| Objective:Glucagon like peptide-1(GLP-1)is a novel drug for the treatment of type 2diabetes mellitus.It can lower blood glucose smoothly,meanwhile also can protect the target organs.For example,protecting the kidney by inhibiting inflammation and oxidative stress in renal tissue,thereby delaying the development of diabetic nephropathy(DN).Some studies have found that GLP-1 can reduce the apoptosis of hepatocytes,cardiomyocytes and pancreatic cells,so we speculated that GLP-1 also can reduce the apoptosis of renal cells in order to protect kidney.In this study,human renal mesangial cells(HRMC)were used as subjects to observe the changes of cell morphology,proliferative activity and apoptosis rate before and after GLP-1 was added to constant or fluctuating high glucose.The mechanism of GLP-1 in reducing apoptosis was discussed.Method:1.Cultivate HMRC in vitro,then do morphological observation through inverted microscope.2.The changes of GRP78 expression were detected by RT-PCR and Western blotting respectively after treating cells by high glucose medium(30mmol/L)persistently for different time(0,3h,6h,12 h,24h,48h).3.The expression of GRP78 was detected by RT-PCR and Western blotting respectively,after HRMC were cultured by high glucose medium and GLP-1jointly for different time(0,3h,6h,12 h,24h,48h),and compared with the GRP78 expression level of simple high glucose cultured.4.HRMC were divided into 6 groups: Normal glucose control group(5.6mmol/L,CON group);Persistent high glucose group(30mmol/L,HG group);Fluctuating high glucose group(30mmol/L for 3h,then 5.6mmol/L for 2h,cycle three times a day,finally overnight in the 5.6mmol/L medium);CON+100nmol/L GLP-1group(CON+G group);HG+100nmol/L GLP-1 group(HG+G group);HNF+100nmol/L GLP-1 group(HNF+G group).Cultured 24 h respectively,then proceed to the following experiment.(1)The proliferation activity of HRMC was detected by MTT assay.(2)The apoptotic rate was measured by flow cytometry.(3)The GRP78 expression level were detected by RT-PCR and Western blotting respectively.Results:1.Under inverted microscope,the number of cells in HNF group was significantly reduced,and the cell morphology was obviously changed too.But the number and morphology obviously changes of HRMC were improved visibly in HNF+G group.2.MTT results: compared with CON group,the cell viability of HG group was significantly increased(P<0.05),but obviously decreased after the addition of GLP-1(P<0.05).The cell viability of HNF group was significantly lower than that of HG group and CON group(P<0.05),but increased evidently after treating with GLP-1 corporately(P<0.05),still lower than the HG+G group.3.The apoptosis rate of HG and HNF group was higher than that of CON group(P<0.05),while treating with GLP-1 jointly,the apoptosis rate was significantly decreased(P<0.05);The apoptosis rate of HNF group was higher than the HG group(P<0.05),regardless of the presence or absence of GLP-1.4.Treating with simple high glucose by different time,the expression level of GRP78 was increased at 3h(P<0.05),reached the peak at 6h(P<0.05),began to decrease in 12 h,but still higher than 0h(P<0.05),after 24 h and 48 h the expression level further declined to the 0h level(P>0.05).There was no significant difference between the two groups(P>0.05).While GLP-1 exists,the GRP78 was obviously increased at 24 h and 48h(P<0.05),and there was no significant change in the other time points(P>0.05).5.Interfere HRMC 24 h by different glucose culture conditions,the expression level of GRP78 in HG group was similar to that in CON group(P>0.05).The GRP78 in HNF group was lower than CON and HG group respectively(P<0.05).The GRP78 of HG+G group was increased compare with HG group(P<0.05).The GRP78 was up-regulated in HNF+G group compared with HNF group(P<0.05).Conclusion:1.A certain degree of high glucose stimulation can promote the proliferation of HRMC,may be involved in the development of DN,and GLP-1 can reduce this effect,the specific mechanism remains to be further studied.2.Sustained high glucose intervening HRMC,early stage can against ERS through upregulation of GRP78 expression,with the duration of stress prolonged,GRP78 expression gradually decreased,it is difficult to fight against ERS and then apoptosis occurred.3.The damage of HRMC in fluctuating blood glucose is more severe compared with sustained high glucose.4.GLP-1 can improve the apoptosis of HRMC induced by high glucose or fluctuating high glucose.The mechanism may be to up-regulate the expression of GRP78 and reduce the damage caused by ERS,thereby protecting renal function. |
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