SMYD3-PARP16 axis accelerates unfolded protein response and mediates neointima formation

Neointimal hyperplasia after vascular injury is a representative complication of restenosis. Endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) is involved in the pathogenesis of vascular intimal hyperplasia. PARP16, a member of the poly(ADP-ribose) polymerases family, is correlated with the nuclear envelope and the ER. Here, we found that PERK and IRE1

Stability of hydroalcoholic extracts of two species of guaco; Mikania glomerata SPRENG. and Mikania laevigata SCHULTZ. (Asteraceae), by UHPLC-MS

It is important to study the stability of plant extracts used as active ingredients in phytotherapic medicine, as degradation of the active principles directly affects the efficacy and safety of these products. Therefore, a stability study of the hydroalcoholic extract of the species: Mikania glomerata and Mikania laevigata was conducted in order to determine the speed of degradation and shelf life of these extracts, which are incorporated in cough syrup in Brazil. Leaves of both species were dried in an oven or by lyophilization (freeze-dried). Hydroalcoholic extracts underwent both accelerated stability study of six months and long-term stability study for 12 months. Samples were stored at different temperatures and every three months were analysed by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) to monitor their chemical profile, quantifying coumarin and chlorogenic acid. For all conditions of the study, a reduction of the content of the chemical marker of this species, coumarin, greater than 5% was observed, so a shelf life of two years cannot be assigned to the hydroalcoholic extracts of these species as observed in commercial extracts.

Effect of Duhuo Jisheng Tang on PERK/Bip Signaling Pathway in Knee Osteoarthritis Model Rats / 中国实验方剂学杂志

Objective: To explore the possible mechanism of Duhuo Jisheng Tang in relieving knee osteoarthritis based on protein kinase R-like endoplasmic reticulum kinase (PERK)/immunoglobulin-binding protein (Bip) signaling pathway.Method: A model of knee osteoarthritis was established by cold stimulation.Rats were randomly divided into blank group,model group,celecoxib group (0.021 g·kg-1),low,medium and high-dose Duhuo Jisheng Tang groups (8.37,16.72,33.48 g·kg-1).Blank group and model group were given equal volume of physiological saline.The changes of knee joint diameter were recorded.The pathological changes of rat articular cartilage were observed by hematoxylin-eosin (HE) staining.The expressions of tumor necrosis factor-alpha (TNF-α),interleukin-1β(IL-1β) and hyaluronic acid (HA) in serum were detected by enzyme-linked immunosorbent assay (ELISA).The mRNA and protein expression levels of PERK,Bip and cysteinyl as parates pecific protein-9(Caspase-9) in cartilage were detected by Real-time PCR and Western blot.Result: The knee joint redn ess and the joint diameter of celecoxib group and high-dose Duhuo Jisheng Tang group were improved,and the joint diameter was reduced significantly (Pα,IL-1β and HA were increased in model group (PPPα,IL-1β and HA in serum of celecoxib group and high-dose Duhuo Jisheng Tang group were decreased (PPPPConclusion: Duhuo Jisheng Tang can alleviate the symptoms of knee osteoarthritis model rats,and its mechanism may be related to the regulation of PERK/Bip signaling pathway in rat cartilage.

Study on mechanism of regulating synthesis of β-sitosterol by SiSQS1 and SiSQS2 in Saussurea involucrata cells / 中草药

Objective: To explore the synthetic mechanism of β-sitosterol by comparing the locus mutation, prokaryotic expression, expression level of SiSQS1 and SiSQS2, and the content of β-sitosterol in three color types of cells. Methods: Firstly, we preformed the cloning and bioinformatic analysis of SiSQS1 and SiSQS2 which were key enzymes involved in the biosynthesis of β-sitosterol in Saussurea involucrata cells. Secondly, we compared the differences of prokaryotic expression between SiSQS1 and SiSQS2, then optimized the expression conditions. Finally, we compared the expression levels of SiSQS1 and SiSQS2 by Real-time PCR and the content of β-sitosterol by GC-MS in three color types of cells, and made the correlative analysis on the expression level and the content of β-sitosterol. Results: There was a locus mutation of amino acid residues in 242E/D between SiSQS1 and SiSQS2. The results of prokaryotic expression analysis and conditions optimization showed that both target proteins had been expressed successfully, but the optimal prokaryotic expression system was different. The results of expression level and quantitative analysis showed a positive correlation to the expression levels of SiSQS1 and SiSQS2 and the content of β-sitosterol, the correlation coefficients were 0.92 and 0.89, respectively. Conclusion: A locus mutation of amino acid residues in 242E/D between SiSQS1 and SiSQS2 may influence the expression of SiSQS, and there may exist the functional differences in catalytic activity and the accumulation of β-sitosterol. The study will provide technical support and lay a theoretical foundation for studying the accumulated mechanism of β-sitosterol regulated by SQS in S. involucrata cells.

Knockdown of interleukin-10 induces the redistribution of sigma1-receptor and increases the glutamate-dependent NADPH-oxidase activity in mouse brain neurons

BACKGROUND: In the central nervous system, interleukin-10 (IL-10) provides trophic and survival effects directly on neurons, modulates neurite plasticity, and has a pivotal importance in the neuronal regeneration in neurodegenerative and neuroinflammatory conditions. This cytokine is primarily produced by glial cells and has beneficial effects on the neuronal viability. However, the mechanisms of IL-10-elicited neuroprotection are not clear. RESULTS: Membrane preparations, isolated from wild-type (Wt) and IL-10 knockout (KO) mice brain were used in this study. It has been shown that compared to wild-type mice, in IL-10 KO mice brain, the amount of immunoglobulin binding protein (BiP) is greatly increased, whereas the content of sigma receptor-1 (SigR1) is not changed significantly. Co-immunoprecipitation experiments have shown that the association of SigR1 with small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1), NR2B subunit of NMDA-receptor (NMDAR) and inositol-3-phosphate receptor (IP3R) is higher in the IL-10 KO mice brain than in the Wt mice brain. Besides, we have found that either glutamate or sigma ligands, separately or together, do not change glutamate-induced NADPH-oxidase (NOX) activity in Wt-type mice brain membrane preparations, whereas in IL-10 KO mice high concentration of glutamate markedly increases the NOX-dependent production of reactive oxygen species (ROS). Glutamate-dependent ROS production was decreased to the normal levels by the action of sigma-agonists. CONCLUSIONS: It has been concluded that IL-10 deprivation, at least in part, can lead to the induction of ER-stress, which causes BiP expression and SigR1 redistribution between components of endoplasmic reticulum (ER) and plasma membrane. Moreover, IL-10 deficiency can change the specific organization of NMDAR, increasing the surface expression of SigR1-sensitive NR2B-containing NMDAR. In these conditions, glutamate-dependent ROS production is greatly increased leading to the initiation of apoptosis. In this circumstances, sigma-ligands could play a preventive role against NMDA receptor-mediated excitotoxicity.

Effect of extract from callus of Saussurea involucrate on proliferation and differentiation of osteosarcoma cell MG-63 / 中草药

Objective: To investigate the effect of extract from callus of Saussurea involucrate (ESI) on the proliferation and differentiation of osteosarcoma cells MG-63 and explore its anti-osteoporosis and mechanism. Methods: Using human osteosarcoma cells MG-63 for the study, the effect of ESI on the proliferation was detected by MTT and LDH methods; The activity of alkaline phosphatase activity (ALP) and the level of hydroxyproline (Hyp) were investigated by related reagent kit; The effect of ESI on mineralized nodules was observed by Alizarin red staining and quantified by CPC, the expression of OPG/RANKL was measured by RT-PCR and Western blotting, and the proliferation, differentiation, mineralization, and OPG/RANKL expression levels of MG-63 were assayed when medium contains both p38 inhibitor SB203580 and ESI. Results: MTT and LDH assay showed that ESI (< 125 μg/mL) was non-toxic to MG-63, and it promoted the proliferation at the concentration of 31.25 and 62.5 μg/mL; ESI could significantly increase the ALP activity, Hyp content, and amount of mineralized nodules; At the mRNA level, ESI could significantly up-regulate OPG expression and down-regulate RANKL expression; At the protein level, ESI could significantly increase the ratio of OPG/RANKL; SB203580 could reverse the acceleration of ESI on the proliferation, differentiation, mineralization, and OPG/RANKL expression of MG-63. Conclusion: ESI has the facilitating effect on the osteoblast proliferation, differentiation, and mineralization; The mechanism may be associated with the expression of OPG and RANKL, as well as the signal transduction pathway of p38 Mitogen-activated potein kinase (MAPK).

The damage of liver cells and the expression of GRP 78 in rats liver tissue after repeated and sustained exposure to different +Gz / 基础医学与临床

Objective To observe the damage of liver cells and to investigate the distribution and expression of glu-cose-regulated protein 78 (Glucose regulated protein78, GRP78/Bip) in liver tissue under the positive acceleration (+Gz) exposure.Methods Totally 24 wistar rats were randomly assigned to four groups:blank control,+6Gz,+9Gz and +12Gz.Each rat was clamped to the centrifuge arm , prone position, with the head of the rat facing the axis of the centrifuge for +Gz orientation.The onset rate was +0.5 Gz/s, which was used trapezoidal acceleration curve effect and controlled by computer .Blank control group rats were placed on the arm of centrifuge and under-went a process similar to that described above , but they were not exposed to acceleration .+6Gz group,+9Gz group and +12Gz group were subjected at peak time 3 min in animal centrifuge , acceleration rate 0.5 G/s, five times with interval 30 min between times.In addition, liver tissue of rats were respectively observed by H .E.staining.Mean while, plasma aspartate aminotransferase ( AST) and alanine aminotransferase ( ALT) were tested determine the damageofliverfunction.Results +GzaccelerationstressinjuryincreasedserumASTandALTlevel.Compared with the stress control, +9Gz group and +12Gz group significantly increased in plasma ALT and AST as compared with control group ( P<0.05 ) .+12 Gz stress induced the highest level in these groups .The level of ALT in+2 Gz group was higher than that in +6 Gz group ( P<0.05 ) .HE staining showed derangement of liver cells , irregular shape, the cell gap is not clear, vacuolar changes in +Gz groups, and with the increase of G value.Compared with the control group, the expression of GRP78/Bip was focused in the cytoplasm;the expression of GRP78 in the experimental group is higher than that in the control group ( P<0.05 ) .+12 Gz group was significantly higher than+6Gz group and the control group (P<0.05).The expression of GRP78/Bip in liver tissue increased with the in-creasing of G value levels;the expression level of GRP78/Bip in +12Gz and +9Gz groups were higher than that in +6Gz and control group (P<0.05).Conclusions There is positively related expression of GRP78/Bip, which was associated with exposure of increasing G values .

Actividad antioxidante y antiinflamatoria del extracto hidroalcohólico de las flores de Tanacetum parthenium L. Sch. Bip. "Santa María". Ayacucho, 2014 / Antioxidant and anti-inflammatory activity of the hydroalcoholic extract of the flowers of Tanacetum parthenium L. Sch. Beep. "Santa Maria". Ayacucho, 2014

Los procesos inflamatorios no controlados producen daño celular donde los radicales libres están presentes y representan un peligro para la vida del paciente, en este contexto la búsqueda de moléculas de origen vegetal son incesantes por su aceptación cultural, una mejor compatibilidad con el cuerpo humano y menos efectos secundarios. El objetivo fue determinar la actividad antioxidante y antiinflamatoria del extracto hidroalcohólico de las flores de Tanacetum parthenium L. Sch. Bip. "santa maria", empleando la técnica in vitro del DPPH y el modelo in vivo de edema plantar inducida por carragenina en ratas Wistar. Se llevó a cabo en los laboratorios de la Escuela de Farmacia y Bioquímica de la Universidad Nacional de San Cristóbal de Huamanga, Ayacucho, Perú. La muestra fue recolectada en la ciudad de Huancayo, región Junin. Se ensayó concentraciones de 1, 2 y 3% en ungüentos base como vehículo semisólido usando el estándar de referencia diclofenaco gel 1%. El extracto hidroalcohólico contiene fenoles y taninos, flavonoides, faetonas y/o cumarinas, triterpenos y/o esteroides y aminoácidos. La concentración de extracto hidroalcohólico de las flores de Tanacetum parthenium L. Sch. Bip. "santa maria" con mayor actividad antiinflamatoria fue al 3%, con un porcentaje de desinflamación del84.6% {porcentaje de inflamación de 15.4%) y con un ABC de 6,214 mllt2. La concentración de extracto hidroalcohólico de las flores de Tanacetum parthenium L. Sch. Bip. "santa maria" con mayor actividad antioxidante fue a 100 ¡Jg/ml, con un porcentaje de 63,9%. Se concluye que el extracto hidroalcohólico de las flores de Tanacetum parthenium L. Sch. Bip. "santa maría" tiene poca actividad antioxidante in vitro pero buena actividad antiinflamatoria in vivo estadísticamente similar al diclofenaco por lo que constituyen una fuente potencial para el tratamiento antiinflamatorio tópico.

Chemical constituents and bioactivity of Kalimeris indica / 中草药

Objective: To study the chemical constituents from Kalimeris indica. Methods: Chemical constituents were isolated by column chromatography and semi-prepared HPLC, and the structures were elucidated by spectral data and physical-chemical properties. Results: Fifthteen compounds were isolated and respectively identified as friedelin (1), epifriedelanol (2), stimasterol (3), β-sitosterol (4), α-spinasterol (5), erythodiol (6), octadecanoic acid (7), fraxinellone (8), β-daucosterol (9), hexacosanol (10), ergoaterol (11), sphondin (12), dibntyl phthalate (13), oleanic acid (14), and dammerdienly acetate (15). Conclusion: Compounds 5, 8-13 are isolated from K. indica for the frist time. Compounds 1, 3, 5, 8, and 12 have inhibitory effects on Cytospora sp. and Rhizoctonia solani.

Chemical constituents of Ainsliaea latifolia / 中草药

Objective: To isolate and identify the chemical constituents of Ainsliaea latifolia. Methods: Compounds were isolated by various kinds of column chromatographies on silica gel, Sephadex LH-20, MPLC, and HPLC, and their structures were elucidated by the physicochemical properties and spectral analyses. Results: Thirteen chemical constituents were obtained and identified as friedelin (1), taraxeryl acetate (2), 3β-hydroxy-11α, 12α-epoxy-friedoolean-14-ene (3), careborin (4), cis-careborin (5), 3α-E-feruloyltaraxerol (6), 3α-Z-feruloyltaraxerol (7), 3-oxo-11α-methoxyolean-12-ene (8), diaspanolide A (9), diaspanolide B (10), ainsliaolide A (11), stigmasterol (12), and β-sitosterol (13). Conclusion: Among the isolated 13 compounds, there are 8 triterpenoids (1-8), 3 sesquiterpenoids (9-11), and 2 steroidals (12-13). All the compounds are isolated from A. latifolia for the first time.

Role of Endoplasmic Reticulum Stress in Rheumatoid Arthritis Pathogenesis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by abnormal proliferation of synoviocytes, leukocyte infiltration, and angiogenesis. The endoplasmic reticulum (ER) is the site of biosynthesis for all secreted and membrane proteins. The accumulation of unfolded proteins in the ER leads to a condition known as ER stress. Failure of the ER's adaptive capacity results in abnormal activation of the unfolded protein response. Recently, we have demonstrated that ER stress-associated gene signatures are highly expressed in RA synovium and synovial cells. Mice with Grp78 haploinsufficiency exhibit the suppression of experimentally induced arthritis, suggesting that the ER chaperone GRP78 is crucial for RA pathogenesis. Moreover, increasing evidence has suggested that GRP78 participates in antibody generation, T cell proliferation, and pro-inflammatory cytokine production, and is therefore one of the potential therapeutic targets for RA. In this review, we discuss the putative, pathophysiological roles of ER stress and GRP78 in RA pathogenesis.

Separation, purification, and preliminary structure analysis of polysaccharides from Kalimeris indica / 中草药

Objective: The polysaccharide from Kalimeris indica (KIP) has been proved to be one of effective parts with anti-gastric ulcer action. In order to accurately define the structure and structure-activity relationship of KIP, the purification and structural analysis of KIP were carried out. Methods: A water-soluble polysaccharide (KIP-1) was obtained from K. indica by hot water extraction, deproteination with enzymolysis and Sevag methods, and fractionation through DEAE cellulose (DE-52) column. The molecular weight of KIP-1 was determined by gel chromatography (Sephadex G-200). The structure was characterized with FTIR, GC, and GC-MS methylation analysis. The microstructure of KIP-1 was observed by atomic force microscope (AFM). Results: The results indicated that KIP-1 had a molecular weight of 11 592 and was mainly composed of mannose, glucose, and galactose with the ratio of 2.523:1.162:1. Methylation analysis showed that KIP-1 had a backbone of 1, 3-linked glucose and 1, 2-linked galactose, of which the branch point was at O-3 composed of a 1, 3, 6-linked mannose residue and terminated with mannose residue. The microstructure study showed that KIP-1 with an average particle size of 1-2 nm was granular, irregular-rounded, and ellipsoidal. Irregularly shaped structure was found together in cubic image of KIP-1 with the height about 1 nm. Conclusion: KIP-1 with branch structure is mainly composed of mannose, glucose, and galactose. It is the first time to get the polysaccharide from K. indica.

Depleted immunoglobulin heavy chain binding protein (BiP) expands the endoplasmic reticulum and the golgi apparatus in dengue virus-infected cells.

Objective: To test whether depleted expression of immunoglobulin heavy chain binding protein (BiP), which is an endoplasmic reticulum (ER) chaperone, in dengue virus (DENV)-infected cells, affected integrities of the ER and the Golgi apparatus of the host cells. Methods: Either siRNA against BiP- or control siRNA-transfected cells was infected with DENV and subjected to electron microscopic evaluation. Results: Depleted expression of BiP affected integrities of the ER and the Golgi apparatus in DENV-infected cells. Conclusion: Integrities of the ER and the Golgi apparatus maintained by BiP in the host cells is necessary for DENV production.

Expression of BiP in rat splenic macrophages with mild heat stress / 中国病理生理杂志

AIM: To investigate the role of mild heat stress towards the immunological function of splenic macrophages and significance of BiP protein expression. METHODS: Primary cultured splenic macrophages were prepared and placed in 41 ℃ thermostat for thermal stress and restored to 37 ℃ after an hour. Heat stressed macrophages were separated into groups at time points of 0 min, 30 min, 60 min, 120 min and 180 min, and their BiP mRNA and BiP protein expressions were detected. At the same time, the phagocytic function, cytotoxicity and chemotaxis of the macrophages were detected. RESULTS: After heat stress, the expressions of BiP mRNA and BiP protein in splenic macrophages remarkably increased, and reached to peak after 30-60 min, still remained higher than control group after 120 min and restored to normal level after 180 min. At the same time, mild heat stress enhanced the phagocytotic, cytotoxicity and chemotactic activities of splenic macrophages, and reached to peak after 60 min then gradually decreased. CONCLUSION: The expression of BiP protein is enhanced after mild heat stress, synchronous changes happen both in BiP protein expression and cellular function. There is close relation between BiP protein expression and increased functions of splenic macrophages induced by mild heat stress.

Progressing growth of tumor cell and synthesis of Bip/GRP78 / 中国药理学通报

AIM: To explore the relationship between tumor progressing growth and synthesis of Bip/GRP78 in vitro. METHOD: Using tumor cell culture, ion exchange chromatography, SDS-PAGE, specific enzymatic, chemical catalysis, mass spectra and so on, the synthesis of Bip/GRP78 of cells growth in exponential, confluent and post-confluent phases was examined,and compared to normal breast epithelial cells. RESULTS: During the progressing growth, tumor cells' synthesis of Bip/GRP78 exhibited growth situation, cell density and malignant degree-dependent. CONCLUSIONS: During the progressing growth, tumor cells can maintain its homeostasis by synthesizing Bip/GRP78. This synthesis is intensely growth situation. Cell density and malignant degree-dependent. By this synthesis, tumor cell establishs its defensive system. Because increasing investigate results have shown that Bip/GRP78 can decrease the sensitivity of tumor cell to be killed by cytotoxic T lymphocytes, increase its tumorigencity and prevent its apoptosis. So aiming at destruction of the synthesis of Bip/GRP78 may point to a new approaches to the therapy of cancer.

Progressing growth of tumor cell and synthesis of Bip/GRP78 / 中国药理学通报

AIM To explore the relationship between tumor progressing growth and synthesis of Bip/GRP78 in vitro. METHOD Using tumor cell culture, ion exchange chromatography, SDS-PAGE, specific enzymatic , chemical catalysis, mass spectra and so on, the synthesis of Bip/GRP78 of cells growth in exponential, confluent and post-confluent phases was examined,and compared to normal breast epithelial cells. RESULTS During the progressing growth, tumor cells' synthesis of Bip/GRP78 exhibited growth situation, cell density and malignant degree-dependent. CONCLUSIONS During the progressing growth, tumor cells can maintain its homeostasis by synthesizing Bip/GRP78. This synthesis is intensely growth situation. Cell density and malignant degree-dependent. By this synthesis, tumor cell establishs its defensive system. Because increasing investigate results have shown that Bip/GRP78 can decrease the sensitivity of tumor cell to be killed by cytotoxic T lymphocytes, increase its tumorigencity and prevent its apoptosis. So aiming at destruction of the synthesis of Bip/GRP78 may point to a new approaches to the therapy of cancer.