WITHDRAWN: Tanshinone-IIA inhibits the inflammatory response and proliferation of PAECs under hypoxic conditions by repressing HMGB1 via the TLR4/NF-κB signalling pathway

Abstract

To explore the potential molecular mechanism of Tanshinone-IIA (Tan-IIA) in pulmonary artery endothelial cells (PAECs) under hypoxic conditions. Rat PAECs were observed under an inverted microscope, and the biomarkers CD31 and factor VIII related antigen (FVIII-Rag) were identified by immunofluorescence (IF). The PAECs were divided into a 20% normoxia group and a 4% hypoxia group, and the cells were subjected to Tan-IIA treatment, high mobility group box 1 (HMGB1) overexpression, or the relevant controls. ELISA and MTT were performed to evaluate proinflammatory factor production and cell proliferation, respectively. RT-qPCR was used to measure HMGB1 mRNA expression, and western blotting was performed to evaluate the protein levels of HMGB1, Toll-like receptor 4 (TLR4) and nuclear factor kappa-B (NF-κB)-p65. Immunocytochemistry (ICC) and western blotting indicated the subcellular distribution of NF-κB-p65. Rat PAECs were round or short spindle-shaped and abundantly expressed CD31 and FVIII-Rag. Compared with the normoxia group, the hypoxia group produced significantly increased levels of proinflammatory factors, which decreased with increasing concentrations of Tan-IIA (p<0.05). The expression of HMGB1 and TLR4 and the phosphorylation of NF-κB-p65 presented gradually decreasing trends in the 4% hypoxia group with increasing concentrations of Tan-IIA. Moreover, ICC indicated that in the 4% hypoxia group, NF-κB-p65 translocated to the nucleus (p<0.05), and western blotting analysis showed that the higher the Tan-IIA concentration was, the lower the NF-κBp65 level in the nucleus was. Compared with the Hypoxia + Vector group, the Hypoxia+ pHMGB1 group exhibited significantly enhanced effects of the hypoxia-induced inflammatory response (p<0.05), and the inhibitory effects of Tan-IIA on the hypoxia-induced inflammatory response were reversed in the Hypoxia+Tan-IIA+pHMGB1 group due to the overexpression of HMGB1 (p<0.05); HMGB1 exerted similar effects on cell proliferation, as illustrated by MTT assay. Tan-IIA inhibits the proliferation and inflammatory response of PAECs by downregulating the expression of HMGB1 via the TLR4/NF-κB signalling pathway under hypoxic conditions.