| Objective:To investigate the protective effect and mechanism of arctiin on T.g.HSP70-induced mice hepatocytes cell injury in T.gondii-infected.Methods:Cultured mouse normal hepatocytes cell NCTC 1469 in vitro,seeded in 6 cm petri dishes at a cell density of 2.5×105.Cells divided into seven groups:normal group(Normal),T.gondii-infected group(T.gondii),T.g.HSP70-induced group(T.g.HSP70),model group(T.gondii+T.g.HSP70)and Arctiin group(ARC 25 ?M,50 ?M,100 ?M).In addition to the normal group and T.gondii-infected group,the remaining groups were infected with RH strain tachyzoites at 5-fold ratio,4 h later,wash away the free T.gondii,change the 5%FBS DMEM and add drugs treatment for 36 h.In addition to Normal group and T.gondii group,the other groups were added to T.g.HSP70 induced 30 min.Observe the cell change in each group,the supernatant and precipitate were collected.The cytotoxicity of ARC and T.g.HSP70 to hepatocytes cell NCTC 1469 was detected by MTT assay;Colorimetric method was adopted to determine the ALT and AST levels in cell supernatant;HE staining method was observed pathological changes in hepatocyte cell with microscope;Western blotting method was used to detect the protein expression of TLR4,MyD88,p-MAPKs,NF-?B,p-cPLA2,PAF and TNF-?;Immunofluorescence assay was observed nuclear transcription of NF-?B gene under confocal laser scanning microscopy;RT-PCR method was used to detect the expression of T.g.HSP70 mRNA and SAG1 mRNA.Results:MTT cytotoxicity test results showed that concentration of ARC(25 ?M,50?M,100 ?M)and T.g.HSP70(1 ?g/mL,2 ?g/mL,3 ?g/mL)were non-toxic in hepatocytes cell NCTC 1469.Biochemical indexes showed that T.gondii-infected group levels of ALT/AST were higher than the normal group;T.g.HSP70 group did not statistically affect levels of ALT/AST;ALT and AST levels were significantly increased in model group and there was a significant difference compared with the T.gondii-infected group.The results show that T.g.HSP70 did not statistically affect levels of ALT/AST compare with normal NCTC 1469 cells.However,T.g.HSP70 could increase ALT/AST levels of T.gondii-infected NCTC 1469 cells.The levels of ALT and AST were dramatically suppressed by ARC treatment in a concentration-dependent manner(P<0.001,P<0.001);The pathology results of liver HE staining of hepatocytes cell NCTC 1469 show that compared with the normal group,there was a large number of T.gondii in the nucleus,so that the cell structure was destroyed and the nucleolus was reduced in the model group.However,treatment with 100?M ARC the number of T.gondii was significantly reduced in the nucleus,the arrangement of liver cells was relatively neat and almost close to the normal group;Western Blotting results show that compared with normal group,the expression of TLR4 in the model group was significantly increased after 30 min of T.g.HSP70-induced(P<0.001),activated the related protein expression of p-MAPKs(p-p38 and p-ERK)and NF-?B nuclear translocation,eventually activated the activation of cPLA2,PAF and the production of inflammatory factors TNF-?(P<0.001),while promoting I?B-? degradation(P<0.001).However ARC group significantly inhibited the protein expression of TLR4,p-MAPKs(p-p38 and p-ERK),p-cPLA2,PAF,TNF-?,NF-?B nuclear translocation and the cytosolic level of I?B-? degradation compared with the model group(P<0.05).Immunofluorescence results show that compared with model group,ARC(100 ?M)exerted inhibition nuclear translocation of NF-?B induced by T.g.HSP70 in NCTC cells.RT-PCR results showed that the expression of T.g.HSP70 mRNA and SAG1 mRNA in the model group was significantly higher than that in the normal group(P<0.001,P<0.001),and ARC successively suppressed T.g.HSP70 mRNA and SAG1 mRNA expression to protect the liver from injury(P<0.001,P<0.001).Conclusion:ARC has a good protective effect on T.g.HSP70-induced T.gondii-infected the liver cells injury.The potential mechanisms were inhibition of T.g.HSP70-induced the production of inflammatory factors via TLR4/MAPK/NF-?B signaling pathway. |
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