Evaluation of Calu-3 cell lines as an in vitro model to study the inhalation toxicity of flavoring extracts.

This study aimed to evaluate the characteristics of Calu-3 cells as a model to examine the toxicological responses of inhalable substances. Calu-3 cells were grown to the confluence at an air-liquid interface (ALI) using a Transwell® permeable support system. The ALI resulted in biomimetic native bronchial epithelium displaying pseudostratified columnar epithelium with more microvilli and secretory vesicles. We further characterized and optimized the Calu-3 cell line model using ALI culturing conditions, immunolabeling of protein expression, ultrastructural analysis using scanning electron microscopy (SEM), and transepithelial electrical resistance (TEER) measurements, and then screened for the cytotoxicity of tobacco flavoring extracts. Calu-3 cells displayed dose-dependent responses when treated with the flavoring extract. Within 8-10 days, cell monolayers developed TEER ≥1000 Ω·cm2. During this time, Calu-3 cells exposed to flavoring extracts X01 and X06 exhibited a loss of cellular integrity and decreased ZO-1 and E-cadherin protein expression. In conclusion, we investigated the Calu-3 cell line culture conditions, culture time, and barrier integrity and tested the effect of six new synthetic tobacco flavoring extracts. Our data demonstrate that the Calu-3 human bronchial epithelial cell monolayer system is a potential in vitro model to assess the inhalation toxicity of inhalable substances.

[Study on early change features of microRNA in the peripheral blood of Sophorae tonkinensis radix et rhizoma induced liver injury rats].

OBJECTIVE: To study early change features of microRNA (miRNA) in the peripheral blood of Sophorae Tonkinensis Radix et Rhizoma induced liver injury rats, and to look for the miRNA biomarkers in the peripheral blood of early liver injury. METHODS: Sixty Wistar rats were randomly divided into the control group and the Sophorae Tonkinensis Radix et Rhizoma (abbreviated as STRR) group, 30 in each group. Rats in the STRR group was administered with STRR decoction at 12 g/kg (2 mL/100 g), while equal volume of the distilled water was given to those in the control group. Rats were anesthetized on day 3, 7, 14, and 28, and 28 days after withdrawal. The serum samples were withdrawn. The alanine aminotransferase (ALT), aspartate transaminase (AST), total bile (TBIL), alkaline phosphatase (ALP), total protein (TP), and albumin (ALB) were detected. The globulin (GLO) level was calculated. HE staining was performed on the liver tissue to observe the pathomorphological changes. The whole blood was collected on day 7, 14, and 28 to perform the microarray test. The differentially expressed miRNAs were screened and verified by RT-PCR. RESULTS: The ALT activity obviously increased on day 7 - 28 in the STRR group (P <0.05). The histopathological results showed the degeneration and swelling of the liver cells on day 28. In the microarray test, there were 11, 22, and 13 up regulated expressed miRNAs on day 7, 14, and 28, respectively. There were 1, 13, 2 down regulated expressed miRNAs on day 7, 14, and 28, respectively. By target gene prediction and pathway analysis of differentially expressed miRNA on day 7, 14, and 28, they involved in regulating and controlling signal transduction, cellular interaction, cytoskeleton. Differentially expressed miRNA might possibly participate in the process of liver injury. The RT-PCR result of the expression of miR-291a-5p with the peak time efficiency on day 7 showed that the expressions of miR-291a-5p in the peripheral blood and the liver tissue were basically identical. CONCLUSION: miR-291a-5p could early indicate the liver injury, which could be taken as one of an early marker in STRR induced liver injury.