Ginsenoside Rg1 attenuates lipopolysaccharide-induced chronic liver damage by activating Nrf2 signaling and inhibiting inflammasomes in hepatic cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng (Panax ginseng C. A. Meyer) is a precious traditional Chinese medicine with multiple pharmacological effects. Ginsenoside Rg1 is a main active ingredient extracted from ginseng, which is known for its age-delaying and antioxidant effects. Increasing evidence indicates that Rg1 exhibits anti-inflammatory properties in numerous diseases and may ameliorate oxidative damage and inflammation in many chronic liver diseases. AIM OF THE STUDY: Chronic inflammatory injury in liver cells is an important pathological basis of many liver diseases. However, its mechanism remains unclear and therapeutic strategies to prevent its development need to be further explored. Thus, our study is to delve the protective effect and mechanism of Rg1 against chronic hepatic inflammatory injuries induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: The chronic liver damage model in mice was build up by injecting intraperitoneally with LPS (200 µg/kg) for 21 days. Serum liver function indicators and levels of IL-1ß, IL-6 and TNF-α were examined by using corresponding Kits. Hematoxylin and Eosin (H&E), Periodic acid-Schiff (PAS), and Masson stains were utilized to visualize hepatic histopathological damage, glycogen deposition, and liver fibrosis. The nuclear import of p-Nrf2 and the generation of Col4 in the liver were detected by IF, while IHC was employed to detect the expressions of NLRP3 and AIM2 in the hepatic. The Western blot and q-PCR were used to survey the expressions of proteins and mRNAs of fibrosis and apoptosis, and the expressions of Keap1, p-Nrf2 and NLRP3, NLRP1, AIM2 inflammasome-related proteins in mouse liver. The cell viability of human hepatocellular carcinoma cells (HepG2) was detected by Cell Counting Kit-8 to select the action concentration of LPS, and intracellular ROS generation was detected using a kit. The expressions of Nuclear Nrf2, HO-1, NQO1 and NLRP3, NLRP1, and AIM2 inflammasome-related proteins in HepG2 cells were detected by Western blot. Finally, the feasibility of the molecular interlinking between Rg1 and Nrf2 was demonstrated by molecular docking. RESULTS: Rg1 treatment for 21 days decreased the levels of ALT, AST, and inflammatory factors of serum IL-1ß, IL-6 and TNF-α in mice induced by LPS. Pathological results indicated that Rg1 treatment obviously alleviated hepatocellular injury and apoptosis, inflammatory cell infiltration and liver fibrosis in LPS stimulated mice. Rg1 promoted Keap1 degradation and enhanced the expressions of p-Nrf2, HO-1 and decreased the levels of NLRP1, NLRP3, AIM2, cleaved caspase-1, IL-1ß and IL-6 in livers caused by LPS. Furthermore, Rg1 effectively suppressed the rise of ROS in HepG2 cells induced by LPS, whereas inhibition of Nrf2 reversed the role of Rg1 in reducing the production of ROS and NLRP3, NLRP1, and AIM2 expressions in LPS-stimulated HepG2 cells. Finally, the molecular docking illustrated that Rg1 exhibits a strong affinity towards Nrf2. CONCLUSION: The findings indicate that Rg1 significantly ameliorates chronic liver damage and fibrosis induced by LPS. The mechanism may be mediated through promoting the dissociation of Nrf2 from Keap1 and then activating Nrf2 signaling and further inhibiting NLRP3, NLRP1, and AIM2 inflammasomes in liver cells.

Phosphoethanolamine cytidylyltransferase ameliorates mitochondrial function and apoptosis in hepatocytes in T2DM in vitro.

Liver function indicators are often impaired in patients with type 2 diabetes mellitus (T2DM), who present higher concentrations of aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase than individuals without diabetes. However, the mechanism of liver injury in patients with T2DM has not been clearly elucidated. In this study, we performed a lipidomics analysis on the liver of T2DM mice, and we found that phosphatidylethanolamine (PE) levels were low in T2DM, along with an increase in diglyceride, which may be due to a decrease in the levels of phosphoethanolamine cytidylyltransferase (Pcyt2), thus likely affecting the de novo synthesis of PE. The phosphatidylserine decarboxylase pathway did not change significantly in the T2DM model, although both pathways are critical sources of PE. Supplementation with CDP-ethanolamine (CDP-etn) to increase the production of PE from the CDP-etn pathway reversed high glucose and FFA (HG&FFA)-induced mitochondrial damage including increased apoptosis, decreased ATP synthesis, decreased mitochondrial membrane potential, and increased reactive oxygen species, whereas supplementation with lysophosphatidylethanolamine, which can increase PE production in the phosphatidylserine decarboxylase pathway, did not. Additionally, we found that overexpression of PCYT2 significantly ameliorated ATP synthesis and abnormal mitochondrial morphology induced by HG&FFA. Finally, the BAX/Bcl-2/caspase3 apoptosis pathway was activated in hepatocytes of the T2DM model, which could also be reversed by CDP-etn supplements and PCYT2 overexpression. In summary, in the liver of T2DM mice, Pcyt2 reduction may lead to a decrease in the levels of PE, whereas CDP-etn supplementation and PCYT2 overexpression ameliorate partial mitochondrial function and apoptosis in HG&FFA-stimulated L02 cells.

Comprehensive Analysis of Long Non-Coding RNAs N4-Acetylcytidine in Alzheimer's Disease Mice Model Using High-Throughput Sequencing.

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 attenuates cerebral ischemia-reperfusion injury due to inhibition of NOX2-mediated calcium homeostasis dysregulation in mice.

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.

SOCE-mediated NFAT1-NOX2-NLRP1 inflammasome involves in lipopolysaccharide-induced neuronal damage and Aß generation.

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 ß-amyloid (Aß) generation. However, the mechanism by which LPS causes neuronal damage has still not been fully clarified. Oxidative stress, neuroinflammation, and Ca2+ overload are regarded as important factors influencing AD. NADPH oxidase 2 (NOX2) and the NOD-like receptor family protein 1 (NLRP1) inflammasome play important roles in promoting oxidative stress and inflammation in neurons. Ca2+ overload can activate calcineurin (CN), which further dephosphorylates nuclear factor of activated T cells (NFAT), leading to its translocation into the nucleus to regulate gene transcription. In the present study, LPS (250 µg/kg) exposure for 14 days was used to induce cognitive dysfunction in mice and LPS (20 µg/ml) exposure for 48 h was used to induce neuronal damage in HT22 cells. The results showed that LPS exposure activated phospholipase C (PLC), CN, and NFAT1; increased the expressions of NOX2- and NLRP1-related proteins; and promoted neuronal damage and Aß deposition in mice and HT22 cells. However, treatment with 2-APB (SOCE inhibitor), apocynin (NOX inhibitor), or tempol (reactive oxygen species scavenger) significantly reversed these LPS-induced changes, and improved neuronal damage and Aß deposition. Moreover, LPS exposure promoted PLC phosphorylation, increased the level of inositol-1,4,5-triphosphate, elevated the intracellular Ca2+ concentration ([Ca2+]i), and disrupted [Ca2+]i homeostasis in HT22 cells. These data indicated that the activation of SOCE-mediated NFAT1-NOX2-NLRP1 inflammasome involves in LPS-induced neuronal damage and Aß generation.

[Protective effect of ginsenoside Rg_1 aganist diabetic retinopathy by inhibiting NLRP3 inflammasome in type 2 diabetic mice].

Ginsenoside Rg_1, one of the main active components of precious traditional Chinese medicine Ginseng Radix et Rhizoma, has the anti-oxidative stress, anti-inflammation, anti-aging, neuroprotection, and other pharmacological effects. Diabetic retinopathy(DR), the most common complication of diabetes, is also the main cause of impaired vision and blindness in the middle-aged and the elderly. The latest research shows that ginsenoside Rg_1 can protect patients against DR, but the protection and the mechanism are rarely studied. This study mainly explored the protective effect of ginsenoside Rg_1 against DR in type 2 diabetic mice and the mechanism. High fat diet(HFD) and streptozotocin(STZ) were used to induce type 2 diabetes in mice, and hematoxylin-eosin(HE) staining was employed to observe pathological changes in the retina of mice. The immunohistochemistry was applied to study the localization and expression of nucleotide-binding oligomerization domain-like receptors 3(NLRP3) and vascular endothelial growth factor(VEGF) in retina, and Western blot was used to detect the expression of nuclear factor-kappa B(NF-κB), p-NF-κB, NLRP3, caspase-1, interleukin-1ß(IL-1ß), transient receptor potential channel protein 6(TRPC6), nuclear factor of activated T-cell 2(NFAT2), and VEGF in retina. The results showed that ginsenoside Rg_1 significantly alleviated the pathological injury of retina in type 2 diabetic mice. Immunohistochemistry results demonstrated that ginsenoside Rg_1 significantly decreased the expression of NLRP3 and VEGF in retinal ganglion cells, middle plexiform layer, and outer plexiform layer in type 2 diabetic mice. According to the Western blot results, ginsenoside Rg_1 significantly lowered the expression of p-NF-κB, NLRP3, caspase-1, IL-1ß, TRPC6, NFAT2, and VEGF in retina of type 2 diabetic mice. These findings suggest that ginsenoside Rg_1 can significantly alleviate DR in type 2 diabetic mice, which may be related to inhibition of NLRP3 inflammasome and VEGF. This study provides experimental evidence for the clinical application of ginsenoside Rg_1 in the treatment of DR.

The variations of endophilin A2-FoxO3a-autophagy signal in angiotensin II-induced dopaminergic neuron injury mouse model and by biochanin A.

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.

Astragaloside IV alleviates liver injury in type 2 diabetes due to promotion of AMPK/mTOR­mediated autophagy.

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 (AS­IV) is a natural saponin isolated from the plant Astragalus membranaceus, which has comprehensive pharmacological effects, such as anti­oxidation, anti­inflammation and anti­apoptosis properties, as well as can enhance immunity. However, whether AS­IV can alleviate diabetic liver injury in T2DM and its underlying mechanisms remain unknown. The present study used high­fat diets combined with low­dose streptozotocin to induce a diabetic liver injury model in T2DM rats to investigate whether AS­IV could alleviate diabetic liver injury and to identify its underlying mechanisms. The results demonstrated that AS­IV 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, AS­IV treatment promoted suppressed autophagy in the liver of T2DM rats and improved IR, dyslipidaemia, oxidative stress and inflammation. In addition, AS­IV activated adenosine monophosphate­activated protein kinase (AMPK), which inhibited mTOR. Taken together, the present study suggested that AS­IV alleviated diabetic liver injury in T2DM rats, and its mechanism may be associated with the promotion of AMPK/mTOR­mediated 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.

Spinal cord NLRP1 inflammasome contributes to dry skin induced chronic itch in mice.

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-1ß, 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.

Inhibition of NOX2-NLRP1 signaling pathway protects against chronic glucocorticoids exposure-induced hippocampal neuronal damage.

Glucocorticoids (GCs) exposure has deleterious alteration on the structure and function in hippocampal neurons. NADPH oxidase 2 (NOX2) is a major contributor to oxidative stress in neurological diseases, and NLRP1 inflammasome can be activated in response to oxidative stress. We hypothesize that inhibition of NOX2-mediated NLRP1 inflammasome activation may protect against chronic GCs exposure-induced neuronal injury. In this study, the lentivirus with NLRP1-siRNA was injected into the hippocampus of male mice which were then treated with dexamethasone (DEX, 5 mg/kg) for 28 d. The data indicated that NLRP1-siRNA treatment down-regulated the NLRP1 expression and significantly improved the exploratory behavior and spatial memory deficits in open field tests and Morris water maze which were deteriorated by chronic DEX treatment in mice. Additionally, inhibition of NLRP1 expression significantly alleviated neuronal degeneration and increased MAP2 expression in the hippocampus in mice. Meanwhile, the results showed that DEX exposure increased NOX2, p22phox and p47phox expression in hippocampus tissue in mice. We further examined the effect of tempol (ROS scavenger) and apocynin (NOX inhibitor) treatment on NLRP1 inflammasome activation in chronic DEX-treated hippocampal neurons. The results revealed that the tempol (50 µM) and apocynin (50 µM) treatment significantly decreased generation of ROS, expression of NOX2 and NLRP1-related protein in DEX-treated hippocampal neurons. These data indicate that NOX2-mediated NLRP1 activation involves in chronic GCs exposure-induced neuronal injury and inhibition of NOX2-NLRP1 signaling pathway protects against GCs-induced neuronal damage.

NADPH oxidase 2-mediated NLRP1 inflammasome activation involves in neuronal senescence in hippocampal neurons in vitro.

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 ß-Galactosidase (ß-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.

Chronic unpredictable mild stress accelerates lipopolysaccharide- induced microglia activation and damage of dopaminergic neurons in rats.

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-1ß (IL-1ß), 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.

Ginsenoside Rg1 protects against H2O2­induced neuronal damage due to inhibition of the NLRP1 inflammasome signalling pathway in hippocampal neurons in vitro.

Oxidative stress and neuroinflammation are important in the pathogenesis of ageing and age­related neurodegenerative diseases, including Alzheimer's disease. NADPH oxidase 2 (NOX2) is a major source of reactive oxygen species (ROS) in the brain. The nucleotide­binding oligomerisation domain (NOD)­like receptor protein 1 (NLRP1) inflammasome is responsible for the formation of pro­inflammatory molecules in neurons. Whether the NOX2­NLRP1 inflammasome signalling pathway is involved in neuronal ageing and age­related 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 H2O2­induced hippocampal neuron damage, which mimics age­related 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 H2O2­treated hippocampal neurons. Furthermore, Rg1 and tempol treatment significantly decreased neuronal apoptosis and the expression of ß­galactosidase, and alleviated the neuronal senescence and damage induced by H2O2. The present study indicates that Rg1 may reduce NOX2­mediated ROS generation, inhibit NLRP1 inflammasome activation, and inhibit neuronal senescence and damage.

Protective effects of Astragaloside IV on endoplasmic reticulum stress-induced renal tubular epithelial cells apoptosis in type 2 diabetic nephropathy rats.

Diabetic nephropathy (DN) has become the leading cause of end-stage renal disease (ESRD) worldwide. Renal tubular injury plays an important role in the development and progression of DN. And apoptosis of renal tubular epithelial cells (RTEC) contribute to the loss of renal function, increased levels of serum creatinine (SCr), blood urea nitrogen (BUN), urine total protein to urine creatinine and microalbuminuria and reduction of creatinine clear rate (CCr). Moreover, recent findings suggested that endoplasmic reticulum (ER) stress may lead to apoptosis of renal cells. Astragalosides IV (AS-IV) has a variety of pharmacological effects such as anti-apoptosis. Thus, in this study we investigated the effects and mechanisms of AS-IV on apoptosis of RTEC in high-fat diets (HFD) and low-dose streptozotocin (STZ)-induced type 2 DN rats. The results showed that AS-IV (40, 80 mg/kg) could alleviate RTEC apoptosis in DN rats. Furthermore, body weight, the majority of biochemical and renal function parameters and histopathological changes in the diabetic kidney were also improved by AS-IV. And AS-IV could reduce the expression of apoptosis-related proteins cleaved caspase-3, Bax/Bcl-2 ratio. ER stress-related proteins GRP78, p-PERK, ATF4 and CHOP were also inhibited by AS-IV in kidney of DN rats. Taken together, our study suggests that the protective effects of AS-IV may be related to inhibit ER stress-induced apoptosis through down-regulating the expression of p-PERK, ATF4 and CHOP. And our study provides a new theoretical basis for the clinical treatment of patients with kidney diseases.

Astragalosides IV protected the renal tubular epithelial cells from free fatty acids-induced injury by reducing oxidative stress and apoptosis.

Renal tubular injury is associated with the development of diabetic nephropathy (DN) and the end-stage renal disease (ESRD). Free fatty acids (FFAs)-associated lipotoxicity contributes to injury of proximal renal tubular epithelial (HK-2) cells in diabetes. Palmitic acid (PA) which is the most abundant saturated fatty acid in FFAs is closely associated with the gradual decline of renal function. Astragalosides IV (AS-IV) has a variety of pharmacological effects such as anti-inflammation and anti-oxidation. In the current study, we investigated the effects of AS-IV on PA-induced apoptosis of HK-2 cells and the underlying mechanisms. The results showed that AS-IV (10, 20, 40 µmol/L) could alleviate PA-induced apoptosis of HK-2 cells. We found that AS-IV reduced the expression of Bax and cleaved-caspase3, but increased the expression of Bcl-2 and phosphorylated Nrf2 in HK-2 cells. Moreover, AS-IV reduced the level of reactive oxygen species (ROS) in the cells. Our study suggests that AS-IV could protect against PA-induced apoptosis in HK-2 cells by inhibiting ROS generation and apoptotic protein expression. This study may provide a new theoretical option for the patients with type 2 diabetes.

Sinomenine exerts anticonvulsant profile and neuroprotective activity in pentylenetetrazole kindled rats: involvement of inhibition of NLRP1 inflammasome.

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-1ß, 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.

Astragaloside IV prevents kidney injury caused by iatrogenic hyperinsulinemia in a streptozotocin­induced diabetic rat model.

Diabetic patients are able to manage their blood glucose with exogenous insulin but, ultimately, remain at risk of diabetic nephropathy (DN). Long­term use of insulin may lead to iatrogenic hyperinsulinemia, which has been suggested to cause kidney injury. However, there are no effective interventions for iatrogenic hyperinsulinemia leading to kidney damage. In the present paper, the hypothesis that astragaloside IV (AS­IV), a novel saponin purified from Astragalus membranaceus (Fisch) Bunge, may prevent DN in iatrogenic hyperinsulinemic diabetic rats through antioxidative and anti­inflammatory mechanisms was investigated. Diabetes was induced with streptozotocin (STZ) (55 mg/kg) by intraperitoneal injection in rats. At 1 week following STZ injection, the diabetic rats were treated with Levemir subcutaneously for 4 weeks. Diabetic rat insulin levels >30 µU/ml were considered as iatrogenic hyperinsulinemia. Rats were divided into six groups (n=8 per group): Iatrogenic hyperinsulinemic rats, and iatrogenic hyperinsulinemic rats treated with Tempol and AS­IV at 2.5, 5 and 10 mg/kg/day, intragastric infusion, for 12 weeks. The normal rats were used as a non­diabetic control group. AS­IV ameliorated albuminuria, mesangial cell proliferation, basement membrane thickening and podocyte foot process effacement in iatrogenic hyperinsulinemic rats. In iatrogenic hyperinsulinemic rat renal tissues, malondialdehyde, interleukin­1ß (IL­1ß), tumor necrosis factor­α (TNF­α), type IV collagen and laminin levels were increased, whereas glutathione peroxidase and superoxide dismutase activity levels were decreased. Nicotinamide adenine dinucleotide phosphate oxidase 4 expression and extracellular signal­regulated kinase 1/2 (ERK1/2) activation were upregulated, and canonical transient receptor potential cation channel 6 (TRPC6) protein expression was downregulated. However, all these abnormalities were attenuated by AS­IV. These findings suggested that AS­IV prevented rat kidney injury caused by iatrogenic hyperinsulinemia by inhibiting oxidative stress, IL­1ß and TNF­α overproduction, downregulating ERK1/2 activation, and upregulating TRPC6 expression.

Chronic glucocorticoid exposure activates BK-NLRP1 signal involving in hippocampal neuron damage.

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-1ß), and large-conductance Ca2+ and voltage-activated K+ channel (BK channels) protein was detected by Western blot. The inflammatory cytokines (IL-1ß and IL-18) were examined by ELISA kit. The mRNA levels of NLRP1, IL-1ß, 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-1ß, L-18, and BK protein and NLRP1, IL-1ß 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.

Biochanin A Protects Against Lipopolysaccharide-Induced Damage of Dopaminergic Neurons Both In Vivo and In Vitro via Inhibition of Microglial Activation.

Neuroinflammation has been reported to be involved in the pathogenesis of Parkinson's disease (PD). Inhibition of microglia-mediated neuroinflammation might be a potential strategy for PD treatment. Biochanin A, is an O-methylated isoflavone, classified as a kind of phytoestrogens due to its chemical structure that is similar to mammalian estrogens. It has been found to possess antifibrotic, antiapoptotic, and antioxidant effects. In the present study, we investigated the neuroprotective effects of biochanin A on lipopolysaccharide (LPS)-induced dopaminergic neurons damage both in vivo and in vitro and the related molecular mechanisms. The results showed that biochanin A treatment for 21 days significantly attenuated the behavioral dysfunction of PD rats, prevented dopaminergic neurons damage, and inhibited activation of microglia in the LPS-induced PD rats. Furthermore, biochanin A decreased the levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the serum, and inhibited the phosphorylation of ERK, JNK, p38 in the substantia nigra of PD rats. In vitro test, biochanin A also inhibited primary microglial activation and protected dopaminergic neurons, decreased the content of nitric oxide, IL-1ß, and TNF-α in supernatants, and inhibited the reactive oxygen species production. Taken together, these results suggest that biochanin A exerts protective effects on LPS-induced PD rats, and the mechanisms may be associated with the inhibition of inflammatory response and the MAPK signaling pathway.

NADPH oxidase-derived production of reactive oxygen species is involved in learning and memory impairments in 16-month-old female rats.

Women undergoing the natural menopause can experience progressive cognitive dysfunction, particularly in the form of memory impairment. However, the mechanisms underlying memory impairments in the menopause remain to be elucidated. There is increasing evidence that oxidative damage caused by excessive reactive oxygen species (ROS) production may correlate with age­associated cognitive impairment. The nicotinamide adenosine dinucleotide phosphate oxidase (NOX) family is important in the generation of ROS in the brain. It has been hypothesized that the accumulation of ROS, derived from NOX, may be involved in menopause­associated learning and memory impairments. The present study investigated whether NOX­derived ROS generation affected the learning and memory ability in 3­month and 16­month­old female rats. The results of a morris water maze assessment revealed that there were significant learning and memory impairments in the 16­month­old female rats. Furthermore, the activity of superoxide dismutase (SOD), level of malondialdehyde (MDA), production of ROS and expression levels of NOX2, p47phox, Ras­related C3 botulinum toxin substrate 1 (RAC1) and protein kinase C α (PKCα) were investigated in the cortex and hippocampus of 3­month and 16­month old female rats. The results demonstrated that the activity of SOD was significantly decreased, whereas the levels of MDA, production of ROS and expression levels of NOX2, p47phox, RAC1 and PKCα were significantly increased in the 16­month old female rats. These results suggested that NOX­mediated oxidative stress may be important in menopause­associated learning and memory impairments.