Berberine Inhibits Advanced Glycation End Products-Induced Human Aortic Vascular Smooth Muscle Cell Calcification Through Suppressing MAPK Signaling

Objective: To explore the impact and mechanism of Berberine(BBR)on calcification of AGEs-induced human aortic vascular smooth muscle cells in vitro through the signaling pathway.This study aims to investigate whether berberine inhibits calcification of HASMCs that are induced by advanced glycation end products(AGEs)through activating the extracellular signal-regulated kinase1/2(ERK1/2)and phosphorylated 38(P38)and c-Jun N-terminal kinase(JNK)mitogen-activated protein kinase(MAPK).And to search whether berberine has the role of down-regulation of ERK1/2,P38 and JNK MAPK signaling pathway,to provide a new theoretical basis for finding new targets for clinical therapeutic drugs in the future.Methods:(1)4-10 generations of human aortic vascular smooth muscle cells were cultured,and cell morphology was observed dynamically and immunofluorescence method was used for cell identification.(2)The effect of berberine on the viability of HASMCs was determined by CCK-8 method.To determine the appropriate concentration of berberine(10,25,50?M/L),which is less than IC50(cell half inhibition concentration).The calcification model of human aortic vascular smooth muscle cells was established via using AGEs,which means,AGEs-BSA(100mg/L)intervention for 7 days.In addition,the total protein of the cells was extracted after 7 days of simultaneous intervention by berberine(10,25,50 ?M/L)respectively and the expressions of osteoprotegerin(OPG),bone morphogentic protein 2(BMP2),osteopontin(OPN)protein were detected by Western Blot.The degree of cell calcification was observed with Von Kossa staining and Alizarin red staining.And AGEs-BSA(100mg/L)were used to intervene for 14 days.35/5000 The calcium content was determined by intracellular calcium content and ELISA was measured to determine the ALP concentration in HASMCs.Immunofluorescence was used to observe the differences in the expression of ?-SMA,F-actin and Runx2 in each group.The experimental groups were as follows: control group,?-GP,DMSO,AGEs,AGEs+(10,25,50?M/L BBR).(3)At first,after 15 mins of treatment of HASMCs by ERK1/2 MAPK specific inhibitor PD98059,P38 MAPK specific inhibitor SB203580,JNK MAPK specific inhibitor SP600125 and processed the total protein of cells.The expressions of phosphorylation-extracellular signal-regulated kinase1/2(P-ERK1/2),extracellular signal-regulated kinase1/2(ERK1/2),phosphorylation –P38 protein(P-P38),and P38 protein were detected by Western Blot Method.To verify the effect of ERK1/2,P38,JNK MAPK signaling pathway on HASMCs calcification.And the effect of different concentration of berberine on the expression of the phosphorylated MAPK signaling pathway induced by AGEs.The experimental group was divided into six groups: control group,100mg/L AGEs,100mg/L AGEs+(10,25,50,100?M/LBBR).The second part is to verify the effect of inhibiting MAPK signaling pathway on HASMCs calcification induced by AGEs.After pretreatment of HASMCs 6 hours with inhibitors(PD98059,SB203580,SP600125),the total protein was extracted after 7 days of intervention with 100mg/L AGEs.The expressions of OPG,BMP2 and OPN were detected by Western Blot.The degree of cell calcification was observed with Von Kossa staining and alizarin red staining.The calcium content was determined by intracellular calcium content and ELISA was determined to determine the ALP concentration in HASMCs.The content of Runx2 in each group was observed by immunofluorescence.The experimental groups are as follows:control group,DMSO,PD98059,SB203580,SP600125,AGEs,AGEs+(50?M/L)BBR? AGEs+PD98059,AGEs+SB203580,AGEs+SP600125,AGEs+PD98059+(50?M/L)BBR,AGEs+SB203580+(50?M/L)BBR,AGEs+SP600125+(50?M/L)BBR?(4)Explore the role of downstream factor ATF4 in AGEs induced HASMCs calcification and the relationship between MAPK and ATF4 factors.After adding ATF4 siRNA to HASMCs for 24 h and then adding 100mg/L AGEs for 7 days,Western Blot was used to detect the expression of OPG,BMP2,OPN,P-ERK1/2,P-P38,P-JNK,ERK1/2,P38 and JNK in each group.Results: 1.In the 2d observation of HASMCs in special culture medium,HASMCs adhered to the growth of the culture plate,and showed the spindle shape,with the typical pattern of "wave of crest and trough".By means of laser confocal microscopy,the cells were stained by staining,and the green fluorescence staining was the HASMCs specific phenotype named ?-smooth muscle actin(?-SMA).2.The effect of berberine on HASMCs 192h(8 days),IC50=129.32?M/L,thus the concentration gradient of berberine are 10,25,50,100?M/L,and the continuous action time is 7 days.3.Berberine can inhibit the calcification of vascular smooth muscle cells induced by AGEs,and it has a dependence of concentration.The content of calcium and ALP in AGEs group was significantly higher than that in the control group(P<0.01).Compared to the AGEs group,in different concentrations of groups of berberine,the calcium content and ALP content of the human aortic vascular smooth muscle cells decreased(P<0.01).The expression of OPG,BMP2 and OPN in AGEs group was significantly higher than that in the control group(P < 0.01).Compared to the AGEs group,in different concentrations of groups of berberine,the expression of OPG,BMP2 and OPN of the human aortic vascular smooth muscle cells decreased(P<0.01).The degree of calcification in the AGEs group was increased compared with that of the control group,however,the degree of calcification of human aortic vascular smooth muscle cells decreased with the concentration of berberine.The fluorescence intensity of F-actin in AGEs group was enhanced,and the intensity of ?-SMA fluorescence decreased.The fluorescence increased intensity of F-actin and decreased increased intensity of ?-SMA can be alleviated by different concentrations of berberine.4.AGEs can activate ERK1/2,P38,JNK MAPK signaling pathway,and berberine can inhibit this effect.ERK1/2 and P38 MAPK were involved in calcification process of HASMCs induced by AGEs,and JNK MAPK had no significant effect on it.ERK1/2 and P38 MAPK inhibitor group(PD98059,SB203580)had significantly lower content of calcium and ALP than the AGEs group(P < 0.01).There was no significant difference in content of calcium and ALP in the JNK MAPK inhibitor group(SP600125)(P>0.05).The expression of OPG,BMP2,OPN,P-ERK1/2,P-P38 and P-JNK in AGEs group were significantly higher than that in the control group(P < 0.01).The expression quantity of OPG,BMP2,OPN,P-ERK1/2,P-P38 and P-JNK protein in the aorta vascular smooth muscle cells is downregulation in different concentrations of berberine of dependence of concentrations(P < 0.01).The expression of OPG,BMP2,OPN protein of ERK1/2,P38 MAPK inhibitor group(PD98059,SB203580)was significantly reduced in the AGEs group(P < 0.01).The expression of OPG,BMP2 and OPN in the JNK MAPK inhibitor group(SP600125)was significantly less than that in the AGEs group(P>0.05).ERK1/2,P38 MAPK inhibitor group(PD98059,SB203580)was significantly less calcified than the AGEs group and JNK MAPK inhibitor group(SP600125)showed no significant difference from the AGEs group.5.ATF4 factor is involved in the HASMCs calcification induced by AGEs,which may be the common downstream factor of ERK1/2 and P38 MAPK.The expression levels of OPG,BMP2 and OPN in AGEs+siATF4 group were significantly decreased than AGEs group(P < 0.01).ERK1/2 and P38 MAPK inhibitor group(PD98059,SB203580)showed a significant decrease in ATF4 protein expression than AGEs group(P < 0.01),but JNK MAPK inhibitor group(SP600125)showed no significant difference from the AGEs group(P > 0.05).There was no significant decrease between the group of siATF4 group and control group in the proteins of P-ERK1/2,P-P38 and P-JNK,ERK1/2,P38,JNK(P>0.05).Conclusion:1.Berberine inhibits the calcification of human aortic vascular smooth muscle cells induced by AGEs;2.ERK1/2 and P38 MAPK were involved in the process of calcification of HASMCs induced by AGEs,and JNK MAPK had no significant effect on it;3.Berberine might reduce calcification of human aortic smooth muscle cells induced by AGEs through the downregulation of ERK1/2,P38 MAPK signal pathway,which may due to the suppression of OPG,BMP2 and OPN expression;4.ATF4 was involved in the process of calcification of HASMCs induced by AGEs.ATF4 may be a common downstream factor for ERK1/2,P38 MAPK signaling pathway.It may be associated with the effect of inhibition of calcification of HASMCs induced by AGEs,which is related to the inhibition of expression of ATF4 by berberine.