LncRNA NEAT1 accelerates renal tubular epithelial cell damage by modulating mitophagy via miR-150-5p-DRP1 axis in diabetic nephropathy.

NEW FINDINGS: What is the central question of this study? Diabetic nephropathy (DN) is a severe complication of diabetes correlated with a higher mortality rate in diabetic patients. Renal tubular injury participates in the pathogenesis of DN. We aimed to uncover the biological function of the NEAT1-miR-150-5p-DRP1 axis in an in vitro model of DN and elaborate the potential mechanisms. What is the main finding and its importance? NEAT1 facilitated high glucose-induced damage in HK-2 cells by reducing mitophagy via the miR-150-5p-DRP1 axis, which sheds light on DN pathogenesis and reveals a potential treatment for DN. ABSTRACT: Diabetic nephropathy (DN) is a severe complication in diabetic patients, with a high mortality rate. Renal tubular injury is involved in the pathogenesis of DN. In this study, we aimed to uncover the regulatory roles of the NEAT1-miR-150-5p-DRP1 axis in an in vitro model of DN and its possible mechanisms. High glucose-challenged HK-2 cells were used as an in vitro DN model. NEAT1, miR-150-5p and DRP1 levels were assessed by RT-qPCR. Cell viability was determined by the MTT assay. MitoSOX Red and JC-1 were used to evaluate intracellular production of reactive oxygen species and mitochondrial membrane potential, respectively. Lactate dehydrogenase release and superoxide dismutase activity were assessed with commercial kits. The protein levels of DRP1, p62, BECN1(beclin 1) and BNIP3 were determined by western blotting. The interaction between NEAT1 (DRP1) and miR-150-5p was verified by a dual-luciferase reporter assay and an RNA immunoprecipitation assay. Our results showed that in response to high glucose the NEAT1 and DRP1 levels were upregulated, whereas the miR-150-5p level was downregulated in HK-2 cells. Knockdown of NEAT1 or DRP1 in high glucose-challenged HK-2 cells inhibited excessive reactive oxygen species production and lactate dehydrogenase release, increased cell viability, mitochondrial membrane potential and superoxide dismutase activity and enhanced mitophagy. Inhibition of miR-150-5p resulted in the opposite results. Mechanistically, NEAT1 sponged miR-150-5p to increase the DRP1 level. Moreover, silencing of NEAT1 or DRP1 could counteract miR-150-5p inhibition-induced deleterious effects. Collectively, our findings indicate that NEAT1 facilitates high glucose-induced damage in HK-2 cells by suppressing mitophagy via the miR-150-5p-DRP 1 axis, which sheds light on a novel mechanism of DN.

[Pharmacokinetics of mercury after oral administration of cinnabaris and Fufang Niuhuang Xiaoyan capsule in rats].

Fufang Niuhuang Xiaoyan capsule was a classical compound prescription with the efficacy of heat-clearing, detoxification, sedation and anti-inflammation, with cinnabaris as one of its active ingredients. The study focuses on the pharmacokinetics of mercury in rats after oral administration of cinnabaris and Fufang Niuhuang Xiaoyan capsule, in order to explore the effect of combined traditional Chinese medicines on mercury metabolism. In this study, the method of nitric-perchloric acid digestion system coupled with cold atomic-atomic fluorescence spectroscopy (CV-AFS) was adopted to accurately determine mercury in whole blood of rats. Fufang Niuhueng Xiaoyan capsule had three dose schemes of oral administration, namely equivalent clinical dose, 3 times of equivalent clinical dose and 10 times of equivalent clinical dose; And the doses of oral administration of cinnabaris was calculated according to that of Fufang Niuhuang Xiaoyan capsule. SPF grade healthy SD rats were fasted overnight before the oral administration with cinnabaris suspension (or Fufang Niuhuang Xiaoyan capsule suspension). After oral administration of different doses of cinnabaris, no obvious changes in tmax and MRT were observed, while Cmax/dose, AUC0-48 h/dose and AUC0-∞/dose decreased with the increase in dose, indicating that total mercury absorption in body was declining. As the dose increased, Ke, CL/F decreased, and t1/2 increased, indicating that the elimination slowed down, and mercury metabolism showed non-linear dynamic characteristics within a certain range of dose (22-220 mg•kg⁻¹). The total mercury metabolism in the whole blood of rats after oral administration with different doses of Fufang Niuhuang Xiaoyan capsule also showed non-linear dynamic characteristics. The results were correlated with the low solubility of cinnabaris in the body. Compared with cinnabaris, Fufang Niuhuang Xiaoyan capsule showed no obvious changes in V/F and MRT, while Ke, CL/F, tmax decreased, and t1/2, Cmax/dose, AUC0-48 h/dose, AUC0-∞/dose increased significantly. The results showed that Fufang Niuhuang Xiaoyan capsule accelerated absorption, slowed down elimination and improved the total absorption of mercury in the whole blood, indicating that Fufang Niuhuang Xiaoyan capsule may contain components for promoting absorption and alleviating elimination of mercury. Fufang Niuhuang Xiaoyan capsule had an impact on the pharmacokinetics of cinnabaris, and long-term administration of cinnabaris (Fufang Niuhuang Xiaoyan capsule) was possible to cause accumulation of mercury in the body. This study could explain changes in efficacy of Fufang Niuhuang Xiaoyan capsule, evaluate the rationality of compound medicines containing toxic elements and provide scientific basis for the rational and safe use of Fufang Niuhuang Xiaoyan capsule.

Toxicity and risk assessment of mercury exposures from cinnabar and Baizi Yangxin Pills based on pharmacokinetic and tissue distribution studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Baizi Yangxin Pills (BZYXP), a popular cinnabar (α-HgS) contained Traditional Chinese Medicines (TCMs) is widely used in clinical trials. However, mercury is one of the most toxic elements. The adverse effects of cinnabar-containing TCMs have been occasionally reported in recent years, leading to the growing concerns about their toxicity and safety. AIM OF THE STUDY: The health risks of BZYXP and cinnabar related to the mercury exposures were evaluated through blood pharmacokinetic and tissue distribution studies in rats. MATERIALS AND METHODS: The distribution of absorbed mercury in rats' blood and tissues were measured by the developed cold-vapor atomic fluorescence spectrometric method. And the tissue damages were determined through the histopathological examinations. For single dose study, the low and high oral doses were equivalent to 1 and 10-fold therapeutic dose, respectively. The multiple doses study was conducted at low and high dose levels every 12 h for 30 consecutive days. RESULTS: Significant differences of mercury blood pharmacokinetic and tissue distribution characteristics were observed between the corresponding BZYXP and cinnabar groups. The herbal ingredients in BZYXP promoted the absorption of bio-accessible mercury of cinnabar and prolonged the elimination process, posing potential health risks. Although mercury was found easily accumulated in kidney, liver and brain tissues, kidney and liver didn't show obvious damages even after 30 days consecutive administration of BZYXP or cinnabar at 10-fold clinically equivalent doses. But brain did show some histopathological changes, and autonomic activities of rats decreased, pointing the potential neurotoxicity. CONCLUSIONS: Mercury tend to be accumulated especially when over-dose or prolonged medication with cinnabar-containing TCMs are given. The mercury exposures even at therapeutic doses of BZYXP or cinnabar do pose health risks from the neurotoxicity point of view.

Evidences of non-reactive mercury-selenium compounds generated from cultures of Pseudomonas fluorescens.

This work was designed to determine chemically inert mercury-selenium (Hg-Se) compounds formed in a culture of Pseudomonas fluorescens exposed to Hg(2+) and Se(IV) (selenite). To isolate these compounds, different digestion methods were studied and sodium dodecyl sulfate (SDS) lysis was selected. The Hg(0) and non-reactive Hg were determined in two series of cultures containing 0.0-6.00 µg L(-1) Se(IV) (0.0-76.0 µmol L(-1)) in combination with low 5.00 µg L(-1) (0.025 µmol L(-1)) or high 100 µg L(-1) (0.500 µmol L(-1)) Hg(2+). It was found that Hg(0) formed in the culture decreased with the increase of initial Se(IV), while the non-reactive Hg increased with the Se(IV). In cultures with low initial [Hg(2+)], a median Se(IV) (2.0 µg L(-1) or 25.3 µmol L(-1)) resulted in about 70% of the added Hg(2+) sequestered as non-reactive Hg, and in culture with high initial Hg(2+), about 40% was sequestered. P. fluorescens was proved to be indispensible for the formation of the non-reactive Hg-Se compounds. The Hg:Se molar ratio in the non-reactive Hg-Se compounds was close to 1, suggesting the existence of mercuric selenide in cells. Mechanisms for the formation of the non-reactive Hg-Se compounds are proposed.

Inverse relationships between selenium and mercury in tissues of young walleye (Stizosedion vitreum) from Canadian boreal lakes.

The concentrations of total mercury (Hg), methylmercury (MeHg) and total selenium (Se) were determined in muscle, liver and brain tissues of young-of-the-year walleye (Stizosedion vitreum) specimens collected from 8 boreal lakes that are located within 107km around the Sudbury smelters in Ontario, Canada. Dry weight basis concentrations of Hg were highest in muscle and lowest in brain (p<0.05), those of MeHg were higher in muscle than in liver and brain but there was no significant difference between liver and brain (p<0.05). The highest Se concentrations were found in liver and the lowest in brain (p<0.05). Considering the biomass of the studied tissues, muscle was the part of the body where most of Hg, MeHg and Se were accumulated. In fish muscle, the percentage of MeHg over Hg was the highest and this percentage was the lowest in liver. The concentrations of Hg, MeHg and Se in the studied tissues were closely related to the concentrations of total dissolved Se in lake waters which vary with the distance of the lakes from the smelters. Thresholds of Se concentrations in tissues were revealed (6.2, 12.0 and 3.5mgkg(-1) dry wt., for muscle, liver and brain, respectively), above which a significant reduction of MeHg concentrations was observed in all studied tissues compared to lower Se levels in the same tissues. Based on the collected information and data analysis, possible mechanisms for the biological processes behind the observed inverse relationships between Se and Hg in fish tissues are discussed.

Improvements of reliability for methylmercury determination in environmental samples.

The determination of methylmercury (MeHg) in environmental samples by ethylation derivation-gas chromatography-atomic fluorescence spectrometry (ED-GC-AFS) is associated with an intimate problem of water moisture accumulation introduced in the ethylation step, which enters the detection system and cause a spectroscopic interference. With a simple modification on the GC-AFS system, this problem was eliminated and the analytical quality of the measurements was significantly improved. The presence of dissolved sulfide in samples can also cause serious chemical interference in the ethylation step resulting in lower or total loss of the MeHg signal. It was found that a masking system of CuSO(4)-Na(2)C(2)O(4) was able to eliminate this interference. With this system, the accurate determination of trace amount of MeHg in high dissolved sulfide containing samples was achieved. Satisfactory analytical results were obtained with the certified reference sediment IAEA405, sulfate reducing bacteria culture and sulfide containing water samples. The limit of detection and quantitation of this masking system is 0.01 and 0.04ngL(-1) respectively. Other factors affecting ethylation are also discussed.