Tofacitinib protects intestinal epithelial cells against oxygen-glucose deprivation/reoxygenation injury by inhibiting the JAK/STAT3 signaling pathway

Abstract

The present study aimed to investigate the role and potential mechanism of action of tofacitinib (Tofa) in intestinal ischemia/reperfusion (I/R) injury. The normal rat small intestine epithelial cell line, IEC-6, was used to establish an I/R injury model by inducing oxygen-glucose deprivation/reoxygenation (OGD/R). Cells were divided into the following five groups: Control, OGD/R, OGD/R with 50, 100 and 200 nM Tofa. Following Tofa administration, cell viability was measured using Cell Counting Kit-8 assay and a lactate dehydrogenase detection kit. The expression levels of cell apoptosis-related proteins, Bcl-2, cleaved-caspase-3 and cleaved-caspase-9 were detected using western blot analysis. Additionally, the levels of oxidative stress-related markers, such as reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD), and inflammatory cytokines, TNF-α, IL-6 and IL-1β were assessed using the colorimetric method. Western blot analysis was also used to measure the expression levels of the Janus kinase (JAK)/STAT3 signaling pathway-related proteins, including phosphorylated (p)-JAK1, p-JAK3 and p-STAT3. Subsequently, colivelin, an agonist of the JAK/STAT3 pathway, was used to investigate whether the effects of Tofa on intestinal I/R injury were mediated by this signaling pathway. The results showed that Tofa dose-dependently elevated cell viability compared with that in the OGD/R group. By contrast, Tofa attenuated cell apoptosis, which was coupled with upregulated Bcl-2 expression, downregulated cleaved-caspase-3 and downregulated cleaved-caspase-9 levels, in OGD/R-induced IEC-6 cells. Furthermore, the contents of ROS and MDA were significantly increased following exposure to OGD/R, which were accompanied by the decreased activity of SOD. These effects were reversed following cell treatment with Tofa. Consistently, Tofa intervention reduced the secretion levels of TNF-α, IL-6 and IL-1β in a dose-dependent manner. Additionally, Tofa markedly downregulated the phosphorylation levels of JAK1, JAK3 and STAT3 in OGD/R-induced IEC-6 cells. However, treatment with colivelin markedly reversed the inhibitory effects of Tofa on cell viability, cell apoptosis, oxidative stress and inflammation. Overall, the findings of the present study suggested that Tofa could protect against intestinal I/R injury by inhibiting the JAK/STAT3 signaling pathway, which may hold promise as a therapeutic agent for intestinal I/R injury.

Keywords: Janus kinase; apoptosis; inflammation; intestinal ischemia/reperfusion; oxidative stress.

Figure 1

Tofa enhances viability and attenuates apoptosis in OGD/R-induced IEC-6 cells. (A) CCK-8 assay was used to detect IEC-6 cell viability after treatment with different doses of Tofa for 12, 24, 48 and 72 h. ^**P<0.01 and ^***P<0.001 vs. Control. (B) Cell viability was assessed by CCK-8 assay after treatment with different doses of Tofa for 24 h in OGD/R-induced IEC-6 cells. (C) LDH activity in the culture supernatant was measured using an LDH assay kit. (D) Western blot analysis was applied to detect the expression levels of apoptosis-related proteins. ^***P<0.001 vs. Control. ^#P<0.05; ^##P<0.01 and ^###P<0.001 vs. OGD/R. Tofa, tofacitinib; OGD/R, oxygen-glucose deprivation/reoxygenation; LDH, lactate dehydrogenase; CCK-8, Cell Counting Kit-8.

Figure 2

Tofa treatment attenuates oxidative stress and inflammation in IEC-6 cells that were exposed to OGD/R. (A) Intracellular ROS production was measured using 2',7'-dichlorodihydrofluorescein diacetate as a fluorescence probe, (B) which was quantified. Magnification, x200. (C) MDA content and (D) SOD activity were assessed in the cell culture supernatant by corresponding MDA and SOD assay kits. Secretion levels of inflammatory factors, namely (E) TNF-α, (F) IL-6 and (G) IL-1β, were measured using ELISA. ^***P<0.001 vs. Control; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. OGD/R. Tofa, tofacitinib; OGD/R, oxygen-glucose deprivation/reoxygenation; ROS, reactive oxygen species; MDA, malondialdehyde; SOD, superoxide dismutase.

Figure 3

Tofa preconditioning inhibits JAK/STAT3 signaling in OGD/R-induced IEC-6 cells. The expression levels of JAK/STAT3 signaling pathway-related proteins were determined by western blot analysis. ^***P<0.001 vs. Control; ^##P<0.01 and ^###P<0.001 vs. OGD/R. Tofa, tofacitinib; JAK, Janus kinase; OGD/R, oxygen-glucose deprivation/reoxygenation; p-, phosphorylated.

Figure 4

Activation of JAK/STAT3 signaling reverses the inhibitory effects of Tofa on OGD/R-induced IEC-6 cell apoptosis. (A) Cell viability was assessed using Cell Counting Kit-8 assay in OGD/R-induced IEC-6 cell apoptosis in the presence or absence of Tofa (200 nM) and colivelin (0.5 µM). (B) LDH activity in the culture supernatant was determined using an LDH assay kit. (C) Western blot analysis was performed to determine the expression levels of apoptosis-related proteins. ^***P<0.001 vs. Control; ^###P<0.001 vs. OGD/R; ^ΔΔP<0.01 and ^ΔΔΔP<0.001 vs. OGD/R + Tofa + vehicle. JAK, Janus kinase; Tofa, tofacitinib; OGD/R, oxygen-glucose deprivation/reoxygenation; LDH, lactate dehydrogenase.

Figure 5

Activation of the JAK/STAT3 signaling reverses the inhibitory effects of Tofa on oxidative stress and inflammation in OGD/R-induced IEC-6 cells. (A) The levels of ROS were measured using 2',7'-dichlorodihydrofluorescein diacetate as a fluorescence probe, (B) which were quantified in OGD/R-induced IEC-6 cell apoptosis in the presence or absence of Tofa (200 nM) and colivelin (0.5 µM). Magnification, x200. The activity of (C) SOD and (D) the content of MDA in the cell culture supernatant were measured using corresponding MDA and SOD assay kits. ELISA was used to measure the concentration of (E) TNF-α, (F) IL-6 and (G) IL-1β. ^***P<0.001 vs. Control; ^###P<0.001 vs. OGD/R; ^ΔP<0.05, ^ΔΔP<0.01 and ^ΔΔΔP<0.001 vs. OGD/R + Tofa + vehicle. JAK, Janus kinase; Tofa, tofacitinib; OGD/R, oxygen-glucose deprivation/reoxygenation; ROS, reactive oxygen species; SOD, superoxide dismutase; MDA, malondialdehyde.

Figure 6

Colivelin treatment activates the JAK/STAT3 signaling pathway in OGD/R-induced IEC-6 cells. Western blotting was used to determine the expression levels of the JAK/STAT3 signaling pathway-related proteins in OGD/R-induced IEC-6 cell apoptosis in the presence or absence of Tofa (200 nM) and colivelin (0.5 µM). ^***P<0.001 vs. Control; ^###P<0.001 vs. OGD/R; ^ΔP<0.05 and ^ΔΔΔP<0.001 vs. OGD/R + Tofa + vehicle. JAK, Janus kinase; OGD/R, oxygen-glucose deprivation/reoxygenation; Tofa, tofacitinib; p-, phosphorylated.