OBJECTIVE: To evaluate the synergy of the Burkholderia signaling molecule cis-2-dodecenoic acid (BDSF) and fluconazole (FLU) or itraconazole (ITRA) against two azole-resistant C. albicans clinical isolates in vitro and in vivo. METHODS: Minimum inhibitory concentrations (MICs) of antibiotics against two azole-resistant C. albicans were measured by the checkerboard technique, E-test, and time-kill assay. In vivo antifungal synergy testing was performed on mice. Analysis of the relative gene expression levels of the strains was conducted by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS: BDSF showed highly synergistic effects in combination with FLU or ITRA with a fractional inhibitory concentration index of ⪕ 0.08. BDSF was not cytotoxic to normal human foreskin fibroblast cells at concentrations of up to 300 μg/mL. The qRT-PCR results showed that the combination of BDSF and FLU/ITRA significantly inhibits the expression of the efflux pump genes CDR1 and MDR1 via suppression of the transcription factors TAC1 and MRR1, respectively, when compared with FLU or ITRA alone. No dramatic difference in the mRNA expression levels of ERG1, ERG11, and UPC2 was found, which indicates that the drug combinations do not significantly interfere with UPC2-mediated ergosterol levels. In vivo experiments revealed that combination therapy can be an effective therapeutic approach to treat candidiasis. CONCLUSION The synergistic effects of BDSF and azoles may be useful as an alternative approach to control azole-resistant Candida infections.
Antifungal Agents ; pharmacology ; Burkholderia cenocepacia ; chemistry ; Candida albicans ; drug effects ; physiology ; Candidiasis ; drug therapy ; Drug Resistance, Fungal ; Fatty Acids, Monounsaturated ; adverse effects ; Fluconazole ; pharmacology ; Humans ; Microbial Sensitivity Tests ; Triazoles ; metabolism

Three new sesquiterpene glycosides with alloaromadendrane and ylangene-derived type aglycones, named dendrofindlayanosides A-C (1-3), one new cyclopacamphane type sesquiterpene named dendrofindlayanobilin A (4), together with five known compounds have been isolated from stems of Dendrobium findlayanum. Their structures were determined on the basis of spectroscopic and chemical methods.

Objective: To investigate and compare enzymatic kinetics of scutellarin,apigenin-7-O-glucronide and paeoniflorin from Xinshao fomula in liver microsomes of sham-operated rats and middle cerebral artery occlusion(MCAO) rats with focal cerebral ischemia-reperfusion injury. Method: Xinshao fomula were incubated respectively with liver microsomes of sham-operated rats and MCAO rats,UPLC-MS and substrate elimination method was employed,Michaelis constant(K_m),maximum velocity of enzymatic reaction(V_max) and intrinsic clearance(CL_int) of these three components from Xinshao fomula in liver microsomes of sham-operated rats and MCAO rats were calculated,these parameters between different groups were evaluated by statistical analysis. Result: The K_m values of scutellarin,apigenin-7-O-glucronide and paeoniflorin in liver microsomes of MCAO rats were (0.798±0.031),(0.213±0.017),(0.499±0.029) μmol·L^-1,which were quite different to these in liver microsomes of sham-operated rats.Compared with the sham-operated group,V_max and CL_int values of scutellarin and paeoniflorin in liver microsomes of MCAO rats were significantly reduced(P<0.05,P<0.01),and V_max of apigenin-7-O-glucronide in liver microsomes of MCAO rat was also significantly reduced(P<0.05). Conclusion: Metabolic rates of these three active components from Xinshao fomula in liver microsomes of MCAO rats with focal cerebral ischemia-reperfusion injury decrease with low elimination rate.

Objective: To establish the quality control methods for the standard decoction of Puerariae Thomsonii Radix.Method:According to the preparation principles for traditional Chinese medicine (TCM) standard decoction,13 batches of Puerariae Thomsonii Radix from different origins were analyzed under the chromatography conditions established in this study and verified with methodology.By referring to Chinese Pharmacopoeia of 2015,puerarin was used as a quantitative indicator to calculate the transfer rate.In this study,the structures of main chromatographic peaks were also identified to clarify the main chemical constituents in the standard decoction.Result:The 13 batches of medicinal herbs were identified as Puerariae Thomsonii Radix,with a recovery rate of 98.0%,and RSD of 1.1%,indicating that the method was accurate and reliable.The transfer rate ranged from 41.4% to 60.0%,and the extraction rate was within the range of 15.7%-34.3%.The corresponding fingerprints were prepared for 13 batches of the standard decoction,and their similarities were all greater than 90.0%.The chemical constituents from Puerariae Thomsonii Radix were identified by mass spectrometry analysis,including citric acid,4'-O-glucosyl puerarin/daidzein-4',7-diglucoside,3'-hydroxy puerarin/genistein puerarin,puerarin apioside,daidzin puerossid A and daidzein,etc.Conclusion: The 13 batches of Puerariae Thomsonii Radix decoction in different origins had consistent properties with the basic properties of medicinal decoction pieces.The established method of quality evaluation can be used to systematically evaluate the standard decoction,providing reference for quality control of related decoction preparations of Puerariae Thomsonii Radix.

Objective:To observe effect of Zeqi Tang in intervening mice with orthotopic lung cancer model, in order to observe its anti-tumor mechanism. Method:An in situ mouse model of non-small cell lung cancer was established through intrapulmonary injection with 1×10⁵ LLC-luc cells. The model mice were intragastrically administered with Zeqi Tang(0.171 g·mL^-1) or normal saline for 35 days. Appearance (spirit, hair, appetite, sleep), survival period and Zeqi Tang anti-tumor effect were observed, weekly vital imaging was performed to detect the fluorescence signal in the lungs of mice. Flow cytometry was used to detect the NK cell content in the spleen of the model mice. CD107α was used to detect the degranulation of NK cells in the spleen of mice after administration of Zeqi Tang. Kromasil 100 5 C₁₈ column was used and eluted with acetonitrile-0.025%phosphoric acid in a gradient mode, with flow rate at 1.0 mL·min^-1, column temperature at 35℃ and detection wavelength of 265 nm, as to establish the fingerprint of Zeqi Tang. The fingerprints of 10 batches of samples was evaluated by using the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Software (2012 Edition) recommended by the Chinese Pharmacopoeia Commission, in order to complete the quality control of Zeqi Tang. Result:Zeqi Tang could significantly inhibit the lung fluorescence signal of lung cancer in situ model mice and prolong the survival of mice(P<0.05, P<0.01). After the intervention with Zeqi Tang, the NK cells in the tumors increased significantly(P<0.01), and the degranulation of CD107α also increased significantly(P<0.01). Moreover, the HPLC-DAD fingerprint of Zeqi Tang showed a significant increase in the fingerprint similarity of 10 batches of lacquer soup aqueous extract. Moreover, the HPLC-DAD fingerprint of Zeqi Tang showed that the fingerprint similarity of 10 batches of lacquer soup aqueous extract was ≥ 0.9, indicating that small differences between the batches. Conclusion:Zeqi Tang may enhance the tumor growth and prolong the survival period of mice by up-regulating the number of NK cells in mice and enhancing their degranulation function. The evaluation of similarity of HPLC fingerprint of Zeqi Tang reflects the quality of lacquer soup to a certain extent, and can provide reference for further study.

Objective:To study the mechanism of aloesin in inducing apoptosis in human non-small cell lung cancer (NSCLC) A549 cells, so as to inhibit its proliferation. Method:A549 cells in logarithmic growth phase were collected, and cell counting kit-8 (CCK-8) was used to detect the effect of different concentrations of aloesin (2, 4, 8, 16, 32, 64, 128 μmol·L^-1) on the proliferation of A549. Effect of aloesin (0, 16 μmol·L^-1) on the number of clones formed in A549 cells and the size of clone formation was determined by crystal violet staining. effect of aloesin on apoptosis of A549 cells was detected by annexin V/propidium iodide(PI)apoptosis kit staining. Hoechst staining was used to detect the phenomenon of apoptotic nuclear pyknosis. Western blot was used to detect aloesin's effect on death-related protein expressions of Bcl-xl/Bcl-2 associated death promoter (Bad), cleaved-Caspase-3,cl-Caspase-3(Asp175), Caspase-3, cleaved poly ADP-ribose polymerase (cl-PARP), poly ADP-ribose polymerase (PARP) in A549 cells. In vivo, 5-week-old nude mice were subcutaneously inoculated with 2×10⁶ A549 cells, and randomly divided into the medication group and the blank group. aloesin or normal saline was intraperitoneally injected for 4 weeks, and the tumor volume of nude mice was measured weekly. The body weight of the mice was observed, and the appearance of the nude mice was observed. Result:Aloesin inhibited the proliferation and cloning of A549 cells in a concentration-dependent manner (P<0.05). After treatment with aloesin, the number of apoptosis and the phenomenon of nuclear pyknosis in A549 cells increased significantly (P<0.01). At the same time, aloesin significantly down-regulated the expression of apoptosis-related protein Bcl-xl (P<0.05), and increased the expression of Bad protein (P<0.01). The expression levels of cl-PARP (P<0.01) and cl-Caspase-3 (P<0.05) were also significantly increased. In addition, in vivo, aloesin significantly shrank the volume of subcutaneous tumors in mice, reduced tumor weight, with a better appearance than that of the control group. Conclusion:Aloesin may inhibit the expression of NSCLC by inducing apoptosis of A549 cells, and is safe to use, with no inhibitory effect on the body weight of mice.

Objective:To explore the hepatoprotective effect and its mechanism of the geraniin on mice with acute liver injury induced by D-galactosamine (D-GalN). Method:A total of 60 Kunming mice were randomly divided into normal group, model group, Silymarin group (positive group 180 mg·kg^-1), and low, medium and high-dose geraniin groups (50, 100, 200 mg·kg^-1). All the mice were given with saline or corresponding dose of drugs (10 mL·kg^-1) by gavage once a day for 10 d. After 2 h of the last administration, except the normal group, the mice of other groups were injected intraperitoneally with D-GalN (500 mg·kg^-1) to induce the acute liver injury. After 16 h, the eye balls of mice were removed to take blood, and all mice were put to death to collect samples of liver. Activity or content of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), malondialdehyde (MDA), total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) in liver were determined by biochemical method. The serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), γ-interferon (IFN-γ) and interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay (ELISA), and expressions of Toll-like receptor-4(TLR-4) and nuclear factor(NF)-κB proteins were detected by Western blot. Liver histopathological changes were observed by hematoxylin-eosin (HE) staining. Result:Compared with the normal group, the serum levels of ALT, AST, ALP, TBIL and liver MDA in the model group were significantly increased (P<0.05, P<0.01). Compared with the model group, geraniin can reduce activities or contents of ALT, AST, ALP, TBIL in serum and MDA in liver of mice (P<0.05, P<0.01), but increase activities of T-SOD and GSH-Px in liver (P<0.05,P<0.01), while inhibiting the contents of TNF-α, IL-1β, IL-6, IFN-γ and the expressions of TLR-4 and NF-κB proteins in serum (P<0.05,P<0.01). HE staining showed that acute liver injury of mice was alleviated obviously by geraniin. Conclusion:Geraniin has an obvious protective effective on acute liver injuries induced by D-GalN in mice. Its mechanism may be correlated with oxidative stress, inflammation and TLR-4/NF-κB signaling pathway.

Objective: To establish the quality control methods for the standard decoction of Zingiberis Rhizoma. Method: DNA barcode primitives were identified for the medicinal materials from different origins; according to the standard of Chinese herbal medicine decoction preparation principle,the identified Zingiberis Rhizoma was prepared into standard decoction for analysis. Meanwhile, the extraction method and analysis method were validated from methodologies, and the transfer rate of 6-gingerol as well as the extraction rate of standard decoction of Zingiberis Rhizoma were calculated. In addition,the quality standard of standard decoction of Zingiberis Rhizoma was also established based. The structures of main chromatographic peaks were identified by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) to clarify the main chemical constituents in the standard decoction of Zingiberis Rhizoma. Result: All the samples were identified as Zingiberis Rhizoma. Under the conditions established in this paper,the standard curve of 6-gingerol was Y=661.56X+2.493 3(r=0.999 3),and the RSD was 0.5%in precision test, indicating that the instrument precision was good. The repeatability test showed that the RSD was 0.3%, indicating that the method had good repeatability. The stability test showed that the RSD was 0.4%, indicating that the test solution had good stability within 24 h. The recovery rate was 97.2%and the RSD was 0.6%,indicating that the method was accurate and reliable. 6-gingerol's transfer rate ranged from 31.8%to 57.4%and the extraction rate was within the range of 9.6%-23.1%. The fingerprint similarity of 12 batches of Zingiberis Rhizoma standard decoction was>90%. Conclusion: The established quality control method for Zingiberis Rhizoma was stable and feasible; meanwhile, the standard preparation method for Zingiberis Rhizoma and its quality evaluation system were also established in this study.

Objective: To observe how glutamine affect the skeletal muscle membrane repair in severely burned mice through promoting the mitsugumin 53 (MG53) dimerization in skeletal muscle and to explore its functional mechanism. Methods: (1) Animal experiments. A total of 179 BALB/c male mice aged 6 to 8 weeks were divided into sham injury group (n=43), burn group (n=73) and burn+ glutamine group (n=63) according to the random number table (the same grouping method below). Mice in sham injury group were sham injured on the back, and mice in burn group and burn+ glutamine group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back. Mice in burn+ glutamine group were intragastrically administered with glutamine (1 mg/kg), and the other two groups were given the same amount of amino acid solution once per day for 14 days. On post burn hour 12, 10 mice from burn group were taken for preparation of burn serum, which is used in the following cell experiments. Blood samples were collected from the hearts to prepare serum from 10 mice in sham injury group immediately after burn and from 10 mice in burn group and burn+ glutamine group on post burn day (PBD) 5, 10, and 14, respectively. And then the whole gastrocnemius muscle was harvested after the mice were sacrificed. On PBD 10, the whole flexor brevis digitorum was harvested from 6 mice in the 3 groups respectively after the mice were sacrificed. On PBD 5, 10, and 14, the whole gastrocnemius muscle tissue was harvested from another 9 mice in the 3 groups respectively after the mice were sacrificed. The mass of the whole gastrocnemius muscle of mice was weighed. The total protein content of gastrocnemius muscle of mice was detected by coomassie brilliant blue method. The repair function of myolemma of flexor brevis digitorum of mice was detected by two-photon laser fiber membrane perforating. The serum content of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) of mice was determined with radioimmunoassay. The expressions of MG53 dimer and monomer in gastrocnemius of mice were determined with non-reductive electrophoresis-Western blotting. The protein expressions of endoplasmic reticulum stress sign proteins CCAAT/enhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) in gastrocnemius of mice were determined with Western blotting. (2) Cell experiments. Mice skeletal muscle precursor cells C2C12 were cultured in vitro, and cells of the second passage were selected for the experiments. The cells were divided into normal control group, burn serum group, and burn serum+ glutamine group, with 3 dishes in each group and 1×10³ cells in each dish. Cells in normal control group were cultured with 1 mL Dulbecco′s modified Eagle medium (DMEM) with fetal bovine serum of volume fraction 10%, cells in burn serum group were cultured with 1 mL DMEM with burn serum of volume fraction 10%, and cells in burn serum+ glutamine group were cultured with 1 mL DMEM with burn serum of volume fraction 10% and 4 μL glutamine with a final molar concentration of 8 mmol/L. After 24 hours of culturing, the repair function of myocyte membrane after differentiation of skeletal muscle precursor cells in mice was detected with the same method before. Another cells were grouped and cultured as before, with 3 wells in each group and 1×10⁵ cells in each well. After 24 hours of culturing, the expressions of MG53 dimer and monomer and endoplasmic reticulum stress marker proteins in the cells were detected as before. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least significant difference t test, and Student Newman Keuls test. Results: Animal experiments. (1) Compared with those in sham injury group, the mass and total protein content of gastrocnemius muscle of mice in burn group were significantly decreased on PBD 5, 10, and 14 (P<0.05). Compared with those in burn group, the mass and total protein content of gastrocnemius muscle of mice in burn+ glutamine group were significantly increased on PBD 5, 10, and 14 (P<0.05). (2) Compared with that in sham injury group (0.9±0.4), the fluorescence intensity of FM1-43 in myofiber of mice in burn group (7.8±0.4) was significantly increased on PBD 10 (t=7.75, P<0.05). Compared with that in burn group, the fluorescence intensity of FM1-43 in myofiber of mice in burn+ glutamine group (4.0±0.4) was significantly decreased on PBD 10 (t=-4.31, P<0.05). (3) Compared with that in sham injury group, the serum content of TNF-α and IL-6 of mice in burn group was significantly increased on PBD 5, 10, and 14 (P<0.05). Compared with that in burn group, the serum content of TNF-α and IL-6 of mice in burn+ glutamine group was significantly decreased on PBD 5, 10, and 14 (P<0.05). (4) Compared with 56.97±2.82, 44.89±4.72, 42.46±1.06, 14.26±0.99, 62.36±2.74, and 29.45±0.84 in sham injury group, the expressions of MG53 dimer and monomer in gastrocnemius of mice were significantly decreased in burn group on PBD 5, 10, and 14 (6.16±0.25, 26.09±1.22, 28.86±1.53, 5.63±0.25, 26.74±0.79, 4.41±0.52, P<0.05). Compared with those in burn group, the expression of MG53 dimer of gastrocnemius of mice in burn+ glutamine group was significantly increased on PBD 10 and 14 (36.79±1.44, 43.96±1.62), and the expression of MG53 monomer of gastrocnemius muscle of mice in burn+ glutamine group was significantly increased on PBD 14 (13.16±2.17, P<0.05). Compared with those in sham injury group, the protein expressions of CHOP and GRP78 in gastrocnemius muscle of mice in burn group were significantly elevated on PBD 5, 10, and 14 (P<0.05). Compared with those in burn group, the protein expressions of CHOP and GRP78 in gastrocnemius of mice in burn+ glutamine group were significantly reduced on PBD 5, 10 (P<0.05). Cell experiments. (1) Compared with that in normal control group (1.76±0.25), the fluorescence intensity of FM1-43 in cells in burn serum group (9.46±1.22) was significantly increased after 24 hours of culturing (t=12.28, P<0.05). Compared with that in burn serum group, the fluorescence intensity of FM1-43 in cells in burn serum+ glutamine group (4.71±0.45) was significantly decreased after 24 hours of culturing (t=-7.59, P<0.05). (2) The expressions of MG53 monomer of cells were similar in normal control group, burn serum group, and burn+ glutamine group after 24 hours of culturing (P>0.05). Compared with 58.5±1.8 in normal control group, the expression of MG53 dimer of cells in burn serum group was significantly decreased after 24 hours of culturing (14.1±1.4, P<0.05). Compared with that in burn serum group, the expression of MG53 dimer of cells in burn serum+ glutamine group was significantly increased after 24 hours of culturing (30.9±0.6, P<0.05). Compared with those in normal control group, the protein expressions of CHOP and GRP78 of cells were significantly elevated in burn serum group after 24 hours of culturing (P<0.05). Compared with those in burn serum group, the protein expressions of CHOP and GRP78 of cells were significantly reduced in burn serum+ glutamine group after 24 hours of culturing (P<0.05). Conclusions Glutamine can promote MG53 dimerization by alleviating endoplasmic reticulum stress in severely burned mice. Thus it can accelerate skeletal muscle membrane repair, reduce the local inflammatory reaction of skeletal muscle and consumption of skeletal muscle.

BACKGROUND: Osteoarthritis (OA) is a kind of chronic bone and joint disease which seriously endangers human health. Cell therapy for OA has aroused widespread concern and gotten rapid development in recent years. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) have the advantages of easy amplification and differentiation, anti-inflammation and recruiting function such as MSCs from other sources. Furthermore, UC-MSCs are young cells that have large quantity, no ethical problems, high proliferative potential and pluripotent differentiation. UC-MSCs have been the most commonly used seed cells in clinical cell therapy. OBJECTIVE: To evaluate the efficacy and safety of UC-MSCs in the treatment of human knee OA to provide theoretical and clinical basis for stem cell therapy of OA. METHODS: The trail will be completed in Arthritis Clinic & Research Center, Beijing, China. Participants will be recruited according to established inclusion/exclusion criteria after obtaining the informed consent and the approval of the Ethics Committee (the first and second parts of the trial have been registered (https://register.clinicaltrials.gov/), with the identifier No. NCT03357770 and NCT03358654, and the third part will be carried out according to the conclusion of the first and second parts). The clinical trial will be divided into three parts: in the first part three groups will be recruited. Each group will contain three participants. The three groups of participants will be treated with high, medium and low dose of MSCs, respectively. Participants will be followed up to evaluate dose-limiting toxicity so as to determine the maximum tolerated dose. The second part will be a single-arm clinical trial. Nine participants will be recruited. The injection dose will be the maximum tolerated dose determined in the first part. Participants will be followed up to evaluate the safety and efficacy of the treatment. The third part will be a randomized controlled trial. Participants will be randomly divided into two groups (n=7 per group) and treated with MSCs and hyaluronic acid, respectively. During the trial, evaluators, participants and interveners will be unaware of grouping information and interventions. Participants will be followed up at designed time points after treatment to evaluate the safety and efficacy of the intervention. The trial will be terminated if there are unexplained local and systemic symptoms or death according to the NCI-CTCAE criteria. EXPECTED RESULTS: With reference to the previous literature, the knee pain will be relieved, the score of knee joint function will increase, and the cartilage defect area will decrease on MRI at 1-2 years after the intervention. The trail is expected to spend 3 years and 6 months.