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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.
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Liver failure （LF）， as a clinical syndrome of severe hepatocyte damage and liver dysfunction， has become a major obstacle to human health due to the triple superposition of high mortality， high morbidity， and high medical resource depletion. It is of great significance to further study the core factors of the disease and supplementary treatment methods to improve the survival rate of patients with LF. The pathogenesis of LF is complex， and mitochondrion is one of the sensitive organelles in hepatocytes and the central link of intracellular energy metabolism. A large number of studies have shown that the structure and function of mitochondria in hepatocytes are changed in LF， and the abnormal structure and function of mitochondria play an important role in the process of LF disease. Among them， multiple factors such as mitochondrial respiratory chain disorder， mitochondrial DNA damage， mitochondrial permeability transition pore opening， mitochondrial quality control imbalance， and mitochondrial oxidative stress are intertwined， forming a complex and unified whole network， which becomes the key node affecting the progression of LF. In recent years， researchers have begun to study drugs that can regulate the function of liver mitochondria to prevent and treat LF. With the deepening of research， traditional Chinese medicine has made breakthroughs in the prevention and treatment of LF. Many studies have confirmed that traditional Chinese medicine can play a role in the prevention and treatment of LF by protecting mitochondrial function， which can be summarized as reducing liver cell damage， inhibiting liver cell death， and promoting liver cell regeneration， so as to effectively compensate for liver function and promote the recovery of liver parenchyma quality and function. This article summarized the structure and function of mitochondria， the relationship between LF and mitochondria， and the research on the intervention of mitochondrial function in the field of traditional Chinese medicine to prevent and treat LF， so as to provide certain ideas and references for the clinical treatment of LF with traditional Chinese medicine.
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Abstract Objective Endometriosis causes a decrease in oocyte quality. However, this mechanism is not fully understood. The present study aimed to analyze the effect of endometriosis on cumulus cell adenosine triphosphate ATP level, the number of mitochondria, and the oocyte maturity level. Methods A true experimental study with a post-test only control group design on experimental animals. Thirty-two mice were divided into control and endometriosis groups. Cumulus oocyte complex (COC) was obtained from all groups. Adenosine triphosphate level on cumulus cells was examined using the Elisa technique, the number of mitochondria was evaluated with a confocal laser scanning microscope and the oocyte maturity level was evaluated with an inverted microscope. Results The ATP level of cumulus cells and the number of mitochondria in the endometriosis group increased significantly (p < 0.05; p < 0.05) while the oocyte maturity level was significantly lower (p < 0.05). There was a significant relationship between ATP level of cumulus cells and the number of mitochondrial oocyte (p < 0.01). There was no significant relationship between cumulus cell ATP level and the number of mitochondrial oocytes with oocyte maturity level (p > 0.01; p > 0.01). The ROC curve showed that the number of mitochondrial oocytes (AUC = 0.672) tended to be more accurate than cumulus cell ATP level (AUC = 0.656) in determining the oocyte maturity level. Conclusion In endometriosis model mice, the ATP level of cumulus cells and the number of mitochondrial oocytes increased while the oocyte maturity level decreased. There was a correlation between the increase in ATP level of cumulus cells and an increase in the number of mitochondrial oocytes.
Subject(s)
Animals , Rats , Oocytes , Adenosine Triphosphate , Endometriosis , Cumulus Cells , Reproductive Health , MitochondriaABSTRACT
SUMMARY: Gastrin plays a vital role in the development and progression of gastric cancer (GC). Its expression is up-regulated in GC tissues and several GC cell lines. Yet, the underlying mechanism remains to be investigated. Here, we aim to investigate the role and mechanism of gastrin in GC proliferation. Gastrin-overexpressing GC cell model was constructed using SGC7901 cells. Then the differentially expressed proteins were identified by iTRAQ analysis. Next, we use flow cytometry and immunofluorescence to study the effect of gastrin on the mitochondrial potential and mitochondria-derived ROS production. Finally, we studied the underlying mechanism of gastrin regulating mitochondrial function using Co-IP, mass spectrometry and immunofluorescence. Overexpression of gastrin promoted GC cell proliferation in vitro and in vivo. A total of 173 proteins were expressed differently between the controls and gastrin- overexpression cells and most of these proteins were involved in tumorigenesis and cell proliferation. Among them, Cox17, Cox5B and ATP5J that were all localized to the mitochondrial respiratory chain were down-regulated in gastrin-overexpression cells. Furthermore, gastrin overexpression led to mitochondrial potential decrease and mitochondria-derived ROS increase. Additionally, gastrin-induced ROS generation resulted in the inhibition of cell apoptosis via activating NF-kB, inhibiting Bax expression and promoting Bcl-2 expression. Finally, we found gastrin interacted with mitochondrial membrane protein Annexin A2 using Co-IP and mass spectrometry. Overexpr ession of gastrin inhibits GC cell apoptosis by inducing mitochondrial dysfunction through interacting with mitochondrial protein Annexin A2, then up-regulating ROS production to activate NF-kB and further leading to Bax/Bcl-2 ratio decrease.
La gastrina juega un papel vital en el desarrollo y progresión del cáncer gástrico (CG). Su expresión está regulada al alza en tejidos de CG y en varias líneas celulares de CG. Sin embargo, el mecanismo subyacente aun no se ha investigado. El objetivo de este estudio fue investigar el papel y el mecanismo de la gastrina en la proliferación de CG. El modelo de células CG que sobre expresan gastrina se construyó usando células SGC7901. Luego, las proteínas expresadas diferencialmente se identificaron mediante análisis iTRAQ. A continuación, utilizamos la citometría de flujo y la inmunofluorescencia para estudiar el efecto de la gastrina en el potencial mitocondrial y la producción de ROS derivada de las mitocondrias. Finalmente, estudiamos el mecanismo subyacente de la gastrina que regula la función mitocondrial utilizando Co-IP, espectrometría de masas e inmunofluorescencia. La sobreexpresión de gastrina promovió la proliferación de células CG in vitro e in vivo. Un total de 173 proteínas se expresaron de manera diferente entre los controles y las células con sobreexpresión de gastrina y la mayoría de estas proteínas estaban implicadas en la tumorigenesis y la proliferación celular. Entre estas, Cox17, Cox5B y ATP5J, todas localizadas en la cadena respiratoria mitocondrial, estaban reguladas a la baja en las células con sobreexpresión de gastrina. Además, la sobreexpresión de gastrina provocó una disminución del potencial mitocondrial y un aumento de las ROS derivadas de las mitocondrias. Por otra parte, la generación de ROS inducida por gastrina resultó en la inhibición de la apoptosis celular mediante la activación de NF-kB, inhibiendo la expresión de Bax y promoviendo la expresión de Bcl-2. Finalmente, encontramos que la gastrina interactuaba con la proteína de membrana mitocondrial Anexina A2 usando Co-IP y espectrometría de masas. La sobreexpresión de gastrina inhibe la apoptosis de las células CG al inducir la disfunción mitocondrial a través de la interacción con la proteína mitocondrial Anexina A2, luego regula el aumento de la producción de ROS para activar NF-kB y conduce aún más a la disminución de la relación Bax/Bcl-2.
Subject(s)
Animals , Mice , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Gastrins/metabolism , Annexin A2/metabolism , Mitochondria/pathology , Mass Spectrometry , NF-kappa B , Fluorescent Antibody Technique , Reactive Oxygen Species , Apoptosis , Cell Line, Tumor , Immunoprecipitation , Cell Proliferation , Carcinogenesis , Flow CytometryABSTRACT
Objective:To investigate whether propofol can cause injury to hippocampal mitochondria in neonatal rats and the regulation of excitatory amino acid receptor AMPA receptor.Methods:Forty-eight Sprague-Dawley (SD) rats aged 7 days were randomly divided into control group, propofol group, propofol+AMPA receptor agonist AMPA group (propofol+AMPA group) and propofol+AMPA receptor inhibitor CNQX group (propofol+CNQX group), with 12 rats in each group. The rats in the propofol groups were intraperitoneally injected with 30 mg/kg propofol, while in control group with 3 mg/kg normal saline. Each group was given 1/2 of the first dose every 20 minutes after the first administration, three times a day, for three consecutive days. The rats in the propofol+AMPA group and the propofol+CNQX group were injected with 1 g/L AMPA or CNQX 5 μL through left ventricle after the first administration. Three days after administration, the rats were sacrificed to obtain brain tissue. Western blotting was used to determine the expression of AMPA receptor glutamate receptors (GluR1, GluR2) subunit totally (T) and on membrane (M) in hippocampus. The expression of dynamin-related protein-1 (DRP-1) and phosphorylated-DRP-1 (p-DRP-1) and mitofusin 2 (Mfn2) related to mitochondrial fission and fusion were determined. The adenosine triphosphate (ATP) content and ATPase activity were determined.Results:Compared with the control group, GluR1 expression and its M/T ratio were significantly increased after treatment of propofol, GluR2 expression and its M/T ratio were significantly decreased, the ATP content and ATP-related enzyme activity were decreased significantly, while the expression of DRP-1 and its phosphorylation was significantly increased, and the expression of Mfn2 was significantly decreased. The changes indicated that repeated intraperitoneal injection of 30 mg/kg propofol leading to the injury of mitochondria in neural cells. Compared with the propofol group, the GluR1 expression and its M/T ratio further increased after AMPA agonist administration [T-GluR1 protein (T-GluR1/β-actin): 2.41±0.29 vs. 1.72±0.11, M-GluR1 protein (M-GluR1/β-actin): 1.18±0.15 vs. 0.79±0.09, M/T ratio: 0.78±0.12 vs. 0.46±0.08, all P < 0.01], GluR2 expression was significantly increased [T-GluR2 protein (T-GluR2/β-actin): 0.65±0.13 vs. 0.30±0.14, P < 0.01; M-GluR2 protein (M-GluR2/β-actin): 0.17±0.05 vs. 0.13±0.07, P > 0.05], but its M/T ratio was further decreased (0.27±0.10 vs. 0.41±0.08, P < 0.05). The ATP-related enzyme activity was further decreased, and the ATP content was further decreased (μmol/g: 0.32±0.07 vs. 0.70±0.10, P < 0.01). Mitochondria DRP-1 expression and its phosphorylation were further increased [DRP-1 protein (DRP-1/GAPDH): 2.75±0.36 vs. 1.70±0.19, p-DRP-1 protein (p-DRP-1/GAPDH): 0.99±0.14 vs. 0.76±0.15, both P < 0.05], and Mfn2 expression was further decreased (Mfn2/GAPDH: 0.23±0.12 vs. 0.54±0.12, P < 0.05). This indicated that the AMPA agonist increased the expression of the AMPA receptor GluR1 subunit on the cell membrane and shifted the GluR2 into the cell, thus increasing the mitochondrial injury caused by propofol. Compared with the propofol group, the GluR1 expression and its M/T ratio decreased significantly after AMPA inhibitor administration [T-GluR1 protein (T-GluR1/β-actin): 0.99±0.14 vs. 1.72±0.11, M-GluR1 protein (M-GluR1/β-actin): 0.21±0.07 vs. 0.79±0.09, M/T ratio: 0.21±0.07 vs. 0.46±0.08, all P < 0.01], the change of GluR2 expression was not significant, but its M/T ratio was significantly increased (0.59±0.09 vs. 0.41±0.08, P < 0.05). The ATP-related enzyme activity was increased significantly, and the ATP content was increased significantly (μmol/g: 0.87±0.12 vs. 0.70±0.10, P < 0.05). Mitochondria DRP-1 expression and its phosphorylation were significantly decreased [DRP-1 protein (DRP-1/GAPDH): 1.18±0.17 vs. 1.70±0.19, p-DRP-1 protein (p-DRP-1/GAPDH): 0.37±0.10 vs. 0.76±0.10, both P < 0.05], and Mfn2 expression was significantly increased (Mfn2/GAPDH: 0.78±0.10 vs. 0.54±0.12, P < 0.05). This indicated that AMPA inhibitor promoted the movement to the cell membrane of GluR2 subunits meanwhile inhibited the expression of GluR1 subunits, thus alleviating the injury of mitochondrial caused by propofol in the brain. Conclusions:Repeated intraperitoneal injection of 30 mg/kg propofol for 3 days can increase the expression of GluR1 subunits of AMPA receptor in 7-day neonatal rats hippocampus mainly distributing in the cell membrane, decrease the expression of GluR2 subunits moving into the cell, thus causing injury of mitochondrial function and dynamics, which can be aggravated by AMPA receptor agonist and alleviated by AMPA receptor inhibitors.
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Cancer cachexia is a multifactorial syndrome characterized by the continuous loss of skeletal muscle. The pathogenic mechanism of cancer cachexia remains unknown, and the effectiveness of routine nutritional therapy is limited. The mitochondrial disorder is demonstrated to play an important role in the mechanism of tumor-induced skeletal muscle atrophy, including alterations to the mitochondrial ultrastructure, biogenesis, dynamics, mitophagy, and functions. Interventions targeting mitochondrial alterations provide a potential solution to cancer cachexia and represent a new focus in this research field.
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@#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.
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Objective To investigate the inhibitory effect of ursolic acid in Hippophae rhamnoides L. on hepatocyte apoptosis in rats with alcoholic liver disease based on the mitochondria-cytochrome c pathway. Methods A total of 50 specific pathogen-free male Wistar rats were divided into normal control group, alcohol model group, and low-, middle-, and high-dose ursolic acid groups using a random number table, with 10 rats in each group. The rats in the normal control group were given normal saline by gavage once a day for 8 weeks; the rats in the alcohol model group were given alcohol at increasing concentrations by gavage for 8 consecutive weeks; the rats in the low-, middle-, and high-dose ursolic acid groups were given ursolic acid at a dose of 50, 100, and 150 mg/kg, respectively, followed by an equal volume of alcohol as the model group 1 hour later. Serum liver function parameters were measured for each group; HE staining was used to observe liver histopathology; an electron microscope was used to observe hepatocyte ultrastructure; the TUNEL method was used to measure hepatocyte apoptosis; Western Blotting was used to measure the protein expression levels of cytochrome c and activated caspase-3 in hepatocyte mitochondria and cytoplasm. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results Compared with the alcohol model group, the middle- and high-dose ursolic acid groups had significant reductions in the serum level of alanine aminotransferase, aspartate aminotransferase, and cholinesterase (all P < 0.05). The rats in the alcohol model group had disordered arrangement of hepatic cords with marked hepatocyte edema and fatty degeneration, while those in the middle- and high- dose ursolic acid groups had basically normal arrangement of hepatic cords and a significant improvement in hepatocyte fatty degeneration, as well as a significant increase in the number of hepatocyte mitochondria and a significant improvement in morphology. Compared with the alcohol model group, the middle- and high-dose ursolic acid groups had significantly lower hepatocyte apoptosis rate and protein expression levels of cytochrome c and caspase-3 in cytoplasm (all P < 0.05). Conclusion Ursolic acid in Hippophae rhamnoides L. can improve the liver function and histomorphology of rats with alcoholic liver disease, possibly by inhibiting the release of cytochrome c in hepatocyte mitochondria, the activation of caspase-3, and the apoptosis of hepatocytes via the mitochondria-cytochrome c pathway.
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ObjectiveTo study the changes of mitochondrial function of ovarian granulosa cells in women of different ages and the effect of Erzhi-Tiangui prescription on in vitro fertilization-embryo transfer （IVF-ET） outcomes for elderly women， so as to verify the connotation of the "Seven-Seven" theory in the Huangdi's Internal Classic （《黄帝内经》）. MethodA total of 150 infertility patients undergoing IVF-ET at the Reproductive and Genetic Center of Integrative Medicine， Affiliated Hospital of Shandong University of Traditional Chinese Medicine were recruited and assigned into "hree-Seven/Four-Seven （30 cases）， Five-Seven （60 cases）， and Six-Seven （60 cases） groups according to the "Seven-Seven" theory. The Five-Seven and Six-Seven groups were further assigned into control and Chinese medicine subgroups using the random number plus envelope method， and the Chinese medicine group was administrated with Erzhi Tiangui prescription from the start day of controlled ovulation stimulation cycle to the trigger day. The IVF outcome was observed， and Western blot was employed to determine the levels of mitofusin 1 （MFN1）， mitofusin 2 （MFN2）， and dynamin-related protein 1 （Drp1） in the ovarian granulosa cells. ResultCompared with the Three-Seven/Four-Seven group， the control subgroups of the Five-Seven and Six-Seven groups showed decreased retrieved oocytes， two pronuclear （2PN） embryos， available embryos， high-quality embryos， clinical pregnancy rate， and live birth rate （P<0.05）. Moreover， the control subgroup of the Six-Seven group showed decreased fresh embryo transfer rate（P<0.05）. Compared with the control subgroup of the Five-Seven group， that of the Six-Seven group showed reduced retrieved oocytes， 2PN embryos， available embryos， high-quality embryos， and clinical pregnancy rate （P<0.05）. The Chinese medicine subgroup had more retrieved oocytes， 2PN oocytes， and available embryos than the control subgroup in the Five-Seven groups （P<0.05）. The Chinese medicine subgroup had more retrieved oocytes， than the control subgroup in the Six-Seven groups （P<0.05）. The control subgroup of the Six-Seven group showed lower expression levels of Mfn1 and Mfn2 and higher level of Drp1 than the control subgroup of the Five-Seven group （P<0.05）， which indicated that the levels of Mfn1 and Mfn2 in ovarian granulosa cells were down-regulated while the expression of Drp1 was up-regulated with aging （P<0.05）. The Chinese medicine subgroup had higher Mfn2 level and lower Drp1 level than the control subgroup in the Five-Seven group （P<0.05）， and the Chinese medicine subgroup had higher Mfn1 and Mfn2 levels and lower Drp1 level than then control subgroup in the Six-Seven group （P<0.05）. ConclusionsThe prognosis of IVF in women after "Five-Seven" became worse with aging， and the mitochondria in ovarian granulosa cells showed decreased fusion ability and increased fission， which verified the connotation of the "Seven-Seven" theory from the mitochondrial function. Erzhi Tiangui prescription can regulate the mitochondrial function of ovarian granulosa cells in elderly women， up-regulate the expression levels of Mfn1 and Mfn2 to promote mitochondrial fusion， and down-regulate the expression of Drp1 to reduce mitochondrial fission， thus alleviating the ovarian hypofunction caused by aging， improve the development potential of oocytes， and improve the IVF outcomes of elderly women. However， this prescription has limited efficacy for the elderly women in the age range of "Six-Seven".
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Liver transplantation is a vital treatment for end-stage liver disease. However, the shortage of donor livers has limited the development of liver transplantation. How to expand the source of donor livers has become a challenge in the academic community. In recent years, the proportion of donors with non-alcoholic fatty liver disease (NAFLD) has been increased. Rational use of steatotic donor livers is a feasible approach to expand the donor pool. Cold ischemia injury during donor liver preservation before liver transplantation increases the risk of postoperative organ dysfunction. Therefore, it is of significance to unravel the mechanism and intervention measures of cold ischemia injury of steatotic donor livers. Cold ischemia injury of steatotic donor livers is characterized as the damage of mitochondria, lysosomes and endoplasmic reticulum at the organelle level, and up-regulated expression of adenosine monphosphate activated protein kinase (AMPK), aldehyde dehydrogenase 2 (ALDH2) and heme oxygenase (HO)-1 at the protein level. In this article, the research progresses on cold ischemia injury of steatotic donor livers and relevant intervention measures were reviewed.
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Mitochondrial diseases are a group of inherited or acquired metabolic disorders caused by mitochondrial dysfunction which may affect almost all the organs in the body and present at any age. However, no satisfactory therapeutic strategies have been available for mitochondrial diseases so far. Mitochondrial transplantation is a burgeoning approach for treatment of mitochondrial diseases by recovery of dysfunctional mitochondria in defective cells using isolated functional mitochondria. Many models of mitochondrial transplantation in cells, animals, and patients have proved effective via various routes of mitochondrial delivery. This review presents different techniques used in mitochondrial isolation and delivery, mechanisms of mitochondrial internalization and consequences of mitochondrial transplantation, along with challenges for clinical application. Despite some unknowns and challenges, mitochondrial transplantation would provide an innovative approach for mitochondrial medicine.
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Myelin-forming oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) are essential for structural and functional homeostasis of nervous tissue. Albeit with certain similarities, the regulation of CNS and PNS myelination is executed differently. Recent advances highlight the coordinated regulation of oligodendrocyte myelination by amino-acid sensing and growth factor signaling pathways. In this review, we discuss novel insights into the understanding of differential regulation of oligodendrocyte and Schwann cell biology in CNS and PNS myelination, with particular focus on the roles of growth factor-stimulated RHEB-mTORC1 and GATOR2-mediated amino-acid sensing/signaling pathways. We also discuss recent progress on the metabolic regulation of oligodendrocytes and Schwann cells and the impact of their dysfunction on neuronal function and disease.
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Amino Acids , Myelin Sheath/metabolism , Schwann Cells/metabolism , Oligodendroglia/metabolism , Signal Transduction , Intercellular Signaling Peptides and Proteins/metabolismABSTRACT
Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.T260A, p.R422W, and p.R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%‒49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%‒17.9%, which was significantly higher than that (6.9%‒7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.
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Humans , Apoptosis Inducing Factor/metabolism , NAD/metabolism , Dimerization , ApoptosisABSTRACT
OBJECTIVE@#To evaluate the apoptosis and cycle arrest effects of Oldenlandia diffusa flavonoids on human gastric cancer cells, determine the action mechanisms in association with the mitochondrial dependent signal transduction pathway that controls production of reactive oxygen species (ROS), and evaluate the pharmacodynamics of a mouse xenotransplantation model to provide a reference for the use of flavonoids in prevention and treatment of gastric cancer.@*METHODS@#Flavonoids were extracted by an enzymatic-ultrasonic assisted method and purified with D-101 resin. Bioactive components were characterized by high-performance liquid chromatography. Cell lines MKN-45, AGS, and GES-1 were treated with different concentrations of flavonoids (64, 96, 128, 160 µg/mL). The effect of flavonoids on cell viability was evaluated by MTT method, and cell nuclear morphology was observed by Hoechst staining. The apoptosis rate and cell cycle phases were measured by flow cytometry, the production of ROS was detected by laser confocal microscope, the mitochondrial membrane potential (MMP) were observed by fluorescence microscope, and the expression of apoptotic proteins related to activation of mitochondrial pathway were measured by immunoblotting. MKN-45 cells were transplanted into BALB/c nude mice to establish a xenograft tumor model. Hematoxylin and eosin staining was used to reveal the subcutaneous tumor tissue. The tumor volume and tumor weight were measured, the expression levels of proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67 were detected by immunohistochemistry, and the expression levels of CA72-4 were measured by enzyme linked immunosorbent assay.@*RESULTS@#Oldenlandia diffusa flavonoids inhibited proliferation of MKN-45 and AGS human gastric cancer cells, arrested the cell cycle in G1/S phase, induced accumulation of ROS in the process of apoptosis, and altered MMP. In addition, flavonoids increased Apaf-1, Cleaved-Caspase-3, and Bax, and decreased Cyclin A, Cdk2, Bcl-2, Pro-Caspase-9, and Mitochondrial Cytochrome C (P<0.05). The MKN-45 cell mouse xenotransplantation model further clarified the growth inhibitory effect of flavonoids towards tumors. The expression levels of PCNA and Ki-67 decreased in each flavonoid dose group, the expression level of CA72-4 decreased (P<0.05).@*CONCLUSION@#Flavonoids derived from Oldenlandia diffusa can inhibit proliferation and induce apoptosis of human gastric cancer cells by activating the mitochondrial controlled signal transduction pathway.
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Humans , Animals , Mice , Oldenlandia/metabolism , Proliferating Cell Nuclear Antigen , Stomach Neoplasms , Flavonoids/pharmacology , Reactive Oxygen Species/metabolism , Mice, Nude , Ki-67 Antigen , Cell Line, Tumor , Apoptosis , Plant Extracts/pharmacology , Caspases , Cell ProliferationABSTRACT
In addition to its own specific functions, an organelle can also interact with other organelles to complete important physiological functions. The disorders of organelle interactions are closely associated the development and progression of various diseases. In recent years, the role of organelle interactions has attracted more attention in the progression of nonalcoholic fatty liver disease, especially the interactions between mitochondria, lipid droplets, and other organelles.
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Objective:To study the characteristics and clinical significance of mitochondrial injury of T lymphocyte subsets in patients with autoimmune hepatitis (AIH).Methods:The clinical data of 57 patients with AIH (AIH group) from June to December 2021 in Hangzhou Xixi Hospital were retrospectively analyzed, while 60 healthy physical examiners were included as healthy group. The peripheral blood T lymphocyte subsets (CD 8+ T lymphocyte count and CD 4+ T lymphocyte count) were detected by flow cytometry, and matched mitochondrial staining value according to certain algorithm was used to determine the mitochondrial damage of helper T lymphocyte (Th cell) and suppressor T lymphocyte (Ts cell). The levels of IgG, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using a Roche E170 automatic electrochemiluminescence immunoassay. Anti-nuclear antibody (ANA) titer was measured by immunofluorescence. Multivariate Logistic regression was used to analyze the independent risk factors of mitochondrial damage of Th cell and Ts cell in patients with AIH. Results:The ALT, AST, IgG, positive rate of ANA titer, CD 4+ T lymphocyte count, CD 8+ T lymphocyte count, rate of Th cell mitochondrial injury and rate of Ts cell mitochondrial injury in AIH group were significantly higher than those in healthy group: (118.90 ± 37.61) U/L vs. (30.96 ± 14.37) U/L, (102.40 ± 36.51) U/L vs. (31.12 ± 14.06) U/L, (18.40 ± 3.71) g/L vs. (13.89 ± 1.98) g/L, 96.49% (55/57) vs. 16.67% (10/60), 438 (323, 637) × 10 6/L vs. 398 (272, 469) × 10 6/L, 296 (211, 296) × 10 6/L vs. 270 (193, 322) × 10 6/L, 61.40% (35/57) vs. 8.33% (5/60) and 82.46% (47/57) vs. 11.67% (7/60), and there were statistical differences ( P<0.01 or <0.05). Multivariate Logistic regression analysis result showed that the AST elevated and CD 8+ T lymphocyte count reduced were the independent risk factors of Ts cell mitochondrial injury in patients with AIH ( OR = 1.06 and 0.99, 95% CI 1.01 to 1.10 and 0.99 to 1.00, P<0.05); the ALT elevated and IgG elevated were the independent risk factors of Th cell mitochondrial injury in patients with AIH ( OR = 1.08 and 1.66, 95% CI 1.02 to 1.14 and 1.11 to 2.48, P<0.05). Conclusions:It is of positive clinical significance to measure the T lymphocyte subtype mitochondrial injury in patients with AIH. The probability of mitochondrial injury of T lymphocyte subtype can be predicted by biochemical indexes such as ALT, AST and IgG, so as to indirectly evaluate the liver cell necrosis.
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Mitochondria are the center of cellular energy metabolism, and their functions are tightly regulated by the nuclear and mitochondria genomes.Potential mechanisms responsible for age-related mitochondrial dysfunction include the accumulation of mitochondrial DNA (mtDNA) damage caused by replication errors or oxidative damage, and the epigenetic changes in mtDNA (mitoepigenetics). These mechanisms are essential for the development and progression of age-related macular degeneration (AMD). Age-related mtDNA damage disrupts energy metabolism and cellular function in the retinal pigment epithelium (RPE) and neuroretinal cells, which further mediates oxidative stress, lysosomal dysfunction and pyroptosis, resulting in RPE degeneration, drusen deposition and retinal inflammation.Mitochondrial genome protection, such as humanin administration, may be a promising preventive or therapeutic target in the early stages of AMD.This review focused on the research progress of the mitochondrial genetic mechanism in AMD pathogenesis and provided new ideas for exploring the prevention and treatment strategies of AMD.
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Objective:To investigate the molecular expression and pathological features of endothelial cell (EC) in a murine model of choroidal neovascularization (CNV) based on single-cell RNA sequencing (scRNA-seq).Methods:Six C57BL/6 mice aged 6-8 weeks were randomly divided into two groups, with 3 mice in each group.Bilateral eyeballs were enucleated.The choroidal tissues from the two groups were isolated by shearing the complex and scraping the choroid, respectively.Single-cell suspension was prepared by continuous digestion with trypsin/type Ⅰ collagenase at 37 ℃, and the cell viability and EC ratio were detected by flow cytometry to determine the preparation method of single-cell suspension.Another 6 mice were randomly assigned into the control group and the CNV group, with 3 mice in each group.The CNV model was induced by laser photocoagulation and single-cell suspensions were prepared 7 days after modeling.Gene expression library construction was performed using the Chromi-um (10x Genomics) instrument.High throughput sequencing was performed using the Illumina Novaseq6000 to obtain the expression matrix.The EC subpopulations were classified according to previous researches and the Cellmarker database.Pseudo-time analysis was performed in EC, revealing the gene expression matrix of different states.CNV-EC were further selected with preliminary analysis of the expression characteristics.Another 6 mice were selected to establish the CNV model and eyeball frozen sections were prepared 7 days after modeling.Expression and distribution as well as the area percentage of EC marker Pecam1, mitochondrial outer membrane proteins Tomm20 and mt-Co1, and capillary markers Kdr and Plvap were observed by immunofluorescence staining, and the vascular diameter was calculated.The use and care of animals followed the ARVO statement.This study protocol was approved by the Experimental Animal Welfare and Ethics Committee of Air Force Military Medical University (No.20200181).Results:The cell viability of the single-cell suspension prepared from choroidal-scleral fragments and choroidal scrapings was 99.4% and 99.1%, respectively, both of which met the sequencing requirements.The percentage of EC detected by flow cytometry was approximately 1.58%.The scRNA-seq result revealed that both the normal control and CNV groups contained 13 choroidal cell clusters.Compared with the normal control group, the proportions of rod/cone photoreceptor cells, EC and hematopoietic cells all increased, while the retinal pigment epithelium (RPE) and Schwan cells reduced in the CNV group.Among all clusters, EC constituted 18.4%.The pseudo-time analysis demonstrated that EC could be further divided into 4 states.The percentage of state 2 EC was 29.1% in the CNV group, which was significantly higher than 9.5% in the normal control group.Differentially expressed gene analysis showed that the expression of mitochondrion-related genes, including mt-Nd4 and mt-Atp6, were upregulated in state 2 EC, while capillary-related genes, including Kdr and Esm1, were downregulated.Immunofluorescent staining revealed that the area of Tomm20 and mt-Co1 in Pecam1-positive EC in the CNV area was (19.50±4.68)% and (4.64±2.82)%, respectively, which were both higher than (3.00±2.09)% and (0.18±0.34)% in normal area ( t=7.88, 3.84; both at P<0.01). The area of Kdr and Plvap in Pecam1-positive EC in the CNV area was (1.50±0.29)% and (0.79±0.97)%, respectively, which were both lower than (31.30±5.44)% and (10.43±2.28)% in the normal area ( t=13.40, 9.48; both at P<0.01). The vascular diameter in the CNV area was (5.52±1.85)μm, which was larger than (4.21±1.84)μm in the normal area ( t=9.57, P<0.001). Conclusions:When CNV occurs, the proportion of EC in choroid increases, and CNV-EC shows pathologic features of mitochondrial metabolic activation and loss of capillary properties, suggesting the mitochondrial activation of EC may play a role in the formation of CNV.
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Objective:To explore the protective effect of zonisamide (ZNS) on oxygen-glucose deprivation (OGD) cell model of traumatic brain injury (TBI), and its underlying mechanism.Methods:Human neuroblastoma cells (SH-SY5Y) were cultured in vitro and divided into the control group, OGD group, and drug administration group (OGD+ZNS group) according to the random number table method. The OGD method was used to establish a TBI cell model. After modeling, the cell activity, the release of lactate dehydrogenase (LDH), and β-galactosidase staining were detected to evaluate cell function and senescence. Additionally, mitochondrial morphology and potential membrane changes were observed using Mito Tracker Red and JC-1 mitochondrial membrane potential staining. ATP concentration was measured, and protein was extracted from SH-SY5Y cells and then subjected to Western blot analysis to detect endoplasmic reticulum stress-related markers, including glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), protein disulfide isomerase (PDI), and β-actin.Results:The OGD group had a significantly lower cell survival rate compared to the control group ( P<0.01), while the OGD+ZNS group had a significant higher cell survival rate than the OGD group ( P<0.01). The LDH release rate was significantly higher in the OGD group than in the control group ( P<0.01), while the OGD+ZNS group had a significant lower LDH release rate compared to the OGD group ( P<0.01). Moreover, the cell staining results indicated that compared to the control and OGD+ZNS groups, the cells in the OGD group exhibited significant damage and senescence with darker staining while the mitochondrial staining results demonstrated a significant reduction in mitochondrial linear junctions and decreased mitochondrial activity in the OGD group compared to the control and OGD+ZNS groups. Compared to the control and OGD+ZNS groups, the OGD group exhibited a significant reduction in mitochondrial staining red fluorescence, a significant increase in green fluorescence, and a significant decrease in mitochondrial membrane potential. The OGD group demonstrated a significant decrease in ATP concentration compared to the control group ( P<0.01), whereas the OGD+ZNS group exhibited a significant higher ATP concentration compared to the OGD group ( P<0.01). Western blot analysis revealed significant upregulation of GRP78, CHOP, and PDI in the OGD group compared to the control group (all P<0.05), while in the OGD+ZNS group, the expression levels of these proteins were significantly downregulated compared to the OGD group (all P<0.05). Conclusions:Zonisamide can protect OGD TBI cell model by preserving mitochondrial activity and inhibiting endoplasmic reticulum stress.
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Objective:To explore the protective effect and underlying mechanism of hyperbaric oxygen (HBO) on delayed encephalopathy after carbon monoxide poisoning (DEACMP) in mice.Methods:Totally 225 adult male Kunming mice were selected to establish CO poisoning model via intraperitoneal injection carbon monoxide (CO), and were randomly divided into the air control group, CO poisoning group, and HBO group. Each group was further divided into five time points group, that was 1, 3, 7, 14 and 21 d. The mice in the air control group were injected intraperitoneally with the same amount of air, and the HBO group received HBO treatment at the same time every day. DEACMP mice model was screened by behaviors using the open field test, new object recognition test and nesting test, and the content of myelin basic protein (MBP) were assayed. The mouse brain tissue and mitochondrial were prepared and malonialdehyde (MDA) and adenosine triphosphate (ATP) content were measured with ultraviolet spectrophotometer. MBP content in brain tissue and cytochrome C (CytC) content in the mitochondrial were measured by ELISA. The mitochondria membrane potential (MMP) was measured by flow cytometry.Results:Compared with the air control group, the content of carboxyhemoglobin (COHB) in blood increased significantly and the content of MBP in brain tissue decreased significantly in CO poisoning mice. CO poisoning mice showed motor ability and cognitive dysfunction. Compared with the air control group, the contents of MMP, CytC and ATP were significantly decreased ( P<0.01) in the CO poisoning group; while the MDA content was significantly increased ( P<0.01). Compared with the CO poisoning group, mice behaviors were improved significantly ( P<0.05), the content of MBP, MMP, CytC and ATP were increased ( P<0.05), while the MDA content decreased significantly ( P<0.01) in the HBO group. Conclusions:The abnormal mitochondrial function might be closely related to the occurrence and development of DEACMP, and HBO therapy plays an effective role in preventing and treating the DEACMP mice model via the mitochondrial pathway.