| To investigate the mechanism of TGF-?1/Smad in osteoporosis rats induced by aluminum chloride(AlCl3),the experiments about osteoporosis rats induced by AlCl3 in vivo and osteoblast(OB)dyed by AlCl3 in vitro were conducted.In vivo experiment,one hundred SD rats(3 weeks old)were randomly divided into two groups.Experimental rats were given drinking the water containing AlCl3·6H2O,and control rats were given distilled water for up to 120 days.The rat bone mineral density(BMD)was detected using the dual-energy X-ray absorptiometry and the weight of rats was recorded every 30 days.The rats were sacrificed at the end of experiments.The levels of Al,calcium(Ca)and phosphorus(P)in serum and bone,the oxidation and antioxidant status of bone,the activity of B-ALP and TRACP-5b,and levels of PINP and NTX-1 in serum,the mRNA expression of B-ALP,TRACP-5b,TGF-?1,,T?R?,TOR?,Smad4 and Smad7,and the protein expression of p-Smad2/3 and p-Smad2/3/4 in bone were detected.Meanwhile,the ultrastructure of bone was observed.In vitro experiment,the skull OB from new born SD rats were exposed in final concentration at 0.12mg/mL AlCl3·6H2O(AlCl3 treated group,GA),4.5ng/mL TGF-?1(TGF-?1 treated group,GT),0.12mg/mL AlCl3·6H2O and 4.5ng/mL TGF-?1(AlCl3+TGF-?1 treated group,GA+T),blank control group(GC).The mRNA expressions of TGF-?1,T?R?,T?R?,Smad4,Smad7,B-ALP and Col ? in OB,the protein expressions of TGF-?1 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 femur,tibia and lumbar(4-6)in all rats were increased with the extension of Experimental time.The BMD in osteoporosis rats on days 120 were significantly lower than those in GC(P<0.05).It indicated that the osteoporotic rats model induced by AlCl3 was established successfully.(2)Under the electron microscope,the number of osteoblasts was reduced,the membrane structure of osteoblasts was damaged,and the nucleus of osteoblasts was pyknosed.The mitochondria was swelled,and the cristae was incomplete,the substrate was dissolved,and appeared physalides.The rough endoplasmic reticulum was decreased,expanded and ruptured,and appeared physalides.It indicated that AlCl3 can damage the osteoblasts ultrastructure structure,and inhibit the formation of bone matrix(3)The activity of GSH-Px and SOD in osteoporosis rats were significantly lower than those in GC(P<0.01),the MDA level was significantly 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 A1 levels were significantly higher(P<0.01),however the levels of Ca and P in the serm and bone of osteoporosis rats were significantly 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 serm,and the B-ALP mRNA expression in bone of osteoporosis rats were significantly lower than those in GC(P<0.01).The TRACP-5b activity in serm and the TRACP-5b mRNA expression in bone of Osteoporosis rats were significantly higher than those in GC(P<0.05).The NTX-? level in serm of osteoporosis rats was significantly higher than that in GC(P<0.01).It indicated that AlCl3 can inhibit the bone formation,strengthen the bone resorption,and then induce the bone transform imbalance.(6)The mRNA expressions of TGF-?1,T?R?,T?R? and Smad4 were significantly lower(P<0.05,P<0.01),and the protein expressions of TGF-?1,p-Smad2/3 and p-Smad2/3/4 were significantly lower(P<0.05),however,the mRNA expression of Smad7 was significantly higher in Osteoporosis rats than that in GC(P<0.05).It indicated that AlCl3 can inhibit the TGF-?1/Smadsignaling pathway in bone.These findings suggested that AlCl3 causes bone damage,at the same time,TGF-?1/Smad signaling pathway is inhibited.The results of the vitro experiment showed as follows:The mRNA expressions of TGF-?1,T?R?,T?R?,Smad4,ALP and Col ?,and the protein expressions of TGF-?1,p-Smad2/3 and p-Smad2/3/4 in GA were significantly lower(P<0.01),however the mRNA expression of Smad7 in GA was significantly higher than that in GC(P<0.01).The mRNA expressions of TGF-?1,T?R?,T?R?,Smad4,ALP and Col ?,and the protein expressions of TGF-?1,p-Smad2/3 and p-Smad2/3/4 in GA were significantly higher(P<0.01),however the mRNA expression of Smad7 in GA was significantly lower than that in GA(P<0.01).It indicated that AlCl3 can inhibit the TGF-?1/Smad signaling pathway and the activity and function of OB,however exogenous TGF-?1 can reverse the inhibitory effects of AlCl3.These findings suggested that AlCl3 inhibits OB osteogenesis through inactivation of TGF-?1/Smad signaling pathway.The above experimental results in vivo and in vitro showed that AlCl3 can induce Osteoporosis through inactivation of TGF-?1/Smad signaling pathway. |
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