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ObjectiveTo study the effect of Xihuangwan extract on mitochondrial energy metabolism in ovarian cancer SKOV3 and HEY cells and to explore the underlying mechanism. MethodSKOV3 and HEY cells were cultured in vitro and treated with different concentrations （0， 5， 10， 15， 20 g·L-1） of Xihuangwan extract. Methyl thiazolyl tetrazolium （MTT） was used to examine the viability of SKOV3 and HEY cells treated with Xihuangwan extract. The adenosine-triphosphate （ATP） levels in SKOV3 and HEY cells were measured by kit. Flow cytometry was employed to measure the content of reactive oxygen species （ROS） in cells. Western blot was employed to determine the protein levels of peroxisome proliferator-activated receptor-γ co-activator 1α （PGC1α）， transcription factor A， mitochondrial （TFAM）， translocase of outer mitochondrial membrane 20 （TOMM20）， and aplasia Ras homologue member Ⅰ （ARHⅠ） in SKOV3 and HEY cells. Mito-Tracker Green staining was used to observe the morphological changes of mitochondria in SKOV3 and HEY cells. ResultCompared with blank group， Xihuangwan extract treatment for 24， 48 h inhibited the viability of SKOV3 and HEY cells in a concentration-dependent manner （P<0.05， P<0.01）. Compared with blank group， Xihuangwan extract （10， 15， 20 g·L-1） groups presented lowered ATP levels （P<0.05， P<0.01）， and the 20 g·L-1 Xihuangwan extract group had lower ATP level than the 10 and 15 g·L-1 Xihuangwan extract groups （P<0.05）. Compared with blank group， Xihuangwan extract increased the content of ROS in SKOV3 and HEY cells in a concentration-dependent manner （P<0.05， P<0.01）， and the 20 g·L-1 Xihuangwan extract group had higher ROS content than the 10 g·L-1 Xihuangwan extract group （P<0.05）. Compared with blank group， Xihuangwan extract up-regulated the expression level of ARHⅠ protein in SKOV3 and HEY cells in a concentration-dependent manner （P<0.01）， and the expression levels of ARHⅠ protein was higher in the 20 g·L-1 Xihuangwan extract group than in the 10 and 15 g·L-1 Xihuangwan extract groups （P<0.05）. Compared with the blank group， Xihuangwan extract down-regulated the protein levels of PGC1α， TFAM， and TOMM20 in SKOV3 and HEY cells in a concentration-dependent manner （P<0.05， P<0.01）， and the protein levels of TFAM and TOMM20 in the HEY cells treated with 20 g·L-1 Xihuangwan extract were lower than those in the HEY cells treated with 10， 15 g·L-1 Xihuangwan extract （P<0.05）. Compared with the blank group， 20 g·L-1 Xihuangwan extract decreased the Mito-Tracker fluorescence intensity of SKOV3 and HEY cells （P<0.05）. ConclusionXihuangwan can compromise the mitochondrial function of ovarian cancer SKOV3 and HEY cells and reduce cell energy metabolism to inhibit the proliferation of SKOV3 and HEY cells by up-regulating ARHⅠ and inhibiting PGC1α/TFAM signaling axis.
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Diabetic kidney disease （DKD） is characterized by the hyperfiltration and albuminuria in the early phase which are followed by progressive renal function decline， renal tubular epithelial cell hypertrophy， and tubulointerstitial fibrosis. Thus， it is one of the leading causes of chronic kidney diseases. The currently available therapies mainly aim to control the primary diseases and reduce the risk of kidney injury. Based on syndrome differentiation， traditional Chinese medicine （TCM） relieves the symptoms by excreting water and alleviating edema and eliminates the root cause by tonifying deficiency and supplementing the original Qi， thereby showing therapeutic effect and delaying the progression of DKD. It excels in comprehensively regulating the constitution of patients with little side effects. Among the Zang-fu organs， kidney takes the second place in the content of mitochondria which participate in the metabolism of water and fluid and are the foundation of kidney Yin and kidney Yang. Mitochondria are energy producers within a cell， which carry out cellular respiration， produce reactive oxygen species， and generate adenosine triphosphate by oxidative phosphorylation. Mitochondrial quality control （MQC） is an effective way to maintain mitochondrial dynamic balance， whose imbalances， such as mitochondrial oxidative stress， mitophagy， mitochondrial dynamic changes， and abnormal calcium regulation， are related to the occurrence and development of DKD. It is generally believed that the destruction of mitochondrial structure in the case of metabolic disorder is the main cause of the disease. In recent years， TCM has attracted the attention of both Chinese and foreign researchers for the unique advantages of treating both symptoms and root cause at the same time and multi-target synergy in the treatment of DKD. However， the specific mechanism is still unclear. It has been frequently verified that mitochondria may be one of the targets of TCM in the treatment of DKD. At the moment， no review on the treatment of DKD by TCM through the intervention of MQC is available. Therefore， this paper aims to summarize the research on TCM treatment of DKD by regulating MQC in the past 10 years， which is expected to provide a new direction for the treatment of DKD by TCM.
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Chronic atrophic gastritis （CAG） is a common and intractable disease in the digestive system characterized by the reduction or disappearance of gastric mucosal glands. The intestinal metaplasia or dysplasia in CAG is called precancerous lesion， which greatly increases the risk of cancerization. Dysactivation of nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） inflammatory corpuscles can release a large number of inflammatory factors， induce inflammatory cascade reactions， and participate in the process of many diseases. As reported， the dysactivation of NLRP3 inflammatory corpuscles can cause long-term chronic inflammatory infiltration of gastric mucosa and induce the development of CAG. Mitochondrial dysfunction plays an important role in the activation of NLRP3 inflammatory corpuscles. The accumulation of reactive oxygen species （ROS） produced by mitochondrial dysfunction is the key to activating NLRP3 inflammatory corpuscles. Professor LIU Youzhang put forward the theory of "spleen-mitochondrion correlation"， which holds that the spleen mainly transports water and grains， generates qi and blood， transports nutrients to the whole body， and supplies energy and materials needed by the body. Adenosine triphosphate （ATP） generated by mitochondria through the circulation of tricarboxylic acid is the main energy source of the human body. The view that both of them serve as human energy processing plants coincides in terms of physiology. Pathologically， spleen deficiency is associated with mitochondrial oxidative phosphorylation dysfunction. Pathological products such as dampness， turbidity， phlegm， and blood stasis due to failure in transportation because of spleen deficiency are consistent with metabolites generated by mitochondrial dysfunction. Based on the theory of "spleen-mitochondrion correlation"， this study discussed the pathogenesis of CAG in traditional Chinese medicine （TCM）， analyzed the relationship between NLRP3 inflammatory corpuscles and the pathogenesis of CAG， and proposed that the activation of NLRP3 inflammatory corpuscles by mitochondrial dysfunction was the modern biological basis of the pathogenesis of spleen deficiency in CAG. The spleen-strengthening method may be related to improving the mitochondrial function and inflammatory response of patients with CAG and alleviating the damage of gastric mucosa， providing a new idea for TCM in the prevention and treatment of CAG.
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【Objective】 To explore the differential expression and functional analysis of circRNA from myocardial mitochondria in diabetes cardiomyopathy (DCM) mice. 【Methods】 The DCM mice model was established in 16-week-old db/db mice, and C57BL/KsJ mice were used as controls. RNA was extracted from the myocardium of two groups of mice, high-throughput sequencing was used to screen mitochondrial circRNA differentially expressed in the two groups, RT-qPCR was used to verify the sequencing results of the first 10 circRNAs with significant differential expression, and functional enrichment analysis was performed on the differentially expressed circRNA target genes, and miRNA target prediction software was used to analyze the circRNA-miRNA co-expression network. 【Results】 There were 147 mitochondrial circRNAs differentially expressed in the myocardium of DCM mice, including 89 highly expressed and 58 low expressed. The expression pattern of differentially expressed circRNAs in tissues was consistent with those of sequencing results. The enrichment analysis of GO and KEGG showed that the differentially expressed circRNA target genes were mainly enriched in cGMP/PKG, glucagon pathways, which were related to mitochondrial energy metabolism and cardiac hypertrophy. circRNA-miRNA co-expression analysis found that the most significantly up-regulated circRNA, chrM:1207-1536+, was associated with miR-491-3p, miR-99a-3p, and miR-99b-3p, and the most significantly down-regulated circRNA, chrM:1453-3205+, was associated with miR-181b-1-3p, miR-181b-2-3p, and miR-672-5p. 【Conclusion】 Compared to the control mice, there is differential expression of circRNAs in myocardial mitochondria of DCM mice. The differentially expressed circRNAs may interact with the corresponding miRNA to affect myocardial fibrosis and hypertrophy through regulation of energy metabolism, apoptosis and other pathways, thus participating in the pathogenesis of DCM.
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Ulcerative colitis （UC）， a chronic inflammatory bowel disease with the accumulation of colorectal mucosa and submucosa， has a risk of developing into cancer. In recent years， the incidence of UC has been on the rise worldwide. However， its pathogenesis has not been fully elucidated by modern medicine， and even the remission rate of the latest drugs is lower than 50%， which seriously affects the patients' work and quality of life. Mitochondria， as the "power station" of eukaryotic cells， are involved in a variety of physiological processes such as the production of reactive oxygen species and the production of adenosine triphosphate by oxidative phosphorylation， and their dysfunction can lead to a series of diseases. Mitochondrial quality control （MQC） is an important way to maintain the stability of mitochondrial form， quantity， and quality. Studies have shown that MQC disorders characterized by low mitochondrial biogenesis， excessive mitochondrial oxidative stress， mitochondrial autophagy defects， mitochondrial dynamics disorders， and calcium regulation abnormalities are closely related to the occurrence and development of UC. Although progress has been achieved in the treatment of UC by traditional Chinese medicine （TCM） which can regulated MQC in a multi-pathway and multi-target manner in recent years， a review on the treatment of UC by TCM via the intervention in MQC remains to be carried out. Therefore， this paper summarized the TCM treatment of UC by regulating MQC， aiming to provide new ideas for the clinical treatment of UC by TCM.
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@#Objective To investigate the effects of a disintegrin and metalloproteinase 17(ADAM17) deletion on the production of reactive oxygen species(ROS) and mitochondrial function in nasopharyngeal carcinoma(NPC) cells.Methods Three groups of ADAM1 7 interfering plasmid ADAM17 shRNA and empty plasmid ADAM17-shRNA-NC were transfected into NPC cell line(CNE1) and detected for the interference efficiency by RT-PCR and Western blot to select shRNA with the best interference effect for the follow-up experiments.The cell proliferation was detected by CCK-8 assay,while the cell growth by clone formation test,the apoptosis and changes in mitochondrial membrane potential(MMP) by flow cytometry,the level of mitochondrial oxidative damage product ROS by fluorescence microscope,the contents of oxidative stress markers MDA and SOD by malondialdehyde(MDA) kit and superoxide dismutase(SOD) kit and the expression of mitochondrial damage markers Bax/Bcl-2,cleaved-caspase 9/caspase 9,cleaved-caspase 3/caspase 3 and c-Myc by Western blot.Results ADAM17-shRNA2 group showed the best interference effect.Compared with shRNA-NC group,the proliferation rate of cell in ADAM17-shRNA 2 group decreased significantly(t=8.964,P=0.036);the number of colonies were significantly reduced(t=10.351,P=0.014);the number of apoptosis increased significantly(t=11.25,P=0.008);the fluorescence intensity representing ROS level in cells increased obviously;the mitochondrial membrane potential decreased significantly(t=9.233,P=0.013);the SOD content decreased(t=7.233,P=0.034) and MDA content increased(t=7.415,P=0.038) significantly;the levels of Bax/Bcl-2,cleaved-caspase 9/caspase 9 and cleaved-caspase 3/caspase 3 significantly increased(t=8.985,9.021 and 7.789,P=0.023,0.011 and 0.031,respectively),while the expression of c-Myc proteins significantly decreased(t=10.352,P=0.004).Conclusion Interfering with ADAM1 7 induced SOD decrease and MDA increase by promoting oxidation,thereby alleviating oxidative damage of cell membrane,which also promoted the expression level of ROS in mitochondrion,reduced MMP,inhibited cell proliferation in vitro,and promoted apoptosis.
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Over the past years, natural products have been explored in order to find biological active substances to treat various diseases. Regarding their potential action against parasites such as trypanosomatids, specially Trypanosoma cruzi and Leishmania spp., much advance has been achieved. Extracts and purified molecules of several species from genera Piper, Tanacetum, Porophyllum, and Copaifera have been widely investigated by our research group and exhibited interesting antitrypanosomal and antileishmanial activities. These natural compounds affected different structures in parasites, and we believe that the mitochondrion is a strategic target to induce parasite death. Considering that these trypanosomatids have a unique mitochondrion, this cellular target has been extensively studied aiming to find more selective drugs, since the current treatment of these neglected tropical diseases has some challenges such as high toxicity and prolonged treatment time. Here, we summarise some results obtained with natural products from our research group and we further highlighted some strategies that must be considered to finally develop an effective chemotherapeutic agent against these parasites.
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The trypanosomatids Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are etiological agents of important neglected tropical diseases, affecting millions of people worldwide, and the drugs available for these diseases present several limitations. Novel efficient and nontoxic drugs are necessary as an alternative to the current chemotherapy. The unique mitochondrion of trypanosomatids and its peculiar features turn this organelle a potential drug target. Several phenotypic studies describe the damage in the parasite mitochondrial ultrastructure, but the molecular target is unknown. Few reports demonstrated the electron transport system (ETS) as a target due to the high similarities to mammalian orthologues, hence ETS is not a good candidate for drug intervention. On the other hand, antioxidant enzymes, such as trypanothione reductase, and an alternative oxidase (AOX) seem to be interesting targets; however no high active inhibitors were developed up to now. Finally, due to the remarkable differences to mammalian machinery, together with the high biological importance for the parasite survival, the mitochondrial import system stands out as a very promising target in trypanosomatids. Archaic translocase of the outer membrane (ATOM) and translocase of the inner membrane (TIM) complexes, which mediate both protein and tRNA import, composed by specific subunits of these parasites, could be excellent candidates, deserving studies focused on the development of specific drugs.
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Hepatocellular carcinoma (HCC) is a type of tumor with a high incidence rate, a low rate of early diagnosis, and poor prognosis, and its development and progression involve many factors. As an important organelle in cells, mitochondria is the "energy factory" of cells and is one of the main sites for the production of reactive oxygen species in vivo, and it also participates in the regulation of cell apoptosis. There are varying degrees of changes in mitochondrial membrane, oxidation respiratory chain, mitochondrial dynamics, mitochondrial DNA, and mitochondrial calcium homeostasis during the development and progression of HCC, and such changes may affect the progression of HCC. This article systematically elaborates on the association between mitochondria and HCC, so as to provide a new direction for the diagnosis and treatment of HCC.
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The remodeling of phospholipid includes two processes: deacylation and reacylation. It realizes the conversion of nascent phospholipids to mature phospholipids by changing the length and types of fatty acids at specific sites of phospholipids, which is a key step in phospholipid metabolism. Phospholipids are not only the basic components of biological membranes, but also participate in the transduction of many molecular signals in cells. Therefore, phospholipid remodeling disorders can affect the structure and function of cell membranes, as well as the activity of membrane proteins, causing a series of intricate signaling cascades, and finally lead to many pathological changes including neurodegeneration. This paper reviews the basic process of phospholipid remodeling and the involvement of its key enzymes, calcium independent group VIA phospholipase A2 (iPLA2β), peroxiredoxin 6 (PRDX6), calcium independent group VIB phospholipase A2 (iPLA2γ) as well as acyl-CoA lysocardiolipin acyltransferase 1 (ALCAT1) in the pathology of Parkinson's disease. The mutations in the gene encoding iPLA2β, PLA2G6, have been widely reported to be directly related to hereditary Parkinson disease-14 (PARK14). Here we focus on the molecular mechanism of iPLA2β in the development of Parkinson's disease, mainly involving phospholipid fatty acid metabolism disorders, mitochondrial physiology abnormalities and α-synuclein aggregate formation and other aspects, which will help to understand the role of phospholipid remodeling in Parkinson's disease, and provide new clues for the development of new Parkinson's disease diagnosis and treatment strategies.
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Objective:To evaluate the role of reactive oxygen species (ROS)-mediated mitochondrial pathway of apoptosis in long-term cognitive impairment induced by multiple exposures to sevoflurane in the neonatal rats.Methods:Sixty SPF healthy neonatal Sprague-Dawley rats, weighing 12-20 g, were divided into 3 groups ( n=20 each) using a random number table method: control group (group C), multiple exposures to sevoflurane for anesthesia group (group S) and ROS inhibitor group (group A). Group S and group A inhaled 3% sevoflurane for 2 h starting from 6, 7 and 8 days after birth, while group C inhaled air.In group A, ROS inhibitor N-acetylcysteine (NAC) 150 mg/kg was intraperitoneally injected before each anesthesia with sevoflurane.The spontaneous activity was evaluated by open field test on day 35 after birth.The cognitive function was determined by Morris water maze test on day 36 after birth.The rats were sacrificed after the end of Morris water maze test, and the hippocampal tissues were obtained for determination of the apoptosis rate of hippocampal neurons, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) (by flow cytometry) and levels of Cyt c and cleaved caspase-9 and caspase-3 (by Western blot). The expression of Bcl-2 and Bax mRNA was detected by real-time polymerase chain reaction.The ultrastructure of mitochondria in hippocampal neurons was observed with a transmission electron microscope. Results:Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the apoptosis rate of hippocampal neurons and levels of ROS and MMP were increased, the expression of Cyt c, cleaved caspase-9, cleaved caspase-3 and Bax mRNA was up-regulated, the expression of Bcl-2 mRNA was down-regulated, the ratio of Bax/Bcl-2 was increased ( P<0.05), mitochondria were swollen, and mitochondrial cristae structure was broken in group S. Compared with group S, the escape latency was significantly shortened, the number of crossing the original platform was increased, the apoptosis rate of hippocampal neurons and levels of ROS and MMP were decreased, the expression of Cyt c, cleaved caspase-9, cleaved caspase-3 and Bax mRNA was down-regulated, the expression of Bcl-2 mRNA was up-regulated, the ratio of Bax/Bcl-2 was decreased ( P<0.05), and the mitochondrial swelling and rupture of cristae structure were improved in group A. Conclusion:The mechanism by which multiple exposures to sevoflurane induce long-term cognitive impairment may be related to activating the ROS-mediated mitochondrial pathway of apoptosis in neonatal rats.
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SIRT3, SIRT4 and SIRT5 are located in mitochondria and also known as mitochondrial sirtuins. They play important roles in regulating many cellular functions including cell survival, cell cycle or apoptosis, DNA repair and metabolism. Mitochondrial sirtuins are involved in the protection of mitochondrial integrity and energy metabolism under stress regulating the expression of neurotransmitter receptors, neurotrophins, extracellular matrix proteins and various transcription factors, thus involved in epileptogenesis triggered by both genetic or acquired factors. Here we review research progress on the actions of mitochondrial sirtuin in epilepsy; and discuss the challenges and perspectives of mitochondrial sirtuin as a potential therapeutic target for epilepsy.
Subject(s)
Humans , Apoptosis , Epilepsy/genetics , Mitochondria/genetics , Sirtuin 3 , SirtuinsABSTRACT
Objective: To investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD +) levels. Methods: The bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H 2O 2 to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H 2O 2 treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H 2O 2 simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD + and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-β-galactosidase (SA-β-gal) staining and TUNEL staining] were detected after 24 hours of treatment. Results: The rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased ( P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H 2O 2, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD + and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-β-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant ( P<0.05). Conclusion: NMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD + levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.
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@# Objective: To investigate the effect of over-expression of paired related homoeobox 1 (PRRX1) on apoptosis of hepatocellular carcinoma SMMC7721 cells, and to explore its detailed mechanism. Methods： ：Lentivirus-mediated PRRX1 over-expression vector (pGMLV-PRRX1) and empty vector (Vector) were respectively infected into SMMC7721 cells, and the mRNA and protein expression levels of PRRX1 in infected cells were detected by qPCR and WB. The effect of PRRX1 over-expression on the cell proliferation and apoptosis of SMMC7721 cells were assessed by CCK-8 assay and Annexin-V FITC/PI double staining flow cytometry assay, respectively. The change of mitochondrial membrane potential of SMMC7721 cells was detected by mitochondrial membrane potential assay kit (JC-10 staining assay). The enzymatic activities of caspase-8 and caspase-9 in infected cells were detected by using caspase activity assay kit (spectrophotometric method). The protein expression levels of p53, Bcl-2, Bax, Fas, Cleaved-caspase-3 and Cty C expressed in mitochondria and cytosol were evaluated by WB. Results： ：PRRX1 over-expressed SMMC7721 cell line was successfully constructed, and the protein and mRNA expression levels of PRRX1 were significantly increased in lentivirus infected cells (all P< 0.01). Compared with control group and vector group, over-expression of PRRX1 significantly inhibited cell proliferation, weakened mitochondrial membrane potential, but increased the rate of apoptosis, elevated the shear level of caspase-3, promoted the release of Cyt C protein from mitochondrial into cytosol and increased the enzymatic activity of caspase-9 (all P<0.05 or P<0.01). In addition, over-expression of PRRX1 also promoted the protein expressions of p53 and Bax but inhibited the protein expression of Bcl-2 (all P< 0.05 or P<0.01); however,it had no significant effect on the expression of Fas protein and the enzymatic activity of caspase-8 (all P＞ 0.05). Conclusion： ：Over-expression of PRRX1 induces apoptosis in hepatocellular carcinoma SMMC7721 cells, which may be related to the activation of p53-mediated mitochondrial apoptotic pathway
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Objective? To? investigate? the? protective? effects? of? Klotho? protein,? a? kind? of? single-pass?transmembrane?protein,?on?acute?kidney?injury?(AKI)?in?septic?mice?and?its?mechanism.? Methods? Sixty?SPF?healthy?male?C57BL/6?mice?(6-8?weeks)?were?randomly?divided?into?sham?operation?group?(Sham?group),?sepsis?model?group?(CLP?group)?and?Klotho?protein?injection?group?(CLP+KL?group),?with?20?in?each?group.?The?septic?AKI?mice?model?was?established?by?cecal?ligation?and?puncture?(CLP);?Sham?group?had?the?same?procedure?except?that?the?cecal?was?not?ligated.?The?CLP+KL?group?was?received?Klotho?protein?(0.02?mg/kg)?by?intraperitoneal?consecutive?injection?for?4?days?after?operation;?Sham?group?and?CLP?group?were?injected?with?the?same?amount?of?saline.?Blood?samples?were?obtained?at?24?hours?after?operation,?the?levels?of?serum?creatinine?(SCr)?and?urea?nitrogen?(BUN)?were?measured?by?sarcosine?oxidase?and?urease?method.?The?mice?were?sacrificed?under?anesthesia?at?5?days?after?operation?to?harvest?renal?tissues,?and?the?pathological?damage?of?the?kidney?was?evaluated?by?hematoxylin-eosin?(HE)?staining.?The?ultrastructure?of?mitochondria?in?mouse?renal?tubular?epithelial?cells?was?observed?under?transmission?electron?microscope.?The?levels?of?reduced? glutathione?hormone?(GSH),?malondialdehyde?(MDA)?and?nitric?oxide?synthase?(NOS)?in?mitochondrion?were?determined?by?micro-enzyme?method,?thiobarbituric?acid?method,?colorimetry?method,?respectively.?The?protein?expressions?of?Klotho,?Bcl-2?and?cytochrome?C?(Cyt?C)?were?detected?by?Western?Blot.? Results? The?pathological?structure?of?the?kidneys?in?the?Sham?group?was?clear?and?intact.?Compared?with?the?Sham?group,?the?renal?tissue?edema?of?the?mice?in?the?CLP?group?was?significant,?and?the?transparent?tube?type?was?observed?in?the?small?lumen,?and?the?interstitial?inflammatory?cells?infiltrated;?the?levels?of?SCr?and?BUN?were?significantly?increased?[SCr?(μmol/L):?182.60±6.97?vs.?47.20±5.37,?BUN?(mmol/L):?53.70±5.12?vs.?18.70±2.62,?both?P?<?0.01];?the?mitochondria?were?swollen?and?deformed,?the?sputum?structure?was?destroyed,?the?matrix?density?was?decreased,?the?outer?membrane?was?lost,?and?the?levels?of?MDA,?GSH?and?NOS?were?significantly?increased?[MDA?(μmol/g):?1.172±0.046?vs.?0.746±0.094,?GSH?(μmol/g):?5.765±0.059?vs.?4.223±0.072,?NOS?(kU/g):?0.91±0.05?vs.?0.68±0.03,?all?P?<?0.01];?the?protein?expressions?of?Klotho?and?Bcl-2??in?renal?tissue?were?decreased,?and?the?protein?expression?of?Cyt?C?was?increased?(Klotho/β-actin:?0.188±0.020?vs.?0.538±0.024,?Bcl-2/β-actin:?0.311±0.010?vs.?0.391±0.015,?Cyt?C/β-actin:?0.226±0.010?vs.?0.135±0.006,?all??P?<?0.01).?Comparing?with?the?CLP?group,?the?glomerular?and?tubular?tissue?epithelial?edema?and?the?small?lumen?in?the?CLP+KL?group?were?reduced;?the?levels?of?SCr?and?BUN?were?significantly?decreased?[SCr?(μmol/L):?85.70±7.23?vs.?182.60±6.97,?BUN?(mmol/L):?35.30±3.50?vs.?53.70±5.12,?both?P?<?0.01];?the?mitochondrial?structure?was?relatively?intact;?the?levels?of?MDA,?GSH?and?NOS?were?significantly?decreased?[MDA?(μmol/g):?0.958±0.072?vs.?1.172±0.046,?GSH?(μmol/g):?4.756±0.107?vs.?5.765±0.059,?NOS?(kU/g):?0.79±0.02?vs.?0.91±0.05,?all?P?<?0.01];?the?protein?expressions?of?Klotho,?Bcl-2?were?significantly?increased,?but?the?protein?expression?of?Cyt?C?was?significantly?decreased?(Klotho/β-actin:?0.336±0.011?vs.?0.188±0.020,?Bcl-2/β-actin:?0.474±0.017?vs.?0.311±0.010,?Cyt?C/β-actin:??0.168±0.006?vs.?0.226±0.010,?all?P?<?0.01).? Conclusion? Klotho?protein?has?significant?protective?effects?on?AKI?in?septic?mice,?and?its?mechanism?is?related?to?maintaining?mitochondrial?structural?integrity?and?oxidative?stress?response.
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OBJECTIVES: Pseudolaric acid B (PAB) has been shown to inhibit the growth of various tumor cells, but the molecular details of its function are still unknown. This study investigated the molecular mechanisms by which PAB induces apoptosis in HeLa cells. METHODS: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to investigate the effect of PAB treatment in various cervical cancer cell lines. Annexin V/propidium iodide staining combined with flow cytometry and Hoechst 33258 staining were used to assess PAB-induced apoptosis. Additionally, we performed bioinformatics analyses and identified a paired box 2 (PAX2) binding site on the BAX promoter. We then validated the binding using luciferase and chromatin immunoprecipitation assays. Finally, western blotting assays were used to investigate PAB effect on the Wnt signaling and the involved signaling molecules. RESULTS: PAB promotes apoptosis and downregulates PAX2 expression in HeLa cells in a time- and concentration-dependent manner. PAX2 binds to the promoter of BAX and inhibits its expression; therefore, PAX2 inhibition is associated with increased levels of BAX, which induces apoptosis of HeLa cells via the mitochondrial pathway. Additionally, PAB inhibits classical Wnt signaling. CONCLUSION: PAB effectively inhibits Wnt signaling and PAX2 expression, and increases BAX levels, which induce apoptosis in HeLa cells. Therefore, PAB is a promising natural molecule for the treatment of cervical cancer.
Subject(s)
Humans , Apoptosis , Binding Sites , Bisbenzimidazole , Blotting, Western , Cell Line , Chromatin Immunoprecipitation , Computational Biology , Flow Cytometry , HeLa Cells , Luciferases , Mitochondria , Uterine Cervical Neoplasms , Wnt Signaling PathwayABSTRACT
OBJECTIVE Diabetes-induced endothelial cell (EC) dysfunction and neovasculariza-tion impairment constitute vascular complications with limited treatment regimens.Transcription factor FOXO1 is a key angiogenic regulator and plays a pathologic role in progression of diabetes.The pres-ent study was designed to determine the involvement of FOXO1 in impaired EC function and post-isch-emic neovascularization in diabetes and investigate underlying mechanisms.RESULTS We found that FOXO1-selective inhibitor AS1842856 improved blood flow recovery and capillary density in ischemic hindlimb,and rescued the delay of wound closure with a concomitant augmentation of mean perfusion rate in diabetic mice. In vitro,treatment with AS1842856 or FOXO1 siRNA abrogated high glucose-in-duced apoptosis and ameliorated capillary tube formation in human umbilical vein endothelial cells(HU-VECs). FOXO1 inhibition relieved alterations in mitochondrial networks and significantly suppressed the over production of mitochondrial reactive oxygen species(mtROS)induced by high glucose in ECs. Expression of dynamin-relatedprotein-1 (Drp1) and phosphorylation at Ser616, a protein required for mitochondrial fission, were enhanced by hyperglycemia, which could be neutralized by FOXO1 inhibition. Moreover, the transcription of Rho-associated coiled-coil containing protein kinase 1 (ROCK1), which phosphorylates Drp1 at Ser616, was shown by luciferase assay to be directly regulated by FOXO1. CONCLUSION These findings suggested that FOXO1 is critical to preserve mitochondrial quantity and func-tion in ECs,and FOXO1 may serve as a therapeutic target for microvascular complications of diabetes.
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Objective To study the effects of hypothermia on learning and memory ability and mRNA expression of fusion gene and fission gene of mitochondria in hippocampus of rats.Methods 32 Wistar rats were randomly divided into 4 groups according to low-temperature exposure time in the test:high-exposure group(low-temperature exposure for 24 h/d),middle-exposure group(low-temperature exposure for 12 h/d),low-exposure group(low-temperature exposure for 6 h/d) and control group.The temperature of low-temperature exposure was 0-5 ℃.The total test time was 45 d.Morris water maze test was performed on each group from the fifth day before the end of the low-temperature test to the first day after the low-temperature test.After the water maze test,the mRNA expressions of mitochondrial fusion genes (Mfn1,Mfn2) and fission genes(Fis1,Drp1) in hippocampus were detected by RT-qPCR.Results Compared with the control group,the escaping latency of the high-exposure group and the middle-exposure group at the first day and the fifth day of water maze test were significantly longer than those in the control group,and the difference was statistically significant(P＜0.05,P＜0.01).The escaping latency of the sixth day and the number of platform crossings at the beginning of the water maze test were significantly different from those in the control group(P ＜0.05,P＜0.01).Compared with the control group,the escaping latency of the high-exposure group and the middle-exposure group was significantly prolonged on the sixth day,and the number of the platform crossings decreased significantly.The difference was statistically significant (P＜ 0.05,P＜ 0.01).The high,middle and low-exposure group of mitochondrial fusion and fission genes mRNA expressions were as follows respectively:Mfn1:4.05 ±0.21,1.51±0.23,1.17 ±0.83;Mfn2:5.38 ±0.74,0.84 ±0.53,0.47 ±0.33;Fis1:1.65 ±0.58,0.49 ± 0.42,0.40±0.32;Drp1:4.11 ±0.37,0.99 ±0.82,0.55 ±0.29.Compared with the control group,the mRNA expression of the fusion and the fission genes in the high-exposure group increased significantly(P＜0.01).Conclusion The abnormalities of mitochondrial fusion and fission gen mRNA expression in the hippocampus may be one of the mechanisms of the decline of learning and memory functions caused by low temperature exposure.
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The incidence and mortality of septic cardiomyopathy were high, which is the common cause of death in the patients with sepsis. The pathogenesis of sepsis induced myocardial injury is still unclear. The mitochondrial dysfunction of myocardial cells plays a very important role in the pathophysiological mechanism. This review attempts to introduce the mitochondrial damage of cardiomyocyte from the regulation of mitochondrial energy metabolism, the mechanism of mitochondrial damage, the role of aquaporin and the adjustment of mitochondrial dynamic, in order to provide help for the early prevention of septic cardiomyopathy.
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OBJECTIVE:To study the inhibitory effect and mechanism of Inula helenium ethyl acetate extract(IHE)on prolif-eration of human pancreatic cancer Capan-2 cells. METHODS:MTT was used to determine the cell proliferation inhibition rate af-ter treated by 0,0.5,1,2,4,8 μg/mL IHE;clone formation test was used to observe the effects of 0,1,2 μg/mL IHE treating for 1 week on cell clone formation;Hoechest 33342 staining was used to observe the changes of nuclear morphology after treated by 0,2,4 μg/mL IHE for 48 h;flow cytometry was used to detect the cell apoptosis rate after treated by 0,4,8,16 μg/mL IHE for 48 h;JC-1 staining was used to observe the changes of intracellular mitochondrial membrane potential after treated by 0,4,8, 16 μg/mL IHE for 24 h;Western blot was used to detect the expressions of mitochondrial apoptosis-related proteins Bcl-2,Bax, Mcl-1,p53 up-regulated modulator of apoptosis (PUMA),and polymerase (PARP) after treated by 0,4,8,16 μg/mL IHE for 48 h. RESULTS:2,4,8 μg/mL IHE had obvious inhibitory effect on cell proliferation,showing concentration-dependent relation-ship,with IC50 of 6.6 μg/mL;1,2 μg/mL IHE can obviously inhibit the clone formation of cells;4 μg/mL IHE can obviously cause cell nuclear condensation;8,16 μg/mL IHE can obviously promote the cell apoptosis,and the cell apoptosis rate reached 45.53% after treated by 16 μg/mL IHE for 48 h;16 μg/mL IHE treating for 24 h can cause the decrease of 82.47% cells'mito-chondrial membrane potential;8 μg/mL IHE can obviously down-regulate the protein expressions of Bcl-2,Mcl-1,PUMA and PARP,and 16 μg/mL IHE can obviously down-regulate the expressions of Mcl-1 and PUMA. CONCLUSIONS:IHE may show its inhibitory effect on proliferation of human pancreatic cancer Capan-2 cells by causing the decrease of mitochondrial mem-brane potential in cells and down-regulating the protein expres-sions of Mcl-1 and PUMA to cause cell apoptosis.