Objective: To investigate the effects and underlying mechanisms of the macrophage migration inhibitory factor(MIF) inhibitor(S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester(ISO-1) on sepsis-induced acute kidney injury(AKI). Methods: Human renal tubular epithelial HK-2 cells were divided into Con group(without any treatment), ISO-1 group(10 μg/ml ISO-1 treatment for 24 h) and LPS group(10 μg/ml LPS treatment for 24 h), LPS+ISO-1 group(10 μg/ml LPS treatment for 24 h followed by 10 μg/ml ISO-1 treatment for 24 h). ELISA was used to measure the levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6) in the cell supernatants. Reactive oxygen species(ROS) levels were assessed using the 6-carboxyl-2 ′,7′-dichlorodihydrofluorescein diacetate fluorescent indicator(DCFH-DA) method. Apoptosis levels were detected by TUNEL staining, and Western blot was employed to analyze the expression of proteins of Kelch like ECH associated protein 1(Keap1), NFE2 like bZIP transcription factor 2(Nrf2), heme oxygenase-1(HO-1), as well as apoptosis-related proteins Bcl-2, Bax, and cleaved Caspase-3(c-Caspase-3). A sepsis mouse model was established using the cecal ligation and puncture(CLP) method, and the mice were divided into four groups: sham-operated(Sham), ISO-1 control(ISO-1), CLP, and ISO-1 treatment(CLP+ISO-1). After the experiment, mouse kidney tissues were collected for HE staining to observe pathological changes. Blood urea nitrogen(BUN), serum creatinine(Scr), myeloperoxidase(MPO) levels in kidney tissues, glutathione(GSH) and superoxide dismutase(SOD) activities were measured. Western blot was also used to detect the expression of MIF and proteins in the Nrf2/Keap1 signaling pathway and apoptosis-related proteins in kidney tissues. Results: Compared to the Con group, the LPS and LPS+ISO-1 groups showed significantly increased levels of TNF-α, IL-1β, IL-6, TUNEL-positive rates, ROS levels, and protein expressions of Keap1, Bax, and c-Caspase-3 in HK-2 cells(P<0.05), while the expressions of Nrf2, HO-1, and Bcl-2 significantly decreased(P<0.05). The ISO-1 group showed no significant changes(P>0.05). Compared to the LPS group, the LPS+ISO-1 group exhibited significantly decreased levels of TNF-α, IL-1β, IL-6, TUNEL-positive rates, ROS levels, and protein expressions of Keap1, Bax, and c-Caspase-3, while the expressions of Nrf2, HO-1, and Bcl-2 significantly increased(P<0.05). In the mouse experiments, compared to the Sham group, the CLP and CLP+ISO-1 groups showed severe kidney tissue damage, increased levels of serum BUN, Scr, and kidney MIF, Keap1, Bax, and c-Caspase-3 protein expressions(P<0.05), while GSH, SOD activities, and protein expressions of Nrf2, HO-1, and Bcl-2 significantly decreased(P<0.05). The ISO-1 group showed no significant changes(P>0.05). Compared to the CLP group, the CLP+ISO-1 group showed significant improvements in the aforementioned indicators(P<0.05). Conclusion The specific MIF inhibitor ISO-1 can ameliorate sepsis-induced AKI by inhibiting oxidative stress, inflammatory response, and apoptosis bothin vitroandin vivo. The mechanism may be through Nrf2/Keap1 signaling pathway.

Ischemic stroke is a major public health problem worldwide. Although the circadian clock is involved in the process of ischemic stroke, the exact mechanism of the circadian clock in regulating angiogenesis after cerebral infarction remains unclear. In the present study, we determined that environmental circadian disruption (ECD) increased the stroke severity and impaired angiogenesis in the rat middle cerebral artery occlusion model, by measuring the infarct volume, neurological tests, and angiogenesis-related protein. We further report that Bmal1 plays an irreplaceable role in angiogenesis. Overexpression of Bmal1 promoted tube-forming, migration, and wound healing, and upregulated the vascular endothelial growth factor (VEGF) and Notch pathway protein levels. This promoting effect was reversed by the Notch pathway inhibitor DAPT, according to the results of angiogenesis capacity and VEGF pathway protein level. In conclusion, our study reveals the intervention of ECD in angiogenesis in ischemic stroke and further identifies the exact mechanism by which Bmal1 regulates angiogenesis through the VEGF-Notch1 pathway.
Rats ; Animals ; Vascular Endothelial Growth Factor A/pharmacology* ; Brain Ischemia/metabolism* ; Ischemic Stroke ; Signal Transduction ; ARNTL Transcription Factors/pharmacology* ; Neovascularization, Physiologic/physiology*