Protective Effect of Liuwei Dihuangwan on Mitochondrial Damage in AD Model of Caenorhabditis Elegans / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of the extract of Liuwei Dihuangwan （LW） on mitochondrial damage in the Alzheimer's disease （AD） model of Caenorhabditis elegans （C. elegans）. MethodC. elegans transfected with human β-amyloid protein （Aβ） 1-42 gene was used as an AD model. The rats were divided into blank group， model group， metformin group （50 mmol·L-1）， and low， medium， and high dose （1.04， 2.08， 4.16 g·kg-1） LW groups. Behavioral methods were used to observe the sensitivity of 5-hydroxytryptamine （5-HT） in nematodes. Western blot was used to detect the expression of Aβ in nematodes. Total ATP content in nematodes was detected by the adenine nucleoside triphosphate （ATP） kit， and mitochondrial membrane potential was detected by the JC-1 method. In addition， the mRNA expression of Aβ expression gene （Amy-1）， superoxide dismutase-1 （SOD-1）， mitochondrial transcription factor A homologous gene-5 （HMG-5）， mitochondrial power-associated protein 1 （DRP1）， and mitochondrial mitoprotein 1 （FIS1） was detected by real-time fluorescence quantitative polymerase chain reaction （RT-PCR）. ResultThe extract of LW could reduce the hypersensitivity of the AD model of nematodes to exogenous 5-HT （P<0.05） and delay the AD-like pathological characteristics of hypersensitivity to exogenous 5-HT caused by toxicity from overexpression of Aβ in neurons of the AD model of nematodes. Compared with the blank group， in the model group， the mRNA expression of Aβ protein and Amy-1 increased （P<0.01）， and the mRNA expression of SOD-1 and HMG-5 decreased （P<0.01）. The mRNA expression of DRP1 and FIS1 increased （P<0.01）， and the level of mitochondrial membrane potential decreased （P<0.05）. The content of ATP decreased （P<0.01）. Compared with the model group， in the positive medicine group and medium and high dose LW groups， the mRNA expression of Aβ protein and Amy-1 decreased （P<0.05，P<0.01）， and the mRNA expression of SOD-1 and HMG-5 increased （P<0.01）. The mRNA expression of DRP1 decreased （P<0.05，P<0.01）， and that of FIS1 decreased （P<0.01）. The level of mitochondrial membrane potential increased （P<0.01）， and the content of ATP increased （P<0.05，P<0.01）. ConclusionThe extract of LW may enhance the antioxidant ability of mitochondria， protect mitochondrial DNA， reduce the fragmentation of mitochondrial division， repair the damaged mitochondria， adjust the mitochondrial membrane potential， restore the level of neuronal ATP， and reduce the neuronal damage caused by Aβ deposition.

Based on Oxidative Stress/NF-κB Exploration of Mechanism of Modified Zhenwutang in Delaying Progression of Chronic Renal Failure Complicated with Cardiac Disease by Inflammatory Factors / 中国实验方剂学杂志

ObjectiveBy observing the effect of modified Zhenwutang on the expression of superoxide dismutase 1（SOD1）， malondialdehyde（MDA）， advanced oxidation protein product（AOPP）， nuclear factor kappa-B（NF-κB） p65，p-p65，IL-1β， TNF-α in serum and renal tissue of adenine-induced chronic renal failure rats and the pathology of heart and kidney tissue， the possible mechanism of modified Zhenwutang delaying the progression of chronic renal failure complicated with heart disease was discussed. MethodFifty SPF male SD rats were divided into normal group 10 and model group 40 according to the random number table method. After one week of adaptive feeding， the experimental chronic renal failure complicated with cardiovascular disease rat model was established by intragastric administration of adenine 150 mg·kg-1·d-1. After the model was completed， 3 rats in the normal group and the model group were randomly selected to detect whether the model was successful. After successful modeling， the rats in the model group were divided into model group ， modified Zhenwutang low-dose group ， modified Zhenwutang medium-dose group， modified Zhenwutang high-dose group and Benazepril hydrochloride group according to the random number table method， with 6 rats in each group. Drugs were administered once a day for 4 weeks. At the end of the 17th week of the experiment， 24-hour urinary total protein（24 h-UTP） and urine creatinine（UCr）were detected. At the end of the 17th week， the rats in each group were anesthetized and the abdominal aorta was taken. After centrifugation， the supernatant was taken to detect triglyceride（TG）， total cholesterol（TC）， serum calcium（Ca）， serum potassium， serum phosphate， serum creatinine（Scr）， blood urea nitrogen（BUN）； the expression levels of serum AOPP， IL-1β and TNF-α were detected by enzyme linked immunosorbent assay（ELISA）. The pathological changes of heart and kidney tissues were observed by hematoxylin-eosin（HE）/Masson method. The ultrastructural changes of proximal renal tubules were observed by transmission electron microscopy . The kidney tissue expressions of SOD1， MDA， AOPP， NF-κB p65，p-p65，IL-1β and TNF-α were observed by immunohistochemistry. The kidney tissue expression levels of SOD1， NF-κB p65， IL-1β and TNF-α mRNA were observed by real-time polymerase chain reaction（Real-time PCR）. The kidney tissue expression levels of SOD1， MDA， NF-κB p65 and p-p65 were detected by Western blot. Result①Compared with the normal group， the experimental rats in the model group showed an increase in 24-hour UTP （P<0.01）and a decrease in UCr（P<0.01）. The experimental rats in the model group showed an increase in Cr， BUN， TG， TC， serum phosphate， and serum potassium（P<0.01）.The levels of AOPP， IL-1β and TNF-α in serum of rats in the model group were significantly increased（P<0.01）. In the model group， the glomerular balloon space was significantly widened， the renal interstitium was significantly widened with a large number of inflammatory cell infiltration， a large number of renal tubular lumens were blocked by brown deposits， and there were a large number of collagen fiber deposition in the renal interstitium. The collagen fibers around the renal vessels， outside the capsule wall of the renal capsule wall， glomerular basement membrane and renal tubular basement membrane were significantly increased， and the cardiac muscle fibers were significantly thickened. There was a small amount of inflammatory cell infiltration around the blood vessels， and a large number of collagen fibers around the cardiac vessels and between the myocardial cells. In the model group， high-density diamond-shaped needle-like crystals were observed in the proximal renal tubular epithelial cells of rats， with increased lysosomes， mitochondrial proliferation， mitochondrial cristae and dense mitochondrial outer membrane. The left ventricular diastolic wall thickness and systolic wall thickness of the experimental rats in the model group was increased in proximal renal tubular epithelial cells and their nuclei.In the model group， the expression of MDA， AOPP， NF-κB p65，p-p65 IL-1β and TNF-α in proximal renal tubular epithelial cells was significantly increased（P<0.01）， the expression of p-p65 in the nucleus of proximal renal tubular epithelial cells was significantly increased（P<0.01）， and the expression of SOD1 in proximal renal tubular epithelial cells was significantly decreased（P<0.01）. The kidney tissue expression of NF-κB p65， IL-1β and TNF-α mRNA in the model group was increased（P<0.01）， and the expression of SOD1 mRNA was decreased（P<0.01）. The kidney tissue expression of SOD1 protein in the model group was significantly decreased（P<0.01）. The kidney tissue expression of MDA， NF-κB p65 and p-p65 protein was increased （P<0.01）. ② Compared with the model group， after the intervention of modified Zhenwutang， 24 h-UTP was decreased （P<0.01）and UCr was increased（P<0.01）. Cr， BUN， TG， TC， serum phosphate， serum potassium was decreased （P<0.01）. Serum AOPP， IL-1β and TNF-α levels were decreased（P<0.01）. Cardiac and Renal pathological damage was reduced； mitochondrial damage in proximal renal tubules was reduced； the expression of MDA， AOPP， NF-κB p65， IL-1β， TNF-α in proximal renal tubular epithelial cells was decreased （P<0.01）， the expression of p-p65 in the nucleus of proximal renal tubular epithelial cells was significantly decreased （P<0.01）， and the expression of SOD1 in proximal renal tubular epithelial cells was significantly increased （P<0.01）. The kidney tissue expression of NF-κB p65， IL-1β， TNF-α mRNA was decreased （P<0.01）， and the expression of SOD1 mRNA was increased（P<0.01）. The kidney tissue expression of SOD1 protein was significantly increased （P<0.01）， and the expression of MDA， NF-κB p65 and p-p65 protein was decreased （P<0.01）. The Chinese medicine group showed a significant dose-effect trend. ConclusionModified Zhenwutang may reduce the production of oxidative stress and mitochondrial damage in proximal renal tubular epithelial cells， thereby reducing oxidative stress products and inhibiting the release of inflammatory factors caused by the activation of NF-κB signaling pathway， reducing the damage to heart and kidney tissues and functions， and delaying the progression of chronic renal failure complicated with heart disease， and the traditional Chinese medicine group has a dose-effect trend.

Bloodletting Acupuncture at Jing-Well Points Alleviates Myocardial Injury in Acute Altitude Hypoxic Rats by Activating HIF-1α/BNIP3 Signaling-Mediated Mitochondrial Autophagy and Decreasing Oxidative Stress / 中国结合医学杂志

OBJECTIVE@#To explore the protective effect and possible mechanisms of bloodletting acupuncture at Jing-well points (BAJP) pre-treatment on acute hypobaric hypoxia (AHH)-induced myocardium injury rat.@*METHODS@#Seventy-five rats were randomly divided into 5 groups by a random number table: a control group (n=15), a model group (n=15), a BAJP group (n=15), a BAJP+3-methyladenine (3-MA) group (n=15), and a BANA (bloodletting at nonacupoint; tail bleeding, n=15) group. Except for the control group, the AHH rat model was established in the other groups, and the corresponding treatment methods were adopted. Enzyme-linked immunosorbent assay (ELISA) was used to detect creatine kinase isoenzyme MB (CK-MB) and cardiac troponins I (CTnI) levels in serum and superoxide dismutase (SOD) and malondialdehyde (MDA) levels in myocardial tissue. Hematoxylin-eosin (HE) staining was used to observe myocardial injury, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was used to observe cell apoptosis. Transmission electron microscopy detection was used to observe mitochondrial damage and autophagosomes in the myocardium. The mitochondrial membrane potential of the myocardium was analyzed with the fluorescent dye JC-1. Mitochondrial respiratory chain complex (complex I, III, and IV) activities and ATPase in the myocardium were detected by mitochondrial respiratory chain complex assay kits. Western blot analysis was used to detect the autophagy index and hypoxia inducible factor-1α (HIF-1α)/Bcl-2 and adenovirus E1B 19k Da-interacting protein 3 (BNIP3) signaling.@*RESULTS@#BAJP reduced myocardial injury and inhibited myocardial cell apoptosis in AHH rats. BAJP pretreatment decreased MDA levels and increased SOD levels in AHH rats (all P<0.01). Moreover, BAJP pretreatment increased the mitochondrial membrane potential (P<0.01), mitochondrial respiratory chain complex (complexes I, III, and IV) activities (P<0.01), and mitochondrial ATPase activity in AHH rats (P<0.05). The results from electron microscopy demonstrated that BAJP pretreatment improved mitochondrial swelling and increased the autophagosome number in the myocardium of AHH rats. In addition, BAJP pretreatment activated the HIF-1α/BNIP3 pathway and autophagy. Finally, the results of using 3-MA to inhibit autophagy in BAJP-treated AHH rats showed that suppression of autophagy attenuated the treatment effects of BAJP in AHH rats, further proving that autophagy constitutes a potential target for BAJP treatment of AHH.@*CONCLUSION@#BAJP is an effective treatment for AHH-induced myocardial injury, and the mechanism might involve increasing HIF-1α/BNIP3 signaling-mediated autophagy and decreasing oxidative stress.

The E3 ubiquitin ligase NEDD4-1 protects against acetaminophen-induced liver injury by targeting VDAC1 for degradation

Acetaminophen (APAP) overdose is a major cause of liver injury. Neural precursor cell expressed developmentally downregulated 4-1 (NEDD4-1) is an E3 ubiquitin ligase that has been implicated in the pathogenesis of numerous liver diseases; however, its role in APAP-induced liver injury (AILI) is unclear. Thus, this study aimed to investigate the role of NEDD4-1 in the pathogenesis of AILI. We found that NEDD4-1 was dramatically downregulated in response to APAP treatment in mouse livers and isolated mouse hepatocytes. Hepatocyte-specific NEDD4-1 knockout exacerbated APAP-induced mitochondrial damage and the resultant hepatocyte necrosis and liver injury, while hepatocyte-specific NEDD4-1 overexpression mitigated these pathological events both in vivo and in vitro. Additionally, hepatocyte NEDD4-1 deficiency led to marked accumulation of voltage-dependent anion channel 1 (VDAC1) and increased VDAC1 oligomerization. Furthermore, VDAC1 knockdown alleviated AILI and weakened the exacerbation of AILI caused by hepatocyte NEDD4-1 deficiency. Mechanistically, NEDD4-1 was found to interact with the PPTY motif of VDAC1 through its WW domain and regulate K48-linked ubiquitination and degradation of VDAC1. Our present study indicates that NEDD4-1 is a suppressor of AILI and functions by regulating the degradation of VDAC1.

Bisphenol A induces testicular oxidative stress in mice leading to ferroptosis / 亚洲男科学杂志(英文版)

Bisphenol A is a common environmental factor and endocrine disruptor that exerts a negative impact on male reproductive ability. By exploring bisphenol A-induced testicular cell death using the Institute of Cancer Research (ICR) mouse model, we found that a ferroptosis phenomenon may exist. Mice were divided into six groups and administered different doses of bisphenol A via intragastric gavage once daily for 45 consecutive days. Serum was then collected to determine the levels of superoxide dismutase and malondialdehyde. Epididymal sperm was also collected for semen analysis, and testicular tissue was collected for ferritin content determination, electron microscope observation of mitochondrial morphology, immunohistochemistry, real-time quantitative polymerase chain reaction, and western blot analysis. Exposure to bisphenol A was found to decrease sperm quality and cause oxidative damage, iron accumulation, and mitochondrial damage in the testes of mice. In addition, bisphenol A was confirmed to affect the expression of the ferroptosis-related genes, glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), cyclooxygenase 2 (COX2), and acyl-CoA synthetase 4 (ACSL4) in mouse testicular tissues. Accordingly, we speculate that bisphenol A induces oxidative stress, which leads to the ferroptosis of testicular cells. Overall, the inhibition of ferroptosis may be a potential strategy to reduce male reproductive toxicity caused by bisphenol A.

Mechanisms of Acupuncture in Improving Alzheimer's Disease Caused by Mitochondrial Damage / 中国结合医学杂志

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases among the elderly and it accounts for nearly 80% of all dementias. The pathogenesis of AD is complicated and enigmatic thus far. The mitochondrial cascade hypothesis assumes that mitochondrial damage may mediate, drive, or contribute to a variety of AD pathologies and may be the main factor in late-onset AD. Currently, there are no widely recognized drugs able to attenuate mitochondrial damage in AD. Notably, increasing evidence supports the efficacy of acupuncture for improving the mitochondrial structure and protecting mitochondrial functions in AD. This review reports the mechanisms by which acupuncture regulates mitochondrial dynamics, energy metabolism, calcium homeostasis and apoptosis. In conclusion, these findings suggest that AD mitochondrial dysfunction represents a reasonable therapeutic target and acupuncture could play a significant role in preventing and treating AD.

Mitochondrial dysfunction on Leishmania (Leishmania) amazonensis induced by ketoconazole: insights into drug mode of action

BACKGROUND Leishmania parasites cause leishmaniasis that range from self-limiting cutaneous lesions to more serious forms of the disease. The search for potential drug targets focusing on biochemical and metabolic pathways revealed the sterol biosynthesis inhibitors (SBIs) as a promising approach. In this class of inhibitors is found ketoconazole, a classical inhibitor of 14α-methysterol 14-demethylase. OBJECTIVE The present study aimed to better understand the biological response of Leishmania (Leishmania) amazonensis promastigotes at the cellular level after ketoconazole treatment. METHODS Herein, techniques, such as fluorimetry, flow cytometry, fluorescence microscopy, electron and scanning microscopy were used to investigate the cellular structures and to identify organelles affected by ketoconazole treatment. FINDINGS The study demonstrated, for the first time, the effect of ketoconazole on mitochondrion functioning and its probable relationship to cell cycle and death on L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8 strain). MAIN CONCLUSIONS Ketoconazole-induced mitochondrial damages led to hyperpolarisation of this single organelle and autophagic vacuoles formation, as a parasite survival strategy. These damages did not reflect directly on the parasite cell cycle, but drove the parasites to death, making them susceptible to ketoconazole treatment in in vitro models.

The protective effect and mechanism of N-acetylcysteine on acute kidney injury by up-regulating Sirtuin3 protein expression in septic mice / 中华急诊医学杂志

Objective:To explore the protective effect and mechanism of the antioxidant N-acetylcysteine (NAC) regulating silent information regulator 3 (Sirt3) on acute kidney injury (AKI) in septic mice.Methods:Male C57BL/6 mice were randomly ( random number) divided into the sham operation group (sham), cecal ligation and perforation group (CLP), CLP + NAC (50 mg/kg) and CLP + NAC (100 mg/kg) groups, with 10 mice in each group. The mice were sacrificed 24 h after CLP, and blood and kidney tissue samples were collected. HE staining was used to evaluate the pathological damage of the kidney tissue of mice in each group. ELISA was used to detect serum creatinine (Scr), urea nitrogen (BUN), kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated apolipoprotein (NGAL) levels. Immunohistochemistry was used to detect the expression of Sirt3 protein in kidney tissue. RT-qPCR was used to detect the level of Sirt3 mRNA. Mitochondrial damage of renal tubular epithelial cells was observed under transmission electron microscope, and the mitochondrial density was calculated. Meanwhile, the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and malondialdehyde (MDA) in the renal cortex were also detected. Results:Compared with the sham group, in the CLP group, the pathological damage of renal tissue was significantly aggravated ( P<0.001), and the levels of renal function indicators (Scr, BUN, KIM-1 and NGAL) were all increased significantly (all P<0.001). The protein and mRNA expression of Sirt3 were all significantly decreased (all P<0.001), the mitochondrial structure damage of renal tubular epithelial cells was increased, and the mitochondrial density was significantly decreased ( P<0.001). The levels of antioxidant enzymes (SOD, GSH-Px and CAT) in the renal cortex were all significantly decreased (all P<0.001), while the lipid peroxide MDA was significantly increased ( P<0.001). Compared with the CLP group, the renal injury score and renal function indexes (Scr, BUN, KIM-1 and NGAL levels) in the 50 mg/kg NAC pretreatment group were decreased, and the levels of SOD, GSH-Px and CAT in renal tissue were increased, but the differences were not significant. However, pretreatment with 100 mg/kg NAC significantly reduced the pathological damage of kidney tissue caused by CLP ( P<0.001), and significantly decreased the levels of Scr, BUN, KIM-1 and NGAL (all P<0.001). The expression of Sirt3 protein [(50.20±2.79) vs.(20.00±0.75), P<0.001] and mRNA [(0.57±0.07) vs. (0.41±0.07), P<0.001] were all significantly increased. The mitochondrial structure of renal tubular epithelial cells was more stable, and the mitochondrial density was significantly increased [(0.60±0.05) vs. (0.43±0.06), P<0.001]. The levels of SOD [(67.37±3.79) U/mg vs. (21.09±0.89) U/mg, P<0.001], GSH-Px [(265.61±9.61) U/mg vs. (180.00±3.31) U/mg, P<0.001] and CAT [(8.58±0.65) U/mg vs. (5.19±0.58) U/mg, P<0.001] were all significantly increased, while the expression level of MDA was significantly reduced [(40.36 ±1.79) vs. (83.81 ±1.70), P<0.001]. Conclusions:NAC can significantly reduce renal pathological damage, improve renal function, maintain mitochondrial structure stability and reduce oxidative stress levels in septic mice by up-regulating Sirt3 protein expression, and has a significant protective effect on CLP-induced AKI.

The protective effect of external diaphragm pacing on ventilator-induced diaphragm dysfunction of rabbits and its mechanism / 中华急诊医学杂志

Objective:To investigate the protective effect of external diaphragm pacing on the prevention of ventilator-induced diaphragm dysfunction (VIDD) in rabbits and its mechanism.Methods:Eighty-five New Zealand white rabbits were randomly (random number) divided into the blank control group (BC, n=5), spontaneous breathing group (SB, n=20), volume control ventilation group (VC, n=20), external diaphragm pacing group (EDP, n=20), external diaphragm pacing and volume control ventilation group (EDP+ VC, n=20). After successful modeling, the rabbits in each group were treated accordingly except for the BC group. Rabbitss in the BC group were not mechanically ventilated, and the diaphragm was removed immediately after anesthetizing. Whole diaphragms of 5 rabbits per time point per other group were also collected after anesthesia at post treatment hour (PTH) 6 and on post treatment day (PTD) 1, 3, and 7. Diaphragm weight/body weight and diaphragm isometric contractile force of each group were measured. The pathological changes of diaphragmatic tissues were observed by HE staining. The protein expressions of Cyt c, RyR1, caspase-3, and p-mTORC1 were measured by Western blot. Repeated measures analysis of variance was used for the comparison between multiple groups of variables at different time points, and LSD- t test was used for the further comparison between two groups at the same time point, a P<0.05 was considered statistically significant. Results:Compared with the BC group, the VC group showed diaphragmatic pathological changes conformed to VIDD: DW/BW was decreased obviously; HE staining revealed obvious changes in diaphragmatic tissue; Diaphragmatic contractility was also significantly decreased; The expression of Cyt c and caspase-3 were increased while the expression of RyR1 and p-mTORC1 were decreased gradually with the extension of treatment time ( P<0.05). Compared the EDP+VC group with the VC group, with the extension of treatment time, DW, DW/BW, pathological damages and diaphragmatic contractility were improved [PTD 1: (0.80±0.05)kg vs (0.56±0.04) kg, PTD 3: (1.06±0.05) kg vs (0.47±0.03) kg, PTD 7: (1.24±0.10) kg vs (0.39±0.07) kg, all P<0.05; PTD 1: (2.05±0.54) vs (1.86±0.72), PTD 3: (2.19±0.61) vs (1.74±0.40), PTD 7: (2.46±0.62) vs (1.53±0.85), all P<0.05; PTD 1: (2.39±0.42) N/cm 2vs (1.91±0.25) N/cm 2, PTD 3: (2.57±0.62) N/cm 2vs (1.72±0.50) N/cm 2, PTD 7: (2.77±0.55) N/cm 2vs (1.54±0.33) N/cm 2, all P<0.05]. The expression of Cyt c and caspase-3 were decreased while the expression of RyR1 and p-mTORC1 were increased gradually in the EDP+VC group ( P<0.05). Conclusions:External diaphragm pacer plays a protective role in ventilator-induced diaphragm dysfunction, which can inhibit mitochondrial damage, reduce oxidative damage, and mitigate diaphragmatic atrophy and injury.

Preclinical models of idiosyncratic drug-induced liver injury (iDILI): Moving towards prediction

Idiosyncratic drug-induced liver injury (iDILI) encompasses the unexpected harms that prescription and non-prescription drugs, herbal and dietary supplements can cause to the liver. iDILI remains a major public health problem and a major cause of drug attrition. Given the lack of biomarkers for iDILI prediction, diagnosis and prognosis, searching new models to predict and study mechanisms of iDILI is necessary. One of the major limitations of iDILI preclinical assessment has been the lack of correlation between the markers of hepatotoxicity in animal toxicological studies and clinically significant iDILI. Thus, major advances in the understanding of iDILI susceptibility and pathogenesis have come from the study of well-phenotyped iDILI patients. However, there are many gaps for explaining all the complexity of iDILI susceptibility and mechanisms. Therefore, there is a need to optimize preclinical human

Overexpression of transcription factor EB regulates mitochondrial autophagy to protect lipopolysaccharide-induced acute lung injury / 中华医学杂志(英文版)

Background@#Acute lung injury (ALI) is characterized by an acute inflammatory process, and oxidative stress in the lung tissue leads to a lack of effective therapeutics. This study aimed to identify whether the overexpression of transcription factor EB (TFEB) regulates mitophagy to protect against lipopolysaccharide (LPS)-induced ALI.@*Methods@#We detected the expression of inflammatory factors, cytochrome c (Cyt.c) and nicotinamide adenine dinucleotide phosphate (NADPH), and autophagy-related proteins and observed the changes in lung histopathology induced by ALI in rats and the changes in the cell ultrastructure of primary alveolar type II epithelial cells induced by changing the expression of TFEB in the context of ALI.@*Results@#The overexpression of TFEB could reduce the expression of proinflammatory factors, such as IL-1 and IL-6, and increase the expression of anti-inflammatory factors, such as IL-10, both in vitro and in vivo. In addition, the overexpression of TFEB could reduce the Cyt.c and NADPH levels both in vivo and in vitro. The overexpression of TFEB could upregulate the expression of autophagy-related proteins, such as lysosomal-associated membrane protein 1 (LAMP1), microtubule-associated protein light chain 3B (LC3B), and Beclin both in vivo and in vitro, and promote mitochondrial autophagy. The overexpression of TFEB significantly improved the histopathologic changes induced by LPS-induced ALI in rats. However, low TFEB expression produced the opposite results.@*Conclusion@#TFEB overexpression can decrease inflammation and mitochondrial damage in the lung tissue and alveolar epithelial cells through regulating mitochondrial autophagy to protect against LPS-induced ALI. Therefore, TFEB is likely a potential therapeutic target in LPS-induced ALI.

Inhibiting miR-186 expression alleviates mitochondrial damage in hypoxic human umbilical vein endothelial cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of miR-186 inhibition on the expression of hypoxia-inducible factor-1α (HIF-α) and mitochondrial function in hypoxic vascular endothelial cells.@*METHODS@#Human umbilical vein endothelial cells (HUVECs) cultured in routine or hypoxic conditions for 6 h were examined for the expression of miR-186. A miR-186 inhibitor was transfected in the HUVECs, and the cells were subsequently cultured in hypoxic condition for 6 h to observe the changes in the mitochondrial structure under an electron microscope. The changes in the mRNA and protein expressions of HIF-1α in response to miR-186 interference were tested using real-time fluorescent quantitative PCR and Western blotting.@*RESULTS@#The expression of miR-18 was mildly increased in HUVECs after hypoxic exposure for 6 h (=0.0188). Interference of miR-186 expression obviously promoted the mRNA and protein expressions of HIF-1α in HUVECs. In hypoxic conditions, miR-186 interference significantly reduced mitochondrial damage in HUVECs as observed under electron microscope (=0.0297).@*CONCLUSIONS@#Inhibition of miR-186 protects vascular endothelial cells against hypoxic injuries by promoting HIF-α expression to lessen mitochondrial damage, suggesting the possibility of targeted miR-186 interference for treatment of hemorrhagic shock.

Mitochondrial damage induced by HTLV-1 infection in host cells / 中华微生物学和免疫学杂志

Objective@#To investigate the effects of human adult T lymphoblastic leukemia virus typeⅠ (HTLV-1) infection on the production of reactive oxygen species (ROS) and mitochondrial damage in host cells.@*Methods@#A cell model of HTLV-1 infection was established by co-culturing HTLV-1-positive cell line MT2 with HeLa cells. ROS, mitochondrial membrane potential (MMP) and total mitochondria were detected using specific fluorescence probe labeling method. Cell apoptosis was detected by Annexin V-FITC/PI method. Western blot was performed to detect viral proteins Tax and p19, as well as mitochondrial proteins TIM23 and TOM20. After the treatment of MT2 cells with different concentrations of reverse transcription inhibitors (ZDV), relative viral loads were detected by quantitative real-time PCR and Western blot, and the mass of mitochondria was analyzed by flow cytometry.@*Results@#After co-culturing HeLa cells with MT2 cells for 24 h, the ROS level in host cells increased without obvious cell apoptosis, while the mitochondrial membrane potential, mitochondrial protein expression and total mitochondria decreased significantly. When the replication of HTLV-1 in MT2 cells was inhibited by ZDV, the ROS level and total mitochondria increased.@*Conclusions@#HTLV-1 infection can cause oxidative stress in host cells, resulting in mitochondrial damage. Autophagy might be activated to degrade mitochondrial damage and maintain cell homeostasis during the infection.

Mitochondrial damage induced by HTLV-1 infection in host cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of human adult T lymphoblastic leukemia virus typeⅠ (HTLV-1) infection on the production of reactive oxygen species (ROS) and mitochondrial damage in host cells. Methods A cell model of HTLV-1 infection was established by co-culturing HTLV-1-positive cell line MT2 with HeLa cells. ROS, mitochondrial membrane potential ( MMP) and total mitochondria were detected using specific fluorescence probe labeling method. Cell apoptosis was detected by Annexin V-FITC/PI method. Western blot was performed to detect viral proteins Tax and p19, as well as mitochondrial pro-teins TIM23 and TOM20. After the treatment of MT2 cells with different concentrations of reverse transcrip-tion inhibitors ( ZDV) , relative viral loads were detected by quantitative real-time PCR and Western blot, and the mass of mitochondria was analyzed by flow cytometry. Results After co-culturing HeLa cells with MT2 cells for 24 h, the ROS level in host cells increased without obvious cell apoptosis, while the mitochon-drial membrane potential, mitochondrial protein expression and total mitochondria decreased significantly. When the replication of HTLV-1 in MT2 cells was inhibited by ZDV, the ROS level and total mitochondria increased. Conclusions HTLV-1 infection can cause oxidative stress in host cells, resulting in mitochon-drial damage. Autophagy might be activated to degrade mitochondrial damage and maintain cell homeostasis during the infection.

Effect of high tidal volume mechanical ventilation on pulmonary autophagy and mitochondrial damage in rats / 中华危重病急救医学

Objective To investigate the relationship between different tidal volume (VT) mechanical ventilation (MV) and autophagy and mitochondrial damage in rats. Methods A total of 120 clean-grade male Sprague-Dawley (SD) rats were divided into five groups (n = 24) by random number table method, and then given 0 (spontaneous breathing), 10, 20, 30, 40 mL/kg VT for MV. The rats in each group were subdivided into four subgroups of 1, 2, 3, and 4 hours according to ventilation time, with 6 rats in each subgroup. The lung tissue and bronchoalveolar lavage fluid (BALF) were harvested, and alveolar macrophages (AMs) and type Ⅱ alveolar epithelial cells (AECⅡ) were cultured in vitro. The mRNA and protein expressions of autophagy-associated protein microtubule-associated protein 1 light chain 3B -Ⅱ (LC3B -Ⅱ) and autophagy-related genes Beclin1 and p62 were determined by reverse transcription-polymerase chain reaction (RT-PCR) or Western Blot. Lung autophagosome formation was observed under transmission electron microscope. The levels of adenosine triphosphate (ATP), reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in lung tissue were determined for assessing mitochondrial damage. Results There were no significant differences in the mRNA and protein expressions of LC3B -Ⅱ, p62 and Beclin1 at 1 hour after ventilation among the groups. With the prolonged ventilation time, the mRNA and protein expressions of LC3B -Ⅱ, p62 and Beclin1 in MV groups were increased gradually, peaked at 2-3 hours, and they were increased significantly in 30 mL/kg VT group as compared with those in spontaneous respiration group with statistical significances [ventilation for 2 hours: LC3B -Ⅱ mRNA (2-ΔΔCt) was 2.44±0.24 vs. 1.12±0.04, LC3B -Ⅱ/LC3B -Ⅰ was 1.42±0.16 vs. 0.57±0.03, p62 mRNA (2-ΔΔCt) was 2.96±0.14 vs. 1.14±0.02, Beclin1 mRNA (2-ΔΔCt) was 2.80±0.13 vs. 1.14±0.02; ventilation for 3 hours: p62/β-actin was 1.14±0.15 vs. 0.55±0.04, Beclin1/β-actin was 1.27±0.06 vs. 0.87±0.04, all P < 0.05]. Autophagosomes and autolysosomes were found in AECⅡ after ventilation for 2 hours at 30 mL/kg VT by transmission electron microscopy, but not in AECⅠ. Compared with spontaneous breathing group, ATP synthesis in AMs was significantly decreased at 2 hours of ventilation in 30 mL/kg VT group (A value: 0.82±0.05 vs. 1.00±0.00, P < 0.05), ROS accumulate in AMs and AECⅡ were significantly increased [ROS in AMs: (33.83±4.00)% vs. (6.90±0.62)%, ROS in AECⅡ: (80.68±0.90)% vs. (2.16±0.19)%, both P < 0.05]. With the increase in VT and the prolongation of ventilation time, ATP and ROS levels in AMs and AECⅡ were gradually decreased, the ATP (A value) in AMs at 4 hours of ventilation in 40 mL/kg VT group was 0.41±0.05, the ROS in AMs was (12.95±0.88)%, and the ROS in AECⅡ was (40.43±2.29)%. With the increase in VT and the prolongation of ventilation time, MMP levels were gradually increased, the MMP (green/red fluorescence intensity ratio) in AMs at 2 hours of ventilation in 30 mL/kg VT group was 1.11±0.17, the MMP in AECⅡwas 0.96±0.04, and the MMP (green/red fluorescence intensity ratio) at 4 hours of ventilation in 40 mL/kg VT group was 0.51±0.07 and 0.49±0.06, respectively. Conclusion The MV with high VT could induce autophagy activation and mitochondrial damage in lung tissue of rats, and the longer the ventilation time, the more obvious autophagy in the lung.

Resveratrol alleviates cerebral ischemia/reperfusion injury in mice by promoting mitophagy / 中国药理学通报

Aim: To investigate the effects of resveratrol (Res) pretreatment on focal cerebral ischemia/reperfusion (I/R) injury and the possible mechanism. Methods: Transient focal cerebral ischemia was introduced into mice by right middle cerebral artery occlusion (MCAO) technique. For Res treatment, C57BL/6J mice were given an intraperitoneal injection with ' Res per day for 5 days. After 24 h of reperfusion, infarct volume, neurological function were assessed. Western blot was used to analyse the expression of silent information regulator 1(SIRT1), mitophagy-relat-ed proteins, and the distribution of cytochrome C (CytC). Flow cytometry assay was introduced to test mitochondrial membrane potential (MMP). A micro-plate reader was used to detect the cellular adenosine triphosphate (ATP) levels. Transmission electron microscopy (TEM) was conducted to observe mitochondrial morphology. Results: Res pretreatment reduced infarct volume and neurological deficit, improved the expression of SIRT1 and mitophagy. Meanwhile, Res attenuated CytC release, recovered MMP, and improved ATP levels. TEM results showed Res could protect the mitochondria from I/R injury-induced destruction. However, as 3-MA was used to inhibit the activation of mitophagy, the protective effects of Res on neurological and mitochondrial function were reversed. Conclusions: Res pretreatment could relieve cerebral I/R injury. The mechanism might be associated with the regulation of mitophagy to maintain mitochondrial structural and functional integrity.

Involvement of FKBP51 in mitochondrial damage induced by high fat diet feeding / 中国比较医学杂志

Objective To investigate the effect of high fat diet feeding on mitochondrial structure and function. Methods Male C57BL/6J mice at the age of 4 weeks were used in this study. After 6 weeks of regular diet(RD)or high-fat diet(HF)feeding, the high fat-induced obesity phenotype was confirmed by body weight measurement and liver histopathology. RNA was isolated from the liver tissue of RD and HF mice and the expression profiles were detected using RNA-seq. Differentially expressed genes between RD and HF mice were analyzed using BRB-ArrayTools. DAVID online tools were applied to analyze the GO and KEGG pathways. Transmission electron microscopy and western blotting were performed to observe the mitochondrial ultrastructure and quantified the expression of function-related proteins. Results Compared with the RD mice,the body weight gain was faster in the HF mice. The size of the lipid droplets was bigger in the HF-fed mouse liver tissue. Multiple pathway analysis all identified that these major gene changes were related to mitochondria. The mitochondrial deformation,enlarged or even destruction was observed in the high fat diet group observed by transmission electron microscopy. This observation was further confirmed by detecting of the expression of genes in the HF liver mitochondria. The levels of MFN1 and PHB1 were significantly increased, while the level of FKBP51 was significantly decreased. Conclusions FKBP51 is involved in the high-fat-induced mitochondrial damage via morphological and structural damages of mitochondria.

Protective effect of MCC950 against lung injury induced by mitochondrial damage-associated molecular patterns / 医学研究生学报

Objective There is still no specific immuno-therapy to acute respiratory distress syndrome induced by severe trau-ma.The article aimed to investigate the effect of MCC 950 on lung in-jury induced by mitochondrial damage-associated molecular patterns ( MTDs) and preliminarily evaluate its molecular mechanism . Methods 40 SD rats were randomly devided into control group , MTDs group, MCC950 group, MTDs+MCC950 group.The rats were were taken MCC950 (20mg/kg) by peritoneal injection pretreatment for 1 hour, followed by tail vein injection of MTDs (5%liver vol-ume) and were killed 12 hours later.ELISA were applied to detect tumor necrosis factor (TNF-α), interleukin-1β( IL-1β) and IL-18 in broncho-alveolar lavage fluid ( BALF) , and BCA method to assess the content of total protein .Lung tissues were weighed to calculate lung wet weight/body weight( LWW/BW) ratio, and stained by HE staining to observe the pathological changes through light micro -scope.Smith lung injury score was used to assess histological lung injury .Western blot was employed to evaluate the protein expression of Pro-Caspase-1 and Caspase-1. Results ①Compared with control group , TNF-α, IL-1βand IL-18 in BALF of MTDs group were significantly increased( all P<0.05), but not in MCC950 group(P>0.05), TNF-αin BALF of MTDs +MCC950 group were signifi-cantly increased( all P<0.05), IL-1βand IL-18 were not(all P>0.05).Compared with MTDs group, IL-1βand IL-18 in BALF of MTDs +MCC950 group were in serious decline (all P<0.05).Compared with control group, the LWW/BW ratio [(4.19±0.36)mg/g vs (6.32±0.54)mg/g, P<0.05] and the content of total protein [(0.12±0.03)g/L vs (0.79±0.07)g/L, P<0.05] were dramatically increased.Compared with MTDs group, the LWW/BW ratio [(4.35±0.29)mg/g, (4.47±0.0.46)mg/g, P<0.05] and the content of total protein [(0.12±0.06)g/L, (0.15±0.06)g/L, P<0.05] were in serious decline.Smith lung injury score revealed that compared with control group the score of MTDs group was elevated (1.00±0.00 vs 8.33±0.58, P<0.05), and the score of MTDs+MCC950 group was significantly decreased than MTDs group ( 8.33±0.58 vs 3.67±0.58, P<0.05) .Compared with control group , the protein expres-sion of Pro-caspase-1 and caspase-1 were markedly improved (all P<0.05).However, the expression of caspase-1 was significantly milder than that in MTDs group ( P<0.05), the protein expression of Pro-caspase-1 was comparable ( P>0.05). Conclusion MCC950 exerts protective effect against lung injury induced by MTDs probably via the inhibition of NLRP 3 inflammasome activation .

Cytotoxicity and mitochondrial injury mechanism of matrine on PC12 cells / 中国药理学与毒理学杂志

OBJECTIVE To study the toxicological effect of matrine (MT) on PC12 cells and the mechanism of mitochondrial damage. METHODS After treatment with MT 2, 4 and 8 mmol·L-1for 8, 16,24 and 48 h,respectively,the viability of PC12 cells was measured with methylthiazolyltetrazolium assay.PC12 cells were treated with MT 2,4 and 8 mmol·L-1for 24 h,before the morphological changes were observed by Hoechst33342 staining. The superoxide dismutase (SOD) activity and malondialde-hyde (MDA) content in cells were detected using hydroxylamine method and thiobarbituric acid method, apoptosis rate and reactive oxygen species (ROS) of PC12 cells were detected with flow cytometry, mitochondrial membrane potential (MMP) was detected with JC-1 staining, and the expressions of procaspase 3, procaspase 9, cleaved-caspase 3, Bax and Bcl-2 were detected with Western blotting. RESULTS MT inhibited the growth of PC12 cells in a time-and concentration-dependent manner.After being treated with MT for 24 h,the nuclei of PC12 cells in MT groups showed chromatin agglutination and partial rupture to different degrees,and compared with normal control group,the apoptosis rates of MT 2,4 and 8 mmol·L-1groups were significantly increased(P<0.01).Intracellular ROS and MDA levels increased (P<0.05, P<0.01), SOD activity decreased (P<0.01), and MMP decreased. The expressions of procaspase 9, procaspase 3 and Bcl-2 were significantly decreased (P<0.01, P<0.05), the expres-sions of cleaved-caspase 3 and Bax were significantly increased(P<0.05,P<0.01),and the ratio of Bcl-2/Bax was significantly decreased (P<0.01). CONCLUSION MT has toxic effect on PC12 cells and induces apoptosis through ROS mediated mitochondrial pathway.

Effects of sub-chronic aluminum-maltolate complex exposure on mitochondrial fission and fusion of hippocampus in SD rats / 中国职业医学

OBJECTIVE: To study effects and mechanism of sub-chronic aluminum-maltolate complex [Al( mal)3]exposure on mitochondrial damage of hippocampus in rats. METHODS: Sixty specific pathogen free healthy adult male SD rats were randomly divided into 5 groups: a blank group,a control group,a low-,a medium- and a high-dose group,with 12 rats in each group. Blank group was not treated. Low-,medium- and high-dose groups were treated with 0. 41,0. 81,1. 62 mg / kg body weigh of Al( mal)3solution respectively. The control group was treated with an equal volume of saline. Al( mal)3exposure was conducted by intraperitoneal injection every other day for 90 days. The activities of Na+-K+-ATPase and Ca2 +-Mg2 +-ATPase in hippocampus were tested by chemical colorimetric technique. Western blot analysis was used to detect the relative expression of cytochrome oxidase Ⅳ( Cox Ⅳ),dynamin-related protein 1( Drp1),optic Atrophy 1( Opa1),mitofusin( Mfn) 1,and Mfn2 in hippocampus. RESULTS: The activity of Na+-k+-ATPase in high-dose group was significantly lower than those in blank-,control-,and low-dose groups( P < 0. 05). The activity of Ca2 +-Mg2 +-ATPase in medium-dose group was significantly lower than those in blank group( P < 0. 05),and that in high-dose group was significantly lower than those in the other four groups( P < 0. 05). The relative expression levels of CoxⅣ in mediumand high-dose groups were lower than those of the other three groups( P < 0. 05). The relative expression levels of Drp1 and Mfn2 in medium-and high-dose groups were significantly higher than those in blank-,control and low dose group( P <0. 05). The relative expression levels of Opa1 in medium- and high-dose groups were significantly higher than those in blank-,control- and low-dose groups( P < 0. 05). The relative expression levels of Mfn1 in medium- and high-groups were significantly higher than that in blank group( P < 0. 05). CONCLUSION: The mitochondria of rat hippocampus was damaged by the sub-chronic aluminum-maltolate exposure. The damage may be related to the change of mitochondrial dynamics.