| Objective1.To observe the anti-SIV or HIV effect of Bushen Jiedu Fang in vitro;2.To explore the effect of HIV-1 infection on ITAMs/ITIMs signaling pathway and its mechanism;3.To observe the effect of HIV-1 infection on the expression of IL-6,IL-18,TNFa and TRAF-4 in cells;4.To observe the effect of Bushen Jiedu Fang on the key receptor of ITAMs/ITIMs signaling pathway and significant change factors of inflammatory response before and after the intervention.Methods1.Systematic solvent separation was used to extract ethanol extracts from YYH,GZ and LW,respectively.The obtained L parts and Y parts of YYli,GZ S parts,and LW S parts all had anti-SIV/HIV effects,and then they were combined into Bushen Jiedu Fang.2.SIVmac251 and BK132 HIV-1 infected CEMx174 cells and BK132 HIV-1 infected M8166 cells were used as experimental models.Through pharmacodynamic experiments in vitro,the number of syncytids induced by virus and real-time fluorescence quanti tative RT-PCR were used to synt hesize CPE effect,expression level of IIIVRNA in cells and vi ral load in supernatant to evaluate the inhibitory effect of Bushen Jiedu Fang on SIV/HIV.Then the pharmacodynamics of Bushen Jiedu Fang and each effective part in the prescription were compared with the same evaluation indexes.3.The mRNA expression of the ITAMs/ITIMs signaling pathway-related genes SHP-1,SHP-2,SHIP1,SHIP2,Syk and Zap70 were detected by Rt-PCR in M8166 infected BK132 HIV-1 cells,and the protein expressions of the ITAMs/ITIMs signaling pathway-related receptors(same as above)were detected by Western blotting.4.The changes of intracellular inflammatory cytokines Il-6,11-18,TNFa and TRAF-4 mRNA were detected by RT-PCR before and after BK132 HIV-1 infection in M8166 cells.5.The M8166 cells model of BK132 HIV-1 infection was interfered by Bushen Jiedu Fang.The mRNA changes of ITAMs/ITIMs pathway key receptors and significantly altered inflammatory factors were detected by Rt-PCR,and the protein expression changes of ITAMs/ITIMs pathway key receptors were detected by Western-blot.Resu ts1.Under the microscope,the maximum non-toxic concentration of Bushen Jiedu Fang on 174 cells and M8166 cells were both 160 ?g/ml.2.The inhibitory effect of Bushen Jiedu Fang on SIV was obvious between 20 ? g/ml 160 ? g/ml,with a dose-dependent,and the effect was particularly well when the concentration was 80 ? g/ml?160 ? g/ml,almost the same as the positive drug.3.Based on the 174 cell model,Bushen Jiedu Fang had a dose-dependent inhibitory effect on HIV in the range of 20?g/ml?160 ?g/ml,and its inhibitory effect was good in the concentration of 40 ?g/ml?160 ? g/ml,almost the same as the positive drug.Based on M8166 cell model,Bushen Jiedu Fang had a dose-dependent inhibi tory effect on HIV in the range of 10?g/ml?160 ?g/ml,and its inhibitory effect was as good as that of the positive drug in the concentration of 40 ?g/ml?160 ?g/ml.4.In the comparison experiment of the inhibitory effect of Bushen Jiedu Fang on SIV/HIV based on 174 cells and every effective parts of the prescription,the compound prescription had better effect,while the anti-HIV efficacy of it on M8166 cells still showed a better effect although its performance was not such outstanding in the 174 cell model.5.After M8166 cells infected with HIV-1,SHP-1 mRNA expression increased significantly,and the difference was statistically significant(P<0.01).SHIP1 mRNA showed a decrease trend,but the difference was not statistically significant(P>0.05).There were no significant changes in SHP-2,SHIP2,Zap70,and Syk mRNA expression(P>0.05).6.After M8166 cells infected with HIV-1,the expression of SHP-1 and PSHP-1 in cells increased significantly,with significant difference(P<0.01).The expression of SHIPl and PSIIIP1 decreased significantly(P<0.05),the total protein expression of Syk increased,and the phosphorylation protein expression of PSyk decreased,but there was no significant difference(P>0.05).SHP-2,SHIP 2 and PSHIP2 protein were all expressed,but there was no significant change(P>0.05).PSHP2,Zap70 and PZap70 were not expressed.7.Before and after infection,the expression of HIV,IL-6,IL-18,TNFa and TRAF-4 in M8166 cells increased significantly(P<0.05).8.After the intervention of Bushen Jiedu Fang,HIV mRNA(P<0.01),SHP-1 mRNA(P<0.05),IL-6 mRNA(P<0.05),IL-18 mRNA(P<0.05),TNFa mRNA(P<0.01),TRAF-4 mRNA(P<0.05)in M8166 cells were significantly down-regulated,with significant differences,and its intervention effect was similar to that of positive drug control group.The protein expression of SHP-1 and PSHP-1 was also lower than that of the non-intervention group,and there was no significant difference compared with the positive drug control group.Conclusion1.The Bushen Jiedu Fang composed of L parts and Y parts of YYH,GZ S parts,and LW S parts had significant anti-SIV/HIV effect,and the inhibitory effect of it on SIV/HIV was better than that of each effective part of the compound alone.The synergistic effect of Bushen Jiedu Fang was well embodied.2.After HIV-1 infected M8166 cells,ITAMs/ITIMs signaling pathway was activated,possibly by promoting the expression of SHP-1 receptor and its phosphorylation level.Increased expression of related inflammatory factors suggested that inflammatory reaction had occured significantly after HIV infection,which was closely related to immune activation.3.The intervention of Bushen Jiedu Fang on immune disorder caused by HIV-1 infection may be related to down-regulation of SHP-1 expression and improvement of inflammation. |
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