Inhibition Of BRD4 Inhibitor JQ1 On High Glucose-induced Mesangial Cells Hypertrophy

Background: Diabetic nephropathy(DN)is a major complication of diabetes and the most common cause of chronic renal failure and end-stage renal disease.However,the pathogenesis of DN remains unclear.Numerous studies suggest that hyperglycemia,oxidative stress injury and subsequent chronic inflammation during diabetes are associated with mesangial cell hypertrophy and matrix expansion,as well as the process of renal fibrosis and glomerulosclerosis.The NF-?B signaling pathway plays a key role in this by promoting proliferation and inflammation.BRD4,a member of bromine domain and extra terminal domain(BET),regulates gene transcription through epigenetics,especially in the process of tumor and inflammation,which is also an important regulatory factor of c-myc and NF-? B.Specific inhibition of BRD4 has a protection in renal disease animal models.BRD4 may become an important therapeutic target for regulating mesangial cell hypertrophy and renal fibrosis during DN.Objective: In this study,by observing the effects of JQ1 on the proliferation and hypertrophic phenotype of human mesangial cells induced by high glucose,the role of BRD4 in the mechanism of high glucose-induced mesangial cell hypertrophy was explored.Methods and Results: 1.Upregulation of BRD4 in human mesangial cell HMCs induced by high glucose: After treatment of HMCS with high glucose(30mm)for 48 hours,Western blot was used to detect the expression level of BRD4 protein.Compared with the normal sugar group(NG),the expression level of BRD4 protein in the high sugar group(HG)was significantly increased.c-Myc expression level was also significantly increased.It's indicated that BRD4 responds to high glucose stimulation and may participate in the pathological changes of mesangial cells during DN.2.JQ1 inhibits proliferation of high glucose-induced HMCs: MTT was used to measure the cell viability.The cell proliferation ability increased significantly in 24 hours and 48 hours after high glucose(30mm)treatment.However,after different doses of JQ1(125,250,500mm)treatment while high glucose induced,it was found that cell growth was significantly inhibited while JQ1 dose increasing,and JQ1(250mm)significantly inhibited cell proliferation and returned to the level of the control group at 48 hours.3.JQ1 significantly inhibit the function of BRD4 and down-regulate the expression of proliferative genes: Western blot showed that the level of c-Myc in HG group was significantly increased compared with NG group,and the level of c-Myc was significantly decreased after the simultaneous administration of JQ1(250n M),suggesting that JQ1 did significantly inhibit the function of BRD4.Akt protein phosphorylation(p-Akt),a proliferation gene closely related to cell proliferation,was also significantly upregulated in the HG group,but down-regulated after JQ1 treatment,suggesting that the inhibitory effect of JQ1 on HMCs proliferation may be related to down-regulation of c-Myc and p-Akt expression.4.JQ1 inhibits hypertrophy of high glucose-induced HMCs: The total protein concentration normalized by the cell number reflects the mesangial epithelial cell hypertrophy.After quantitative analysis,it was found that the total protein concentration of HMCs was significantly increased after 48 hours of high glucose treatment,and JQ1 can inhibit this hypertrophic phenotype.The expression of ?-SMA increased in the glomerular mesangial cell hypertrophy,which was closely related to interstitial fibrosis.Western blot detection revealed that the ?-SMA level in the HG group was significantly up-regulated,and JQ1 inhibited this up-regulation,which further confirmed that the inhibition of BRD4 has a significant inhibitory effect on the hyperglycemia-induced hypertrophy of HMCs.5.JQ1 inhibits collagen-IV synthesized by high glucose-induced HMCs: Collagen-IV secreted by human mesangial hypertrophy increases in DN,and matrix metalloproteinase MMP2,which clears the matrix,is the main cause of glomerulosclerosis.After 48 hours of high glucose treatment,the expression of Collagen-IV in HMCs cells increased significantly,while the expression of MMP2 decreased.It is indicated that HMCs hypertrophy causes interstitial fibrosis of DN.Meanwhile,JQ1 significantly reverse this change,leading to the down-regulation of Collagen-IV and the recovery of MMP2.It is suggested that inhibition of BRD4 may have a therapeutic effect on glomerulosclerosis induced by high glucose.6.JQ1 inhibits NF-?B in high glucose-induced HMCs.High-glucose treatment for 48 hours increases the level of I?B phosphorylation in HMCs cells and the expression of P65 and P50 subunits in the nucleus(which in the NF-?B classical activation pathway),indicating that high glucose promoted the activation of NF-?B in HMCs cells.This is the main mechanism of mesangial cell proliferation and hypertrophy.Although the level of I?B phosphorylation was not significantly reduced after JQ1 treatment,P65 nuclear translocation was significantly reduced.It is suggested that JQ1 inhibit NF-?B activation,which is involved in inhibition of cell hypertrophy phenotype in high glucose-induced HMCs.Conclusion: In this study,JQ1 can significantly inhibit the function of BRD4 and inhibit the proliferation and hypertrophy of HMCs by reducing the activation of NF-? B and Akt induced by high glucose.It provides new experimental data and clues for the prevention and treatment of glomerulosclerosis in DN.