Paeoniflorin protects PC12 cells from oxygen-glucose deprivation/reoxygenation-induced injury via activating JAK2/STAT3 signaling

Abstract

Ischemic stroke is the most common type of stroke, and it has become a major health issue as it is characterized by high mortality and morbidity rates. Paeoniflorin (PF) is a natural compound and the main active ingredient of Radix Paeoniae. The aim of the present study was to investigate the role of PF in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury of PC12 cells and its association with the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway. An in vitro model of OGD/R injury was established in PC12 cells. Subsequently, Cell Counting Kit-8 assay and ELISA were used to evaluate cell viability and the secretion of inflammatory factors, respectively, in PC12 cells subjected to OGD/R and treated with PF. The levels of oxidative stress indicators and inflammatory factors were measured using corresponding commercial kits. In addition, the apoptosis rate of PC12 cells subjected to OGD/R and treated with PF was determined by flow cytometry, and the expression of apoptosis-related proteins was analyzed by western blotting. Additionally, the expression levels of JAK2/STAT3 pathway-related proteins were also evaluated. The cell viability, levels of oxidative stress, inflammation and apoptosis were also measured in OGD/R-induced PC12 cell injury models following co-treatment of cells with PF and FLLL32, a specific inhibitor of JAK2/STAT3 signaling. Cell viability was reduced, while oxidative stress and inflammation were increased after OGD/R-induced injury. However, the treatment of cells with PF significantly enhanced cell viability, and alleviated oxidative stress, inflammation and apoptosis of OGD/R-treated PC12 cells. Furthermore, PF activated the JAK2/STAT3 signaling pathway. Following FLLL32 intervention, the effects of PF on oxidative stress, inflammation and apoptosis of OGD/R-treated PC12 cells were reversed. In conclusion, the findings of the present study suggested that PF may protect PC12 cells from OGD/R-induced injury via activating the JAK2/STAT3 signaling pathway, thus providing novel insight into the mechanism through which PF may alleviate ischemic stroke and indicating a potential strategy for ischemic stroke treatment.

Keywords: apoptosis; inflammation; ischemic stroke; oxygen-glucose deprivation/reoxygenation; paeoniflorin.

Figure 1

PF enhances the viability of OGD/R-treated PC12 cells. (A) Chemical structure of PF. (B) CCK-8 assay was used to determine the viability of PC12 cells treated with increasing doses of PF. (C) Following establishment of OGD/R-induced PC12 cell injury model, the apoptosis rate of cells treated with increasing doses of PF was determined using CCK-8 assay. ^***P<0.001 vs. the control group; ^##P<0.01, ^###P<0.001 vs. the OGD/R group. PF, paeoniflorin; OGD/R, oxygen-glucose deprivation/reoxygenation; CCK-8, Cell Counting Kit-8.

Figure 2

PF alleviates oxidative stress and inflammation in OGD/R-treated PC12 cells. Activity of (A) LDH, (B) MPO and (C) SOD in PC12 cells subjected to OGD/R and treated with PF was measured using commercial kits. ELISA was used to determine the secretion levels of the inflammatory factors (D) TNF-α (E) IL-6 and (F) IL-10. ^***P<0.001 vs. the control group; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. the OGD/R group. PF, paeoniflorin; OGD/R, oxygen-glucose deprivation/reoxygenation; LDH, lactate dehydrogenase; MPO myeloperoxidase; SOD, superoxide dismutase.

Figure 3

PF attenuates the apoptosis of OGD/R-treated PC12 cells. (A and B) Flow cytometry was used to evaluate the apoptosis of PC12 cells subjected to OGD/R and treated with PF. (C) Western blot analysis was used to determine the expression levels of apoptosis-related proteins in OGD/R-treated PC12 cells. ^***P<0.001 vs. the control group; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. the OGD/R group. PF, paeoniflorin; OGD/R, oxygen-glucose deprivation/reoxygenation.

Figure 4

PF activates the JAK2/STAT3 signaling pathway in OGD/R-treated PC12 cells. The expression levels of the JAK2/STAT3 signaling-related proteins were detected by western blotting. ^***P<0.001 vs. the control group; ^#P<0.05, ^###P<0.001 vs. the OGD/R group. PF, paeoniflorin; OGD/R, oxygen-glucose deprivation/reoxygenation; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3.

Figure 5

FLLL32, a specific inhibitor of Janus kinase 2/signal transducer and activator of transcription 3 signaling, abrogates the antioxidant and anti-inflammatory effects of PF on OGD/R-treated PC12 cells. (A) Cell viability was assessed using Cell Counting Kit-8 assay. The expression levels of the oxidative stress indicators (B) LDH, (C) MPO and (D) SOD were measured using commercial kits. The secretion levels of the inflammatory factors (E) TNF-α (F) IL-6 and (G) IL-10 in PC12 cells subjected to OGD/R and treated with PF and FLLL32 were determined using ELISA. ^**P<0.01 ^***P<0.001 vs. the control group; ^#P<0.05, ^###P<0.001 vs. the OGD/R group; ^$P<0.05, ^$$P<0.01 vs. the OGD/R + PF group. PF, paeoniflorin; OGD/R, oxygen-glucose deprivation/reoxygenation; LDH, lactate dehydrogenase; MPO myeloperoxidase; SOD, superoxide dismutase.

Figure 6

FLLL32, a specific inhibitor of Janus kinase 2/signal transducer and activator of transcription 3 signaling, attenuates the anti-apoptotic effects of PF on OGD/R-induced PC12 cells. (A and B) Flow cytometry was used to evaluate the apoptosis of PC12 cells subjected to OGD/R and co-treated with PF and FLLL32. (C) Western blot analysis was used to determine the expression levels of apoptosis-related proteins in PC12 cells subjected to OGD/R and treated with PF and FLLL32. ^***P<0.001 vs. the control group; ^###P<0.001 vs. the OGD/R group; ^$$P<0.01, ^$P<0.05, ^$$$P<0.001 vs. the OGD/R + PF group. PF, paeoniflorin; OGD/R, oxygen-glucose deprivation/reoxygenation; PARP, poly(adenosine diphosphate-ribose) polymerase.