Pleural mesothelial cell migration into lung parenchyma by calpain contributes to idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia. It is unknown why fibrosis in IPF distributes in the peripheral or named sub-pleural area. Migration of pleural mesothelial cells (PMC) should contribute to sub-pleural fibrosis. Calpain is known to be involved in cell migration, but the role of calpain in PMC migration has not been investigated. In this study, we found that PMCs migrated into lung parenchyma in patients with IPF. Then using Wt1tm1(EGFP/Cre)Wtp /J knock-in mice, we observed PMC migration into lung parenchyma in bleomycin-induced pleural fibrosis models, and calpain inhibitor attenuated pulmonary fibrosis with prevention of PMC migration. In vitro studies revealed that bleomycin and transforming growth factor-ß1 increased calpain activity in PMCs, and activated calpain-mediated focal adhesion (FA) turnover as well as cell migration, cell proliferation, and collagen-I synthesis. Furthermore, we determined that calpain cleaved FA kinase in both C-terminal and N-terminal regions, which mediated FA turnover. Lastly, the data revealed that activated calpain was also involved in phosphorylation of cofilin-1, and p-cofilin-1 induced PMC migration. Taken together, this study provides evidence that calpain mediates PMC migration into lung parenchyma to promote sub-pleural fibrosis in IPF.

Lethal (2) giant larvae regulates pleural mesothelial cell polarity in pleural fibrosis.

Pleural fibrosis is barely reversible and the underlying mechanisms are poorly understood. Pleural mesothelial cells (PMCs) which have apical-basal polarity play a key role in pleural fibrosis. Loss of cell polarity is involved in the development of fibrotic diseases. Partition defective protein (PAR) complex is a key regulator of cell polarity. However, changes of PMC polarity and PAR complex in pleural fibrosis are still unknown. In this study, we observed that PMC polarity was lost in fibrotic pleura. Next we found increased Lethal (2) giant larvae (Lgl) bound with aPKC and PAR-6B competing against PAR-3A in PAR complex, which led to cell polarity loss. Then we demonstrated that Lgl1 siRNA prevented cell polarity loss in PMCs, and Lgl1 conditional knockout (ER-Cre+/-Lgl1flox/flox) attenuated pleural fibrosis in a mouse model. Our data indicated that Lgl1 regulates cell polarity of PMCs, inhibition of Lgl1 and maintenance of cell polarity in PMCs could be a potential therapeutic treatment approach for pleural fibrosis.

miR-18a-5p Inhibits Sub-pleural Pulmonary Fibrosis by Targeting TGF-ß Receptor II.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease that typically leads to respiratory failure and death within 3-5 years of diagnosis. Sub-pleural pulmonary fibrosis is a pathological hallmark of IPF. Bleomycin treatment of mice is a an established pulmonary fibrosis model. We recently showed that bleomycin-induced epithelial-mesenchymal transition (EMT) contributes to pleural mesothelial cell (PMC) migration and sub-pleural pulmonary fibrosis. MicroRNA (miRNA) expression has recently been implicated in the pathogenesis of IPF. However, changes in miRNA expression in PMCs and sub-pleural fibrosis have not been reported. Using cultured PMCs and a pulmonary fibrosis animal model, we found that miR-18a-5p was reduced in PMCs treated with bleomycin and that downregulation of miR-18a-5p contributed to EMT of PMCs. Furthermore, we determined that miR-18a-5p binds to the 3' UTR region of transforming growth factor ß receptor II (TGF-ßRII) mRNA, and this is associated with reduced TGF-ßRII expression and suppression of TGF-ß-Smad2/3 signaling. Overexpression of miR-18a-5p prevented bleomycin-induced EMT of PMC and inhibited bleomycin-induced sub-pleural fibrosis in mice. Taken together, our data indicate that downregulated miR-18a-5p mediates sub-pleural pulmonary fibrosis through upregulation of its target, TGF-ßRII, and that overexpression of miR-18a-5p might therefore provide a novel approach to the treatment of IPF.

Crosstalk between calpain activation and TGF-ß1 augments collagen-I synthesis in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease of unknown cause that typically leads to respiratory failure and death within 3-5years of diagnosis. TGF-ß1 is considered a major profibrotic factor. However, TGF-ß1 is necessary but not sufficient to the pathogenesis of fibrotic lesion of the lungs. Recent observations have revealed that calpain, a calcium dependent protease, plays a pivotal role in tissue remodeling and fibrosis. However, the mechanism of calpain mediating pulmonary fibrosis is not understood. Calpain conditional knockout (ER-Cre(+/-)capns1(flox/flox)) mice and primary human lung fibroblasts (HLFs) were used here to investigate the relationship between calpain and TGF-ß1. Calpain knockout mice were protected from fibrotic effects of bleomycin. Bleomycin induced increases in TGF-ß1 via calpain activation in HLFs. Moreover, TGF-ß1 also activated calpain. This crosstalk between calpain activation and TGF-ß1 triggered the downstream signaling pathway including TGF-ß1 Smad2/3 and non-Smad (Akt) pathways, as well as collagen-I synthesis. Taken together, our data indicate that the crosstalk between calpain activation and TGF-ß1 augments collagen-I synthesis in HLFs and in pulmonary fibrosis. Intervention in the crosstalk between calpain activation and TGF-ß1 is a novel potential strategy to prevent pulmonary fibrosis.

[Comparison of the brain pharmacokinetics of nasal tetramethylpyrazine phosphate pH-sensitive in situ gel in normal rats and model rats].

The study is to investigate the brain pharmacokinetics change of nasal tetramethylpyrazine phosphate (TMPP) pH-sensitive in situ gel in normal and model rats. Acute cerebral ischemia rat model was successfully established by middle cerebral artery occlusion (MCAO) method. Both normal and model rats were given nasal TMPP pH-sensitive in situ gel (10 mg x kg(-1)). Perfusates of brain striatum area were collected at each time point by microdialysis. The content of TMPP was determined by HPLC. The pharmacokinetics parameters were calculated by Kinetica 4.4 software at each time point of the brain drug concentration. The main pharmacokinetics parameters of TMPP were fitted with compartments 2. After nasal TMPP pH-sensitive in situ gel the values of C(max) and AUC of both components in brain showed as follows: the value of model group > that of normal group. Significant difference can be observed in the process of brain pharmacokinetics in normal and model rats after giving nasal TMPP pH-sensitive in situ gel.

Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic disease with high mortality. Currently, pirfenidone and nintedanib are the only approved drugs for IPF by the U.S. Food and Drug Administration (FDA), but their efficacy is limited. The activation of multiple phosphotyrosine (pY) mediated signaling pathways underlying the pathological mechanism of IPF has been explored. A Src homology-2 (SH2) superbinder, which contains mutations of three amino acids (AAs) of natural SH2 domain has been shown to be able to block phosphotyrosine (pY) pathway. Therefore, we aimed to introduce SH2 superbinder into the treatment of IPF. Methods: We analyzed the database of IPF patients and examined pY levels in lung tissues from IPF patients. In primary lung fibroblasts obtained from IPF patient as well as bleomycin (BLM) treated mice, the cell proliferation, migration and differentiation associated with pY were investigated and the anti-fibrotic effect of SH2 superbinder was also tested. In vivo, we further verified the safety and effectiveness of SH2 superbinder in multiple BLM mice models. We also compared the anti-fibrotic effect and side-effect of SH2 superbinder and nintedanib in vivo. Results: The data showed that the cytokines and growth factors pathways which directly correlated to pY levels were significantly enriched in IPF. High pY levels were found to induce abnormal proliferation, migration and differentiation of lung fibroblasts. SH2 superbinder blocked pY-mediated signaling pathways and suppress pulmonary fibrosis by targeting high pY levels in fibroblasts. SH2 superbinder had better therapeutic effect and less side-effect compare to nintedanib in vivo. Conclusions: SH2 superbinder had significant anti-fibrotic effects both in vitro and in vivo, which could be used as a promising therapy for IPF.

[The pharmacokinetics study on Sinomenine transdermal patch on anaesthetized Beagle dogs].

OBJECTIVE: To explore the pharmacokinetic of Sinomenine transdermal patch. METHODS: The plasma drug concentration of Beagle dogs was determined after administration with HPLC-UVD as analysis tools. The pharmacokinetics parameters were fitted with kinetica software package. RESULTS: The exclusive analysis method was established with the following pharmacokinetics parameters: T (peak) = 8 h, Cmax = 366 ng/mL, MRT = 13 h. The pharmacokinetic characteristics was accordance with one-order rate and two-compartment model. CONCLUSION: The method is preferable to be applied to the pharmacokinetics research and further applied pharmacological study which will play a reference role in clinical application.

[Determination of plasma protein binding rate of tetramethylpyrazine phosphate by ultrafiltration].

OBJECTIVE: To study the plasma protein binding rate of Tetramethylpyrazine Phosphate. METHODS: The ultrafiltration was employed to determine the plasma protein binding rate of Tetramethylpyrazine Phosphate. The plasma concentrations of Tetramethylpyrazine Phosphate were measured by RP-HPLC. RESULTS: The plasma protein binding rate of Tetramethylpyrazine Phosphate with HSA, human plasma and rat plasma were (64.64 +/- 0.68)%, (65.85 +/- 7.35)% and (73.65 +/- 2.35)%, respectively. CONCLUSION: The binding rate of Tetramethylpyrazine Phosphate with plasma protein is middling strength.

The preparation and characterization of nanocomposite film reinforced by modified cellulose nanocrystals.

In the present study, in order to improve the properties of nanostarch-based nanocomposite film for food packaging, a type of nanocomposite film based on corn nanostarch (CNS) as the matrix and modified cellulose nanocrystals (modified-CNCs) as the reinforcement was prepared using a solution casting method. The cellulose nanocrystals (CNCs) were modified by a two-step method in which they were initially crosslinked with citric acid, and subsequently amidated with chitosan. Then, a type of CNS/modified-CNCs nanocomposite film with different content levels of modified-CNC were prepared and characterized using Fourier Transform Infrared spectroscopy (FTIR); X-ray Photoelectron Spectroscopy (XPS); X-Ray Diffraction (XRD); Differential Scanning Calorimetry (DSC); and Scanning Electron Microscopy (SEM). It was observed that when compared with the pure CNS film, the 8.0 wt% modified-CNCs loaded nanostarch-based nanocomposite film had displayed a 230.0% increase in tensile strength. And the moisture absorption ability had decreased by 25.6%; water vapor permeability had decreased by 87.4%; and the water contact angle value had increased by 18.1%. Also the results of this experimental study had revealed that the CNS/modified-CNCs nanocomposite film had displayed better antimicrobial activities against E. coli and S. aureus bacteria when compared with the pure CNS film.

Cigarette smoking aggravates bleomycin-induced experimental pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease that typically leads to respiratory failure and death. The cause of IPF is poorly understood. Although several environmental and occupational factors are considered as risk factors in IPF, cigarette smoking seems to be the most strongly associated risk factor. Here firstly, we treated mice with cigarette (16 mg tar, 1.0 mg nicotine in each cigarette) smoking and tried to explore the role of cigarette smoking in pulmonary fibrosis. Mice were continuously subjected to smoke for about 1 h each day (12 cigarettes per day, 5 days per week) during 40 days. Bleomycin was administrated by intraperitoneal injection at a dose of 40 mg/kg on days 1, 5, 8, 11 and 15. We found bleomycin induced pulmonary fibrosis in mice, and cigarette smoking augmented bleomycin-induced fibrosis reflected by both in fibrotic area and percentages of collagen in the lungs. Then we prepared and employed cigarette smoke extract (CSE) in cell models and found that CSE could induce the activation of p-Smad2/3 and p-Akt, as well as collagen-I synthesis and cell proliferation in lung fibroblasts and pleural mesothelial cells (PMCs). TGF-ß1 signaling mediated CSE-induced PMCs migration. Moreover, in vitro studies revealed that CSE had superimposed effect on bleomycin-induced activation of TGF-ß-Smad2/3 and -Akt signaling. TGF-ß-Smad2/3 and -Akt signaling were further augmented by cigarette smoking in the lung of bleomycin-treated mice. Taken together, these findings represent the first evidence that cigarette smoking aggravated bleomycin-induced pulmonary fibrosis via TGF-ß1 signaling.

[Dynamic determination of sinomenine in skin by microdialysis].

OBJECTIVE: To explore the feasibility and advantages of using microdialysis as sampling method for dynamic determination of sinomenine in topical skin. METHODS: In this study, sinomenine was administered to the rats by transdermal drug delivery system and celiac injection. With microdialysis technique for sampling, the concentration of sinomenine in dialysate was determined by high performance liquid chromatography (HPLC). RESULTS: Under existing determination condition, topical drug concentration in the skin of rats was hard to be detected after sinomenine administered to the skin, but it could be detected after celiac injection. CONCLUSION: Microdialysis sampling method can be used to determine topical drug concentration in the skin dynamically, and this method is better than traditional methods obviously.

[Bioequivalence study of sinomenine patch by microdialysis].

OBJECTIVE: To study bioequivalence of Sinomenine patch made by different preparation process, and to testify feasibility and superiority of microdialysis as a new method in topical bioequivalence study. METHOD: Normal gel patch and liposome gel patch of sinomenine were prepared by different preparation, nude mouse served as the experimental subjects sampling method of drug in the skin was tissue homogenization microdialysis, and drug concentration in dialysate was determined by HPLC. RESULT: Results of tissue homogenization showed that liposome gel patch leads more remainder drug in the skin of nude mouse than normal gel patch, and results of microdialysis showed that liposome gel patch led higher instantaneous drug concentration than normal gel patch. Concentration-time curve of sinomenine in the skin accorded with the results of most dermal delivery systems studies over the world. CONCLUSION: Topical bioequivalence of liposome gel patch of sinomenine is higher than that of normal gel patch of sinomenine. Microdialysis can be used to study bioavailability and bioequivalence of different preparation.

[Determination in vitro of rat plasma protein binding rate of sinomenine by using microdialysis method].

AIM: To determine in vitro the rat plasma protein binding rate by using microdialysis method. METHODS: The binding rate was determined by using microdialysis probe as sampling tools and zero-net flux method as calibrating method. The regression equation was made by the difference of concentrations between the dialysis sample and the perfusate. The x-intercept of regression equation was the free drug concentration (Cf). The plasma protein binding rate was calculated by using the following equation: f = ( C0 - Cf)/C0. RESULT: The binding rate was kept relatively stable in the studied concentration range. CONCLUSION: It is feasible that the plasma protein binding rate can be determined by using microdialysis method.

[Study on determination of entrapment efficiency of sinomenine liposomes].

OBJECTIVE: To establish an HPLC method for the determination of entrapment efficiency of sinomenine liposomes. METHOD: The liposomes and dissociated drugs were separated by sephadex filtration, mini-column centrifugation and dialysis. The methodology study and the optimization of determining condition were carried out at the same time. RESULT: Sephadex filtration could effectively separate the sinomenine liposomes from dissociated sinomenine. The column recovery was 98.8%, the average entrapment efficiency of three tests was64.9%, RSD 2.67%. CONCLUSION: The method was simple, exact, and had a good reappearance. It can be used to examine the entrapment efficiency of sinomenine liposomes.

[A study on the percutaneous penetration of artesunate from various reservoir vehicles].

OBJECTIVE: To determine the main factors which affect the percutaneous penetration of artesunate and provide efficient data for the artesunate transdermal delivery system. METHOD: Transdermal speed constant and accumulative amount of 12 hours were used for the estimations of various reservior vehicles, and the supplement orthodox design was used to study the effect of pH, various proportion of IPA/Water/IPM, and drug concentration. RESULT: Drug concentration and pH were the main factors which affected the percutaneous penetration of artesunate. CONCLUSION: The suitable reservior vehicle can prompt the percutaneous penetration of artesunate, and artesunate TTS will be made with further studies.

Effect of Xiongbing compound on the pharmacokinetics and brain targeting of tetramethylpyrazine.

OBJECTIVES: To investigate the effect of the Xiongbing compound (XBC) on the pharmacokinetics and brain targeting of tetramethylpyrazine (TMP). METHODS: Three microemulsions containing the same TMP concentration were prepared. XBC microemulsions were made from Rhizoma ligustric Chuanxiong extracts, borneol and TMP. TMP microemulsions were made with TMP only. Borneol microemulsions contained borneol and TMP. Microdialysis with high performance liquid chromatography (HPLC) was used to measure the concentration of TMP in the blood and striatum after intravenous (i.v.) or intragastric (i.g.) administration of the three different microemulsions. KEY FINDINGS: The pharmacokinetics of free TMP concentration in the blood and the striatum fit a first-order rate, open two-compartment model after intravenous and intragastric microemulsion administration. The maximal concentration (C(max) ) and area under curve (AUC) values in the XBC microemulsion i.v. group were significantly higher than that in the TMP microemulsion and borneol microemulsion i.v. groups. After XBC microemulsion i.g. administration, the t(½), mean residence time (MRT) and AUC of TMP in both plasma and brain tissues were greater than those with TMP microemulsion and borneol microemulsion administration. The relative brain targeting efficiency of TMP for the XBC microemulsion i.v and i.g. groups relative to the TMP microemulsion and borneol microemulsion groups were greater than 1. CONCLUSION: XBC microemulsion can enhance TMP oral bioavailability, brain targeting and tissue distribution, mainly through a synergistic action of Rhizoma ligustric Chuanxiong extracts and borneol.