Effect of diosmetin on ferroptosis of hippocampal cells in rats with bacterial meningitis and the mechanism of SIRT1-Nrf2 signaling pathway / 中华行为医学与脑科学杂志

Objective:To explore the effect and mechanism of diosmetin (Dio) on neuronal ferroptosis in rats with bacterial meningitis (BM).Methods:Male SD rats aged 6-7 weeks of SPF grade were selected for the experiment. The BM model was established by injecting group B hemolytic streptococcus into the cisterna magna of cerebellum. Sixty BM model rats were successfully modeled and divided into model group, low-dose Dio group, medium-dose Dio group, high-dose Dio group and inhibitor group according to the random number table method, with 12 rats in each group. Another 12 weight-matched rats were taken as the control group.The rats in the low-dose Dio group, medium-dose Dio group, high-dose Dio group and the inhibitor group were intragastrically administered with Dio at 50 mg/kg, 100 mg/kg, 200 mg/kg and 200 mg/kg, respectively. The rats in the control group were intragastrically administered with an equal volume of 0.9 % sodium chloride solution. On the day of intragastric administration, the rats in the inhibitor group were intraperitoneally injected with SIRT1 pathway inhibitor EX527 (10 mg/kg), and the rats in the other groups were injected with an equal volume of 0.9% sodium chloride solution. The above interventions were performed once a day for 28 consecutive days. Loeffler neurological score was used to evaluate the neurological impairment in rats. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cerebrospinal fluid of rats were detected by ELISA. The number of white blood cells in cerebrospinal fluid was detected by a blood cell analyzer. Glutathione (GSH) was detected by micro-enzyme labeling method, malondialdehyde (MDA) was detected by thiobarbituric acid colorimetric method, reactive oxygen species(ROS) was detected by colorimetry, and Fe 2+ level was detected by ferrozine method. Hematoxylin-eosin staining, Prussian blue staining and TUNEL staining were used to observe the pathological damage, iron accumulation and apoptosis in the hippocampus, respectively.Western blot was applied to measure the expression of transferrin (Tf), proliferating cell nuclear antigen (PCNA), Bcl-2-associated X protein (Bax), caspase-3 and SIRT1/Nrf2/HO-1/Gpx4 signaling pathway proteins. Graphpad Prism 9.0 was used for data analysis. One-way ANOVA was used for statistical analysis, and SNK- q test was used for further pairwise comparisons. Results:(1) There was a statistically significant difference in neurological function scores among the 6 groups of rats ( F=125.451, P<0.001). The neurological function score of the model group was lower than that of control group, while the neurological function scores of the low-dose Dio group, medium-dose Dio group, and high-dose Dio group were higher than those of the model group (all P<0.05). The neurological function score of the inhibitor group ((2.57±0.26)) was lower than that of high-dose Dio group ((4.34±0.48)) ( P<0.05). (2) There were statistically significant differences in the levels of IL-6, TNF-α and the number of white blood cells in the cerebrospinal fluid of rats among the 6 groups ( F=127.817, 102.413, 180.967, all P<0.001). The levels of IL-6, TNF-α and the number of white blood cells in model group were higher than those of control group(all P<0.05). The levels of IL-6, TNF-α and the number of white blood cells in low-dose Dio group, medium-dose Dio group and high-dose Dio group were lower than those of model group (all P<0.001), and those in inhibitor group were all higher than those in high-dose Dio group(all P<0.001). (3) There were statistically significant differences in iron deposition rate and neuronal apoptosis rate among the 6 groups of rats ( F=90.857, 88.835, both P<0.001). The iron deposition rate ((18.37±3.14)%) and neuronal apoptosis rate ((27.58±2.63)%) in the inhibitor group were higher than those in the high-dose Dio group ((6.35±1.08)%, (14.02±1.87)%) (both P<0.05). (4) The levels of GSH, ROS, MDA, and Fe 2+ in the hippocampus of the 6 groups of rats showed statistically significant differences ( F=54.465, 106.453, 55.969, 105.457, all P<0.001). The GSH content in the inhibitor group ((103.48±8.76) mmol/g) was lower than that in the high-dose Dio group ((133.97±10.54) mmol/g), while the contents of ROS, MDA, Fe 2+ ((225.17±16.32) μmol/mg, (10.73±1.58) μmol/mg, (62.71±5.43) μg/g) were higher than those of the high-dose Dio group ((131.87±11.67) μmol/mg, (4.35±0.87) μmol/mg, (34.86±2.95) μg/g) (all P<0.05). (5)There were statistically significant differences in the protein levels of Tf, PCNA, Bax, caspase-3, SIRT1, Nrf2, HO-1 and Gpx4 in the hippocampus of the 6 groups of rats ( F=120.179, 107.568, 157.265, 98.031, 90.932, 52.283, 59.424, 114.539, all P<0.001). The protein levels of Tf, Bax and caspase-3 in the hippocampus of inhibitor group were higher than those of the high-dose Dio group, while the protein levels of PCNA, SIRT1, Nrf2, HO-1, Gpx4 were lower than those of the high-dose Dio group (all P<0.05). Conclusion:Diosmetin can activate SIRT1/Nrf2/HO-1/Gpx4 signaling pathway, thereby inhibiting neuronal ferroptosis in BM rats.

Differential transcription of mating-type genes during sexual reproduction of natural Cordyceps sinensis / 中国中药杂志

Natural Cordyceps sinensis as an insect-fungal complex, which is developed after Ophiocordyceps sinensis infects a larva of Hepialidae family. Seventeen genotypes of O. sinensis have been identified in natural C. sinensis. This paper summarized the literature reports and GenBank database regarding occurrence and transcription of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in natural C. sinensis, in Hirsutella sinensis(GC-biased Genotype #1 of O. sinensis), to infer the mating pattern of O. sinensis in the lifecycle of natural C. sinensis. The mating-type genes and transcripts of MAT1-1 and MAT1-2 idiomorphs were identified in the metagenomes and metatranscriptomes of natural C. sinensis. However, their fungal sources are unclear because of co-colonization of several genotypes of O. sinensis and multiple fungal species in natural C. sinensis. The mating-type genes of MAT1-1 and MAT1-2 idiomorphs were differentially present in 237 H. sinensis strains, constituting the genetic control of the O. sinensis reproduction. Transcriptional control of the O. sinensis reproduction includes: differential transcription or silencing of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs, and the MAT1-2-1 transcript with unspliced intron I that contains 3 stop codons. Research on the H. sinensis transcriptome demonstrated differential and complementary transcriptions of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in Strains L0106 and 1229, which may become mating partners to accomplish physiological heterothallism. The differential occurrence and transcription of the mating-type genes in H. sinensis are inconsistent with the self-fertilization hypothesis under homothallism or pseudohomothallism, but instead indicate the need of mating partners of the same H. sinensis species, either monoecious or dioecious, for physiological heterothallism, or heterospecific species for hybridization. Multiple GC-and AT-biased genotypes of O. sinensis were identified in the stroma, stromal fertile portion(densely covered with numerous ascocarps) and ascospores of natural C. sinensis. It needs to be further explored if the genome-independent O. sinensis genotypes could become mating partners to accomplish sexual reproduction. S. hepiali Strain FENG experienced differential transcription of the mating-type genes with a pattern complementary to that of H. sinensis Strain L0106. Additional evidence is needed to explore a hybridization possibility between S. hepiali and H. sinensis, whether they are able to break the interspecific reproductive isolation. Genotypes #13～14 of O. sinensis feature large DNA segment reciprocal substitutions and genetic material recombination between 2 heterospecific parental fungi, H. sinensis and an AB067719-type fungus, indicating a possibility of hybridization or parasexuality. Our analysis provides important information at the genetic and transcriptional levels regarding the mating-type gene expression and reproduction physiology of O. sinensis in the sexual life of natural C. sinensis and offers crucial reproductive physiology evidence, to assist in the design of the artificial cultivation of C. sinensis to supplement the increasing scarcity of natural resource.

Astaxanthin attenuates contrast-induced acute kidney injury in rats by inhibiting dynamin-related protein 1-mediated mitochondrial fission through activating SIRT1 pathway / 中华肾脏病杂志

Objective:To investigate whether astaxanthin (AST) down-regulates dynamin-related protein 1 (Drp1) through activating the silent mating type information regulation 2 homolog-1 (SIRT1) signaling pathway, thereby attenuating contrast-induced acute kidney injury.Methods:Forty adult male Sprague-Dawley rats weighing 160-180 g were randomly divided into five groups: sham surgery group (Sham group), contrast medium injury group (CM group), astaxanthin-intervention group (AST+CM group), SIRT1 inhibitor Ex527 intervention group (Ex527+CM group), and astaxanthin combined with Ex527 intervention group (AST+Ex527+CM group). After 72 hours of modeling, heart blood was removed and kidney tissues were collected for follow-up testing. Serum creatinine (Scr), blood urea nitrogen (BUN), and oxidative stress-related indexes total superoxide dismutase (T-SOD) and malondialdehyde (MDA) were measured by biochemistry; hematoxylin and eosin staining was performed to observe the pathological changes in the kidney; mitochondrial morphology and number were observed by transmission electron microscopy; reactive oxygen species (ROS) levels were detected by ROS staining in frozen sections; TUNEL staining was performed to detect apoptosis level. The expression levels of SIRT1, p53, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), Drp1 and apoptosis-related proteins Bcl-2 and Bax were detected by Western blotting.Results:(1) Compared with the CM group, Scr and BUN level were significantly lower, T-SOD level was higher and MDA level was lower in the AST+CM group, while T-SOD level decreased and MDA level increased after the combination of Ex527 (all P<0.05). (2) ROS expression was lower in the AST+CM group compared to the CM group and higher after the combination of Ex527 (both P<0.05). (3) The number of apoptotic cells was significantly reduced in the AST+CM group compared to the CM group and increased after the combination of Ex527 (both P<0.05). (4) The protein expression levels of SIRT1, PGC-1α and Bcl-2 were increased and the protein expression levels of p53, Drp1 and Bax were decreased (all P<0.05) in the AST+CM group compared with the CM group, and the protein expression levels of SIRT1, PGC-1α and Bcl-2 were decreased and the protein expression levels of p53, Drp1 and Bax were increased when Ex527 was combined (all P<0.05). Conclusion:Astaxanthin can inhibit Drp1-mediated mitochondrial fission by activating the SIRT1 pathway, thereby reducing contrast-induced acute kidney injury in rats.

Mechanism of Blood-activating and Toxin-removing Method in Treatment of Coronary Heart Disease and Cognitive Dysfunction Based on Treating Different Diseases with Same Method / 中国实验方剂学杂志

As China is about to enter an era of deep aging， the coexistence of multiple diseases is gradually increasing. Coronary heart disease （CHD） and cognitive dysfunction also show increasing incidence year by year. The two diseases affect and cause each other， becoming the major chronic diseases harmful to the health of the elderly. It is of great clinical significance to explore the methods integrating traditional Chinese and western medicine for the prevention and treatment of the two diseases. The relationship between CHD and cognitive dysfunction in traditional Chinese medicine （TCM） was first recorded in Huangdi’s Internal Classic （Huang Di Nei Jing）. As the understanding of CHD and cognitive dysfunction is deepening， the influences of stasis and toxin on both diseases have attracted increasing attention. According to the theories of TCM， CHD and cognitive dysfunction have common points in the etiology and pathogenesis. Therefore， the theory of treating different diseases with same method provides a theoretical basis for the clinical treatment of different diseases with the same pathogenesis. Moreover， this theory conforms to the principle of integrated and individualized prevention and treatment of multi-disease coexistence in modern medicine. This paper systematically proposed that the coexistence of stasis and toxin is a major pathogenesis of CHD and cognitive dysfunction. We then explored the possible mechanisms of the blood-activating and toxin-removing method in the treatment of CHD and cognitive dysfunction based on the theory of treating different diseases with same method. The mechanisms include the regulation of ceramide metabolism， activation of silent mating-type information regulation 2 homolog 1 （SIRT1）， inhibition of pyroptosis， regulation of mitogen-activated protein kinase/nuclear factor-κB （MAPK/NF-κB） signaling pathway， inhibition of mitochondrial division， and regulation of DNA methylation. We hope this paper will provide an idea for the future research on the prevention and treatment of CHD and cognitive dysfunction with TCM.

Effects of sodium arsenite on mitochondrial function and expression of SIRT1/PGC-1α pathway-related proteins in human normal liver cell / 环境与职业医学

Background Long-term exposure to arsenic can cause liver injury of varying degrees. Mitochondrial damage may be an early key event of arsenic-induced liver injury. Silent mating type information regulation 2 homolog 1 (SIRT1)/ recombinant peroxisome proliferators-activated receptor gamma coactivator 1 alpha (PGC-1α) is an important pathway regulating mitochondrial mass and function. However, whether arsenic-induced liver injury is related to mitochondrial dysfunction mediated by SIRT1/PGC-1α pathway remains unclear. Objective To investigate potential effects of sodium arsenite (NaAsO2) on mitochondrial function and expressions of SIRT1/PGC-1α pathway-related proteins in human normal liver cell. Methods Human normal liver cells (MIHA cells) were used as the research object. MIHA cells were treated with different concentrations of NaAsO2 (0, 5, 10 and 20 μmol·L−1) for 24 h, and the cells were collected for study. The ultrastructure of mitochondria was observed by transmission electron microscopy, adenosine triphosphate (ATP) concentration by fluorescence method, mitochondrial membrane potential (MMP) level by flow cytometry, and SIRT1, PGC-1α and their downstream nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) protein expression levels by Western blotting. One-way analysis of variance and trend test were used for data statistical analysis. Results The viability of MIHA cells decreased gradually with the increase of NaAsO2 concentration (F=6495.47, P<0.001). The transmission electron microscope observation showed that the size of mitochondria in the 10 μmol·L−1 NaAsO2 treatment group was different, and the mitochondria were swollen or elongated in a rod-like shape. The mitochondria in the 20 μmol·L−1 NaAsO2 treatment group swelled like air spheres or vacuoles. The ATP concentration and MMP level of MIHA cells gradually decreased with the increase of NaAsO2 concentration (Ftrend of ATP=172.28, Ftrend of MMP=59.91, both Ps<0.001). Compared with the control group, the protein expression levels of SIRT1, PGC-1α, NRF1, and TFAM were not significantly changed in the 5 μmol·L−1 NaAsO2 treatment group, while the protein expression levels of SIRT1, PGC-1α, and TFAM were decreased in the 10 μmol·L−1 NaAsO2 treatment group, and the protein expression levels of SIRT1, PGC-1α, and NRF1 were decreased in the 20 μmol·L−1 NaAsO2 treatment group. The results of trend test showed that the protein expression levels of SIRT1, PGC-1α, NRF1, and TFAM decreased gradually with the increase of NaAsO2 concentration (Ftrend of SIRT1=47.07, P<0.001; Ftrend of PGC-1α=15.17, P<0.01; Ftrend of NRF1=13.54, P<0.01; F trend of TFAM=4.20, P<0.05). Conclusion The down-regulation of SIRT1/PGC-1α and its downstream NRF1 and TFAM may be involved in NaAsO2-induced mitochondrial dysfunction in liver cells.

Jiangzhi Tongluo Soft Capsules Regulate SIRT1/FoxO3 Pathway to Affect Cell Apoptosis in Rats with Membranous Nephropathy / 中国实验方剂学杂志

ObjectiveTo observe the effect of Jiangzhi Tongluo soft capsule on the protein levels of silent mating-type information regulation 2 homolog 1 （SIRT1） and forkhead transcription factor FoxO3 and podocyte apoptosis in the renal tissue of rats with membranous nephropathy and to reveal the underlying molecular mechanisms for the treatment of MN. MethodSixty male SD rats were randomly assigned into 6 groups with 10 rats each. The six groups included a normal group， a model group， benazepril hydrochloride group， and Jiangzhi Tongluo soft capsule groups of low， medium and high doses （25， 50， 100 mg·kg-1， respectively）. The model rats were established by injection with cationized bovine serum albumin into the tail vein. After modeling， the rats were administrated with corresponding agents by gavage for 4 weeks. At the end of the 4th week， an electron microscope was used to observe the pathological changes in the kidney. Western blot was employed to detect the protein levels of SIRT1 and FoxO3 protein in rat kidney， and immunohistochemistry to detect the expression of B lymphocytoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， Bcl-2-associated death promoter （Bad）， and podocyte split diaphragm proteins nephrin and podocin. ResultCompared with normal group， the expression of pro-apoptotic factors Bax， Bad， and FoxO3 in the kidney was up-regulated （P<0.05）， while that of anti-apoptotic factors Bcl-2， SIRT1， nephrin， and podocin was down-regulated （P<0.05） after modeling. Compared with the model group， the treatments down-regulated the expression of Bax， Bad， and FoxO3 （P<0.05） and up-regulated that of Bcl-2， SIRT1， nephrin， and podocin （P<0.05）. ConclusionJiangzhi Tongluo soft capsule may regulate the SIRT1/FoxO3 pathway to reduce podocyte apoptosis and maintain podocyte structure stability， thereby exerting the renal protection effect.

Electroacupuncture promotes recovery from disuse muscular atrophy through the miR-133a/SIRT1 pathway / 中华物理医学与康复杂志

Objective:To explore the role of microRNA-133a (miR-133a) and silent mating information regulation 2 homolog 1 (SIRT1) in the effects of electroacupuncture on persons with disuse muscular atrophy.Methods:Thirty C57BL/6 mice were randomly divided into a control group, an experimental control group and an experimental group, each of 10. Disuse muscular atrophy was induced in the mice of the experimental and experimental control groups using tail suspension. The mice in the electroacupuncture group were given 15 minutes of electroacupuncture over the Yanglingquan and Zusanli points every day for 14 days. Wet weight ratio and the cross-sectional area of the gastrocnemius and soleus were tracked, and the structure of the mitochondria in the skeletal muscles was observed using a transmission electron microscope. The protein expressions of SIRT1, peroxisome proliferator-activated receptor γ coactivator-1a (PGC-1a), nicotinamide phosphoribosyl transferase (NAMPT), adenosine 5-monophosphate-activated protein kinase-a (AMPK-a) and phospho-AMPK-a (P-AMPK-a) were detected using western blotting. The expressions of the muscle atrophy F-box (Atrogin-1), muscle ring finger1 (MuRF1), miR-133a, SIRT1, paired box gene 7 (Pax7), myogenic determination (MyoD) and myogenin (MyoG) genes were detected through polymerase chain reactions. The concentration of Niacylamide adenine dinucleotide (NAD+ ) and the ratio of NAD+ to reduced nicotinamide adenine dinucleotide were also measured.Results:Compared with the experimental control group, the average wet weight increased by 21% and the cross-sectional area of the soleus increased by 30% in the experimental group. The average wet weight of the gastrocnemius increased by 5% and the area by 17%. The average expressions of Atrogin-1, MuRF1, SIRT1, PGC-1a and NAMPT, the concentration of NAD+ , as well as the average value of P-AMPK-a/AMPK-a and NAD+ /NADH were significantly lower in the experimental group than in the experimental control group, while the average expression of miR-133a in the experimental group was significantly (163%) higher. The average expressions of Pax7 and MyoD were significantly up-regulated in the experimental control group compared with the other two groups, while MyoG was highly expressed in the experimental group compared with the other 2 groups.Conclusions:The SIRT1 pathway is one of the reflexive protective mechanisms that mediate in the natural recovery of skeletal muscles. Electroacupuncture enhances myoblast differentiation, improves energy metabolism in the mitochondria, and thus promotes recovery from disuse muscular atrophy.

Frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes in Argentina / Frecuencia y distribución geográfica de genotipos y tipos sexuales de los complejos de especies Cryptococcus neoformans y Cryptococcus gattii en Argentina

Abstract The aim of this work was to know the frequency and geographical distribution of genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii species complexes isolated from human infections in Argentina during the period from April 2009 to April 2011. A multicenter study was conducted, in which 372 isolates were obtained from 61 laboratories throughout the country. Of those, 98.8% of the isolates belonged to the C. neoformans species complex and 1.1% to the C. gattii species complex. Genotype VNI (MATa) was the most frequently isolated (n = 326, 87.6%), followed by VNII (MATa) (n = 22, 5.9%), the recently described VNII-VNIV (aADa) hybrid (n = 14, 3.8%), VNIV (MATa) (n=4, 1.1%), VNIII (aADa) hybrid (n = 1, 0.3%), and VNIII (aADa) hybrid (n = 1, 0.3%). The Argentine Central region showed the greatest number of cases and genotype diversity. Interestingly, a relative high frequency was observed in genotype VNII (MATa) in the Cuyo, Northeast and Northwest regions and, also in VNII-VNIV (aADa) hybrids in the Northwest region. C. gattii species complex was isolated at a low rate; 3 VGI (MATa) and 1 VGII (MATa) isolates were obtained from the Northwest and Central regions. In conclusion, this study shows that genotype frequencies seem to vary among regions in Argentina and reveals a relatively high frequency of rare hybrids in the Northwest region. Further regional clinical and environmental studies may help to elucidate if those varia-tions in frequencies are associated with the existence of regional ecological niches or any other regional factors.

Resumen El objetivo de! trabajo fue conocer la frecuencia y la distribución geográfica de genotipos y tipos sexuales de aislados pertenecientes a los complejos de especies Cryptococcus neoformansy Cryptococcus gattii obtenidos de infecciones humanas en Argentina. Entre abril de 2009 y abril de 2011 se realizó un estudio multicéntrico del que se obtuvieron 372 aislados de 61 laboratorios de diferentes zonas del país. El 98,8% de los aislados pertenecieron al complejo C. neoformansy el 1,1% al complejo C. gattii. El genotipo VNI (MATa) fue el más frecuente (n = 326; 87,6%), le siguieron VNII (MATa) (n = 22; 5,9%), el híbrido VNII-VNIV (aADa) (n = 14; 3,8%), VNIV (MATa) (n =4; 1,1%) y los híbridos VNIII (aADa) (n = 1; 0,3%) y VNIII (aADa) (n = 1; 0,3%). La región Centro mostró el mayor número de casos y la mayor diversidad de genotipos. Cabe destacar que el genotipo VNII (MATa) tuvo una frecuencia relativamente alta en las regiones de Cuyo, Noreste y Noroeste. En esta última región, también fue alta la frecuencia del híbrido VNII-VNIV (aADa). La frecuencia de aislamiento de miembros del complejo C. gattii fue baja: se obtuvieron 3 aislados VGI (MATa) y 1 VGII (MATa) de las regiones Centro y Noroeste. En conclusión, este estudio muestra que las frecuencias de genotipos varían entre las distintas regiones de Argentina y señala la presencia de híbridos poco comunes en una frecuencia relativamente alta dentro de la región Noroeste. Contar con mayor número de estudios clínicos y ambientales regionales podría ayudar a elucidar si tales variaciones están asociadas a la existencia de nichos ecológicos particulares o a algún otro factor regional.

Isorhamnetin activates Sirt1/PGC-1α signaling pathway to inhibit MPP+-induced SH-SY5Y cell injury / 药学学报

We studied the protective effect and mechanism of isorhamnetin (ISO) on 1-methyl-4-phenylpyridiniumion (MPP+)-induced SH-SY5Y cells injury. MPP+-induced SH-SY5Y cell injury model was established, and cell viability was measured by MTT and LDH methods. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in cells were determined to investigate the level of oxidative stress. DCFH-DA and MitoSOX fluorescence probes were used to detect the levels of intracellular reactive oxygen species (ROS) and mitochondria superoxide, respectively. JC-1 fluorescence probe was used to detect the changes of mitochondrial membrane potential. Western blot and immunofluorescence methods were used to determine the expressions of Sirt1 and PGC-1 proteins, as well as the expression levels of apoptosis-related proteins Bax and Bcl-2. MPP+ at the dose of 500 μmol·L-1 significantly reduced SH-SY5Y cells viability to 52.46% and increased LDH release to 417.63%. ISO at 5 and 15 μmol·L-1 significantly increased the expression of Sirt1 and PGC-1α, inhibited LDH release, reduced intracellular ROS and mitochondria superoxide, inhibited the decline of mitochondrial membrane potential and increased cell viability to 61.61% and 67.55%. In addition, ISO could downregulate the expression of Bax and upregulate the expression of Bcl-2 to reduce cell apoptosis. ISO-mediated inhibition of apoptosis could be reversed by Sirt1 specific inhibitor Sirtinol. Through activating Sirt1/PGC-1α signaling pathway, ISO could reduce oxidative stress injury and inhibit cell apoptosis to protect cells from MPP+ injury.

Development of a Molecular Marker Linked to the A4 Locus and the Structure of HD Genes in Pleurotus eryngii

Allelic differences in A and B mating-type loci are a prerequisite for the progression of mating in the genus Pleurotus eryngii; thus, the crossing is hampered by this biological barrier in inbreeding. Molecular markers linked to mating types of P. eryngii KNR2312 were investigated with randomly amplified polymorphic DNA to enhance crossing efficiency. An A4-linked sequence was identified and used to find the adjacent genomic region with the entire motif of the A locus from a contig sequenced by PacBio. The sequence-characterized amplified region marker 7-2299 distinguished A4 mating-type monokaryons from KNR2312 and other strains. A BLAST search of flanked sequences revealed that the A4 locus had a general feature consisting of the putative HD1 and HD2 genes. Both putative HD transcription factors contain a homeodomain sequence and a nuclear localization sequence; however, valid dimerization motifs were found only in the HD1 protein. The ACAAT motif, which was reported to have relevance to sex determination, was found in the intergenic region. The SCAR marker could be applicable in the classification of mating types in the P. eryngii breeding program, and the A4 locus could be the basis for a multi-allele detection marker.

Effect of Capsaicin in Inhibiting Migration and Invasion of Breast Cancer MCF-7 Cells by Down-regulating SIRT1 / 中国实验方剂学杂志

Objective: To investigate the effect of capsaicin on the migration and invasion of human breast cancer MCF-7 cells and the underlying molecular mechanism. Method: Three capsaicin intervention groups of different concentrations (25, 50, 75 μmol·L-1) and a blank group were set up. After MCF-7 cells were treated with different concentrations of capsaicin (25, 50, 75 μmol·L-1) for 24 h, the cell migration and invasion abilities were assessed by Transwell migration and invasion assay, respectively. Meanwhile, the mRNA level of silent information regulator 2 homolog 1 (SIRT1) and DNA polymerase δ catalytic subunit p125 encoding gene POLD1 (POLD1) were detected by Real-time polymerase chain reaction (Real-time PCR). The protein levels of SIRT1 and DNA polymerase δ catalytic subunit p125 (p125) were detected by Western blot. Result: Compared with the blank group, the number of transmembrane cells was significantly reduced, and the mobility was significantly decreased (P-1) in MCF-7 cells for 24 h. Capsaicin (25, 50, 75 μmol·L-1) significantly down-regulated the mRNA and protein expressions of SIRT1 (P-1) in MCF-7 cells for 24 h. Furthermore, capsaicin (25, 50, 75 μmol·L-1) also significantly down-regulated the mRNA expression of POLD1 and the protein expression of p125 (P-1) in MCF-7 cells for 24 h. Conclusion: Capsaicin remarkably inhibits the cell migration and invasion of breast cancer MCF-7 cells, and the possible mechanism may be related to the down-regulation of SIRT1 and POLD1 mRNA expression levels and SIRT1 and p125 protein expression levels.

SIRT1 signaling pathway in bone metabolism / 上海交通大学学报（医学版）

Osteoporosis is a bone disease characterized by low bone mass and deteriorated bone microstructure, which could be related to the disorders of energy metabolism and bone senescence. Silent mating-type information regulator 2 homolog 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that regulates cell senescence, energy metabolism and bone remodeling. SIRT1 could be activated not only by adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), c-Jun N-terminal kinase 1 (JNK1) and casein kinase 2 (CK2), but also by small-molecular drugs such as resveratrol. All these kinases and drugs can affect bone metabolism. Recent findings indicate that SIRT1 signaling pathway plays a direct role in bone metabolism, but the underlying mechanism remains unclear. This paper reviews the structure and function of SIRT1, and the role of SIRT1 in bone metabolism, and discusses the potential of SIRT1 signaling pathway as a new therapeutic target in osteoporosis.

Role of silent mating type information regulation 2 homolog 1 on lipopolysacchride-induced mitochondrial injury in insulinoma cell lines-1 / 中华急诊医学杂志

Objective To investigate the role of silent mating type information regulation 2 homolog 1(SIRT-1) in lipopolysaccharide (LPS)-induced mitochondrial injury on INS-1(insulinoma cell lines-1)cells. Methods INS-1 cells were divided into 5 groups: control group, 1 mg/L LPS group, LPS group with 10 μmol/L RSV (resveratrol) pretreatment for 1 h, LPS group with 20 μmol/L EX527 pretreatment for 1 h, LPS group together with EX527 pretreatment for 1 h, then incubated these INS-1 cells with RSV and LPS for 24 h. The cell viability and ATP generation were detected, then total, cytoplasmic and mitochondrial protein were isolated from INS-1 cells. The protein expression of SIRT1, TLR4, acetylated FoxO1, and cytochrome C (CytC), Mfn1 (mitofusion1), Mfn2 (mitofusion2), and Fis1 (fission1) were tested by Western-blot. Mfn1, Mfn2, and Fis1 genes expression were detected by real-time PCR. The comparison of multiple groups was performed by one-way ANOVA. LSD-t method was used to compare between every two groups. Results After 1 mg/L LPS stimulation for 24 h, there was decreased cell viability (n=4, F=13.98, P<0.01) and ATP generation (n=4, F=27.92, P<0.05) in INS-1 cells. RSV pretreatment could maintain ATP production, but it could not reverse the EX527-induced ATP decrease (P>0.05). Furthermore, RSV upregulated gene and protein expression of SIRT1, Mfn1 and Mfn2, whereas decreased TLR4 and Fis1 expression. LPS-induced CytC released to cytoplasm was alleviated by RSV (P<0.01), but there was no significant change about FoxO1 protein expression (P>0.05). Conclusions RSV may regulate FoxO1 acetylation followed by its effects on SIRT1 activity, which may be partly the mechanism of mitochondrial damage induced by LPS on INS-1 cells.

Activation of the Mating Pheromone Response Pathway of Lentinula edodes by Synthetic Pheromones

Pheromone (PHB)-receptor (RCB) interaction in the mating pheromone response pathway of Lentinula edodes was investigated using synthetic PHBs. Functionality of the C-terminally carboxymethylated synthetic PHBs was demonstrated by concentration-dependent induction of a mating-related gene (znf2) expression and by pseudoclamp formation in a monokaryotic strain S1-11 of L. edodes. Treatment with synthetic PHBs activated the expression of homeodomain genes (HDs) residing in the A mating type locus, and of A-regulated genes, including znf2, clp1, and priA, as well as genes in the B mating type locus, including pheromone (phb) and receptor (rcb) genes. The synthetic PHBs failed to discriminate self from non-self RCBs. PHBs of the B4 mating type (B4 PHBs) were able to activate the mating pheromone response pathway in both monokaryotic S1-11 and S1-13 strains, whose B mating types were B4 (self) and B12 (non-self), respectively. The same was true for B12 PHBs in the B4 (non-self) and B12 (self) mating types. The synthetic PHBs also promoted the mating of two monokaryotic strains carrying B4-common incompatible mating types (A5B4 × A1B4). However, the dikaryon generated by this process exhibited abnormally high content of hyphal branching and frequent clamp connections and, more importantly, was found to be genetically unstable due to overexpression of mating-related genes such as clp1. Although synthetic PHBs were unable to discriminate self from non-self RCBs, they showed a higher affinity for non-self RCBs, through which the mating pheromone response pathway in non-self cells may be preferentially activated.

Multiplex polymerase chain reaction as an improved method for screening Histoplasma capsulatum mating types

Histoplasmosis is a systemic mycosis infection caused by Histoplasma capsulatum, a heterothallic ascomycete. The sexual reproduction of this fungus is regulated by the mating type (MAT1) locus that contains MAT1-1 and MAT1-2 idiomorphs, which were identified by uniplex polymerase chain reaction (PCR). This study aimed to optimise single-step multiplex PCR for the accurate detection of the distinct mating types of H. capsulatum. Among the 26 isolates tested, 20 had MAT1-1 genotype, while six showed MAT1-2 genotype, in agreement with the uniplex PCR results. These results suggest that multiplex PCR is a fast and specific tool for screening H. capsulatum mating types.

Expression of Nampt and bone morphogenetic protein 7 in spontaneous diabetic mice and effect of nicotinamide mononucleotide on fibrosis of rats' glomerular cells HBZY-1 in high glucose culture / 中国药理学与毒理学杂志

OBJECTIVE To investigate the mechanism of nicotinamide mononucleotide (NMN) on inflammation and fibrosis between endogenous nicotinamide phosphoribosyltransferase (NAMPT) and bone morphogenetic protein 7 (BMP-7) in diabetic glomerular cells.METHODS ① In vivo,spontaneous diabetic C57/BL6 mice and wild C57/BL6 mice were divided into two groups.When blood glucose was above (34.2±1.9) mmol· L-1,renal histology of diabetic mice became obvious.The protein expressions of Nampt and nuclear transcription factors-kappa B p65 (NF-κB p65),silent mating type information regulation 2 homolog 1 (SIRT1) and BMP7 were analyzed by lengths of immunofluorescence.② In vitro,rats' glomerular cells HBZY-1 were incubated with glucose 200 mmol· L-1 for different lengths of time (0,24,48 and 72 h) and at different concentrations of NMN (0,50,100 and 200 iμmol· L-1).The protein levels of Nampt and BMP7 were detected by Western blotting and the protein expressions of NF-κB p65 and α-SMA were measured by immunofluorescence assay.The protein levels of Nampt,BMP7 and NF-κB p65 were detected by Western blotting after HBZY-1 cells were treated with NMN 100 μmol· L-1 and FK866 10 μmol· L-1 for 24 h.RESULTS ① In vivo,the glomeruli of diabetic C57/BL6 mice showed obvious atrophy.Fluorescence intensity of Nampt was increased (P＜0.05),but that of BMP7 and SIRT1 in renal glomeruli cells was decreased compared with the wild type (P＜0.01).② In vitro,HBZY-1 cells were cultured in glucose 200 mmol· L-1 for 48 and 72 h.The protein expression of NAMPT was increased,but that of BMP7 was decreased (P＜0.05,P＜0.01).Expressions of NF-κB p65 and α-SMA were increased (P＜0.01) by immunofluorescence.The expression of BMP7 was increased after treatment with glucose 200 mmol· L-1,followed by NMN 50,100 and 200 μmol · L-1 for 24 h (P＜0.01).The expressions of NAMPT and NF-κB p65 were decreased (P＜0.01).The expressions of Nampt and NF-κB p65 in glucose 5.6 mmol· L1 +FK866 and glucose 5.6 mmol· L-1+ NMN groups were increased (P＜0.01),but the expression of BMP7 did not change.CONCLUSION Upregulation of endogenous Nampt obviously intervenes in BMP7 expression in the process of glomerular inflammatory fibrosis in severe diabetes.NMN can affect the protein expression of BMP7 via a special Nampt signaling pathway.

Protective effects of tetrahydroxystilbeneglucoside on cardiac dysfunction in experimental diabetic cardiomyopathy / 中国药理学通报

Aim To study the protective effect of tetra-hydroxystilbeneglucoside ( TSG ) on cardiac injury and the mechanism involved in silent mating type informa-tion regulation 2 homolog 1 ( SIRT1 ) and adenosine monophosphate-activated protein kinase( AMPK) in the diabetic rats. Methods Type 2 diabetic rats were sac-rificed after administration with TSG for 8 weeks. Blood glucose, blood lipids, liverfunction,creatine ki-nase ( CK ) , lactate dehydrogenase ( LDH ) as well as myocardial nonesterified fatty acids( NEFA) were deter-mined by using biochemical test. The concentration of myocardial fatty acid transport proteins ( FATPs ) and-fatty acid β-oxidase ( FA-β-oxidase ) , and the levels of tumor necrosis factor alpha ( TNF-α) , interleukin -6 ( IL-6 ) , interleukin-1β( IL-1β) in serum were also measured by ELISA method and radio immunoassay re-spectively. The protein expressions of TNF-α, IL-6, IL-1β, SIRT1 and AMPK were detected by Western blot. Results Treatment of TSG reduced the contentof blood lipids, NEFA and collagen without affecting the content of blood glucose and insulin. The levels of TNF-α, IL-6 and IL-1βin serum as well as the protein expressions of TNF-α, IL-6 and IL-1β of cardia were also inhibited by administration with TSG. Treatment of TSG caused a significantly increased concentration of myocaidial FATPs and FA-β-oxidase, and dramatically restored the decreased protein expressions of SIRT1 and pAMPK in diabetic rats. Conclusion The protec-tive mechanisms of TSG against diabetic rats are in-volved in the alleviation of inflammatory mediator injury and improving energy metabolism.

Hypermethylation of HIC1 and aberrant expression of HIC1/SIRT1 contribute to papillary thyroid carcinoma / 中华内分泌代谢杂志

Objective To explore promoter methylation of HIC1 gene and the expression of HIC1/SIRT1 related to the occurrence, development, and metastasis of papillary thyroid carcinoma. Methods Using Bisulfite sequencing PCR to analyze the promoter methylation of HIC1 gene. Using quantitative real-time PCR and Western blot to analyze expression differences of HIC1 and SIRT1 genes in tissues of papillary thyroid carcinoma(40 cases) and in adjacent normal thyroid(40 cases), of which datas were analyzed by statistics. Results The degree of HIC1 gene promoter methylation was significantly higher than that in adjacent normal tissues(P<0. 01). The degree of HIC1 gene promoter methylation in papillary thyroid carcinoma was related to lymph node metastasis, age, and the tumor-node-metastasis stages(P<0. 01). Compared with the expression of HIC1 mRNA and protein in adjacent normal thyroid tissue, that in papillary thyroid carcinoma was significantly lower(P<0. 01), while the expression of SIRT1 mRNA and protein in papillary thyroid carcinoma was significantly higher(P<0. 01). The lower expression of HIC1 mRNA and protein in the tumor tissues was related to the stage of lymph node metastasis, age, and the tumor-node-metastasis stages(P<0. 05). There was a strong negative correlation between the degree of HIC1 gene promoter methylation and expression of HIC1 in papillary thyroid carcinoma(P<0. 05). The expression of HIC1 mRNA and protein between that of SIRT1 also showed a strong negative correlation(P<0. 01). Conclusion Promoter hypermethylation of HIC1 and aberrant expression of HIC1/SIRT1 in papillary thyroid carcinoma may play a significant role in the oncogenesis and progress of papillary thyroid carcinoma. HIC1 is expected to become a new marker for prevention and treatment of papillary thyroid carcinoma.

Analysis of the MAT1-1 and MAT1-2 Gene Ratio in Black Koji Molds Isolated from Meju

Aspergillus luchuensis is known as an industrially important fungal species used for making fermented foods such as awamori and shochu in Japan, makgeolli and Meju in Korea, and Pu-erh tea in China. Nonetheless, this species has not yet been widely studied regarding mating-type genes. In this study, we examined the MAT1-1 and MAT1-2 gene ratio in black koji molds (A. luchuensis, Aspergillus niger, and Aspergillus tubingensis) and in Aspergillus welwitschiae isolated from Meju, a fermented soybean starting material for traditional soy sauce and soybean paste in Korea. The number of strains with the MAT1-1 locus was 2 of 23 (A. luchuensis), 6 of 13 (A. tubingensis), 21 of 28 (A. niger), and 5 of 10 (A. welwitschiae). Fungal species A. tubingensis and A. welwitschiae showed a 1 : 1 ratio of MAT1-1 and MAT1-2 mating-type loci. In contrast, A. luchuensis revealed predominance of MAT1-2 (91.3%) and A. niger of MAT1-1 (75%). We isolated and identified 2 A. luchuensis MAT1-1 strains from Meju, although all strains for making shochu in Japan are of the MAT1-2 type. These strains may be a good resource for breeding of A. luchuensis to be used in the Asian fermented-food industry.

Silent mating-type information regulator 2 homolog 1 (SIRT1) in Alzheimer’ s disease:an update on potential mechanisms / 中国药理学通报

Alzheimer ’ s disease ( AD ) is characterized by pro-gressive loss of memory and other cognitive functions .With re-cent discoveries , activation of silent mating-type information reg-ulator 2 homolog 1 ( SIRT1 ) could attenuate the cognitive dys-function of AD via reducing amyloid-βaggregation and tau pro-tein phosphorylation , inhibiting inflammatory reaction , and regu-lating synaptic plasticity .This review aims to highlight the in-volvement of these new discoveries of SIRT 1, and Akt/protein kinase B(PKB) signaling pathways, for their potential therapeu-tic effect against AD .