To explore the effect and mechanism of Gegen Qinlian Decoction(GQD)in inhibiting M1 polarization of macrophages under inflammatory hypoxia by simulating intestinal hypoxia microenvironment in vitro.A tri-gas incubator was used to simulate normal physiological hypoxia of the colon and inflammatory hypoxia microenvironments of ulcerative colitis(UC).RAW264.7 macrophages were divided into 18.5％O2(normoxia group),4％O2(physiological hypoxia group),and 1％O2(inflammatory hypoxia group),and they were induced by lipopolysaccharide(LPS)for 24 h.M1 polarization was detected by flow cytometry.Under the condition of 1％inflammatory hypoxia,they were divided into blank group,model group,and GQD-containing serum low,medium,and high dose groups.Flow cytometry was used to detect M1 polarization marker CD86,and ELISA was used to detect the expression of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in cell supernatant.The mRNA expression of hypoxia-inducible factor-1α(HIF-1α),TNF-α,and IL-1β was detected by qRT-PCR.Western blot was used to detect the expression of HIF-1α/nuclear transcription factor-KB(NF-κB)signaling pathway-related proteins.The nuclear translocation of NF-κB p65 was detected by immunofluorescence.The results showed that the positive rate of CD86 in the 1％O2 group was the highest.Under the condition of 1％inflammatory hypoxia,compared with the blank group,the expression of CD86,TNF-α,IL-1β,and HIF-1α in the model group increased.Compared with the model group,each group of GQD could reduce the expression of CD86,TNF-α,IL-1β,and HIF-1α.Compared with the blank group,the protein expression of HIF-1α,NF-κB p65,p-IKKα/β,and p-IκBα in the model group increased.Compared with the model group,the protein expression of HIF-1α,NF-κB p65,p-IKKα/β,and p-IKBα in GQD groups was significantly decreased.Compared with the blank group,NF-κB p65 in the model group entered the nucleus significantly.Compared with the model group,the nuclear expression of NF-κB p65 was decreased in each GQD group.Studies have shown that GQD may protect the intestine by down-regulating the HIF-1α/NF-κB signaling pathway to inhibit M1 polarization of macrophages and secretion of related inflammatory factors under 1％inflammatory hypoxia.
Gegen Qinlian Decoctionulcerative colitisinflammatory hypoxia microenvironmentpolarization of macrophage
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