[Mechanism of Tanreqing Injection in treatment of acute lung injury based on network pharmacology and molecular docking].

This study aimed to explore the mechanism of Tanreqing Injection in the treatment of acute lung injury(ALI) based on network pharmacology and molecular docking. The active components and action targets of Tanreqing Injection were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), PubChem, and SwissTargetPrediction databases, as well as available literature reports. The ALI-related targets were obtained from the GeneCards database and then mapped with Tanreqing Injection targets. Following the construction of "drug-component-potential target" network with Cytoscape 3.6.1, the potential targets were input into STRING to yield the protein-protein interaction(PPI) network, which was plotted using Cytoscape 3.6.1. Then the screened key targets were subjected to gene ontology(GO) and Kyoto encyclopedia of genes and genomes(KEGG) enrichment analysis based on DAVID database. The top three key targets RAC-alpha serine/threonine-protein kinase(AKT1), albumin(ALB) and interleukin-6(IL6) were docked to the top three key compounds by PyMOL and AutoDock vina. A total of 58 active components of Tanreqing Injection, 597 corresponding targets and 503 common targets shared by Tanreqing Injection and ALI were fi-gured out, with the key targets AKT1, ALB and IL6 involved. GO and KEGG enrichment analysis yielded 1 445 biological processes and 148 signaling pathways, respectively. Molecular docking verified a good binding ability of the top three key targets to the top three key compounds. The analysis based on network pharmacology and molecular docking uncovered that Tanreqing Injection directly or indirectly regulated the pulmonary capillary endothelial cells and alveolar epithelial cells via anti-inflammation, thus alleviating ALI.

Influence of vegetation type and temperature on the performance of constructed wetlands for nutrient removal.

In this study, the influence of vegetation type and environmental temperature on performance of constructed wetlands (CWs) was investigated. Results of vegetation types indicated that the removal of most nutrients in polyculture was greater than those in monoculture and unplanted control. The greatest removal percentages of NH4+-N, total nitrogen (TN) and total phosphorus (TP) in polyculture were 98.7%, 98.5%, and 92.6%, respectively. In experiments of different temperatures, the removal percentages of NH4+-N, NO3--N, TN and TP in all CWs tended to decrease with the decline of temperature. Especially, a sharp decline in the removal percentages of NO3--N (decreased by above 13.8%) and TN (decreased by above 7.9%) of all CWs was observed at low temperature (average temperature of 8.9 °C). Overall, the performance of CWs was obviously influenced by temperature, and the polyculture still showed best performance in the removal of nitrogen when the average temperature dropped to 19.8 °C. Additionally, the variations of urease activities in rhizosphere soil tended to decrease with the decreasing temperature. Overall, a substantial enhancement for nitrogen and TP removal in polyculture (Canna indica + Lythrum salicaria) was observed. In conclusion, CW cultivated with polyculture was a good strategy for enhancing nutrient removal when temperature was above 19.8 °C.

[Expression and characterization of Kringle 1-5 domains of human plasminogen].

The cDNA encoding Kringle 1-5 domains of human plasminogen (designated as K1-5), obtained from HepG2 by RT-PCR, was cloned into expression vector pHIL-S1. The recombinant plasmid pHIL-K1-5 was transformed into Pichia pastoris GS115 and the recombinant yeast was induced by methanol to express the recombinant protein. The expressed protein was purified by lysine affinity chromatography. The recombinant K1-5 inhibited the growth of bovine aortic endothelial cells (BAEC) stimulated by the basic fibroblast growth factor (bFGF), in a dosage-dependent manner, with a half maximal concentration of 14 mg/L. And rhK1-5 inhibited 47% of the BAEC migration stimulated by bFGF at the concentration of 50 mg/L. rhK1-5 also affected the cell cycle of BAEC and caused G(0)-G(1) arrest at the concentration of 14 mg/L.

Expression of Human Epiregulin in E.coli.

Human epiregulin cDNA was amplified from the lung cancer cell line A549 using RT-PCR. After adding 6 His codon to its 3' end, it was cloned into a high efficient secretive Escherichia coli system with alkaline phosphatase promoter(phoA promoter)constructed in our lab and induced for expression. The product was purified one-step by Ni-NTA column. Amino acid sequence analysis revealed the identity of our product with that previously reported. The product showed strong proliferative effect on fibroblast cell line Balb/c3T3 and growth inhibitory effect on epithelial carcinoma cell line A431.

[Expression and characterization of Kringle 1-4.5 domains of human plasminogen].

The cDNA encoding Kringle 1-4 and part of Kringle 5 domains of human plasminogen (K1-4.5), obtained from HepG2 by RT-PCR, was cloned into expression vector pHIL-S1. The recombinant plasmid pHIL-K1-4.5 was transformed into Pichi pastoris GS115 and the recombinant yeast was induced to express the recombinant proteins by methanol. The expressed proteins were purified by lysine affinity chromatography to a purity of 95%. The recombinant K1-4.5 inhibited the growth of bovine capillary endothelial cells (BAEC) stimulated by the basic fibroblast growth factor (bFGF), in a dosage-dependent manner with a half maximal concentration of 2 mg/L. rhK1-4.5 also inhibited 40% of the BAEC migration stimulated by bFGF in the concentration of 1 mg/L.

Functional Difference between N Domain and C Domain of hEGF and hTGF-alpha.

By exchanging the N domain and C domain of hEGF and hTGF-alpha genes by PCR, two chimeras E-TGF(EGF(1-32)-TGF-alpha(34-50))and T-EGF(TGF-alpha(1-33)-EGF(33-53))were constructed. The wild and chimeric molecules were expressed in E.coli under phoA system. The expressed hEGF, hTGF-alpha and two chimeras were purified. The EGF receptor competitive binding affinity of the four molecules was hEGF > hTGF-alpha and E-TGF > T-EGF and the cell proliferation stimulating activity of them was hTGF-alpha and E-TGF > T-EGF > hEGF. The result suggests that the N domain of hEGF and hTGF-alpha may play a major role in receptor binding activity and C domain of them may be responsible for stimulating cell proliferation.

Apoptotic effects of psiRNA-STAT3 on 4T1 breast cancer cells in vitro.

BACKGROUND: The aim of this study was to investigate the effect of a Lipofectamine2000 (Life2000) Transfection Reagent transfected psiRNA-STAT3 plasmid on 4T1 breast cancer cells. MATERIALS AND METHODS: MTT was used to detect the cell proliferation of breast cancer 4T1 cells at different periods (0h, 6h, 8h, 10h); the cell cycle was assessed by flow cytometry; variation of apoptosis and mitochondrial membrane potential was observed under a fluorescence microscope; immunohistochemical staining was used to determine the expression of caspase-3 and cyclin-D1 protein. RESULTS: An obvious effect of inhibition to 4T1 cancer cells could be observed at 8h after the psiRNA-STAT3 was transfected. Typical alterations of apoptotic morphological features were visible in the psiRNA-STAT3 treatment group. Mitochondrial membrane potential decreased significantly, the number of cells was increased in G0/G1 phase, and the number of cells was decreased in S phase, and the data were statistically significant (p<0.05), compared with the Scramble and Mock groups. Expression of caspase-3 protein was increased significantly, while that of cyclin D1 was significantly decreased. CONCLUSIONS: Life2000 transfected psiRNA-STAT3 plasmid can inhibit 4T1 tumor cell proliferation and promote apoptosis of 4T1 tumor cells, which process depends on the regulation of expression of cyclin D1 and caspase-3 protein.

Rapid and sensitive detection of vinorelbine in the urine of tumor patients by capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II)-based electrochemiluminescence assay.

A capillary electrophoresis method coupled with electrochemiluminescence detection for the analysis of vinorelbine (VNB) in the urine of tumor patients was established in this research. Complete determination of VNB was achieved in 8 min using a background electrolyte of 50 mmol/L phosphate buffer (pH 9.0) and a separation voltage of 15 kV. The calibration curves showed a linear range from 2.8 × 10(-10) to 1.6 × 10(-8) mol/L. The relative standard derivation for VNB was below 3.4%. The linear relationships were good and the correlation factor of VNB exceeded 0.985. The detection limits were 1.0 × 10(-11) mol/L under the optimal conditions. The developed method was applied to the sensitive determination of VNB in the urine of tumor patients.

[Moxibustion on the Governor Vessel for lung and kidney qi deficiency type in chronic obstructive pulmonary disease: a randomized controlled trial].

OBJECTIVE: To observe the difference between the therapeutic effect of Du-moxibustion (Moxibustion on the Governor Vessel) combined with western medicine and that of simple western medication for the remission stage of chronic obstructive pulmonary disease. METHODS: Two hundred and ten cases were randomly divided into an observation group (108 cases)and a control group (102 cases). The observation group was treated by routine treatment of western medicine combined with herb-partitioned spread moxibustion on the Governor Vessel between Dazhui (GV 14)and Yaoshu (GV 2). Simple western medicine was used in control group. The therapeutic effects of two groups were compared with the changes of symptom scores and pulmonary function before and after treatment. RESULTS: The total effective rate of observation group (90.7%, 98/108) was higher than that of control group (74.5%, 76/102) (P < 0.01). The symptom scores and some pulmonary function indices such as forced vital capacity (FVC), forced expiratory volume in one second (FEV1), the percentage of force expiratory volume in one second in predicted value(FEV1%) and maximal expiratory flow(PEF) after treatment were improved obviously than those before treatment in observation group (P < 0.05, P < 0.01), and the improvement degree was better than that of control group (all P < 0.01). CONCLUSION: Moxibustion on the Governor Vessel combined with western medicine can improve the clinical symptoms and pulmonary function of chronic obstructive pulmonary disease of lung and kidney qi deficiency type effectively,and the effect is better than that of simple western medication.

[BTF performance treating a chlorobenzene-contaminated gas stream].

In this study, biotrickling filter (BTF) inoculated with acclimated sludge was established to treat waste gas containing chlorobenzene. The BTF performance, average well color development (AWCD) values and microbial community were examined in steady state. Results revealed BTF achieved removal efficiency more than 80% of chlorobenzene under the conditions of < 0.6 g x m(-3) inlet concentration and > 45 s EBRT. Therefore, BTF have an advantage in treating low-concentration waste gas containing chlorobenzene (< or = 0.6 g x m(-3)). The overall chlorobenzene elimination capacity reached a maximum of 70 g x (m3 x h)(-1) at an inlet load of 80 g x (m3 x h)(-1). The mass ratio of carbon dioxide produced to the BTo-X removed was approximately 1.92, which confirms complete degradation of chlorobenzene, given that some of the organic carbon consumed is also used for the microbial growth. The degradation of chlorobenzene in the BTF followed Michaelis-Menten kinetic model, the maximum specific degradation rate (r(max)) was 35.6 g x (m3 x h)(-1). The AWCD values indicated that the microorganisms in the BTF showed high the microbial metabolic activity. The PCR-DGGE fingerprinting analysis on biofilm samples in the BTF indicated that the microbial community had a relative stability and complexity during the steady-state phase. The stability and complexity of microbial community could contribute to the degradation and mineralization of chlorobenzene in BTF.

[Feeding of mixed-carbon-resource during the expression phase in cultivation of recombinant Pichia pastoris expressing angiostatin].

A recombinant strain of Pichia pastoris with a phenotype of Muts was used to produce angiostatin in a 5-L fermentor. The methanol utilization ability of the present strain was weak, which resulted in extremely low growth rate and angiostatin productivity during the expression phase with methanol as the sole carbon source. To enhance the cell density and angiostatin expression level, mixed-carbon-source of glycerol-methanol was used in the expression phase. The methanol concentration was well controlled at 5 g/L by a methanol sensor and control system, and glycerol was continuously fed into the fermentor to achieve a higher cell density. 120 g/L of cells and 39 mg/L of angiostatin were reached at the end of fermentation which lasted 110 h. The mean specific cell growth rate in the expression phase was 0.01 h(-1), and the mean specific angiostatin productivity was 0.006 mg/(g x h). According to the data obtained in several runs of fermentation in which glycerol was fed at different rates, a higher mean specific angiostatin productivity was reached at the mean specific cell growth rate of 0.012 h(-1). To avoid the repression of angiostatin expression caused by residual glycerol and ethanol accumulation due to overfeeding of glycerol, glycerol addition was controlled to produce continuous oscillations in dissolved oxygen, because the change of dissolved oxygen concentration could deliver the information of available carbon source in the fermentation broth. Controlled glycerol feeding also avoided the problem of oxygen limitation brought by high cell density, and thus decreased the cooling requirement of the fermentor. Cell density reached 150 g/L at the end of fermentation, and angiostatin level reached 108 mg/L after an expression period of 96 h when the mean specific growth rate was maintained at 0.012 h(-1) by using the glycerol feeding strategy to result in the oscillations in dissolved oxygen. The mean specific angiostatin productivity was improved to 0.02 mg/(g x h). The apparent cell yield on glycerol and methanol were respectively 0.69 g/g and 0.93 g/g, higher than those in the fermentation without using the feeding strategy with dissolved oxygen as the indicator of metabolism.