Study On The Active Components And Molecular Mechanism Of Bletilla Striata On Suppressing Pulmonary Fibrosis Based On RAW264.7 Macro-Phage Inflammation Model

Objective To separate,purify and identify the anti-inflammation active components from Bletilla striata guided by lipopolysaccharide(LPS)stimulated the mouse monocyte-macrophage leukemia cells(RAW264.7)inflammation cell model combined with all kinds of colum purification technique.Meanwhile,the inhibitory effect and molecular mechanism of the active components were discussed as well.Methods RAW264.7 cells were cultured in vitro.The macrophage inflammatory model was established by using LPS stimulated method,and the treatment concentration and time of LPS was optimized by IL-1β mRNA expression level detection with fluorescence quantitative PCR technique.The extract of Bletilla striata was prepared by ethanol reflux extraction method and coupled with polyamide chromatography method.The macrophage inflammatory model was used to evaluate the drug effect and guide the separation and purification the active components of Bletilla striata,and finally identify the monomer components.The suitable treatment concentration active monomer of Bletilla striata were determined by MTS method.The morphological changes of RAW264.7 were recorded by optical microscope.The levels of IL-6,TNF-α and MCP-1 in RAW264.7 cells were detected by flow cytometry and the mRNA expression level of IL-1β,IL-6,iNOS and COX2 were determined by RT-PCR method.The expression of MAPK pathway,NF-κB pathway,COX2,iNOS,p-IRF3/IRF3 protein in RAW264.7 cells was detected by Western Blot.Results ① The relative expression of IL-1β mRNA increased with the rise of LPS concentration and increased time treatment,and the relative expression of IL-1β mRNA was increased by nearly 1000 times in the concentration of 200 ng/mL LPS stimulated for 6h.②HPLC results indicated that there were only few overlapping peaks among the five crude fractions of the Bletilla striata,which showed good separation effect by polyamide column chromatography.The crude fractions could dramatically down regulate the relative expression of IL-1β mRNA in a dose-dependent manner compared with the model group(P<0.01),however except that the crude fraction of 80%ethanol(E80),and among which,the 40%ethanol eluted fraction(E40)showed the most significant effect.③The sub-fractions(E40-(1-6))of E40 were obtained by silica gel column method.RAW264.7 cells inflammatory model analyzed that except the sub-fraction of E40-6,all others sub-fractions showed dose dependent inhibitory activity on IL-1β mRNA expression level compared with the model group(P<0.01).And the sub-fractions of E40-3 and E40-4 with remarkable activity.④The two active sub-components of E40-3 and E40-4 were isolated and purified by semi-preparative liquid chromatography,and two active components were purified,and identified,namely the RT16.6 is Fritillaria(2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene),RT21.0 is batatasin III(3’,3"-dihydroxy-5’-methoxybibenzyl).⑤The effect of different concentrations of Bletilla striata monomer with and without LPS on the survival rate of RAW264.7 cells was detected by MTS method.When without LPS,the monomer RT16.6 and RT21.0 could promote proliferation in the low dose,but it had toxic effects when the concentration was higher than 15 μg/mL compared with the control group.While coupled with LPS treatment,the toxic concentration of RT16.6 and RT21.0 were elevated,which was 20 μg/mL and 25 μg/mL respectively.⑥The effects of different concentrations of Bletilla striata monomer on the cell morphology of RAW264.7 were observed by the light microscopy.The number of cells increased slightly while the morphology did not change significantly compared with normal cells when the dose of RT16.6 ranged from 1 μg/mL to 5 μg/mL and RT21.0 ranged from 2.5 μg/mL to 10 pg/mL.⑦Flow cytometry results verified that the expression of MCP-1,TNF-α and IL-6 increased significantly in RAW264.7 cells treated by LPS 200 ng/mL compared with the control group(P<0.01).However the Bletilla striata monomer RT16.6 could significantly reduced the contents of MCP-1 and IL-6 with dose dependent manner compared with the model group(P<0.01).While the TNF-a secretion was decreased in low doses,but promoted in high doses.Bletilla striata monomer RT21.0 could also reduce the secretion of MCP-1,TNF-α and IL-6 in a dose-dependent manner(P<0.01).⑧RT-PCR results indicated that the LPS treatment could dramatically increase IL-1β,IL-6,iNOS,COX2 and TNF-α mRNA expression(P<0.01),as well as TGF-β mRNA level(P<0.05)compared with the control group.The monomer RT16.6 decrease the mRNA expression of inflammation gene relatively with dose dependent.However,at low dose of 1 μg/mL,the relative expression of COX2,TNF-α and TGF-β mRNA was higher than that of the model group,which may be related to cell proliferation effect at the low dose.Except TNF-a level,the monomer RT21.0 could dose dependently decrease the mRNA expression of inflammation genes compared with the model group(P<0.01).⑨The protein expression of MAPK pathway,NF-κB pathway,COX2,iNOS,p-IRF3/IRF3 in LPS-induced RAW264.7 cells was detected by Western Blot.The results showed that the phosphorylation levels of IKK,IKBa,NF-κB,ERK1/2,JNK,P38 and IRF3 were significantly increased in the model group compared with the control group(P<0.01),and the levels of COX2 and iNOS protein increased significantly as well(P<0.01).Compared with the model group,the phosphorylation level of IKK,IKBα,NF-κB,JNK and IRF3 were significantly decreased in both RT16.6 and RT21.0 treatment groups(P<0.01).The expression of iNOS and COX2 protein was also inhibited in dose-dependent manner.In addition,RT16.6 significantly increased the phosphorylation of ERK and P38(P<0.01).While RT21.0 significantly reduced P38 phosphorylation(P<0.01)and increased ERK phosphorylation level.Conclusion ①The inflammation gene mRNA expression in RAW264.7 cells dramatically increased stimulated by 200 ng/mL LPS for 6 h,which could be used as a model to screen anti-inflammatory active drugs as well as uncover their molecular mechanism.②Two monomer components were purified and identified from the Bletilla striata extract with the guiding of the RAW264.7 inflammatory cell model.That were RT16.6 and RT21.0,which named as Fritillaria(2,7-dihydroxy-4-methoxy-9,10-dihydro-phenanthrene)and batatasin III(3’,3"-dihydroxy-5’-methoxybibenzyl)respectively.③ The molecular mechanism of Bletilla striata active monomers on treating pulmonary fibrosis disease maybe target on multiple signaling pathways,such as NF-κB,p-IRF3/IRF3,MAPK,iNOS and COX2,which could decrease IL-1β,IL-6,iNOS,COX2,TNF-a,TGF-β inflammatory gene expression and inhibit inflammatory factor secretion such as MCP-1,TNF-a and IL-6.