AlCl₃ Inhibits The TGF-β₁/Smad Pathway Of Osteoblasts In Rats

The osteoblasts(OB) from 3 days old SD rats were cultured in vitro to investigate the mechanism of TGF-β1/Smad in OB treated with Aluminum(Al). The OB were isolated from SD rats’ skull and cultured in DEME medium. The third generation of OB were exposed to Al Cl3·6H2O in final concentration of 0(control group, CG), 0.06 mg/m L(low-dose group, LG), 0.12 mg/mL(middle-dose group, MG), and 0.24 mg/mL(high-dose group, HG), respectively. The m RNA expressions of ALP、Col I、TGF-β1、TβR I、TβR II、Smad4 and Smad7, the protein expressions of TGF-β1 、 p-Smad2/3 、 p-JNK 、 p-ERK and p-p38, the p-Smad2/3/4 protein expressions in OB cytoplasm and nucleus were detected by real-time fluorescence quantitative PCR(q RT-PCR), Western blot(WB), co-immunoprecipitation(Co-IP), respectively. The results showed as follows:(1) The OB ALP and Col I m RNA expressions in all Al-treated groups were significantly lower than those in CG(p<0.05, p<0.01). It indicated that Al Cl3 can inhibit the activity and function of OB.(2) The mRNA expressions of TGF-β1、TβR I、TβR II and Smad4, the protein expressions of TGF-β1 and p-Smad2/3 were significantly lower, while the Smad7 mRNA expression were significantly higher in all Al-treated groups than those in CG(p<0.05, p<0.01). It indicated that Al Cl3 can disorder the TGF-β1/Smad pathway in OB.(3) The OB p-ERK 、 p-JNK and p-p38 protein expressions, the p-Smad2/3/4 protein expressions of OB cytoplasm and nucleus in all Al-treated groups were significantly lower than those in CG(p<0.05, p<0.01. In Al-treated groups, the p-Smad2/3/4 protein expression in OB nucleus were significantly lower than that in cytoplasm(p<0.05, p<0.01), but there is no significantly deference in CG(P>0.05). It indicated that Al can inhibit the phosphorylation of JNK、ERK and p38 in OB, and then inhibit the formation and transfer into nucleus of p-Smad2/3/4, strengthen the inhibition of TGF-β1/Smad pathway induced by Al Cl3.The above results showed that Al Cl3 can inhibit the TGF-β1/Smad pathway in OB, at the same time, inhibit the phosphorylation of JNK、ERK and p38, then inhibit the p-Smad2/3/4 transfer into nucleus and decrease the ALP and Col I mRNA expressions, causing the cytotoxicity in OB finally.