[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.

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.

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.

Acid-sensing ion channel 1a contributes to the effect of extracellular acidosis on NLRP1 inflammasome activation in cortical neurons.

BACKGROUND: Acid-sensing ion channels (ASICs) are cation channels which were activated by extracellular acidosis and involved in various physiological and pathological processes in the nervous system. Inflammasome is a key component of the innate immune response in host against harmful and irritable stimuli. As the first discovered molecular platform, NLRP1 (nucleotide-binding oligomerization domain (NOD)-like receptor protein 1) inflammasome is expressed in neurons and implicated in many nervous system diseases such as brain injury, nociception and epilepsy. However, little is known about the effect of ASICs on NLRP1 inflammasome activation under acidosis. METHODS: The expression of inflammasome complex protein (NLRP1, ASC (apoptosis-associated speck-like protein containing a caspase-activating recruitment domain) and caspase-1), inflammatory cytokines (IL-1ß and IL-18), and apoptosis-related protein (Bax, Bcl-2, and activated caspase-3) was detected by Western blot. Large-conductance Ca(2+) and voltage-activated K(+) (BK) channel currents were recorded by whole-cell patch-clamp technology. Measurement of [K(+)] i was performed by fluorescent ion imaging system. Co-expression of ASICs and BK channels was determined by dual immunofluorescence. Cell viability was assessed by MTT and LDH kit. RESULTS: ASICs and BK channels were co-expressed in primary cultured cortical neurons. Extracellular acidosis increased the expression of NLRP1, ASC, caspase-1, IL-1ß, and IL-18. Further mechanistic studies revealed that acidosis-induced ASIC1a activation results in the increase of BK channel currents, with the subsequent K(+) efflux and a low concentration of intracellular K(+), which activated NLRP1 inflammasome. Furthermore, these effects of acidosis could be blocked by specific ASIC1a inhibitor PcTX1 and BK channel inhibitor IbTX. The data also demonstrated neutralization of NLRP1-protected cortical neurons against injury induced by extracellular acidosis. CONCLUSIONS: Our data showed that NLRP1 inflammasome could be activated by extracellular acidosis though ASIC-BK channel K(+) signal pathway and was involved in extracellular acidosis-induced cortical neuronal injury.

[Effects of astragalosides on PC12 cells injury and APP expression induced by dexamethasone and Abeta(25-35)].

OBJECTIVE: To study the effects of Astragaloside (AST) on PC12 cells injury and APP expression induced by dexamethasone (DEX) and beta-amyloid protein 25-35 (Abeta(25-35). METHODS: Logarithmic growth phase of the PC12 cells were seeded in culture plates. DEX 5 micromol/L and Abeta(25-35) 1 micromol/L were used to induce PC12 cells injury. MTT assay was used to detect the PC12 cells activity. RT-PCR was used to detect the APP, alpha-secretase and beta-secretase mRNA level of PC12 cells. Western bloting was used to detect APP protein expression of PC12 cells. RESULTS: MTT results showed that, DEX and Abeta(35-35) co-application could significantly decrease PC12 cells activity (P5 < 0.01). AST (10.20 mg/L) and Ginsenoside Rg1 (16 micromol/L) could increase PC12 cells activity. RT-PCR analysis showed that DEX and Abeta(25-35) co-application could significantly increase the beta-secretase mRNA levels and APP770, lower alpha-secretase mRNA levels. AST (10.20 mg/L) and Rg1 could decrease the elevated APP770, beta-secretase mRNA levels and increase the alpha-secretase mRNA level of PCl2 cells. Western bloting analysis result showed that AST (10.20 mg/L) and Rg1 could decrease the APP expression of PC12 cells induced by DEX and Abeta(25-35). CONCLUSION: AST has protective effects on PC12 cell injury induced by DEX and Abeta(25-35). The mechanism may be associated with decreasing the beta-secretase mRNA levels and APP expression, increasing the alpha-secretase mRNA levels.

[Protective effect and mechanism of astragaloside IV on oxidative stress injury of mesangial cells].

OBJECTIVE: To study the protective effect of astragaloside IV (AS IV) on H2O2 induced human mesangial cells (HMC), and further explore its molecular mechanism. METHOD: The cultured mesangial cells were divided into 5 groups: the normal control group, the H2O2 model group, the AS IV (12.5, 100 nmol x L(-1)) group and the Tempol (1 x 10(5) nmol x L(-1)) group. The MTT method was used to observe cell viability. Hoechst 33258 staining was used to observe the HMC apoptosis and DHE staining was used to detect the generation of reactive oxygen species (ROS). The flow cytometry was used to detect the changes in cell cycle. Western blot was used to detect the expression of Cyclin D1, CyclinA, p38, and T-p38. RESULT: H2O2 (1 x 10(5), 2 x 10(5), 3 x 10(5), and 4 x 10(5) nmol x L(-1)) could induce HMC oxidative stress injury, with significant decrease in the cell survival rate. AS IV (100 nmol x L(-1)) could significantly inhibit HMC oxidative stress injury induced by H2O2 (3 x 10(5) nmol x L(-1)), increase the survival rate of HMC cells, inhibit cell apoptosis, and decrease intracellular ROS production. AS IV could also increase the expression of Cyclin D1, recover normal cell proliferation, and decrease the expression of p38. CONCLUSION: AS IV can protect H2O2 induced oxidative stress injury in mesangial cells. Its mechanisms may be related to inhibiting the p38/MAPK signaling pathway, increasing the expression of Cyclin D1 and decreasing the intracellular ROS oxidative stress injury.

Interruption of TRPC6-NFATC1 signaling inhibits NADPH oxidase 4 and VSMCs phenotypic switch in intracranial aneurysm.

Intracranial aneurysm (IA) is a frequent cerebrovascular disorder with unclear pathogenesis. The vascular smooth muscle cells (VSMCs) phenotypic switch is essential for IA formation. It has been reported that Ca2+ overload and excessive reactive oxygen species (ROS) are involved in VSMCs phenotypic switch. The transient receptor potential canonical 6 (TRPC6) and NADPH oxidase 4 (NOX4) are the main pathway to participate in Ca2+ overload and ROS production in VSMCs. Ca2+ overload can activate calcineurin (CN), leading to nuclear factor of activated T cell (NFAT) dephosphorylation to regulate the target gene's transcription. We hypothesized that activation of TRPC6-NFATC1 signaling may upregulate NOX4 and involve in VSMCs phenotypic switch contributing to the progression of IA. Our results showed that the expressions of NOX4, p22phox, p47phox, TRPC6, CN and NFATC1 were significantly increased, and VSMCs underwent a significant phenotypic switch in IA tissue and cellular specimens. The VIVIT (NFATC1 inhibitor) and BI-749327 (TRPC6 inhibitor) treatment reduced the expressions of NOX4, p22phox and p47phox and the production of ROS, and significantly improved VSMCs phenotypic switch in IA rats and cells. Consistent results were obtained from IA Trpc6 knockout (Trpc6-/-) mice. Furthermore, the results also revealed that NFATC1 could regulate NOX4 transcription by binding to its promoter. Our findings reveal that interrupting the TRPC6-NFATC1 signaling inhibits NOX4 and improves VSMCs phenotypic switch in IA, and regulating Ca2+ homeostasis may be an important therapeutic strategy for IA.

[Effects of ginkgolide on hemodynamics in myocardial ischemic dogs].

OBJECTIVE: To investigate the effects of Ginkgolide on hemodynamics and myocardial oxygen metabolism in acute myocardial ischemic dogs. METHODS: Acute myocardial ischemic model dogs were established by ligating left anterior descending coronary artery (LAD). Some hemodynamic parameters and cardiac oxygen consumption were observed after Ginkgolide were given to dogs through the duodenum. RESULTS: Ginkgolide could increase LVSP, + dp/dt(max), LVWI, SW, CO and CBF, decrease TPVR, CVR, MVO2 and MOUR of Acute myocardial ischemic model dogs at different time points after ligation of LAD. CONCLUSION: Ginkgolide can improve the hemodynamic conditions and oxygen metabolism of myocardial ischemic dogs.

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 Shexiang Xingnaonin on focal cerebral ischemia/reperfusion injury and mechanism].

OBJECTIVE: To study the protective effects of Shexiang Xingnaonin (SXN) on focal cerebral ischemia/reperfusion injury and its mechanism. METHOD: Middle cerebral artery occlusion (MCAO) was used to make focal cerebral ischemia/reperfusion model by in travascular nylon filament occlusion. The protective effects of SXN at different doses were evaluated by investigating neurological function score, pathomorphology of brain, the ultrastructure of neuron, expression of tumor necrosis factor-alpha, thrombogenesis in vitro, platelet aggregation and lysing effect of blood clot in vitro. RESULT: Compared with model group, SXN (0.08, 0.16 g x kg(-1)) could decrease the neurological score, improve pathomorphology and neuron ultrastructure of brain, inhibit the expression of TNF-alpha, decrease the length, wet weight and dry weight of thromb and inhibit platelet aggregation. And SXN (0.16, 0.32 g x L(-1)) could dissolve blood clot in vitro. CONCLUSION: SXN has protective effects on focal cerebral ischemia/reperfusion injury. The role of inhibit the expression of TNF-alpha, inhibit thrombogenesis and platelet aggregation might contribute to its neuroprotective 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.

[Effects of AST and ASI on metabolism of free radical in senescent rats treated by HC].

OBJECTIVE: To study the effects of Astragaloside (AST) and Astragalus Saponin I (ASI) on metabolism of free radical and immune function in senescent rats treated by HC. METHOD: Hydrocorisone (HC) was used to estabilsh the aging model in rats. The content of molondialdehyde (MDA), glutathoine (GSH) and oxidized glutathoine (GSSG) in liver and brain was detected according to kit. The activity of Mn superoride dismulase (Mn-SOD) and catalase (CAT) was also surveyed by kit. Concanavalin (ConA) was used to detect the proliferation and interleukin-2 (IL-2) production of splenocytes. RESULT: Compared with HC control, AST and ASI could decrease the content of MDA, GSH and GSSG in liver and brain, increase the activity of Mn-SOD and CAT, and promote the proliferation and interleukin-2 (IL-2) activity of splenocytes. CONCLUSION: AST and ASI could delay the aging effect in rats treated by HC, and its mechanism maybe the antioxidant and regulating immunity.

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.

Biochanin A protects dopaminergic neurons against lipopolysaccharide-induced damage and oxidative stress in a rat model of Parkinson's disease.

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.

Biochanin A attenuates LPS-induced pro-inflammatory responses and inhibits the activation of the MAPK pathway in BV2 microglial cells.

Inflammation in the brain, characterized by the activation of microglia, is believed to participate in the pathogenesis of Parkinson's disease. Biochanin A, an O-methylated isoflavone, is a natural organic compound and is classified as a phytoestrogen. In this study, using murine BV2 microglial cells, we investigated the anti-inflammatory effects of biochanin A and the possible mechanisms involved. BV2 microglial cells were treated with lipopolysaccharide (LPS) to induce pro-inflammatory responses and the cells were then treated with biochanin A. Cell viability was examined by MTT assay. The production of nitric oxide (NO) was examined using Griess reagent and intracellular reactive oxygen species (ROS production) was measured by DCFH-DA assay. The mRNA expression of interleukin-1ß (IL-1ß), inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) was examined by RT-PCR. The expression of p-ERK, p-JNK, p-p38 and iNOS was measured by western blot analysis. In addition, the protein and mRNA and phosphorylation levels of pro-inflammatory cytokines were determined by western blot analysis and RT-PCR, respectively. The results revealed that biochanin A attenuated LPS-induced microglial activation and the production of TNF­α, IL-1ß, nitric oxide and reactive oxygen species in a dose-dependent manner. Biochanin A significantly decreased the LPS-induced mRNA expression of TNF-α and IL-1ß, and inhibited iNOS mRNA and protein expression. Furthermore, biochanin A significantly inhibited the LPS-induced phosphorylation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38. These findings suggest that the inhibitory effects of biochanin A on LPS-induced proinflammatory responses may be associated with the inhibition of mitogen-activated protein kinase (MAPK) signaling pathways in BV2 microglial cells.

Chronic restraint stress promotes learning and memory impairment due to enhanced neuronal endoplasmic reticulum stress in the frontal cortex and hippocampus in male mice.

Chronic stress has been implicated in many types of neurodegenerative diseases, such as Alzheimer's disease (AD). In our previous study, we demonstrated that chronic restraint stress (CRS) induced reactive oxygen species (ROS) overproduction and oxidative damage in the frontal cortex and hippocampus in mice. In the present study, we investigated the effects of CRS (over a period of 8 weeks) on learning and memory impairment and endoplasmic reticulum (ER) stress in the frontal cortex and hippocampus in male mice. The Morris water maze was used to investigate the effects of CRS on learning and memory impairment. Immunohistochemistry and immunoblot analysis were also used to determine the expression levels of protein kinase C α (PKCα), 78 kDa glucose-regulated protein (GRP78), C/EBP-homologous protein (CHOP) and mesencephalic astrocyte-derived neurotrophic factor (MANF). The results revealed that CRS significantly accelerated learning and memory impairment, and induced neuronal damage in the frontal cortex and hippocampus CA1 region. Moreover, CRS significantly increased the expression of PKCα, CHOP and MANF, and decreased that of GRP78 in the frontal cortex and hippocampus. Our data suggest that exposure to CRS (for 8 weeks) significantly accelerates learning and memory impairment, and the mechanisms involved may be related to ER stress in the frontal cortex and hippocampus.

Anti­fibrotic effects of Acremoniumterricola milleretal mycelium on immunological hepatic fibrosis in rats.

Acremoniumterricola milleretal mycelium (AMM) exerts numerous protective effects on organs, and has been used in Chinese herb prescriptions to treat refractory diseases. The aim of this study was to investigate the effects of AMM on immunological hepatic fibrosis induced by porcine serum (PS) in rats. Male Sprague Dawley rats were administered 0.5 ml sterile PS by intraperitoneal injections twice a week for 18 weeks. AMM (175, 350 or 700 mg/kg) and colchicine (0.1 mg/kg) were administered intragastrically each day until the rats were sacrificed. PS administration resulted in marked hepatic fibrosis, as assessed by increased oxidative stress and hepatic collagen content, as well as α­smooth muscle actin (α­SMA) expression. AMM significantly reduced liver damage and fibrosis. In addition, AMM decreased the elevation in hydroxyproline, hyaluronic acid, laminin and procollagen type III; increased the activity of superoxide dismutase and glutathione peroxidase; decreased α­SMA expression; and eliminated hepatic collagen deposits. Furthermore, AMM inhibited Smad2/3 phosphorylation and Smad7 expression. These results indicate that AMM is able to reduce oxidative stress, inhibit collagen synthesis and block the transforming growth factor­ß/Smad signaling pathway in a dose­dependent manner.

Astragalosides attenuate learning and memory impairment in rats following ischemia­reperfusion injury.

Astragalosides (ASTs) have been traditionally used in the treatment of various cardiovascular and cerebrovascular diseases. The aim of the present study was to investigate the neuroprotective effects of AST on learning and memory following focal cerebral ischemia/reperfusion in a rat model. A Morris water maze was used to measure the effect of AST on learning and memory impairments. A histological examination and Hoechst 33258 staining was used to observe the neuronal changes and apoptosis in the hippocampus. The activity of phospho-extracellular signal­regulated kinases (p­ERK), p­c-Jun N-terminal kinases (JNK) and p­Akt was measured by western blotting. The data revealed that AST improved the rats learning and memory abilities, attenuated neuronal cells apoptosis, increased the expression of p­ERK and p­Akt, and decreased the expression of p­JNK. These findings indicated that AST has protective effects that may be correlated with the inhibition of neuronal cell apoptosis and the regulation of p­ERK, p­Akt and p­JNK expression.