The Intervening Effects Of Pioglitazone On The CXCL10-induced Impairment And TLR4/NF-κB Gene Expression In βTc6 Cells

Objective:1 To investigate the changes of TLR4/NF-κB expression, morphological change,proliferation,and apoptosis induced by CXCL10 inβcells.2 To evaluate the intervening effects of Pioglitazone on the CXCL10 mediated-changes and TLR4/NF-κB expression inβTc6 cells.Methods:1 Insulinoma cell lineβTc6 cells were cultured in vitro,treating with CXCL10(0.1ng/ml,1.0ng/ml,10.0ng/ml)for 30 minute to 48 hours, then the expression of TLR4/NF-κB,theβTc6 cells'proliferation and cell apoptosis were determined by RT-PCR, Western Blot,CCK-8 viability assay,DNA laddering assay and Flow cytometry respectively. In addition the morphological change ofβTc6 cells was observed.2βTc6 cells were treated with Pioglitazone(0.1umol/L,1.0umol/L,10.0umol/L)for 24h prior to 24 hours CXCL10 intervention,then the expression of TLR4/NF-κB,theβTc6 cells'proliferation and apoptosis were determined by RT-PCR, Western Blot, CCK-8 viability assay, DNA laddering assay and Flow cytometry respectively.In addition the morphological change ofβTc6 cells was observed.Results:1 After treating with CXCL10 0.1ng/ml～1.0ng/ml for 12～48 hours,theβTc6 cells didn't appear to be apoptosis in morphology. But after treating with CXCL10 10.0ng/ml for 24～48 hours,βTc6 cells'proliferation decreased gradually. 2 The expression of TLR4 mRNA and protein didn't change after intervening with 0.1～1.0 ng/ml CXCL10 for 12 hours, but it was increased when CXCL10 was 10.0 ng/ml.After treating with CXCL10 1.0ng/ml for 24 hours,TLR4 mRNA and the protein expression was also increased.When the intervening duration prolonged to 48 hours, the expression of TLR4 mRNA and the protein expression was further increased,espectially with 10.0 ng/ml CXCL10 for 48 hours.CXCL10 mRNA and protein expression was increased inβTc6 cells in a dose- and time-dependent manner after cultivation with CXCL10.3 The activation of NF-κB was increased after intervening with 1.0～10.0 ng/ml CXCL10 for 30 minute.TheβTc6 cells'NF-κB activation continues to 24 hours after intervening with 10.0ng/ml CXCL10. When the intervening duration prolonged to 48 hours,theβTc6 cells treating with CXCL10 0.1～10.0ng/ml didn't appear aforesaid similar changes. NF-κB activation inβTc6 cells had a dose- and time-dependent manner after cultivation with CXCL10.4 After treating with CXCL10 0.1～1.0ng/ml for 24 hours, CXCL10 didn't induce the formation of the DNA ladder and apoptosis.Flow cytometry and DNA ladder analysis show that theβTc6 cells tended to be apoptosis after treating with CXCL10 1.0ng/ml for 48 hours and CXCL10 10.0ng/ml for 24～48 hours.5 The protection effects of Pioglitazone on the cell apoptosis ofβTc6 cells in CXCL10 state as below:(1)CXCL10 inducedβTc6 cells apoptosis and proliferation inhibition would be prevented by treating with 1.0umol/L～10.0umol/L Pioglitazone for 24 hours in advance,but 0.1umol/L Pioglitazone would not preventβTc6 cells from apoptosis. (2)βTc6 cells treating with 1.0umol/L～10.0umol/L Pioglitazone for 24 hours in advance would reduce the expression of TLR4/NF-κB in CXCL10 state,but 0.1umol/L Pioglitazone would not change the CXCL10 induced the cell TLR4/NF-κB signal conduction.Conclusions:1 CXCL10 with finite concentration can change NF-κB activation, but can't affect expression of TLR4,βcells'proliferation and apoptosis in a short time.2 CXCL10 with high concentration can change the expression of TLR4,βcells'proliferation and apoptosis in a long time.3 Treating with Pioglitazone in optimal concentration and duration beforehand, the TLR4 expression and NF-κB activation inβcells is reduced,βcells apoptosis is prevented, and its proliferation is improved obviously.