Caloric Restriction Induces MicroRNAs to Improve Mitochondrial Proteostasis.

Both caloric restriction (CR) and mitochondrial proteostasis are linked to longevity, but how CR maintains mitochondrial proteostasis in mammals remains elusive. MicroRNAs (miRNAs) are well known for gene silencing in cytoplasm and have recently been identified in mitochondria, but knowledge regarding their influence on mitochondrial function is limited. Here, we report that CR increases miRNAs, which are required for the CR-induced activation of mitochondrial translation, in mouse liver. The ablation of miR-122, the most abundant miRNA induced by CR, or the retardation of miRNA biogenesis via Drosha knockdown significantly reduces the CR-induced activation of mitochondrial translation. Importantly, CR-induced miRNAs cause the overproduction of mtDNA-encoded proteins, which induces the mitochondrial unfolded protein response (UPRmt), and consequently improves mitochondrial proteostasis and function. These findings establish a physiological role of miRNA-enhanced mitochondrial function during CR and reveal miRNAs as critical mediators of CR in inducing UPRmt to improve mitochondrial proteostasis.

Quantitative proteomics reveals mitochondrial respiratory chain as a dominant target for carbon ion radiation: Delayed reactive oxygen species generation caused DNA damage.

Heavy ion radiotherapy has shown great promise for cancer therapy. Understanding the cellular response mechanism to heavy ion radiation is required to explore measures of overcoming devastating side effects. Here, we performed a quantitative proteomic analysis to investigate the mechanism of carbon ion irradiation on human AHH-1 lymphoblastoid cells. We identified 4602 proteins and quantified 4569 proteins showing high coverage in the mitochondria. Data are available via ProteomeXchange with identifier PXD008351. After stringent filtering, 290 proteins were found to be significantly up-regulated and 16 proteins were down-regulated. Functional analysis revealed that these up-regulated proteins were enriched in the process of DNA damage repair, mitochondrial ribosome, and particularly mitochondrial respiratory chain, accounting for approximately 50% of the accumulated proteins. Bioinformatics and functional analysis demonstrated that these up-regulated mitochondrial respiratory chain proteins enhanced ATP production and simultaneously reactive oxygen species release. More importantly, increased reactive oxygen species led to secondary organelle injury and lagged DNA double-strand breaks. Consistently, the expression of antioxidant enzymes was up-regulated for free radical scavenging. The mechanism of lagged secondary injury originated from disturbances in the mitochondrial respiratory chain. Our results provided a novel target for cell self-repair against heavy ion radiation-induced cellular damage.

A new anti-fibrinolytic hemostatic compound 8-O-acetyl shanzhiside methylester extracted from Lamiophlomis rotata.

BACKGROUND: Fibrinolysis prevents blood clots from growing and becoming problematic. Antifibrinolytics are used as inhibitors of fibrinolysis. Aprotinin was doubted after identification of major side effects, especially on kidney. Lysine analogues has their own defects and whether they are adequate substitutes for aprotinin is still under doubt. Lamiophlomis rotata (Benth.) Kudo. was previous found to have hemostatic activity. But the active compound in L. rotata and its hemostatic mechanism were unknown. OBJECTIVES: To find the major hemostatic compound in L. rotata and identify its haemostasis mechanism. METHODS: Traumatic hemorrhage model and coagulant activity assays were monitored in mice and platelets in drug treatment group and control group. Hyperfibrinolysis model was established by intravenous administration of urokinase in mice. Capillary blood clotting time (CBCT), activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen and euglobulin clot lysis time (ECLT) were measured. RESULTS: The anti-fibrinolytic activity come from 8-O-Acetyl shanzhiside methylester (ASM) one of the highest iridoid glycosides contents in TIG extracted from L. rotata. ASM significantly (P<0.05) shorten CBCT and reduced blood loss volume in vivo, but did not influence mice APTT, PT or TT. In particular, it significantly prolonged ECLT in hyperfibrinolysis mice. It indicated that ASM could inhibit fibrinolysis. ASM was also effective in CBCT, traumatic bleeding volume and ECLT in hyperfibrinolysis mice model. CONCLUSIONS: ASM was the major hemostatic compound in L. rotata. The haemostasis mechanism of ASM was achieved by anti-fibrinolytic activity. ASM was a new fibrinolysis inhibitor as iridoid glycoside compound.

[Protective Effect of Saussurea involucrata Alcohol Extract on Liver Mitochondria in Mice Under Hypoxia Condition].

OBJECTIVE: To study the protective effect of Saussurea involucrata alcohol extract on liver mitochondria in mice under hypoxia condition. METHODS: The hypoxia mice model was established, the BALB/c mice were randomly divided into four groups:normal group ,hypoxia model group, positive control group and Saussurea involucrata alcohol extract group. Mice were put into low pressure oxygen chamber and decompressed, adapted to hypobaric hypoxia environment of simulated altitude of 8,000 m for 12 h, and then recovered to normal altitude. The mice were sacrificed and the liver mitochondria was isolated, the mitochondrial membrane potential and the activity of malate dehydrogenase, aconitase, α-ketoglutarate dehydrogenase, pyruvate dehydrogenase and mitochondrial complex I, II and V were measured. RESULTS: Compared with hypoxia model group, Saussurea involucrata alcohol extract protected mitochondrial membrane potential, sustained the activities of aconitase, α-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and mitochondrial complex I, II and V under hypoxia condition. CONCLUSION: Saussurea involucrata alcohol extract can protect the liver mitochondrial function in mice under hypoxia condtion.

Anti-hypoxia Activity of the Novel NO Donor Acetyl Ferulic Isosorbide Mononitrate in Acute High-Altitude Hypoxia Mice.

Nitric oxide (NO) may act as either a pro-oxidant or an antioxidant in biological systems. Previous work has found inhalation of NO improved survival in a high altitude rat model. NO donor isosorbide mononitrate derivants might have a protective effect against hypoxia. We synthesized a series of isosorbide mononitrate derivant compounds to test their anti-hypoxia activities. Normobaric hypoxia and hypobaric hypoxia models were used to study the protective role of NO donor in mice. The results showed isosorbide mononitrate derivants had protective effects in hypoxia mice. Among those compounds, acetyl ferulic isosorbide mononitrate (AFIM) was the most effective. It prolonged the survival time during the normobaric hypoxia test. It decreased malondialdehyde (MDA) and H2O2 in hypobaric hypoxia mice. The antioxidase activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) remained in normal ranges in the AFIM group. As a sign of mitochondrial dysfunction, the activities of ATPase were down regulated in mice under hypobaric hypoxia conditions. AFIM also protected ATPase activities. The protective effects of AFIM might come from a sustained NO supply and the release of acetyl ferulic acid with anti-oxidant activity.

[Chemical Constituents with Anti-hypoxia Activity from Saussurea involucrata].

OBJECTIVE: To investigate the chemical constituents with anti-hypoxia activity from Saussurea involucrata. METHODS: The chemical constituents, isolated and purified by column chromatography from Saussurea involucrata, were identified by several spectroscopic methods. The anti-hypoxic activities of these compounds were examined using the normobaric hypoxic model of mice. RESULTS: Twelve compounds were isolated from petroleum ether extract of Saussurea involucrata and identified as n-octacosane (1), 1-undecanol (2), heptadecan-l-ol(3), heptacosan-1-ol(4), myristicin (5), apiol(6), ß-sitosterol(7), lupeol(8), moslosooflavone (9), mosloflavone (10), negletein(11), and 5, 6-dihydroxy-7, 8-dimethoxyflavone(12). CONCLUSION: All compounds except 7 and 8 are isolated from this plant for the first time. Compound 1, 5 and 8 - 12 can significantly prolong the survival time of hypoxic mice.

Convergent synthesis of moslosooflavone, isowogonin and norwogonin from chrysin.

A convergent synthesis route of moslooflavone, isowogonin and norwogoninis reported,starting from chrysin, an easily available flavone, by methylation, bromination, methoxylation and demethylation procedures. This synthetic route is convenient and can give the three rare flavones in good yield.

[Protective effect and action mechanism of petroleum ether extracts from Saussurea involucrate on brain tissues of hypoxia rats].

OBJECTIVE: To investigate the protective effect and action mechanism of petroleum ether extracts from Saussurea involucrate on brain tissues of hypoxia rats under constant pressure and closed conditions. METHOD: The PESI dosage-dependent experiment for hypoxia rats was conducted under constant pressure and closed conditions by intraperitoneally injecting 125, 250, 500 mg x kg(-1) to finalize that the optimum dosage is the high dose of PESI. Afterwards, 90 Wistar rats were randomly divided into the hypoxic model group, the acetazolamide 250 mg x kg(-1) group and the PESI high dose group. Each group was further divided into three subgroups according to different hypoxia times, with 10 rats in each subgroup. Under the same hypoxia and administration conditions, the rats were sacrificed after 0, 3, 6 h respectively. Their brain samples were collected for common pathological observation and immunohistochemical staining of HIF-1alpha. Real-time RT-PCR was used to detect HIF-1alpha, EPO, HO-1 and Caspase-3 gene expressions. And the Western blot assay was adopted to detect HIF-1alpha protein expression. RESULT: The brain tissues of the hypoxia model group were severely damaged with the increase in the hypoxia time. The acetazolamide group and the PESI high does group were damaged in a much lower degree. According to the gene expression and the Western blot assay, high dose of PESI could inhibit HIF-1alpha expression. According to the pure gene expression test, high dose of PESI could increase EPO and HO-1 mRNA expressions, but inhibit Caspase-3 mRNA expression. CONCLUSION: PESI's protective mechanism for brain tissues of hypoxia rats under constant pressure and closed conditions may be related to its effects in inhibiting HIF-1alpha expression, increasing EPO expression and resisting cell apoptosis.

Liquid Chromatography Tandem Mass/Mass Spectrometry for the Quantification of Fudosteine in Human Serum without Precolumn Derivatization.

A quantitative analysis method for fudosteine in human serum by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI/MS/MS) was established, which shows high sensitivity and selectivity. The mobile phase composition was 75% 20 mM acetic acid and 25% acetonitril, which was pumped at a flow rate of 0.40 mL/min. The overall chromatographic run time was approximately 7 min. The autosampler was set with an injection volume of 10 µL. The calibration curve was linear in the concentration range of 0.1~15.0 µg/mL. The coefficient of determination (r) was greater than 0.9998. This method has been fully validated and shown to be specific, accurate and precise. The method was simple, rapid and the sample preparation was minimal. It was successfully applied to the analysis of healthy volunteer.

[Effect of ethanol extract from Saussurea involucrata on biochemical indicators of simulated high-altitude hypoxia induced mice].

OBJECTIVE: To estimate the effect of ethanol extract from Saussurea involucrata (EES) on biochemical indicators of simulated high-altitude hypoxia induced mice and its mechanism. METHODS: The oxidative stress indicator( MDA content, SOD activity) and metabolism parameters (LD content, LDH activity, ATP content, Na+ -K+ -ATPase and Ca2+ -Mg2+ -ATPase activity) in both brain and heart of the simulated high-altitude hypoxia induced mice were detected. RESULTS: Compared with the model group, the ESS group could significantly increase the activity of SOD and LDH and decrease the content of MDA and LD in both brain and heart, the content of ATP and the activity of Na+ -K -ATPase and Ca2+ -Mg2+ -ATPase were also elevated. CONCLUSION: The results demonstrate that EES can increase the antioxidant ability, decrease the injury of free radical and ease the disfunction of energy metabolism caused by hypoxia.

5,6-Dihy-droxy-7,8-di-meth-oxy-flavone.

THE TITLE COMPOUND (SYSTEMATIC NAME: 5,6-dihy-droxy-7,8-dimeth-oxy-2-phenyl-chromen-4-one), C17H14O6, is a flavone that was isolated from the petroleum ether-soluble fraction of the rare traditional Chinese medicinal herb Saussurea involucrata. The flavone mol-ecule is almost planar, with a dihedral angle between the planes of the benzo-pyran-4-one group and the attached phenyl group of 1.89 (6)°. The 5-hy-droxy group forms a strong intra-molecular hydrogen bond with the carbonyl group, resulting in a six-membered hydrogen-bonded ring. The 6-hy-droxy group also forms an intra-molecular O-H⋯O contact. In the crystal, the molecules are linked by O-H⋯O and C-H⋯O hydrogen bonds and π-π inter-actions [3.37 (2)-3.39 (2) Å], which build up a three-dimensional network.

The antioxidative effect of a novel free radical scavenger 4'-hydroxyl-2-substituted phenylnitronyl nitroxide in acute high-altitude hypoxia mice.

Acute mountain sickness is caused by sub-acute hypoxia in healthy individuals going rapidly to altitude. Both tissue hypoxia in vitro and whole-body hypoxia in vivo have been found to promote the release of reactive oxygen species. Nitronyl nitroxide can trap free radicals such as ·NO or ·OH, and may therefore be efficient protective agents. This study assessed the ability of nitronyl nitroxide to against acute mountain sickness as a free radical scavenger in acute high-altitude hypoxia mice model. Normobaric hypoxia and hypobaric hypoxia model were used to estimate the protect effects of nitronyl nitroxide against acute mountain sickness. Low pressure oxygen compartment system was used to stimulate high-altitude hypobaric hypoxia environment. Mice in nitronyl nitroxide groups survived longer than acetazolamide group in normobaric hypoxia test. Hydrogen peroxide (H2O2) and malondialdehyde (MDA) increased in both cerebrum and myocardium in vehicle group. The results indicated more radicals were generated during high-altitude hypobaric hypoxia environment. In therapeutic groups H2O2 and MDA were significantly reduced while the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were similar to normal group. These results demonstrated that nitronyl nitroxide was an efficient tissue radical scavenger and a potential protective agent for acute mountain sickness.

Simultaneous determination of four shanzhiside methylester derivatives in rabbit plasma by liquid chromatography/tandem mass spectrometry.

A simple and sensitive high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for simultaneous determination of shanzhiside methylester and its three derivatives in rabbit plasma. The method showed good linearity and no endogenous material interfered with the marked compounds and internal standard (IS) capatol peaks. Samples were processed by acetonitrile precipitation. Chromatography was performed using a C18 column (150 × 3.9 mm i.d., 4 µm). The mobile phase consisted of methanol and water (60:40, v/v) during a total run time of 7 min. The main mass parent ions and daughter ions pairs (m/z) for monitoring were: shanzhiside methylester, 429.0/267.4; 8-O-acetyl shanzhiside methylester, 470.9/411.3; loganin, 413.2/251.4; phloyoside II, 479.2/281.3; and IS 385.2/203.3. Finally, the method was applied to a pharmacokinetic study of rabbits following intravenous administration of iridoid glycosides extracted from traditional herb Lamiophlomis rotata.

2-[3-Hy-droxy-4-(2-hy-droxy-eth-oxy)phen-yl]-4,4,5,5-tetra-methyl-2-imidazoline-1-oxyl 3-oxide.

In the title compound, C(15)H(21)N(2)O(5), the imidazoline ring displays a twisted conformation. The mean plane of the imidazoline ring makes a dihedral angle of 22.55 (5)° with the benzene ring. In the crystal, O-H⋯O and C-H⋯O hydrogen bonds link the mol-ecules into a layer parallel to the bc plane.

2-[2-(2-Hy-droxy-eth-oxy)phen-yl]-4,4,5,5-tetra-methyl-2-imidazoline-1-oxyl 3-oxide.

In the title compound, C(15)H(21)N(2)O(4), the nitronyl nitroxide unit displays a twisted conformation. The crystal structure is stabilized by non-classical C-H⋯O and C-H⋯π hydrogen bonds, which build up a three-dimensional network.

Anti-hypoxic activity at simulated high altitude was isolated in petroleum ether extract of Saussurea involucrata.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhodiola algida, Saussurea involucrata, and other herbs grown in Qinghai-Tibetan plateau have long been used to prevent and treat acute mountain sickness. AIM OF THE STUDY: To screen and identify the anti-hypoxic constituents in the herbs grown in Qinghai-Tibetan plateau of Northwestern China. MATERIALS AND METHODS: The anti-hypoxic activities of 20 selected plateau herbs were examined against two positive controls, Rhodiola algida and acetazolamide, using the normobaric hypoxia model of mice. The herb with the highest activity was successively extracted with 70% ethanol, petroleum ether, chloroform, ethyl acetate and n-butanol. The extract with the highest activity was identified by comparing the survival time of mice under normobaric hypoxia condition after being subjected to different extracts. The identified extract was further tested by simulating high altitudes through an acute decompression model and a chronic decompression model for mice. RESULTS: The herb found to have the highest anti-hypoxic activity was Saussurea involucrate (Kar. et Kir.) Sch.-Bip, and the most effective fraction was in the petroleum ether extract. Administration of petroleum ether extract of Saussurea involucrata (PESI) to mice at 50mg/kg significantly decreased the mortality of animals under acute decompression conditions. Changes in biochemical indicators for glycometabolism and energy metabolism, including adenosine triphosphate (ATP) content and adenosine triphosphatase (ATPase) activity in brain and cardiac muscle, lactic acid (LAC) and lactate dehydrogenase (LDH) in blood and cardiac muscles, blood sugar, and glycogen content in liver and skeletal muscle were reversed under chronic decompression conditions. CONCLUSIONS: Saussurea involucrata (Kar. et Kir.) Sch.-Bip exhibits high anti-hypoxic activity that may be effective in preventing acute mountain sickness, and the active constituents are mainly in the petroleum ether extract.

Phytochemical and biological studies of plants from the genus Meconopsis.

In this review, the literature data on the phytochemical and biological investigations on the genus of Meconopsis are summarized from 49 references. Up to now, more than 95 compounds were isolated from 19 Meconopsis plant species. The chemical constituents are mostly alkaloids, flavonoids, phenols, steroids, and terpenes, together with minor constituents of essential oil, and others. The crude extracts and metabolites have been found to possess various bioactivities including antitumor activity, central action, cardiovascular system effects, antibiosis, antiviral activity, anti-inflammatory effects, and other biological activities.

HPLC-DAD and HPLC-ESI-MS separation, determination and identification of the spin-labeled diastereoisomers of podophyllotoxin.

Spin-labeled nitroxide derivatives of podophyllotoxin had better antitumor activity and less toxicity than that of the parent compounds. However, the 2-H configurations of these spin-labeled derivatives cannot be determined by nuclear magnetic resonance (NMR) methods. In the present paper, a high-performance liquid chromatography-diode array detection (HPLC-DAD) and a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI/MS/MS) method were developed and validated for the separation, identification of four pairs of diastereoisomers of spin-labeled derivatives of podophyllotoxin at C-2 position. In the HPLC-ESI/MS spectra, each pair of diastereoisomers of the spin-labeled derivatives in the mixture was directly confirmed and identified by [M+H](+) ions and ion ratios of relative abundance of [M-ROH+H](+) (ion 397) to [M+H](+). When the [M-ROH+H](+) ions (at m/z 397) were selected as the precursor ions to perform the MS/MS product ion scan. The product ions at m/z 313, 282, and 229 were the common diagnostic ions. The ion ratios of relative abundance of the [M-ROH+H](+) (ion 397) to [M+H](+), [A+H](+) (ion 313) to [M-ROH+H](+), [A+H-OCH(3)](+) (ion 282) to [M-ROH+H](+) and [M-ROH-ArH+H](+) (ion 229) to [M-ROH+H](+) of each pair of diastereoisomers of the derivatives specifically exhibited a stereochemical effect. Thus, by using identical chromatographic conditions, the combination of DAD and MS/MS data permitted the separation and identification of the four pairs of diastereoisomers of spin-labeled derivatives of podophyllotoxin at C-2 in the mixture.

Separation, determination and identification of the diastereoisomers of podophyllotoxin and its esters by high-performance liquid chromatography/tandem mass spectrometry.

A rapid and stable high-performance liquid chromatography-diode array detection (HPLC-DAD) and a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI/MS/MS) method were developed and validated for the separation, determination, and identification of eight pairs of diastereoisomers of podophyllotoxin and its esters at C-2 position. The separation was carried out on BDS Hypersil C18 column with CH3OH-CH3CN-H2O as the mobile phase in a gradient program. Interestingly, every 2alpha-H compound migrated before its corresponding 2beta-H epimer under optimum conditions. Also, the [M+NH(4)](+) of all eight pairs of compounds was observed in the HPLC-ESI/MS spectra. The characteristic elimination from the precursor protonated ions and the product ions at m/z 397, 313, 282, and 229 were the common diagnostic masses. The ion ratios of relative abundance [M-ROH+H](+) (ion 397) to [M+NH(4)](+), [A+H](+) (ion 313) to [M-ROH+H](+), and [M-ROH-ArH+H](+) (ion 229) to [M-ROH+H](+) in the ESI/MS/MS spectra of each pair of diastereoisomers of the lignans specifically exhibited a stereochemical effect. Thus, by using identical sample solutions and chromatographic conditions (including the same columns and gradient programs), the combination of DAD and MS/MS data permitted the separation and identification of the eight pairs of diastereoisomers of the podophyllotoxin and its esters in the mixture. The method could be used in rapidly identifying the purity and monitoring of the epimerization of 2-H of podophyllotoxin and its analogues from natural products, chemical reactions, and pharmaceutical metabolism.