Ulcerative colitis (UC) is a chronic inflammatory disorder with a complex etiology, characterized by intestinal inflammation and barrier dysfunction. Platycodon grandiflorus polysaccharides (PGP), the primary component of Platycodon grandiflorus, and hesperidin (Hesp), a prominent active component in Citrus aurantium L. (CAL), have both demonstrated anti-inflammatory properties. This study aims to elucidate the underlying mechanism of the synergistic effect of PGP combined with Hesp on UC, focusing on the coordinated interaction between the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathways. A mouse model of UC induced by dextran sulfate sodium (DSS) and a cell model using lipopolysaccharide (LPS)-induced RAW264.7/IEC6 cells were employed to investigate the in vitro and in vivo anti-inflammatory effects of PGP combined with Hesp on UC and its potential mechanism of action. The results indicated that compared to the effects of either drug alone, the combination of PGP and Hesp significantly modulated inflammatory factor levels, inhibited oxidative stress, regulated colonic mucosal immunity, suppressed apoptosis, and restored intestinal barrier function in vitro and in vivo. Further in vitro studies revealed that PGP significantly inhibited the PI3K/AKT signaling pathway, while Hesp significantly inhibited the JAK2/STAT3 signaling pathway. The use of inhibitors and activators targeting both pathways validated the synergistic effects of PGP combined with Hesp on the PI3K/AKT and JAK2/STAT3 signaling pathways. These findings suggest that PGP combined with Hesp exhibits a synergistic effect on DSS-induced colitis, potentially mediated through the phosphatase and tensin homolog (PTEN)/PI3K/AKT and interleukin-6 (IL-6)/JAK2/STAT3 signaling pathways.
Animals ; STAT3 Transcription Factor/genetics* ; Janus Kinase 2/genetics* ; Polysaccharides/administration & dosage* ; Colitis, Ulcerative/chemically induced* ; Mice ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/genetics* ; Drug Synergism ; Male ; Hesperidin/administration & dosage* ; Platycodon/chemistry* ; Phosphatidylinositol 3-Kinases/genetics* ; Disease Models, Animal ; RAW 264.7 Cells ; Mice, Inbred C57BL

Objective:To investigate the effect and underlying mechanism of isoliquiritigenin (ISL) on chondrocyte ferroptosis in a rat model of knee osteoarthritis (KOA).Methods:Sixty male SD rats were randomly divided into a sham group, KOA group, celecoxib group, ISL low-dose group, and ISL high-dose group. Except for the sham group, KOA models were induced in the other groups using the modified Hulth method. The ISL low-dose and high-dose groups received intraperitoneal injections of 10 mg/kg and 40 mg/kg ISL, respectively; the celecoxib group was orally administered 24 mg/kg celecoxib; the sham and KOA groups received equivalent doses of saline via intraperitoneal injection. Interventions were administered once daily for 8 weeks. Behavioral changes in the open field test, histopathological observations of cartilage, inflammatory cytokine detection, ferroptosis-related indicators, and Sirt1/Nrf2/GPX4 pathway protein expression were measured to determine the optimal ISL dose. Another 60 male SD rats were randomly divided into sham, KOA, ISL high-dose, Sirt1 inhibitor (EX-527), and ISL high-dose + EX-527 groups. KOA models were established in all groups except the sham group. The ISL high-dose group received 40 mg/kg ISL, the EX-527 group received 10 mg/kg EX-527, and the combination group received both. The sham and KOA groups were given saline. Interventions lasted 8 weeks. Histopathological staining evaluated cartilage damage and scoring; ELISA measured tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 levels in synovial fluid; iron deposition, Fe 2+, malonaldehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione (GSH) levels were assessed; Western blot analyzed Sirt1/Nrf2/GPX4 pathway proteins; immunohistochemistry detected Collagen II, Aggrecan, MMP-3, and MMP-13 expression. Results:The joint cartilage tissue damage in the ISL low-dose and high-dose rat groups was alleviated compared to the KOA group. The OARSI score, levels of TNF-α, IL-1β and IL-6 in joint fluid, iron deposition in cartilage tissue, Fe 2+, MDA and ROS levels were 8.33±1.86 and 4.50±1.52, respectively. 67.24±7.25 pg/ml, 42.06±5.12 pg/ml; 37.97±4.9 pg/ml, 23.75±4.12 pg/ml; 31.67±4.16 pg/ml, 20.91±3.28 pg/ml; 2.00±0.20, 1.53±0.14; 2.84±0.19 μmol/mg, 1.87±0.16 μmol/mg; 9.11±1.08 nmol/ml, 5.49±1.05 nmol/ml; 759.15±59.80 μmol/ml and 610.85±44.23 μmol/ml were lower than those in KOA group ( P<0.05), and the serum SOD and GSH contents were 12.12±1.52 U/ml and 16.79±2.14 U/ml, respectively. Compared with KOA group, the protein expressions of Sirt1, Nrf2, GPX4 and SLC7A11 were 0.70±0.11 and 0.96±0.13, 0.69±0.10 and 0.95±0.14, 0.51±0.06 and 0.87±0.12, 0.56±0.06 and 0.83±0.10, which were higher than those in KOA group ( P<0.05). The expressions of Acetyl-H4K16, ACSL4, MMP-3 and MMP-13 were 1.68±0.17 and 1.30±0.10, 1.39±0.12 and 0.97±0.10, 1.70±0.14 and 1.10±0.10, 1.64±0.15 and 1.28±0.10, which were lower than those of KOA group ( P<0.05). And the changes of these indexes were higher in Sirt1 inhibitor group. Compared with the ISL high-dose group, the ferroptosis-related indexes were significantly increased in the ISL high-dose+Sirt1 inhibitor group. Conclusion:ISL alleviates articular cartilage injury in KOA rats, and its mechanism is related to activating the Sirt1/Nrf2/GPX4 pathway and inhibiting ferroptosis.

Objective:To investigate the effect and underlying mechanism of isoliquiritigenin (ISL) on chondrocyte ferroptosis in a rat model of knee osteoarthritis (KOA).Methods:Sixty male SD rats were randomly divided into a sham group, KOA group, celecoxib group, ISL low-dose group, and ISL high-dose group. Except for the sham group, KOA models were induced in the other groups using the modified Hulth method. The ISL low-dose and high-dose groups received intraperitoneal injections of 10 mg/kg and 40 mg/kg ISL, respectively; the celecoxib group was orally administered 24 mg/kg celecoxib; the sham and KOA groups received equivalent doses of saline via intraperitoneal injection. Interventions were administered once daily for 8 weeks. Behavioral changes in the open field test, histopathological observations of cartilage, inflammatory cytokine detection, ferroptosis-related indicators, and Sirt1/Nrf2/GPX4 pathway protein expression were measured to determine the optimal ISL dose. Another 60 male SD rats were randomly divided into sham, KOA, ISL high-dose, Sirt1 inhibitor (EX-527), and ISL high-dose + EX-527 groups. KOA models were established in all groups except the sham group. The ISL high-dose group received 40 mg/kg ISL, the EX-527 group received 10 mg/kg EX-527, and the combination group received both. The sham and KOA groups were given saline. Interventions lasted 8 weeks. Histopathological staining evaluated cartilage damage and scoring; ELISA measured tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 levels in synovial fluid; iron deposition, Fe 2+, malonaldehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione (GSH) levels were assessed; Western blot analyzed Sirt1/Nrf2/GPX4 pathway proteins; immunohistochemistry detected Collagen II, Aggrecan, MMP-3, and MMP-13 expression. Results:The joint cartilage tissue damage in the ISL low-dose and high-dose rat groups was alleviated compared to the KOA group. The OARSI score, levels of TNF-α, IL-1β and IL-6 in joint fluid, iron deposition in cartilage tissue, Fe 2+, MDA and ROS levels were 8.33±1.86 and 4.50±1.52, respectively. 67.24±7.25 pg/ml, 42.06±5.12 pg/ml; 37.97±4.9 pg/ml, 23.75±4.12 pg/ml; 31.67±4.16 pg/ml, 20.91±3.28 pg/ml; 2.00±0.20, 1.53±0.14; 2.84±0.19 μmol/mg, 1.87±0.16 μmol/mg; 9.11±1.08 nmol/ml, 5.49±1.05 nmol/ml; 759.15±59.80 μmol/ml and 610.85±44.23 μmol/ml were lower than those in KOA group ( P<0.05), and the serum SOD and GSH contents were 12.12±1.52 U/ml and 16.79±2.14 U/ml, respectively. Compared with KOA group, the protein expressions of Sirt1, Nrf2, GPX4 and SLC7A11 were 0.70±0.11 and 0.96±0.13, 0.69±0.10 and 0.95±0.14, 0.51±0.06 and 0.87±0.12, 0.56±0.06 and 0.83±0.10, which were higher than those in KOA group ( P<0.05). The expressions of Acetyl-H4K16, ACSL4, MMP-3 and MMP-13 were 1.68±0.17 and 1.30±0.10, 1.39±0.12 and 0.97±0.10, 1.70±0.14 and 1.10±0.10, 1.64±0.15 and 1.28±0.10, which were lower than those of KOA group ( P<0.05). And the changes of these indexes were higher in Sirt1 inhibitor group. Compared with the ISL high-dose group, the ferroptosis-related indexes were significantly increased in the ISL high-dose+Sirt1 inhibitor group. Conclusion:ISL alleviates articular cartilage injury in KOA rats, and its mechanism is related to activating the Sirt1/Nrf2/GPX4 pathway and inhibiting ferroptosis.