An experimental study of acute toxicity and pharmacology of fermented Platycodonis Radix / 中国中药杂志

This study investigated the acute toxicity of fermented Platycodonis Radix on mice and its effect on coughing in mice infected with Mycoplasma pneumoniae. The maximum dosage(MAD) was used in the acute toxicity experiment on mice to observe the signs of mice. After 14 days, dissection, blood biochemical examination, and pathological tissue section observation were conducted. In the pharmacological experiment of fermented Platycodonis Radix, 60 healthy BALB/c mice, 30 males and 30 females, were randomly divided into a blank group, a model group, a carbetapentane group(0.013 g·kg~(-1)·d~(-1)), and high-, medium-, and low-dose fermented Platycodonis Radix groups(5.2, 2.6, and 1.3 g·kg~(-1)·d~(-1)), with 10 mice in each group. Except for the blank group, the mice in the other five groups underwent model induction by intranasally instilling 20 μL of 1×10~6 CCU M. pneumoniae for 3 days, and the mice in each group were orally administered the corresponding drugs for 7 days. Cough induction experiment was conducted to observe and record the cough latency and total cough count within 3 min for each group. Hematoxylin-eosin(HE) staining and Masson staining were used to observe the pathological changes in lung tissues. Immunohistochemistry was performed to observe the protein expression of transient receptor potential A1(TRPA1), calcitonin gene-related peptide(CGRP), and substance P(SP) in the lung tissues of mice in each group. Real-time fluorescence-based quantitative polymerase chain reaction(qRT-PCR) was used to elucidate the changes in the mRNA levels of cough-related factors TRPA1, CGRP, and SP in mice treated with fermented Platycodonis Radix. No mice died in the acute toxicity experiment, and there were no changes in general behavior and major organ histopathological examinations. Compared with the blank group, there were no statistically significant differences in blood biochemical indexes. In the pharmacological experiment of fermented Platycodonis Radix, compared with the model group, the high-and medium-dose fermented Platycodonis Radix groups showed improved lung tissue structure of mice, with clear structure and regular tissue morphology. The qRT-PCR and immunohistochemical detection showed a decrease in the expression of TRPA1, CGRP, and SP in the fermented Platycodonis Radix groups. Fermented Platycodonis Radix can exert an inhibitory effect on cough by suppressing the expression of TRPA1, CGRP, and SP in lung tissues, thereby identifying the target of the drug.

Preparation of purified proteins from fresh Pheretima and their inhibitory effect against pulmonary fibrosis in mice / 南方医科大学学报

OBJECTIVE@#To develop a convenient method for rapid purification of fresh Pheretima proteins and assess the inhibitory effect of these proteins against pulmonary fibrosis.@*METHODS@#The crude extract of fresh Pheretima was obtained by freeze-drying method and then purified by size exclusion chromatography. The composition of the purified proteins was analyzed by mass spectrometry. MRC-5 cells were treated with 5 ng/mL TGF-β1 alone (model group) or in combination with SB431542 (2 μmol/L) or the purified proteins (13.125 μg/mL), and the cytotoxicity of purified proteins and their inhibitory effects on cell proliferation were detected with CCK8 assay. Flow cytometry was used to detect the changes in cell apoptosis, and the cellular expressions of α-SMA, Vimentin, E-cadherin, collagen I, Smad2/3 and P-Smad2/3 were detected using RT-PCR and Western blotting. In the animal experiment, adult male C57BL/6 mice were subjected to intratracheal instillation of bleomycin followed by treatment with the purified proteins (5 mg/mL) for 21 days, after which HE and Masson staining was used to observe the pathological changes in the lung tissue of the mice.@*RESULTS@#We successfully obtained purified proteins from fresh Pheretima protein by size exclusion chromatography. Treatment with the purified proteins significantly inhibited TGF-β1-induced proliferation of MRC-5 cells (P < 0.01), reduced the cellular expressions of α-SMA, Vimentin and collagen I (P < 0.001 or P < 0.01), increased the expression of E-cadherin (P < 0.01), and inhibited the expressions of Smad2/3 and P-Smad2/3 (P < 0.001 or P < 0.01). In male C57BL/6 mice models of bleomycin-induced pulmonary fibrosis, treatment with the purified proteins obviously reduced the number of inflammatory cells and fibrotic area in the lungs.@*CONCLUSION@#The purified proteins from fresh Pheretima obtained by size exclusion chromatography can inhibit pulmonary fibrosis in mice by regulating the TGF-β/ Smad pathway.

Effect of baicalin on expression of fibrogenic factors TGF-β1, mmp2 and timp2 in tissue of mice with pulmonary fibrosis / 中国中药杂志

To investigate the effect of baicalin extracted from Qinbai Qingfei Concentrated Pills on the expressions of TGF-β1, mmp2 and timp2 in mice with pulmonary fibrosis induced by bleomycin. The Biacore technique was used to detect the specific binding between Qinbai Qingfei Concentrated Pills and TGF-β1, and the affinity components were enriched, regenerated and recovered by Biacore fishing. Then ultra-performance liquid chromatography and quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS) were used to determine whether the monomer was baicalin. Biacore was used to verify the affinity kinetics of baicalin, which was validated by pharmacodynamics in vivo. Totally 30 BALB/C mice were randomly divided into three groups: baicalin group, blank group and model group. The blank group was given sodium chloride injection(0.08 mL·kg~(-1)), while the model group and the baicalin group were injected with 4 mg·kg~(-1) bleomycin. The localization of TGF-β1, mmp2 and timp2 protein in the cells and the mRNA expressions of TGF-β1, mmp2 and timp2 were detected by RT-PCR 14 days later. The results of Biacore affinity analysis showed that the peak of binding response between Qinbai Qingfei Concentrated Pills and TGF-β1 protein reached 1 524.0 RU, with specific binding. The affinity constant K_D of baicalin and TGF-β1 was 1.620 06 μmol·L~(-1), which was determined by SPR kinetic analysis, suggesting a stable binding between baicalin and TGF-β1, which verified the results of angulation. The results of immunohistochemistry showed that the deposition of cellulose in baicalin group was significantly less than that in model group, the mRNA expressions of TGF-β1, mmp2 and timp2 were decreased in baicalin solution compared with the model group. Baicalin combined with TGF-β1 could inhibit the expressions of mmp2 and timp2 and delay the progress of pulmonary fibrosis.

Effect of Dilong on expression of fibrogenic factors TGF-β1 and α-SMA in lung tissue of mice with pulmonary fibrosis / 中国中药杂志

The aim of this paper was to investigate the effect of Dilong( geosaurus) on the expressions of fibrotic factors TGF-β1 and α-SMA in bleomycin-induced pulmonary fibrosis mice. The binding ability of Dilong to fibrotic factor TGF-β1 was initially detected by Biacore technology and verified by in vivo pharmacodynamics. A total of 60 SPF C57 mice were randomly divided into 6 groups. Except the blank group( injecting 0. 08 m L·kg-1 sodium chloride in the trachea),the other five groups were given bleomycin( 4 mg·kg-1) to replicate the pulmonary fibrosis model. After 14 days of drug treatment,the expressions of TGF-β1 and α-SMA were detected by Masson staining,immunohistochemistry and RT-PCR. The results of Biacore experiment showed that the extract of Dilong was well bound to TGF-β1 protein in vitro,and the binding value reached 619. 3. Compared with the model group,Masson's results showed that cellulose deposition in high-dose,medium-dose and low-dose Dilong groups decreased to varying degrees. RT-PCR results showed that different doses of Dilong could reduce protein and mRNA expressions of TGF-β1 and α-SMA to a certain extent in a dose-dependent manner. In conclusion,Dilong could delay the process of pulmonary fibrosis by binding to target protein TGF-β1 and inhibiting the expression of α-SMA.

Levels of P27Kip1 expression and apoptosis in HL-60 cells after treatment with TGF-ß1 and/or arsenic trioxide / 中国实验血液学杂志

This study was purposed to investigate the effects of TGF-beta1 and arsenic trioxide (As₂O₃) on cell apoptosis, cell cycle and changes of P27(Kip1), endogenous TGF-ß1, cyclin E and BCL-2 in HL-60 cells, and to explore the relationship between expression of P27(Kip1) and apoptosis induced by As₂O₃ and/or TGFß1. Cell apoptosis and cell cycle changes of HL-60 cells treated with As₂O₃ and/or TGFß1 were detected by cytomorphologic observation and flow cytometry, the protein expressions of P27(Kip1), TGF-ß1, cyclin E and BCL-2 were measured by immunohistochemistry. The results showed the effect of 5 μmol/L of As₂O₃ was the most strong among the different concentration of As₂O₃ ,and the effect on apoptosis at 48 hour was more strong than that at 24 hours (p < 0.05). The TGF-beta1 (5 ng/ml) induced arrest of cells in G₁ phase (p < 0.05) compared with As₂O₃ alone and As₂O₃ combined with TGF-ß1, while there was no difference with control. P27(Kip1) expression was up regulated (p < 0.05), cyclin E and BCL-2 expression was down-regulated (p < 0.05) in TGFß1-treated group. BCl-2 expression was down regulated, endogenesis TGFß1 expression was up regulated (p < 0.05), and the level of P27(kip1) and cyclin E were not changed in As₂O₃-treated group (p > 0.05). The down-regulating effect of TGF-ß1 combined with As₂O₃ on BCL-2 protein was more strong than that in single factor treated group (p < 0.05). It is concluded that TGFß1 induces cell cycle arrest and apoptosis in HL-60 cells, while the P27(kip1) expression is up regulated. P27 protein is the key effector of TGFß-induced cell cycle arrest. The effect of TGF-ß1 combined with As₂O₃ on apoptosis as well as the down-regulation of BCL-2 protein in HL-60 cells is more strong than that in single factor-treated groups, that indicates the passages linking up each other.

Oriented immobilization of human IgG by protein A on imaging ellipsometry biosensor / 生物工程学报

The biosensor based on optical imaging ellipsometry, can be used to detect directly, without labeling, the surface concentration of biomolecules on solid surface. The feasibility of using protein A to immobilize antibody on the silicon surface of the imaging ellipsometry biosensor was investigated in this study. The results showed that the anti-IgG immobilized by the protein A on silicon surface could bind effectively human IgG, and the human IgG immobilized on silicon surface by protein A bound more polyclonal antibody molecules than that immobilized on silicon surface directly, suggesting that protein A might block the surface to prevent the absorption of human IgG on surface directly, which might compromise its native configuration. The silicon surface modified with protein A is expected to be used to immobilize a variety of antibodies, as protein A can bind selectively the Fc regions of many mammalian IgG. The combination of imaging ellipsometry and the protein A surface modification has the potential to be developed into immunoassays of high sensitivity.