The Effects Of Baicalin On The Apoptosis Of Human Embryo Lung Fibroblasts Infected With Human Cytomegalovirus

Objectives：1The cytotoxic of baicalin and the anti-HCMV median efficacious concentration ofbaicalin were detected.2The effect of baicalin on the changes of apoptosis was investigated comprehensivly.Methods：1The vitro model of HCMV infection was created:The vitro model was infected with the standard laboratory strain AD169; the multiplicity ofinfection was determined accordancing to international standards: high multiplicity ofinfection (MOI=2.5), low multiplicity of infection (MOI=0.25).2The cytotoxic of baicalin and the anti-HCMV median efficacious concentrations ofbaicalin were measured:CCK-8kit was used to determinate the maximum tolerated dose (MTC) of HEL cell tobaicalin while the anti-HCMV median efficacious concentration (EC50) of baicalin wasdeterminted by standard plaque reduction method.3Apoptosis of cells infected by HCMV were ivtestigated at different time:1) The rates of apoptosis cell were detected by Flow cytometry method: The rate ofapoptosis cell which treated with low or high MOI(0.25and2.5) HCMV were detected byAnnexin-V/PI double staining kit at24h,48h,72h,96h respectively.2) The expression of pro-caspase-3which was the key protein of apoptosis were analyzedby Western-blot: Firstly, the cells were infected with HCMV，then were collected at36h, 48h,60h,72h,84h to analyze the expression of pro-caspase-3protein using western-blotmethod, setting β-actin as internal reference. The correction value of Κwas used tosemin-quantitative detection.4The effect of baicalin on apoptosis of cell infected by cytomegalovirus：we designed thebaicalin experiment dose as high-dose (20μg/ml) and small-dose (10μg/ml) based on themaximum tolerated dose (20.6μg/ml).1) The rates of apoptosis cell were detected by Flow cytometry method：Experimentalgroups were divided into nine groups according to the amount of virus infection and doseof baicalin:①the normal control group;②t he small-dose baicalin treatment group;③thehigh-dose baicalin treatment group;④t he low MOI HCMV infected group;⑤the low MOIHCMV infected group with small-dose baicalin treatment;⑥the low MOI HCMV infectedgroup with high-dose baicalin treatment;⑦t he high MOI HCMV infected group;⑧thehigh MOI HCMV infected group with small-dose baicalin treatment;⑨t he highMOIHCMV infected group with high-dose baicalin treatment. Each group was collected toanalyze the apoptosis ratio by Annexin-V/PI double staining kit at24h,48h,72h,96h,respectively2) Western-blot method: Experimental groups were divided into four groups:①the normalcontrol group;②t he high MOI HCMV infected group;③the high MOI HCMV infectedgroup with small-dose baicalin treatment;④the high MOI HCMV infected group withhigh-dose baicalin treatment. Four groups were collected to determine the expression ofpro-caspase-3protein at72h by western-blot method, setting β-actin as internal reference.The correction value of Κwas used to semin-quantitative detection.Results:1The vitro infection model of HCMV was established successfully;2The maximum tolerated concentration (MTC) of baicalin was20.6μg/ml;3The anti-HCMV median efficacious concentration of baicalin was16.13μg/ml;4The apoptosis rate of HEL which teated with HCMV was investigated at diferent phase: 1) The results of the apoptosis cell rates detected by Flow cytometry: The apoptosis ratio ofnormal control group remained at low level; while the apoptosis ratio increased gradually inthe group with low multiplicity HCMV infection and reached the peak (28.75%) at96h;the ratio of apoptotic cells had been rasing in the group with high multiplicity HCMVinfection at early time, reaching the peak (36.36%) at72h, while the ratio of apoptotic cellswas going to decrese at96h.2) Western-blot results: The expression of pro-caspase-3was increased gradually with theinfection time going on at early time in the group with high multiplicity HCMV infection,but it began to decrease at60h and reached the lowest value at72h.5The effect of baicalin on the apoptosis ratio of infected cell:1) The results of flow cytometry: The ratio of apoptosis had no significantly differecebetween the normal control group and the group treated with both does of baicalin. But theapoptosis ratio of infectd cells with both does baicalin declined at early time, showingsignificant dose-dependent manner, in addition to the ratio of apoptotic cells raised in largedoes group of high multiplicity infection at96h.2) The results of western-blot: The expression of pro-caspase-3were higher in high-dosebaicalin treatment group than the infection control group, showing significantly difference.Conclusions:1) The HEL cell began to apoptotic when treated with HCMV by influenced theexpression levels of pro-caspase-3, showing significantly infection dose-dependent andtime-dependent manner;2) Baicalin had not only a direct anti-HCMV effect, but also could significantlydown-regulated the apoptosis ratio of infected cells. And baicalin antagonisticpro-caspase-3of activation may be one of the mechanisms for inhibition of apoptosis.