Effect Of Oxymatrine On TLR4/NF-κB Signal Pathway In Pancreatic Stellate Cells Induced By LPS

Objective: To investigate the effects of oxymatrine on the expression of TLR4 signal pathway in pancreatic stellate cells(LTC-14) induced by Lipopolysaccharide(LPS). The protein and mRNA levels of TLR4, MyD88, NF-κB and TNF-α were observed in pancreatic stellate cells(LTC-14) to explore the possible mechanism of OM in chronic pancreatitis.Methods:The LTC-14 cells were divided into four groups: control group, LPS group, OM + LPS group and OM Group. Control group cultured in serum-free medium without double antibiotics for 3h. LPS group cultured in serum-free medium containing a concentration of 10μg/ml LPS for 3h; OM + LPS group incubated with 500μg/ml OM for 30 min before administration 10μg/ml LPS; OM Group simply incubated with 500μg/ml of OM 3h. The following five techniques were used in this experiment:(1) The expression of TLR4 were detected by immunofluorescence staining.(2)The expression of intracellular and nucleus NF-κB were observed with immunohistochemical staining method.(3) The protein expression of TLR4、MyD88 and NF-κB were measured by Western blot.(4)The mRNA expression of TLR4、MyD88 and NF-κB was evaluated by Q-PCR.(5) ELISA was performed to detect the concentrations of TNF-α in cell supernatant.Results: Compared with control group, the expression of TLR4、NF-κB mRNA and protein were significantly increased when induced by LPS, which could be down regulated by OM. The translocation of NF-κB to the nucleus could be observed by immunohistochemical staining in LPS group. Compared with LPS group, the translocation of NF-κB was decreased significantly in OM+LPS group. The increaced protein expression of TNF-αinduced by LPS in cell supernatant were also decreased by OM. As for the protein expression and mRNA level of MyD88, there are no significant difference among four groups.Conclusion: The reduction of TLR4、NF-κB mRNA and protein as well as the inhibition of NF-κB translocation to the nucleus in LPS treated pancreatic stellate cells might be involved in the treatment effects of OM on pancreatic fibrosis.