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Background The incidence of ischemic cerebrovascular disease is increasing in recent years and has been one of the leading causes of neurological dysfunction and death. Ginsenoside Rg1 has been found to protect against neuronal damage in many neurodegenerative diseases. However, the effect and mechanism by which Rg1 protects against cerebral ischemia-reperfusion injury (CIRI) are not fully understood. Here, we report the neuroprotective effects of Rg1 treatment on CIRI and its possible mechanisms in mice. Methods A bilateral common carotid artery ligation was used to establish a chronic CIRI model in mice. HT22 cells were treated with Rg1 after OGD/R to study its effect on [Ca2+]i. The open-field test and pole-climbing experiment were used to detect behavioral injury. The laser speckle blood flowmeter was used to measure brain blood flow. The Nissl and H&E staining were used to examine the neuronal damage. The Western blotting was used to examine MAP2, PSD95, Tau, p-Tau, NOX2, PLC, p-PLC, CN, NFAT1, and NLRP1 expression. Calcium imaging was used to test the level of [Ca2+]i. Results Rg1 treatment significantly improved cerebral blood flow, locomotion, and limb coordination, reduced ROS production, increased MAP2 and PSD95 expression, and decreased p-Tau, NOX2, p-PLC, CN, NFAT1, and NLRP1 expression. Calcium imaging results showed that Rg1 could inhibit calcium overload and resist the imbalance of calcium homeostasis after OGD/R in HT22 cells. Conclusion Rg1 plays a neuroprotective role in attenuating CIRI by inhibiting oxidative stress, calcium overload, and neuroinflammation.

Biochanin A (Bioch A) is a natural plant estrogen, with various biological activities such as anti-apoptosis, anti-oxidation, and suppression of inflammation. In this study, we investigated the protective effects of Bioch A on angiotensin II (AngII) – induced dopaminergic (DA) neuron damage in vivo and on molecular mechanisms. Spontaneous activity and motor ability of mice among groups was detected by open-field test and swim-test. The expression of TH, microtubule-associated proteins light chain 3B II (LC3BII)/LC3BI, beclin-1, P62, forkhead box class O3 (FoxO3), phosphorylated (p) FoxO3a/FoxO3a, FoxO3, and endophilin A2 were determined by Western blot and immunohistochemistry or immunofluorescence staining. Our results showed that AngII treatment significantly increased the behavioral dysfunction of mice and DA neuron damage. Meanwhile, AngII treatment increased the expression of LC3BII/LC3BI, beclin-1, P62, and FoxO3a and decreased the expression of endophilin A2 and p-FoxO3a/FoxO3a, however, Bioch A treatment alleviate these changes. In summary, these results suggest that Bioch A exerts protective effects on AngII-induced mouse model may be related to regulating endophilin A2, FoxO3a, and autophagy-related proteins; however, the specific mechanism is not yet clear and needs further study.

Alzheimer's disease (AD) is a common neurodegenerative disorder. Amyloid β (Aβ) deposition is considered an important pathological feature of AD. Growing evidence has linked neuroinflammation and autophagy to Aβ deposition in the progression of AD. However, there are few drug options for inhibiting neuroinflammation and autophagy to prevent AD. Ginsenoside Rg1 (Rg1), a steroidal saponin extracted from ginseng, has been reported to possess multiple neuroprotective effects. The present study aimed to evaluate whether Rg1 treatment could attenuate cognitive disorders and neuronal injuries by inhibiting NLRP1 inflammasome and autophagy dysfunction in an AD model of APP/PS1 mice. The results of behavioral tests indicated that Rg1 treatment for 12 weeks could significantly improve olfactory dysfunction as well as learning and memory impairments. The results of histopathological tests indicated that Rg1 treatment could reduce Aβ deposition and neuronal damages in APP/PS1‑9M mice. Additionally, the results of immunoblot, reverse transcription‑quantitative PCR or immunohistochemistry demonstrated that Rg1 treatment significantly downregulated the expression levels of inflammation‑related proteins of NLRP1, caspase1, IL‑1β and TNF‑α, as well as autophagy‑related proteins of p‑AMPK/AMPK, Beclin1 and LC3 II/LC3 I, and increased the expression levels of p‑mTOR/mTOR and P62 in APP/PS1‑9M mice. In addition, the molecular docking analysis showed that there was favorable binding result between Rg1 and NLRP1. The present study suggested that Rg1 may alleviate learning and memory impairments and Aβ disposition by inhibiting NLRP1 inflammasome and improving autophagy dysfunction, suggesting that Rg1 may be a potential therapeutic agent for delaying AD.

Background: N4-acetylcytidine (ac4C), an important posttranscriptional modification, is involved in various disease processes. Long noncoding RNAs (lncRNAs) regulate gene expression mainly through epigenetic modification, transcription, and posttranscriptional modification. Alzheimer’s disease (AD) is a neurodegenerative disease characterized by amyloidosis of the brain. However, the role of lncRNA ac4C modification in AD remains unclear. Objective: In this study, we investigated the association between ac4C modification and AD, and the underlying mechanisms of ac4C modification in AD. Methods: The male 9-month-old APP/PS1 double transgenic mice, age- and sex-matched wild type (WT) mice were used in this study. Then, ac4C-RIP-seq and RNA-seq were used to comprehensively analyze lncRNA ac4C modification in AD mice. The lncRNA-miRNA-mRNA regulatory networks using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed the regulatory relationships among these three lncRNAs and AD. Results: The results showed that there were 120 significantly different ac4C peaks located on 102 lncRNAs in AD, of which 55 were hyperacetylated and 47 were hypoacetylated. Simultaneously, 231 differentially expressed lncRNAs were identified, including 138 upregulated lncRNAs and 93 downregulated lncRNAs. Moreover, 3 lncRNAs, lncRNA Gm26508, lncRNA A430046D13Rik, and lncRNA 9530059O14Rik, showed significant changes in both the ac4C and RNA levels using conjoint analysis. Conclusion: The abundance of lncRNA ac4C modification is significantly different in AD and indicates that lncRNA ac4C is associated with the occurrence and development of AD, which could provide a basis for further exploration of the related regulatory mechanisms.

Ginsenoside Rg1 (Rg1) is one of the main active components in Panax ginseng C. A. Meyer (ginseng), which has been widely used to delay senescence or improve health conditions for more than 2000 years. Increasing studies have revealed that Rg1 could regulate cell proliferation and differentiation, as well as anti-inflammatory and anti-apoptotic effects, and might have protective effects on many chronic kidney diseases.Diabetic nephropathy (DN) is one of the most dangerous microvascular complications of diabetes and is the leading cause of end-stage renal disease worldwide. However, the role and mechanism of Rg1 against high-glucose and high-fat-induced glomerular fibrosis in DN are not clear. This study aimed to investigate the protective effect of Rg1 on DN and its possible mechanism.The type 2 diabetes mellitus (T2DM) mice models were established with a high-fat diet (HFD) combined with an intraperitoneal injection of streptozotocin (STZ). Urine protein and serum biochemical indexes were detected by corresponding kits. The kidney was stained with HE, PAS, and Masson to observe the pathological morphology, glycogen deposition, and fibrosis. The expression of CD36 and p-PLC in the kidney cortex was detected by IHC. The expressions of FN and COL4 were detected by IF. Western blot and PCR were performed to examine protein and mRNA expressions of kidney fibrosis and TRPC6/NFAT2-related pathways in DN mice. Calcium imaging was used to examine the effect of Rg1 on [CaRg1 treatment for 8 weeks could prominently decrease urinary protein, serum creatinine, and urea nitrogen and downgrade blood lipid levels and renal lipid accumulation in T2DM mice. The pathological results indicated that Rg1 treatment attenuated renal pathological injury and glomerular fibrosis. The further results demonstrated that Rg1 treatment remarkably decreased the expressions of CD36, TRPC6, p-PLC, CN, NFAT2, TGF-β, p-Smad2/3, COL4, and FN in renal tissues from T2DM mice. Calcium imaging results found that Rg1 downgraded the base levels of [CaThese results revealed that Rg1 could ameliorate renal lipid accumulation, pathological damage, and glomerular fibrosis in T2DM mice. The mechanism may be involved in reducing the overexpression of CD36 and inhibiting the TRPC6/NFAT2 signaling pathway in renal tissues of T2DM mice.

Objectives 2,4-Dinitrofluorobenzene (DNFB) induced allergic contact dermatitis model is widely used in the research of inflammation, immune response and pruritus. However, there are several modeling cycles of DNFB model in many literatures, which brings some confusion for choice in the experiments. Here, we established two-weeks and five-weeks models of DNFB and aimed to compare and evaluate them. Methods Two-week and five-week DNFB models were established in male SPF C57BL/6 mice. Results The results showed that the scratching bouts of the ACD mice in two groups both significantly increased. Following the modeling process, the skin of the neck and back of the modeling site of the ACD mice in two-weeks group gradually appeared erythema, edema, ulceration, scabbing, scaling and dryness. The skin surface features of ACD mice in five-weeks group obviously showed erythema, edema, ulceration, scabbing, scaling, re-scabbing, re-scaling, harden and dryness.The whole modeling process are both accompanied by severe scratching reaction after the skin repeated exposure to DNFB. Moreover, the results of hematoxylin-eosin (HE) staining showed that the two modeling methods both had thickened epidermis (p < 0.001) and hyperkeratosis. The toluidine blue (TB) staining revealed obvious inflammatory cell infiltration (p < 0.0001) in two groups. Conclusion In our opinion, the two modeling methods both are feasible. The two-weeks ACD model had a shorter modeling period, and while the 5-weeks model could have enough time to observe the effect of drug. Our results provided further experimental basis and evidence for animal models of skin pruritus and inflammation.

Glucocorticoid (GC) exposure can lead to deterioration of the structure and function of hippocampal neurons and is closely involved in Alzheimer's disease (AD). Amyloid-β (Aβ) overproduction is an important aspect of AD pathogenesis. Our study mainly investigated the mechanism of chronic GC exposure in accelerating Aβ production in primary cultured hippocampal neurons from APP/PS1 mice. The results indicated that chronic dexamethasone (DEX, 1 μM) significantly accelerated neuronal damage and Aβ accumulation in hippocampal neurons from APP/PS1 mice. Meanwhile, DEX exposure markedly upregulated APP, NCSTN, BACE1 and p-Tau/Tau expression in hippocampal neurons from APP/PS1 mice. Our study also indicated that chronic DEX exposure significantly increased intracellular Ca

Aging is often accompanied by liver injury and fibrosis, eventually leading to the decline in liver function. However, the mechanism of aging‑induced liver injury and fibrosis is still not fully understood, to the best of our knowledge, and there are currently no effective treatment options available for liver aging. Ginsenoside Rg1 (Rg1) has been reported to exert potent anti‑aging effects due to its potential antioxidant and anti‑inflammatory activity. The present study aimed to investigate the protective effect and underlying mechanism of action of Rg1 in aging‑induced liver injury and fibrosis in senescence‑accelerated mouse prone 8 (SAMP8) mice treated for 9 weeks. The histopathological results showed that the arrangement of hepatocytes was disordered, vacuole‑like degeneration occurred in the majority of cells, and collagen IV and TGF‑β1 expression levels, that were detected via immunohistochemistry, were also significantly upregulated in the SAMP8 group. Rg1 treatment markedly improved aging‑induced liver injury and fibrosis, and significantly downregulated the expression levels of collagen IV and TGF‑β1. In addition, the dihydroethylene staining and western blotting results showed that Rg1 treatment significantly reduced the levels of reactive oxygen species (ROS) and IL‑1β, and downregulated the expression levels of NADPH oxidase 4 (NOX4), p47phox, p22phox, phosphorylated‑NF‑κB, caspase‑1, apoptosis‑associated speck‑like protein containing a C‑terminal caspase recruitment domain and the NLR family pyrin domain containing 3 (NLRP3) inflammasome, which were significantly upregulated in the liver tissues of elderly SAMP8 mice. In conclusion, the findings of the present study suggested that Rg1 may attenuate aging‑induced liver injury and fibrosis by reducing NOX4‑mediated ROS oxidative stress and inhibiting NLRP3 inflammasome activation.

The level of lipopolysaccharide (LPS) is higher in the blood and brains of patients with Alzheimer's disease (AD), and this phenomenon is strongly linked to AD-related neuronal damage and β-amyloid (Aβ) generation. However, the mechanism by which LPS causes neuronal damage has still not been fully clarified. Oxidative stress, neuroinflammation, and Ca

Glucocorticoid (GC), secreted by adrenal cortex, plays important roles in regulating many physiological functions, while chronic stress level of GC exposure has many adverse effects on the structure and function of hippocampal neurons, and is closely implicated to the deterioration of Alzheimer's disease (AD). Oxidative stress and neuroinflammation play an important role in the occurrence and development of AD. However, it is still unclear whether chronic GC exposure promotes beta-amyloid (Aβ) accumulation and neuronal injury by increasing oxidative stress and neuroinflammation. In this study, we investigated the effects of chronic GC exposure on NOX2-NLRP1 inflammasome activation and the protective effects of NLRP1-siRNA against GC-induced neuronal injury in primary hippocampal neurons of APP/PS1 mice. The results showed that chronic dexamethasone (DEX, 1 µM) exposure 72 h had no significant effect on the primary hippocampal neurons of WT mice, but significantly increased Aβ

Diabetic liver injury is a serious complication of type 2 diabetes mellitus (T2DM), which is often irreversible in the later stage, and affects the quality of life. Autophagy serves an important role in the occurrence and development of diabetic liver injury. For example, it can improve insulin resistance (IR), dyslipidaemia, oxidative stress and inflammation. Astragaloside IV (AS-IV) is a natural saponin isolated from the plant Astragalus membranaceus, which has comprehensive pharmacological effects, such as anti-oxidation, anti-inflammation and anti-apoptosis properties, as well as can enhance immunity. However, whether AS-IV can alleviate diabetic liver injury in T2DM and its underlying mechanisms remain unknown. The present study used high-fat diets combined with low-dose streptozotocin to induce a diabetic liver injury model in T2DM rats to investigate whether AS-IV could alleviate diabetic liver injury and to identify its underlying mechanisms. The results demonstrated that AS-IV treatment could restore changes in food intake, water intake, urine volume and body weight, as well as improve liver function and glucose homeostasis in T2DM rats. Moreover, AS-IV treatment promoted suppressed autophagy in the liver of T2DM rats and improved IR, dyslipidaemia, oxidative stress and inflammation. In addition, AS-IV activated adenosine monophosphate-activated protein kinase (AMPK), which inhibited mTOR. Taken together, the present study suggested that AS-IV alleviated diabetic liver injury in T2DM rats, and its mechanism may be associated with the promotion of AMPK/mTOR-mediated autophagy, which further improved IR, dyslipidaemia, oxidative stress and inflammation. Thus, the regulation of autophagy may be an effective strategy to treat diabetic liver injury in T2DM.

Sex is an important biological variable that impacts diverse physiological and pathological processes, including the progression of diabetic nephropathy. Diabetic nephropathy is one of the most common complications of diabetes mellitus and is the leading cause of end-stage renal disease. The endothelial nitric oxide synthase-deficient (eNOS−/−) db/ db mouse is an appropriate and valuable model to study mechanisms in the development of diabetic nephropathy because of the similarities of the features of diabetic kidney disease in this model to those in humans. The aim of the present study was to determine whether there was a sex difference in renal injury in eNOS−/− db/ db mice. Both male and female eNOS−/− db/ db mice showed hyperglycemia, obesity, and renal hypertrophy. However, there was no significant difference in those variables between male and female mice. Furthermore, both male and female diabetic mice showed progressive albuminuria and significantly greater levels of serum creatinine and blood urea nitrogen compared with the same sex of wild-type mice (nondiabetic controls). Although all three variables in female eNOS−/− db/ db mice had a tendency to be greater than those in male eNOS−/− db/ db mice, those sex differences were not statistically significant. Moreover, both male and female eNOS−/− db/ db mice showed significant mesangial expansion, higher glomerular injury scores, profound renal fibrosis, and substantial accumulation of fibronectin and collagen type IV proteins. However, sex differences in those structural changes were not observed. Similarly, survival rates of male and female eNOS−/− db/ db mice were comparable. Taken together, the results from the present study suggest no sex difference in renal structural and functional damage in eNOS−/− db/ db mice.

Oxidative stress and inflammation are closely related to neuron ageing. NADPH oxidase 2 (NOX2) is a major source of reactive oxygen species (ROS) generation in brain. The nucleotide-binding oligomerisation domain (NOD)-like receptor protein 1 (NLRP1) inflammasome is responsible for the formation of proinflammatory molecules in neurons. We hypothesize that NOX2-derived ROS accumulation mediates activation of NLRP1 inflammasome, which is involved in age-related neuronal damage. In the present study, we investigated the changes of NOX2-NLRP1 signaling pathway in primary hippocampal neurons cultured for different time (6, 9 and 12 days, d). Meanwhile, we further examined the effect of ROS inhibitor and NLRP1-siRNA on neuronal senescence. The results showed that, compared with 6 d group, the neuronal apoptosis and β-Galactosidase (β-Gal) expression were significantly increased, and the microtubule-associated protein 2 (MAP2) expression significantly decreased in primary hippocampal neurons cultured for 12 d. In addition, the results also showed that the production of ROS, the expressions of NOX2 and NLRP1 inflammasome were significantly increased with the prolongation of culture time in hippocampal neurons. Moreover, the NOX inhibitor (apocynin) and ROS scavenger (tempol) significantly decreased ROS production and alleviated neuronal damage. Meanwhile, the tempol and apocynin treatment significantly decreased the expression of NLRP1 inflammasome in hippocampal neurons. Furthermore, the NLRP1-siRNA and caspase-1 inhibitor treatment also alleviated neuronal damage. These results suggest that NOX2-derived ROS generation may induce brain inflammation via NLRP-1 inflammasome activation and lead to age-related neuronal damage. The NADPH oxidase and NLRP1 inflammasome may be important therapeutic targets for age-related neuronal damage.

Parkinson's disease (PD) is a neurodegenerative disorder, which is characterized by microglia activation and dopaminergic neurons affected by inflammatory processes. Inflammation has been recognized to be necessary for initiation and progress of PD. Emerging evidence indicates that NLRP3 inflammasome complex is involved in the recognition and execution of host inflammatory response. Stress is acknowledged to be a predisposing and precipitating factor in some neurodegenerative diseases. However, it is unknown whether chronic unpredictable mild stress (CUMS) sensitized microglia to pro-inflammatory stimuli. In this study, in vivo experiments are used to evaluate the effects of CUMS on lipopolysaccharide (LPS)-induced microglia activation and NLRP3 inflammasome activation. The results showed that CUMS pretreatment for 14 days significantly aggravated the behavioral dysfunction of PD rats, increased the activation of microglia. Pretreatment with CUMS for 14 days increased the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a) in the serum, and increased the expression of NLRP-3, ASC, Casepase-1 in the substantia nigra of PD rats. Our data showed that pretreatment with CUMS for 14 days increased the microglia activation and the DA neurons damage, and the mechanisms may be associated with the acceleration of the inflammatory response and activation of NLRP3 inflammasome.

BackgroundDiabetic nephropathy (DN) is a serious complication of type 2 diabetes mellitus (T2DM). Hyperlipidemia plays a key role in occurrence and development of DN. The main saturated fatty acids of palmitic acid (PA) is closely related to glomeruli and tubules injury in T2DM. Astragaloside IV (AS-IV) has comprehensive pharmacological effects, such as anti-inflammation, anti-oxidation and anti-apoptosis. However, whether AS-IV can attenuate PA-induced renal tubular epithelial cells damages and its underlying mechanisms remain unclear.MethodsIn vitro, the HK-2 cells were stimulated with PA (200 μM) for 6, 9, 12, and 24 h respectively, then treated with AS-IV (10, 20 or 40 μM) and Apo (100 μM) for 24 h. In vivo, we investigated the effects of AS-IV in high-fat diets (HFD) and low-dose streptozotocin (STZ)-induced type 2 DN rats. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay kit. The lipid deposition was detected by ORO Staining assay kit. Annexin V-FITC/PI staining was used to detect the cell apoptosis. Immunofluorescence staining and Immunohistochemistry were used to measure the expressions of NLRP3 and NOX4. The ELISA kits were conducted to assess the levels of IL-1β, IL-18, GSH and SOD. Western blotting was used to assess the expressions of NLRP3, caspase-1, ASC, 1L-1β, NOX4, p22phox and p47phox proteins.ResultsOur results indicated that PA exposure not only significantly decreased cell viability, GSH and SOD expressions, and increased lipids deposition and apoptosis, but also increased the level of ROS and the expressions of IL-1β and IL-18. Moreover, PA exposure significantly increased the expressions of NADPH oxidase 4 (NOX4), NLRP3, ASC and caspase-1. However, all these abnormalities were significantly ameliorated by AS-IV and apocynin (Apo) treatment. In addition, our results indicated that AS-IV (80 mg/kg) could attenuate the renal tubular pathological injury and lipids deposition, and decrease the expressions of NLRP3, ASC, caspase-1, NOX4, p22phox and p47phox in renal tubules. Conclusions The study suggested that AS-IV could improve PA-induced HK-2 cells injury and renal tubular damage in T2DM rats, and the mechanism may be related to inhibition of NOX4-mediated NLRP3 inflammasome activation.

Lipopolysaccharide (LPS) is a toxic component of cell walls of Gram-negative bacteria that are widely present in gastrointestinal tracts. Increasing evidence showed that LPS plays important roles in the pathogeneses of neurodegenerative disorders, such as Alzheimer's disease (AD). NADPH oxidase s2 (NOX2) is a complex membrane protein that contributes to the production of reactive oxygen species (ROS) in several neurological diseases. The NLRP1 inflammasome can be activated in response to an accumulation of ROS in neurons. However, it is still unknown whether LPS exposure can deteriorate neuronal damage by activating NOX2-NLRP1 inflammasomes. Ginsenoside Rg1 (Rg1) has protective effects on neurons, although whether Rg1 alleviates LPS-induced neuronal damage by inhibiting NOX2-NLRP1 inflammasomes remains unclear. In the present study, the effect of concentration gradients and different times of LPS exposure on neuronal damage was investigated in HT22 cells, and further observed the effect of Rg1 treatment on NOX2-NLPR1 inflammasome activation, ROS production and neuronal damage in LPS-treated HT22 cells. The results demonstrated that LPS exposure significantly induced NOX2-NLRP1 inflammasome activation, excessive production of ROS, and neuronal damage in HT22 cells. It was also shown that Rg1 treatment significantly decreased NOX2-NLRP1 inflammasome activation and ROS production and alleviated neuronal damage in LPS-induced HT22 cells. The present data suggested that Rg1 has protective effects on LPS-induced neuronal damage by inhibiting NOX2-NLRP1 inflammasomes in HT22 cells, and Rg1 may be a potential therapeutic approach for delaying neuronal damage in AD.

Background Ginsenoside Rg1 (Rg1), an active ingredient in ginseng, may be a potential agent for the treatment of Alzheimer’s disease (AD). However, the protective effect of Rg1 on neurodegeneration in AD and its mechanism of action are still incompletely understood. Methods Wild type (WT) and APP/PS1 AD mice, from 6 to 9 months old, were used in the experiment. The open field test (OFT) and Morris water maze (MWM) were used to detect behavioral changes. Neuronal damage was assessed by hematoxylin and eosin (H&E) and Nissl staining. Immunofluorescence, western blotting, and quantitative real-time polymerase chain reaction (q-PCR) were used to examine postsynaptic density 95 (PSD95) expression, amyloid beta (Aβ) deposition, Tau and phosphorylated Tau (p-Tau) expression, reactive oxygen species (ROS) production, and NAPDH oxidase 2 (NOX2) expression. Results Rg1 treatment for 12 weeks significantly ameliorated cognitive impairments and neuronal damage and decreased the p-Tau level, amyloid precursor protein (APP) expression, and Aβ generation in APP/PS1 mice. Meanwhile, Rg1 treatment significantly decreased the ROS level and NOX2 expression in the hippocampus and cortex of APP/PS1 mice. Conclusions Rg1 alleviates cognitive impairments, neuronal damage, and reduce Aβ deposition by inhibiting NOX2 activation in APP/PS1 mice., Graphical abstract Image 1, Highlights • Rg1 treatment significantly alleviated cognitive dysfunction and neuronal damage in APP/PS1 mice. • Rg1 treatment significantly reduced APP expression and Aβ deposition in APP/PS1 mice. • The expression of NOX2 and ROS production were significantly increased in APP/PS1 mice. • Rg1 treatment significantly decreased NOX2 expression and ROS accumulation in APP/PS1 mice.

Background: Oxidative stress and neuroinflammation play important roles in the whole pathogenesis of Alzheimer's disease (AD). NADPH oxidase 2 (NOX2) is an important enzyme that is responsible for ROS generation in many neurodegenerative diseases. The nucleotide-binding oligomerization domain (NOD)-like receptor protein 1 (NLRP1) inflammasome is responsible for the formation of pro-inflammatory molecules in neurons. However, it is still unclear whether inhibition of NOX2 and NLRP1 inflammasome decreases amyloid-beta (Aβ) generation and deposition in APP/PS1 mice. Ginsenoside Rg1 (Rg1) is an active component in ginseng, and maybe a potential agent for neurodegenerative diseases. In this study, we investigated the effects and mechanisms of Rg1 treatment on cognitive performance, neuronal damage, Aβ deposition and NOX2-NLRP1 inflammasome activation in APP/PS1 mice.Methods: WT and APP/PS1 mice were used in the experiment from 6 months (M) old to 9M old, and 6M APP/PS1 mice were used as pre-treatment controls. The open field test (OFT) and Morris water maze (MWM) were used to detect behavioral change and cognitive function. The H&E and Nissl staining were used to assess neuronal damage. We further examined PSD95 expression, Aβ generation and deposition, Tau and p-Tau expression, NOX2-mediated ROS generation and NLRP1 inflammasome activation by using immunofluorescence, western blot analysis, and real time q-PCR.Results: Rg1 treatment for 12 weeks alleviated learning and memory impairments and neuronal damage, decreased p-Tau level, APP expression and Aβ deposition in APP/PS1 mice. Meanwhile, Rg1 treatment significantly decreased the levels of ROS and IL-1β, and reduced the expressions of NOX2 and NLRP1 inflammasome in the brain cortex and hippocampus in APP/PS1 mice. Furthermore, apocynin (a NOX inhibitor) and NLRP1-siRNA treatment also alleviated cognitive impairments, neuronal damage and Aβ deposition, and reduced the expression of NLRP1 inflammasome in brain cortex and hippocampus in APP/PS1 mice.Conclusions: Rg1 treatment could alleviate learning and memory impairment, neuronal damage, and reduce Aβ generation and deposition by inhibiting NLRP1 inflammasome activation in APP/PS1 mice.

Astragaloside IV (AS-IV), a natural product derived from Radix Astragali (Astragalus membranaceus), is beneficial for the treatment of Alzheimer's disease (AD), but the mechanisms underlying this benefit are not completely understood. Peroxisome proliferator-activated receptor gamma (PPARγ) and brain-derived neurotrophic factor (BDNF) are potential therapeutic targets for AD. In this study, we found that amyloid β protein fragment 1-42 oligomers (AβO) suppressed BDNF and PPARγ expression, and inhibited tyrosine receptor kinase B (TrkB) phosphorylation in cultured hippocampal neurons; these changes were ameliorated by treatment with AS-IV. Inhibition of PPARγ by genetic and pharmacological methods also blocked the effect of AS-IV on BDNF expression in AβO-treated cells. Importantly, exogenous BDNF protected against neurotoxicity and apoptosis induced by AβO, whereas inhibition of PPARγ reversed protective effects of AS-IV against these outcomes. In vivo data further revealed that AS-IV improved AβO-induced memory impairment and reduced apoptosis of hippocampal neurons. Moreover, AS-IV suppressed the AβO-induced reduction in BDNF by promoting PPARγ activation in the hippocampus. Taken together, these results indicate that AS-IV prevents AβO-induced memory impairment and hippocampal neuronal apoptosis, probably by promoting the PPARγ/BDNF signaling pathway.

Background Dry skin itch is one of the most common skin diseases and elderly people are believed to be particularly prone to it. The inflammasome has been suggested to play an important role in chronic inflammatory disorders including inflammatory skin diseases such as psoriasis. However, little is known about the role of NLRP1 inflammasome in dry skin-induced chronic itch. Methods Dry skin-induced chronic itch model was established by acetone-ether-water (AEW) treatment. Spontaneous scratching behavior was recorded by video monitoring. The expression of nucleotide oligomerization domain (NOD)-like receptor protein 1 (NLRP1) inflammasome complexes, transient receptor potential vanilloid type 1 (TRPV1), and the level of inflammatory cytokines were determined by western blot, quantitative real-time PCR, and enzyme-linked immunosorbent assay (ELISA) kits. Nlrp1a knockdown was performed by an adeno-associated virus (AAV) vector containing Nlrp1a-shRNA-eGFP infusion. H.E. staining was used to evaluate skin lesion. Results AEW treatment triggers spontaneous scratching and significantly increases the expression of NLRP1, ASC, and caspase-1 and the levels of IL-1β, IL-18, IL-6, and TNF-α in the spinal cord and the skin of mice. Spinal cord Nlrp1a knockdown prevents AEW-induced NLRP1 inflammasome assembly, TRPV1 channel activation, and spontaneous scratching behavior. Capsazepine, a specific antagonist of TRPV1, can also inhibit AEW-induced inflammatory response and scratching behavior. Furthermore, elderly mice and female mice exhibited more significant AEW-induced scratching behavior than young mice and male mice, respectively. Interestingly, AEW-induced increases in the expression of NLRP1 inflammasome complex and the levels of inflammatory cytokines were more remarkable in elderly mice and female mice than in young mice and male mice, respectively. Conclusions Spinal cord NLRP1 inflammasome-mediated inflammatory response contributes to dry skin-induced chronic itch by TRPV1 channel, and it is also involved in age and sex differences of chronic itch. Inhibition of NLRP1 inflammasome may offer a new therapy for dry skin itch.

Aging is closely related to the progress of renal fibrosis, which eventually results in renal dysfunction. Ginsenoside Rg1 (Rg1) has been reported to have an extensive anti-aging effect. However, the role and mechanism of Rg1 in aging-related renal fibrosis remain unclear. The present study aimed to evaluate the protective effect and mechanism of Rg1 in renal fibrosis during kidney aging in a model of SAMP8 mice. Taking SAMR1 mice as the control group, SAMP8 mice were administered Apocynin (50 mg/kg), Tempol (50 mg/kg), or Rg1 (5, 10 mg/kg) intragastrically for 9 weeks as treatment groups. The results showed that the elevated levels of blood urea nitrogen, serum creatinine and senescence-associated β-galactosidase (β-Gal) were markedly decreased, the glomerular mesangial proliferation was significantly alleviated and the increased levels of collagen IV and TGF-β1 were significantly downregulated by Rg1 in SAMP8 mice. In addition, the generation of ROS and the expression of NADHP oxidase 4 (NOX4) in the renal cortex were significantly reduced by Rg1 treatment. The expression levels of NLRP3 inflammasome-related proteins and the inflammation-related cytokine IL-1β were also inhibited by Rg1 treatment in the SAMP8 mice. These results suggested that Rg1 could delay kidney aging and inhibit aging-related glomerular fibrosis by reducing NOX4-derived ROS generation and downregulating NLRP3 inflammasome expression.

Background Epilepsy is a common neurological disorder and is not well controlled by available antiepileptic drugs (AEDs). Inflammation is considered to be a critical factor in the pathophysiology of epilepsy. Sinomenine (SN), a bioactive alkaloid with anti-inflammatory effect, exerts neuroprotective activity in many nervous system diseases. However, little is known about the effect of SN on epilepsy. Methods The chronic epilepsy model was established by pentylenetetrazole (PTZ) kindling. Morris water maze (MWM) was used to test spatial learning and memory ability. H.E. staining and Hoechst 33258 staining were used to evaluate hippocampal neuronal damage. The expression of nucleotide oligomerization domain (NOD)-like receptor protein 1 (NLRP1) inflammasome complexes and the level of inflammatory cytokines were determined by western blot, quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA) kits. Results SN (20, 40, and 80 mg/kg) dose-dependently disrupts the kindling acquisition process, which decreases the seizure scores and the incidence of fully kindling. SN also increases the latency of seizure and decreases the duration of seizure in fully kindled rats. In addition, different doses of SN block the hippocampal neuronal damage and minimize the impairment of spatial learning and memory in PTZ kindled rats. Finally, PTZ kindling increases the expression of NLRP1 inflammasome complexes and the levels of inflammatory cytokines IL-1β, IL-18, IL-6, and TNF-α, which are all attenuated by SN in a dose- dependent manner. Conclusions SN exerts anticonvulsant and neuroprotective activity in PTZ kindling model of epilepsy. Disrupting the kindling acquisition, which inhibits NLRP1 inflammasome-mediated inflammatory process, might be involved in its effects. Electronic supplementary material The online version of this article (10.1186/s12974-018-1199-0) contains supplementary material, which is available to authorized users.

The levels of lipopolysaccharide (LPS) are increased in the brain and blood of Alzheimer’s disease (AD) patients and contribute to neuroinflammation and neurodegeneration in AD. Ginsenoside Rg1 (Rg1) is reported to possess neuroprotective effects on neurodegenerative diseases by improving oxidative stress and inflammation. However, the protective effect and mechanism of Rg1 in neuroinflammation and neuronal damage in LPS-induced mice remain unclear. In this study, the results demonstrated that exposure to LPS for 14 days significantly induced cognitive impairment, neuronal injury and NLRP1 inflammasome activation, significantly increased ROS generation and NADPH oxidase 2 (NOX2) expression and elevated the levels of p-PLC, calcineurin (CN) and nuclear factor of activated T cells 1 (NFAT1) in the hippocampus. Treatment with Rg1, tempol (a superoxide dismutase mimic) and apocynin (an inhibitor of NOX) significantly improved cognitive impairment and neuronal damage, inhibited the activation of NLRP1 inflammasome, reduced ROS generation and NOX2 expression and decreased the levels of p-PLC, CN and NFAT1 in the hippocampus. Additionally, the calcium imaging results suggested that Rg1 administration significantly inhibits [Ca2+]i overload and maintains calcium homeostasis in LPS-induced HT22 cells. These results suggest that Rg1 treatment can improve LPS-induced activation of the NLRP1 inflammasome and neuronal damage through inhibiting NOX2, [Ca2+]i overload and CN-NFAT1 activation.

Our previous study demonstrated that the abundance of extracellular matrix proteins was suppressed by store-operated Ca2+entry (SOCE) in mesangial cells (MCs). The present study was conducted to investigate the underlying mechanism focused on the transforming growth factor-β1 (TGF-β1)/Smad3 pathway, a critical pathway for ECM expansion in diabetic kidneys. We hypothesized that SOCE suppressed ECM protein expression by inhibiting this pathway in MCs. In cultured human MCs, we observed that TGF-β1 (5 ng/ml for 15 h) significantly increased Smad3 phosphorylation, as evaluated by immunoblot. However, this response was markedly inhibited by thapsigargin (1 µM), a classical activator of store-operated Ca2+channels. Consistently, both immunocytochemistry and immunoblot showed that TGF-β1 significantly increased nuclear translocation of Smad3, which was prevented by pretreatment with thapsigargin. Importantly, the thapsigargin effect was reversed by lanthanum (La3+; 5 µM) and GSK-7975A (10 µM), both of which are selective blockers of store-operated Ca2+channels. Furthermore, knockdown of Orai1, the pore-forming subunit of the store-operated Ca2+channels, significantly augmented TGF-β1-induced Smad3 phosphorylation. Overexpression of Orai1 augmented the inhibitory effect of thapsigargin on TGF-β1-induced phosphorylation of Smad3. In agreement with the data from cultured MCs, in vivo knockdown of Orai1 specific to MCs using a targeted nanoparticle small interfering RNA delivery system resulted in a marked increase in abundance of phosphorylated Smad3 and in nuclear translocation of Smad3 in the glomerulus of mice. Taken together, our results indicate that SOCE in MCs negatively regulates the TGF-β1/Smad3 signaling pathway.

Neuroinflammation mediated by NLRP-1 inflammasome plays an important role in the pathogenesis of neurodegeneration diseases such as Alzheimer's disease (AD). Chronic glucocorticoids (GCs) exposure has deleterious effect on the structure and function of neurons and was found to be correlated with development and progression of AD. We hypothesize that chronic glucocorticoids may down-regulate the expression of glucocorticoids receptor (GR) and activate NLRP-1 inflammasome in hippocampal neurons, which may promote neuroinflammation and induce neuronal injury. The present results showed that chronic DEX exposure significantly increased LDH release and apoptosis, decreased MAP2 and GR expression in hippocampal neurons. DEX (5μΜ) exposure for 3d significantly increased the expression of NLRP-1, ASC, caspase-1 and IL-1β in the hippocampal neurons and the release of IL-1β and IL-18 in the supernatants. Moreover, DEX (1, 5μΜ) treatment for 3d significantly increased the expression of NF-κB in hippocampal neurons. The GR antagonist, mifepristone (RU486), had protective effects on chronic DEX induced hippocampal neurons injury and NLRP1 inflammasome activation. The results suggest that chronic GCs exposure can decrease GR expression and increase neuroinflammation via NLRP1 inflammasome and promote hippocampal neurons degeneration, which may play an important role in the progression and development of AD.

Background Neuroinflammation mediated by NLRP1 (nucleotide-binding oligomerization domain (NOD)-like receptor protein 1) inflammasome plays an important role in many neurological diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD). Our previous studies showed that chronic glucocorticoid (GC) exposure increased brain inflammation via NLRP1 inflammasome and induce neurodegeneration. However, little is known about the mechanism of chronic GC exposure on NLRP1 inflammasome activation in hippocampal neurons. Methods Hippocampal neurons damage was assessed by LDH kit and Hoechst 33258 staining. The expression of microtubule-associated protein 2 (MAP2), inflammasome complex protein (NLRP1, ASC and caspase-1), inflammatory cytokines (IL-1β), and large-conductance Ca2+ and voltage-activated K+ channel (BK channels) protein was detected by Western blot. The inflammatory cytokines (IL-1β and IL-18) were examined by ELISA kit. The mRNA levels of NLRP1, IL-1β, and BK were detected by real-time PCR. BK channel currents were recorded by whole-cell patch-clamp technology. Measurement of [K+]i was performed by ion-selective electrode (ISE) technology. Results Chronic dexamethasone (DEX) treatment significantly increased LDH release and neuronal apoptosis and decreased expression of MAP2. The mechanistic studies revealed that chronic DEX exposure significantly increased the expression of NLRP1, ASC, caspase-1, IL-1β, L-18, and BK protein and NLRP1, IL-1β and BK mRNA levels in hippocampal neurons. Further studies showed that DEX exposure results in the increase of BK channel currents, with the subsequent K+ efflux and a low concentration of intracellular K+, which involved in activation of NLRP1 inflammasome. Moreover, these effects of chronic DEX exposure could be blocked by specific BK channel inhibitor iberiotoxin (IbTx). Conclusion Our findings suggest that chronic GC exposure may increase neuroinflammation via activation of BK-NLRP1 signal pathway and promote hippocampal neurons damage, which may be involved in the development and progression of AD.

Significance: Regulation of Ca2+ signaling cascade by reactive oxygen species (ROS) is becoming increasingly evident and this regulation represents a key mechanism for control of many fundamental cellular functions. Canonical transient receptor potential (TRPC) 6, a member of Ca2+-conductive channel in the TRPC family, is widely expressed in kidney cells, including glomerular mesangial cells, podocytes, tubular epithelial cells, and vascular myocytes in renal microvasculature. Both overproduction of ROS and dysfunction of TRPC6 channel are involved in renal injury in animal models and human subjects. Although regulation of TRPC channel function by ROS has been well described in other tissues and cell types, such as vascular smooth muscle, this important cell regulatory mechanism has not been fully reviewed in kidney cells. Recent Advances: Accumulating evidence has shown that TRPC6 is a redox-sensitive channel, and modulation of TRPC6 Ca2+ signaling by altering TRPC6 protein expression or TRPC6 channel activity in kidney cells is a downstream mechanism by which ROS induce renal damage. Critical Issues: This review highlights how recent studies analyzing function and expression of TRPC6 channels in the kidney and their response to ROS improve our mechanistic understanding of oxidative stress-related kidney diseases. Future Directions: Although it is evident that ROS regulate TRPC6-mediated Ca2+ signaling in several types of kidney cells, further study is needed to identify the underlying molecular mechanism. We hope that the newly identified ROS/TRPC6 pathway will pave the way to new, promising therapeutic strategies to target kidney diseases such as diabetic nephropathy. Antioxid. Redox Signal. 25, 732–748.

Oxidative stress and neuroinflammation are important in the pathogenesis of ageing and age‑related neurodegenerative diseases, including Alzheimer's disease. NADPH oxidase 2 (NOX2) is a major source of reactive oxygen species (ROS) in the brain. The nucleotide‑binding oligomerisation domain (NOD)‑like receptor protein 1 (NLRP1) inflammasome is responsible for the formation of pro‑inflammatory molecules in neurons. Whether the NOX2‑NLRP1 inflammasome signalling pathway is involved in neuronal ageing and age‑related damage remains to be elucidated. Ginsenoside Rg1 (Rg1) is a steroidal saponin found in ginseng. In the present study, the primary hippocampal neurons were treated with H2O2 (200 µM) and Rg1 (1, 5 and 10 µM) for 24 h to investigate the protective effects and mechanisms of Rg1 on H2O2‑induced hippocampal neuron damage, which mimics age‑related damage. The results showed that H2O2 treatment significantly increased ROS production and upregulated the expression of NOX2 and the NLRP1 inflammasome, and led to neuronal senescence and damage to hippocampal neurons. Rg1 decreased ROS production, reducing the expression of NOX2 and the NLRP1 inflammasome in H2O2‑treated hippocampal neurons. Furthermore, Rg1 and tempol treatment significantly decreased neuronal apoptosis and the expression of β‑galactosidase, and alleviated the neuronal senescence and damage induced by H2O2. The present study indicates that Rg1 may reduce NOX2‑mediated ROS generation, inhibit NLRP1 inflammasome activation, and inhibit neuronal senescence and damage.

Background Our previous study suggested that palmitate (PA) induces human glomerular mesangial cells (HMCs) fibrosis. However, the mechanism is not fully understood. Recent studies suggested that transient receptor potential canonical channel 6 (TRPC6)/nuclear factor of activated T cell 2 (NFAT2) played an important role in renal fibrosis. Moreover, cluster of differentiation 36 (CD36) regulated the synthesis of TPRC6 agonist diglyceride. In the present study, we investigated whether PA induced HMCs fibrosis via TRPC6/ NFAT2 mediated by CD36. Methods A type 2 diabetic nephropathy (DN) model was established in Sprague Dawley rats, and HMCs were stimulated with PA. Lipid accumulation and free fatty acid (FFA) uptake were measured. The expression levels of TGF-β1, p-Smad2/3, FN, TRPC6, NFAT2 and CD36 were evaluated. The intracellular calcium concentration ([Ca2+]i) was assessed. Results FFA were elevated in type 2 DN rats with kidney fibrosis in addition to NFAT2 and CD36 expression. In vitro, PA induced HMCs fibrosis, [Ca2+]i elevation and NFAT2 activation. SKF96365 or TRPC6-siRNA could attenuate PA-induced HMCs damage. By contrast, the TRPC6 activator showed the opposite effect. Moreover, NFAT2-siRNA also suppressed PA-induced HMCs fibrosis. CD36 knockdown inhibited the PA-induced [Ca2+]i elevation and NFAT2 expression. In addition, long-term treatment with PA decreased TRPC6 expression in HMCs. Conclusion The results of this study demonstrated that PA could induce the activation of the [Ca2+]i/NFAT2 signaling pathway through TRPC6, which led to HMCs fibrosis. Although activation of TRPC6 attributed to CD36-mediated lipid deposition， long-term stimulation of PA may lead to negative feedback on the expression of TPRC6.

Astragaloside IV (AS-IV), a natural product derived from Radix Astragali (Astragalus membranaceus), is beneficial for the treatment of Alzheimer's disease (AD), but the mechanisms underlying this benefit are not completely understood. Peroxisome proliferator-activated receptor gamma (PPARγ) and brain-derived neurotrophic factor (BDNF) are potential therapeutic targets for AD. In this study, we found that amyloid β protein fragment 1-42 oligomers (AβO) suppressed BDNF and PPARγ expression, and inhibited tyrosine receptor kinase B (TrkB) phosphorylation in cultured hippocampal neurons; these changes were ameliorated by treatment with AS-IV. Inhibition of PPARγ by genetic and pharmacological methods also blocked the effect of AS-IV on BDNF expression in AβO-treated cells. Importantly, exogenous BDNF protected against neurotoxicity and apoptosis induced by AβO, whereas inhibition of PPARγ reversed protective effects of AS-IV against these outcomes. In vivo data further revealed that AS-IV improved AβO-induced memory impairment and reduced apoptosis of hippocampal neurons. Moreover, AS-IV suppressed the AβO-induced reduction in BDNF by promoting PPARγ activation in the hippocampus. Taken together, these results indicate that AS-IV prevents AβO-induced memory impairment and hippocampal neuronal apoptosis, probably by promoting the PPARγ/BDNF signaling pathway.

Parkinson's disease (PD) is the second most common neurodegenerative disease, which is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Accumulated evidences have suggested that oxidative stress is closely associated with the dopaminergic neurodegeneration of PD that can be protected by antioxidants. Biochanin A that is an O-methylated isoflavone in chickpea is investigated to explore its protective mechanism on dopaminergic neurons of the unilateral lipopolysaccharide (LPS)-injected rat. The results showed that biochanin A significantly improved the animal model's behavioral symptoms, prevented the loss of dopaminergic neurons and inhibited the deleterious microglia activation in the LPS-induced rats. Moreover, biochanin A inhibited nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) activation and malondialdehyde (MDA) production, increased superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in the rat brain. These results suggested that biochanin A might be a natural candidate with protective properties on dopaminergic neurons against the PD.

Diabetic cardiomyopathy (DCM) is one of the main complications of type 2 diabetes mellitus (T2DM), and it is also one of the main causes of heart failure and death in advanced diabetes. The myocardial lipotoxic injury induced by abnormal lipid metabolism plays an important role in the occurrence and development of DCM, such as myocardial inflammation and fibrosis, ultimately leading to myocardial remodeling and cardiac insufficiency. Astragaloside IV (AS-IV) has many pharmacological effects such as anti-oxidation, anti-inflammatory, immune regulation, and anti-ischemic brain damage. This study was performed to investigate whether AS-IV could prevent T2DM-induced cardiomyopathy and regulate the abnormal myocardial lipid metabolism in diabetes. In this study, the T2DM model was induced by feeding with high-fat food and injected with low-dose STZ in rats. Then the model rats were treated with AS-IV and metformin (Met) for 8 weeks. The results showed that AS-IV improved cardiac systolic and diastolic function, and ameliorated the cardiac histopathological changes in the T2DM rats. Moreover, AS-IV significantly improved circulating TC, TG and HDL levels and cardiac lipid accumulation in T2DM rats as well as in high-fat diet (HFD) rats. Furthermore, AS-IV significantly inhibited the expressions of TNF-α, IL-6 and IL-1β and myocardial fibrosis in T2DM rats, which might be attributed to the improvement of myocardial lipid metabolism, ultimately improving cardiac function in T2DM rats. Taken together, these data suggested that AS-IV has protective effects on T2DM-induced myocardial injury in rats, and its mechanism may be related to the improvement of lipid metabolism in cardiomyocytes.