Experimental Study On The Effect Of Anti-endotoxin Of Forsythoside A And Its Mechanism

Objective:To observe the effects of forsythoside A (FA) on the cell viability，the expressionof TLR4，MyD88，NF-κB and TNF-α in macrophage RAW264.7induced by LPSafter12hours, and to observe the effects of FA on the expression of TLR4, MyD88and NF-κB on lung tissue and levels of ET and TNF-α in peripheral blood of lunginjury mice induced by LPS, in order to determine the anti-endotoxin effect of FA andits possible mechanisms.Methods:1Effects of FA on TLR4signal-transducing pathway in macrophageinduced by LPS1.1Experimental groups: Cultured RAW264.7cells have been randomlydivided into six groups: control group: without any treatment; LPS group: addingLPS at final concentration of100ng/ml for12h; FA1、FA2、FA3group:2h of FA(320ug/ml、80ug/ml、20ug/ml in final concentration) preconditioning before addingLPS for12h; PMB group:2h of Ploy B (10ug/ml in final concentration)preconditioning before adding LPS for12h.1.2Indicators of detection:1.Cell viability assay by MTT analysis；2.Theexpression of TLR4and MyD88mRNA by RT-PCR；3.The expression of TLR4,MyD88and NF-κB protein were measured by western blot；4The TNF-aconcentration was detected by ELISA.2.Protective effect of forsythoside A on acute lung injure induced by LPS inmice2.1The established endotoxemia mouse model of acute lung injury was inducedby LPS at dosage of10mg/kg;2.2The experiment were carried out in six groups: A) control group: without anytreatment (n=8); B) acute lung injury model group: mice were given LPS at dosage of10mg/kg (n=8); C) antibody group: mice were given Anti-TLR4/MD2antibody (50ug/20g)12hours before modeling (n=8); D) high FA group: mice were given FAat dosage of80mg/kg (n=8); E) medium FA group: mice were given FA at dosage of20mg/kg (n=8); F) low FA group: mice were given FA at dosage of5mg/kg (n=8).Mice in all FA treatment groups were given FA at respective dosage once a day till7days before modeling. All treatment agents were given through ip. Blood and lungtissue specimens were taken4hours after modeling.2.3Amount of endotoxin in plasma was measured by kinetic turbidimetric assay.Degree of lungs damage was graded by the histopathology score via HE stain.Expression of TLR4were measured at both mRNA and protein level by RT-PCR andWestern blot. Expression of MyD88and NF-κB were detected byimmunohistochemical. Amount of TNF-α in serum were detected by ELISA.Results:1Effects of FA on TLR4signal-transducing pathway in macrophageinduced by LPS1.1MTT assay results: Compared with control group, the cell viability of LPSgroup significantly decreased (P<0.01); FA1, FA2, FA3group significantly enhancedthe cell viability(P<0.01), which was dose-dependent; there was no significantdifference between FA1group and PMB group.1.2TLR4mRNA and protein expression: It was found that TLR4mRNA andprotein had a basic expression in RAW264.7cells under normal condition; and it wasincreased significantly after LPS acted on RAW264.7cells than that of control groupat12h; adding FA before the presence of LPS, the expressions of TLR4in FA1, FA2,FA3group decreased remarkably than that of LPS group, which was dose-dependent.1.3MyD88and NF-κB protein expression:Compared with the control group,the expressions of MyD88and NF-κB protein in LPS group raised significantly (p<0.01), FA1, FA2and FA3group could reduce the above changes.1.4TNF-α level: Administration of LPS stimulation, TNF-α content in theculture supernatant was significantly increased(P<0.01), FA pretreatment can reduceTNF-α secretion, which was dose-dependent.2Protective effects of forsythoside A on acute lung injure induced by LPS inmice 2.1General changes: After modeling, mice apathetic, lazy move, anorexia,incontinence. Compared with model group, FA group had obvious improvement aftermedication treatment.2.2Results of the detection indicators: When compared to control group,endotoxin and TNF-α in model group were significantly increased (P<0.01), withobvious pathological damage in lung tissue. When compared to model group,expression of TLR4, MyD88-and NF-κB in the lung in groups treated with FA wereup-regulated markedly (P<0.01). In addition, detectable level of endotoxin andTNF-α in plasma were decreased significantly (P<0.01). Damages in lung tissue werealso attenuated. The protective effect of FA seemed to be dosage-dependent.Conclusion:FA preconditioning can enhance the cell viability of RAW264.7macrophagesinduced by LPS, and FA has protective effect on acute lung injury induced by LPS inmice. It was verified that FA had the effect of anti-endotoxin from the level of cellsand entire animal, its underlying mechanism may due to interference in LPS-TLR4-MyD88-NF-κB signaling pathway.