Pretreatment with Liposome-Encapsulated Shrimp Shell Extract Attenuated Neuronal Damage and Death in Aß1-42-Induced Memory Deficits in Rats.

The accumulation of amyloid-beta (Aß) peptides is a crucial factor in the neuronal degeneration of Alzheimer's disease (AD). The current study investigated the underlying neuroprotective mechanisms of shrimp shell extract (SSE) and liposome-encapsulated SSE (SSE/L) against Aß1-42-induced neuronal damage and death in rats. Intracerebroventricular infusion of Aß1-42 effectively induced memory decline, as observed in a reduction of the rat's discriminating ability in the novel object recognition and novel object location tasks. Oral pretreatment with 100 mg/kg of SSE demonstrated no preventive effect on the memory decline induced by Aß1-42 infusion. However, treatment with SSE/L 100 mg/kg BW effectively attenuated memory deficits in both behavioral assessments following two and four weeks after Aß1-42 infusion. Moreover, SSE/L exerted neuroprotective effects by reducing lipid peroxidation and increasing Nrf2/HO-1 expression. There was a significant decrease in Iba1 and GFAP (biomarkers of microglia and astrocyte activity, respectively), as well as a decrease in the levels of NF-κB expression and the inflammatory cytokines TNF-α and IL-6 in the cortical and hippocampal tissues. Treatment with SSE/L also reduced the pro-apoptotic proteins Bax and cleaved caspase-3 while raising the anti-apoptotic protein Bcl2. In addition, the beneficial effects of SSE/L were along with the effects of a positive control commercial astaxanthin (AST). The findings of this study indicated that SSE/L provided neuroprotective effects on Aß1-42-induced AD rats by ameliorating oxidative stress, neuroinflammation and apoptotic cell death. Therefore, SSE/L might be employed to prevent and mitigate Aß accumulation-induced neurotoxicity in AD.

Effect of kaolin impregnated with calico plant extract on properties of starch films.

Starch film has poor tensile properties and poor water resistance. We aimed to improve these properties by adding kaolin impregnated with calico plant extract (CP-Kaolin). UV-Vis spectrophotometry showed that the calico plant extract (CPE) contained 4867.52 mg/L of total phenolic compounds and betacyanins were the predominant constituents. CP-Kaolin was characterized by Fourier transform infrared spectroscopy (FTIR), zeta potential, scanning electron microscopy (SEM) and x-ray diffraction (XRD) analysis. FTIR analysis showed that betacyanins were adsorbed on kaolin via hydrogen bonding. Zeta potential analysis confirmed the adsorption of betacyanins on kaolin. The intercalation of betacyanins between kaolin platelets was observed by XRD. SEM revealed that CP-Kaolin was well dispersed and embedded within the starch matrix. It was found that the addition of 10 wt% of CP-Kaolin increased the water resistance, tensile strength and thermal stability of starch film. Moreover, starch film containing 10 wt% of CP-Kaolin was sensitive to the change in pH of the fish during storage. Therefore, the addition of CP-Kaolin improved the properties of starch film and starch film composite with CP-Kaolin could be applied as a smart packaging in the food industry.

Chitooligosaccharide from Pacific White Shrimp Shell Chitosan Ameliorates Inflammation and Oxidative Stress via NF-κB, Erk1/2, Akt and Nrf2/HO-1 Pathways in LPS-Induced RAW264.7 Macrophage Cells.

Chitooligosaccharide (COS), found in both insects and marine sources, has several bioactivities, such as anti-inflammation and antioxidant activities. However, the mechanism of shrimp shell COS on retardation of inflammatory and antioxidant effects is limited. Therefore, the aim of this study is to examine the mechanism of the aforementioned activities of COS in LPS-activated RAW264.7 macrophage cells. COS significantly improved cell viability in LPS-activated cells. COS at the level of 500 µg/mL could reduce the TNF-α, NO and IL-6 generations in LPS-activated cells (p < 0.05). Furthermore, COS could reduce ROS formation, NF-κB overactivation, phosphorylation of Erk1/2 and Akt and Nrf2/HO-1 in LPS-exposed cells. These results indicate that COS manifests anti-inflammatory activity and antioxidant action via NF-κB, Erk1/2, Akt and Nrf2/HO-1 signaling with an increasing relevance for inflammatory disorders.

Effects of collagen, chitosan and mixture on fibroblast responses and angiogenic activities in 2D and 3D in vitro models.

Despite accumulating evidences have demonstrated the potential of collagen and chitosan on tissue repair, it remains unclear on their combination effects. Here, we examined the regenerative effects of single collagen, chitosan and their mixture on fibroblasts and endothelial cells at cellular levels. The results showed that fibroblast responses, as indicated by high proliferative rate, increased spheroid diameter and migrated area existing from spheroid edge, and decreased wound area, were significantly promoted by either collagen or chitosan stimulation. Similarly, both collagen and chitosan resulted in increased endothelial cell proliferation and migration with accelerated tube-like network formation and upregulated VE-cadherin expression, although collagen strongly provided this effect. While the 1:1 mixture (100:100 µg/mL of chitosan to collagen) treatment caused a reduction in fibroblast viability, the lower ratio of chitosan (1:10 mixture; 10:100 µg/mL) did not produce any impact on both fibroblast and endothelial cell viabilities. The 1:10 mixture also significantly enhanced the additional effects on fibroblast responses and angiogenic activities as shown by higher endothelial growth, proliferation and migration with accelerated capillary-like network formation than those treated with the single substance. Further investigation of signaling proteins found that collagen significantly increased expressions of p-Fak, p-Akt and Cdk5 whereas chitosan upregulated p-Fak and Cdk5 expressions. Comparing to the single treatments, p-Fak, p-Akt and Cdk5 were higher expressed in the 1:10 mixture. These observations indicate that proper collagen-chitosan mixture provides the combination effects on fibroblast responses and angiogenic activities when a high concentration of collagen is used, possibly through Fak/Akt and Cdk5 signaling pathways. Therefore, this study helps to define the clinical use of collagen and chitosan as promising biomaterials for tissue repair.

Chitooligosaccharide prevents vascular endothelial cell apoptosis by attenuation of endoplasmic reticulum stress via suppression of oxidative stress through Nrf2-SOD1 up-regulation.

CONTEXT: Endoplasmic reticulum (ER) stress contributes to endothelium pathological conditions. Chitooligosaccharides (COS) have health benefits, but their effect on endothelial cells is unknown. We demonstrate for the first time a protective effect of COS against ER-induced endothelial cell damage. OBJECTIVE: To evaluate the protective effect of COS on ER stress-induced apoptosis in endothelial cells. MATERIAL AND METHODS: Endothelial (EA.hy926) cells were pre-treated with COS (250 or 500 µg/mL) for 24 h, and then treated with 0.16 µg/mL of Tg for 24 h and compared to the untreated control. Apoptosis and necrosis were detected by Annexin V-FITC/propidium iodide co-staining. Reactive oxygen species (ROS) were measured with the DCFH2-DA and DHE probes. The protective pathway and ER stress markers were evaluated by reverse transcription-polymerase chain reaction, western blot, and immunofluorescence analyses. RESULTS: COS attenuated ER stress-induced cell death. The viability of EA.hy926 cells treated with Tg alone was 44.97 ± 1% but the COS pre-treatment increased cells viability to 74.74 ± 3.95% in the 250 µg/mL COS and 75.34 ± 2.4% in the 500 µg/mL COS treatments. Tg induced ER stress and ROS, which were associated with ER stress-mediated death. Interestingly, COS reduced ROS by upregulating nuclear factor-E2-related factor 2 (Nrf2), and the oxidative enzymes, superoxide dismutase1 (SOD1) and catalase. COS also suppressed up-regulation of the ER-related apoptosis protein, CHOP induced by Tg. CONCLUSIONS: COS protected against ER stress-induced apoptosis in endothelial cells by suppressing ROS and up-regulation Nrf2 and SOD1. These findings support the use of COS to protect endothelial cells.

Cordycepin attenuates migration and invasion of HSC-4 oral squamous carcinoma cells through autophagy-dependent FAK/Akt and MMP2/MMP9 suppression.

Background/purpose: Cordycepin has been proposed anti-cancer effects, however, it is unclear whether and how cordycepin affects oral squamous carcinoma cell (OSCC) migration and invasion. This study aimed to investigate the effect of cordycepin on migration and invasion of OSCC (HSC-4 cells), and its underlying mechanism. Materials and methods: Cell viability was measured with MTT assay. Migrative and invasive abilities were determined by scratch wound healing, agarose spot and transwell invasion assays, respectively. Monodasylcadaverine (MDC) staining, immunofluorescence staining of LC3 and RT-PCR evaluated the gene expression of LC3 and p62 were applied to investigate autophagy. MMP2 and MMP9 gene expression and activity were examined by RT-PCR and gelatin zymography. Expression of caspase 3, cleaved caspase 3, FAK, p-FAK, Akt and p-Akt was determined by Western blot. Results: Cordycepin significantly inhibited HSC-4 cell migration and invasion in a concentration-dependent manner. Cordycepin treatment caused an induction of autophagy, as evidenced by increased MDC fluorescence intensity and MDC positive cells, and upregulated expression level of LC3 gene. In addition, inhibition of autophagy by chloroquine (CQ) significantly abolished cordycepin-inhibited HSC-4 cell migration and invasion, demonstrating that cordycepin-inhibited migration and invasion was mediated by autophagy. Mechanistic studies showed that cordycepin significantly suppressed FAK and Akt phosphorylation, and MMP2 and MMP9 activities. Conversely, CQ pre-incubation significantly restored its expression and activity in cordycepin-treated cells. Conclusion: Cordycepin induces autophagy to suppress FAK and Akt phosphorylation, and MMP2 and MMP9 activity, which responsible for the attenuation of HSC-4 cell migration and invasion.

Oral Administration of Ethanolic Extract of Shrimp Shells-Loaded Liposome Protects against Aß-Induced Memory Impairment in Rats.

Alzheimer's disease is characterized by a progressive loss of memory and cognition. Accumulation of amyloid-beta (Aß) in the brain is a well-known pathological hallmark of the disease. In this study, the ethanolic extract of white shrimp (Litopenaous vannamei) shells and the ethanolic extract-loaded liposome were tested for the neuroprotective effects on Aß1-42-induced memory impairment in rats. The commercial astaxanthin was used as a control. Treatment with the ethanolic extract of shrimp shells (EESS) at the dose of 100 mg/kg BW showed no protective effect in Aß-treated rats. However, treatment with an EESS-loaded liposome at the dose of 100 mg/kg BW significantly improved memory ability in Morris water maze and object recognition tests. The beneficial effect of the EESS-loaded liposome was ensured by the increase of the memory-related proteins including BDNF/TrkB and pre- and post-synaptic protein markers GAP-43 and PSD-95 as well as pErk1/2/Erk1/2 in the cortex and hippocampus. These findings indicated the neuroprotective effects of the EESS-loaded liposome on Aß-induced memory impairment in rats. It produced beneficial effects on learning behavior probably through the function of BDNF/TrkB/pErk1/2/Erk1/2 signaling pathway and subsequently the upregulation of synaptic proteins. The present study provided evidence that the neuroprotective property of the ESSE-loaded liposome could be a promising strategy for AD protection.

Investigation of mechanisms underlying the inhibitory effects of metformin against proliferation and growth of neuroblastoma SH-SY5Y cells.

Besides being anti-diabetic drug, metformin also has anti-proliferation and growth in several tumors; however, details of possible mechanism have not been elucidated. Here, we investigated the effects of metformin in neuroblastoma which has been termed as extra-cranial solid tumor that is due to a differentiation block with more stemness. The results showed that 5 mM metformin inhibited cell cycle progression at G0/G1 phase. Metformin also induced morphological differentiation of neuroblastoma into neuron-like phenotypes by which upregulation of MAP2, ß-tubulin III and tyrosine hydroxylase expressions with no significant difference to retinoic acid (RA)-treated cells. We also tested proliferative, growth and self-renewal ability after neuroblastoma being differentiated by metformin for 24 h. The proliferative rate, sizes and numbers of colonies and spheroids were significantly reduced in differentiated neuroblastoma compared to undifferentiated neuroblastoma. A significant increase of ROS and ADP/ATP ratio with decreased mitochondrial membrane potential (MMP) were observed in metformin-treated cells, indicating mitochondrial biogenesis and metabolic change during metformin-mediated differentiation. The further studies exhibited that p-Erk1/2 and Cdk5 levels were reduced in metformin treatment whereas using PD98095 and roscovitine, selectively inhibited Erk1/2 and Cdk5, respectively, significantly increased neurite length and MAP2 expression. In addition, cell proliferation was decreased by cell cycle arrested at G0/G1 phase. Taken together, this study suggests the inhibitory effects of metformin against proliferation and growth of neuroblastoma due to induced morphological differentiation may be through Erk1/2 and Cdk5 pathways. Therefore, metformin might be eventually considered as a differentiation agent for neuroblastoma treatment in term of differentiation therapy.

Impact of Hydrolyzed Collagen from Defatted Sea Bass Skin on Proliferation and Differentiation of Preosteoblast MC3T3-E1 Cells.

Osteoporosis is a serious problem affecting health of the elderly. Drugs (bisphosphonates) applied for treatment are often accompanied by adverse side effects. Thus, fish byproduct-derived peptides, particularly hydrolyzed collagen (HC) from defatted sea bass skin, could be a safe source of anti-osteoporosis agents. This study aimed to examine the effects of HC on proliferation and differentiation of preosteoblast cells. HC prepared using papain before Alcalase hydrolysis was determined for molecular weight (MW) distribution. Thereafter, the resulting HC (50-800 µg/mL) was added to the cell. Proliferation, alkaline phosphatase activity (AP-A) and mineralization of cells were investigated. Moreover, the expression of runt-related transcription factor 2 (RUNX2) and the p-Akt/Akt pathway were also determined using Western blot. The results showed that HC had an MW < 3 kDa. HC (50-200 µg/mL) could promote cell proliferation. Nevertheless, HC at 100 µg/mL (HC-100) had enhanced AP-A and increased mineralization during the first 7 days of culture. Moreover, HC-treated cells had higher calcium depositions than the control (p < 0.05). Additionally, cells treated with HC-100 had higher levels of RUNX2 and p-Akt expressions than control (p < 0.05). Therefore, HC could be a promising functional ingredient to promote osteoblast proliferation and differentiation, which could enhance bone strength.

Effects of sonication and ultrasound on properties and bioactivities of liposomes loaded with hydrolyzed collagen from defatted sea bass skin conjugated with epigallocatechin gallate.

Hydrolyzed collagen (HC) from defatted sea bass skin conjugated with 3% epigallocatechin gallate (EGCG) was prepared and the resulting HC-EGCG conjugate at various levels (0.25%-2%, w/v) was loaded into liposome. The obtained liposomes were subjected to sonication (S). Liposome loaded with 1% conjugate showed the highest encapsulation efficiency (EE) (p < .05). When the ultrasound-assisted process (UAP) at different amplitudes (20% and 40%) and times (2, 5, 10, and 15 min) were implemented, the highest EE of conjugate-loaded liposome was found at 20% amplitude for 2 min (p < .05). When S-liposome and UAP-liposome were lyophilized, decreasing EE of both samples was observed (p < .05). Lyophilized UAP-liposome had higher stability than lyophilized S-liposome during storage at 25℃ for 28 days. Additionally, antioxidant activity in the gastrointestinal track model system (GIMs) and digest obtained from GIMs were higher for UAP-liposome (p < .05). Therefore, liposome can be used for the delivery of conjugate. PRACTICAL APPLICATIONS: HC from defatted sea bass skin is considered to possess several bioactivities, especially skin nourishment and bone strengthening. Nevertheless, antioxidant activity, related to the treatment of several ailments, is still low for HC. Thus, grafting of HC with polyphenol such as EGCG via free radical method can be used for the enhancement of the antioxidant activity of HC. Although the resulting conjugate has augmented activity, it is unstable during storage and in the gastrointestinal digestion system. Liposome is a promising means to stabilize the conjugate under harsh condition, especially with the aid of the UAP. Thus, liposome loaded with conjugate having the reduced size has higher antioxidant activity with increased stability, which can have a wider range of applications.

Suppressing Cdk5 Activity by Luteolin Inhibits MPP+-Induced Apoptotic of Neuroblastoma through Erk/Drp1 and Fak/Akt/GSK3ß Pathways.

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neurons. The cause of PD is still unclear. Oxidative stress and mitochondrial dysfunction have been linked to the development of PD. Luteolin, a non-toxic flavonoid, has become interested in an alternative medicine, according to its effects on anti-oxidative stress and anti-apoptosis, although the underlying mechanism of luteolin on PD has not been fully elucidated. This study aims to investigate whether luteolin prevents neurotoxicity induction by 1-methyl-4-phenylpyridinium iodide (MPP+), a neurotoxin in neuroblastoma SH-SY5Y cells. The results reveal that luteolin significantly improved cell viability and reduced apoptosis in MPP+-treated cells. Increasing lipid peroxidation and superoxide anion (O2-), including mitochondrial membrane potential (Δψm) disruption, is ameliorated by luteolin treatment. In addition, luteolin attenuated MPP+-induced neurite damage via GAP43 and synapsin-1. Furthermore, Cdk5 is found to be overactivated and correlated with elevation of cleaved caspase-3 activity in MPP+-exposed cells, while phosphorylation of Erk1/2, Drp1, Fak, Akt and GSK3ß are inhibited. In contrast, luteolin attenuated Cdk5 overactivation and supported phosphorylated level of Erk1/2, Drp1, Fak, Akt and GSK3ß with reducing in cleaved caspase-3 activity. Results indicate that luteolin exerts neuroprotective effects via Cdk5-mediated Erk1/2/Drp1 and Fak/Akt/GSK3ß pathways, possibly representing a potential preventive agent for neuronal disorder.

In vitro antioxidant and wound-healing activities of hydrolyzed collagen from defatted Asian sea bass skin as influenced by different enzyme types and hydrolysis processes.

Hydrolyzed collagen (HC) from defatted Asian sea bass skin was prepared by different enzymatic hydrolysis processes. For one-enzyme hydrolysis, papain (0.3 unit per g dry matter, DM) at 40 °C for 90 min or Alcalase (0.2 or 0.3 unit per g DM) at 50 °C for 90 min were used. The two-enzyme hydrolysis was accomplished with papain at 0.3 unit per g DM (P0.3), followed by Alcalase hydrolysis at 0.2 or 0.3 units per g DM (A0.2 or A0.3, respectively). HC prepared using the P0.3 + A0.3 process showed higher peptide yield, recovery and imino acid content in addition to stronger ABTS, DPPH radical scavenging activities and ferric reducing antioxidant power than other hydrolysis processes. HC obtained from the P0.3 + A0.3 process (at 125-500 µg mL-1) induced MRC-5 fibroblast proliferation and augmented migration and lamellipodia formation in the cells. Peptides with average molecular weight of 750 Da exhibited the highest ABTS radical scavenging activity while the 4652 Da fraction had the lowest. Thus, HC can be considered as a suitable ingredient to formulate functional products for skin nourishment and wound healing.

Luteolin attenuates migration and invasion of lung cancer cells via suppressing focal adhesion kinase and non-receptor tyrosine kinase signaling pathway.

BACKGROUND/OBJECTIVES: Non-small cell lung cancer is mostly recognized among other types of lung cancer with a poor prognosis by cause of chemotherapeutic resistance and increased metastasis. Luteolin has been found to decrease cell metastasis. However, its underlying mechanisms remain unresolved. The objective of this study was to examine the effect (and its mechanism) of luteolin on the migration and invasion of human non-small cell lung cancer A549 cells. MATERIALS/METHODS: Cell viability was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Wound healing and transwell assays were evaluated to assess migration and invasion, respectively. Western blot analysis and immunofluorescence were further performed to investigate the role of luteolin and its mechanisms of action. RESULTS: Administration with up to 40 µM luteolin showed no cytotoxic activity on lung cancer A549 cells or non-cancer MRC-5 cells. Additionally, luteolin at 20-40 µM significantly suppressed A549 cells' migration, invasion, and the formation of filopodia in a concentration-dependent manner at 24 h. This is similar with western blot analysis, which revealed diminished the phosphorylated focal adhesion kinase (pFAK), phosphorylated non-receptor tyrosine kinase (pSrc), Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division control protein 42 (Cdc42), and Ras homolog gene family member A (RhoA) expression levels. CONCLUSIONS: Overall, our data indicate that luteolin plays a role in controlling lung cancer cells' migration and invasion via Src/FAK and its downstream Rac1, Cdc42, and RhoA pathways. Luteolin might be considered a promising candidate for suppressing invasion and metastasis of lung cancer cells.

Self-assembled polydopamine nanoparticles improve treatment in Parkinson's disease model mice and suppress dopamine-induced dyskinesia.

Although Levodopa (l-DOPA), a dopamine precursor, exhibits a high risk of dyskinesia, it remains the primary treatment in Parkinson's disease (PD), a progressive neurodegenerative disorder. In this study, we designed poly(l-DOPA)-based self-assembled nanodrug (NanoDOPA) from amphiphilic block copolymer possessing poly(l-DOPA(OAc)2), which is a precursor of l-DOPA as a hydrophobic segment, for treatment in a PD model mouse. Under physiological enzyme treatment, the poly(l-DOPA(OAc)2) in the block copolymer was hydrolyzed to liberate l-DOPA gradually. Using the MPTP-induced PD mouse model, we observed that mice treated with NanoDOPA demonstrated a significant improvement of PD symptoms compared to the l-DOPA treatment. Interestingly, the NanoDOPA treatment did not cause the dyskinesia symptoms, which was clearly observed in the l-DOPA-treated mice. Furthermore, NanoDOPA exhibited remarkably lower toxicity in vitro compared to l-DOPA, in addition with no noticeable NanoDOPA toxicity observed in the treated mice. These results suggested that self-assembled NanoDOPA is a promising therapeutic in the treatment of PD. STATEMENT OF SIGNIFICANCE: In this study, we proposed a therapeutic approach for the effective treatment of Parkinson's disease (PD) using newly designed poly(l-DOPA)-based self-assembled nanodrug (NanoDOPA) prepared from amphiphilic block copolymers possessing poly(l-DOPA(OAc)2), which is a precursor of l-DOPA as a hydrophobic segment, for treatment in a PD model mouse. Under physiological enzyme treatments, NanoDOPA was hydrolyzed to liberate l-DOPA gradually, improving the pharmacokinetic value of l-DOPA. The mice treated with NanoDOPA significantly improved PD symptoms compared to the l-DOPA treatment in a neurotoxin-induced PD mouse model. Interestingly, NanoDOPA treatment did not cause dyskinesia symptoms, which was observed in the l-DOPA-treated mice. The obtained results in this study suggested that self-assembled NanoDOPA is a promising therapeutic in the treatment of PD.

Metformin Inhibit Cervical Cancer Migration by Suppressing the FAK/Akt Signaling Pathway.

BACKGROUND: Metformin, an antidiabetic drug, has been previously reported to have anti-cancer activities. However, its role in the control of cancer cell migration remains elusive. METHODS: To examine the possible effect of metformin on migration of cervical cancer cells. The related mechanisms were further determined by immunocytochemistry and Western's blotting assay. RESULTS: The results showed that metformin treatment substantially inhibited the migration ability of cervical cancer cells. Consistently, the filopodia and lamellipodia formation were depleted after exposure to metformin. The suppression of migration mediated through the regulatory proteins such as focal adhesion kinase (FAK), ATP-dependent tyrosine kinase (Akt), Rac1 and RhoA after metformin treatment. CONCLUSION: Metformin displays antimigration effects in cervical cancer cells by inhibiting filopodia and lamellipodia formation through the suppression of FAK, Akt and its downstream Rac1 and RhoA protein. We propose that metformin could be a novel potential candidate as an antimetastatic cancer drug in the cervical cancer management.

Effects of Astaxanthin from Shrimp Shell on Oxidative Stress and Behavior in Animal Model of Alzheimer's Disease.

This study aimed to investigate the effect of astaxanthin (ASX) extracted and ASX powder from shrimp (Litopenaeus vannamei) shells on Wistar rats with Alzheimer's disease, induced by amyloid-ß (1-42) peptides. In this task, the rats were divided into eight groups: (1) Control, (2) sham operate, (3) negative control (vehicle) + Aß1-42, (4) ASX extract+Aß1-42, (5) commercial ASX + Aß1-42, (6) ASX powder + Aß1-42, (7) blank powder + Aß1-42, and (8) vitamin E + Aß1-42. All treatments were orally administrated for 30 days. At 14- and 29-days post injection, animals were observed in behavioral tests. On the 31st day, animals were sacrificed; the hippocampus and cortex were collected. Those two brain areas were then homogenized and stored for biochemical and histological analysis. The results showed that the Aß1-42 infused group significantly reduced cognitive ability and increased memory loss, as assessed by the Morris water maze test, novel object recognition test, and novel object location test. Moreover, the Aß1-42 infused group exhibited a deterioration of oxidative markers, including glutathione peroxidase enzymes (GPx), lipid peroxidation (MDA), products of protein oxidation, and superoxide anion in the cortex and the hippocampus. Meanwhile, ASX powder (10 mg/kg body weight) showed a significant reduction in cognitive and memory impairments and oxidative stress which is greater than ASX extract in the same dose of compound or vitamin E (100 mg/kg body weight). Our study indicates the beneficial properties of ASX in alleviation of cognitive functions and reducing neurodegeneration in Wistar rats induced by amyloid-ß (1-42) peptides.

ECa 233 Suppresses LPS-Induced Proinflammatory Responses in Macrophages via Suppressing ERK1/2, p38 MAPK and Akt Pathways.

A current anti-inflammatory agent often targets the prevention of inflammatory disorder development. The standardized Centella asiatica ECa 233 extract has been previously reported for anti-inflammatory effect. This study aimed to investigate its anti-inflammatory effect and mechanisms of ECa 233 in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nitric oxide (NO) assay, reactive oxygen species (ROS) production assay, enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Our results found that ECa 233 significantly inhibited LPS-stimulated pro-inflammatory mediators production including ROS, NO and prostaglandin E2 (PGE2), and pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-1ß without cytotoxicity. In addition, ECa 233 downregulated not only the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), but also the activation of nuclear factor-kappa B (NF-κB), activated protein kinase B (Akt), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (MAPK) induced by LPS. The inhibition of LPS-induced inflammation due to ECa 233 offered an opportunity as a tentatively potential candidate for the prevention and treatment of inflammatory diseases.

Metformin Promotes Neuronal Differentiation via Crosstalk between Cdk5 and Sox6 in Neuroblastoma Cells.

Metformin has recently emerged as a key player in promotion of neuroblastoma differentiation and neurite outgrowth. However, molecular mechanisms of how metformin promotes cellular differentiation have not yet been fully elucidated. In this study, we investigated how metformin promotes cell differentiation, via an inhibition of cell proliferation, by culturing SH-SY5Y neuroblastoma cells with or without metformin. Pretreatment with reactive oxygen species (ROS) scavenger, NAC, revealed that ROS plays a crucial role in induction of cell differentiation. Cell differentiation was observed under various morphological criteria: extension of neuritic processes and neuronal differentiation markers. Treatment with metformin significantly increased neurite length, number of cells with neurite, and expression of neuronal differentiation markers, ß-tubulin III and tyrosine hydroxylase (TH) compared with untreated control. Further investigation found that metformin significantly decreased Cdk5 but increased Sox6 during cell differentiation. Analysis of the mechanism underlying these changes using Cdk5 inhibitor, roscovitine, indicated that expressions of Cdk5 and Sox6 corresponded to metformin treatment. These results suggested that metformin produces neuronal differentiation via Cdk5 and Sox6. In addition, phosphorylated Erk1/2 was decreased while phosphorylated Akt was increased in metformin treatment. Taken together, these findings suggest that metformin promotes neuronal differentiation via ROS activation through Cdk5/Sox6 crosstalk, relating to Erk1/2 and Akt signaling.

Induction of keratinocyte migration by ECa 233 is mediated through FAK/Akt, ERK, and p38 MAPK signaling.

Centella asiatica is widely considered the most important medicinal plant for treating and relieving skin diseases. Recently developed standardized extract of Centella asiatica ECa 233 has demonstrated positive effects on wound healing of incision and burn wound in rats. However, knowledge associated with wound healing mechanism of ECa 233 was scare. Therefore, this study aimed to investigate the effect and underlying molecular mechanisms of ECa 233 on the migration of a human keratinocyte cell line (HaCaT) using scratch wound healing assay. Formation of filopodia, a key protein in cell migration as well as signaling pathways possibly involved were subsequently assessed. It was found that HaCaT cell migration was significantly enhanced by ECa 233 in a concentration- and time-dependent manner. The filopodia formations were accordingly increased in exposure to ECa 233 at concentrations of 0.1-100 µg/ml. Furthermore, ECa 233 was found to significantly upregulate the expression of Rac1 and RhoA and to induce phosphorylation of FAK and Akt as well as ERK and p38 MAPK. Taken all together, it is suggestive that ECa 233 induces cell migration and subsequently promotes wound healing activity, through the activation of FAK, Akt, and MAPK signaling pathways thereby supporting the role of ECa 233 to be further developed for the clinical treatment of wound.

The antioxidant and neurochemical activity of Apium graveolens L. and its ameliorative effect on MPTP-induced Parkinson-like symptoms in mice.

BACKGROUND: Apium graveolens L. is a traditional Chinese medicine prescribed as a treatment for hypertension, gout, and diabetes. This study aimed to determine the neuroprotective effects of A. graveolens extract against a Parkinson's disease (PD) model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57BL/6 mice. METHODS: Male C57BL/6 mice treated with MPTP were orally dosed with A. graveolens extract daily for 21 days. Behavioral tests, including a rotarod apparatus, a narrow beam test, a drag test, a grid walk test, a swimming test, and a resting tremor evaluation, were performed. Thereafter, the mice were sacrificed, and monoamine oxidase A and B activity, lipid peroxidation activity, and superoxide anion levels were measured. Immunohistochemical staining of tyrosine hydroxylase was performed to identify dopaminergic neurons. RESULTS: We found that treatment with A. graveolens at dose of 375 mg/kg demonstrated the highest effect and led to significant improvements in behavioral performance, oxidative stress parameters, and monoamine oxidase A and B activity compared with the untreated group (p < 0.05). Moreover, the extract increased the number of neurons immunopositive for tyrosine hydroxylase expression compared with MPTP alone or MPTP with a positive control drug (p < 0.05). CONCLUSIONS: We speculated that A. graveolens ameliorated behavioral performance by mediating neuroprotection against MPTP-induced PD via antioxidant effects, related neurotransmitter pathways and an increase in the number of dopaminergic neurons.