Effects of Guben Fangxiao Beverage （固本防哮饮） on Lung Tissue Oxidative Stress and AMPK/Nrf2/HO-1 Pathway in Mice with Bronchial Asthma in Remission / 中医杂志

ObjectiveTo investigate the possible mechanism of Guben Fangxiao Beverage （固本防哮饮） for the prevention and treatment of chronic airway inflammation during asthma remission. MethodsThirty-six female Balb/c mice were randomly divided into normal group， model group， low-， medium-， and high-dose of Guben Fangxiao Beverage group and montelukast sodium group， with 6 mice in each group. Except for the normal group， ovalbumin and respiratory syncytial virus were used in other groups to establish a mouse model of bronchial asthma in remission stage. After molding， the low-， medium-， and high-dose groups of Guben Fangxiao Beverage were respectively given 12， 24， and 36 g/（kg·d）， the montelukast sodium group was given montelukast sodium granule 2.6 mg/（kg·d）， and the mice in the normal group and model group were given 20 ml of double-distilled water， all by gavage， once a day for 28 days. The levels of interleukin 4 （IL-4） and interleukin 5 （IL-5） in the lung tissue of mice were detected； HE staining was used to observe the pathology of the lung tissue and to score the inflammation； DHE staining was used to observe the level of reactive oxygen species （ROS） in the lung tissue， and the activities of mitochondrial respiratory chain complexes Ⅰ， Ⅱ， Ⅲ， Ⅳ， and Ⅴ in the lung tissue were detected； the levels of serum superoxide dismutase （SOD）， catalase （CAT）， malondialdehyde （MDA） and adenosine triphosphate （ATP） were detected； the protein expression levels of phosphorylated adenosine monophosphate-activated protein kinase （p-AMPK）， nuclear factor erythroid 2-related factor 2 （Nrf2）， haem oxygenase 1 （HO-1） and cAMP responsive element binding protein （CREB） in the lung tissues of the model group were detected by Western blot. ResultsCompared with the normal group， the histopathological results of the lungs of mice in the model group showed an increase in inflammatory cells around the airways and an increase in inflammatory score； DHE staining showed an increase in the level of ROS， and an increase in the levels of IL-4 and IL-5 in the lung tissues； the levels of serum SOD， CAT， and ATP were reduced， and the level of MDA was elevated； the activities of the mitochondrial respiratory chain complexes Ⅰ， Ⅱ， Ⅲ， Ⅳ， and Ⅴ of the lung tissues were reduced， and the activities of p-AMPK， Nrf2， CREB protein expression decreased （P＜0.05）. Compared with the model group， the lung tissue inflammatory cells and inflammation scores of mice in each Guben Fangxiao Beverage dose group and montelukast sodium group were reduced； the levels of ROS， IL-4 and IL-5 in the lung tissue were reduced； the levels of CAT and ATP in the serum increased， and the content of MDA was reduced； and the activities of mitochondrial respiratory chain complexes Ⅰ and Ⅱ， as well as the expression of CREB protein， were elevated in the lung tissue （P＜0.05）. Compared with the high-dose group， the MDA level of the medium-dose Guben Fangxiao Beverage group decreased （P＜0.05）. The activity of mitochondrial respiratory chain complex V in the medium-dose group was higher than that in the montelukast sodium group， and the activity of mitochondrial respiratory chain complex Ⅳ in the medium- and high-dose groups was higher than that in the low-dose group （P＜0.05）. ConclusionGuben Fangxiao Beverage can inhibit oxidative stress and improve mitochondrial function to relieve chronic airway inflammation in bronchial asthma model mice during asthma remission， and its mechanism may be related to the activation of AMPK/Nrf2/HO-1 signaling pathway.

Overexpression of miR-431-5p impairs mitochondrial function and induces apoptosis in gastric cancer cells via the Bax/Bcl-2/caspase3 pathway / 南方医科大学学报

OBJECTIVE@#To investigate the expression of microRNA miR-431-5p in gastric cancer (GC) tissues and its effects on apoptosis and mitochondrial function in GC cells.@*METHODS@#The expression level of miR-431-5p in 50 clinical samples of GC tissues and paired adjacent tissues was detected using real-time fluorescence quantitative PCR, and its correlation with the clinicopathological features of the patients was analyzed. A cultured human GC cell line (MKN-45 cells) were transfected with a miR-431-5p mimic or a negative control sequence, and the cell proliferation, apoptosis, mitochondrial number, mitochondrial potential, mitochondrial permeability transition pore (mPTP), reactive oxygen species (ROS) production and adenosine triphosphate (ATP) content were detected using CCK-8 assay, flow cytometry, fluorescent probe label, or ATP detection kit. The changes in the expression levels of the apoptotic proteins in the cells were detected with Western blotting.@*RESULTS@#The expression level of miR-431-5p was significantly lower in GC tissues than in the adjacent tissues (P < 0.001) and was significantly correlated with tumor differentiation (P=0.0227), T stage (P=0.0184), N stage (P=0.0005), TNM stage (P=0.0414) and vascular invasion (P=0.0107). In MKN-45 cells, overexpression of miR-431-5p obviously inhibited cell proliferation and induced cell apoptosis, causing also mitochondrial function impairment as shown by reduced mitochondrial number, lowered mitochondrial potential, increased mPTP opening, increased ROS production and reduced ATP content. Overexpression of miR-431-5p significantly downregulated the expression of Bcl-2 and increased the expressions of pro-apoptotic proteins p53, Bcl-2 and cleaved caspase-3 protein.@*CONCLUSION@#The expression of miR-431-5p is down-regulated in GC, which results in mitochondrial function impairment and promotes cell apoptosis by activating the Bax/Bcl-2/caspase3 signaling pathway, suggesting the potential role of miR-431-5p in targeted therapy for GC.

Neuron stem cell NLRP6 sustains hippocampal neurogenesis to resist stress-induced depression

Neurogenesis decline in hippocampal dentate gyrus (DG) participates in stress-induced depressive-like behaviors, but the underlying mechanism remains poorly understood. Here, we observed low-expression of NOD-like receptor family pyrin domain containing 6 (NLRP6) in hippocampus of stress-stimulated mice, being consistent with high corticosterone level. NLRP6 was found to be abundantly expressed in neural stem cells (NSCs) of DG. Both Nlrp6 knockout (Nlrp6-/-) and NSC-conditional Nlrp6 knockout (Nlrp6CKO) mice were susceptible to stress, being more likely to develop depressive-like behaviors. Interestingly, NLRP6 was required for NSC proliferation in sustaining hippocampal neurogenesis and reinforcing stress resilience during growing up. Nlrp6 deficiency promoted esophageal cancer-related gene 4 (ECRG4) expression and caused mitochondrial dysfunction. Corticosterone as a stress factor significantly down-regulated NLRP6 expression, damaged mitochondrial function and suppressed cell proliferation in NSCs, which were blocked by Nlrp6 overexpression. ECRG4 knockdown reversed corticosterone-induced NSC mitochondrial function and cell proliferation disorders. Pioglitazone, a well-known clinical drug, up-regulated NLRP6 expression to inhibit ECRG4 expression in its protection against corticosterone-induced NSC mitochondrial dysfunction and proliferation restriction. In conclusion, this study demonstrates that NLRP6 is essential to maintain mitochondrial homeostasis and proliferation in NSCs, and identifies NLRP6 as a promising therapeutic target for hippocampal neurogenesis decline linked to depression.

Up-regulation of p21 activated kinase 4 expression in the retina of diabetes mice and its effects on the behavior and mitochondrial function in retinal vascular endothelial cells / 中华眼底病杂志

Objective:To observe the effects of p21 activated kinase 4 (PAK4) on the mitochondrial function and biological behavior in retinal vascular endothelial cells.Methods:The experimental study was divided into two parts: in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 12 healthy C57BL/6J male mice were randomly divided into normal control group and diabetes group, with 6 mice in each group. Diabetes mice were induced by streptozotocin to establish diabetes model. Eight weeks after modeling, quantitative real-time polymerase chain reaction and Western blots were performed to detect the expression of PAK4 in diabetic retinas. In vitro cell experiments: the human retinal microvascular endothelial cells (hRMEC) were divided into three groups: conventional cultured cells group (N group), empty vector transfected (Vector group); pcDNA-PAK4 eukaryotic expression plasmid transfected group (PAK4 group). WB and qPCR were used to detect transfection efficiency, while scratching assay, cell scratch test was used to detect cell migration in hRMEC of each group. In vitro white blood cell adhesion experiment combined with 4 ', 6-diamino-2-phenylindole staining was used to detect the number of white blood cells adhering to hRMEC in each group. The Seahorse XFe96 cell energy metabolism analyzer measures intracellular mitochondrial basal respiration, adenosine triphosphate (ATP) production, maximum respiration, and reserve respiration capacity. The t-test was used for comparison between the two groups. Single factor analysis of variance was used for comparison among the three groups. Results:In vivo animal experiments: compared with normal control group, the relative expression levels of PAK4 mRNA and protein in retina of diabetic mice were significantly increased, with statistical significance ( t=25.372, 22.419, 25.372; P<0.05). In vitro cell experiment: compared with the N group and Vector group, the PAK4 protein, mRNA relative expression and cell mobility in the hRMEC of PAK4 group were significantly increased, with statistical significance ( F=36.821, 38.692, 29.421; P<0.05). Flow cytometry showed that the adhesion number of leukocytes on hRMEC in PAK4 group was significantly increased, and the difference was statistically significant ( F=39.649, P<0.01). Mitochondrial pressure measurement results showed that the capacity of mitochondrial basic respiration, ATP production, maximum respiration and reserve respiration in hRMEC in PAK4 group was significantly decreased, with statistical significance ( F=27.472, 22.315, 31.147, 27.472; P<0.05). Conclusion:Over-expression of PAK4 impairs mitochondrial function and significantly promotes leukocyte adhesion and migration in retinal vascular endothelial cells.

Study on the effect of polysaccharide from stigma maydison on ATP synthesis in renal mitochondria and its mechanism in D-galactose-induced aging mice / 中华老年医学杂志

Objective:To investigate the effect of Stigma Maydis Palysaccharide(SMPS)on ATP synthesis in kidney mitochondria of D-galactose-induced aging mice, and to clarify its possible mechanism.Methods:The aging mouse model was established by subcutaneous injection of D-galactose solution in the back of the neck.The 48 SPF male mice were randomly divided into normal control group(control group), D-galactose model group(D-Gal group), SMPS low-dose group and SMPS high-dose group(n=12 for each). The control group was subcutaneously injected with 150 mg/kg normal saline on the back of the neck every day, while the other three groups were subcutaneously injected with 150 mg/kg of D-gal solution on the back of the neck every day.SMPS-L and-H dose groups were given 30 mg/kg and 60 mg/kg of SMPS solution by gavage at the same day of D-Gal injection.The control group and D-GAL group were given the same volume of normal saline daily by gavage for 42 days.Blood samples were collected from the eyeball under general anesthesia after 42 days of intervention for the detection of serum levels of superoxide dismutase(SOD), glutathione peroxidase(GSH-Px)and MDA.After harvesting the kidney tissue, various tests were used to detect ATP content, the mRNA expression levels and protein expression levels in kidney.Luciferase assay was used to detect ATP content in renal tissue.Real-time fluorescent quantitative PCR was used to detect the mRNA expression levels of succinate dehydrogenase(SDH)of complex Ⅱ, cytochrome C reductase(Cycs)of complex Ⅲ, complex Ⅳ(COXⅣ)and ATP5b in ATP synthase in mitochondrial oxidative respiratory chain.Western blot was used to detect the expression levels of mitochondrial fusion protein 2(MFN2), dynamin-related protein1(DRP1)and mitochondrial autophagy related protein P62 in renal tissues of each group.Results:Compared with control group, the activities of serum of SOD(116.53±10.01)U/mg and GSH-Px(127.58±8.74)μmol/L were significantly decreased in D-GAL group(both P< 0.01), and serum MDA content(15.42±0.91)μmol/L increased significantly in D-GAL group( P<0.01). Compared with D-GAL group, the activities of SOD(152.80±9.29)U/mg and GSH-Px(274.07±10.73)μmol/L were significantly increased in SMPS intervention group( P< 0.01), while the MDA content(8.10±0.66)μmol/L decreased significantly in SMPS intervention group( P< 0.01). Compared with control group, the content of ATP(178±4)10 -4 μmol in D-gal group was significantly decreased( P<0.01), the mRNA expression levels of SDH, Cycs and COXⅣ were not significantly changed in D-gal group, and the mRNA expression level of ATP5b(0.67±0.01)was down-regulated in D-gal group( P<0.01), the expression of MFN2 protein(0.29±0.02)was significantly decreased in D-gal group( P<0.01), and the expression of DRP1 and P62 protein(0.31±0.02 and 0.21±0.01)was significantly increased in D-gal group(both P<0.01). Compared with the D-gal group, the ATP content(193±1)10 -4 μmol in the kidney tissue of the mice was significantly increased in SMPS intervention group( P< 0.01), and the ATP5b mRNA expression and MFN2 protein expression(0.87±0.05 and 0.71±0.08)were significantly increased in SMPS intervention group(both P< 0.01), DRP1 and P62 protein expressions(0.20±0.01 and 0.10±0.01)were significantly down-regulated in in SMPS intervention group(both P< 0.01). Conclusions:SMPS can improve the mitochondrial dynamic homeostasis disorder in aging mice by increasing the activity of antioxidant enzymes, up-regulating the expression of ATP5b mRNA and MFN2 protein, down-regulating the expression of DRP1 and P62 protein, and promoting the generation of mitochondrial ATP in D-gal-induced aging mice kidney tissue.

Comparative study of the radiosensitivity of hematopoietic stem/progenitor cells derived from fetal liver and bone marrow / 中华放射医学与防护杂志

Objective:To investigate the difference in the radiation sensitivity of hematopoietic stem and progenitor cells (HSPCs) derived from fetal liver and bone marrow.Methods:HSPCs from fetal liver of 14.5 d embryo or bone marrow of 8 week-old mice were isolated to receive a single dose of 5 or 10 Gy irradiation in vitro using a 60Co irradiator. Twelve hours later, the cell apoptosis, mitochondrial reactive oxygen species (ROS) level, colony formation ability and DNA damage in HSPCs were detected. Freshly isolated HSPCs were injected into lethally irradiated CD45.1 + C57BL/6J mice (4.5 Gy+ 5 Gy with an interval of 30 min) Chimerism rate, lineage constitution, and cell cycle were analyzed 12 weeks after transplantation. Results:Compared with bone marrow HSPCs after irradiation, the percentage of apoptosis in fetal liver HSPCs was significantly higher ( t=16.21, 12.27, P<0.05), the level of ROS was dramatically elevated ( t=68.72, 18.89, P<0.05). At 10 Gy, fetal liver HSPCs could not form colonies at all ( t=12.41, 15.67, 9.46, P<0.05). γ-H2AX immunofluorescence staining showed that the DNA damage of fetal liver HSPCs was more severe after irradiation, and the number of Foci formed was significantly higher than that of bone marrow HSPCs ( t=2.27, 2.03, P< 0.05), which indicated that fetal liver HSPCs were more sensitive to radiation. The chimerism rate of transplanted fetal liver HSPCs was lower than that of bone marrow cells ( t=5.84, P<0.05) with a higher proportion of myeloid lineage, suggesting that fetal liver HSPCs had lower in vivo reconstitution capacity than bone marrow HSPCs and were more prone to myeloid differentiation. The cell cycle of bone marrow HSPCs from transplanted chimeric mice was examined, and the proportion of S-phase was significantly higher in the fetal liver group than that in the bone marrow group ( t=2.89, P<0.05). Mitochondrial stress results showed that fetal liver HSPCs had higher basal respiratory capacity ( t=39.19, P<0.05), proton leakage ( t=6.64, P<0.05), ATP production ( t=9.33, P<0.05), and coupling efficiency ( t=7.10, P<0.05) than bone marrow c-Kit + cells, while respiratory reserve capacity ( t=5.53, P< 0.05) was lower than that of bone marrow c-Kit + cells. Conclusions:HSPCs derived from fetal liver display higher radiosensitivty compared with bone marrow HSPCs, laying the foundation for an in-depth illustration of the effects of radiation on hematopoietic stem cells at different developmental stages.

Protective effects and mechanisms of L-carnitine on cognitive dysfunction in rats with chronic cerebral hypoperfusion / 中华行为医学与脑科学杂志

Objective:To explore the protective effects and mechanisms of L-carnitine (LCAR) on cognitive dysfunction in chronic cerebral hypoperfusion rats.Methods:Totally 90 SD male rats (SPF class) aged 3-4 months were divided into four groups according to random number talbe: sham operated control group (SHAM group, n=15), sham operated with L-carnitine treatment group (LCAR group, n=25), 2-vessel occlusion group (2VO group, n=25), and 2-vessel occlusion with L-carnitine treatment group (2VO+ LCAR group, n=25). The chronic cerebral hypoperfusion model was established by bilateral common carotid artery ligation, and the carotid arteries from SHAM group and LCAR group were only separated without ligation.L-carnitine was administered intraperitoneally (300 mg·kg -1·d -1) for 30 days after surgery in the LCAR and 2VO+ LCAR groups.After 30 days of L-carnitine intervention, Morris water maze was performed to test the spatial cognitive function of the rats, the ATP level of hippocampal tissue was detected by chemiluminescence, the mitochondrial structure and synaptic structure of hippocampal neurons were observed by transmission electron microscopy, the degree of mitochondrial damage was scored, the vesicle density was counted and measured, the level of N-methyl-D-aspartate receptor subunit 2A or 2B(NR2A/B) and postsynaptic density 95(PSD95) in hippocampal tissue were detected by Western blot.The expression and distribution levels of transcription factor cAMP response element-binding protein(CREB) in brain tissues were observed by immunofluorescence.SPSS 16.0 software was used for statistical analysis.The escape latency data of repeated learning training in Morris water maze was conducted by repetitive measurement ANOVA, while other data were adopted by one-way ANOVA, and Dunnett's t test was used for further pairwise comparison. Results:(1)Morris water maze results showed that the time and group interaction of escape latency was not significant among the 4 groups of rats ( F=1.4, P>0.05), but the time main effect and group main effect were significant( F=21.6, 15.2, both P<0.05). Morris water maze results showed that platform position learning from 3rd to 7th day, the escape latencies in 2VO group were longer than those in SHAM group and 2VO+ LCAR group (all P<0.05). The results of short-term memory showed that the escape latency in 2VO group was longer than those in SHAM group and 2VO+ LCAR group (all P<0.05). Meanwhile, the retention time and crossing times in the platform area of 2VO group were less than those in SHAM group and 2VO+ LCAR group (all P<0.05). (2) The absolute and relative levels of ATP in hippocampus showed that the difference among the 4 groups were statistically significant ( F=14.6, 13.2, both P<0.05). ATP level of hippocampus in 2VO group was lower than those in SHAM group and 2VO+ LCAR group (both P<0.05). Electron microscopic observation of mitochondrial morphology showed that the Flameng score of mitochondrial damage in the hippocampus of rats in 2VO group (2.82±0.17) was higher than those in SHAM group (0.25±0.07) and 2VO+ LCAR group (1.76±0.09) (both P<0.05). (3) The density of synaptic vesicles in the hippocampus of rats in 2VO group ((289.09±22.41)/μm 2)was lower than those in SHAM group ((497.49±28.89)/μm 2)and 2VO+ LCAR group ((401.23±45.09)/μm 2) (both P<0.01). Western blot results showed that the relative levels of synaptic proteins NR2A/B, PSD95 and CREB in 2VO group were lower than those in SHAM group and 2VO+ LCAR group (all P<0.05). Immunofluorescence results showed that the relative level of CREB expression in hippocampal subregions and cortex in 2VO group was lower than those in SHAM group and 2VO+ LCAR group (both P<0.01). Conclusion:L-carnitine can improve spatial learning and memory dysfunction in rats with chronic cerebral hypoperfusion, which are related with promoting ATP production and protecting mitochondrial morphology, and promoting synaptic vesicle synthesis and synaptic protein expression.

Synergistic activation of AMPK by AdipoR1/2 agonist and inhibitor of EDPs-EBP interaction recover NAFLD through enhancing mitochondrial function in mice

Nonalcoholic fatty liver disease (NAFLD), especially nonalcoholic steatohepatitis (NASH), is a common hepatic manifestation of metabolic syndrome. However, there are no effective therapy to treat this devastating disease. Accumulating evidence suggests that the generation of elastin-derived peptides (EDPs) and the inhibition of adiponectin receptors (AdipoR)1/2 plays essential roles in hepatic lipid metabolism and liver fibrosis. We recently reported that the AdipoR1/2 dual agonist JT003 significantly degraded the extracellular matrix (ECM) and ameliorated liver fibrosis. However, the degradation of the ECM lead to the generation of EDPs, which could further alter liver homeostasis negatively. Thus, in this study, we successfully combined AdipoR1/2 agonist JT003 with V14, which acted as an inhibitor of EDPs-EBP interaction to overcome the defect of ECM degradation. We found that combination of JT003 and V14 possessed excellent synergistic benefits on ameliorating NASH and liver fibrosis than either alone since they compensate the shortage of each other. These effects are induced by the enhancement of the mitochondrial antioxidant capacity, mitophagy, and mitochondrial biogenesis via AMPK pathway. Furthermore, specific suppression of AMPK could block the effects of the combination of JT003 and V14 on reduced oxidative stress, increased mitophagy and mitochondrial biogenesis. These positive results suggested that this administration of combination of AdipoR1/2 dual agonist and inhibitor of EDPs-EBP interaction can be recommended alternatively for an effective and promising therapeutic strategy for the treatment of NAFLD and NASH related fibrosis.

The role of Drp1 mediated mitochondrial dynamics balance in sepsis induced cardiomyopathy / 中华急诊医学杂志

Objective:To analyze the effect of inhibiting excessive mitochondrial fission mediated by dynamic related protein 1 (Drp1) on the function of injured cells and mitochondria in the septic myocardium, and to explore the protective effect of maintaining mitochondrial dynamic balance in the pathogenesis of sepsis induced cardiomyopathy(SIC).Methods:Rat H9C2 cardiomyocytes were cultured and stimulated with lipopolysaccharide (LPS) to establish a model of SIC. Mitochondrial division inhibitor 1 (Mdivi-1) was given 30 min before LPS stimulation. They were divided into the control group, LPS stimulated group (LPS), Mdivi-1 control group (Mdivi-1), and LPS+Mdivi-1 intervention group (LPS+Mdivi-1). CCK-8 was used to detect the cell viability, and lactate dehydrogenase (LDH) was used to detect cellular damage. A MitoTracker probe was used to observe mitochondrial morphology by laser scanning confocal microscopy, JC-1 staining was used to detect mitochondrial membrane potential level, a DCFH-DA probe was used to detect total ROS level, and an AnnexinV-FITC/PI probe was used to detect the cell apoptosis ratio. The expression levels of mitochondrial fission protein Drp1 and fusion proteins Optic Atrophy 1(Opa1) and Mitofusin2 (Mfn2) were detected by real-time PCR and Western blot. One-way ANOVA was used to compare the differences between groups, and the LSD- t test was used for pairwise comparisons between groups. Results:Compared with the control group, cell viability, the average length of mitochondria and the mitochondrial membrane potential were decreased, and ROS production, the cell apoptosis rate and LDH were increased in the LPS group (all P<0.05). After Mdivi-1 intervention, compared with the LPS-stimulated group, the cell viability was increased, myocardial cell damage was reduced, the average length of mitochondria was prolonged, mitochondrial dysfunction was alleviated, and the cell apoptosis rate was inhibited in the LPS+Mdivi-1 group (all P<0.05). Conclusions:Mdivi-1 might inhibit mitochondrial fission mediated by Drp1, maintain mitochondrial dynamic balance, alleviate mitochondrial dysfunction and protect myocardial cells from LPS-induced injury.

Isobavachalcone induces cell death through multiple pathways in human breast cancer MCF-7 cells / 南方医科大学学报

OBJECTIVE@#To explore the effects of isobavachalcone (IBC) on cell death of human breast cancer MCF-7 cells and explore the possible mechanism.@*METHODS@#MCF-7 cells were treated with different concentrations of IBC, and the changes in cell proliferation were assessed using MTT assay. Apoptosis of MCF-7 cells following treatment with 10, 20, and 40 μmol/L IBC was analyzed using flow cytometry with annexin V-FITC/PI double staining and fluorescence microscopy, and the expressions of apoptosis- and autophagy-related proteins (Bax, Bcl-2, Akt, p-Akt, p62, and LC3) were detected with Western blotting. Electron microscopy was used to observe the changes in submicrostructure of the cells following treatment with 40 μmol/L IBC. JC-1 assay kit, ATP assay kit, and reactive oxygen species (ROS) kit were used to determine the effect of IBC on mitochondrial function of the cells.@*RESULTS@#MTT assay showed that IBC significantly inhibited the proliferation of MCF-7 cells in a concentration- and time-dependent manner, with IC50 values of 38.46, 31.31, and 28.26 μmol/L at 24, 48, and 72 h, respectively. IBC also concentration-dependently induced apoptosis of MCF-7 cells. IBC-induced cell death was inhibited by z-VAD-fmk, a caspase inhibitor (P < 0.05), but not by the necroptosis inhibitor necrostatin-1 (Nec-1). Western blotting showed that IBC-induced MCF-7 cell apoptosis by increasing Bax expression and down-regulating the expressions of Bcl-2, Akt and p-Akt-473 (all P < 0.05). With the increase of IBC concentration, the expression of autophagy-related protein p62 and the LC3-II/I ratio increased progressively. Electron microscopy revealed the presence of autophagic bodies in IBC-treated MCF-7 cells. IBC treatment also resulted in decreased mitochondrial membrane potential and intracellular ATP level and increased ROS accumulation in MCF-7 cells (P < 0.05).@*CONCLUSION@#IBC is capable of inducing both apoptosis and autophagy in MCF-7 cells, suggesting the potential value of IBC as a lead compound in the development of anti-breast cancer agents.

Effect of Naozhenning on Hippocampal Mitochondrial Function in Model Rats with Post-concussion Syndrome / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Naozhenning on learning and memory ability and neuron damage in hippocampal CA1 region of post-concussion syndrome model rats based on mitochondrial function. MethodMultiple cerebral concussion （MCC） was induced in SPF Wistar rats with the free-fall impact method. Then the model rats were randomly classified into model group （equivalent volume of distilled water）， piracetam （0.43 g·kg-1， ig） group， and low-， medium-， and high-dose NZN （5.4， 10.8， 21.6 g·kg-1， respectively， ig） groups， with 10 rats in each group， and another 10 normal rats were included in the normal control group （equivalent volume of distilled water）. The administration lasted 14 days and then relevant indexes were detected. Morris water maze test was used to observe the changes of learning and memory ability in each group， such as escape latency， residence time in primary quadrant， and times of crossing platform. The pathological changes of hippocampal CA1 region were observed based on hematoxylin-eosin （HE） staining and Nissl staining. The ultrastructure of mitochondria was observed under the transmission electron microscope （TME） and the activity of mitochondrial respiratory chain complex Ⅰ was detected by colorimetry. The content of adenosine triphosphate （ATP） was determined by fluorescence probe and mitochondrial membrane potential （MMP） by fluorescein enzyme-linked fluorescence immunoassay. ResultCompared with the normal control group， the model group showed long escape latency， short residence time in target quadrant， few times of crossing the platform， significant decrease in counts of neurons and Nissl bodies in hippocampal CA1 region， damage of neuronal morphology and mitochondrial structure， and significant reduction of MMP and the content of mitochondrial ATP and respiratory chain complex I （P<0.05， P<0.01）. The NZN groups demonstrated short escape latency， long residence time in target quadrant， increased times of crossing the platform， small number of neurons and Nissl bodies in hippocampal CA1 region， alleviated damage of neuronal morphology and mitochondrial structure， and increase in MMP and the content of mitochondrial ATP and respiratory chain complex I （P<0.05， P<0.01）. ConclusionNZN can improve the learning and memory ability of MCC rats by improving mitochondrial structure and function and alleviating hippocampal neuron injury.

Regulation of Wenjingtang on HIF-1α Expression and Mitochondrial Function of Ectopic Endometrial Stromal Cells in Patients with Endometriosis / 中国实验方剂学杂志

ObjectiveThis study investigated the mechanism of Wenjingtang in the prevention and treatment of endometriosis （EMT） from the perspective of regulating hypoxia stress and mitochondrial function. MethodPrimary human endometrial stromal cells （ESCs） form ectopic endometrial tissues were isolated and cultured， the cells were divided into control group （Control）， 5% control serum group （5% KBXQ）， 10% control serum group （10% KBXQ）， 5% Wenjingtang serum group （5% WJTXQ） and 10% Wenjingtang serum group （10% WJTXQ）. ESCs in different groups were detected for proliferation by methyl thiazolyl tetrazolium （MTT） assay， mRNA and protein expression of hypoxia inducible factor-1α （HIF-1α） by real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） and Western blot analysis， mitochondrial ultrastructure by transmission electron microscope， mitochondrial function ［mitochondrial membrane potential， mitochondrial membrane potential（MMP） and cytochrome C（Cyt C） content］ and apoptosis （cell membrane permeability， nuclear fluorescence intensity， nuclear size and cell counts） by high content screening （HCS） assay， apoptosis rate by flow cytometry， and proteins of B-cell lymphoma-2 （Bcl-2） associated X （Bax）， Bcl-2 and cleaved cysteinyl aspartate specific proteinase-3 （cleaved Caspase-3） by Western blot. ResultCompared with Control group， the 5% KBXQ and 10% KBXQ groups showed increased cell viability （P<0.01）， there was no significant change in HIF-1α mRNA and protein expression， transmission electron microscopy showed that the mitochondrial cristae were obvious and the inner and outer membranes of mitochondria were clear， HCS multichannel fluorescence staining showed that there were no significant changes in the expression of MMP， Cyt C and cell membrane permeability， and the nuclei showed uniform light staining， there were no significant changes in apoptosis rate， cleaved Caspase-3 protein expression and Bax/Bcl-2 ratio. Compared with Control group and corresponding concentration KBXQ group， the 5% WJTXQ and 10% WJTXQ group showed decreased cell viability （P<0.01） and HIF-1α mRNA and protein levels （P<0.05，P<0.01）， the ultrastructure of mitochondria was destroyed， some mitochondria were swollen and the cristae were blurred， moreover， decreased MMP and up-regulated Cyt C release （P<0.05，P<0.01）， increased cell membrane permeability （P<0.01）， and apoptosis characteristics included nuclear pyknosis， DNA agglutination in nucleus and decrease of cell numbers were observed （P<0.05，P<0.01）， increased apoptosis rate （P<0.01）， which was consistent with the results of HCS analysis， and up-regulated expression levels of cleaved Caspase-3 protein and Bax/Bcl-2 ratio （P<0.05，P<0.01）. ConclusionIn conclusion， the results suggest that Wenjingtang can improve hypoxia stress via down-regulating HIF-1α expression in ectopic ESCs， and inhibit cell proliferation， reduce mitochondrial biological activity and induce apoptosis， which might be the internal mechanism of Wenjingtang in preventing and treating EMT.

MicroRNA-145 plays a role in mitochondrial dysfunction in alveolar epithelial cells in lipopolysaccharide-induced acute respiratory distress syndrome / 世界急诊医学杂志（英文）

@#BACKGROUND: Acute respiratory distress syndrome (ARDS) causes substantial mortalities. Alveolar epithelium is one of the main sites of cell injuries in ARDS. As an important kind of microRNAs (miRNAs), microRNA-145 (miR-145) has been studied in various diseases, while its role in ARDS has not been investigated. METHODS: Lipopolysaccharide (LPS) was intratracheally instilled to establish a rat ARDS model. Cytokines from bronchoalveolar lavage fluid (BALF) were measured using rat tumor necrosis factor-α and interleukin-6 enzyme-linked immunosorbent assay kits (R&D Systems), and the pathological structures were evaluated using hematoxylin and eosin (H&E) staining and transmission electron microscope; the lung miR-145 messenger RNA (mRNA) was detected using quantitative polymerase chain reaction. Bioinformatics focused on the target genes and possible pathways of gene regulation. RESULTS: A rat model of LPS-induced ARDS was successfully established. The miR-145 was down-regulated in the LPS-induced ARDS lung, and mitochondrial dysfunction was observed in alveolar epithelial cells, most obviously at 72 hours after LPS. TargetScan and miRDB databases were used to predict the target genes of miR-145. A total of 428 overlapping genes were identified, seven genes were associated with mitochondrial function, and Ogt, Camk2d, Slc8a3, and Slc25a25 were verified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched in the mitogen-activated protein kinase (MAPK) signaling pathway, and Gene Ontology (GO) biological process was mainly enriched in signal transduction and transcription regulation. CONCLUSIONS: The miR-145 is down-regulated in LPS-induced ARDS, and affects its downstream genes targeting mitochondrial functions.

Effect of Chaihu Shugansan on Mitochondrial Function and Mitophagy in Gastric Tissue of Rats with Functional Dyspepsia / 中国实验方剂学杂志

Objective:To observe the effects of Chaihu Shugansan on gastric motility and mitochondrial function and mitophagy in rats with functional dyspepsia （FD） and to preliminarily reveal its mechanism in preventing and treating FD. Method:Thirty-two SD rats of SPF grade were randomly divided into the normal group， model group， Chaihu Shugansan （4.8 g·kg^-1） group， and domperidone group （4.5 mg·kg^-1） after one week of adaptive feeding， with eight rats in each group. Rats in all groups except for the normal group were exposed to modified tail-clamping stimulation for inducing FD. Four weeks later， the semi-solid nutritive medium was used to observe the gastric emptying rate of FD rats， and the serum citrate synthase （CS）， motilin （MTL）， and gastrin （GAS） were detected by enzyme-linked immunosorbent assay （ELISA）. The pathological changes in gastric tissue were observed by hematoxylin-eosin （HE） staining， the mitochondrial characteristics by transmission electron microscopy， and the expression of microtubule-associated protein light chain 3 （LC3） and voltage-dependent anion-selective channel 1 （VDAC1） by immunofluorescence co-localization. The mitochondria were extracted from fresh gastric tissue for measuring the reactive oxygen species （ROS）， malondialdehyde （MDA）， and superoxide dismutase （SOD） levels using the biochemical kits. The mitochondrial LC3， yeast Atg6 homologous（Beclin1）， and p62 protein expression was assayed by Western blot. Result:Compared with the normal group， the model group exhibited significantly reduced gastric emptying rate （<italic>P</italic><0.01） and serum CS， MTL， and GAS levels （<italic>P</italic><0.01）. HE staining showed no pathological changes like erosion or ulcer in the gastric tissue of rats in each group. However， the mitochondria in the gastric tissue were observed swollen and dilated under the transmission electron microscope， and vacuolar lesions appeared. The co-expression of LC3 with VDAC1 increased significantly （<italic>P</italic><0.01）. The mitochondrial ROS and MDA levels were elevated significantly （<italic>P</italic><0.01）， while SOD content was significantly reduced （<italic>P</italic><0.01）. The protein expression levels of LC3 and Beclin1 were significantly up-regulated （<italic>P</italic><0.05， <italic>P</italic><0.01）， whereas p62 protein expression was down-regulated significantly （<italic>P</italic><0.01）. Compared with the model group， Chaihu Shugansan and domperidone elevated the gastric emptying rate （<italic>P</italic><0.05） and serum CS， MTL， and GAS levels （<italic>P</italic><0.05， <italic>P</italic><0.01）. It was observed under the transmission electron microscope that the mitochondrial nuclear membrane of the gastric tissue was intact， with clear structure and high density of mitochondrial cristae， and mitochondrial fission and fusion were observed in some parts. The co-localization area of LC3 and VDAC1 was significantly reduced （<italic>P</italic><0.01）. The mitochondrial ROS and MDA levels declined significantly （<italic>P</italic><0.05， <italic>P</italic><0.01）， whereas SOD level rose （<italic>P</italic><0.05）. The protein expression levels of LC3 and Beclin1 were significantly down-regulated （<italic>P</italic><0.05， <italic>P</italic><0.01）， while that of p62 was obviously up-regulated （<italic>P</italic><0.01）. Conclusion:The mechanism of Chaihu Shugansan in preventing and treating FD may be related to the improvement of mitochondrial function and the inhibition of mitophagy in gastric tissue.

Molecular mechanism study of Shouhui Tongbian Capsules on promoting energy metabolism of gastrointestinal stromal cells / 中国中药杂志

Mechanism study was performed to explore how Shouhui Tongbian Capsules promotes energy metabolism of gastrointestinal stromal cells. In this study, gastrointestinal stromal cells line GIST-882 was used as the model to explore energy metabolism regulation effects of Shouhui Tongbian Capsules extract(10, 20, 50 and 100 μg·mL~(-1)) by measuring the cell proliferation, ATP level, mitochondrial membrane potential, and mitochondrial isocitrate dehydrogenase activity. Meanwhile, Western blot was used to detect the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Our results showed that Shouhui Tongbian Capsules promoted cell proliferation and increased ATP level of gastrointestinal stromal cells. In addition, Shouhui Tongbian Capsules obviously improved mitochondrial structural integrity, and increased mitochondrial membrane potential in GIST-882 cells. Mechanism study revealed that Shouhui Tongbian Capsules increased mitochondrial isocitrate dehydrogenase activity and up-regulated the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Collectively, our study indicated that Shouhui Tongbian Capsules promoted the energy metabolism for gastrointestinal stromal cells proliferation by activating mitochondrial isocitrate dehydrogenase to induce ATP production, as well as activating SCF/c-Kit and CDK2/cyclin A signaling pathways.

Apamin-induced alterations in J774 1. 6 macrophage metabolism / [Alterações induzidas por apamina no metabolismo de macrófagos J774 1. 6]

Among the immune system cells, macrophages have an important role. Apamin, a bee venom constituent, is important in the defense of these insects. Thus, we aimed to evaluate the metabolism of J774 1.6 macrophage cell line when exposed to isolated and purified apamin, using cytotoxicity tests by MTT reduction and analysis by flow cytometry (apoptosis / necrosis, production of reactive oxygen species (ROS), membranous lipoperoxidation (LPO), electrical potential of the mitochondrial membrane (mMP) and DNA fragmentation). None of the tested concentrations (10 to 100µg/mL) were cytotoxic according to MTT reductions. Apoptosis rates decreased at concentrations of 2.5, 5.0, and 10.0µg/mL (P<0.05), while necrosis rates increased (P<0.05). However, rates of healthy cells at the highest tested concentration (10µg/mL) did not differ from control (P>0.05). Apamin did not alter ROS, LPO, or DNA fragmentation. Therefore, all analyzed concentrations (1.25 to 10µg/mL) decreased mMP. Such decrease in apoptosis might be due to a suppression of mitochondrial pro-apoptotic messengers, as this peptide causes no oxidative stress, lipid peroxidation, and DNA damage. Highly sensitive techniques are majorly important for proper interpretation of cellular toxicity mechanisms, combined with routine laboratory methods.(AU)

Das células do sistema imunológico, macrófagos desempenham um papel fundamental. Apamina, constituinte do veneno de abelhas, é importante na defesa destas. Objetivou-se avaliar o metabolismo da linhagem de macrófagos J774 1.6 expostos à apamina isolada e purificada, avaliando-se citotoxicidade por redução de MTT e análise por citometria de fluxo (apoptose / necrose, produção de espécies reativas de oxigênio (EROs), lipoperoxidação membranosa (LPO), potencial elétrico da membrana mitocondrial (MMP) e fragmentação do DNA). Nenhuma concentração testada (10 a 100µg / mL) foi citotóxica. As taxas de apoptose diminuíram nas concentrações 2,5, 5,0 e 10,0µg / mL (P<0,05), enquanto as de necrose aumentaram (P<0,05). Entretanto, as taxas de células saudáveis na maior concentração testada (10µg / mL) não diferiram do controle (P>0,05). A apamina não alterou as ERO, a LPO nem a fragmentação do DNA. Portanto, todas as concentrações analisadas (1,25 a 10µg / mL) diminuíram a mMP. Tal diminuição na apoptose pode ser por uma supressão de mensageiros pró-apoptóticos mitocondriais, já que este peptídeo não causa estresse oxidativo, peroxidação lipídica nem dano ao DNA. Técnicas altamente sensíveis são importantes para adequada interpretação dos mecanismos de citotoxicidade.(AU)

Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Resveratrol (3,5,4'-trihydroxystilbene, RSV) has been widely used in mammalian cells, but whether it can be used during freezing boar semen is still unknown. The effects of RSV treatment during boar semen freezing on its anti-freezing ability, apoptosis, and possible apoptotic pathways were observed in this study. Sperm motility, mitochondrial membrane potential (ΔΨm), adenosine triphosphate (ATP) content, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic state, and messenger RNA (mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested. The results showed that: (1) Compared with fresh sperm, the motility, normal acrosome rate, and plasma membrane integrity rate of frozen boar sperm decreased significantly (P0.05), but it did significantly improve the normal acrosome rate (57.65% vs. 47.00%, P<0.05) and plasma membrane integrity rate (46.67% vs. 38.85%, P<0.05). (2) After freezing, most boar sperm showed low mitochondrial ΔΨm. RSV treatment could increase the rate of high mitochondrial ΔΨm of boar sperm. (3) RSV treatment significantly decreased reactive oxygen species (ROS) levels (58.65% vs. 88.41%, P<0.05) and increased the ATP content (0.49 μmol/L vs. 0.25 μmol/L, P<0.05) of boar sperm during freezing. (4) The apoptotic rate of the freezing group (80.41%) with TUNEL detection increased significantly compared to the fresh group (9.70%, P<0.05), and RSV treatment greatly decreased the apoptotic rate (68.32%, P<0.05). (5) Real-time polymerase chain reaction (RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway (tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and Caspase-8), but also the genes from the mitochondria-mediated apoptotic pathway (manganese superoxide dismutase (MnSOD), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-9) were both significantly changed after freezing. RSV treatment during freezing greatly changed their expression levels. Although RSV treatment during boar semen freezing did not significantly increase motility after thawing, it still played an efficient antioxidant role, which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor- and mitochondria-mediated apoptotic pathways.

Mechanism and experimental verification of antioxidant action of Heyan Kuntai Capsule based on systematic pharmacology / 中草药

Objective: To explore and verify the potential antioxidation mechanism of traditional Chinese medicine Heyan Kuntai Capsule (HKC). Methods: Gene targets of 21 ingredients of HKC were searched for through Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis with Database for Annotation, Visualization and Integrated Discovery (DAVID) tools. Further, the anti-oxidative function of HKC was validated in an in vitro H2O2-induced oxidative stress model in MII oocytes. Results: A total of 151 genes were obtained as targets to 21 components of HKC, whose GO were further significantly enriched in mitochondrion, superoxide dismutase activity and energy pathways, while KEGG showed the enrichment in PI3K/AKT, mTOR and ErbB1 signaling pathways. Afterwards, it was confirmed that HKC increased ATP production in H2O2-treated MII oocytes by improving mitochondrial function and reduced the dissipation of mitochondrial membrane potential (ΔΨm) to exert the antioxidation effect. Conclusion:s HKC exerts anti-oxidative effect by improving mitochondrial function.

Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Resveratrol (3,5,4'-trihydroxystilbene, RSV) has been widely used in mammalian cells, but whether it can be used during freezing boar semen is still unknown. The effects of RSV treatment during boar semen freezing on its anti-freezing ability, apoptosis, and possible apoptotic pathways were observed in this study. Sperm motility, mitochondrial membrane potential (ΔΨ), adenosine triphosphate (ATP) content, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic state, and messenger RNA (mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested. The results showed that: (1) Compared with fresh sperm, the motility, normal acrosome rate, and plasma membrane integrity rate of frozen boar sperm decreased significantly (P0.05), but it did significantly improve the normal acrosome rate (57.65% vs. 47.00%, P<0.05) and plasma membrane integrity rate (46.67% vs. 38.85%, P<0.05). (2) After freezing, most boar sperm showed low mitochondrial ΔΨ. RSV treatment could increase the rate of high mitochondrial ΔΨ of boar sperm. (3) RSV treatment significantly decreased reactive oxygen species (ROS) levels (58.65% vs. 88.41%, P<0.05) and increased the ATP content (0.49 μmol/L vs. 0.25 μmol/L, P<0.05) of boar sperm during freezing. (4) The apoptotic rate of the freezing group (80.41%) with TUNEL detection increased significantly compared to the fresh group (9.70%, P<0.05), and RSV treatment greatly decreased the apoptotic rate (68.32%, P<0.05). (5) Real-time polymerase chain reaction (RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway (tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and Caspase-8), but also the genes from the mitochondria-mediated apoptotic pathway (manganese superoxide dismutase (MnSOD), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-9) were both significantly changed after freezing. RSV treatment during freezing greatly changed their expression levels. Although RSV treatment during boar semen freezing did not significantly increase motility after thawing, it still played an efficient antioxidant role, which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor- and mitochondria-mediated apoptotic pathways.

Hypoglycemic activity of puerarin through modulation of oxidative stress and mitochondrial function via AMPK / 中国天然药物

Hyperglycemia is the dominant phenotype of diabetes and the main contributor of diabetic complications. Puerarin is widely used in cardiovascular diseases and diabetic vascular complications. However, little is known about its direct effects on diabetes. The aim of our study is to investigate its antidiabetic effect in vivo and in vitro, and explore the underlying mechanism. We used type I diabetic mice induced by streptozotocin to observe the effects of puerarin on glucose metabolism. In addition, oxidative stress and hepatic mitochondrial respiratory activity were evaluated in type I diabetic mice. In vitro, glucose consumption in HepG2 cells was assayed along with the qPCR detection of glucogenesis genes expression. Moreover, ATP production was examined and phosphorylation of AMPK was determined using Western blot. Finally, the molecular docking was performed to predict the potential interaction of puerarin with AMPK utilizing program LibDock of Discovery Studio 2018 software. The results showed that puerarin improved HepG2 glucose consumption and upregulated the glucogenesis related genes expression. Also, puerarin lowered fasting and fed blood glucose with improvement of glucose tolerance in type I diabetic mice. Further mechanism investigation showed that puerarin suppressed oxidative stress and improved hepatic mitochondrial respiratory function with enhancing ATP production and activating phosphorylation of AMPK. Docking study showed that puerarin interacted with AMPK activate site and enhancing phosphorylation. Taken together, these findings indicated that puerarin exhibited the hypoglycemic effect through attenuating oxidative stress and improving mitochondrial function via AMPK regulation, which may serve as a potential therapeutic option for diabetes treatment.