Myeloid gasdermin D drives early-stage T cell immunity and peripheral inflammation in a mouse model of Alzheimer's disease.

BACKGROUND: It is now realized that peripheral inflammation and abnormal immune responses, especially T cells, contribute to the development of Alzheimer's disease (AD). Gasdermin D (GSDMD) -mediated pyroptosis has been associated with several neuroinflammatory diseases, but whether GSDMD is involved in the peripheral inflammation and T cell immunity during AD remains unclear. METHODS: We dynamically investigated GSDMD activation in the peripheral and central nervous system of 5×FAD mouse model and dissected the role of myeloid GSDMD using genetic knockout mice, especially its influence on peripheral T cell responses and AD inflammation. RNA sequencing and in vitro coculture were used to elucidate the underlying immune mechanisms involved. Targeted inhibitor experiments and clinical correlation analysis were used to further verify the function of GSDMD in AD. RESULTS: In the present study, caspase activated GSDMD in the spleen of 5×FAD mice earlier than in the brain during disease progression. Loss of myeloid cell GSDMD was shown to impair early-stage effector T cell activation in the periphery and prevent T cell infiltration into the brain, with an overall reduction in neuroinflammation. Furthermore, myeloid cell GSDMD induced T cell PD-1 expression through the IL-1ß/NF-κB pathway, restricting regulatory T cells. The administration of a GSDMD inhibitor combined with an anti-PD-1 antibody was found to mitigate the development of AD-associated inflammation. In some AD patients, plasma sPD-1 is positively correlated with IL-Iß and clinical features. CONCLUSIONS: Our study systematically identified a role for GSDMD in the AD-related peripheral inflammation and early-stage T cell immunity. These findings also suggest the therapeutic potential of targeting GSDMD for the early intervention in AD.

Microglial AIM2 alleviates antiviral-related neuro-inflammation in mouse models of Parkinson's disease.

Inflammasome involvement in Parkinson's disease (PD) has been intensively investigated. Absent in melanoma 2 (AIM2) is an essential inflammasome protein known to contribute to the development of several neurological diseases. However, a specific role for AIM2 in PD has not been reported. In this study, we investigated the effect of AIM2 in the N-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced PD model by use of various knockout and bone marrow chimeric mice. The mechanism of action for AIM2 in PD was assessed by RNA-sequencing and in vitro primary microglial transfection. Results were validated in the A30P transgenic mouse model of PD. In the MPTP mouse model, AIM2 activation was found to negatively regulate neuro-inflammation independent of the inflammasome. Microglial AIM2 deficiency exacerbated behavioral and pathological features of both MPTP-induced and transgenic PD mouse models. Mechanistically, AIM2 reduced cyclic GMP-AMP synthase (cGAS)-mediated antiviral-related inflammation by inhibition of AKT-interferon regulatory factor 3 (IRF3) phosphorylation. These results demonstrate microglial AIM2 to inhibit the antiviral-related neuro-inflammation associated with PD and provide for a foundation upon which to identify new therapeutic targets for treatment of the disease.

Systemic inflammasome activation and pyroptosis associate with the progression of amnestic mild cognitive impairment and Alzheimer's disease.

BACKGROUND: Growing evidence indicates that inflammasome-mediated inflammation plays important roles in the pathophysiology of amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD). Pyroptosis induced by inflammasome, and Gasdermin D (GSDMD) is involved in several neurodegenerative disorders. However, it is not clear whether peripheral inflammasome and pyroptosis are activated in aMCI and AD patients, influencing on neuroinflammation. The aim of this study was to examine the association between systemic inflammasome-induced pyroptosis and clinical features in aMCI and AD. METHODS: A total of 86 participants, including 33 subjects with aMCI, 33 subjects with AD, and 20 cognitively normal controls, in this study. The Mini Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA) scale were used for cognitive assessment. Levels of inflammasome-related genes/proteins in peripheral blood mononuclear cells (PBMCs) were determined using quantitative polymerase chain reaction and Western blotting. The levels of IL-1ß, Aß1-42, Aß1-40, p-tau, and t-tau in cerebrospinal fluid (CSF), as well as the plasma IL-1ß level, were measured by enzyme-linked immunosorbent assay. Finally, lipopolysaccharides (LPS) were used to investigate the effects of systemic inflammasome-induced pyroptosis in an AD mice model. RESULTS: Several genes involved in the inflammatory response were enriched in PBMCs of AD patients. The mRNA and protein levels of NLRP3, caspase-1, GSDMD, and IL-1ß were increased in PBMCs of aMCI and AD patients. The IL-1ß level in plasma and CSF of aMCI and AD patients was significantly higher than that in controls and negatively correlated with the CSF Aß1-42 level, as well as MMSE and MoCA scores. Furthermore, there was a positive correlation between the IL-1ß level in plasma and CSF of aMCI or AD patients. In vivo experiments showed that systemic inflammasome-induced pyroptosis aggravated neuroinflammation in 5 × FAD mice. CONCLUSIONS: Our findings showed that canonical inflammasome signaling and GSDMD-induced pyroptosis were activated in PBMCs of aMCI and AD patients. In addition, the proinflammatory cytokine IL-1ß was strongly associated with the pathophysiology of aMCI and AD. As such, targeting inflammasome-induced pyroptosis may be a new approach to inhibit neuroinflammation in aMCI and AD patients.

Baicalein Attenuates Neuroinflammation by Inhibiting NLRP3/caspase-1/GSDMD Pathway in MPTP Induced Mice Model of Parkinson's Disease.

BACKGROUND: Inflammasome-induced neuroinflammation is a major pathogenic mechanism underlying the degeneration of nigral dopaminergic neurons in Parkinson's disease (PD). Baicalein is a flavonoid isolated from the traditional Chinese medicinal herbal Scutellaria baicalensis Georgi with known anti-inflammatory and neuroprotective efficacy in models of neurodegenerative diseases, including PD. However, its effects on inflammasome-induced neuroinflammation during PD remain unclear. METHODS: We used N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce PD-like pathology in mice. Behavioral assessments including the pole test, rotarod test and open filed test were conducted to evaluate the effects of baicalein on MPTP-induced motor dysfunction. The efficacies of baicalein against MPTP-induced dopaminergic neuron loss and glial cell activation in the substantia nigra compact (SNc) were examined by immunohistochemistry, effects on proinflammatory cytokines by qPCR and enzyme-linked immunosorbent assay (ELISA), effects on inflammasome pathway activation by immunoblotting and flow cytometry. RESULTS: Administration of baicalein reversed MPTP-induced motor dysfunction, loss of dopaminergic neurons, and pro-inflammatory cytokine elevation. Baicalein also inhibited NLRP3 and caspase-1 activation and suppressed gasdermin D (GSDMD)-dependent pyroptosis. Additionally, baicalein inhibited the activation and proliferation of disease-associated proinflammatory microglia. CONCLUSIONS: These findings suggest that baicalein can reverse MPTP-induced neuroinflammation in mice by suppressing NLRP3/caspase-1/GSDMD pathway. Our study provides potential insight of baicalein in PD therapy.

Nuclear Factor Erythroid 2-Related Factor 2 Deficiency Results in Amplification of the Liver Fat-Lowering Effect of Estrogen.

Transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates multiple biologic processes, including hepatic lipid metabolism. Estrogen exerts actions affecting energy homeostasis, including a liver fat-lowering effect. Increasing evidence indicates the crosstalk between these two molecules. The aim of this study was to evaluate whether Nrf2 modulates estrogen signaling in hepatic lipid metabolism. Nonalcoholic fatty liver disease (NAFLD) was induced in wild-type and Nrf2-null mice fed a high-fat diet and the liver fat-lowering effect of exogenous estrogen was subsequently assessed. We found that exogenous estrogen eliminated 49% and 90% of hepatic triglycerides in wild-type and Nrf2-null mice with NAFLD, respectively. This observation demonstrates that Nrf2 signaling is antagonistic to estrogen signaling in hepatic fat metabolism; thus, Nrf2 absence results in striking amplification of the liver fat-lowering effect of estrogen. In addition, we found the association of trefoil factor 3 and fatty acid binding protein 5 with the liver fat-lowering effect of estrogen. In summary, we identified Nrf2 as a novel and potent inhibitor of estrogen signaling in hepatic lipid metabolism. Our finding may provide a potential strategy to treat NAFLD by dually targeting Nrf2 and estrogen signaling.

Compound Astragalus and Salvia miltiorrhiza extracts suppress hepatocarcinogenesis by modulating transforming growth factor-ß/Smad signaling.

BACKGROUND AND AIM: Previous studies showed Compound Astragalus and Salvia miltiorrhiza extract (CASE), extract from Astragalus membranaceus and Salvia miltiorhiza, significantly suppresses hepatocellular carcinoma (HCC) in rats induced by diethylinitrosamine (DEN), and in vitro experiments further demonstrated that CASE's anti-HepG2 cell invasion is associated with transforming growth factor-ß (TGF-ß). We hypothesized that CASE's suppression of HCC is modulated by TGF-ß/Smad signaling, and we conducted this in vivo study to test this hypothesis. METHODS: Rats were divided into the normal control, the DEN group, and three CASE (60, 120, and 240 mg/kg) treatment groups. The expression of phosphorylation(p) Smad both at C-terminal and linker region, plasminogen activator inhibitor 1, and Smad4 and Smad7 of liver tissues were measured and compared across the five groups. RESULTS: The positive staining of pSmad2L and pSmad3L increased both in hepatoma nodule areas and adjacent relatively normal liver tissues in rats treated with DEN, while the positive staining of pSmad2C and pSmad3C increased only in relatively normal liver tissues adjacent to hepatoma tissues. The elevated expression of pSmad2C, pSmad2L, pSmad3L, Smad4, and plasminogen activator inhibitor 1 proteins were suppressed by CASE in a dose-dependent manner. CASE treatment also significantly reduced the intranuclear amounts of pSmad2L and pSmad3L, and upregulated the elevation of pSmad3C positive cells and protein expression in a dose-dependent manner. CONCLUSION: The results suggest that CASE significantly suppresses HCC progression by mediating TGF-ß/Smad signaling, especially by modulating Smad3 phosphorylation both at the C-terminal and linker region.

Baicalin attenuates chronic unpredictable mild stress-induced hippocampal neuronal apoptosis through regulating SIRT1/PARP1 signaling pathway.

Baicalin (BA), a flavonoid glycoside extracts from Scutellaria baicalensis Georgi, has been reported to exert antidepressant effects. Emerging evidence indicates that neuronal apoptosis plays a crucial role in the pathogenesis of depression. Poly (ADP-ribose) polymerase-1 (PARP1) is established as a key regulator of the cellular apoptosis. In the present study, we explored whether BA exerts antidepressant effects by regulating PARP1 signaling pathway and elucidated the underlying mechanisms. We found that administration of BA (30 mg/kg, 60 mg/kg) alleviated chronic unpredictable mild stress (CUMS)-induced depressive-like behaviors by increasing sucrose consumption in sucrose preference test (SPT), improving activity status in open field test (OFT) and reducing rest time in tail suspension test (TST). Hematoxylin and eosin (HE) staining and Nissl staining showed that BA ameliorated CUMS-induced neuronal damage in the hippocampus. Moreover, BA significantly upregulated anti-apoptotic protein Bcl-2, downregulated pro-apoptotic protein Bax and cleaved-caspase-3 after CUMS in hippocampal of mice. Intriguingly, western blot and immunohistochemistry (IHC) results showed that the protein level of PARP1 was significantly increased in hippocampal tissue after CUMS, which was reversed by BA treatment. In primary hippocampal neurons (PHNs), BA abrogated the neuronal apoptosis caused by PARP1 overexpression. Meanwhile, BA significantly increased the protein level of SIRT1, SIRT1 inhibitor (EX-527) treatment reversed the effect of BA on reducing the protein level of PARP1 and neuronal apoptosis in CUMS-induced mice. Overall, our results indicated that BA attenuated the CUMS-induced hippocampal neuronal apoptosis through regulating the SIRT1/PARP1 signaling pathway.

Quercetin alleviates chronic unpredictable mild stress-induced depressive-like behaviors by promoting adult hippocampal neurogenesis via FoxG1/CREB/ BDNF signaling pathway.

Quercetin, a naturally occurring flavonoid, has been reported to exert antidepressant effects, however, the underlying mechanisms are still uncertain. Recent studies have demonstrated that Forkhead box transcription factor G1 (FoxG1) regulates the process of adult hippocampal neurogenesis (AHN) and exerts neuroprotective effects. In this study, we explored whether quercetin plays an anti-depressant role via regulation of FoxG1 signaling in mice and revealed the potential mechanisms. To explore the antidepressant effects of quercetin, mice were subjected to behavioral tests after a chronic unpredictable mild stress (CUMS) exposure. We found that chronic quercetin treatment (15 mg/kg, 30 mg/kg) obviously restored the weight loss of mice caused by CUMS and alleviated CUMS-induced depression-like behaviors, such as increased sucrose consumption, improved locomotor activity and shorten immobility time. In addition, to clarify the relationship between quercetin and AHN, we detected neurogenesis markers in the dentate gyrus (DG) of the hippocampus. Furthermore, FoxG1-siRNA was employed and then stimulated with quercetin to further investigate the mechanism by which FoxG1 participates in the antidepressant effects of quercetin. Our results indicate that chronic quercetin treatment dramatically increased the number of doublecortin (DCX)-positive and BrdU/NeuN-double positive cells. Besides, the expression levels of FoxG1, p-CREB and Brain-derived neurotrophic factor (BDNF) were also enhanced by quercetin in the DG. Strikingly, quercetin failed to reverse the levels of p-CREB and BDNF after FoxG1-siRNA was performed in SH-SY5Y cells and Neural Progenitor Cells (NPCs). Our results thus far suggest that quercetin might exert antidepressant effects via promotion of AHN by FoxG1/CREB/ BDNF signaling pathway.

Compound Astragalus and Salvia miltiorrhiza extract suppresses hepatocellular carcinoma progression by inhibiting fibrosis and PAI-1 mRNA transcription.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus membranaceus and Salvia miltiorrhiza have been used for centuries in China to treat liver diseases. Previous studies have shown that these herbs and their extracts inhibit the development of liver fibrosis and the proliferation and invasion of human hepatoma HepG2 cells. Further study of their pharmacological effects on hepatocellular carcinoma (HCC) is needed. To investigate the effects of Compound Astragalus and Salvia miltiorrhiza Extract (CASE) on diethylinitrosamine (DEN)-induced hepatocarcinogenesis in rats. MATERIALS AND METHODS: Male rats were divided into five groups, with the first group serving as normal control, the second group receiving 0.2% DEN solution five times a week for 14 weeks, and the third to fifth group receiving the same DEN as in the second group together with CASE at the doses of 60, 120, and 240 mg/kg per day for 16 weeks, respectively. Hepatoma incidence, serum enzymes levels, degree of fibrosis and hydroxyproline content were evaluated and compared across the five groups to determine CASE's suppression of fibrosis and HCC progression. In addition, an in vitro experiment using HepG2 cells was conduct to verify CASE's effect on the transcription of plasminogen activator inhibitor-1 (PAI-1) mRNA. RESULTS: CASE treatment significantly reduced the incidence and multiplicity of DEN-induced HCC development in a dose-dependent manner. It significantly suppressed the elevation of alanine transaminase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, hyaluronic acid, direct bilirubin and total bilirubin, and significantly lessened the depression of serum total protein in DEN-induced HCC rats. CASE treatment also significantly suppressed the elevated expression of GST-P and α-SMA. The in vitro experiment confirmed that CASE inhibits the transcription of PAI-1 mRNA in HepG2 cells induced by TGF-ß1 in a dose-dependent manner. CONCLUSIONS: CASE suppresses DEN-induced hepatocarcinogenesis by inhibiting fibrosis and PAI-1 mRNA transcription, suggesting its potential clinical application in preventing and treating human HCC.

Nrf2 is involved in maintaining hepatocyte identity during liver regeneration.

Nrf2, a central regulator of the cellular defense against oxidative stress and inflammation, participates in modulating hepatocyte proliferation during liver regeneration. It is not clear, however, whether Nrf2 regulates hepatocyte growth, an important cellular mechanism to regain the lost liver mass after partial hepatectomy (PH). To determine this, various analyses were performed in wild-type and Nrf2-null mice following PH. We found that, at 60 h post-PH, the vast majority of hepatocytes lacking Nrf2 reduced their sizes, activated hepatic progenitor markers (CD133, TWEAK receptor, and trefoil factor family 3), depleted HNF4α protein, and downregulated the expression of a group of genes critical for their functions. Thus, the identity of hepatocytes deficient in Nrf2 was transiently but massively impaired in response to liver mass loss. This event was associated with the coupling of protein depletion of hepatic HNF4α, a master regulator of hepatocyte differentiation, and concomitant inactivation of hepatic Akt1 and p70S6K, critical hepatocyte growth signaling molecules. We conclude that Nrf2 participates in maintaining newly regenerated hepatocytes in a fully differentiated state by ensuring proper regulation of HNF4α, Akt1, and p70S6K during liver regeneration.