| ObjectiveObserve the degree of endoplasmic reticulum stress response and the apoptosis level of normal human trabecular meshwork cells(HTMCs)after treatment with tunicamycin(TM),and explore the effect and mechanism of sodium 4-phenylbutyrate(4-PBA)on HTMCs endoplasmic reticulum stress-dependent apoptosis pathway.MethodsHTMCs were cultured in vitro.When the cells grew to the logarithmic growth phase,they were digested and resuspended,and then evenly plated in 6-well plates or96-well plates.When the cells were close to confluence,they were cultivated with serum-free medium for 24 h.Then the experiment is divided into two parts:Part one: TM-induced Expression of Endoplasmic Reticulum Stress-related Genes in HTMCsHTMCs were treated with different concentrations of tunicamycin(0mg/L,1.25mg/L,2.5mg/L,5mg/L,10mg/L,20mg/L)for different durations(0h,6h,12 h,24h,36 h,48h),and cell viability was detected by CCK-8 method.Then,Cells were treated with 10mg/L tunicamycin at different time points(0h,6h,12 h,24h),and the apoptosis rate was detected by flow cytometry,the expression of GRP78,CHOP,Bcl-2and Caspase-3 were measured by RT-PCR and Western blot,respectively.Part two: The Protective Effect of 4-PBA on TM-induced Apoptosis of HTMCs and Its MechanismCells were pretreated with different concentrations of 4-PBA(0mmol/L,0.2mmol/L,0.5 mmol/L,1.0 mmol/L,2.0 mmol/L,5.0 mmol/L,10.0mmol/L)for 2 hours,and then 5mg/L tunicamycin was added for 12 hours.Cell viability was detected by CCK-8.The HTMCs were divided into the following four groups: blank control group(Ctrl group),sodium 4-phenylbutyrate group(4-PBA group),tunicamycin group(TM group),tunicamycin + 4-phenylbutyric acid Sodium group(TM + 4-PBA group).Ctrl group was cultured with medium for 2h,and then cultured with new medium for 12h;4-PBA group was pretreated with 4-PBA for 2h,and then cultured with new medium for 12h;TM group was cultured with medium for 2h Then,intervention with tunicamycin for 12h;TM + 4-PBA group was pretreated with 4-PBA for 2h,and then intervention with tunicamycin for 12 h.Flow cytometry was used to detect the apoptosis rate.RT-PCR and Western blot were used to detect the expression of GRP78,CHOP,Bcl-2 and Caspase-3.Results Part one: TM-induced Expression of Endoplasmic Reticulum Stress-related Genes in HTMCsCCK-8 results showed that the inhibition rate of HTMCs increased with the increase of tunicamycin concentration and duration of intervention.When the concentration of tunicamycin was 1.25 mg / L and 2.5mg / L,there was no significant difference in cell inhibition rate compared with the control group(P> 0.05).When the concentration of tunicamycin was 5mg / L and the duration of action was 12 h or more,the cells showed significant inhibition,of which the cell inhibition rates at 12 h,24h,36 h and 48 h were(21.11 ± 1.23)% and(31.34 ± 0.86,respectively))%,(44.37 ± 1.01)%,(51.47 ± 0.36)%,compared with the 0h group,the differences were statistically significant(P <0.05).The results of flow cytometry suggest that with the increase of the time of action of tunicamycin,the early apoptosis rate and the late apoptosis rate of the cells increase significantly.Compared with the control group,the experimental group with different intervention durations had significantly higher early cell apoptosis rate and late cell apoptosis rate,and the difference was statistically significant(P <0.05).RT-PCR test results indicated that the expression levels of GRP-78,CHOP,and Caspase-3 m RNA in the experimental groups(6h,12 h,and 24h)with different intervention durations were all increased compared with the control group,and the difference was statistically significant(P <0.05);BCL-2 m RNA expression in the experimental groups(6h,12 h,24h)with different intervention durations was reduced compared with the control group,and the difference was statistically significant(P<0.05).Western blot test results showed that compared with the control group,GRP-78 and CHOP protein expressions of the experimental groups(6h,12 h,24h)with different intervention durations increased,the difference was statistically significant(P <0.05).Compared with the control group,the expression level of Caspase-3 protein increased in the 12 h group and the difference was statistically significant(P <0.05).The increase in the expression level of the remaining experimental group was not obvious,and the difference was not statistically significant(P> 0.05);24h Compared with the control group,the expression level of BCL-2 protein was significantly reduced,the difference was statistically significant(P <0.05),the expression level of BCL-2 protein in the remaining experimental group was not significantly reduced,and the difference was not statistically significant(P> 0.05).Part two: The Protective Effect of 4-PBA on TM-induced Apoptosis of HTMCs and Its MechanismThe results of CCK-8 showed that when the concentration of 4-PBA was 0.2mmol/L,the cells had the highest survival rate.Compared with the control group(the concentration of 4-PBA was 0 mmol/L),the difference was significantly increased,and the difference was statistically significant(P <0.05).Flow cytometry results showed that the TM + 4-PBA group compared with the TM group,the early apoptosis rate and the late apoptosis rate were significantly reduced(P<0.05);the TM group compared with the Ctrl group,early cell apoptosis The rate and late apoptosis rate increased significantly(P <0.05).RT-PCR test results showed that compared with TM group,the expression of CHOP,GRP78,and Caspase3 m RNA in TM + 4-PBA group decreased significantly(P<0.05),and the expression of BCL-2 m RNA increased significantly(P <0.05);Compared with the Ctrl group,the expression of CHOP,GRP78,and Caspase3 m RNA increased significantly(P <0.05),and the expression of BCL-2 m RNA decreased significantly(P <0.05).ConclusionTunicamycin can cause endoplasmic reticulum stress in HTMCs,activate the CHOP pathway which leads to apoptosis.4-PBA can inhibit the activation of CHOP and reduce the endoplasmic reticulum stress response,thereby reducing the apoptosis of HTMCs induced by tunicamycin and playing a protective role. |
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