Anti-fibrotic effect of rosmarinic acid on inhibition of pterygium epithelial cells.

AIM: To investigate the anti-fibrosis effect of rosmarinic acid (RA) in pterygium epithelial cells (PECs) to determine if RA is a potent agent for treating pterygium. METHODS: The PECs (1×104 cells/mL) were treated with 100 µmol/L of RA for 1, 3 and 6h. After RA treatment, the cell viability was determined by staining with acridine orange/DAPI and analysis via a NucleoCounter NC-3000. The protein expression levels of type I collagen, transforming growth factor beta-1 (TGF-ß1), TGF-ß type II receptor (TGF-ßRII), p-Smad1/5, p-Smad2, p-Smad3, and Smad4 of the cell lysates were measured by Western blot analysis. RESULTS: The cell viability of PECs was significantly decreased after RA treatment (P<0.01). As the result, RA reduced the protein expression of type I collagen and TGF-ß1 of PECs. Additionally, RA also inhibited TGF-ß1/Smad signaling by decreasing the protein expressions of TGF-ßRII, p-Smad1/5, p-Smad2, p-Smad3, and Smad4. CONCLUSION: This study demonstrate that RA could inhibit fibrosis of PECs by down-regulating type I collagen expression and TGF-ß1/Smad signaling. Therefore, RA is a potent therapeutic agent for the treatment of pterygium.

Inhibitory effects of rosmarinic acid on pterygium epithelial cells through redox imbalance and induction of extrinsic and intrinsic apoptosis.

Pterygium is a common tumor-like ocular disease, which may be related to exposure to chronic ultraviolet (UV) radiation. Although the standard treatment for pterygium is surgical intervention, the recurrence rate of pterygium is high when no effective inhibitory drug is used after surgery. Rosmarinic acid (RA) is a polyphenol antioxidant with many biological activities, including anti-UV and anti-tumor properties. This study aimed to examine the inhibitory effects of RA on pterygium epithelial cells (PECs). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was used to examine the cell cytotoxicity of PECs after RA treatment. A fluorescent probe, DCFH-DA (2',7'-dichlorofluorescin diacetate), was stained with PECs to measure intracellular reactive oxygen species (ROS) levels. Antioxidant activity assays were used to measure the levels of superoxide dismutase (SOD) and catalase (CAT) in PECs. Western blot analysis was used to determine the protein expression of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), quinone acceptor oxidoreductase 1 (NQO1), and apoptosis-associated proteins. RA significantly reduced the cell viability of the PECs. Treatment with RA remarkably increased the Nrf2 protein expression levels in the nucleus, HO-1 and NQO1 protein expression levels, and the activities of SOD and CAT. As a result, intracellular ROS levels in PECs were decreased. Additionally, the induction of extrinsic apoptosis on PECs by RA was associated with increasing expressions levels of Fas, Fas-associated protein with death domain (FADD), tumor necrosis factor-alpha (TNF-α), and caspase 8 protein. Moreover, the induction of intrinsic apoptotic cell death in PECs was confirmed through upregulation of cytochrome c, Bax, caspase 9, and caspase 3 and downregulation of Bcl-2 and pro-caspase 3. Our study demonstrated that RA could inhibit the viability of PECs through regulation of extrinsic and intrinsic apoptosis pathways. Therefore, RA may have potential as a therapeutic medication for pterygium.

PAL31 may play an important role as inflammatory modulator in the repair process of the spinal cord injury rat.

Functional regeneration in a complete T8 transection model Cheng et al. (1996) and most recently, acidic fibroblast growth factor (aFGF; also known as FGF-1) involved in the repair process of the spinal cord injury (SCI) rat Tsai et al. (2008) have been reported. To further reveal the mechanism of the repair process of SCI, in additionally, we have identified a 30 kDa specific protein kinase A substrate induced at 6 days after SCI. However, the induction of the transducing signal was reduced in samples treated with aFGF. The 30 kDa protein was purified and identified by mass spectrometry as a novel protein, PAL31. The results of immunohistochemical study showed that PAL31 is abundantly expressed in the epicenter of the injured spinal cord and colocalizes with ED1-positive cells (macrophages) and CD8 T lymphocytes. Over-expression of PAL31 in RAW 264.7 cells resulted in the down-regulation of macrophage chemoattractant protein 1, inducible nitric oxide synthase, and signal transducer and activator of transcription-1. However, knockdown of PAL31 by small interfering RNA seems to lead to apoptosis when the cells were treated with inflammatory inducers. These experimental results suggest that PAL31 may involve in the modulation of the inflammatory response and, at the same time, prevent apoptosis process of macrophage after SCI.

Involvement of acidic fibroblast growth factor in spinal cord injury repair processes revealed by a proteomics approach.

Acidic fibroblast growth factor (aFGF; also known as FGF-1) is a potent neurotrophic factor that affects neuronal survival in the injured spinal cord. However, the pathological changes that occur with spinal cord injury (SCI) and the attribution to aFGF of a neuroprotective effect during SCI are still elusive. In this study, we demonstrated that rat SCI, when treated with aFGF, showed significant functional recovery as indicated by the Basso, Beattie, and Bresnahan locomotor rating scale and the combined behavior score (p < 0.01-0.001). Furthermore proteomics and bioinformatics approaches were adapted to investigate changes in the global protein profile of the damaged spinal cord tissue when experimental rats were treated either with or without aFGF at 24 h after injury. We found that 51 protein spots, resolvable by two-dimensional PAGE, had significant differential expression. Using hierarchical clustering analysis, these proteins were categorized into five major expression patterns. Noticeably proteins involved in the process of secondary injury, such as astrocyte activation (glial fibrillary acidic protein), inflammation (S100B), and scar formation (keratan sulfate proteoglycan lumican), which lead to the blocking of injured spinal cord regeneration, were down-regulated in the contusive spinal cord after treatment with aFGF. We propose that aFGF might initiate a series of biological processes to prevent or attenuate secondary injury and that this, in turn, leads to an improvement in functional recovery. Moreover the quantitative expression level of these proteins was verified by quantitative real time PCR. Furthermore we identified various potential neuroprotective protein factors that are induced by aFGF and may be involved in the spinal cord repair processes of SCI rats. Thus, our results could have a remarkable impact on clinical developments in the area of spinal cord injury therapy.

Dissection of cry gene profiles of Bacillus thuringiensis isolates in Taiwan.

On the basis of the newly revised nomenclature system of cry genes, the PCR amplification method has been adopted to resolve the cry gene combinations of 294 Bacillus thuringiensis isolates from five selected areas of Taiwan. Our results indicate that cry1 (especially cry1A + 1B + 1F) and cry2 were the most abundant cry genes in Taiwan. In contrast, cry3 and cry6 genes were detected only on Yang Ming Mountain, while the cry13 gene was found only on Snow Mountain. In addition, some distinctive combinations of cry genes were detected in distinct areas of Taiwan, such as cry1C, cry1D, cry1C + 1D, cry4, cry1 + 4, cry1 + 11, cry4 + 11, and cry1 + 4 + 11 in the Taipei area; cry1A + 1C + 1F in the Taichung area; cry1E and cry1A + 1B + 1I on Yang Ming Mountain; cry1 + 13, cry1 + 2 + 11, and cry1 + 2 + 13 on Snow Mountain; and cry1 + 5 and cry1 + 2 + 5 on Jade Mountain. These data clearly indicate that the distribution of cry gene combinations of B. thuringiensis isolates seems to be geographically related.

The IspA protease's involvement in the regulation of the sporulation process of Bacillus thuringiensis is revealed by proteomic analysis.

We have observed that the process of sporulation of the ispA-deficient mutant was delayed under phase-contrast microscopy. The protein profiles of the ispA-deficient mutant have been analyzed using two-dimensional gel electrophoresis. The results of a proteomic analysis using MALDI-TOF MS indicated that a sporulation-associated protein, pro- [Formula: see text], was upregulated, while two other sporulation-associated proteins, SpoVD and SpoVR, were downregulated in the ispA-deficient mutant. It has been known that pro- [Formula: see text] is a precursor of [Formula: see text] and is required for gene expression related to the late stage of sporulation. Moreover, SpoVD and SpoVR are known to be involved in the formation of the spore cortex. Based on these observations, we propose that the delay in the sporulation process observed in the ispA-deficient mutant may be due to a failure of [Formula: see text] to signal sporulation. This phenomenon may be further enhanced by insufficient amount of SpoVD and SpoVR for cortex formation. In this study, we have revealed for the first time a possible pathway for the regulation of sporulation-associated proteins via IspA.