Effects Of Bradykinin On Expression Of CTGF And FN In Human Mesangial Cells Cultured In High Glucose And The Correlation With JNK Signal Pathway

Objective:Using the eternal life human mesangial cells(HMC) cultured in high glucose in Vitro,we investigated the effects of bradykinin(BK)on connective tissue growth factor(CTGF),fibronectin(FN) and the signaling pathway of JNK.Using the BK-B2 recepter antagonist HOE-140 or the JNK signal pathway blockade SP600125 respectively,we studied the correlation between the signaling pathway of JNK and the expression of CTGF and FN that regulated by BK in mesangial cells cultured in high glucose.The main aim of our experiment was to provide a new method for prevention and treatment in clinical renal fibrosis.Method:1,Hunman mesangial cells cultured in Vitro were pretreated by BK with different concentration(0,10,100,1000ng/ml) respectively for 15 minutes and then were co-cultivated with glucose(30mmol/l) for 48 hours.Cells were treated with glucose(5mmol/l) and were used for control group.The expression of CTGFmRNA and FNmRNA was detected by RT-PCR.2,Hunman mesangial cells cultured in Vitro were pretreated by BK with the same concentration(100ng/ml) for 15 minutes and then were co- cultivated with glucose(30mmol/l) for 24,48,72 hours respectively.The expression of CTGFmRNA and FNmRNA was detected by RT-PCR.3,Humman mesangial cells cultured in Vitro were pretreated by BK with the same concentration(100ng/ml) for 15 minutes,and then were co- cultivated with glucose(30mmol/l) for 30 minutes.Instant expression of JNK phosphorylation protein was detected by Western Blotting.4,Humman mesangial cells were pretreated by BK-B2 antagonistHOE-140(1umol/l) and SP-600125(10umol/l) respectively for 15minutes, then treated with the same concentration bradykinin(100ng/ml) for 15 minutes, and then co-cultivated with high glucose (30mmol/l)for 30 minutes and 48 hours.Instant expression JNK phosphorylation was detected by Western Blotting and the expression of CTGFmRNA and FNmRNA was detected respectively by RT-PCR.Cultured mesangial cells were divided into five groups:the control group,the high glucose(30mmol/l)group,the glucose(30mmol/l) and the BK co-cultivated group,BK-B2 antagonist(HOE-140)pretreated group and JNK specific blockade(SP600125)pretreated group.Results:1.Bradykinin significantly reduced the expression of CTGF mRNA in HMC treated with high glucose and that effect was dose-dependent (p<0.01).BK-B2 recepter blockade(HOE-140) could partly abolish the inhibitory effects of BK on the expression of CTGFmRNA in mesangial cells(HMC) cultured in high glucose(p<0.05).2.Compared with the expression of CTGFmRNA in human mesangial cells treated with glucose(30mmol/l),that in the cells co-cultivated with BK(100ng/ml) and glucose(30mmol/l) significantly decreased after 48h,72h(p<0.01).The expression of CTGF decreased time-dependently in human mesangial cells co-cultivated with BK(100ng/ml) and glucose(30mmol/l) (p<0.01).3.Compared with the expression of FNmRNA in human mesangial cells cultured in glucose(30mmol/l),that did not significantly reduced in the cells co-cultivated with the BK of different concentration and glucose (30mmol/l)for 48h(p>0.05).BK-B2 recpter blockade(HOE-140)could not abolish the inhibitory effects of BK on the expression of FNmRNA in HMC cultured in high glucose for 48h(p>0.05).4.Compared with the expression of FNmRNA in the cells treated with glucose(30mmol/l),that in the cells co-cultivated with glucose(30mmol/l) and BK(100ng/ml) did not significantly reduced after 24h,48h (p>0.05).There was less expression of FNmRNA after 72h than after 48h,24h in human mesangial cells co-cultivated with glucose(30mmol/l) and BK(100ng/ml) (p<0.05).BK-B2 recpter blockade(HOE-140) could partly abolish theinhibitory effects of BK on the expression of FNmRNA in human mesangial cells treated with high glucose(p<0.05).5,Compared with the expression of JNK phosphorylated protein in the cells treated with glucose(5mmol/l) for 30min,that in human mesangial cells cultured in glucose(30mmol/l) significantly increased(p<0.01);There was less expression of JNK phosphorylated protein in human mesangial cells co-cultivated with glucose(30mmol/l) and BK(100ng/ml) than that in cells cultured in glucose(30mmol/l)(p<0.01).BK-B2 recpter blockade could partly abolish the inhibitory effects of BK on the expression of JNK phosphory- lated protein induced by high glucose.JNK signal pathway blockade(SP600- 125) could reduce the expression of JNK phosphorrylated protein in human mesangial cells co-cultivated with glucose(30mmol/l) and BK (100ng/ml), which was the lest in all the tested groups(p<0.01).Conclusion:1,Bradykinin reduced the expression of CTGFmRNA in HMC treated with high glucose that was dose-dependent and time-dependent.BK-B2 recpter blockade could partly abolish the inhibitory effects of BK on the expression of CTGFmRNA induced by high glucose.2,Bradykinin reduced the expression of FNmRNA in HMC treated with high glucose,which is maybe associated with time-dependence.3,Bradykinin reduced the expression of JNK phosphorylated protein in HMC treated with high glucose.4,The inhibitory effect of bradykinin on the expression of CTGF mRNA or FNmRNA in HMC treated with high glucose was maybe associated with the inhibitory expression of JNK phosphorylated protein.