Fasting improves tolerance to acute hypoxia in rats.

Acute high-altitude illness seriously threatens the health and lives of people who rapidly ascend to high altitudes, but there is currently no particularly effective method for the prevention or treatment of acute high-altitude illness. In the present study, we found that fasting preconditioning effectively improved the survival rate of rats exposed to a simulated altitude of 7620 m for 24 h, and a novel animal model of rapid adaptation to acute hypoxia was established. Compared with control treatment, fasting preconditioning activated AMPK, induced autophagy, decreased ROS levels, and inhibited NF-κB signaling in the cardiac tissues of rats. Our results suggested that fasting effectively improved the acute hypoxia tolerance of rats, which was gradually enhanced with prolongation of fasting. In addition, the acute hypoxia tolerance of young rats was significantly higher than that of adult rats. These experimental results lay the foundation for achieving rapid adaptation to acute hypoxia in humans.

Elevated ROS depress mitochondrial oxygen utilization efficiency in cardiomyocytes during acute hypoxia.

Mitochondria are important sites for the production of ATP and the generation of ROS in cells. However, whether acute hypoxia increases ROS generation in cells or affects ATP production remains unclear, and therefore, monitoring the changes in ATP and ROS in living cells in real time is important. In this study, cardiomyocytes were transfected with RoGFP for ROS detection and MitGO-Ateam2 for ATP detection, whereby ROS and ATP production in cardiomyocytes were respectively monitored in real time. Furthermore, the oxygen consumption rate (OCR) of cardiomyocytes was measured. Similar results were produced for adult and neonatal rat cardiomyocytes. Hypoxia (1% O2) reduced the basal OCR, ATP-linked OCR, and maximal OCR in cardiomyocytes compared with these OCR levels in the cardiomyocytes in the normoxic group (21% O2). However, ATP-linked OCR, normalized to maximal OCR, was increased during hypoxia, indicating that the electron leakage of complex III exacerbated the increase of ATP-linked oxygen consumption during hypoxia and vice versa. Combined with the result that cardiomyocytes expressing MitGO-Ateam2 showed a significant decrease in ATP production during hypoxia compared with that of normoxic group, acute hypoxia might depress the mitochondrial oxygen utilization efficiency of the cardiomyocytes. Moreover, cardiomyocytes expressing Cyto-RoGFP or IMS-RoGFP showed an increase in ROS generation in the cytosol and the mitochondrial intermembrane space (IMS) during hypoxia. All of these results indicate that acute hypoxia generated more ROS in complex III and increased mitochondrial oxygen consumption, leading to less ATP production. In conclusion, acute hypoxia depresses the mitochondrial oxygen utilization efficiency by decreasing ATP production and increasing oxygen consumption as a result of the enhanced ROS generation at mitochondrial complex III.

Comparison of hypoxic effects induced by chemical and physical hypoxia on cardiomyocytes.

The degree and duration of chemical hypoxia induced by sodium dithionite (Na2S2O4) have not been reported. It is not yet clear how much reduction in the O2 concentration (physical hypoxia) can lead to hypoxia in cultured cardiomyocytes. In this study, oxygen microelectrodes were used to measure changes in the O2 concentration in media containing different concentrations of Na2S2O4. Then, hypoxic effects of 0.8, 1.0, and 2.0 mM Na2S2O4 or 1%, 3%, and 5% O2 in cultured cardiomyocytes from neonatal rats were observed and compared. The results showed that the O2 concentration failed to remain constant by Na2S2O4 treatment during the 180-minute observation period. Only the 2.0 mM Na2S2O4 group significantly increased the expression of hypoxia-inducible factor 1α (HIF-1α) and hypoxic responses. Notably, 3% O2 only significantly increased the expression of HIF-1α in cardiomyocytes, while 1% O2 not only increased the expression of HIF-1α but also increased the apoptotic rate in cardiomyocytes. These results suggest that Na2S2O4 is not suitable for establishing a hypoxic model in cultured neonatal rat cardiomyocytes, and neonatal rat cardiomyocytes cultured at or below 1% O2 induced significant hypoxic effects, which can be used as a starting O2 concentration for establishing a hypoxic cell model.

Vasorelaxation effect of 18ß-glycyrrhetinic acid on the thoracic aorta of rats: proposed mechanism.

18ß-Glycyrrhetinic acid (18ß-GA) is an effective component extracted from the traditional Chinese medicine Radix glycyrrhizae (Leguminosae) and has various biological activities. This study was performed to investigate the vasodilatory effects of 18ß-GA on isolated rat thoracic aortic rings and explore the underlying mechanisms. The rings were obtained from normal Sprague-Dawley rats and then precontracted with norepinephrine (NE) (1 µM) or KCl (60 mM). 18ß-GA (1.883-11.297mg/L) was added successively by cumulative dosing to observe and record the changes in the tension of the vascular ring. The effects of NG-nitro-l-arginine methylester (L-NAME), indomethacin (INDO), barium chloride (BaCl2), 4-aminopyridine(4-AP), tetraethylammonium (TEA), and glibenclamide on the vascular diastolic function of 18ß-GA were determined. 18ß-GA substantially exhibited a dose-dependent vasorelaxant effect on the NE-induced and KCl-induced contractions of the rings. The integrity of the vascular endothelium had no influence on the 18ß-GA-induced vasorelaxation effect in the rings. L-NAME and IDON showed no significant differences in their effects on this vasorelaxation process in the rings precontracted with NE. This result suggests that the vasorelaxation mechanism of 18ß-GA may be independent of the vascular endothelium . BaCl2 and 4-AP antagonized the vasorelaxation effect of 18ß-GA, but TEA and glibenclamide showed no remarkable effect on the vasodilation of 18ß-GA. Findings suggest that 18ß-GA induces vasorelaxation in thoracic aortic rings via the receptor-operated Ca2+ channels and voltage-operated Ca2+ channels and the opening of inward rectifier potassium channels and voltage-operated potassium.

Antinociceptive effect of matrine on vincristine-induced neuropathic pain model in mice.

Chemotherapy drugs treatment causes neuropathic pain, hyperalgesia and allodynia are common components of neuropathic pain, so effectively therapeutic strategy is required. In this study, we evaluated the antinociceptive effects of matrine on vincristine-induced neuropathic pain in mice. Vincristine (100 µg/kg i.p.) was administered once per day for 7 days (day 0-6) in mice. Matrine (15, 30, 60 mg/kg, i.p.) was repeated administration in early phase (day 0-6) or late phase (day 7-13). Hyperalgesia and allodynia were evaluated by withdrawal response using von Frey filaments, plantar and cold-plate on 7, 14 and 21 day. Injection of vincristine produced mechanical hyperalgesia and cold allodynia. Matrine was found to produce a protective role in both von Frey filaments and cold-plate test. The analysis of the effect supports the hypothesis that matrine is useful in therapy of vincristine-induced neuropathic pain. In conclusion, this study demonstrates that administration of matrine is associated with antinociceptive effect on mechanical and cold stimuli in a mice model of vincristine-induced neuropathy pain.

Anti-apoptotic and neuroprotective effects of oxysophoridine on cerebral ischemia both in vivo and in vitro.

In this study, we investigated the neuroprotective effect of oxysophoridine on ischemia and ischemia-like insults. Protection by oxysophoridine was studied at the in vivo level using a model of middle cerebral artery occlusion in mice and at the in vitro level using primary rat hippocampal neuronal cultures exposed to oxygen-glucose deprivation, a model of ischemia-like injury. The behavioral test was performed by using the neurological scores. The infarction volume of brain was assessed in the brain slices stained with 2,3,5-triphenyl tetrazolium chloride. The neuron apoptosis was evaluated by Hoechst 33342 staining. The morphological change in the neurons was examined using a Transmission Electron Microscope (TEM or EM). To evaluate neuron apoptosis, caspase-3, -9, and - 8 activities were measured using assay kits with an ELISA reader. The Western blotting assay was used to evaluate the release of cytochrome c and expression of caspase-3, Bcl-2, and Bax proteins. The quantitative real-time PCR assay was used to evaluate the release of cytochrome c and the expression of caspase-3 mRNA. Oxysophoridine-treated groups (62.5, 125, 250 mg/kg) markedly reduced neurological deficit scores and infarct volumes. Treatment with oxysophoridine (5, 20, 80 µmol/L) significantly attenuated neuronal damage, with evidence of decreased cell apoptosis and decreased cell morphologic impairment. Furthermore, treatment with oxysophoridine could effectively downregulate the expression of cytochrome c and caspase-3 in both mRNA and protein levels, and Bax in the protein level, and induce an increase of Bcl-2 in the protein level. The caspase-3, -9, and -8 activities were also inhibited. These findings suggested that oxysophoridine may be a potential neuroprotective agent for cerebral ischemia injury.

Analysis of Risk Factors for Hypoparathyroidism After Total Thyroidectomy.

Objective: To analyze the risk factors of hypoparathyroidism after total thyroidectomy. Methods: Clinical data of patients who undergo total thyroidectomy in the Luwan Branch of Ruijin Hospital Affiliated to Medical College of Shanghai Jiaotong University was collected from January 2015 to December 2018, retrospectively. Logistic regression was used to analyze the risk factors associated with transient and long-term hypoparathyroidism. Results: A total of 537 patients were collected. The patients' average age included in the study was 47.3 ± 12.7 years old, including 135 males (25.1%) and 702 females (74.9%). There were 194 patients (36.1%) with transient postoperative hypoparathyroidism, and 21 patients (3.9%) had long-term postoperative hypoparathyroidism. After multivariate analysis, the main risk factors related to postoperative transient hypoparathyroidism were gender (P = 0.038, OR 0.686), combined lymph node dissection (P = 0.008, OR 1.569), and the maximum diameter of the thyroid (P = 0.011, OR 1.192), second operation (P = 0.001, OR 1.974), preoperative blood calcium (P < 0.001, OR 0.028). The main risk factors associated with long-term postoperative hypoparathyroidism are combined with lymph node dissection (P = 0.011, OR 1.594), maximum thyroid diameter (P = 0.032, OR 1.254), and PTH on the first day after surgery (P < 0.001, OR 1.199). Conclusions: Gender, combined lymph node dissection, maximum thyroid diameter, a second surgery, and preoperative blood calcium are risk factors for transient hypoparathyroidism after thyroid surgery. The combined lymphatic dissection and the thyroid gland's maximum diameter are risk factors for long-term hypoparathyroidism after thyroid surgery. PTH on the first day after surgery has a predictive effect on patients with long-term hypoparathyroidism.

Mitochondrial electron transport chain, ROS generation and uncoupling (Review).

The mammalian mitochondrial electron transport chain (ETC) includes complexes I­IV, as well as the electron transporters ubiquinone and cytochrome c. There are two electron transport pathways in the ETC: Complex I/III/IV, with NADH as the substrate and complex II/III/IV, with succinic acid as the substrate. The electron flow is coupled with the generation of a proton gradient across the inner membrane and the energy accumulated in the proton gradient is used by complex V (ATP synthase) to produce ATP. The first part of this review briefly introduces the structure and function of complexes I­IV and ATP synthase, including the specific electron transfer process in each complex. Some electrons are directly transferred to O2 to generate reactive oxygen species (ROS) in the ETC. The second part of this review discusses the sites of ROS generation in each ETC complex, including sites IF and IQ in complex I, site IIF in complex II and site IIIQo in complex III, and the physiological and pathological regulation of ROS. As signaling molecules, ROS play an important role in cell proliferation, hypoxia adaptation and cell fate determination, but excessive ROS can cause irreversible cell damage and even cell death. The occurrence and development of a number of diseases are closely related to ROS overproduction. Finally, proton leak and uncoupling proteins (UCPS) are discussed. Proton leak consists of basal proton leak and induced proton leak. Induced proton leak is precisely regulated and induced by UCPs. A total of five UCPs (UCP1­5) have been identified in mammalian cells. UCP1 mainly plays a role in the maintenance of body temperature in a cold environment through non­shivering thermogenesis. The core role of UCP2­5 is to reduce oxidative stress under certain conditions, therefore exerting cytoprotective effects. All diseases involving oxidative stress are associated with UCPs.

Clinical and immunological features of primary Sjögren's syndrome patients with positive anti-centromere protein B antibody / 北京大学学报(医学版)

OBJECTIVE@#To investigate the clinical and immunological features of primary Sjögren's syndrome (pSS) patients with positive anti-centromere protein B (CENP-B) antibody.@*METHODS@#In this cross-sectional study, the general clinical data, radiographic examination and labial salivary gland biopsy data, and serum immunological and biochemical data of patients diagnosed with pSS from January 2016 to August 2022 were evaluated. The included patients were divided into the anti-CENP-B antibody positive and negative groups. Intergroup differences were analyzed with SPSS 23.0 software. Subgroup analysis was further performed by dividing the anti-CENP-B antibody positive group into the single anti-CENP-B antibody positive and with other auto-antibodies positive groups to determine the characters related to anti-CENP-B antibody.@*RESULTS@#In this study, 288 patients with pSS were evaluated, including 75 patients with anti-CENP-B antibody positive and 213 with anti-CENP-B antibody negative. Univariate analysis showed that compared with the anti-CENP-B antibody negative group, the patients of the anti-CENP-B antibody positive group were older, had lower proportion of the patients with salivary gland enlargement and higher proportion of autoimmune liver disease. As for immunological indicators, the positive proportions of anti-SSA/Ro60, anti-Ro52, and anti-SSB antibodies were significantly lower. Moreover, the immunoglobulin (Ig) G and rheumatoid factor levels were significantly lower, while the IgM level was significantly higher in the patients of the anti-CENP-B antibody positive group. As for serum biochemical indicators, for the patients of the anti-CENP-B antibody positive group, the level of total protein (TP) was lower, the albumin/globulin ratio was higher, and the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), lactate dehydrogenase (LDH) were higher. Subgroup analysis showed that the levels of TP and IgA in the patients of the single anti-CENP-B antibody positive group were significantly lower than those of the patients with other autoantibodies positive group.@*CONCLUSION@#The pSS patients with anti-CENP-B antibody positive have unique clinical and immunological features of lower disease activity, less likely to involve salivary gland, higher risk for autoimmune liver disease, and higher levels of liver function indicators. Anti-CENP-B antibody may be a marker for a distinct subset of polyautoimmunity in Sjögren's syndrome.

Betulinic Acid Improves Cardiac Function in Septic Rats Through AKT / mTOR and AKT / AMPK -modulated Autophagy / 中国生物化学与分子生物学报

Betulinic acid (BA) exerts protective effects on organs in septic animals. However, whether BA can improve cardiac function in sepsis and the underlying mechanism remain unclear. Here, male Sprague-Dawley rats were pretreated with BA (25 mg/ kg/ d, i. g.) for 5 days and then intraperitoneally injected with lipopolysaccharide (LPS, 10 mg/ kg). The rats were anesthetized to determine transthoracic echocardiography using a high-resolution imaging system for small animals after they were treated with LPS for 6 h. Histopathologic alterations were examined by HE staining. Myocardial injury markers (cTnI and CK-MB) and inflammatory factors (TNF-α, IL-1β and IL-6) in the serum were measured by the enzyme-linked immunosorbent assay. Autophagy-related proteins (p62 and LC3 Ⅱ) and AKT-modulated autophagy pathways in the myocardium were determined by Western blotting. Pretreatment with BA markedly improved left ventricular ejection fraction (EF) and fraction shortening (FS) (P<0. 05), improved myocardial histomorphology, and significantly inhibited cTnI, CK-MB, TNF-α, IL-1β and IL-6 (P<0. 05) in the septic rat serum. BA markedly decreased p62 (P<0. 01), increased LC3 Ⅱ (P< 0. 001), and significantly down-regulated p-AKT (Thr308), p-AMPKα (Ser485/ 491), p-mTOR (Ser2448) and p-S6K (Thr389) (P<0. 05), while markedly up-regulated p-AMPKα (Thr172) and pULK1 (Ser317) (P<0. 01) in septic rat hearts. The findings indicate that BA can attenuate sepsis-induced myocardial dysfunctions associated with down-regulating autophagy inhibiting pathways mediated by AKT/ mTOR and AKT/ AMPK pathways.

The antidepressant effect of albiflorin based on TSPO and mechanism / 中国药理学通报

Aim To study the antidepressant effects of albiflorin and its relationship with TSPO(translocator protein 18 ku). Methods Mice were divided into eight groups(control group, chronic unpredictable stress group, fluoxetine group, albiflorin low, medium, high dose groups, PK11195 group, PK11195+ albiflorin high dose group)based on the data of the behavioral tests conducted to assess the antidepressant-like effects of albiflorin. After the behavioral tests Western blot and ELISA were conducted to evaluate the TSPO expression, progesterone and allopregnanolone level in hippocampus of mice. Results In the behavioral tests, there were significant differences between the model group and the control group, which indicated that the model was successfully established. The positive drug and albiflorin in different dose groups could reverse the effects of the model group, and PK11195 could reverse the effects of paeoniflorin in high dose group. The results of Western blot and ELISA showed that the TSPO expression, progesterone and allopregnanolone level in the model group significantly decreased. The positive drug and albiflorin groups with different doses could reverse the effects of the model group, and PK11195 could reverse the effects of the high dose group. Conclusions Albiflorin has significant antidepressant and antianxiety effects on CUS mice by TSPO, which provides experimental basis for the further study on the antidepressant effects and mechanism of albiflorin in the future.

Cloning and function analysis of chalcone isomerase gene and chalcone synthase gene in Lonicera macranthoides / 中国中药杂志

In order to explore the functions of genes of key rate-limiting enzymes chalcone isomerase(CHI) and chalcone synthase(CHS) in the biosynthesis of flavonoids in Lonicera macranthoides, this study screened and cloned the cDNA sequences of CHI and CHS genes from the transcriptome data of conventional variety and 'Xianglei' of L. macranthoides. Online bioinformatics analysis software was used to analyze the characteristics of the encoded proteins, and quantitative reverse-transcription polymerase chain reaction(qRT-PCR) to detect the expression of CHI and CHS in different parts of the varieties at different flowering stages. The content of luteo-loside was determined by high performance liquid chromatography(HPLC) and the correlation with the expression of the two genes was analyzed. The results showed that the CHI and CHS of the two varieties contained a 627 bp and 1170 bp open reading frame(ORF), respectively, and the CHI protein and CHS protein were stable, hydrophilic, and non-secretory. qRT-PCR results demonstrated that CHI and CHS of the two varieties were differentially expressed in stems and leaves at different flowering stages, particularly the key stages. Based on HPLC data, luteoloside content was in negative correlation with the relative expression of the genes. Thus, CHI and CHS might regulate the accumulation of flavonoids in L. macranthoides, and the specific functions should be further studied. This study cloned CHI and CHS in L. macranthoides and analyzed their expression for the first time, which laid a basis for investigating the molecular mechanism of the differences in flavonoids such as luteoloside in L. macranthoides and variety breeding.

Application of Methylprednisolone Sodium Succinate Combined with Tropisetron in Prevention of Nausea and Vomiting under Microvascular Decompression of Hemifacial Spasm / 中国医学科学院学报

Objective To evaluate the effect of methylprednisolone sodium succinate combined with tropisetron on postoperative nausea and vomiting(PONV)under microvascular decompression of hemifacial spasm.Methods From January to June 2019,485 patients undergoing microvascular decompression for facial spasm at Department of Neurosurgery,Peking University People's Hospital were randomly assigned into two groups with random number table method.For group A(n=242),2 ml saline was administrated by intravenous drip before induction and 5 mg tropisetron after operation.For group B(n=243),40 mg methylprednisolone sodium succinate was administrated by intravenous drip before induction and 5 mg tropisetron after operation.The anesthesia time,operation time,and incidence of PONV in 0-24 h and 24-48 h were recorded for the comparison of the remedial treatment rate of nausea and vomiting between the two groups.Results There was no significant difference in age,gender,smoking history,body mass index value,American Society of Anesthesiologists score,medical history,surgical side,PONV history,operation time or anesthesia time between the two groups(all P > 0.05).The incidence of PONV in group A was 35.5% and 18.2% during 0-24 h and 24-48 h,respectively,which was significantly higher than that(18.5%,χ

Management of non-compressible torso hemorrhage: An update / 中华创伤杂志(英文版)

With the widespread adoption of advanced tourniquets, the mortality rate of limb wound hemorrhage has decreased significantly, and non-compressible torso hemorrhage has gradually occupied the leading position of potentially preventable death, both in military and civilian circumstances. With the emergence of novel hemostatic devices and materials, strategies for the management of non-compressible torso hemorrhage have changed significantly. This review summarizes the current treatment strategies and types of equipment for non-compressible torso hemorrhage and suggests future research directions, hoping to provide a comprehensive review for the medical personnel and researchers engaging in this field.

Evaluation Effectiveness of in Vitro Cultivation of Bezoar on Mouse Model Combining Disease with Syndrome of Coronavirus Pneumonia with Yidu Xifei Syndrome / 中国实验方剂学杂志

Objective:To determine the therapeutic effect of <italic>in vitro</italic> cultivation of bezoar on a mouse model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. Method:BALB/c mice were randomly divided into six groups according to their weight grade： normal group， HCoV-229E infection group， cold and damp group， a mouse model combining disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome， and high and low dose group of <italic>in vitro</italic> cultivation of bezoar. The combination model of human coronavirus pneumonia with Yidu Xifei syndrome mice was established by the method of cold dampness condition stimulation+coronavirus HCoV-229E infection. <italic>In vitro</italic> cultivation of bezoar （0.128，0.064 g·kg^-1） was administrated by gavage for 3 days from the day of infection. The observation indexes included： general state observation of mice， inhibition rate of lung index and lung index of mice. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the viral load in the lung tissues of mice. Serum levels of motilin（MTL）， gastrin （GAS）， and cytokines interleukin（IL）-10，IL-6， tumor necrosis factor-<italic>α</italic>（TNF-<italic>α</italic>）and interferon-<italic>γ</italic>（IFN-<italic>γ</italic>） in lung tissue of mice were determined by enzyme-linked immunosorbent assay（ELISA）. The percentages of CD4⁺ T lymphocytes，CD8⁺ T lymphocytes and B lymphocytes in the blood of mice were determined by flow cytometry. Result:The high and low dose group of <italic>in vitro</italic> cultivation of bezoar can significantly improve the general condition of model mice. Compared with blank group， model group mice lung index increased significantly （<italic>P</italic><0.01）， nucleic acids significantly increased expression of lung tissue in mice （<italic>P</italic><0.01）， significantly higher serum MTL content in mice， GAS content significantly decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）， lung tissue cells in the immune factor TNF-<italic>α</italic>， IL-10 and IL-6 were significantly increased （<italic>P</italic><0.01）， peripheral blood lymphocyte CD4⁺ T cells in mice， The percentages of CD8⁺ T cells and B cells were significantly decreased （<italic>P</italic><0.01）. Compared with model group， <italic>in vitro</italic> cultivation bezoar mice lung index of high and low dose group were significantly lower （<italic>P</italic><0.01）， the lung tissue of mice express nucleic acid decreased significantly （<italic>P</italic><0.01）， MTL content decreased significantly （<italic>P</italic><0.01）， the lung tissue of mice in the IL-6， IL-10， the TNF-<italic>α</italic>， IFN-<italic>γ</italic> levels were significantly lower （<italic>P</italic><0.01）， <italic>in vitro</italic> cultivation bezoar high dose group can significantly increase the CD4⁺ T cell percentage （<italic>P</italic><0.05）， <italic>in vitro</italic> cultivation bezoar can to a certain extent reduce model mice lung inflammatory exudation， pulmonary interstitial edema， as well as blood stasis symptoms. Conclusion:<italic>In vitro</italic> cultivation of bezoar has a significant therapeutic effect on a mice model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. It can be treated by reducing the lung index of the model mice， improving the pathological damage of the lung tissue， adjusting the immune effective and inhibiting the clearing of inflammatory factors， and to provide a laboratory basis for clinical medication.

A strategy for screening antioxidants in Ginkgo biloba extract by comprehensive two-dimensional ultra high performance liquid chromatography.

Recently, screening of bioactive compounds by on-line ultra-high performance liquid chromatography (UHPLC) draws increasing attentions for the advantages of rapidity and intuition. Nevertheless, most on-line methods were limited to the shortcoming like low resolution and peak capacity, which could interfere the active ingredient identification. Comprehensive two-dimensional UHPLC (LC×LC) has revealed to be a powerful tool to separate complex mixtures. Herein, a strategy based on LC×LC analysis coupled with pre-column 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was proposed to screen the antioxidants from the extract of Ginkgo biloba (EGB). A total of 61 compounds were identified in EGB, and 25 of them showed appreciable radical scavenging capacity. This work may offer pharmacodynamics base for further research about EGB, also the strategy is likelihood to be applied in screening antioxidant in other herbal medicine.

Accurate analysis of ginkgolides and their hydrolyzed metabolites by analytical supercritical fluid chromatography hybrid tandem mass spectrometry.

Hydrolysis plays an important role in the metabolic transformations of lactones. Analysis of lactones and their hydrolyzed metabolites in biological samples is challenging, because unexpected hydrolysis or reversed dehydration may occur depending on the test environments. In this work, a supercritical fluid chromatography hybrid triple-quadrupole mass spectrometry (SFC-QQQ MS) method has been proposed for simultaneous analysis of ginkgolides and hydrolyzed metabolites. The SFC utilizes carbon dioxide as the mobile phase, avoiding hydrolysis of ginkgolides that always happens during reversed phase liquid chromatographic detection. Compared with normal phase liquid chromatography, the SFC provides good resolutions especially for ginkgolide with similar structures. This SFC-QQQ MS method was validated in linearity, precision, accuracy, and stability for ginkgolides and metabolites. Then this method was successfully applied to pharmacokinetic study of 3 ginkgolides and their 6 hydrolyzed metabolites after intravenous administration of total ginkgolide extract. Ginkgolides and metabolites showed different clear rates and excluded in 2-4h. The developed SFC-QQQ MS method allows accurate determination of ginkgolides and metabolites with high resolutions, and can be extended to analysis of other water-unstable compounds.

Rapid preparation of rare ginsenosides by acid transformation and their structure-activity relationships against cancer cells.

The anticancer activities of ginsenosides are widely reported. The structure-activity relationship of ginsenosides against cancer is not well elucidated because of the unavailability of these compounds. In this work, we developed a transformation method to rapidly produce rare dehydroxylated ginsenosides by acid treatment. The optimized temperature, time course, and concentration of formic acid were 120°C, 4 h and 0.01%, respectively. From 100 mg of Rh1, 8.3 mg of Rk3 and 18.7 mg of Rh4 can be produced by acid transformation. Similarly, from 100 mg of Rg3, 7.4 mg of Rk1 and 15.1 mg of Rg5 can be produced. From 100 mg of Rh2, 8.3 mg of Rk2 and 12.7 mg of Rh3 can be generated. Next, the structure-activity relationships of 23 ginsenosides were investigated by comparing their cytotoxic effects on six human cancer cells, including HCT-116, HepG2, MCF-7, Hela, PANC-1, and A549. The results showed that: (1) the cytotoxic effect of ginsenosides is inversely related to the sugar numbers; (2) sugar linkages rank as C-3 > C-6 > C-20; (3) the protopanaxadiol-type has higher activities; (4) having the double bond at the terminal C20-21 exhibits stronger activity than that at C20-22; and (5) 20(S)-ginsenosides show stronger effects than their 20(R)-stereoisomers.

Pharmacokinetic studies of ginkgolide K in rat plasma and tissues after intravenous administration using ultra-high performance liquid chromatography-tandem mass spectrometry.

Ginkgolide K (GK), a derivative compound of ginkgolide B, has been recently isolated from the leaves of Ginkgo biloba. It is a powerful natural platelet activate factor (PAF) antagonist, and also has obvious protect effects for cerebral ischemia. However, no reports have been described for the pharmacokinetic study of GK. In this study, a simple, sensitive and reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed for the determination of GK in rat plasma and tissues. Biological samples were pretreated by an efficient liquid-liquid extraction with ethyl acetate. The chromatographic separation was achieved on an Agilent ZORBAX SB-Aq column (4.6 mm × 50 mm, 1.8 µm) with a mobile phase of 0.5% aqueous formic acid (A)-menthol (B). Quantitation was carried out on a triple quadruple mass spectrometry using positive electrospray ionization in multiple reaction monitoring mode. Diazepam was used as internal standard (IS). The ion transitions monitored were set at m/z 407.10 → 389.20 and m/z 285.08 → 193.10 for GK and IS, respectively. The developed method was fully validated and successfully applied to the pharmacokinetics and tissue distribution study of GK after intravenous administration. The current results have indicated that pharmacokinetic parameters of GK vary in a dose-dependent manner with rapid elimination in 4h. The major distribution tissues of GK in rats were liver and kidney. This study would provide critical information to promote the future study of GK.

Hypoxic preconditioning attenuates hypoxia/reoxygenation injury of cardiomyocytes in SD rats via activating AMPK / 解放军医学杂志

[Abstract] Objective To investigate the protective effects of hypoxic preconditioning (HPC) on cardiomyocytes after hypoxia/reoxygenation (H/R) by activating AMP-activated protein kinase (AMPK). Methods The primary cardiomyocytes were prepared from neonatal SD rats. The model of H/R of cardiomyocytes was established and randomly divided into control group, H/R group, HPC group, H/R+A-769662 (AMPK agonist) group and HPC+compound C (AMPK inhibitor) group. The survival rate of cardiomyocytes was detected by chromatometry with Cell Counting Kit-8 (CCK-8); the reactive oxygen species (ROS) and ATP contents in the cells were detected respectively by dihydroethidium (DHE) dyeing and ATP Detection Assay Kit measured by fluorescence microplate reader; the phosphorylation level of AMPK and activation level of caspase-3 were detected by Western blotting; the concentration of free Ca2+ in cardiomyocytes was detected by Fluo-3AM dyeing using laser scanning confocal microscopy; the apoptotic rate of cardiomyocytes was detected by TUNEL staining. Results CCK-8 assay showed that compared with H/R group, the survival rate of cardiomyocytes in HPC group and H/R+A-769662 group increased obviously (P0.05); compared with H/ R group, the AMPK phosphorylation levels elevated significantly in HPC group and H/R+A-769662 group respectively (P<0.05, P<0.01). Meanwhile, the activation levels of caspase-3 in H/R group and HPC+compound C group were increased obviously compared with that in control group (P<0.01), but in HPC group and H/R+A-769662 group were significantly lower than that in H/R group (P<0.01, P<0.05). The results of Fluo-3AM staining showed that compared with control group, the concentrations of intracellular free Ca2+ increased obviously in H/R group and HPC+compound C group (P<0.01); the concentrations of free Ca2+ in cardiomyocytes of HPC group and H/R+A-769662 group were less than that in H/R group (P<0.01), while the concentrations of free Ca2+ in HPC+compound C group much more increased than in HPC group (P<0.01). The results of TUNEL staining showed that compared with H/R group, the apoptotic rate of cardiomyocytes decreased obviously in HPC group and H/R+A-769662 group (P<0.01, P<0.05); while compared with HPC group, the apoptotic rate of cardiomyocytes increased significantly in HPC+compound C group (P<0.05). Conclusions H/R may lead to the increase of ROS production, the decrease of ATP contents, and increase the levels of free Ca2+ in cardiomyocytes, and thus activate caspase-3, lead to apoptosis eventually. While HPC may reduce the production of ROS by activating AMPK to ensure the energy supply of cardiomyocytes after H/R treatment, prevent apoptosis of cardiomyocytes, and then reduce the H/R injury to cardiomyocytes.