The TGF-β₁/Smad Signal Transmission Mechanism For Osteoporosis In Rats Induced By AlCl₃

To investigate the mechanism of TGF-β₁/Smad in osteoporos is rats induced by aluminum chlor ide（ Al Cl₃）, the exper iments about osteopor os is rats induced by AlCl₃ in vivo and osteoblast（OB） dyed by AlCl₃ in vitro were conducted.In vivo exper iment, one hundr ed SD rats（3 weeks old） were randomly divided into two groups. Exper imental rats were given dr inking the water containing Al Cl₃·6H2O, and contr ol rats were given distilled water for up to 120 days. The rat bone miner al dens ity（ BMD）was detected using the dual- energy X-r ay absor ptiometr y and the weight of rats was recorded ever y 30 days. The rats were sacrificed at the end of experiments.The levels of Al, calc ium（Ca） and phos phorus（ P） in s erum and bone, the oxidat ion and antioxidant status of bone, the activity of B- ALP and TRACP-5b, and levels of PI NP and NT X-I in serum, the m RNA express ion of B- ALP, TRACP- 5b, TGF-β₁, TβRI, TβRII, Smad4 and Smad7, and the protein express ion of p-Smad2/3 and p-Smad2/3/4 in bone were detected. Meanwhile, the ultrastructure of bone w as observed.In vitr o exper iment, the s kull OB from new born SD r ats were expos ed in f inal concentr ation at 0.12mg/m L Al Cl₃·6H2O（ AlCl₃ treated group, GA）, 4.5ng/m L T GF-β₁（TGF- β₁ tr eated gr oup, GT）, 0.12mg/m L AlCl₃·6H2O and 4.5ng/m L TGF- β₁（ AlCl₃+TGF- β₁ tr eated group, GA+T）, blank control group（GC）. The m RNA express ions of TGF-β₁, TβRI, TβRII, Smad4, Smad7, B-ALP and Col I in OB, the protein express ions of TGF- β₁ and p-Smad2/3 in OB, and the p-Smad2/3/4 protein expressions in OB cytoplasm and nucleus were detected.The results of the vivo experiment showed as follows:（1） The BMD of f emur, tibia and lumbar（ 4-6） in all rats wer e increased with the extens ion of Exper imental time. The BMD in osteoporos is r ats on days 120 were s ignif ic antly lower than those in GC（P<0. 05）. It indicated that the osteoporotic rats model induced by Al Cl₃ was established successfully.（2） Under the electron microscope, the number of osteoblasts was reduced, the membr ane structure of osteoblasts was damaged, and the nuc leus of osteoblasts was pyknosed. T he mitochondr ia was swelled, and the cristae was incomplete, the substr ate was dissolved, and appear ed physalides. The rough endoplasmic reticulum was decreased, expanded and r uptur ed, and appear ed physalides. It indicated that Al Cl₃ can damage the osteoblasts ultr astructur e structure, and inhibit the formation of bone matrix（3） The activity of GS H-P x and SOD in osteoporos is rats were s ignif ic antly lower than those in GC（P<0.01）, the MDA level was s ignif icantly higher than that in GC（P<0.01）. It indicated that AlCl₃ can induce the bone oxidative stress, and then cause the bone oxidative damage.（4） The Al levels wer e s ignif icantly higher（P<0.01）, however the levels of Ca and P in the serm and bone of osteopor os is rats were signif icantly lower than those in GC（P<0.01,P<0.05）. It indicated that AlCl₃ can accumulate in the bone and induce Ca and P metabolic disorders.（5） The B- ALP activity and PINP level in ser m, and the B- ALP mRNA express ion in bone of osteoporos is rats were s ignif icantly lower than those in GC（P<0.01）. The TRACP- 5b activity in serm and the TRACP-5b mRNA expr ession in bone of Osteoporos is rats were signif icantly higher than those in GC（P<0.05）.The NT X-I level in s erm of osteoporos is rats was signif ic antly higher than that in GC（P<0.01）. It indic ated that AlCl₃ can inhibit the bone formation, strengthen the bone resorption, and then induce the bone transform imbalance.（6） The mRNA express ions of TGF-β₁, TβRI, TβRII and Smad4 were significantly lower（P<0.05, P<0.01）, and the protein expr essions of TGF-β₁, p-Smad2/3 and p- Smad2/3/4 were signif icantly lower（P<0.05）, however, the m RNA express ion of Smad7 was s ignif icantly higher in Osteoporos is r ats than that in GC（P<0. 05）. It indicated that AlCl₃ can inhibit the TGF- β₁/Smad signaling pathway in bone.These f indings suggested that Al Cl₃ c auses bone damage, at the same time, TGF-β₁/Smad signaling pathway is inhibited.The results of the vitro experiment showed as follows:The m RNA expr essions of T GF-β₁, TβRI, TβRII, Smad4, ALP and Col I, and the protein expr essions of TGF-β₁, p-Smad2/3 and p-Smad2/3/4 in GA wer e signif icantly lower（P<0.01）, however the m RNA expr ess ion of Smad7 in GA was s ignif icantly higher than that in GC（P<0.01）. The m RNA expr ess ions of TGF-β₁, TβRI, TβRII, Smad4, ALP and Col I, and the protein expr essions of T GF-β₁, p-S mad2/3 and p-Smad2/3/4 in GA wer e signif ic antly higher（P <0.01）, however the m RNA expr ess ion of Smad7 in G A was s ignif ic antly lower than that in GA（P <0.01）. It indicated that AlCl₃ can inhibit the TGF-β₁/Smad signaling pathway and the activity and function of OB, however exogenous TGF- β₁ can r everse the inhibitory effects of Al Cl₃. These findings suggested th at AlCl₃ inhibits OB osteogenes is through inactivation of TGF-β₁/Smad signaling pathway.The above exper imental results in vivo and in vitr o showed that Al Cl₃ can induce Osteoporosis through inactivation of TGF-β₁/Smad signaling pathway.