Effect and mechanism of CXC chemokine receptor 3 on autophagy and inflammation of thyroid cells and its mechanisms
Objective To investigate the effect of CXC chemokine receptor 3(CXCR3)on interferon-γ(IFN-γ)-induced autophagy and inflammation of thyroid cells and the underlying mechanisms.Methods Altogether 500 U/mL of IFN-γ was administered to Nthy-ori3-1 cells for 24 hours toestablish the injury modelof thyroid follicular epithelial cells.The cells were divided as follows:control group(non-transfected Nthy-ori3-1 cells),IFN-γ group(IFN-γ treated Nthy-ori3-1 cells),si-NC group(negative control small interfering RNA trans-fected cells),si-CXCR3 group(CXCR3 siRNA transfected cells),si-CXCR3+si-NC group(CXCR3 siRNA and negative control siRNA co-transfected cells),si-CXCR3+si-Beclin1 group(CXCR3 siRNA and Beclin1 siRNA co-transfected cells)and si-CXCR3+3-MA group(CXCR3 siRNA transfected and 3-MA treated cells).The mRNA expression of CXCR3 and C-X-C motifligand 9(CXCL9)were detected by real-time quantitative PCR(RT-qPCR).The protein expression of CXCR3,CXCL9,microtubule-associated protein 1 light chain 3(LC3I),LC3II,vacuolar protein sorting 34(Vps34)and Beclin1 was detected using Western blot.The number of LC3 puncta in Nthy-ori3-1 cells was assessed using immunofluorescence staining inflammatory cytokines interleukin-6(IL-6),tumor necrosis factor α(TNF-α)and interleukin-1β(IL-1β)were detected using ELISA.The interaction between CXCR3 and beclin1 was identified by co-immunoprecipitation.Results CXCR3 and CXCL9 expression was significantly increased in IFN-γ group when compared with the control group(P<0.01).Compared with the si-NC group,the expression of beclin1,Vps34,LC3 II/LC3 I and LC3 puncta markedly increased in the si-CXCR3 group(P<0.05).Further-more,compared with the si-NC group,IL-6,TNF-α and IL-1β levels were significantly decreased in the si-CXCR3 group(P<0.01).The co-immunoprecipitation assay reveled that CXCR3 interacted with beclin1.Compared with the si-CXCR3+si-NC group,the expression of beclin1,Vps34,LC3 II/LC3 I and LC3 puncta was significantly decreased,while levels of IL-6,TNF-α and IL-1β were significantly increased in the si-CXCR3+si-beclin1 group and si-CXCR3+3-MA group(P<0.01).Conclusions CXCR3 inhibited the Beclin1/Vps34 signaling pathway,reducing autophagy and promoting inflammation of thyroid follicular epithelial cells.
Hashimoto's thyroiditisCXC chemokine receptor 3Beclin1Vacuolar protein sorting 34Autophagy
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