Protective effects of melatonin on mitochondrial biogenesis and mitochondrial structure and function in the HEK293-APPswe cell model of Alzheimer's disease.

OBJECTIVE: The effects and mechanisms of melatonin on Alzheimer's disease (AD) are still not researched thoroughly. 20E2 cells (HEK293-APPswe cells) are a cellular model of AD. The modulation effects of melatonin on the structure and function of mitochondria in 20E2 cells need to be studied. MATERIALS AND METHODS: The Alzheimer's disease (AD) cell model was assessed for cell viability, expression levels of mitochondrial biogenesis factors (peroxisome proliferator-activated receptor gamma coactivator 1-alpha [PGC-1α], nuclear respiratory factor 1 [NRF1], nuclear respiratory factor 2 [NRF2], mitochondrial transcription factor A [TFAM]), mitochondrial membrane potential, Na+-K+-adenosine triphosphatase (ATPase) and cytochrome C oxidase activity, adenosine triphosphate (ATP) level, mitochondrial DNA/nuclear DNA (mtDNA/nDNA) ratio, and mitochondrial structure with and without melatonin. RESULTS: Melatonin improved 20E2 cell viability, expression of mitochondrial biogenesis factors (PGC-1α, NRF1, NRF2, TFAM), mitochondrial membrane potential, Na+-K+-ATPase, and cytochrome C oxidase activity, ATP level, mtDNA/nDNA ratio, mitochondrial structure, and decreased amyloidogenic amyloid precursor protein processing. CONCLUSIONS: Mitochondrial biogenesis disorder is associated with the pathogenesis of AD through PGC-1α-NRF-TFAM pathway, and melatonin improves the mitochondrial structure and function by enhancing mitochondrial biogenesis and decreasing amyloidogenic APP processing in Alzheimer's disease.

[Killing effect of Robo1 targeted Chimeric Antigen Receptor modified NK92 cells against glioma and neuroblastoma cells].

Objective: To study the cytotoxicity of Robo1-CAR-NK92 cells against U87-MG and SH-SY5Y cells, to explore the effects of IL-15, IL-21 and dexamethasone on the proliferation, survival and cytotoxicity of Robo1-CAR-NK92 cells and to optimize the culture protocol. Methods: Robo1-CAR-NK92 cells were constructed by lentivirus transfection.The Robo1 car positive cells were sorted, expanded and detected by flow cytometry.The levels of Robo1 expression in SH-SY5Y and U87-MG cells were examined by flow cytometry.The cytotoxicity of Robo1-CAR-NK92 or NK92 cells against target cells was tested by CCK-8 and live cell imaging. The levels of cytokines in the supernatant of cultured cells during the cytotoxicity assay were quantified by the multiplex bead-array assay.NK92 and Robo1-CAR-NK92 cells (4×10(4)/ml) were treated with 25 ng/ml of IL-15, 25 ng/ml of IL-21 and/or 50 nmol/L dexamethasone for 3 days and were stained with trypan blue to acquire the viable cell numbers and survival rates. Results: Robo1-CAR-NK92 cells were constructed and tested 98.89% positive after sorting and expansion. While 88.14% of U87-MG cells were Robo1 positive, there were 99.75% of Robo1 positive SH-SY5Y cells.The specific lysis of Robo1-CAR-NK92 cells against target cells was significantly higher than that of NK92 cells (P<0.05). Robo1-CAR-NK92 cells obviously secreted more cytokines including IL-6, IL-10, TNF-α and IFN-γ than parental NK92 cells during cytotoxic activity against U87-MG cells (P<0.05). IL-15 significantly increased the proliferation and survival of Robo1-CAR-NK92 cells, but IL-21 played the opposite role.Remarkably, IL-21 and IL-15+ IL-21 enhanced the cytotoxicity of NK92 and Robo1-CAR-NK92 cells.The combination of dexamethasone and interleukins dramatically promoted the proliferation and survival but obviously impaired the cytotoxicity of NK92 and Robo1-CAR-NK92 cells (except that IL+ 15 and dexamethasone have no effect on the cytotoxicity of Robo1-CAR-NK92 cells). Conclusions: Compared to parental NK92 cells, Robo1-CAR-NK92 cells exhibited more potent targeted killing against glioma and neuroblastoma cells.Collectively, treatment of IL-15 and dexamethasone was the optimized protocol for culture of Robo1 CAR NK cells during our experimental time.

Doubts and difficulties in studying blood pressure variability.

The blood pressure (BP) fluctuation was first noticed in the 18th century. However, its clinical significance did not get attention until recent years. The increase in BP variability (BPV) is possibly more valuable than the increase in BP level for predicting damages in target organs. Moreover, attenuating BPV is more important than decreasing the BP level. However, the concept of BPV was not used in any related guideline for diagnosing, defining, and grading the risk of hypertension, which is due to the understanding of BPV is not profound, and blind areas and misunderstanding still exist in the definition, features, and classification of BPV. In this paper, the doubts and difficulties in studying BPV are analyzed, which may conduce to understand BPV and thus help for the clinical diagnosis and treatment of hypertension.

Mitophagy in APPsw/PS1dE9 transgenic mice and APPsw stably expressing in HEK293 cells.

OBJECTIVE: To observe mitophagy in APPsw/PS1dE9 transgenic mice and APPsw stably expressing HEK293 cell. MATERIALS AND METHODS: The APPsw/PS1dE9 transgenic mice and 20E2 cell (HEK293 cell with APPsw) was used to be the model of Alzheimer's disease. We dynamically observed the behavior, mitochondrial structure and mitophagy in brain of APP/PS1 transgenic mice in different age groups. Mitochondrial structure and mitophagy of HEK293 and 20E2 cell in vitro were also recorded. RESULTS: The mitochondrion was changed significantly in APP/PS1 transgenic mice and 20E2 cell. LC3 protein, associated with autophagy, was up-regulated. Mitophagy related protein PINK1 and PARKIN were up-regulated. CONCLUSIONS: Mitophagy disorder may be associated with the pathogenesis of Alzheimer's Disease through PINK1/Parkin pathway.

Granulocyte colony-stimulating factor attenuates chronic neuroinflammation in the brain of amyloid precursor protein transgenic mice: an Alzheimer's disease mouse model.

Recent evidence suggests that inflammatory mechanisms contribute significantly to the progression of Alzheimer's disease. Granulocyte colony-stimulating factor (G-CSF) is an anti-inflammatory immunomodulator, but the mechanism of its anti-inflammatory effect is unclear. This study was designed to investigate whether G-CSF could inhibit inflammation in a mouse model of Alzheimer's disease through an α7 nicotinic acetylcholine receptor (α7 nAChR) pathway. Mice transgenic for the V171I mutant amyloid precursor protein (APP) were injected subcutaneously with G-CSF 50 µg/kg per day or phosphate-buffered saline (PBS; control group) for 7 days, and wild-type C57/BL6 mice were injected with PBS daily for 7 days. Mice were killed on days 7, 14 and 28 after treatment began. Levels of α7 nAChR protein were significantly increased and levels of interleukin-1ß, tumour necrosis factor-α and nuclear factor-κB (NF-κB) protein were significantly decreased in the brain of APP transgenic mice in response to G-CSF. Levels of α7 nAChR protein correlated negatively with NF-κB levels. It is concluded that G-CSF might attenuate inflammation by down-regulating NF-κB and up-regulating α7 nAChR in the brain of APP transgenic mice, indicating a potential new therapeutic approach to Alzheimer's disease.