decreased significantly, and the expression of pro-apoptosis protein--Bax, cleaved caspase 3, cytochrome C, caspase 8 was up regulated while the anti-apoptosis protein Bcl-2 was down regulated. However, the treatment of LIG (0.1, 1.0, 2.5, 5.0 microg/mL) significantly restored these changes. CONCLUSION: LIG has the protective effect against cytotoxicity induced by in SH-SY5Y cells via inhibit the apoptosis induced by Abeta25-35.

Neuroprotective effect of alpha-kinase 1 knockdown against cerebral ischemia through inhibition of the NF-κB pathway and neuroinflammation.

BACKGROUND: Activation of the nuclear factor B (NF-κB) signaling pathway by pattern recognition receptors (PRRs) is regarded as a crucial mechanism of neuroinflammation and brain injury after acute ischemic stroke. The stimulation of alpha-kinase 1 (ALPK1), a newly identified PRR, triggers NF-κB activation and an inflammatory response. Longitudinal population-based genetic epidemiological studies suggest that the ALPK1 gene is a susceptible site to ischemic stroke. However, the function of ALPK1 in the central nervous system remains unclear. The present study explored the role of ALPK1 in acute ischemic stroke. METHODS: BV2 microglial cells were stimulated with conditioned medium (CM) that was collected from oxygen and glucose deprivation (OGD)-treated HT22 neurons, and a murine brain ischemia model was established to detect the changes of ALPK1 expression. We used lentivirus to knockdown ALPK1 to explore the effects of ALPK1 in cerebral ischemia models in vitro and in vivo. RESULTS: We observed a significant increase of ALPK1 expression in BV2 cells that were stimulated with OGD CM. The knockdown of ALPK1 inhibited the phosphorylation of tumor necrosis factor receptor associated factor-interacting protein with a forkhead-associated domain (TIFA), the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), the activation of NF-κB, and the levels of proinflammatory factors in the BV2 cells. We also verified a neuroprotective effect of ALPK1 knockdown against ischemic brain injury through inhibition of the TIFA/TRAF6/NF-κB pathway and neuroinflammation in mice. CONCLUSIONS: This study demonstrates that ALPK1 is implicated in sterile inflammatory injury after acute brain ischemia, which provides first evidence for the therapeutic potential of ALPK1 inhibition in ischemic stroke.

Gut Microbiota in Tumor Microenvironment: A Critical Regulator in Cancer Initiation and Development as Potential Targets for Chinese Medicine.

Cancer is a disease with a high mortality and disability rate. Cancer consists not only of cancer cells, but also of the surrounding microenvironment and tumor microenvironment (TME) constantly interacting with tumor cells to support tumor development and progression. Over the last decade, accumulating evidence has implicated that microbiota profoundly influences cancer initiation and progression. Most research focuses on gut microbiota, for the gut harbors the largest collection of microorganisms. Gut microbiota includes bacteria, viruses, protozoa, archaea, and fungi in the gastrointestinal tract, affecting DNA damage, host immune response and chronic inflammation in various types of cancer (i.e., colon cancer, gastric cancer and breast cancer). Notably, gut dysbiosis can reshape tumor microenvironment and make it favorable for tumor growth. Recently, accumulating studies have attached the importance of traditional Chinese medicine (TCM) to cancer treatments, and the bioactive natural compounds have been considered as potential drug candidates to suppress cancer initiation and development. Interestingly, more recent studies demonstrate that TCM could potentially prevent and suppress early-stage cancer progression through the regulation of gut microbiota. This review is on the purpose of exhausting the significance of gut microbiota in the tumor microenvironment as potential targets of Chinese medicine.

Age-related cognitive decline is associated with microbiota-gut-brain axis disorders and neuroinflammation in mice.

Age-related cognitive decline is associated with chronic low grade neuroinflammation that may result from a complex interplay among many factors, such as bidirectional communication between the central nervous system (CNS) and gut microbiota. The present study used 2-month-old (young group) and 15-month-old (aged group) male C57BL/6 mice to explore the potential association between age-related cognitive decline and the microbiota-gut-brain axis disorder. We observed that aged mice exhibited significant deficits in learning and memory, neuronal and synaptic function compared with young mice. Aged mice also exhibited significant dysbiosis of the gut microbiota. Disruptions of the intestinal barrier and blood-brain barrier were also observed, including increases in intestinal, low-grade systemic and cerebral inflammation. Furthermore, plasma and brain levels of lipopolysaccharide (LPS) were significantly higher in aged mice compared with young mice, with increasing expression of Toll-like receptor 4 (TLR4) and myeloid differential protein-88 (MyD88) and the nuclear translocation of nuclear factor κB (NF-κB) in intestinal and brain tissues. These findings showed that microbiota-gut-brain axis dysfunction that occurs through LPS-induced activation of the TLR4/NF-κB signaling pathway is implicated in age-related neuroinflammation and cognitive decline.

Nrf2 signalling pathway and autophagy impact on the preventive effect of green tea extract against alcohol-induced liver injury.

OBJECTIVES: To explore the potential molecular mechanism underlying the effect of green tea extract (TE), rich in tea polyphenols (TPs), on improving alcohol-induced liver injury. METHODS: Mice were intragastrically treated with 50% (v/v) alcohol administration (15 ml/kg BW) with or without three doses of TE (50, 120 and 300 mg TPs/kg BW) daily for 4 weeks, and biological changes were tested. KEY FINDINGS: The TE improved the functional and histological situations in the liver of the mice accepted alcohol administration, including enzymes for alcohol metabolism, oxidative stress and lipid accumulation. Interestingly, the TE increased the nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2), with the decreasing expression of kelch-like ECH-associated protein 1 (Keap1), indicating the association between the effect of TE with Nrf2-mediated antioxidant signalling. Moreover, the TE restored the activity of autophagy, showing as lifted Beclin-1 expression, LC3B-II/LC3B-I ratio, and decreased p62 expression. Importantly, all these effects were dose-dependent. CONCLUSIONS: These findings provide a new notion for the first time that the TE preventing against alcohol-induced liver injury is closely related to accelerated metabolism of alcohol and relieved oxidative stress, which is associated with Nrf2 signalling activation and autophagy restoration, thus the reduction of lipid accumulation in liver.

Neuroprotective Effect of Phthalide Derivative CD21 against Ischemic Brain Injury:Involvement of MSR1 Mediated DAMP peroxiredoxin1 Clearance and TLR4 Signaling Inhibition.

The macrophage scavenger receptor 1 (MSR1)-induced resolution of neuroinflammation may be a novel therapeutic strategy for ischemic stroke. Our previous study showed that the neuroprotective and anti-inflammatory effects of phthalide are associated with the inhibition of the post-ischemic damage-associated molecular pattern (DAMP)/Toll-like receptor 4 (TLR4) pathway. This study investigated the effects of the phthalide derivative CD21 on ischemic brain injury and the mechanism underlying MSR1-induced resolution of neuroinflammation. Using a rat model of 2 h transient middle cerebral artery occlusion (MCAO), MSR1-induced peroxiredoxin1 (PRX1) clearance in RAW264.7 macrophages were investigated. We show here that CD21 significantly ameliorated infarct volumes and neurological deficits in a dose-dependent manner with a ≥ 12 h therapeutic time window. Moreover, administration of 5 mg/kg/day CD21 over 24 h significantly reduced pathological damages, with associated inhibition of PRX1 expression, reduced TLR4/nuclear factor-κB activation and the suppression of the inflammatory response in MCAO rats. Furthermore, the expression of MAFB/MSR1 in the ischemic brain was elevated and the phagocytosis of PRX1 in CD68-positive macrophages isolated from the ischemic brain was enhanced. Further in vitro studies show that CD21 (20 µM) strongly enhanced the Msr1 mRNA and MSR1 protein levers in RAW264.7 cells and PRX1 internalization in cellular lysosomes, which were significantly reversed by N-acetylcysteine treatment. These results suggest that CD21 may exert neuroprotective and anti-inflammatory effects with a wide time window for the treatment of ischemic stroke. The anti-stroke effects of CD21 appear to be mediated partially via the induction of MSR1-promoted DAMP (PRX1) clearance, TLR4/nuclear factor-κB pathway inhibition, and the resolution of inflammation. Graphical Abstract The neuroprotective action of CD21 was associated with the resolution of neuroinflammation through enhancement of the MAFB-MSR1 pathway that leads to DAMP (PRX1) phagocytosis and TLR4 pathway inhibition. Red solid arrows represent promotion, red dotted arrow represents the positive correlation, green arrows represent inhibition.

Z-ligustilide attenuates lipopolysaccharide-induced proinflammatory response via inhibiting NF-kappaB pathway in primary rat microglia.

AIM: To investigate the anti-inflammatory effect of Z-ligustilide (LIG) on lipopolysaccharide (LPS)-activated primary rat microglia. METHODS: Microglia were pretreated with LIG 1 h prior to stimulation with LPS (1 microg/mL). After 24 h, cell viability was tested with MTT, nitric oxide (NO) production was assayed with Griess reagent, and the content of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and monocyte chemoattractant protein (MCP-1) was measured with ELISA. Protein expression of the nuclear factor-kappaB (NF-kappaB) p65 subunit, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) was detected with immunocytochemistry 1 h or 24 h after LPS treatment. RESULTS: LIG showed a concentration-dependent anti-inflammatory effect in LPS-activated microglia, without causing cytotoxicity. Pretreatment with LIG at 2.5, 5, 10, and 20 micromol/L decreased LPS-induced NO production to 75.9%, 54.4%, 43.1%, and 47.6% (P<0.05 or P< 0.01), TNF-alpha content to 86.2%, 68.3%, 40.1%, and 39.9% (P<0.01, with the exception of 86.2% for 2.5 micromol/L LIG), IL-1beta content to 31.5%, 27.7%, 0.6%, and 0% (P<0.01), and MCP-1 content to 84.4%, 50.3%, 45.1%, and 42.2% (P<0.05 or P<0.01), respectively, compared with LPS treatment alone. LIG (10 micromol/L) significantly inhibited LPS-stimulated immunoreactivity of activated NF-kappaB, COX-2, and iNOS (P<0.01 vs LPS group). CONCLUSION: LIG exerted a potent anti-inflammatory effect on microglia through inhibition of NF-kappaB pathway. The data provide direct evidence of the neuroprotective effects of LIG and the potential application of LIG for the treatment of the neuroinflammatory diseases characterized by excessive microglial activation.

Phthalide derivative CD21 ameliorates ischemic brain injury in a mouse model of global cerebral ischemia: involvement of inhibition of NLRP3.

The activation of NLRP3 inflammasome is closely related to ischemic brain injury and inhibition of NLRP3 inflammasome activation may be a new therapeutic strategy for ischemic stroke. Our previous studies showed that ligustilide (LIG) had a dose-dependent neuroprotective effect on various models of cerebral ischemia and dementia in vivo and in vitro. CD21, a kind of phthalide derivative, was modified from LIG. In this study, we established a global cerebral ischemia-reperfusion model in mice by bilateral common carotid artery ligation (2VO), and explored the neuroprotective effect of CD21 and its anti-inflammatory mechanism on cerebral ischemia mice. CD21 significantly improved weight loss, neurobehavioral deficits and neurons loss in hippocampal CA1 and caudate putamen (CPu) subregions, which were induced by 2VO in mice. CD21 significantly inhibited the overactivation of astrocyte and microglia, and decreased the mRNA level of IL-6, TNF-α and IL-1ß. Moreover, CD21 significantly inhibited the activation of TLR4/NF-κB signaling pathway mediated by HMGB1 and NLRP3/ASC/Caspase-1 signaling pathway mediated by Cathepsin B, thus inhibiting the activation of NLRP3 inflammasome. Our results demonstrated that CD21 may exert a neuroprotection by inhibiting NLRP3 inflammasome activation after cerebral ischemia. These findings provide a new strategy for the treatment of ischemic stroke.

Chronic periodontitis induces microbiota-gut-brain axis disorders and cognitive impairment in mice.

Epidemiological studies suggest that chronic periodontitis (CP) is closely associated with the incidence and progression of cognitive impairment. The present study investigated the causal relationship between CP and cognitive decline and the underlying mechanism in mice. Long-term ligature around the left second maxillary molar tooth was used to induce CP in mice. Severe alveolar bone loss and inflammatory changes were observed in gingival tissues, accompanied by progressive cognitive deficits during a 12-month period. We also observed cerebral neuronal and synaptic injury and glial activation in this mouse model of CP. Furthermore, CP mice exhibited significant dysbiosis of the oral and gut microbiota, disruption of the intestinal barrier and blood-brain barrier, increases in the serum contents of proinflammatory cytokines and lipopolysaccharide (LPS), and increases in brain LPS levels, Toll-like receptor 4 (TLR4) expression, nuclear factor-κB (NF-κB) nuclear translocation and proinflammatory cytokine mRNA levels. These results indicate that CP may directly induce progressive cognitive decline and its mechanism is probably related to microbiota-gut-brain axis disorders, LPS/TLR4/NF-κB signaling activation and neuroinflammatory responses in mice. Therefore, the microbiota-gut-brain axis may provide the potential strategy for the prevention and treatment of CP-associated cognitive impairment.

Klotho overexpression improves amyloid-ß clearance and cognition in the APP/PS1 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent type of dementia, characterized by the presence of amyloid-ß (Aß) plaques. We previously reported that Klotho lowered Aß levels in the brain and protected against cognitive deficits in amyloid precursor protein/presenilin 1(APP/PS1) mice. However, the underlying mechanism remains unclear. In this study, we induced intracerebral Klotho overexpression in 13-month-old APP/PS1 mice by injecting lentivirus that carried full-length mouse Klotho cDNA in the lateral ventricle of the brain. We examined the effects of Klotho overexpression on cognition, Aß burden, Aß-related neuropathology, microglia transformation, and Aß transport systems in vivo. Additionally, we investigated the effects of Klotho on Aß transport at the blood-cerebrospinal fluid barrier by knocking down Klotho in primary human choroid plexus epithelial cells (HCPEpiCs). The upregulation of Klotho levels in the brain and serum significantly ameliorated Aß burden, neuronal and synaptic loss and cognitive deficits in aged APP/PS1 mice. Klotho treatment significantly inhibited NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and the subsequent transformation of microglia to the M2 type that may enhance microglia-mediated Aß clearance. Meanwhile, Klotho overexpression also regulated Aß transporter expression, which may promote Aß transporter-mediated Aß clearance. Moreover, the ability of HCPEpiCs to transport Aß in vitro was also significantly impaired by Klotho knockdown. Given the neuroprotective effect of Klotho overexpression, the present findings suggest that Klotho should be further investigated as a potential therapeutic target for AD.