Embryo development is impaired by sperm mitochondrial-derived ROS

Background Basal energetic metabolism in sperm, particularly oxidative phosphorylation, is known to condition not only their oocyte fertilising ability, but also the subsequent embryo development. While the molecular pathways underlying these events still need to be elucidated, reactive oxygen species (ROS) could have a relevant role. We, therefore, aimed to describe the mechanisms through which mitochondrial activity can influence the first stages of embryo development. Results We first show that embryo development is tightly influenced by both intracellular ROS and mitochondrial activity. In addition, we depict that the inhibition of mitochondrial activity dramatically decreases intracellular ROS levels. Finally, we also demonstrate that the inhibition of mitochondrial respiration positively influences sperm DNA integrity, most likely because of the depletion of intracellular ROS formation. Conclusion Collectively, the data presented in this work reveals that impairment of early embryo development may result from the accumulation of sperm DNA damage caused by mitochondrial-derived ROS.

The mechanism of action of mitochondrial dysfunction in the development of non-alcoholic fatty liver disease / 临床肝胆病杂志

Nonalcoholic fatty liver disease （NAFLD） has gradually become the main reason affecting human liver health， and many factors are involved in the development and progression of NAFLD. Mitochondria， as the “energy factory” of cells， plays an important role in maintaining normal physiological functions. Studies have shown that hepatic mitochondrial dysfunction promotes the development and progression of NAFLD. This article briefly introduces the latest research advances in the basic characteristics and physiological function of liver mitochondria and reviews new research findings in the association of mitochondrial dysfunction with obesity， simple fatty liver disease， and nonalcoholic steatohepatitis， in order to provide new ideas for the research on targeted mitochondrial therapy for NAFLD.

Research on BHLHE40 targets HMGA2 to reduce the sensitivity of thyroid cancer cells to cisplatin through activating the oxidative phosphorylation pathway / 国际肿瘤学杂志

Objective:To explore whether BHLHE40 can affect the sensitivity of thyroid cancer (TC) cells to cisplatin by activating oxidative phosphorylation (OXPHOS) pathway by targeting high mobility group A2 (HMGA2) .Methods:The mRNA expression of HMGA2 and its upstream transcription factor BHLHE40 in TC tissues was analyzed by TCGA-THCA and hTFtarget online databases. The si-HMGA2, oe-HMGA2, oe-BHLHE40, negative control si-NC and oe-NC were transfected into TC cells (K1 and SW579) by liposome transfection method. The mRNA expression levels of BHLHE40 and HMGA2 in TC cells (SW579, FTC-133, and K1) and normal thyroid cells (Nthy ori3-1) were detected by real-time quantitative PCR (qRT-PCR). The cell viability was detected by MTT assay, the half inhibitory concentration (IC 50) value of cisplatin was calculated by CCK-8 assay, the apoptosis level was detected by flow cytometry, and the expression of OXPHOS complex was detected by Western blotting. Seahorse XFe 96 was used to analyze the oxygen consumption rate of the TC cells. Dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay were used to analyze the binding relationship between BHLHE40 and HMGA2. Results:TCGA database results showed that the mRNA expression levels of HMGA2 and BHLHE40 in TC tissues (10.57±2.58, 13.89±1.13) were higher than those in normal thyroid tissues (4.82±1.69, 12.28±1.01), with statistically significant differences ( t=16.69, P<0.001; t=10.43, P<0.001). The results of qRT-PCR showed that the relative mRNA expression levels of HMGA2 in normal thyroid cells (Nthy ori3-1) and TC cells (SW579, FTC-133, and K1) were 1.00±0.13, 2.94±0.23, 4.71±0.41 and 6.29±0.49, while those of BHLHE40 were 1.00±0.12, 2.60±0.23, 3.39±0.35 and 6.18±0.51 respectively, both with statistically significant differences ( F=130.50, P<0.001; F=125.20, P<0.001). Further pairwise comparison showed that mRNA expression levels of HMGA2 and BHLHE40 in TC cells were significantly higher than those in normal thyroid cells (all P<0.001). According to MTT experimental results, si-HMGA2 treatment significantly reduced the cell viability of K1 cells compared to the si-NC group (all P<0.05). In addition, compared to the oe-NC group, oe-HMGA2 treatment significantly increased the cell viability of SW579 cells (all P<0.05). Compared to the oe-NC+DMSO group, the oe-HMGA2+DMSO group showed enhanced cell viability of SW579 cells, while the OXPHOS pathway inhibitor Gboxin was able to reverse the effect of overexpressing HMGA2 on cell viability (all P<0.05). The results of flow cytometry and CCK-8 experiments showed that compared to the si-NC group (apoptosis level: 6.19%±0.28%; cisplatin IC 50 value: 17.47 μmol/L), knocking down HMGA2 could increase the apoptosis level (11.96%±0.32%; t=19.17, P<0.001) and cisplatin sensitivity (IC 50 value: 1.49 μmol/L) of K1 cells. In addition, compared to the oe-NC group (apoptosis level: 9.98%±0.32%; cisplatin IC 50 value: 8.17 μmol/L), overexpressing HMGA2 significantly decreased the apoptosis level (4.32%±0.25%; t=19.65, P<0.001) and cisplatin sensitivity (IC 50 value: 34.95 μmol/L) of SW579 cells. The results of dual-luciferase assay showed that compared with the si-NC group, knocking down the expression of BHLHE40 in human kidney epithelial 293T cells significantly reduced the luciferase activity of wild-type HMGA2 (0.31±0.02 vs. 1.00±0.11; t=10.69, P=0.004). However, there was no significant effect on the luciferase activity of mutant-type HMGA2 (1.06±0.11 vs. 1.00±0.07; t=0.80, P=0.470). ChIP results showed that the mRNA expression level of HMGA2 in K1 cells was significantly increased in the anti-BHLHE40 group (6.57±0.62) compared with the IgG group (1.00±0.10; t=15.36, P<0.001). Compared to the oe-NC+DMSO group, the oe-HMGA2+DMSO group showed decreased apoptosis level ( P<0.05) and cisplatin sensitivity of SW579 cells, with a significant increase in the expression of OXPHOS complexes Ⅰ-Ⅴ and cellular oxygen consumption rates (all P<0.05). The effect of overexpressing HMGA2 was reversed by treatment with oe-HMGA2+Gboxin (all P<0.05). The recovery experiment showed that compared to the oe-NC+si-NC group, overexpression of BHLHE40 in SW579 cells increased cell viability and the expression of OXPHOS complexes Ⅰ-Ⅴ, while decreasing apoptosis levels and increasing cellular oxygen consumption rates and cisplatin IC 50 values (all P<0.05). However, simultaneous knockdown of HMGA2 reversed the effect of overexpressing BHLHE40 (all P<0.05) . Conclusion:BHLHE40 can activate the OXPHOS pathway by targeting and regulating the expression of HMGA2, thereby affecting the sensitivity of TC cells to cisplatin.

Mitochondrial combined oxidative phosphorylation deficiency type 1 in childrena case report and literature review / 中国热带医学

@#Objective　To summarize the phenotypic and genotypic characteristics of mitochondrial combined oxidative phosphorylation deficiency type 1 (COXPD1), and to improve the clinicians' awareness of this mitochondrial encephalomyopathy. Methods　The clinical characteristics, physical examination, laboratory examination and other data of a child with COXPD1 were analyzed retrospectively. The diagnosis was confirmed by clinical whole exon sequencing and high-precision mitochondrial genome full-length PLUS gene detection, and the phenotype and genotype were analyzed by reviewing relevant literature. Results　A one-year and five-month-old boy mainly presented with hyperlactacidemia and abnormal liver function. Clinical whole exon sequencing showed that the child had homozygous variation of c. 688G>A(p.G230S) in the GFM1 gene. Sanger sequencing verified that the variation was respectively inherited from the parents of the child (both were heterozygous) with the autosomal recessive inheritance pattern. The high-precision mitochondrial genome full-length PLUS detection also did not find pathogenic mutations related to clinical phenotypes. The child was diagnosed with COXPD1. After "cocktail" therapy and liver protection therapy, the patient's condition improved. Conclusions　The phenotype of COXPD1 is complicated and variable, mainly liver type and brain type. The mutation of GFM1 gene affects mitochondrial translation system function, and early gene detection is helpful for definite diagnosis.

Isoliquiritigenin alleviates energy metabolism imbalance in type 2 diabetic mice / 药学学报

Isoliquiritigenin (ISL) is a flavonoid compound isolated from licorice. It possesses excellent antioxidant and anti-diabetic activities. This study aims to investigate the molecular mechanism underlying the alleviatory effect of ISL on energy metabolism imbalance caused by type 2 diabetes mellitus (T2DM). 8-week-old male C57BL/6J mice were used in in vivo experiments. The high-fat-high-glucose diet combined with intraperitoneal injection of streptozotocin was applied to establish T2DM animal model. All animal experiments were performed in accordance with the Institutional Guidelines of Laboratory Animal Administration issued by the Committee of Ethics at Beijing University of Chinese Medicine. HepG2 cells were used in in vitro experiments. Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative polymerase chain reaction (RT-qPCR) were used to examine the protein and mRNA levels of mitochondrial function-related targets. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in HepG2 cells were measured by the flow cytometry. Additionally, the molecular docking of ISL and key target proteins was analyzed. It was found that ISL significantly inhibited the activity of mitochondrial respiratory chain complex I and increased the protein levels of uncoupling protein 2 (UCP2) in the livers of mice and HepG2 cells. It also obviously decreased the ROS levels and increased the MMP levels in cultured HepG2 cells. In addition, ISL promoted mitochondrial biogenesis by activating proliferator-activated receptor gamma co-activator 1α (PGC-1α) and enhanced mitophagy by upregulating Parkin. It also improved mitochondrial fusion by increasing the mRNA and protein levels of mitofusin 2 (MFN2). In conclusion, ISL alleviates energy metabolism imbalance caused by T2DM through suppression of excessive mitochondrial oxidative phosphorylation and promotion of mitochondrial biogenesis, mitophagy, and fusion.

Combined oxidative phosphorylation deficiency type 4: clinical features and TUFM gene mutation analysis / 中华神经科杂志

Objective:To investigate the clinical characteristics of patients with combined oxidative phosphorylation deficiency type 4 (COXPD4) related to TUFM gene variation, in order to improve clinicians′ understanding of the disease. Methods:A case of COXPD4 with cystic leukodystrophy admitted to the Children′s Hospital of Zhengzhou University in June 2021 was taken as the study subject, and her clinical characteristics and genetic testing results were retrospectively analyzed. The "combined oxidative phosphorylation deficiency type 4" " TUFM gene" "cystic leukodystrophy" "combined oxidative phosphorylation deficiency 4" "COXPD 4" " TUFM" and "cystic leukodystrophy" were used as keywords, and the documents on COXPD4 related to TUFM gene mutations were reviewed from Wanfang Data Knowledge Service Platform, CNKI, PubMed Document Database, and National Center for Biotechnology Information (NCBI) until August 2021. The COXPD4 patients that have been reported internationally were analyzed for clinical features and variant types. Results:The patient was a 2-month-old girl with clinical manifestations of delayed development and progressive aggravation, elevated lactic acid in serum and cerebrospinal fluid, and diffuse white matter dysplasia with multiple cystic lesions in cerebral magnetic resonance imaging (MRI). Whole exome sequencing showed TUFM gene complex heterozygous variants c.684_684+4delGGTGA and c.1105C>T, which had not been reported in the past. A total of 5 cases of COXPD4 were reported in 4 English literatures. Together with 1 case in this study, there were 4 cases with detailed clinical history data, including 1 male and 3 females. The clinical manifestations were severe early-onset lactic acidosis and developmental lag, and 3 cases were accompanied by progressive infantile encephalopathy. Among them, 3 cases underwent head MRI examination, all of which showed diffuse white matter signal with multiple cystic lesions, 2 cases with basal ganglia involvement and multiple cerebellar gyri deformity. Genetic test indicated different types of TUFM gene variation. Conclusions:COXPD4 is a rare hereditary mitochondrial disease. For cases with COXPD4 clinical and imaging features, TUFM gene mutations can be screened first.

Targeting metabolic vulnerability in mitochondria conquers MEK inhibitor resistance in KRAS-mutant lung cancer

MEK is a canonical effector of mutant KRAS; however, MEK inhibitors fail to yield satisfactory clinical outcomes in KRAS-mutant cancers. Here, we identified mitochondrial oxidative phosphorylation (OXPHOS) induction as a profound metabolic alteration to confer KRAS-mutant non-small cell lung cancer (NSCLC) resistance to the clinical MEK inhibitor trametinib. Metabolic flux analysis demonstrated that pyruvate metabolism and fatty acid oxidation were markedly enhanced and coordinately powered the OXPHOS system in resistant cells after trametinib treatment, satisfying their energy demand and protecting them from apoptosis. As molecular events in this process, the pyruvate dehydrogenase complex (PDHc) and carnitine palmitoyl transferase IA (CPTIA), two rate-limiting enzymes that control the metabolic flux of pyruvate and palmitic acid to mitochondrial respiration were activated through phosphorylation and transcriptional regulation. Importantly, the co-administration of trametinib and IACS-010759, a clinical mitochondrial complex I inhibitor that blocks OXPHOS, significantly impeded tumor growth and prolonged mouse survival. Overall, our findings reveal that MEK inhibitor therapy creates a metabolic vulnerability in the mitochondria and further develop an effective combinatorial strategy to circumvent MEK inhibitors resistance in KRAS-driven NSCLC.

Berberine targets the electron transport chain complex I and reveals the landscape of OXPHOS dependency in acute myeloid leukemia with IDH1 mutation / 中国天然药物

Metabolic reprogramming, a newly recognized trait of tumor biology, is an intensively studied prospect for oncology medicines. For numerous tumors and cancer cell subpopulations, oxidative phosphorylation (OXPHOS) is essential for their biosynthetic and bioenergetic functions. Cancer cells with mutations in isocitrate dehydrogenase 1 (IDH1) exhibit differentiation arrest, epigenetic and transcriptional reprogramming, and sensitivity to mitochondrial OXPHOS inhibitors. In this study, we report that berberine, which is widely used in China to treat intestinal infections, acted solely at the mitochondrial electron transport chain (ETC) complex I, and that its association with IDH1 mutant inhibitor (IDH1mi) AG-120 decreased mitochondrial activity and enhanced antileukemic effect in vitro andin vivo. Our study gives a scientific rationale for the therapy of IDH1 mutant acute myeloid leukemia (AML) patients using combinatory mitochondrial targeted medicines, particularly those who are resistant to or relapsing from IDH1mi.

Association of serum lactate levels among patients with Type 2 diabetes mellitus of a tertiary care hospital, Uttar Pradesh

Background: Serum lactate levels are a direct quantification of gap in between energy-expenditure and oxidative capacity. Variation in the lactate levels among the resting individuals could be exigent, requiring attention often and raised lactate levels can be observed among the individuals with mitochondrial impairments in the oxidative phosphorylation pathways. Aim and Objectives: Aim and objectives of the study was to evaluate the association of serum lactate levels among Type 2 diabetes mellitus patients. Materials and Methods: This present observational cross-sectional study involved 300 diabetic patients who presented to the tertiary care hospital of Uttar Pradesh. The patients were stratified according to their random blood sugar, Glycated hemoglobin and lactate levels. Student t-test was used for assessing the significance, a probability of <0.05 is considered significant. Results: A total of 300 patients have been included in this study, of which 150 are males and 150 are females, the median age was 48 years. The highest age of patient included was 84 years, and the lowest of patient included in the study was 34 years. The patients with high lactate levels are found to have poor glycaemic control, which may further proceed to worst outcome of the disease. Conclusion: Serum lactate levels could be the potential predictor and reliable indicator of poor glycemic control/status of diabetic patients.

Markers of mitochondrial energy metabolism and their potential relationships with fatigue in human adults: a scoping review

This scoping review aimed to synthesize the best available evidence of the associations between molecular and genetic markers of mitochondrial metabolism and fatigue in human adults. The research question guiding this review was, "Are there potential relationships between mitochondrial metabolism markers and fatigue?" The literature search used three terms (mitochondria; fatigue; energy metabolism), which yielded 263 manuscripts and 22 theses/dissertations. The studies included in the review had to meet three criteria: (1) Include adult participants (≥18 years of age); (2) Show a relationship between mitochondrial energy metabolism and fatigue; (3) Be published in English, Spanish, or Portuguese. Of the 17 articles included for a full-text review, some had a cross-sectional design (6/17, 35%), and more than half (12/17, 70%) were published between 2015 and 2020. The predominant population studied were patients diagnosed with chronic fatigue syndrome (9/17, 53%). Most studies (15/17, 88%) assessed fatigue with validated instruments. Mitochondrial markers associated with fatigue are a) mitochondrial transport pathways and respiratory chain, b) mutations in mitochondrial DNA, and c) energy disorders in cells of the immune system, such as natural killer cells. Mitochondrial metabolic activities, such as the production and transport of ATP, are significant components that may help understand the etiology of fatigue. Future directions should include longitudinal study designs, characterization of fatigue phenotypes, and the identification of markers involved in production and transport pathways. The clinical relevance in this field can lead to interventions targeting mitochondrial markers to reduce or prevent fatigue.

Case report of compound oxidative phosphorylation deficiency type 10 caused by a new site mutation of MTO1 gene / 中华实用儿科临床杂志

The clinical data of a case of compound oxidative phosphorylation deficiency type 10 (COXPD10) caused by a new site mutation of MTO1 gene in the Department of Pediatrics, Affiliated Hospital of Southwest Medical University on December 29, 2020 were retrospectively analyzed.The patient was a 2 months and 19 days old boy of Han nationality.The main clinical manifestations were shortness of breath, hyperlactic acidemia, hyperammonemia and brain damage.Cardiac hypertrophy was not obvious.Heterozygous mutations at c. 344delA and c. 1055C>T sites in the MTO1 gene have not been reported in domestic and foreign literature.COXPD10 caused by MTO1 gene mutations may result in diversified clinical manifestations due to inconsistent mutation sites.For hyperlactic acidemia with unknown predisposing factors, early genetic examination should be conducted to confirm the possibility of COXPD10.

Oxidative phosphorylation in septic organ injury / 国际儿科学杂志

Sepsis is a systemic inflammatory response syndrome caused by pathogenic microorganisms that infect the host.If treated improperly, it can progress to severe sepsis or even septic shock.As such, it′s one of the main reasons for the death of children in PICU.The inflammatory response of sepsis exerts great influence on a series of basic physiological functions of cells, including the oxidative phosphorylation of the mitochondria.Oxidative phosphorylation is a process during which oxygen is reduced to generate high-energy phosphate bonds in the form of adenosine triphosphate(ATP), which supplies energy for cells and produces a series of functional by-products.During sepsis, the process of oxidative phosphorylation in mitochondria undergoes a series of complex alterations, which in turn can further promote the development of septic organ injury.The present review aims to clarify the relationship between changes in oxidative phosphorylation and the impairment of various organs in sepsis.

Combined oxidative phosphorylation deficiency type 28: a case report and literature review / 中华实用儿科临床杂志

Objective:To summarize the clinical manifestations of a case of combined oxidative phosphorylation deficiency 28 (COXPD-28) caused by the mutations of the SLC25A26 gene, thus providing references for the diagnosis and genetic counseling of the disease. Methods:Clinical data of a case of COXPD-28 treated in the Third Affiliated Hospital of Zhengzhou University in October 2020 were retrospectively analyzed.In addition, The retrieval words " Combined oxidative phosphorylation deficiency 28, SLC25A26 gene" were used to search domestic and foreign databases.The clinical characteristics of combined phosphorylation deficiency 28 and the variation characteristics of SLC25A26 gene were summarized. Results:(1) A female patient full-term delivered after 30 min presented with groaning breath was admitted.Her main manifestations included pale complexion, groaning breathing, metabolic acidosis, and high lactate and pyruvate levels.Symptomatic support treatment like anti-infection and assisted ventilation were given, but her condition gradually worsened and died of respiratory and circulatory failure on the day of admission.The child was compound heterozygous mutation of SLC25A26 gene, the terminating mutation of exon 5 c. 403G>T caused the protein change to p. E135, and the non-synonymous mutation of exon 4 c. 212A>G caused the protein change to p. Y71C.(2) A total of 3 cases of COXPD-28 were searched in online databases, and no cases were reported in China.Through literature review, clinical features of COXPD-28 mainly included respiratory and circulatory fai-lure, elevations of lactate and pyruvate, and reductions of complexes Ⅰ, Ⅲ and Ⅳ in muscle biopsy.Two types of mutations in the SLC25A26 gene were detected, including 3 cases of missense mutations and 1 case of splicing mutation. Conclusions:COXPD-28 is an autosomal recessive genetic disease involving multiple systems and mitochondrial dysfunction.Mutations in the SLC25A26 gene is the pathological cause of COXPD-28.

Hexokinase inhibitor 2-deoxyglucose combined with phenformin induces cell apoptosis of triple-negative breast cancer / 药学实践杂志

Objective To investigate the effect of phenformin combined with hexokinase inhibitor 2-deoxyglucose (2-DG) on the treatment of triple-negative breast cancer cell lines 4T1 and MDA-MB-231. Methods Following treatment with phenformin, 2-DG or phenformin combined with 2-DG on 4T1 and MDA-MB-231 cells for 48 h, the cell proliferation in each group was detected by SRB and the apoptosis of cells was detected by flow cytometry. The concentration of glucose and lactic acid in cell culture supernatant was detected by ELISA. The activity of mitochondrial respiratory chain complex Ⅰ was detected by FlexStation3 and the mitochondrial oxygen consumption (OCR) was assayed with the Seahorse X Fe Analyzer. Results The hexokinase expression (4.6±0.17,3.73±0.21), glucose consumption (356±31，397±42) μg/105 cells , Lactic acid concentration (5.59±0.52, 7.83±0.78) μmol/L in the supernatant of 4T1 and MDA-MB-231 cells in Phenformin group were higher than that in control group ( 1±0.15，1±0.12 ) , ( 289±25，301±32) μg/105cells , ( 2.37±0.18，4.01±0.45) μmol/L (P < 0.01). Even if the dose was reduced by 90%, the cell viability of phenformin combined with 2-DG group (64.63±2.28, 51.97±2.29) % was still higher than that of phenformin group (86.70±1.83, 85.53±1.46) % (P<0.001). The combination of the two drugs significantly promoted the apoptosis of 4T1 and MDA-MB-231. In addition, compared with the phenformin group (5.59±0.52, 7.83±0.78) μmol/L, the phenformin combined with 2-DG group (3.46±0.37, 5.18±0.62) μmol/L cell lactic acid production also greatly reduced (P<0.01). Compared with the phenformin or 2-DG single-drug group, the phenformin combined with 2-DG group can significantly inhibit the growth rate of tumors in tumor-bearing mice (P<0.01). The median survival time of tumor-bearing mice in the phenformin combined with 2-DG group was 72.5 d, which was higher than that in the phenformin group 57 d and 2-DG group 55.5 d (P<0.01). Conclusion Hexokinase inhibitor 2-DG significantly enhances the therapeutic effects of phenformin on triple-negative breast cancer cells.

(+)-corynoline Regulates the Proliferation,Stemness and Apoptosis of Triple Negative Breast Cancer Cells / 中国医学科学院学报

Objective To explore the performance and mechanism of(+)-corynoline in treating triple negative breast cancer MDA-MB-436 cells and thus provide an option for the development of drugs against this cancer. Methods The viability,proliferation,apoptosis and migration/invasion of MDA-MB-436 cells treated with(+)-corynoline were detected by CCK-8 assay,colony formation assay,flow cytometry and Transwell assay,respectively.Furthermore,Western blotting was employed to determine the expression of related proteins,and RNA-Seq was performed for the MDA-MB-436 cells treated with(+)-corynoline. Results (+)-corynoline inhibited the proliferation and stemness and promoted the apoptosis of MDA-MB-436 cells.Further,(+)-corynoline may activate the oxidative phosphorylation pathway to play a role in inhibiting triple negative breast cancer. Conclusion (+)-corynoline can inhibit triple negative breast cancer cells,which helps to address the poor efficacy of existing chemotherapeutics and facilitate the development of drugs against this cancer.

Identification and functional analysis of combined oxidative phosphorylation deficiency 28 gene mutation / 中华内科杂志

Objective:To report a case of combined oxidative phosphorylation deficiency 28 (COXPD28) in China, identified the pathogenic mutation and explored the pathogenic mechanism preliminarily.Methods:The clinical characteristics of a patient with COXPD28 were retrospectively analyzed and the pathogenic mutations were identified by mitochondrial gene sequencing and whole exome sequencing. The wild-type and mutant plasmids of pathogenic genes were constructed, and effect of mutation on protein expression by quantitative real-time PCR (qPCR) and Western blot were evaluated. Statistical methods mainly used one-way ANOVA and LSD test.Results:A 21 year old female patient presented with lactic acid poisoning due to repeated chest distress and wheezing since childhood. The sequencing of the whole exon group gene found that solute carrier family 25 member 26 (SLC25A26) gene had a compound heterozygous mutation (c.34G>C, p.A12P; c.197C>A, p.A66E), which was the first report in China. In vitro function test showed that the expression levels of SLC25A26 mRNA and S-adenosylmethionine carrier (SAMC) protein in cells transfected with SLC25A26 mutant plasmid were significantly lower than those transfected with wild type plasmid. The p.A66E mutant plasmid reduced the expression level of SLC25A26 mRNA and SAMC protein to 6% and 26% of wild type plasmids respectively (both P<0.001), while p.A12P mutant plasmid decreased to 62% and 82% of wild type plasmids respectively ( P<0.001, P=0.044). When the double mutant (p.A66E+p.A12P) plasmids were co-transfected, the expression levels of SLC25A26 mRNA and SAMC protein decreased to 47% and 57% of the wild type plasmids, respectively ( P<0.001, P=0.001). Conclusion:The pathogenic mutation gene of this patient with COXPD28 is SLC25A26 gene mutation (p.A66E, p.A12P), which causes the decrease of SLC25A26 expression level, mitochondrial oxidative phosphorylation dysfunction, and induces COXPD28.

Effect of Dihydroartemisinin on Oxidative Damage and Energy Metabolism of HepG2 Cells and Its Synergistic Effect with Sorafenib / 中国实验方剂学杂志

Objective:To explore the inhibitory effect of dihydroartemisinin （DHA） on the proliferation of HepG2 cells， elucidate the mechanism from the perspectives of oxidative damage and energy metabolism， and discuss the possibility of combined use of DHA with sorafenib （Sora）. Method:Cell counting kit-8 （CCK-8） assay was used to obtain the 50% inhibitory concentration （IC₅₀） of DHA and Sora on HepG2 and SW480 cells and Chou-Talalay method was used to obtain the combination index （CI） of DHA and Sora. HepG2 cells were classified into the control group， DHA group （10 µmol·L^-1）， Sora group （5 µmol·L^-1）， and DHA + Sora group （DHA 10 µmol·L^-1， Sora 5 µmol·L^-1） and then incubated with corresponding drugs for 8-12 h. Seahorse XF glycolytic rate assay kit and cell mito stress test kit were employed to respectively detect the glycolysis function of cells and oxidative phosphorylation function of mitochondria. DCFH-DA and lipid peroxidation MDA assay kit were separately used to analyze the intracellular levels of reactive oxygen species （ROS） and malondialdehyde （MDA）. Western blot was applied to determine the intracellular levels of heme oxygenase-1 （HO-1） and glutamate-cysteine ligase catalytic subunit （GCLC）. Result:Compared with the control group， DHA alone inhibited the ATP synthesis in mitochondrial oxidative phosphorylation and glycolysis （<italic>P</italic><0.01）， increased the levels of intracellular ROS and MDA （<italic>P<</italic>0.05）， and decreased the levels of HO-1 and GCLC （<italic>P<</italic>0.05） in HepG2 cells. DHA and Sora had synergistic inhibitory effect on proliferation of HepG2 and SW480 cells， with CI < 0.90. The DHA + Sora group showed stronger suppression of ATP synthesis in mitochondrial oxidative phosphorylation and glycolysis （<italic>P</italic><0.01）， higher levels of intracellular ROS and MDA （<italic>P<</italic>0.01）， and lower levels of intracellular antioxidation-related proteins HO-1 and GCLC in HepG2 cells （<italic>P<</italic>0.01） than the DHA group. Conclusion:DHA may increase the level of MDA by reducing HO-1 and GCLC and increasing ROS in HepG2 cells， which results in mitochondria oxidative damage， restricts cell glycolysis and mitochondrial oxidative phosphorylation， and thus finally inhibits the proliferation of HepG2 cells. DHA and Sora have synergistic inhibitory effect on the proliferation of HepG2 and SW480 cells， and the mechanism may be related to the synergistic oxidative damage that affects the mitochondrial electron transport chain and suppresses cell energy metabolism.

Effect of Echinococcus multilocularis infections on mitochondrial functions of macrophages / 中国血吸虫病防治杂志

Objective To investigate the changes of mitochondrial metabolic functions of macrophages following Echinococcus multilocularis infections, so as to provide insights into the pathogenesis of alveolar echinococcosis. Methods Two groups were assigned according to different treatment methods. In the culture group, mouse leukemic monocyte macrophage RAW264.7 cells were cultured with 2 000 E. multilocularis at a ratio of 500∶1, while RAW264.7 cells in the control group were given no treatment. Then, both the culture and control groups were further divided into the 24 h and 72 h subgroups. Mitochondria were stained with MitoTracker® Deep Red FM and the mean fluorescence intensity of macrophage mitochondria was measured with the Cytation 5 Cell Imaging Multi-Mode Reader. The mitochondrial DNA copy number was quantified using the quantitative real-time PCR (qPCR) assay, and the mitochondrial energy metabolism was monitored using the Seahorse XF assay. In addition, the mitochondrial reactive oxygen species and mitochondrial membrane potential were detected using flow cytometry. Results The mean fluorescence intensities of macrophage mitochondria were significantly lower in the 24 h (15 341 ± 2 532 vs. 17 823 ± 3 429; t = 6.379, P < 0.01) and 72 h (18 102 ± 3 505 vs. 21 511 ± 5 144; t = 17.680, P < 0.01) culture subgroups than in the corresponding control subgroups, and lower mitochondrial DNA copy numbers were measured in the 72 h culture subgroup than in the 72 h control group [(3.23 × 109 ± 1.78 × 107) vs. (4.39 × 109 ± 3.70 × 107); t = 8.85, P < 0.001]. The oxygen consumption rates were significantly greater in the 24 h [(241.70 ± 73.13) pmol/min vs. (69.05 ± 52.30) pmol/min; t = 7.89, P < 0.01] and 48 h culture groups [(249.50 ± 42.06) pmol/min vs. (60.28 ± 40.66) pmol/min; t = 8.64, P < 0.01] than in the corresponding control groups, and a higher extracellular acidification rate was seen in the 48 h culture group than in the 48 h control group ([ 111.6 ± 17.49) mpH/min vs. (35.05 ± 7.57) mpH/min; t = 16.90, P < 0.01]. In addition, flow cytometry detected higher mean fluorescence intensity of mitochondrial reactive oxygen species (58 264 ± 10 087 vs. 4 307 ± 97; t = 12.930, P < 0.01) and lower mitochondrial membrane potential (9.833% ± 2.285% vs. 2.667% ± 0.208%; t = 6.645, P < 0.01) in the 72 h culture group than in the control group. Conclusions E. multilocularis infection may impair mitochondrial functions and inhibit oxidative phosphorylation of macrophages, resulting in increased macrophage glycolysis. It is speculated that the alteration of macrophage metabolic states may contribute to the mechanisms underlying the development and progression of alveolar echinococcosis.

Altered Energy Metabolism During Early Optic Nerve Crush Injury: Implications of Warburg-Like Aerobic Glycolysis in Facilitating Retinal Ganglion Cell Survival / 神经科学通报·英文版

Neurons, especially axons, are metabolically demanding and energetically vulnerable during injury. However, the exact energy budget alterations that occur early after axon injury and the effects of these changes on neuronal survival remain unknown. Using a classic mouse model of optic nerve-crush injury, we found that traumatized optic nerves and retinas harbor the potential to mobilize two primary energetic machineries, glycolysis and oxidative phosphorylation, to satisfy the robustly increased adenosine triphosphate (ATP) demand. Further exploration of metabolic activation showed that mitochondrial oxidative phosphorylation was amplified over other pathways, which may lead to decreased retinal ganglion cell (RGC) survival despite its supplement to ATP production. Gene set enrichment analysis of a microarray (GSE32309) identified significant activation of oxidative phosphorylation in injured retinas from wild-type mice compared to those from mice with deletion of phosphatase and tensin homolog (PTEN), while PTEN-/- mice had more robust RGC survival. Therefore, we speculated that the oxidation-favoring metabolic pattern after optic nerve-crush injury could be adverse for RGC survival. After redirecting metabolic flux toward glycolysis (magnifying the Warburg effect) using the drug meclizine, we successfully increased RGC survival. Thus, we provide novel insights into a potential bioenergetics-based strategy for neuroprotection.