Investigation Of Aikeqing On Inflammatione After HAART Based On ADAM17

Objective:To investigate the effect on inflammatory Aikeqing HIV/AIDS patients after HAART from ADAM17 point of view,looking for a new perspective of Chinese medicine intervention of AIDS and the new strategy from the perspective of inflammatory regulation.Method:Experiment 1.Clinical trials.123 patients of HIV/AIDS in Dongguan People's Hospital were included in the experimental study.123 patients were divided into ZDV group and TDV group.ZDV group has 55 persons,using the ZDV+3TC+EFV/NVP;TDF group has 68 persons,using TDF+3TC+EFV/NVP.123 patients were drawn from venous blood,and 1ml was sent to the laboratory within three hours,plasma sCD163 and sCD14 were detected using ELISA.Blood routine test,viral load and CD4+T cell count were detected in hospital.Each group of 14cases?a total of 28 cases?were collected 5ml blood samples within three hours sent to the laboratory,too.The blood was separated of peripheral blood mononuclear cells and CD14+cells were purified.CD3,CD14,CD19,CD56,CD16,CD34,CD45,CD235a,HLA-DR and CD163 were deceted by flow cytometry;20 kinds of factors including CD163,CCR2,CCR5,CX3CR1,MCP-1,MIP-1?,HLA-DR,CD86,iNOS,HMOX-1,IL-6,IL-10,IL-1?,TNF-?,ABCA1,ABCG1 and PPAR?/?1/?2/?were detected by real-time PCR.Experiment 2.Cell experiment.The human monocyte THP-1 was cultured in the RPMI1640 medium containing 10%FBS and 1%double resistance,and was placed in the incubator of 37 and 5%CO₂.According to the results of pre experiment,the THP-1 cells were stimulated with LPS for 18 hours,then GolgiPlug was added for 6 hours.ATZ?6uM or 30uM?was added.After 24 hours,the cells were collected,and ADAM17 was detected by Western Blot,immunohistochemistry staining and by flow cytometry.Experiment 3.Clinical trials.In the Eighth People's Hospital of Guangzhou,20 patients of HIV/AIDS enter into the experimental study.20 patients were divided into two groups,HAART group?TDF+3TC+EFV/NVP/LPV/r?,not taking Aikeqing;Aikeqing group?HAART+Aikeqing?.Collect the general data of patients and draw from venous blood sending to the laboratory in three hours.plasma sCD163,LPS was detected by ELISA;blood routine examination of blood more than viral load and CD4+T cell count,liver function,renal function,blood lipid and blood glucose were detected in hospital.According to the exclusion criteria and rejection criteria set by the experiment,16 patients were included in the study at the end of the experiment.Experiment 4.Cell experiment.The human monocyte THP-1 was cultured in the RPMI1640 medium which containing 10%FBS and 1%double resistance,and was placed in the incubator of 37 and 5%CO2.According to the results of preliminary experiments,LPS?1ug/ml?and aikeqing?2ug/ml and 10ug/ml?were put in cells.After 0.5 hours and 2 hours,the cytokines including CD163,ADAM17,IL-1?,IL-6 and TNF-?were determined by real-time PCR.After 12 hours,the remaining cells were collected supernatant and cell pellet,the expression of sCD163 was determined by ELISA and the expression of ADAM17 was determined by WesternBlot.Result:Experiment 1.Clinical trials.1.the incidence of anemia in ZDV group and TDF group was not significant?P>0.05?.But a series of hematological parameters related to red blood cells were significantly different.Compared with the TDF group,the erythrocyte count in the ZDV group was significantly lower?P<0.01?,and MCV was significantly increased?P<0.01?.2.PBMC was extracted from the blood of 28 patients,and flow cytometry proved that there was a red cell formation disorder in group ZDV.Compared with the TDF group,the CD34+subgroup in the cycle decreased significantly in the ZDV group?P<0.05?,which confirmed the side effects of ZDV myelosuppression.At the same time,the proportion of erythroid progenitor cells found in group ZDV was higher than that in group TDF?P<0.05?,which suggests that the development of erythroid progenitor cells is impaired.3.There was no significant difference in plasma sCD14 level between group ZDV and group TDF,but the level of sCD163 in group ZDV was significantly lower than that in group TDF?P<0.001?.The level of sCD163 was positively correlated with the red blood cell count,and was negatively correlated with MCV,but had no correlation with the concentration of hemoglobin.4.EFV/NVP had no significant influence on red blood cell related indexes.The effect of EFV/NVP on the level of sCD163 was significantly weaker than that of AZT/TDF?P=0.076?,so the synergistic effect of NVP/EFV and AZT/TDF on sCD163 level could not be concluded.5.The proportion of CD14+CD16+in patients receiving ZDV treatment is significantly higher than that in patients receiving TDF treatment.There is no significant difference in the proportion of CD14++CD16-and non classical CD14lowCD16+.Compared with TDF group,ZDV group of cell surface CD163 expression was significantly increased,especially on the surface of CD14++CD16.The expression of monocyte?CD163+ratio:P<0.01;average fluorescence intensity:CD163 P<0.05?.There was no difference in the CD163 expression on the surface of CD14++CD16+and CD14lowCD16+mononuclear cells.In addition,the sCD163 level was negatively correlated with the proportion of CD163 expressed in CD14++CD16-mononuclear cells.6.A total of 20 factors were detected by real time quantitative PCR.In the purified CD14+mononuclear cells of group ZDV and group TDF,there were five cytokines expression have differences,including:iNOS,IL-6,,CX3CR1,MIP-1?and PPAR?-1.Experiment 2.Cell experiment.1.Western blot:two hours after LPS stimulation,the ADAM17 in all groups appeared glycosylation?120 kDa?.But after the action of AZT?30M?for 24 hours,the glycosylation of ADAM17 was significantly reduced.Under the synergistic effect of GolgiPlug,the ADAM17strip of glycosylation was significantly reduced and replaced by a non glycosylated strip.2.Immunocytochemical staining:compared with untreated cells,the expression of ADAM17 on the surface of THP-1 cells treated by AZT?30M?was reduced.3.Flow cytometry:compared with untreated cells,the expression of ADAM17 on the surface of THP-1 cells treated by AZT?30M?was reduced.AZT could enhance the inhibitory effect of GolgiPlug on ADAM17 glycosylation,but detection showed that the proportion of ADAM17+on the surface of THP-1 cells did not decrease.Experiment 3.Clinical trials.1.HAART group and Aikeqing group in CD4+T and CD8+T lymphocyte count,liver function,renal function,blood glucose and blood lipid data were no statistically significant difference?P>0.05?.2,ELISA:the cytokines LPS and sCD163 were statistically significant ?P<0.05?between the HAART group and the Aikeqing group.Experiment 4.Cell experiment.1.Real-time PCR:?1?CD163:all groups have statistically significant differences with the blank group?P<0.05?;Blank+LPS?0.5hours?group and Blank+LPS?2hours?group has statistically significant?P<0.05?;Bland+LPS?0.5 hours?group and LPS+Aikeqing?10ug/ml,0.5 hours?group has statistically significant?P<0.05?;LPS+Aikeqing?2ug/ml?group and LPS+Aikeqing Aikeqing?10ug/ml?group,0.5 hours and 2 hours were statistically significant?P<0.05?.?2?ADAM17:all groups have statistically significant differences with the blank group?P<0.05?;Black+LPS?0.5 hours?and LPS+Aikeqing?10ug/ml,0.5 hours?group has not statistically significant?P>0.05?;others were statistically significant differences?P<0.05?.?3?IL-1?:all groups have statistically significant differences with the blank group?P<0.05?;Blank+LPS?0.5hours?and LPS+Aikeqing?10ug/ml,0.5 hours?group has not statistically significant?P>0.05?;Black+LPS?0.5hours?and LPS+Aikeqing?10ug/ml,0.5 hours?group had no statistical significance?P>0.05?,others were statistically significant differences?P<0.05?.?4?IL-6:all groups have statistically significant differences with the blank group?P<0.05?;Blank+LPS?0.5hours?and LPS+Aikeqing?10ug/ml,0.5 hours?group has not statistically significant?P>0.05?;others were statistically significant differences?P<0.05?.?5?TNF-?:all groups have statistically significant differences with the blank group?P<0.05?;Bland+LPS?0.5hours?and Bland+LPS+Aikeqing?10ug/ml,0.5 hours?group has not statistically significant?P>0.05?;others have statistically significant differences?P<0.05?.2.ELISA?sCD163?:All groups have statistically significant differences eath other?P<0.05?.3,WesternBlot?ADAM17?:compared with the blank group and LPS+blank group,2ug/ml Aikeqing after 12 hours,the glycosylation level decreased significantly,Aikeqing?10ug/ml?can also inhibit glycosylation of ADAM17,but the effect is not as good as Aikeqing?2ug/ml?.Conclusion:1.Clinical experiment.ZDV and TDF affect the level of sCD163 by regulating the fall of CD163.Compared with the TDF group,the CD14 cells in peripheral blood of the ZDV group had lower levels of CD163 and higher level of proliferation and activation.2.Cell experiment.ZDV can reduce the expression of ADAM17 on the surface of THP-1 cells stimulated by LPS,and inhibit the glycosylation of ADAM17.3.Clinical experiment.The combination of AiKeqing and HAART can reduce the expression of LPS and inhibit the exfoliate of CD163.4.Cell experiment.Aikeqing can decreased the expression of IL-1?,IL-6and TNF-?in THP-1 cells,inhibition of ADAM17 and reduce the shedding of CD163.