Combining RNAi and in vivo confocal microscopy analysis of the photoconvertible fluorescent protein Dendra2 to study a DNA repair protein

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of "cytosolic" enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state.

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Suppressive Effect of Huzhentongfeng on Experimental Gouty Arthritis: An In Vivo and In Vitro Study

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2019/2969364 ◽

2019 ◽

Vol 2019 ◽

pp. 1-15

Author(s):

Zi-cong Wu ◽

Qiang Xue ◽

Zhen-ling Zhao ◽

Peng-jun Zhou ◽

Qun Zhou ◽

...

Keyword(s):

Confocal Microscopy ◽

Antioxidant Capacity ◽

Radical Scavenging ◽

Neutrophil Infiltration ◽

Control Group ◽

Raw264.7 Macrophages ◽

Microscopy Analysis ◽

Confocal Microscopy Analysis

Background. Huzhentongfeng (HZTF) is an extract from four Chinese medical herbs for treating gout. This study aims to evaluate its antigout activity and preliminary explore its mechanism in vivo and in vitro. Methods. The rats were intragastrically administered with HZTF for 5 days and then injected 0.1 ml (10 mg) of MSU crystals to their joints for generating a gout model to analyze the paw volume and histopathology of joint synovial tissues of rats with different doses. We also investigated the antioxidant capacity of HZTF in vitro using indication including lipid peroxidation, DPPH·, and ABTS+ radical-scavenging capacity; besides, we used qRT-PCR to measure the effect of HZTF on interleukin (IL)-1β, caspase-1, NLRP3, and NQO1 expression in hydrogen peroxide-stimulated RAW264.7 macrophages and IL-1β, IL-6, and tumor necrosis factor (TNF)-α in MSU crystal-induced THP-1 monocytes. Confocal microscopy analysis was used to observe the dimerization of ASC adapter proteins. In addition, we also established quality standard of HZTF by using the high-performance liquid chromatography (HPLC) method. Results. HZTF could significantly suppress the paw swelling and neutrophil infiltration induced by MSU intra-articular injection in rats compared with the control group. HZTF also showed inhibition effects of inflammatory cytokines (IL-1β, IL-6, and TNF-α) secretion at 25.00 and 50.00 μg/ml in MSU-induced THP-1 cells but showed no effects of IL-1β, IL-6, and TNF-α mRNA expression in MSU-induced THP-1 cells. Furthermore, confocal microscopy analysis showed that HZTF could prevent the oligomerization of ASC. Moreover, HZTF also showed effects in cell-free and cell-base tests of antioxidant capacity. Conclusion. The results prove that HZTF possessed the potential preventive effect against gout arthritis, and the effect may be attributed to its preventing effect on neutrophil infiltration and proinflammatory cytokines secretion such as IL-1β, IL-6, and TNF-α which were caused by the activation of inflammasome.

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