Baicalein protects renal tubular epithelial cells againsthypoxia-reoxygenation injury

Abstract

Background: To investigate the protective effects and mechanism of baicalein (BAI), a naturally occurring flavonoid, against hypoxia-reoxygenation (HR) injury in renal tubular epithelial cells (HK-2).

Methods: Cultured human renal proximal tubular cell line HK-2 was exposed to 24 h of hypoxia (5% CO₂, 1% O₂, and 94% N₂), followed by 12 h of reoxygenation (5% CO₂, 21% O₂, and 74% N₂). HK-2 cells were divided into three groups: control, HR, and HR-BAI (0.3 µg/ml). Reactive oxygen species (ROS) were measured and cell apoptosis was analyzed by flow cytometry and morphology. ELISAs were performed to determine the levels of IL-1, intercellular adhesion molecule-1 (ICAM-1), and monocyte chemotactic protein-1 (MCP-1). IL-1β, ICAM-1, and MCP-1 mRNA levels were determined by real-time quantitative PCR.

Results: HK-2 cells that underwent HR exhibited increases in IL-1β expression by 0.94%, ROS by 0.59%, ICAM-1 expression by 0.8%, and MCP-1 expression by 1.2%. Moreover, HK-2 cell apoptosis was increased after HR (p < .05). Compared with the HR group, BAI treatment reduced the elevation of oxidative stress (ROS) by 0.76%, as well as HR-mediated induction of IL-1β and apoptosis of HK2 cells. Protein and mRNA levels of ICAM-1 and MCP-1 were also reduced.

Conclusions: BAI protects renal tubular epithelial cells from HR injury by reducing inflammatory cytokine expression and oxidative stress.

Keywords: Baicalein; hypoxia-reoxygenation; inflammation; oxidative stress; renal tubular epithelial cell.

Figure 1.

(A) Effect of BAI at various concentration on the proliferation of HK-2 cells under normoxic conditions. Results are expressed as the mean ± SD (n = 9). *p < .05 vs. control. (B) Effect of BAI at various concentration on the viability of HR-treated HK-2 cells. Results are expressed as the mean ± SD (n = 9). *p < .05 vs. HR group without treatment; #p < .05 vs. other HR + BAI groups.

Figure 2.

BAI reduces HR-induced IL-1β levels in HK-2 cells. (A) Determination of IL-1β levels in culture supernatants by ELISA. (B) IL-1β mRNA levels in HK-2 cells. ***p < .001.

Figure 3.

BAI reduces ICAM-1 levels in HK-2 cells following HR. (A) Determination of ICAM-1 levels in culture supernatants by ELISA. (B) ICAM-1 mRNA levels in HK-2 cells. ***p < .001, *p < .05.

Figure 4.

BAI reduces MCP-1 levels in HR-exposed HK-2 cells. (A) Determination of MCP-1 levels in culture supernatants by ELISA. (B) MCP-1 mRNA level in HK-2 cells. ***p < .001. BAI reduces the ROS level in HK-2 cells after HR.

Figure 5.

BAI reduces elevated ROS levels in HK-2 cells following HR. ***p < .001.

Figure 6.

BAI treatment decreases cell apoptosis after HR. (A) Observation under an inverted microscope (×100). HK-2 cells in the HR group showed cytoplasmic blebbing, necrosis, and nuclear pyknosis, whereas BAI protected cells from these events. (B) Observation under a fluorescence inverted microscope (×400). The nuclei of apoptotic cells stained with Hoechst 33258 showed blue fluorescence. Control and BAI groups showed less blue fluorescence than the HR group. This was the typical morphological changes of apoptosis. (C) Flow cytometry of HK-2 cells following the various treatments. (D) Histogram of HK-2 cell apoptosis. ***p < .001.