The mechanism of stimulating the secretion of IL-17A from bile duct epithelial cells by Clonorchis sinensis to activate hepatic stellate cells to promote hepatic fibrosis
Objective To investigate the mechanism of HSC activation through IL-17A production by bile duct epithelial cells stimulated by Clonorchis sinensis infection.Methods IL-17A expression level in sera of patients infected with C.sinensis from endemic areas was detected by ELISA.New Zealand white rabbits and BALB/c mice were infected by gavage with C.sinensis metacercariae isolated from freshwater fish.Liver tissue and peripheral blood were collected aseptically.ELISA and western blot were applied to detect the IL-17A expression in mouse sera and liver respectively.Immunohistochemical staining was used to detect the expression of IL-17A in the bile duct epithelium.C.sinensis adults were obtained from rabbits'liver and cultured in PBS.The culture supernatant was saved as ESP.After RBE cells were stimulated with ESP,IL-17A level was analyzed by qRT-PCR,western blot,and ELISA respectively.Subsequently,LX-2 cells were cultured by adding the culture supernatant of RBE cells.The expression levels of α-SMA and Collagen-Ⅰ were assessed by qRT-PCR and western blot.Results The IL-17A levels were significantly elevated in patients serum infected with C.sinensis than in healthy individuals(t=6.536,P＜0.01).The expression of IL-17A in the peripheral blood and liver of infected mice was also significantly increased compared with normal mice(t=12.98,P＜0.01;t=4.155,P＜0.05).Immunohistochemical staining showed that more IL-17A signals were detected in the epithelium of intrahepatic bile ducts of infected mice(t=10.80,P＜0.01).The IL-17A levels in RBE cells were significantly increased after ESP stimulation(P＜0.01).The mRNA and protein levels of α-SMA and Collagen-I in LX-2 cells were significantly increased after cultured with ESP-stimulated RBE cells supernatant(P＜0.01).Conclusion C.sinensis may promote the activation of HSC and hepatic fibrosis by stimulating bile duct epithelial cells to produce IL-17A and consequent inflammation.
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