Berberine ameliorates lipid accumulation of mouse primary hepatocytes by decreasing fatty acids uptake / 中国药理学通报

Aim To investigate the role of berberine in mouse primary hepatocytes steatosis and whether aden-osine monophosphate-activated protein kinase(AMPK) is essential in this process in order to explore the mech-anism of non-alcoholic fatty liver disease treatment. Methods Different concentrations of oleic acid(OA) were used in mouse primary hepatocytes to determine the appropriate dose inducing steatosis. Subsequently, hepatocytes were treated with berberine and OA at the same time for 24 h serving metformin as positive con-trol. Lactic dehydrogenase (LDH) release test was performed to investigate cell viability. Lipid level was determined by oil red staining and triglyceride assay. Western blot measured the phosphorylation level of AMPK and Acetyl CoA carboxylase. An AMPK inhibi-tor compound C(CC) pre-treated hepatocytes for 1 h followed by berberine 24 h-treatment. The relationship between free fatty acid(FFA) uptake and mitochondri-al inhibition was evaluated by measuring FFA in the supernatant of OA,berberine and rotenone (mitochon-drial complex I inhibitor) group. Results Berberine could significantly reduce primary hepatocytes steatosis induced by oleic acid and stimulate AMPK and ACC phosphorylation at a non-toxic dose. In addition, CC obviously inhibited AMPK activity,but failed to dimin-ish the lipid dysregulation improvement of berberine. Berberine and rotenone intervention reduced OA up-take by 31.2% and 23.6%,respectively. Conclusion Berberine ameliorates hepatocytes lipid accumulation by suppressing fatty acid uptake,which is probably re-sulted from inhibition of mitochondrial respiratory chain complex I,independently of AMPK activation.

Effects of High Intensity Interval Training on the Activity of the Myocardial Mitochondrial Respiratory Chain Complex of Rats / 中国运动医学杂志

Objective To explore the heart function and activity of the mitochondrial respiratory chain complex of rats with myocardial hypertrophy induced by the long-term high-intensity interval training(HIIT).Methods Ninety Wistar rats were randomly divided into an HIIT group,a moderate intensity continuous training(MICT) group and a rest control(RC) group,with each group allocated three subgroups according to the observation time(2,6 and 10 weeks),9 groups altogether(n=10 in each group).Each group was given intervention as their names implied.Then,the heart function was measured using the ultrasoundcardiogram,and the body weight as well as the weight of the heart was weighted.The myocardium mitochondria were extracted using the differential centrifugation after homoge nation to detect the activity of the myocardial and mitochondrial citrate synthase (CS),the activities of the respiratory chain complex C Ⅰ ～CⅣ as well as myocardial protein expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1 α),α-myosin heavy chain (α-MHC),β-MHC,atrial natriuretic factor(ANF) and brain natriuretic peptide(BNP).Results The myocardial hypertrophy was found in HIIT and MICT groups after 1-week and 9-week intervention respectively.At the 2nd and 10th week,no significant differences were found in the heart function,respiratory chain complex activity and protein expression of all three groups(P＞0.05).At the 6th week,the left ventricular ejection fraction,left ventricular fractional shortening,myocardial α-MHC protein expression,and the activities of respiratory chain complex C Ⅰ,C Ⅲ and C Ⅳ of HIIT group were significantly lower while the myocardial β-MHC and BNP protein expression were significantly higher than those of RC and MICT groups(P＜0.05).Conclusion Long-term HIIT but not MICT can induce temporarily pathological myocardial hypertrophy and reduced heart function in Wistar rats,and the mechanism might be related to the downregulation of the myocardial mitochondrial respiratory chain complex activity.

The mitochondrial toxicity of bentysrepinine on HepG2 cells / 中国药理学通报

Aim To provide references for clinical trials dose and rational drug use by evaluating mitochondrial toxicity of bentysrepinine on HepG2 cells.Methods Mitochondrial toxicity of bentysrepinine on HepG2 cells was cmomprehensively evaluated by measuring proliferation inhibition rate, lactic acid content in culture supernatant, reactive oxygen species(ROS) content, mitochondrial membrane potential (MMP) variation and the activity of mitochondrial respiratory chain complex enzymes Ⅰ to Ⅳ.Results The half inhibitory concentration of bentysrepinine of HepG2 cells was 359 μmol·L-1.Compared with the control group, bentysrepinine could reduce the MMP, raise the level of lactic acid, increase the content of ROS and lower the activity of mitochondrial respiratory chain complex enzymes Ⅰ to Ⅲ with the concentration of 400 μmol·L-1(196 mg·L-1), showing an obvious mitochondrial toxicity.Compared with lamivudine and adefovir dipivoxil, bentysrepinine exerted no influence on indexes above with the same concentration 100 μmol·L-1.Conclusions Bentysrepinine shows an obvious mitochondrial toxicity on HepG2 cells with the concentration of 400 μmol·L-1.This mitochondrial toxicity is not presented with the concentration of 200 μmol·L-1.It shows that the safety range of bentysrepinine about mitochondrial toxicity is relatively wide.The test plays a guiding role in clinical trial dose design as well as clinical treatment.

TNF-α induced RIP1-dependent apoptosis in L929-A cells by interrupting mitochondrial respiratory chain complex Ⅲ / 军事医学

Objective To explore the mechanism by which tumor necrosis factor alpha(TNF-α) induces RIP1 kinase-dependented apoptosis in L929-A fibroblastoma cells.Methods The sub-mitochondrial localization of receptor-interacting protein 1(RIP1),caspase-8 and Bid proteins was detected by dose-gradient trypsin digestion and Western blotting.The levels of reactive oxygen species (ROS),intracellular calcium concentration,mitochondrial membrane potential (MMP),and cellular adenosine triphosphate(ATP) content were determined by fluorescent probe labeling and flow cytometry assay.The mitochondrial respiratory chain complex Ⅰ and Ⅲ activities were detected by commercial kits.Nec-1,A RIP1 kinase specific inhibitor,and RIP1-/-or Bid-/-L929-A cells were used to detect the roles of RIP1 kinase and Bid protein in cell death.Results RIP1,caspase-8 and Bid proteins were co-located in the outer membrane of mitochondrial.TNF-α exposure for 3 h could induce Bid cleavage,inhibit mitochondrial respiratory chain complex Ⅲ activity and reduce MMP.Following these changes and after TNF-α exposure for 6-12 h,the intracellular calcium concentration and ROS were increased,whereas the ATP concentration was decreased,and the cells were killed.Inhibiting RIP1 kinase or knockdown RIP1 or Bid protein could suppress all the cytotoxic effects of TNF-α.Conclusion TNF-α treatment can result in RIP1 kinase-mediated Bid cleavage and inhibit mitochondrial respiratory chains and cell energy metabolism,which ultimately leads to the death of L929-A cells.

Ultrastructural Changes in Skeletal Muscle of Infants with Mitochondrial Respiratory Chain Complex I Defects

BACKGROUND AND PURPOSE: The pathogenesis of mitochondrial disease (MD) involves the disruption of cellular energy metabolism, which results from defects in the mitochondrial respiratory chain complex (MRC). We investigated whether infants with MRC I defects showed ultrastructural changes in skeletal muscle. METHODS: Twelve infants were enrolled in this study. They were initially evaluated for unexplained neurodegenerative symptoms, myopathies, or other progressive multiorgan involvement, and underwent muscle biopsies when MD was suspected. Muscle tissue samples were subjected to biochemical enzyme assays and observation by transmission electron microscopy. We compared and analyzed the ultrastructure of skeletal muscle tissues obtained from patients with and without MRC I defects. RESULTS: Biochemical enzyme assays confirmed the presence of MRC I defects in 7 of the 12 patients. Larger mitochondria, lipid droplets, and fused structures between the outer mitochondrial membrane and lipid droplets were observed in the skeletal muscles of patients with MRC I defects. CONCLUSIONS: Mitochondrial functional defects in MRC I disrupt certain activities related to adenosine triphosphate synthesis that produce changes in the skeletal muscle. The ultrastructural changes observed in the infants in this study might serve as unique markers for the detection of MD.

Zhibai Dihuang Decoction improves the activity of sperm mitochondrial respiratory chain complex in rats with Ureaplasma urealyticum infection / 中华男科学杂志

<p><b>Objective</b>To investigate the effect of Zhibai Dihuang Decoction (ZDD) on the sperm mitochondrial respiratory chain complex (MRCC) in rats with Ureaplasma urealyticum (UU) infection.</p><p><b>METHODS</b>Ninety male SD rats were randomly divided into five groups, sham operation, UU infection model control, ZDD (crude drug at 8.56 g per kg of the body weight per day), doxycycline (DC, at 20 mg per kg of the body weight per day), and ZDD+DC. The model of UU infection was established by injecting UU into the bladder of all the rats except those of the sham operation group. After modeling, the rats were treated intragastrically with respective drugs for 21 days and then executed and their epididymides harvested for examination of sperm quality and determination of the activities of sperm MRCCs I, II, III and IV by spectrophotometry.</p><p><b>RESULTS</b>At 10 days after modeling, the UU-positive rates in the model control, sham operation, ZDD, DC and ZDD+DC groups were 92.9%, 0%, 33.3%, 26.7% and 20.0%, respectively, significantly higher in the model control than in the other groups (P<0.05). The epididymal sperm concentrations in the five groups were (0.97±0.23), (3.02±0.52), (1.21±0.35), (1.02±0.31) and (1.52±0.28) ×106 ml, the sperm motilities were (58.62±15.36), (80.45±7.21), (75.52±8.78), (68.43±10.25) and (78.25±7.67)%, and rates of grade a+b sperm were (6.15±1.02), (10.32±1.14), (10.12±1.08), (9.01+1.27) and (10.74±1.03)%, respectively, all remarkably lower in the model control than in the sham operation group (P<0.01), but markedly higher in the ZDD and ZDD+DC groups than in the model controls (P<0.05). The activities of MRCC I in the model control, sham operation, ZDD, DC and ZDD+DC groups were (31.54±16.25), (136.86±6.34), (100.68±14.41), (81.68±6.78) and (124.06±5.54) μmol/(min·mg), those of MRCC II were (9.50±3.86), (20.34±0.37), (10.88±1.04), (12.93±1.07) and (16.23±0.60) μmol/(min·mg), those of MRCC III were (5.58±1.79), (19.60±0.61), (11.34±1.35), (13.87±1.23) and (15.96±0.69) μmol/(min·mg), and those of MRCC IV were (9.54±1.34), (28.98±3.33), (17.02±2.04), (18.41±2.67) and (21.66±2.93) μmol/(min·mg), respectively, all significantly lower in the model control than in the sham operation group (P<0.01), with the activities of MRCCs I, III and IV remarkably higher in the ZDD, DC and ZDD+DC groups (P<0.01) and that of MRCC II higher in the DC and ZDD+DC groups than in the model control (P<0.05).</p><p><b>CONCLUSIONS</b>ZDD can improve the epididymal sperm quality and the activity of the sperm MRCC in UU-infected rats, which may be one of the mechanisms of ZDD acting on male infertility caused by UU infection.</p>

Effect of inhibiting mitochondrial respiratory chain complex I on migration and invasion capacity of colon cancer cell line Caco2 / 第二军医大学学报

Objective To investigate the effect of inhibiting mitochondrial respiratory chain complex I on the migration and invasion capacity of colon cancer cell line Caco2, and to explore the possible molecular mechanism. Methods Human colon cancer cell line Caco2 was treated with 1 μmol/L rotenone in vitro. Then the relative activity of mitochondrial respiratory chain complex I was examined by chromatometry, the capacity of cell migration and invasion was determined by trans well assay, and the reactive oxygen species (ROS) level in cells was determined using flow cytometry. Results The activity of mitochondrial respiratory chain complex I of Caco2 cells treated with 1 μmol/L rotenone was significantly lower than that of the untreated cells(P<0. 01). In addition, Transwell assay showed that the cell migration rate and invasive rate in Caco2 cells treated with rotenone were significantly higher than those in untreated Caco2 cells after 48 h (migrant rate [30. 4±1. 4]% vs [22. 6 ± 1. 4]%, invasive rate [20. 3 ± 1. 0]% vs [15. 2 ± 1. 3] %, P<0. 01). Furthermore, the ROS level in the rotenone treated cells was significantly higher than that in untreated cells ([5. 68 ± 0. 44] Y vs [3. 46 ± 0. 30]%, P<0. 01). Conclusion Our data suggest that inhibiting the activity of mitochondrial respiratory chain complex I may promote cell migration and invasion by increasing ROS production in colon cancer cells.

Mitochondrial Respiratory Complex I Deficiency Simulating Spinal Muscular Atrophy / 대한소아신경학회지

Two female patients with clinical features resembling spinal muscular atrophy are introduced. Patient 1 presented with hypotonia and proximal weakness of extremities at the age of 4 months. The electromyography revealed motor neuronopathy suggestive of spinal muscular atrophy. Patient 2 presented with severe hypotonia, motor weakness, and joint contractures since birth. The muscle biopsy finding was consistent with spinal muscular atrophy. However, deletions in the survival motor neuron genes and the neuronal apoptosis inhibitor protein genes were not found in both the patients. They finally showed the clinical features against spinal muscular atrophy; epileptic seizures, cardiomyopathy, and spasticity. We measured the mitochondrial respiratory chain complex enzyme activities in cultured skin fibroblasts, whose results were suggestive of isolated complex I deficiency in both the patients. In conclusion, for the patients who have clinical features resembling SMA without any deletions in the SMA genes it should be considered a possibility of the mitochondrial respiratory chain complex I deficiency.

Changes of ND1, ND2 and mtTFA gene expressions in myocardial cells and intestinal epithelial cells of rats after hemorrhagic shock / 第三军医大学学报

Objective To explore the transcription pattern of ND1, ND2 and mtTFA gene in the myocardial cells and intestinal epithelial cells of rats after hemorrhagic shock. Methods Total RNA of myocardial cells and intestinal epithelial cells in rats were extracted after hemorrhagic shock. ND1, ND2, and mtTFA gene transcription levels were measured by reverse transcription and polymerase chain reaction (RT PCR). Results During the period from 1 to 2 h after hemorrhagic shock, the ND1 gene transcription levels in myocardial cells in the hemorrhagic shock groups were higher than that in the normal control group, but the levels in intestinal epithelial cells were lower than that in the normal control group. The pattern for the changes of ND2 gene transcription in myocardial cells and intestinal epithelial cells was basically similar. Conclusion There might exist certain tissue differences in the changes of ND1 gene transcripts in myocardial cells and intestinal epithelial cells of rats with hypoxic and ischemic damage.