The Role Of Cx43in PDGF-BB-induced Rat VSMCs Proliferation

Objectives:By using Cx43siRNA to silencing the expression of Cx43and using ERK1/2blocker PD98059, to explore the effect of Cx43on PDGF-BB-induced rat VSMCsproliferation and its signaling pathway mechanisms.Methods:1. Cx43siRNA was transfected into VSMCs by liposome to silence theexpression of VSMCs Cx43protein.2. VSMCs activity was detected by MTT Assay. VSMCs were randomly dividedinto6groups.①PDGF-BB, Cx43siRNA+PDGF-BB, ScrambleRNA+PDGF-BB,④PD98059+PDGF-BB,⑤Cx43siRNA+PD98059+PDGF-BB,⑥Scramble RNA+PD98059+PDGF-BB.3. The cell proliferation index of VSMCs was detected by flow cytometry.4. Expression changes of Cx43, p-Cx43, p-ERK1/2and cyclin E protein weredetected by Western blot.Results:1. Western blot showed that Cx43protein expression of Cx43siRNA group was47%that of control, which indicated silencing effect was qualified（P＜0.01）.2. MTT assay showed the absorbance value of Cx43siRNA+PDGF-BB groupwas significantly decreased compared with PDGF-BB group(P<0.01); the absorbancevalue of Cx43siRNA+PD98059+PDGF-BB group was significantly reducedcompared with Cx43siRNA+PDGF-BB group(P<0.05).3. Flow cytometry assay showed proliferation rate of Cx43siRNA+PDGF-BBgroup was significantly decreased compared with PDGF-BB group(P<0.01); theproliferation rate of Cx43siRNA+PD98059+PDGF-BB group was significantly reduced compared with Cx43siRNA+PDGF-BB group(P<0.05), which indicated thatCx43siRNA can significantly inhibit PDGF-BB-induced VSMCs proliferation, andPD98059can significantly decrease PDGF-BB-induced VSMCs proliferation too.4. Western blot showed：①Compared with PDGF-BB group, Cx43protein expression of Cx43siRNA+PDGF-BB group was significantly reduced(P<0.05); Cx43proteinexpression of Cx43siRNA+PD98059+PDGF-BB group was significantly reducedcompared with Cx43siRNA+PDGF-BB group(P<0.05）, which suggested Cx43siRNA silencing effect was good, and if ERK1/2pathway was blocked, Cx43proteinexpression would be much more decreased.②p-Cx43protein expression of PD98059+PDGF-BB group was significantlydecreased significantly compared with PDGF-BB group(P<0.05), which showed thatwhen ERK1/2pathway was blocked, the p-Cx43protein expression would besignificantly decreased.p-ERK1/2protein expression of Cx43siRNA+PDGF-BB group wassignificantly reduced compared with PDGF-BB group (P<0.05); p-ERK1/2proteinexpression of Cx43siRNA+PD98059+PDGF-BB group was significantly reducedcompared with Cx43siRNA+PDGF-BB group(P<0.05).The results demonstrated thatlow expression of Cx43would promote obvious down regulation of p-ERK1/2protein level.④cyclin E protein expression of Cx43siRNA+PDGF-BB group wassignificantly reduced compared with PDGF-BB group(P<0.01); cyclin E proteinexpression of Cx43siRNA+PD98059+PDGF-BB group was significantly reducedcompared with Cx43siRNA+PDGF-BB group(P<0.05).The results indicated Cx43protein and cyclin E protein expression was positively correlated and if ERK1/2pathway was blocked, cyclin E protein expression would be much moredown-regulated.Conclusions:1. Cx43siRNA can specifically silence the expression of Cx43protein,significantly inhibited PDGF-BB-induced proliferation of VSMCs and significantlyreduce p-ERK1/2and cyclin E protein expression. 2. Treatment VSMCs with PD98059can significantly increase the inhibitoryeffect of low expression of Cx43on PDGF-BB-induced VSMCs proliferation, andcan significantly reduce Cx43, p-Cx43, and cyclin E protein expression.3. The above results indicated that Cx43may increase VSMCs proliferationthrough ERK1/2pathway and then promote atherosclerosis pathological process.