Potential Therapeutic Activity of Berberine in Thyroid-Associated Ophthalmopathy: Inhibitory Effects on Tissue Remodeling in Orbital Fibroblasts.

Purpose: Berberine (BBR), an alkaloid produced by a traditional Chinese plant, was recently attributed multiple effects on lipometabolism, inflammation, and fibrosis. Thyroid-associated ophthalmopathy (TAO) is highly associated with these pathologic changes. Thus, we aimed to examine the potential therapeutic effect of BBR in an in vitro model of TAO. Methods: Orbital fibroblasts (OFs) obtained from control donors (n = 6) or patients with TAO (n = 6) were cultured. The CCK-8 assay was conducted for assessing the optimal concentration range. Oil Red O staining, Western blotting, and quantitative RT-PCR (qRT-PCR) were conducted to assess adipogenesis in OFs. RNA sequencing (RNA-seq) was used to screen the key pathways of the antiadipogenic effect mediated by BBR. Along with incremental concentrations of BBR, IL-1ß-induced expression of proinflammatory molecules was determined by ELISA and qRT-PCR. In addition, TGF-ß-induced hyaluronan (HA) production and fibrosis were evaluated by ELISA, qRT-PCR, and Western blotting. Results: TAO-OFs, but not control fibroblasts (CON-OFs), were readily differentiated into adipocytes with the commercial medium. Intracellular lipid accumulation was dose-dependently decreased by BBR, and adipogenic markers were also downregulated. Moreover, the PPARγ and AMPK pathways were screened out by RNA-seq and their downstream effectors were suppressed by BBR. Besides, BBR attenuated IL-1ß-induced expression of proinflammatory molecules in both TAO-OFs and CON-OFs by blocking nuclear factor-κB signaling. BBR's inhibitory effect on TGF-ß-mediated tissue remodeling was also confirmed in OFs. Conclusions: These findings demonstrate BBR has outstanding capabilities of controlling adipogenesis, inflammation, HA production, and fibrosis in OFs, highlighting its potential therapeutic role in TAO management.

Signaling pathways involved in HSP32 induction by hyperbaric oxygen in rat spinal neurons.

Spinal cord injury (SCI) is a debilitating disease, effective prevention measures are in desperate need. Our previous work found that hyperbaric oxygen (HBO) preconditioning significantly protected rats from SCI after stimulated diving, and in vitro study further testified that HBO protected primary cultured rat spinal neurons from oxidative insult and oxygen glucose deprivation injury via heat shock protein (HSP) 32 induction. In this study, underlying molecular mechanisms were further investigated. The results showed that a single exposure to HBO significantly increased intracellular levels of reactive oxygen species (ROS) and nitric oxide (NO) and activated MEK1/2, ERK1/2, p38 MAPK, CREB, Bach1 and Nrf2. The induction of HSP32 by HBO was significantly reversed by pretreatment neurons with ROS scavenger N-Acetyl-L-cysteine, p38 MAPK inhibitor or Nrf2 gene knockdown, enhanced by MEK1/2 inhibitors or gene knockdown but not by ERK1/2 inhibitor. CREB knockdown did not change the expression of HSP32 induced by HBO. N-Acetyl-L-cysteine significantly inhibited the activation of MEK1/2, ERK1/2, p38 MAPK, and Nrf2. Activation of Nrf2 was significantly inhibited by p38 MAPK inhibitor and the nuclear export of Bach1 was significantly enhanced by MEK1/2 inhibitor. The results demonstrated that HBO induces HSP32 expression through a ROS/p38 MAPK/Nrf2 pathway and the MEK1/2/Bach1 pathway contributes to negative regulation in the process. More importantly, as we know, this is the first study to delineate that ERK1/2 is not the only physiological substrates of MEK1/2.