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Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response
Published on: September 15, 2017
ROCK inhibitor fasudil reduces the expression of inflammatory factors in LPS-induced rat pulmonary microvascular
Huanlong Liu1, Zhenhua Pan2, Xindi Ma3
1Pharmaceutical Department of the Second Hospital of Hebei Medical University, Shijiazhuang, PR China.
Background:
Inflammation plays a major role in the pulmonary artery hypertension (PAH) and the acute lung injury (ALI) diseases. The common feature of these complications is the dysfunction of pulmonary microvascular endothelial cells (PMVECs). Fasudil, the only Rho kinase (ROCK) inhibitor used in clinic, has been proved to be the most promising new drug for the treatment of PAH, with some anti-inflammatory activity. Therefore, in the present study, the effect of fasudil on lipopolysaccharide (LPS)-induced inflammatory injury in rat PMVECs was investigated.
Methods:
LPS was used to make inflammatory injury model of rat PMVECs. Thereafter, the mRNA and protein expression of pro-inflammatory factors was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) assay respectively. Intracellular reactive oxygen species (ROS) levels were measured by the confocal laser scanning system. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) were determined by using commercial kits according to the manufacturer's instructions. Western blot assay was used to detect the protein expression of nuclear factor kappa B (NF-κB) p65.
Results:
Fasudil effectively prevented inflammatory injury induced by LPS, which is manifested by the decrease of pro-inflammatory cytokines interleukin-6 (IL-6) and monocyte chenotactic protein-1 (MCP-1). Meanwhile, fasudil dramatically reduced the levels of ROS and MDA, and also elevated the activities of SOD and GSH-Px. Furthermore, the nuclear translocation of NF-κB p65 induced by LPS was also suppressed by fasudil. Additionally, the ROS scavengers N-Acetylcysteine (N-Ace) was also found to inhibit the nuclear translocation of NF-κB and the mRNA expression of IL-6 and MCP-1 induced by LPS, which suggested that ROS was essential for the nuclear translocation of NF-κB.
Conclusions:
The present study revealed that fasudil reduced the expression of inflammatory factors, alleviated the inflammatory and oxidative damage induced by LPS in rat PMVECs via ROS-NF-κB signaling pathway.
Insights
Fasudil, a Rho kinase inhibitor, effectively combats inflammation and oxidative damage in rat pulmonary microvascular endothelial cells by inhibiting the ROS-NF-κB pathway. This study highlights fasudil
Area of Science:
- Cell Biology
- Pharmacology
- Biochemistry
Background:
- Inflammation is a key factor in pulmonary artery hypertension (PAH) and acute lung injury (ALI).
- Pulmonary microvascular endothelial cell (PMVEC) dysfunction is common in these conditions.
- Fasudil, a Rho kinase (ROCK) inhibitor, shows anti-inflammatory properties and potential for PAH treatment.
Purpose of the Study:
- To investigate the effects of fasudil on lipopolysaccharide (LPS)-induced inflammatory injury in rat PMVECs.
- To elucidate the underlying molecular mechanisms, particularly the role of the ROS-NF-κB signaling pathway.
Main Methods:
- Establishment of an inflammatory injury model in rat PMVECs using LPS.
- Quantification of pro-inflammatory factors (IL-6, MCP-1) via RT-PCR and ELISA.
- Measurement of intracellular reactive oxygen species (ROS), antioxidant enzyme activities (SOD, GSH-Px), and lipid peroxidation (MDA).
- Western blot analysis to assess NF-κB p65 nuclear translocation.
Main Results:
- Fasudil significantly reduced LPS-induced expression of IL-6 and MCP-1.
- Fasudil decreased ROS and MDA levels while increasing SOD and GSH-Px activities.
- Fasudil inhibited LPS-induced nuclear translocation of NF-κB p65.
- N-Acetylcysteine (N-Ace) demonstrated that ROS is crucial for NF-κB activation and inflammatory cytokine expression.
Conclusions:
- Fasudil effectively mitigates LPS-induced inflammatory and oxidative damage in rat PMVECs.
- The protective effects of fasudil are mediated through the ROS-NF-κB signaling pathway.
- Fasudil represents a promising therapeutic agent for inflammatory lung conditions involving PMVEC dysfunction.
